Anti-adrenomedullin (ADM) antibodies, or anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds for use in the treatment and therapy of hemostasis in patients in need thereof

Abstract

The subject of the present invention is an anti-adrenomedullin (ADM) antibody, or an anti-adrenomedullin antibody fragment, or an anti-ADM non-Ig scaffold for use in the treatment and therapy of hemostasis in patients in need thereof.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The subject of the present invention is an anti-adrenomedullin (ADM) antibody, or an anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold for use in the treatment and therapy of hemostasis in patients in need thereof. [Background technology]

[0002] background The adrenomedullin (ADM) peptide was first reported in 1993 as a novel hypotensive peptide containing 52 amino acids (Kitamura et al., 1993, Biochem Biophys Res Comm 192(2):553-560), but had previously been isolated from a human pheochromocytoma cell line (SEQ ID NO: 20). In the same year, a cDNA encoding a 185-amino acid precursor peptide and its complete amino acid sequence were also reported. This precursor peptide contains a 21-amino acid signal sequence, particularly at its N-terminus, and is referred to as "pre-proadrenomedullin" (pre-proADM). In this specification, all amino acid positions specified generally refer to the 185-amino acid pre-proADM. The adrenomedullin (ADM) peptide is a 52-amino acid peptide (SEQ ID NO: 20) that includes amino acids 95-146 of pre-proADM, from which the peptide is formed by proteolytic cleavage. Currently, only a few of the peptide fragments formed by cleavage of pre-proADM have been characterized in detail. These include the physiologically active peptide ADM and the "PAMP" peptide, which contains the 20 amino acids (22-41) following the 21 amino acids of the signal peptide in pre-proADM. The discovery and characterization of ADM in 1993 initiated an intensive research effort, the results of which have been summarized in various review articles. In the present context, reference is made in particular to the articles published in the special issue of "Peptides" on ADM (Takahashi 2001, Peptides 22:1691; Eto 2001, Peptides 22:1693-1711). Another review article is that of Hinson et al., 2000 (Hinson et al., 2000, Endocrine Reviews 21(2):138-167). Scientific research to date has shown that ADM can be considered, inter alia, as a multifunctional regulatory peptide.ADM is released into the circulation in an inactive form prolonged by glycine (Kitamura et al. 1998, Biochem Biophys Res Comm 244(2):551-555). Specific binding proteins for ADM also exist (Pio et al. 2001, The Journal of Biological Chemistry 276(15):12292-12300), likely modulating the effects of ADM as well. The most important physiological effect of ADM and PAMP studied to date is its effect on blood pressure.

[0003] Therefore, because ADM is an effective vasodilator, its blood pressure-lowering effect can be linked to a specific peptide compartment within the C-terminal portion of ADM. Furthermore, the physiologically active peptide PAMP formed from pre-proADM has been found to exhibit similar blood pressure-lowering effects, although it appears to have a different mechanism of action than ADM (see the above-mentioned review articles Eto et al., 2001 and Hinson et al., 2000, as well as Kuwasaki et al., 1997, FEBS Lett. 414(1):105-110; Kuwasaki et al., 1999, Ann. Clin. Biochem. 36:622-628; Tsuruda et al., 2001, Life Sci. 69(2):239-245; European Patent Application Publication No. EP-A2 0 622 458). Furthermore, concentrations of ADM that can be measured in the circulation and other body fluids have been found to be significantly greater in many pathological conditions than those found in healthy control subjects. Thus, ADM levels are significantly elevated, albeit to varying degrees, in patients with congestive heart failure, myocardial infarction, renal disease, hypertension, diabetes, and in the acute stage of shock, sepsis, and septic shock. PAMP concentrations are also elevated in some of the above pathological conditions, although plasma levels are lower than ADM (Eto 2001, Peptides 22:1693-1711). Abnormally high concentrations of ADM have been observed in sepsis, with the highest concentrations reported in septic shock (Eto 2001, Peptides 22:1693-1711; Hirata et al. J. Clinical Endocrinology and Metabolism 81(4):1449-1453; Ehlenz et al. 1997, Exp. Clin. Endocrinol. Diabetes 105:156-162; Tomoda et al. 2001, Peptides 22:1783-1794; Ueda et al. 1999, Am. J. Respir. Crit. Care Med. 160:132-136; Wang et al. 2001, Peptides 22:1835-1840).

[0004] Plasma concentrations of ADM are elevated in patients with heart failure and correlate with the severity of the disease (Hirayama et al. 1999, J Endocrinol 160:297-303; Yu et al. 2001, Heart 86:155-160). High plasma ADM is an independent negative prognostic indicator in these subjects (Poyner et al. 2002, Pharmacol Rev 54:233-246).

[0005] The role of MR-proADM (SEQ ID NO: 33) in heart failure has been investigated in several studies, including the BACH trial (Maisel et al. 2010, J. Am. Coll. Cardiol. 55:2062-2076), which found that MR-proADM was a strong prognostic factor for death at 90 days, adding prognostic value beyond that of diuretic peptides. Data from the PRIDE trial (Shah et al. 2012, Eur. Heart J. 33:2197-2205) subsequently solidified the potential prognostic role of MR-proADM. Across patient populations, MR-proADM had the best area under the curve (AUC) for 1-year mortality. Similarly, levels of MR-proADM in patients with chronic heart failure (CHF) correlate strongly with the severity of the disease, and elevated levels of this peptide are strongly associated with an increased risk of death over a 12-month follow-up period (van Haehling et al. 2010, European Journal of Heart Failure 12:484-491; Adlbrecht et al. 2009, European Journal of Heart Failure 11:361-366).

[0006] MR-proADM was examined during treatment of patients with acute decompensated heart failure (Boyer et al. 2012, Congest Heart Failure 18(2):91-97). Patients who tended to have elevated MR-proADM levels during acute treatment had findings associated with persistent congestion. Patients with elevated MR-proADM had increased peripheral edema during the 12- to 24-hour period following treatment. Kaiser et al. measured MR-proADM in patients with a single ventricle (Kaiser et al. 2014, Europ J Heart Failure 16:1082-1088). Levels were significantly higher in patients with impaired Fontan circulation (ascites and peripheral edema) compared with patients without Fontan circulation disruption. Furthermore, Eisenhut speculated whether treatments that reduce adrenomedullin levels could reduce the severity and extent of alveolar edema in pneumonia and sepsis (Eisenhut 2006, Crit Care 10:418).

[0007] Further known in the art are methods for determining the immunoreactivity of adrenomedullin in body fluids for diagnostic purposes, particularly for the diagnosis of sepsis, heart disease, and cancer. According to the present invention, a partial peptide (SEQ ID NO: 33) located in the central region of pro-adrenomedullin and containing amino acids 45-92 of the entire pre-adrenomedullin is measured by immunoassay using at least one labeled antibody that specifically recognizes the central-pro-ADM sequence (WO 2004 / 090546).

[0008] WO 2004 / 097423 describes the use of antibodies against adrenomedullin for the diagnosis, prognosis, and treatment of cardiovascular disorders. Treatment of diseases by blocking the ADM receptor has also been reported in the art (e.g., WO 2006 / 027147, PCT / EP2005 / 012844), including sepsis, septic shock, cardiovascular diseases, infectious diseases, skin diseases, endocrine diseases, metabolic diseases, gastrointestinal diseases, cancer, inflammation, blood diseases, respiratory diseases, musculoskeletal diseases, neurological diseases, and urinary diseases.

[0009] During the early stages of sepsis, ADM has been reported to improve cardiac function and blood supply in the liver, spleen, kidneys, and small intestine. Anti-ADM neutralizing antibodies neutralize these effects during the early stages of sepsis (Wang et al. 2001, Peptides 22:1835-1840).

[0010] Blocking ADM may be somewhat effective in other diseases. However, complete neutralization of ADM may be harmful, since some physiological functions require certain amounts of ADM. Many reports have emphasized that administration of ADM may be effective in some diseases. Conversely, other reports have demonstrated that ADM can be life-threatening when administered in certain conditions.

[0011] WO 2013 / 072510 describes the use of non-neutralizing anti-ADM antibodies in treating a patient with a severe chronic or acute disease or condition to reduce the patient's risk of death.

[0012] WO 2013 / 072511 describes the use of non-neutralizing anti-ADM antibodies in the treatment of severe chronic or acute diseases or conditions in patients with the aim of preventing or reducing organ dysfunction or organ failure.

[0013] WO 2013 / 072512 describes the half-life (t 1 / 2 A non-neutralizing anti-ADM antibody has been described that stabilizes ADM by increasing the retention time (half the value of the retention time). This ADM-stabilizing antibody blocks the biological activity of ADM to less than 80%.

[0014] WO 2013 / 072513 describes non-neutralizing anti-ADM antibodies for use in treating acute diseases or conditions in patients to stabilize circulation.

[0015] WO 2013 / 072514 describes non-neutralizing anti-ADM antibodies for regulating fluid balance in patients with chronic or acute diseases or conditions. Summary of the Invention

[0016] Description of the Invention According to the present invention, an anti-ADM antibody or anti-ADM antibody fragment that binds to ADM, or an anti-ADM non-Ig scaffold that binds to ADM, can be administered to treat and cure a patient in need of treatment and therapy for hemostasis. [Brief explanation of the drawings]

[0017] [Figure 1a] Diagram of antibody formats - Fv and scFv variants. [Figure 1b] Diagram of antibody formats - heterofusions and bifunctional antibodies. [Figure 1c] Diagram of antibody forms - bivalent and bispecific antibodies. [Figure 2a] a: Dose-response curve of human ADM. Maximal cAMP stimulation was defined as 100% activation. [Figure 2b] Dose / inhibition curve of human ADM 22-52 (ADM receptor antagonist) in the presence of 5.63 nM hADM. [Figure 2c] Dose / inhibition curve of CT-H in the presence of 5.63 nM hADM. [Figure 2d] Dose / inhibition curve of MR-H in the presence of 5.63 nM hADM. [Figure 2e] Dose / inhibition curve of NT-H in the presence of 5.63 nM hADM. [Figure 2f] Dose-response curve of mouse ADM. Maximum cAMP stimulation was defined as 100% activation. [Figure 2g] Dose / inhibition curve of human ADM 22-52 (ADM receptor antagonist) in the presence of 0.67 nM mADM. [Figure 2h]Dose / inhibition curve of CT-H in the presence of 0.67 nM mADM. [Figure 2i] Dose / inhibition curve of MR-H in the presence of 0.67 nM mADM. [Figure 2j] Dose / inhibition curve of NT-H in the presence of 0.67 nM mADM. [Figure 2k] This shows the inhibition of ADM by F(ab)2 NT-M. [Figure 2l] 1 shows the inhibition of ADM by Fab NT-M. [Figure 3] The figure shows a typical hADM dose / signal curve and the hADM dose / signal curve in the presence of 100 μg / ml of antibody NT-H. [Figure 4] This figure shows the stability of hADM in human plasma (citrate) in the absence and presence of NT-H antibodies. [Figure 5] Alignment of Fab with homologous human framework sequences. [Figure 6] Extravascular albumin accumulation 18 hours after performing CLP and applying NT-M at different doses. [Figure 7] VEGF expression 18 hours after CLP was performed and NT-M was applied at different doses. [Figure 8] Angiopoietin-1 expression 18 hours after performing CLP and applying NT-M at different doses. [Figure 9] ADM concentrations in healthy human subjects up to 60 days after application of different doses of NT-M. [Figure 10] Timetable of treatment and blood sample collection in the porcine two-hit model. [Figure 11] Concentrations (mean and SEM) of ADM in the vehicle group (squares) and treatment groups (dots) (p=0.003 for interaction; t=7 to 19 hours, multivariate, time × group). [Figure 12] Heart rates (mean and SEM) for vehicle group (squares) and treatment group (dots) (p=0.097 for interaction; from t=7 h to t=19 h, multivariate, time × group). [Figure 13] Cardiac output (mean and SEM) of vehicle group (squares) and treatment groups (dots) (t-test: p<0.05 for t=9, 10, and 11 hours). [Figure 14] Fluid replacement required / applied (mean and SEM) in vehicle (squares) and treatment groups (dots) (t-test at 17 hours: p=0.034; t-test at 19 hours: p=0.045). [Figure 15] Cumulative fluid replacement required / applied (mean and SEM) in vehicle (squares) and treatment groups (dots) (t-test at 19 hours: p=0.039; Mann-Whitney at 19 hours: p=0.036). [Figure 16] Noradrenaline required / applied (mean and SEM) in vehicle group (squares) and treatment group (dots). [Figure 17] Need for vasodilator support in vehicle group (squares) versus treatment group (dots) (χ test at t = 19 h: p = 0.014 for interaction (t = 7 h to 19 h, multivariate, time × group: 0.019)). [Figure 18] Systemic vascular resistance (mean and SEM) of the vehicle group (squares) and the treatment group (dots) (t-test: 15 h: p = 0.069; 17 h: p = 0.037; 19 h: p = 0.066). [Figure 19] Concentration of rADM in an LPS-induced endotoxemia rat model. [Figure 20] Vascular permeability in an endotoxemic rat model. DETAILED DESCRIPTION OF THE INVENTION

[0018] Throughout this specification, an "antibody" or "antibody fragment" or "non-Ig scaffold" according to the present invention can be referred to as an "anti-ADM antibody," "anti-ADM antibody fragment," or "anti-ADM non-Ig scaffold," because it is capable of binding to and directed against ADM.

[0019] The advantage of administering an anti-ADM antibody or anti-ADM antibody fragment that binds to ADM, or an anti-ADM non-Ig scaffold that binds to ADM, is that it has a renal protective effect, unlike, for example, the administration of diuretics. Because the anti-ADM antibody or anti-ADM antibody fragment that binds to ADM, or the anti-ADM non-Ig scaffold that binds to ADM, does not harm the kidney, no side effects are expected in this regard.

[0020] According to the present invention, administration of an anti-ADM antibody or anti-ADM antibody fragment that binds to ADM, or administration of an anti-ADM non-Ig scaffold that binds to ADM, is preferably systemic administration.

[0021] In one particular embodiment, an anti-ADM antibody or anti-ADM antibody fragment that binds to ADM, or an anti-ADM non-Ig scaffold that binds to ADM, can be administered to a patient with vascular barrier dysfunction or endothelial dysfunction that may lead to hemostasis.

[0022] Vascular barrier dysfunction, or endothelial dysfunction, is a systemic pathological condition of the endothelium (the inner lining of blood vessels) and can be broadly defined as an imbalance between vasodilators and vasoconstrictors produced by or acting on the endothelium (Deanfield et al. 2005, J Hypertens 23(1):7-17). Normal functions of endothelial cells include mediating coagulation, platelet adhesion, immune function, and regulating the volume and electrolyte content of the intravascular and extravascular spaces. The endothelium is a monolayer of cells lining the entire cardiovascular system and regulates many processes, including vascular tone, thrombosis, angiogenesis, and inflammation. Endothelial cells are known to be phenotypically variable, transitioning between quiescent and activated states in response to various local and systemic stimuli (Colombo et al. 2015, Curr Heart Fail Rep. 12(3):215-222). In recent years, emerging research has demonstrated that endothelial dysfunction is a major contributing factor to cardiovascular disease, including hypertension, atherosclerosis, and congestive heart failure (Gutierrez et al. 2013, European Heart Journal 34:3175-3181). The endothelium tightly regulates fluid exchange from the circulatory system to peripheral tissues, and dysfunction of this barrier can lead to uncontrolled fluid exudation, resulting in congestion and / or edema. One common feature of edema (e.g., pulmonary edema) is increased permeability of small solutes to water (Rocker et al. 1987, Thorax 42:620-623).

[0023] Endothelial dysfunction can result from and / or contribute to several disease processes, such as occurs in hypertension, hypercholesterolemia, diabetes, septic shock, etc. Endothelial dysfunction is a major pathophysiological mechanism leading to coronary artery disease and other atherosclerotic diseases.

[0024] Preclinical studies in sepsis / septic shock models have shown that administration of anti-ADM antibodies induces an increase in plasma bio-ADM concentrations (Example 8, Figure 9), which coincides with an increase in survival rates (Struck et al. 2013, Intensive Care Med Exp 1(1):22). The mechanism behind this effect is thought to be as follows: When administered intravenously, this antibody, due to its size, is unable to cross the endothelial barrier and enter the interstitium, remaining in the blood circulation. Therefore, when administered in molar amounts significantly exceeding the endogenous ADM, this antibody binds to virtually all ADM in the plasma, reaching a binding equilibrium. A simple consequence is that ADM moves from the interstitium into the blood circulation. ADM located in the interstitium can bind to vascular smooth muscle, inducing relaxation and resulting vasodilation. This is reduced by administration of this antibody. Meanwhile, ADM in the plasma binds to endothelial cells, thereby stabilizing the blood vessels and even restoring them to an intact state. Thus, the elevated plasma ADM levels resulting from administration of this non-neutralizing antibody enhance this function. Finally, the binding of this antibody to ADM reduces its proteolysis.

[0025] Surprisingly, in the PROTECT study (Example 6) and the BIOSTAT study (Example 7), we observed that bio-ADM concentrations increased in patients with heart failure in response to the presence and severity of congestion, despite treatment with diuretics. Thus, the increase in bio-ADM levels in these patients represents a patient-mediated countermeasure to tissue congestion. However, this natural increase is insufficient to effectively achieve this countermeasure. Tissue congestion also occurs in sepsis. Examples 5, 9, and 10 demonstrate that administration of anti-ADM antibodies in an animal model of sepsis restores damaged blood vessels to an intact state. Because the mechanisms of tissue congestion are common in both sepsis and heart failure, experts in the field can confidently conclude that administration of anti-ADM antibodies should be beneficial in treating congestion in heart failure as well as in sepsis / septic shock.

[0026] In a particular embodiment, for use in the treatment and therapy of hemostasis, an anti-ADM antibody or anti-ADM antibody fragment that binds ADM, or an anti-ADM non-Ig scaffold that binds ADM, can be administered to a patient in conjunction with a diagnostic method, which is described below.

[0027] Pro-adrenomedullin or a fragment thereof consisting of at least five amino acids can be used as an early surrogate marker of hemostasis and thus guide the treatment or management of hemostasis. determining a level of pro-adrenomedullin, or a fragment thereof consisting of at least five amino acids, in a body fluid obtained from the subject; a) correlating the level of pro-adrenomedullin or a fragment thereof with the degree of congestion in a subject or diagnosing congestion (elevated levels above a predetermined threshold are indicative of congestion or the degree of congestion); or b) correlating the level of pro-adrenomedullin or a fragment thereof with the need for or success of treatment of congestion or the need for or success of treatment and therapy in the subject (a level below a predetermined threshold predicts successful treatment of congestion or successful treatment and therapy, and a level above a predetermined threshold indicates the need for treatment of congestion or the need for treatment and therapy); or c) correlating the level of pro-adrenomedullin or a fragment thereof with a prediction of whether congestion will be cleared or remain after treating congestion or after treatment and therapy (elevated levels above a predetermined threshold predict whether congestion will remain after treating congestion or after treatment and therapy, while levels below a predetermined threshold predict whether congestion will be cleared after treating congestion or after treatment and therapy); or d) correlating the level of pro-adrenomedullin or a fragment thereof with whether congestion is relieved or remains after treatment or a procedure and treatment (elevated levels above a predetermined threshold indicate residual congestion after treatment or a procedure and treatment, whereas levels below a predetermined threshold indicate relief of congestion after treatment or a procedure and treatment); or e) correlating the level of pro-adrenomedullin or a fragment thereof with an assessment of discharge decision (elevated levels above a predetermined threshold mean that the subject cannot be discharged, and levels below a predetermined threshold mean that the subject may be discharged), The pro-adrenomedullin or fragment thereof is selected from the group comprising pro-adrenomedullin of SEQ ID NO: 31, PAMP of SEQ ID NO: 32, MR-proADM of SEQ ID NO: 33, ADM-NH2 of SEQ ID NO: 20, ADM-Gly of SEQ ID NO: 34, CT-proADM of SEQ ID NO: 35.

[0028] Such methods are described in detail in European Patent Applications EP 16199092 and EP 16178725, the contents of which are incorporated herein by reference. The therapies and treatments referred to in the above diagnostic methods are the administration of anti-ADM antibodies or anti-ADM antibody fragments that bind to ADM, or the administration of anti-ADM non-Ig scaffolds that bind to ADM.

[0029] The terms congestion severity, degree of congestion, level of congestion, congestion grade, and the like are used synonymously throughout this application.

[0030] Mature ABM, BioADM, ADM-NH2 are synonymous terms throughout this application and refer to the molecule of SEQ ID NO:20.

[0031] Pro-adrenomedullin or a fragment thereof is a quantitative, accurate, and early surrogate marker of congestion in the setting of acute heart failure and heart failure, particularly in subjects with acute heart failure and / or subjects with heart failure showing signs of worsening and / or subjects with symptoms of heart failure or acute heart failure. An accurate, early surrogate marker of congestion in the setting of acute heart failure or heart failure means that its concentration and / or level of immunoreactivity reflects the degree of congestion.

[0032] If the level of pro-adrenomedullin or a fragment thereof exceeds a predetermined threshold level, an anti-ADM antibody or anti-ADM antibody fragment that binds to ADM, or an anti-ADM non-Ig scaffold that binds to ADM, is administered as a therapy or treatment for congestion.

[0033] In a particular embodiment of the present invention, this means that if a sample of body fluid taken from a patient shows elevated levels of pro-ADM and / or fragments thereof having at least 5 amino acids above a predetermined threshold, an anti-ADM antibody or anti-ADM antibody fragment that binds ADM, or an anti-ADM non-Ig scaffold that binds ADM, is used to treat and cure hemostasis in that patient. This diagnostic method using pro-ADM and / or fragments thereof thus serves as a companion diagnostic method.

[0034] In a particular embodiment of the diagnostic method, the selection of pro-ADM and / or fragments thereof having at least 5 amino acids comprises: SEQ ID NO: 31 (proADM): 164 amino acids (amino acids 22 to 185 of preproADM) ARLDVASEF RKKWNKWALS RGKRELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL, SEQ ID NO: 32 (proadrenomedullin N-20 terminal peptide, PAMP): amino acids 22 to 41 of prepro-ADM ARLDVASEF RKKWNKWALS R, SEQ ID NO: 33 (central region pro-adrenomedullin, MR-proADM): amino acids 45-92 of pre-proADM ELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RV, SEQ ID NO: 20 (mature adrenomedullin (mature ADM); amidated ADM; bioADM): amino acids 95-146-CONH2 YRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGY-CONH2, SEQ ID NO: 34 (Adrenomedullin 1-52-Gly (ADM 1-52-Gly)): amino acids 95 to 147 of prepro-ADM YRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYG, SEQ ID NO: 35 (C-terminal pro-adrenomedullin, CT-proADM): amino acids 148 to 185 of pre-proADM RRR RRSLPEAGP RTLVSSKPQA HGAPAPPSGS APHFL It is made from a group including

[0035] In a particular embodiment of the diagnostic method, proADM and / or a fragment thereof having at least 5 amino acids is selected from the group comprising mature ADM-NH2 (SEQ ID NO: 20), ADM 1-52-Gly (SEQ ID NO: 34), MR-proADM (SEQ ID NO: 33), CT-proADM (SEQ ID NO: 35).

[0036] In a particular embodiment of the diagnostic method, the level of mature ADM-NH2 (SEQ ID NO:20) immune activity, and / or the level of ADM 1-52-Gly (SEQ ID NO:34) immune activity, or the level of MR-proADM (SEQ ID NO:33) immune activity, or the level of CT-proADM (SEQ ID NO:35) immune activity is determined and correlated with a patient's need for therapy or treatment, wherein the patient is identified as having such a need if the level of mature ADM-NH2 (SEQ ID NO:20) immune activity, and / or the level of ADM 1-52-Gly (SEQ ID NO:34) immune activity, or the level of MR-proADM (SEQ ID NO:33) immune activity, or the level of CT-proADM (SEQ ID NO:35) immune activity in a body fluid is above a threshold value.

[0037] In one particular embodiment of the diagnostic method, the level of pro-ADM and / or its fragments is determined using at least one binding agent selected from the group consisting of a binding agent that binds to a region within the sequence of mature ADM-NH2 (SEQ ID NO: 20) and / or ADM 1-52-Gly (SEQ ID NO: 34) and a second binding agent that binds to a region within the sequence of mature ADM-NH2 (SEQ ID NO: 20) and / or ADM 1-52-Gly (SEQ ID NO: 34).

[0038] In a particular embodiment of the diagnostic method, the level of proADM and / or fragments thereof is determined using at least one binding agent selected from the group consisting of a binding agent that binds to a region within the sequence of MR-proADM (SEQ ID NO: 33) and a second binding agent that binds to a region within the sequence of MR-proADM (SEQ ID NO: 33).

[0039] In a particular embodiment of the diagnostic method, the level of proADM and / or fragments thereof is determined using at least one binding agent selected from the group consisting of a binding agent that binds to a region comprised in the sequence of CT-proADM (SEQ ID NO: 35) and a second binding agent that binds to a region comprised in the sequence of CT-proADM (SEQ ID NO: 35).

[0040] In a particular embodiment of the diagnostic method of the invention, the subject is a method of the invention in which the fragment can be chosen from MR-proADM of SEQ ID NO: 33 or from mature ADM-NH2 of SEQ ID NO: 20.

[0041] The subject of the diagnostic method of the present invention is a method according to the diagnostic method of the present invention, in which the level of pro-adrenomedullin and / or its fragments having at least 5 amino acids is determined using a binding agent for pro-adrenomedullin and / or its fragments having at least 5 amino acids.

[0042] The subject of this diagnostic method is a method according to the diagnostic method of the invention, in which the binding agent is selected from the group comprising antibodies, antibody fragments, non-Ig scaffolds that bind to pro-ADM and / or fragments thereof having at least 5 amino acids.

[0043] In a particular embodiment, the body fluid of the present invention is a blood sample. The blood sample can be selected from the group consisting of whole blood, serum, and plasma. In a particular embodiment of the diagnostic method, the sample is selected from the group consisting of human citrated plasma, heparinized plasma, and EDTA plasma.

[0044] In a particular embodiment of the invention, an anti-ADM antibody or anti-ADM antibody fragment that binds to ADM, or an anti-ADM non-Ig scaffold that binds to ADM, according to any embodiment of the invention, is used to treat and cure congestion in patients who are diuretic-resistant or unresponsive to diuretic therapy.

[0045] Another specific embodiment of the present invention relates to an anti-adrenomedullin antibody, or an anti-adrenomedullin antibody fragment, or an anti-ADM non-Ig scaffold for use in the treatment and therapy of hemostasis in a patient in need thereof, wherein the anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold comprises the N-terminal portion of adrenomedullin (amino acids 1-21): YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 22) binds to Patients are diuretic resistant or do not respond to diuretic therapy).

[0046] The term "diuretic resistance" is generally defined as the inability to reduce extracellular fluid volume despite adequate diuretic use (Ravnan et al. 2002, CHF 8:80-85). Epstein et al. defined diuretic resistance as the inability to excrete at least 90 mmol of sodium within 72 hours after oral administration of furosemide at a dose of 160 mg twice daily (Epstein et al. 1977, Curr Ther Res. 21:656-667).

[0047] Diuretic adaptation and diuretic resistance may occur through similar mechanisms. Adaptation to diuretics can be classified as adaptations that occur during the diuretic's action, adaptations that cause short-term sodium retention (leading to "post-diuretic NaCl retention"), and adaptations that chronically increase sodium retention (the "brake phenomenon"). The kidneys adapt to long-term diuretic therapy in the following ways. First, as the NaCl load increases during diuretic administration, nephron segments downstream of the site of diuretic action increase NaCl reabsorption. Second, as the concentration of diuretic decreases in the renal tubules, the renal tubules function to retain NaCl until the next diuretic administration. Third, the ability of diuretics to increase renal NaCl excretion decreases over time, an effect resulting from both extracellular fluid volume deficiency and changes in the structure and function of the renal tubules themselves. All of these adaptations increase the rate of NaCl reabsorption, thereby counteracting the effects of diuretic therapy. For reviews see Ellison 1999, Semin Nephrol. 19(6):581-597 and De Bruyne 2003, Postgrad Med J 79:268-271.

[0048] Although precise figures are difficult to pinpoint, diuretic resistance is thought to occur in one in three patients with congestive HF. Heart failure is the most common clinical setting in which diuretic resistance is observed. In mild congestive HF, diuretic resistance is generally not encountered as long as renal function is preserved. However, in patients with moderate and severe congestive HF, diuretic resistance occurs more frequently and is often a clinical problem (Brater 1985, Drugs 30:427-443; Taylor 2000, Cardiol Rev. 8:104-114).

[0049] In a particular embodiment of the diagnostic method, the level of pro-ADM and / or fragments thereof having at least five amino acids is determined using an assay having an assay sensitivity capable of quantifying mature ADM-NH2 in healthy subjects of less than 70 pg / ml, preferably less than 40 pg / ml, and more preferably less than 10 pg / ml, and these concentrations can be used as thresholds in the methods of the invention.

[0050] In a particular embodiment of the diagnostic method, the level of proADM and / or its fragments having at least 5 amino acids is determined using an assay having an assay sensitivity capable of quantifying MR-proADM in healthy subjects of less than 0.5 nmol / l, preferably less than 0.4 nmol / l, more preferably less than 0.2 nmol / l, and these concentrations can be used as threshold values in the methods of the invention.

[0051] In a particular embodiment of the diagnostic method, the levels of proADM and / or its fragments having at least 5 amino acids are determined using an assay having an assay sensitivity capable of quantifying CT-proADM in healthy subjects of less than 100 pmol / L, preferably less than 75 pmol / L, more preferably less than 50 pmol / L, and these concentrations can be used as threshold values in the methods of the invention.

[0052] In a particular embodiment of the diagnostic method, the binding agent has at least 10 7 M -1 , preferably 10 8 M -1 The preferred affinity is 10 9 M -1 More than 10, most preferably 10 M -1 Those skilled in the art know that a lower affinity can be considered to be compensated for by applying a higher dose of the compound and that this measure is not considered to fall outside the scope of the present invention.

[0053] To determine the affinity of the antibody for adrenomedullin, the binding kinetics of adrenomedullin to the immobilized antibody was determined by label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). The antibody was reversibly immobilized using an anti-mouse Fc antibody covalently coupled to a CM5 sensor surface at high density (Mouse Antibody Capture Kit; GE Healthcare) according to the manufacturer's instructions (Lorenz et al. 2011, Antimicrob Agents Chemother. 55(1):165-173).

[0054] In one particular embodiment of the diagnostic method, the binding agent is selected from the group comprising antibodies, or antibody fragments, or non-Ig scaffolds, that bind to pro-ADM and / or fragments thereof.

[0055] In one particular embodiment of the diagnostic method, an assay is used to determine the level of pro-ADM and / or fragments thereof having at least 5 amino acids. Such an assay is a sandwich assay, preferably a fully automated assay.

[0056] In one embodiment of the present invention, a testing technology that can be performed near the patient within an hour without the need for a fully automated assay system would be a so-called POC (point-of-care) test. An example of this technology is an immunochromatographic testing technology.

[0057] In one embodiment of the diagnostic method, the assay is a sandwich immunoassay utilizing any type of detection technology (non-limiting examples of which include enzyme-labeled, chemiluminescent-labeled, and electrochemiluminescent-labeled), with fully automated assays being preferred. In one embodiment of the diagnostic method, the assay is an enzyme-labeled sandwich assay. Examples of automated or fully automated assays include those available in one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, BiomerieuxVidas®, and Alere Triage®.

[0058] A wide variety of immunoassays are known and can be utilized in the assays and methods of the present invention, including radioimmunoassays ("RIA"), homogeneous enzyme multiplexed immunoassays ("EMIT"), enzyme-linked immunosorbent assays ("ELISA"), apoenzyme reactivation immunoassays ("ARIS"), dipstick immunoassays, and immunochromatographic assays.

[0059] In one particular embodiment of the diagnostic method, at least one of the two binding agents is labeled and detected.

[0060] The subject of the present invention is an anti-adrenomedullin (ADM) antibody, or an anti-adrenomedullin antibody fragment, or an anti-ADM non-Ig scaffold for use in the treatment and therapy of congestion in patients with a disease or condition selected from the group consisting of congestive hypertension, swelling or fluid retention (edema), heart failure (particularly acute heart failure), kidney disease, and liver disease.

[0061] The subject of the present invention is an anti-adrenomedullin (ADM) antibody, or an anti-adrenomedullin antibody fragment, or an anti-ADM non-Ig scaffold for use in the treatment and therapy of congestion in patients with a disease or condition selected from the group consisting of congestive hypertension, swelling or fluid retention (edema), and heart failure (particularly acute heart failure).

[0062] Heart failure (HF) is a cardiac condition that occurs when problems with the heart's structure or function impair its ability to provide adequate blood flow to meet the body's needs. HF can cause a variety of symptoms, particularly shortness of breath at rest and during exercise (SOB), signs of fluid retention (such as pulmonary congestion and ankle swelling), and objective evidence of abnormalities in cardiac structure or function at rest.

[0063] Heart failure (HF) is a clinical syndrome characterized by a constellation of symptoms and signs resulting from cardiac dysfunction. It is one of the leading causes of morbidity and mortality in developed countries, affecting 1–2% of people. HF can be classified as chronic HF or acute HF. Patients with chronic HF can be classified as having stable chronic HF, chronic HF with worsening signs and symptoms, or acutely decompensated chronic HF. Acute heart failure (AHF) is defined as the sudden onset of signs and symptoms of heart failure resulting in the need for emergency treatment or hospitalization. AHF can manifest as acute de novo HF (new onset of AHF in a patient with no previous cardiac dysfunction) or acutely decompensated chronic HF. AHF is the leading cause of hospitalization in adults over 65 years of age. Despite significant improvements in the prognosis for patients with chronic HF over the past few decades, primarily due to advances in treatment, both short-term and long-term outcomes remain very poor when patients are hospitalized with decompensated HF. Nearly 25% of patients hospitalized with AHF require readmission within 30 days of discharge, and fewer than 50% survive beyond 5 years after hospitalization. In addition to significantly reduced survival and quality of life for affected patients, the financial burden of AHF on health care systems is enormous. The total cost of caring for heart failure was estimated to reach $31 billion in 2012 in the United States alone, with the majority of this cost associated with hospital care. This cost is projected to increase to $70 billion by 2030 due to an aging population.

[0064] Heart failure encompasses a wide range of patients, from those with a normal left ventricular ejection fraction (LVEF) (also known as HF with preserved EF (HFpEF)), typically considered to be ≥50%, to those with a reduced LVEF, typically considered to be <40%. Patients with an LVEF in the 40-49% range represent a "gray area" defined as HF with an intermediate EF (Ponikowski et al. 2016, European Heart Journal 18(8):891-975).

[0065] The primary goals of AHF treatment in the hospital setting are decongestion (simply the removal of excess intracellular and extracellular fluid accumulation) and relief of the symptoms and signs of congestion. Diuretics remain the primary decongestant in AHF, and nearly all hospitalized patients receive this class of medication. Other classes of medications (e.g., inotropes, vasodilators) that increase cardiac output and decrease filling pressures are given to selected groups of patients. Ultrafiltration may also be considered in some patients, especially those who do not respond adequately to diuretic therapy.

[0066] Although patients (generally) respond well to diuretic therapy, a significant proportion of patients are discharged without achieving adequate levels of decongestion and normal blood volume (i.e., residual congestion). This is primarily related to the fact that current approaches to the clinical assessment of congestion are inadequate. There is consistent evidence that the presence of residual congestion at discharge is associated with worse post-discharge outcomes, particularly readmission. Therefore, there is a great unmet need for more accurate and reliable surrogate markers of congestion that facilitate objective and optimal decisions regarding the adequacy of the achieved level of decongestion and the timing of discharge.

[0067] In a particular aspect of the present invention, the subject is a subject with heart failure. In another particular aspect of the present invention, the subject is a subject with acute heart failure, and / or a subject with heart failure showing signs of worsening, and / or a subject with heart failure or symptoms of acute heart failure. In a particular aspect of the present invention, the subject has acute heart failure, i.e., new-onset AHF or acute decompensated HF. In another particular aspect of the present invention, the subject has acute decompensated chronic HF or worsening signs / symptoms of chronic heart failure. In a particular aspect of the present invention, the subject has acute heart failure, particularly new-onset AHF.

[0068] The term "acute" is used to indicate a sudden onset or to describe worsening or decompensated heart failure, and refers to episodes that can characterize a patient as needing emergency treatment or hospitalization as a result of changes in the signs and symptoms of heart failure.

[0069] The term "chronic" means persistent over time. Chronic heart failure is a long-term condition that is usually kept stable by treating the condition (stable chronic HF).

[0070] Stable chronic HF is characterized by: 1. There is a structural or functional disorder of the heart that impairs its ability to supply sufficient blood flow to meet the body's needs; 2. There is no fluid overload (manifesting as pulmonary and / or systemic congestion) and / or a significant decrease in cardiac output (manifesting as hypotension and / or renal failure and / or shock syndrome), Patients will not require emergency treatment or care coordination and will not require hospitalization.

[0071] Chronic HF is characterized by worsening signs and symptoms: 1. There is a structural or functional disorder of the heart that impairs its ability to supply sufficient blood flow to meet the body's needs; 2. There is fluid overload (manifesting as pulmonary and / or systemic congestion) and / or a significant decrease in cardiac output (manifesting as hypotension and / or renal failure and / or shock syndrome), The patient does not require emergency treatment or hospitalization, but may require adjustment of care.

[0072] Chronic heart failure can also be decompensated (called acutely decompensated heart failure or acutely decompensated chronic heart failure). This most commonly results from intercurrent illness (e.g., pneumonia), myocardial infarction, arrhythmia, untreated hypertension, or the patient's inability to maintain fluid restriction, diet, or medication. After treatment, patients with acutely decompensated chronic HF can revert to a stable chronically decompensated state (stable chronic HF).

[0073] New-onset acute HF and acutely decompensated chronic HF are characterized by: 1. There is a structural or functional disorder of the heart that impairs its ability to supply sufficient blood flow to meet the body's needs; 2. There is fluid overload (manifesting as pulmonary and / or systemic congestion) and / or a significant decrease in cardiac output (manifesting as hypotension and / or renal failure and / or shock syndrome), The patient is in need of urgent treatment or adjustment of care and requires hospitalization.

[0074] [Table 1]

[0075] The above definition of acute heart failure as new-onset AHF, acute decompensated HF, acute decompensated chronic HF, or signs / symptoms of worsening chronic HF is consistent with Voors et al., European Journal of Heart Failure (2016), 18, 716-726.

[0076] Deteriorating renal function is common in both acute and chronic heart failure (HF) settings and has recently been described as the "cardiorenal syndrome." Renal congestion (RC) is increasingly recognized as a potential contributing factor to the cardiorenal syndrome, and it has been proposed that adequate control of congestion while simultaneously improving / preserving renal function should be the primary goal in managing patients with HF (Aronson 2012, Expert Rev Cardiovasc Ther 10:177-189).

[0077] Congestion in HF is defined as high left ventricular diastolic pressure accompanied by signs and symptoms of HF (dyspnea, and / or Russell's syndrome, and / or edema). These congestion-related signs and symptoms are the leading reason for HF-related hospitalization.

[0078] Relief of congestion (and its associated signs / symptoms) and achievement of normal blood volume remain the primary goals of inpatient AHF therapy, yet no standard algorithm or clinical tool exists for the assessment of congestion. The current clinical assessment of congestion focuses on signs and symptoms. Physical examination findings (elevated jugular venous pressure (JVP), peripheral edema, orthopnea, S3 heart sound, hepatomegaly, etc.) or chest radiographic findings (cardiomegaly) and interstitial / pulmonary bubble edema are used as surrogate markers of congestion. Beyond careful assessment of JVP, it is important to note that the predictive value of these parameters for detecting congestion is moderate. There is a great unmet need for reliable and accurate surrogate markers of congestion. There is a great unmet need in medicine to quantitatively and qualitatively determine and / or predict and / or assess and / or monitor congestion and decongestion. There is a need to determine and / or predict and / or assess and / or monitor the degree of congestion, ie, the grade of congestion.

[0079] In the context of the present invention, the degree of congestion can also be expressed as a grade of congestion severity, which is determined as follows: However, those skilled in the art will recognize that the degree of congestion can also be expressed by other scores or surrogate markers, such as the score used by Ambrosy et al. (Ambrosy et al. 2013, European Heart Journal 34(11):835-843).

[0080] As explained above, congestion can be classified in many different ways. Those skilled in the art are aware that the degree of congestion can be expressed by other scores or surrogate markers. Clinical classification can be based on a physician's bedside examination to detect clinical symptoms / signs of congestion ("wet" if present, "dry" if absent) and / or the presence of peripheral hypoperfusion ("cold" if present, "warm" if absent) (for a review, see Ponikowski et al. 2016, Eur Heart J. ehw128). Combinations of these options identify four groups: the most commonly present warm and moist (well-perfused and congested); cold and moist (hypoperfused and congested); cold and dry (hypoperfused without congestion); and warm and dry (compensated and well-perfused without congestion). This classification can be useful in guiding treatment at an early stage and contains prognostic information.

[0081] Typically, the symptoms and signs of AHF reflect fluid overload (pulmonary congestion and / or peripheral edema) or, less frequently, reduced cardiac output with peripheral hypoperfusion. Chest radiography can be a useful test for diagnosing AHF. Pulmonary venous congestion, pleural effusion, interstitial or alveolar edema, and cardiomegaly are the most specific findings of AHF, although chest radiographs are nearly normal in up to 20% of patients with AHF.

[0082] Symptoms / signs of left-sided congestion are defined as orthopnea, paroxysmal nocturnal dyspnea, pulmonary Russell syndrome (bilateral), and peripheral edema (bilateral). Symptoms / signs of right-sided congestion are defined as jugular venous distention, peripheral edema (bilateral), congestive liver enlargement, hepatojugular reflux, ascites, and symptoms of intestinal congestion (for review, see Table 12.2 in Ponikowski et al. 2016, Eur Heart J. ehw128).

[0083] Edema is the accumulation of fluid in intercellular tissues resulting from an abnormal increase in interstitial fluid volume. Fluid flow between the interstitial and intravascular spaces is regulated by gradients of capillary hydrostatic pressure and osmotic pressure across the capillaries (Trayes et al. 2013, Am Fam Physician 88(2):102-110). Fluid accumulation occurs when local or systemic conditions disrupt this equilibrium, leading to increased capillary hydrostatic pressure, increased plasma volume, decreased plasma oncotic pressure (hypoalbuminemia), increased capillary permeability, or lymphatic obstruction.

[0084] Clinically, edema manifests as swelling. The volume of interstitial fluid is determined by the balance of fluid homeostasis, and edema can occur due to increased fluid secretion into the interstitium or ineffective fluid removal. Increased hydrostatic pressure leads to heart failure. Causes of edema that are generalized throughout the body can cause edema in multiple organs and the periphery. For example, severe heart failure can lead to pulmonary edema, pleural effusion, ascites, and peripheral edema.

[0085] Pulmonary edema is the accumulation of fluid in the alveoli and lung parenchyma. This can impair gas exchange and lead to respiratory failure. It can result from either a failure of the left ventricle to adequately clear blood from the pulmonary circulation ("cardiogenic pulmonary edema") or damage to the lung parenchyma or vasculature ("noncardiogenic pulmonary edema") (Ware and Matthay 2005, N. Engl. J. Med. 353(26):2788-2796). Treatment focuses on three aspects: first, improving respiratory function, second, treating the underlying cause, and third, avoiding further lung damage. Pulmonary edema, especially acute pulmonary edema, can lead to life-threatening respiratory distress or cardiac arrest due to hypoxia, a crucial feature of congestive heart failure.

[0086] The most common symptom of pulmonary edema is difficulty breathing, but symptoms can also include hematemesis (classically seen as pink, frothy sputum), excessive sweating, anxiety, and pale skin. Shortness of breath can manifest as dyspnea (inability to lie down due to shortness of breath) and / or paroxysmal nocturnal dyspnea (episodes of sudden, severe shortness of breath at night). These are common symptoms of chronic pulmonary edema and result from left ventricular failure. Progression of pulmonary edema can be accompanied by symptoms and signs of "fluid overload." This is a lay term describing the manifestation of left ventricular failure in the rest of the body and includes peripheral edema (swelling in the legs (commonly of various "pitting" types) that is slow to return to normal when the skin over those areas is pressed), elevated jugular venous pressure, and hepatomegaly (an enlarged liver that may be tender or pulsatile). Other signs include an inspiratory crackle on auscultation (a sound heard at the end of a deep breath) and the presence of a third heart sound.

[0087] As already emphasized, clinical surrogate markers do not come close to providing optimal predictive value for detecting congestion. In the so-called PROTECT trial (O'Connor et al. 2012, European Journal of Heart Failure 14:605-612), three of the strongest clinical surrogate markers of congestion (i.e., JVP, peripheral edema, and orthopnea) were combined to improve accuracy and developed a Composite Clinical Congestion Score (CCS) using the following scheme:

[0088] [Table 2]

[0089] The scores for each of these three parameters were then added together to obtain a composite congestion score, which ranged from 0 to 8.

[0090] The following algorithm is then used to rank the severity of congestion: CCS 0, no clinical congestion CCS 1-3, clinically mild congestion CCS 4-5, clinically moderate congestion CCS ≥ 6, clinically severe congestion.

[0091] Conditions that affect kidney structure and function can be considered acute or chronic depending on their duration (chronic kidney disease (CKD), acute kidney disease (AKD), acute kidney injury (AKI)).

[0092] AKD is characterized by structural renal damage for less than 3 months and functional criteria also seen in AKI, i.e., a GFR of 1.73 m 2 It is characterized by a GFR of less than 60 ml / min per day for less than three months, or a decline in GFR of 35% or more, or an increase in serum creatinine (SCr) of more than 50% for less than three months (Kidney International Supplements, Vol. 2, No. 1, March 2012, pp. 19-36).

[0093] AKI is one of many acute kidney diseases and disorders (AKD) that can occur with or without other acute or chronic kidney diseases and disorders.

[0094] AKI is defined as a decline in renal function, including a decline in glomerular filtration rate (GFR) and renal failure. The criteria for diagnosing AKI and the stages of its severity are based on changes in SCr and urine output. AKI does not require structural criteria (although they may be present), but it is characterized by a 50% increase in serum creatinine (SCr) or an increase of 0.3 mg / dL (26.5 micromol / L) within 7 days, or a decrease in urine output. AKD can occur in patients with trauma, stroke, sepsis, SIRS, septic shock, acute myocardial infarction (MI), post-MI, local and systemic bacterial and viral infections, autoimmune diseases, burns, surgery, cancer, liver disease, and lung disease, as well as those receiving nephrotoxins (e.g., cyclosporine), antibiotics (including aminoglycosides), and anticancer drugs (e.g., cisplatin).

[0095] Renal failure is one stage of AKI and affects a body surface area of 1.73 m 2 defined as a GFR less than 15 ml / min per year or the need for renal replacement therapy (RRT).

[0096] CKD is defined as a glomerular filtration rate (GFR) of 1.73 m or greater for a period of more than 3 months. 2 Kidney damage is characterized by a blood flow rate of less than 60 ml / min per minute and a duration of kidney damage greater than 3 months (Kidney International Supplements, 2013; Vol. 3:19-62).

[0097] Chronic expansion of extracellular volume is the most common and classic problem and constitutes the syndrome set of end-stage renal disease (ESRD). Moderate to mild volume expansion may go undetected or overlooked in ESRD, but significant fluid overload in these patients is a medical emergency that ultimately requires hospitalization and additional dialysis. Both pulmonary edema and congestive heart failure are common in ESRD (Zoccali et al. 2013 Blood Purif 36:184-191).

[0098] Liver disease (also called hepatic disease) is a type of liver damage or disease. Liver disease can occur through several mechanisms. One common form of liver disease is viral infection, for example, caused by hepatitis viruses. Cirrhosis is the formation of fibrous tissue in place of liver cells that have died from a variety of causes, including viral hepatitis, excessive alcohol consumption, and other forms of liver toxicity that lead to chronic liver failure.

[0099] Congestive hepatopathy refers to a variety of chronic liver injury resulting from passive hepatic congestion in the setting of right-sided heart failure and any cause of elevated central venous pressure, including severe pulmonary hypertension (Shah and Sass 2015, Liver Res Open J. 1(1):1-10). Cardiohepatic dysfunction is frequently observed in patients with acutely decompensated HF, and cardiohepatic syndromes share several common pathophysiological mechanisms with cardiorenal syndromes, including increased venous congestion (Nikolaou et al. 2013, European Heart Journal 34:742-749). End-stage liver disease results in significant salt and water retention. Most of this fluid accumulation manifests as ascites in the peritoneal cavity, but peripheral edema may become prominent in later stages, especially if severe hypoalbuminemia is present (Cho and Atwood 2002, Am J Med. 113:580-586).

[0100] The treatment or therapy of congestion in a patient according to the present invention may be combined with current therapies, which may be selected from the group comprising administration of diuretics, administration of cardiac agents, administration of vasodilators, ultrafiltration, with diuretics being particularly preferred.

[0101] Furthermore, in one embodiment of the invention, the anti-adrenomedullin (ADM) antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold is monospecific.

[0102] A monospecific anti-adrenomedullin (ADM) antibody, or monospecific anti-adrenomedullin antibody fragment, or monospecific anti-ADM non-Ig scaffold means that the antibody, antibody fragment, or non-Ig scaffold binds to one specific region of the target ADM that contains at least five amino acids. A monospecific anti-adrenomedullin (ADM) antibody, or monospecific anti-adrenomedullin antibody fragment, or monospecific anti-ADM non-Ig scaffold is an anti-adrenomedullin (ADM) antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold that all have affinity for the same antigen.

[0103] In another specific and preferred embodiment, the anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold that binds to ADM is a monospecific antibody, or monospecific antibody fragment, or monospecific non-Ig scaffold, respectively. Monospecificity means that the antibody, antibody fragment, or non-Ig scaffold binds to one specific region of the target ADM that contains at least four amino acids. The monospecific antibodies, or monospecific fragments, or monospecific non-Ig scaffolds of the present invention are antibodies, fragments, or non-Ig scaffolds that all have affinity for the same antigen. While monoclonal antibodies are monospecific, monospecific antibodies can also be generated by means other than generation from a common germline.

[0104] The anti-ADM antibody or antibody fragment that binds to ADM, or the non-Ig scaffold that binds to ADM, can be a non-neutralizing anti-ADM antibody or non-neutralizing antibody fragment that binds to ADM, or a non-neutralizing non-Ig scaffold that binds to ADM.

[0105] In a particular embodiment, the anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold is a non-neutralizing antibody, or non-neutralizing fragment, or non-neutralizing non-Ig scaffold. A neutralizing anti-ADM antibody, or neutralizing anti-ADM antibody fragment, or neutralizing anti-ADM non-Ig scaffold will block nearly 100%, at least more than 90%, and preferably at least more than 95% of the biological activity of ADM.

[0106] Conversely, a non-neutralizing anti-ADM antibody, or a non-neutralizing anti-ADM antibody fragment, or a non-neutralizing anti-ADM non-Ig scaffold blocks the biological activity of ADM to less than 100%, preferably less than 95%, preferably less than 90%, preferably less than 80%, and even more preferably less than 50%. This means that the biological activity of ADM is reduced to less than 100%, by 95%, by 90%, by 80%, or by 50%. This means that the residual biological activity of ADM bound to a non-neutralizing anti-ADM antibody, or a non-neutralizing anti-ADM antibody fragment, or a non-neutralizing anti-ADM non-Ig scaffold is greater than 0%, preferably greater than 5%, preferably greater than 10%, more preferably greater than 20%, and more preferably greater than 50%.

[0107] In this context, (a) a molecule is an antibody, or antibody fragment, or non-Ig scaffold, having "non-neutralizing anti-ADM activity," collectively referred to herein for simplicity as a "non-neutralizing" anti-ADM antibody, or "non-neutralizing" antibody fragment, or "non-neutralizing" non-Ig scaffold, e.g., that blocks the biological activity of ADM to less than 80%. This molecule is defined as: - one or more molecules that bind to ADM, which, when added to a culture of a eukaryotic cell line expressing a functional human recombinant ADM receptor consisting of CRLR (calcitonin receptor-like receptor) and RAMP3 (receptor-activity-modifying protein 3), reduce the amount of cAMP produced by the cell line through the action of a human synthetic ADM peptide added in parallel (wherein the human synthetic ADM peptide is added in an amount that provides half-effective stimulation of cAMP synthesis in the absence of the non-neutralizing antibody being assayed), and the degree of cAMP reduction by the ADM-binding molecule does not exceed 80%, even when the non-neutralizing ADM-binding molecule being assayed is added in an amount 10-fold greater than the amount required to maximally reduce cAMP synthesis using the non-neutralizing antibody being assayed.

[0108] The same definition applies to other ranges such as 95%, 90%, 50%, etc.

[0109] The antibodies or fragments of the present invention are proteins comprising one or more polypeptides substantially encoded by immunoglobulin genes that specifically bind to an antigen. Recognized immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma (IgG1, IgG2, IgG3, IgG4), delta (IgD), epsilon (IgE), and mu (IgM) constant region genes, as well as numerous immunoglobulin variable region genes. Full-length immunoglobulin light chains are generally approximately 25 kD, or 214 amino acids in length.

[0110] Full-length immunoglobulin heavy chains are generally about 50 kD, or 446 amino acids in length. Light chains are encoded by a variable region gene (about 110 amino acids in length) at the NH2-terminus and a kappa or lambda constant region gene at the COOH-terminus. Heavy chains are similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.

[0111] The basic structural unit of an antibody is generally a tetramer consisting of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. Within each pair, the light and heavy chain variable regions bind to antigen, and the constant regions mediate effector functions. Immunoglobulins exist in a variety of other forms, including, for example, Fv, Fab, (Fab')2, as well as bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al. 1987, Eur. J. Immunol. 17:105; Huston et al. 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al. 1988, Science 242:423-426; Hood et al. 1984, Immunology, Benjamin, New York, 2nd ed.; Hunkapiller and Hood 1986, Nature 323:15-16). The variable region of an immunoglobulin's light or heavy chain contains a framework region interrupted by three hypervariable regions, also called complementarity-determining regions (CDRs) (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., 1983, U.S. Department of Health and Human Services). As noted above, the CDRs are primarily responsible for binding to an epitope of an antigen. The resulting immune complex is an antibody (such as a monoclonal antibody, chimeric antibody, humanized antibody, or human antibody) or functional antibody fragment that specifically binds to the antigen.

[0112] Chimeric antibodies are antibodies in which the light and heavy chain genes are constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. For example, the variable region of a gene from a mouse monoclonal antibody can be joined to a human constant region (κ, γ1, γ3). In one example, a therapeutic chimeric antibody is thus a hybrid protein consisting of a variable or antigen-binding domain from a mouse antibody and a constant or effector domain from a human antibody, although other mammalian species can be used, and the variable regions can be engineered by molecular techniques. Methods for producing chimeric antibodies are well known in the art; see, for example, U.S. Patent No. 5,807,715. A "humanized" immunoglobulin is an immunoglobulin containing a human framework region and one or more CDRs from a non-human (mouse, rat, synthetic, etc.) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor," and the human immunoglobulin providing the framework is referred to as the "acceptor." In one embodiment, all CDRs are derived from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if present, they should be substantially identical to those of human immunoglobulins, i.e., at least about 85-90%, e.g., about 95% or more. Thus, all portions of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding portions of a natural human immunoglobulin sequence. A "humanized antibody" is an antibody containing a humanized light chain immunoglobulin and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or humanized antibody may contain a limited number of substitutions with amino acids taken from the donor framework. A humanized or other monoclonal antibody may contain additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Examples of conservative substitutions include gly, ala, val, ile, leu, asp, glu, asn, gln, ser, thr, lys, arg, phe, and tyr.Humanized immunoglobulins can be constructed by genetic engineering (see, e.g., U.S. Patent No. 5,585,089). Human antibodies are antibodies whose light and heavy chain genes are of human origin. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells secreting the antibody of interest. Immortalization can be achieved, for example, by EBV infection or by fusing human B cells with myeloma or hybridoma cells to generate trioma cells. Human antibodies can also be produced by phage display (see, e.g., WO 91 / 17271; WO 92 / 001047; WO 92 / 20791) or selected from human combinatorial monoclonal antibody libraries (see the MorphoSys website). Human antibodies can also be produced using transgenic animals carrying human immunoglobulin genes (see, eg, WO 93 / 12227; WO 91 / 10741).

[0113] Thus, anti-ADM antibodies can have any format known in the art, including human, monoclonal, humanized, chimeric, and CDR-grafted antibodies. In a preferred embodiment, the antibodies of the invention are recombinantly produced antibodies (e.g., IgG, a typical full-length immunoglobulin) or antibody fragments containing at least the F variable domains of the heavy and / or light chains (e.g., chemically coupled antibodies (fragment antigen binding), non-limiting examples of which include Fab fragments (Fab minibodies, single-chain Fab antibodies, monovalent Fab antibodies with epitope tags (e.g., Fab-V5Sx2)); bivalent Fab (miniantibodies) dimerized with CH3 domains; bivalent Fab or multivalent Fab (e.g., formed by multimerization with the aid of heterologous domains (e.g., Fab-V5Sx2 by dimerization of dHLX domains)). dHLX-FSx2); F(ab')2 fragments, scFv fragments, multimerized multivalent and / or multispecific scFv fragments, bivalent and / or bispecific dimers, BITE® (bispecific T cell engagers), trifunctional antibodies, multivalent antibodies (e.g., from classes other than G); single domain antibodies (e.g., nanobodies derived from camel or fish immunoglobulins), and many other antibodies.

[0114] In addition to anti-ADM antibodies, other biopolymer scaffolds are well known in the art to form complexes with target molecules and have been used to generate highly target-specific biopolymers. Examples include aptamers, spiegelmers, anticalins, and conotoxins. See Figures 1a, 1b, and 1c for schematic diagrams of antibody formats.

[0115] In a preferred embodiment, the anti-ADM antibody format is selected from the group consisting of Fv fragments, scFv fragments, Fab fragments, scFab fragments, F(ab)2 fragments, and scFv-Fc fusion proteins. In another preferred embodiment, the antibody format is selected from the group consisting of scFab fragments, Fab fragments, scFv fragments, and bioavailability-optimized conjugates thereof (e.g., PEGylated fragments). One of the most preferred formats is the scFab format.

[0116] Non-Ig scaffolds can be protein scaffolds that can bind to ligands or antigens and thus can be used as antibody mimics. The choice of non-Ig scaffold may include tetranectin-based non-Ig scaffolds (e.g., as described in U.S. Patent Application Publication No. 2010 / 0028995), fibronectin scaffolds (e.g., as described in EP 1266025), lipokine-based non-Ig scaffolds (e.g., as described in WO 2011 / 154420), ubiquitin scaffolds (e.g., as described in WO 2011 / 073214), transferrin scaffolds (e.g., as described in U.S. Patent Application Publication No. 2004 / 0023334), protein A scaffolds (e.g., as described in EP 2231860), ankyrin repeat-based scaffolds (e.g., as described in WO 2010 / 060748), microprotein (preferably cysteine knot-forming microprotein) scaffolds (e.g., as described in EP 2314308), Fyn SH3 domain-based scaffolds (e.g., as described in WO 2011 / 023685), EGFR-A domain based scaffolds (e.g. as described in WO 2005 / 040229), Kunitz domain based scaffolds (e.g. as described in EP 1941867).

[0117] In one embodiment of the invention, anti-ADM antibodies of the invention can be generated by synthesizing a fragment of ADM as an antigen, as outlined in Example 1. Binders to the fragment are then identified using the methods described below or other methods known in the art.

[0118] Humanization of mouse antibodies can be performed according to the following procedure: To humanize an antibody of mouse origin, the antibody sequence is analyzed to determine the structural interactions between the framework regions (FRs) with the complementarity-determining regions (CDRs) and the antigen. Based on structural modeling, appropriate FRs of human origin are selected, and the mouse CDR sequences are grafted into the human FRs. By introducing variations in the amino acid sequences of the CDRs or FRs, the structural interactions that were lost due to species switching with respect to the FR sequences can be regained. This restoration of structural interactions can be achieved by a random approach using phage display libraries or by a direct approach guided by molecular modeling (Almagro and Fransson 2008, "Antibody Humanization," Front Biosci. 2008, January 1; Vol. 13: 1619-1633).

[0119] In a preferred embodiment, the anti-ADM antibody format is selected from the group consisting of Fv fragments, scFv fragments, Fab fragments, scFab fragments, F(ab)2 fragments, and scFv-Fc fusion proteins. In another preferred embodiment, the antibody format is selected from the group consisting of scFab fragments, Fab fragments, scFv fragments, and bioavailability-optimized conjugates thereof (e.g., PEGylated fragments). One of the most preferred formats is the scFab format.

[0120] In another preferred embodiment, the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold is a full-length antibody, antibody fragment, or non-Ig scaffold.

[0121] In a preferred embodiment, the anti-adrenomedullin antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold is directed against and capable of binding to an epitope contained within ADM and consisting of at least 5 amino acids in length.

[0122] In a more preferred embodiment, the anti-adrenomedullin antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold is directed against and capable of binding to an epitope contained within ADM and consisting of at least four amino acids in length.

[0123] In one particular embodiment of the present invention, an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment that binds to adrenomedullin, or an anti-ADM non-Ig scaffold that binds to adrenomedullin and is not ADM binding protein-1 (complement factor H), is provided for use in treating or preventing an acute disease or condition in a patient.

[0124] In one particular embodiment of the invention, an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment that binds to adrenomedullin, or an anti-ADM non-Ig scaffold that binds to adrenomedullin, is provided comprising the sequence of amino acids 1-42 of mature human ADM: SEQ ID NO: 23 YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA Antibodies or fragments or scaffolds that bind to preferably at least four or at least five regions within are provided for use in treating or preventing acute diseases or conditions in patients.

[0125] In one particular embodiment of the invention, an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment that binds to adrenomedullin, or an anti-ADM non-Ig scaffold that binds to adrenomedullin, is provided comprising the sequence of amino acids 1 to 21 of mature human ADM: SEQ ID NO: 22 YRQSMNNFQGLRSFGCRFGTC Antibodies or fragments or scaffolds that bind to preferably at least four or at least five regions within are provided for use in treating or preventing acute diseases or conditions in patients.

[0126] In a preferred embodiment of the invention, the anti-ADM antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold binds to a region or epitope located within the N-terminal portion of adrenomedullin (amino acids 1-21).

[0127] In another preferred embodiment, the anti-ADM antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold recognizes and binds to amino acids 1-14 of adrenomedullin (SEQ ID NO: 25), i.e., a region or epitope located within the N-terminal portion (amino acids 1-14) of adrenomedullin. In another preferred embodiment, the anti-ADM antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold recognizes and binds to amino acids 1-10 of adrenomedullin (SEQ ID NO: 26), i.e., a region or epitope located within the N-terminal portion (amino acids 1-10) of adrenomedullin.

[0128] Amino acids 1-14 of ADM YRQSMNNFQGLRSF (SEQ ID NO: 25)

[0129] Amino acids 1-10 of ADM YRQSMNNFQG (SEQ ID NO: 26)

[0130] In another preferred embodiment, the anti-ADM antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold recognizes and binds to amino acids 1-6 of adrenomedullin (SEQ ID NO:27), a region or epitope of ADM located within the N-terminal portion of adrenomedullin (amino acids 1-6). As noted above, this region or epitope preferably comprises at least four or five amino acids in length.

[0131] Amino acids 1-6 of ADM YRQSMN (SEQ ID NO: 27)

[0132] In another preferred embodiment, the anti-ADM antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold recognizes and binds to the N-terminus (amino acid 1) of adrenomedullin. The N-terminus (amino acid 1) of adrenomedullin means that amino acid 1, i.e., "Y" in SEQ ID NOs: 20, 22, and 23, is essential for antibody binding. It is believed that this antibody, fragment, or scaffold does not bind to N-terminally extended, N-terminally modified, or N-terminally degraded adrenomedullin. This means that, in another preferred embodiment, when the N-terminus of ADM is free, the anti-ADM antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold binds only to a region within the sequence of mature ADM. In this embodiment, the anti-ADM antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold is not expected to bind to a region within the sequence of mature ADM when that sequence is contained within, for example, pro-ADM.

[0133] For clarity, numbers in parentheses referring to a particular region of ADM, such as "N-terminal portion (amino acids 1-21)," will be understood by those skilled in the art to mean that the N-terminal portion of ADM consists of amino acids 1-21 of the mature ADM sequence.

[0134] In another particular embodiment according to the invention, the anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold provided herein does not bind to the C-terminus of ADM, i.e., amino acids 43-52 of ADM.

[0135] (SEQ ID NO: 24) PRSKISPQGY-NH2

[0136] In a particular embodiment, it is preferable to use an anti-ADM antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold of the present invention, which increases the level of ADM or ADM immunoreactivity in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably more than 50%, and most preferably more than 100%.

[0137] In a particular embodiment, it is preferable to use an anti-ADM antibody, or an anti-adrenomedullin antibody fragment, or an anti-ADM non-Ig scaffold of the present invention, and this anti-ADM antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold has a half-life (t 1 / 2 an ADM-stabilized antibody, or an adrenomedullin-stabilized antibody fragment, or an adrenomedullin-stabilized non-Ig scaffold that increases the retention time (i.e., half of the retention time) by at least 10%, preferably at least 50%, more preferably more than 50%, and most preferably more than 100%.

[0138] The half-life (half of the retention time) of ADM can be determined using immunoassays to quantify ADM in human serum, blood, and plasma in the absence and presence of ADM-stabilizing antibodies, or adrenomedullin-stabilizing antibody fragments, or adrenomedullin-stabilized non-Ig scaffolds, respectively.

[0139] The following steps can be performed: ADM can be diluted in human citrated plasma in the absence and presence of an ADM stabilizing antibody or an adrenomedullin stabilizing antibody fragment or an adrenomedullin stabilizing non-Ig scaffold, respectively, and incubated at 24°C. - At a selected time point (eg, within 24 hours), an aliquot can be taken and frozen at -20°C to arrest the degradation of the ADM in that aliquot. - If the hADM immunoassay of choice is not affected by the stabilizing antibody, the amount of ADM can be determined directly by the assay. Alternatively, an aliquot can be treated with a denaturing agent (e.g., HCl), and after removing debris from the sample (e.g., by centrifugation), the pH can be neutralized and ADM quantified by an ADM immunoassay. Alternatively, ADM can be quantified using a non-immunoassay technique (e.g., RP-HPLC). - Calculate half-lives for ADM incubated in the absence and presence of ADM-stabilizing antibodies, or adrenomedullin-stabilizing antibody fragments, or adrenomedullin-stabilized non-Ig scaffolds, respectively. - The stabilized ADM will have an increased half-life calculated compared to ADM incubated in the absence of the ADM-stabilizing antibody, or adrenomedullin-stabilizing antibody fragment, or adrenomedullin-stabilized non-Ig scaffold.

[0140] A two-fold increase in the half-life of ADM is a 100% increase in the half-life.

[0141] Half-life (half the retention time) is defined as the period of time it takes for the concentration of a particular chemical or drug to fall to half of its baseline concentration in a particular body fluid or blood.

[0142] An assay that can be used to determine the half-life (half retention time) of adrenomedullin in serum, blood, and plasma is described in Example 3.

[0143] In a preferred embodiment, the anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold is a non-neutralizing antibody, or non-neutralizing fragment, or non-neutralizing scaffold. A neutralizing anti-ADM antibody, or neutralizing anti-ADM antibody fragment, or neutralizing anti-ADM non-Ig scaffold is considered to block the biological activity of ADM by nearly 100%, at least more than 90%, and preferably at least more than 95%. In other words, this means that a non-neutralizing anti-ADM antibody, or non-neutralizing anti-ADM antibody fragment, or non-neutralizing anti-ADM non-Ig scaffold blocks the biological activity of ADM by less than 100%, preferably less than 95%, and preferably less than 90%. In one embodiment, where a non-neutralizing anti-ADM antibody, or non-neutralizing anti-ADM antibody fragment, or non-neutralizing anti-ADM non-Ig scaffold blocks the biological activity of ADM to less than 95%, an anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold that would block the biological activity of ADM to greater than 95% is considered outside the scope of this embodiment, which in one embodiment means that the biological activity is reduced by up to 95%, preferably by 90%, more preferably by 80%, and more preferably by 50%.

[0144] In one embodiment of the present invention, the non-neutralizing antibody is an antibody that binds to a region of at least five amino acids in the sequence of amino acids 1 to 42 of mature human ADM (SEQ ID NO: 23), preferably in the sequence of amino acids 1 to 32 of mature human ADM (SEQ ID NO: 28), or an antibody that binds to a region of at least five amino acids in the sequence of amino acids 1 to 40 of mature mouse ADM (SEQ ID NO: 29), preferably in the sequence of amino acids 1 to 31 of mature human ADM (SEQ ID NO: 30).

[0145] In another preferred embodiment of the present invention, the non-neutralizing antibody is an antibody that binds to a region of at least four amino acids in the sequence of amino acids 1 to 42 of mature human ADM (SEQ ID NO: 23), preferably in the sequence of amino acids 1 to 32 of mature human ADM (SEQ ID NO: 28), or an antibody that binds to a region of at least four amino acids in the sequence of amino acids 1 to 40 of mature mouse ADM (SEQ ID NO: 29), preferably in the sequence of amino acids 1 to 31 of mature human ADM (SEQ ID NO: 30).

[0146] Amino acids 1-32 of mature human ADM YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQ (SEQ ID NO: 28)

[0147] Amino acids 1-40 of mature mouse ADM YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQLTDKDKDGMA (SEQ ID NO: 29)

[0148] Amino acids 1-31 of mature mouse ADM YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQL (sequence number 30).

[0149] In a specific embodiment of the present invention, a non-neutralizing anti-ADM antibody, a non-neutralizing anti-adrenomedullin antibody fragment, or a non-neutralizing anti-ADM non-Ig scaffold is used that inhibits ADM biological activity to less than 80% (of baseline values), preferably less than 50%. It should be understood that this limit of inhibition (meaning reduction in biological activity) of ADM biological activity occurs even when the antibody, fragment, or scaffold is at an excess concentration (the excess of antibody, fragment, or scaffold relative to ADM). This limit of inhibition is an inherent property of the ADM binding agent itself in this specific embodiment. This means that the maximum inhibition of the antibody, fragment, or scaffold is 80% or 50%, respectively. In a preferred embodiment, the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold is believed to inhibit / reduce anti-ADM biological activity by at least 5%. This means that approximately 20%, 50%, or even 95% residual ADM activity remains, respectively.

[0150] Thus, according to the present invention, the anti-ADM antibodies, anti-ADM antibody fragments, and anti-ADM antibody non-Ig scaffolds provided do not neutralize the respective ADM biological activity.

[0151] Biological activity is defined as the effect of a substance on an organism, tissue, organ, or functional unit in vivo or in vitro (e.g., assay) after interaction. In the case of ADM biological activity, the biological activity can be the effect of ADM in a human recombinant adrenomedullin receptor cAMP functional assay. Therefore, according to the present invention, biological activity is defined through an adrenomedullin receptor cAMP functional assay. To determine the biological activity of ADM in such an assay, the following steps can be performed: - To generate a dose-response curve using ADM in the adrenomedullin receptor cAMP functional assay. - The half-effective ADM concentration for cAMP stimulation can be calculated. - At a constant ADM concentration at half-maximal effect of cAMP stimulation, a dose-response curve is performed with either the ADM-stabilized antibody, or the adrenomedullin-stabilized antibody fragment, or the adrenomedullin-stabilized non-Ig scaffold (up to a final concentration of 100 μg / ml).

[0152] A maximal inhibition of 50% in the ADM bioassay means that the anti-ADM antibody, anti-adrenomedullin antibody fragment, or anti-adrenomedullin non-Ig scaffold each inhibits ADM biological activity to 50% of the baseline value. A maximal inhibition of 80% in the ADM bioassay means that the anti-ADM antibody, anti-adrenomedullin antibody fragment, or anti-adrenomedullin non-Ig scaffold each inhibits ADM biological activity to 80% of the baseline value. This means that ADM biological activity is not inhibited below 80%. This means that approximately 20% residual ADM biological activity remains.

[0153] However, according to this specification and in the above context, the expression "blocking the biological activity of ADM" in relation to the anti-ADM antibodies, anti-ADM antibody fragments, and anti-ADM antibody non-Ig scaffolds disclosed herein should be understood simply as reducing the biological activity of ADM from 100% to a maximum of 20% residual ADM biological activity, preferably from 100% to 50% residual ADM biological activity, in any case remaining ADM biological activity which can be determined as detailed above.

[0154] The biological activity of ADM can be determined in a human recombinant adrenomedullin receptor cAMP functional assay (adrenomedullin bioassay) according to Example 2.

[0155] In a preferred embodiment, the regulatory antibody or regulatory anti-adrenomedullin antibody fragment, or the regulatory anti-adrenomedullin non-Ig scaffold is used to treat or prevent a chronic or acute disease or condition in a patient to stabilize circulation, particularly systemic circulation.

[0156] The "modulating" anti-ADM antibody, or the modulating anti-adrenomedullin antibody fragment, or the modulating anti-adrenomedullin non-Ig scaffold, can be used to shorten the half-life (t 1 / 2 ; half-retention time) by at least 10%, preferably at least 50%, more preferably more than 50%, and most preferably more than 100%, while inhibiting ADM biological activity to less than 80%, preferably less than 50%, and this anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold is considered to inhibit ADM biological activity by at least 5%. It should be understood that these values for half-life and inhibition of biological activity relate to the assays described above for determining these values. This means that ADM biological activity will not be inhibited to less than 80% or less than 50%, respectively.

[0157] Such regulatory anti-ADM antibodies, regulatory anti-adrenomedullin antibody fragments, or regulatory anti-adrenomedullin non-Ig scaffolds offer the advantage of ease of administration. Partially blocking or partially reducing the biological activity of adrenomedullin combined with extending its in vivo half-life (increasing the biological activity of adrenomedullin) offers the advantage of simplicity in the development of anti-adrenomedullin antibodies, anti-adrenomedullin antibody fragments, or anti-adrenomedullin non-Ig scaffolds. In situations where endogenous adrenomedullin is in excess (maximal stimulation, late sepsis, shock, and debilitating stages), the activity-reducing effect of the antibody, fragment, or scaffold is the primary influence, limiting the negative effects of adrenomedullin. When endogenous adrenomedullin concentrations are low or normal, the biological effect of anti-adrenomedullin antibodies, or anti-adrenomedullin antibody fragments, or anti-ADM non-Ig scaffolds is a combination of reduction (by partial blockage) and enhancement by prolonging the half-life of adrenomedullin. Thus, non-neutralizing and regulatory anti-adrenomedullin antibodies, or anti-adrenomedullin antibody fragments, or anti-adrenomedullin non-Ig scaffolds act like an ADM bioactivity buffer, maintaining the bioactivity of ADM within a physiological range.

[0158] In a particular embodiment of the invention, the antibody is a monoclonal antibody or a fragment thereof. In one embodiment of the invention, the anti-ADM antibody or anti-ADM antibody fragment is or is derived from a human or humanized antibody. In a particular embodiment, one or more (murine) CDRs are grafted into a human antibody or human antibody fragment.

[0159] In one aspect, the subject of the present invention is a human CDR-grafted antibody or antibody fragment thereof that binds to ADM. This human CDR-grafted antibody or antibody fragment thereof comprises: SEQ ID NO: 1 GYTFSRYW, and / or SEQ ID NO: 2 ILPGSGST, and / or SEQ ID NO: 3 TEGYEYDGFDY and / or an antibody heavy chain comprising SEQ ID NO:4 QSIVYSNGNTY, and / or Sequence "RVS" (not part of the sequence listing) RVS, and / or SEQ ID NO:5 FQGSHIPYT The antibody further comprises an antibody light chain (L chain) comprising:

[0160] In one particular embodiment of the invention, the subject of the invention is a human monoclonal antibody that binds to ADM, or an antibody fragment thereof that binds to ADM, in which the heavy chain comprises SEQ ID NO: 1 GYTFSRYW, SEQ ID NO: 2 ILPGSGST, SEQ ID NO: 3 TEGYEYDGFDY and the light chain comprises at least one CDR selected from the group comprising: SEQ ID NO:4 QSIVYSNGNTY, Sequence "RVS" (not part of the sequence listing) RVS, SEQ ID NO:5 FQGSHIPYT and (iii) a CDR selected from the group consisting of:

[0161] In one more particular embodiment of the invention, the subject of the invention is a human monoclonal antibody that binds to ADM, or an antibody fragment thereof that binds to ADM, in which the heavy chain has the following sequence: SEQ ID NO: 1 GYTFSRYW, SEQ ID NO: 2 ILPGSGST, SEQ ID NO: 3 TEGYEYDGFDY and the light chain comprises the following sequence: SEQ ID NO:4 QSIVYSNGNTY, Sequence "RVS" (not part of the sequence listing) RVS, SEQ ID NO:5 FQGSHIPYT Contains:

[0162] In one very particular embodiment, the anti-ADM antibody has a sequence selected from the group comprising SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13.

[0163] The anti-ADM antibodies, or anti-adrenomedullin antibody fragments, or anti-ADM non-Ig scaffolds of the present invention exhibit affinity for human ADM, with affinity constants of 10 -7 More than M, preferably 10 -8 M, with a preferred affinity of 10 -9 More than M, most preferably 10 -10 The affinity constant is greater than M. Those skilled in the art will know that a higher dose of the compound will compensate for lower affinity, and this indication is not considered to fall within the scope of the present invention. The affinity constant can be determined according to the method described in Example 1.

[0164] The subject of the present invention is a human monoclonal antibody or fragment thereof that binds to ADM, for use in the treatment and therapy of hemostasis in patients according to the invention, said antibody or fragment comprising: Sequence number 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTT LTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH, SEQ ID NO: 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH, SEQ ID NO: 8 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH, SEQ ID NO: 9 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH, SEQ ID NO: 10 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH, SEQ ID NO: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, SEQ ID NO: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, SEQ ID NO: 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC It comprises a sequence selected from the group comprising:

[0165] A subject of the present invention is a pharmaceutical formulation comprising an antibody or fragment or scaffold of the invention for use in the treatment and therapy of hemostasis in a patient.

[0166] A subject of the present invention is a pharmaceutical formulation comprising an antibody or fragment or scaffold of the invention for use in the treatment and therapy of congestion in a patient, the patient having a disease or condition selected from the group comprising congestive hypertension, swelling or fluid retention (edema), heart failure (especially acute heart failure), kidney disease, liver disease.

[0167] A subject of the present invention is a pharmaceutical formulation comprising an antibody or fragment or scaffold of the invention for use in the treatment and therapy of hemostasis in a patient, said pharmaceutical formulation being a solution, preferably a ready-to-use solution.

[0168] A subject of the present invention is a pharmaceutical formulation comprising an antibody or fragment or scaffold of the invention for use in the treatment and therapy of hemostasis in a patient, said pharmaceutical formulation being in lyophilized form.

[0169] A subject of the present invention is a pharmaceutical preparation comprising an antibody or fragment or scaffold of the invention for use in the treatment and therapy of hemostasis in a patient, said pharmaceutical preparation being administered intramuscularly.

[0170] A subject of the present invention is a pharmaceutical preparation comprising an antibody or fragment or scaffold of the invention for use in the treatment and therapy of hemostasis in a patient, said pharmaceutical preparation being administered intravascularly.

[0171] A subject of the present invention is a pharmaceutical preparation comprising an antibody or fragment or scaffold of the invention for use in the treatment and therapy of hemostasis in a patient, said pharmaceutical preparation being administered by infusion.

[0172] A subject of the present invention is a pharmaceutical preparation comprising an antibody or fragment or scaffold of the invention for use in the treatment and therapy of hemostasis in a patient, said pharmaceutical preparation being administered systemically.

[0173] The following embodiments are the subject of the present invention:

[0174] 1. An anti-adrenomedullin (ADM) antibody, or an anti-adrenomedullin antibody fragment, or an anti-ADM non-Ig scaffold for use in the treatment and therapy of hemostasis in a patient in need thereof.

[0175] 2. The anti-adrenomedullin (ADM) antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold described in paragraph 1 for use in the treatment and therapy of congestion in a patient, wherein the patient has a disease or condition selected from the group consisting of congestive hypertension, swelling or fluid retention (edema), heart failure (particularly acute heart failure), kidney disease, and liver disease.

[0176] 3. The anti-adrenomedullin (ADM) antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold according to paragraph 1 or 2 for use in the treatment and therapy of congestion in a patient, wherein the patient has a disease or condition selected from the group consisting of congestive hypertension, swelling or fluid retention (edema), and heart failure (particularly acute heart failure).

[0177] 4. A monospecific anti-adrenomedullin (ADM) antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold according to any one of paragraphs 1 to 3 for use in the treatment and therapy of hemostasis in a patient.

[0178] 5. At least 10 for ADM -7 5. The anti-adrenomedullin (ADM) antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold of any one of paragraphs 1 to 4, which exhibits binding affinity for M, for use in the treatment and therapy of hemostasis in a patient.

[0179] 6. Sequence of amino acids 1-42 of mature human ADM: YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA (SEQ ID NO: 23) 6. An anti-adrenomedullin (ADM) antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold according to any one of items 1 to 5, which binds to preferably at least four or at least five regions of the above for use in the treatment and therapy of congestion in a patient.

[0180] 7. N-terminal portion of adrenomedullin (amino acids 1-21): YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 22) 7. The anti-ADM antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold according to any one of items 1 to 6, which is bound to

[0181] 8. An anti-ADM antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold according to any one of paragraphs 1 to 7, which recognizes and binds to the N-terminus (amino acid 1) of adrenomedullin, for use in the treatment and therapy of congestion in a patient.

[0182] 9. The sequence of amino acids 43-52 of ADM: PRSKISPQGY-NH2 (SEQ ID NO: 24) Item 9. The anti-adrenomedullin (ADM) antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold according to any one of Items 1 to 8, for use in the treatment and therapy of congestion in a patient, characterized in that it does not bind to the C-terminal portion of ADM having the structure:

[0183] 10. An anti-ADM antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold according to any one of paragraphs 1 to 9, for use in the treatment and therapy of congestion in a patient, which blocks the biological activity of ADM to 80% or less, preferably 50% or less.

[0184] 11. The anti-ADM antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold according to any one of paragraphs 1 to 10, for use in the treatment and therapy of congestion in a patient, wherein the patient is an ICU patient.

[0185] 12. A human monoclonal antibody or fragment thereof that binds to ADM, wherein the heavy chain has the sequence: SEQ ID NO: 1 GYTFSRYW, SEQ ID NO: 2 ILPGSGST, SEQ ID NO: 3 TEGYEYDGFDY and the light chain comprises the sequence: SEQ ID NO:4 QSIVYSNGNTY, Sequence "RVS" (not part of the sequence listing) RVS, SEQ ID NO:5 FQGSHIPYT 12. The anti-ADM antibody or anti-adrenomedullin antibody fragment according to any one of items 1 to 11, for use in treating and curing congestion in a patient.

[0186] 13.Sequence number 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTT LTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH, SEQ ID NO: 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH, SEQ ID NO: 8 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH, SEQ ID NO: 9 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH, SEQ ID NO: 10 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH, SEQ ID NO: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, SEQ ID NO: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, SEQ ID NO: 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 13. A human monoclonal antibody or fragment thereof that binds to ADM according to claim 12, or an antibody fragment thereof, for use in the treatment and therapy of hemostasis in a patient, comprising a sequence selected from the group comprising:

[0187] 14. An anti-ADM antibody or anti-ADM antibody fragment that binds to ADM, or an anti-ADM non-Ig scaffold that binds to ADM, according to any one of paragraphs 1 to 13, for use in the treatment and therapy of congestion in a patient, wherein the level of pro-ADM and / or the level of a fragment thereof having at least five amino acids is higher than a predetermined threshold in a sample of body fluid taken from the patient.

[0188] 15. An anti-ADM antibody or anti-ADM antibody fragment that binds to ADM, or an anti-ADM non-Ig scaffold that binds to ADM, according to any one of paragraphs 1 to 14, for use in the treatment and therapy of congestion in a patient, wherein the patient is diuretic-resistant or does not respond to diuretic therapy.

[0189] 16. A pharmaceutical formulation for use in the treatment and therapy of hemostasis in a patient, comprising the antibody or fragment or scaffold of any one of paragraphs 1 to 15.

[0190] 17. A pharmaceutical formulation comprising the antibody or fragment or scaffold of any one of paragraphs 1 to 15 for use in the treatment and therapy of congestion in a patient, wherein the patient has a disease or condition selected from the group consisting of congestive hypertension, swelling or fluid retention (edema), heart failure (especially acute heart failure), kidney disease, and liver disease.

[0191] 18. A pharmaceutical formulation according to paragraph 16 or 17 for use in the treatment and therapy of blood congestion, which is a solution, preferably a ready-to-use solution.

[0192] 19. A pharmaceutical preparation for use in the treatment and therapy of blood congestion according to item 18, which is in a freeze-dried state.

[0193] 20. A pharmaceutical preparation for use in the treatment and therapy of congestion according to any one of items 18 to 19, which is administered intramuscularly.

[0194] 21. A pharmaceutical preparation for use in the treatment and therapy of congestion according to any one of items 18 to 19, which is administered intravascularly.

[0195] 22. A pharmaceutical preparation for use in the treatment and therapy of blood congestion according to paragraph 21, administered via infusion.

[0196] 23. A pharmaceutical preparation for use in the treatment and therapy of blood congestion according to any one of items 18 to 22, which is administered systemically. [Example]

[0197] It should be emphasized that the antibodies, antibody fragments and non-Ig scaffolds in the Examples section according to the present invention should be considered as anti-ADM antibodies / antibody fragments / non-Ig scaffolds, since they bind to ADM.

[0198] Example 1 Preparation of antibodies and determination of their affinity constants Several human and mouse antibodies were generated and their affinity constants determined (see Tables 1 and 2).

[0199] Peptides / conjugates for immunization The peptides for immunization were synthesized with an additional N-terminal cysteine residue (if no cysteine was present in the selected ADM sequence) to conjugate the peptide to bovine serum albumin (BSA) (see Table 1) (JPT Technologies, Berlin, Germany). The peptides were covalently coupled to BSA using Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the Perbio manual.

[0200] The mouse antibody was produced according to the following method.

[0201] Balb / c mice were immunized with 100 μg of peptide-BSA conjugate (emulsified in 100 μl of complete Freund's adjuvant) on days 0 and 14 and with 50 μg (in 100 μl of incomplete Freund's adjuvant) on days 21 and 28. Three days before the fusion experiment, mice were given one intraperitoneal injection and one intravenous injection of 50 μg of the conjugate dissolved in 100 μl of saline.

[0202] Spleen cells from immunized mice were fused with myeloma cell line SP2 / 0 cells using 1 ml of 50% polyethylene glycol at 37°C for 30 seconds. After washing, the cells were seeded into 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium (RPMI 1640 medium supplemented with 10% fetal bovine serum and HAT supplement). After 2 weeks, the HAT medium was replaced with HT medium for three passages before returning to normal cell culture medium.

[0203] Three weeks after fusion, cell culture supernatants were initially screened for antigen-specific IgG antibodies. Microcultures that tested positive were transferred to 24-well plates and expanded. After retesting, selected cultures were cloned, recloned, and isotyped using limiting dilution techniques (see also Lane, RD 1985, J. Immunol. Meth. 81:223-228; Ziegler et al. 1996, Horm. Metab. Res. 28:11-15).

[0204] Mouse monoclonal antibody production: Antibodies were produced by standard antibody production methods (Marx et al., 1997, Monoclonal Antibody Production, ATLA 25:121) and purified with protein A. The purity of the antibodies was greater than 95% based on SDS gel electrophoresis analysis.

[0205] Human antibodies: Human antibodies were generated by phage display according to the following procedure.

[0206] Using the human naive antibody gene library HAL7 / 8, we isolated recombinant single-chain F variable domains (scFv) against adrenomedullin peptides. The antibody gene library was screened using a panning strategy involving the use of peptides containing a biotin tag linked to the adrenomedullin peptide sequence via two different spacers. Panning with nonspecifically binding antigens and panning with streptavidin-binding antigens was used in combination to minimize background due to nonspecific binding. Phages eluted from the third panning round were used to generate E. coli strains expressing monoclonal scFvs. Supernatants from the cultures of these clonal strains were directly used in antigen ELISA tests (see also Hust et al. 2011, Journal of Biotechnology 152:159–170; Schutte et al. 2009, PLoS One 4:e6625).

[0207] Positive clones were selected based on positive ELISA signals on antigen-coated microtiter plates and negative ELISA signals on streptavidin-coated microtiter plates. For further characterization, the scFv open reading frame was cloned into the expression plasmid pOPE107 (Hust et al., J. Biotechn. 2011), captured from culture supernatants via immobilized metal ion affinity chromatography, and purified by size-exclusion chromatography.

[0208] Affinity constant: To determine the affinity of the antibody for adrenomedullin, the binding kinetics of adrenomedullin to the immobilized antibody was determined by label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). The antibody was reversibly immobilized using high-density covalently coupled anti-mouse Fc antibody (Mouse Antibody Capture Kit; GE Healthcare) to the CM5 sensor surface according to the manufacturer's instructions (Lorenz et al. 2011, Antimicrob Agents Chemother. 55(1):165-173).

[0209] Monoclonal antibodies were generated against the following ADM regions in human and mouse ADM. The table below lists the antibodies obtained for use in further experiments. Selection was based on the target region.

[0210] [Table 3]

[0211] Below is a list of additional monoclonal antibodies that have been obtained.

[0212] [Table 4]

[0213] Generation of antibody fragments by enzymatic digestion Fab and F(ab)2 fragments were generated by enzymatic digestion of the mouse full-length antibody NT-M. Antibody NT-M was digested using a) a pepsin-based F(ab)2 preparation kit (Pierce, 44988) and b) a papain-based Fab preparation kit (Pierce, 44985). The fragmentation procedures were performed according to the instructions provided by the supplier. Digestion was performed for 8 hours at 37°C for F(ab)2 fragmentation. Digestion for 16 hours for Fab fragmentation.

[0214] Fab generation and purification procedures The immobilized papain was equilibrated by washing the resin with 0.5 ml of digestion buffer, after which the column was centrifuged at 5000 × g for 1 minute. The buffer was then discarded. A desalting column was prepared by removing the storage solution, washing it with digestion buffer, and then centrifuging it at 1000 × g for 2 minutes each time. 0.5 ml of the prepared IgG sample was added to the spin column tube containing the equilibrated immobilized papain. The digestion reaction was incubated at 37°C on a benchtop rocker for 16 hours. The digest was separated from the immobilized papain by centrifuging the column at 5000 × g for 1 minute. The resin was then washed with 0.5 ml of PBS and centrifuged at 5000 × g for 1 minute. The wash fraction was added to the digested antibody. The total sample volume was 1.0 ml. The NAb Protein A column was equilibrated with PBS and IgG elution buffer at room temperature. The column was centrifuged for 1 minute to remove the storage solution (containing 0.02% sodium azide), equilibrated by adding 2 ml of PBS, centrifuged again for 1 minute, and the flow-through discarded. The sample was applied to the column and resuspended by inversion. It was incubated for 10 minutes at room temperature with inversion mixing. The column was centrifuged for 1 minute, and the flow-through containing the Fab fragments was saved. (References: Coulter and Harris 1983, J. Immunol. Meth. 59, 199-203; Lindner et al. 2010, Cancer Res. 70, 277-287; Kaufmann et al. 2010, PNAS. 107, 18950-18955; Chen et al. 2010, PNAS. 107, 14727-14732; Uysal et al. 2009, J. Exp. Med. 206, 449-462; Thomas et al. 2009, J. Exp. Med. 206, 1913-1927; Kong et al. 2009, J. Cell Biol. 185, 1275-1284).

[0215] F(ab)2 fragment generation and purification procedure The immobilized pepsin was equilibrated by washing the resin with 0.5 ml of digestion buffer, and then the column was centrifuged at 5000 × g for 1 minute. The buffer was then discarded. A desalting column was prepared by removing the storage solution, washing it with digestion buffer, and then centrifuging it at 1000 × g for 2 minutes each time. 0.5 ml of the prepared IgG sample was added to the spin column tube containing the equilibrated immobilized pepsin. The digestion reaction was incubated at 37°C on a benchtop rocker for 16 hours. The digest was separated from the immobilized pepsin by centrifuging the column at 5000 × g for 1 minute. The resin was then washed with 0.5 ml of PBS and centrifuged at 5000 × g for 1 minute. The wash fraction was added to the digested antibody. The total sample volume was 1.0 ml. The NAb Protein A column was equilibrated with PBS and IgG elution buffer at room temperature. The column was centrifuged for 1 minute to remove the storage solution (containing 0.02% sodium azide), equilibrated by adding 2 ml of PBS, centrifuged again for 1 minute, and the flow-through was discarded. The sample was applied to the column and resuspended by inversion. It was incubated for 10 minutes at room temperature with inversion mixing. The column was centrifuged for 1 minute, and the flow-through containing the F(ab)2 fragments was saved.(References: Mariani et al. 1991, Mol. Immunol. 28:69-77; Beale 1987, Exp Comp Immunol 11:287-296; Ellerson et al. 1972, FEBS Letters 24(3):318-322; Kerbel and Elliot 1983, Meth Enzymol 93:113-147; Kulkarni et al. 1985, Cancer Immunol Immunotherapy 19:211-214; Lamoyi 1986, Meth Enzymol 121:652-663; Parham et al. 1982, J Immunol Meth 53:133-173; Raychaudhuri et al. 1985, Mol. Immunol 22(9):1009-1019; Rousseaux et al. 1980, Mol Immunol 17:469-482; Rousseaux et al. 1983, J Immunol Meth 64:141-146; Wilson et al. 1991, J Immunol Meth 138:111-119).

[0216] Humanization of NT-H antibody fragment Antibody fragments have been humanized by the CDR grafting method (Jones et al. 1986, Nature 321:522-525).

[0217] The following steps were performed to achieve the humanized sequence: Total RNA extraction: Total RNA was extracted from NT-H hybridoma using a Qiagen kit.

[0218] Initial RT-PCR: QIAGEN® OneStep RT-PCR Kit (Cat. No. 210210) was used. RT-PCR was performed using primer sets specific for the heavy and light chains. For each RNA sample, 12 individual heavy chains and 11 light chains were RT-PCRed using a degenerate forward primer mixture covering the leader sequences of the variable regions. Reverse primers were located in the constant regions of the heavy and light chains. No restriction sites were engineered into the primers.

[0219] Reaction composition: 5.0 μl 5x QIAGEN® OneStep RT-PCR buffer, 0.8 μl dNTP Mix (containing 10 mM each dNTP), 0.5 μl primer set, 0.8 μl QIAGEN® OneStep RT-PCR Enzyme Mix, 2.0 μl template RNA, and RNase-free water (remaining until a total volume of 20.0 μl was reached). PCR conditions: reverse transcription: 50°C for 30 min; initial PCR activation: 95°C for 15 min; cycling: 94°C for 25 s; 54°C for 30 s; 72°C for 30 s (20 cycles); final extension: 72°C for 10 min. Second semi-nested PCR: The RT-PCR products from the first reaction were further amplified in a second PCR. 12 individual heavy chains and 11 light chains were RT-PCR-reacted using semi-nested primer sets specific for antibody variable regions.

[0220] Reaction composition: 10 μl of 2x PCR mix; 2 μl of primer set; 8 μl of initial PCR product; total volume 20 μl; PCR conditions reported for hybridoma antibody cloning: initial denaturation at 95°C for 5 minutes; 25 cycles of 95°C for 25 seconds, 57°C for 30 seconds, and 68°C for 30 seconds; final extension at 68°C for 10 minutes.

[0221] After PCR was completed, PCR reaction samples were loaded onto an agarose gel to visualize the amplified DNA fragments. After sequencing the more than 15 DNA fragments cloned and amplified by nested RT-PCR, the heavy and light chains of several mouse antibodies were cloned and visualized correctly. Protein sequence alignment and CDR analysis identified one heavy chain and one light chain. The humanized sequence obtained for the variable heavy chain after alignment with the homologous human framework sequence is as follows: see Figure 5. Because amino acids at positions 26, 40, and 55 of the variable heavy chain and at position 40 of the variable light chain are crucial for binding properties, these may have been reverted to their mouse counterparts. The resulting candidates are shown below. (Padlan 1991. Mol. Immunol. 28, 489-498; Harris and Bajorath 1995. Protein Sci. 4, 306-310)

[0222] Annotation of antibody fragment sequences (SEQ ID NOs: 7-14): CDRs 1, 2, and 3 are in bold and underlined, arranged in order of appearance. Constant regions are in italics. The hinge region is highlighted in bold, and the histidine tag is in bold and italics. Framework point mutations have a grey text background.

[0223] Sequence number 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISCKAT GYTFSRYW IEWVKQRPGHGLEWIGE ILPGSGST NYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYC TEGYEYDGFDY WGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK HHHHHH

[0224] SEQ ID NO: 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKAS GYTFSRYW ISWVRQAPGQGLEWMGR ILPGSGST NYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH

[0225] SEQ ID NO: 8 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKAS GYTFSRYW IEWVRQAPGQGLEWMGR ILPGSGST NYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH

[0226] SEQ ID NO: 9 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKAT GYTFSRYW ISWVRQAPGQGLEWMGE ILPGSGST NYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH

[0227] SEQ ID NO: 10 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKAT GYTFSRYW IEWVRQAPGQGLEWMGE ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH

[0228] SEQ ID NO: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSS QSIVYSNGNTY LEWYLQKPGQSPKLLIY RVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHIPYT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0229] SEQ ID NO: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSS QSIVYSNGNTY LNWFQQRPGQSPRRLIY RVS NRDSGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC FQGSHIPYT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0230] SEQ ID NO: 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSS QSIVYSNGNTY LEWFQQRPGQSPRRLIY RVS NRDSGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC FQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0231] Example 2 Effect of selected anti-ADM antibodies on anti-ADM biological activity The effects of selected anti-ADM antibodies on anti-ADM bioactivity were examined using a human recombinant adrenomedullin receptor cAMP functional assay (adrenomedullin bioassay).

[0232] Testing antibodies targeting human and mouse adrenomedullin in a human recombinant adrenomedullin receptor cAMP functional assay (adrenomedullin bioassay)

[0233] material: Cell line: CHO-K1 Receptor: Adrenomedullin (CRLR+RAMP3) Receptor registration number cell line: CRLR: U17473; RAMP3: AJ001016

[0234] Prior to testing, CHO-K1 cells expressing the human recombinant adrenomedullin receptor (FAST-027C) grown in antibiotic-free medium were detached by gentle flushing with PBS-EDTA (5 mM EDTA), collected by centrifugation, and resuspended in assay buffer (KRH: 5 mM KCl, 1.25 mM MgSO, 124 mM NaCl, 25 mM HEPES, 13.3 mM glucose, 1.25 mM KHPO, 1.45 mM CaCl, 0.5 g / L BSA).

[0235] Dose-response curves were obtained in parallel using a reference agonist (hADM or mADM).

[0236] Antagonist Test (96 wells): To test for antagonists, 6 μl of a reference agonist (human adrenomedullin (5.63 nM) or mouse adrenomedullin (0.67 nM)) was mixed with 6 μl of test sample containing different dilutions of the antagonist or with 6 μl of buffer. After incubation at room temperature for 60 minutes, 12 μl of cells (2,500 cells / well) were added. The plate was incubated at room temperature for 30 minutes. After adding lysis buffer, the percentage of DeltaF was assessed using an HTRF kit from Cis-Bio International (catalog number 62AM2 PEB) according to the manufacturer's instructions. hADM 22-52 was used as the reference antagonist.

[0237] cAMP-HTRF assay to test antibodies The antagonist activity of anti-h-ADM antibodies (NT-H, MR-H, and CT-H) was examined in the human recombinant adrenomedullin receptor (FAST-027C) cAMP functional assay in the presence of 5.63 nM human ADM 1-52 at final antibody concentrations of 100 μg / ml, 20 μg / ml, 4 μg / ml, 0.8 μg / ml, and 0.16 μg / ml.

[0238] The antagonist activity of anti-m-ADM antibodies (NT-M, MR-M, and CT-M) was examined in the presence of 0.67 nM mouse ADM 1-50 in a human recombinant adrenomedullin receptor (FAST-027C) cAMP functional assay. The final antibody concentrations were as follows: 100 μg / ml, 20 μg / ml, 4 μg / ml, 0.8 μg / ml, and 0.16 μg / ml. The data were plotted to obtain the relative inhibition versus antagonist concentration (see Figures 2a-l). The maximum inhibition by each antibody is shown in Table 3.

[0239] [Table 5]

[0240] Example 3 Data on stabilization of hADM by anti-hADM antibodies The effect of human ADM stabilization by human ADM antibodies was investigated using a hADM immunoassay.

[0241] Immunoassay for quantifying human adrenomedullin The technique used was a sandwich-coated tube luminescence immunoassay based on an acridinium ester label.

[0242] Compound to be labeled (tracer): 100 μg (100 μl) of CT-H (1 mg / ml in PBS, pH 7.4, AdrenoMed AG, Germany) was mixed with 10 μl of acridinium NHS-ester (1 mg / ml in acetonitrile, InVent GmbH, Germany) (EP 0353971) and incubated at room temperature for 20 minutes. The labeled CT-H was purified by gel filtration HPLC on a Bio-Sil® SEC 400-5 (Bio-Rad Laboratories, Inc., USA). The purified CT-H was diluted in (300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, 5 g / L bovine serum albumin, pH 7.0). The final concentration was approximately 800,000 relative light units (RLU) per 200 μl of labeled compound (approximately 20 ng of labeled antibody). Chemiluminescence of the acridinium ester was measured using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

[0243] Solid phase: Polystyrene tubes (Austria) were coated with MR-H (AdrenoMed AG, Germany) (1.5 μg MR-H / 0.3 ml 100 mmol / L NaCl, 50 mmol / L TRIS / HCl, pH 7.8) for 18 h at room temperature. After blocking with 5% bovine serum arbutin, the tubes were washed with PBS, pH 7.4, and dried in vacuo.

[0244] calibration: The assay was calibrated using hADM (BACHEM AG, Switzerland) diluted in 250 mmol / l NaCl, 2 g / l Triton X-100, 50 g / l bovine serum albumin, and 20 tablets / l protease inhibitor cocktail (Roche Diagnostics AG, Switzerland).

[0245] hADM Immunoassay: 50 μl of sample (or calibrator) was pipetted into the coated tube, and after adding 200 μl of labeled CT-H, the tube was incubated for 4 h at 4° C. Unbound tracer was removed by washing five times (1 ml each time) with washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100).

[0246] Tube-bound chemiluminescence was measured using LB 953:

[0247] Figure 3 shows a typical hADM dose / signal curve and the dose-signal curve of hADM in the presence of 100 μg / ml of antibody NT-H. NT-H had no effect on the hADM immunoassay described.

[0248] Stability of human adrenomedullin: Human ADM was diluted in human citrated plasma (final concentration 10 nM) and incubated at 24°C. Degradation of hADM was stopped at selected time points by freezing to -20°C. Incubations were performed in the absence and presence of NT-H (100 μg / ml). Remaining hADM was quantified using the hADM immunoassay described above.

[0249] Figure 4 shows the stability of hADM in human plasma (citrate) in the absence and presence of NT-H antibodies. The half-life of hADM was 7.8 hours alone and 18.3 hours in the presence of NH-H (2.3-fold greater stability).

[0250] Example 4 Measurement of in vivo side effects of antibody NT-H Male C57B1 / 6 mice (Charles River Laboratories, Germany) aged 12–15 weeks were used in the study. Six mice were treated with NT-M at a dose of 0.2 mg / ml (10 μl per gram of body weight). As a control, six mice were treated with PBS (10 μl per gram of body weight). Survival and physical condition were monitored for 14 days. There were no deaths in either group, and there was no difference in physical condition between the NT-M and control groups.

[0251] Example 5 Dose-dependence of the effect of NT-H The dose-dependence of the effects of NT-H in a mouse CLP model was investigated based on renal barrier dysfunction as revealed by immunohistochemical staining of the kidney. Male C57B1 / 6 mice (Charles River Laboratories, Germany; n = 6 / group, 4 groups) aged 12–15 weeks were used in this study. After light anesthesia with isoflurane (and surgical subcutaneous injection of rimadyl at 0.5 mg / kg), peritonitis was surgically induced. An incision was made in the upper left quarter of the abdominal cavity (the normal location of the cecum). The cecum was exposed and tightly ligated around it, with the stitches positioned distal to the entrance to the small intestine. A single puncture wound was made in the cecum with a 24-gauge needle, and a small amount of cecal contents was expelled through the wound. The cecum was returned to the abdominal cavity, and the abdominal incision was closed. Finally, the mice were returned to their cages with free access to food and water. 500 μl of saline was injected subcutaneously instead of body fluids. Eighteen hours after CLP and test compound application, the mice were euthanized and the kidneys were removed.

[0252] Mice were treated with different concentrations of vehicle and compounds. The vehicles and compounds were supplied by the sponsor as "ready-to-use" solutions in test tubes labeled A, B, C, and D. Five minutes before CLP, intravenous injections were administered via tail vein injection in a volume of 5 μl per gram of body weight at doses of 0.1, 2, or 20 mg per kg of body weight. The vehicle was 20 mM His / HCl, pH 6.0.

[0253] Mice that survived for 18 hours were subjected to terminal bleeding. Six hours after CLP, 500 μl of blood was obtained by terminal bleeding from three additional mice per group. EDTA-plasma samples were frozen one hour after sampling.

[0254] Kidney processing for immunohistochemistry: Mice were euthanized by exsanguination. Because the kidneys were therefore not filled with blood, they were immediately removed. After removal, the kidneys were cut into sagittal sections, yielding two intact halves. The two kidney halves were placed in at least 10 volumes of 10% formalin (4% formaldehyde neutral buffered; Fischer 639 3113). The two halves were placed separately (not stuck together) in 5 ml cups filled with 10% formalin and fixed at room temperature for 6 days (allowing samples to be mailed to us during fixation) (dehydrated and paraffin embedded overnight: 2 hour washes in dH2O, 1 hour in 40% ethanol, 2 x 1 hour in 70% ethanol, 1 hour in 80% ethanol, 1 hour in 90% ethanol, 2 x 1 hour in 100% ethanol, 1 hour in xylene at 40°C, 1.5 hours in xylene at 45°C, 3 x 1 hour in paraffin at 60°C, embedded in cassettes). Left kidneys were dissected immediately upon receipt, fixed in formalin for 6 days, embedded in paraffin, and deparaffinized from 5 μm sections. They were exposed to HIER, 10% goat or donkey serum, 1° antibodies (VEGF, Alb, Ang1), or 2° anti-rabbit or anti-goat IgG AP, then exposed to Dako REAL chromogen and counterstained with hematoxylin. Slides were analyzed with Axio Vision (rel. 4.8) software (Zeiss, Jena, Germany) and expressed as mean density sum readings. Density assessment of kidneys stained for albumin revealed significantly lower extravascular albumin accumulation at all three doses tested, with a slightly less effective dose at 20 mg / kg (Figure 6).

[0255] VEGF is known to increase endothelial vascular permeability and therefore serves as an auxiliary biomarker for the status of renal barrier function. As shown in Figure 7, VEGF expression was significantly reduced at all doses tested, with no dose-dependent effect.

[0256] Angiopoietin 1 is known to protect against this VEGF-induced plasma leakage and should therefore be inversely related to VEGF expression levels, as shown significantly at all doses tested in Figure 8.

[0257] This study evaluated the effects of three different doses of NT-H administered intravenously 5 minutes before surgery in a mouse model of CLP-induced peritonitis. NT-H significantly improved renal vascular integrity in septic mice at all doses tested compared with the placebo group. NT-H showed beneficial effects over a wide dose range, with a tendency for the effect to be slightly smaller at 20 mg / kg.

[0258] Furthermore, the results of this study indicate that application of NT-H antibodies has a positive effect by reducing endothelial vascular permeability, thereby preventing or protecting against vascular fluid exudation and ultimately preventing or protecting against congestion and / or edema.

[0259] Example 6 PROTECT Study Study population and measurements Details of this trial have been published (Massie et al. 2010, N Engl J Med. 363:1419-1428; Weatherley et al. 2010, J Card Fail. 16:25-35; Voors et al. 2011, J Am Coll Cardiol. 57:1899-1907). Briefly, 2,033 patients with acute heart failure and impaired renal function (estimated creatinine clearance of 20-80 ml / min by the Cockcroft-Gault formula) were included and randomly assigned to receive rolofylline or placebo. The PROTECT trial protocol was approved by the institutional review boards of each participating institution, and written informed consent was obtained from all participants.

[0260] BioADMs were measured in plasma collected during baseline assessment in 1572 hospitalized AHF patients included in the PROTECT trial (all available baseline samples). PROTECT (which stands for "Placebo-controlled Randomized Study of the Selective A1 Adenosine Receptor Antagonist Rolofylline for Patients Hospitalized with Acute Decompensated Heart Failure and Volume Overload to Assess Treatment Effect on Congestion and Renal Function") is a multicenter, randomized, double-blind trial that compared rolofylline with placebo in 2033 patients hospitalized with AHF.

[0261] Test results As already emphasized, clinical surrogate markers do not provide optimal predictive value for detecting congestion. In this analysis, three of the strongest clinical surrogate markers of congestion (i.e., JVP, peripheral edema, and orthopnea) were combined to improve accuracy and develop a composite clinical congestion score (CCS) using the scheme shown below.

[0262] [Table 6]

[0263] The scores for each of these three parameters were then added together to obtain a composite congestion score ranging from 0 to 8. A similar scheme was previously used by Ambrosy et al. (Ambrosy et al. 2013, European Heart Journal 34(11):835-843).

[0264] The following algorithm is then used to rank the severity of congestion: CCS 0, no clinical congestion CCS 1-3, mild clinical congestion CCS 4-5, moderate clinical congestion CCS ≥ 6, severe clinical congestion.

[0265] Diuretic response was defined as weight loss by day 4 per 40 mg diuretic dose.

[0266] Hemoconcentration was coded as 0 (if hemoglobin levels decreased or remained unchanged compared with baseline by day 4) or 1 (if hemoglobin levels increased compared with baseline by day 4).

[0267] Significant residual congestion was defined as a CCS of ≥2 based on JVP, orthopnea, and edema assessment by day 7.

[0268] statistical analysis Baseline clinical variables and biomarkers (including bioADMs) were summarized by severity of clinical congestion at baseline (scheme above). Multivariable logistic regression models were utilized to identify baseline factors independently associated with severity of clinical congestion at baseline (CCS variable was recoded as a two-level binary output; 0 = mild / moderate (CCS < 6) and 1 = severe (CCS ≥ 6)).

[0269] Linear regression analysis was used to assess the relationship between baseline bioADM levels and diuretic response (continuous variables). Binary logistic regression analysis was performed for the outcomes of hemoconcentration and significant residual congestion. Multivariable models were used to assess the adjusted relationship between bioADM levels and these outcomes.

[0270] result Table 4 shows that the concentration of bio-ADM increases with the severity of congestion.

[0271] [Table 7-1] [Table 7-2]

[0272] Furthermore, multivariable logistic regression revealed that bioADM was an independent predictor of congestion severity, the strongest predictor compared with all other available variables (Table 5).

[0273] [Table 8]

[0274] The area under the curve (AUC) for the overall model was 0.69, with individual AUCs of bioADM=0.66, BMI=0.61, serum albumin=0.58, and previous HF hospitalization=0.54.

[0275] Only a few baseline clinical variables or biomarkers are associated with the severity of clinical congestion at baseline, with Bio-ADM appearing to be by far the strongest.

[0276] We further analyzed whether Bio-ADM predicted decongestion. Table 6 shows that Bio-ADM was an independent predictor of significant residual congestion by day 7.

[0277] [Table 9]

[0278] Baseline levels of BioADM independently predict significant residual congestion by day 7.

[0279] Bio-ADM concentrations were greater with increasing amounts of diuretic therapy, as would be expected given that it is a marker of congestion (Tables 7 and 8).

[0280] [Table 10]

[0281] [Table 11]

[0282] MR-proADM was also determined in the same sample set. Baseline variables by tertiles of MR-proADM are shown in Table 9: Similar to bioADM, increasing levels of MR-proADM were associated with increasing degrees of edema.

[0283] [Table 12-1] [Table 12-2] [Table 12-3]

[0284] Example 7 BIOSTAT study Additional analyses were performed in the BIOSTAT (Biological Study for Personalized Treatment in Chronic Heart Failure) trial, which has been reported in detail (WWW.BIOSTAT-CHF.EU; Voors et al. 2016, Eur J Heart Fail. June; 18(6):716–726). The Biostat-CHF trial included 2,516 patients from 11 European countries with signs and / or symptoms of worsening heart failure who were considered to be receiving suboptimal medical care. Another 1,738 patients from Scotland were included in the validation cohort. Overall, both patient cohorts were well matched. The majority of patients were hospitalized with acute heart failure, and the remainder presented with signs and / or symptoms of worsening heart failure in outpatient clinics. Nearly half of the patients were New York Heart Association class III, and 7% of the index cohort and 34% of the validation cohort had heart failure with preserved ejection fraction. According to the study design, all patients were receiving diuretics, but due to the inclusion criteria for both cohorts, patients were not receiving optimal evidence-based care. During the follow-up phase, patients were encouraged to increase their diuretic dose to the guideline-recommended dose.

[0285] Study population Patients met the following inclusion criteria: Age 18 or older with new-onset or worsening heart failure symptoms, There is objective and documented evidence of cardiac dysfunction, which is either: Left ventricular ejection fraction is 40% or less, Plasma concentrations of BNP and / or N-terminal pro-brain natriuretic peptide (NT-proBNP) greater than 400 pg / ml or greater than 2000 pg / ml, respectively; At the time of inclusion, patients were receiving oral or intravenous furosemide ≤40 mg / day or equivalent treatment. · not previously receiving evidence-based therapy [angiotensin-converting enzyme (ACE) inhibitors / angiotensin receptor antagonists (ARBs) and β-blockers] or receiving 50% or less of the target dose of these medications at the time of inclusion; Your doctor has scheduled you to be started on or increase the dose of an ACE inhibitor / ARB and / or a beta-blocker.

[0286] Patients were recruited as inpatients or from outpatient clinics: approximately two-thirds were inpatients and one-third were outpatients.

[0287] A subset of patients (n=1806) was analyzed in this study, including all patient types included in the study (for whom baseline biomarker measurements were available). Similar to the PROTECT study (Example 6), increasing levels of bio-ADM in the BIOSTAT study correlated with increasing edema severity (Table 10).

[0288] [Table 13]

[0289] MR-proADM was also determined in the same sample set. Baseline variables by tertiles of MR-proADM are shown in Table 11: Similar to bioADM, increasing levels of MR-proADM were associated with increasing degrees of edema.

[0290] [Table 14-1] [Table 14-2] [Table 14-3]

[0291] Example 8 Administration of NT-H to healthy humans The study was conducted as a randomized, double-blind, placebo-controlled trial in healthy male subjects, with three sequential groups (each consisting of eight healthy male subjects) receiving increasing doses of NT-H antibody as an intravenous (iv) infusion (0.5 mg / kg in the first group, 2 mg / kg in the second group, and 8 mg / kg in the third group) (n=6 active agent, n=2 placebo in each group).

[0292] The main inclusion criteria were written informed consent, age between 18 and 35 years, agreement to reliably use contraception, and a BMI between 18 and 30 kg / m 2 It was that.

[0293] Subjects received a single dose of NT-H antibody (0.5 mg / kg; 2 mg / kg; 8 mg / kg) or placebo intravenously by slow infusion over 1 hour in the laboratory.

[0294] There were no differences in baseline ADM levels among the four groups: median ADM levels were 7.1 pg / ml in the placebo group, 6.8 pg / ml in the first treatment group (0.5 mg / kg), 5.5 pg / ml in the second treatment group (2 mg / kg), and 7.1 pg / ml in the third treatment group (8 mg / kg).

[0295] The results show that ADM levels increased rapidly within the first 1.5 hours after administration of NT-H antibody to healthy individuals, then reached a plateau and slowly declined (Figure 9).

[0296] Example 9 Administration of NT-H in a porcine two-hit model of sepsis Heart failure was induced using an established two-hit septic shock model in pigs (Simon TP et al., Crit Care. 2012 Jan 25; 16(1):R16) to examine the effects of antibody NT-H on hemodynamic and clinical parameters, including heart failure.

[0297] Sixteen German Landrace sows were anesthetized and ventilated (n = 16; mean ± standard deviation (SD) 33 ± 1.5 kg body weight (BW)) following standard procedures for laboratory animal care. The study was approved by the Institutional Local Committee for Animal Care and Use (Landesamt fur Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany, 84-02.04.2015.A037).

[0298] After premedicating the pig with azaperone (1–2 mg / kg body weight) and ketamine (10 mg / kg body weight), general anesthesia was induced by intravenous injection of propofol (1–2 mg / kg body weight). The pig was orally intubated and positioned supine. General anesthesia was maintained with infusion of propofol and fentanyl. Pressure-controlled ventilation was selected, with an inspired oxygen fraction of 0.5, an inspiration / expiration ratio of 1:1.5, and PEEP of 5 cm H2O, and the pig was ventilated with a tidal volume of 8–10 ml / kg body weight. The inspiration rate was set to maintain a PaCO2 of 3.5–4.5 kPa. Core body temperature was maintained above 37.5°C using a heating blanket. Two central venous catheters were inserted into the external jugular vein, and an arterial PICCO catheter was inserted into the femoral artery via percutaneous puncture.

[0299] At the end of the study, pigs still in a deep coma were euthanized with a lethal dose of Narcoren® (Merial, Hallbergmoos, Germany) in the presence of a veterinarian.

[0300] In this model, 7–9 × 10 per kg of body weight 11 Septic shock was induced using thrombi containing colony-forming units (CFU) of E. coli.

[0301] Hemodynamic measurements: All venous pressure measurements were performed at the mid-thoracic level, with measurements taken at the end of expiration. Heart rate, mean arterial pressure (MAP), central venous pressure (CVP), and stroke volume variation (SVV) were continuously recorded. Cardiac output (CO) was measured using transpulmonary thermodilution (PICCO, Pulsion medical systems, Feldkirchen, Germany). Extravascular lung water (EVLW), intrathoracic blood volume (ITBV), and end-diastolic volume (GEDV) were calculated using standard formulas.

[0302] Experimental Protocol: During catheterization, pigs were administered a balanced crystalloid solution at 10 ml / h per kg body weight. Hemorrhagic shock was induced in the pigs by bleeding through the femoral vein catheter. The pigs were allowed to bleed until half of their baseline mean arterial pressure was reached. After maintaining hemorrhagic shock for 45 minutes, resuscitation fluid was administered with balanced crystalloid solution to restore baseline mean arterial pressure. Two hours after hemorrhagic shock, the blood collected during hemorrhagic shock was again transfused. Six hours after hemorrhagic shock, a second shock was administered, inducing sepsis by placing a clot containing Escherichia coli into the peritoneal cavity. Pigs were randomly assigned to receive either an adrenomedullin antibody or a vehicle solution. Treatment with the antibody or vehicle solution began immediately after sepsis induction. 2 mg / kg body weight of the antibody / vehicle solution was infused over 30 minutes. Four hours after sepsis induction, treatment of septic shock began with balanced crystalloid and norepinephrine as needed. As recommended by the Surviving Sepsis Campaign, fluid replacement and vasodilators were titrated to maintain central venous pressure at 8–12 mmHg, mean arterial pressure at >65 mmHg, and central venous oxygen saturation at 70%. Sepsis treatment was continued for another 8 hours. For several measurements, EDTA-plasma and serum samples were obtained before hemorrhagic shock, before sepsis induction, and at 1, 2, 3, 4, 6, 8, 10, and 12 hours after sepsis induction and stored at -80°C until measurement. Hemorrhagic shock and septic shock were not administered to the sham group of pigs; however, they otherwise received the same treatment (including intravascular catheterization, midline laparotomy, and random application of antibody / vehicle solutions), and blood samples were collected in the same manner as for the septic pigs. A timeline of treatment and blood sample collection is shown in Figure 10.

[0303] As expected, ADM plasma concentrations began to increase in both groups after sepsis induction. This increase was accelerated by administering NT-H antibody concomitantly with sepsis induction. In the vehicle group, ADM concentrations increased to approximately 30 pg / ml 1 hour after sepsis induction, while in the treatment group, they reached 265 pg / ml at the same time point. Treatment reached a plateau of approximately 1,100 pg / ml 3 hours after NT-H antibody administration, while the vehicle group showed a steady increase in ADM concentrations, reaching 700 pg / ml at the end of the experiment (Figure 11). Application of NT-H antibody also induced an increase in plasma ADM in sham control animals, similar to the results observed in healthy humans (Example 8).

[0304] Heart rate (HR) increased to compensate for volume loss during hemorrhagic shock, but returned to baseline after rehydration with crystalloid solution and blood. After sepsis induction, HR remained constant at 60–65 beats per minute for the first hour and then began to increase. The rate of increase was greater in the vehicle group than in the antibody-treated group, ultimately reaching 125 beats per minute (vehicle group) and 98 beats per minute (treated group) (Figure 12).

[0305] Stroke volume (CO) describes the volume of blood pumped by the heart, particularly the left or right ventricle, per unit time. CO values ​​can be expressed using many physical units (e.g., 1 / min). In this study, stroke volume was significantly lower in the NT-H antibody-treated group than in the vehicle group (Figure 13).

[0306] The amount of fluid replacement (Figures 14 and 15) and noradrenaline (Figure 16) required to maintain a constant mean arterial pressure was significantly less in the NT-H antibody-treated group compared with the vehicle group. Importantly, only one-third of the NT-H-treated animals went into shock (e.g., required vasodilators to maintain target MAP), whereas all vehicle controls required vasodilators (Figure 17). Urine output did not differ between the NT-H antibody-treated and vehicle groups. The reduced fluid requirements with NT-H antibody application, especially when combined with the reduced noradrenaline requirements, suggest that less fluid escaped from the circulation, and therefore less congestion occurred in the NT-H antibody-treated group.

[0307] Vascular resistance refers to the resistance to blood flow provided by all systemic vascular systems except the pulmonary vasculature. Systemic vascular resistance (SVR) is used in calculations of blood pressure, blood flow, and cardiac function. SVR (units: dyne-seconds / cm) 5 ) was calculated from other measurements using the formula: SVR = 80 × (MAP - CVP) / CO. A greater SVR strengthens the arterial vasculature to maintain blood pressure. As shown in Figure 18, systemic vascular resistance in this animal model was greater in the NT-H antibody-treated group than in the vehicle group.

[0308] Example 10 Administration of NT-H in LPS-induced endotoxemia in rats The aim of this study was to investigate the effect of HAM8101 on vascular permeability in the liver and kidney after LPS-induced endotoxemia in rats.

[0309] Male Wistar rats (n=8 / group) were intravenously administered 0.02 mg, 0.1 mg, 0.5 mg, or 2.5 mg / kg body weight of HAM8101 or PBS (see Table 12). Five minutes later, 2.5 mg or 5 mg / kg body weight of LPS was administered to induce endotoxemia (the 2.5 mg LPS / kg group was used only to determine the appropriate LPS dose and was not further evaluated). Blood samples were collected 3, 6, and 24 hours after LPS and HAM8101 administration.

[0310] Twenty-four hours after LPS application, Evans Blue was slowly administered via tail vein injection. 15 minutes later, rats were sacrificed and perfused with heparinized saline (50 IU / ml). The kidneys and livers were removed, weighed, dissected, and further manipulated to determine the concentration of Evans Blue in the tissues (absorbance at 620 nm), which indicates vascular permeability. Additionally, urine was collected from the bladder before perfusion.

[0311] [Table 15]

[0312] The time course of rADM plasma concentrations in the placebo (NaCl + LPS) group increased during the first 6 hours after LPS application, reaching a peak plasma concentration of 140 pg / ml, and then decreased to 64 pg / ml after 24 hours.

[0313] Total rADM levels further increased in a dose-dependent manner with HAM8101 treatment, reaching peak rADM concentrations of 550 pg / ml at 2.5 pg / ml and 270 pg / ml at 0.5 pg / ml 3 h after LPS and HAM8101 application. The lower doses of 0.1 mg / kg and 0.2 mg / kg did not show any further increases in rADM compared with the LPS-induced increase (Figure 19A). When total rADM levels were normalized to the levels achieved in the placebo group at each time point, the maximum peak levels increased 5.3-fold and 2.7-fold in the 2.5 mg / kg and 0.5 mg / kg groups, respectively. This increase in total plasma ADM concentrations compared to baseline was greater than in healthy rats (3-fold at 2.5 mg / kg) and septic mice (2-fold). The lower doses of 0.1 mg / kg and 0.2 mg / kg did not increase total rADM levels above those achieved with LPS alone (FIG. 19B).

[0314] Rats were treated with 5 mg of NaCl (healthy, green) or LPS (placebo, red) per kg of body weight. They were further treated by a single intravenous bolus injection of different doses of NaCl (placebo, red) or HAM8101 (blue) 5 min before LPS application. rADM levels were determined 3, 6, and 24 h after LPS application and expressed as (A) mean ± SEM or (B) x-fold increase compared to the placebo group. Vascular permeability significantly increased after LPS challenge. Treatment with HAM8101 at doses of 0.1 to 2.5 mg / kg resulted in a clear and significant reduction in vascular permeability in the kidney (see Figure 20A). The 0.1 mg / kg dose was significantly more effective at restoring permeability to a healthy normal state than the 0.5 mg / kg dose, and even the 2.5 mg / kg dose (p<0.05). The reliability of this effect requires further testing. In contrast, 0.02 mg / kg had little beneficial effect on vascular permeability. Liver vascular permeability data showed a similar trend, but were not statistically significant (Figure 20B).

[0315] Rats were treated with 5 mg of NaCl (control, green) or LPS (placebo, red) per kg of body weight. They were further treated with a single intravenous bolus injection of different doses of NaCl (placebo, red) or HAM8101 (blue) 5 min before LPS application. Vascular permeability was measured by determining the concentration of Evans Blue in (A) kidney tissue or (B) liver tissue 24 h after LPS challenge and treatment. Values ​​are given as mean ± SEM. A p value of less than 0.05 was considered statistically significant.

[0316] In conclusion, in this rat endotoxemia model, treatment with HAM8101, starting at a dose of 0.1 mg / kg, significantly restored vascular integrity in the kidney. A comparable effect was observed in the liver, but it was not statistically significant. It is unclear whether this was a real effect or due to the detection method used. A significant increase in total plasma rADM levels was observed with HAM8101 at doses above 0.1 mg / kg.

[0317] array SEQ ID NO: 1 GYTFSRYW

[0318] SEQ ID NO: 2 ILPGSGST

[0319] SEQ ID NO: 3 TEGYEYDGFDY

[0320] SEQ ID NO:4 QSIVYSNGNTY

[0321] Sequence "RVS" (not part of the sequence listing) RVS

[0322] SEQ ID NO:5 FQGSHIPYT

[0323] Sequence number 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGT TLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH

[0324] SEQ ID NO: 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH

[0325] SEQ ID NO: 8 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH

[0326] SEQ ID NO: 9 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH

[0327] SEQ ID NO: 10 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH

[0328] SEQ ID NO: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0329] SEQ ID NO: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0330] SEQ ID NO: 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0331] SEQ ID NO: 14 (human ADM 1-21) YRQSMNNFQGLRSFGCRFGTC

[0332] SEQ ID NO: 15 (human ADM 21-32) CTVQKLAHQIYQ

[0333] SEQ ID NO: 16 (human ADM C-42-52) CAPRSKISPQGY-CONH2

[0334] SEQ ID NO: 17 (mouse ADM 1-19) YRQSMNQGSRSNGCRFGTC

[0335] SEQ ID NO: 18 (mouse ADM 19-31) CTFQKLAHQIYQ

[0336] SEQ ID NO: 19 (Mouse ADM C-40-50) CAPRNKISPQGY-CONH2

[0337] SEQ ID NO: 20 (mature human adrenomedullin (mature ADM); amidated ADM; bioADM): amino acids 1-52, or amino acids 95-146 of proADM) YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY-CONH2

[0338] SEQ ID NO: 21 (amino acids 1-50 of mouse ADM) YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQLTDKDKDGMAPRNKISPQGY-CONH2

[0339] SEQ ID NO: 22 (amino acids 1-21 of human ADM) YRQSMNNFQGLRSFGCRFGTC

[0340] SEQ ID NO: 23 (amino acids 1-42 of human ADM) YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA

[0341] SEQ ID NO: 24 (amino acids 43-52 of human ADM) PRSKISPQGY-NH2

[0342] SEQ ID NO: 25 (amino acids 1-14 of human ADM) YRQSMNNFQGLRSF

[0343] SEQ ID NO: 26 (amino acids 1-10 of human ADM) YRQSMNNFQG

[0344] SEQ ID NO: 27 (amino acids 1-6 of human ADM) YRQSMN

[0345] SEQ ID NO: 28 (amino acids 1-32 of human ADM) YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQ

[0346] SEQ ID NO: 29 (amino acids 1-40 of mouse ADM) YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQLTDKDKDGMA

[0347] SEQ ID NO: 30 (amino acids 1-31 of mouse ADM) YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQL

[0348] SEQ ID NO: 31 (proADM: 164 amino acids (amino acids 22 to 185 of preproADM)) ARLDVASEF RKKWNKWALS RGKRELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

[0349] SEQ ID NO: 32 (proadrenomedullin N-20 terminal peptide, PAMP: amino acids 22 to 41 of preproADM) ARLDVASEF RKKWNKWALS R

[0350] SEQ ID NO: 33 (central region pro-adrenomedullin, MR-proADM: amino acids 45-92 of pre-proADM) ELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RV

[0351] SEQ ID NO: 34 (Adrenomedullin 1-52-Gly (ADM 1-52-Gly): amino acids 95 to 147 of prepro-ADM) YRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYG

[0352] SEQ ID NO: 35 (C-terminal pro-adrenomedullin, CT-proADM: amino acids 148-185 of pre-proADM) RRR RRSLPEAGP RTLVSSKPQA HGAPAPPSGS APHFL

Claims

1. 1. A pharmaceutical composition comprising an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment for the treatment and therapy of blood congestion in a patient in need thereof, wherein the anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment is an N-terminal portion of adrenomedullin (amino acids 1-21): YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 22) wherein said patient is diuretic resistant or unresponsive to diuretic therapy.

2. 10. The pharmaceutical composition of claim 1, wherein the patient has a disease or condition selected from the group consisting of congestive hypertension, swelling or fluid retention (edema), heart failure, kidney disease, and liver disease.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the patient has a disease or condition selected from the group consisting of congestive hypertension, swelling or fluid retention (edema), and heart failure.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the antibody or antibody fragment is monospecific.

5. The antibody or antibody fragment has a specific activity against ADM of at least 10 -7 The pharmaceutical composition of any one of claims 1 to 4, which exhibits binding affinity for M.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the antibody or antibody fragment recognizes and binds to the N-terminus (amino acid 1) of adrenomedullin.

7. The antibody or antibody fragment comprises the sequence of amino acids 43 to 52 of ADM: PRSKISPQGY-NH 2 (SEQ ID NO: 24) The pharmaceutical composition according to any one of claims 1 to 6, characterized in that it does not bind to a C-terminal portion having the formula:

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the antibody or antibody fragment inhibits the biological activity of ADM by 80% or less.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the patient is an ICU patient.

10. The antibody or antibody fragment is a human monoclonal antibody or human monoclonal antibody fragment that binds to ADM, or an antibody fragment thereof, wherein the heavy chain has the sequence: SEQ ID NO: 1 GYTFSRYW, SEQ ID NO: 2 ILPGSGST, SEQ ID NO: 3 TEGYEYDGFDY and the light chain comprises the sequence: SEQ ID NO:4 QSIVYSNGNTY, Sequence: RVS, SEQ ID NO:5 FQGSHIPYT The pharmaceutical composition according to any one of claims 1 to 9, comprising:

11. the antibody or antibody fragment Sequence number 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTT LTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH, SEQ ID NO: 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH, SEQ ID NO: 8 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH, SEQ ID NO: 9 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH, SEQ ID NO: 10 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH, SEQ ID NO: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, SEQ ID NO: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, SEQ ID NO: 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 11. The pharmaceutical composition of claim 10, comprising a sequence selected from the group consisting of:

12. 12. The pharmaceutical composition according to any one of claims 1 to 11, wherein in a sample of body fluid taken from the patient, the level of pro-ADM and / or the level of fragments thereof having at least 5 amino acids is higher than a predetermined threshold.

13. The pharmaceutical composition according to any one of claims 1 to 12, which is a solution.

14. The pharmaceutical composition of claim 13, which is in a lyophilized form.

15. 15. The pharmaceutical composition of claim 13 or 14, which is administered intramuscularly.

16. The pharmaceutical composition of claim 13 or 14, which is administered intravascularly.

17. 17. The pharmaceutical composition of claim 16, which is administered via infusion.

18. The pharmaceutical composition according to any one of claims 13 to 17, which is administered systemically.

Images