DPP3 for nt-ADM therapy guidance, monitoring, and stratification in patients suffering from shock

Abstract

The present application is directed to methods of therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from and / or in a shock state.SOLUTION: In particular, the method comprises providing a sample from said patient and determining the level of dipeptidyl peptidase 3 (DPP3) in said sample, wherein the level of DPP3 in said sample is indicative for the need of treatment with an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold. In a preferred embodiment of the invention, the method further comprises measuring the level of ADM-NH2 in a sample from said patient.SELECTED DRAWING: FIG. 1a

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock. In particular, the method comprises providing a sample from the patient and measuring the level of dipeptidyl peptidase 3 (DPP3) in the sample, wherein the DPP3 level in the sample indicates whether treatment with an anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold is necessary. In a preferred embodiment of the present invention, the method further comprises measuring the level of ADM-NH2 in the sample from the patient. Furthermore, the present invention also relates to a kit for carrying out the method of the present invention. [Background technology]

[0002] Dipeptidyl peptidase 3 (also known as dipeptidyl aminopeptidase III, dipeptidyl arylamidase III, dipeptidyl peptidase III, enkephalinase B, or red blood cell angiotensinase; abbreviated DPP3 or DPPIII) is a metallopeptidase that removes dipeptides from physiologically active peptides (e.g., enkephalins and angiotensin). DPP3 was first identified and its activity measured in purified bovine anterior pituitary extracts by Ellis and Nuenke in 1967. This enzyme is listed as EC 3.4.14.4, has a molecular weight of approximately 83 kDa, and is highly conserved in eukaryotes and prokaryotes (Prajapati & Chauhan 2011). The amino acid sequence of the human variant is shown in SEQ ID NO: 1. Dipeptidyl peptidase III is a ubiquitously expressed, primarily cytosolic peptidase. Although it lacks a signal sequence, several studies have reported membrane activity (Lee & Snyder 1982).

[0003] DPP3 is a zinc-dependent exopeptidase belonging to the peptidase family M49. DPP3 has broad substrate specificity for oligopeptides of 3, 4, or 10 amino acids of various compositions and can also cleave after proline. DPP3 is known to hydrolyze dipeptides from the N-terminus of its substrates, including angiotensin II, III, and IV; Leu-enkephalin and Met-enkephalin; and endomorphin 1 and 2. The metallopeptidase DPP3 has optimal activity at pH 8.0–9.0 and is cleaved by divalent metal ions (Co). 2+ and Mg 2+ It can be activated by adding

[0004] Structural analysis of DPP3 revealed the catalytic motifs HELLGH (hDPP3 450–455) and EECRAE (hDPP3 507–512), as well as the amino acids Glu316, Tyr318, Asp366, Asn391, Asn394, His568, Arg572, Arg577, Lys666, and Arg669 that are important for substrate binding and hydrolysis (Prajapati & Chauhan 2011; Kumar et al. 2016; numbers refer to the human DPP3 sequence; see SEQ ID NO: 1). Considering all known amino acids or sequence regions involved in substrate binding and hydrolysis, the active site of human DPP3 can be limited to the region between amino acids 316 and 669.

[0005] The most important substrate of DPP3 is angiotensin II (Ang II), a major effector of the renin-angiotensin system (RAS). The RAS is activated in cardiovascular disease (Dostal et al. 1997. J Mol Cell Cardiol 29:2893-902; Roks et al. 1997. Heart Vessels. Suppl 12:119-24) and sepsis and septic shock (Correa et al. 2015. Crit Care 19:98). Ang II, in particular, has been shown to alter many cardiovascular functions, including blood pressure control and cardiac remodeling.

[0006] Recently, two assays have been devised, characterized, and validated to specifically detect DPP3 in human biological fluids (e.g., blood, plasma, and serum): a fluorescent immunoassay (LIA) to measure DPP3 protein concentration and an enzyme capture activity assay (ECA) to detect specific DPP3 activity (Rehfeld et al. 2019. JALM 3(6): 943-953). After removing all interfering substances through a washing step, DPP3 activity is actually detected. Both methods are highly specific and therefore allow for reproducible detection of DPP3 in blood samples.

[0007] Blood DPP3 levels have been shown to be elevated in patients with cardiogenic shock and were associated with a higher risk of early mortality and severe organ dysfunction (Deaniau et al. 2020. Eur J Heart Fail. 22(2):290-299). Furthermore, DPP3 measured at enrollment distinguished between cardiogenic shock patients who developed refractory versus non-refractory shock, and DPP3 concentrations ≥ 59.1 ng / mL were associated with a higher risk of mortality (Takagi et al. 2020. Eur J Heart Fail. 22(2):279-286).

[0008] The peptide adrenomedullin (ADM) 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 precursor peptide containing 185 amino acids and the complete amino acid sequence of this precursor peptide were also reported. This precursor peptide contains a 21-amino acid signal sequence, particularly at the 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 peptide adrenomedullin (ADM) 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-pro-ADM have been characterized in detail. These include the physiologically active peptide ADM and the 20 amino acids (22–41) in pre-pro-ADM that follow the 21 amino acids of the signal peptide. 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, we refer 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 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 Commun 244(2): 551-555). Specific binding proteins for ADM also exist (Pio et al. 2001. The Journal of Biological Chemistry 276(15): 12292-12300), which likely regulate the effects of ADM as well. The most important physiological effect of ADM and PAMP studied to date is its effect on blood pressure.

[0009] Therefore, because ADM is an effective vasodilator, its hypotensive 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 a similar hypotensive effect, although it appears to have a different mechanism of action than ADM (see the above-mentioned reviews, 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, and EP-A2 0 622 458). Furthermore, the 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, kidney 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. Journal of 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 and Wang et al. 2001. Peptides 22: 1835-1840).

[0010] 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).

[0011] 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.

[0012] During the early stage 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 the above effects during the early stage of sepsis (Wang et al. 2001. Peptides 22: 1835-1840).

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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%.

[0017] WO 2013 / 072513 describes non-neutralizing anti-ADM antibodies for use in treating acute diseases or conditions in patients to stabilize circulation.

[0018] WO 2013 / 072514 describes non-neutralizing anti-ADM antibodies for regulating fluid balance in patients suffering from chronic or acute diseases or conditions.

[0019] WO2017 / 182561 describes a method for measuring total or active DPP3 in a patient sample for the diagnosis of diseases associated with necrotic processes. It further describes a method for treating necrosis-associated diseases with antibodies against DPP3. Summary of the Invention

[0020] It is a surprising finding of the present invention that in patients suffering from shock and in a shock state, DPP3 levels in body fluid samples can be used for therapy guidance and / or therapy monitoring and / or therapy stratification using anti-ADM antibodies and / or anti-ADM antibody fragments and / or anti-ADM non-Ig scaffolds. Moreover, the results of the present invention clearly show that patients suffering from shock will benefit most from therapy with anti-ADM antibodies when DPP3 levels in body fluid samples are below a threshold.

[0021] The subject of the present invention is a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, comprising: measuring the level of dipeptidyl peptidase 3 (DPP3) in a body fluid sample from said patient; comparing the measured DPP3 level to a predetermined threshold; and the level of DPP3 in the sample indicates whether treatment with an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold is necessary; and Here, in this method, the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold binds to the N-terminal portion of ADM (amino acids 1 to 21): YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14). [Brief explanation of the drawings]

[0022] [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 adjusted to 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 for 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] 1 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] ADM concentrations in healthy human subjects up to 60 days after application of different doses of NT-M. [Figure 7] Kaplan-Meier survival plots in relation to low (<40.5 ng / mL) and high (≥40.5 ng / mL) DPP3 concentrations. (A) 7-day survival rate of patients with sepsis in relation to DPP3 plasma concentration. (B) 7-day survival rate of patients with cardiogenic shock in relation to DPP3 plasma concentration. (C) 7-day survival rate of patients with septic shock in relation to DPP3 plasma concentration. [Figure 8] Kaplan-Meier survival plot for all patients (14-day mortality in patients treated with placebo (Plac) or the N-terminal ADM antibody adrecizumab (Adz)). [Figure 9] Kaplan-Meier survival plot for patients with DPP3 <50 ng / mL (14-day mortality in patients treated with placebo (Plac) or N-terminal ADM antibody adrecizumab (Adz)). [Figure 10] Kaplan-Meier survival plot for patients with DPP3 >50 ng / mL (14-day mortality in patients treated with placebo (Plac) or N-terminal ADM antibody adrecizumab (Adz)). DETAILED DESCRIPTION OF THE INVENTION

[0023] The subject of the present invention is a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, comprising: measuring the level of dipeptidyl peptidase 3 (DPP3) in a body fluid sample from said patient; comparing the measured DPP3 level to a predetermined threshold; and administering to the patient an anti-adrenomedullin (ADM) antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold, wherein if the measured DPP3 level is below a predetermined threshold, the patient is treated with the anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold; and Here, in this method, the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold binds to the N-terminal portion of ADM (amino acids 1 to 21): YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

[0024] The subject of the present application is a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, comprising: measuring the level of dipeptidyl peptidase 3 (DPP3) in a body fluid sample from said patient; comparing the measured DPP3 level to a predetermined threshold; and administering to said patient an anti-adrenomedullin (ADM) antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold; Including, wherein the patient is treated if the measured DPP3 level is below a predetermined threshold; and Here, in this method, the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold binds to the N-terminal portion of ADM (amino acids 1 to 21): YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

[0025] One embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from and / or in a state of shock, wherein said shock is selected from the group comprising hypovolemic shock, cardiogenic shock, vaso-occlusive shock and distributive shock, in particular cardiogenic shock or septic shock.

[0026] A preferred embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, comprising: In the case of cardiogenic shock, the patient may be suffering from an acute coronary syndrome (e.g., acute myocardial infarction), or the patient may be suffering from heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or In the case of hypovolemic shock, the patient may have a bleeding disorder, including gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor invasion into major blood vessels) and spontaneous bleeding during anticoagulation, or a non-hemorrhagic disorder, including vomiting, diarrhea, renal losses, cutaneous / insensible losses (e.g., burns, heat stroke), or third-space losses during pancreatitis, liver cirrhosis, intestinal obstruction, and trauma; or In the case of vaso-occlusive shock, the patient may be suffering from cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis; or In the case of distributive shock, the patient may be suffering from septic shock, neurogenic shock, anaphylactic shock or shock due to adrenal crisis.

[0027] Another embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in a patient suffering from shock and / or in a state of shock, wherein the predetermined threshold value of DPP3 in a body fluid sample of the subject is 20-120 ng / mL, more preferably the threshold value is 30-80 ng / mL, even more preferably the threshold value is 40-60 ng / mL, and most preferably the threshold value is 50 ng / mL.

[0028] Another specific embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein either the level of DPP3 protein and / or the level of active DPP3 is measured and compared with a predefined threshold.

[0029] Another preferred embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein the level of DPP3 is measured by contacting said body fluid sample with a capture binder that specifically binds to DPP3.

[0030] One embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein said measuring comprises the use of a capture binder that specifically binds to full-length DPP3, wherein said capture binder may be selected from the group comprising an antibody, an antibody fragment, or a non-IgG scaffold.

[0031] A further embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state, wherein the amount of DPP3 protein and / or DPP3 activity is measured in a body fluid sample of said subject, and wherein said measuring comprises the use of a capture binder that specifically binds to full-length DPP3, wherein said capture binder is an antibody.

[0032] One embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein the amount of DPP3 protein and / or DPP3 activity is measured in a body fluid sample of said subject, and wherein said measuring comprises the use of a capture binder that specifically binds to full-length DPP3, wherein said capture binder is immobilized on a surface.

[0033] Another embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state, wherein the amount of DPP3 protein and / or DPP3 activity is measured in a body fluid sample of said subject, and wherein said separating step is a washing step that removes sample components that are not bound to said capture binder from the captured DPP3.

[0034] Another embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in a patient suffering from shock and / or in a state of shock, wherein the method for measuring DPP3 activity in a body fluid sample of said subject comprises the following steps: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3; quantifying DPP3 activity by measuring and quantifying the conversion of a DPP3 substrate; Includes:

[0035] Another specific embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein DPP3 activity is measured in a body fluid sample of said subject, and wherein DPP3 substrate conversion is detected by a method selected from the group comprising: fluorescence of a fluorogenic substrate (e.g., Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate linked to aminoluciferin (Promega Protease-Glo™ Assay), mass spectrometry, HPLC / FPLC (reverse-phase chromatography, size-exclusion chromatography), thin-layer chromatography, capillary zone electrophoresis, gel electrophoresis after activity staining (fixed, active DPP3) or Western blotting (cleavage products).

[0036] Another preferred embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein DPP3 activity is measured in a body fluid sample of said subject, and wherein said substrate may be selected from the group comprising: angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, chromophore or aminoluciferin, wherein said dipeptide is Arg-Arg.

[0037] Another embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein DPP3 activity is measured in a body fluid sample of said subject, and wherein said substrate may be selected from the group comprising: a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, wherein said dipeptide is Arg-Arg.

[0038] One embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein the patient is further characterized by having an ADM-NH2 level above a threshold.

[0039] A specific embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein the threshold value for ADM-NH2 in a body fluid sample of said patient is 40-100 pg / mL, more preferably the threshold value is 50-90 pg / mL, even more preferably the threshold value is 60-80 pg / mL, and most preferably the threshold value is 70 pg / mL.

[0040] A preferred embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein the level of ADM-NH2 is measured by contacting said body fluid sample with a capture binder that specifically binds ADM-NH2.

[0041] Another embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in a patient suffering from shock and / or in a state of shock, wherein the patient's body fluid sample is selected from the group of blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.

[0042] Another specific embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein the levels of DPP3 and ADM-NH2 are measured in combination.

[0043] Another preferred embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state, wherein the levels of DPP3 and ADM-NH2 are measured simultaneously.

[0044] Another embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein the levels of DPP3 and ADM-NH2 are measured using a point-of-care device.

[0045] Another preferred embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein said point-of-care device is a microfluidic device.

[0046] As used herein, a microfluidic device has multiple chambers connected in parallel and arranged at different locations into which a measured amount of fluid can be efficiently dispensed without the use of a separate driving source, wherein the device includes a platform having a center of rotation and includes at least one microfluidic structure. Microfluidic devices are used to perform biological or chemical reactions by manipulating small amounts of fluid.

[0047] Another embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein said anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to the N-terminal end (first amino acid) of ADM.

[0048] A further embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein the antibody, antibody fragment or non-Ig scaffold does not bind to the C-terminal portion of ADM having amino acids 43 to 52 of the sequence: PRSKISPQGY-NH2 (SEQ ID NO: 24).

[0049] Another embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein said antibody or fragment is a monoclonal antibody or fragment that binds to ADM or an antibody fragment thereof, wherein the heavy chain has the following sequence: CDR1: SEQ ID NO: 1 GYTFSRYW CDR2: SEQ ID NO: 2 ILPGSGST CDR3: SEQ ID NO: 3 TEGYEYDGFDY and wherein the light chain comprises the following sequence: CDR1: SEQ ID NO: 4 QSIVYSNGNTY CDR2: RVS CDR3: SEQ ID NO: 5 FQGSHIPYT Includes:

[0050] Another embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein said antibody or fragment comprises as a VH region the following: Sequence number 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 8 (AM-VH2E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 9 (AM-VH3-T26E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 10 (AM-VH4-T26E40E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK and as the VL region the following sequence: SEQ ID NO: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC The sequence comprises a sequence selected from the group comprising:

[0051] Another embodiment of the present application relates to a method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, wherein said antibody or fragment comprises as heavy chain the following sequence: SEQ ID NO: 32 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK or a sequence >95% identical thereto, and having as the light chain the following sequence: SEQ ID NO: 33 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC or a sequence that is >95% identical thereto.

[0052] In one embodiment of the present invention, either the level of DPP3 protein and / or the level of active DPP3 is measured and compared to a threshold level.

[0053] In a specific embodiment of the present invention, the threshold value of DPP3 in the body fluid sample of the patient is 20 to 120 ng / mL, more preferably 30 to 80 ng / mL, even more preferably 40 to 60 ng / mL, and most preferably 50 ng / mL.

[0054] In a specific embodiment of the present invention, the threshold level of DPP3 is 5 times the median concentration of a normal healthy population, preferably 4 times the median concentration, more preferably 3 times the median concentration, and most preferably 2 times the median concentration.

[0055] The level of DPP3 as the amount of DPP3 protein and / or DPP3 activity in the body fluid sample of the subject can be measured by various methods, such as immunological assays, activity assays, mass spectroscopy, and the like.

[0056] DPP3 activity can be measured by detecting cleavage products of DPP3-specific substrates. Known peptide hormone substrates include Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH (adrenocorticotropic hormone), and MSH (melanocyte-stimulating hormone; Abramic et al. 2000, Barsun et al. 2007, Dhanda et al. 2008). Cleavage of the mentioned peptide hormones, as well as other untagged oligopeptides (e.g., Ala-Ala-Ala-Ala; Dhanda et al. 2008), can be monitored by detecting the respective cleavage products. Non-limiting examples of detection methods include HPLC analysis (e.g., Lee & Snyder 1982), mass spectrometry (e.g., Abramic et al. 2000), and H-lysine cleavage. 1-NMR analysis (e.g., Vandenberg et al. 1985), capillary zone electrophoresis (CE; e.g., Barsun et al. 2007), thin-layer chromatography (e.g., Dhanda et al. 2008), or reversed-phase chromatography (e.g., Mazocco et al. 2006).

[0057] Detection of fluorescence resulting from hydrolysis of fluorescent substrates by DPP3 is a standard procedure for monitoring DPP3 activity. These substrates are specific dipeptides or tripeptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) coupled to fluorophores. Non-limiting examples of fluorophores include β-naphthylamide (2-naphthylamide, βNA, 2NA), 4-methoxy-β-naphthylamide (4-methoxy-2-naphthylamide), and 7-amido-4-methylcoumarin (AMC, MCA; Abramic et al. 2000, Ohkubo et al. 1999). Cleavage of these fluorescent substrates releases fluorescent β-naphthylamide or 7-amido-4-methylcoumarin, respectively. In a liquid-phase assay (ECA), the substrate and DPP3 are incubated in a 96-well plate format, for example, and fluorescence is measured using a fluorescence detector (Ellis & Nuenke 1967). In addition, samples containing DPP3 can be immobilized on the surface of a gel and resolved by electrophoresis. The gel can be stained with a fluorescent substrate (e.g., Arg-Arg-βNA) and Fast Garnet GBC, and the fluorescent protein bands can be detected using a fluorescence reader (Ohkubo et al. 1999). The same peptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) can be coupled to a chromophore (e.g., p-nitroanilide diacetate). Detection of the color change resulting from hydrolysis of the chromogenic substrate can be used to monitor DPP3 activity.

[0058] Another option for detecting DPP3 activity is the Protease-Glo™ assay (commercially available from Promega). In this embodiment of the method, DPP3-specific dipeptides or tripeptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) are coupled to aminoluciferin. Upon cleavage by DPP3, the aminoluciferin is released and serves as a substrate for reaction with the bound luciferase, emitting detectable luminescence.

[0059] In a preferred embodiment, DPP3 activity is measured by adding the fluorescent substrate Arg-Arg-βNA and monitoring fluorescence in real time.

[0060] In one particular embodiment of the method for determining active DPP3 in a body fluid sample of a subject, the capture binder that reacts with DPP3 is immobilized on the surface of a solid phase.

[0061] A test sample is passed over the immobile binder, whereupon DPP3, if present, binds to the binder and is immobilized and detected. Substrate is then added, allowing the reaction product to be detected, indicating the presence or amount of DPP3 in the test sample. For purposes herein, the term "solid phase" includes any material or vessel in or on which an assay can be performed, including, but not limited to, porous or non-porous materials, test tubes, wells, slides, agarose resin (e.g., Sepharose from GE Healthcare Life Sciences), and magnetic particles (e.g., Dynabeads™ or Pierce™ magnetic beads from Thermo Fisher Scientific).

[0062] In another embodiment of the invention, the level of DPP3 is measured by contacting said body fluid sample with a capture binder that specifically binds DPP3.

[0063] In another preferred embodiment of the present invention, said capture binder for measuring the level of DPP3 may be selected from the group of antibodies, antibody fragments or non-IgG scaffolds.

[0064] In a specific embodiment of the invention, the capture binder is an antibody.

[0065] The amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject can be determined, for example, by one of the following methods.

[0066] 1. Fluorescence immunoassay (LIA) for quantifying DPP3 protein levels (Rehfeld et al., 2019 JALM 3(6): 943-953)

[0067] The LIA is a single-step chemiluminescent sandwich immunoassay using high-binding polystyrene microtiter plates as the solid phase. These plates are coated with the monoclonal anti-DPP3 antibody AK2555 (capture antibody). The tracer, anti-DPP3 antibody AK2553, is conjugated with MA70-acridinium-NHS ester and used at a concentration of 20 ng per well. 20 μl of sample (e.g., serum, heparin-plasma, citrate-plasma, or EDTA-plasma from patient blood) and calibrators are pipetted into the coated white microtiter plate. After adding the tracer antibody AK2553, the microtiter plate is incubated at 600 rpm for 3 hours at room temperature. Unbound tracer is then removed by four washing steps (350 μl per well). Residual chemiluminescence is measured for 1 second per well using a microtiter plate luminometer. DPP3 concentrations are determined using a six-point calibration curve. Calibrators and samples are preferably measured in duplicate.

[0068] 2. Enzyme Capture Activity Assay (ECA) for Quantifying DPP3 Activity (Rehfeld et al., 2019 JALM 3(6): 943-953)

[0069] ECA is a DPP3-specific activity assay that uses black high-binding polystyrene microtiter plates as the solid phase. These plates are coated with the monoclonal anti-DPP3 antibody AK2555 (capture antibody). 20 μl of sample (e.g., serum, heparin-plasma, citrate-plasma, EDTA-plasma, cerebrospinal fluid, and urine from patient blood) and calibrators are pipetted into the coated black microtiter plates. After adding assay buffer (200 μl), the microtiter plates are incubated at 22°C and 600 rpm for 2 hours. DPP3 present in the sample is immobilized by binding to the capture antibody. Unbound components of the sample are removed by four washing steps (350 μl per well). The specific activity of the immobilized DPP3 is measured by adding the fluorescent substrate Arg-Arg-β-naphthylamide (Arg2-βNA) to the reaction buffer and incubating at 37°C for 1 hour. DPP3 specifically cleaves Arg2-βNA to form Arg-Arg dipeptide and fluorescent β-naphthylamine. Fluorescence is measured using a fluorometer with an excitation wavelength of 340 nm and emission detection at 410 nm. DPP3 activity is determined using a six-point calibration curve. Calibrators and samples are preferably measured in duplicate.

[0070] 3. Liquid-phase assay (LAA) for quantifying DPP3 activity (modified from Jones et al., Analytical Biochemistry, 1982)

[0071] The LAA is a liquid-phase assay that measures DPP3 activity using black, non-binding polystyrene microtiter plates. 20 μl of sample (e.g., serum, heparin-plasma, citrate-plasma) and calibrator are pipetted into a coated, black, non-binding microtiter plate. After adding the fluorescent substrate Arg2-βNA to the assay buffer (200 μl), the initial βNA fluorescence (T = 0) is measured using a fluorometer with an excitation wavelength of 340 nm and emission detection at 410 nm. The plate is then incubated at 37°C for 1 hour. The final fluorescence (T = 60) is measured. The difference between the initial and final fluorescence is calculated. DPP3 activity is determined using a six-point calibration curve. Calibrators and samples are preferably measured in duplicate.

[0072] In a specific embodiment, an assay is used to measure the level of DPP3, wherein the assay sensitivity of said assay is capable of quantifying DPP3 in healthy subjects and is <20 ng / ml, preferably <30 ng / ml, more preferably <40 ng / ml.

[0073] In a specific embodiment, the binder comprises at least 10 7 M -1 exhibits a binding affinity for DPP3 of 10 8 M -1 and a more preferred affinity of 10 9 M -1 and the most favorable affinity is 10 10 M -1 Those skilled in the art will know that applying a higher dose of the compound will compensate for the lower affinity, so this determination will not fall outside the scope of the present invention.

[0074] In another embodiment of the invention, the body fluid sample is selected from the group of whole blood, plasma, and serum. Mature ADM, bio-ADM and ADM-NH2 are used interchangeably throughout this application and are molecules according to SEQ ID NO:20.

[0075] In one particular embodiment, the body fluid according to the present invention is a blood sample. The blood sample may be selected from the group comprising whole blood, serum and plasma. In a specific embodiment of the method, the sample is selected from the group comprising human citrated plasma, heparinized plasma and EDTA plasma.

[0076] In a specific embodiment, an assay is used to measure levels of ADM-NH2, wherein the assay sensitivity of the assay is capable of quantifying mature ADM-NH2 in healthy subjects and is <70 pg / ml, preferably <40 pg / ml, and more preferably <10 pg / ml.

[0077] In a specific embodiment of the present invention, the threshold value for ADM-NH2 is 40-100 pg / mL, a more preferred threshold value is 50-90 pg / mL, an even more preferred threshold value is 60-80, and a most preferred threshold value of 70 pg / mL is applied.

[0078] In a specific embodiment of the invention, the threshold value for plasma ADM-NH2 is 5 times the median concentration of a normal healthy population, preferably 4 times the median concentration, more preferably 3 times the median concentration, and most preferably 2 times the median concentration.

[0079] In a specific embodiment, the binder has a molecular weight of at least 10 with respect to ADM-NH2. 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.

[0080] To determine the affinity of the antibody for adrenomedullin, the binding rate 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).

[0081] In a specific embodiment, the binder is selected from the group comprising an antibody, or antibody fragment, or a non-Ig scaffold that binds to ADM-NH2.

[0082] In a specific embodiment, an assay is used to measure levels of ADM-NH2, wherein such assay is a sandwich assay, preferably a fully automated assay.

[0083] In one embodiment, the assay for measuring the level of biomarkers (DPP3 and / or ADM-NH2) is a sandwich immunoassay utilizing any type of detection technology (non-limiting examples of which include enzyme-labeled, chemiluminescent-labeled, or 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®.

[0084] A wide variety of immunoassays are known and can be utilized in the assays and methods of the present invention, including mass spectrometry (MS), luminescence immunoassays (LIA), radioimmunoassays ("RIA"), homogeneous enzyme multiplexed immunoassays ("EMIT"), enzyme-linked immunosorbent assays ("ELISA"), apoenzyme reactivation immunoassays ("ARIS"), luminescence-based bead arrays, magnetic bead-based arrays, protein microarray assays, rapid assay formats such as dipstick immunoassays, immunochromatographic strip tests, rare cryptate assays, and automated systems / analyzers.

[0085] In one embodiment of the present invention, such assays are sandwich immunoassays utilizing any type of detection technology (non-limiting examples of which include enzyme, chemiluminescent, and electrochemiluminescent labels), and are preferably fully automated. In one embodiment of the present invention, such assays are enzyme-labeled sandwich assays. Examples of automated or fully automated assays include those available for one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, and Alere Triage®.

[0086] In one embodiment of the present invention, the assay can be a so-called point-of-care (POC) assay. This is an assay technology that allows the assay to be performed near the patient within an hour, without the need for a fully automated assay system. An example of this technology is an immunochromatographic assay technology, e.g., a microfluidic device.

[0087] In a preferred embodiment, the label is selected from the group comprising a chemiluminescent label, an enzyme label, a fluorescent label, a radioactive iodine label.

[0088] Assays can be homogeneous or heterogeneous, competitive or non-competitive. In one embodiment, the assay is in the form of a sandwich assay, a non-competitive immunoassay, in which the molecule to be detected and / or quantified is bound to a first antibody and a second antibody. The first antibody can be bound to a solid phase (e.g., a bead, a surface of a well or other container, a chip, or a strip), and the second antibody is labeled, for example, with a dye, a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody that binds to the analyte is then measured by an appropriate method. The general structure and procedures involved in "sandwich assays" are well defined and known to those skilled in the art (The Immunoassay Handbook, Ed. David Wild, Elsevier LTD, Oxford; 3rd ed. (May 2005), ISBN-13: 978-0080445267; Hultschig C et al., Curr Opin Chem Biol. 2006 Feb;10(1):4-10. PMID: 16376134).

[0089] In another embodiment, the assay comprises two capture molecules (preferably antibodies), both of which are dispersed in a liquid reaction mixture, in which a first label component, part of a fluorescence- or chemiluminescence-quenching or amplification-based labeling system, is attached to the first capture molecule, and a second label component of the labeling system is attached to the second capture molecule, such that a measurable signal is generated when both capture molecules bind to the analyte, thereby allowing detection of the sandwich complex formed in the sample-containing solution.

[0090] In another embodiment, the labeling system comprises a rare earth cryptate or rare earth chelate in combination with a fluorescent or chemiluminescent dye, particularly a cyanine-type dye.

[0091] In the context of the present invention, fluorescence-based assays involve the use of dyes, such as FAM (5-carboxyfluorescein or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes (CY3, CY5, CY3.5, CY5.5, Cy7, etc.), xanthene, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), ), TET, 6-carboxy-4',5'-dichloro-2',7'-dimethodifluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes (such as BODIPY TMR), Oregon Green, coumarins (such as umbelliferone), benzimides (such as Hoechst 33258); phenanthridines (such as Texas Red), Yakima Yellow, Alexa Fluor, PET, ethidium bromide, acridinium dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, and the like.

[0092] In the context of the present invention, chemiluminescence-based assays involve the use of dyes based on physical principles. The physical principles of chemiluminescent materials are described in Kirk-Othmer, Encyclopedia of Chemical Technology, 4th ed., executive editor: J.I. Kroschwitz; editor: M. Howe-Grant, John Wiley & Sons, 1993, vol. 15, pp. 518-562 (incorporated herein by reference, including the citations on pages 551-562). Preferred chemiluminescent dyes are acridinium esters.

[0093] As used herein, an "assay" or "diagnostic assay" can be of any type used in the field of diagnostics. Such assays can be based on the binding of the analyte to be detected to one or more capture probes with a given affinity. The affinity constant for the interaction between the capture molecule and the target molecule or molecule of interest is 10 8 M -1 It is preferable that it is greater than .

[0094] In a specific embodiment, at least one of the two binders is labeled so as to be detectable.

[0095] The ADM-NH2 levels of the present invention were measured using the ADM-NH2 assay (Weber et al. 2017. JALM 2(2):1-4), as described. The DPP3 levels of the present invention were measured using the DPP3 assay (Rehfeld et al. 2019. JALM 3(6):943-953), as described and outlined in the Examples. The thresholds may be different for other assays if they are calibrated differently from the assay system used in the present invention. Therefore, the cutoff values ​​will be adjusted to account for the calibration differences and tailored to such differently calibrated assays. One possibility for quantifying the calibration differences is to perform a method comparison (correlation) between the assay in question and each biomarker assay used in the present invention by measuring each biomarker (e.g., bio-ADM, DPP3) in samples used in both methods. Another possibility is to use the assay in question (assuming the test has sufficient analytical sensitivity) to determine the median biomarker level for each normal population, compare the results with the median biomarker levels reported in the literature, and recalculate the calibration value based on the difference obtained from this comparison. Using the calibration values ​​used in this invention, samples from normal (healthy) subjects were measured: the median plasma bio-ADM (mature ADM-NH2) was 24.7 pg / ml, with a nadir of 11 pg / ml and a 99th percentile of 43 pg / ml (Marino et al. 2014. Critical Care 18:R34). The calibration used in this invention measured samples from 5,400 normal (healthy) subjects (a Swedish single-center candidate population-based study (MPP-RES)): the median plasma DPP3 (interquartile range) was 14.5 ng / ml (11.3 ng / ml to 19 ng / ml).

[0096] As used herein, the term "therapy guidance" refers to the application of a particular therapy or medical intervention based on the value of one or more biomarkers, clinical parameters and / or clinical scores.

[0097] The term "therapy monitoring" in the context of the present invention refers to the monitoring and / or adjustment of the therapeutic treatment of said patient, for example by obtaining feedback of the effectiveness of the therapy.

[0098] The term "therapy stratification" particularly relates to grouping or categorizing patients into various groups, such as treatment populations that will or will not receive therapeutic treatment according to their classification.

[0099] A particular advantage of the methods of the present invention is that patients suffering from or in a state of shock are stratified with regard to the treatment they require, where the treatment is administration of an anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold that binds to the N-terminal portion (amino acids 1-21) of ADM (SEQ ID NO: 14). The stratified patient groups can include patients who require treatment initiation and patients who do not require treatment initiation.

[0100] Another particular advantage of the present invention is that the method distinguishes between patients who are less likely to benefit from the therapy and those who are more likely to benefit from the therapy.

[0101] In preferred embodiments, treatment is initiated or modified immediately upon receipt of sample analysis results indicating the level of DPP3 and / or ADM-NH2 in the sample. In further embodiments, treatment may be initiated within 12 hours, preferably 6, 4, 2, 1, 0.5, 0.25 hours, or immediately thereafter, of receiving the sample analysis results.

[0102] In some embodiments, the method comprises or consists of single and / or multiple measurements of DPP3 and / or ADM-NH2 in samples from the patient in a single sample and / or in multiple samples obtained at substantially the same time point to guide, monitor and / or stratify therapy, wherein said therapy is administration of an anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold that binds to the N-terminal portion (amino acids 1-21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

[0103] In the context of the methods of the present invention, it is particularly desirable to measure the level of DPP3 in the same sample as the level of ADM-NH2. In this embodiment, both biomarkers DPP3 and ADM-NH2 can be measured in the same sample simultaneously in a multiplex assay format, or at various time points in a multiplex assay format or a single assay format. The multiplex assay can be a duplicate assay for measuring both markers, where the assay can be a point-of-care assay that can be performed in the patient's presence immediately after sample isolation.

[0104] The present invention further relates to a kit for carrying out the method of the present invention, comprising detection reagents for determining the level of DPP3 in a sample from a patient, and further for determining the level of ADM-NH2.

[0105] It may also be preferably determined as a point-of-care assay that can be performed directly where the patient encounters medical personnel, such as in an emergency department or primary care unit. Furthermore, the assays for the detection of DPP3 and also ADM-NH2 may be single assays, preferably duplicate assays, and / or automated or semi-automated point-of-care assays.

[0106] In a preferred embodiment, the present invention relates to methods and kits for measuring the level of DPP3 in a sample from a patient, and further for measuring the level of ADM-NH2 and optionally further biomarkers.

[0107] The further biomarker may be selected from the group comprising procalcitonin (PCT), C-reactive protein (CRP), lactate.

[0108] The present invention further relates to a kit for carrying out the method of the present invention, comprising detection reagents for measuring DPP3 and further for measuring the level of ADM-NH2 in a sample from a patient, and reference data, such as reference and / or threshold levels, corresponding to a DPP3 level in said sample of 20 to 120 ng / mL, more preferably 30 to 80 ng / mL, even more preferably 40 to 60 ng / mL, and most preferably 50 ng / mL, wherein said reference data is preferably stored on a computer-readable medium and / or is available in the form of computer-executable code configured to compare the measured levels of DPP3 and further ADM-NH2 with said reference data.

[0109] In one embodiment of the methods described herein, the method further comprises comparing the measured levels of DPP3 and also ADM-NH2 in the patient suffering from shock and / or in a state of shock to reference and / or threshold levels, wherein said comparison is performed in a computer processor using computer executable code.

[0110] The method of the present invention can be partially implemented by a computer. For example, the step of comparing the detected level of a marker, such as DPP3 and / or ADM-NH2, with a reference and / or threshold level can be performed by a computer system. For example, the measured value can be input into the computer system (manually by a medical professional or automatically from a device that measures the level of each marker(s)). The computer system can be located locally at the clinical site (e.g., in the primary care department or ED), or it can be located remotely and connected via a computer network (e.g., via the Internet, or a specialized medical cloud system that can optionally connect to other IT systems or platforms, such as a hospital information system (HIS)). Alternatively or additionally, relevant treatment guidance and / or treatment stratification can be displayed and / or printed for the user (typically a medical professional, such as a physician).

[0111] In a specific embodiment of the invention, said shock is selected from the group comprising hypovolemic shock, cardiogenic shock, vaso-occlusive shock, and distributive shock.

[0112] In another specific embodiment of the invention, said shock is selected from the group comprising hypovolemic shock, cardiogenic shock, vaso-occlusive shock and distributive shock, in particular cardiogenic or septic shock.

[0113] In a specific embodiment of the invention, the shock is one of the following: In the case of cardiogenic shock, the patient is suffering from an acute coronary syndrome (e.g., acute myocardial infarction) or heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture or massive pulmonary embolism, or In the case of hypovolemic shock, the patient may have a bleeding disorder, including gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor invasion into major blood vessels) and spontaneous bleeding during anticoagulation, or a non-hemorrhagic disorder, including vomiting, diarrhea, renal losses, cutaneous / insensible losses (e.g., burns, heat stroke), or third-space losses during pancreatitis, liver cirrhosis, intestinal obstruction, and trauma; or In the case of vaso-occlusive shock, the patient may be suffering from cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis; or In the case of distributive shock, the patient is suffering from septic shock, neurogenic shock, anaphylactic shock or shock due to adrenal crisis; is selected from the group comprising:

[0114] Shock is characterized by cellular and tissue hypoxia due to decreased oxygen supply and / or increased oxygen consumption, or inadequate oxygen utilization. Shock is a life-threatening condition of circulatory failure, most commonly manifesting as hypotension (systolic blood pressure less than 90 mm Hg or MAP less than 65 mmHg). Based on the underlying cause, shock can be divided into four types: hypovolemic, cardiogenic, vaso-occlusive, and distributive (Vincent and De Backer 2014. N. Engl. J. Med. 370(6): 583).

[0115] Hypovolemic shock is characterized by a decrease in intravascular volume and can be divided into two broad subtypes: hemorrhagic and nonhemorrhagic. Common causes of hemorrhagic hypovolemic shock include gastrointestinal bleeding, trauma, vascular etiologies (e.g., ruptured abdominal aortic aneurysm, tumor invasion of major blood vessels), and spontaneous bleeding in the setting of anticoagulant use. Common causes of nonhemorrhagic hypovolemic shock include vomiting, diarrhea, renal losses, cutaneous / insensible losses (e.g., burns, heatstroke), or interstitial losses in the setting of pancreatitis, liver cirrhosis, intestinal obstruction, and trauma. For a review, see Koya and Paul 2018. Shock. StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2019-2018 Oct 27.

[0116] Cardiogenic shock (CS) is defined as a state of critical end-organ hypoperfusion due to reduced cardiac output. CS manifests as a spectrum ranging from mild hypoperfusion to severe shock. Established diagnostic criteria for CS include: (i) a systolic blood pressure of ≤90 mmHg for more than 30 minutes or the need for vasopressors to achieve a blood pressure of ≥90 mmHg; (ii) pulmonary congestion or elevated left ventricular filling pressure; and (iii) signs of impaired organ perfusion, accompanied by at least one of the following: (a) altered mental status; (b) cold, clammy skin; (c) oliguria (<0.5 mL / kg / h or <30 mL / h); and (d) elevated serum lactate (Reynolds and Hochman 2008. Circulation 117: 686-697). Acute myocardial infarction (AMI) and subsequent ventricular dysfunction are the most common causes of CS, accounting for approximately 80% of cases. Mechanical complications (e.g., ventricular septum (4%) or free wall rupture (2%)) and acute severe mitral regurgitation (7%) are less frequent causes of CS after AMI (Hochman et al. 2000. J Am Coll Cardiol 36: 1063-1070). Non-AMI-related CS can be caused by decompensated valvular heart disease, acute cardiomyopathy, or arrhythmias, and multiple treatment options are available. This translates into 40,000-50,000 patients per year in the United States and 60,000-70,000 patients in Europe. Despite advances in primary treatment with early revascularization, with subsequent reductions in mortality, CS remains the leading cause of death in AMI, with recent registry and randomized trials still indicating mortality rates approaching 40-50% (Goldberg et al. 2009. Circulation 119: 1211-1219).

[0117] Vaso-occlusive shock is due to physical obstruction of a large blood vessel or the heart itself. Several conditions can result in this form of shock (e.g., cardiac tamponade, tension pneumothorax, pulmonary embolism, aortic stenosis). For a review, see Koya and Paul 2018. Shock. StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2019-2018 Oct 27.

[0118] There are four types of distributive shock, depending on the cause: neurogenic shock (decreased sympathetic stimulation resulting in decreased vascular tone), anaphylactic shock, septic shock, and shock due to adrenal crisis. Distributive shock can result from sepsis as well as systemic inflammatory response syndrome (SIRS) caused by noninfectious causes (e.g., pancreatitis, burns, trauma). Other causes include toxic shock syndrome (TSS), anaphylaxis (a sudden, severe allergic reaction), adrenal insufficiency (acute worsening of chronic adrenal insufficiency, destruction or removal of the adrenal glands, adrenal suppression due to exogenous steroids, hypopituitarism, and inability to metabolize hormones produced), drug or toxin reactions, heavy metal poisoning, liver failure, and damage to the nervous system. For a review, see Koya and Paul 2018. Shock. StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2019-2018 Oct 27.

[0119] Refractory shock was defined as the requirement for a norepinephrine infusion of >0.5 μg / kg / min despite adequate volume resuscitation. Mortality in these patients can be as high as 94%, and the evaluation and management of these patients requires a more aggressive approach to survival. The term "refractory shock" is used when tissue perfusion cannot be restored with initial corrective measures (e.g., vasopressors); therefore, it is also referred to as "highly vasopressor-dependent" or "vasopressor-resistant" shock (Udupa and Shetty 2018. Indian J Respir Care 7: 67-72). Patients suffering from refractory shock may have features of inadequate perfusion due to hypoxia and acidemia, such as hypotension (meaning arterial pressure <65 mmHg), tachycardia, hypothermic peripheral blood, short capillary refill times, and tachypnea. Fever may also be seen in septic shock. Other signs of hypoperfusion, such as paresthesia, hyperlactemia, and oliguria, may also be present. These familiar signs of shock are not useful for identifying whether the problem is in the pump (heart) or the circuit (blood vessels and tissues). Different types of shock can coexist, and all forms of shock can be refractory, as evidenced by unresponsiveness to high-dose vasopressors (Udupa and Shetty 2018. Indian J Respir Care 7: 67-72).

[0120] Septic shock is a potentially fatal condition that occurs when sepsis, a condition resulting from organ damage or infection, leads to dangerously low blood pressure and abnormalities in cellular metabolism. The Third International Consensus Definition of Sepsis and Septic Shock (Sepsis-3) defines septic shock as a subset of sepsis, particularly when severe cardiovascular, cellular, and metabolic abnormalities pose a higher risk of death than sepsis alone. Patients with septic shock require vasopressors to maintain a mean arterial pressure of 65 mm Hg or higher and can be clinically identified by serum lactate levels above 2 mmol / L (>18 mg / dL) in the absence of hypovolemia. This combination is associated with a hospital mortality rate of over 40% (Singer et al. 2016. JAMA. 315 (8): 801-10). The primary infection is most commonly caused by bacteria, but can also be caused by fungi, viruses, or parasites. Although it can be present in any part of the body, it is most commonly present in the lungs, brain, urinary tract, skin, or abdominal organs. It can cause multiple organ dysfunction syndrome (formerly known as multiple organ failure) and death. People with septic shock are often treated in intensive care units. It most commonly affects children, immunocompromised individuals, and the elderly because their immune systems cannot fight infection as effectively as those of healthy adults. The mortality rate from septic shock is approximately 25-50%.

[0121] In one embodiment of the invention, the patient is a critically ill patient suffering from or in a state of shock at the time the patient's body fluid sample is taken.

[0122] A "patient in shock" is defined as a critically ill patient who is not suffering from shock at the time fluid is taken from said patient, but who is at high risk of developing shock.

[0123] In a specific embodiment, the shock is septic shock or cardiogenic shock.

[0124] The efficacy of non-neutralizing antibodies targeted against the N-terminus of ADM was investigated in survival studies in mice with CLP-induced sepsis. Pretreatment with the non-neutralizing antibody resulted in lower catecholamine infusion rates, renal dysfunction, and significantly improved survival (Struck et al. 2013. Intensive Care Med Exp 1(1):22; Wagner et al. 2013. Intensive Care Med Exp 1(1):21).

[0125] Because of these positive results, a humanized version of the N-terminal anti-ADM antibody, designated adrecizumab, was developed for further clinical development. The beneficial effects of adrecizumab on vascular barrier function and survival were recently demonstrated in preclinical models of systemic inflammation and sepsis (Geven et al. 2018. Shock 50(6):648-654). In this study, pretreatment with adrecizumab reduced renal vascular leakage in endotoxemic rats and mice suffering from CLP-induced sepsis, which simultaneously resulted in enhanced renal expression of the protective peptide Ang-1 and decreased expression of the toxic peptide vascular endothelial growth factor. Pretreatment with adrecizumab also improved 7-day survival in mice with CLP-induced sepsis by 10-50% after single doses and 0-40% after multiple doses. Moreover, a phase 1 study demonstrated good safety and tolerability (see Example 6): no serious adverse events were observed, no indications of more frequent adverse events were detected in adrecizumab-treated subjects, and no relevant changes in other safety parameters were noted (Geven et al. 2017. Intensive Care Med Exp 5 (Suppl 2): ​​0427). Of particular interest is the proposed mechanism of action of adrecizumab. Both animal and human data demonstrate a strong dose-dependent increase in circulating ADM after administration of this antibody. Based on pharmacokinetic data and the lack of an increase in MR-proADM (an inactive peptide fragment derived from the same prohormone as ADM), the higher circulating ADM levels cannot be explained by enhanced production.

[0126] A mechanistic explanation for this increase is that because ADM is small enough to cross the endothelial barrier, whereas antibodies are not, excess circulating antibodies may expel ADM from the interstitium into the circulation (Geven et al. 2018. Shock. 50(2):132-140). Additionally, antibody binding to ADM leads to an extended half-life of ADM. Although NT-ADM antibodies partially inhibit ADM-mediated signaling, the large increase in circulating ADM results in an overall "net" increase in ADM activity in the blood compartment, where it exerts beneficial effects on endothelial cells (ECs; predominant barrier stabilization), while the deleterious effects of ADM on vascular smooth muscle cells (VSMCs; vasodilatory responses) in the interstitium are reduced.

[0127] Throughout the specification, an "antibody," "antibody fragment," or "non-Ig scaffold" according to the present invention is capable of binding to ADM and is thereby directed against ADM, and can thus be referred to as an "anti-ADM antibody," "anti-ADM antibody fragment," or "anti-ADM non-Ig scaffold."

[0128] The term "antibody" generally includes monoclonal and polyclonal antibodies and their binding fragments (especially Fc fragments), as well as so-called "single-chain antibodies" (Bird et al. 1988), chimeric antibodies, humanized antibodies (especially CDR-grafted antibodies), and dibodies or tetrabodies (Holliger et al. 1993). It also includes immunoglobulin-like proteins selected through techniques including, for example, phage display, that specifically bind to a molecule of interest contained in a sample. In this context, the phrase "specifically binds" refers to an antibody raised against the molecule of interest or a fragment thereof. An antibody is considered specific if its affinity for the molecule of interest or said fragment thereof is preferably at least 50-fold greater, more preferably 100-fold greater, and most preferably at least 1000-fold greater than for other molecules contained in the sample containing the molecule of interest. Methods for generating antibodies and selecting antibodies with a given specificity are well known in the art.

[0129] Furthermore, in one embodiment of the present invention, the anti-adrenomedullin (ADM) antibody, or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold is monospecific.

[0130] 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. While monoclonal antibodies are monospecific, monospecific antibodies can also be generated by means other than generation from a common germline.

[0131] 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.

[0132] In a specific 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 is believed to block nearly 100%, at least more than 90%, and preferably at least more than 95% of the biological activity of ADM.

[0133] 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%.

[0134] 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.

[0135] The same definition applies to other ranges such as 95%, 90%, 50%, etc.

[0136] 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.

[0137] 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.

[0138] 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 also 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, NY, 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, US 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 a functional antibody fragment that specifically binds to the antigen.

[0139] 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. Pat. 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 called the "donor," and the human immunoglobulin providing the framework is called the "acceptor." In one embodiment, all CDRs are derived from the donor immunoglobulin in the 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. Pat. 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).

[0140] 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 present 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 bearing 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.

[0141] 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 create highly target-specific biopolymers. Examples include aptamers, spiegelmers, anticalins, and conotoxins. See Figure 1a, Figure 1b, and Figure 1c for schematic diagrams of antibody formats.

[0142] 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.

[0143] Non-Ig scaffolds can be protein scaffolds that can bind to ligands or antigens and thus can be used as antibody mimics. Non-Ig scaffolds 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).

[0144] In one embodiment of the present 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 that fragment are then identified using the methods described below or other methods known in the art.

[0145] 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 a phage display library or by a direct approach guided by molecular modeling (Almagro and Fransson 2008. Humanization of antibodies. Front Biosci. 2008 Jan 1;13:1619-33).

[0146] 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.

[0147] 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.

[0148] In a preferred embodiment, the anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold is directed against and capable of binding to an epitope contained within ADM that is at least 5 amino acids in length.

[0149] In a more preferred embodiment, the anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold is directed against and capable of binding to an epitope contained within ADM that is at least four amino acids in length.

[0150] In a specific embodiment of the present invention, an anti-ADM antibody or anti-ADM antibody fragment that binds to adrenomedullin, or an anti-ADM non-Ig scaffold that binds to adrenomedullin, is provided for use in treating or preventing shock in a patient, wherein the antibody or fragment or scaffold is not ADM binding protein-1 (complement factor H).

[0151] In a specific 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, is provided for use in treating or preventing shock in a patient, wherein the antibody or fragment or scaffold binds to a region of preferably at least 4 or at least 5 amino acids within the sequence of amino acids 1 to 21 of mature human ADM: YRQSMNNFQGLRSFGCRFGTC SEQ ID NO: 14.

[0152] In a preferred embodiment of the present invention, the anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold binds to a region or epitope located within the N-terminal portion (amino acids 1 to 21) of adrenomedullin.

[0153] In another preferred embodiment, the anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold recognizes and binds to amino acids 1 to 14 of adrenomedullin: YRQSMNNFQGLRSF (SEQ ID NO: 25), i.e., a region or epitope located within the N-terminal portion (amino acids 1 to 14) of adrenomedullin.

[0154] In another preferred embodiment, the anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold recognizes and binds to the 1st to 10th amino acids of adrenomedullin: YRQSMNNFQG (SEQ ID NO: 26), i.e., a region or epitope located within the N-terminal portion (1st to 10th amino acids) of adrenomedullin.

[0155] In another preferred embodiment, the anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold recognizes and binds to a region or epitope of ADM located within the first six amino acids of adrenomedullin: YRQSMN (SEQ ID NO: 27), i.e., the N-terminal portion (amino acids 1-6) of adrenomedullin. As noted above, this region or epitope preferably comprises at least four or five amino acids in length.

[0156] In another preferred embodiment, the anti-ADM antibody, or anti-ADM 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, 14, 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, the anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold only binds to a region within the sequence of mature ADM when the N-terminus of ADM is free. In this embodiment, it is believed that the anti-ADM antibody, or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold does not bind to a region within the sequence of mature ADM when the sequence is contained in, for example, pro-ADM.

[0157] 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.

[0158] In another specific embodiment according to the present 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 to 52 of ADM: PRSKISPQGY-NH2 (SEQ ID NO: 24).

[0159] An epitope, also known as an antigenic determinant, is the part of an antigen that is specifically recognized by an antibody and by the immune system. For example, an epitope is the specific piece of an antigen to which an antibody binds. The part of the antibody that binds to the epitope is called the paratope. Epitopes of protein antigens are divided into two categories based on their structure and interaction with the paratope: conformational epitopes and linear epitopes.

[0160] Conformational and linear epitopes interact with a paratope based on the 3-D structure adopted by the epitope, which is determined by the surface features and shape of the involved epitope residues or the tertiary structure of other segments of the antigen. Conformational epitopes are formed by the 3-D structure adopted by the interaction of non-contacting amino acid residues. A linear or continuous epitope is an epitope recognized by an antibody by its linear sequence or primary structure of amino acids and formed by the 3-D structure adopted by the interaction of adjacent amino acid residues.

[0161] In a specific embodiment, it is preferred to use an anti-ADM antibody, or anti-ADM 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 >50%, and most preferably >100%.

[0162] In a specific embodiment, it is preferable to use an anti-ADM antibody, an anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold of the present invention, and this anti-ADM antibody, an anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold has a half-life (t 1 / 2 ; half-retention time) by at least 10%, preferably at least 50%, more preferably >50%, and most preferably >100%.

[0163] The half-life (half-residence time) of ADM can be determined using immunoassays to quantify ADM in human serum, blood, and plasma in the absence and presence of an ADM-stabilized antibody, or an ADM-stabilized antibody fragment, or an ADM-stabilized non-Ig scaffold, respectively.

[0164] The following steps may be performed: ADM can be diluted in human citrated plasma in the absence and presence of an ADM-stabilizing antibody or ADM-stabilizing antibody fragment or an ADM-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 (such as 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, non-immunoassay techniques (e.g., RP-HPLC) can be used to quantify ADM. - Calculate the half-life for ADM incubated in the absence and presence of an ADM-stabilized antibody, or an ADM-stabilized antibody fragment, or an ADM-stabilized non-Ig scaffold, respectively. - For stabilized ADM, an increased half-life is calculated compared to ADM incubated in the absence of the ADM-stabilizing antibody, or ADM-stabilizing antibody fragment, or ADM-stabilized non-Ig scaffold.

[0165] A two-fold increase in the half-life of ADM is a 100% increase in the half-life.

[0166] Half-life (residence half-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.

[0167] An assay that can be used to determine the half-life (half-life residence time) of adrenomedullin in serum, blood, or plasma is described in Example 3.

[0168] 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 more 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%.

[0169] 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 21 of mature human ADM (SEQ ID NO: 14), or an antibody that binds to a region of at least five amino acids in the sequence of amino acids 1 to 19 of mature mouse ADM (SEQ ID NO: 17).

[0170] 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 21 of mature human ADM (SEQ ID NO: 14), or an antibody that binds to a region of at least four amino acids in the sequence of amino acids 1 to 19 of mature mouse ADM (SEQ ID NO: 17).

[0171] In a specific embodiment of the present invention, a non-neutralizing anti-ADM antibody, or a non-neutralizing anti-ADM 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 to inhibition (meaning reduction in biological activity) of ADM biological activity occurs even when the antibody, fragment, or scaffold is at an excessive concentration (the excess of antibody, fragment, or scaffold relative to ADM). This limit to inhibition is an inherent property of the ADM binder itself in this particular 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, or 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.

[0172] 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.

[0173] 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 for half-maximal effect of cAMP stimulation, a dose-response curve is performed with either the ADM-stabilized antibody, or the ADM-stabilized antibody fragment, or the ADM-stabilized non-Ig scaffold (up to a final concentration of 100 μg / ml).

[0174] A maximal inhibition of 50% in the ADM bioassay means that the anti-ADM antibody or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold, respectively, inhibits the biological activity of ADM to 50% of the baseline value. A maximal inhibition of 80% in the ADM bioassay means that the anti-ADM antibody or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold, respectively, inhibits the biological activity of ADM to 80% of the baseline value. This means that ADM biological activity is not inhibited to below 80%. This means that approximately 20% residual ADM biological activity remains.

[0175] 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.

[0176] The biological activity of ADM can be determined in a human recombinant adrenomedullin receptor cAMP functional assay (adrenomedullin bioassay) according to Example 2.

[0177] In a preferred embodiment, the regulatory antibody or regulatory anti-ADM antibody fragment, or regulatory anti-ADM non-Ig scaffold, is used to treat or prevent shock in a patient.

[0178] The "modulating" anti-ADM antibody, or the modulating anti-ADM antibody fragment, or the modulating anti-ADM non-Ig scaffold, reduces the half-life (t 1 / 2 and half-life (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 by 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 are relative to the assays described above for determining these values. This means that ADM biological activity will not be inhibited by less than 80% or less than 50%, respectively.

[0179] Such regulatory anti-ADM antibodies, or regulatory anti-ADM antibody fragments, or regulatory anti-ADM non-Ig scaffolds offer the advantage of ease of administration. The combination of partially blocking or partially reducing the biological activity of adrenomedullin and extending its in vivo half-life (increasing the biological activity of adrenomedullin) offers the advantage of simplifying the anti-ADM antibodies, or anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds. In situations where endogenous adrenomedullin is in excess (maximal stimulation, late sepsis, shock, and debilitating stages), the activity-reducing effect is the primary impact of the antibody, fragment, or scaffold, limiting the (negative) effects of ADM. When endogenous ADM concentrations are low or normal, the biological effect of the anti-ADM antibodies, or anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds is a combination of a reduction (by partial blocking) and an increase (by extending the half-life of ADM). Thus, non-neutralizing and regulatory anti-ADM antibodies, or anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds act like ADM bioactivity buffers, maintaining the bioactivity of ADM within a physiological range.

[0180] In a specific 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 specific embodiment, one or more (murine) CDRs are grafted into a human antibody or human antibody fragment.

[0181] In one aspect, the subject of the present invention is a human or humanized CDR-grafted antibody or antibody fragment thereof that binds to ADM. This human or humanized CDR-grafted antibody or antibody fragment thereof has the following structure: GYTFSRYW (SEQ ID NO: 1), ILPGSGST (SEQ ID NO: 2) and / or TEGYEYDGFDY (SEQ ID NO: 3) and / or an antibody heavy chain comprising QSIVYSNGNTY (SEQ ID NO: 4), RVS (not part of the sequence listing) and / or FQGSHIPYT (SEQ ID NO: 5), The antibody further comprises an antibody light chain (L chain) comprising:

[0182] In a specific embodiment of the present invention, the subject of the present invention is a human monoclonal antibody that binds to ADM, or an antibody fragment thereof that binds to ADM, in which the heavy chain is one of the following: GYTFSRYW (SEQ ID NO: 1), ILPGSGST (SEQ ID NO: 2), TEGYEYDGFDY (SEQ ID NO: 3), and the light chain comprises at least one CDR selected from the group comprising: QSIVYSNGNTY (SEQ ID NO: 4), RVS (not part of the sequence listing), FQGSHIPYT (SEQ ID NO: 5), and wherein the CDR is selected from the group comprising:

[0183] In a more specific embodiment of the present invention, the subject of the present 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: GYTFSRYW (SEQ ID NO: 1), ILPGSGST (SEQ ID NO: 2), TEGYEYDGFDY (SEQ ID NO: 3), and the light chain comprises the following sequence: QSIVYSNGNTY (SEQ ID NO: 4), RVS (not part of the sequence listing), FQGSHIPYT (SEQ ID NO: 5), Contains:

[0184] In very specific embodiments, the anti-ADM antibody has a sequence selected from the group comprising SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 32 and 33.

[0185] The anti-ADM antibodies, or anti-ADM 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.

[0186] A subject of the present invention is a human or humanized monoclonal antibody or fragment thereof that binds to ADM, for use in the treatment or prevention of shock in a patient according to the invention, said antibody or fragment being: Sequence number 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 7 (AM-H1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 8 (AM-H2E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 9 (AM-VH3-26E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 10 (AM-VH4-26E40E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 11 (AM-VL-) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 12 (AM-L1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13 (AM-VL2-40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC The nucleic acid sequence comprises a sequence selected from the group comprising:

[0187] Another embodiment of the present invention relates to a human or humanized monoclonal antibody or fragment that binds to ADM or an antibody fragment thereof for use in treating or preventing shock in a patient, wherein said antibody or fragment has as its heavy chain the following sequence: SEQ ID NO: 32 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFScSVMHEALHNHY TQKSLSLSPGK and as the light chain the following sequence: SEQ ID NO: 33 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC Includes:

[0188] In a specific embodiment of the invention, the antibody has the following sequence as the heavy chain: SEQ ID NO: 32 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK or a sequence that is >95%, preferably >98%, preferably >99% identical thereto, and which has as its light chain the following sequence: SEQ ID NO: 33 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC or a sequence that is >95%, preferably >98%, preferably >99% identical thereto, wherein the heavy chain comprises the following sequence: CDR1: SEQ ID NO: 1 GYTFSRYW CDR2: SEQ ID NO: 2 ILPGSGST CDR3: SEQ ID NO: 3 TEGYEYDGFDY and wherein the light chain comprises the following sequence: CDR1: SEQ ID NO: 4 QSIVYSNGNTY CDR2:RVS CDR3: SEQ ID NO: 5 FQGSHIPYT Includes: This means that in one embodiment of the present invention, the sequences of the CDRs do not exhibit any sequence variations, and any variations of the above sequences are outside the scope of the CDR sequences in said embodiment.

[0189] To assess identity between two amino acid sequences, a pairwise alignment is performed, where identity is defined as the percentage of amino acids that directly match in the alignment.

[0190] In an embodiment of the invention, an anti-ADM antibody or anti-ADM antibody fragment for use in treating or preventing shock in a patient may be administered at a dose of at least 0.5 mg / kg body weight, particularly at least 1.0 mg / kg body weight, more particularly 1.0 to 20.0 mg / kg body weight, for example, 2.0 to 10 mg / kg body weight, 2.0 to 8.0 mg / kg body weight, or 2.0 to 5.0 mg / kg body weight.

[0191] The term "pharmaceutical formulation" means a pharmaceutical ingredient in combination with at least one pharmaceutically acceptable excipient, and which is in a form that allows the biological activity of the pharmaceutical ingredient contained therein to be effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation will be administered. The term "pharmaceutical ingredient" means a therapeutic composition that can be optionally combined with a pharmaceutically acceptable excipient to provide a pharmaceutical formulation or dosage form.

[0192] A subject of the present invention is a pharmaceutical formulation comprising an antibody or a fragment or a scaffold according to the invention, for use in the treatment or prevention of shock in a patient.

[0193] A subject of the present invention is a pharmaceutical formulation comprising an antibody or fragment or scaffold according to the invention, for use in the treatment or prevention of shock in a patient, wherein said shock is selected from the group comprising hypovolemic shock, cardiogenic shock, vaso-occlusive shock and distributive shock, in particular cardiogenic shock or septic shock.

[0194] A subject of the present invention is a pharmaceutical preparation for use in the treatment or prevention of shock in patients according to the invention, wherein said pharmaceutical preparation is a solution, preferably a ready-to-use solution.

[0195] A subject of the present invention is a pharmaceutical preparation for use in the treatment or prevention of shock in a patient according to the invention, wherein said pharmaceutical preparation is in lyophilized form.

[0196] A subject of the present invention is a pharmaceutical preparation for use in the treatment or prevention of shock in a patient according to the invention, wherein said pharmaceutical preparation is administered intramuscularly.

[0197] A subject of the present invention is a pharmaceutical preparation for use in the intervention and treatment of hemostasis in a patient according to the invention, wherein said pharmaceutical preparation is administered intravascularly.

[0198] A subject of the present invention is a pharmaceutical preparation for use in the intervention and treatment of hemostasis in patients according to the invention, wherein said pharmaceutical preparation is administered by infusion.

[0199] A subject of the present invention is a pharmaceutical preparation for use in the treatment or prevention of shock in a patient according to the invention, wherein said pharmaceutical preparation is administered systemically.

[0200] In accordance with the above relationships, the following consecutively numbered embodiments provide further specific aspects of the present invention:

[0201] 1. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, comprising: measuring the level of dipeptidyl peptidase 3 (DPP3) in a body fluid sample from said patient; comparing the measured DPP3 level to a predetermined threshold; and administering to said patient an anti-adrenomedullin (ADM) antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold; Including, wherein the patient is treated if the measured DPP3 level is below a predetermined threshold; and Here, the method comprises the step of: the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold binding to the N-terminal portion (amino acids 1 to 21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

[0202] 2. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, as described in embodiment 1, wherein the shock is selected from the group comprising hypovolemic shock, cardiogenic shock, vaso-occlusive shock and distributive shock, in particular cardiogenic shock or septic shock.

[0203] 3. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to embodiment 1 or 2, comprising: In the case of cardiogenic shock, the patient may be suffering from an acute coronary syndrome (e.g., acute myocardial infarction), or the patient may be suffering from heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or In the case of hypovolemic shock, the patient may have a bleeding disorder, including gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor invasion into major blood vessels) and spontaneous bleeding during anticoagulation, or a non-hemorrhagic disorder, including vomiting, diarrhea, renal losses, cutaneous / insensible losses (e.g., burns, heat stroke), or third-space losses during pancreatitis, liver cirrhosis, intestinal obstruction, and trauma; or In the case of vaso-occlusive shock, the patient may be suffering from cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis; or In the case of distributive shock, the patient may be suffering from septic shock, neurogenic shock, anaphylactic shock, or shock due to adrenal crisis.

[0204] 4. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of embodiments 1 to 3, wherein the predetermined threshold value for DPP3 in a body fluid sample of the subject is 20 to 120 ng / mL, more preferably 30 to 80 ng / mL, even more preferably 40 to 60 ng / mL, and most preferably 50 ng / mL.

[0205] 5. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state, according to any one of embodiments 1 to 4, wherein either the level of DPP3 protein and / or the level of active DPP3 is measured and compared with a predetermined threshold value.

[0206] 6. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, according to any one of embodiments 1 to 5, wherein the level of DPP3 is measured by contacting the body fluid sample with a capture binder that specifically binds to DPP3.

[0207] 7. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of embodiments 1 to 6, wherein said measuring comprises the use of a capture binder that specifically binds to full-length DPP3, wherein said capture binder may be selected from the group comprising an antibody, an antibody fragment, or a non-IgG scaffold.

[0208] 8. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state, according to any one of embodiments 1 to 7, wherein the amount of DPP3 protein and / or DPP3 activity is measured in a body fluid sample from the subject, and said measuring comprises the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody.

[0209] 9. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state, as described in any one of embodiments 1 to 8, wherein the amount of DPP3 protein and / or DPP3 activity is measured in a body fluid sample of the subject, and said measuring comprises the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is immobilized on a surface.

[0210] 10. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state, as described in any one of embodiments 1 to 9, wherein the amount of DPP3 protein and / or DPP3 activity is measured in a body fluid sample of the subject, and the separation step is a washing step that removes sample components that are not bound to the capture binder from the captured DPP3.

[0211] 11. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of embodiments 1 to 10, wherein the method for measuring DPP3 activity in a body fluid sample of said subject comprises the following steps: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3; quantifying DPP3 activity by measuring and quantifying the conversion of a DPP3 substrate; A method comprising:

[0212] 12. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of embodiments 1 to 11, wherein the DPP3 activity is measured in a body fluid sample of the subject and the DPP3 substrate conversion is detected by a method selected from the group comprising: fluorescence of a fluorogenic substrate (e.g., Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate linked to aminoluciferin (Promega Protease-Glo™ Assay), mass spectrometry, HPLC / FPLC (reverse-phase chromatography, size-exclusion chromatography), thin-layer chromatography, capillary zone electrophoresis, gel electrophoresis after activity staining (fixed, active DPP3) or Western blotting (cleavage products).

[0213] 13. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of embodiments 1 to 12, wherein the DPP3 activity is measured in a body fluid sample of the subject, and the substrate may be selected from the group comprising: angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or aminoluciferin, wherein the dipeptide is Arg-Arg.

[0214] 14. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, according to any one of embodiments 1 to 13, wherein the DPP3 activity is measured in a body fluid sample of the subject, and the substrate can be selected from the group comprising: a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, wherein the dipeptide is Arg-Arg.

[0215] 15. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state, as described in any one of embodiments 1 to 14, further characterized in that the patient has an ADM-NH2 level above a threshold.

[0216] 16. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state, as described in embodiment 15, wherein the threshold value for ADM-NH2 in a body fluid sample of the patient is 40 to 100 pg / mL, more preferably the threshold value is 50 to 90 pg / mL, even more preferably the threshold value is 60 to 80 pg / mL, and most preferably the threshold value is 70 pg / mL.

[0217] 17. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state, as described in embodiment 15 or 16, wherein the level of ADM-NH2 is measured by contacting the body fluid sample with a capture binder that specifically binds to ADM-NH2.

[0218] 18. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in a patient suffering from shock and / or in a shock state, as described in any one of embodiments 1 to 17, wherein the patient's body fluid sample is selected from the group of blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.

[0219] 19. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state, according to any one of embodiments 1 to 18, wherein the levels of DPP3 and ADM-NH2 are measured in combination.

[0220] 20. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state, as described in embodiment 19, wherein the level of DPP3 and the level of ADM-NH2 are measured simultaneously.

[0221] 21. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state according to embodiment 19 or 20, wherein the levels of DPP3 and ADM-NH2 are measured using a point-of-care device.

[0222] 22. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state, as described in embodiment 21, wherein the point-of-care device is a microfluidic device.

[0223] 23. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, according to any one of embodiments 1 to 22, wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to the N-terminal end (first amino acid) of ADM.

[0224] 24. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state, according to any one of embodiments 1 to 23, wherein the antibody, antibody fragment or non-Ig scaffold does not bind to the C-terminal portion of ADM having amino acids 43 to 52 of the sequence: PRSKISPQGY-NH2 (SEQ ID NO: 24).

[0225] 25. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of embodiments 1 to 24, wherein the antibody or fragment is a monoclonal antibody or fragment that binds to ADM or an antibody fragment thereof, wherein the heavy chain has the following sequence: CDR1: SEQ ID NO: 1 GYTFSRYW CDR2: SEQ ID NO: 2 ILPGSGST CDR3: SEQ ID NO: 3 TEGYEYDGFDY and wherein the light chain comprises the following sequence: CDR1: SEQ ID NO: 4 QSIVYSNGNTY CDR2: RVS CDR3: SEQ ID NO: 5 FQGSHIPYT A method comprising:

[0226] 26. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of embodiments 1 to 25, wherein the antibody or fragment comprises as a VH region one of the following: Sequence number 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 8 (AM-VH2E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 9 (AM-VH3-T26E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 10 (AM-VH4-T26E40E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK and as the VL region the following sequence: SEQ ID NO: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC The method of claim 1, wherein the sequence is selected from the group comprising:

[0227] 27. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of embodiments 1 to 25, wherein the antibody or fragment has as its heavy chain the following sequence: SEQ ID NO: 32 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK or a sequence >95% identical thereto, and having as the light chain the following sequence: SEQ ID NO: 33 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC or a sequence that is >95% identical thereto. [Example]

[0228] Example 1 – Generation of antibodies and determination of their affinity constants Several human and mouse antibodies were generated and their affinity constants were measured (see Tables 1 and 2). It should be emphasized that the antibodies, antibody fragments, and non-Ig scaffolds in the Examples section according to the present invention are bound to ADM and should therefore be considered as anti-ADM non-antibody / antibody fragment / Ig scaffolds.

[0229] Peptides / conjugates for immunization: Peptides for immunization were synthesized with an additional N-terminal cysteine ​​residue (if no cysteine ​​was present in the selected ADM sequence) for conjugation to bovine serum albumin (BSA) (see Table 1) (JPT Technologies, Berlin, Germany). The peptides were covalently linked to BSA by using Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the Perbio manual.

[0230] Mouse monoclonal antibody production: 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, the animals received 50 μg of the conjugate dissolved in 100 μl of saline, administered intraperitoneally and intravenously. Splenocytes from the immunized mice and cells of the myeloma cell line SP2 / 0 were fused with 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 culturing in HAT medium (RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement). After two weeks, the HAT medium was replaced with HT medium for three passages, followed by a return to normal cell culture medium. Cell culture supernatants were first screened for antigen-specific IgG antibodies three weeks after fusion. Positively tested microcultures were transferred to 24-well plates for propagation. After retesting, selected cultures were cloned and recloned using limiting dilution, and their isotypes were determined (see also Lane, RD 1985. J. Immunol. Meth. 81: 223-228; Ziegler et al. 1996. Horm. Metab. Res. 28: 11-15).

[0231] Antibodies were produced by standard antibody production methods (Marx et al., 1997. Monoclonal Antibody Production, ATLA 25, 121) and purified via protein A. Antibody purity was >95% based on SDS gel electrophoresis analysis.

[0232] Human antibodies Human antibodies were generated by phage display according to the following procedure: the human native antibody gene library HAL7 / 8 was used to isolate 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. A mixture of panning steps using nonspecific binding antigens and streptavidin-binding antigens was used to minimize the background of nonspecific binders. Lytic phage from the third panning step were used to generate monoclonal scFv-expressing E. coli strains. Supernatants from the culture 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). Positive clones were selected based on a positive ELISA signal against the antigen and a negative signal 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 by immobilized metal ion affinity chromatography, and purified by size-exclusion chromatography.

[0233] Affinity constant: To measure the affinity of the antibody for ADM, the binding kinetics of ADM to immobilized antibody were measured by label-free surface plasmon resonance using a Biacore2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibody was performed using an anti-mouse Fc antibody covalently coupled to a CM5 sensor surface at high density according to the manufacturer's instructions (Mouse Antibody Capture Kit; GE Healthcare) (Lorenz et al. 2011. Antimicrob Agents Chemother. 55(1): 165-173).

[0234] Monoclonal antibodies were raised against the ADM regions of human and mouse ADM shown below, respectively. The table below represents a panel of the resulting antibodies used in further experiments. Selection was based on target region:

[0235] Table 1: Immunizing peptides [Table 1]

[0236] Below is a list of further monoclonal antibodies obtained. Table 2: [Table 2-1] [Table 2-2] [Table 2-3]

[0237] Production of antibody fragments by enzymatic digestion: Fab and F(ab)2 fragments were produced by enzymatic digestion of the mouse full-length antibody NT-M. The 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 carried out at 37°C for 8 hours for F(ab)2 fragmentation. Fab fragmentation digestions were carried out for 16 hours each.

[0238] Fab preparation and purification procedure: The immobilized papain was equilibrated by washing the resin with 0.5 ml of digestion buffer and centrifuging the column 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 incubation time for the digestion reaction was 16 hours on a tabletop rocker at 37°C. The column was centrifuged at 5000 × g for 1 minute to separate the immobilized papain and the digestion product. 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 in a total sample volume of 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 poured off to waste. The sample was applied to the column and resuspended by inversion. Incubation was carried out at room temperature for 10 minutes with end-over-end mixing. The column was centrifuged for 1 minute to prevent the Fab fragments from flowing through. (Citations: Coulter and Harris 1983. J. Immunol. Meth. 59, 199-203.; Lindner et al. 2010. Cancer Res. 70, 277-87; Kaufmann et al. 2010. PNAS. 107, 18950-5.; Chen et al. 2010. PNAS. 107, 14727-32; Uysal et al. 2009 J. Exp. Med. 206, 449-62; Thomas et al. 2009. J. Exp. Med. 206, 1913-27; Kong et al. 2009 J. Cell Biol. 185, 1275-840).

[0239] F(ab')2 Production and Purification Procedure: Immobilized pepsin was equilibrated by washing the resin with 0.5 ml of digestion buffer and centrifuging the column at 5000 x 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 x 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 incubation time for the digestion reaction was 16 hours on a tabletop rocker at 37°C. The column was centrifuged at 5000 x g for 1 minute to separate the immobilized papain and the digestion product. The resin was then washed with 0.5 mL of PBS and centrifuged at 5000 x g for 1 minute. The wash fraction was added to the digested antibody in a total sample volume of 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 poured off to waste. The sample was applied to the column and resuspended by inversion. Incubation was carried out at room temperature for 10 minutes with end-over-end mixing. The column was centrifuged for 1 minute to prevent the Fab fragments from flowing through.(Citations: Mariani et al. 1991. Mol. Immunol. 28: 69-77; Beale 1987. Exp Comp Immunol 11:287-96; Ellerson et al. 1972. FEBS Letters 24(3):318-22; Kerbel and Elliot 1983. Meth Enzymol 93:113-147; Kulkarni et al. 1985. Cancer Immunol Immunotherapy 19:211-4; Lamoyi 1986. Meth Enzymol 121:652-663; Parham et al. 1982. J Immunol Meth 53:133-73; Raychaudhuri et al. 1985. Mol Immunol 22(9):1009-19; Rousseaux et al. 1980. Mol Immunol 17:469-82; Rousseaux et al. 1983. J Immunol Meth 64:141-6; Wilson et al. 1991. J Immunol Meth 138:111-9).

[0240] Humanization of NT-H antibody fragments: The antibody fragments were humanized by the CDR grafting method (Jones et al. 1986. Nature 321, 522-525).

[0241] The following steps were performed to obtain the humanized sequence: Total RNA extraction: Total RNA was extracted from NT-H hybridomas using a Qiagen kit. First-stage RT-PCR: The QIAGEN® OneStep RT-PCR Kit (Cat. No. 210210) was used. RT-PCR was performed using heavy- and light-chain-specific primer sets. For each RNA sample, 12 individual heavy-chain and 11 light-chain RT-PCR reactions were set up using a degenerate forward primer mixture spanning the leader sequences of the variable regions. The reverse primers were located in the heavy- and light-chain constant regions. No restriction sites were designed into the primers.

[0242] Reactions were prepared using 5.0 μl of 5x QIAGEN® OneStep RT-PCR buffer, 0.8 μl of dNTP Mix (containing 10 mM of each dNTP), 0.5 μl of primer set, 0.8 μl of QIAGEN® OneStep RT-PCR Enzyme Mix, 2.0 μl of template RNA, and RNase-free water up to 20.0 μl, for a total volume of 20.0 μl. PCR conditions were: reverse transcription at 50°C for 30 min; initial PCR activation at 95°C for 15 min; cycling at 94°C for 25 s; 54°C for 30 s; and 72°C for 30 s for 20 cycles; final extension at 72°C for 10 min. Second-stage semi-nested PCR: The RT-PCR products from the first-stage reactions were further amplified in a second-stage PCR. 12 individual heavy chain and 11 light chain RT-PCR reactions were prepared using semi-nested primer sets specific for antibody variable regions.

[0243] Reaction set up: 10 μl 2x PCR mix; 2 μl primer set; 8 μl first-stage PCR product; total volume 20 μl; Hybridoma Antibody Cloning Report. PCR conditions: 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.

[0244] After PCR was completed, PCR reaction samples were run on an agarose gel to visualize the amplified DNA fragments. After sequencing more than 15 cloned DNA fragments amplified by nested RT-PCR, several mouse antibody heavy and light chains were cloned and appeared to be correct. Protein sequence alignment and CDR analysis identified one heavy chain and one light chain. After alignment with homologous human framework sequences, the resulting humanized variable heavy chain sequence was as follows: see Figure 5 (amino acids 26, 40, and 55 in the variable heavy chain and 40 in the variable light chain are important for binding properties, so they may be reverting to murine origin. The resulting candidates are shown below) (Padlan 1991. Mol. Immunol. 28, 489-498; Harris and Bajorath 1995. Protein Sci. 4, 306-310).

[0245] Annotation for antibody fragment sequences (SEQ ID NOs: 6-13; 32 and 33): bold and underlined are CDRs 1, 2, 3 arranged in chronological order; italics are constant regions; hinge regions are highlighted in bold; framework point mutations are in gray background. Sequence number 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 8 (AM-VH2E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 9 (AM-VH3-T26E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 10 (AM-VH4-T26E40E55) VQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 32 (Addressizumab heavy chain) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 33 (Addressizumab light chain) DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0246] Example 2 - Effect of selected anti-ADM antibodies on anti-ADM bioactivity The effects of selected ADM antibodies on ADM bioactivity were tested in a human recombinant adrenomedullin receptor cAMP functional assay (Adrenomedullin Bioassay).

[0247] Testing of antibodies targeting human or mouse adrenomedullin in the human recombinant adrenomedullin receptor cAMP functional assay (Adrenomedullin Bioassay)

[0248] Materials: Cell line: CHO-K1, Receptor: Adrenomedullin (CRLR + RAMP3), Receptor cell line accession numbers: CRLR: U17473; RAMP3: AJ001016

[0249] Prior to testing, CHO-K1 cells (FAST-027C) expressing human recombinant adrenomedullin receptors, cultured 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).

[0250] Dose-response curves were run in parallel with standard agonists (hADM or mADM).

[0251] Antagonist test (96-well): For antagonist testing, 6 μl of standard agonist (human (5.63 nM) or mouse (0.67 nM) adrenomedullin) was mixed with 6 μl of test sample at different antagonist dilutions; or 6 μl of buffer. After 60 minutes of incubation at room temperature, 12 μl of cells (2,500 cells / well) were added. The plate was incubated for 30 minutes at room temperature. After adding lysis buffer, the percentage of DeltaF was estimated using an HTRF kit (cat no. 62AM2 PEB) from Cis-Bio International according to the manufacturer's specifications. hADM22-52 was used as the standard antagonist.

[0252] cAMP-HTRF assay to test antibodies Anti-h-ADM antibodies (NT-H, MR-H, CT-H) were tested for antagonist activity in the human recombinant adrenomedullin receptor (FAST-027C) cAMP functional assay in the presence of 5.63 nM human ADM1-52 at the following final antibody concentrations: 100 μg / ml, 20 μg / ml, 4 μg / ml, 0.8 μg / ml, and 0.16 μg / ml.

[0253] Anti-m-ADM antibodies (NT-M, MR-M, and CT-M) were tested for antagonist activity in the human recombinant adrenomedullin receptor (FAST-027C) cAMP functional assay in the presence of 0.67 nM mouse ADM1-50 at the following final antibody concentrations: 100 μg / ml, 20 μg / ml, 4 μg / ml, 0.8 μg / ml, and 0.16 μg / ml. Data were plotted as relative inhibition versus antagonist concentration (see Figures 3a–3l). Maximum inhibition by individual antibodies is shown in Table 3.

[0254] Table 3: Maximum inhibition of bio-ADM activity [Table 3]

[0255] Example 3 – Stabilization of hADM by anti-ADM antibodies The stabilization effect of human ADM by human ADM antibodies was tested using a hADM immunological assay.

[0256] Immunoassay for quantification of human adrenomedullin The technology used was a sandwich-coated tube luminescent immunoassay based on acridinium ester labeling.

[0257] Labeled compound (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) (EP0353971) and incubated at room temperature for 20 minutes. The labeled CT-H was purified by gel filtration HPLC using 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) of labeled compound (approximately 20 ng labeled antibody) per 200 μL. Acridinium ester chemiluminescence was measured using an AutoLumat LB953 (Berthold Technologies GmbH & Co. KG).

[0258] Solid phase: Polystyrene tubes (Greiner Bio-1 International AG, Austria) were coated with MR-H (AdrenoMed AG, Germany) (1.5 μg of 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 albumin, the tubes were washed with PBS, pH 7.4, and then vacuum dried.

[0259] Calibration: The assay was calibrated using a dilution of hADM (BACHEM AG, Switzerland) in 250 mmol / L NaCl, 2 g / L Triton X-100, 50 g / L bovine serum albumin, 20 tab / L protease inhibitor cocktail (Roche Diagnostics AG, Switzerland).

[0260] hADM immunoassay: After adding labeled CT-H (200 μl), 50 μl of sample (or calibrator) was pipetted into the coated tube, and the tube was incubated for 4 hours at 4° C. Unbound tracer was removed by washing five times (1 ml each) with wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100).

[0261] Tube-bound chemiluminescence was measured using LB953: Figure 3 shows a typical hADM dose / signal curve and the hADM dose-signal curve in the presence of 100 μg / mL of antibody NT-H. NT-H did not affect the hADM immunoassay described.

[0262] Stability of human adrenomedullin: Human ADM was diluted in human citrated plasma (final concentration 10 nM) and incubated at 24°C. At selected time points, degradation of hADM was stopped by freezing at -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 previously.

[0263] Figure 4 shows the stability of hADM in human plasma (citrate) in the absence and presence of NT-H antibody. The half-life of hADM alone was 7.8 h, and in the presence of NT-H, the half-life was 18.3 h (2.3-fold greater stability).

[0264] Example 4 – Sepsis Mortality a) Early treatment of sepsis Animal Model: Male C57Bl / 6 mice (Charles River Laboratories, Germany), aged 12 to 15 weeks, were used in the study. Peritonitis was surgically induced under light isoflurane anesthesia. An incision was made in the upper left quadrant of the abdominal cavity (the normal location of the cecum). The cecum was exposed, and a tight ligature was placed around the cecum distal to its attachment 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 expressed through the wound. The cecum was returned to the abdominal cavity, and the laparotomy site was closed. Finally, the animals were returned to their cages with free access to food and water. 500 μl of saline was given subcutaneously for hydration.

[0265] Application and dosage of compounds (NT-M, MR-M, CT-M): Mice were treated immediately after CLP (early treatment), where CLP is the abbreviation for cecal ligation and puncture (CLP).

[0266] Study groups: Three compounds were tested against vehicle and against control compound treatment. Each group included five mice for blood collection one day later for BUN (serum blood urea nitrogen test) measurement. An additional 10 mice per group were followed over a four-day period.

[0267] Group Treatment (10 μl / g body weight) Dose / Follow-up: 1 NT-M, 0.2mg / ml, survival for 4 days 2 MR-M, 0.2mg / ml Survival over 4 days 3 CT-M, 0.2mg / ml Survival over 4 days 4 Non-specific mouse IgG, 0.2 mg / ml, survival over 4 days 5 Control - PBS, 10 μl / g body weight Survival over 4 days

[0268] Clinical Chemistry: Blood urea nitrogen (BUN) concentrations related to renal function were measured at baseline and on day 1 after CLP. Blood samples were collected from the cavernous sinus using a capillary tube under light ether anesthesia. Measurements were performed using an AU 400 Olympus Multianalyser. Four-day mortality is shown in Table 4. Mean BUN concentrations are shown in Table 4.

[0269] Table 4: 4-day mortality and BUN concentration [Table 4]

[0270] Table 4 shows that the NT-M antibody significantly reduced mortality. After four days, 70% of the mice treated with the NT-M antibody survived. After four days, 30% of the animals treated with the MR-M antibody survived, and 10% of the animals treated with the CT-M antibody survived. In contrast, when treated with nonspecific mouse IgG, all mice died after four days. The same results were obtained in the control group, in which mice were administered phosphate-buffered saline (PBS). The blood urea nitrogen (BUN) test is used to measure kidney function, to help diagnose kidney disease, and to monitor patients with acute or chronic kidney dysfunction or failure. The results of the S-BUN test revealed that the NT-M antibody was most effective in protecting the kidneys.

[0271] b) Late-stage treatment of sepsis Animal Model: Male C57Bl / 6 mice (Charles River Laboratories, Germany), aged 12 to 15 weeks, were used in the study. Peritonitis was surgically induced under light isoflurane anesthesia. An incision was made in the upper left quadrant of the abdominal cavity (the normal location of the cecum). The cecum was exposed, and a tight ligature was placed around the cecum distal to its attachment 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 expressed through the wound. The cecum was returned to the abdominal cavity, and the laparotomy site was closed. Finally, the animals were returned to their cages with free access to food and water. 500 μl of saline was given subcutaneously for hydration.

[0272] Compound (NT-M FAB2) application and dosage: NT-M FAB2 was tested against vehicle and control compound treatment. Treatment was performed after the full development of sepsis, i.e., 6 hours after CLP (late treatment). Each group contained 4 mice and was followed for a period of 4 days.

[0273] Group Treatment (10 μl / g body weight) Dose / Follow-up: 1 NT-M, FAB2 0.2mg / ml survived for 4 days 2 Control: Non-specific mouse IgG, 0.2 mg / ml, survival over 4 days 3 Vehicle: PBS 10 μl / g body weight Survival over 4 days

[0274] Table 5: 4-day mortality rate [Table 5]

[0275] Table 5 shows that the NT-M FAB2 antibody significantly reduced mortality. After 4 days, 75% of the mice treated with the NT-M FAB2 antibody survived. In contrast, when treated with nonspecific mouse IgG, all mice died after 4 days. The same results were obtained in the control group in which mice were administered PBS (phosphate-buffered saline).

[0276] Example 5 – Administration of NT-H to healthy humans The study was a randomized, double-blind, placebo-controlled trial conducted 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). The main inclusion criteria were written informed consent, age between 18 and 35 years, agreement to reliably practice contraception, and a BMI between 18 and 30 kg / m. 2 The objective of this study was to determine whether the ADM levels were significantly higher than those of the placebo group. Subjects received a single intravenous dose of NT-H antibody (0.5 mg / kg; 2 mg / kg; 8 mg / kg) or placebo administered by slow infusion over 1 hour in the laboratory. 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). The results show that ADM levels rapidly increased within the first 1.5 hours after administration of NT-H antibody to healthy individuals, then reached a plateau and slowly declined (Figure 6).

[0277] Example 6 – Methods for measuring DPP3 protein and DPP3 activity Generation of antibodies and measurement of DPP3 binding ability: Several mouse antibodies were generated and screened for their binding ability to human DPP3 in a specific binding assay (see Table 6).

[0278] Peptides / conjugates for immunization: DPP3 peptides for immunization were synthesized with an additional N-terminal cysteine ​​residue (if no cysteine ​​was present in the selected DPP3 sequence) for conjugation with bovine serum albumin (BSA), see Table 6 (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. Recombinant GST-hDPP3 was produced by USBio (United States Biological, Salem, MA, USA).

[0279] Immunization of mice, immune cell fusion, and screening: Balb / c mice were injected intraperitoneally (i.p.) with 84 μg of GT-hDDP3 or 100 μg of DPP3-peptide-BSA conjugate (emulsified in TiterMax Gold adjuvant) on day 0, 84 μg or 100 μg of DPP3-peptide-BSA conjugate (emulsified in complete Freund's adjuvant) on day 14, and 42 μg or 50 μg of DPP3-peptide-BSA conjugate (emulsified in incomplete Freund's adjuvant) on days 21 and 28. On day 49, mice were injected intravenously (i.v.) with 42 μg of GT-hDDP3 or 50 μg of DPP3-peptide-BSA conjugate dissolved in saline. Three days later, mice were euthanized and immune cell fusion was performed.

[0280] Spleen cells from immunized mice were fused with cells from the myeloma cell line SP2 / 0 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 in HAT medium (RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement). One week later, the HAT medium was replaced with HT medium for three passages, after which the cells were returned to normal cell culture medium. Two weeks after the fusion, cell culture supernatants were first screened for IgG antibodies that bind to recombinant DPP3. Therefore, GST-tagged recombinant hDPP3 (USBiologicals, Salem, USA) was immobilized (100 ng / well) on a 96-well plate and incubated with 50 μl of cell culture supernatant per well at room temperature for 2 hours. After washing the plate, 50 μl / well of POD-rabbit anti-mouse IgG was added and incubated at room temperature for 1 hour. After a subsequent washing step, 50 μl of chromophore solution (3.7 mM o-phenylenediamine in citrate / hydrogen phosphate buffer, 0.012% H2O2) was added to each well and incubated for 15 min at room temperature. The color reaction was then stopped by adding 50 μl of 4 N sulfuric acid. Absorbance was detected at 490 nm.

[0281] Positive microcultures were transferred to 24-well plates for expansion, and after retesting, selected cultures were cloned and recloned using limiting dilution techniques and isotyped.

[0282] Production of mouse monoclonal antibodies Antibodies against GST-tagged human DPP3 or DPP3 peptides were produced according to standard antibody production methods (Marx et al. 1997) and purified through protein A. The purity of the antibodies was ≥90% based on SDS gel electrophoresis analysis.

[0283] Antibody Characterization—Binding to hDPP3 and / or Immunizing Peptide Binding assays were performed to analyze the ability of different antibodies and antibody clones to bind to the DPP3 / immunizing peptide.

[0284] Solid phase: GST-tagged recombinant hDPP3 (SEQ ID NO: 34) or DPP3 peptide (immunizing peptide, SEQ ID NO: 35) was immobilized on the surface of a high-binding microtiter plate (96-well polystyrene microplate, Greiner Bio-One international AG, Austria, 1 μg per well in coupling buffer [50 mM Tris, 100 mM NaCl, pH 7.8] at room temperature for 1 hour). After blocking with 5% bovine serum albumin, the microplate was dried under vacuum.

[0285] Labeling procedure (tracer): 100 μg (100 μl) of various anti-DPP3 antibodies (1 mg / ml detection antibody in PBS, pH 7.4) were mixed with 10 μl of acridinium NHS ester (1 mg / ml in acetonitrile, InVent GmbH, Germany; EP 0 353 971) and incubated at room temperature for 30 minutes. The labeled anti-DPP3 antibodies were purified by gel filtration HPLC on Shodex Protein 5 μm KW-803 (Showa Denko, Japan). The purified labeled antibodies were diluted in assay buffer (50 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na2-EDTA, 5 g / L bovine serum albumin, 1 g / L mouse IgG, 1 g / L bovine IgG, 50 μmol / L amastatin, 100 μmol / L leupeptin, pH 7.4). The final concentration of labeled compound (approximately 20 ng of labeled antibody) was approximately 5-7 × 10 per 200 μl. 6 The chemiluminescence of the acridinium ester was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG) in relative light units (RLU).

[0286] hDPP3 binding assay: Plates were filled with 200 μl of labeled and diluted detection antibody (tracer) and incubated for 2–4 h at 2–8°C. Unbound tracer was removed by washing four times with 350 μl of wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Chemiluminescence of well-bound antibody was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

[0287] Antibody Characterization - hDPP3 Inhibition Assay To analyze the DPP3 inhibitory potential of various antibodies and antibody clones, a DPP3 activity assay was performed according to a previously described procedure (Jones et al., 1982). GST-tagged recombinant hDPP3 was diluted in assay buffer (25 ng / ml GST-DPP3 in 50 mM Tris-HCl, pH 7.5, and 100 μM ZnCl2), and 200 μl of this solution was incubated with 10 μg of each antibody at room temperature. After 1 h of preincubation, the fluorescent substrate Arg-Arg-βNA (20 μl, 2 mM) was added to the solution, and the generation of free βNA was monitored at 37°C using a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG). βNA fluorescence was detected by excitation at 340 nm and emission at 410 nm. The slope of increasing fluorescence (in RFU / min) was calculated for different samples. The gradient of native human DPP3 with buffer control is taken as 100% activity. The inhibitory activity of a potential capture binder is defined as the reduction (in %) of GST-hDPP3 activity upon incubation with said capture binder.

[0288] The table below lists the resulting antibodies and their binding rates (expressed in relative light units (RLU)) as well as their relative inhibitory potency (%; Table 6). The monoclonal antibodies against the following DPP3 regions were selected for their ability to bind to recombinant DPP3 and / or the immunizing peptide and their inhibitory potency.

[0289] All antibodies against the GST-tagged full-length form of recombinant hDPP3 showed strong binding to immobilized GST-tagged hDPP3. Antibodies against the peptide of SEQ ID NO: 35 also bind to GST-hDPP3. The antibody of SEQ ID NO: 35 also binds strongly to the immunizing peptide.

[0290] Table 6: List of antibodies against full-length hDPP3 or hDPP3 sequences and their binding ability (in RLU) to hDPP3 (SEQ ID NO: 34) or the immunizing peptide (SEQ ID NO: 35), as well as the maximum inhibition rate of recombinant GST-hDPP3. [Table 6]

[0291] The development of a fluorescent immunoassay (DPP3-LIA) for quantifying DPP3 protein concentration and an enzyme capture activity assay (DPP3-ECA) for quantifying DPP3 activity has recently been reported (Rehfeld et al. 2019. JALM 3(6): 943-953), which is incorporated herein by reference in its entirety.

[0292] Example 7 – DPP3 in shock DPP3 concentrations were measured in the plasma of patients suffering from sepsis / septic shock and cardiogenic shock and were associated with early mortality in the patients.

[0293] a) Study Cohort - Sepsis / Septic Shock 574 plasma samples from patients in the Adrenomedullin and Outcomes in Severe Sepsis and Septic Shock (AdrenOSS) study were measured for DPP3. AdrenOSS is a prospective, observational, multinational study including 583 patients admitted to intensive care units with sepsis or septic shock (Hollinger et al., 2018). 292 patients were diagnosed with septic shock.

[0294] b) Study cohort - cardiogenic shock Plasma samples from 108 patients diagnosed with cardiogenic shock were screened for DPP3. Blood was collected within 6 hours of detection of cardiogenic shock. Mortality was tracked over 7 days.

[0295] hDPP3 immunoassay: DPP3 levels in patient plasma were determined using immunoassays to detect human DPP3 levels (LIA) or activity assays to detect human DPP3 activity (ECA). Antibody immobilization, labeling, and incubation were performed as described by Rehfeld et al. (2019. JALM 3(6): 943-953).

[0296] result Early patient survival in sepsis patients was associated with DPP3 plasma concentrations at admission. Patients with DPP3 plasma concentrations above 40.5 ng / ml (third quartile) had an increased risk of death compared with patients with DPP3 plasma concentrations below this threshold (Figure 7A). Applying the same cutoff value to a subcohort of septic shock patients revealed an even more pronounced risk of early death associated with high DPP3 plasma concentrations (Figure 7B). When applying the same cutoff value to patients with cardiogenic shock, an increased risk of early death within 7 days was also observed in patients with high DPP3 levels (Figure 7C).

[0297] Example 8 - NT-ADM antibody (AdrenOSS-2) in patients suffering from septic shock AdrenOSS-2 is a double-blind, placebo-controlled, randomized, multicenter, proof-of-concept, and dose-ranging phase II clinical trial to investigate the safety, tolerability, and efficacy of an N-terminal ADM antibody called adrecizumab in patients with septic shock and elevated adrenomedullin (Geven et al. BMJ Open 2019;9:e024475). In total, 301 patients with septic shock and bio-ADM concentrations >70 pg / mL were randomized (2:1:1) to receive either placebo (n=152), adrecizumab 2 ng / kg (n=72), or adrecizumab 4 ng / kg (n=77) via a single intravenous infusion over approximately 1 hour. All-cause mortality within 28 (90) days after enrollment was 25.8% (34.8%). The mean age was 68.4 years, and 61% were male. For the per-protocol analysis, n=294 patients remained eligible, and the 14-day all-cause mortality rate was 18.5%.

[0298] A trend toward lower early mortality (14 days after admission) was observed in patients treated with adrecizumab (both doses combined, per protocol population) compared with placebo (hazard ratio (HR) 0.701 [0.408-1.21], p=0.100) (Figure 8). Surprisingly, the treatment effect was more pronounced in patients with DPP3 concentrations below 50 ng / mL on admission (n=244, HR 0.426, p=0.007) (Figure 9). However, in patients with elevated DPP3 (>50 ng / mL, n=44), outcomes were comparable between adrecizumab and placebo (HR 1.69, p=0.209) (Figure 10). The treatment effect (14-day mortality) for various DPP3 thresholds is summarized in Table 7.

[0299] Table 7. Hazard ratios (HR) for 14-day mortality using various DPP3 concentrations [Table 7]

[0300] array SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDY SEQ ID NO:4 QSIVYSNGNTY Sequence "RVS" (not part of the sequence listing): RVS SEQ ID NO:5 FQGSHIPYT

[0301] Sequence number 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0302] SEQ ID NO: 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0303] SEQ ID NO: 8 (AM-VH2E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0304] SEQ ID NO: 9 (AM-VH3-T26E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0305] SEQ ID NO: 10 (AM-VH4-T26E40E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0306] SEQ ID NO: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC

[0307] SEQ ID NO: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC

[0308] SEQ ID NO: 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC

[0309] SEQ ID NO: 14 (positions 1 to 21 of human ADM) YRQSMNNFQGLRSFGCRFGTC SEQ ID NO: 15 (positions 21 to 32 of human ADM) CTVQKLAHQIYQ SEQ ID NO: 16 (human ADM C-42 to C-52) CAPRSKISPQGY-CONH2 SEQ ID NO: 17 (positions 1 to 19 of mouse ADM) YRQSMNQGSRSNGCRFGTC SEQ ID NO: 18 (positions 19 to 31 of mouse ADM) CTFQKLAHQIYQ SEQ ID NO: 19 (mouse ADM C-40 to C-50) CAPRNKISPQGY-CONH2

[0310] SEQ ID NO: 20 (mature human adrenomedullin (mature ADM); amidated ADM; bio-ADM): amino acids 1 to 52 or 95 to 146 of pro-ADM YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY-CONH2 SEQ ID NO: 21 (positions 1 to 50 of mouse ADM) YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQLTDKDKDGMAPRNKISPQGY-CONH2 SEQ ID NO: 22 (positions 1 to 21 of human ADM): YRQSMNNFQGLRSFGCRFGTC SEQ ID NO: 23 (positions 1 to 42 of human ADM): YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA

[0311] SEQ ID NO: 24 (aa 43-52 of human ADM) PRSKISPQGY-NH2 SEQ ID NO: 25 (aa1-14 of human ADM) YRQSMNNFQGLRSF SEQ ID NO: 26 (aa1-10 of human ADM) YRQSMNNFQG SEQ ID NO: 27 (aa1 to 6 of human ADM) YRQSMN SEQ ID NO: 28 (aa1-32 of human ADM) YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQ

[0312] SEQ ID NO: 29 (aa 1 to 40 of mouse ADM) YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQLTDKDKDGMA SEQ ID NO: 30 (aa1 to 31 of mouse ADM) YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQL

[0313] SEQ ID NO: 31 (proADM: 164 amino acids (positions 22 to 185 of preproADM) ARLDVASEF RKKWNKWALS RGKRELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

[0314] SEQ ID NO: 32 (Addressizumab heavy chain) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0315] SEQ ID NO: 33 (Addressizumab light chain) DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC

[0316] SEQ ID NO: 34 - Human DPP3 (amino acids 1-737) MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAWYGGLAVLLQTSPEAPYIYALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTKFVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLRHLGLGKEGITTYF SGNCTMEDAKLAQDFLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDSEVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESFTQGSIEAHKRGSRFWIQDKGPIVESYIGFIESYRDPFGSRGEFFEGFVAVVNKAMSAKFE RLVASAEQLLKELPWPPTFEKDKFLTPDFTSLDVLTFAGSGIPAGINIPNYDDLRQTEGFKNVSLGNVLAVAYATQREKLTFLEEDDKDLYILWKGPSFDVQVGLHELLGHGSGKLFVQDEKGAFNFDQETVINPETGEQIQSWYRSGETWDSKFSTIASSYEECRAESVGL YLCLHPQVLEIFGFEGADAEDVIYVNWLNMVRAGLLALEFYTPEAFNWRQAHMQARFVILRVLLEAGEGLVTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTGDVAGGRALYEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLE YEASAAGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA

[0317] SEQ ID NO: 35 - Human DPP3 (amino acids 474-493 (N-Cys)) - immunizing peptide with an additional N-terminal Cysteine CETVINPETGEQIQSWYRSGE

[0318] SEQ ID NO: 36 - IGHV1-69*11 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYYYYYGMDVWGQGTTVTVSS

[0319] SEQ ID NO: 37 - HB3 QVQLQQSGAELMKPGASVKISKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSS

Claims

1. 1. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock, said method comprising: measuring the level of dipeptidyl peptidase 3 (DPP3) in a body fluid sample from said patient; comparing the measured DPP3 level to a predetermined threshold; and - the level of DPP3 in the sample indicates whether treatment with an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold is necessary; and wherein the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold binds to the N-terminal portion (amino acids 1 to 21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

2. 10. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to claim 1, said method comprising: measuring the level of dipeptidyl peptidase 3 (DPP3) in a body fluid sample from said patient; comparing the measured DPP3 level to a predetermined threshold; and administering to the patient an anti-adrenomedullin (ADM) antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold, wherein if the measured DPP3 level is below a predetermined threshold, the patient is treated with the anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold; and wherein the anti-ADM antibody, anti-ADM fragment, or anti-ADM non-Ig scaffold binds to the N-terminal portion (amino acids 1 to 21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 14).

3. 3. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to claim 1 or 2, wherein the shock is selected from the group comprising hypovolemic shock, cardiogenic shock, vaso-occlusive shock and distributive shock, in particular cardiogenic shock or septic shock.

4. 4. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 3, comprising: In the case of cardiogenic shock, the patient may be suffering from an acute coronary syndrome (e.g., acute myocardial infarction), or the patient may be suffering from heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or In the case of hypovolemic shock, the patient may have a bleeding disorder, including gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor invasion into major blood vessels) and spontaneous bleeding during anticoagulation, or a non-bleeding disorder, including vomiting, diarrhea, renal losses, cutaneous / insensible losses (e.g., burns, heat stroke), or third-space losses during pancreatitis, liver cirrhosis, intestinal obstruction, and trauma; or In the case of vaso-occlusive shock, the patient may be suffering from cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or In the case of distributive shock, the patient may be suffering from septic shock, neurogenic shock, anaphylactic shock or shock due to adrenal crisis.

5. 5. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 4, wherein the predetermined threshold value for DPP3 in a body fluid sample of the subject is 20 to 120 ng / mL, more preferably said threshold value is 30 to 80 ng / mL, even more preferably said threshold value is 40 to 60 ng / mL, and most preferably said threshold value is 50 ng / mL.

6. 6. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 5, wherein either the level of DPP3 protein and / or the level of active DPP3 is measured and compared with a predefined threshold value.

7. 7. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 6, wherein the level of DPP3 is measured by contacting the body fluid sample with a capture binder that specifically binds to DPP3.

8. 8. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 7, wherein said measuring comprises the use of a capture binder that specifically binds to full-length DPP3, wherein said capture binder may be selected from the group comprising an antibody, an antibody fragment, or a non-IgG scaffold.

9. 9. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state according to any one of claims 1 to 8, wherein the amount of DPP3 protein and / or DPP3 activity is measured in a body fluid sample of the subject, and said measuring comprises the use of a capture binder that specifically binds to full-length DPP3, wherein said capture binder is an antibody.

10. 10. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 9, wherein the amount of DPP3 protein and / or DPP3 activity is measured in a body fluid sample of the subject, and said measuring comprises the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is immobilized on a surface.

11. 11. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state according to any one of claims 1 to 10, wherein the amount of DPP3 protein and / or DPP3 activity is measured in a body fluid sample of the subject, and the separation step is a washing step that removes sample components that are not bound to the capture binder from the captured DPP3.

12. 12. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 11, wherein the method for measuring DPP3 activity in a body fluid sample of said subject comprises the following steps: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; - adding a DPP3 substrate to the separated DPP3; Quantifying DPP3 activity by measuring and quantifying the conversion of a DPP3 substrate; A method comprising:

13. 13. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 12, wherein the DPP3 activity is measured in a body fluid sample of the subject and the DPP3 substrate conversion is detected by a method selected from the group comprising: fluorescence of a fluorogenic substrate (e.g., Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate linked to aminoluciferin (Promega Protease-Glo™ Assay), mass spectrometry, HPLC / FPLC (reverse-phase chromatography, size-exclusion chromatography), thin-layer chromatography, capillary zone electrophoresis, gel electrophoresis after activity staining (fixed, active DPP3) or Western blotting (cleavage products).

14. 14. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 13, wherein the DPP3 activity is measured in a body fluid sample of the subject and the substrate can be selected from the group comprising: angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, wherein the dipeptide is Arg-Arg.

15. 15. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 14, wherein the DPP3 activity is measured in a body fluid sample of the subject and the substrate can be selected from the group comprising: a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, wherein the dipeptide is Arg-Arg.

16. 16. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 15, wherein said patients have an ADM-NH above a threshold. 2 The method further characterized by having levels.

17. 17. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to claim 16, comprising detecting ADM-NH in a body fluid sample of said patient. 2 wherein the threshold is 40-100 pg / mL, more preferably 50-90 pg / mL, even more preferably 60-80 pg / mL, and most preferably 70 pg / mL.

18. 18. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to claim 16 or 17, wherein said ADM-NH 2 The level of ADM-NH 2 wherein the antibody is measured by contacting the antibody with a capture binder that specifically binds to the antibody.

19. 19. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in a patient suffering from shock and / or in a state of shock according to any one of claims 1 to 18, wherein the patient's body fluid sample is selected from the group of blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.

20. 20. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 19, comprising comparing the levels of DPP3 and ADM-NH 2 The levels of are measured in combination.

21. 21. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to claim 20, wherein the level of DPP3 and ADM-NH 2 The levels of

22. 22. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to claim 20 or 21, comprising comparing the levels of DPP3 and ADM-NH 2 wherein the level of is measured using a point-of-care device.

23. 23. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a shock state according to claim 22, wherein the point-of-care device is a microfluidic device.

24. 24. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 23, wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to the N-terminal end (first amino acid) of ADM.

25. 25. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 24, wherein said antibody, antibody fragment or non-Ig scaffold is a C-terminal part of ADM having the sequence amino acids 43 to 52 of ADM: PRSKISPQGY-NH 2 (SEQ ID NO: 24).

26. 26. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 25, wherein said antibody or fragment is a monoclonal antibody or fragment that binds to ADM or an antibody fragment thereof, wherein its heavy chain has the following sequence: CDR1: SEQ ID NO: 1 GYTFSRYW CDR2: SEQ ID NO: 2 ILPGSGST CDR3: SEQ ID NO: 3 TEGYEYDGFDY and wherein the light chain comprises the following sequence: CDR1: SEQ ID NO: 4 QSIVYSNGNTY CDR2: RVS CDR3: SEQ ID NO: 5 FQGSHIPYT A method comprising:

27. 27. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 26, wherein said antibody or fragment comprises as a VH region: Sequence number 6 (AM-VH-C) QVQLQQSGAELMKPGASVKISKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 7 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 8 (AM-VH2E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 9 (AM-VH3-T26E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SEQ ID NO: 10 (AM-VH4-T26E40E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK and as a VL region the following sequence: SEQ ID NO: 11 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 12 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC The method of claim 1, wherein the sequence is selected from the group comprising:

28. 27. A method for therapy guidance and / or therapy monitoring and / or therapy stratification in patients suffering from shock and / or in a state of shock according to any one of claims 1 to 26, wherein said antibody or fragment comprises as heavy chain the following sequence: SEQ ID NO: 32 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK or a sequence >95% identical thereto, and having as the light chain the following sequence: SEQ ID NO: 33 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC or a sequence that is >95% identical thereto, wherein the heavy chain has the following sequence: CDR1: SEQ ID NO: 1 GYTFSRYW CDR2: SEQ ID NO: 2 ILPGSGST CDR3: SEQ ID NO: 3 TEGYEYDGFDY and wherein the light chain comprises the following sequence: CDR1: SEQ ID NO: 4 QSIVYSNGNTY CDR2: RVS CDR3: SEQ ID NO: 5 FQGSHIPYT A method comprising:

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