Predicting increase in DPP3 in patients with sepsis shock

Abstract

The present invention relates to a method for predicting an increase in dipeptidyl peptidase 3 (DPP3) in critical patients. Specifically, the method comprises providing a sample from the patient, determining a dipeptidyl peptidase 3 (DPP3) level in the sample, comparing the level to a predetermined threshold, where if the DPP3 level is above a predetermined threshold level, the DPP3 level in the sample indicates an increase in DPP3, the predetermined threshold level being in a range between 22 ng / ml and 40 ng / ml. Furthermore, the present invention relates to a method of preventing an increase in DPP3 in a critically ill patient wherein a DPP3 inhibitor is administered to the patient if the DPP3 level is above a threshold between 40 ng / ml and 22 ng / ml, and the DPP3 inhibitor is an anti-DPP3 antibody and / or an anti-DPP3 antibody fragment and / or an anti-DPP3 scaffold. In addition, the present invention relates to a DPP3 inhibitor for use in the prevention of an increase in DPP3 in critically ill patients.

Description

Technical Field

[0001] The present invention relates to a method for predicting an increase in dipeptidyl peptidase 3 (DPP3) in a critically ill patient. Furthermore, the present invention also relates to a method for preventing an increase in DPP3 in a critically ill patient, wherein if the DPP3 level is above a threshold between 40 ng / ml and 22 ng / ml, a DPP3 inhibitor is administered to the patient, and the DPP3 inhibitor is an anti-DPP3 antibody and / or an anti-DPP3 antibody fragment and / or an anti-DPP3 scaffold. Furthermore, the present invention also relates to a DPP3 inhibitor for preventing an increase in DPP3 in a critically ill patient. Background Art

[0002] Dipeptidyl peptidase 3 - also known as dipeptidyl aminopeptidase III, dipeptidyl arylamidase III, dipeptidyl peptidase III, enkephalinase B or erythrocyte angiotensin; abbreviations: DPP3, DPPIII - is a metallopeptidase that removes dipeptides from physiologically active peptides such as enkephalins and angiotensin. DPP3 was first identified by Ellis and Nuenke in 1967 and its activity was measured in purified bovine anterior pituitary extracts. The enzyme, classified as EC 3.4.14.4, has a molecular mass of approximately 83 kDa and is highly conserved in prokaryotes and eukaryotes ( Prajapati and Chauhan 2011 The amino acid sequence of the human variant is depicted in SEQ ID NO 36. Dipeptidyl peptidase III is a ubiquitously expressed major cytoplasmic peptidase. Despite the lack of a signal sequence, some studies have reported membrane activity ( Lee and Snyder 1982 ).

[0003] DPP3 is a zinc-dependent exopeptidase belonging to the peptidase family M49. It has a broad substrate specificity for oligopeptides of three or four to ten amino acids of varying composition and is also able to cleave behind proline. DPP3 is known to hydrolyze dipeptides from the N-terminus of its substrates, including angiotensin II, III and IV; Leu- and Met-enkephalins; endomorphins 1 and 2. The metallopeptidase DPP3 has its optimal activity at pH 8.0-9.0 and can be activated by the addition of divalent metal ions such as Co 2+ and Mg 2+ And activated.

[0004] Structural analysis of DPP3 revealed the catalytic motifs HELLGH (hDPP3 450-455) and EECRAE (hDPP3 507-512), as well as the following amino acids important for substrate binding and hydrolysis: Glu316, Tyr318, Asp366, Asn391, Asn394, His568, Arg572, Arg577, Lys666, and Arg669 ( Prajapati & Chauhan 2011; Kumar et al. 2016; Numbering refers to the sequence of human DPP3, see SEQ ID NO. 36). Taking into account all known amino acids or sequence regions involved in substrate binding and hydrolysis, the active site of human DPP3 can be defined as the region between amino acids 316 and 669.

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

[0006] Recently, two assays were generated, characterized, and validated to specifically detect DPP3 in human body fluids (e.g., blood, plasma, serum): a luminescent immunoassay (LIA) to detect DPP3 protein concentration and an enzyme capture activity assay (ECA) to detect specific DPP3 activity ( Rehfeld et al. 2019. JALM 3(6): 943-953 ). Before the actual detection of DPP3 activity is performed, a washing step removes all interfering substances. Both methods are highly specific and allow reproducible detection of DPP3 in blood samples.

[0007] Circulating DPP3 levels were shown to be increased in patients with cardiogenic shock and were associated with an increased risk of short-term mortality and severe organ dysfunction ( Deaniau et al. 2020. Eur J Heart Fail. 22(2):290-299 In addition, DPP3 measured at inclusion distinguished cardiogenic shock patients who developed refractory shock from those who did not, and DPP3 concentrations ≥59.1 ng / mL were associated with a greater risk of death ( Takagi et al. 2020. Eur J Heart Fail. 22 (2):279-286 ).

[0008] The peptide adrenomedullin (ADM) was first described in 1993 ( Kitamura et al., 1993. Biochem Biophys Res Comm 192 (2): 553-560) as a novel hypotensive peptide comprising 52 amino acids, which has been isolated from a human pheochromocytoma cell line (SEQ ID No.: 20). In the same year, a cDNA encoding a precursor peptide comprising 185 amino acids and the complete amino acid sequence of the precursor peptide were also described. The precursor peptide, which contains a signal sequence of 21 amino acids in particular at the N-terminus, is called "pre-pro-adrenomedullin" (pre-proADM). In the present specification, all amino acid positions specified are generally related to pre-proADM comprising 185 amino acids. Peptide ADM is a peptide comprising 52 amino acids (SEQ ID No: 20) and including amino acids 95 to 146 of pre-proADM, which is formed from pre-proADM by proteolytic cleavage. To date, only a few of the peptide fragments formed in the cleavage of pre-proADM have been studied more precisely, in particular the physiologically active peptide ADM and the "PAMP", a peptide comprising 20 amino acids (22-41) that follows the 21 amino acids of the signal peptide in pre-proADM. The discovery and characterization of ADM in 1993 triggered an intensive research campaign, the results of which have been summarized in various review articles, in the context of the present specification, reference is made in particular to the articles found in the publication "Peptides" which focus in particular on ADM ( Takahashi 2001. Peptides 22: 1691; Eto 2001. Peptides 22: 1693-1711 A further review is by Hinson et al. 2000 ( Hinson et al. 2000. Endocrine Reviews 21(2):138-167 In scientific research to date, it has been found that ADM can be considered a multifunctional regulatory peptide. It is released into the circulation in an inactive form extended by glycine ( Kitamura et al. 1998. Biochem Biophys Res Commun 244(2): 551-555 There is also a binding protein ( Pio et al. 2001. The Journal of Biological Chemistry 276(15): 12292-12300 ), which binding protein is specific for ADM and may also regulate the effects of ADM. In the studies so far, the primary physiological effects of ADM and PAMPs are those that affect blood pressure.

[0009] Therefore, ADM is a potent vasodilator, and therefore the hypotensive effect can be associated with a specific peptide segment in the C-terminal portion of ADM. It has also been found that the above-mentioned physiologically active peptide PAMP formed from pre-proADM also exhibits a hypotensive effect, even though it appears to have a different mechanism of action from ADM (in addition to the above-mentioned review article, there is Eto et al. 2001 and Hinson et al. 2000 , see also 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). It has also been found that ADM concentrations, which can be measured in the circulation and other biological fluids, are in many pathological states significantly higher than those found in healthy control subjects. Thus, in patients with congestive heart failure, myocardial infarction, kidney disease, hypertensive conditions, diabetes, in the acute phase of shock, and in sepsis and septic shock, ADM levels are significantly increased, although to varying degrees. PAMP concentrations are also increased in some of the pathological states, but plasma levels are lower relative to ADM ( Eto 2001. Peptides 22: 1693- 1711 Abnormally high ADM concentrations have been reported in sepsis, with the highest concentrations observed 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 disease severity ( Hirayama et al. 1999. J Endocrinol 160: 297–303; Yu et al. 2001. Heart 86: 155–160 High plasma ADM was an independent negative prognostic indicator in these subjects ( Poyner et al. 2002. Pharmacol Rev 54: 233–246 ).

[0011] WO2004 / 097423 describes the use of antibodies to ADM for the diagnosis, prognosis and treatment of cardiovascular disorders. The art also describes the treatment of diseases by blocking ADM receptors (e.g., WO2006 / 027147, PCT / EP2005 / 012844), which can be sepsis, septic shock, cardiovascular disease, infection, skin disease, endocrine disease, metabolic disease, gastrointestinal disease, cancer, inflammation, blood disease, respiratory disease, musculoskeletal disease, neurological disease, urinary disease).

[0012] It has been reported that ADM improves cardiac function and blood supply to the liver, spleen, kidneys, and small intestine in the early stages of sepsis. Anti-ADM neutralizing antibodies neutralize the aforementioned effects in the early stages of sepsis ( Wang et al. 2001. Peptides 22: 1835-1840 ).

[0013] For other diseases, blocking ADM may be beneficial to a certain extent. However, it may also be harmful if ADM is completely neutralized, because there are several physiological functions that may require a certain amount of ADM. It is emphasized in many reports that the administration of ADM may be beneficial in certain diseases. In contrast, in other reports, it is reported that ADM is life-threatening when administered under certain conditions.

[0014] WO2013 / 072510 describes a non-neutralizing anti-ADM antibody for use in treating a serious chronic or acute disease or acute condition in a patient to reduce the risk of death in the patient.

[0015] WO2013 / 072511 describes a non-neutralizing anti-ADM antibody for use in treating a chronic or acute disease or acute condition in a patient to prevent or reduce organ dysfunction or organ failure.

[0016] WO2013 / 072512 describes a non-neutralizing anti-ADM antibody that enhances the half-life (t 1 / 2 The ADM stabilizing antibody has a half retention time of 0.5-1.0. This ADM stabilizing antibody blocks the biological activity of ADM to less than 80%.

[0017] WO2013 / 072513 describes a non-neutralizing anti-ADM antibody for use in treating an acute disease or condition in a patient to stabilize circulation.

[0018] WO2013 / 072514 describes a non-neutralizing anti-ADM antibody for use in regulating fluid balance in patients suffering from a chronic or acute disease or acute condition.

[0019] WO2017 / 182561 describes a method for determining the total amount of active DPP3 in a patient sample to diagnose a disease associated with a necrotic process. It further describes a method for treating a necrosis-related disease by an antibody against DPP3.

[0020] WO2021 / 170838 describes a method for treatment guidance and / or treatment monitoring and / or treatment stratification in patients with shock and patients developing shock by determining DPP3 levels and administering anti-ADM antibodies if the level is below a threshold of preferably 50 ng / ml.

[0021] High DPP3 blood levels are associated with higher organ dysfunction scores, the need for cardiovascular support, and the development of myocardial dysfunction, refractory shock, acute kidney injury, and increased short-term mortality. Based on these clinical associations, it seems plausible that active DPP3 released in the blood of shock patients is a factor that contributes to the deterioration of vascular tone in shock syndrome by abrogating the vasopressor effects of endogenous Ang II ( Malovan et al. 2023 FEBS Journal 290(9):2246- 2262). In patients with shock, the presence of endothelial dysfunction is detected with the aid of the biomarker bio-ADM, while cardiac dysfunction is detected by measuring DPP3. DPP3 above a threshold has been used as an exclusion criterion for the use of drugs targeting pathways other than DPP3, such as anti-ADM antibodies, in particular the anti-ADM antibody Adrecizumab, which targets the N-terminus of ADM. Adrecizumab therapy shows better efficacy if patients with DPP3 above 50 ng / ml are excluded from treatment (WO2021 / 170838). However, in patients with initial DPP3 levels below 50 ng / ml, the DPP3 level may increase to above 50 ng / ml in the following time, which would indicate an exclusion for the use of drugs targeting pathways other than DPP3. Therefore, the surprising finding of the present invention is that if in critically ill patients DPP3 is well below a threshold of 50 ng / ml, preferably below a threshold of ≤ 40 ng / ml or in a range between 22 ng / ml and 40 ng / ml, the patient will be unlikely to experience an increase in DPP3 during follow-up. On the other hand, this means that if in critically ill patients DPP3 is above a threshold of 40 ng / ml, preferably above a threshold in a range between 40 ng / ml and 22 ng / ml, the patient will be more likely to experience an increase in DPP3 during follow-up. Furthermore, the inventors found that this increase is a short-term increase that will occur within a few hours or at most 7 days, respectively.

[0022] This would therefore mean that patients whose DPP3 levels are below a threshold in the range between 22 ng / ml and 40 ng / ml can be treated with drugs targeting pathways other than DPP3, such as anti-ADM antibodies, in particular the anti-ADM antibody adrezumab, and patients whose DPP3 levels are above a threshold in the range between 40 ng / ml and 22 ng / ml cannot be treated with drugs targeting pathways other than DPP3, such as anti-ADM antibodies, in particular the anti-ADM antibody adrezumab.

[0023] The purpose of the present invention is to reduce the number of critically ill patients who will experience a short-term increase in DPP3 levels (e.g., within a few hours to 7 days) to above a critical cutoff value (e.g., above 40ng / ml or 50ng / ml), which means pursuing a high sensitivity of the test for this endpoint. Another surprising discovery of the present invention is that DPP3 is predicted not to increase above the critical cutoff value in critically ill patients during follow-up, wherein the DPP3 level is below a predetermined threshold level, and the predetermined threshold level is in the range between 22ng / ml and 40ng / ml. In this way, it is possible to identify patients who are suitable for treatment with drugs for pathways other than DPP3, such as anti-ADM antibodies, especially anti-ADM antibody aderezumab. Surprisingly, in critically ill patients during short-term follow-up, the "irrecoverable point" when the DPP3 level will further increase to above the critical cutoff value has reached a much lower DPP3 level than known in the art. Identifying patients, particularly as early as possible, who will not experience such short-term increases in DPP3 levels above a critical cutoff, significantly improves the overall efficacy of treatment for the patient population, which treatment may be standard of care treatment, or drugs that target pathways other than DPP3, such as anti-ADM antibodies, particularly the anti-ADM antibody aderizumab.

[0024] Description of the Invention

[0025] The subject of the present invention is a method for predicting an increase in dipeptidyl peptidase 3 (DPP3) in a critically ill patient, the method comprising:

[0026] Determining the level of DPP3 in a body fluid sample of said patient,

[0027] - comparing said determined DPP3 level with a predetermined threshold value,

[0028] wherein the predetermined threshold value of DPP3 is within the range between 40 ng / ml and 22 ng / ml,

[0029] wherein the level of DPP3 in the sample being above the predetermined threshold indicates an increase in DPP3 in the patient.

[0030] One embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein the patient is a patient with severe infection, sepsis, heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, a patient with organ dysfunction or organ failure (e.g., liver, kidney, lung dysfunction or failure), a patient undergoing major surgery, a patient with trauma (e.g., burns, multiple trauma), a patient with shock and / or a patient developing shock.

[0031] One embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein the patient suffers from shock or is developing shock, wherein the shock is selected from the group consisting of shock caused by hypovolemia, cardiogenic shock, obstructive shock and distributive shock, in particular cardiogenic shock or septic shock.

[0032] A preferred embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein the patient suffers from shock and / or is developing shock, wherein

[0033] In the case of cardiogenic shock, the patient may have suffered an acute coronary syndrome (e.g., acute myocardial infarction) or in which the patient has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmias, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or

[0034] In the setting of hypovolemic shock, the patient may have had a bleeding disorder including gastrointestinal bleeding, trauma, vascular etiologies (e.g., ruptured abdominal aortic aneurysm, tumor erosion into a major vessel), and spontaneous bleeding in the setting of anticoagulant use, or a non-bleeding disorder including vomiting, diarrhea, renal loss, skin loss / insensitivity (e.g., burns, heat stroke) or pancreatitis, cirrhosis, intestinal obstruction, third space loss in the setting of trauma, or

[0035] In the case of obstructive shock, the patient may have suffered cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or

[0036] • In cases of distributive shock, the patient may be suffering from septic shock, neurogenic shock, anaphylactic shock, or shock caused by adrenal crisis.

[0037] Another embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein the predetermined threshold value of DPP3 in a body fluid sample of the subject is within a range between 40 ng / ml and 22 ng / mL.

[0038] Another embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein the predetermined threshold value of DPP3 in a body fluid sample of the subject is within a range between 30 ng / ml and 22 ng / ml.

[0039] Another embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein the predetermined threshold value of DPP3 in a body fluid sample of the subject is within a range between 25 ng / ml and 22 ng / ml.

[0040] Another embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein the predicted increase is an increase to a DPP3 level equal to or higher than 40 ng / ml, preferably equal to or higher than 50 ng / ml.

[0041] Another embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein the predicted increase in DPP3 level is equal to or higher than 10%, more preferably equal to or higher than 20%, even more preferably equal to or higher than 40%, even more preferably equal to or higher than 50%, even more preferably equal to or higher than 75%, even more preferably equal to or higher than 100%. Specifically, this relates to an increase in DPP3 level relative to the DPP3 level determined in a sample.

[0042] Another embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein the predicted increase in DPP3 level is equal to or higher than 2.5 ng / ml, more preferably equal to or higher than 5 ng / ml, even more preferably equal to or higher than 10 ng / ml, even more preferably equal to or higher than 15 ng / ml, even more preferably equal to or higher than 20 ng / ml, even more preferably equal to or higher than 25 ng / ml. Specifically, this relates to an increase in DPP3 level relative to the DPP3 level determined in the sample.

[0043] Another embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein if the DPP3 level in the sample is below the predetermined threshold, the patient is selected for therapy / treatment, wherein the therapy is selected from the group consisting of alkaline phosphatase, immunosuppressants, corticosteroids, vasopressors, fluids, anti-ADM antibodies or antibody fragments, or scaffolds.

[0044] Another embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein if the DPP3 level in the sample is above the predetermined threshold, the patient is selected for therapy / treatment with a DPP3 inhibitor, wherein the DPP3 inhibitor is selected from the group of anti-DPP3 antibodies or anti-DPP3 antibody fragments or anti-DPP3 scaffolds.

[0045] Another specific embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein the level of DPP3 protein and / or the level of active DPP3 is determined and compared to a predetermined threshold.

[0046] Another preferred embodiment of the present application relates to a method for predicting an increase in DPP3 in critically ill patients, wherein the DPP3 level is determined by contacting the body fluid sample with a capture binder that specifically binds to DPP3.

[0047] One embodiment of the present application relates to a method for predicting an increase in DPP3 in critically ill patients, wherein the assay comprises the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder can be selected from the group of antibodies, antibody fragments or non-IgG scaffolds.

[0048] Another embodiment of the present application relates to a method for predicting an increase in DPP3 in critically ill patients, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and wherein the determination comprises the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody.

[0049] One embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and wherein the determination comprises the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is immobilized on a surface.

[0050] Another embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein the DPP3 level is DPP3 activity, wherein the method for determining the DPP3 activity in a body fluid sample of the subject comprises the following steps:

[0051] ● contacting the sample with a capture binder that specifically binds full-length DPP3,

[0052] ● separating DPP3 bound to the capture binder,

[0053] ● adding a DPP3 substrate to the isolated DPP3,

[0054] - Quantification of the DPP3 activity by measuring and quantifying the conversion rate of the DPP3 substrate.

[0055] Another embodiment of the present application relates to a method for treatment guidance and / or treatment monitoring and / or treatment stratification in critically ill patients, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and wherein the separation step is a washing step for removing sample components that are not bound to the capture binder from the captured DPP3.

[0056] Another specific embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein DPP3 activity is determined in a body fluid sample of the subject, and wherein the DPP3 substrate conversion rate is detected by a method selected from the group consisting of: fluorescence of a fluorescent substrate (e.g., Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate coupled to aminofluorescein (Promega Protease-Glo ™ assay), mass spectrometry, HPLC / FPLC (reversed phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis followed by activity staining (fixed active DPP3) or western blot (lysate).

[0057] Another preferred embodiment of the present application relates to a method for predicting an increase in DPP3 in critically ill patients, wherein DPP3 activity is determined in a body fluid sample of the subject and wherein the substrate can be selected from the group comprising angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide coupled to a fluorophore, a chromophore or aminofluorescein, wherein the dipeptide is Arg-Arg.

[0058] Another embodiment of the present application relates to a method for predicting an increase in DPP3 in critically ill patients, wherein DPP3 activity is determined in a body fluid sample of the subject and wherein the substrate may be selected from the group comprising: a dipeptide coupled to a fluorophore, a chromophore or aminofluorescein, wherein the dipeptide is Arg-Arg.

[0059] Another embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein the body fluid sample of the patient is selected from the group consisting of whole blood, serum and plasma.

[0060] The level of DPP3 as the amount of DPP3 protein and / or DPP3 activity in the body fluid sample of the subject can be determined by various methods, such as immunoassay, activity assay, mass spectrometry, and the like.

[0061] DPP3 activity can be measured by detecting the cleavage products of DPP3-specific substrates. Known peptide hormone substrates include Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, β-casomorphin, dynorphin, protoenterin, ACTH (adrenocorticoid hormone), and MSH (melanocyte stimulating hormone; Abramić et al. 2000, Baršun et al. 2007, Dhanda et al. 2008The peptide hormones mentioned, as well as other unlabeled oligopeptides (e.g. Ala-Ala-Ala-Ala, Dhanda et al. 2008 ) can be monitored by detecting the respective cleavage products. Detection methods include, but are not limited to, HPLC analysis (e.g. Lee and Snyder 1982 ), mass spectrometry (e.g. Abramić et al. 2000 ), H1-NMR analysis (e.g. Vandenberg et al. 1985 ), capillary zone electrophoresis (CE; e.g. Baršun et al. 2007 ), thin layer chromatography (e.g. Dhanda et al. 2008 ) or reverse phase chromatography (e.g. Mazocco et al. 2006 ).

[0062] Detection of fluorescence due to hydrolysis of fluorescent substrates by DPP3 is a standard procedure for monitoring DPP3 activity. Those 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. Fluorophores include, but are not limited to, β-naphthylamide (2-naphthylamide, βNA, 2NA), 4-methoxy-β-naphthylamide (4-methoxy-2-naphthylamide), and 7-amido-4-methylcoumarin (AMC, MCA; Abramić et al. 2000, Ohkubo et al. 1999 ). Cleavage of these fluorescent substrates results in the release of fluorescent β-naphthylamide or 7-amino-4-methylcoumarin, respectively. In a liquid phase assay, the ECA substrate and DPP3 are incubated in, for example, a 96-well plate format and the fluorescence is measured using a fluorescence detector ( Ellis and Nuenke 1967 Alternatively, samples carrying DPP3 can be fixed by electrophoresis and separated on a gel, the gel is stained with a fluorescent substrate (e.g., Arg-Arg-βNA) and Fast Garnet GBC and the fluorescent protein bands are detected by a fluorescence reader ( Ohkubo et al.1999 The same peptide (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 such as Asp-nitroaniline diacetate. Detection of a color change due to hydrolysis of the chromogenic substrate can be used to monitor DPP3 activity.

[0063] Another option for detecting DPP3 activity is Protease-Glo ™ Assay (commercially available from Promega). In this embodiment of the method, a DPP3-specific dipeptide or tripeptide (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) is coupled to aminoluciferin. Upon cleavage by DPP3, aminoluciferin is released and serves as a substrate for a coupled luciferase reaction that emits detectable luminescence.

[0064] In a preferred embodiment, DPP3 activity is measured by adding the fluorescent substrate Arg-Arg-βNA and monitoring fluorescence in real time.

[0065] In a specific embodiment of said method for determining active DPP3 in a bodily fluid sample of a subject, said capture binder reactive with DPP3 is immobilized on a solid phase.

[0066] The test sample is passed over the immobilized conjugate, and DPP3, if present, binds to the conjugate and itself is immobilized for detection. Substrate may then be added, and the reaction product may be detected to indicate the presence or amount of DPP3 in the test sample. For the purposes of this specification, the term "solid phase" may be used to include any material or container in or on which an assay may be performed, and includes, but is not limited to: porous materials, non-porous materials, test tubes, wells, slides, agarose resins (e.g., Sepharose from GE Healthcare Life Sciences), magnetic particles (e.g., Dynabeads from Thermo Fisher Scientific), ™ or Pierce ™ Magnetic beads), etc.

[0067] In another embodiment of the invention, the level of DPP3 is determined by contacting said body fluid sample with a capture binder that specifically binds to DPP3.

[0068] In another preferred embodiment of the present invention, the capture binder used for determining the level of DPP3 may be selected from the group of antibodies, antibody fragments or non-IgG scaffolds.

[0069] In a specific embodiment of the present invention, the capture conjugate is an antibody.

[0070] The DPP3 level may be the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of the subject and may be determined, for example, by one of the following methods:

[0071] 1. Luminescent Immunoassay for Quantification of DPP3 Protein Concentration (LIA) Rehfeld et al., 2019 JALM 3 (6): 943-953 ).

[0072] LIA is a one-step chemiluminescent sandwich immunoassay that uses white high-binding polystyrene microtiter plates as solid phase. The plates are coated with the monoclonal anti-DPP3 antibody AK2555 (capture antibody). The tracer anti-DPP3 antibody AK2553 is labeled with MA70-acridinium-NHS-ester and used at a concentration of 20 ng / well. Twenty microliters of sample (e.g., serum, heparin-plasma, citrate-plasma, or EDTA-plasma derived from patient blood) and calibrators are pipetted into the coated white microtiter plates. After adding the tracer antibody AK2553, the microtiter plates are incubated at room temperature and 600 rpm for 3 hours. Unbound tracer is then removed by 4 washing steps (350 µL per well). The residual chemiluminescence is measured for 1 second per well by using a microtiter plate photometer. The concentration of DPP3 is determined using a 6-point calibration curve. Calibrators and samples are preferably run in duplicate.

[0073] 2. Enzyme capture activity assay for quantification of DPP3 activity (ECA) Rehfeld et al., 2019 JALM 3 (6): 943-953 ).

[0074] ECA is a DPP3-specific activity assay using black high-binding polystyrene microtiter plates as solid phase. The plates are coated with the monoclonal anti-DPP3 antibody AK2555 (capture antibody). Twenty microliters of sample (e.g., serum, heparin-plasma, citrate-plasma, EDTA-plasma) and calibrators are pipetted into the coated black microtiter plates. After addition of 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 sample components are removed by 4 washing steps (350 µL per well). The specific activity of the immobilized DPP3 is measured by addition of the fluorescent substrate Arg-Arg-β-naphthylamide (Arg2-βNA) to the reaction buffer followed by incubation at 37°C for 1 hour. DPP3 specifically cleaves Arg2-βNA into Arg-Arg dipeptide and fluorescent β-naphthylamine. Fluorescence was measured with a fluorimeter using an excitation wavelength of 340 nm and emission detected at 410 nm. DPP3 activity was determined using a 6-point calibration curve. Calibrators and samples were preferably run in duplicate.

[0075] 3. Liquid phase assay (LAA) for quantification of DPP3 activity (modified from Jones et al., Analytical Biochemistry, 1982 ).

[0076] LAA is a liquid phase assay that measures DPP3 activity using black non-binding polystyrene microtiter plates. Twenty microliters of sample (e.g., serum, heparin-plasma, citrate-plasma) and calibrators are pipetted into non-binding black microtiter plates. After addition of the fluorescent substrate Arg2-βNA in assay buffer (200µL), the initial βNA fluorescence (T=0) is measured in a fluorimeter using an excitation wavelength of 340nm and emission detection at 410nm. The plate is then incubated at 37°C for 1 hour. The final fluorescence is measured (T=60). The difference between the final fluorescence and the initial fluorescence is calculated. DPP3 activity is determined using a 6-point calibration curve. Calibrators and samples are preferably run in duplicate.

[0077] In a specific embodiment, an assay is used to determine DPP3 levels, wherein the assay sensitivity of the assay is capable of quantifying DPP3 in healthy subjects and is <20 ng / ml, preferably <30 ng / ml and more preferably <40 ng / ml.

[0078] In a specific embodiment, the binder exhibits a binding affinity for DPP3 of at least 10 7 M -1 , preferably 10 8 M -1 , preferably with an affinity greater than 10 9 M -1 , most preferably greater than 10 10 M -1 Those skilled in the art will appreciate that compensating for lower affinity by administering a higher dose of the compound may be considered, and such measures would not be beyond the scope of the present invention.

[0079] In a particular embodiment, the body fluid according to the present invention is a blood sample. The blood sample can 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 citrate plasma, heparin plasma and EDTA plasma.

[0080] In one embodiment, such assays for determining DPP3 levels are sandwich immunoassays using any type of detection technology (including but not limited to enzyme labels, chemiluminescent labels, electrochemiluminescent labels), preferably fully automated assays. In one embodiment of the diagnostic method, such assays are enzyme-labeled sandwich assays. Examples of automated or fully automated assays include assays that can be used in one of the following systems: Roche Elecsys ® 、Abbott Architect ® 、Siemens Centauer ® 、Brahms Kryptor ®、BiomerieuxVidas ® 、Alere Triage ® .

[0081] A variety of immunoassays are known and can be used in the assays and methods of the present invention, these include: mass spectrometry (MS), luminescent immunoassay (LIA), radioimmunoassay ("RIA"), homogeneous enzyme multiplication immunoassay ("EMIT"), enzyme-linked immunosorbent assay ("ELISA"), apoenzyme reactivation immunoassay ("ARIS"), luminescent-based bead arrays, magnetic bead-based arrays, protein microarray assays, rapid test formats such as dipstick immunoassays, immunochromatographic strip tests, rare cryptate assays, and automated systems / analyzers.

[0082] In one embodiment of the invention, it may be a so-called POC (point-of-care) test, which is a testing technology that allows testing to be performed in less than 1 hour near the patient without the need for a fully automated assay system. An example of this technology is an immunochromatographic testing technology, such as a microfluidic device.

[0083] In a preferred embodiment, the label is selected from the group consisting of a chemiluminescent label, an enzyme label, a fluorescent label, and a radioiodine label.

[0084] The assay can be a homogeneous or heterogeneous assay, a competitive and non-competitive assay. In one embodiment, the assay is in the form of a sandwich assay, which is 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 an antibody labeled, for example, with a dye, a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general compositions and procedures involved in "sandwich assays" are well-known and known to the skilled artisan ( The Immunoassay Handbook , Compiled by David Wild ed., Elsevier LTD, Oxford; 3rd edition (May 2005), ISBN-13: 978-0080445267; Hultschig C et al., Curr Opin Chem Biol. 2006 Feb;10(1):4-10. PMID: 16376134 ).

[0085] In another embodiment, the assay comprises two capture molecules, preferably antibodies, both capture molecules being present as a dispersion in a liquid reaction mixture, wherein a first labeling component is attached to the first capture molecule, wherein said first labeling component is part of a labeling system based on fluorescence or chemiluminescence quenching or amplification, and a second labeling component of said labeling system is attached to the second capture molecule, such that when both capture molecules bind to the analyte, a measurable signal is generated, which allows detection of a sandwich complex formed in a solution containing the sample.

[0086] In another embodiment, the labeling system comprises a rare earth cryptate or a rare earth chelate in combination with a fluorescent dye or a chemifluorescent dye, in particular a cyanine-type dye.

[0087] In the context of the present invention, fluorescence-based assays include the use of dyes, which may be selected, for example, from the group consisting of FAM (5-carboxyfluorescein or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes such as CY3, CY5, CY3.5, CY5.5, Cy7, xanthene, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), T ET, 6-carboxy 4',5'-dichloro-2',7'-dimethoxyfluorescein (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 BODIPYTMR), Oregon Green, coumarins (such as umbelliferone), benzimides (such as Hoechst 33258); phenanthridines such as Texas Red, Yakima Yellow, Alexa Fluor, PET, ethidium bromide, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrine dyes, polymethine dyes, etc.

[0088] In the context of the present invention, chemiluminescence-based assays involve the use of dyes based on the physical principles described for chemiluminescent materials in the following references ( Kirk-Othmer, Encyclopedia of chemicals technology, 4th edition, Executive Editor: JI Kroschwitz Editors: M. Howe-Grant, John Wiley & Sons, 1993, Vol. 15, pp. 518-562, incorporated herein by reference, including citations to pages 551-562 ). Preferred chemiluminescent dyes are acridinium esters.

[0089] As referred to herein, an "assay" or "diagnostic assay" may be of any type that is applied in the field of diagnostics. Such assays may be based on the binding of an analyte to be detected to one or more capture probes with a certain affinity. With respect to the interaction between a capture molecule and a target molecule or a target molecule, the affinity constant is preferably greater than 10 8 M -1 .

[0090] In a specific embodiment, at least one of the two binders is labeled for detection.

[0091] The ADM-NH2 levels of the present invention have been determined using the described ADM-NH2 assay ( Weber et al. 2017. JALM 2 (2):1-4The DPP3 levels of the present invention have been determined using the DPP3-assay as described as outlined in the Examples ( Rehfeld et al. 2019. JALM 3(6): 943-953 ). If the assay system used in the present invention has been calibrated differently, the thresholds mentioned above may be different in other assays. Therefore, taking into account the differences in calibration, the cutoff values ​​mentioned above should be applied to such differently calibrated assays accordingly. One possibility of quantitative calibration differences is to conduct a method comparison analysis (correlation) of the assay discussed with the corresponding biomarker assay used in the present invention by using two methods to measure the corresponding biomarkers (e.g., bio-ADM, DPP3) in the sample. Another possibility is to measure the median biomarker levels of a representative normal population with the assay discussed in view of the sufficient analytical sensitivity of the test, compare the results with the median biomarker levels described in the literature, and recalculate the calibration values ​​based on the differences obtained by this comparison. With the calibration values ​​used in the present invention, samples from normal (healthy) subjects have been measured: the median plasma bio-ADM (mature ADM-NH2) is 24.7pg / ml, the lowest value is 11pg / ml and the 99th percentile is 43pg / ml ( Marino et al. 2014. Critical Care 18:R34 ). With the calibration values ​​used in the present invention, samples from 5,400 normal (healthy) subjects (Swedish single center prospective population-based study (MPP-RES)) have been measured: median (interquartile range) plasma DPP3 was 14.5 ng / ml (11.3 ng / ml-19 ng / ml). DPP3 concentration has been shown to strongly correlate with DPP3 activity in blood ( Rehfeld et al. 2019. J Appl Lab Med 3: 943–953; Deniau et al. 2019. Eur J Heart Fail 22: 290–299 ). Thus, the level of active DPP3 can be determined using corresponding threshold values ​​and threshold ranges that correspond to the threshold values ​​and threshold ranges used to determine the level of DPP3 protein.

[0092] In a preferred embodiment, treatment is started or changed immediately after providing a sample analysis result indicating the level of DPP3 in the sample. In a further embodiment, treatment can be started within 12 hours, preferably 6 hours, 4 hours, 2 hours, 1 hour, 0.5 hours, 0.25 hours, or immediately after receiving the sample analysis result.

[0093] In certain embodiments of the invention, the increase in DPP3 in said patient is during the follow-up time.

[0094] In certain embodiments of the present invention, the follow-up time is at most 12 hours, preferably at most 24 hours, 48 ​​hours, 72 hours, 96 hours, more preferably at most 5 days, even more preferably at most 6 days, most preferably at most 7 days.

[0095] In certain embodiments of the invention, the DPP3 in the patient is increased within 12 hours, preferably at most 24 hours, 48 ​​hours, 72 hours, 96 hours, more preferably at most 5 days, even more preferably at most 6 days, most preferably at most 7 days. In other embodiments of the invention, the follow-up time is 2 days, or 3 days, or 7 days.

[0096] In certain embodiments of the invention, the DPP3 in said patient is increased within at most 12 hours, preferably at most 24 hours, 48 ​​hours, 72 hours, 96 hours, more preferably at most 5 days, even more preferably at most 6 days, most preferably at most 7 days.

[0097] In other embodiments of the invention, the DPP3 in said patient is increased within 2 days, or 3 days, or 7 days.

[0098] The present invention further relates to a kit for implementing the method of the present invention, the kit comprising a detection reagent for determining DPP3 in a sample from a patient, and reference data corresponding to a DPP3 level between 22 ng / mL and 40 ng / mL in said sample, such as a reference level and / or a threshold level, wherein said reference data is preferably stored on a computer readable medium and / or used in the form of a computer executable code configured for comparing the determined DPP3 with said reference data.

[0099] In one embodiment of the methods described herein, the method further comprises comparing the DPP3 level determined in the patient having shock or developing shock to a reference level and / or a threshold level, wherein the comparison is performed in a computer processor using computer executable code.

[0100] The method of the present invention may be partially computer-implemented. For example, the step of comparing the detected DPP3 level with a reference level and / or a threshold level may be performed in a computer system. For example, the measured value may be input (manually input by a health care professional or automatically input from one or more devices in which the corresponding one or more marker levels have been determined) into a computer system. The computer system may be directly at a care point (e.g., a primary care unit or ED), or it may be at a remote location connected via a computer network (e.g., via the Internet, or optionally in a dedicated medical cloud system combined with other IT systems or platforms such as a hospital information system (HIS)). Alternatively or additionally, relevant treatment guidance and / or treatment stratification will be displayed and / or printed for a user (typically a health care professional such as a physician).

[0101] The term "critically ill patient" refers to a patient suffering from an acute disease or acute condition, such as an intensive care unit (ICU) patient who requires continuous and / or intense observation of the patient's health status. The patient may suffer from severe infection, sepsis, heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, organ dysfunction or organ failure (e.g., liver, kidney, lung dysfunction or failure), undergo major surgery, trauma (e.g., burns, multiple injuries), shock and / or is developing into shock. In other embodiments, the patient may suffer from severe infection, sepsis, heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, organ dysfunction or organ failure (e.g., liver, kidney, lung dysfunction or failure), undergo major surgery, trauma (e.g., burns, multiple injuries), shock and / or is developing into shock, or ARDS.

[0102] Patients with chronic heart failure (HF) can include patients with worsening signs and symptoms of chronic heart failure as well as patients with acute decompensation of chronic heart failure.

[0103] Chronic HF with worsening signs and symptoms is particularly characterized by:

[0104] (i) the presence of structural or functional failure of the heart that impairs its ability to supply adequate blood flow to meet the body's needs,

[0105] (ii) Volume overload (manifested by pulmonary and / or systemic congestion) and / or profound depression of cardiac output (manifested by hypotension, renal insufficiency, and / or shock syndrome), where the patient does not require urgent treatment and does not require hospitalization but requires modification of therapy.

[0106] Chronic heart failure may also decompensate (called acute decompensated heart failure or acute decompensated chronic heart failure), most often due to an intercurrent disorder (such as pneumonia), myocardial infarction, arrhythmias, uncontrolled high blood pressure, or the patient's inability to maintain fluid restrictions, diet, or drugs.

[0107] New-onset acute HF and acute decompensated chronic HF are characterized by:

[0108] (i) the presence of structural or functional failure of the heart that impairs its ability to supply adequate blood flow to meet the body's needs,

[0109] (ii) Volume overload (manifested by pulmonary and / or systemic congestion) and / or profound depression of cardiac output (manifested by hypotension, renal insufficiency, and / or shock syndrome), requiring urgent treatment or modification of treatment and hospitalization.

[0110]

[0111] The above definition of acute heart failure (new-onset AHF or acute decompensated HF or acute decompensated chronic HF or worsening signs / symptoms of chronic heart failure) is consistent with Voors et al., European Journal of Heart Failure (2016), 18, 716–726.

[0112] The patients described herein may be in an emergency department (ED) or intensive care unit (ICU), or in other point-of-care settings, such as in an emergency transport facility (such as an ambulance), or at a general practitioner who is confronted with a patient suffering from one of the diseases detailed herein.

[0113] The term "ICU-patient" relates to, but is not limited to, a patient who has been admitted to an intensive care unit. An intensive care unit (also known as an intensive care unit or intensive treatment unit (ITU) or critical care unit (CCU)) is a special department of a hospital or healthcare facility that provides critical care medicines. ICU-patients typically suffer from serious and life-threatening illnesses and injuries that require continuous, close monitoring and support from specialized equipment and / or medicines in order to ensure normal body function. Common conditions treated in an ICU include, but are not limited to, acute respiratory distress syndrome (ARDS), trauma, organ dysfunction or failure, sepsis, and shock.

[0114] In another specific embodiment of the present invention, the shock is selected from the group comprising shock caused by hypovolemia, cardiogenic shock, obstructive shock and distributive shock, in particular cardiogenic shock or septic shock.

[0115] In another specific embodiment of the present invention, the shock is selected from the group comprising:

[0116] In the setting of cardiogenic shock, the patient has had an acute coronary syndrome (e.g., acute myocardial infarction) or heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmias, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or

[0117] In the setting of hypovolemic shock, the patient may have had a bleeding disorder including gastrointestinal bleeding, trauma, vascular etiologies (e.g., ruptured abdominal aortic aneurysm, tumor erosion into a major vessel), and spontaneous bleeding in the setting of anticoagulant use, or a non-bleeding disorder including vomiting, diarrhea, renal loss, skin loss / insensitivity (e.g., burns, heat stroke) or pancreatitis, cirrhosis, intestinal obstruction, third space loss in the setting of trauma, or

[0118] In the case of obstructive shock, the patient may have suffered cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or

[0119] - In cases of distributive shock, the patient suffers from septic shock, neurogenic shock, anaphylactic shock, or shock caused by adrenal crisis.

[0120] Shock is characterized by decreased oxygen delivery and / or increased oxygen consumption or inadequate oxygen utilization, resulting in cellular and tissue hypoxia. It is a life-threatening condition of circulatory failure that most often presents with hypotension (systolic blood pressure less than 90 mm Hg or MAP less than 65 mm Hg). Shock is divided into four main types based on the underlying cause: hypovolemic shock, cardiogenic shock, obstructive shock, and distributive shock ( Vincent and De Backer 2014. N. Engl. J. Med. 370(6): 583 ).

[0121] Hypovolemic shock is characterized by a decrease in intravascular volume and can be divided into two broad subtypes: hemorrhagic shock and nonhemorrhagic shock. Common causes of hemorrhagic hypovolemic shock include gastrointestinal bleeding, trauma, vascular etiologies (eg, ruptured abdominal aortic aneurysm, tumor erosion into a major vessel), and spontaneous bleeding in the setting of anticoagulant use. Common causes of nonhemorrhagic hypovolemic shock include vomiting, diarrhea, renal loss, skin loss / insensitivity (eg, burns, heat stroke) or third space loss in the setting of pancreatitis, cirrhosis, intestinal obstruction, or trauma. For review, see Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019-October 27, 2018 .

[0122] Cardiogenic shock (CS) is defined as a state of severe intrinsic hypoperfusion due to decreased cardiac output. Of note, CS forms a spectrum ranging from mild hypoperfusion to profound shock. Established diagnostic criteria for CS are: (i) systolic blood pressure ≤90 mmHg for >30 min or vasopressor-needed blood pressure ≥90 mmHg; (ii) pulmonary congestion or elevated left ventricular filling pressures; (iii) signs of impaired organ perfusion with at least one of the following criteria: (a) altered mental status; (b) clammy skin; (c) oliguria (<0.5 mL / kg / h or <30 mL / h); (d) increased serum lactate ( Reynolds and Hochman 2008. Circulation 117: 686–697 ). Ventricular dysfunction following acute myocardial infarction (AMI) is the most common cause of CS, accounting for approximately 80% of cases. Mechanical complications such as ventricular septal (4%) or free wall rupture (2%) and acute severe mitral regurgitation (7%) are less common 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 myocarditis, arrhythmias, etc., with different treatment options. This translates into 40 000 to 50 000 patients per year in the United States and 60 000 to 70 000 patients in Europe. Despite advances in treatment, mainly through early revascularization and subsequent reduction in mortality, CS remains the leading cause of death in AMI, with a mortality rate still approaching 40%-50% ( ) according to recent registries and randomized trials. Goldberg et al. 2009. Circulation 119: 1211–1219 ).

[0123] Obstructive shock is caused by physical obstruction of a large vessel or the heart itself. Several conditions can cause this form of shock (eg, cardiac tamponade, tension pneumothorax, pulmonary embolism, aortic stenosis). Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019-October 27, 2018 .

[0124] There are four types of distributive shock, depending on the cause: neurogenic shock (decreased sympathetic stimulation leading to decreased vascular tone), anaphylactic shock, septic shock, and shock due to adrenal crisis. In addition to sepsis, distributive shock can be caused by a systemic inflammatory response syndrome due to conditions other than infection, such as pancreatitis, burns, or trauma. Other causes include toxic shock syndrome (TSS), anaphylaxis (a sudden, severe allergic reaction), adrenal insufficiency (acute exacerbation of chronic adrenal insufficiency, destruction or removal of the adrenal glands, adrenal suppression due to exogenous steroids, hypopituitarism, or metabolic disorders of hormone production), reactions to drugs or toxins, heavy metal poisoning, hepatic (liver) insufficiency, and damage to the central nervous system. For a review, see Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019-October 27, 2018 .

[0125] Refractory shock has been defined as the requirement for >0.5 µg / kg / min of norepinephrine infusion 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 calibration measures (e.g., vasopressors), and may therefore be referred to as "high vasopressor-dependent" or "vasopressor-resistant" shock ( Udupa and Shetty 2018. Indian J Respir Care 7: 67–72). Patients with refractory shock may have features of hypoperfusion such as hypotension (mean arterial blood pressure <65 mmHg), tachycardia, peripheral coolness, prolonged capillary refill time, and tachypnea caused by hypoxia and acidosis. Fever may be seen in septic shock. Other signs of hypoperfusion may also be seen, such as altered central sensorium, hyperlactatemia, and oliguria. These well-known signs of shock are not helpful in identifying whether the problem is with the pump (heart) or with the circuit (vasculature and tissue). Different types of shock can coexist, and all forms of shock can become refractory, as evidenced by lack of response to high doses of vasopressors ( Udupa and Shetty 2018. Indian J Respir Care 7: 67–72 ).

[0126] Septic shock is a potentially fatal medical condition that occurs when sepsis (which is organ injury or damage in response to 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 subcategory of sepsis in which particularly profound circulatory, cellular, and metabolic abnormalities are associated with a higher risk of mortality than sepsis alone. Patients with septic shock can be clinically identified by the requirement for vasopressors to maintain a mean arterial pressure of 65 mm Hg or higher and a serum lactate level above 2 mmol / L (>18 mg / dL) in the absence of hypovolemia. This combination is associated with a hospital mortality rate of more than 40% ( Singer et al. 2016. JAMA. 315 (8): 801–10. ). The primary infection is most often caused by bacteria, but can also be caused by fungi, viruses, or parasites. It can be located anywhere in the body, but is most often in the lungs, brain, urinary tract, skin, or abdominal organs. It can cause multiple organ dysfunction syndrome (formerly called multiple organ failure) and death. Often, people with septic shock are cared for in an intensive care unit. It most commonly affects children, immunocompromised individuals, and the elderly because their immune systems cannot fight the infection as effectively as healthy adults. The mortality rate from septic shock is approximately 25%-50%.

[0127] In one embodiment of the present invention, the patient is a critically ill patient who has shock or is developing shock when the body fluid sample of the patient is obtained.

[0128] A "patient developing shock" is defined as a critically ill patient who is not in shock at the time the body fluid is removed from the patient but who has an increased risk of developing shock.

[0129] In a specific embodiment, the shock is septic shock or cardiogenic shock.

[0130] The term "therapy" or "treatment" refers to a critical care drug. The critical care drug is selected from the group consisting of alkaline phosphatase, immunosuppressants, corticosteroids, vasopressors, fluids, anti-ADM antibodies, anti-ADM antibody fragments, anti-ADM stents, anti-DPP3 inhibitors (e.g., anti-DPP3 antibodies, anti-DPP3 antibody fragments, anti-DPP3 stents).

[0131] Another preferred embodiment of the present application is further a method for predicting an increase in DPP3 in critically ill patients, wherein if the DPP3 level in the sample is below the predetermined threshold, the patient is selected for therapy / treatment, wherein the therapy is selected from the group consisting of alkaline phosphatase, immunosuppressants, corticosteroids, vasopressors, fluids, anti-ADM antibodies or antibody fragments.

[0132] Another preferred embodiment of the present application is further a method for predicting DPP3 increase in critically ill patients, wherein the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM scaffold is directed against the N-terminal part (amino acids 1-21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 14).

[0133] Another embodiment of the present application relates to a method for predicting increased DPP3 in critically ill patients, wherein the anti-ADM antibody or anti-ADM antibody fragment recognizes and binds to the N-terminus (amino acid 1) of ADM.

[0134] Another embodiment of the present application relates to a method for predicting increased DPP3 in critically ill patients, wherein the antibody, antibody fragment or non-Ig scaffold does not bind to the C-terminal portion of ADM, which has the amino acid 43-52 sequence of ADM: PRSKISPQGY-NH2 (SEQ ID NO: 24).

[0135] Another embodiment of the present application relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein the antibody or fragment is a monoclonal antibody or fragment thereof that binds to ADM, wherein the heavy chain comprises the sequence:

[0136] CDR1: SEQ ID NO: 1

[0137] GYTFSRYW

[0138] CDR2: SEQ ID NO: 2

[0139] ILPGSGST

[0140] CDR3: SEQ ID NO: 3

[0141] TEGYEYDGFDY

[0142] And wherein the light chain comprises the following sequence:

[0143] CDR1: SEQ ID NO: 4

[0144] QSIVYSNGNTY

[0145] CDR2:

[0146] RVS

[0147] CDR3: SEQ ID NO: 5

[0148] FQGSHIPYT.

[0149] Another embodiment of the present application relates to a method for predicting an increase in DPP3 in critically ill patients, wherein the antibody or fragment comprises as a VH region a sequence selected from the group consisting of:

[0150] SEQ ID NO: 6 (AM-VH-C)

[0151] SEQ ID NO: 7 (AM-VH1)

[0152] SEQ ID NO: 8 (AM-VH2-E40)

[0153] SEQ ID NO: 9(AM-VH3-T26-E55)

[0154] SEQ ID NO: 10(AM-VH4-T26-E40-E55)

[0155] and comprising as a VL region a sequence selected from the group consisting of:

[0156] SEQ ID NO: 11 (AM-VL-C)

[0157] SEQ ID NO: 12 (AM-VL1)

[0158] SEQ ID NO: 13 (AM-VL2-E40)

[0159] Another embodiment of the present application relates to a method for predicting an increase in DPP3 in critically ill patients, wherein the antibody or fragment comprises the following sequence as a heavy chain:

[0160] SEQ ID NO: 32

[0161] or sequences >95% identical to them,

[0162] and contains the following sequence as a light chain:

[0163] SEQ ID NO: 33

[0164] Or a sequence that is >95% identical thereto.

[0165] A specific embodiment of the present invention relates to a method for predicting an increase in DPP3 in critically ill patients, wherein the anti-ADM or anti-ADM antibody fragment can 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 mg / kg body weight to 20.0 mg / kg body weight, for example 2.0 mg / kg body weight to 10 mg / kg body weight, 2.0 mg / kg body weight to 8.0 mg / kg body weight or 2.0 mg / kg body weight to 5.0 mg / kg body weight.

[0166] The efficacy of a non-neutralizing antibody targeting the N-terminus of ADM was investigated in a survival study of CLP-induced sepsis in mice. Pretreatment with the non-neutralizing antibody resulted in a reduced catecholamine infusion rate, renal dysfunction, and ultimately improved survival ( Struck et al. 2013. Intensive Care Med Exp 1(1):22; Wagner et al. 2013. Intensive Care Med Exp 1(1):21 ).

[0167] As a result of these positive results, an N-terminal humanized form of the anti-ADM antibody, termed aderezumab, has been developed for further clinical development. The beneficial effects of aderezumab on vascular barrier function and survival have recently been demonstrated in preclinical models of systemic inflammation and sepsis ( Geven et al. 2018. Shock 50(6):648-654 ). In this study, pretreatment with aderezumab attenuated renal vascular leakage in endotoxemic rats as well as in mice with CLP-induced sepsis, consistent with increased renal expression of the protective peptide Ang-1 and decreased expression of the harmful peptide vascular endothelial growth factor. In addition, pretreatment with aderezumab increased the 7-day survival rate of mice in CLP-induced sepsis by 10%-50% (single administration) and 0%-40% (repeated administration). In addition, in the Phase I study, excellent safety and tolerability were demonstrated (see Example 6): no serious adverse events were observed, no signals of adverse events occurring more frequently in subjects treated with aderezumab were detected, and no relevant changes in other safety parameters were found ( Geven et al. 2017. Intensive Care Med Exp 5 (Supplement 2): 0427 ). Of particular interest is the proposed mechanism of action of aderezumab. Both animal and human data reveal potent dose-dependent increases in circulating ADM following administration of the antibody. Based on pharmacokinetic data and the lack of increases in MR-proADM (an inactive peptide fragment derived from the same prohormone as ADM), the higher circulating ADM levels cannot be explained by increased production.

[0168] A possible mechanistic explanation for this increase is that excess circulating antibodies may excrete ADM from the interstitial tissue into the circulation, since ADM are small enough to cross the endothelial barrier, whereas antibodies are not ( Geven et al. 2018. Shock. 50(2):132- 140 ). In addition, binding of the antibody to ADM results in an extension of the half-life of ADM. Even though NT-ADM antibodies partially inhibit ADM-mediated signaling, the large increase in circulating ADM causes an overall “net” increase in ADM activity in the blood compartment, where it exerts beneficial effects on endothelial cells (EC; primarily barrier stabilization), while the deleterious effects of ADM on vascular smooth muscle cells (VSMC; vasodilation) in the interstitial tissue are reduced.

[0169] Throughout the specification, an "antibody" or "antibody fragment" or "non-Ig scaffold" according to the present invention is capable of binding to ADM, and is therefore directed against ADM, and may therefore be referred to as an "anti-ADM antibody", "anti-ADM antibody fragment" or "anti-ADM non-Ig scaffold".

[0170] The term "antibody" generally includes monoclonal and polyclonal antibodies and their binding fragments, especially Fc fragments and so-called "single-chain antibodies" ( Bird et al. 1988 ), chimeric antibodies, humanized antibodies, especially CDR-grafted antibodies, and double-chain antibodies or four-chain antibodies ( Holliger et al. 1993 ). Also included are immunoglobulin-like proteins that specifically bind to a target molecule contained in a sample, selected by techniques including, for example, phage display. In this context, the term "specific binding" refers to antibodies raised against a target molecule or a fragment thereof. An antibody is considered specific if its affinity for the target molecule or its aforementioned fragment is at least preferably 50 times, more preferably 100 times, and most preferably at least 1000 times greater than its affinity for other molecules contained in a sample containing the target molecule. How to prepare antibodies and select antibodies with a given specificity is well known in the art.

[0171] In one embodiment of the invention, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is monospecific.

[0172] A monospecific anti-ADM antibody or monospecific anti-adrenomedullin antibody fragment or monospecific anti-ADM non-Ig scaffold means that the antibody or antibody fragment or non-Ig scaffold binds to a specific region covering at least 5 amino acids within the target ADM. A monospecific anti-ADM antibody or monospecific anti-adrenomedullin antibody fragment or monospecific anti-ADM non-Ig scaffold is an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold that all have affinity for the same antigen. Monoclonal antibodies are monospecific, but monospecific antibodies can also be produced by other means except producing them from common germ cells.

[0173] The anti-ADM antibody or antibody fragment that binds to ADM or the non-Ig scaffold that binds to ADM may be a non-neutralizing anti-ADM antibody or antibody fragment that binds to ADM or the non-Ig scaffold that binds to ADM.

[0174] In a specific embodiment, the anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold is a non-neutralizing antibody, fragment or non-Ig scaffold. Neutralizing anti-ADM antibodies, anti-ADM antibody fragments or anti-ADM non-Ig scaffolds will block the biological activity of ADM by nearly 100%, at least more than 90%, and preferably at least more than 95%.

[0175] In contrast, the non-neutralizing anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the biological activity of ADM by less than 100%, preferably less than 95%, preferably less than 90%, more 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%, reduced to 95% or less but not more than, reduced to 90% or less but not more than, reduced to 80% or less but not more than, reduced to 50% or less but not more than. This means that the residual biological activity of ADM bound to the non-neutralizing anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold will be greater than 0%, preferably greater than 5%, preferably greater than 10%, more preferably greater than 20%, and more preferably greater than 50%.

[0176] In this context, one or more molecules that, for example, block the biological activity of ADM to less than 80%, i.e., an antibody or antibody fragment or non-Ig scaffold having "non-neutralizing anti-ADM activity", for simplicity, are collectively referred to herein as "non-neutralizing" anti-ADM antibodies, antibody fragments or non-Ig scaffolds, are defined as:

[0177] - one or more molecules that bind to ADM and which, after addition 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 added human synthetic ADM is added in an amount that results in a half-maximal stimulation of cAMP synthesis in the absence of the non-neutralizing antibody to be analyzed, wherein the reduction of cAMP by said molecules that bind to ADM occurs to an extent of no more than 80%, even when the non-neutralizing molecules that bind to ADM to be analyzed are added in an amount that is 10 times the amount required to obtain the maximal reduction in cAMP synthesis obtainable with the non-neutralizing antibodies to be analyzed.

[0178] The same definition applies to other ranges: 95%, 90%, 50%, etc.

[0179] The antibodies or fragments according to the present invention are proteins comprising one or more polypeptides substantially encoded by immunoglobulin genes that specifically bind to an antigen. The recognized immunoglobulin genes include 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. The length of a full-length immunoglobulin light chain is generally about 25Kd or 214 amino acids.

[0180] The length of a full-length immunoglobulin heavy chain is usually about 50Kd or 446 amino acids. The light chain is 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. The heavy chain is similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.

[0181] The basic structural unit of an antibody is usually a tetramer composed of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions bind to the antigen, and the constant regions mediate effector functions. Immunoglobulins also exist in a variety of other forms, including, for example, Fv, Fab, and (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 ). An immunoglobulin light or heavy chain variable region comprises 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 described above, CDR is mainly responsible for binding to antigen epitopes. Immune complexes are antibodies that specifically bind to antigens, such as monoclonal antibodies, chimeric antibodies, humanized antibodies or human antibodies, or functional antibody fragments.

[0182] Chimeric antibodies are antibodies whose light chain and heavy chain genes have been constructed from immunoglobulin variable and constant region genes belonging to different species, typically by genetic engineering. For example, the variable segment of the gene from a mouse monoclonal antibody can be connected to a human constant segment, such as κ and γ1 or γ3. In one example, therapeutic chimeric antibodies are therefore a hybrid protein consisting of a variable domain or antigen-binding domain from a mouse antibody and a constant domain or effector domain from a human antibody, but other mammalian species can be used, or variable regions can be produced by molecular techniques. Methods for preparing chimeric antibodies are well known in the art, for example, referring to U.S. Patent No. 5,807,715. "Humanized" immunoglobulins are immunoglobulins including human framework regions and one or more CDRs from non-human (such as mouse, rat or synthetic) immunoglobulins. The non-human immunoglobulins providing CDRs are referred to as "donors", while the human immunoglobulins providing frameworks are referred to as "acceptors". In one embodiment, all CDRs in humanized immunoglobulins are from donor immunoglobulins. Constant regions do not need to be present, but if present, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85%-90% identical, such as about 95% or more identical. Thus, except for possible CDRs, all parts of humanized immunoglobulins are substantially identical to corresponding parts of natural human immunoglobulin sequences. "Humanized antibodies" are antibodies comprising humanized light chains and humanized heavy chain immunoglobulins. Humanized antibodies bind to the same antigen as the donor antibody providing the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of amino acid substitutions taken from the donor framework. Humanized antibodies or other monoclonal antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are those such as the following: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed by genetic engineering (see, e.g., U.S. Pat. No. 5,585,089). Human antibodies are antibodies in which the light chain 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 that secrete the antibody of interest. Immortality can be achieved, for example, by EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce trioma cells. Human antibodies can also be produced by phage display methods (see, e.g., WO91 / 17271 ; WO92 / 001047; WO92 / 20791 ), or selected from a human combinatorial monoclonal antibody library (see the Morphosys website). Human antibodies can also be prepared by using transgenic animals carrying human immunoglobulin genes (see, for example, WO93 / 12227 ; WO 91 / 10741).

[0183] Therefore, the anti-ADM antibody may have a form known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies. In a preferred embodiment, the antibody according to the invention is a recombinantly produced antibody, such as an IgG, a typical full-length immunoglobulin, or an antibody fragment containing at least the F variable domain of a heavy chain and / or a light chain, such as a chemically coupled antibody (antigen-binding fragment), including but not limited to Fab fragments, including Fab miniantibodies, single-chain Fab antibodies, monovalent Fab antibodies with epitope tags (e.g. Fab-V5Sx2); divalent Fab (miniantibodies) dimerized with a CH3 domain; divalent Fab or multivalent Fab, for example formed by multimerization with the aid of a heterologous domain, for example formed by dimerization of a dHLX domain, such as Fab-dHLX-FSx2; F(ab')2-fragment, scFv-fragment, multimerized multivalent or / and multispecific scFv-fragment, divalent and / or bispecific diabodies, BITE ® (bispecific T-cell engagers), trifunctional antibodies, multivalent antibodies (e.g. from classes other than G); single domain antibodies such as nanobodies derived from camelid or fish immunoglobulins and many others.

[0184] In addition to anti-ADM antibodies, other biopolymer scaffolds are well known in the art to complex with target molecules and have been used to generate highly target-specific biopolymers. Examples are aptamers, spiegelmers, anticalins, and conotoxins. For illustration of antibody formats, see Figure 1a , Figure 1b and Figure 1c .

[0185] In a preferred embodiment, the anti-ADM antibody form is selected from the group including Fv fragments, scFv fragments, Fab fragments, scFab fragments, F(ab)2 fragments and scFv-Fc fusion proteins. In another preferred embodiment, the antibody form is selected from the group including scFab fragments, Fab fragments, scFv fragments and their bioavailability optimized conjugates (such as pegylated fragments). One of the most preferred forms is the scFab form.

[0186] The non-Ig scaffolds may be protein scaffolds and may be used as antibody mimics as they are able to bind to ligands or antigens. The non-Ig scaffolds may be selected from the group comprising: tetranectin-based non-Ig scaffolds (e.g. US 2010 / 0028995 fibronectin scaffolds (e.g., in EP 1 266 025); lipocalin-based scaffolds (e.g., in WO 2011 / 154420 ); ubiquitin scaffolds (e.g., in WO 2011 / 073214 transferrin scaffolds (e.g., in US 2004 / 0023334 ); Protein A scaffolds (e.g., in EP 2 231 860 ); ankyrin repeat-based scaffolds (e.g., in WO 2010 / 060748 ); microprotein (preferably cysteine ​​knot-forming microprotein) scaffolds (e.g., in EP 2314308 ); FynSH3 domain-based scaffolds (e.g., in WO 2011 / 023685 Scaffolds based on the EGFR-A domain (e.g., in WO 2005 / 040229 ) and Kunitz domain-based scaffolds (e.g., in EP 1 941 867 ).

[0187] In one embodiment of the invention, anti-ADM antibodies according to the invention can be produced by synthesizing ADM fragments as antigens as outlined in Example 1. Thereafter, binders of said fragments are identified using the methods described below or other methods known in the art.

[0188] Humanization of mouse antibodies can be performed according to the following procedure:

[0189] For humanization of antibodies of murine origin, the framework regions (FR) of the antibody sequence are analyzed for structural interactions with the complementarity determining regions (CDRs) and the antigen. Based on structural modeling, appropriate FRs of human origin are selected and the murine CDR sequences are grafted into the human FRs. Variations in the amino acid sequences of the CDRs or FRs can be introduced to regain structural interactions that were eliminated by species conversion of the FR sequences. This restoration of structural interactions can be achieved by random approaches using phage display libraries or by directed approaches guided by molecular modeling ( Almagro and Fransson 2008. Humanization of antibodies. Front Biosci. 2008 Jan 1;13:1619-33 ).

[0190] In a preferred embodiment, the ADM antibody form is selected from the group comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, F(ab)2 fragment and scFv-Fc fusion protein. In another preferred embodiment, the antibody form is selected from the group comprising scFab fragment, Fab fragment, scFv fragment and their bioavailability optimized conjugate (such as pegylated fragment). One of the most preferred forms is the scFab form.

[0191] 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.

[0192] In a preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed against and can bind to an epitope contained in ADM that is at least 5 amino acids in length.

[0193] In a more preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed against and can bind to an epitope contained in ADM that is at least 4 amino acids in length.

[0194] In a specific embodiment of the invention, the anti-ADM or anti-ADM antibody fragment that binds to adrenomedullin or the anti-ADM non-Ig scaffold that binds to adrenomedullin is not ADM binding protein-1 (complement factor H).

[0195] In a specific embodiment of the present invention, the anti-ADM antibody or anti-ADM antibody fragment that binds to adrenomedullin or the anti-ADM non-Ig scaffold that binds to adrenomedullin binds to a region of preferably at least 4 or at least 5 amino acids within the sequence of amino acids 1-21 of mature human ADM: YRQSMNNFQGLRSFGCRFGTC SEQ ID No.: 14.

[0196] 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 of ADM located in the N-terminal portion (amino acids 1-21) of adrenomedullin.

[0197] 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 within amino acids 1-14 of adrenomedullin: YRQSMNNFQGLRSF (SEQ ID No.: 25), meaning the N-terminal portion of adrenomedullin (amino acids 1-14).

[0198] 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 within amino acids 1-10 of adrenomedullin: YRQSMNNFQG (SEQ ID No.: 26); meaning the N-terminal portion (amino acids 1-10) of adrenomedullin.

[0199] 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 within amino acids 1-6 of adrenomedullin: YRQSMN (SEQ ID No.: 27); meaning the N-terminal portion of adrenomedullin (amino acids 1-6). As described above, the region or epitope is preferably at least 4 or at least 5 amino acids in length.

[0200] 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-terminal portion (amino acid 1) of adrenomedullin. The N-terminus means amino acid 1, i.e. "Y" of SEQ ID No. 20, 14 or 23, respectively, and is necessary for binding. The antibody or fragment or scaffold will neither bind to adrenomedullin with N-terminal extension nor to adrenomedullin with N-terminal modification nor to adrenomedullin with N-terminal degradation. This means that in another preferred embodiment, if the N-terminus of ADM is free, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold will only bind to regions within the sequence of mature ADM. In the embodiment, if the sequence is, for example, contained within pro-ADM, the anti-ADM antibody or anti-ADM antibody fragment or non-Ig scaffold will not bind to regions within the sequence of mature ADM.

[0201] For the sake of clarity, numbers in brackets for a specific region of ADM such as "N-terminal portion (amino acids 1-21)" are 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.

[0202] In another specific embodiment according to the present invention, the anti-ADM or anti-ADM antibody fragment or anti-ADM non-Ig scaffold provided herein does not bind to the C-terminal portion of ADM, ie, amino acids 43-52 of ADM: PRSKISPQGY-NH2 (SEQ ID No.: 24).

[0203] In a specific embodiment, it is preferred to use an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold according to the present invention, wherein the anti-ADM antibody or the anti-ADM antibody fragment or the anti-ADM non-Ig scaffold causes an increase in ADM levels or ADM immunoreactivity in serum, blood, or plasma by at least 10%, preferably at least 50%, more preferably >50%, and most preferably >100%.

[0204] In a specific embodiment, it is preferred to use an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold according to the present invention, wherein the anti-ADM antibody or the anti-ADM antibody fragment or the anti-ADM non-Ig scaffold is an ADM stable antibody or an ADM stable antibody fragment or an ADM stable non-Ig scaffold, so that the half-life (t 1 / 2 ; half retention time) is enhanced by at least 10%, preferably at least 50%, more preferably>50%, and most preferably>100%.

[0205] The half-life (half-retention time) of ADM can be determined in human serum, blood or plasma using an immunoassay for ADM quantification in the absence and presence of ADM stabilizing antibodies or ADM stabilizing antibody fragments or ADM stabilizing non-Ig scaffolds, respectively.

[0206] You can do the following steps:

[0207] - ADM can be diluted in human citrated plasma in the absence and presence of ADM stabilizing antibodies or ADM stabilizing antibody fragments or ADM stabilizing non-Ig scaffolds, respectively, and can be incubated at 24°C.

[0208] - Aliquots are taken at selected time points (eg within 24 hours) and degradation of ADM in the aliquots can be stopped by freezing at -20°C.

[0209] - If the assay chosen is not affected by stabilizing antibodies, the amount of ADM can be determined directly by hDM immunoassay. Alternatively, an aliquot can be treated with a denaturing agent such as HCl, and after clearing the sample (e.g., by centrifugation), the pH can be neutralized and ADM quantified by ADM immunoassay. Alternatively, non-immunoassay techniques such as RP-HPLC can be used for ADM quantification.

[0210] - Calculation of the ADM half-life of ADM incubated in the absence and presence of ADM stabilizing antibodies or ADM stabilizing antibody fragments or ADM stabilizing non-Ig scaffolds, respectively.

[0211] - The enhancement in the half-life of the stabilized ADM is calculated compared to ADM that has been incubated in the absence of the ADM stabilizing antibody or ADM stabilizing antibody fragment or ADM stabilizing non-Ig scaffold.

[0212] A two-fold increase in the half-life of ADM is a 100% half-life enhancement. The half-life (half retention time) is defined as the period of time required for the concentration of a given chemical or drug to drop to half of its baseline concentration in a given fluid or blood. An assay that can be used to determine the half-life (half retention time) of adrenomedullin in serum, blood, or plasma is described in Example 3.

[0213] In a preferred embodiment, the anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold is a non-neutralizing antibody, fragment or scaffold. Neutralizing anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold will block the biological activity of ADM to nearly 100%, to at least more than 90%, preferably to at least more than 95%. In other words, this means that the non-neutralizing anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the biological activity of ADM to less than 100%, preferably less than 95%, preferably less than 90%. In embodiments where the non-neutralizing anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the biological activity of ADM to less than 95%, the anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold that blocks the biological activity of ADM to more than 95% will be outside the scope of the embodiment. This means that in one embodiment the biological activity is reduced to 95% or less but not more than, preferably to 90% or less, more preferably to 80% or less, more preferably to 50% or less but not more than.

[0214] In one embodiment of the present invention, the non-neutralizing antibody is an antibody that binds to a region of at least 5 amino acids within the sequence of amino acids 1-21 of mature human ADM (SEQ ID No.: 14), or an antibody that binds to a region of at least 5 amino acids within the sequence of amino acids 1-19 of mature mouse ADM (SEQ ID No.: 17).

[0215] In another embodiment of the present invention, the non-neutralizing antibody is an antibody that binds to a region of at least 4 amino acids within the sequence of amino acids 1-21 of mature human ADM (SEQ ID No.: 14), or an antibody that binds to a region of at least 5 amino acids within the sequence of amino acids 1-19 of mature mouse ADM (SEQ ID No.: 17).

[0216] In a specific embodiment according to the present invention, a non-neutralizing anti-ADM antibody or anti-ADM antibody fragment or ADM non-Ig scaffold is used, wherein the anti-ADM antibody or anti-ADM antibody fragment blocks the biological activity of ADM to less than 80% (of the baseline value), preferably less than 50%. It should be understood that the limited blocking of the biological activity of ADM (meaning a reduction in biological activity) will occur even at an excess antibody, fragment or scaffold concentration (meaning an excess of antibody, fragment or scaffold relative to ADM). In the specific embodiment, the limited blocking is an inherent property of the ADM conjugate itself. This means that the antibody, fragment or scaffold has a maximum inhibition of 80% or 50%, respectively. In a preferred embodiment, the anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold will block the biological activity of anti-ADM / reduce the biological activity of anti-ADM to at least 5%. The above content means that about 20% or 50% or even 95% of the residual ADM biological activity still exists.

[0217] Thus, according to the present invention, provided anti-ADM antibodies, anti-ADM antibody fragments and anti-ADM non-Ig scaffolds do not neutralize the corresponding ADM biological activity.

[0218] Biological activity is defined as the effect of a substance on a living organism or tissue or organ or functional unit after its interaction in vivo or in vitro (e.g. in an assay). In the case of ADM biological activity, this may be the effect of ADM in a human recombinant ADM receptor cAMP functional assay. Therefore, according to the present invention, biological activity is defined by an ADM receptor cAMP functional assay. In order to measure the biological activity of ADM in such an assay, the following steps may be performed:

[0219] - Obtain a dose response curve with ADM in the human recombinant ADM receptor cAMP functional assay.

[0220] - The ADM concentration that gives half maximal cAMP stimulation can be calculated.

[0221] - Dose response curves were obtained with ADM-stabilized antibodies or ADM-stabilized antibody fragments or ADM-stabilized non-Ig scaffolds at a constant half-maximal cAMP-stimulating ADM concentration (up to 100 µg / ml final concentration).

[0222] In the ADM bioassay, 50% of the maximum inhibition means that the anti-ADM antibody or the anti-ADM antibody fragment or the anti-ADM non-Ig scaffold blocks the biological activity of ADM to 50% of the baseline value respectively. In the ADM bioassay, 80% of the maximum inhibition means that the anti-ADM antibody or the anti-ADM antibody fragment or the anti-ADM non-Ig scaffold blocks the biological activity of ADM to 80% respectively. This is no more than 80% in the sense of blocking the biological activity of ADM. This means that about 20% of the residual ADM biological activity still exists.

[0223] However, through this specification and in the above context, the expression "blocking the biological activity of ADM" in connection with the anti-ADM antibodies, anti-ADM antibody fragments and anti-ADM non-Ig scaffolds disclosed herein should be understood as only reducing the biological activity of ADM from 100% to at most 20% remaining ADM biological activity, preferably reducing the biological activity of ADM from 100% to 50% remaining ADM biological activity; but in any case, there is still ADM biological activity remaining that can be determined as detailed above. The biological activity of ADM can be determined in the human recombinant ADM receptor cAMP functional assay (adrenomedullin bioassay) according to Example 2.

[0224] In a preferred embodiment, the regulatory anti-ADM antibody or regulatory anti-ADM antibody fragment or regulatory anti-ADM non-Ig scaffold is used to treat or prevent shock in a patient.

[0225] A "regulatory" anti-ADM antibody or a regulatory anti-ADM antibody fragment or a regulatory anti-ADM non-Ig scaffold is a protein that converts the half-life (t 1 / 2 The half-retention time (half retention time) of the present invention is an antibody or antibody fragment or non-Ig scaffold that enhances at least 10%, preferably at least 50%, more preferably>50%, most preferably>100% and blocks the biological activity of ADM to less than 80%, preferably less than 50%, and the anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold will block the biological activity of ADM to at least 5%. These values ​​related to the blocking of the half-life and biological activity must be understood relative to the aforementioned assay method to measure these values. This is no more than 80% or no more than 50% in the sense of blocking the biological activity of ADM.

[0226] Such regulatory anti-ADM antibodies or regulatory anti-ADM antibody fragments or regulatory anti-ADM non-Ig scaffolds provide the advantage of convenient quantitative administration. The combination of partial blocking or partial reduction of ADM bioactivity and increased in vivo half-life (increased ADM bioactivity) leads to the beneficial simplicity of administration of anti-ADM antibodies or anti-ADM antibody fragments or anti-ADM non-Ig scaffolds. In the case of excess endogenous ADM (maximum stimulation, late sepsis stage, shock, low support stage), the effect of reducing activity is the main effect of the antibody or fragment or scaffold, limiting the (negative) effect of ADM. In the case of low or normal endogenous ADM concentrations, the biological effect of anti-ADM antibodies or anti-ADM antibody fragments or anti-ADM non-Ig scaffolds is a combination of reduction (by partial blocking) and increase by increasing ADM half-life. Therefore, non-neutralizing and regulatory anti-ADM antibodies or anti-ADM antibody fragments or anti-ADM non-Ig scaffolds act like ADM bioactivity buffers to keep the bioactivity of ADM within a certain physiological range.

[0227] In a specific embodiment of the present invention, antibody is a monoclonal antibody or its fragment. In an embodiment of the present invention, anti-ADM antibody or anti-ADM antibody fragment is a human antibody or a humanized antibody or is derived from a human antibody or a humanized antibody. In a specific embodiment, one or more (mouse) CDRs are transplanted into a human antibody or an antibody fragment.

[0228] The subject of the present invention is, in one aspect, a human or humanized CDR-grafted antibody or an antibody fragment thereof that binds to ADM, wherein the human or humanized CDR-grafted antibody or an antibody fragment thereof comprises: an antibody heavy chain (H chain) comprising:

[0229] GYTFSRYW (SEQ ID No.: 1),

[0230] ILPGSGST (SEQ ID No.: 2) and / or

[0231] TEGYEYDGFDY (SEQ ID No.: 3)

[0232] And / or further comprising: an antibody light chain (L chain) comprising:

[0233] QSIVYSNGNTY (SEQ ID No.: 4),

[0234] RVS (not part of the sequence listing) and / or

[0235] FQGSHIPYT (SEQ ID No.: 5).

[0236] In a specific embodiment of the invention, the subject of the invention is a human or humanized monoclonal antibody binding to ADM or an antibody fragment thereof binding to ADM, wherein the heavy chain comprises at least one CDR selected from the group comprising:

[0237] GYTFSRYW (SEQ ID No.: 1),

[0238] ILPGSGST (SEQ ID No.: 2),

[0239] TEGYEYDGFDY (SEQ ID No.: 3)

[0240] and wherein the light chain comprises at least one CDR selected from the group comprising:

[0241] QSIVYSNGNTY (SEQ ID No.: 4),

[0242] RVS (not part of the sequence listing),

[0243] FQGSHIPYT (SEQ ID No.: 5).

[0244] In a more specific embodiment of the present invention, the subject of the present invention is a human monoclonal antibody binding to ADM or an antibody fragment thereof binding to ADM, wherein the heavy chain comprises the following sequence:

[0245] GYTFSRYW (SEQ ID No.: 1),

[0246] ILPGSGST (SEQ ID No.: 2),

[0247] TEGYEYDGFDY (SEQ ID No.: 3)

[0248] And wherein the light chain comprises the following sequence:

[0249] QSIVYSNGNTY (SEQ ID No.: 4),

[0250] RVS (not part of the sequence listing),

[0251] FQGSHIPYT (SEQ ID No.: 5).

[0252] In a very specific embodiment, the anti-ADM antibody has a sequence selected from the group consisting of SEQ ID No. 6, 7, 8, 9, 10, 11, 12, 13, 32 and 33.

[0253] The anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold according to the present invention exhibits affinity for human ADM such that the affinity constant is greater than 10 -7 M, preferably 10 -8 M, preferably with an affinity greater than 10 -9 M, most preferably above 10 -10 M. Those skilled in the art will appreciate that it may be considered to compensate for the lower affinity by administering a higher dose of the compound, and such measures would not be beyond the scope of the present invention. The affinity constant may be determined according to the method described in Example 1.

[0254] The subject of the present invention is a human or humanized monoclonal antibody or fragment thereof binding to ADM for use in the treatment or prevention of shock in a patient according to the invention, wherein said antibody or fragment comprises a sequence selected from the group comprising:

[0255] SEQ ID NO: 6 (AM-VH-C)

[0256] SEQ ID NO: 7 (AM-VH1)

[0257] SEQ ID NO: 8 (AM-VH2-E40)

[0258] SEQ ID NO: 9(AM-VH3-T26-E55)

[0259] SEQ ID NO: 10(AM-VH4-T26-E40-E55)

[0260] SEQ ID NO: 11 (AM-VL-C)

[0261] SEQ ID NO: 12 (AM-VL1)

[0262] SEQ ID NO: 13 (AM-VL2-E40)

[0263] Another embodiment of the present invention relates to a human or humanized monoclonal antibody or fragment thereof that binds to ADM for use in treating or preventing shock in a patient, wherein the antibody or fragment comprises the following sequence as a heavy chain:

[0264] SEQ ID NO: 32

[0265] and contains the following sequence as a light chain:

[0266] SEQ ID NO: 33

[0268] In a specific embodiment of the invention, the antibody comprises the following sequence as heavy chain:

[0269] SEQ ID NO: 32

[0270] or a sequence that is >95% identical, preferably >98% identical, preferably >99% identical thereto and comprises the following sequence as a light chain:

[0271] SEQ ID NO: 33

[0272] or a sequence that is >95% identical, preferably >98% identical, preferably >99% identical thereto, wherein the heavy chain comprises the following sequence:

[0273] CDR1: SEQ ID NO: 1

[0274] GYTFSRYW

[0275] CDR2: SEQ ID NO: 2

[0276] ILPGSGST

[0277] CDR3: SEQ ID NO: 3

[0278] TEGYEYDGFDY

[0279] And wherein the light chain comprises the following sequence:

[0280] CDR1: SEQ ID NO: 4

[0281] QSIVYSNGNTY

[0282] CDR2:

[0283] RVS

[0284] CDR3: SEQ ID NO: 5

[0285] FQGSHIPYT.

[0286] This means that, in one embodiment of the invention, the CDRs do not exhibit any sequence variations. In said embodiment any variations in the above sequences are outside the CDRs.

[0287] To assess the identity between two amino acid sequences, a pairwise alignment is performed. Identity is defined as the percentage of amino acids that have direct matches in the alignment.

[0288] Epitopes, also known as antigenic determinants, are parts of antigens that are recognized by the immune system, especially antibodies. For example, an epitope is a specific fragment of an antigen to which an antibody binds. The part of the antibody that binds to an epitope is called a paratope. Based on their structure and interaction with the paratope, epitopes of protein antigens are divided into two categories, namely conformational epitopes and linear epitopes.

[0289] Conformational and linear epitopes are based on the 3-D conformation adopted by the epitope in interaction with the paratope, which is determined by the surface features of the epitope residues involved and the shape or tertiary structure of other segments of the antigen. Conformational epitopes are formed by the 3-D conformation adopted by the interaction of discontinuous amino acid residues. Linear or continuous epitopes are epitopes that are recognized by antibodies through their linear sequence of amino acids or primary structure and are formed by the 3-D conformation adopted by the interaction of continuous amino acid residues.

[0290] One embodiment of the present invention relates to a method for predicting an increase in DPP3 in a critically ill patient, wherein if the DPP3 level in said sample is above said predetermined threshold, the patient is selected for therapy / treatment with a DPP3 inhibitor.

[0291] One embodiment of the present invention relates to a method for preventing an increase in DPP3 in a critically ill patient, wherein a DPP3 inhibitor is administered if the DPP3 level in said sample is above a predetermined threshold.

[0292] One embodiment of the invention relates to a DPP3 inhibitor for use in preventing an increase in DPP3 in a critically ill patient, wherein the patient has a DPP3 level above a threshold.

[0293] In another specific embodiment of the present invention, the DPP3 inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 scaffold.

[0294] In another specific embodiment of the present invention, the inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 scaffold that binds to a DPP3 region or epitope comprised in SEQ ID No. 36 and having a length of at least 4 to 5 amino acids.

[0295] In another specific embodiment of the present invention, the inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 scaffold that binds to a DPP3 region or epitope comprised in SEQ ID No. 37 and having a length of at least 4 to 5 amino acids.

[0296] In another preferred embodiment of the present invention, the inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 scaffold that binds to the DPP3 region or epitope located in SEQ ID No. 38.

[0297] In another preferred embodiment of the present invention, the anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 scaffold binds to the DPP3 region or epitope located in SEQ ID No. 39.

[0298] In another specific embodiment of the present invention, the inhibitor is an inhibitor that exhibits a minimum binding affinity for DPP3 equal to or less than 10 -7 M.

[0299] In another specific embodiment of the present invention, the inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 scaffold, and inhibits DPP3 activity by at least 10%, or at least 50%, more preferably at least 60%, even more preferably more than 70%, even more preferably more than 80%, even more preferably more than 90%, even more preferably more than 95%.

[0300] In another specific embodiment of the present invention, the antibody is a monoclonal antibody or a monoclonal antibody fragment.

[0301] In another specific embodiment of the present invention, the inhibitor is a monoclonal anti-DPP3 antibody or an anti-DPP3 antibody fragment, wherein the complementarity determining region (CDR) in the heavy chain comprises the following sequence:

[0302] SEQ ID NO.: 42, SEQ ID NO.: 43 and / or SEQ ID NO.: 44

[0303] And the complementarity determining region (CDR) in the light chain contains the following sequence:

[0304] SEQ ID NO.: 45, KVS and / or SEQ ID NO.: 46.

[0305] In another specific embodiment of the present invention, the inhibitor is a monoclonal anti-DPP3 antibody or an anti-DPP3 antibody fragment, wherein the monoclonal antibody or antibody fragment is a humanized monoclonal antibody or a humanized monoclonal antibody fragment.

[0306] In another specific embodiment of the present invention, the inhibitor is a humanized monoclonal anti-DPP3 antibody or an anti-DPP3 antibody fragment, wherein the heavy chain comprises the following sequence:

[0307] SEQ ID NO:40

[0308] And wherein the light chain comprises the following sequence:

[0309] SEQ ID NO.: 41.

[0310] In another specific embodiment of the present invention, the inhibitor is a humanized monoclonal anti-DPP3 antibody or an anti-DPP3 antibody fragment, the heavy chain of which consists of the following sequence:

[0311] SEQ ID NO:47

[0312] And the light chain consists of the following sequence:

[0313] SEQ ID NO.: 48.

[0314] Procizumab, a humanized monoclonal IgG1 antibody having a heavy chain consisting of the sequence SEQ ID NO.: 47 and a light chain consisting of the sequence SEQ ID NO.: 48, specifically binds to circulating DPP3, targeting and modulating DPP3 activity, which is an essential regulator of cardiovascular function. Its mode of action is associated with acute disease, which is associated with massive cell death and uncontrolled release of intracellular DPP3 into the bloodstream. Translocated DPP3 remains active in the circulation, where it cleaves bioactive peptides in an uncontrolled manner. Procizumab is able to block circulating DPP3, inhibiting the degradation of bioactive peptides in the bloodstream. This blockade results in stabilization of cardiovascular and renal function and a reduction in short-term mortality. As shown in the Examples section, preclinical studies of Procizumab in animal models of cardiovascular failure have shown impressive and immediate efficacy. For example, injection of Procizumab in rats with shock-induced cardiovascular failure resulted in an immediate normalization of shortening. In several preclinical models of cardiovascular failure, Procizumab has been shown to improve all in vivo clinically relevant endpoints. It normalizes ejection fraction and renal function and reduces mortality.

[0315] Throughout the specification, an "antibody" or "antibody fragment" or "scaffold" according to the present invention is capable of binding to DPP3, and is therefore directed against DPP3, and may therefore be referred to as an "anti-DPP3 antibody", "anti-DPP3 antibody fragment" or "anti-DPP3 scaffold".

[0316] The term "antibody" generally includes monoclonal antibodies and polyclonal antibodies and their binding fragments, especially Fc fragments and so-called "single-chain antibodies" (Bird et al. 1988), chimeric antibodies, humanized antibodies, especially CDR-grafted antibodies, and double-chain antibodies or four-chain antibodies (Holliger et al. 1993). Also included are immunoglobulin-like proteins that specifically bind to a target molecule contained in a sample, selected by techniques including, for example, phage display. In this context, the term "specific binding" refers to antibodies raised against a target molecule or a fragment thereof. An antibody is considered specific if its affinity for the target molecule or its aforementioned fragment is at least preferably 50 times, more preferably 100 times, and most preferably at least 1000 times greater than its affinity for other molecules contained in a sample containing the target molecule. How to prepare antibodies and select antibodies with a given specificity is well known in the art.

[0317] In one embodiment of the invention, the anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold is monospecific.

[0318] A monospecific anti-DPP3 antibody or monospecific anti-DPP3 antibody fragment or monospecific anti-DPP3 non-Ig scaffold means that the antibody or antibody fragment or non-Ig scaffold binds to one specific region (SEQ ID No. 36) within the target DPP3 covering at least 5 amino acids. A monospecific anti-DPP3 antibody or monospecific anti-DPP3 antibody fragment or monospecific anti-DPP3 non-Ig scaffold is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that all have affinity for the same antigen. Monoclonal antibodies are monospecific, but monospecific antibodies can also be produced by other means than producing them from common germ cells.

[0319] The antibodies or fragments according to the present invention are proteins comprising one or more polypeptides substantially encoded by immunoglobulin genes that specifically bind to an antigen. The recognized immunoglobulin genes include 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. The length of a full-length immunoglobulin light chain is generally about 25Kd or 214 amino acids.

[0320] The length of a full-length immunoglobulin heavy chain is usually about 50Kd or 446 amino acids. The light chain is 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. The heavy chain is similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.

[0321] The basic structural unit of an antibody is usually a tetramer composed of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions bind to the antigen, and the constant regions mediate effector functions. Immunoglobulins also exist in a variety of other forms, including, for example, Fv, Fab, and (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 ). An immunoglobulin light or heavy chain variable region comprises 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 described above, CDR is mainly responsible for binding to antigen epitopes. Immune complexes are antibodies that specifically bind to antigens, such as monoclonal antibodies, chimeric antibodies, humanized antibodies or human antibodies, or functional antibody fragments.

[0322] Chimeric antibodies are antibodies whose light chain and heavy chain genes have been constructed from immunoglobulin variable and constant region genes belonging to different species, typically by genetic engineering. For example, the variable segment of the gene from a mouse monoclonal antibody can be connected to a human constant segment, such as κ and γ1 or γ3. In one example, therapeutic chimeric antibodies are therefore a hybrid protein consisting of a variable domain or antigen-binding domain from a mouse antibody and a constant domain or effector domain from a human antibody, but other mammalian species can be used, or variable regions can be produced by molecular techniques. Methods for preparing chimeric antibodies are well known in the art, for example, referring to U.S. Patent No. 5,807,715. "Humanized" immunoglobulins are immunoglobulins including human framework regions and one or more CDRs from non-human (such as mouse, rat or synthetic) immunoglobulins. The non-human immunoglobulins providing CDRs are referred to as "donors", while the human immunoglobulins providing frameworks are referred to as "acceptors". In one embodiment, all CDRs in humanized immunoglobulins are from donor immunoglobulins. Constant regions do not need to be present, but if present, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85%-90% identical, such as about 95% or more identical. Thus, except for possible CDRs, all parts of humanized immunoglobulins are substantially identical to corresponding parts of natural human immunoglobulin sequences. "Humanized antibodies" are antibodies comprising humanized light chains and humanized heavy chain immunoglobulins. Humanized antibodies bind to the same antigen as the donor antibody providing the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of amino acid substitutions taken from the donor framework. Humanized antibodies or other monoclonal antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are those such as the following: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed by genetic engineering (e.g., see U.S. Patent No. 5,585,089). Human antibodies are antibodies in which the light chain 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 that secrete the target antibody. Immortality can be achieved, for example, by EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce trioma cells. Human antibodies can also be produced by phage display methods (see, e.g., WO91 / 17271; WO92 / 001047; WO92 / 20791), or selected from human combinatorial monoclonal antibody libraries (see Morphosys website). Human antibodies can also be prepared by using transgenic animals carrying human immunoglobulin genes (e.g., see WO93 / 12227; WO 91 / 10741).

[0323] Thus, the anti-DPP3 antibody may have a form known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies. In a preferred embodiment, the antibody according to the invention is a recombinantly produced antibody, such as an IgG, a typical full-length immunoglobulin, or an antibody fragment containing at least the F variable domain of a heavy chain and / or a light chain, such as a chemically coupled antibody (antigen-binding fragment), including but not limited to a Fab fragment, including a Fab miniantibody, a single-chain Fab antibody, a monovalent Fab antibody with an epitope tag (e.g. Fab-V5Sx2); a divalent Fab (miniantibody) dimerized with a CH3 domain; a divalent Fab or a multivalent Fab, for example formed by multimerization with the aid of a heterologous domain, for example formed by dimerization of a dHLX domain, for example Fab-dHLX-FSx2; a F(ab')2-fragment, a scFv-fragment, a multimerized multivalent or / and multispecific scFv-fragment, a divalent and / or bispecific diabody, a BITE ® (bispecific T-cell engagers), trifunctional antibodies, multivalent antibodies (e.g. from classes other than G); single domain antibodies such as nanobodies derived from camelid or fish immunoglobulins and many others.

[0324] In a preferred embodiment, the anti-DPP3 antibody form is selected from the group including Fv fragments, scFv fragments, Fab fragments, scFab fragments, F(ab)2 fragments and scFv-Fc fusion proteins. In another preferred embodiment, the antibody form is selected from the group including scFab fragments, Fab fragments, scFv fragments and their bioavailability optimized conjugates (such as pegylated fragments). One of the most preferred forms is the scFab form.

[0325] In one embodiment of the invention, anti-DPP3 antibodies according to the invention can be produced by synthesizing DPP3 fragments or full-length DPP3 as antigen as outlined in Example 1. Thereafter, binders of said fragments are identified using the methods described below or other methods known in the art.

[0326] Humanization of mouse antibodies can be performed according to the following procedure:

[0327] For humanization of antibodies of murine origin, the framework regions (FR) of the antibody sequence are analyzed for structural interactions with the complementarity determining regions (CDRs) and the antigen. Based on structural modeling, appropriate FRs of human origin are selected and the murine CDR sequences are grafted into the human FRs. Variations in the amino acid sequences of the CDRs or FRs can be introduced to regain structural interactions that were eliminated by species conversion of the FR sequences. This restoration of structural interactions can be achieved by random approaches using phage display libraries or by directed approaches guided by molecular modeling ( Almagro and Fransson 2008. Humanization of antibodies. Front Biosci. 13:1619-33 ).

[0328] Methods for obtaining monoclonal antibodies

[0329] In all of the following embodiments, the term monoclonal antibody is intended to include monoclonal antibodies as well as fragments of monoclonal antibodies, such as those described in detail herein, more particularly monoclonal antibodies.

[0330] Hybridoma

[0331] In another aspect, the antibody according to the invention is a monoclonal antibody obtainable by a method comprising the steps of:

[0332] i) fusing antibody-secreting cells from an animal previously immunized with an antigen with myeloma cells to obtain a large number of hybridomas,

[0333] ii) isolating hybridomas producing the desired monoclonal antibody from the plurality of hybridomas.

[0334] In certain embodiments, the antibodies according to the present invention are monoclonal antibodies obtainable by isolating a hybridoma producing the desired monoclonal antibody from a plurality of hybridomas produced by fusing antibody-secreting cells from an animal previously immunized with an antigen with myeloma cells to obtain a plurality of hybridomas.

[0335] Desirable monoclonal antibodies are particularly those that bind to the antigen, particularly with a binding affinity of at least 10 7 M -1 , preferably 10 8 M -1 Monoclonal antibodies, preferably with an affinity greater than 10 9 M -1 , most preferably greater than 10 10 M -1 .

[0336] In certain embodiments of the method for obtaining an antibody, in step i), the animal is a mammal, particularly a rabbit, a mouse or a rat, more particularly a mouse, more particularly a Balb / c mouse.

[0337] In certain embodiments of the method for obtaining antibodies, in step i), the antibody-secreting cells are spleen cells, more particularly activated B cells.

[0338] In certain embodiments of the method for obtaining an antibody, in step i), the fusion involves the use of polyethylene glycol.

[0339] In certain embodiments of the method for obtaining an antibody, in step i), the myeloma is derived from a mammal, in certain embodiments from the same species as the mammal from which the antibody-secreting cells are obtained in large quantities. In certain specific embodiments of the method for obtaining an antibody, in step i), the myeloma cell is of the cell line SP2 / 0.

[0340] In certain embodiments of the method for obtaining an antibody, the fusion in step i) comprises PEG-assisted fusion, Sendai virus-assisted fusion or electric current-assisted fusion.

[0341] In certain embodiments of the method for obtaining antibodies, said isolating in step ii) comprises performing an antibody capture assay, an antigen capture assay and / or a functional screen.

[0342] In certain embodiments of the method for obtaining an antibody, isolating hybridomas producing the desired monoclonal antibody in step ii) may involve cloning and recloning the hybridomas using limiting dilution techniques.

[0343] In one embodiment, the antibody capture assay comprises

[0344] a) binding the antigen to a substrate, in particular a solid substrate,

[0345] b) allowing the produced antibody to bind to the antigen,

[0346] c) removing unbound antibodies by washing,

[0347] d) Detecting bound antibodies.

[0348] In one embodiment, the antigen capture assay comprises

[0349] a) binding the produced antibody to a substrate, in particular a solid substrate,

[0350] b) allowing the antigen to bind to the antibody,

[0351] c) removing unbound antigen by washing,

[0352] d) detecting bound antigen;

[0353] Or the antigen capture assay comprises

[0354] a) allowing the antigen to bind to the produced antibody to form an antibody-antigen complex,

[0355] b) binding the antibody-antigen complex to a substrate, in particular a solid substrate,

[0356] c) removing unbound antigen by washing,

[0357] d) Detection of bound antigen.

[0358] In one embodiment, said isolating of step ii) comprises performing enzyme-linked immunosorbent assay, fluorescence activated cell sorting, cell staining, immunoprecipitation and / or Western blotting.

[0359] In one embodiment, said detection of an antibody or antigen is accomplished using an immunoassay.

[0360] In one embodiment, the animal is a transgenic animal, particularly a transgenic mouse (in which the mouse immunoglobulin (Ig) loci have been replaced by human loci, particularly within the genome of the transgenic animal), such as a HuMabMouse or a XenoMouse.

[0361] In one embodiment, the antigen comprises a peptide as described in Table 1 or Table 6 herein, respectively, which peptides may be conjugated to proteins, particularly serum proteins, more particularly serum albumin, more particularly BSA, in certain embodiments (particularly for immunization).

[0362] In a preferred embodiment, the antibody according to the invention is a monoclonal antibody obtainable by a method comprising the following steps:

[0363] i) fusing spleen cells from Balb / c mice previously immunized with the peptides described in Table 1 or Table 6 herein with SP2 / 0 myeloma cells using polyethylene glycol to obtain a large number of hybridomas,

[0364] ii) isolating a hybridoma producing a desired monoclonal antibody from the plurality of hybridomas;

[0365] More preferably, the method comprises

[0366] ● Grow the hybridomas in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement] for a first period of time (typically 2 weeks)

[0367] ●Subsequently, the HAT medium was replaced with HT medium for multiple passages (especially 3 times)

[0368] ● Then return to normal cell culture medium for a second period of time, typically until the end of three weeks post-confluency

[0369] ● Preliminary screening of antigen-specific IgG antibodies in cell culture supernatants

[0370] Proliferate a microculture of cells that tested positive in 4)

[0371] ● Retest the cell culture supernatant of the microculture for antigen-specific IgG antibodies

[0372] ● Using limiting dilution techniques, clone and reclon the cultures that tested positive in 6).

[0373] ● Optionally determine the isotype of the clones obtained from 7)

[0374] ●Optional purification of antibodies by protein A

[0375] Phage display

[0376] In another aspect, the antibody according to the invention is a monoclonal antibody obtainable by a method comprising the steps of:

[0377] i) isolating at least one antibody having affinity for the antigen from the antibody gene library;

[0378] ii) producing at least one cell line expressing said at least one antibody;

[0379] iii) isolating the at least one antibody from the culture of the at least one cell line obtained in step ii).

[0380] An antibody having an affinity for an antigen, particularly one having a binding affinity of at least 10 7 M -1 , preferably 10 8 M -1 Antibodies with an affinity greater than 10 9 M -1 , most preferably greater than 10 10 M -1 .

[0381] In a certain embodiment, the antibody according to the invention is a monoclonal antibody obtainable by isolating at least one antibody from a culture derived from at least one cell strain expressing at least one antibody having affinity for an antigen from an antibody gene library.

[0382] In one embodiment, the antigen comprises a peptide as described herein in Table 1 or Table 6, respectively, which peptide may be bound to a solid phase in certain embodiments.

[0383] In certain embodiments of the method for obtaining antibodies, in step i), the antibody gene library is a primary antibody gene library, particularly a human primary antibody gene library, more particularly in which the antibodies are presented by phage display, i.e., presented on phage comprising a nucleotide sequence encoding such corresponding antibody; more particularly, it is library HAL 7, HAL8 or HAL 9, more particularly a library comprising the human primary antibody gene library HAL7 / 8.

[0384] In certain embodiments of the method for obtaining antibodies, the screening in step i) comprises the use of an antigen, in particular an antigen containing a tag linked thereto via two different spacers, more particularly a biotin tag. In a particular embodiment, such a panning strategy comprises a mixture of panning rounds with non-specifically bound antigen and antigen specifically bound to streptavidin via a tag (in the case of a biotin tag). In this way, the background of non-specific binders can be minimized.

[0385] In certain embodiments of the method for obtaining an antibody, in step i), in embodiments wherein the library is a phage display library, the antibody is isolated by isolating phage presenting the antibody (and comprising a nucleotide sequence encoding the antibody).

[0386] In certain embodiments of the method for obtaining an antibody, in step ii), the cell strain is produced by introducing a nucleotide sequence encoding the antibody; in embodiments in which the library in step i) is a phage display library, the phage isolated from step i) can be used to produce a bacterial strain, such as an E. coli strain, that expresses the antibody.

[0387] In certain embodiments of the method for obtaining antibodies, in step iv): In embodiments wherein the library in step i) is a phage display library and wherein in step ii) a bacterial strain is produced, the antibody may be isolated from the supernatant of the culture.

[0388] It should be understood that, as used in describing a method for obtaining an antibody, the term "an antibody" in the expression "at least one antibody" may particularly include more than one antibody molecule of an antibody having the same amino acid sequence. This understanding applies, mutatis mutandis, to the term "a cell line".

[0389] In certain embodiments of the method for obtaining antibodies, more than one antibody (respectively, multiple antibodies with different amino acid sequences) is isolated in step i), and thus more than one cell line is produced in step ii). Such methods may involve, for example, selecting clones that are positive for binding to an antigen by a binding assay (e.g., an ELISA assay involving the antigen), and cells that are positive for binding to the antigen may be isolated to produce a monoclonal cell line.

[0390] In a preferred embodiment, the antibody according to the invention is a monoclonal antibody obtainable by a method comprising the following steps:

[0391] i) isolating at least one antibody having affinity for the antigen from an antibody gene library comprising a human primary antibody gene library HAL7 / 8 by eluting phage carrying the antibody from the library;

[0392] ii) producing at least one E. coli cell strain expressing said at least one antibody;

[0393] iii) isolating the at least one antibody from the supernatant of the culture of the at least one E. coli cell strain obtained in step ii).

[0394] In yet another aspect, antibody fragment according to the present invention is produced by involving enzyme digestion antibody. In certain embodiments, the method produces for example Fab or F (ab) 2 antibody fragment. In certain embodiments, the method involves digestion with pepsin or papain, and pepsin or papain are optionally fixed on the surface.

[0395] In certain embodiments, antibodies may be humanized by CDR grafting, particularly by a method involving the following steps:

[0396] - extracting RNA from a hybridoma expressing an antibody of interest (e.g., a hybridoma obtained by the methods described herein);

[0397] - amplifying the extracted RNA by RT-PCR, in particular using a primer set specific for the heavy and light chains of the antibody of interest, to obtain a DNA product;

[0398] - further amplifying the DNA product by PCR, in particular using a semi-nested primer set specific for the antibody variable region;

[0399] - Determine the sequence of the DNA product;

[0400] - Aligning the sequences with homologous human framework sequences to determine the humanized sequences of the variable heavy and variable light chain sequences (of the desired antibody).

[0401] In certain embodiments, the antibody can be humanized by aligning the sequence of a DNA product obtained by amplifying RNA extracted from a hybridoma expressing the antibody of interest by RT-PCR, particularly using a primer set specific for the heavy and light chains of the antibody of interest, and further amplifying DNA obtained from the extracted RNA by PCR, particularly using a semi-nested primer set specific for the variable region of the antibody, to determine the humanized sequence of the variable heavy chain and variable light chain sequences (of the desired antibody).

[0402] In certain embodiments, antibodies can be humanized by:

[0403] - Determine the complementarity determining regions (CDRs), which can be done by analyzing the structural interactions between the framework regions (FRs) and the complementarity determining regions (CDRs) and the antigen;

[0404] - Grafting the CDR sequences into human framework regions.

[0405] In certain embodiments, antibodies can be humanized by grafting CDR sequences, which preferably have been determined by analyzing the structural interactions of the framework regions (FRs) with the complementarity determining regions (CDRs) and antigen, into human framework regions.

[0406] In certain embodiments, variations in the amino acid sequence of the CDRs or FRs can be introduced to maintain structural interactions with the antigen (which otherwise can be abolished by introducing human FR sequences), for example, by random approaches using phage display libraries or by directed approaches guided by molecular modeling.

[0407] The DNA sequences encoding the antibodies determined as detailed herein can be transferred into cells by known genetic engineering techniques and used to produce the antibodies.

[0408] Producing antibodies

[0409] In another aspect, the antibody according to the invention is a monoclonal antibody obtainable by the method described herein, produced by a method comprising:

[0410] - cultivating a cell line comprising a nucleotide sequence encoding the antibody;

[0411] - isolating the antibody from said culture.

[0412] In another certain aspect, the antibody according to the present invention is a monoclonal antibody obtainable by the method described herein, produced by isolating the antibody from a culture of a cell line comprising a nucleotide sequence encoding the antibody.

[0413] In certain embodiments of the method, the cell strain is produced as described above and may comprise a bacterial cell, such as a Gram-negative bacterium, e.g., Escherichia coli, Proteus mirabilis, or Pseudomonas putidas; a Gram-positive bacterium, e.g., Bacillus brevis, Bacillus subtilis, Bacillus megaterium; a Lactobacillus, such as Lactobacillus zeae / Lactobacillus casei, or Lactobacillus paracasei; or a Streptomyces, such as Streptomyces lividans; a eukaryotic cell, such as a yeast, e.g., Pichia pastoris, Saccharomyces cerevisiae. cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis or Yarrowialipolytica; a fungus, for example a filamentous fungus, for example Trichoderma or Aspergillus, such as A. niger (e.g. subgenus awamori) and Aspergillus oryzae, Trichoderma reesei, a Chrysosporium, such as C. lucknowense; a protozoan, such as a Leishmania, for example L. tarentolae; an insect cell, such as an insect cell transfected with a baculovirus, such as AcNPV, such as from Spodoptera frugiperda. frugiperda) (e.g. Sf-9 or Sf-21), Drosophila melanogaster (e.g. DS2) or Trichopulsia ni, for example High Five cells (BTI-TN-5B1-4); mammalian cells, such as hamster cells, for example Chinese hamster ovary cells, such as K1-, DukX B11-, DG44, Lec13 or BHK; mouse, for example mouse myeloma, such as NS0; modern human, for example Per.C6, AGE1.HN, HEK293. .

[0414] In certain embodiments of the methods, the cell can be a hybridoma cell, such as a hybridoma cell described herein.

[0415] In certain embodiments of the method, the cultivation can be carried out in a static suspension culture, a stirred suspension culture, a membrane-based culture, a matrix-based culture, or a high cell density bioreactor; the container for such cultivation can be selected from the group consisting of: a T-flask, a roller culture, a rotary culture, a stirred tank bioreactor, an airlift bioreactor, a static membrane-based or matrix-based culture system, a suspension bioreactor, a fluidized bed bioreactor, a ceramic bioreactor, a perfusion system, a hollow fiber bioreactor.

[0416] In certain embodiments of the methods, the cells may be immobilized on a matrix.

[0417] A high cell density bioreactor is specifically a culture system capable of producing cell densities greater than 10^8 cells / ml.

[0418] In another aspect, the antibody according to the invention is a monoclonal antibody obtainable by the method described herein, produced by a method comprising:

[0419] - Producing a transgenic plant or animal comprising a nucleotide sequence encoding the antibody;

[0420] - isolating the antibody from said plant or animal or from a secretion or product of said plant or animal.

[0421] In certain further aspects, the antibody according to the invention is a monoclonal antibody obtainable by the method described herein, by isolating the antibody from a transgenic plant or transgenic animal having a nucleotide sequence encoding the antibody or from a secretion or product of the transgenic plant or transgenic animal.

[0422] The animal may be, for example, selected from chicken, mouse, rat, rabbit, cow, goat, sheep, pig; the secretion or product may be, for example, milk or eggs. The plant may be, for example, selected from tobacco (N. tabacum or N. benthamiana), duckweed (Lemna minor), Chlamydomonas reinhardtii, rice, Arabidopsis thaliana, alfalfa (Medicago sativa), lettuce, corn.

[0423] In certain embodiments, the antibodies can be separated by physicochemical fractionation, such as size exclusion chromatography, precipitation (e.g., using ammonium sulfate), ion exchange chromatography, immobilized metal chelate chromatography, gel filtration, zone electrophoresis; can be separated based on their classification, such as binding to bacterial proteins A, G or L, jacalin; can be separated by antigen-specific affinity purification of immobilized ligand / antigen; if necessary, low molecular weight components can be removed by methods such as dialysis, desalting and diafiltration.

[0424] In some embodiments, the antibody is encoded by a nucleotide sequence, wherein the nucleotide sequence is a reverse transcript of an amino acid sequence from an antibody produced by one of the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0425] Figure 1a : Description of antibody formats - Fv and scFv - variants.

[0426] Figure 1b : Antibody formats - description of heterologous fusions and bifunctional antibodies.

[0427] Figure 1c : Antibody formats - description of bivalent and bispecific antibodies.

[0428] Figure 2a : Dose response curve of human ADM. Maximum cAMP stimulation was adjusted to 100% activation.

[0429] Figure 2b : Dose / inhibition curve of human ADM 22-52 (ADM-receptor antagonist) in the presence of 5.63 nM hADM.

[0430] Figure 2c : Dose / inhibition curve of CT-H in the presence of 5.63 nM hADM.

[0431] Figure 2d : Dose / inhibition curve of MR-H in the presence of 5.63 nM hADM.

[0432] Figure 2e : Dose / inhibition curve of NT-H in the presence of 5.63 nM hADM.

[0433] Figure 2f : Dose response curve of mouse ADM. Maximum cAMP stimulation was adjusted to 100% activation.

[0434] Figure 2g : Dose / inhibition curve of human ADM 22-52 (ADM-receptor antagonist) in the presence of 0.67 nM mADM.

[0435] Figure 2h : Dose / inhibition curve of CT-M in the presence of 0.67 nM mADM.

[0436] Figure 2i : Dose / inhibition curve of MR-M in the presence of 0.67 nM mADM.

[0437] Figure 2j : Dose / inhibition curve of NT-M in the presence of 0.67 nM mADM.

[0438] Figure 2k : Shows the inhibition of ADM by F(ab)2 NT-M and Fab NT-M.

[0439] Figure 2l : Shows the inhibition of ADM by F(ab)2 NT-M and Fab NT-M.

[0440] Figure 3 : The figure shows a typical hADM dose / signal curve. And the hADM dose-signal curve in the presence of 100 µg / mL antibody NT-H.

[0441] Figure 4 : The figure shows the stability of hADM in human plasma (citrate) in the absence and presence of NT-H antibody.

[0442] Figure 5 : Alignment of Fab with homologous human framework sequences.

[0443] Figure 6 : ADM concentrations in healthy human subjects after administration of NT-H at different doses for up to 60 days.

[0444] Figure 7 : Inhibition curve of native DPP3 from blood cells with the inhibitory antibody AK1967. The inhibition of DPP3 by the specific antibody is concentration-dependent, with an IC of 50 is ~15ng / ml.

[0445] Figure 8 : Association and dissociation curves of AK1967-DPP3 binding assay using Octet. AK1967 loaded biosensors were immersed in a series of recombinant GST-tagged human DPP3 dilutions (100 nM, 33.3 nM, 11.1 nM, 3.7 nM) and association and dissociation were monitored.

[0446] Fig. 9 : Western blot of blood cell lysate dilutions and detection of DPP3 using AK1967 as primary antibody.

[0447] Fig.10 : Protuzumab significantly improved fractional shortening (A) and mortality (B) in rats with sepsis-induced heart failure.

[0448] Fig.11 : Experimental design - Isoproterenol induced cardiac stress in mice followed by protuzumab treatment (B) and control (A).

[0449] Fig.12 : In mice with isoproterenol-induced heart failure, protuzumab improved fractional shortening (A) and reduced renal resistive index (B) at 1 and 6 h after administration, respectively.

[0450] Fig.13 : High DPP3 levels 24 hours after admission were associated with the worst SOFA scores in patients with sepsis.

[0451] Fig.14 : High DPP3 plasma levels are associated with organ dysfunction in patients with sepsis. Bar graph of SOFA scores in AdrenOSS-1 according to the evolution of DPP3 levels during ICU stay. HH: DPP3 above median on admission and at 24 h; HL: above median on admission but below median at 24 h; LL: below median on admission and at 24 h; LH: below median on admission but above median at 24 h.

[0452] Figure 15: High DPP3 levels 24 hours after admission were associated with the worst SOFA scores in patients with sepsis, by organ. (A) Heart, (B) kidney, (C) respiratory system, (D) liver, (E) coagulation, and (F) central nervous system SOFA score values ​​were calculated based on the DPP3 dynamics between admission and 24 hours (HH: high / high, HL: high / low, LH: low / high, LL: low / low).

[0453] Fig.16 : Kaplan-Meier survival plots associated with low (<40.5 ng / mL) and high (≥40.5 ng / mL) DPP3 concentrations. (A) 7-day survival of patients with sepsis associated with DPP3 plasma concentrations; (B) 7-day survival of patients with cardiogenic shock associated with DPP3 plasma concentrations; (C) 7-day survival of patients with septic shock associated with DPP3 plasma concentrations.

[0454] Fig.17 : Kaplan-Meier survival plot for all patients (14-day mortality in patients treated with placebo (Plac) or the N-terminal ADM antibody aderizumab (Adz))

[0455] Fig.18 : Kaplan-Meier survival plot for patients with DPP3 < 50 ng / mL (14-day mortality in patients treated with placebo (Plac) or the N-terminal ADM antibody aderizumab (Adz))

[0456] Fig.19 : Kaplan-Meier survival plot for patients with DPP3>50ng / mL (14-day mortality in patients treated with placebo (Plac) or the N-terminal ADM antibody aderizumab (Adz))

[0457] Figure 20: Efficacy of aderezumab treatment using different DPP3 thresholds: Kaplan-Meier survival plot (28-day mortality) of patients treated with placebo or the N-terminal ADM antibody aderezumab, including patients with (A) DPP3 < 50 ng / mL, (B) DPP3 < 40 ng / ml, (C) DPP3 < 30 ng / ml and (D) DPP3 < 22 ng / ml, respectively.

[0458] Fig.21 : Efficacy of adeleizumab treatment using different DPP3 thresholds: Kaplan-Meier survival plot (28-day mortality) of patients treated with placebo or the N-terminal ADM antibody adeleizumab, including patients with baseline DPP3 values ​​below 50 ng / ml and DPP3 < 50 ng / mL on the following day.

[0459] sequence

[0460] SEQ ID No.: 1 (anti-ADM CDR 1 heavy chain)

[0461] GYTFSRYW

[0462] SEQ ID No.: 2 (anti-ADM CDR 2 heavy chain)

[0463] ILPGSGST

[0464] SEQ ID No.: 3 (anti-ADM CDR 3 heavy chain)

[0465] TEGYEYDGFDY

[0466] SEQ ID No.: 4 (anti-ADM CDR 1 light chain)

[0467] QSIVYSNGNTY

[0468] Sequence "RVS" (anti-ADM CDR 2 light chain, not part of the sequence listing):

[0469] RVS

[0470] SEQ ID No.: 5 (anti-ADM CDR 3 light chain)

[0471] FQGSHIPYT

[0472] SEQ ID No.: 6 (AM-VH-C)

[0473] QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0474] SEQ ID No.: 7 (AM-VH1)

[0475] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0476] SEQ ID No.: 8 (AM-VH2-E40)

[0477] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0478] SEQ ID No.: 9(AM-VH3-T26-E55)

[0479] QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0480] SEQ ID No.: 10(AM-VH4-T26-E40-E55)

[0481] QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK

[0482] SEQ ID No.: 11(AM-VL-C)

[0483] DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0484] SEQ ID No.: 12 (AM-VL1)

[0485] DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0486] SEQ ID No.: 13 (AM-VL2-E40)

[0487] DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0488] SEQ ID No.: 14 (Human ADM 1-21)

[0489] YRQSMNNFQGLRSFGCRFGTC

[0490] SEQ ID No.: 15 (Human ADM 21-32)

[0491] CTVQKLAHQIYQ

[0492] SEQ ID No.: 16 (human ADM C-42-52)

[0493] CAPRSKISPQGY-CONH2

[0494] SEQ ID No.: 17 (murine ADM 1-19)

[0495] YRQSMNQGSRSNGCRFGTC

[0496] SEQ ID No.: 18 (rat ADM 19-31)

[0497] CTFQKLAHQIYQ

[0498] SEQ ID No.: 19 (rat ADM C-40-50)

[0499] CAPRNKISPQGY-CONH2

[0500] SEQ ID No.: 20 (mature human adrenomedullin (mature ADM); amidated ADM; bio-ADM): amino acids 1-52 or amino acids 95-146 of pro-ADM

[0501] YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY-CONH2

[0502] SEQ ID No.: 21 (rat ADM 1-50)

[0503] YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQLTDKDKDGMAPRNKISPQGY-CONH2

[0504] SEQ ID No.: 22 (1-21 of human ADM):

[0505] YRQSMNNFQGLRSFGCRFGTC

[0506] SEQ ID No.: 23 (1-42 of human ADM):

[0507] YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA

[0508] SEQ ID No.: 24 (amino acids 43-52 of human ADM)

[0509] PRSKISPQGY-NH2

[0510] SEQ ID No.: 25 (amino acids 1-14 of human ADM)

[0511] YRQSMNNFQGLRSF

[0512] SEQ ID No.: 26 (amino acids 1-10 of human ADM)

[0513] YQS

[0514] SEQ ID No.: 27 (amino acids 1-6 of human ADM)

[0515] YQ

[0516] SEQ ID No.: 28 (amino acids 1-32 of human ADM)

[0517] YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQ

[0518] SEQ ID No.: 29 (amino acids 1-40 of mouse ADM)

[0519] YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQLTDKDKDGMA

[0520] SEQ ID No.: 30 (amino acids 1-31 of mouse ADM)

[0521] YRQSMNQGSRSNGCRFGTCTFQKLAHQIYQL

[0522] SEQ ID No.: 31 (proADM: 164 amino acids (22-185 of preproADM))

[0523] APHFL

[0524] SEQ ID NO: 32 (Adrelizumab heavy chain)

[0525] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0526] SEQ ID NO: 33 (Aderizumab light chain)

[0527] DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0528] SEQ ID No. 34 - IGHV1-69*11

[0529] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYYYYYGMDVWGQGTTVTVSS

[0530] SEQ ID No. 35 - HB3

[0531] QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSS

[0532] SEQ ID No. 36 - Human DPP3 (amino acids 1 - 737)

[0533] MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSPEAPYIYALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTKFVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLRHLGLGKEGITTYFSGNCTMEDAKLAQDFLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDSEVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESFTQGSIEAHKRGSRFWIQDKGPIVESYIGFIESYRDPFGSRGEFEGFVAVVNKAMSAKFERLVASAEQLLKELPWPPTFEKDKFLTPDFTSLDVLTFAGSGIPAGINIPNYDDLRQTEGFKNVSLGNVLAVAYATQREKLTFLEEDDKDLYILWKGPSFDVQVGLHELLGHGSGKLFVQDEKGAFNFDQETVINPETGEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLYLCLHPQVLEIFGFEGADAEDVIYVNWLNMVRAGLLALEFYTPEAFNWRQAHMQARFVILRVLLEAGEGLVTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTGDVAGGRALYEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEYEASAAGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA

[0534] SEQ ID No. 37 - Human DPP3 (amino acids 474-493 (N-Cys)) - Immunizing peptide with an additional N-terminal cysteine

[0535] CETVINPETGEQIQSWYRSGE

[0536] SEQ ID No. 38 - hDPP3 amino acids 477-482 - epitope of AK1967

[0537] INPETG

[0538] SEQ ID No. 39—hDPP3 amino acids 480-483

[0539] ETGE

[0540] SEQ ID No. 40 - Variable region of mouse AK1967 in the heavy chain

[0541] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPSGKGLEWLAHIWWNDNKSYNPALKSRLTISRDTSNNQVFLKIASVVTADTGTYFCARNYSYDYWGQGTTLTVSS

[0542] SEQ ID No. 41 - Variable region of mouse AK1967 in the light chain

[0543] DVVVTQTPLSLSVSLGDPASISCRSSRSLVHSIGSTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

[0544] SEQ ID No. 42 - CDR1 of mouse AK1967 in the heavy chain

[0545] GFSLSTSGMS

[0546] SEQ ID No. 43 - CDR2 of mouse AK1967 in the heavy chain

[0547] IWWNDNK

[0548] SEQ ID No. 44 - CDR3 of mouse AK1967 in the heavy chain

[0549] ARNYSYDY

[0550] SEQ ID No. 45 - CDR1 of mouse AK1967 in the light chain

[0551] RSLVHSIGSTY

[0552] CDR2 of mouse AK1967 in the light chain (no sequence ID)

[0553] KVS

[0554] SEQ ID No. 46 - CDR3 of mouse AK1967 in the light chain

[0555] SQSTHVPWT

[0556] SEQ ID No. 47 - Humanized AK1967 - Heavy chain sequence (IgG1κ backbone)

[0557] MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQVVLTMTNMDPVDTG TYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0558] SEQ ID No. 48 - Humanized AK1967 - Light chain sequence (IgG1κ main chain)

[0559] METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHV PWTTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0560] For the avoidance of doubt, references to the C-terminal groups "NH2" and "CONH2" refer equally to the C-terminal amide group.

[0561] In the above context, the following consecutively numbered embodiments provide further specific aspects of the present invention:

[0562] 1. A method for predicting an increase in dipeptidyl peptidase 3 (DPP3) in a critically ill patient, the method comprising:

[0563] Determining the level of DPP3 in a body fluid sample of said patient,

[0564] ● comparing the determined DPP3 level to a predetermined threshold, wherein the threshold is in the range between 40 ng / ml and 22 ng / ml

[0565] wherein the level of DPP3 in the sample being above the predetermined threshold indicates an increase in DPP3 in the patient.

[0566] In certain embodiments, the method is used to predict an increase in dipeptidyl peptidase 3 (DPP3) in a critically ill patient, the method comprising:

[0567] Determining the level of DPP3 in a body fluid sample of said patient,

[0568] ● comparing the determined DPP3 level to a predetermined threshold, wherein the threshold is in the range between 40 ng / ml and 22 ng / ml

[0569] wherein the level of DPP3 in the sample being above the predetermined threshold indicates an increase in DPP3 in the patient during the follow-up time.

[0570] 2. The method for predicting an increase in DPP3 in critically ill patients according to embodiment 1, wherein the predetermined threshold is between 30 ng / ml and 22 ng / ml.

[0571] 3. The method for predicting an increase in DPP3 in critically ill patients according to embodiment 1 or 2, wherein the predetermined threshold is between 25 ng / ml and 22 ng / ml.

[0572] 4. The method for predicting an increase in DPP3 in a critically ill patient according to any one of embodiments 1 to 3, wherein the predicted increase is an increase to a DPP3 level equal to or higher than 40 ng / ml, preferably equal to or higher than 50 ng / ml.

[0573] 5. The method for predicting an increase in DPP3 in critically ill patients according to embodiments 1 to 3, wherein the predicted increase in DPP3 level is equal to or higher than 10%, more preferably equal to or higher than 20%, even more preferably equal to or higher than 40%, even more preferably equal to or higher than 50%, even more preferably equal to or higher than 75%, even more preferably equal to or higher than 100%.

[0574] 6. The method for predicting an increase in DPP3 in critically ill patients according to embodiments 1 to 5, wherein the DPP3 increase is within at most 12 hours, preferably at most 24 hours, 48 ​​hours, 72 hours, 96 hours, more preferably at most 5 days, even more preferably at most 6 days, most preferably at most 7 days.

[0575] 7. A method for predicting an increase in DPP3 in a critically ill patient according to embodiments 1 to 6, wherein the patient is a patient with severe infection, sepsis, heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, a patient with organ dysfunction or organ failure (e.g., liver, kidney, lung dysfunction or failure), a patient undergoing major surgery, a patient with trauma (e.g., burns, multiple trauma), a patient with shock and / or a patient developing shock, or alternatively a patient with ARDS.

[0576] 8. The method for predicting an increase in DPP3 in a critically ill patient according to embodiment 7, wherein the shock is selected from the group consisting of shock caused by hypovolemia, cardiogenic shock, obstructive shock and distributive shock.

[0577] 9. The method for predicting an increase in DPP3 in a critically ill patient according to embodiment 8, wherein

[0578] In the case of cardiogenic shock, the patient may have suffered an acute coronary syndrome (e.g., acute myocardial infarction) or in which the patient has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmias, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or

[0579] In the setting of hypovolemic shock, the patient may have had a bleeding disorder, including spontaneous bleeding from gastrointestinal bleeding, trauma, vascular etiologies (e.g., ruptured abdominal aortic aneurysm, tumor erosion into a major vessel), and anticoagulant use, or a non-bleeding disorder, including vomiting, diarrhea, renal loss, skin loss / insensate loss (e.g., burns, heat stroke), or third space loss in the setting of pancreatitis, cirrhosis, or intestinal obstruction, or

[0580] In the case of obstructive shock, the patient may have suffered cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or

[0581] • In cases of distributive shock, the patient may be suffering from septic shock, neurogenic shock, anaphylactic shock, or shock caused by adrenal crisis.

[0582] 10. The method for predicting an increase in DPP3 in a critically ill patient according to any one of embodiments 7 to 9, wherein the shock is selected from the group comprising cardiogenic shock or septic shock.

[0583] 11. A method for predicting an increase in DPP3 in a critically ill patient according to any one of embodiments 1 to 10, wherein if the DPP3 level in the sample is below the predetermined threshold, the patient is selected for therapy / treatment, wherein the therapy is selected from the group of alkaline phosphatase, immunosuppressants, corticosteroids, vasopressors, fluids, anti-adrenomedullin antibodies or antibody fragments or scaffolds.

[0584] 12. The method for predicting an increase in DPP3 in critically ill patients according to embodiment 11, wherein the anti-adrenomedullin antibody or anti-adrenomedullin antibody fragment or anti-adrenomedullin scaffold is directed against the N-terminal portion (amino acids 1-21) of adrenomedullin (ADM): YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 14).

[0585] 13. A method for predicting an increase in DPP3 in a critically ill patient according to any one of embodiments 1 to 12, wherein if the DPP3 level in the sample is above the predetermined threshold, the patient is selected for therapy / treatment with a DPP3 inhibitor, wherein the DPP3 inhibitor is selected from the group of anti-DPP3 antibodies or anti-DPP3 antibody fragments or anti-DPP3 scaffolds.

[0586] 14. The method for predicting an increase in DPP3 in a critically ill patient according to any one of embodiments 1 to 13, wherein the DPP3 level is the amount of DPP3 protein and / or the level of active DPP3.

[0587] 15. The method for predicting an increase in DPP3 in critically ill patients according to any one of embodiments 1 to 14, wherein the DPP3 level is determined by different methods including immunoassay, activity assay or mass spectrometry.

[0588] 16. The method for predicting an increase in DPP3 in critically ill patients according to embodiment 15, wherein the immunoassay is a sandwich immunoassay.

[0589] 17. The method for predicting an increase in DPP3 in a critically ill patient according to any one of embodiments 1 to 16, wherein the body fluid is selected from whole blood, serum or plasma.

[0590] 18. A method for preventing an increase in DPP3 in a critically ill patient, the method comprising:

[0591] ● determining the level of DPP3 in a body fluid sample of said patient,

[0592] comparing the determined DPP3 level to a predetermined threshold, wherein the predetermined threshold is between 40 ng / ml and 22 ng / ml, and wherein a DPP3 level in the sample above the predetermined threshold is indicative of an increased DPP3 in the patient, and

[0593] if said determined DPP3 level is above said predetermined threshold, administering a DPP3 inhibitor,

[0594] The DPP3 inhibitor is an anti-DPP3 antibody and / or an anti-DPP3 antibody fragment and / or an anti-DPP3 scaffold.

[0595] 19. The method for preventing an increase in DPP3 in critically ill patients according to embodiment 18, wherein the predetermined threshold is between 30 ng / ml and 22 ng / ml.

[0596] 20. The method for preventing an increase in DPP3 in a critically ill patient according to embodiment 18 or 19, wherein the predetermined threshold is between 25 ng / ml and 22 ng / ml.

[0597] 21. The method for preventing an increase in DPP3 in a critically ill patient according to any one of embodiments 18 to 20, wherein the increase is to a DPP3 level equal to or higher than 40 ng / ml, preferably equal to or higher than 50 ng / ml.

[0598] 22. A method for preventing an increase in DPP3 in a critically ill patient according to embodiments 18 to 21, wherein the patient is a patient with severe infection, sepsis, heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, a patient with organ dysfunction or organ failure (e.g., liver, kidney, lung dysfunction or failure), a patient undergoing major surgery, a patient with trauma (e.g., burns, multiple trauma), a patient with shock and / or a patient developing shock, or alternatively a patient with ARDS.

[0599] 23. The method for preventing an increase in DPP3 in a critically ill patient according to embodiment 22, wherein the shock is selected from the group consisting of shock caused by hypovolemia, cardiogenic shock, obstructive shock and distributive shock.

[0600] 24. The method for preventing an increase in DPP3 in a critically ill patient according to embodiment 23, wherein

[0601] In the case of cardiogenic shock, the patient may have suffered an acute coronary syndrome (e.g., acute myocardial infarction) or in which the patient has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmias, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or

[0602] In the setting of hypovolemic shock, the patient may have had a bleeding disorder, including spontaneous bleeding from gastrointestinal bleeding, trauma, vascular etiologies (e.g., ruptured abdominal aortic aneurysm, tumor erosion into a major vessel), and anticoagulant use, or a non-bleeding disorder, including vomiting, diarrhea, renal loss, skin loss / insensate loss (e.g., burns, heat stroke), or third space loss in the setting of pancreatitis, cirrhosis, or intestinal obstruction, or

[0603] In the case of obstructive shock, the patient may have suffered cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or

[0604] • In cases of distributive shock, the patient may be suffering from septic shock, neurogenic shock, anaphylactic shock, or shock caused by adrenal crisis.

[0605] 25. The method for preventing an increase in DPP3 in a critically ill patient according to any one of embodiments 22 to 24, wherein the shock is selected from the group comprising cardiogenic shock or septic shock.

[0606] 26. A DPP3 inhibitor for use in preventing an increase in DPP3 in a critically ill patient, wherein the patient has a DPP3 level above a threshold, wherein the threshold is between 40 ng / ml and 22 ng / ml, and wherein the DPP3 inhibitor is an anti-DPP3 antibody and / or an anti-DPP3 antibody fragment and / or an anti-DPP3 scaffold. Example

[0607] Example 1 - Generation of anti-ADM antibodies and determination of their affinity constants

[0608] Several human and murine antibodies were generated and their affinity constants determined (see Table 1 and Table 2). It should be emphasized that the antibodies, antibody fragments and non-Ig scaffolds according to the Examples section of the present invention bind to ADM and should therefore be considered anti-ADM antibodies / antibody fragments / non-Ig scaffolds.

[0609] Peptides / conjugates for immunization :

[0610] The peptides used for immunization, see Table 1 (JPT Technologies, Berlin, Germany), were synthesized with an additional N-terminal cysteine ​​(if cysteine ​​was not present in the selected ADM-sequence) residue for conjugation of the peptide to bovine serum albumin (BSA). 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.

[0611] Mouse monoclonal antibody generation :

[0612] Balb / c mice were immunized with 100 µg of peptide-BSA-conjugate (emulsified in 100 µl complete Freund's adjuvant) on days 0 and 14 and with 50 µg of the conjugate (in 100 µl incomplete Freund's adjuvant) on days 21 and 28. Three days before fusion experiments, animals received 50 µg of the conjugate dissolved in 100 µl saline as one intraperitoneal and one intravenous injection. Splenocytes from immunized mice and cells of the myeloma cell line SP2 / 0 were fused with 1 ml 50% polyethylene glycol at 37°C for 30 seconds. After washing, cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium [RPMI 1640 medium supplemented with 20% fetal calf serum and HAT supplement]. After two weeks, the HAT medium was changed to HT medium, passaged three times, and then returned to normal cell culture medium. Three weeks after fusion, cell culture supernatants are initially screened for antigen-specific IgG antibodies. Positive test microcultures are transferred to 24-well plates for expansion. After retesting, selected cultures are cloned and recloned using limiting dilution techniques and isotyped (see also Lane, RD 1985. J. Immunol. Meth. 81: 223-228; Ziegler et al. 1996. Horm. Metab. Res. 28: 11-15 ).

[0613] Antibodies were generated using standard antibody production methods ( Marx et al., 1997. Monoclonal Antibody Production, ATLA 25, 121 ) and purified by protein A. The antibody purity was >95% based on SDS gel electrophoresis analysis.

[0614] Human Antibodies :

[0615] Human antibodies were produced by phage display according to the following procedure: The human primary antibody gene library HAL7 / 8 was used to isolate recombinant single-chain F-variable domains (scFv) against the adrenomedullin peptide. The antibody gene library was screened with a panning strategy involving the use of peptides containing a biotin tag linked to the adrenomedullin peptide sequence via two different spacers. A mix of panning rounds using nonspecifically bound antigen and streptavidin-bound antigen was used to minimize the background of nonspecific binders. Phage eluted from the third round of panning have been used to generate E. coli strains expressing monoclonal scFvs. Supernatants from the cultivation of these clonal strains have been used directly for antigen ELISA tests (see also Hust et al. 2011. Journal of Biotechnology 152, 159–170; Schütte et al. 2009. PLoS One 4, e6625 Positive clones were selected based on positive ELISA signals for antigen and negative ELISA signals for 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.

[0616] Affinity constant : To determine the affinity of the antibodies for ADM, the binding kinetics of ADM to the immobilized antibodies were determined by label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibodies was performed according to the manufacturer's instructions using an anti-mouse Fc antibody covalently coupled to the CM5 sensor surface at high density (Mouse Antibody Capture Kit; GE Healthcare). Lorenz et al. 2011. Antimicrob Agents Chemother. 55(1): 165–173 ).

[0617] Monoclonal antibodies were generated against the ADM region of human and mouse ADM described below, respectively. The following table shows the selection of antibodies obtained for further experiments. Selection was based on the target region:

[0618] Table 1: ADM immune peptides

[0619]

[0620]

[0621] Table 2: Further obtained monoclonal anti-ADM antibodies

[0622]

[0623]

[0624] Generation of antibody fragments by enzymatic digestion : Generation of Fab and F(ab)2 fragments by enzymatic digestion of the full-length murine antibody NT-M. Antibody NT-M was digested using a) a pepsin-based F(ab)2 preparation kit (Pierce 44988) and b) a papain-based Fab preparation kit (Pierce 44985). The fragmentation procedure was performed according to the instructions provided by the supplier. In the case of F(ab)2-fragmentation, digestion was performed at 37°C for 8 hours. Fab-fragmentation digestion was performed for 16 hours, respectively.

[0625] Fab production and purification procedures: The immobilized papain was balanced by washing the resin with 0.5 ml digestion buffer and centrifuging the column at 5000 × g for 1 minute. The buffer was then discarded. The desalting column was prepared by removing the storage solution and washing it with digestion buffer, followed by centrifugation 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 balanced immobilized papain. The incubation time of the digestion reaction was 16 hours at 37 ° C on a tabletop shaker. The column was centrifuged at 5000 × g for 1 minute to separate the digest from the immobilized papain. The resin was then washed with 0.5 ml PBS and centrifuged at 5000 × g for 1 minute. The wash fraction was added to the digested antibody, with a total sample volume of 1.0 ml. The NAb protein A column was balanced 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) and balanced by adding 2 ml PBS, centrifuged again for 1 minute and the flow-through was discarded. The sample was applied to the column and resuspended by inversion. Incubate at room temperature with end-over-end mixing for 10 minutes. Centrifuge the column for 1 minute and save the flow-through with the Fab fragment. (Reference: 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 ).

[0626] F(ab´)2 fragment generation and purification procedures : The immobilized pepsin was equilibrated by washing the resin with 0.5 ml digestion buffer and centrifuging the column at 5000 × g for 1 minute. The buffer was then discarded. The desalting column was prepared by removing the storage solution and washing it with digestion buffer, followed by centrifugation at 1000 × g for 2 minutes each time. 0.5 ml of the prepared IgG sample was added to the spin column tube containing the equilibrated immobilized pepsin. The incubation time of the digestion reaction was 16 hours at 37 ° C on a tabletop shaker. The column was centrifuged at 5000 × g for 1 minute to separate the digest from the immobilized papain. The resin was then washed with 0.5 mL PBS and centrifuged at 5000 × g for 1 minute. The wash fraction was added to the digested antibody, with 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) and equilibrated by adding 2 mL PBS, centrifuged again for 1 minute and the flow-through was discarded. The sample was applied to the column and resuspended by inversion. Incubate at room temperature with end-over-end mixing for 10 minutes. Centrifuge the column for 1 minute and save the flow-through with the Fab fragment. (Reference: 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 ).

[0627] NT-H-Antibody Fragment Humanization: Humanization of antibody fragments by CDR grafting method ( Jones et al. 1986. Nature 321, 522–525 ).

[0628] Perform the following steps to obtain the humanized sequence :

[0629] Total RNA extraction: Total RNA was extracted from NT-H hybridoma using Qiagen kit. First round RT-PCR: Total RNA was extracted from NT-H hybridoma using Qiagen kit. ® One-step RT-PCR kit (Catalog No. 210210). RT-PCR was performed with primer sets specific for the heavy and light chains. For each RNA sample, 12 separate heavy chain and 11 light chain RT-PCR reactions were set up using a degenerate forward primer mix covering the variable region leader sequence. The reverse primers were located in the constant region of the heavy and light chains. No restriction sites were engineered into the primers.

[0630] Reaction setup: 5x QIAGEN ® One-step RT-PCR buffer 5.0µl, dNTP mixture (containing 10mM of each dNTP) 0.8µl, primer set 0.5µl, QIAGEN ® One-step RT-PCR enzyme mix 0.8µl, template RNA 2.0µl, RNase-free water to 20.0µl, total volume 20.0µl. PCR conditions: Reverse transcription: 50℃, 30 minutes; Initial PCR activation: 95℃, 15 minutes; Cycling: 20 cycles: 94℃, 25 seconds; 54℃, 30 seconds; 72℃, 30 seconds; Final extension: 72℃, 10 minutes; Second round of semi-nested PCR: RT-PCR products from the first round of reaction were further amplified in the second round of PCR. 12 separate heavy chain and 11 light chain RT-PCR reactions were set up using semi-nested primer sets specific for the variable regions of the antibody.

[0631] Reaction setup: 2× PCR mix 10µl; primer set 2µl; first round PCR product 8µl; total volume 20µl; hybridoma antibody clone reporter PCR conditions: initial denaturation at 95°C for 5 min; 25 cycles of 95°C for 25 sec, 57°C for 30 sec, and 68°C for 30 sec; final extension at 68°C for 10 min.

[0632] After PCR was completed, the PCR reaction samples were electrophoresed on 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 had been cloned and appeared to be correct. Protein sequence alignment and CDR analysis identified one heavy chain and one light chain. After alignment with the homologous human framework sequence, the resulting humanized sequence of the variable heavy chain was as follows: See Figure 5 Since the amino acids at positions 26, 40, and 55 in the variable heavy chain and the amino acid at position 40 in the variable light chain are critical for binding properties, they can be reverted to their original state in the mouse. The resulting candidates are depicted below. ( Padlan 1991. Mol. Immunol. 28, 489–498; Harris and Bajorath. 1995. Protein Sci. 4, 306–310 ).

[0633] Annotation of antibody fragment sequences (SEQ ID No.: 6-13; 32 and 33): In bold and underlined are CDRs 1, 2, 3 in chronological order; in italics are constant regions; hinge regions are highlighted with bold letters; framework point mutations have a grey letter background.

[0634]

[0635]

[0636]

[0637] Example 2 - Effect of Selected Anti-ADM Antibodies on Anti-ADM Biological Activity

[0638] Selected ADM antibodies were tested for their effects on ADM bioactivity in a human recombinant adrenomedullin receptor cAMP functional assay (adrenomedullin bioassay).

[0639] Tested in the human recombinant adrenomedullin receptor cAMP functional assay (adrenomedullin bioassay) Antibodies targeting human or mouse adrenomedullin

[0640] Material :Cell line CHO-K1, receptor adrenomedullin (CRLR+RAMP3), receptor accession number Cell line: CRLR: U17473; RAMP3: AJ001016

[0641] CHO-K1 cells expressing human recombinant adrenomedullin receptor (FAST-027C) grown in antibiotic-free medium prior to testing were detached by gentle washing with PBS-EDTA (5 mM EDTA), recovered by centrifugation and resuspended in assay buffer (KRH: 5 mM KCl, 1.25 mM MgSO4, 124 mM NaCl, 25 mM HEPES, 13.3 mM glucose, 1.25 mM KH2PO4, 1.45 mM CaCl2, 0.5 g / l BSA).

[0642] Dose response curves were obtained in parallel with a reference agonist (hADM or mADM).

[0643] Antagonist test (96-well): For antagonist testing, 6µl of reference agonist (human (5.63nM) or mouse (0.67nM) adrenomedullin) was mixed with 6µl of test sample at different antagonist dilutions; or with 6µl of buffer. After incubation at room temperature for 60 minutes, 12µl of cells (2,500 cells / well) were added. The plate was incubated at room temperature for 30 minutes. After adding lysis buffer, the percentage of DeltaF was estimated using the HTRF kit from Cis-Bio International (Cat. No. 62AM2 PEB) according to the manufacturer's instructions. hADM 22-52 was used as a reference antagonist.

[0644] Testing antibodies for cAMP-HTRF assay

[0645] Antagonistic activity of anti-h-ADM antibodies (NT-H, MR-H, CT-H) was tested in the human recombinant adrenomedullin receptor (FAST-027C) cAMP functional assay in the presence of 5.63nM human ADM 1-52 at the following final antibody concentrations: 100µg / ml, 20µg / ml, 4µg / ml, 0.8µg / ml, 0.16µg / ml.

[0646] The antagonist activity of anti-m-ADM antibodies (NT-M, MR-M, CT-M) was tested in the presence of 0.67nM mouse ADM 1-50 in a human recombinant ADM receptor (FAST-027C) cAMP functional assay at the following final antibody concentrations: 100µg / ml, 20µg / ml, 4µg / ml, 0.8µg / ml, 0.16µg / ml. Data were plotted as relative inhibition versus antagonist concentration (see Figure 2a to Figure 2l ). The maximum inhibition of individual antibodies is given in Table 3.

[0647] Table 3: Maximum inhibition of bio-ADM activity

[0648]

[0649] Example 3 - Stabilization of hADM by anti-ADM antibodies

[0650] The hADM immunoassay was used to test the stabilization effect of human ADM antibodies on human ADM.

[0651] Immunoassay for the Quantification of Human Adrenomedullin

[0652] The technique used was a sandwich-coated tube luminescence immunoassay based on acridinium ester labeling.

[0653] Labeled compounds (tracers): 100 µg (100 µl) CT-H (1 mg / ml in PBS, pH 7.4, AdrenoMed AG Germany) was mixed with 10 µl acridine NHS-ester (1 mg / ml in acetonitrile, InVent GmbH, Germany) (EP0353971) and incubated at room temperature for 20 min. ® Labeled CT-H was purified by gel filtration HPLC on SEC 400-5 (Bio-Rad Laboratories, Inc., USA). 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 by using AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

[0654] Solid Phase Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 h at room temperature) with MR-H (AdrenoMed AG, Germany) (1.5 µg MR-H / 0.3 mL 100 mmol / L NaCl, 50 mmol / L TRIS / HCl, pH 7.8). After blocking with 5% bovine serum albumin, the tubes were washed with PBS (pH 7.4) and dried in vacuo.

[0655] calibration : The assay was calibrated using dilutions of hADM (BACHEM AG, Switzerland) in 250 mmol / L NaCl, 2 g / L Triton X-100, 50 g / L bovine serum albumin, 20 tablets / L protease inhibitor cocktail (Roche Diagnostics AG, Switzerland).

[0656] 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 h at 4 °C. Unbound tracer was removed by washing 5 times (1 ml each) with washing solution (20 mM PBS (pH 7.4), 0.1% Triton X-100).

[0657] By using LB 953 tubes to measure the chemiluminescence: Figure 3 A typical hDM dose / signal curve is shown. As well as a hADM dose-signal curve in the presence of 100 µg / mL antibody NT-H. NT-H does not affect the hADM immunoassay described.

[0658] 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 terminated by freezing at -20°C. Incubation was performed in the absence and presence of NT-H (100 µg / ml). Remaining hADM was quantified by using the hADM immunoassay described above.

[0659] Figure 4 The stability of hADM in human plasma (citrate) in the absence and presence of NT-H antibody is shown. The half-life of hADM alone is 7.8 hours and in the presence of NT-H the half-life is 18.3 hours. (2.3-fold stability).

[0660] Example 4 - Sepsis mortality in mice treated with anti-ADM antibodies

[0661] 4) Early treatment of sepsis

[0662] Animal Model: Male C57Bl / 6 mice (Charles River Laboratories, Germany) aged 12-15 weeks were used for the study. Peritonitis had been induced surgically under mild isoflurane anesthesia at 79°C. An incision was made in the left upper abdomen (normal location of the cecum) in the peritoneal cavity. The cecum was exposed and sutured distal to the insertion of the small intestine, with a tight ligature around the cecum. A puncture wound was created in the cecum with a 24-gauge needle, and a small amount of cecal contents was squeezed out through the wound. The cecum was placed in the peritoneal 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 as rehydration.

[0663] Administration and Dosage of Compounds (NT-M, MR-M, CT-M) : Treatment of mice immediately after CLP (early treatment). CLP is the abbreviation for cecal ligation and puncture (CLP).

[0664] Research Group : Three compounds were tested relative to vehicle and relative to control compound treatment. Each group contained 5 mice, and blood was collected after 1 day for BUN determination (serum blood urea nitrogen test). Another 10 mice per group were followed up for 4 days.

[0665] Treatment Group (10µl / g body weight) Dosage / Follow-up:

[0666] 1NT-M, 0.2mg / ml survival for 4 days

[0667] 2MR-M, 0.2mg / ml survival for 4 days

[0668] 3CT-M, 0.2mg / ml, survival for 4 days

[0669] 4 Non-specific mouse IgG, 0.2mg / ml survival for 4 days

[0670] 5. Control - PBS 10µl / g body weight Survival for 4 days

[0671] Clinical Chemistry : Blood urea nitrogen (BUN) concentrations for renal function were measured at baseline and on day 1 after CLP. Blood samples were obtained from the cavernous sinus with a capillary tube under light ether anesthesia. Measurements were performed using an AU 400 Olympus Multianalyser. The 4-day mortality and mean BUN concentrations are given in Table 4.

[0672] Table 4: 4-day mortality and BUN concentration

[0673]

[0674] As can be seen in Table 4, NT-M antibody greatly reduced mortality. After 4 days, 70% of the mice were alive when treated with NT-M antibody. When treated with MR-M antibody, 30% of the animals survived, and when treated with CT-M antibody, 10% of the animals survived after 4 days. In contrast, when treated with nonspecific mouse IgG, all mice died after 4 days. The same results were obtained in a control group in which PBS (phosphate buffered saline) was administered to the mice. The blood urea nitrogen or BUN test is used to evaluate kidney function, help diagnose kidney disease, and monitor patients with acute or chronic renal dysfunction or failure. The results of the S-BUN test revealed that NT-M antibody was most effective in protecting the kidneys.

[0675] b) Late-stage treatment of sepsis

[0676] Animal models : Studies were performed using male C57Bl / 6 mice (Charles River Laboratories, Germany) aged 12-15 weeks. Peritonitis had been induced surgically under 80% mild isoflurane anesthesia. An incision was made in the left upper abdomen (normal location of the cecum) in the peritoneal cavity. The cecum was exposed and sutured distal to the insertion of the small intestine, with a tight ligature around the cecum. A puncture wound was created in the cecum with a 24-gauge needle, and a small amount of cecal contents was squeezed out through the wound. The cecum was placed in the peritoneal 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 as rehydration.

[0677] Administration and dosage of compound (NT-M FAB2): NT-M FAB2 was tested relative to vehicle and relative to control compound treatment. Treatment was performed 6 hours after CLP, after sepsis had fully developed (late treatment). Each group contained 4 mice and was followed up for 4 days.

[0678] Treatment Group (10µl / g body weight) Dosage / Follow-up:

[0679] 1NT-M, FAB2 0.2mg / ml survival for 4 days

[0680] 2. Control nonspecific mouse IgG, 0.2mg / ml survival for 4 days

[0681] 3. Vehicle: -PBS 10µl / g body weight for 4 days

[0682] Table 5: 4-day mortality

[0683]

[0684] As can be seen from Table 5, NT-M FAB 2 antibody greatly reduced mortality. After 4 days, 75% of mice were alive when treated with NT-M FAB 2 antibody. In contrast, all mice died after 4 days when treated with nonspecific mouse IgG. The same results were obtained in a control group in which PBS (phosphate buffered saline) was administered to mice.

[0685] Example 5 - Administration of NT-H in Healthy Humans

[0686] The study was conducted as a randomized, double-blind, placebo-controlled study in healthy male subjects, where single ascending doses of NT-H antibody were administered as intravenous (iv) infusions in 3 consecutive groups of 8 healthy male subjects (0.5 mg / kg in group 1, 2 mg / kg in group 2, and 8 mg / kg in group 3) (n=6 active treatment and n=2 placebo treatment in each group). The main inclusion criteria were written informed consent, age 18-35 years, agreement to use reliable contraception and BMI below 18 kg / m 2 With 30kg / m 2between. Subjects received a single intravenous dose of NT-H antibody (0.5 mg / kg; 2 mg / kg; 8 mg / kg) or placebo, slowly infused over 1 hour in the study unit. Baseline ADM values ​​did not differ among the 4 groups. The median ADM value was 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 / mL). The results showed that in healthy human individuals, ADM values ​​increased rapidly within the first 1.5 hours after administration of NT-H antibody, then reached a stable level and slowly decreased ( Figure 6 ).

[0687] Example 6 - Methods for measuring DPP3 protein and DPP3 activity

[0688] Antibody Generation and Determination of DPP3 Binding Ability: Several murine antibodies were generated and screened by their ability to bind human DPP3 in a specific binding assay (see Table 6).

[0689] Peptides / conjugates for immunization : Synthesized DPP3 peptides for immunization, see Table 6 (JPT Technologies, Berlin, Germany), with an additional N-terminal cysteine ​​(if cysteine ​​is not present in the selected DPP3-sequence) residue for conjugation of the peptide to bovine serum albumin (BSA). The peptide was covalently linked to BSA by 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).

[0690] Mouse immunization, immune cell fusion and screening: Balb / c Mice were injected intraperitoneally (ip) with 84µg GST-hDPP3 or 100µg DPP3-peptide-BSA-conjugate (emulsified in TiterMax Gold adjuvant) on day 0, 84µg or 100µg conjugate (emulsified in complete Freund's adjuvant) on day 14, and 42µg or 50µg conjugate (in incomplete Freund's adjuvant) on days 21 and 28. On day 49, animals received an intravenous (iv) injection of 42µg GST-hDPP3 or 50µg DPP3-peptide-BSA-conjugate dissolved in saline. Three days later, mice were sacrificed and immune cell fusion was performed.

[0691] Splenocytes from 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 in 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement]. After one week, the HAT medium was changed to HT medium, passaged three times, and then returned to normal cell culture medium. Two weeks after fusion, cell culture supernatants were initially screened for recombinant DPP3-binding IgG antibodies. Therefore, recombinant GST-tagged hDPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng / well) and incubated with 50 µl of cell culture supernatant per well for 2 hours at room temperature. After the plates were washed, 50 µl / well of POD-rabbit anti-mouse IgG was added and incubated for 1 hour at room temperature. After the next washing step, 50 µl of chromogen solution (3.7 mM o-phenylenediamine in citrate / hydrogen phosphate buffer, 0.012% H2O2) was added to each well, incubated for 15 min at room temperature, and the color reaction was stopped by adding 50 µl 4N sulfuric acid. The absorbance was measured at 490 mm. Positive test microcultures were transferred to 24-well plates for expansion. After retesting, selected cultures were cloned and recloned using the limiting dilution technique and isotyped.

[0692] Mouse monoclonal antibody generation

[0693] Antibodies raised against GST-tagged human DPP3 or DPP3-peptide were generated by standard antibody production methods ( Marx et al. People1997 ) and purified by protein A. Based on SDS gel electrophoresis analysis, the antibody purity was ≥90%.

[0694] Characterization of antibodies - binding to hDPP3 and / or immunizing peptides

[0695] To analyze the ability of DPP3 / immunizing peptides to bind to different antibodies and antibody clones, binding assays were performed:

[0696] Solid phase: Recombinant GST-tagged hDPP3 (SEQ ID NO.36) or DPP3 peptide (immune peptide, SEQ ID NO.37) was immobilized on the surface of a high-binding microtiter plate (96-well polystyrene microtiter plate, Greiner Bio-Oneinternational AG, Austria, 1 µg / well in coupling buffer [50 mM Tris, 100 mM NaCl, pH 7.8], 1 h at room temperature). After blocking with 5% bovine serum albumin, the microtiter plate was vacuum dried.

[0697] Marking procedures (tracers) : 100 µg (100 µl) of different anti-DPP3 antibodies (detection antibodies, 1 mg / ml in PBS, pH 7.4) were mixed with 10 µl of acridine NHS-ester (1 mg / ml in acetonitrile, InVent GmbH, Germany; EP 0 353 971) and incubated for 30 min at room temperature. 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 is about 5-7*10 per 200µl 6 The acridinium ester chemiluminescence was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

[0698] hDPP3 binding assay : The plate was filled with 200 µl of labeled and diluted detection antibody (tracer) and incubated for 2-4 hours at 2-8°C. Unbound tracer was removed by washing 4 times with 350 µl of washing solution (20 mM PBS (pH 7.4), 0.1% Triton X-100). Well-bound chemiluminescence was measured by using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

[0699] Characterization of antibodies - hDPP3 inhibition assay

[0700] To analyze the ability of different antibodies and antibody clones to inhibit DPP3, a DPP3 activity assay was performed using known procedures (Jones et al., 1982). Recombinant GST-tagged 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 the corresponding antibody at room temperature. After a 1-h preincubation, the fluorescent substrate Arg-Arg-βNA (20 µl, 2 mM) was added to the solution and the generation of free βNA was monitored over time at 37 °C using a Twinkle LB970 microplate fluorimeter (Berthold Technologies GmbH & Co. KG). The fluorescence of βNA was detected by excitation at 340 nm and measurement of emission at 410 nm. The slope of the fluorescence increase (in RFU / min) of the different samples was calculated. The slope of GST-hDPP3 with buffer control was assigned as 100% activity. The inhibitory capacity of a potential capture-binder was defined as the reduction in percentage of GST-hDPP3 activity by incubation with said capture-binder.

[0701] The table below represents the selection of antibodies obtained and their binding rate in relative light units (RLU) as well as their relative inhibitory capacity (%; Table 6). The monoclonal antibodies generated against the DPP3 regions described below were selected by their ability to bind recombinant DPP3 and / or immunizing peptides as well as their inhibitory potential.

[0702] All antibodies raised against the GST-tagged full-length form of recombinant hDPP3 showed strong binding to immobilized GST-tagged hDPP3. Antibodies raised against the SEQ ID NO.: 37 peptide also bound to GST-hDPP3. The SEQ ID NO.: 37 antibody also bound strongly to the immunizing peptide.

[0703] The development of a luminescent immunoassay for quantifying DPP3 protein concentration (DPP3-LIA) as well as an enzyme capture activity assay for quantifying DPP3 activity (DPP3-ECA) have been recently described ( Rehfeld et al. 2019. JALM 3(6): 943-953 ), which is incorporated herein by reference in its entirety.

[0704] Table 6: Antibodies raised against the full length or sequence of hDPP3 and their binding to hDPP3 (SEQ ID NO.: 36) or the immune peptide (SEQ ID NO.: 37), and the maximum inhibitory effect of recombinant GST-hDPP3 .

[0705]

[0706] Example 7 - Development of Protuzumab

[0707] The antibodies raised against SEQ ID No.: 37 were characterized in more detail (epitope mapping, binding affinity, specificity, inhibitory potential). The results for clone 1967 (AK1967; "Protuzumab") of SEQ ID No.: 37 are shown here as an example.

[0708] Determination of the AK1967 epitope on DPP3 :

[0709] For epitope mapping of AK1967, a number of N- or C-terminally biotinylated peptides were synthesized (peptides &elephants GmbH, Hennigsdorf, Germany). These peptides included the sequence of the full immunizing peptide (SEQ ID No. 37) or fragments thereof, with one amino acid removed stepwise from the C- or N-terminus (for a complete list of peptides, see Table 8).

[0710] High binding 96-well plates were coated with 2 µg of avidin (Greiner Bio-One international AG, Austria) per well in coupling buffer (500 mM Tris-HCl (pH 7.8), 100 mM NaCl). The plates were then washed and filled with a specific solution of biotinylated peptides (10 ng / well; buffer - 1× PBS with 0.5% BSA).

[0711] According to Example 6, the anti-DPP3 antibody AK1967 was labeled with a chemiluminescent label.

[0712] The plate was filled with 200 µl of labeled and diluted detection antibody (tracer) and incubated for 4 h at room temperature. Unbound tracer was removed by washing 4 times with 350 µl of washing solution (20 mM PBS (pH 7.4), 0.1% Triton X-100). Well-bound chemiluminescence was measured by using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). Binding of AK1967 to the corresponding peptides was determined by evaluating relative light units (RLU). Any peptide that showed a significantly higher RLU signal than nonspecific binding of AK1967 was defined as an AK1967 binder. Combined analysis of binding and non-binding peptides revealed a specific DPP3 epitope for AK1967.

[0713] Binding affinity determination using Octet :

[0714] Experiments were performed using Octet Red96 (ForteBio). AK1967 was captured on a kinetic grade anti-human Fc (AHC) biosensor. The loaded biosensor was then immersed in a series of recombinant GST-tagged human DPP3 dilutions (100 nM, 33.3 nM, 11.1 nM, 3.7 nM). Association was observed for 120 seconds followed by dissociation for 180 seconds. The buffers used for the experiments are depicted in Table 7. Kinetic analysis was performed using a 1:1 binding model and global fit.

[0715] Table 7: Buffers used for Octet measurements

[0716]

[0717] Western blot analysis of AK1967 binding specificity :

[0718] Blood cells from human EDTA-blood were washed (3 times in PBS), diluted in PBS and lysed by repeated freeze-thaw cycles. The total protein concentration of blood cell lysate was 250µg / ml and the DPP3 concentration was 10µg / ml. Dilutions of blood cell lysate (1:40, 1:80, 1:160 and 1:320) and dilutions of purified recombinant human His-DPP3 (31.25ng / ml-500ng / ml) were subjected to SDS-PAGE and Western blot analysis. Blots were incubated in 1.) blocking buffer (1× PBS-T with 5% nonfat dry milk), 2.) primary antibody solution (AK1967 at 1:2.000 in blocking buffer) and 3.) HRP-labeled secondary antibody (goat anti-mouse IgG at 1:1.000 in blocking buffer). Bound secondary antibodies were detected using Amersham ECL Western blotting detection reagents and AmershamImager 600 UV (both from GE Healthcare).

[0719] DPP3 inhibition assay To analyze the ability of AK1967 to inhibit DPP3, a DPP3 activity assay with known procedures was performed as described in Example 6 ( Jones et al., 1982 ). The AK1967 inhibitory capacity was defined as the reduction in percentage of GST-hDPP3 activity by incubation with the antibody. The resulting reduced DPP3 activity is shown in Figure 7 in the inhibition curve.

[0720] Epitope mapping : Analysis of peptides that bind and do not bind to AK1967 revealed that the DPP3 sequence INPETG (SEQ ID No.: 38) is an essential epitope for AK1967 binding (see Table 8).

[0721] Binding affinity: AK1967 with 2.2*10 -9 M binds to recombinant GST-hDPP3 with affinity (kinetic curves see Figure 8 ).

[0722] Table 8: Peptides used for AK1967 epitope mapping

[0723]

[0724]

[0725] Specificity and inhibitory potential :

[0726] The only protein detected in blood cell lysates using AK1967 as the primary antibody was DPP3 of 80 kDa ( Fig. 9 ). The total protein concentration of the lysate was 250µg / ml, while the estimated DPP3 concentration was approximately 10µg / ml. Even though 25-fold more nonspecific proteins were present in the lysate, AK1967 specifically bound and detected DPP3, and no other nonspecific binding occurred.

[0727] AK1967 inhibits 15 ng / ml DPP3 in a specific DPP3 activity assay with an IC50 of approximately 15 ng / ml ( Figure 7 ).

[0728] Chimerism / humanization :

[0729] The monoclonal antibody AK1967 ("provizumab"), which is able to inhibit DPP3 activity by 70%, was selected as a potential therapeutic antibody and also used as a template for chimerization and humanization.

[0730] Humanization of mouse antibodies can be performed according to the following procedure :

[0731] For humanization of antibodies of murine origin, the framework regions (FR) of the antibody sequence are analyzed for structural interactions with the complementarity determining regions (CDRs) and the antigen. Based on structural modeling, appropriate FRs of human origin are selected and the murine CDR sequences are grafted into the human FRs. Variations in the amino acid sequences of the CDRs or FRs can be introduced to regain structural interactions that were eliminated by species conversion of the FR sequences. This restoration of structural interactions can be achieved by random approaches using phage display libraries or by directed approaches guided by molecular modeling ( Almagro and Fransson, 2008. Humanization of antibodies. Front Biosci. 13:1619-33 ).

[0732] In the above context, the variable region can be linked to a constant region of any subclass (IgG, IgM, IgE, IgA), or to a scaffold only, Fab fragment, Fv, Fab and F(ab)2. Murine antibody variants contain an IgG2a backbone. For chimerization and humanization, a human IgG1κ backbone is used.

[0733] For epitope binding, only the complementarity determining regions (CDRs) are important. The CDRs of the heavy and light chains of the murine anti-DPP3 antibody (AK1967; "Prolizumab") are shown in SEQ ID No. 42, SEQ ID No. 43 and SEQ ID No. 44 for the heavy chain and SEQ ID No. 45, sequence KVS and SEQ ID No. 46 for the light chain, respectively.

[0734] Sequencing of the anti-DPP3 antibody (AK1967; "Prolizumab") revealed the antibody heavy chain variable region (H chain) according to SEQ ID No.: 47 and the antibody light chain variable region (L chain) according to SEQ ID No.: 48.

[0735] Example 8 - Effect of Protuzumab in Sepsis-Induced Heart Failure

[0736] In this experiment, the effect of prolizumab injection in rats with sepsis-induced heart failure ( Rittirsch et al. 2009 ) was studied by monitoring the shortening fraction.

[0737] CLP model of septic shock :

[0738] Male Wistar rats (2-3 months, 300 g to 400 g, group size see Table 9) from Centre d'élevage Janvier (France) were randomly assigned to one of three groups. All animals were anesthetized intraperitoneally (ip) with ketamine hydrochloride (90 mg / kg) and xylazine (9 mg / kg). To induce polymicrobial sepsis, cecal ligation and puncture (CLP) was performed using a Rittirsch protocol with slight modifications. A midline abdominal incision (1.5 cm) was made to allow exteriorization of the cecum. The cecum was then ligated just below the ileocecal valve and punctured once with an 18-gauge needle. The abdominal cavity was then closed in two layers, followed by fluid resuscitation (3 ml / 100 g body weight of saline subcutaneously) and the animals were returned to their cages. Sham-operated animals underwent surgery, but their cecum was not punctured. CLP animals were randomized between placebo and therapeutic antibodies.

[0739] Study Design :

[0740] The research process Figure 8Depicted in . After CLP or sham surgery, animals were allowed to rest for 20 hours with free access to water and food. They were then anesthetized, tracheotomized, and arterial and venous lines were placed. AK1967 or vehicle (saline) was administered at 5 mg / kg bolus injection 24 hours after CLP surgery, followed by a 3-hour infusion of 7.5 mg / kg. As a safety measure, hemodynamics were monitored invasively and continuously from t=0 to 3 hours.

[0741] At t = 0 (baseline), all CLP animals were in septic shock and developed reduced cardiac function (hypotension, low fractional shortening). At this time point, either Protuzumab or vehicle (PBS) was injected (iv) and saline infusion was started. There was 1 control group and 2 CLP groups, summarized in the table below (Table 9). At the end of the experiment, animals were euthanized and organs were harvested for subsequent analysis.

[0742] Table 9: List of experimental groups (sepsis-induced heart failure)

[0743]

[0744] Invasive blood pressure :

[0745] Hemodynamic variables were obtained using the AcqKnowledge system (BIOPAC Systems, Inc., USA). It provides a fully automated blood pressure analysis system. The catheter was connected to the BIOPAC system via a pressure transducer.

[0746] For this procedure, rats were anesthetized (ketamine and xylazine). The animals were moved to a heating pad to achieve the desired body temperature of 37°C-37.5°C. A temperature feedback probe was inserted into the rectum. The rats were placed on the operating table in a supine position. The trachea was opened and a catheter (16G) was inserted for external ventilator without damaging the carotid artery and vagus nerve. An arterial catheter was inserted into the right carotid artery. The carotid artery was separated from the vagus nerve before ligation. A central venous catheter was inserted through the left jugular vein, allowing the administration of PCZ or PBS. After surgery, the animals were allowed to rest to reach a stable state before hemodynamic measurements. Baseline blood pressure (BP) was then recorded. During data collection, saline infusion via the arterial line was stopped.

[0747] Echocardiography :

[0748] Animals were anesthetized using ketamine hydrochloride. The chest was shaved and the rats were placed in the recumbent position. For transthoracic echocardiography (TTE) examinations, a commercial GE Healthcare Vivid 7 ultrasound system equipped with a high-frequency (14-MHz) linear probe and a 10-MHz cardiac probe was used. All examinations were digitally recorded and stored for subsequent offline analysis.

[0749] Grayscale images were recorded at a depth of 2 cm. Two-dimensional examinations were started in the parasternal long-axis view to measure the aortic annulus diameter and the pulmonary artery diameter. Left ventricular (LV) dimensions were also measured and fractional shortening (FS%) was assessed using M-mode. LVFS was calculated as LV end-diastolic diameter-LV end-systolic diameter / LV end-diastolic diameter and expressed in %. Therefore, the time of end-diastole was defined as the maximum diameter of the LV. Therefore, end-systole was defined as the minimum diameter in the same cardiac cycle. All parameters were measured manually. Three cardiac cycles were averaged for each measurement.

[0750] From the same parasternal long-axis view, pulmonary artery flow was recorded using pulsed-wave Doppler. The velocity time integral of pulmonary artery outflow was measured. From the apical five-chamber view, mitral flow was recorded using pulsed-wave Doppler at the level of the mitral valve tip.

[0751] result :

[0752] Sepsis-induced heart failure rats treated with PBS (CLP+PBS) showed reduced fractional shortening ( Fig.10 A). The CLP+PBS group also showed a high mortality rate ( Fig.10 B). In contrast, administration of prolizumab to rats with sepsis-induced heart failure improved the fractional shortening ( Fig.10 A) and significantly reduced mortality ( Fig.10 B).

[0753] Example 9 - Effects of Protuzumab on Cardiac and Renal Function

[0754] The effect of protuzumab in isoproterenol-induced heart failure in mice was investigated by monitoring fractional shortening and renal resistive index.

[0755] Isoproterenol-induced cardiac stress in mice :

[0756] Acute heart failure was induced in 3-month-old male mice by subcutaneous injection of 300 mg / kg isoproterenol, a nonselective ß-adrenergic agonist (DL-isoproterenol hydrochloride, Sigma Chemical Co) (ISO) twice daily for two days. Vergaro et al., 2016 ISO dilutions were performed in NaCl 0.9%. Isoproterenol-treated mice were randomly divided into two groups (Table 10) and baseline echocardiography (Gao et al., 2011) and renal resistance index measurements ( Lubas et al., 2014; Dewitte et al., 2012 ) followed by intravenous injection of PBS or protuzumab (10 mg / kg) ( Fig.11 A and Fig.11 B). At 1 hour, 6 hours and 24 hours ( Fig.11 A and Fig.11 B) by echocardiography ( Gao et al., 2011 ) and renal resistance index ( Lubas et al., 2014; Dewitte et al., 2012 ) to assess cardiac function. The group of mice injected with vehicle (PBS) instead of isoproterenol received no further drug treatment and served as a control group (Table 10).

[0757] Table 10: Experimental group list (heart failure induced by isoproterenol)

[0758]

[0759] result :

[0760] Administration of protuzumab to mice with isoproterenol-induced heart failure restored cardiac function within the first hour after administration ( Fig.12 A). At 6 hours after PCZ injection, renal function in diseased mice showed significant improvement and was comparable to that of sham-operated animals at 24 hours ( Fig.12 B).

[0761] Example 10 - DPP3 and organ dysfunction in sepsis

[0762] The AdrenOSS-1 study was a prospective, multicenter, observational study in patients with severe sepsis and septic shock (ClinicalTrials.gov NCT02393781). Twenty-four centers in five European countries (France, Belgium, the Netherlands, Italy, and Germany) contributed to the trial achievement, which enrolled 583 patients (from June 2015 to May 2016). Of the 583 patients enrolled, DPP3 plasma levels were measured in 581 patients. The study protocol was approved by the local ethics committee and was conducted in accordance with the Declaration of Helsinki. The study enrolled patients aged 18 years and older who (1) were admitted to the ICU due to sepsis or septic shock or (2) were transferred from another ICU with sepsis and septic shock within less than 24 hours of admission. The included patients were stratified by severe sepsis and septic shock according to the 2001 definition of sepsis and organ failure ( Levy et al. 2003. 2001 SCCM / ESICM / ACCP / ATS / SIS International Sepsis Definitions Conference. Crit Care Med. 31(4):1250– 6). Patients were treated according to local practice, and treatments and procedures were registered. The primary endpoint was 28-day mortality. Secondary endpoints involved organ failure (as defined by the Sequential Organ Failure Assessment [SOFA] score) and organ support, use of vasopressors / inotropes, fluid balance, and use of renal replacement therapy (RRT).

[0763] After admission, demographics (age, sex), body-mass index, presence of septic shock, type of ICU admission, organ dysfunction score (SOFA, Acute Physiology Assessment and Chronic Health Evaluation II [APACHE II]), origin of sepsis, preexisting comorbidities (ie, treated within the past year), past medical history, laboratory values, and organ support were recorded, and blood was drawn for measurement of bio-ADM and other markers. After recruitment, the following data were collected daily during the first week: SOFA score, antimicrobial therapy, fluid balance, ventilation status, Glasgow Coma Scale score, central venous pressure, need for RRT, invasive procedures for sepsis control, and vasopressor / inotropic therapy. In addition, discharge status and mortality were recorded on day 28 after ICU admission. Blood was sampled for the central laboratory within 24 hours after ICU admission and on day 2 after the first sampling (mean, 47 hours, SD, 9 hours). The samples were subsequently processed and stored at −80°C.

[0764] DPP3 measurement : Immunoassay (LIA) or enzymatic activity assay (ECA) for detecting the amount of human DPP3 (LIA) or the activity of human DPP3 (ECA), respectively, was used to determine the level of DPP3 in patient plasma. Antibody fixation, labeling and incubation were performed as described by Rehfeld et al. ( Rehfeld et al. 2019. JALM 3(6): 943-953 ).

[0765] The AdrenOSS-1 study was designed to evaluate the association between circulating DPP3, organ dysfunction (e.g., cardiovascular and renal dysfunction) in patients admitted for sepsis and septic shock. The median DPP3 measured on admission in all AdrenoSS-1 patients was 45.1 ng / mL (interquartile range, 27.5-68.6). High DPP3 levels measured on admission were associated with worse metabolic parameters, renal and cardiac function, and SOFA scores: patients with DPP3 levels below the median had a median SOFA score (score) of 6 (IQR 4-9) compared to patients with DPP3 levels above the median 45.1 ng / mL with a median SOFA score of 8 (IQR 5-11) ( Fig.13 ).

[0766] Regardless of DPP3 levels at admission, high DPP3 levels 24 hours later were associated with the worst SOFA scores, both overall ( Fig.14 ) or by organ (Figure 15A-Figure 15F).

[0767] In conclusion, these data show that high levels of DPP3 are associated with survival and the degree of organ dysfunction in a large international cohort of patients with sepsis or septic shock. This study found a significant association between DPP3 <45.1 ng / ml on admission and short-term survival in sepsis and septic shock, as well as a prognostic cutoff value of 45.1 pg / ml. Regarding organ dysfunction, a positive correlation was found between DPP3 and SOFA score on ICU admission. More importantly, the relationship between DPP3 levels and the degree of organ dysfunction seen on ICU admission also held during the recovery period. In fact, patients with high DPP3 levels on admission who showed a decline towards normal DPP3 values ​​on day 2 were more likely to recover all organ functions, including cardiovascular, renal, pulmonary, and liver function.

[0768] Example 11 - DPP3 in septic shock and cardiogenic shock

[0769] DPP3 concentrations were measured in plasma of patients with sepsis / septic shock and cardiogenic shock and were associated with short-term mortality.

[0770] a) Study Cohort - Sepsis / Septic Shock

[0771] The same study (AdrenOSS-1) as in Example 10 was analyzed. In this study, 292 of 583 patients were diagnosed with septic shock.

[0772] b) Study Cohort - Cardiogenic Shock

[0773] Plasma samples from 108 patients diagnosed with cardiogenic shock were screened for DPP3. Blood was drawn within 6 hours of detection of cardiogenic shock. Mortality was followed for 7 days.

[0774] result : Short-term patient survival in patients with sepsis 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 ( Fig.16 A). Applying this cutoff value to a subcohort of patients with septic shock revealed an even more pronounced short-term risk of mortality associated with high DPP3 plasma concentrations ( Fig.16 B). When the same cutoff value was applied to patients with cardiogenic shock, an increased risk of short-term mortality within 7 days was also observed in patients with high DPP3 ( Fig.16 C).

[0775] Example 12 - Prediction of DPP3 Increase in Sepsis and Septic Shock (AdrenOSS-1)

[0776] The AdrenOSS-1 study as described in Example 10 was used to analyze whether DPP3 levels at baseline could predict In the following days, DPP3 increased .

[0777] result :

[0778] Statistical analysis of DPP3 plasma levels at baseline (day 1, DPP3.d1) in patients with septic shock (n=292) was performed with the goal of determining a threshold value predicting an increase in DPP3 plasma concentrations above 50 ng / ml in the following days. A DPP3 concentration of 50 ng / ml reflects a threshold above which patients a) exceed the upper normal limit of DPP3, b) have high organ dysfunction and mortality (see Example 11; Blet et al. 2021. Crit Care 25(1): 61 ) and c) the N-terminal ADM antibody aderezumab has been shown to have a lower therapeutic effect ( WO2021 / 170838 ).

[0779] Table 11: DPP3 plasma levels in patients with septic shock

[0780]

[0781] The ability of different DPP3 thresholds at baseline (d1) to predict the percentage of patients whose DPP3 plasma concentration increased to above 50ng / ml in the following days (days 2 and 3) was analyzed. Table 11 shows that the lower the DPP3 plasma concentration (DPP3.d1) at baseline, the lower the percentage of patients whose DPP3 increased to above 50ng / ml in the following days. In this septic shock population, 223 and 156 patients had DPP3 concentrations below <50ng / ml or <30ng / ml, respectively, at baseline. For 156 patients below 30ng / ml at baseline, 7 (4%) patients showed an increase in DPP3 concentration to a DPP3 threshold above 50ng / ml in the following days. On the other hand, among 67 septic shock patients whose DPP3 plasma concentration was between 30ng / ml and 50ng / ml at baseline, 15 (22.4%) patients increased their DPP3 plasma levels to above 50ng / ml in the following days. Therefore, a low DPP3 threshold (in the range between 22 ng / ml and 40 ng / ml) is suitable for predicting a later increase in DPP3, which can be used for treatment decisions at baseline, such as the use of anti-ADM antibody (adrezumab) therapy in patients with septic shock. The low DPP3 concentration threshold at baseline (d1) ensures that the DPP3 pathological pathway (which is associated with high short-term organ dysfunction and mortality) is not the main pathway in the selected septic shock population. Therefore, this septic shock population with a DPP3 concentration below the above threshold range at baseline can obtain a higher treatment effect from, for example, anti-ADM antibody therapy (adrezumab).

[0782] In the second step, we analyzed the septic shock population with bio-ADM plasma concentrations above 70 pg / ml. Bio-ADM concentrations above 70 pg / ml are associated with sepsis severity and the development of organ dysfunction, including vasopressor / inotrope dependence ( Marino et al. 2014. Critical Care 18: R34; Caironi et al. 2017. Chest 152(2):312-320; Mebazaa et al. 2018. Crit Care 22: 354 ). The ability of different DPP3 thresholds at baseline (d1) to predict the percentage of patients whose DPP3 plasma concentration increased to above 50 ng / ml in the following days was analyzed. Table 12 again shows that the lower the DPP3 plasma concentration at baseline (DPP3.d1), the lower the percentage of septic shock patients with high bio-ADM whose DPP3 increased to above 50 ng / ml in the following days. In the septic shock and high bio-ADM populations, 154 and 100 patients had DPP3 concentrations below <50 ng / ml or <30 ng / ml, respectively, at baseline. For the 100 patients with less than 30 ng / ml at baseline, 4 (4%) patients showed an increase in DPP3 concentration to above the DPP3 threshold of 50 ng / ml in the next 2 days. On the other hand, among 54 septic shock patients whose DPP3 plasma concentrations were between 30 ng / ml and 50 ng / ml at baseline, 13 (24.1%) patients increased their DPP3 plasma levels to above 50 ng / ml in the following days.

[0783] Table 12: DPP3 plasma levels in septic shock patients with bio-ADM levels above 70 pg / ml

[0784]

[0785] Example 13 - NT-ADM Antibody (AdrenOSS-2) in Septic Shock Patients

[0786] AdrenOSS-2 is a double-blind, placebo-controlled, randomized, multicenter, proof-of-concept and dose-finding Phase II clinical trial to investigate the safety, tolerability, and efficacy of an N-terminal ADM antibody called aderizumab 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 treatment with a single intravenous infusion of placebo (n=152), aderezumab 2 ng / kg (n=72), or aderezumab 4 ng / kg (n=77) over approximately 1 hour. The all-cause mortality rate within 28 (90) days of inclusion 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%.

[0787] A trend toward lower short-term mortality (14 days after admission) was observed in patients treated with aderezumab (both dose combinations, per-protocol population) compared with placebo (hazard ratio (HR) 0.701 [0.408-1.21], p=0.100) ( Fig.17 Surprisingly, the treatment effect was more pronounced in patients whose DPP3 concentration was less than 50 ng / mL at admission (n=244, HR 0.426, p=0.007) ( Fig.18 ), while in patients with elevated DPP3 (greater than 50 ng / mL, n=44), results were comparable between adeleizumab and placebo (HR 1.69, p=0.209) ( Fig.19 ).

[0788] The treatment effects at different DPP3 thresholds (14-day mortality) are summarized in Table 13 .

[0789] Table 13: Hazard risk (HR) for 14-day mortality with different DPP3 concentrations

[0790]

[0791] Example 14 - Lower DPP3 Threshold in AdrenOSS-2 and Efficacy of NT-ADM Antibody Therapy

[0792] To validate the findings from Example 9 and perform proof of concept, the efficacy of anti-ADM antibody (adrezumab) therapy at different lower thresholds of DPP3 at baseline (Day 1) was evaluated in a septic shock population with high bio-ADM from the AdrenOSS-2 study cohort, and patients with DPP3 plasma values ​​above the different lower thresholds were excluded from the analysis. The efficacy of anti-ADM antibody therapy was specifically evaluated in relation to the mortality endpoint in the placebo arm and the treatment arm.

[0793] result :

[0794] Statistical analysis of DPP3 plasma levels at baseline and over the next 144 hours in patients (n=298) with high bio-ADM (above 70 pg / ml) in septic shock, with the aim of determining the DPP3 threshold at baseline to assess the efficacy of anti-ADM antibody therapy. Patients above the corresponding DPP3 plasma threshold were excluded from the analysis. The 28-day all-cause mortality was evaluated using different DPP3 plasma concentration thresholds using a Kaplan-Meier plot comparing anti-ADM antibody therapy with placebo. The log-rank test was selected to show the difference in mortality between treatment groups. The hazard ratio (HR) for each DPP3 plasma concentration threshold was calculated to estimate the reduction in the risk of death imposed by anti-ADM antibody therapy relative to placebo.

[0795] The DPP3 plasma concentration thresholds used at baseline were 50 ng / ml, 40 ng / ml, 30 ng / ml, and 22 ng / ml, respectively. For each threshold, the number of patients excluded from the all-cause mortality analysis was determined. For the 50 ng / ml, 40 ng / ml, 30 ng / ml, and 22 ng / ml thresholds, 16%, 24%, 35%, and 51% of the patients were excluded from the analysis, respectively.

[0796] The HR for each DPP3 plasma concentration threshold at baseline was also determined to estimate the reduction in mortality with anti-ADM antibody therapy relative to placebo. For 50 ng / ml, 40 ng / ml, 30 ng / ml, and 22 ng / ml thresholds, the HRs were 0.606, 0.568, 0.309, and 0.258, respectively. This analysis showed that the lower the DPP3 plasma concentration threshold, the higher the reduction in mortality in the treatment group. Similarly, the all-cause mortality analysis using Kaplan-Meier indicated that the lower the DPP3 plasma concentration threshold, the more obvious and significant the reduction in mortality in the treatment arm group (Figure 20A- Fig.20D ).

[0797] For each DPP3 plasma concentration threshold at baseline, the percentage of patients showing an increase in DPP3 plasma concentration to above 50ng / ml within the next 144 hours is also estimated. Similar to the results of the AdrenOSS-1 study in Example 12, it is shown that: the lower the DPP3 plasma concentration threshold at baseline, the lower the percentage of patients showing an increase in their DPP3 plasma levels to above 50ng / ml threshold in the following days. In this septic shock and high bio-ADM population, 249 and 195 patients had a DPP3 concentration lower than <50ng / ml or <30ng / ml, respectively, at baseline. For 195 patients lower than 30ng / ml at baseline, 16 (8%) patients showed an increase in DPP3 concentration to above 50ng / ml DPP3 thresholds in the following 6 days. On the other hand, among the 54 septic shock patients whose DPP3 plasma concentrations were between 30 ng / ml and 50 ng / ml at baseline, 11 (20.4%) patients increased their DPP3 plasma levels to above 50 ng / ml in the following days. These results suggest that lower DPP3 thresholds (well below 50 ng / ml) are appropriate to guide use and select patients who are likely to benefit from anti-ADM antibody therapy.

[0798] Different thresholds of DPP3 levels at baseline (day 1) were further used for subgroup analysis, comparing anti-ADM antibody therapy (adrezumab) with placebo using Kaplan-Meier plots for 28-day all-cause mortality in the treatment and placebo arms. The baseline DPP3 plasma concentration thresholds used were 50 ng / ml and 30 ng / ml.

[0799] When evaluating all-cause mortality in the septic shock population with DPP3 levels below the 30 ng / ml threshold (n=195), the mortality rate was surprisingly significantly lower in the anti-ADM antibody (adrezumab) therapy arm compared with the placebo arm ( Fig.21 C). The same results were observed when only patients with DPP3 values ​​below 50 ng / ml were included, with lower mortality in the treatment arm than in the placebo arm. Finally, when evaluating all-cause mortality from admission to 28 days in patients with septic shock who had DPP3 levels below 50 ng / ml at baseline and whose DPP3 levels remained low (< 50 ng / ml) during the following 144 hours, the mortality rate in the treatment arm was significantly lower than that in the placebo arm ( Fig.21 ). Therefore, a low DPP3 threshold at baseline (well below 50 ng / ml) is most appropriate for selecting patients who will benefit most from anti-ADM antibody therapy.

[0800] In general, patients with DPP3 levels above the 30ng / ml threshold at baseline have a higher chance of showing an increase in DPP3 plasma concentration in the following days. A subsequent increase in DPP3 plasma concentration to above 50ng / ml is associated with a lower efficacy of anti-ADM antibody therapy. Therefore, in order to stratify patients on anti-ADM antibody therapy, a lower threshold should be used, much lower than 50ng / ml, preferably in the range of between 22ng / ml and 40ng / ml, with a threshold of 30ng / ml being most preferred.

Claims

1. A method for predicting an increase in dipeptidyl peptidase 3 (DPP3) in a critically ill patient, the method comprising: Determining the level of DPP3 in a body fluid sample of said patient, ● comparing the determined DPP3 level to a predetermined threshold, wherein the threshold is in the range between 40 ng / ml and 22 ng / ml wherein the level of DPP3 in the sample being above the predetermined threshold indicates an increase in DPP3 in the patient. 2 . The method for predicting an increase in DPP3 in critically ill patients according to claim 1 , wherein the predetermined threshold is between 30 ng / ml and 22 ng / ml.

3. The method for predicting an increase in DPP3 in critically ill patients according to claim 1 or 2, wherein the predetermined threshold is between 25 ng / ml and 22 ng / ml.

4. The method for predicting an increase in DPP3 in a critically ill patient according to any one of claims 1 to 3, wherein the predicted increase is an increase to a DPP3 level equal to or higher than 40 ng / ml, preferably equal to or higher than 50 ng / ml.

5. The method for predicting an increase in DPP3 in critically ill patients according to claims 1 to 4, wherein the predicted increase in the DPP3 level is equal to or higher than 10%, more preferably equal to or higher than 20%, even more preferably equal to or higher than 40%, even more preferably equal to or higher than 50%, even more preferably equal to or higher than 75%, even more preferably equal to or higher than 100%.

6. The method for predicting an increase in DPP3 in critically ill patients according to claims 1 to 5, wherein the DPP3 increase is within at most 12 hours, preferably at most 24 hours, 48 ​​hours, 72 hours, 96 hours, more preferably at most 5 days, even more preferably at most 6 days, most preferably at most 7 days.

7. A method for predicting an increase in DPP3 in a critically ill patient according to claims 1 to 6, wherein the patient is a patient with severe infection, sepsis, heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, a patient with organ dysfunction or organ failure (e.g., liver, kidney, lung dysfunction or failure), a patient undergoing major surgery, a patient with trauma (e.g., burns, multiple trauma), a patient with shock and / or a patient developing shock, or alternatively a patient with ARDS.

8. The method for predicting an increase in DPP3 in critically ill patients according to claim 7, wherein the shock is selected from the group consisting of shock caused by hypovolemia, cardiogenic shock, obstructive shock and distributive shock.

9. The method for predicting an increase in DPP3 in critically ill patients according to claim 8, wherein In the case of cardiogenic shock, the patient may have suffered an acute coronary syndrome (e.g., acute myocardial infarction) or in which the patient has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmias, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or In the setting of hypovolemic shock, the patient may have had a bleeding disorder, including spontaneous bleeding from gastrointestinal bleeding, trauma, vascular etiologies (e.g., ruptured abdominal aortic aneurysm, tumor erosion into a major vessel), and anticoagulant use, or a non-bleeding disorder, including vomiting, diarrhea, renal loss, skin loss / insensate loss (e.g., burns, heat stroke), or third space loss in the setting of pancreatitis, cirrhosis, or intestinal obstruction, or In the case of obstructive shock, the patient may have suffered cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or • In cases of distributive shock, the patient may be suffering from septic shock, neurogenic shock, anaphylactic shock, or shock caused by adrenal crisis.

10. The method for predicting an increase in DPP3 in a critically ill patient according to any one of claims 7 to 9, wherein the shock is selected from the group comprising cardiogenic shock or septic shock.

11. A method for predicting an increase in DPP3 in a critically ill patient according to any one of claims 1 to 10, wherein if the DPP3 level in the sample is below the predetermined threshold, the patient is selected for a therapy / treatment, wherein the therapy is selected from the group of alkaline phosphatase, immunosuppressants, corticosteroids, vasopressors, fluids, anti-adrenomedullin antibodies or antibody fragments or scaffolds.

12. The method for predicting an increase in DPP3 in critically ill patients according to claim 11, wherein the anti-adrenomedullin antibody or anti-adrenomedullin antibody fragment or anti-adrenomedullin scaffold is directed against the N-terminal portion (amino acids 1-21) of adrenomedullin (ADM): YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 14).

13. A method for predicting an increase in DPP3 in a critically ill patient according to any one of claims 1 to 12, wherein if the DPP3 level in the sample is above the predetermined threshold, the patient is selected for therapy / treatment with a DPP3 inhibitor, wherein the DPP3 inhibitor is selected from the group of anti-DPP3 antibodies or anti-DPP3 antibody fragments or anti-DPP3 scaffolds.

14. The method for predicting an increase in DPP3 in a critically ill patient according to any one of claims 1 to 13, wherein the DPP3 level is the amount of DPP3 protein and / or the level of active DPP3.

15. The method for predicting an increase in DPP3 in critically ill patients according to any one of claims 1 to 14, wherein the DPP3 level is determined by different methods, including immunoassays, activity assays or mass spectrometry.

16. The method for predicting an increase in DPP3 in critically ill patients according to claim 15, wherein the immunoassay is a sandwich immunoassay.

17. The method for predicting an increase in DPP3 in critically ill patients according to any one of claims 1 to 16, wherein the body fluid is selected from whole blood, serum or plasma.

18. A method for preventing an increase in DPP3 in a critically ill patient, the method comprising: Determining the level of DPP3 in a body fluid sample of the patient, comparing the determined DPP3 level to a predetermined threshold, wherein the predetermined threshold is between 40 ng / ml and 22 ng / ml, and wherein a DPP3 level in the sample above the predetermined threshold is indicative of an increased DPP3 in the patient, and if said determined DPP3 level is above said predetermined threshold, administering a DPP3 inhibitor, The DPP3 inhibitor is an anti-DPP3 antibody and / or an anti-DPP3 antibody fragment and / or an anti-DPP3 scaffold.

19. The method for preventing an increase in DPP3 in critically ill patients according to claim 18, wherein the predetermined threshold is between 30 ng / ml and 22 ng / ml.

20. The method for preventing an increase in DPP3 in critically ill patients according to claim 18 or 19, wherein the predetermined threshold is between 25 ng / ml and 22 ng / ml.

21. Method for preventing an increase in DPP3 in critically ill patients according to any one of claims 18 to 20, wherein the increase is to a DPP3 level equal to or higher than 40 ng / ml, preferably equal to or higher than 50 ng / ml.

22. A method for preventing an increase in DPP3 in a critically ill patient according to claims 18 to 21, wherein the patient is a patient with severe infection, sepsis, heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, a patient with organ dysfunction or organ failure (e.g., liver, kidney, lung dysfunction or failure), a patient undergoing major surgery, a patient with trauma (e.g., burns, multiple trauma), a patient with shock and / or a patient developing shock, or alternatively a patient with ARDS.

23. The method for preventing an increase in DPP3 in critically ill patients according to claim 22, wherein the shock is selected from the group consisting of shock caused by hypovolemia, cardiogenic shock, obstructive shock and distributive shock.

24. The method for preventing an increase in DPP3 in a critically ill patient according to claim 23, wherein In the case of cardiogenic shock, the patient may have suffered an acute coronary syndrome (e.g., acute myocardial infarction) or in which the patient has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmias, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or In the setting of hypovolemic shock, the patient may have had a bleeding disorder, including spontaneous bleeding from gastrointestinal bleeding, trauma, vascular etiologies (e.g., ruptured abdominal aortic aneurysm, tumor erosion into a major vessel), and anticoagulant use, or a non-bleeding disorder, including vomiting, diarrhea, renal loss, skin loss / insensate loss (e.g., burns, heat stroke), or third space loss in the setting of pancreatitis, cirrhosis, or intestinal obstruction, or In the case of obstructive shock, the patient may have suffered cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or • In cases of distributive shock, the patient may be suffering from septic shock, neurogenic shock, anaphylactic shock, or shock caused by adrenal crisis.

25. The method for preventing an increase in DPP3 in a critically ill patient according to any one of claims 22 to 24, wherein the shock is selected from the group comprising cardiogenic shock or septic shock.

26. A DPP3 inhibitor for preventing an increase in DPP3 in a critically ill patient, wherein the patient has a DPP3 level above a threshold, wherein the threshold is between 40 ng / ml and 22 ng / ml, and wherein the DPP3 inhibitor is an anti-DPP3 antibody and / or an anti-DPP3 antibody fragment and / or an anti-DPP3 scaffold.

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