Methods of treating initial episode of TTP with immunoglobulin single variable domains

Abstract

To provide methods of treating human patients with a thrombotic thrombocytopenic purpura (TTP) episode, e.g., an initial episode and / or recurrent episode of TTP.SOLUTION: A polypeptide comprises two anti-human von Willebrand factor (vWF) immunoglobulin single variable domains (ISVDs). The polypeptide is for use in treating (the symptoms of) an initial episode of a vWF-related disease in a human, by administering to the human a dose of 1-80 mg, preferably 5-40 mg, further preferably 10 mg or 11 mg of the polypeptide.SELECTED DRAWING: None

Description

[Technical field]

[0001] 1. Field of the invention The present invention is based on the discovery that administration of a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) against von Willebrand factor (vWF) to a human patient having an episode of thrombotic thrombocytopenic purpura (TTP), such as a first episode and / or a recurrent episode of TTP, results in improved outcomes, including a shorter time to platelet count response, a lower proportion of patients who die, relapse or have a severe thromboembolic event (TE) during treatment, a lower relapse rate, and prevention of refractory states. The present invention provides a polypeptide comprising at least one ISVD against vWF for use in treating a vWF-related disease, preferably TTP, in a human in need of treatment. The present invention further relates to a unit dosage form, a kit, and a medical use for treating TTP. [Background technology]

[0002] 2. Background of the invention 2.1 The role of vWF in platelet aggregation The multimeric plasma protein vWF is essential for the recruitment of circulating platelets to the damaged vessel wall upon vascular injury, a recruitment that is mediated by binding of the vWF A1-domain to the platelet receptor glycoprotein GPIb-IX-V.

[0003] When expressed by endothelial cells, vWF is secreted into the circulation as ultra-large or ultra-large vWF (ULvWF). These multimers are processed into smaller normal-sized multimers via enzymatic cleavage by disintegrin-like and metalloprotease with thrombospondin repeats 13 (ADAMTS13). In these normal-sized multimers of vWF, the GPIb-IX-V platelet receptor binding site in the A1 domain is hidden and does not spontaneously react with platelets. Conformational activation of the GPIb-IX-V platelet receptor binding site in the A1 domain is induced by immobilization or under conditions of shear stress, leading to platelet adhesion and subsequent thrombus formation.

[0004] 2.2 Role of vWF and vWF processing in the pathophysiology of TTP TTP is a rare, life-threatening disorder of the blood coagulation system, thought to be associated with the accumulation of ULvWF multimers, which leads to an increased risk of thrombus formation in small vessels due to excessive platelet aggregation. The condition is characterized by systemic platelet aggregation in the microcirculation, which leads to fluctuating ischemia in many organs. If sustained, this can lead to profound thrombocytopenia and tissue infarction associated with red blood cell fragmentation.

[0005] ULvWF multimers have the natural ability to spontaneously interact with the platelet receptor GPIb-IX-V. ADAMTS13 activity was found to be severely deficient in hereditary TTP as well as acquired idiopathic TTP (aTTP). The majority of TTP patients have autoantibodies against ADAMTS13, which leads to impaired processing of ULvWF multimers. As a result, the A1 domain of ULvWF is constitutively active and readily interacts with the GPIb-IX-V platelet receptor. This ultimately leads to the formation of the characteristic blood clots found in the TTP patient population.

[0006] Current therapies for TTP using plasma exchange (abbreviated herein as "PE" or "PEX") and transfusions gradually result in normalization of ULvWF processing by replenishing ADAMTS13 and removing antibodies to the enzyme. However, this treatment requires multiple exchanges and transfusions over many days, during which the active process of ULvWF-mediated platelet aggregation is not directly pharmacologically targeted.

[0007] Although the introduction of PE and transfusions has significantly reduced the mortality rate from TTP over the past 30 years, this condition still carries a significant risk of mortality and morbidity. In patients under the control of current therapies, the mortality rate from acute episodes of acute idiopathic TTP ranges from approximately 10% to 30% (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3). In the case of secondary TTP, the efficacy of PE and transfusions is recognized to be relatively low, and the mortality rate is significantly higher. In cases where the disease is secondary to pregnancy (in which case PE is considered to be moderately effective), the mortality rate from acute episodes of TTP is approximately 25%, rising to over 40% in cases with coexisting preeclampsia (Non-Patent Document 4). However, in cases secondary to, for example, underlying malignancies or bone marrow transplantation, the mortality rate ranges from 40% to 60%, despite the use of such treatment regimens (Non-Patent Document 3; Non-Patent Document 5; Non-Patent Document 6).

[0008] Given the still substantial level of mortality from TTP and the observed complications of PE and transfusion, there is a clear need for the development of additional therapeutic approaches that may complement or reduce the need for current treatment methods.

[0009] Research into TTP conducted over the past 30 years has improved our understanding of the pathophysiology of the disease and has led to the possibility of developing new drugs that target the underlying disease process.

[0010] Immunoglobulin single variable domains (ISVDs) against / binding to vWF are described, for example, in US Pat. No. 5,399,413, US Pat. No. 5,499,621, US Pat. No. 5,523,636, US Pat. No. 5,611,625, US Pat. No. 5,711,433, US Pat. No. 5,711,625, and US

[0011] US Patent No. 5,999,999 in the name of Ablynx describes, inter alia, the unexpected discovery that administration of a polypeptide comprising at least one ISVD that binds to vWF to human TTP patients results in a reduction in the time to response, as objectified by a platelet recovery of 150,000 / μL or more. This is reflected in a hazard ratio (or platelet count normalization ratio) of 2.2, which means that subjects treated with caplacizumab were 2.2 times more likely to achieve a platelet count response at any time point. This reduces the thrombotic process initiated by the platelet-vWF complex characteristic of the disease. Thus, the proof of concept of the polypeptide of the invention was achieved by a statistically significant and clinically meaningful reduction in the time to confirmed platelet response. Furthermore, the number of exacerbations was reduced from 11 in the placebo arm to 3 in the treatment arm. There were no deaths in the treatment arm compared to 2 deaths in the placebo arm. Indeed, ISVDs against vWF (eg, ALX0081) have been shown to be effective and safe in treating patients with TTP in Phase II (TITAN) and Phase III (HERCULES) trials.

[0012] However, US Pat. No. 5,399,633 and the information on the results of TITAN and HERCULES do not mention patients with a first episode of aTTP.

[0013] Untreated, TTP has a mortality rate of 90%, but this can be reduced by prompt plasma exchange. Nevertheless, early deaths still occur: approximately half of deaths in UK community registries occur within 24 hours of symptom onset, mainly in women. (Non-Patent Document 7). It is therefore crucial to make an accurate diagnosis of an aTTP episode (see Non-Patent Document 8). However, diagnosis can be difficult because of the clinical overlap with hemolytic uremic syndrome (HUS), autoimmune diseases, and a range of pregnancy-related problems (see Non-Patent Document 9; Non-Patent Document 10, Non-Patent Document 11). In fact, the guidelines for diagnosing TTP (and associated microangiopathy) have changed several times in recent years, from a diagnosis based on clinical history, patient examination, and blood smears, using a pentad of thrombocytopenia, MAHA (microangiopathic hemolytic anemia), labile neurological signs, renal dysfunction, and fever (often of insidious onset) (Non-Patent Document 9), to a diagnosis including (i) isolated MAHAT (microangiopathic hemolytic anemia and thrombocytopenia), (ii) new focal neurological symptoms, seizures, or myocardial infarction (MI) with MAHAT of unknown etiology, and (iii) a history of TTP (Non-Patent Document 11). The ADAMTS13 assay helps to confirm the diagnosis, monitor the course of the disease, and even direct further treatment options.

[0014] Due to the low incidence of TTP in the population, accurate diagnosis is complicated, especially when a subject presents with a first episode of TTP, because most physicians are not familiar with the symptoms. In fact, the first episode of TTP is often diagnosed after a recurrent episode. In the latter case, the patient and medical staff are aware of the medical history and recognize the symptoms more easily. As a result, patients with a first episode of TTP have a later onset of symptoms and a higher severity of the disease when visiting the clinic than patients with recurrent disease. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] WO2004 / 015425 [Patent Document 2] WO2004 / 062551 [Patent Document 3] WO2006 / 074947 [Patent Document 4] WO2006 / 122825 [Patent Document 5] WO2009 / 115614 [Patent Document 6] WO2011 / 067160 [Patent Document 7] WO2015 / 193326 [Non-patent literature]

[0016] [Non-Patent Document 1] Vesely et al., Blood 2003;102:60-68 [Non-Patent Document 2] Allford et al., Br. J. Haematol. 2003;120:556-573 [Non-Patent Document 3] Sadler et al., Hematology. Am. Soc. Hematol. Educ. Program. 2004; 407-423 [Non-Patent Document 4] Martin et al., Am. J. Obstet. Gynecol. 2008;199:98-104 [Non-Patent Document 5] Elliott et al., Mayo Clin. Proc. 2003;78:421-430 [Non-Patent Document 6] Kremer Hovinga and Meyer Curr. Opin. Hematol. 2008;15:445-450 [Non-Patent Document 7] Scully et al., 2008, BJH 142:819-826 [Non-Patent Document 8] Bindi et al., 2010, Transf Aph Sci 43:167-170 [Non-Patent Document 9] Scully et al., 2012, BJH 158:323-335 [Non-Patent Document 10] George and Al-Nouri, 2012, Hematology, 604-609 [Non-Patent Document 11] Scully et al., 2017, J Thromb Haem 15:312-322 Summary of the Invention [Problem to be solved by the invention]

[0017] Therefore, it remains to be elucidated whether polypeptides having at least one ISVD against vWF, such as ALX0081 or ALX0081-A, are beneficial in patients with a first episode of TTP, whether polypeptides comprising at least one ISVD against vWF, such as ALX0081 or ALX0081-A, have favorable effects, and what effective treatments and dosage regimens would be.

[0018] There is a need for improved therapies for patients with TTP, especially when patients present with a first episode of TTP. [Means for solving the problem]

[0019] 3. Overview of the Invention The present invention is based on the unexpected discovery that administration of a polypeptide comprising at least one ISVD against vWF to human TTP patients improves the outcome of patients with a first episode of TTP, who have a later onset of symptoms and a more severe disease at baseline than patients with recurrent disease (i.e., patients with recurrent episodes of TTP) (see Example 7.11).

[0020] Furthermore, the present invention also demonstrates that the polypeptides of the present invention (e.g., ALX0081 or ALX0081-A) resulted in a faster time to platelet count normalization, prevented exacerbations, and prevented patients from becoming refractory to treatment, as reflected, inter alia, in reduced plasma exchange parameters, hospital stays, and intensive care unit (ICU) stays (see Example 7.12).

[0021] Furthermore, the present invention demonstrates that open-label (OL) treatment with a polypeptide of the present invention (e.g., ALX0081 or ALX0081-A) was effective in patients experiencing an exacerbation of aTTP (see Example 7.13).

[0022] Thus, the currently available data demonstrates that reduction in PE and transfusion and their associated complications is achieved from the use of the polypeptide of the present invention itself, without any serious adverse events, which is a clear safety advantage of using the polypeptide of the present invention in treating TTP patients.

[0023] Thus, administration of a polypeptide comprising at least one ISVD against vWF to a human TTP patient results in an unexpected reduction in time to response, sustained and prolonged efficacy, reduced exacerbations, reduced hospitalizations including ICU stays, reduced morbidity, reduced mortality, and / or reduced PE in patients having an episode of TTP, e.g., a first episode and / or a recurrent episode of TTP.

[0024] To support the dosing regimen, various scenarios were developed based on extensive mechanism-based pharmacokinetic-pharmacodynamic (PKPD) modeling. The model fully described the drug-vWF complex interactions over time, including effects dependent on disease progression and PE treatment in patients with aTTP. The model demonstrated the effect of caplacizumab on vWF in the target population. It has been successfully applied to facilitate understanding of the PKPD interrelationships between the covariates and the pharmacokinetics of caplacizumab, and the use of simulations supported the dosing rationale in both adult and pediatric patients, allowing bridging to Japanese aTTP patients (see Example 7.18). Population pharmacokinetic analysis in aTTP patients showed that age, sex, race, and blood type did not affect the pharmacokinetics of caplacizumab. Body weight and renal function as expressed by creatinine clearance (CrCL) had a statistically significant effect on pharmacokinetics, with higher expected exposures in patients with lower body weight and CrCL. However, in patient populations with extreme values ​​of these covariates, the expected exposure ranges largely overlapped, and no specific dose adjustments were deemed necessary. Although baseline vWF levels had a statistically significant effect on drug exposure, increased drug exposure for patients with increased vWF did not result in a different pharmacodynamic effect, and no separate dose adjustments were deemed necessary (see Example 7.16.5).

[0025] Because no pediatric patients were enrolled in clinical trials of caplacizumab, this PKPD model was also used to recommend dosing in adolescents and children. Regardless, the recommended dose for adolescents aged 12-18 years weighing 40 kg or more is 10 mg, and for those weighing less than 40 kg, it is 5 mg. Because no differences in vWF:Ag inhibition based on age were expected, the same recommended doses apply to children aged 2-12 years: 10 mg if weighing 40 kg or more, and 5 mg if weighing less than 40 kg (see Example 7.19).

[0026] Current therapy of TTP using PE and transfusions gradually normalizes ULvWF processing by replenishing ADAMTS13 and removing antibodies to this enzyme, however, this treatment requires multiple exchanges and transfusions over many days, during which the active process of ULvWF-mediated platelet aggregation is not directly pharmacologically targeted.

[0027] During clinical trials in aTTP patients, caplacizumab was administered as a 10 mg intravenous (iv) bolus before PE, followed by a 10 mg subcutaneous (sc) dose daily during the daily PE period and for at least 30 days thereafter. The potential effect of different time intervals between the first iv bolus and subsequent PE, as well as the effect of different PE schedules, were also investigated. Surprisingly, effective drug levels are expected up to 5 hours after the first iv dose of caplacizumab is administered until PE begins. For longer delays, an additional 10 mg or 11 mg subcutaneous dose before PE may be envisaged. If twice-daily (bid) PE is administered for 7 days, the subcutaneous dosing schedule of caplacizumab may be adjusted with caplacizumab twice-daily after each PE procedure (see Example 7.17).

[0028] During the managed access program (MAP), patients received caplacizumab as frontline therapy or for the treatment of refractory aTTP. Any safety reports were without new safety signals and were consistent with the safety profile observed in clinical trials. The first real-world evidence with caplacizumab in patients with aTTP confirms the important benefits of caplacizumab observed in clinical trials, especially when initiated as frontline therapy.

[0029] The polypeptide of the present invention does not interfere with the enzymes that are recruited by plasma transfusion. The polypeptide of the present invention (e.g., ALX0081 or ALX0081-A) can be used in combination with PE and transfusion to directly inhibit the formation of continuous small thrombi and platelet consumption in the microvasculature. This has the advantage that the underlying thrombotic process and the associated platelet consumption can be controlled more quickly, reducing the severity of ischemic and hemorrhagic complications. This also leads to a faster clinical recovery and reduced morbidity with shorter duration and fewer PE and transfusions. In fact, it is specific and clinically relevant Analysis of organ damage biomarkers LDH, troponin T or I, and creatinine suggested that there is clinical utility in more rapidly suppressing ischemia of microvascular tissues. Furthermore, the inhibition of ULvWF-mediated platelet interaction and observed antithrombotic effect shown by the polypeptides of the present invention (e.g., ALX0081 or ALX0081-A) indicates a favorable effect of preventing recurrence and / or exacerbation of the disease when the polypeptides of the present invention are used for a longer period after the patient has recovered from an acute episode of TTP, e.g., a first episode and / or a recurrent episode of TTP. Reducing the frequency of acute TTP episodes, e.g., a first episode and / or a recurrent TTP episode, is of great benefit in that it reduces the mortality and morbidity associated with TTP and may further reduce the need for PE and blood transfusions over the patient's lifetime.

[0030] Although faster recovery from TTP and reduced exacerbations and recurrences are obvious clinical benefits in terms of efficacy of the procedure, reduced duration and frequency of PE and transfusions also provide additional benefits in terms of patient safety. Although PE and transfusions are currently considered standard procedures in the management of TTP (Scully et al., Br. J. Haem. 2012;158:323-335), these procedures carry significant risk of complications. PE procedures require high fluid volumes and flow rates and require the use of a central venous double lumen hemodialysis catheter. Complications of this procedure include bleeding from catheter insertion, sepsis, catheter-related 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). Anaphylactoid reactions occur in 0.25% to 0.5% of procedures (Allford et al., 2003, supra; George 2007, supra). Furthermore, transfusion of plasma containing blood products can cause non-infectious transfusion-associated acute lung injury (TRALI). This condition is recognized as one of the most common causes of transfusion-associated deaths, with an estimated incidence of 0.02% to 0.05% per plasma-containing unit. With a daily average of 17 plasma units, the daily risk can be calculated to range from 0.34% to 0.85%. Most TTP patients require multiple PEs and transfusions. Patients with acute idiopathic TTP require daily treatments, with an average of approximately 16 treatments required to achieve remission (Allford et al., 2003, supra). In refractory cases, the frequency of treatments can be increased to twice daily (Allford et al., 2003, supra). For patients with familial TTP, regular prophylactic plasma infusions at 2- to 3-week intervals are recommended (Lammle et al., J. Thromb. Haemost. 2005;3:1663-1675).Anaphylaxis and TRALI are therefore obvious risks for TTP patients whose treatment requires such frequency and regularity of PE and transfusions. It is believed that this risk may be reduced if solvent / detergent (S / D) treated plasma is used instead of fresh frozen plasma, but the use of large volumes of S / D plasma may be associated with an increased risk of venous thromboembolism (Allford et al., 2003, supra; Fontana et al., 2004, supra). Overall, it is estimated that approximately 30% to 40% of patients experience adverse events from PE and transfusions, and the mortality rate from these procedures is approximately 2% to 3% (George et al., Semin. Hematol. 2004; Issue 41: 60-67; George 2007, supra; George and Al-Nouri 2012, supra). Thus, a reduction in the duration and frequency of PE and transfusions also offers additional benefits in terms of patient safety.

[0031] After recovery from a TTP episode, e.g., initial and / or recurrent TTP episodes, many patients report having cognitive abnormalities for many years, affecting memory, concentration, and activity. Patients report bothersome problems with reduced strength and fatigue. These symptoms negatively impact the quality of daily life of patients. Moreover, these reduced quality of life may occur in all patients with TTP, regardless of etiology and severity (Lewis et al., Transfusion 2009;49:118-124). It is believed that these symptoms may reflect the residual effects of tissue ischemia. Based on this, it can be reasonably proposed that the accelerated recovery from TTP and the limited thrombus formation in the microvasculature provided by the polypeptide of the present invention, e.g., ALX0081 or ALX0081-A, will result in improved long-term outcomes for patients in terms of their quality of life.

[0032] Surprisingly, various in vitro experiments, comparative nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) studies showed that caplacizumab and C-terminally extended caplacizumab had similar characteristics and similar binding profiles to the target vWF, although C-terminally extended caplacizumab had relatively low pre-existing antibody binding. Considering the similar characteristics and profiles between caplacizumab and C-terminally extended caplacizumab, it is expected that such C-terminally extended caplacizumab compounds can also be used to treat and / or prevent aTTP, similar to caplacizumab (see Example 7.21).

[0033] Thus, the present invention provides a method for treating or alleviating a vWF-related disease, such as TTP, in a subject by administering to the subject a polypeptide comprising at least one ISVD against vWF, wherein the amount of the administered polypeptide is effective for reducing the time to response, reducing exacerbations, reducing relapses, reducing hospitalization, including ICU stay, reducing ischemia, reducing the number of deaths, and / or reducing the number of PEs required. The present invention provides specific dose ranges and dosing schedules of the polypeptides of the present invention that provide one or more of these beneficial effects on vWF-related diseases, such as TTP. In particular, the present invention provides pharmacologically active agents, compositions, methods, and / or dosing schedules that have certain advantages, including a relatively low frequency of need to perform PE, compared to agents, compositions, methods, and / or dosing schedules currently in use and / or known in the art. These advantages will become clear from the further description below.

[0034] In a preferred embodiment, the present invention relates to a polypeptide comprising two anti-human vWF immunoglobulin single variable domains (ISVDs), for use in treating a vWF-related disease in a human, preferably a symptom of TTP, by administering to said human a dose of 10 mg or 11 mg of said polypeptide if said human has a body weight of 40 kg or more, or 5 mg if said human has a body weight of less than 40 kg.

[0035] In one aspect, the invention relates to a polypeptide comprising two anti-human von Willebrand factor (vWF) immunoglobulin single variable domains (ISVDs), for use in treating a first episode (symptoms of) a vWF-related disease in a human, preferably TTP, by administering to the human a dose of 1-80 mg, preferably 5-40 mg, even more preferably 10 mg or 11 mg of said polypeptide.

[0036] In one aspect the invention relates to a polypeptide comprising two anti-human von Willebrand Factor (vWF) immunoglobulin single variable domains (ISVDs), for use in the frontline treatment of a vWF-related disease, preferably TTP, in a human by administering to the human a dose of 1 to 80 mg, preferably 5 to 40 mg, even more preferably 10 mg or 11 mg of said polypeptide.

[0037] In a further aspect, the present invention relates to a method for treating a subject, comprising administering a polypeptide of the present invention to a subject comprising administering to said subject a subject a polypeptide of the present invention; The present invention relates to a polypeptide as described herein for use in treating a first episode (symptoms of) a vWF-related disease in a human, preferably TTP, for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or even longer than 10 days, such as 20 days, preferably longer than 30 days, such as 2 months, 3 months, 4 months, 5 months, 6 months, or even more (treatment period).

[0038] In a further aspect, the invention relates to a polypeptide as described herein for use in treating a first episode (symptoms of) a vWF-related disease in a human, preferably TTP, wherein said treatment results in a shorter time to platelet count response, a lower percentage of patients who die, relapse or have a severe TE event during the treatment period, a lower relapse rate, and / or prevention of refractory state.

[0039] In a further aspect, the present invention relates to a polypeptide as described herein for use in treating a first episode (symptoms of) a vWF-related disease in a human, preferably TTP, wherein said polypeptide comprises at least one ISVD that binds to human vWF (SEQ ID NO: 20).

[0040] In a further aspect, the present invention relates to a polypeptide as described herein for use in treating (symptoms of) a vWF-related disease in a human, preferably TTP, wherein said polypeptide is at least 90% identical to SEQ ID NO:1 and comprises a C-terminal extension (X)n, where n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4, or 5 (preferably 1 or 2, such as 1); each X is an independently selected (preferably naturally occurring) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I); even more preferably alanine.

[0041] In a further aspect, the present invention relates to a polypeptide as described herein for use in treating a first episode (symptoms of) vWF-related disease in humans, wherein at least one ISVD is represented by SEQ ID NO: 19 (12A02H1).

[0042] In a further aspect, the present invention relates to a polypeptide as described herein for use in treating a first episode (symptoms of) a vWF-related disease in a human, preferably TTP, wherein said polypeptide is at least 90% identical to SEQ ID NO:1.

[0043] In a further aspect, the present invention relates to a polypeptide as described herein for use in treating a first episode (symptoms of) a vWF-related disease in a human, preferably TTP, wherein said polypeptide is ALX0081 (SEQ ID NO: 1) or ALX0081-A (SEQ ID NO: 24).

[0044] In a further aspect, the present invention relates to a polypeptide as described herein for use in treating a first episode (symptoms of) a vWF-related disease in a human, preferably TTP, wherein said dose is administered once daily or twice daily.

[0045] In a further aspect, the present invention relates to a polypeptide as described herein for use in treating a first episode (symptoms of) a vWF-related disease, preferably TTP, in a human, comprising repeating administration of said polypeptide until the platelet count in said human is at least 150,000 / μl.

[0046] In a further embodiment, the present invention relates to a method for treating a platelet count disorder comprising administering the polypeptide to a patient in need thereof, the method comprising the steps of: repeating administration of the polypeptide until the platelet count in the human is at least 150,000 / μl on at least two consecutive measurements; The present invention also relates to a polypeptide as described herein for use in treating a first episode (symptoms of) a vWF-related disease in said human, comprising administering to said human a therapeutically effective amount of the polypeptide.

[0047] In a further embodiment, the present invention relates to a polypeptide as described herein for use in treating a first episode (symptoms of) a vWF-related disease, preferably TTP, in a human, wherein said step of administering a polypeptide of the invention is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, or even for a period longer than 10 days, such as 20 days, preferably for a period longer than 30 days or more, after said platelet count is at least 150,000 / μl in at least two consecutive measurements.

[0048] In a further embodiment, the present invention relates to a polypeptide as described herein for use in treating a first episode (symptoms of) a vWF-related disease in a human, wherein said two successive measurements are spaced at least 3 days apart, such as at least 24 hours, more preferably 48 hours apart, or even more than 4, 5, 6 or even 7 days apart, preferably one week apart.

[0049] In a further aspect, the present invention relates to a polypeptide as described herein for use in treating a first episode (symptoms of) a vWF-related disease, preferably TTP, in a human, comprising repeating administration of the polypeptide until the ADAMTS13 activity in the human is at least 10%, such as at least 15%, 20%, 25%, 30%, 35%, 45% or even 50% of the baseline ADAMTS13 activity.

[0050] In a further aspect, the present invention relates to a polypeptide as described herein for use in treating a first episode (symptoms of) a vWF-related disease, preferably TTP, in a human, comprising repeating administration of the polypeptide until the levels of organ damage markers, such as LDH levels, troponin T levels, troponin I levels and / or creatinine levels in said human return to at least 40%, or even at least 50%, such as 60%, 70%, 80%, 90% or even 100% of normal levels.

[0051] In a further aspect, the present invention relates to a polypeptide as described herein for use in treating a first episode (symptoms of) a vWF-related disease in a human, preferably TTP, comprising performing plasma exchange.

[0052] In a further aspect, the present invention relates to a polypeptide as described herein for use in treating a first episode (symptoms of) a vWF-related disease in a human, wherein said 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), preferably TTP.

[0053] In one aspect, the present invention relates to a polypeptide comprising two anti-human von Willebrand Factor (vWF) immunoglobulin single variable domains (ISVDs), for use in reducing hospital stay and / or intensive care unit (ICU) stay during the treatment of an acute episode of a vWF-related disease, preferably TTP, in a human in need of such treatment comprising administering to the human a dose of 1 to 80 mg, preferably 5 to 40 mg, even more preferably 10 mg or 11 mg of said polypeptide.

[0054] In a further aspect, the present invention relates to a method for treating a subject, comprising administering a polypeptide of the present invention to a subject comprising administering to said subject a subject a polypeptide of the present invention; The present invention relates to a polypeptide as described herein for use in reducing the number of hospital days and / or intensive care unit (ICU) stays during the treatment of an acute episode of a vWF-related disease, preferably TTP, in a human in need of such treatment, repeated over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or even longer than 10 days, such as 20 days, preferably longer than 30 days, such as 2 months, 3 months, 4 months, 5 months, 6 months, or even more (treatment period).

[0055] In a further aspect, the invention relates to a polypeptide as described herein for use in reducing the number of hospital days and / or intensive care unit (ICU) stays during the treatment of an acute episode of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein said treatment results in a shorter time to platelet count response, a lower percentage of patients who die, relapse or have a severe TE event during the treatment period, a lower relapse rate, and / or prevention of refractory state.

[0056] In a further aspect, the present invention relates to a polypeptide as described herein for use in reducing hospital stay and / or intensive care unit (ICU) stay during treatment of an acute episode of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein said polypeptide comprises at least one ISVD that binds to SEQ ID NO: 20.

[0057] In a further aspect, the present invention relates to a polypeptide as described herein for use in reducing the number of hospital days and / or intensive care unit (ICU) stays during the treatment of an acute episode of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein at least one ISVD is represented by SEQ ID NO: 19 (12A02H1).

[0058] In a further aspect, the present invention relates to a polypeptide as described herein for use in reducing hospital stay and / or intensive care unit (ICU) stay during treatment of an acute episode of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein said polypeptide is at least 90% identical to SEQ ID NO:1.

[0059] In a further aspect, the present invention relates to a polypeptide as described herein for use in reducing hospital stay and / or intensive care unit (ICU) stay during treatment of an acute episode of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein said polypeptide is ALX0081 (SEQ ID NO: 1) or ALX0081-A (SEQ ID NO: 24).

[0060] In a further aspect, the present invention relates to a polypeptide as described herein for use in reducing the number of hospital days and / or the number of intensive care unit (ICU) stays during the treatment of an acute episode of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein said dose is administered once daily or twice daily.

[0061] In a further aspect, the present invention relates to a polypeptide as described herein for use in reducing the number of hospital days and / or intensive care unit (ICU) stays during the treatment of an acute episode of a vWF-related disease, preferably TTP, in a human in need of such treatment, comprising repeating administration of said polypeptide until the platelet count in said human is at least 150,000 / μl.

[0062] In a further aspect, the present invention provides a method for reducing the number of hospital days and / or intensive care unit (ICU) stays during the treatment of an acute episode of a vWF-related disease, preferably TTP, in a human in need of such treatment, comprising repeating the administration of said polypeptide until said human has a platelet count of at least 150,000 / μl in at least two consecutive measurements. In particular, the present invention relates to a polypeptide as described herein for use in a method for treating a disease.

[0063] In a further embodiment, the present invention relates to a polypeptide as described herein for use in reducing the number of hospital days and / or intensive care unit (ICU) stays during the treatment of an acute episode of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein said step of administering a polypeptide of the invention is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, or even for a period longer than 10 days, such as 20 days, preferably for a period longer than 30 days or more, after said platelet count is at least 150,000 / μl in at least two consecutive measurements.

[0064] In a further embodiment the present invention relates to a polypeptide as described herein for use in reducing hospital stay and / or intensive care unit (ICU) stay during the treatment of an acute episode of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein said two successive measurements are spaced at least 24 hours, more preferably 48 hours apart, such as at least 3 days apart, or even more than 4, 5, 6 or even 7 days apart, preferably one week apart.

[0065] In a further aspect, the present invention relates to a polypeptide as described herein for use in reducing the number of hospital days and / or intensive care unit (ICU) stays during the treatment of an acute episode of a vWF-related disease, preferably TTP, in a human in need of such treatment, comprising repeating administration of the polypeptide until the ADAMTS13 activity in said human is at least 10%, such as at least 15%, 20%, 25%, 30%, 35%, 45% or even 50% of the baseline ADAMTS13 activity.

[0066] In a further aspect, the present invention relates to a polypeptide as described herein for use in reducing the number of hospital days and / or intensive care unit (ICU) stays during the treatment of an acute episode of a vWF-related disease, preferably TTP, in a human in need of such treatment, comprising repeating administration of said polypeptide until the levels of organ damage markers, such as LDH levels, troponin T levels, troponin I levels and / or creatinine levels in said human return to at least 40%, or even at least 50%, such as 60%, 70%, 80%, 90% or even 100% of normal levels.

[0067] In a further aspect, the present invention relates to a polypeptide as described herein for use in reducing the number of hospital days and / or intensive care unit (ICU) stays during the treatment of an acute episode of a vWF-related disease, preferably TTP, in a human in need of such treatment, including by performing plasma exchange.

[0068] In a further aspect, the present invention relates to a polypeptide as described herein for use in reducing the number of hospital days and / or intensive care unit (ICU) stays during the treatment of an acute episode of a vWF-related disease in a human in need of such treatment, wherein said 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), preferably TTP.

[0069] In one aspect, the present invention relates to a polypeptide comprising two anti-human von Willebrand factor (vWF) immunoglobulin single variable domains (ISVDs), comprising the treatment of an acute episode, such as a primary episode and / or a recurrent episode, of a vWF-related disease, preferably TTP, in a human in need thereof, comprising administering to the human a dose of 1-80 mg, preferably 5-40 mg, even more preferably 10 mg or 11 mg of said polypeptide. The present invention relates to a polypeptide for use in preventing a patient from becoming refractory to a cancer.

[0070] In a further embodiment, the present invention relates to a polypeptide as described herein for use in preventing a patient from becoming refractory to treatment of an acute episode, such as a primary episode and / or a recurrent episode, of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein said step of administering a polypeptide of the invention is repeated for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or even longer than 10 days, such as 20 days, preferably longer than 30 days, such as 2 months, 3 months, 4 months, 5 months, 6 months, or even more (treatment period).

[0071] In a further aspect, the invention relates to a polypeptide as described herein for use in preventing a patient from becoming refractory to treatment of an acute episode, such as a primary episode and / or a recurrent episode, of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein said treatment results in a shorter time to platelet count response, a lower percentage of patients who die, relapse or have a severe TE event during the treatment period, a lower relapse rate, and / or prevention of refractory state.

[0072] In a further aspect, the present invention relates to a polypeptide as described herein for use in preventing a patient from becoming refractory to treatment of an acute episode, such as a primary episode and / or a recurrent episode, of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein said polypeptide comprises at least one ISVD that binds to SEQ ID NO: 20.

[0073] In a further aspect, the present invention relates to a polypeptide as described herein for use in preventing a patient from becoming refractory to treatment of an acute episode, such as a primary episode and / or a recurrent episode, of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein at least one ISVD is represented by SEQ ID NO: 19 (12A02H1).

[0074] In a further aspect, the present invention relates to a polypeptide as described herein for use in preventing a patient from becoming refractory to treatment of an acute episode, such as a primary episode and / or a recurrent episode, of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein said polypeptide is at least 90% identical to SEQ ID NO:1.

[0075] In a further aspect, the present invention relates to a polypeptide as described herein for use in preventing a patient from becoming refractory to treatment of an acute episode, such as a primary episode and / or a recurrent episode, of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein said polypeptide is ALX0081 (SEQ ID NO: 1) or ALX0081-A (SEQ ID NO: 24).

[0076] In a further aspect, the present invention relates to a polypeptide as described herein for use in preventing a patient from becoming refractory to the treatment of an acute episode, such as a primary episode and / or a recurrent episode, of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein said dose is administered once daily or twice daily.

[0077] In a further aspect, the invention relates to a method for preventing a patient from becoming refractory to treatment of acute episodes, such as initial and / or recurrent episodes, of a vWF-related disease, preferably TTP, in a human in need of such treatment, comprising repeating administration of said polypeptide until the platelet count in said human is at least 150,000 / μl. The present invention relates to a polypeptide as described herein for use in

[0078] In a further aspect, the present invention relates to a polypeptide as described herein for use in preventing a patient from becoming refractory to treatment of acute episodes, such as initial and / or recurrent episodes, of a vWF-related disease, preferably TTP, in a human in need of such treatment, comprising repeating administration of said polypeptide until the platelet count in said human is at least 150,000 / μl in at least two consecutive measurements.

[0079] In a further embodiment, the present invention relates to a polypeptide as described herein for use in preventing a patient from becoming refractory to the treatment of an acute episode, such as a primary episode and / or a recurrent episode, of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein said step of administering a polypeptide of the invention is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, or even for a period longer than 10 days, such as 20 days, preferably for a period longer than 30 days or more, after said platelet count is at least 150,000 / μl in at least two consecutive measurements.

[0080] In a further embodiment the present invention relates to a polypeptide as described herein for use in preventing a patient from becoming refractory to the treatment of an acute episode, such as a primary episode and / or a recurrent episode, of a vWF-related disease, preferably TTP, in a human in need of such treatment, wherein said two successive measurements are spaced at least 3 days apart, such as at least 24 hours, more preferably 48 hours apart, or even more than 4, 5, 6 or even 7 days apart, preferably one week apart.

[0081] In a further aspect, the present invention relates to a polypeptide as described herein for use in preventing a patient from becoming refractory to treatment of acute episodes, such as initial and / or recurrent episodes, of a vWF-related disease, preferably TTP, in a human in need of such treatment, comprising repeating administration of the polypeptide until the ADAMTS13 activity in said human is at least 10%, such as at least 15%, 20%, 25%, 30%, 35%, 45% or even 50% of the baseline ADAMTS13 activity.

[0082] In a further aspect, the present invention relates to a polypeptide as described herein for use in preventing a patient from becoming refractory to treatment of acute episodes, such as initial and / or recurrent episodes, of a vWF-related disease, preferably TTP, in a human in need of such treatment, comprising repeating administration of the polypeptide until the levels of organ damage markers, such as LDH levels, troponin T levels, troponin I levels and / or creatinine levels in said human return to at least 40%, or even at least 50%, such as 60%, 70%, 80%, 90% or even 100% of normal levels.

[0083] In a further aspect, the present invention relates to a polypeptide as described herein for use in preventing a patient from becoming refractory to treatment of an acute episode, such as a primary episode and / or a recurrent episode, of a vWF-related disease, preferably TTP, in a human in need of such treatment, including performing plasma exchange.

[0084] In a further aspect, the present invention relates to a polypeptide as described herein for use in preventing a patient from becoming refractory to treatment of an acute episode, such as a primary episode and / or a recurrent episode, of a vWF-related disease in a human in need of such treatment, Here, the vWF-associated disease is selected from acute coronary syndrome (ACS), transient ischemic attack, unstable or stable angina, stroke, myocardial infarction, or thrombotic thrombocytopenic purpura (TTP), preferably TTP.

[0085] The present invention therefore provides a polypeptide comprising at least one ISVD, preferably two ISVDs against vWF, even more preferably ALX0081 or ALX0081-A, for use in treating and / or preventing a vWF-related disease, preferably TTP, in a human in need of such treatment and / or prevention as described herein, comprising administering to said human a first dose of 1 to 80 mg, such as 5 to 40 mg, preferably 10 mg or 11 mg, of said polypeptide.

[0086] The present invention provides a polypeptide as described herein for use in treating and / or preventing a vWF-associated disease, preferably TTP, in a human in need of such treatment and / or prevention, wherein said administration of said polypeptide is followed by a first PE within 5 minutes to 8 hours.

[0087] The present invention provides a polypeptide as described herein for use in treating and / or preventing a vWF-related disease, preferably TTP, in a human in need of such treatment and / or prevention, wherein said administration of said first dose is preceded by a previous PE, preferably within 36 hours, such as within 32 hours, within 30 hours, within 28 hours, within 26 hours, within 24 hours, within 22 hours, within 20 hours, within 18 hours, within 16 hours, within 14 hours, within 12 hours, within 10 hours, within 8 hours, such as within 7 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, within 1 hour, within 45 minutes, within 30 minutes, within 20 minutes, within 15 minutes, within 10 minutes, or even within 5 minutes, of said first PE.

[0088] The present invention provides a polypeptide as described herein for use in treating and / or preventing a vWF associated disease, preferably TTP, in a human in need of such treatment and / or prevention as described herein, wherein said first PE is followed by administration of a second dose of 1 to 80 mg, such as 5 to 40 mg, preferably 10 mg or 11 mg, of said polypeptide, preferably by subcutaneous injection, preferably within 1 to 60 minutes, more preferably within 30 minutes of said first PE.

[0089] The present invention provides a polypeptide as described herein for use in treating and / or preventing a vWF-associated disease, preferably TTP, in a human in need of such treatment and / or prevention as described herein, wherein said prior 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 said first PE.

[0090] The present invention provides a polypeptide as described herein for use in treating and / or preventing a vWF-associated disease, preferably TTP, in a human in need of such treatment and / or prevention, wherein said polypeptide is administered parenterally, preferably by subcutaneous, intraperitoneal, intravenous or intramuscular injection, preferably by intravenous (iv) bolus push injection.

[0091] The present invention relates to a method for treating and / or preventing a subject in need of the treatment and / or prevention described herein, wherein administration of said polypeptide is followed by PE within 5 minutes to 8 hours, such as 10 minutes to 6 hours or 15 minutes to 4 hours, such as within 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes or even 5 minutes, preferably less than 5 hours. The present invention provides a polypeptide as described herein for use in treating and / or preventing a vWF-associated disease in humans, preferably TTP.

[0092] The present invention relates to a polypeptide as described herein for use in treating and / or preventing a vWF-related disease, preferably TTP, in a human in need of such treatment and / or prevention, wherein said treatment of a vWF-related disease such as TTP, preferably TTP, in a human in need of such treatment comprises: (i) carrying out a PE; (followed by) (ii) administering a dose of 1 to 80 mg, e.g., 5 to 40 mg, of the polypeptide 5 minutes to 4 hours after the PE of step (i); (iii) optionally measuring the patient's platelet count and / or ADAMTS13 activity; Further comprising: Here, steps (i) and (ii) are repeated once a day, preferably until the patient's platelet count is 150,000 / μl or more and / or the ADAMTS13 activity is at least 10%, such as at least 15%, 20%, 25%, 30%, 35%, 45% or even 50% of the baseline ADAMTS13 activity.

[0093] The present invention provides a polypeptide as described herein for use in treating and / or preventing a vWF associated disease, preferably TTP, in a human in need of such treatment and / or prevention, further comprising administering a dose of 1 to 80 mg, such as 5 to 40 mg, preferably 10 mg or 11 mg, of said polypeptide once per day for at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 90 or even 120 days after said patient's platelet count first reaches ≥ 150,000 / μl.

[0094] The present invention provides a polypeptide as described herein for use in treating and / or preventing a vWF-related disorder, preferably TTP, in a human in need of such treatment and / or prevention as described herein, further comprising administering a dose of 1 to 80 mg, such as 5 to 40 mg, preferably 10 mg or 11 mg, of said polypeptide once daily until said human goes into remission.

[0095] The present invention provides a polypeptide as described herein for use in treating and / or preventing a vWF-associated disease, preferably TTP, in a human in need of such treatment and / or prevention, comprising administering the polypeptide until ADAMTS13 activity is at least 10%, such as at least 15%, 20%, 25%, 30%, 35%, 45% or even 50% of baseline ADAMTS13 activity.

[0096] The invention provides a polypeptide as described herein, wherein the dose is about 1-80 mg, or 5-40 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 or 11 mg, of the polypeptide.

[0097] The present invention provides a polypeptide as described herein for use in treating and / or preventing a vWF-related disease, preferably TTP, in a human in need of such treatment and / or prevention as described herein, said human suffering from a TTP episode, such as a first TTP episode and / or a recurrent TTP episode, an exacerbation of TTP, and / or a recurrence of TTP.

[0098] In a preferred embodiment, the present invention relates to a polypeptide comprising at least one ISVD against vWF as described herein for use in treating a vWF-related disease such as TTP, preferably TTP, in a human in need of such treatment, said treatment comprising: (1) carrying out an earlier permanent establishment, if applicable; (2) administering to said human a first dose of 1 to 80 mg, such as 5 to 40 mg, preferably 10 mg or 11 mg, of said polypeptide, if step (1) has been performed, preferably within 36 hours, such as within 32 hours, within 30 hours, within 28 hours, within 26 hours, within 24 hours, within 22 hours, within 20 hours, within 18 hours, within 16 hours, within 14 hours, within 12 hours, within 10 hours, within 8 hours, such as within 7 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, within 1 hour, within 45 minutes, within 30 minutes, within 20 minutes, within 15 minutes, within 10 minutes, or even within 5 minutes of (the end of) step (1); (3) optionally performing PE within 5 minutes to 8 hours, e.g., within 10 minutes to 6 hours or within 15 minutes to 4 hours, e.g., within 8 hours, within 7 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, within 1 hour, within 45 minutes, within 30 minutes, within 20 minutes, within 15 minutes, within 10 minutes, or even within 5 minutes, of step (2); (4) administering a further dose of 1 to 80 mg, such as 5 to 40 mg, preferably 10 mg or 11 mg, of said polypeptide, preferably within 5 minutes to 8 hours, such as within 10 minutes to 6 hours or within 15 minutes to 4 hours, such as within 8 hours, within 7 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, within 1 hour, within 45 minutes, within 30 minutes, within 20 minutes, within 15 minutes, within 10 minutes, or even within 5 minutes, of (the end of) step (3); (5) repeating steps (3) and (4) once a day; optionally until the patient's platelet count is 150,000 / μl or greater and / or the ADAMTS13 activity is at least 10%, such as at least 15%, 20%, 25%, 30%, 35%, 45%, or even 50% of baseline ADAMTS13 activity; (6) optionally administering a dose of 1-80 mg, e.g., 5-40 mg, preferably 10 mg or 11 mg, of the polypeptide once daily for at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, or even 120 days after the patient's platelet count first reaches 150,000 / μl or more, or until the ADAMTS13 activity is at least 10%, such as at least 15%, 20%, 25%, 30%, 35%, 45%, or even 50% of baseline ADAMTS13 activity; The present invention provides a polypeptide comprising:

[0099] When step (3) is performed 5 hours after step (2), a further dose of 1 to 80 mg, for example 5 to 40 mg, preferably 10 mg or 11 mg, of said polypeptide is administered.

[0100] In another preferred embodiment, the present invention relates to a polypeptide comprising at least one ISVD against vWF as described herein for use in treating a vWF-related disease such as TTP, preferably TTP, in a human in need of such treatment, said treatment comprising: (1) PE should be performed twice a day; (2) administering to the human a dose of 1 to 80 mg, for example 5 to 40 mg, preferably 10 mg or 11 mg, of the polypeptide after each PE of step (1); The present invention provides a polypeptide comprising:

[0101] Furthermore, the present invention relates to a polypeptide comprising two anti-human vWF ISVDs, and by administering to a human a dose of 1 to 80 mg, for example 5 to 40 mg, preferably 10 mg or 11 mg, of the polypeptide, recurrence (symptoms) of a vWF-related disease in a human can be prevented. and for use in

[0102] The present invention provides a polypeptide as described herein, wherein said ISVD against vWF comprises at least one immunoglobulin single variable domain that binds to SEQ ID NO:20.

[0103] The present invention provides a polypeptide as described herein, wherein said ISVD against vWF comprises a heavy chain variable domain derived from a conventional four-chain antibody, or a heavy chain variable domain derived from a heavy chain antibody or a Nanobody.

[0104] The present invention provides a polypeptide as described herein, wherein said Nanobody is a VHH.

[0105] The present invention relates to a method for the preparation of a nucleic acid sequence for a vWF comprising the steps of: a) CDR1 is: - the amino acid sequence YNPMG; or - an amino acid sequence that has two or only one amino acid difference from the amino acid sequence YNPMG Consists of or consists essentially of; b) CDR2 is: - the amino acid sequence AISRTGGSTYYPDSVEG; or - an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the amino acid sequence AISRTGGSTYYPDSVEG; or - an amino acid sequence that has two or only one amino acid difference from the amino acid sequence AISRTGGSTYYPDSVEG Consists of or consists essentially of; c) CDR3 is: - the amino acid sequence AGVRAEDGRVRTLPSEYTF; or - an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the amino acid sequence AGVRAEDGRVRTLPSEYTF; or - an amino acid sequence that has two or only one amino acid difference from the amino acid sequence AGVRAEDGRVRTLPSEYTF In accordance with another aspect of the present invention, there is provided a polypeptide as described herein, comprising or consisting essentially of:

[0106] The present invention relates to a polypeptide as described herein: a) CDR1 is YNPMG (SEQ ID NO:21); b) CDR2 is AISRTGGSTYYPDSVEG (SEQ ID NO: 22); c) a polypeptide is provided in which CDR3 is AGVRAEDGRVRTLPSEYTF (SEQ ID NO: 23).

[0107] The present invention provides a polypeptide as described herein, wherein the ISVD for vWF is represented by SEQ ID NO: 19 (12A02H1).

[0108] The present invention provides a polypeptide as described herein comprising or consisting of at least two ISVDs for vWF.

[0109] The present invention relates to a method for determining whether or not each of the at least two ISVDs for vWF is a four-frame ISVD. and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: a) CDR1 is: - the amino acid sequence YNPMG; or - an amino acid sequence that has two or only one amino acid difference from the amino acid sequence YNPMG Consists of or consists essentially of; b) CDR2 is: - the amino acid sequence AISRTGGSTYYPDSVEG; or - an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the amino acid sequence AISRTGGSTYYPDSVEG; or - an amino acid sequence that has two or only one amino acid difference from the amino acid sequence AISRTGGSTYYPDSVEG Consists of or consists essentially of; c) CDR3 is: - the amino acid sequence AGVRAEDGRVRTLPSEYTF; or - an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the amino acid sequence AGVRAEDGRVRTLPSEYTF; or - an amino acid sequence that has two or only one amino acid difference from the amino acid sequence AGVRAEDGRVRTLPSEYTF In accordance with another aspect of the present invention, there is provided a polypeptide as described herein, comprising or consisting essentially of:

[0110] According to the present invention, each ISVD for vWF essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: a) CDR1 is YNPMG (SEQ ID NO:21); b) CDR2 is AISRTGGSTYYPDSVEG (SEQ ID NO: 22); c) A polypeptide as described herein is provided, wherein CDR3 is AGVRAEDGRVRTLPSEYTF (SEQ ID NO: 23).

[0111] The present invention provides a polypeptide as described herein, comprising or consisting of SEQ ID NO: 1-18 or 24, preferably SEQ ID NO: 1 or 24.

[0112] The present invention provides a polypeptide as described herein, wherein said ISVD against vWF is a single-chain polypeptide comprising one or more immunoglobulin single variable domains.

[0113] The present invention provides a polypeptide as described herein, wherein said ISVD for vWF is monovalent or multivalent.

[0114] The present invention provides a polypeptide as described herein, wherein said ISVD for vWF is monospecific or multispecific.

[0115] The present invention provides polypeptides as described herein, in which one or more immunoglobulin single variable domains are CDR-grafted, humanized, camelized, deimmunized or selected by phage display.

[0116] The present invention provides a polypeptide as described herein, wherein said ISVD for vWF comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1.

[0117] The present invention provides a polypeptide as described herein that comprises two anti-human vWF immunoglobulin single variable domains (ISVDs) and an anti-human serum albumin (HSA) ISVD.

[0118] The present invention provides a polypeptide as described herein formulated in a pharma- ceutically acceptable formulation.

[0119] The invention provides a polypeptide as described herein, wherein the formulation comprises a citrate or phosphate buffer having a pH in the range of 5.0 to 7.5.

[0120] The invention provides a polypeptide as described herein, wherein the formulation is suitable for parenteral administration, e.g., one or more selected from intravenous injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection.

[0121] The invention provides a polypeptide as described herein, wherein the formulation is in a liquid form, a lyophilized form, a spray-dried form, a reconstituted lyophilized form, or a frozen form.

[0122] The invention provides a kit or article of manufacture comprising a container containing a polypeptide as described herein or a formulation as described herein, and instructions for use.

[0123] The invention provides a kit or article of manufacture as described herein, wherein the formulation is present in a vial or a syringe for injection.

[0124] The invention provides a kit or article of manufacture as described herein, wherein the formulation is present in a pre-filled syringe for injection.

[0125] The invention provides a kit or article of manufacture as described herein, wherein the syringe or vial is made from a polymeric material selected from glass, plastic, or a cyclic olefin polymer or copolymer.

[0126] The present invention relates to a formulation comprising: (a) a polypeptide described herein at a concentration of about 0.1 mg / mL to about 80 mg / mL; (b) an excipient selected from sucrose, glycine, mannitol, trehalose, or NaCl at a concentration of about 1% to about 15% (w / v); (c) Tween-80 at a concentration of about 0.001% to 0.5% (v / v); (d) a buffer selected from a citrate buffer at a concentration of about 5 mM to about 200 mM such that the pH of the formulation is about 6.0 to 7.0, and a phosphate buffer at a concentration of about 10 mM to about 50 mM such that the pH of the formulation is about 6.5 to 7.5; Including, The present invention provides a formulation for use in treating a vWF associated disease, preferably TTP, in a human in need of treatment by administering to the human a dose of 1 to 80 mg, such as a 5 to 40 mg dose, preferably 10 mg or 11 mg, of said polypeptide, optionally followed by a first plasma exchange (PE) within 5 minutes to 8 hours, such as 15 minutes to 4 hours.

[0127] The present invention relates to a method for the preparation of a polyclonal antibody as described herein, which is suitable for parenteral administration to a patient, preferably a human patient. Pharmaceutical unit dosage forms are provided that include a peptide or a formulation described herein.

[0128] The present invention provides a polypeptide as described herein, wherein said vWF-related disease is selected from acute coronary syndrome (ACS), transient ischemic attack, unstable or stable angina, stroke, myocardial infarction, or (acquired and / or congenital) thrombotic thrombocytopenic purpura (TTP), preferably TTP.

[0129] The present invention provides a method for treating a human patient susceptible to or diagnosed with a von Willebrand factor (vWF)-associated disease, such as TTP, comprising administering to the human patient an effective amount of a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) against vWF.

[0130] The present invention provides a method for treating or preventing a vWF-associated disease, such as TTP, comprising administering to a human a dose of 1 to 80 mg, such as 5 to 40 mg, preferably 10 mg or 11 mg, of a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) against von Willebrand factor (vWF), thereby reducing one or more symptoms associated with the vWF-associated disease.

[0131] The invention provides for the treatment described herein, wherein said administration of a polypeptide described herein is followed by a first plasma exchange (PE) within 5 minutes to 8 hours, for example within 15 minutes to 4 hours.

[0132] The present invention provides for the treatment as described herein, wherein said administration of a polypeptide as described herein is preceded by a prior plasma exchange (PE) within 36 hours, preferably within 32, 30, 28, 26, 24, 22, 20, 18, or 16 hours, preferably within about 24 hours, of said first PE.

[0133] The invention provides a treatment as described herein, wherein said first PE is followed by administration of a second dose of 1 to 80 mg, such as 5 to 40 mg, preferably 10 mg or 11 mg, of a polypeptide as described herein within 5 minutes to 8 hours, such as 10 minutes to 6 hours or 15 minutes to 4 hours, such as within 8 hours, within 7 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, within 1 hour, within 45 minutes, within 30 minutes, within 20 minutes, within 15 minutes, within 10 minutes, or even within 5 minutes (e.g. said second dose of said polypeptide is administered within 1 to 60 minutes, such as within 30 minutes, preferably by subcutaneous injection) of said first PE.

[0134] The present invention relates to (i) carrying out a PE; (followed by) (ii) administering a dose of 1 to 80 mg, such as 5 to 40 mg, preferably 10 mg or 11 mg, of a polypeptide as described herein 15 minutes to 4 hours after said PE of step (i); (iii) optionally measuring the patient's platelet count and / or ADAMTS13 activity; Further comprising: Here, steps (i) and (ii) are provided as described herein, wherein steps (i) and (ii) are repeated once a day, optionally until the patient's platelet count is 150,000 / μl or greater and / or until ADAMTS13 activity is at least 10%, such as at least 15%, 20%, 25%, 30%, 35%, 45%, or even 50% of baseline ADAMTS13 activity.

[0135] The invention also provides a treatment as described herein further comprising administering a dose of 1 to 80 mg, such as 5 to 40 mg, preferably 10 mg or 11 mg, of a polypeptide as described herein once daily for at least 5, 10, 15, 20, 25, or even 30 days after said patient's platelet count is 150,000 / μl or greater.

[0136] The invention provides a treatment as described herein further comprising administering a dose of 1 to 80 mg, such as 5 to 40 mg, preferably 10 mg or 11 mg, of a polypeptide as described herein once daily until the human goes into remission.

[0137] The present invention provides treatments as described herein, comprising administering the polypeptide until ADAMTS13 activity is at least 10%, such as at least 15%, 20%, 25%, 30%, 35%, 45%, or even 50% of baseline ADAMTS13 activity.

[0138] In one embodiment, the present invention relates to a method for reducing and / or preventing the risk of acute episodes of vWF-related diseases, such as episodes of TTP, e.g. initial and / or recurrent episodes of TTP, in a human in need thereof, said method comprising or consisting of: (i) administering to said human a dose of 5-40 mg, preferably 10 mg or 11 mg, of a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) against von Willebrand factor (vWF); wherein administration of said polypeptide reduces and / or prevents the risk of acute episodes of vWF-related diseases, such as episodes of TTP, e.g. initial and / or recurrent episodes of TTP. Preferably, said risk is reduced by a factor of 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 or more, e.g. 3, 4, 5, 6, 7, 8, 9 or even 10 or even more, e.g. 20, 50 or even 100. Preferably, the risk is reduced by 10% or even more, such as 20%, 30%, 40%, 50%, 60% or more, such as 80% or even 100%.

[0139] In one embodiment, the present invention relates to a method as described herein, wherein said step (i) of administering a polypeptide of the present invention is repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or even more than 10 times, such as 20 times, preferably more than 30 times or even more.

[0140] In one embodiment, the present invention relates to a method as described herein, wherein said step (i) of administering a polypeptide of the present invention is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or even for a period of more than 10 days, such as 20 days, preferably for a period of more than 30 days, such as 2 months, 3 months, 4 months, 5 months, 6 months, or even more.

[0141] In one embodiment, the present invention relates to the methods described herein, wherein said dose is administered once daily or twice daily (bid).

[0142] In one embodiment, the present invention relates to a method according to the present disclosure, comprising the steps of: (ii) optionally measuring ADAMTS13 activity in said patient; (iii) optionally comparing the ADAMTS13 activity of the patient with a reference ADAMTS13 activity; (iv) if the ADAMTS13 activity is less than 30%, e.g., 20%, 15%, or 10%, of the reference ADAMTS13 activity, administering the polypeptide of the present invention ( Repeating i) and The present invention relates to a method comprising the steps of:

[0143] In one embodiment, the present invention relates to a method as described herein, wherein said ADAMTS13 activity in said patient is measured daily, or every 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, preferably at least once a week.

[0144] In one embodiment, the present invention relates to a method described herein, wherein the step of administering a polypeptide of the present invention is repeated until the ADAMTS13 activity is at least 10%, 15%, for example 20%, or even 30% or more of the baseline ADAMTS13 activity.

[0145] In one embodiment, the present invention relates to a method as described herein, wherein step (i) is repeated until said ADAMTS13 activity is at least 10%, 15%, such as 20% or 30% of said baseline ADAMTS13 activity in at least two consecutive measurements. Preferably, said two consecutive measurements are separated by at least 24 hours, more preferably 48 hours, such as at least 3 days, or even longer, such as 4, 5, 6 or even 7 days, preferably one week.

[0146] In one embodiment, the present invention relates to a method as described herein, wherein said step (i) of administering a polypeptide of the present invention is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or even for a period longer than 10 days, such as 20 days, preferably longer than 30 days or even more, after said ADAMTS13 activity is at least 10% or 15%, such as 20% or 30%, of said baseline activity in at least two consecutive measurements.

[0147] In one embodiment, the present invention relates to a method according to the present disclosure, comprising the steps of: - optionally measuring the ADAMTS13 activity in said patient; - optionally comparing the ADAMTS13 activity of said patient with a reference ADAMTS13 activity; - if the ADAMTS13 activity is 10% or more, such as more than 15%, or more than 20% or 30% of the baseline ADAMTS13 activity, repeating step (i) of administering a polypeptide of the invention for up to 30 days, such as up to 20 days, or even for 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, or even for 1 day. The present invention relates to a method comprising the steps of:

[0148] In one embodiment, the present invention relates to a method for reducing the risk of and / or preventing an acute episode of a vWF-related disease, such as an episode of TTP, e.g. a first and / or recurrent episode of TTP, in a human in need thereof, said method comprising at least the following steps: (i) measuring ADAMTS13 activity in said patient; (ii) comparing the ADAMTS13 activity to a reference ADAMTS13 activity; (iii) if the ADAMTS13 activity is less than 30%, 20%, 15%, or 10% of the baseline activity, administering to the human a dose of 5 to 40 mg, e.g., 10 mg or 11 mg, of a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) directed against von Willebrand factor (vWF); Includes.

[0149] In one embodiment, the present invention relates to a method according to the present disclosure, comprising the steps of: - 10% to 20% risk of organ damage, ischemic injury, and / or microthrombus formation , by 30%, preferably by at least 40%, or even by at least 50%, such as by 60%, 70%, 80%, 90%, or even by 100%; - the risk of organ damage, ischemic damage and / or microthrombus formation is reduced by a factor of 1.2, 1.3, 1.4, 1.5, 1.75, 2 or more, such as 3, 4, 5, 6, 7, 8, 9, or even 10, or even more, such as 20, 50, or even 100; - organ damage, ischemic damage and / or microthrombus formation is preferably reduced by at least 10%, 20%, 30%, 40% or even at least 50%, such as by 60%, 70%, 80%, 90% or even up to 100%; - organ damage, ischemic damage, and / or microthrombus formation is reduced by a factor of 2 or more, such as 3, 4, 5, 6, 7, 8, 9, or even 10, or even more, such as 20, 50, or even 100; - markers of organ damage, such as LDH levels, troponin T, troponin I levels, and / or creatinine levels, return to at least 40% of normal levels, or even at least 50%, such as 60%, 70%, 80%, 90%, or even 100%; - markers of organ damage such as LDH levels, troponin T, troponin I levels and / or creatinine levels are improved by at least 20%, such as 30%, or even more such as 40%, or even by at least 50%, such as 60%, 70%, 80%, 90% or even 100% of normal levels (preferably, said markers of organ damage such as LDH levels, troponin T, troponin I levels and / or creatinine levels are improved within less than 30 days of treatment, preferably within less than 20 days of treatment, such as within 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 days or even within 1 day), - Platelet count is maintained at 150,000 / μl or higher. - the risk of progression is reduced by at least 10%, 20%, 30%, 40%, or even at least 50%, such as by 60%, 70%, 80%, 90%, or even 100%; - the risk of progression is reduced by a factor of 2 or more, e.g. 3, 4, 5, 6, 7, 8, 9, or even 10, or even more, e.g. 20, 50, or even 100; - the mortality rate attributable to said vWF-associated disease is reduced by 10%, 20%, 30%, preferably by at least 40%, or even by at least 50%, such as by 60%, 70%, 80%, 90%, or even by 100%; - the mortality rate attributable to said vWF-associated disease is reduced by a factor of 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 or more, such as 3, 4, 5, 6, 7, 8, 9, or even 10 or even more, such as 20, 50, or even 100; - the refractory state is reduced, preferably by at least 10%, 20%, 30%, 40%, or even by at least 50%, such as by 60%, 70%, 80%, 90%, or even by 100%; and / or - refractory state is reduced by a factor of 2 or more, e.g. 3, 4, 5, 6, 7, 8, 9, or even 10, or even more, e.g. 20, 50, or even 100; It concerns the method.

[0150] In one embodiment, the present invention relates to a method as described herein, further comprising measuring platelet count; and repeating said step of administering a polypeptide of the present invention if said platelet count is below 150,000 / μl.

[0151] In one embodiment, the present invention relates to a method as described herein, wherein said platelet count of said patient is measured daily, or every 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, preferably at least weekly.

[0152] In one embodiment, the present invention relates to a method as described herein, wherein said step of administering a polypeptide of the present invention is repeated until said platelet count is at least 150,000 / μl.

[0153] In one embodiment, the present invention relates to a method as described herein, wherein the step of administering a polypeptide of the present invention is repeated until the platelet count is at least 150,000 / μl for at least two consecutive measurements, preferably at least 24 hours apart, such as at least 3 days apart, more preferably 48 hours apart, or even longer, such as 4, 5, 6 or even 7 days apart, preferably one week apart.

[0154] In one embodiment, the present invention relates to a method as described herein, wherein said step of administering a polypeptide of the present invention is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or even for a period longer than 10 days, such as 20 days, preferably longer than 30 days or even more, after said platelet count is at least 150,000 / μl in at least two consecutive measurements. Preferably, said two consecutive measurements are spaced at least 24 hours apart, more preferably 48 hours apart, such as at least 3 days apart, or even longer, such as 4, 5, 6, or even 7 days apart, preferably one week apart.

[0155] In one embodiment, the present invention relates to a method as described herein, further comprising measuring the platelet count of said patient; and if said platelet count is equal to or greater than 150,000 / μl, repeating said step (i) of administering a polypeptide of the present invention for up to 30 days, such as up to 20 days, or even 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, or even 1 day.

[0156] In one embodiment, the present invention relates to a method for reducing the risk and / or preventing an acute episode of a vWF-related disease, such as an episode of TTP, e.g. a first episode and / or a recurrent episode of TTP, preferably TTP, in a human in need of such reduction and / or prevention, the method comprising at least the following steps: (i) determining the patient's platelet count; (ii) administering to the human a dose of 5 to 40 mg, e.g., 10 mg or 11 mg, of a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) directed against von Willebrand factor (vWF), if the platelet count is less than 150,000 / μl; Including, Here, administration of said polypeptide reduces the risk of and / or prevents acute episodes of a vWF-related disease, such as episodes of TTP, for example a first episode and / or a recurrent episode of TTP, preferably TTP.

[0157] In one embodiment, the present invention relates to a method for treating a TTP episode, such as a first TTP episode and / or a recurrent TTP episode, preferably TTP, in a human in need of such treatment, said method comprising at least the following steps: (i) 5 to 40 mg, preferably 10 mg or less, of a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) against von Willebrand factor (vWF) administering to the human a first dose of 11 mg or 15 mg of the compound; (ii) carrying out a first plasma exchange (PE) preferably within 5 minutes to 8 hours, preferably within 5 hours, of step (i); Includes.

[0158] In one embodiment, the present invention relates to a method for treating a TTP episode, such as a first TTP episode and / or a recurrent TTP episode, preferably TTP, in a human in need of treatment, wherein step (i), i.e. administering a polypeptide of the present invention to said human, is preceded by performing a preceding PE, preferably within 24 hours of step (ii), i.e. performing the first PE.

[0159] In one embodiment, the present invention relates to a method for treating a TTP episode, such as a first TTP episode and / or a recurrent TTP episode, preferably TTP, in a human in need thereof, comprising at least the following steps: (i) performing plasma exchange (PE); and (ii) administering to said human a dose of 5-40 mg, preferably 10 mg or 11 mg, of a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) against von Willebrand factor (vWF). Preferably, said step (i), i.e. performing PE, and said step (ii), i.e. administering to said human said polypeptide of the invention, are repeated once or twice daily for up to 1, 2, 3, 4, 5, 6, or 7 days.

[0160] In one embodiment, the present invention relates to a method for treating a TTP episode, such as a first TTP episode and / or a recurrent TTP episode, in a human in need of treatment, as described in the present invention, wherein step (ii), i.e. administering said polypeptide of the present invention to said human, is performed within 15 minutes to 4 hours of step (i), i.e. performing PE.

[0161] In one embodiment, the present invention relates to a method for treating a TTP episode, such as a primary TTP episode and / or a recurrent TTP episode, in a human in need of treatment as described in the present invention, which method further comprises measuring the platelet count of said human, preferably after step (ii), i.e. administering said polypeptide of the present invention to said human; if said platelet count is below 150,000 / μl, repeating said step (i), i.e. performing PE, and said step (ii), i.e. administering said polypeptide to said human.

[0162] In one embodiment, the present invention relates to a method for treating a TTP episode, such as a first TTP episode and / or a recurrent TTP episode, in a human in need of treatment, as described herein, which further comprises measuring the platelet count of said human (preferably after step (ii), i.e. administering said polypeptide of the present invention to said human); repeating step (i), i.e. performing PE, and step (ii), i.e. administering said polypeptide to said human [once / twice daily] until said platelet count is at least 150,000 / μl for at least two consecutive measurements. Preferably, said two consecutive measurements are at least 24 hours apart, more preferably 48 hours apart, such as at least 3 days apart, or even longer, such as 4, 5, 6 or even 7 days apart, preferably one week apart.

[0163] In one embodiment, the present invention relates to a method for treating a TTP episode, such as a first TTP episode and / or a recurrent TTP episode, in a human in need of such treatment, comprising administering 5-40 mg of said polypeptide, preferably 100 mg of said polypeptide to said patient, for at least 1-30 days after said human's platelet count reaches 150.000 / μl for the first time. or 10 mg or 11 mg once daily.

[0164] In one embodiment, the present invention relates to a method for treating a TTP episode, such as a first TTP episode and / or a recurrent TTP episode, in a human in need of treatment as described in the present invention, which method further comprises measuring the ADAMTS13 activity of said human, preferably after step (ii), i.e. administering said polypeptide to said human.

[0165] In one embodiment, the present invention relates to a method for treating a TTP episode, such as a first TTP episode and / or a recurrent TTP episode, in a human in need of treatment, 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, are repeated until ADAMTS13 activity is [for the first time] greater than 15%, or 20%, or even 30% of baseline ADAMTS13 activity.

[0166] In one embodiment, the present invention relates to a method for reducing and / or preventing the risk of ischemic damage, organ damage, refractory state, and / or microthrombus formation [caused by a vWF-related disease such as TTP, an episode of TTP, such as a first episode and / or a recurrent episode of TTP] in a human in need of such reduction and / or prevention, the method comprising at least the following steps: (i) administering to said human a dose of 5-40 mg / day, preferably 10 mg / day or 11 mg / day of a polypeptide comprising at least one ISVD against vWF; wherein administration of said polypeptide reduces and / or prevents the risk of ischemic damage, organ damage, refractory state, and / or microthrombus formation by 10%, 20%, 30%, preferably by at least 40%, or even by at least 50%, such as by 60%, 70%, 80%, 90%, or even by 100%. Preferably, administration of the polypeptide reduces and / or prevents the risk of ischemic damage, organ damage, refractory state, and / or microthrombus formation by a factor of 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 or more, such as 3, 4, 5, 6, 7, 8, 9, or even 10 or even more, such as 20, 50, or even 100.

[0167] In one embodiment, the present invention relates to a method, wherein said step of administering said polypeptide is repeated for at least 1, 2, 3, 4, 5, 6, 7 days, or even longer, such as 1 week, 2 weeks, 3 weeks, or even longer, such as 1 month, or even 2 months.

[0168] In one embodiment, the invention relates to a method further comprising measuring the ADAMTS13 activity in said patient, preferably once a week.

[0169] In one embodiment, the invention relates to a method, wherein the step of administering the polypeptide is repeated for at least 1, 2, 3, 4, 5, 6, 7 days, or even longer, such as 1 week, 2 weeks, 3 weeks, or even longer, such as 1 month, or even 2 months, when the ADAMTS13 activity is [for the first time] 10% or more, such as more than 15%, or even more than 20% of the baseline ADAMTS13 activity.

[0170] In one embodiment, the present invention relates to a method for treating a symptom of a vWF-related disease, such as TTP, in a human suffering from said disease, comprising administering to a subject a polypeptide of the present invention in an amount effective to treat the symptom of a vWF-related disease in a human suffering from said disease.

[0171] In one embodiment, the present invention relates to a method for preventing or reducing the onset of a vWF-associated disease, such as TTP, in humans. and a method for inhibiting the onset or progression of disease in a human, the inhibition being brought about by binding of a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) to von Willebrand factor (vWF) to vWF, the method comprising administering an inhibitory effective amount of said polypeptide to a human at predetermined intervals, wherein each administration of the polypeptide delivers to the human 0.1 mg to 25 mg per kg of body weight of the human, preferably 10 mg or 11 mg of said polypeptide per kg of body weight of the human, so as to inhibit the onset or progression of disease in the human.

[0172] In one embodiment, the invention relates to a method of reducing the likelihood that a human will suffer ischemic organ damage from a von Willebrand factor (vWF) associated disease, comprising administering to the human a predetermined dose of a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) against vWF, wherein each administration of the antibody delivers to the human 0.1 mg to 25 mg of said polypeptide per kg of body weight of the human, preferably 10 mg or 11 mg of said polypeptide if said human has a body weight of 40 kg or more, and 5 mg if said human has a body weight of less than 40 kg, so as to reduce the likelihood that the human will suffer ischemic organ damage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0173] 4. Detailed Description Unless otherwise indicated, all methods, steps, techniques and operations that are not specifically described can be carried out in a manner essentially known, as is clear to those skilled in the art, and have been carried out in this manner.For example, reference is again made to the standard handbooks and general background art mentioned in this specification, and the further references cited therein; besides, for example, the following reviews: Scully et al., 2017, see above, and 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, Supplement A, S106-12; Gonzales et al., Tumour Biol., 2005, vol. 26(1), pp. 31-43, which describe techniques for protein engineering, such as affinity maturation, and other techniques for improving the specificity and other desired properties of proteins, such as immunoglobulins. Unless otherwise indicated, all terms not specifically defined herein are known in the relevant art and will be clear to those of skill in the art; for example, Scully et al. (2017, supra) describe a consensus on standardization of terminology in TTP and related thrombotic microangiopathies.

[0174] As used herein, it should be noted that the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a reagent" includes one or more of such various reagents, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art that can be modified or substituted for the method described herein.

[0175] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0176] As used herein, the term "and / or" necessarily implies "and," "or," "or " and "all or any other combination of the elements connected by said term."

[0177] As used herein, the term "about" or "approximately" means within 20%, preferably within 15%, more preferably within 10%, and most preferably within 5% of a given value or range.

[0178] Throughout this specification and the claims which follow, unless the context otherwise requires, the word "comprise," and variations such as "comprises" and "comprising," are understood to imply the inclusion of a recited integer or step or group of integers or steps, but not the exclusion of any other integers or steps or group of integers or steps. As used herein, the term "comprising" may be replaced with the terms "containing" or "including," or, as the case may be, with the term "having" when used herein.

[0179] The therapeutic potential of the polypeptides of the invention, in particular ALX0081, in the setting of TTP was further evaluated and demonstrated in a Phase III (Hercules) trial.

[0180] It has been demonstrated that patients with first aTTP episodes have a later onset of symptoms and a higher disease severity at baseline than patients with disease recurrence episodes.Even in this setting of relatively severe disease, it has been shown that treatment with caplacizumab improves outcomes (Example 7.11).In particular, it has been demonstrated that the polypeptides of the present invention, such as ALX0081 and ALX0081-A, improve outcomes, including a shorter time to platelet count response, a lower proportion of patients who die, relapse, or have severe TE events during treatment, a lower relapse rate during the entire treatment period, and prevention of refractory disease, compared to placebo.

[0181] TTP is an acute disease with recurrent bouts or bouts, i.e., individual recurrent disease events that require immediate treatment for each episode. As used herein, a "first episode of TTP" (also referred to as a "first TTP episode") is the first time a subject has a bout or bout of TTP. As used herein, a "recurrent episode of TTP" (also referred to as a "recurrent TTP episode") is any TTP bout after the first TTP episode.

[0182] The present invention is also based, at least in part, on the discovery that rapid blockade of vWF-mediated platelet adhesion by the polypeptides of the invention, e.g., ALX0081 or ALX0081-A, provides a new treatment option for aTTP. In particular, treatment with caplacizumab resulted in improved treatment outcomes reflected in meaningful reductions in healthcare resource utilization, including days of PE, length of hospital stay, and ICU stay (Example 7.12).

[0183] Furthermore, it was demonstrated that administration of a polypeptide of the present invention, such as ALX0081 or ALX0081-A, comprising at least one ISVD that binds to vWF, was effective in patients experiencing an exacerbation of aTTP (Example 7.13).

[0184] Furthermore, it was demonstrated that administration of a polypeptide of the present invention, such as ALX0081 or ALX0081-A, which comprises at least one ISVD that binds to vWF, was effective in reducing the refractory state of patients.

[0185] Although current therapies for TTP using PE and blood transfusions have significantly reduced mortality from TTP, frontline therapy with the administration of a polypeptide according to the invention comprising at least one ISVD that binds to vWF, such as ALX0081 or ALX0081-A, has been shown to be particularly advantageous. For example, the use of caplacizumab as frontline therapy can buy valuable time.

[0186] Thus, the present invention provides unexpected sustained and prolonged efficacy, reduced exacerbations, reduced hospitalizations, reduced morbidity, reduced number of required PEs, reduced ischemia, reduced refractory states, reduced organ damage, and / or reduced mortality, even in disease settings where the severity of the initial TTP episode is relatively high.

[0187] Thus, the present invention relates to the use of the polypeptide of the present invention for treating or ameliorating vWF-related diseases such as TTP in patients, by the unexpectedly large reduction in time to response, as demonstrated by the promotion of platelet recovery.The present invention also leads to a reduction in the frequency of PE, while unexpectedly maintaining platelet recovery in human patients for a long period of time.Therefore, a method is provided for reducing the time to response in human patients by administering the polypeptide of the present invention to the patient, wherein the amount of the polypeptide administered is effective to change one or more disease markers of TTP, such as platelet count, thrombocytopenia, neurocognitive function, ADAMTS13 level and anti-ADAMTS13 antibody titer, ADAMTS13 activity level, cardiac markers (Troponin T (TnT or cTnT) or Troponin I (TnI or cTnI)), BNP (brain natriuretic peptide) or N-terminal pro-brain natriuretic peptide (NT proBNP), and brain injury markers (such as NSE (neuron specific enolase) and Sβ100 (S100 beta)), preferentially increasing the number of platelets.

[0188] Furthermore, the polypeptides of the invention were safe when administered to human TTP patients as assessed by safety laboratory markers such as RICO, vWF and FVIII chromogen, with a possible increased bleeding risk that was entirely manageable.

[0189] These markers can be measured using standard methods known and used by those of skill in the art, such as various immunologically-based assays, including enzyme-linked immunosorbent assay (ELISA; also known as enzyme immunoassay (EIA)), radioimmunoassay, or immunoenzymatic assays. Chemical, colorimetric, and enzyme-based assays can also be used, if suitable.

[0190] Thus, the present invention provides a polypeptide, preferably ALX0081 or ALX0081-A, comprising at least one ISVD against vWF, for use in treating a vWF-related disease, such as TTP, in a human in need of such treatment by repeatedly administering 5 to 40 mg doses of said polypeptide to the human, wherein said dose is followed by a first PE within 15 minutes to 4 hours.

[0191] The polypeptides of the present invention are administered at a particular time relative to the PE procedure as an adjunctive treatment to treat or prevent (e.g., reduce or ameliorate one or more symptoms associated with) a vWF-associated disease, such as TTP.

[0192] The term "treat" refers to administering a therapeutic agent in an amount, manner, and / or mode effective to improve a condition, symptom, or parameter associated with a disease, either to a statistically significant extent or to a degree detectable by one of skill in the art, or to prevent the progression of a disease. In therapeutic use, treatment refers to the administration of a therapeutic agent to improve a disease or condition in a subject. It may improve, cure, maintain, or reduce its duration. In therapeutic use, the symptoms of the subject may be partially or completely manifested. In typical cases, the treatment improves the subject's disease or condition to a degree detectable by a physician, or prevents the disease or condition from worsening. For example, the clinical features and signs of acute episodes of TTP are improved, as shown in Table 1, or as presented in the TTP treatment guidelines (Scully et al., 2012, supra). For example, the treatment results in normalization of platelet count, reduction in ADAMTS13 autoantibody titer, and / or increase in ADAMTS13 activity, all of which are known in the art and / or further detailed herein (see below). The effective amount, manner, or mode may vary depending on the subject and may be tailored to the subject.

[0193] The term "treatment period" refers to the interval of time during which a patient is treated, which may include administration of PE, and / or a polypeptide of the invention, and optionally other drugs, such as, for example, steroids or rituximab. In a preferred embodiment, a treatment period refers to administering a polypeptide of the invention to a patient in need thereof, wherein said administration is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or even for a period longer than 10 days, such as 20 days, preferably for a period longer than 30 days, such as 2 months, 3 months, 4 months, 5 months, 6 months, or even more.

[0194] As used herein, the term "prevent" refers to alleviating the symptoms of the disorder referred to. In particular, the term encompasses the full range of therapeutically beneficial effects of administering to a subject a polypeptide of the present invention, including the reduction, alleviation, and amelioration of vWF-related disorders, such as TTP, and their symptoms. The term "prevent" includes preventing or postponing the onset of disease, preventing or postponing the onset of symptoms, and / or reducing the severity of symptoms that will or are expected to occur. These further include ameliorating existing symptoms, preventing further symptoms, and ameliorating or preventing the underlying cause of symptoms.

[0195] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the terms "subject" and "subjects" refer to animals, such as mammals, including non-primates (e.g., cows, pigs, horses, donkeys, goats, camels, cats, dogs, guinea pigs, rats, mice, sheep) and primates (e.g., monkeys such as cynomolgus monkeys, gorillas, chimpanzees, and humans). "Patient" preferably refers to humans. The patient may include elderly, adults, adolescents, and children of any age, such as children in the range of 2 to less than 12 years, adolescents in the range of 12 to less than 18 years, adults in the range of 18 to less than 65 years, and elderly people aged 65 years or older.

[0196] Non-limiting examples of vWF-associated 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.

[0197] PE procedures for treating or preventing vWF-related diseases, such as TTP, are described in consensus and guidelines for the diagnosis and management of TTP and other thrombotic microangiopathies (Scully et al., 2017, supra), which are expressly incorporated herein by reference. Complete remission is defined as normal platelet counts, i.e., 150,000 / μl or greater, and, optionally, the absence of exacerbations (Scully et al., 2012, 2017, supra).

[0198] As used herein, "time to response" refers to the time to response after an acute episode of TTP, e.g. The time between the first treatment of a patient with an initial and / or recurrent episode of TTP and a platelet count of 150,000 / μl or greater, wherein the first treatment is administration of PE or a polypeptide of the invention, or both, whichever occurs first.

[0199] As used herein, the term "refractory state" or "refractory" refers to persistent thrombocytopenia, lack of persistent platelet count increase, or a platelet count increase of 50% or more despite 5 rounds of PE and steroid treatment. * 10 9 L -1 Persistently elevated LDH levels (>1.5 ULN) refers to subjects who fail to achieve remission or whose platelet counts and LDH levels improve but who worsen despite continued treatment.

[0200] The term "plasma exchange" ("PE" or "PEX") refers to a therapeutic procedure used to treat various diseases, including TTP, by bulk removal of plasma, i.e., removing a large amount of plasma (usually 1-1.5 plasma volumes) and replacing it with replacement fluid (Winters, 2012, Hematology ASH Education Book 1:7-12). Through bulk removal and replacement of plasma, PE removes pathological materials such as autoantibodies against ADAMTS13 and ULvWF, but also removes some platelets. Plasma is used as replacement fluid to replenish ADAMTS13 in treating TTP (McLeod Best Pract Res Clin Haematol. 2006;19:157-167). Bulk removal and replacement of plasma also affects laboratory tests, complicating patient testing.

[0201] Because PE involves the bulk removal of plasma, everything circulating in the plasma is removed, and thus the procedure is nonselective, removing both normal and pathological plasma components, as well as any drugs administered prior to PE to treat TTP.

[0202] Despite the advantages of PE in the treatment of TTP, the present invention shows that caplacizumab is beneficial when it is started as a frontline therapy. The term "frontline" therapy (or frontline treatment) refers to the first treatment (also called "first-line" treatment) given for a disease, such as TTP. Following frontline therapy, additional treatments such as PE and supplementary immunosuppressive treatment (e.g., corticosteroids such as (methyl)-prednisolone or (methyl)-prednisone; or rituximab), antiplatelet agents (e.g., aspirin), supportive therapy using red blood cell transfusion or folic acid supplementation, vincristine or cyclosporine, anti-autologous ADAMTS13 antibody, or treatment using ADAMTS13 may be added or used instead.

[0203] Those skilled in the art are familiar with determining the number of platelets. Platelet counts can be done by any method known in the art, such as manual counting using a hemocytometer or by automated analyzer methods, such as electronic counting. Counts can also be estimated during blood smear examination. In this microscopic method, a phase contrast microscope is used to view the blood on a hemocytometer slide. Electronic counting of platelets is the most common method. There are two types of electronic counting: voltage pulse counting systems and electro-optical counting systems. For example, the ADVIA® hematology analyzer can be used to obtain a platelet count and the obtained count can be verified by estimating the count in a Wright stained blood smear. ADVIA measures platelets by flow cytometry, which is based on the principle of light scattering. For example, platelets are identified by their size (<30 FL, low angle light scatter) and refractive index (n=1.35 to n=1.40, or high angle light scatter).

[0204] In various patients following an acute episode of TTP, e.g., a first TTP episode and / or a recurrent TTP episode, at least one antibody to vWF is detected after the patient has undergone PE ("prior PE"; PE preceding administration of a first dose of a polypeptide of the invention). The subjects were administered a polypeptide of the invention comprising one ISVD, such as ALX0081 or ALX0081-A.

[0205] Thus, the present invention relates to administering a PE (preceding PE) to a patient in need of PE, e.g., a patient having an acute episode of TTP, e.g., a first TTP episode and / or a recurrent TTP episode, followed by a subsequent PE within 24 hours of said preceding PE, and administering a polypeptide of the present invention ("first dose") about 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 even 5 minutes before the start of said subsequent PE, e.g., 6 hours to 15 minutes before the start of said subsequent PE ("first PE"). In the present invention, the term "first dose" refers to the first administration of a polypeptide of the present invention to a patient in need of administration, e.g., after an acute episode of TTP, e.g., a first TTP episode and / or a recurrent TTP episode.

[0206] In one embodiment, administration of a polypeptide of the invention to a patient, preferably the first dose, is followed by PE within 5 minutes to 8 hours, such as 10 minutes to 6 hours or 15 minutes to 4 hours, such as within 8 hours, within 7 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 3 hours, within 1 hour, within 45 minutes, within 30 minutes, within 20 minutes, within 15 minutes, within 10 minutes, or even within 5 minutes.

[0207] In the present invention, the term "first PE" refers to the first PE that is performed after (or optionally simultaneously with) administration of the first dose of a polypeptide of the present invention to a patient.

[0208] The polypeptide of the present invention can be administered in the form of a solution (e.g., injection and infusion solution) or can be used for administration in the form of a solution. Such compositions can be administered parenterally (e.g., subcutaneous, intraperitoneal, or intramuscular injection) or by inhalation. As used herein, the terms "parenteral administration" and "administered parenterally" refer to administration modes other than enteral and topical administration, usually by injection, and include subcutaneous (sc) or intramuscular administration, as well as intravenous (iv), intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcuticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Preferably, the second or further dose of the polypeptide of the present invention described herein is administered subcutaneously.

[0209] Preferably, the administration of the first dose of the polypeptide of the present invention after an acute episode of TTP, such as an initial TTP episode and / or a recurrent TTP episode, is an intravenous bolus injection administered once over 1 or 2 minutes, e.g., delivering the polypeptide through an intravenous line. Even more preferably, the administration of the first dose of the polypeptide of the present invention after an acute episode of TTP, such as an initial TTP episode and / or a recurrent TTP episode, is an intravenous push injection administered once over a period of less than about 30 seconds, e.g., delivering the polypeptide through an intravenous line.

[0210] Considering that the polypeptides of the invention are safe to use as demonstrated in previous studies in healthy volunteers and in this study in TTP patients (see Examples), that TTP can be difficult to diagnose, especially in a first TTP episode, and that time lost before treatment begins can be a difficult situation, the inventors concluded that this discovery has the advantage that treatment with the polypeptides of the invention can be started in a timely manner, even before the patient enters the hospital, for example, immediately in the ambulance. Preferably, the polypeptides of the invention, e.g., ALX0081 or ALX0081-A, are administered by intravenous push injection, which can be easily performed outside of a hospital, however. This will save you valuable time.

[0211] Thus, the present invention relates to administering a polypeptide of the invention to a patient in need thereof, such as, for example, a patient having an acute episode (acute attack) of TTP, e.g., a first TTP episode and / or a recurrent TTP episode, about 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 even 5 minutes, for example 6 hours to 15 minutes, prior to the onset of PE (the "first dose").

[0212] In one embodiment, the administration of a first dose of a polypeptide of the invention after an acute episode of TTP, such as a first TTP episode and / or a recurrent TTP episode, is followed by a PE ("first PE"). This first PE, whether preceded by a previous PE or not, is followed by the administration of a second or further dose of a polypeptide of the invention ("second dose" or "further dose"). Preferably, the second or further dose is administered within 120, 90, or 60 minutes, such as within 1 to 60 minutes, such as within 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or even 1 minute, after the first PE. In some cases, it may be advantageous to administer the second or further dose together with or simultaneously with replacement fluid, such as plasma of the PE.

[0213] In a further embodiment, the first dose, second dose or further dose of the polypeptide of the 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 5-40 mg, even more preferably 10 mg or 11 mg, which can be administered to a patient in need thereof, preferably daily. For administration to younger patients, such as children and adolescents, the dose may be adjusted to the patient's body weight. In certain embodiments, the dose is about 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 for a 70 kg adult. In other embodiments, if the child or adolescent weighs less than 40 kg, the dose is about 5 mg.

[0214] In one embodiment, the invention relates to administration of about 5 to 40 mg, preferably 10 mg or 11 mg, of a polypeptide of the invention, e.g., ALX0081 or ALX0081-A, within 1 to 60 minutes after a PE procedure, e.g., a first PE, a second PE, or a further PE.

[0215] In one embodiment, a polypeptide of the invention, e.g., ALX0081 or ALX0081-A, is administered once or twice daily to a TTP patient in need thereof, preferably a patient having a platelet count below 100,000 per μl of plasma and / or an ADAMTS13 activity below 10%, e.g. below 5%.

[0216] In a further embodiment, a TTP patient in need of treatment is treated with (i) PE; and (ii) a dose of 5 to 40 mg, preferably 10 mg or 11 mg, of said polypeptide 60 minutes to 1 minute after said PE of step (i), wherein steps (i) and (ii) are repeated once or twice daily until said patient's platelet count is at least 50,000 per μl of plasma, such as 75,000, 100,000, 125,000, or even 150,000 per μl of plasma.

[0217] In some cases, it may be advantageous to repeat steps (i) and (ii) after complete remission (platelet count of 150,000 or more per μl of plasma) for a minimum of 2 days.

[0218] In one embodiment, 5 to 40 mg of the polypeptide of the invention is administered once or twice daily for at least 5, 10, 15, 20, 25, 30, 60, 90 or even 120 days after the patient's platelet count is 150,000 or more per μl of plasma, in particular after the patient's ADAMTS13 activity is 10% or less, such as 5% or less, or after the last PE.

[0219] Thus, administration of a polypeptide comprising at least one ISVD against vWF, e.g., ALX0081 or ALX0081-A, to a human TTP patient following an acute episode of TTP, e.g., a first TTP episode and / or a recurrent TTP episode, results in an unexpected reduction in the time to response, regardless of the order of administration of the polypeptide and the PE, e.g., whether PE is performed before or after administration of the first dose of a polypeptide of the invention.

[0220] Surprisingly, it was further found that the number of exacerbations was reduced when patients were switched to open-label ("OL") caplacizumab during the double-blind (DB) treatment period, in conjunction with resumption of daily plasma exchange (PEX) and immunosuppression, while maintaining blinding to the initial treatment assignment (see Example 7.13).

[0221] As used herein, the term "exacerbation" refers to recurrent thrombocytopenia that occurs after a confirmed platelet response and requires reinitiation of daily PE treatment more than 1 day and up to 30 days after the last daily PE session.

[0222] This indicates that the polypeptides of the invention, such as ALX0081 or ALX0081-A, may be the sole cause of treatment and / or alleviation of (symptoms of) TTP.

[0223] Thus, the present invention relates to a polypeptide, such as ALX0081 or ALX0081-A, comprising at least one ISVD against vWF, for use in treating a vWF-related disease such as TTP in a human in need of such treatment, by administering to said human a dose of 1 to 80 mg or 5 to 40 mg of said polypeptide, such as 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 or 11 mg, if said human has a body weight of 40 kg or more, or 5 mg if said body weight is less than 40 kg.

[0224] Based on the surprising observations herein, the inventors have designed a further optimized treatment protocol that is essentially based on the idea that the distribution of platelet response confirmation times is shorter in the CAP (caplacizumab) arm compared to the placebo arm, without skew and bias to the right (longer time to response). In this further optimized treatment protocol, all subjects were treated with a constant PE treatment period set for 3 to 5 days, such as 3 days or 4 days or 5 days, preferably 3 days. In this case, the PE treatment period can be independent of platelet recovery (150,000 / μl or more). In the further optimized treatment protocol, the burden and cost of the patient is reduced.

[0225] Thus, the present invention relates to a method for the treatment of a polypeptide comprising at least one ISVD against vWF, e.g., ALX0081 or ALX0081-A, comprising: (i) performing PE; and (ii) administering a dose of 5 to 40 mg, e.g., 10 mg or 11 mg, of the polypeptide of the present invention 15 minutes to 4 hours after said PE in step (i), wherein steps (i) and (ii) are administered for a period of 3 to 5 days, such as 3 days, 4 days, or 5 days, preferably. or once daily for at least 3 days; followed by administration of a dose of 5-40 mg, e.g., 10 mg or 11 mg, of said polypeptide once daily for at least 10 days, e.g., at least 20 days or at least 30 days, and / or for at least 10 days, e.g., at least 20 days or at least 30 days, after said patient's platelet count first reaches 150,000 / μl or greater, The present invention relates to a polypeptide for use in treating a vWF-associated disease in a human in need of such treatment.

[0226] In this study, TTP patients were followed for remission for up to one year. As used herein, the term "remission" refers to confirmed platelet response and absence of exacerbation. As used herein, the term "confirmed platelet response" refers to the time to treatment response defined as recovery of ≥150,000 / μL platelets, which must be confirmed 48 hours after the first documented platelet recovery of >150,000 / μL with a new measurement of ≥150,000 / μL platelets and LDH preferably ≤2×ULN.

[0227] As mentioned above, platelet count is the main means to evaluate remission. Measurement of ADAMTS13 activity in patients with a history of classical TTP is important because low levels have been shown to predict relapse. However, it is currently unclear (and data is conflicting) as to 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 of ADAMTS13 are performed under static conditions and do not necessarily accurately reflect the physiological changes that occur in vivo (http: / / practical-haemostasis.com / Miscellaneous / Miscellaneous%20Tests / adamts13_assays.html).

[0228] Amelioration appears to be more pronounced in the subgroup of subjects with low baseline ADAMTS13 activity (i.e., less than 10%, e.g., less than 5%) at the start of treatment (e.g., administration of a first dose of a polypeptide of the invention, e.g., ALX0081 or ALX0081-A).

[0229] Thus, the present invention relates to a polypeptide comprising at least one ISVD against vWF for use in treating a vWF-related disease in a human in need of such treatment by administering to said human a first dose of 1-40 mg, preferably 10 mg or 11 mg, of said polypeptide until said human has a platelet count of 150000 / μl or more. In a preferred embodiment, said human has less than 10%, such as less than 5%, of ADAMTS13 activity when administered said polypeptide.

[0230] As used herein, the term "relapse" refers to a new event of TTP occurring more than 30 days after the last daily PE.

[0231] ADAMTS13 activity is a predictive marker for TTP recurrence and possible treatment decisions. ADAMTS13 activity can predict recurrences occurring shortly after cessation of caplacizumab treatment. Such recurrences are considered as recurrent symptomatic TTP episodes (unresolved disease activity based on consistently low ADAMTS13 activity). It has been demonstrated that the duration of treatment with caplacizumab for up to 30 days (post-PE) has a significant impact on the number of exacerbations. Thus, extending the caplacizumab treatment period in patients at risk of recurrence (i.e., patients with underlying disease activity based on ADAMTS13 activity) maintains the protective effect of caplacizumab until the underlying disease is adequately treated and resolved. Conversely, caplacizumab Prophylaxis with izumab reduces the risk of (new) acute episodes of TTP.

[0232] Thus, treatment with the polypeptide of the invention, e.g., ALX0081 or ALX0081-A, should be continued for a longer period of time compared to patients with high ADAMTS13 activity. The polypeptide of the invention should be administered to TTP patients to reduce the risk of recurrence and / or prevent the chance of recurrence until the ADAMTS13 activity is at least 10%, e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, or even 50% compared to normal or baseline activity.

[0233] Thus, the present invention relates to a polypeptide comprising at least one ISVD against vWF for use in reducing and / or preventing the risk of acute episodes of TTP, such as a first TTP episode and / or a recurrent TTP episode, in a human in need thereof, comprising step (i): administering to said human a dose of 5-40 mg, preferably 10 mg or 11 mg, of said polypeptide. Preferably, said risk is reduced by at least a factor of 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 or more, such as 3, 4, 5, 6, 7, 8, 9 or even 10 or even more, such as 20, 50 or even 100. Preferably, said risk is reduced by 10% or even more, such as 20%, 30%, 40%, 50%, 60% or more, such as 80% or even 100%.

[0234] Thus, the present invention relates to a polypeptide as described herein for use in treating and / or preventing a vWF-associated disease in a human in need of such treatment and / or prevention as described herein, wherein said step of administering said polypeptide to said human is repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or even more than 10 times, such as 20 times, preferably more than 30 times or even more.

[0235] Thus, the present invention relates to a polypeptide as described herein for use in treating and / or preventing a vWF-associated disease in a human in need of such treatment and / or prevention, wherein said step of administering said polypeptide to said human is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or even for a period longer than 10 days, such as 20 days, preferably for a period longer than 30 days, such as 2 months, 3 months, 4 months, 5 months, 6 months, or even more.

[0236] Thus, the present invention relates to a polypeptide as described herein for use in treating and / or preventing a vWF-associated disease in a human in need of such treatment and / or prevention, wherein said dose is administered once daily or twice daily.

[0237] The present invention therefore relates to a polypeptide as described herein for use in treating and / or preventing a vWF-related disease in a human in need of such treatment and / or prevention, said treatment and / or prevention comprising: (ii) comparing the ADAMTS13 activity to a baseline ADAMTS13 activity; (iii) repeating step (i) of administering the polypeptide to the human if the ADAMTS13 activity is less than 30%, e.g., 20%, 15%, 10%, or 5%, of the baseline ADAMTS13 activity. Further includes:

[0238] Thus, the present invention provides a method for treating and / or preventing a vWF-associated disease in a human in need of such treatment and / or prevention, comprising administering to the human anthracycline or medicament ... With respect to polypeptides, wherein said ADAMTS13 activity in said patient is measured and / or compared daily, or every 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, preferably at least once a week.

[0239] Thus, the present invention relates to a polypeptide as described herein for use in treating and / or preventing a vWF-related disease in a human in need of the treatment and / or prevention described herein, wherein the step of administering a polypeptide of the present invention to the human is repeated until the ADAMTS13 activity is at least 5%, 10%, 15%, for example 20%, or even 30% or more of the baseline ADAMTS13 activity.

[0240] The present invention therefore relates to a polypeptide comprising two anti-human von Willebrand factor (vWF) immunoglobulin single variable domains (ISVDs), for use in treating (a symptom of) an exacerbation of a vWF-related disease in said human, comprising administering to said human a dose of 1 to 80 mg, preferably 5 to 40 mg, even more preferably 10 mg or 11 mg of said polypeptide once daily or twice daily until the ADAMTS13 activity is greater than 10% of the baseline ADAMTS13 activity; optionally repeating said administration step for a period of at least 2 days, such as at least 5 days, 7 days or even more, such as 14 days, 21 days or even at least 1 month, until the ADAMTS13 activity is greater than 10% of the baseline ADAMTS13 activity.

[0241] The present invention therefore relates to a polypeptide comprising two anti-human von Willebrand factor (vWF) immunoglobulin single variable domains (ISVDs), for use in treating (symptoms of) a recurrence of a vWF-related disease in said human, comprising administering to said human a dose of 1 to 80 mg, preferably 5 to 40 mg, even more preferably 10 mg or 11 mg of said polypeptide once daily or twice daily until the ADAMTS13 activity is greater than 10% of the baseline ADAMTS13 activity; optionally repeating said administration step for a period of at least 2 days, such as at least 5 days, 7 days or even more, such as 14 days, 21 days or even at least 1 month, until the ADAMTS13 activity is greater than 10% of the baseline ADAMTS13 activity.

[0242] Thus, the present invention relates to a polypeptide as described herein for use in treating and / or preventing a vWF-associated disease in a human in need of such treatment and / or prevention as described herein, wherein the step of administering said polypeptide to said human is repeated until said ADAMTS13 activity is at least 5%, 10%, 15%, such as 20% or 30% of said baseline ADAMTS13 activity in at least two consecutive measurements. Preferably, said two consecutive measurements are separated by at least 24 hours, more preferably 48 hours, such as at least 3 days, or even longer, such as 4, 5, 6 or even 7 days, preferably one week.

[0243] Thus, the present invention relates to a polypeptide as described herein for use in treating and / or preventing a vWF-associated disease in a human in need of such treatment and / or prevention as described herein, wherein the step 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 even for a period longer than 10 days, such as 20 days, preferably longer than 30 days or even longer, after the ADAMTS13 activity is at least 5%, at least 10%, at least 15%, such as 20% or at least 30% of the baseline activity in at least two consecutive measurements.

[0244] Therefore, the present invention relates to a polypeptide comprising at least one ISVD for vWF, comprising: step (i): administering 5 to 40 mg, preferably 10 mg or 11 mg, of said polypeptide; 5. The method of claim 1, wherein the polypeptide is administered to a human in need of such treatment at a dose of about 100 mg / kg or more, and / or about 100 mg / kg or more, and / or about 150 mg / kg or more, and / or about 150 mg / kg or more, wherein the reduction and / or prevention is - measuring ADAMTS13 activity in said patient; - comparing said ADAMTS13 activity to a reference ADAMTS13 activity; - if the ADAMTS13 activity is 5% or more, such as 10% or more, or even 15% or more, or 20% or 30% or more of the reference ADAMTS13 activity, repeating step (i) for up to 30 days, such as up to 20 days, or even 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, or even 1 day. Further includes:

[0245] The present invention therefore relates to a polypeptide comprising at least one ISVD for vWF, comprising at least the following steps: (i) optionally measuring ADAMTS13 activity in said patient; (ii) optionally comparing said ADAMTS13 activity to a reference ADAMTS13 activity; (iii) administering to the human a dose of 5 to 40 mg, preferably 10 mg or 11 mg, of the polypeptide comprising at least one ISVD for vWF, if the ADAMTS13 activity is less than 30%, 20%, 15%, 10%, or 5% of the baseline activity. The present invention relates to a polypeptide comprising:

[0246] As used herein, reducing the risk or incidence includes a decrease in the probability or incidence of a sign, symptom, or consequence of a vWF-related disease, such as TTP, in a subject compared to a relevant (e.g., untreated) control population, or to the same subject prior to treatment according to the present invention.

[0247] As used herein, signs, symptoms, or outcomes of vWF-related diseases such as TTP include organ damage, ischemic damage, microthrombus formation, exacerbation, mortality, relapse, refractory state, one or more disease markers of vWF-related diseases such as TTP, such as platelet count, thrombocytopenia, neurocognitive function, ADAMTS13 levels and anti-ADAMTS13 antibody titers, ADAMTS13 activity levels, cardiac markers (Troponin T or Troponin I), BNP (brain natriuretic peptide) or N-terminal pro-brain natriuretic peptide (NT proBNP), creatinine, and brain damage markers (e.g., NSE (neuron specific enolase) and Sβ100 (S100 beta)), preferentially any one of LDH levels, Troponin T and / or Troponin I levels, and / or creatinine levels.

[0248] The reduction of risk or incidence can include delaying or preventing the occurrence of signs, symptoms, or consequences of vWF-related diseases such as TTP.The risk or incidence can also be reduced when the severity of signs, symptoms, or consequences of vWF-related diseases such as TTP is reduced to a level that is not clinically relevant.That is, signs, symptoms, or consequences of vWF-related diseases such as TTP may exist, but at a level that does not endanger the life, activity, and / or health of the subject.In some situations, the occurrence of vWF-related diseases such as TTP is reduced to a level that the subject does not show any signs of vWF-related diseases such as TTP during and / or after the treatment period.

[0249] It is understood that when a treatment is provided, there is no actual evidence of a reduced individual risk, since it is impossible to say whether signs, symptoms, or consequences of vWF-related diseases such as TTP would have occurred or would have occurred relatively quickly in the absence of such treatment. Therefore, the concept of risk, and the increased or reduced risk, are merely references to statistics. Furthermore, the reduced risk of signs, symptoms, or consequences of vWF-related diseases such as TTP may be reflected in the reduced severity of signs, symptoms, or consequences of vWF-related diseases such as TTP, as well as the absence or delayed observation of signs, symptoms, or consequences of vWF-related diseases such as TTP.

[0250] It is understood that the polypeptide of the present invention reduces the risk and / or prevents acute episodes of TTP, such as first TTP episode and / or recurrent TTP episode.Therefore, it also reduces the signs, symptoms or consequences of acute episodes of TTP, such as first TTP episode and / or recurrent TTP episode.In view of the pathophysiology of acquired TTP, in which ULvWF strings consume platelets in forming microthrombi, it is determined that the recovery of platelet count is an indirect measure of the prevention of further microthrombi formation.The morbidity and acute mortality associated with acquired TTP are the result of these microthrombi.

[0251] Indeed, this inference 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 receiving the polypeptide of the present invention, for example, ALX0081 or ALX0081-A, than in subjects receiving placebo (see examples).

[0252] Thus, the results suggest that an increased rate of normalization of these organ injury markers is associated with improved clinical outcomes, i.e., reduced risk and extent of organ injury due to microthrombus-induced organ ischemia, and reduced refractory state.

[0253] Accordingly, the present invention provides a method according to the present invention, comprising the steps of: - the risk of organ damage, ischemic damage and / or microthrombus formation is reduced by 10%, 20%, 30%, preferably at least 40%, or even at least 50%, such as 60%, 70%, 80%, 90%, or even 100% (e.g. to the absence of organ damage, ischemic damage and / or microthrombus formation due to vWF-related diseases); - the risk of organ damage, ischemic damage, 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 or more, such as 3, 4, 5, 6, 7, 8, 9, or even 10, or even more, such as 20, 50, or even 100; - organ damage, ischemic damage and / or microthrombus formation is preferably reduced by at least 10%, 20%, 30%, 40% or even at least 50%, such as by 60%, 70%, 80%, 90% or even up to 100%; - organ damage, ischemic damage, 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 or more, such as 3, 4, 5, 6, 7, 8, 9, or even 10, or even more, such as 20, 50, or even 100; - markers of organ damage, such as LDH levels, troponin T, troponin I levels, and / or creatinine levels, return to at least 40% of normal levels, or even at least 50%, such as 60%, 70%, 80%, 90%, or even 100%; - markers of organ damage such as LDH levels, troponin T, troponin I levels, and / or creatinine levels are at least 20%, e.g. 30%, or lower than normal levels or even more, such as 40%, or even 60%, 70%, 80%, 90%, or even 100% (preferably, said markers of organ damage, such as LDH levels, troponin T, troponin I levels, and / or creatinine levels, are improved within less than 30 days of treatment, preferably within less than 20 days of treatment, e.g., within 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or even within 1 day), - Platelet count is maintained at 150,000 / μl or higher. - the time to platelet normalization (>150000 / μl) is reduced by at least 10%, 20%, 30%, 35%, 39%, preferably by at least 40%, or even by at least 50%, for example by 60%, 70%, 80%, - the risk of progression is reduced by at least 10%, 20%, 30%, 40%, or even at least 50%, such as by 60%, 70%, 80%, 90%, or even 100%; - the risk of progression is reduced by a factor of 2 or more, e.g. 3, 4, 5, 6, 7, 8, 9, or even 10, or even more, e.g. 20, 50, or even 100; - the mortality rate attributable to said vWF-associated disease is reduced by 10%, 20%, 30%, preferably by at least 40%, or even by at least 50%, such as by 60%, 70%, 80%, 90%, or even by 100%; - the mortality rate attributable to said vWF-associated disease is reduced by a factor of 2 or more, such as 3, 4, 5, 6, 7, 8, 9, or even 10, or even more, such as a factor of 20, 50, or even 100; - remission is increased by a factor of 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 or more, such as 3, 4, 5, 6, 7, 8, 9, or even 10 or even more, such as 20, 50 or even 100, and refractory status is reduced, preferably by at least 10%, 20%, 30%, 40% or even by at least 50%, such as by 60%, 70%, 80%, 90% or even by 100%; and / or - refractory state is reduced by a factor of 2 or more, e.g. 3, 4, 5, 6, 7, 8, 9, or even 10, or even more, e.g. 20, 50, or even 100; It concerns the method.

[0254] The term "reference activity" as used herein refers to the average ADAMTS13 activity of 5 healthy subjects in the assay carried out, which is set to 100%.For example, in the fluorescence resonance energy transfer (FRET)-vWF73 assay, a normal human plasma pool is used to generate a calibration curve, where the slope of the regression curve is calculated for each calibration sample and used to generate a calibration curve (trend line: y=ax+b; where x=ADAMTS13(%) and y=delta RFU / delta time).Then, as explained by Kokame et al. (Br J Haematol. 2005, vol. 129:93-100), the ADAMTS13 activity (%) of the sample is calculated as follows: (yb)×1 / a.In fact, relapsed patients generally have lower ADAMTS13 activity than non-relapsed patients.

[0255] The present invention therefore relates to a polypeptide for reducing and / or preventing the risk of ischemic damage, organ damage and / or microthrombus formation, e.g. caused by a vWF-related disease such as TTP, an episode of TTP, e.g. a first TTP episode and / or a recurrent TTP episode, in a human in need of such reduction and / or prevention, said reduction and / or prevention being achieved by the administration of at least one immunoglobulin single variable domain against von Willebrand factor (vWF) ( The method comprises the step (i) of administering to said human a dose of 5-40 mg / day, preferably 10 mg / day or 11 mg / day, of a polypeptide comprising an ISVD; wherein administration of said 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 even at least 50%, such as by 60%, 70%, 80%, 90%, or even up to 100%. Preferably, administration of said polypeptide reduces and / or prevents the risk of ischemic injury, organ damage, refractory state, and / or microthrombus formation by a factor of 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 or more, such as 3, 4, 5, 6, 7, 8, 9, or even 10 or even more, such as 20, 50, or even 100.

[0256] Thus, the present invention relates to a polypeptide as described herein for reducing the risk of and / or preventing ischemic damage, organ damage, refractory states and / or microthrombus formation, wherein said step of administering said polypeptide is repeated for at least 1, 2, 3, 4, 5, 6, 7 days, or even longer periods such as 1 week, 2 weeks, 3 weeks, or even longer periods such as 1 month, or even 2 months.

[0257] Thus, the present invention relates to a polypeptide for reducing the risk of and / or preventing ischemic damage, organ damage, refractory states and / or microthrombus formation as described herein, further comprising measuring the ADAMTS13 activity in said patient, preferably once a week.

[0258] Thus, the present invention relates to a polypeptide for reducing the risk of and / or preventing ischemic damage, organ damage, refractory state, and / or microthrombus formation as described herein, wherein said step (i) of administering said polypeptide is repeated for at least 1, 2, 3, 4, 5, 6, 7 days, or even longer periods such as 1 week, 2 weeks, 3 weeks, or even longer periods such as 1 month, or even 2 months, when ADAMTS13 activity is [for the first time] 5% or more, such as 10% or more, or even 15% or more of baseline ADAMTS13 activity.

[0259] Thus, the present invention relates to a polypeptide of the present invention for treating a symptom of a vWF-related disease, such as TTP, in a human suffering from said disease, said treatment comprising administering to a subject a polypeptide of the present invention in an amount effective to treat a symptom of a vWF-related disease in a human suffering from said disease.

[0260] Thus, the present invention relates to a polypeptide of the present invention for inhibiting the onset or progression of a vWF-associated disease such as TTP in a human, as described herein, which inhibition is brought about by a polypeptide comprising at least one ISVD for vWF binding to vWF, and which comprises administering an inhibitory effective amount of said polypeptide to a human at predetermined intervals, wherein each administration of the antibody delivers to the human 0.1 mg to 25 mg of said polypeptide per kg body weight of the human, preferably 10 mg or 11 mg of said polypeptide if said human has a body weight of 40 kg or more, and 5 mg if said human has a body weight of less than 40 kg, so as to inhibit the onset or progression of the disease in the human.

[0261] Accordingly, the present invention relates to a polypeptide for reducing the likelihood that a human will suffer ischemic organ damage due to a vWF-associated disease, comprising administering to the human a dose of a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) against von Willebrand factor (vWF) as described herein, wherein each administration of the antibody is from 0.1 mg / kg to 1.0 mg / kg body weight of the human such that the likelihood of the human suffering ischemic organ damage is reduced. In one embodiment, the polypeptide is delivered to a human from the amount of 10 to 25 mg, preferably 10 or 11 mg if the human weighs 40 kg or more, and 5 mg if the human weighs less than 40 kg.

[0262] Modeling based on these results indicates that sustained administration of the polypeptides of the invention over a long period of time is effective in preventing acute episodes. This favorable profile results in reduced adverse health outcomes. It can therefore be concluded that the polypeptides of the invention prevent relapse.

[0263] Thus, the present invention relates to administering the polypeptide of the present invention at a dose ranging from 1 to 80 mg, such as 5 to 40 mg, every 1, 2, 3, 4, 5, 6, 7 days, or even every 2, 4, 6, or 8 weeks, preferably in preventing acute episodes of TTP. A particular effective dose is 10 to 20 mg. In a particular embodiment, 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 or 11 mg, of a polypeptide comprising at least one ISVD for vWF, such as ALX0081 or ALX0081-A. A particular effective dose in humans weighing less than 40 kg, such as children and adolescents, is 2 to 10 mg. In certain embodiments, the dose comprises about 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg, preferably 5 mg, of a polypeptide comprising at least one ISVD against vWF, such as ALX0081 or ALX0081-A.

[0264] In one embodiment, the present invention provides a method for preventing relapse in a TTP patient, comprising: (1) optionally measuring ADAMTS13 activity from a TTP patient by an assay, such as a direct assay or an indirect assay; (2) optionally comparing ADAMTS13 activity in TTP patients with a reference value (normal value); (3) administering a polypeptide of the invention, e.g., ALX0081 or ALX0081-A, if the TTP patient has ADAMTS13 activity less than 15%, e.g., less than 10% and less than 5%, of the baseline value; and thereby preventing recurrence.

[0265] Preliminary results suggest that simply administering a first dose of the polypeptide of the invention prior to the first PE results in an increase in platelet count.

[0266] Thus, the present invention relates to administering to a patient in need of a polypeptide of the present invention, e.g. a patient experiencing an acute episode of TTP, e.g. a first and / or recurrent episode of TTP, a polypeptide comprising at least one ISVD against vWF, e.g. ALX0081 or ALX0081-A, at 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 or 11 mg.

[0267] A polypeptide of the present invention, e.g., ALX0081 or ALX0081-A, comprising at least one ISVD against vWF, may be administered alone or in combination with a second agent, e.g., a therapeutically or pharmacologically active second agent, to a subject (e.g., a human subject) to treat or prevent (e.g., reduce or ameliorate one or more symptoms associated with) a vWF-associated disease, e.g., TTP.

[0268] The polypeptides of the invention, which can be used as therapeutically or pharmacologically active second agents or which contain at least one ISVD against vWF, e.g. ALX008 Non-limiting examples of agents that can be combined with ALX0081-A or ALX0081-1 include, for example, adjunctive immunosuppressive treatment (e.g., corticosteroids such as (methyl)prednisolone or (methyl)-prednisone; or rituximab), antiplatelet agents (e.g., aspirin), supportive therapy using red blood cell transfusions or folic acid supplementation, vincristine or cyclosporine, anti-self ADAMTS13 antibodies, or treatment with ADAMTS13. Such combination therapies can advantageously utilize lower dosages of the administered therapeutic agents, thereby avoiding potential toxicities or complications associated with various monotherapies.

[0269] In one embodiment, the present invention relates to a combination therapy of the polypeptide of the present invention in combination with an immunosuppressive treatment, in particular rituximab, to effectively prevent relapse in TTP patients. Preferably, the combination therapy is provided until ADAMTS13 activity is at least 5% or more of normal activity, such as 10% or more, more than 15%, more than 20%, 25%, 30%, 35%, 40%, 45%, or even 50% or more.

[0270] The diagnosis of TTP remains based on clinical history, patient examination, and blood smears. The ADAMTS13 assay helps to confirm the diagnosis and monitor the course of the disease and the possible need for further treatment. Acute episodes of TTP can be diagnosed according to Table 1 and the guidelines of, for example, Scully et al. (2012, 2017, see above).

[0271] [Table 1]

[0272] The efficacy of any particular polypeptide of the present invention or dosing regimen can be determined by methods available to those skilled in the art.In brief, during clinical trials, patients are observed by medical personnel and disease status is evaluated by any combination of criteria.Based on these criteria, the improvement of the patient's condition is evaluated at multiple time points, and the combination of these evaluations in a patient population is plotted to evaluate the efficacy of treatment.

[0273] In an exemplary embodiment, efficacy assessment may be measured by any or all of the following criteria: · Time to treatment response, defined by recovery of platelets ≥ 150,000 / μL. This response must be confirmed 48 hours after the first documented platelet recovery above 150,000 / μL with a new measurement of 150,000 / μL or greater and an LDH preferably below 2×ULN. Number of subjects who achieved complete remission Number of TTP exacerbations (subjects with exacerbations) and time to first TTP exacerbation. Exacerbations are defined as recurrent thrombocytopenia occurring after a response and requiring resumption of daily PE treatment ≥ 1 day but ≤ 30 days after the last daily PE. Number of subjects with recurrence of TTP (defined as a new event of TTP occurring more than 30 days after the last daily PE) for up to 1 year and time to first recurrence of TTP Daily PE data, including serious adverse events (SAEs) associated with daily PE procedures Neurocognitive function as measured by a neurocognitive test battery at complete remission and at one-year follow-up. This test will be preceded by measuring the subject's state of consciousness by the Glasgow Coma Score. -Improvement of organ dysfunction and signs and symptoms associated with TTP All-cause mortality during daily PE treatment and subsequent study drug treatment (including tapering) Assessment of biomarkers of TTP, including but not limited to levels of disintegrin-like domain and thrombospondin repeat-containing metalloprotease 13 (ADAMTS13) and anti-ADAMTS13 antibody titers

[0274] Those of skill in the art are familiar with determining efficacy.

[0275] For example, ADAMTS13 activity can be assessed using electrophoresis of vWF multimers to detect ultra-large multimers that are not cleaved by proteases (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 may be tested using FRETS-vWF73, a fragment of vWF that has been chemically modified to fluoresce when cleaved by ADAMTS13. In this assay, FRETS-vWF73 is added to a patient's plasma sample and the change in fluorescence is measured over time to determine ADAMTS13 activity. If an inhibitor is present, it is often a neutralizing IgG antibody against ADAMTS13, which can be measured by ELISA (Kokame et al. (2005), British journal of haematology vol. 129, pp. 93-100). Alternatively, or in addition, ADAMTS13 activity can be determined, for example, as described in Vesely et al. (2003, supra), Fontana et al. (2004, supra), or Remuzzi et al. (Blood 2002; vol. 100: 778-785 2002). For example, an indirect ADAMTS13 activity assay involves detecting cleavage products, either full-length VWF molecules or VWF fragments encompassing the ADAMTS13 cleavage site within the A2 domain of VWF. (1) Collagen binding assay. BaCl, which denatures VWF, is used to detect cleavage products. 2Normal plasma or purified VWF is incubated with test plasma samples in the presence of 1M urea and 1.5M urea. VWF is cleaved by ADAMTS13 and the remaining VWF is measured by its binding to type III collagen. Bound VWF is quantified using an ELISA assay with a conjugated anti-VWF antibody. (2) Ristocetin-induced aggregation. This is similar to the collagen binding assay above, but the remaining VWF is measured by ristocetin-induced platelet aggregation using an aggregometer. (3) Functional ELISA assay. In this assay, recombinant VWF fragments are immobilized on ELISA plates using antibodies against tags on VWF. The VWF fragments encode the A2 domain and the ADAMTS13 cleavage site at Tyr1605-Met1606 and are tagged with glutathione-S-transferase [GST]-histidine [GST-VWF73-His]. Immobilized GST-VWF73-His fragment When plasma is added to the plate, cleavage of the immobilized fragment occurs at the ADAMTS13 cleavage site. The remaining cleaved VWF fragments are measured by using a second monoclonal antibody that recognizes only the cleaved VWF fragments and not the intact fragments. Thus, ADAMTS13 activity is inversely proportional to the residual substrate concentration. This method forms the basis of the TECHNOZYM® ADAMTS13 Activity ELISA.

[0276] Those skilled in the art are familiar with the determination of autoantibodies against ADAMTS13; for example, anti-ADAMTS13 autoantibodies can be determined by ELISA, such as TECHNOZYM® ADAMTS13 INH ELISA (Technoclone).

[0277] Those skilled in the art are familiar with determining ristocetin cofactor activity in human samples, for example, ristocetin cofactor can be determined in an aggregometer, PAP-8E analyzer (Bio / Data corp.), using Bio / Data corp's vW Select (registered trademark).

[0278] Those skilled in the art are familiar with determining factor VIII in human samples using, for example, Coamatic Factor VIII (Chromogenix) in a STA-R evolution analyzer (Diagnostica Stago).

[0279] The skilled artisan can, for example, perform the assay using an immunoturbidimetric assay (e.g., STA Lia test vWF:Ag) to determine von Willebrand factor antigen in human samples.

[0280] Those skilled in the art are familiar with the determination of LDH levels. Most methods are based on enzymatic assays based on lactate dehydrogenase in a spectrophotometer. A convenient review is presented in 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 (cat. no. KA1653), which measures the catalysis by LDH of the interconversion of lactate and pyruvate, a non-radioactive colorimetric LDH assay based on the reduction of the tetrazolium salt MTT to its reduced form, which has an absorption maximum at 565 nm, in an NADH-coupled enzymatic reaction. The intensity of the resulting purple color is directly proportional to the enzymatic activity. Similarly, Sigma offers an assay for the determination of LDH levels using the 565 nm LDH-based colorimetric assay, which is based on the reduction of the tetrazolium salt MTT to its reduced form, which has an absorption maximum at 565 nm. In the Aldrich kit (MAK066-1KT), LDH reduces NAD to NADH, which is specifically detected by a colorimetric (450 nm) assay. Normal levels are presented in Table 1.1 below.

[0281] Those skilled in the art are familiar with the determination of 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 (3rd generation). A convenient review is presented in 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. July 1999; 45(7): 1104-21. Normal levels This is presented in Table 1.1 below.

[0282] Those skilled in the art are familiar with the determination of creatinine. A convenient review is provided in Peake and Whiting, "Measurement of Serum Creatinine-Current Status and Future Goals," Clin Biochem Rev. November 2006;27(4):173-184. For example, creatinine levels can be measured using Abcam's Creatinine Assay Kit (ab65340) or BioVision's Creatinine Assay Kit (ab65340). The assay can be determined by the creatinine concentration assay kit. In this assay, creatinine is converted to creatine by creatininase, which converts creatine to sarcosine, which is specifically oxidized to produce a product that reacts with the probe to produce a red color (λmax=570 nm) and fluorescence (Ex / Em=538 / 587 nm). Normal levels are presented in Table 1.1 below. Men usually have higher creatinine levels than women because the amount of creatinine in the blood increases with muscle mass.

[0283] [Table 2]

[0284] It will be appreciated that the normal levels provided in Table 1.1 may vary from laboratory to laboratory, between males and females, and by age, although the skilled artisan will consider that, depending on the assay used, the normal levels provided by the manufacturer, or alternatively, those assessed by the clinician in a particular setting, can usually be used as a reference.

[0285] The polypeptide of the present invention typically comprises at least one ISVD for vWF. The ISVD of the present invention binds to vWF and / or has affinity for vWF ("for vWF"). In the scope of the present invention, "vWF" includes but is not limited to cynomolgus monkey, baboon, pig, guinea pig, mouse and / or human vWF, and is most preferably human vWF, i.e., SEQ ID NO: 20 or GenBank entry: NP_000543.

[0286] Preferably, the ISVD for vWF consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: a) CDR1 is: - the amino acid sequence YNPMG; or - an amino acid sequence that has two or only one amino acid difference from the amino acid sequence YNPMG Consists of or consists essentially of; b) CDR2 is: - the amino acid sequence AISRTGGSTYYPDSVEG; or - at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least an amino acid sequence having at least 99% sequence identity; or - an amino acid sequence that has two or only one amino acid difference from the amino acid sequence AISRTGGSTYYPDSVEG Consists of or consists essentially of; c) CDR3 is: - the amino acid sequence AGVRAEDGRVRTLPSEYTF; or - an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the amino acid sequence AGVRAEDGRVRTLPSEYTF; or - an amino acid sequence that has two or only one amino acid difference from the amino acid sequence AGVRAEDGRVRTLPSEYTF Comprise or consist essentially of.

[0287] Even more preferably, the ISVD for vWF consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: a) CDR1 is YNPMG (SEQ ID NO:21); b) CDR2 is AISRTGGSTYYPDSVEG (SEQ ID NO: 22); c) CDR3 is AGVRAEDGRVRTLPSEYTF (SEQ ID NO: 23).

[0288] Even more preferably, the ISVD for vWF is represented by SEQ ID NO: 19 (12A02H1).

[0289] Preferably, the polypeptide of the invention comprises or consists of at least two ISVDs for vWF.

[0290] Even more preferably, the polypeptide of the invention comprises or consists of two ISVDs against vWF as defined by SEQ ID NO: 1-18 or 24, most preferably SEQ ID NO: 1 (ALX0081; INN "Caplacizumab") or SEQ ID NO: 24. ALX0081 is a bivalent Nanobody consisting of two identical monovalent components that target vWF. ALX0081-A is ALX0081 with a C-terminal alanine.

[0291] Polypeptides comprising at least one ISVD against vWF, such as SEQ ID NO: 1-19 or 24, can be used to treat vWF-associated diseases, in particular thrombotic thrombocytopenic purpura (TTP).

[0292] The terms "polypeptide" and "amino acid sequence" are used interchangeably herein.

[0293] Thus, for example, suitable polypeptides for use in the present invention may include compounds having 80% or more, more preferably 85% or more, and most preferably 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to a compound in Table A-1, such as SEQ ID NOs: 1-19 or 24, or a compound defined by any of SEQ ID NOs: 1-19 or 24 in Table A-1 (see definitions section for "sequence identity").

[0294] Preferably, the ISVD for vWF used in the polypeptide of the present invention is a 12A02H1-like compound. For the purposes of this description, a 12A02H1-like compound is a compound that contains 12A02H1 (i.e., SEQ ID NO: 19) or is a compound that is similar to 12A02H1 (SEQ ID NO: 19) and a compound having 80% or more, more preferably 85% or more, and most preferably 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity with ALX0081 (SEQ ID NO: 1) or ALX0081-A (SEQ ID NO: 24). Particularly preferred polypeptides containing two ISVDs for vWF are ALX0081 (SEQ ID NO: 1) or ALX0081-A (SEQ ID NO: 24).

[0295] Immunoglobulin single variable domains, such as camelid VHH domains, camelized VH domains or humanized VHH domains, are a rapidly growing class of therapeutics. For example, immunoglobulin single variable domains against vWF are described in WO2004 / 015425, WO2004 / 062551, WO2006 / 074947, WO2006 / 122825, WO2009 / 115614 and WO2011 / 067160. Further preferred immunoglobulin single variable domains for use in the polypeptides of the invention include the improved Nanobodies described in WO06 / 122825.

[0296] Unless otherwise indicated, the term "immunoglobulin sequence" is used herein as a general term to include full-sized antibodies, their individual chains as well as all parts, domains, or fragments thereof (including but not limited to antigen-binding domains or fragments such as VHH domains or VH / VL domains, respectively), whether used to refer to heavy chain antibodies or traditional four-chain antibodies. Furthermore, the term "sequence" as used herein (e.g., 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 as well as the nucleic acid or nucleotide sequence encoding same, unless the context requires a more limited interpretation.

[0297] The term "immunoglobulin single variable domain" ("ISVD"), used interchangeably with "single variable domain", defines a molecule in which an antigen-binding site resides in and is formed by a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins or fragments thereof, in which two immunoglobulin domains (particularly two variable domains) interact to form the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity determining regions (CDRs) of both the VH and the VL contribute to the antigen-binding site. That is, a total of six CDRs are involved in the formation of the antigen-binding site.

[0298] In contrast, the binding site of an immunoglobulin single variable domain is formed by a single VH or VL domain and thus the antigen-binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.

[0299] Thus, the term "immunoglobulin single variable domain" does not include conventional immunoglobulins or fragments thereof, which require the interaction of at least two variable domains for the formation of an antigen-binding site. This also applies to embodiments of the invention which "comprise" or "contain" an immunoglobulin single variable domain. Within the scope of the present invention, such embodiments exclude conventional immunoglobulins or fragments thereof. Thus, a polypeptide or composition which "comprises" or "contains" an immunoglobulin single variable domain may for example relate to a construct comprising two or more immunoglobulin single variable domains. Alternatively, further components other than immunoglobulin single variable domains may be present, for example various kinds of auxiliary agents, protein tags, colorants, dyes, etc. However, these terms do not include the term "immunoglobulin single variable domain", which is intended to include the antigen-binding portion. The present invention includes fragments of conventional immunoglobulins in which the antibody fragment is formed by a single variable domain.

[0300] Generally, a single variable domain is an amino acid sequence that essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively). Such single variable domains and fragments are most preferably such that they comprise an immunoglobulin fold or are capable of forming an immunoglobulin fold under suitable conditions. Thus, a single variable domain may comprise, for example, 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 a VHH sequence) or a suitable fragment thereof; provided that said domain is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit that essentially consists of a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit, as is for example the case for the variable domains present in conventional antibodies and scFv fragments (single chain variable fragments), which need to interact with another variable domain (e.g., by VH / VL interaction) to form a functional antigen-binding domain).

[0301] In one embodiment of the invention, the immunoglobulin single variable 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 particularly, the immunoglobulin single variable domain can be a heavy chain variable domain sequence derived from a traditional four-chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody (e.g. a VHH).

[0302] For a general description of heavy chain antibodies and their variable domains, reference is made, inter alia, to the prior art cited herein, as well as to the prior art mentioned on page 59 of WO08 / 020079, and to the list of references mentioned on pages 41-43 of International Application WO06 / 040153. These prior art and references are incorporated herein by reference. As described in these references, Nanobodies (in particular VHH sequences, and partially humanized Nanobodies) may be characterized, in particular, by the presence of one or more "hallmark residues" in one or more of the framework sequences. Further description of Nanobodies, including humanized and / or camelized Nanobodies, as well as other modified forms, portions or fragments, derivatives or "Nanobody fusions", multivalent constructs (including some non-limiting examples of linker sequences), and various modifications that increase the half-life of Nanobodies and their preparations, can be found, for example, in WO08 / 101985 and WO08 / 142164.

[0303] For example, the single variable domain or immunoglobulin single variable domain (or suitable amino acid sequence for use as an immunoglobulin single variable domain) may be a (single) domain antibody (or suitable amino acid sequence for use as a (single) domain antibody), a "dAb" or dAb (or suitable amino acid sequence for use as a dAb) or Nanobody (as defined herein, including but not limited to VHH sequences); other single variable domains, or any suitable fragment of any one of these. For a general review of (single) domain antibodies, reference is made to the prior art cited herein as well as to EP0368684. For the term "dAb" see, for example, Ward et al., 1989 (Nature 341(6242):544-6), Holt et al., 2003 (Trends Biotechnol. 21(11):484-490); and, for example, WO04 / 068820, WO06 / 030220, WO06 / 003388 and other published patent applications of Domantis Ltd. It should also be noted that single variable domains may be derived from certain species of sharks (e.g. the so-called "I gNAR domain" (see, e.g., WO05 / 18629).

[0304] In particular, the immunoglobulin single variable domain may be a Nanobody® (as defined herein) or a suitable fragment thereof. [Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx NV.] For a general description of Nanobodies, reference is made to the further description below as well as to the prior art cited herein, e.g. as described in WO08 / 020079 (page 16).

[0305] The amino acid sequence and structure of an immunoglobulin sequence, and in particular an immunoglobulin single variable domain, can be considered in the art and herein as consisting of four framework regions or "FRs", referred to respectively in the art and in the present specification as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4"; these framework regions can be considered (but are not limited to) as being interrupted by three complementarity determining regions or "CDRs", referred to in the art as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively.

[0306] The total number of amino acid residues in an immunoglobulin single variable domain may be in the region of 110-120, preferably 112-115, and most preferably 113. It should be noted, however, that parts, fragments, analogs or derivatives of immunoglobulin single variable domains are not particularly limited with regard to their length and / or size, so long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are preferably suitable for the purposes described herein.

[0307] Thus, in the sense of the present invention, the term "immunoglobulin single variable domain" or "single variable domain" includes peptides derived from a non-human source, preferably a camelid, preferably a camelid heavy chain antibody. These may be humanized, as previously described, for example in WO08 / 101985 and WO08 / 142164. Furthermore, the term includes polypeptides derived from a non-camelid source, for example mouse or human, which have been "camelized", as previously described, for example in WO08 / 101985 and WO08 / 142164.

[0308] The term "immunoglobulin single variable domain" encompasses immunoglobulin sequences of various origins, including mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. Also included are fully human, humanized or chimeric immunoglobulin sequences. For example, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences or camelidized immunoglobulin single variable domains, such as the camelidized dAbs described by Ward et al. (see, for example, WO 94 / 04678 and Davies and Riechmann 1994, Febs Lett. 339:285 and 1996, Protein Engineering 9:531).

[0309] All the above mentioned ISVDs (or vWF binding agents) for vWF are known from the literature, including their preparation (see, in particular, for example, WO2006 / 122825, but also WO2004 / 062551). For example, ALX0081 or ALX0081-A is prepared as described, for example, in WO2006 / 122825 or WO2009 / 115614.

[0310] The immunoglobulin single variable domains provided by the invention are preferably in isolated or essentially isolated form or form part of a protein or polypeptide of the invention, which may comprise or essentially consist of one or more immunoglobulin single variable domains, optionally further comprising one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, but not limited to, in such a protein or polypeptide, one or more immunoglobulin single variable domains may be used as binding units, which may optionally comprise one or more further amino acid sequences that may function as binding units (i.e. for one or more targets other than cell-associated antigens), so as to result in a monovalent, multivalent or multispecific polypeptide of the invention, respectively (all as described herein). Such a protein or polypeptide may be in isolated or essentially isolated form. Thus, according to the invention, immunoglobulin single variable domains include constructs comprising two or more antigen binding units in the form of a single domain, as outlined above. For example, two (or more) immunoglobulin single variable domains with the same or different antigen specificities can be linked to form, for example, bivalent, trivalent or multivalent constructs. Immunoglobulin single variable domains of two or more specificities can be combined to form bispecific, trispecific etc. constructs. For example, a polypeptide according to the invention may comprise two immunoglobulin single variable domains against target A and one immunoglobulin single variable domain against target B, such that it is bivalent for A and monovalent for B. All such constructs, and modifications thereof that can be easily envisaged by the skilled artisan, are encompassed by the present invention. In a particular embodiment, the present invention relates to biparatopic constructs comprising at least two immunoglobulin single variable domains against different epitopes within the same target antigen.

[0311] All such molecules are referred to as "polypeptides of the invention", which is synonymous with the "immunoglobulin sequences" or "immunoglobulin single variable domains" of the invention.

[0312] Additionally, as used herein (e.g., "immunoglobulin sequences," "antibody sequences," "variable domain sequences," "V HH The term "sequence" as used herein (in terms such as "protein sequence" or "protein sequence") should generally be understood to include both the relevant amino acid sequence as well as the nucleic acid or nucleotide sequence encoding same, unless the context requires a more limited interpretation.

[0313] According to one non-limiting embodiment of the invention, the immunoglobulin sequence, Nanobody®, or polypeptide of the invention is glycosylated. According to another non-limiting embodiment of the invention, the immunoglobulin sequence, Nanobody®, or polypeptide of the invention is non-glycosylated.

[0314] As mentioned above, the present invention relates to a polypeptide comprising typically at least one, such as two or more, ISVDs against vWF, i.e. ISVDs that bind to and / or have affinity for an antigen as defined herein, such as von Willebrand factor (vWF), preferably human vWF (SEQ ID NO: 20).

[0315] Within the scope of the present invention, "binding to and / or having affinity for" a particular antigen has its ordinary meaning as understood in the art, e.g., in the context of antibodies and their respective antigens.

[0316] In certain embodiments of the invention, the term "binds to and / or has affinity for" refers to an immunoglobulin sequence that specifically interacts with an antigen. , are used interchangeably with immunoglobulin sequences "against" said antigen.

[0317] 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 (e.g., 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, represented by the equilibrium constant (KD) of dissociation of an antigen with an antigen-binding protein, is a measure of the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein: the lower the value of KD, the higher the binding strength between an antigenic determinant and an antigen-binding molecule (alternatively, affinity can be expressed as an affinity constant (KA), which is 1 / KD). As will be clear to the skilled artisan (e.g., based on the further disclosures herein), affinity can be determined in a manner known per se, depending on the particular antigen of interest. Avidity is a measure of the strength of binding between an antigen-binding molecule (e.g., Nanobody® or a polypeptide of the invention) and the appropriate antigen. Avidity relates to both the affinity between an antigenic determinant and its antigen-binding site on the antigen-binding molecule and the number of suitable binding sites present on the antigen-binding molecule.

[0318] Typically, the immunoglobulin sequences of the invention (e.g., the amino acid sequences, Nanobodies®, and / or polypeptides of the invention) are -5 From 10 -12 moles / liter or less, preferably 10 -7 From 10 -12 moles / liter or less, more preferably 10 -8 From 10 -12 Dissociation constant (KD) in moles / liter (i.e., 10 5 From 10 12 liters / mole or more, preferably 10 7 From 10 12 liters / mole or more, more preferably 10 8 From 10 12 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 10 ...2 M -1 s -1 About 10 7 M -1 s -1 Between 10 and 20 3 M -1 s -1 From 10 7 M -1 s -1 More preferably between 10 4 M -1 s -1 From 10 7 M -1 s -1 Between, for example, 10 5 M -1 s -1 From 10 7 M -1 s -1 and / or binds to a cell-associated antigen as defined herein at a k on rate between 1 s -1 (t1 / 2=0.69s) to 10 -6 s -1 (resulting in nearly irreversible complexes with t1 / 2 of several days), preferably between 10 -2 s -1 From 10 -6 s -1 More preferably between 10 -3 s -1 From 10 -6 s -1 Between, for example, 10 -4 s -1 From 10 -6 s -1 The antibody binds to a cell-associated antigen as defined herein with a koff rate between

[0319] 10 -4 Any KD value above M (or 10 4 M -1 Any K A value less than 0.05 is generally considered to indicate nonspecific binding.

[0320] Preferably, the monovalent immunoglobulin sequences of the invention 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.

[0321] Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and their different variations known per se in the art; as well as other techniques mentioned herein.

[0322] The dissociation constant (KD) may be an actual or apparent dissociation constant, as will be apparent to one of skill in the art. Methods for determining dissociation constants will be apparent to one of skill in the art and include, for example, the techniques mentioned herein. In this regard, -4 moles / liter or 10 -3 More than 10 moles per liter (e.g., 10 -2 Measure the dissociation constant (in moles / liter) It will also be apparent that it may not be possible to determine the dissociation constant. In some cases, as will also be apparent to one of skill in the art, the (actual or apparent) dissociation constant can be calculated based on the (actual or apparent) association constant (KA) using the following relationship [KD=1 / KA].

[0323] Affinity describes the strength or stability of a molecular interaction. Affinity is typically given as the KD or dissociation constant in 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) in units of kD. Herein, the stability of an interaction between two molecules (e.g., an amino acid sequence, immunoglobulin sequence, Nanobody®, or polypeptide of the invention and its intended target) is primarily expressed in terms of the KD value of those interactions; considering the relationship KA=1 / KD, it is clear to the skilled artisan that specifying the strength of a molecular interaction by its KD value can also be used to calculate the corresponding KA value. The KD value also characterizes the strength of a molecular interaction in a thermodynamic sense, since it is related to the free energy of binding (DG) by the well-known relationship DG=RT.ln(KD) (or 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.

[0324] The KD for a biological interaction, such as the binding of an immunoglobulin sequence of the invention to a cell-associated antigen as defined herein, that is considered meaningful (e.g., specific) is typically greater than or equal to 10 -10 M(0.1nM) to 10 -5 M (10,000 nM). The stronger the interaction, the lower its KD.

[0325] KD can also be expressed as the ratio of the dissociation rate constant of the complex, expressed as koff, to its association rate, expressed as kon (hence KD=koff / kon and KA=kon / koff). The off-rate koff is calculated as s -1 The unit is 1 / s (where s is the SI unit of second). The on-speed kon is M -1 s -1 is the unit.

[0326] For the immunoglobulin sequences of the present invention, the on-rate is 10 2 M -1 s -1 About 10 7 M -1 s -1The off-rate can vary between 10 and 100, approaching the diffusion-limited association rate constant of the bimolecular interaction. The off-rate is related to the half-life of a given molecular interaction by the relationship t1 / 2=ln(2) / koff. The off-rates of the immunoglobulin sequences of the present invention range from 10 -6 s -1 (a nearly irreversible complex with a t1 / 2 of several days) to 1s -1 (t1 / 2=0.69s).

[0327] The affinity of a molecular interaction between two molecules can be measured by various techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, for example, Ober et al., Intern. Immunology, vol. 13, pp. 1551-1559, 2001), in which one molecule is immobilized on a biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions to obtain k, k measurements and thus KD (or K) values. This can be done, for example, using the well-known Biacore instrument.

[0328] It will be clear to those skilled in the art that the measured KD may also correspond to the apparent KD if the measurement process somehow affects the inherent binding affinity of the molecules implied, for example, due to artifacts associated with coating one molecule on the biosensor. The apparent KD may also be measured when one molecule contains two or more recognition sites for the other molecule. In such a situation, the measured affinity may be affected by the avidity of the interaction of the two molecules.

[0329] Another approach that can be used to assess affinity is described by Friguet et al. J. Immunol. Methods, vol. 77, pp. 305-19, 1985) is a two-step ELISA (enzyme-linked immunosorbent assay) procedure. This method establishes a solution-phase binding equilibrium measurement and avoids possible artifacts associated with the adsorption of one of the molecules on a support such as plastic.

[0330] However, accurate measurement of KD often requires a significant amount of effort, and as a result, apparent KD values are often determined to evaluate the binding strength of two molecules. As long as all measurements are performed in a consistent manner (e.g., without changing assay conditions), the apparent KD measurement can be used as an approximation of the true KD, and thus it should be noted that in this document, KD and apparent KD should be treated with equal importance or relevance.

[0331] Finally, it should be noted that in many situations, an experienced scientist may find it convenient to determine the relative binding affinity for some reference molecule. For example, to evaluate the binding strength between molecules A and B, a reference molecule C can be used that is preferably labeled with, for example, a fluorophore or chromophore group that facilitates detection in ELISA or FACS (fluorescence-activated cell sorting) or other formats, or another chemical moiety such as biotin (a fluorophore for fluorescence detection, a chromophore for light absorption detection, biotin for streptavidin-mediated ELISA detection). Typically, the reference molecule C is kept at a constant concentration, and the concentration of A varies for a given concentration or amount of B. As a result, an IC 50 value corresponding to the concentration of A at which the signal measured for C in the absence of A is halved is obtained. Given that the KD of the reference molecule, KDref, and the total concentration of the reference molecule, cref ref are known, the apparent KD for the interaction between A - B can be obtained from the following equation: KD = IC50 / (1 + cref / KDref). Note that if cref << KDref, then KD ≈ IC50. Given that the measurement of IC50 is performed in a consistent manner (e.g., by fixing cref) for the binding agents being compared, throughout this article, the strength or stability of a molecular interaction can be evaluated by IC50, and this measurement is judged to be equivalent to KD or apparent KD.

[0332] The present invention relates to immunoglobulin single variable domains as described in or obtainable by the methods disclosed in WO2004 / 015425, WO2004 / 062551, WO2006 / 074947, WO2006 / 122825, WO2009 / 115614 or WO2011 / 067160 (all in the name of the present applicant).

[0333] The present invention also encompasses optimized variants of these amino acid sequences. In general, an "optimized variant" of an amino acid sequence according to the present invention is a variant that contains one or more beneficial substitutions, such as substitutions that increase i) the degree of "humanization", ii) chemical stability, and / or iii) the level of expression; and still has a potency (e.g., measured by the potency assay described in the Examples section of WO2006 / 122825) equivalent (i.e., within 10% deviation) to wild-type 12A02 (as defined in WO2006 / 122825) or equivalent to variant 12A02H1 (SEQ ID NO: 19) (also as defined in WO2006 / 122825). Preferably, the amino acid sequence of the present invention contains at least one such substitution, preferably at least two such substitutions, and preferably at least three humanizing substitutions, preferably at least 10 such humanizing substitutions, compared to the wild-type sequence of 12A02.

[0334] In a particular embodiment, the amino acid sequence of the invention has a total of between 1 and 15, preferably between 2 and 14, for example between 9 and 13, amino acid residues compared to the wild-type sequence 12A02. , for example 10, 11 or 12 amino acid substitutions. As mentioned, such differences preferably include at least one, preferably at least two, for example 3, 4 or 5, or 10 humanizing substitutions, and may optionally include one or more additional substitutions (e.g., any one of the additional substitutions (a) to (c) mentioned herein, or any suitable combination of any two or more). Again, a person skilled in the art will be able to select one or more such suitable humanizing substitutions and / or additional substitutions (suitable combinations) based on the disclosure herein, and optionally after some trial and error.

[0335] The present invention encompasses polypeptide sequences that are highly similar to any of the specific examples presented herein or any of the specific examples defined by the above references. Highly similar means at least 90%, for example 95, 97, 98, or 99% amino acid identity. Highly similar polypeptide sequences, such as ALX0081-A derived from ALX0081, have the same function as the sequences from which they are derived, i.e., bind to vWF, more particularly bind to vWF and inhibit the interaction between vWF and platelets.

[0336] In a particular embodiment, the invention relates to any one of SEQ ID NOs: 1-19 or 24, in particular sequences highly similar to SEQ ID NO: 1. However, as the invention specifically refers to variants or highly similar sequences that are stable in the formulations defined herein, each variant sequence needs to be assessed for stability in the formulations defined herein.

[0337] 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 bacterial or yeast host cells. Those skilled in the art are also familiar with suitable purification techniques and protocols.

[0338] The present invention also provides formulations of polypeptides comprising at least one immunoglobulin single variable domain against vWF, e.g., ALX0081 or ALX0081-A, that are stable and preferably suitable for pharmaceutical uses, including the manufacture of a medicament (also referred to as "pharmaceutical formulations of the invention" or "formulations of the invention").

[0339] In certain embodiments, the formulation comprises one or more polypeptides selected from SEQ ID NOs: 1-19 or 24, preferably SEQ ID NO: 1.

[0340] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient (the polypeptide of the present invention) to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is to be administered. Such formulations are sterile. A "pharmaceutical acceptable" excipient (vehicle, additive) is one that can be reasonably administered to a mammalian subject to provide an effective amount of the active ingredient employed.

[0341] The term "excipient" as used herein refers to an inert substance that is commonly used as a diluent, vehicle, preservative, lyoprotectant, surfactant, binder, carrier, or stabilizer for a compound that confers beneficial physical properties to the formulation. Those skilled in the art are familiar with excipients suitable for pharmaceutical purposes that may have specific functions in the formulation, such as lyoprotection, stabilization, preservation.

[0342] A "sterile" formulation is aseptic or free or essentially free of all live microorganisms and their spores, which is readily accomplished by filtration through sterile filtration membranes.

[0343] A "stable" formulation is one in which the protein therein essentially retains its physical and / or chemical stability and / or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended expiration date of the formulation. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, pp. 247-301, edited by Vincent Lee, published by Marcel Dekker, Inc. (New York, NY) (1991), and Jones, A. Adv. Drug Delivery Rev. vol. 10: pp. 29-90 (1993). Stability can be measured at a selected temperature for a selected time. In certain embodiments, the formulation is stable at about 40° C. for at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or more. Additionally, the formulation is preferably stable after freezing (e.g., to -20°C or -70°C) and thawing the formulation, e.g., after 1, 2, 3, 4, or 5 cycles of freezing and thawing. Stability can be assessed qualitatively and / or quantitatively in a variety of different ways known to those skilled in the art. Stability studies have shown that ALX0081 or ALX0081-A is stable at -20°C for at least 3 years.

[0344] The formulation comprises an aqueous carrier, which is particularly a buffer.

[0345] As used herein, "buffer" refers to a buffered solution that resists pH change due to the action of its acid-base conjugate components.The formulation of the present invention comprises a buffer selected from at least one of citrate buffer or phosphate buffer, preferably citrate buffer.As previously determined, these buffers improve the stability of vWF binding agent.

[0346] The formulation according to the invention comprises a citrate buffer at a concentration of 5-200 mM, preferably 7.5-80 mM, even more preferably within the range of 10-50, e.g. 10, 15, 20, 25 or 30 mM, most preferably 20 mM, where each value is understood to optionally include a range of ±5 mM. Alternatively, the formulation according to the invention may comprise a phosphate buffer at a concentration of 5-200 mM, preferably 5-80 mM, more preferably 7.5-60 mM, even more preferably within the range of 10-40, e.g. 10, 15, 20, 25 or 30 mM, most preferably 10 mM, where each value is understood to optionally include a range of ±5 mM. It is understood that a relatively low concentration of buffer will affect the final osmolality and accordingly any further solutes that may need to be added.

[0347] The pH of the formulation of the present invention is in the range of 5.0 to 7.5, where each value is understood to include a range of ±0.2. The most favorable pH depends on the buffer contained in the formulation. Thus, the present invention is particularly directed to formulations containing phosphate buffer, preferably having 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 significantly more suitable for storage and use. Thus, the present invention is directed to formulations containing citrate buffer, preferably having a pH between 6.0 and 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 each value is understood to include a range of ±0.2, if applicable.

[0348] The formulations of the invention contain a polypeptide of the invention, in particular an immunoglobulin single variable domain, or at least one immunoglobulin single variable domain against vWF, at a concentration suitable for clinical purposes, including the concentration used in a stock solution for dilution prior to use in a patient. In addition to improved stabilization, the formulations of the present invention allow for high concentrations of polypeptides that comprise at least one ISVD for vWF, such as ALX0081 or ALX0081-A.

[0349] Typical concentrations of active agents in the formulations of the invention, e.g., polypeptides comprising at least one ISVD for vWF, e.g., ALX0081 or ALX0081-A, include non-limiting examples within 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 or 11 mg / mL, where each value is understood to optionally encompass a range of ±20% (e.g., a value of 10 optionally encompasses a range of 8 to 12 mg / mL).

[0350] In a further embodiment of the invention, the formulation according to any aspect of the invention may further comprise a surfactant or surface active agent.

[0351] As used herein, "surfactant" refers to a surfactant, preferably a non-ionic surfactant. Examples of surfactants herein include polysorbates; poloxamers (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; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl taurate, or disodium methyl oleyl taurate; and the MONAQUAT® series (Mona Polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.); and the like. In one embodiment, the surfactant herein is polysorbate 80. Preferred suitable surfactants or surfactants for use in 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 will know further non-limiting examples of surfactants, such as those listed in WO2010 / 077422. In a preferred embodiment, the surfactant is a non-ionic surfactant. More particularly, the surfactant is polysorbate-80, hereafter also referred to as Tween-80. Those skilled in the art can easily determine the suitable concentration of surfactant for the formulation of the present invention.Typically, this concentration is as low as possible while maintaining the beneficial effects of the surfactant, such as its stabilizing effect under conditions of shear stress, such as stirring, which reduces the aggregation of the formulated polypeptide of the present invention. In exemplary, non-limiting embodiments, the concentration of the surfactant may be within the range of 0.001 to 0.5%, such as 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 between 0.01 and 0.05%, more preferably between 0.01 and 0.02%, such as a concentration of 0.01% (v / v).

[0352] The formulations of the present invention may further comprise excipients, such as preservatives.

[0353] A "preservative" is an agent that essentially reduces bacterial activity in a formulation, e.g., a multi-purpose formulation. Preservatives are compounds that can be optionally included in the formulation to facilitate the production of the agent.Examples of possible preservatives include octadecyldimethylbenzyl ammonium 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 alcohol, butyl alcohol, and benzyl alcohol, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.In one embodiment, the preservative herein is benzyl alcohol.

[0354] The formulations of the present invention may further include stabilizers such as polyols.

[0355] A "polyol" is a substance that has multiple hydroxyl groups, and includes sugars (reducing 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). A "reducing sugar" is one that contains a hemiacetal group that can reduce metal ions or covalently react with lysine and other amino groups in proteins, and a "non-reducing sugar" is one that does not have these properties of a reducing sugar. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, and glucose. Non-reducing sugars include sucrose, trehalose, sorbose, melezitose, and raffinose. Mannitol, xylitol, erythritol, threitol, sorbitol, and glycerin are examples of sugar alcohols. With respect to sugar acids, these include L-gluconic acid and its metal salts. If it is desired that the formulation be freeze-thaw stable, the polyol is preferably one that does not crystallize at freezing temperatures (e.g., -20°C) which would destabilize the antibody in the formulation. In certain embodiments, non-reducing sugars such as sucrose and trehalose are examples of polyols, with sucrose being preferred here, despite the solution stability of trehalose.

[0356] The therapeutic compounds of the invention used in accordance with the present invention are prepared for storage in the form of a lyophilized formulation or aqueous solution by mixing the polypeptide having the desired purity with an optional pharma- ceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th ed., Osol, A., Ed.

[1980] ). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed. Thus, the formulations of the present invention may optionally include one or more excipients.

[0357] 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 such compositions include 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, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, gelatin, polyethylene polyoxypropylene block polymers, polyethylene glycol and wool fat, antioxidants including ascorbic acid and methionine; preservatives; low molecular weight (less than about 10 residues) polypeptides; proteins; and glycine, glutamine, asparagine, histidine, alginate, glycine ... Examples of suitable excipients include, but are not limited to, amino acids such as guanine or lysine. In an advantageous embodiment, the excipient may be one or more selected from the list consisting of NaCl, trehalose, sucrose, mannitol, or glycine.

[0358] The active ingredient can also be encapsulated in microcapsules, such as hydroxymethylcellulose microcapsules or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, prepared by coacervation techniques or interfacial polymerization, in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980).

[0359] The polypeptide of the present invention can be formulated into any pharma- ceutically acceptable formulation.The formulation can be liquid or dry.The formulation can be produced by mixing, drying, lyophilization, vacuum drying, or any known method for formulating pharmaceutical compositions.

[0360] A preferred formulation of the present invention comprises a polypeptide comprising at least one ISVD for vWF, such as ALX0081 or ALX0081-A, in a phosphate buffer solution (pH 7.1). Even more preferably, the formulation of the present invention comprises a polypeptide comprising at least one ISVD for vWF, such as ALX0081 or ALX0081-A, in a phosphate buffer solution (pH 7.1), glycine (0.2M), and polysorbate 80 (0.02% v / v).

[0361] The polypeptides of the invention may be further formulated as described in WO2014 / 184352.

[0362] Particularly preferred formulations are: (a) a polypeptide comprising at least one ISVD against vWF, e.g., ALX0081 or ALX0081-A, at a concentration of about 0.1 mg / mL to about 80 mg / mL, preferably 5 mg or 10 mg or 11 mg; (b) an excipient selected from sucrose, glycine, mannitol, trehalose, or NaCl at a concentration of about 1% to about 15% (w / v); (c) Tween-80 at a concentration of about 0.001% to 0.5% (v / v); (d) a citrate buffer at a concentration of about 5 mM to about 200 mM such that the pH of the formulation is about 6.0 to 7.0; Includes.

[0363] Further preferred formulations of the invention comprise a polypeptide comprising at least one ISVD for vWF, such as ALX0081 or ALX0081-A, preferably at a concentration of 5 mg / ml, 10 mg / ml or 11 mg / ml, and citrate buffer at a concentration of 20 mM (pH 6.5), further comprising 7% sucrose (w / v), and Tween-80 at a concentration of 0.01% (v / v).

[0364] In some embodiments, the formulation is stored as a liquid. In other embodiments, the formulation is manufactured as a liquid and then dried, for example by lyophilization or spray drying, before storage. The dried formulation may be used as a dry compound, for example as an aerosol or powder, or may be reconstituted to its original concentration or another concentration, for example with water, buffer, or other suitable liquid.

[0365] The invention also relates to vials filled with lyophilizate containing 12.5 mg caplacizumab and excipients for injection. Excipients (per mL of reconstituted solution): 0.21 mg citric acid, 5.58 mg trisodium citrate dihydrate, 70 mg sucrose, 0.11 mg polysorbate-80 (pH 6.5+ / -0.5) per vial. After reconstitution with 1 mL water for injection (WFI), the strength is 12.5 mg caplacizumab / mL (for a nominal dose of 10 mg or 11 mg).

[0366] The present invention also encompasses products that can be obtained by further processing of liquid formulations, such as frozen, lyophilized, or spray-dried products. Upon reconstitution, these solid products can become liquid formulations as described herein (but are not limited thereto). Thus, the term "formulation" in its broadest sense encompasses both liquid and solid formulations. However, it is understood that solid formulations can be derived from liquid formulations (e.g., by freezing, freeze-drying, or spray-drying) and therefore have various characteristics defined by the features described herein for liquid formulations. The present invention does not exclude reconstitution that leads to the composition deviating from its original composition, e.g., before lyophilization or spray-drying. Thus, lyophilized formulations may be reconstituted to result in a formulation with a concentration different from the original concentration (i.e., before lyophilization), depending on the amount of water or diluent added to the lyophilized body relative to the volume of the original freeze-dried liquid. Suitable formulations can be identified by assaying one or more parameters of antibody integrity.

[0367] In a preferred embodiment, the formulation of the present invention is isotonic with respect to human blood. An isotonic solution has the same osmotic pressure as blood plasma, and therefore can be injected into a subject's vein without changing the osmotic pressure of the subject's blood plasma. Osmolality can be expressed in terms of osmolality, which can be a theoretical osmolality or preferably an experimentally determined osmolality. Typically, osmolality is within the range of 290±60 mOsm / kg, preferably 290±20 mOsm / kg.

[0368] The formulation of the present invention may contain a compound that is particularly useful for protecting the polypeptide of the present invention during freeze-drying. Such compounds are also known as lyoprotectants and are well known to those skilled in the art. Specific examples include, but are not limited to, sugars such as sucrose, sorbitol or trehalose; amino acids such as glutamic acid, especially sodium glutamate, or histidine; betaine, magnesium sulfate, sugar alcohols, propylene glycol, polyethylene glycol, and combinations thereof. By understanding the present invention, those skilled in the art can easily determine the necessary amount of such compounds to be added, taking into account the stability of the formulation when in liquid form and when freeze-drying is performed. Formulations that are particularly suitable for freeze-drying may further contain a bulking agent. Suitable agents are widely known to those skilled in the art. It has been shown that formulations that contain sucrose are particularly suitable not only for maintaining the physical stability of vWF binding agents, for example during storage and freeze-thawing, but also as lyoprotectants.

[0369] As outlined above, any of the above formulations can be further processed, for example by lyophilization, spray drying, or freezing, for example bulk freezing. The resulting processed product has the characteristics derived from the liquid starting formulation, as defined above. If necessary, additional agents, such as lyoprotectants, can be included for further processing.

[0370] The formulations of the present invention have the advantage, after lyophilization, of maintaining the chemical and physical integrity of the polypeptides of the present invention, particularly ALX0081 or ALX0081-A. Even after extended storage (e.g., at temperatures between -70°C and +40°C for durations as defined above), the purity / impurity profile of the product remains essentially unchanged. For example, extended storage after lyophilization did not significantly affect the reversed-phase high performance liquid chromatography (RP-HPLC), size-exclusion high performance liquid chromatography (SE-HPLC), or capillary isoelectric focusing (cIEF) profiles.

[0371] The polypeptide 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 polypeptide of the present invention is subjected to a suitable purification regimen before being formulated according to the present invention.

[0372] Generally, the polypeptides of the present invention are produced by living host cells that are genetically engineered to produce the polypeptides. Methods for genetically engineering cells to produce proteins are well known in the art. See, for example, Current Protocols in Molecular Biology (Wiley, New York), eds. Ausubel et al. (1990). Such methods include introducing a nucleic acid that encodes and allows the expression of the polypeptide into a living host cell. These host cells may be bacterial, fungal, or animal cells grown in culture. Bacterial host cells include, but are 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. Fungal host cells that can be used include, but are 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. New animal cell lines can be established (e.g., by transformation, viral infection, and / or selection) using methods well known to those skilled in the art. Optionally, the polypeptide can be secreted into the medium by the host cell.

[0373] In some embodiments, the polypeptide can be produced in a bacterial cell, such as an E. coli cell. For example, if the polypeptide is encoded by a sequence in a phage display vector that contains a suppressible stop codon between the display entity and a bacteriophage protein (or a fragment thereof), the vector nucleic acid can be transferred into a bacterial cell that cannot suppress the stop codon. In this case, the polypeptide is not fused to the gene III protein and is secreted into the periplasm and / or medium.

[0374] Polypeptides can also be produced in eukaryotic cells, hi one embodiment, the polypeptides are expressed in yeast bacteria such as Pichia (see, e.g., Powers et al., 2001, J Immunol Methods 251:123-35), Hansenula, or Saccharomyces.

[0375] In one embodiment, the polypeptide is produced in a mammalian cell. Exemplary mammalian host cells for expressing a clonal antibody or antigen-binding fragment thereof include Chinese Hamster Ovary cells (CHO cells) (e.g., as described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with the DHFR selection marker, as described in Kaufman and Sharp, 1982, Mol. Biol. 159:601-621). Examples of suitable cells include dhfr-CHO cells, which are derived from dhfr-expressing IgG4-associated ...

[0376] In addition to the nucleic acid sequence encoding a polypeptide, recombinant expression vector may carry additional sequences, such as sequences that control the replication of vector in host cells (e.g., origin of replication), and selection marker genes.Selection marker genes facilitate the selection of host cells that vector is introduced into (see, for example, U.S. Patent Nos. 4,399,216; 4,634,665; and 5,179,017).For example, selection marker genes typically provide resistance to drugs, such as G418, hygromycin, or methotrexate, to host cells that vector is introduced into.

[0377] Standard molecular biology techniques can be used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody molecules from the culture medium. For example, the polypeptides of the invention can be isolated by affinity chromatography.

[0378] In one embodiment, the polypeptides of the invention are purified as described in WO10 / 056550. In an exemplary embodiment, the polypeptides are purified from one or more contaminants by: contacting a mixture of the polypeptide and contaminants with a Protein A based support and / or an ion exchange support under conditions that allow the polypeptide to bind or adsorb to the support; washing the bound support under conditions that maintain the polypeptide bound to the support to remove the one or more contaminants; and selectively eluting the polypeptide from the support by eluting the adsorbed polypeptide molecules with an elution buffer.

[0379] The polypeptide of the present invention can also be produced by transgenic animals. For example, US Patent No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and a nucleic acid that codes for an antibody molecule and a signal sequence for secretion. The milk produced by the female of such a transgenic mammal contains the single domain of interest secreted therein. The antibody molecule may be purified from the milk or, in some applications, may be used directly.

[0380] The present invention includes methods of producing the formulations defined herein.

[0381] The purification and formulation steps may be performed simultaneously, for example, when the polypeptide of the invention is eluted from a column using a buffer according to the invention. Alternatively, the formulation of the invention may be prepared by exchanging the buffer by any suitable means, such as dialysis, ultrafiltration, etc., as are widely used in the art.

[0382] In some embodiments, the method of producing a formulation of the invention may also involve the reconstitution of a lyophilized or spray-dried formulation, for example, by the addition of water or a suitable buffer, which may optionally contain additional excipients.

[0383] The methods for producing the formulations of the present invention may include further steps, such as filling the formulation into a vial suitable for clinical use, such as a sealed container, and / or compounding the formulation into a unit dosage form. These methods may include further steps, such as spray drying, lyophilization, or freezing, e.g., bulk freezing. The present invention relates to containers, unit dosage forms, or other products obtainable by any of the methods enumerated herein. Also includes.

[0384] The formulations of the invention can be used to store the polypeptides of the invention, such as polypeptides comprising at least one ISVD for vWF, such as ALX0081 or ALX0081-A, as defined herein. Thus, the invention encompasses a method for storing the polypeptides of the invention as used herein, characterized by the use of a formulation as defined herein. More particularly, the invention encompasses a method for stabilizing the polypeptides of the invention for storage, including, for example, the manufacture of a formulation as described herein. Storage can be for a period of 1 to 36 months, such as 1, 1.5, 3, 6, 9, 12, 18, 24, 30 or 36 months, such as at least 12 months, optionally at a temperature between -70°C and +40°C, such as -70°C, -20°C, +5°C, +25°C or +40°C, preferably at a temperature between -70°C and +25°C, more preferably at a temperature between -20°C and +5°C. Thus, storage can encompass freezing, freeze-drying (lyophilization), and / or spray-drying. The storage method may further include evaluation of the physical and chemical integrity of the vWF binding agent, as defined herein.

[0385] The present invention also relates to a method for analyzing a preparation comprising at least one of the vWF binding agents defined herein.The preparation can be analyzed for signs of chemical or physical instability of the vWF binding agent defined herein.For example, the preparation can be evaluated for the presence of degradation products, such as low molecular weight derivatives, such as proteolytic fragments; and / or chemical derivatives, such as pyroglutamic acid variants; and / or high molecular weight derivatives, such as aggregates, aggregates, etc.The preparation can also be evaluated for total protein content and / or potency.Each of the various assay methods mentioned herein can be used in the analysis method of the present invention.

[0386] Thus, the present invention also relates to methods for monitoring and / or evaluating the quality and / or stability of the formulation, e.g., during one or more of manufacture, storage, and use. The present invention also relates to methods for quality control of the formulation, e.g., evaluating that the formulation meets product specifications as further described herein. In any of these embodiments, the present invention includes one or more selected from comparison to one or more reference samples, analysis of batch-to-batch variability, and continuous monitoring of the production process.

[0387] The present invention relates, without being limited in any way, to any product that is related to the formulation of the invention, for example by containing the formulation of the invention or by being necessary for its production or preparation.

[0388] For example, the present invention relates to articles of manufacture, such as sealed containers, that contain one or more of the formulations of the present invention.

[0389] The present invention also relates to pharmaceutical unit dosage forms, e.g., dosage forms suitable for parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous) to a patient, preferably a human patient, comprising one or more of the formulations according to any of the embodiments described herein.

[0390] The unit dosage forms may be in the format of, for example, prefilled syringes, ampoules, cartridges, or vials.

[0391] Also provided is a kit or article of manufacture comprising a formulation of the invention and instructions for use, e.g., by a health care practitioner. The kit or article of manufacture may include a vial or syringe containing a formulation of the invention as described herein.

[0392] The vial or syringe is preferably made of a polymeric material selected from glass, plastic, or cyclic olefin polymer or copolymer. The syringe, ampoule, cartridge, or vial can be made of any suitable material, such as glass or plastic, and may include rubber materials, such as rubber stoppers for vials and rubber plungers and rubber seals for syringes and cartridges. The present invention also relates to kits that include one or more of the formulations of the present invention. The kits may further include instructions for use and / or clinical leaflets. In any embodiment of the product defined herein, the present invention also encompasses the presence of packaging materials, instructions for use, and / or clinical leaflets, for example as required by regulatory aspects.

[0393] For purposes 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.

[0394] Alternatively, the degree of sequence identity between two amino acid sequences may be calculated using known computer algorithms, such as those mentioned above, for determining the degree of sequence identity of nucleotide sequences, again using standard settings.

[0395] Typically, for purposes of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the greatest number of amino acid residues is referred to as the "first" amino acid sequence and the other amino acid sequence is referred to as the "second" amino acid sequence.

[0396] In addition, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art may take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue with a similar chemical structure, and which have little or no effect on the function, activity, or other biological properties of a polypeptide.Such conservative amino acid substitutions are well known in the art, for example, from WO04 / 037999, GB-A-3357768, WO98 / 49185, WO00 / 46383, and WO01 / 09300; such (preferred) types and / or combinations of substitutions can be selected based on the appropriate teachings in WO04 / 037999 and WO98 / 49185 and the further references cited therein. Such conservative substitutions are preferably those in which one amino acid residue in the following groups (a) to (e) is replaced with another amino acid residue in the same group: (a) small aliphatic non-polar 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 non-polar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative substitutions are: 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 T yr; Tyr to Trp; and / or Phe to Val, Ile or Leu. Any amino acid substitutions applied to the polypeptides described herein may be based on the analysis of the frequency of amino acid differences between homologous proteins of different species developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978, or the analysis of structure forming ability developed by Chou and Fasman, Biochemistry 13:211, 1974 and Adv. Enzymol. 47:45-149, 1978, or the analysis of structure forming ability developed by Eisenberg et al., Proc. Natl. Acad. Sci. USA 81:140-144, 1984; Kyte & Doolittle; J. Analysis of hydrophobicity patterns in proteins may be based on the analysis developed by Molec. Biol. 157:105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15:321-353, 1986, all of which are incorporated herein by reference in their entirety. Information regarding the primary, secondary and tertiary structures of the Nanobodies® are presented in the description herein and in the general background art cited above. For this purpose, the V. llama-derived HH Crystal structures of domains are presented, for example, in Desmyter et al., Nature Structural Biology, vol. 3, no. 9, p. 803 (1996); Spinelli et al., Natural Structural Biology (1996); vol. 3, pp. 752-757; and Decaniere et al., Structure, vol. 7, no. 4, p. 361 (1999). H V in Domain H / V L Further information regarding the amino acid residues which form the interface, and some of the possible camelizing substitutions at these positions, can be found in the prior art cited above.

[0397] The present invention also relates to a method for treating or preventing vWF-related disease, such as acute coronary syndrome (ACS), transient ischemic attack, unstable or stable angina, stroke, myocardial infarction, or thrombotic thrombocytopenic purpura (TTP); the method comprises administering to a subject a pharmaceutical composition comprising the preparation of the present invention, thereby reducing one or more symptoms associated with the vWF-related disease. In particular, the vWF-related disease is TTP.

[0398] In another embodiment of the present invention, an article of manufacture is provided that contains materials useful for treating diseases as described above. The article of manufacture includes a container, a label, and a package insert. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers can be made of a variety of materials, such as glass or plastic. The container holds a composition that is effective for treating a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag, or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a polypeptide of the invention, such as ALX0081 or ALX0081-A. A label on or associated with the container indicates that the composition is used to treat a particular condition. The article of manufacture may further include a second container that contains a pharma- ceutically acceptable buffer, such as phosphate buffered saline or citrate buffered saline, as described herein. The article of manufacture may further include other materials desirable from a user or commercial standpoint, such as other buffers, diluents, filters, needles, and syringes.

[0399] The invention provides a kit or article of manufacture comprising a container containing a polypeptide as described herein or a formulation as described herein, and instructions for use.

[0400] The invention provides a kit or article of manufacture as described herein, wherein the formulation is present in a vial or a syringe for injection.

[0401] The invention provides a kit or article of manufacture as described herein, wherein the formulation is present in a pre-filled syringe for injection.

[0402] The invention provides a kit or article of manufacture as described herein, wherein the syringe or vial is made from a polymeric material selected from glass, plastic, or a cyclic olefin polymer or copolymer.

[0403] The embodiments shown and described herein are intended only to teach those skilled in the art the best way known to the inventors how to make and use the invention. As will be appreciated by those skilled in the art in light of the above teachings, modifications and variations of the above-described embodiments of the invention are possible without departing from the invention. It is therefore understood that, within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described.

[0404] The present invention will now be further described by means of the following non-limiting preferred embodiments, examples and figures.

[0405] The entire contents of all references cited throughout this specification (including literature references, issued patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference, particularly for the teachings referenced above.

[0406] 5. Abbreviations A list of explanations for commonly used abbreviations herein is provided in Table 5. [Brief description of the drawings]

[0407] [Figure 1] Time to confirmed normalization of platelet counts in the intention-to-treat population. Data from the double-blind daily plasma exchange period up to the cutoff point were used for the analysis of the primary endpoint. The data cutoff point was defined by: 45 days of daily plasma exchange from the start of study drug, cessation of daily plasma exchange, or cessation of study drug treatment; whichever occurred first. [Diagram 2]ADAMTS13 activity by relapse. Panels A and B show the categories of ADAMTS13 activity for individual patients the week after the end of daily plasma exchange and at the end of study drug treatment, respectively. Each panel presents the ADAMTS13 categories (suppression ≦10% or normalization >10%) for patients in different treatment groups. The left part of each panel shows data for patients who did not have a relapse, and the right part of each panel shows patients who had a relapse and when the relapse occurred relative to the end of daily PE or the end of treatment. [Diagram 3] Mean vWF:Ag levels over time profile during repeated daily subcutaneous administration of caplacizumab 10 mg for 7 days in healthy volunteers (upper panel) and during repeated daily subcutaneous administration of caplacizumab 10 mg for 7 days in patients with aTTP (lower panel). aTTP: acquired thrombotic thrombocytopenic purpura; PE: plasma exchange; FU: observation; vWF: von Willebrand factor. [Figure 4] Mean (±SD) RICO activity in phase II ALX-0681-2.1 / 10 ("TITAN", top panel) and phase III ALX0681-C301 ("HERCULES", bottom panel). RICO values ​​below 20% represent the threshold for pharmacological activity of caplacizumab; for the purposes of this graph, values ​​below the lower limit of quantification of 15% were set to 15% and values ​​above the upper limit of quantification of 120% were set to 120%. Graphs show mean values ​​± standard error of the mean. PE: plasma exchange; FU: observation; RICO: ristocetin cofactor; SD: standard deviation; vWF: von Willebrand factor. [Diagram 5] Plasma concentration of caplacizumab versus time profile after administration of single ascending subcutaneous doses (A) and after single and repeated daily subcutaneous doses of 10 mg over 7 days (B) in healthy volunteers. EXAMPLES

[0408] 7. Working Example 7.1 Applicable Regulations All human samples used in the Examples section were obtained either from commercial sources or from human volunteers (after all necessary consents and approvals were obtained) and were used in accordance with applicable legal and regulatory requirements (including those regarding medical confidentiality and patient privacy).

[0409] The clinical trials were conducted in accordance with applicable laws and regulations (including the Declaration of Helsinki and the principles of medical confidentiality and patient privacy) and after all necessary approvals (including approval by the relevant ethical committees) and consents (including informed consent of the subjects involved) were obtained.

[0410] The purpose and content of this clinical trial and its results have been kept confidential and inaccessible to third parties. Employees who participated in the study were bound by confidentiality obligations. All unused drugs were returned to the applicant or destroyed.

[0411] 7.2 Eligibility Criteria In the Phase III trial, patients had to meet all of the following criteria to be eligible for study enrollment: Inclusion criteria 1. Adult male or female aged 18 years or older at the time of signing the Informed Consent Form (ICF) 2. Clinical diagnosis of acquired TTP, including thrombocytopenia and microscopic evidence of red blood cell fragmentation (e.g., schistocytes). 3. Patients must start daily PE treatment and have received one PE treatment prior to randomization

[0412] Exclusion criteria 100×10 9 Platelet count of / L or higher Platelet count 30×10 9 Serum creatinine level >200 μmol / L if >200 μmol / L (to exclude possible cases of atypical hemolytic uremic syndrome [atypical HUS]) Other known causes of thrombocytopenia, including but not limited to: - Clinical evidence of intestinal infection with E. coli 0157 or related organisms - Atypical HUS - Thrombotic microangiopathy associated with hematopoietic stem cell, bone marrow, or solid organ transplantation - History or suspected sepsis - Diagnosis of disseminated intravascular coagulation Congenital TTP (known at study entry) ·Pregnant or breastfeeding Clinically significant active bleeding or high risk of bleeding (excluding thrombocytopenia) Known chronic treatment with anticoagulant medication that cannot be safely stopped (interrupted), including but not limited to: - Vitamin K antagonists - Heparin or low molecular weight heparin (LMWH) - Non-acetylsalicylic acid nonsteroidal anti-inflammatory molecules Malignant arterial hypertension ·Clinical conditions other than those related to TTP, such as terminal malignancies, with a life expectancy of less than 6 months. ·Subjects who have previously enrolled in a clinical trial of caplacizumab and received caplacizumab or whose assigned treatment arm is unknown.

[0413] 7.3 Study Design This study was designed as a Phase III double-blind, placebo-controlled randomized trial to evaluate the efficacy and safety of treatment with caplacizumab administered in addition to standard of care treatment in subjects with an acute episode of acquired TTP (Hercules). The study evaluated the efficacy of caplacizumab in speeding up the recovery of normal platelet counts during study drug treatment, and the effect of treatment with caplacizumab on the composite endpoint of TTP-related mortality, prevention of recurrent symptomatic TTP episodes, and prevention of severe thromboembolic events (TEs). After confirming eligibility for study participation (see Example 7.2) and initiating PE treatment, subjects were randomized to receive either caplacizumab or placebo in a 1:1 ratio in addition to standard of care therapy. Randomization was stratified by the severity of neurological symptoms (Glasgow Coma Scale [GCS]).

[0414] The duration of the study per subject ranged from approximately 2 months to a maximum of approximately 6 months if there was treatment extension during the 30-day period following daily PE and progression or recurrence during treatment extension.

[0415] Patients were followed at various stages during the study: - Screening period: from signing the Informed Consent Form (ICF) to randomization; - Study drug treatment period: covering the daily PE period (variable duration) and the 30-day post-daily PE period; - Treatment extension period: up to 28 days with extension every 7 days, i.e. 4 x 7 days; - Open label: If progression during the 30-day treatment period or relapse during the treatment extension period occurred (first progression or relapse), subjects received open-label (OL) caplacizumab in conjunction with reinitiated daily PE and optimized immunosuppressive treatment. The caplacizumab treatment schedule and visit schedule were the same as for the initial study drug treatment period (covering daily PE [variable duration] and the 30-day period after daily PE) and the potential treatment extension period; - 4-week follow-up (FU) period: the first FU visit is 7 days after the last day of study drug administration and the final FU visit is 28 days later.

[0416] Patients received optimal medical care and treatment as deemed appropriate by the investigators at each site and in accordance with guidelines for the treatment of TTP.

[0417] The study medication was administered as an adjunctive treatment at specific times relative to the PE procedure and consisted of 10 mg caplacizumab ("treatment group" or "CAPLA") or placebo ("placebo group") once or twice daily.

[0418] 7.4 Treatment of Study Drugs in the Hercules Trial: Intravenous loading dose: Subjects received a single loading dose of study drug of 10 mg by intravenous bolus injection 6 hours to 15 minutes before their first PE after randomization; they also received a 10 mg intravenous bolus before their first PE for treatment of their first exacerbation or relapse. Daily subcutaneous (sc) dose: After completing each daily PE session, 10 mg subcutaneous injections of study drug were administered daily for the entire duration of the PE treatment. Daily subcutaneous administration of 10 mg of study drug was continued for 30 days after cessation of PE, with no adjustments made to this period for tapering of PE. This extension of study drug treatment beyond 30 days was guided by a number of risk factors for recurrence of symptomatic TTP episodes, including the ADAMTS13 activity profile and other signs and symptoms of ongoing underlying disease activity. If the first exacerbation of a symptomatic TTP episode occurred, subjects were randomized to open-label He received caplacizumab. If a subject experienced a first TTP relapse while still receiving study drug during the treatment extension period, daily PE was initiated as part of standard of care treatment along with appropriate immunosuppressive treatment.If a subject experienced a first or subsequent TTP relapse after completing study drug treatment (i.e., during the FU period), standard of care treatment of daily PE and appropriate immunosuppressive treatment was initiated according to institutional practice.

[0419] 7.5 Endpoints The primary endpoint of this Phase III study was cessation of daily PE within 5 days, 150 × 10 9 The primary endpoints were time to platelet response, defined as an initial platelet count ≥ 1 / L. 1. Proportion of subjects with TTP-related death, recurrence of TTP, or at least one treatment-emergent severe thromboembolic event (e.g., myocardial infarction, cerebrovascular accident, pulmonary embolism, or deep vein thrombosis [DVT]) during study drug treatment (including extension). 2. The proportion of subjects who experienced a recurrence of TTP during the entire study period (including the 4-week FU period). 3. The proportion of subjects in this study with refractory TTP, defined as failure of platelet count to double after 4 days of standard treatment and LDH>ULN. 4. Time to normalization of all three of the following organ damage marker levels: LDH ≤ 1 x upper limit of normal (ULN), and cTnI ≤ 1 x ULN, and serum creatinine ≤ 1 x ULN. 5. Other endpoints included duration and volume of plasma exchange, duration of hospitalization and intensive care unit stay, mortality, pharmacodynamic and pharmacokinetic parameters, and immunogenicity. 6. Safety assessments were performed during study drug treatment and follow-up, including vital signs, physical examination, laboratory tests, and 12-lead electrocardiogram. Reported adverse events were coded using preferred terms from the Medical Dictionary for Regulatory Activities, version 20.0.

[0420] 7.6 Statistical analysis The Hercules trial specified a planned sample size of 132 patients to provide 80% power to detect a 40% reduction in the median time to platelet count normalization with caplacizumab, assuming a dropout rate of 10%, using a two-sided log-rank test at a significance level of 5%. The sample size of 132 subjects was also set to provide 83% power to detect a 20% reduction in the first major secondary endpoint, using a two-sided chi-square test with large sample approximation and a significance level of 5%. All efficacy analyses were performed in the intention-to-treat population (consisting of all patients randomized), and safety and immunogenicity analyses were performed in the safety population (consisting of all patients who received at least one dose of study drug). Time to platelet count response in the caplacizumab and placebo groups was compared by performing a two-sided stratified log-rank test based on Kaplan-Meier analysis, using the severity of neurological involvement as a stratification factor.

[0421] A fixed sequence approach was applied for the analysis of the main secondary endpoints: TTP-related death, severe thromboembolic events, and TTP recurrence (exacerbation), adjusted for the severity of neurological involvement, were analyzed using the Cochran-Mantel-Haenszel test, and fourth, time to normalization of organ damage markers, adjusted for the severity of neurological involvement and baseline LDH, were analyzed using a stratified log-rank test based on Kaplan-Meier analysis.

[0422] 7.7 Determination of ADAMTS13 activity Fluorogenic assay using FRETS-VWF73 substrate (Kokame et al., 200 5, Br J Haematol vol. 129 (issue 1): pp. 93-100; Kremer ADAMTS13 activity and functional inhibitor activity were measured according to Hovinga et al., 2006, J Thromb Haemost 4(5):1146-8.

[0423] Briefly, the FRETS-VWF73 assay was performed essentially as described (Kokame et al., 2005, see above), with the following modifications: Pefabloc SC (Boehringer, Mannheim, Germany) was added to the 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. Calibration of the assay was obtained using a normal human plasma pool (NHP; Swiss Red Cross Blood Services, Bern, Switzerland) diluted 1:25 (100%) in assay buffer. Further calibration samples were obtained by pre-diluting NHP in heat-inactivated NHP in a stepwise manner to 3:4 (75%), 1:2 (50%), 1:4 (25%), 1:10 (10%), 1:20 (5%), 1:50 (2%), and 1:100 (1%), and were incubated at 56°C for 30 minutes, followed by centrifugation at 15000×g for 15 minutes to correct for plasma matrix effects in the low activity range of the standard curve. These standard samples, as well as heat-inactivated NHP (0% ADAMTS13 activity), and all test samples were then diluted 1:25 in assay buffer. 25 μL of each diluted standard sample or patient sample was then incubated at 37°C in a 384-well white plate (NUNC, Roskilde, Denmark). After 10 min, 25 μl of 4 μmol L-1 FRETS-VWF73 peptide substrate dissolved in assay buffer was added to each well and fluorescence emission was recorded at 37 °C in 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 min). Reaction rates were calculated by linear regression analysis (Passing-Bablok) of the fluorescence emission over time from 5 min (cycle 2) to 60 min (cycle 13). The slope of the regression curve was calculated for each calibration sample and used to generate a calibration curve (trend line: y = ax + b; where x = ADAMTS13 (%) and y = delta RFU / delta time).The ADAMTS13 activity (%) of the samples was then calculated as follows: (yb) × 1 / a.

[0424] The activity of ADAMTS13 functional inhibitors was measured by the same fluorogenic FRETS-VWF73 method by determining the residual ADAMTS13 activity in normal human plasma after 1:1 (v:v) incubation with heat-inactivated patient plasma (56°C for 30 min) for 2 h at 37°C.

[0425] For each analytical batch, a normal human plasma pool (NHP; Swiss Red Cross Blood Services, Bern, Switzerland) diluted 1:25 (100%) in assay buffer was used to generate a calibration curve. Further calibration samples were obtained by serially pre-diluting NHP in heat-inactivated NHP to 1:2 (50%), 1:4 (25%), 1:10 (10%), 1:20 (5%), 1:50 (2%), and 1:100 (1%). All calibration points were applied to singlicates. Acceptance criteria: (1) the slope of the final regression of the standard curve must be greater than 6.0; (2) the R of the regression of the final plot must be greater than 0.05; 2 must be greater than 0.98 (or R > 0.9899), otherwise the assay was rejected.

[0426] 7.8 Study population In the Hercules trial, 145 patients were randomly assigned to receive caplacizumab (n=72) or placebo (n=73). All patients received study drug, except for one patient who withdrew consent before the first dose. Overall, 108 patients completed the study (i.e., completed all scheduled treatment visits and attended their final follow-up visit), but 37 patients discontinued (14 in the caplacizumab group and 23 in the placebo group). The most common reasons for discontinuation were adverse events, withdrawal of consent, and investigator decision.

[0427] Demographic and baseline disease characteristics were generally similar in the two study arms, except for previous TTP episodes and ADAMTS13 activity (Table 7.8). In total, 97% (140 / 145) of patients received glucocorticoids, and of these, 24% (35 / 145) initiated treatment with rituximab during daily plasma exchange (17% or 12 / 72 in the caplacizumab arm and 32% or 23 / 73 in the placebo arm).

[0428] [Table 3]

[0429] 7.9 Primary and Key Secondary Endpoints Based on Kaplan Meier analysis and stratified log-rank test, There was a significant reduction in the time to platelet response in the caplacizumab group (Figure 1). At any time point, patients receiving caplacizumab were 1.55 times more likely to achieve a platelet response compared with placebo-treated patients (platelet count normalization ratio, 1.55; 95% CI, 1.09 to 2.19; P=0.01). During the study drug treatment period, treatment with caplacizumab resulted in a 74% reduction in the number of subjects who had a TTP-related death, a recurrence of TTP, or a severe thromboembolic event (P<0.0001, Table 7.9). Over the entire study period, including the 28-day treatment-free follow-up period, 28 patients in the placebo group experienced a recurrence compared with 9 in the caplacizumab group, a 67% reduction (P<0.001, Table 7.9). In all six patients in the caplacizumab group who experienced a recurrence of TTP during follow-up (i.e., recurrence >30 days after the end of daily plasma exchange), ADAMTS13 activity levels were <10% at the end of study drug treatment, suggesting that the underlying disease was still active at the time the study drug was stopped. None of the caplacizumab-treated patients were refractory to therapy, whereas three patients in the placebo group were (P=0.057). Caplacizumab treatment was also associated with a trend toward more rapid normalization of three organ damage markers: LDH, cTnI, and serum creatinine.

[0430] [Table 4]

[0431] 7.10 ADAMTS13 activity in relation to relapse Exacerbations: Of the 145 patients randomized in the Hercules trial, 129 achieved a platelet count response and completed the daily plasma exchange period (65 in the caplacizumab arm and 64 in the placebo arm). The week following completion of daily plasma exchange, ADAMTS13 activity had returned to >10% in 42% (54 / 129) of patients, while the other 58% (75 / 129) remained suppressed below 10%. A total of 31 patients experienced exacerbations, of which 2 had normalized ADAMTS13 activity and 29 had unresolved underlying disease as evidenced by ADAMTS13 activity levels below 10% (Figure 2, Panel A).

[0432] Relapse: ADAMTS13 activity levels at the end of study drug treatment were available for 120 patients (60 at the end of double-blind caplacizumab treatment, 34 at the end of double-blind placebo treatment, and 26 at the end of open-label caplacizumab treatment). Of these patients, 74% (89 / 120) had normalized ADAMTS13 activity levels by the time treatment was stopped. None of these patients suffered a relapse during the 28-day follow-up after the end of study drug treatment. The other 26% (31 / 120) of patients still had suppressed ADAMTS13 activity at the time study drug treatment was stopped. A total of 9 of these 31 patients suffered a relapse during the subsequent 28 days, 6 of the 9 after completion of double-blind caplacizumab treatment and 3 of the 9 after completion of open-label caplacizumab treatment (Figure 2, Panel B) (see Example 7.13).

[0433] 7.11 Characterization and Outcome of aTTP Patients with Primary or Recurrent Disease Context: Acquired thrombotic thrombocytopenic purpura (aTTP) is a life-threatening autoimmune coagulation disorder. Patients are at risk of significant morbidity and mortality with each episode. Efficacy and safety data are available from the phase III Hercules trial of caplacizumab in patients with aTTP (Scully et al. Blood 2017;130:LBA-1).

[0434] Aims: To characterize disease manifestations and evaluate treatment outcomes in patients enrolled in the HERCULES trial with a first or recurrent aTTP episode.

[0435] Methods: Descriptive summaries were used to assess demographics, baseline disease characteristics, and treatment outcomes (time to platelet count response, mortality, relapse, severe thromboembolic events (TE), and refractory status) for both subgroups. Platelet counts, LDH levels, and cardiac troponin I levels were determined according to standard methods (see detailed description).

[0436] Results: A total of 145 patients were randomized: 82 with a first aTTP episode and 63 with recurrent disease. Demographic characteristics were generally balanced between groups, but baseline disease characteristics were more severe in first episodes than in recurrent episodes: mean platelet count (28.8 × 10 9 / L vs 44.4 × 10 9 / L), mean LDH (598 U / L vs. 523 U / L), and median cardiac troponin I (0.119 μg / L vs. 0.036 μg / L). The time from first symptom to diagnosis was also longer in those experiencing a first episode (6.5 days) versus relapsed patients (3.9 days). More patients in the caplacizumab group (66.7%) had a first aTTP episode compared with the placebo group (46.6%). Treatment with caplacizumab improved outcomes compared with placebo in both subgroups [i.e., shorter time to platelet count response, lower proportion of patients who died, relapsed, or had a severe TE event during treatment, lower relapse rate over the entire study period, and prevention of refractory disease] (see Table 7.11 for detailed results). (See ).

[0437] CONCLUSIONS: Patients with a first aTTP episode have later onset of symptoms and more severe disease at baseline than patients with recurrent disease. Treatment with caplacizumab improves outcomes in both subgroups.

[0438] [Table 5]

[0439] 7.12 Treatment of acquired TTP with anti-vWF Nanobody significantly reduces healthcare resource utilization 284 Background: The efficacy and safety of caplacizumab, an anti-von Willebrand factor (vWF) nanobody, for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) was evaluated (Scully et al., Blood 2017;130:LBA-1).

[0440] Aims: To investigate the effect of treatment with caplacizumab on healthcare resource utilization: plasma exchange (PE) parameters, hospital stay, and intensive care unit (ICU) stay.

[0441] Methods: Amount and days of PE, hospital stay, and ICU stay were summarized and compared between the caplacizumab and placebo arms over the entire study treatment period using the Wilcoxon rank sum test with normal approximation.

[0442] RESULTS: The Hercules trial randomized 145 patients, 73 to placebo and 72 to caplacizumab. Treatment with caplacizumab expedited normalization of platelet counts, prevented progression, and prevented patients from becoming refractory to treatment. This was reflected in a 38% reduction in mean days (±SE) of PE in the caplacizumab (n=71) vs. placebo (n=73) groups: 5.8 (±0.51) vs. 9.4 (±0.81) days (p<0.001). The mean (±SE) total volume of plasma exchanged was also reduced by 41%: 21.3 (±1.6) L vs. 35.9 (±4.2) L (p<0.001). The mean duration of hospitalization (±SE) was reduced by 31% in the caplacizumab (n=71) vs. placebo group (n=73): 9.9 (±0.7) days vs. 14.4 (±0.7) days (p=0.0025).

[0443] One-third of patients were admitted to the ICU (28 patients in the caplacizumab group and and 27 patients in the placebo group. The caplacizumab group reduced the mean (±SE) length of ICU stay by 65%: 3.4 (±0.4) days vs. 9.7 (±2.1) days (p=0.0098).

[0444] CONCLUSIONS: Caplacizumab represents a novel treatment for aTTP by rapidly blocking vWF-mediated platelet adhesion. Treatment with caplacizumab results in improved outcomes reflected in a meaningful reduction in healthcare resource utilization.

[0445] 7.13 Efficacy and Safety of Open-Label Caplacizumab in Patients with Exacerbations of aTTP Background: The efficacy and safety of caplacizumab in patients with acquired thrombotic thrombocytopenic purpura (aTTP) has been demonstrated in a single-blind phase 2 study (Peyvandi et al. 2016 N Engl J Med 374:511-522) and confirmed in a double-blind phase 3 study (Scully et al. Blood 2017 130:LBA-1).

[0446] Aims: Here, we present efficacy and safety results for patients who received open-label (OL) caplacizumab.

[0447] Methods: If relapse occurred during the double-blind (DB) treatment period, patients were switched to OLcaplacizumab in conjunction with resumption of daily plasma exchange (PEX) and immunosuppression while maintaining blindness to the initial treatment assignment. Platelet counts were determined according to standard methods (see detailed description). ADAMTS13 activity was determined as described in Example 7.7.

[0448] Results: During DB, 31 patients experienced progression, 28 in the placebo group and 3 in the caplacizumab group. Of these, 28 switched to OL treatment with caplacizumab (26 of 28 placebo-treated patients and 2 of 3 caplacizumab-treated patients). By day 6, 81% of patients receiving OL caplacizumab had a confirmed platelet count response (i.e., ≥150 × 10 confirmed by cessation of daily PE within 5 days).9 All patients achieved a platelet count of ≥ 10 / L. There were no deaths. During the OL treatment period, one patient (3.6%) experienced a TTP exacerbation and one patient (3.6%) experienced a severe thromboembolic event (vena cava thrombosis). After completion of OL treatment, three patients experienced a recurrence of TTP, and all three had ADAMTS13 activity < 10% at the time of treatment cessation. The safety profile in the OL caplacizumab group was consistent with that observed in the DB caplacizumab group. A total of 25 patients (89.3%) reported at least one treatment-emergent adverse event.

[0449] Conclusions: Open-label treatment with caplacizumab was effective in patients experiencing exacerbations of aTTP. The safety profile was consistent with that observed during the double-blind period (see Example 7.14).

[0450] 7.14 Overall Study Period: Safety and Immunogenicity. Because of the design of the Hercules trial (which included switching to open-label caplacizumab treatment if TTP recurred while on double-blind study drug) and because nearly all recurrences occurred in the placebo group (see Example 7.13), the median duration of exposure was longer in the caplacizumab group than in the placebo group: 35 days (range 1 to 65 days) vs. 23 days (range 2 to 66 days).

[0451] During the entire study, 69 patients (97.2%) in the caplacizumab group and 71 patients (97.3%) in the placebo group reported at least one adverse event. Adverse events were reported by the investigator in 41 patients (57.7%) in the caplacizumab group and Events were considered at least possibly related to study drug in 32 patients (43.8%) in the placebo group.

[0452] During the study, four patients had adverse events leading to death: one in the caplacizumab group (1.4%) (deemed by the investigator to be unrelated to study drug during the treatment-free follow-up period) and three in the placebo group (4.1%) (all during the study drug treatment period).

[0453] Serious adverse events were reported by 28 patients (39.4%) in the caplacizumab group and 39 patients (53.4%) in the placebo group during the entire study period. According to the protocol, relapses had to be reported as serious adverse events and were numerically the main driver in the placebo group. Serious adverse events were considered by the investigators to be at least possibly related to study drug in 10 patients (14.1%) in the caplacizumab group and 4 patients (5.5%) in the placebo group.

[0454] Five patients in the caplacizumab group and nine subjects in the placebo group reported adverse events leading to discontinuation of study drug.

[0455] Bleeding-related adverse events were reported in 46 patients (64.8%) in the caplacizumab group and 35 patients (47.9%) in the placebo group. The most common were nosebleeds and gingival bleeding; all of these resolved mostly without intervention. These events were mild or moderate in the majority of patients and severe in three patients in the caplacizumab group and one patient in the placebo group. Severe bleeding adverse events were reported in eight patients (11.3%) in the caplacizumab group and one patient (1.4%) in the placebo group. The most frequently reported serious bleeding adverse event was nosebleed in four patients in the caplacizumab group.

[0456] Drug-induced anti-drug antibodies developed in 3.1% of patients treated with caplacizumab. No impact on clinical efficacy was observed in these patients, and no serious adverse events were reported (see Example 7.21).

[0457] 7.15: Pharmacodynamics of Caplacizumab The interaction of caplacizumab with vWF is highly specific, and the binding of caplacizumab to the vWF A1 domain does not affect the ability of vWF to interact with coagulation factor VIII (FVIII), for which vWF has a carrier function. Similarly, the selective binding of caplacizumab does not affect the ability of vWF to interact with fibrillar collagen, type VI collagen, or ADAMTS13. Furthermore, caplacizumab does not cross-react with red blood cells or platelets. Because of this high specificity, off-target effects are not expected and have not been observed in preclinical and clinical trials.

[0458] Levels of total (free + drug complexed) vWF, ristocetin cofactor activity (RICO), and FVIII levels were measured in healthy volunteers after receiving a single ascending intravenous or subcutaneous dose, and multiple daily 10 mg subcutaneous doses, and in patients with aTTP who received an initial 10 mg intravenous bolus or placebo followed by multiple 10 mg once daily subcutaneous doses or placebo.

[0459] 7.15.1 vWF antigen (vWF:Ag) Baseline vWF:Ag levels were higher in aTTP patients than in healthy subjects. In clinical trials conducted with caplacizumab, the mean (±standard deviation [SD]) plasma vWF:Ag levels were 38.5±10.9 nM in healthy subjects and 70.5±30.0 nM in aTTP patients. Caplacizumab treatment improved the pharmacokinetics of vWF. , resulting in a transient reduction in total circulating vWF:Ag levels. On average, this effect was reversed within 2 to 7 days after the last dose in healthy volunteers and aTTP patients (Figure 3).

[0460] The transient decrease in total vWF:Ag levels was likely due to the faster clearance of the drug-target complex compared with the free target.

[0461] 7.15.2 RIPA / RICO Ristocetin platelet aggregation (RIPA) and RICO assays are in vitro assays to evaluate the platelet-binding ability of vWF present in blood. The methodology of the RIPA and RICO assays is based on the fact that the addition of ristocetin to plasma in the presence of platelets results in platelet aggregation. Ristocetin, an antibiotic, activates vWF to an extent similar to high shear blood flow conditions, and as a consequence, modulates the binding of vWF to the platelet receptor GP1b. These methods were chosen to evaluate the activity of caplacizumab during treatment, as the interaction of caplacizumab with vWF can block in vitro platelet aggregation. A reduction in RIPA or RICO activity below 10% or 20%, respectively, indicated that vWF-mediated platelet adhesion was completely inhibited by caplacizumab. In healthy volunteers, complete and stable target inhibition for at least 24 hours was observed after a single subcutaneous dose of 10 mg or more. This daily administered 10 mg subcutaneous dose also induced complete inhibition of vWF-mediated platelet adhesion in aTTP patients over the entire treatment period (Figure 4). In all clinical trials, RICO activity returned to baseline values ​​within 7 days of discontinuation of study drug.

[0462] 7.15.3 FVIII vWF acts as a carrier for FVIII. Modulation of total vWF levels by caplacizumab also resulted in a transient reduction in FVIII levels, with a return of FVIII to the normal range observed within 2 to 7 days after administration of the final dose in healthy volunteers and aTTP patients with vWF.

[0463] 7.15.4 Thorough QT / QTc Studies No cardiovascular effects have been observed in nonclinical studies, and there was no evidence of clinically relevant electrocardiogram (ECG) findings in completed human studies (see Example 7.13).

[0464] Given caplacizumab's molecular structure and size, its target specificity, and lack of cardiovascular liability in vivo, it was not expected that caplacizumab would prolong the QT interval. This has not been observed.

[0465] 7.16 Pharmacokinetics of Caplacizumab Total (free + target-bound) caplacizumab concentration levels were measured in plasma of healthy volunteers and aTTP patients. A complete pharmacokinetic profile was obtained in healthy subjects after a single ascending intravenous infusion, as well as single and repeated subcutaneous doses (Figure 5). Sparse plasma samples were obtained in aTTP patients in phase II and III trials. The pharmacokinetic profile of caplacizumab has been investigated by standard noncompartmental analysis (NCA) in healthy volunteers and in population pharmacokinetic analyses in healthy volunteers and aTTP patients.

[0466] Following repeated daily subcutaneous administration of 10 mg, steady state was rapidly achieved at the time of the second drug administration, with limited accumulation of caplacizumab depending on the expression of the target vWF:Ag.

[0467] 7.16.1 Absorption The main exposure parameters, estimated by standard noncompartmental methods after a single dose in healthy volunteers or model predicted at steady state in aTTP patients, are reported in Tables 7.16.1A and 7.16.1B. After single dose administration, the extent of exposure (area under the curve; AUC) and rate of exposure (maximum concentration; Cmax) increased with the administered dose, but not proportionally. After a 10 mg subcutaneous dose, the Cmax of caplacizumab was achieved 4 hours after administration. Absolute bioavailability, determined in a population pharmacokinetic analysis, was estimated to be 90% in aTTP patients and close to 100% in healthy volunteers.

[0468] [Table 6]

[0469] [Table 7]

[0470] 7.16.2 Distribution and metabolism The pharmacokinetics of caplacizumab demonstrated a biphasic plasma profile. Population pharmacokinetic modeling estimated central and peripheral compartment volumes of distribution of 5.35 and 6.33 L, respectively, in healthy volunteers and patients with aTTP.

[0471] Preclinical studies in cynomolgus monkeys showed that caplacizumab bound and neutralized vWF activity in the systemic circulation within 5 minutes. The major form of caplacizumab in the circulation, the caplacizumab-vWF complex, was distributed to the liver as well as to unbound vWF, which was rapidly catabolized by the reticuloendothelial system. Preclinical studies suggested that excess unbound caplacizumab was distributed to other well-perfused organs / tissues, where it was degraded by bulk nonspecific catabolism. Free caplacizumab, with a MW of 28 KDa, was filtered in the glomerulus, although not freely. The contribution of the kidney to the overall loss of small proteins depended on proteolytic activity in other body regions. For caplacizumab, a negligible proportion of the administered dose was recovered in the urine (<0.5%).

[0472] 7.16.3 Disappearance The apparent clearance of caplacizumab varied with the administered dose. After a single intravenous dose of 10 mg in healthy volunteers, a mean clearance of 769 ± 343 mL / h and a mean terminal half-life of 19.2 ± 7.5 h were estimated using model-dependent methods. The pharmacokinetics of caplacizumab after subcutaneous dosing appears to be absorption-controlled. After a single subcutaneous dose of 10 mg in healthy volunteers, the mean apparent clearance was 386 ± 160 mL / h and the mean terminal half-life was 38.5 ± 22.2 h.

[0473] 7.16.4 Dose proportionality In healthy subjects, with escalating subcutaneous caplacizumab doses from 2 to 16 mg, the increases in Cmax and AUC were less than dose proportional. With dose increasing ratios of 1.0:2.0:4.0:5.0:8.0, the mean Cmax ratios were 1.0:1.8:2.0:2.4:2.8 and the mean AUC increased in the ratios 1.0:3.9:4.1:5.3:6.1.

[0474] The total clearance of caplacizumab depended on drug and target levels and was the sum of linear (catabolic) and nonlinear (target-mediated) clearance. The terminal half-life of caplacizumab also depended on drug and target levels. In healthy volunteers, the mean apparent terminal half-life increased from 13 to 40 hours after a single intravenous dose (0.5 to 12 mg) and from 11 to 53 hours after a single subcutaneous dose (2 to 16 mg).

[0475] 7.16.5 Effects of demographic factors and body size A population pharmacokinetic analysis in patients with aTTP showed that age, sex, race, and blood type did not affect the pharmacokinetics of caplacizumab. Body weight and renal function as measured by creatinine clearance (CrCL) had a statistically significant effect on pharmacokinetics, with predicted exposure being higher in patients with lower body weight and CrCL.

[0476] However, as shown in Table 7.16.5, in patient populations with extreme values ​​of these covariates, the expected exposure ranges largely overlap and no specific dose adjustment was deemed necessary. Although baseline vWF levels have a statistically significant effect on drug exposure, increased drug exposure for patients with elevated vWF did not result in a different pharmacodynamic effect (change from baseline vWF levels) and no separate dose adjustment was deemed necessary (see also Examples 7.16.6 and 7.18).

[0477] [Table 8]

[0478] 7.16.6 Pharmacokinetics in Special Populations No formal trials have been performed with caplacizumab in patients with severe acute or chronic liver injury, and no data are available regarding its use in these populations. The risks associated with the use of caplacizumab in patients with mild and moderate liver injury were thought to be similar to those in the overall patient population, because caplacizumab-vWF complexes are expected to be cleared by activated Kupffer cells rather than by damaged hepatocyte parenchyma. However, caplacizumab should be used with caution in patients with severe liver injury who present a high risk of bleeding.

[0479] The effect of renal impairment on the pharmacokinetics of caplacizumab has not been formally studied. In population pharmacokinetic / pharmacodynamic models, renal function (baseline CrCL, range: 11.9 to >120 mL / min) had a statistically significant effect, resulting in a limited increase in predicted exposure (AUCss) in severe renal impairment. In clinical trials in patients with aTTP, those with reduced renal function did not pose an additional risk of adverse events.

[0480] 7.16.7 Drug-Drug Interactions Nanobodies are single variable domain antibody fragments that are ubiquitously protected against proteolytic degradation. No in vitro drug-drug interaction studies were performed with caplacizumab, as it is expected to be enzymatically catabolized and does not directly interact with cytochrome P450 isoforms or other metabolic enzymes or transporters. Although cytokine modulation may be an indirect mechanism by which Nanobodies may alter CYP expression, cytokine-mediated CYP-related drug-drug interactions are unlikely for caplacizumab: the Nanobody® selectively targets vWF and is therefore not expected to have immunomodulatory properties.

[0481] Treatment of aTTP often involves the use of corticosteroids and rituximab, and in some cases aspirin and low molecular weight heparin are used as thromboprophylaxis. Less frequently, vincristine, cyclophosphamide, or cyclosporine are used for refractory disease. All of these compounds except rituximab are known to be largely eliminated by the liver through CYP-mediated pathways, with renal clearance pathways being only a minor pathway, and no interactions between drugs and Nanobodies are expected when these treatments are initiated in combination with caplacizumab. As with other monoclonal antibodies, the degradation of rituximab occurs by nonspecific bulk proteolysis, and no potential drug-drug interactions are expected.

[0482] Caplacizumab selectively and specifically inhibits the A1 domain of vWF. In vitro, caplacizumab only partially competed with heparin binding to vWF. In vivo, pharmacodynamic interactions between coadministered heparin and caplacizumab were not expected to be clinically significant because the primary on-target effect of heparin is mediated by binding to the enzyme inhibitor antithrombin III.

[0483] 7.16.8 Dose-Response Relationships Studies in healthy volunteers after single ascending intravenous doses and single and repeated subcutaneous doses confirmed the expected pharmacological responses and safety of caplacizumab.

[0484] 7.17 Effect of PE on the pharmacokinetics and pharmacodynamics of caplacizumab 284 Background: During clinical trials in patients with acquired thrombotic thrombocytopenic purpura, caplacizumab was administered as a 10 mg intravenous (iv) bolus before plasma exchange (PE), followed by daily subcutaneous (sc) doses of 10 mg throughout the PE period and for at least 30 days thereafter.

[0485] Aims: We investigated the potential effects of different time intervals between the first intravenous bolus and subsequent PE, as well as the effects of different PE schedules.

[0486] Methods: Using nonlinear mixed-effects modeling, we developed an integrated pharmacokinetic (PK) / pharmacodynamic (PD) model to describe the interaction between caplacizumab and vWF and used it to simulate the resulting PK and PD for the following different given scenarios: (1) PE was initiated 3, 5, 11, and 23 hours after a 10 mg intravenous bolus of caplacizumab (2) PE schedule: once daily and twice daily (bid) on the first day or for 7 days

[0487] RESULTS: Data from clinical trials showed complete neutralization of vWF activity at caplacizumab plasma concentrations ≥ 500 ng / mL. Simulations suggested that median caplacizumab plasma levels remained above this threshold if PE was initiated up to 5 hours after an intravenous bolus of caplacizumab. For longer delays, caplacizumab plasma levels The median value may fall below 500 ng / mL and an additional 10 mg subcutaneously administered prior to PE can be used to maintain effective drug exposure.

[0488] Simulation of the effect of twice-daily PE for 7 days suggested that the subcutaneous caplacizumab dosing schedule during PE could be adjusted to 10 mg caplacizumab subcutaneously twice daily after each PE treatment during the twice-daily PE treatment.

[0489] Conclusions: Effective drug levels are expected if PE is initiated up to 5 hours after the first intravenous dose of caplacizumab. For longer delays, an additional 10 mg subcutaneous dose may be envisaged before PE.

[0490] If twice daily PE is administered for 7 days, the subcutaneous caplacizumab administration schedule may be adjusted with twice daily caplacizumab following each PE treatment.

[0491] 7.18 Rationale for Caplacizumab Dosing in Patients with aTTP Supported by Mechanism-Based PKPD Modeling Objective: To describe the correlation between caplacizumab concentrations and its target, von Willebrand factor antigen (vWF:Ag), after treatment in various adult populations. The developed model should be utilized for simulation of hypothetical scenarios to support dosing regimens.

[0492] METHODS: This analysis included 10 phase I to III trials of caplacizumab in healthy volunteers (n=100), patients undergoing percutaneous coronary intervention (PCI) (n=225), and patients with acquired thrombotic thrombocytopenic purpura (n=216). 1-10 The data were based on a total of 3629 PK observations and 6295 PD observations from 2016-2019. The majority of patients with aTTP received plasma exchange (PE) and immunosuppressant treatment as standard of care. A wide range of dose levels, treatments, and PE schedules were represented. Data after both intravenous and subcutaneous administration were included.

[0493] Population PKPD analysis was performed by nonlinear mixed-effects modeling using NONMEM, version 7.3.0. The model was developed in stages. First, a subset of the dataset including data on healthy volunteers and PCI patients was used for model development. The model was then updated to account for specific characteristics associated with aTTP disease state and standard of care PE in the subset of the dataset including aTTP patients. The effects of age, sex, race, blood type, weight, creatinine clearance, and concomitant treatment were assessed based on graphical evaluation using stratified prediction-corrected visual prediction checks and univariate assessments in NONMEM.

[0494] Simulations using the final model were performed for patients with aTTP to evaluate the effects of changes in dose, patient weight, and need for dose adjustments in pediatric patients.

[0495] RESULTS: The interaction between caplacizumab and vWF:Ag was fully described by a complete target-mediated pharmacokinetic model. This model included a two-compartment pharmacokinetic model with parallel slow and fast first-order absorption processes and first-order linear elimination of the free drug. This model explained the formation of drug-vWF complexes with the ability to form both dimers and trimers. The generation and maturation of vWF was explained by storage in Weibel-Palade bodies in the endothelium, as well as storage of vWF in the transit and pool compartments, mimicking the subsequent rapid release and elimination of free vWF. The half-life of free vWF was fixed at 16 hours, the literature value (Lenting et al., 2015, Blood 26:2019-2028; Favalo et al., 2015). (Ro et al., 2007, Thromb Haemost 97:922-930; Dobrkovska et al., 1998, Haemophilia 4:33-39; Goudemand et al., 2005, J Thromb Haemost 3:2219-2227). A dual feedback mechanism was involved, stimulating the rate of production and release of vWF from the pool when vWF decreased below the subject's baseline levels.

[0496] For patients with aTTP, disease progression was captured as a transient increase in vWF:Ag over time, and the effect of PE was explained by the parallel clearance of free vWF, free drug, and drug-vWF complexes. The typical overall clearance rates of the population under PE were estimated to be 3.7-fold higher for free drug, 3.5-fold higher for free vWF, and 1.7-fold higher for drug-vWF complexes.

[0497] Body weight was non-proportionally included in the model (fixed index), and creatinine clearance was identified as a statistically significant covariate, as clearance was slightly reduced for patients with CRCL below the median CRCL (100 ml / min) in patients with aTTP.

[0498] This model was successfully applied to simulate hypothetical scenarios to support dosing regimens, dosing in special populations, and how to handle missed doses. Simulations were also performed to obtain information on dosing regimens in pediatric patients and to predict PKPD behavior in Japanese aTTP patients based on differences in body size. Simulations were also performed to gain more knowledge on the impact of baseline vWF:Ag concentrations and the effect of PE schedules in terms of timing, intensity, and duration.

[0499] Conclusions: A semi-mechanistic population PKPD model was developed to describe the interaction between caplacizumab and vWF (based on vWF:Ag observations). The model fully described the drug-vWF complex interactions over time, including effects dependent on disease progression and PE treatment in aTTP patients. The model was successfully applied to improve understanding of the PKPD interrelationship between caplacizumab and vWF in the target population, supporting the dosing rationale in both adult and pediatric patients through the use of simulations, and enabling bridging to Japanese aTTP patients.

[0500] 7.19 Model-Based Dosing Recommendations for Caplacizumab in Pediatric Patients with Acquired Thrombotic Thrombocytopenic Purpura Context: Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare, life-threatening autoimmune blood clotting disorder with a significantly lower incidence in children compared with adults. Caplacizumab is being evaluated in phase 2 and 3 randomized clinical trials in adult patients with aTTP.

[0501] Aims: No pediatric patients were enrolled in clinical trials of caplacizumab, so model-based simulations were used to develop dose recommendations for this population.

[0502] Methods: Nonlinear mixed-effects modeling was used to develop a semimechanistic pharmacokinetic-pharmacodynamic (PKPD) population model describing the interaction between caplacizumab and von Willebrand factor antigen (vWF:Ag) following intravenous and subcutaneous administration of caplacizumab at various dose levels in various adult populations. Simulations based on the nonproportional scaled PKPD model were performed to establish the preferred dosing regimen in adolescents and children over 2 years of age. Eight age categories with 1000 individuals in each category were defined, and the weights of the corresponding individuals were sampled from the National Health and Nutrition Examination Survey database. Simulated caplacizumab exposure levels in different age categories were compared with those expected in adults.

[0503] Results: Exposure simulations show that a flat daily dose of 10 mg results in relatively high exposures in low-weight children, mainly those under 10 years of age. Adjusting the daily dose to 5 mg for children weighing less than 40 kg would result in similar exposures on average across age and weight groups. Weight-adjusted dosing is also predicted to result in similar vWF:Ag suppression across different age groups.

[0504] Conclusions: The recommended dose for adolescents aged 12-18 years weighing ≥40 kg is 10 mg, and for those weighing <40 kg it is 5 mg. As no age-based differences in vWF:Ag suppression are expected, the same recommended doses apply to children aged 2-12 years: ≥40 kg, 10 mg, and for those weighing <40 kg, 5 mg.

[0505] 7.20 Real-World Experience in Patients with aTTP Treated with Caplacizumab Through a Managed Access Program Context: Acquired thrombotic thrombocytopenic purpura (aTTP) is a life-threatening immune-mediated thrombotic microangiopathy (TMA). The efficacy and safety of caplacizumab in patients with aTTP have been demonstrated in randomized clinical trials.

[0506] Aims: To descriptively report the first real-world experience with caplacizumab in patients with aTTP in a managed access program (MAP).

[0507] Methods: Eligibility criteria for MAP were: (i) patients with an episode of aTTP, (ii) not satisfactorily treated with approved therapy; (iii) patients had no known hypersensitivity to any of the active substances or excipients, and (iv) were not pregnant. Access to caplacizumab was permitted in accordance with the laws and regulations in force in the country where the product was requested. Safety-related events were to be reported by the requesting physician in accordance with all pharmacovigilance laws of the country.

[0508] Results: From May 14, 2018 to January 22, 2019, 118 MAP requests were received, of which 75 were approved, 7 were rejected, 35 were not pursued / withdrawn by the requester, and 1 was under review. Of the approved requests, 67 patients were treated with caplacizumab, and 8 did not ultimately start therapy. Patients received caplacizumab as frontline therapy or for the treatment of aTTP with a refractory course. Therapy was successfully completed in 37 patients, ongoing in 13 patients, and discontinued in 17 patients (of which 5 died). All deaths occurred in patients in whom caplacizumab was initiated late due to a severe refractory course, and none were considered related to caplacizumab. Any safety reports were consistent with the safety profile observed in clinical trials without any new safety signals.

[0509] Conclusions: First real-world evidence with caplacizumab in patients with aTTP confirms the important benefits of caplacizumab observed in clinical trials, especially when initiated as frontline therapy.

[0510] 7.21 New Format To detect drug-induced treatment-induced ADA, a modified anti-drug antibody (mADA) assay was used. mADA uses a modified caplacizumab molecule, caplacizumab-ALA variant (SEQ ID NO: 24), as a detection reagent in a crosslinking format. The C-terminal alanine-extended caplacizumab does not bind to any pre-existing antibodies. There wasn't.

[0511] In various in vitro experiments, caplacizumab and C-terminal alanine-extended caplacizumab behaved comparably.

[0512] Comparative Nuclear Magnetic Resonance (NMR) analysis of 12A2H1 (a component of Caplacizumab; SEQ ID NO: 19) was used to demonstrate that the C-terminal extension (alanine) did not affect the overall conformation of the Nanobody.

[0513] Furthermore, surface plasmon resonance (SPR) demonstrated similar binding profiles to the target (vWF) for caplacizumab and C-terminal alanine-extended caplacizumab.

[0514] Given the similar characteristics and profiles between caplacizumab and C-terminally extended caplacizumab, it is expected that such C-terminally extended caplacizumab compounds, similar to caplacizumab, can also be used to treat and / or prevent aTTP.

[0515] 7.22 Conclusion. Over the past two decades, despite improved understanding of the pathophysiology of aTTP and the use of relatively new immunosuppressants, treatment outcomes have remained unchanged, with high morbidity and reported mortality rates reaching 20% ​​(Joly et al., 2017, Blood 129:2836-2846). Caplacizumab, a nanobody against the A1 domain of vWF, blocks vWF-platelet adhesion and aggregation, preventing further consumption of platelets in microthrombi that cause severe thrombocytopenia and tissue and organ damage in aTTP.

[0516] The clinical diagnosis of aTTP remains based on the patient's medical history, physical examination, and examination of peripheral blood smears. Indeed, patients with a first aTTP episode have a later onset of symptoms and a higher disease severity at baseline than patients with recurrent episodes of the disease.

[0517] Patients were eligible to enroll in the study if they had a clinical diagnosis of aTTP and had confirmatory ADAMTS13 activity testing performed at randomization. The clinical diagnosis was confirmed by ADAMTS13 activity of less than 10% in 86% of those randomized. In addition, 13 of the 20 patients with baseline ADAMTS13 activity >10% had evidence of a TTP diagnosis, such as a history of TTP or suppressed ADAMTS13 activity at other times during the study.

[0518] In the phase 2 TITAN trial, treatment with caplacizumab accelerated normalization of platelet counts and prevented disease progression. However, early relapse occurred in a subset of patients with unresolved underlying disease activity. Thus, the study encouraged investigators to extend blinded study drug treatment in conjunction with optimization of immunosuppression in patients with evidence of persistent autoimmune activity.

[0519] Results from the phase 3 HERCULES trial supported the hypothesis that treatment with caplacizumab reduces the time to platelet count response by halting platelet consumption in microthrombi. The results also demonstrated a clinically meaningful impact of caplacizumab on TTP-related death, TTP recurrence, or severe thromboembolic events during study drug treatment, and on overall recurrence. The validity of the approach of extending study drug treatment and optimizing immunosuppression in patients with evidence of unresolved underlying disease was validated by the results of this study, as demonstrated by the low number of recurrences after study drug treatment compared with those observed in the phase 2 trial. Indeed, all recurrences observed after discontinuation of study drug occurred in patients with ADAMTS <10%. 13 In a real-world clinical setting, to protect such patients from recurrence of aTTP, treatment with caplacizumab should be extended until the underlying disease has resolved.

[0520] Even in the setting of relatively severe disease in patients experiencing a first aTTP episode, treatment with caplacizumab demonstrated improved outcomes compared with placebo, including faster time to platelet count response, lower proportion of patients who died, relapsed, or had a severe TE event during the treatment period, lower relapse rates over the entire treatment period, and prevention of refractory disease.

[0521] In both phase 2 and 3 trials, no deaths were reported during treatment with caplacizumab, a favorable outcome in terms of preventing death. Furthermore, none of the patients treated with caplacizumab were refractory to plasma exchange, suggesting that caplacizumab has the potential to prevent refractory disease and the worse outcomes reported in this subpopulation. There was also a trend toward more rapid normalization of markers associated with organ damage in patients treated with caplacizumab.

[0522] The impact of treatment with caplacizumab on time to platelet response and prevention of relapse was associated with a significant reduction in healthcare resources.

[0523] Caplacizumab interferes with vWF, a key protein in hemostasis, and based on this pharmacological effect, is associated with a high risk of mucocutaneous bleeding similar to that observed in von Willebrand's disease (Leebeek & Eikenboom, 2016, N Engl J Med 375:2067-2080). Safety results were consistent with those previously reported.

[0524] ADAMTS13 activity is gaining increasing relevance in the management of aTTP, as it is a predictive marker of recurrence risk (Westwood et al., 2013, J Thromb Haemost 11:481-490; Peyvandi et al., 2008, Haematologica 93:232-239; Lammle et al., 2008, Haematologica 93:172-177; Knovich et al., 2012, Eur J Haematol 88:518-525; Kremer Hovinga et al., 2010, Blood 115:1500-1511; Quiz 662; Bresin et al., 2009, Thromb Haemost 101:233-238; Cataland et al., 2009, Eur J Haematol 83:559-564; Goyal et al., 2013, J Clin Apher 28:390-394). In this study, almost all relapsed patients had suppressed ADAMTS13 activity, confirming its value. In this group, more than 50% of patients still had not achieved normalization of ADAMTS13 activity at the end of the plasma exchange period, and exacerbations occurred up to 25 days after the end of plasma exchange, supporting the need to continue treatment with caplacizumab for at least 30 days after a platelet count response had been achieved.

[0525] Caplacizumab rapidly blocks vWF-mediated platelet adhesion and thus represents an important addition to the treatment armamentarium for patients with aTTP.

[0526] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13]

[0527]

Table 14

Table 15

[0528]

Table 16

Table 17

Claims

1. 1. A pharmaceutical composition comprising a polypeptide comprising two anti-human von Willebrand factor (vWF) immunoglobulin single variable domains (ISVDs) for use in treating a first episode (symptoms of) a vWF-associated disease in a human, said treatment comprising administering to the human a dose of 5 mg of said polypeptide; the human is between 12 and 18 years old and weighs less than 40 kg; Each anti-human vWF ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), The pharmaceutical composition, wherein at least one anti-human vWF ISVD is represented by SEQ ID NO:

19.

2. 2. The pharmaceutical composition of claim 1, 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 even for a period longer than 10 days, such as 20 days, preferably for a period longer than 30 days, such as 2 months, 3 months, 4 months, 5 months, 6 months, or even more (treatment period).

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the treatment results in a shorter time to platelet count response, a lower proportion of patients who die, relapse, or have a severe thromboembolic event (TE) during treatment, a lower relapse rate, and / or prevention of refractory state.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the polypeptide comprises at least one ISVD that binds to SEQ ID NO:

20.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the polypeptide is at least 90% identical to SEQ ID NO:

1.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the polypeptide is ALX0081 (SEQ ID NO: 1) or ALX0081-A (SEQ ID NO: 24).

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the dose is administered once a day or twice a day.

8. The pharmaceutical composition according to any one of claims 1 to 7, comprising repeating administration of the polypeptide until the platelet count in a human is at least 150,000 / µl.

9. 9. The pharmaceutical composition of claim 8, comprising repeating administration of the polypeptide until the platelet count in the human is at least 150,000 / μl on at least two consecutive measurements.

10. 10. The pharmaceutical composition of claim 9, 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 even for a period longer than 10 days, such as 20 days, preferably longer than 30 days or more, after the platelet count is at least 150,000 / μl in at least two consecutive measurements.

11. 11. The pharmaceutical composition according to claim 9 or 10, wherein the two successive measurements are spaced at least 24 hours, more preferably 48 hours apart, such as at least 3 days apart, or even more, such as 4, 5, 6 or even 7 days apart, preferably 1 week apart.

12. 12. The pharmaceutical composition of any one of claims 1 to 11, wherein the treatment comprises repeated administration of the polypeptide until ADAMTS13 activity in a human is at least 10%, such as at least 15%, 20%, 25%, 30%, 35%, 45%, or even 50% of baseline ADAMTS13 activity.

13. 13. The pharmaceutical composition of any one of claims 1 to 12, wherein the treatment comprises repeating administration of the polypeptide until the level of the organ damage marker in the human returns to at least 50%, such as at least 40%, or even 60%, 70%, 80%, 90%, or even 100%, of normal levels.

14. 14. The pharmaceutical composition of claim 13, wherein the organ damage marker is LDH level, troponin T level, troponin I level, and / or creatinine level.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the treatment comprises performing plasma exchange.

16. The pharmaceutical composition of claim 15, wherein plasma exchange is performed within 5 hours after administration of the polypeptide.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the vWF-associated disease is selected from acute coronary syndrome (ACS), transient ischemic attack, unstable or stable angina, stroke, myocardial infarction, or thrombotic thrombocytopenic purpura (TTP).

18. The pharmaceutical composition according to claim 17, wherein the vWF-associated disease is thrombotic thrombocytopenic purpura (TTP).

19. A kit or product comprising a container containing the pharmaceutical composition of any one of claims 1 to 18 and instructions for use.

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