Therapy guidance and / or therapy monitoring for treatment with angiotensin-receptor-agonist and / or precursor thereof

Abstract

To provide a therapy guidance and / or therapy monitoring for treatment with an angiotensin-receptor-agonist and / or a precursor thereof.SOLUTION: Subject matter of the present invention is an angiotensin-receptor-agonist and / or a precursor thereof for use in the treatment of a disease in a subject, wherein the disease is selected from the group consisting of heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burn injuries, traumatic injuries, severe infection (microbial, viral (e.g. AIDS), parasitic diseases (e.g. Malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. seizures, neurodegenerative diseases), autoimmune diseases, vascular diseases, hypotension and shock, and wherein the subject has an amount of DPP3 protein and / or DPP3 activity in a sample of bodily fluid that is above a predetermined threshold.SELECTED DRAWING: None

Description

[Technical field]

[0001] The subject of the present invention is an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject, the disease is selected from the group consisting of heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial, viral (e.g. AIDS), parasitic (e.g. malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. stroke, neurodegenerative diseases), autoimmune diseases, vascular diseases, hypotension, and shock; said subject has an amount of DPP3 protein and / or DPP3 activity in a bodily fluid sample that exceeds a pre-determined threshold. [Background technology]

[0002] Dipeptidyl peptidase 3 (also known as dipeptidyl aminopeptidase III, dipeptidyl arylamidase III, dipeptidyl peptidase III, enkephalinase B, or red blood cell angiotensinase, abbreviated DPP3 or DPPIII) is a metallopeptidase that removes dipeptides from physiologically active peptides, such as enkephalins and angiotensin.

[0003] DPP3 was first identified in 1967 by Ellis and Nuenke from purified bovine anterior pituitary extracts and its activity was measured. The enzyme is listed as EC 3.4.14.4, has a molecular weight of approximately 83 kDa, and is highly conserved in eukaryotes and prokaryotes (Prajapati and Chauhan 2011). The amino acid sequence of the human variant is shown in SEQ ID NO:1. Dipeptidyl peptidase III is a ubiquitously expressed, primarily cytosolic peptidase. Although it lacks a signal sequence, several studies have reported membrane activity (Lee and Snyder 1982).

[0004] DPP3 is a zinc-dependent exopeptidase that belongs to the peptidase family M49. DPP3 has broad substrate specificity towards oligopeptides of 3 / 4-10 amino acids of various compositions and can also cleave after proline. DPP3 is known to hydrolyze dipeptides from the N-terminus of its substrates, which include angiotensin II, III, and IV; Leu-enkephalin and Met-enkephalin; and endomorphins 1 and 2. Metallopeptidase DPP3 has optimal activity at pH 8.0-9.0 and is highly selective for divalent metal ions (Co 2+ and Mg 2+ It can be activated by adding

[0005] 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 and Chauhan 2011; Kumar et al. 2016; numbers are for the sequence of human DPP3; see SEQ ID NO:1). Considering all known amino acids or sequence regions involved in substrate binding and hydrolysis, the active site of human DPP3 can be limited to the region between amino acids 316 and 669.

[0006] The most important substrate of DPP3 is angiotensin II (Ang II), the main effector of the renin-angiotensin system (RAS), which is activated in cardiovascular disease (Dostal et al. 1997 J Mol Cell Cardiol 29:2893-902; Roks et al. 1997 Heart Vessels. Suppl 12:119-24) and in sepsis and septic shock (Correa et al. 2015 Crit Care 19:98). Ang II in particular has been shown to alter many cardiovascular functions, including blood pressure control and cardiac remodeling.

[0007] Although the exact biological function of DPPIII in cell physiology is unknown, recent findings suggest a role in protein metabolism as well as in pain modulation and inflammatory processes (Prajapati and Chauhan 2011). In addition, DPP3 reduced blood pressure in hypertensive mice infused with Ang II without changing heart rate (Pang et al. 2016). However, normotensive mice showed no change in blood pressure when injected with DPP3.

[0008] Administration of an angiotensin receptor antagonist in combination with DPP3 in Ang II-infused mice reduced blood pressure similar to that achieved by injection of DPP3 alone or angiotensin receptor antagonist alone (Pang et al. 2016 Hypertension 68:630-41).

[0009] Angiotensin II (Ang II) is a peptide hormone produced naturally by the body that regulates blood pressure through vasoconstriction and sodium reabsorption. The hemodynamic effects of Ang II administration have been the subject of numerous clinical studies, which have demonstrated significant effects on systemic and renal blood flow (Harrison-Bernard 2009 Adv. Physiol Edu. 33(4):270).

[0010] The beneficial effect of Ang II treatment in vasodilatory and septic shock is currently being debated (Khanna, A. et al., 2017; Antonucci, E. et al., 2017; Tumlin, JA et al., 2018). Patients with vasodilatory shock (80% of septic shock) treated with angiotensin II were more likely to survive to 28 days and showed a significant correlation with hypotension (Khanna, A. et al., 2017; Tumlin, JA et al., 2018).

[0011] It has been speculated that angiotensin-converting enzyme (ACE) is highly dysregulated in patients with shock, leading to altered angiotensin I / II ratios, and that infusion of angiotensin III can compensate for such dysregulation (Tumlin, JA et al., 2018).

[0012] Recently, two assays have been devised, characterized and validated to specifically detect DPP3 in human body fluids (e.g. blood, plasma and serum): a fluorescent immunoassay (LIA) to measure the concentration of DPP3 protein and an enzyme capture activity assay (ECA) to detect specific DPP3 activity (Rehfeld et al. 2018 JALM in press). In both methods, the actual DPP3 protein or DPP3 activity is detected after removing all interfering substances in a washing step. Both methods are highly specific and therefore allow reproducible detection of DPP3 in blood samples. Summary of the Invention

[0013] The subject of the present invention is an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject, the disease is selected from the group consisting of heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial, viral (e.g. AIDS), parasitic (e.g. malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. stroke, neurodegenerative diseases), autoimmune diseases, vascular diseases, hypotension, and shock; said subject has an amount of DPP3 protein and / or DPP3 activity in a bodily fluid sample that exceeds a pre-determined threshold. [Brief description of the drawings]

[0014] [Figure 1] Kaplan-Meier survival curves associated with low (<68.6 ng / ml) and high (≥68.6 ng / ml) DPP3 plasma concentrations. (A) 7-day survival rate vs. DPP3 plasma concentration in patients with sepsis / septic shock (cut-off value 68.6 ng / ml); (B) 7-day survival rate vs. DPP3 plasma concentration in patients with cardiogenic shock (cut-off value 68.6 ng / ml); (C) 7-day survival rate vs. DPP3 plasma concentration in patients with acute myocardial infarction (cut-off value 68.6 ng / ml); (D) 3-month survival rate vs. DPP3 plasma concentration in patients with respiratory distress; (E) 4-week survival rate vs. DPP3 plasma concentration in burn patients; (F) 7-day survival rate vs. DPP3 plasma concentration in patients with septic shock. [Diagram 2] SDS-PAGE on gradient gel (4-20%) of native hDPP3 purified from human erythrocyte lysate. Molecular weight markers are indicated by arrows. [Diagram 3] Experimental Design—Effects of native hDPP3 in animal models. [Figure 4] (A) Injection of DPP3 leads to a decrease in fractional shortening and therefore to a deterioration in cardiac function. (B) A decrease in renal function is also observed through an increased renal resistive index. [Diagram 5]Association-dissociation curves of AK1967-DPP3 binding assay using Octet. AK1967-loaded biosensors were soaked in serial dilutions of GST-tagged recombinant human DPP3 (100, 33.3, 11.1, 3.7 nM) and dissociation and association were monitored. [Figure 6] Western blot of dilutions of hemocyte lysates and detection of DPP3 using AK1967 as the primary antibody. [Figure 7] Inhibition curve of natural DPP3 from blood cells using the inhibitory antibody AK1967. The inhibition of DPP3 by the specific antibody was concentration-dependent, and the IC50 was approximately 15 ng / ml when analyzed against 15 ng / ml of DPP3. [Figure 8] Experimental Setting -Effect of Procizumab on Sepsis-Induced Heart Failure. [Figure 9] Procizumab dramatically improves fractional shortening (A) and mortality (B) in mice with sepsis-induced heart failure. [Figure 10] Experimental setup - Mice were induced with cardiac stress by isoproterenol followed by treatment with procizumab (B) or control (A). [Figure 11] In mice with isoproterenol-induced heart failure, procizumab improved fractional shortening within 1 hour (A) and reduced renal resistive index within 6 hours (B) of administration. [Figure 12] Experimental Setup – Effect of Valsartan in healthy mice injected with DPP3. [Figure 13] The decrease in fractional shortening induced by DPP3 was rescued by treatment with valsartan. [Figure 14] Higher DPP3 concentration levels 24 hours after admission in septic patients were associated with the worst SOFA scores. [Figure 15]High cDPP3 plasma levels correlate with organ dysfunction in septic patients.Bar graph of SOFA score calculated from changes in DPP3 levels during ICU stay in AdrenOSS1.HH: DPP3 greater than median at admission and 24 hours;HL: greater than median at admission but less than median at 24 hours;LL: less than median at admission and 24 hours;LH: less than median at admission but greater than median at 24 hours. [Figure 16] Higher cDPP3 concentration levels 24 hours after admission in septic patients were associated with the worst organ-specific SOFA scores. SOFA scores for (A) heart, (B) kidney, (C) respiratory system, (D) liver, (E) coagulation, and (F) central nervous system (HH: high / high, HL: high / low, LH: low / high, LL: low / low) were calculated from the fluctuating cDPP3 levels from admission to 24 hours later. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Initiation of treatment with angiotensin receptor agonists and / or precursors thereof is indicated when the amount of DPP3 protein and / or DPP3 activity in a body fluid sample in a patient exceeds a predetermined threshold level.

[0016] The patient may be a patient with a fatal illness. This means a patient suffering from a fatal illness, which is any disease process that causes physiological instability leading to incapacity or death within minutes or hours. Disturbances of the nervous and cardiorespiratory systems generally have the most immediate lethal effect. Such instability can be reliably detected by deviations from normal ranges in simple clinical observations (level of consciousness, respiratory rate, heart rate, blood pressure, urine output, etc.). Such measurements can be compiled into score systems for assessing the severity of many common diseases (e.g., CRB-65 score for pneumonia, Glasgow score for pancreatitis, etc.) (Frost 2007 British Journal of Hospital Medicine 68(10):M180-183).

[0017] The disease may be selected from the group including heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial diseases, viral diseases (e.g. AIDS), parasitic diseases (e.g. malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. stroke, neurodegenerative diseases), autoimmune diseases, vascular diseases, hypotension, and shock.

[0018] Thus, the condition may be shock, which may be selected from the group including hypovolemic shock, cardiogenic shock, vaso-occlusive shock, and distributive shock.

[0019] Shock is characterized by cellular and tissue hypoxia due to reduced oxygen supply and / or increased oxygen consumption, or inadequate oxygen utilization. Shock is a life-threatening condition of circulatory failure, most commonly manifesting as hypotension (systolic blood pressure < 90 mm Hg or MAP < 65 mmHg). Based on the underlying cause, shock is divided into four types: hypovolemic, cardiogenic, vaso-occlusive, and distributive (Vincent and De Backer 2014 N. Engl. J. Med. 370(6):583).

[0020] Hypovolemic shock is characterized by a decrease in intravascular volume and can be divided into two broad subtypes: hemorrhagic and nonhemorrhagic. Common causes of hemorrhagic hypovolemic shock include spontaneous hemorrhage in the setting of gastrointestinal hemorrhage, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor invasion of major blood vessels), and anticoagulant use. Common causes of nonhemorrhagic hypovolemic shock include vomiting, diarrhea, renal losses, cutaneous / insensible losses (e.g., burns, heat stroke), or interstitial losses in the setting of pancreatitis, liver cirrhosis, ileus, and trauma. For a review, see Koya and Paul 2018, Shock. StatPearls [Internet]. Treasure Island, FL: StatPearls Publishing Inc.; October 27, 2019-2018.

[0021] Cardiogenic shock (CS) is defined as a state of critical end-organ hypoperfusion due to reduced cardiac output. In particular, CS forms a spectrum ranging from mild hypoperfusion to severe shock. Established criteria for the diagnosis of CS are (i) systolic blood pressure <90 mmHg for >30 min or the need for vasopressors to achieve blood pressure ≥90 mmHg; (ii) pulmonary congestion or elevated left ventricular filling pressure; (iii) signs of impaired organ perfusion accompanied by at least one of the following criteria: (a) altered mental status; (b) cold, clammy skin; (c) oliguria (urine output <30 ml / hr); and (d) elevated serum lactate (Reynolds and Hochman 2008 Circulation 117:686-697). Acute myocardial infarction (AMI) and subsequent ventricular dysfunction are the most common 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 frequent causes of CS after AMI (Hochman et al., 2000 J Am Coll Cardiol 36:1063-1070). Non-AMI-related CS can result from decompensated valvular heart disease, acute cardiomyopathy, or arrhythmias and has multiple treatment options.

[0022] Vaso-occlusive shock is due to physical obstruction of the large blood vessels or the heart itself. Several conditions can result in this form of shock (e.g., cardiac tamponade, tension pneumothorax, pulmonary embolism, aortic stenosis). For review, see Koya and Paul 2018, Shock. StatPearls [Internet]. Treasure Island, FL: StatPearls Publishing; 2019-2018 Oct. 27.

[0023] 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. Distributive shock can result from sepsis as well as systemic inflammatory response syndrome (SIRS) with non-infectious causes (pancreatitis, burns, trauma, etc.). Other causes include toxic shock syndrome (TSS), anaphylaxis (sudden severe allergic reaction), adrenal insufficiency (acute worsening of chronic adrenal insufficiency, destruction or removal of the adrenal glands, suppression of adrenal function due to exogenous steroids, hypopituitarism, and inability to metabolize hormones produced), reaction to drugs or toxins, heavy metal poisoning, liver failure, and damage to the nervous system. For review, see Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island, FL: StatPearls Publishing; 2019-27 October 2018.

[0024] In one embodiment of the invention, the patient is a shock patient. In yet another embodiment of the invention, the shock can be selected from the group of hypovolemic shock, cardiogenic shock, vaso-occlusive shock and distributive shock. In another particular embodiment of the invention, the distributive shock is septic shock.

[0025] An agonist is a chemical that binds to a receptor and activates that receptor, resulting in a biological response. Receptors can be activated by endogenous agonists (such as hormones and neurotransmitters) or exogenous agonists (such as drugs), resulting in a biological response. A full agonist binds to a receptor and activates it with the maximum response that an agonist can elicit at that receptor. A partial agonist is a drug that binds to and activates a given receptor but has only a partial effect at that receptor compared to a full agonist. Potency is the amount of agonist needed to elicit the desired response. The potency of an agonist is determined by its EC 50 There is an inverse relationship with the EC value. 50 The EC can be measured for a given agonist by determining the concentration of agonist required to elicit half of the maximal biological response of that agonist. 50 EC values ​​are useful for comparing the potencies of drugs that are similar in potency and produce similar physiological effects. 50 The lower the value, the more potent the agonist is and the lower the concentration of drug required to elicit the maximal biological response.

[0026] Treatment with angiotensin receptor agonists and / or precursors thereof can be continued as long as the patient has an amount of DPP3 protein and / or DPP3 activity in a body fluid sample above a pre-determined threshold level.

[0027] The amount of DPP3 protein and / or DPP3 activity is measured at least every 24 hours, preferably every 12 hours, more preferably every 8 hours, even more preferably every 6 hours, even more preferably every 4 hours, even more preferably every 2 hours, even more preferably every hour, and most preferably every 30 minutes.

[0028] Treatment with angiotensin receptor agonists and / or precursors thereof can be discontinued when the patient has an amount of DPP3 protein and / or DPP3 activity in a body fluid sample that is below a predetermined threshold level.

[0029] In one embodiment, the angiotensin receptor agonist and / or precursor thereof is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, and angiotensin IV.

[0030] Angiotensin Therapeutics:

[0031] Angiotensin I, also called proangiotensin, is formed by the action of renin on angiotensinogen. Renin cleaves the peptide bond between leucine (Leu) and valine (Val) residues, resulting in the decapeptide (10 amino acids) (des-Asp) angiotensin I. Angiotensin I is converted to angiotensin II by the removal of two C-terminal residues by angiotensin-converting enzyme (ACE), primarily in the lungs (but also present in endothelial cells, renal epithelial cells, and the brain).

[0032] Angiotensin II, angiotensin III, and angiotensin IV are peptide hormones produced naturally by the body that regulate blood pressure through vasoconstriction and sodium reabsorption. The hemodynamic effects of angiotensin II administration have been the subject of numerous clinical studies that have demonstrated significant effects on systemic and renal blood flow (Harrison-Bernard, LM, The Renal Renin-Angiotensin System, Adv Physiol Educ, 33(4):270 (2009)).

[0033] Angiotensin II is a hormone produced by the renin-angiotensin-aldosterone system (RAAS) that regulates blood pressure through regulation of vascular smooth muscle tone and extracellular fluid homeostasis. Angiotensin II mediates its effects on the vasculature by inducing vasoconstriction and sodium retention, and is therefore the target of many hypertension treatments. In addition to its systemic effects, angiotensin II has profound effects on the renal efferent arteriole, maintaining glomerular filtration when blood flow is reduced. Angiotensin II inhibits the upregulation of Na in the proximal tubule. + / H + It also regulates sodium reabsorption in the kidney by stimulating renin exchange and inducing the release of aldosterone and vasopressin (Harrison-Bernard, LM, The Renal Renin-Angiotensin System, Adv Physiol Educ, 33(4):270 (2009)).

[0034] An angiotensin II therapeutic agent that can be used in the compositions and methods of the present disclosure can be Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 13), also known as 5-isoleucine angiotensin II. SEQ ID NO: 13 is an octapeptide that occurs naturally in humans and other species (horses, pigs, etc.). The isoleucine can be replaced with valine, resulting in 5-valine angiotensin II, i.e., Asp-Arg-Val-Tyr- Val-His-Pro-Phe (SEQ ID NO: 14). Other angiotensin II analogs, such as [Asn 1 -Phe 4 ]-angiotensin II (SEQ ID NO: 15), hexapeptide Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 16), nonapeptide Asn-Arg-Val-Tyr-Tyr-Val-His-Pro-Phe (SEQ ID NO: 17), [Asn 1 Ile 5 Ile 8 ]-angiotensin II (SEQ ID NO: 18), [Asn 1 -lle 5 -Ala 8]-angiotensin II (SEQ ID NO: 19), and [Asn 1 -Jinde Tyr 4 -Ile 5 -angiotensin II (SEQ ID NO: 20) can also be used. Angiotensin II can be synthesized, for example, by solid phase peptide synthesis, incorporating modifications such as C-terminal amidation. The term "angiotensin II" is intended to mean any of these various forms, as well as combinations thereof, unless further specified.

[0035] In some aspects, the composition comprising angiotensin II can be selected from 5-valine angiotensin III, 5-valine angiotensin III amide, 5-L-isoleucine angiotensin III, and 5-L-isoleucine angiotensin III amide, or pharma- ceutically acceptable salts thereof, which are preferably manufactured under current good manufacturing practice (cGMP). In some aspects, the composition can contain different forms of angiotensin II in different proportions (e.g., a mixture of hexapeptide angiotensin II and nonapeptide angiotensin II). The composition comprising angiotensin II can be suitable for parenteral administration (e.g., injection or intravenous infusion).

[0036] The angiotensin II sequences used in the compositions and methods disclosed herein can be homologous to the above angiotensin II sequences. In some aspects, the invention includes isolated, synthetic, or recombinant amino acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, and / or 20. Any of these variant sequences can be used in place of the angiotensin II described in the previous paragraph.

[0037] For all sequences, sequence identity is determined according to the following method: percent identity is calculated by multiplying the number of matches in a pair by 100 and dividing by the length of the aligned region (including gaps) (percent identity = [number of matches x 100] / length of aligned region (including gaps)).

[0038] Angiotensin III is a metabolite of angiotensin II and has approximately 40% of the activity of angiotensin II. Angiotensin III therapeutic agent that can be used in the compositions and methods of the present disclosure can be Arg-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 21). SEQ ID NO: 21 is a heptapeptide that occurs naturally in humans and other species (horses, pigs, etc.). Isoleucine can be replaced with valine, resulting in Arg-Val-Tyr-Val-His-Pro-Phe (SEQ ID NO: 22). Other angiotensin III analogs, such as [Phe 3 ]-angiotensin III (SEQ ID NO: 23), [Ile 4 -Ala 7 ]-angiotensin III (SEQ ID NO: 24), and [diindoTyr 3 -Ile 4 ]-angiotensin III (SEQ ID NO: 25) can also be used. Angiotensin III can be synthesized, for example, by solid phase peptide synthesis, incorporating modifications such as C-terminal amidation. The term "angiotensin III" is intended to mean any of these various forms, as well as combinations thereof, unless further specified.

[0039] In some aspects, the composition comprising angiotensin III can be selected from 4-valine angiotensin III, 4-valine angiotensin III amide, 4-L-isoleucine angiotensin III, and 4-L-isoleucine angiotensin III amide, or a pharma- ceutically acceptable salt thereof, preferably manufactured under current good manufacturing practice (cGMP).The composition comprising angiotensin III can be suitable for parenteral administration (e.g., injection or intravenous infusion).

[0040] Angiotensin III sequences used in the compositions and methods disclosed herein can be homologous to the above angiotensin III sequences. In some aspects, the invention includes isolated, synthetic, or recombinant amino acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 21, 22, 23, 24, and / or 25. Any of these variant sequences can be used in place of the angiotensin II described in the previous paragraph.

[0041] Angiotensin IV is a metabolite of angiotensin III and has less activity than angiotensin II. Angiotensin IV therapeutic agent that can be used in the compositions and methods of the present disclosure can be Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 26). SEQ ID NO: 26 is a hexapeptide that occurs naturally in humans and other species. Isoleucine can be replaced with valine, resulting in Val-Tyr-Val-His-Pro-Phe (SEQ ID NO: 27). Other angiotensin IV analogs, such as [Phe 2 ]-angiotensin III (SEQ ID NO: 28), [Ile 3 -AIa 6 ]-angiotensin IV (SEQ ID NO: 29), and [diindoTyr 2 -Ile 3]-angiotensin IV (SEQ ID NO: 30) can also be used. Angiotensin IV can be synthesized, for example, by solid phase peptide synthesis, incorporating modifications such as C-terminal amidation. The term "angiotensin IV" is intended to mean any of these various forms, as well as combinations thereof, unless further specified.

[0042] In some aspects, the composition comprising angiotensin IV can be selected from 3-valine angiotensin IV, 3-valine angiotensin IV amide, 3-L-isoleucine angiotensin IV, and 3-L-isoleucine angiotensin IV amide, or a pharma- ceutical acceptable salt thereof, which is preferably manufactured under current good manufacturing practice (cGMP).The composition comprising angiotensin IV can be suitable for parenteral administration (e.g., injection or intravenous infusion).

[0043] The angiotensin IV sequences used in the compositions and methods disclosed herein can be homologous to the above angiotensin IV sequences. In some aspects, the invention includes isolated, synthetic, or recombinant amino acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 25, 26, 27, 28, 29, and / or 30. Any of these variant sequences can be used in place of the angiotensin IV described in the previous paragraph.

[0044] The angiotensin II, angiotensin III, or angiotensin IV therapeutic agents can be used in any suitable salt (e.g., acetate), deprotected, acetylated, deacetylated, and / or prodrug form of the peptides described above, including PEGylated forms of the peptides or conjugates as disclosed in U.S. Patent Application Publication No. 2011 / 0081371, incorporated herein by reference. The term "prodrug" refers to a precursor compound capable of generating or releasing the peptides described above under physiological conditions. Such prodrugs or precursors can be larger peptides that are selectively cleaved to form the peptides of the invention. For example, in some aspects, the prodrugs or precursors can be angiotensinogen, angiotensin I, or homologs thereof that become angiotensin II through the action of some endogenous or exogenous enzyme. Further prodrugs or precursors include peptides having protected amino acids, for example with protecting groups at one or more carboxylic acid and / or amino groups. Suitable protecting groups for amino groups include benzyloxycarbonyl, t-butyloxycarbonyl (BOC), fluorenylmethyloxycarbonyl (FMOC), formyl, acetyl, or acyl groups. Suitable protecting groups for carboxylic acid groups include esters, such as benzyl esters and t-butyl esters. The present invention also contemplates the use of angiotensin II, angiotensin III, angiotensin IV, and / or precursor peptides that have amino acid substitutions, deletions, additions (substitutions and additions include standard D- and L-amino acids), and modified amino acids (e.g., amidated and acetylated amino acids), while maintaining the therapeutic activity of the basic peptide sequence at a pharmacologically useful level.

[0045] In a preferred embodiment, the angiotensin II is angiotensin II acetate. Angiotensin II acetate is L-aspartyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine acetate. The counter ion acetate is present in a non-stoichiometric ratio. The molecular formula of angiotensin II acetate is C 50 H 71 N 13 O 12 (C2H4O2) n (n is the number of acetate molecules; theoretical n=3) and the average molecular weight is 1046.2 (as the free base).

[0046] One embodiment of the present invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, said subject having an amount of DPP3 protein and / or DPP3 activity in a body fluid sample above a pre-determined threshold, said amount of DPP3 protein and / or DPP3 activity having been determined at least once in a body fluid sample of said subject prior to and / or during treatment with said angiotensin receptor agonist and / or precursor thereof.

[0047] One embodiment of the present invention is an angiotensin receptor agonist and / or precursors thereof for use in the treatment of a disease in a subject, said subject having an amount of DPP3 protein and / or DPP3 activity in a body fluid sample above a pre-determined threshold, and wherein the determination of the amount of DPP3 protein and / or DPP3 activity determined in the body fluid sample is used as a therapeutic guide and / or therapeutic monitoring of said treatment with an angiotensin receptor agonist and / or precursors thereof.

[0048] In one embodiment of the invention, the amount of DPP3 protein and / or DPP3 activity in a body fluid sample is determined before the treatment of the subject with an angiotensin receptor agonist and / or its precursor. In one embodiment of the invention, the amount of DPP3 protein and / or DPP3 activity in a body fluid sample is determined at least once, preferably at least twice, during the treatment of the subject with an angiotensin receptor agonist and / or its precursor, or preferably at least once every day during the treatment. In one embodiment of the invention, the amount of DPP3 protein and / or DPP3 activity in a body fluid sample is determined after the treatment of the subject with an angiotensin receptor agonist and / or its precursor. In one embodiment of the invention, the amount of DPP3 protein and / or DPP3 activity in a body fluid sample is determined before, during, and / or after the treatment of the subject with an angiotensin receptor agonist and / or its precursor.

[0049] One embodiment of the present invention is an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject, said subject having an amount of DPP3 protein and / or DPP3 activity in a body fluid sample above a pre-determined threshold, said body fluid sample of said subject being selected from whole blood, plasma and serum.

[0050] One embodiment of the invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, wherein said angiotensin receptor agonist and / or precursor thereof is administered to said subject in a pharmaceutical formulation.

[0051] One embodiment of the present invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, said angiotensin receptor agonist and / or precursor thereof being angiotensin II and said pharmaceutical formulation being a solution, preferably a ready-to-use solution.In another embodiment, the subject of the present invention is also a pharmaceutical formulation according to the invention, said pharmaceutical formulation being in a dry state to be reconstituted before use.

[0052] The pharmaceutical compositions of the present invention may also contain diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutical acceptable carrier" refers to a non-toxic carrier that can be administered to a patient together with a therapeutically effective substance of the present invention (such as angiotensin II) and does not destroy the pharmacological activity of the therapeutically effective substance. The term "pharmaceutical acceptable" refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient.

[0053] The characteristics of the carrier will depend on the route of administration. The term "excipient" means an additive in a formulation or composition that is not a medicamentously active ingredient.

[0054] Those skilled in the art will recognize that the selection of any one excipient may affect the selection of any other excipient. For example, the selection of a particular excipient may preclude the use of one or more additional excipients, because the combination of excipients may cause undesirable effects. Non-limiting examples of excipients of the present invention include co-solvents, solubility enhancers, buffers, pH adjusters, bulking agents, surfactants, sealants, tonicity adjusters, stabilizers, protectants, and viscosity modifiers.

[0055] In some aspects, it may be advantageous to include a pharma- ceutically acceptable carrier in the composition of the invention.

[0056] In some aspects, it may be advantageous to include a solubilizer in the composition of the present invention. The solubilizer may be useful for increasing the solubility of any component of the formulation or composition, including therapeutically effective substances (e.g., angiotensin II, angiotensin III, angiotensin IV) or excipients. The solubilizers described herein are not intended to constitute an exhaustive list, but merely provide representative solubilizers that can be used in the composition of the present invention. In some aspects, non-limiting examples of solubilizers include ethyl alcohol, t-butyl alcohol, polyethylene glycol, glycerol, methylparaben, propylparaben, polyvinylpyrrolidone, and any pharma- ceutically acceptable salts and / or combinations thereof.

[0057] In some aspects, it may be advantageous to adjust the pH of the composition by including a pH adjusting agent in the composition of the present invention. Changing the pH of the formulation or composition may have beneficial effects, for example, on the stability or solubility of a therapeutically effective substance, or in making the formulation or composition suitable for parenteral administration. pH adjusting agents are well known in the art. Thus, the pH adjusting agents described herein are not intended to constitute an exhaustive list, but merely provide representative pH adjusting agents that can be used in the compositions of the present invention. pH adjusting agents may include, for example, acids and bases. In some aspects, non-limiting examples of pH adjusting agents include acetic acid, hydrochloric acid, phosphoric acid, sodium hydroxide, sodium carbonate, and combinations thereof.

[0058] The pH of the compositions of the invention can be any pH that provides the desired properties for the formulation or composition. Desirable properties can include, for example, stability of the therapeutically effective substance (e.g., angiotensin II, angiotensin III, or angiotensin IV), increased retention of the therapeutically effective substance compared to compositions at other pHs, and improved filtration efficiency. In some aspects, the pH of the compositions of the invention can be about 3.0 to about 9.0, such as about 5.0 to about 7.0. In particular aspects, the pH of the compositions of the invention can be 5.5±0.1, 5.6±0.1, 5.7±0.1, 5.8±0.1, 5.9±0.1, 6.0±0.1, 6.1±0.1, 6.2±0.1, 6.3±0.1, 6.4±0.1, or 6.5±0.1.

[0059] In some aspects, it may be advantageous to include one or more buffers in the composition to reduce pH fluctuations. In some aspects, the buffers can have a pKa of, for example, about 5.5, about 6.0, or about 6.5. One of skill in the art will recognize that appropriate buffers can be selected for inclusion in the compositions of the invention based on their pKa and other properties.

[0060] Buffers are well known in the art. Thus, the buffers described herein are not intended to constitute an exhaustive list, but merely provide representative buffers that can be used in the compositions of the present invention. In some aspects, the buffer can include one or more of Tris, TrisHCl, potassium phosphate, sodium phosphate, sodium citrate, sodium ascorbate, a combination of sodium and potassium phosphate, Tris / TrisHCl, sodium bicarbonate, arginine phosphate, arginine hydrochloride, histidine hydrochloride, cacodylate, succinate, 2-(N-morpholino)ethanesulfonic acid (MES), maleate, bis-Tris, phosphate, carbonate, and any pharma- ceutically acceptable salt and / or combination thereof.

[0061] In some aspects, it may be advantageous to include a surfactant in the compositions of the present invention. Surfactants generally reduce the surface tension of liquid compositions. Surfactants may provide advantageous properties such as improved ease of filtration. Surfactants may also function as emulsifiers and / or solubilizers. Surfactants are well known in the art. Thus, the surfactants described herein are not intended to constitute an exhaustive list, but merely provide representative surfactants that may be used in the compositions of the present invention. Non-limiting examples of surfactants that may be included include sorbitan esters (such as polysorbates (e.g., polysorbate 20 and polysorbate 80)), lipopolysaccharides, polyethylene glycols (e.g., PEG 400 and PEG 3000), poloxamers (i.e., Pluronics), ethylene oxides and polyethylene oxides (e.g., Triton X-100), saponins, phospholipids (e.g., lecithin), and combinations thereof.

[0062] In some aspects, it may be advantageous to include a tonicity adjuster in the composition of the present invention. The tonicity of a liquid composition is an important consideration when administering the composition to a patient, for example, by parenteral administration. Thus, a tonicity adjuster can be used to help make the formulation or composition suitable for administration. Tonicity adjusters are well known in the art. Thus, the tonicity adjusters described herein are not intended to constitute an exhaustive list, but merely provide representative tonicity adjusters that can be used in the composition of the present invention. Tonicity adjusters can be ionic or non-ionic, non-limiting examples of which include inorganic salts, amino acids, carbohydrates, sugars, sugar alcohols, and carbohydrates. Representative inorganic salts can include sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate. One representative amino acid is glycine. Representative sugars can include sugar alcohols (such as glycerol), propylene glycol, glucose, sucrose, lactose, and mannitol.

[0063] In some aspects, it may be advantageous to include a stabilizer in the compositions of the present invention. The stabilizer helps to increase the stability of the therapeutically effective substance contained in the compositions of the present invention. This may occur, for example, by reducing the degradation of the therapeutically effective substance or by preventing aggregation of the therapeutically effective substance. Without wishing to be bound by theory, mechanisms of increasing stability may include sequestering the therapeutically effective substance from the solvent or inhibiting free radical oxidation of the anthracycline compound. Stabilizers are well known in the art. Thus, the stabilizers described herein are not intended to constitute an exhaustive list, but merely provide representative stabilizers that can be used in the compositions of the present invention. Non-limiting examples of stabilizers include emulsifiers and surfactants.

[0064] The compositions of the invention can be administered in a variety of conventional ways. In some aspects, the compositions of the invention are suitable for parenteral administration. These compositions can be administered, for example, intraperitoneally, intravenously, intrarenally, or intrathecally. In some aspects, the compositions of the invention are injected intravenously. Those skilled in the art will recognize that the method of administering a therapeutically effective agent formulation or composition according to the invention will depend on factors such as the age, weight, and physical condition of the patient being treated, and the disease or condition being treated. Thus, those skilled in the art will be able to select the most suitable method of administration for the patient in each case.

[0065] The angiotensin receptor agonist and / or its precursor can be administered in any suitable manner, but is typically administered by continuous infusion. Thus, the increase or decrease in administration rate can be achieved by changing the flow rate of the intravenous drip, changing the concentration of the drug contained in the intravenous drip, etc. However, the manner of changing the administration rate will depend on the method of administration of the therapeutic agent. If the therapeutic agent is administered transmucosally or transdermally, the rate can be increased, for example, by changing to a patch or transdermal composition that releases at a faster rate. If the therapeutic agent is administered orally, the rate can be increased, for example, by switching to a higher dose form, administering additional doses, or administering a controlled release dosage form that releases at a faster rate.

[0066] If the therapeutic agent is administered by inhalation, the rate can be increased, for example, by administering an additional, more concentrated, or faster releasing bolus. Other methods of administration (via subcutaneous infusion pumps, suppositories, etc.) can be similarly modified, and a decrease in the rate of administration can be achieved by performing the opposite of the operation that would increase the rate of administration of the therapeutic agent.

[0067] One embodiment of the invention is an angiotensin receptor agonist and / or precursor thereof for use in treating a disease in a subject, wherein said angiotensin receptor agonist and / or precursor thereof is angiotensin II administered at a rate of 0.1-200 ng / kg / min, preferably 1-100 ng / kg / min, more preferably 2-80 ng / kg / min, even more preferably 5-60 ng / kg / min, even more preferably 10-50 ng / kg / min, even more preferably 15-40 ng / kg / min, and most preferably at a rate of 20 ng / kg / min.

[0068] In one particular embodiment of the invention, the starting dose (initial rate) of angiotensin II is 80 ng / kg / min, more preferably 40 ng / kg / min, and most preferably 20 ng / kg / min by continuous intravenous infusion.

[0069] In another particular embodiment of the invention for titrating angiotensin II, blood pressure response (e.g., mean arterial pressure; MAP) is monitored. Angiotensin II titration can be performed every 60 minutes, more preferably every 45 minutes, even more preferably every 30 minutes, even more preferably every 15 minutes, even more preferably every 10 minutes, and most preferably every 5 minutes. Angiotensin II titration can be performed incrementally up to 40 ng / kg / min, more preferably up to 20 ng / kg / min, and most preferably up to 15 ng / kg / min as needed to achieve or maintain the target blood pressure. In another preferred embodiment, the dose of angiotensin II should not exceed 80 ng / kg / min for the first 3 hours of treatment. Most preferably, the maintenance dose does not exceed 40 ng / kg / min, and doses as low as 1.25 ng / kg / min can be used.

[0070] In some embodiments, the patient has an initial mean arterial pressure (MAP) prior to administration of the composition of about 40 mm Hg, about 45 mm Hg, about 50 mm Hg, about 55 mm Hg, about 60 mm Hg, about 65 mm Hg, about 70 mm Hg, or about 75 mm Hg. Treatment methods can include measuring the patient's mean arterial pressure and increasing the rate of administration of angiotensin II if the mean arterial pressure is less than about 40 mm Hg, about 45 mm Hg, about 50 mm Hg, about 55 mm Hg, about 60 mm Hg, about 65 mm Hg, about 70 mm Hg, or about 75 mm Hg.

[0071] In one embodiment of the invention, the patient may be administered a vasopressor (e.g., norepinephrine, norepinephrine equivalents, epinephrine, dopamine, catecholamines such as phenylephrine, or combinations thereof. In some embodiments, the vasopressor is vasopressin (terlipressin, argipressin, desmopressin, felypressin, lypressin, or ornipressin).

[0072] One embodiment of the invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, said angiotensin receptor agonist and / or precursor thereof being administered in combination, in particular angiotensin II, with an inhibitor of DPP3.

[0073] One embodiment of the present invention is a combination of an angiotensin receptor agonist and / or a precursor thereof with an inhibitor of DPP3 for use in the treatment of a disease in a subject, said inhibitor of DPP3 being selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

[0074] According to the present invention, an "anti-DPP3 antibody" is an antibody that specifically binds to DPP3, an "anti-DPP3 antibody fragment" is a fragment of said anti-DPP3 antibody, said fragment specifically binds to DPP3, and an "anti-DPP3 non-Ig scaffold" is a non-Ig scaffold that specifically binds to DPP3.

[0075] One embodiment of the invention is a combination of an angiotensin receptor agonist and / or a precursor thereof with an inhibitor of DPP3 for use in the treatment of a disease in a subject, said inhibitor of DPP3 being an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold binding to SEQ ID NO: 1, in particular SEQ ID NO: 2.

[0076] One embodiment of the present invention is a combination of an angiotensin receptor agonist and / or a precursor thereof with an inhibitor of DPP3 for use in the treatment of a disease in a subject, said inhibitor of DPP3 being at least 10 -7 The antibody or fragment or scaffold exhibits a minimum binding affinity to DPP3 of M or less.

[0077] According to the present invention, those skilled in the art are well aware that the binding affinity of the DPP3 binder disclosed herein to DPP3 can be measured by various suitable assays known in the art.Representative examples are shown below, but these examples should not be interpreted as limiting the possibility of measuring the binding affinity of the DPP3 binder disclosed herein to DPP3.

[0078] For example, the binding affinity of the DPP3 binders to the epitope can be determined. Binding assays can be performed to detect and / or quantify antibodies that bind, for example, to the immunizing peptide of each binder. For example, the immunizing peptide can be immobilized on the surface of a solid phase. A test sample (e.g., an antibody solution) can be passed over the immobilized immunizing peptide to detect bound antibodies. For purposes of this specification, the term "solid phase" can include any material or container in or on which an assay can be performed, and non-limiting examples of solid phases include porous and non-porous materials, test tubes, wells, slides, magnetic beads, and the like.

[0079] Typical detection methods include: - Labeling the antibody before contacting it with the solid phase and detecting the respective label (fluorescent, chemiluminescent, enzymatic, etc.); - using a labeled secondary antibody against the specific Fc portion of the sample antibody: the antibody bound to the solid phase is incubated with the secondary antibody (e.g. anti-human IgG, anti-mouse IgG) and the respective label (fluorescent, chemiluminescent, enzymatic, etc.) is detected; - using a labeled antibody (e.g. labeled AK1967) as a competitor for binding to the solid phase; - The binding affinity is quantified by the reduction in signal.

[0080] As another method to determine the affinity of an antibody for DPP3, the kinetics of binding of DPP3 to immobilized antibody can be determined by label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibody can be performed using anti-mouse Fc antibodies covalently coupled at high density to the surface of a CM5 sensor (Mouse Antibody Capture Kit; GE Healthcare) (Lorenz et al., 2011 Antimicrob Agents Chemother. 55(1):165-173).

[0081] One embodiment of the present invention is a combination of an angiotensin receptor agonist and / or precursor thereof with an inhibitor of DPP3 for use in treating a disease in a subject, said inhibitor of DPP3 being a monospecific antibody or fragment or scaffold. In one embodiment, said inhibitor of DPP3 is a monoclonal antibody or fragment or scaffold.

[0082] Monospecific antibodies or fragments or non-Ig scaffolds according to the present invention are antibodies or fragments or non-Ig scaffolds that all have affinity for the same antigen. Although monoclonal antibodies are monospecific, monospecific antibodies can be generated by means other than from a common germline.

[0083] In a particular embodiment, said capture binders that bind full-length DPP3 specifically inhibit DPP3 activity by less than 50%, preferably less than 40%, more preferably less than 30% in a solution-phase assay. See above for the definition of solution-phase assay. In a particular embodiment for preventing inhibition of DPP3, the capture binder should not bind to DPP3 in the region around the active center and substrate binding region (amino acids 316-669 of SEQ ID NO: 1).

[0084] One embodiment of the invention is a combination of an angiotensin receptor agonist and / or precursor thereof with an inhibitor of DPP3 for use in treating a disease in a subject, said inhibitor of DPP3 being an antibody or fragment or scaffold that binds to full-length DPP3 and inhibits the activity of DPP3 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%.

[0085] The inhibition of DPP3 activity by binders in a solution phase assay can be determined as follows: in a solution phase assay, potential DPP3 capture binders are incubated with recombinant DPP3 or purified native DPP3 and a specific DPP3 substrate. It is preferable to select the capture binder for ECA with the least inhibitory capacity. The capture binder should inhibit DPP3 activity by less than 50%, preferably less than 40%, preferably less than 30%. A special solution phase DPP3 activity assay for determining the inhibitory capacity of potential DPP3 capture binders includes the following steps: · 25 ng / ml native human DPP3 was incubated with 5 μg / ml of each capture binder and buffer control in 50 mM Tris-HCl, pH 7.5 and 100 μM ZnCl2 for 1 h at room temperature. Add the fluorescent substrate Arg-Arg-βNA (20 μl, 2 mM). Incubate at 37°C and monitor the production of free βNA over 1 h in a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH). βNA fluorescence is detected by exciting at 340 nm and measuring emission at 410 nm. The slope of the fluorescence increase (in RFU / min) of the different samples is calculated. The slope of native human DPP3 with the buffer control is taken as 100% activity. The inhibitory activity of a possible capture binder is defined as the reduction (in %) of native human DPP3 activity by incubation with said capture binder.

[0086] In the liquid phase assay, the body fluid sample is directly exposed to a fluorescent substrate (e.g., Arg-Arg-β-NA). Since many different aminopeptidases are present in plasma (Sanderink et al., 1988), the substrate used will be cleaved by peptidases other than DPP3. To circumvent this problem, one preferred method for detecting specific DPP3 activity is the use of an enzyme capture activity assay.

[0087] In one particular embodiment, a method for determining active DPP3 in an enzyme capture assay comprises the steps of: contacting the sample with a capture binder that binds full-length DPP3 but inhibits DPP3 activity by less than 50%, preferably less than 40%, more preferably less than 30% in a solution phase assay. To prevent inhibition of DPP3, the capture binder should not bind DPP3 in the region surrounding the active center and substrate binding region (amino acids 316-669 of SEQ ID NO: 1). separating the DPP3 bound to the capture binder from the body fluid sample; adding a DPP3 substrate to the isolated DPP3; Quantifying DPP3 activity by measuring the conversion of a substrate of DPP3.

[0088] For purposes of the present invention, an "antibody" is a protein (including one or more polypeptides) substantially encoded by immunoglobulin genes that specifically binds to an antigen. Known immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma (IgG1, IgG2, IgG3, IgG4), delta (IgD), epsilon (IgE), and mu (IgM) constant region genes, as well as a number of immunoglobulin variable region genes. Full-length immunoglobulin light chains are generally about 25 kDa, or 214 amino acids in length.

[0089] Full-length immunoglobulin heavy chains are generally about 50 kDa, or 446 amino acids in length. Light chains are encoded by a variable region gene at the NH2-terminus (about 110 amino acids in length) and a kappa or lambda constant region gene at the COOH-terminus. Heavy chains are similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.

[0090] The basic structural unit of an antibody is generally a tetramer of two identical immunoglobulin chain pairs, each pair having one light and one heavy chain, in which the light and heavy chain variable regions bind antigen and the constant regions mediate effector functions. Immunoglobulins exist in a variety of alternative forms, such as Fv, Fab, and F(ab')2, as well as bifunctional hybrid antibodies and single-chain antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17:105, 1987; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; Bird et al., Science 242:423-426, 1988; Hood et al., Immunology, Benjamin, New York, 2nd ed., 1984; Hunkapiller and Hood, Nature 323:15-16, 1986).

[0091] The light or heavy chain variable region of an immunoglobulin contains one framework region separated by three hypervariable regions, also called complementarity determining regions (CDRs) (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., U.S. Department of Health and Human Services, 1983). As noted above, the CDRs are primarily responsible for binding to an epitope of an antigen. The immune complex is an antibody (e.g., a monoclonal, chimeric, humanized, or human antibody) or a functional antibody fragment that specifically binds to an antigen.

[0092] A "chimeric antibody" is an antibody whose light and heavy chain genes are constructed by genetic engineering from immunoglobulin variable and constant region genes, typically belonging to different species. For example, the variable section of the genes from a mouse monoclonal antibody can be joined to human constant sections (such as kappa and gamma 1 or gamma 3). In one example, a therapeutic chimeric antibody is thus a hybrid protein consisting of the variable or antigen binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species can be used or the variable regions can be generated by molecular techniques. Methods for making chimeric antibodies are well known in the art, see for example U.S. Pat. No. 5,807,715. A "humanized" immunoglobulin is an immunoglobulin that contains a human framework region and one or more CDRs from a non-human (e.g. mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor".

[0093] In one embodiment of the invention, all CDRs in a humanized immunoglobulin are derived from a donor immunoglobulin. Constant regions may be absent, but if present, should be substantially identical to human immunoglobulin constant regions, i.e. at least about 85-90% (e.g., about 95% or more) identical. Thus, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to the corresponding parts of a natural humanized immunoglobulin sequence.

[0094] A "humanized antibody" according to the present invention is an antibody that comprises a humanized light chain immunoglobulin and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or humanized antibody can have a limited number of substitutions with amino acids taken from the donor framework. A humanized or other monoclonal antibody can have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are 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, for example, U.S. Pat. No. 5,585,089). A human antibody is an antibody whose light and heavy chain genes are of human origin. Human antibodies can be generated using methods known in the art. Human antibodies can be produced by immortalizing human B cells secreting the antibody of interest. Immortalization can be achieved, for example, by infection with EBV or fusing human B cells with myeloma cells or hybridoma cells to generate trioma cells. Human antibodies can also be produced by phage display (see, for example, Dower et al., PCT Publication No. WO 91 / 17271; McCafferty et al., PCT Publication No. WO 92 / 001047; and Winter, PCT Publication No. WO 92 / 20791) or by selecting from human combinatorial monoclonal antibody libraries (see Morphosys website). Human antibodies can also be prepared by using transgenic animals carrying human immunoglobulin genes (see, e.g., Lonberg et al., PCT Publication No. WO 93 / 12227; and Kucherlapati, PCT Publication No. WO 91 / 10741).

[0095] Thus, the anti-DPP3 antibody or anti-DPP3 antibody fragment according to the present invention can have any format known in the art, including, but not limited to, a human antibody, a monoclonal antibody, a humanized antibody, a chimeric antibody, a CDR-grafted antibody, or an antibody fragment thereof.

[0096] In a particular embodiment of the present invention, the anti-DPP3 antibody is a monoclonal antibody or a fragment thereof. In one embodiment of the present invention, the anti-DPP3 antibody or anti-DPP3 antibody fragment is or is derived from a human or humanized antibody. In a particular embodiment, one or more (murine) CDRs are grafted onto a human antibody or human antibody fragment.

[0097] In a preferred embodiment, the antibodies of the invention are recombinantly produced antibodies (e.g., IgG, a typical full-length immunoglobulin) or antibody fragments containing at least the F variable domains of the heavy and / or light chains (e.g., chemically conjugated antibodies (fragment antigen binding). Non-limiting examples of chemically conjugated antibodies include Fab fragments (including Fab minibodies), single chain Fab antibodies, monovalent Fab antibodies with epitope tags (e.g., Fab-V5Sx2); bivalent Fab dimerized with a CH3 domain (mini antibodies); and antibodies with heterologous domain support (e.g., Fab-V5Sx2). bivalent or multivalent Fab formed by multimerizing the dHLX domains (e.g. by dimerization of dHLX domains) using only the Fab domains (e.g. Fab-dHLX-FSx2); F(ab')2 fragments, scFv fragments, multimerized multivalent and / or multispecific scFv fragments, bivalent and / or bispecific diabodies, BITE® (bispecific T cell engagers), trifunctional antibodies, multivalent antibodies (e.g. antibodies derived from classes different from G); single domain antibodies (e.g. nanobodies derived from camelid or fish immunoglobulins) and many others.

[0098] In addition to anti-DPP3 antibodies or anti-DPP3 antibody fragments, other biopolymer scaffolds (so-called non-Ig scaffolds) are known to be complexed with target molecules and have been used to generate highly target-specific biopolymers, examples of which are aptamers, spiegelmers, anticullins, and conotoxins.

[0099] A non-Ig scaffold in the context of the present invention can be a protein scaffold and can be used as an antibody mimic, since the non-Ig scaffold can bind a ligand or antigen. Non-Ig scaffolds include tetranectin-based non-Ig scaffolds (e.g., as described in U.S. Patent Application Publication No. 2010 / 0028995), fibronectin scaffolds (e.g., as described in EP 1266 025); lipocalin-based scaffolds (e.g., as described in WO 2011 / 154420); ubiquitin scaffolds (e.g., as described in WO 2011 / 073214), transferring scaffolds (e.g., as described in U.S. Patent Application Publication No. 2004 / 0023334), protein A scaffolds (e.g., as described in EP 2231860), ankyrin repeat-based scaffolds (e.g., as described in WO 2010 / 060748), microprotein (preferably cystine-knot forming microprotein) scaffolds (e.g., as described in EP 2314308), Fyn SH3 domain-based scaffolds (e.g., as described in WO 2011 / 023685), EGFR-A domain based scaffolds (e.g. as described in WO 2005 / 040229), and Kunitz domain based scaffolds (e.g. as described in EP 1941867). Non-Ig scaffolds can be peptide or oligonucleotide aptamers. Aptamers are usually generated by selection from a large pool of random sequences and are short strands of oligonucleotides (DNA, RNA, or XNA; Xu et al. 2010, Deng et al. 2014) or short variable peptide domains attached to a protein scaffold (Li et al. 2011).

[0100] In an alternative embodiment, the format of the anti-DPP3 antibody is selected from the group comprising Fv fragments, scFv fragments, Fab fragments, scFab fragments, F(ab)2 fragments, and scFv-Fc fusion proteins. In another preferred embodiment, the format of the antibody is selected from the group comprising scFab fragments, Fab fragments, scFv fragments, and conjugates thereof optimized for bioavailability (such as PEGylated fragments).

[0101] In the context of the present invention, the term "antibody" generally includes monoclonal and polyclonal antibodies and their binding fragments (especially Fc fragments), as well as so-called "single-chain antibodies" (Bird et al. 1988), chimeric antibodies, humanized antibodies (especially CDR-grafted antibodies), and di- or tetrabodies (Holliger et al. 1993). It also includes immunoglobulin-like proteins that are selected, for example through techniques including phage display, and that specifically bind to a molecule of interest contained in a sample. In this context, the expression "specifically binds" refers to an antibody that is raised against the molecule of interest or a fragment thereof. An antibody is considered specific if its affinity for the molecule of interest or said fragment thereof is preferably at least 50 times greater, more preferably 100 times greater, and most preferably at least 1000 times greater, than for other molecules contained in the sample containing the molecule of interest. Methods for generating antibodies and selecting antibodies with a given specificity are well known in the art.

[0102] In a particular embodiment of the present invention, the anti-DPP3 antibody or anti-DPP3 antibody fragment that binds to the epitope of SEQ ID NO: 2 contained in the DPP3 protein or functional derivative thereof is a monoclonal antibody or a monoclonal antibody fragment thereof. In one embodiment of the present invention, the anti-DPP3 antibody or anti-DPP3 antibody fragment that binds to the epitope of SEQ ID NO: 2 contained in the DPP3 protein or functional derivative thereof is a human antibody, or a humanized antibody, or an antibody derived from that antibody, or a humanized antibody fragment, or a fragment derived from that humanized antibody fragment.

[0103] In a particular embodiment, one or more (murine) CDRs are grafted onto a human antibody or antibody fragment.

[0104] In another aspect of the present invention, the subject matter provided is a human CDR-grafted anti-DPP3 antibody, or an anti-DPP3 antibody fragment thereof, directed against and binding to the epitope of SEQ ID NO: 2 contained in a DPP3 protein or a functional derivative thereof, the human CDR-grafted anti-DPP3 antibody, or an anti-DPP3 antibody fragment thereof, comprising an antibody heavy chain variable region (H chain) comprising SEQ ID NO: 4 and / or further comprising an antibody light chain variable region (L chain) comprising SEQ ID NO: 5.

[0105] A further subject of the present invention in another aspect is a human CDR-grafted anti-DPP3 antibody or an anti-DPP3 antibody fragment thereof, directed against and binding to the epitope of SEQ ID NO: 2 contained in a DPP3 protein or a functional derivative thereof, said human CDR-grafted anti-DPP3 antibody or an anti-DPP3 antibody fragment comprising an antibody heavy chain variable region (H chain) comprising SEQ ID NO: 11 and / or further comprising an antibody light chain variable region (L chain) comprising SEQ ID NO: 12.

[0106] In one particular embodiment of the invention, the subject of the invention is a human monoclonal anti-DPP3 antibody or a monoclonal anti-DPP3 antibody fragment directed against and binding to the epitope of SEQ ID NO: 2 contained in the DPP3 protein or a functional derivative thereof, wherein the heavy chain comprises at least one CDR of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 and the light chain comprises at least one CDR of SEQ ID NO: 9, KVS or SEQ ID NO: 10.

[0107] The amount of DPP3 protein and / or DPP3 activity in a body fluid sample from said subject can be determined by a variety of methods (eg, immunoassays, activity assays, mass spectrometry, etc.).

[0108] The amount of DPP3 protein and / or DPP3 activity in a body fluid sample from said subject can be determined, for example, by one of the following methods.

[0109] 1. Fluorescence immunoassay (LIA) to quantify DPP3 protein concentration (Rehfeld et al., JALM, 2018)

[0110] The LIA is a one-step chemiluminescence sandwich immunoassay that uses high-binding polystyrene microtiter plates as solid phase. These 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 per well. 20 μl of sample (e.g. serum, heparin-plasma, citrate-plasma, or EDTA-plasma from the patient's blood) and calibrators are pipetted into the coated white microtiter plate. After addition of the tracer antibody AK2553, the microtiter plate is incubated at room temperature and 600 rpm for 3 hours. Unbound tracer is then removed by 4 washing steps (350 μl per well). Residual chemiluminescence is measured for 1 second per well using a microtiter plate luminometer. The concentration of DPP3 is determined using a 6-point calibration curve. Calibrators and samples are preferably run in duplicate.

[0111] 2. Enzyme Capture Activity Assay (ECA) to Quantify DPP3 Activity (Rehfeld et al., JALM, 2018)

[0112] ECA is a DPP3 specific activity assay that uses black high-binding polystyrene microtiter plates as solid phase. These plates are coated with monoclonal anti-DPP3 antibody AK2555 (capture antibody). 20 μl of sample (e.g. serum, heparin-plasma, citrate-plasma, EDTA-plasma, cerebrospinal fluid, and urine from patient blood) and calibrators are pipetted into the coated black microtiter plates. After addition of assay buffer (200 μl), the microtiter plates are incubated for 2 h at 600 rpm at 22°C. DPP3 present in the samples is immobilized by binding to the capture antibody. Unbound components of the samples are removed by 4 washing steps (350 μl per well). The specific activity of the immobilized DPP3 is measured by adding the fluorescent substrate Arg-Arg-β-naphthylamide (Arg2-βNA) in the reaction buffer, followed by incubation for 1 h at 37°C. DPP3 specifically cleaves Arg2-βNA into Arg-Arg dipeptide and fluorescent β-naphthylamine. Fluorescence is measured using a fluorometer with an excitation wavelength of 340 nm and emission detected at 410 nm. DPP3 activity is determined using a six-point calibration curve. Calibrators and samples are preferably measured in duplicate.

[0113] 3. Liquid-phase assay (LAA) for quantifying DPP3 activity (modified from Jones et al., Analytical Biochemistry, 1982)

[0114] LAA is a liquid-phase assay that measures DPP3 activity using black non-binding polystyrene microtiter plates. 20 μl of sample (e.g. serum, heparin-plasma, citrate-plasma) and calibrators are pipetted into coated black non-binding microtiter plates. After addition of the fluorescent substrate Arg2-βNA in assay buffer (200 μl), the initial βNA fluorescence (T=0) is measured using a fluorometer with an excitation wavelength of 340 nm and emission detected at 410 nm. The plate is then incubated for 1 h at 37°C. The final fluorescence (T=60) is measured. The difference between the initial and final fluorescence is calculated. The activity of DPP3 is determined using a 6-point calibration curve. Calibrators and samples are preferably measured in duplicate.

[0115] The above cut-off values ​​may be different for other assays if they are calibrated differently from the assay system used in the present invention. The above cut-off values ​​will therefore be adapted to such differently calibrated assays, taking into account the differences in calibration. One possibility to quantify the calibration difference is to perform a method comparison analysis (correlation) of the assay in question and each biomarker assay used in the present invention by measuring each biomarker (e.g. DPP3) in samples used in both methods. Another possibility is to determine the median biomarker level of each normal population using the assay in question (assuming that this test has sufficient analytical sensitivity), compare the results with the median biomarker levels reported in the literature, and recalculate the calibration value based on the difference obtained by this comparison. Using the calibration value used in the present invention, samples from normal (healthy) subjects were measured, and the median plasma DPP3 (interquartile range) was 24.6 ng / ml (19.2 ng / ml to 32.4 ng / ml).

[0116] A wide variety of immunoassays are known and can be utilized in the assays and methods of the present invention, including radioimmunoassays ("RIA"), homogeneous enzyme multiplexed immunoassays ("EMIT"), enzyme-linked immunosorbent assays ("ELISA"), apoenzyme reactivation immunoassays ("ARIS"), chemiluminescent and fluorescent immunoassays, Luminex-based bead assays, protein microarray assays, and rapid test formats such as immunochromatographic strip tests ("dipstick immunoassays"), and immunochromatographic assays.

[0117] In one embodiment of the invention, such an assay is a sandwich immunoassay utilizing any type of detection technology (non-limiting examples of which include enzyme labeling, chemiluminescent labeling, electrochemiluminescent labeling), preferably a fully automated assay. In one embodiment of the invention, such an assay is an enzyme-labeled sandwich assay. Examples of automated or fully automated assays include assays available for one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®.

[0118] In one embodiment of the invention, the assay can be a so-called point-of-care (POC) test, which is a testing technique that allows the test to be performed close to the patient within an hour without the need for a fully automated assay system. An example of this technique is the immunochromatographic testing technique.

[0119] In one embodiment of the invention, at least one of the binders is labeled to allow detection.

[0120] In a preferred embodiment, the label is selected from the group comprising a chemiluminescent label, an enzyme label, a fluorescent label, a radioactive iodine label.

[0121] The assays can be homogeneous or heterogeneous, competitive and non-competitive. 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, e.g., with a dye, a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody that binds to the analyte is then measured by an appropriate method. The general format and procedures involved in "sandwich assays" are well defined and known to those skilled in the art (The Immunoassay Handbook, edited by David Wild, Elsevier LTD, Oxford; 3rd Edition (May 2005); ISBN-13: 978-0080445267; Hultschig C et al., Curr Opin Chem Biol. February 2006; 10(1):4-10; PMID: 16376134).

[0122] In another embodiment, the assay comprises two capture molecules, preferably antibodies, both of which are present in dispersion in a liquid reaction mixture, in which a first label component is part of a fluorescence- or chemiluminescence-quenching or amplification-based labeling system attached to the first capture molecule, and a second label component of said labeling system attached to the second capture molecule, thereby allowing detection of the sandwich complex formed in the sample-containing solution by generating a measurable signal when both capture molecules bind to the analyte.

[0123] In another embodiment, the labeling system comprises a rare earth cryptate or rare earth chelate in combination with a fluorescent or chemiluminescent dye, particularly a cyanine type dye.

[0124] In the context of the present invention, a fluorescence-based assay involves the use of dyes, such as FAM (5-carboxyfluorescein or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes (CY3, CY5, CY3.5, CY5.5, Cy7, etc.), xanthene, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HE X), TET, 6-carboxy-4',5'-dichloro-2',7'-dimethodifluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes (such as BODIPY TMR), Oregon Green, coumarins (such as umbelliferone), benzimides (such as Hoechst 33258); phenanthridines (such as Texas Red), Yakima Yellow, Alexa Fluor, PET, ethidium bromide, acridinium dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, and the like.

[0125] In the context of the present invention, chemiluminescence-based assays include the use of dyes based on physical principles. The physical principles of chemiluminescent materials are described in Kirk-Othmer, Encyclopedia of chemical technology, 4th Edition, Editor in Chief JI Kroschwitz; Editor M. Howe-Grant, John Wiley & Sons, Inc., 1993, Vol. 15, pp. 518-562 (hereby incorporated by reference, including the citations on pp. 551-562). Preferred chemiluminescent dyes are acridinium esters.

[0126] As used herein, an "assay" or "diagnostic assay" can be of any type used in the field of diagnostics. Such an assay can be based on the binding of the analyte to be detected to one or more capture probes with a given affinity. The affinity constant for the interaction between the capture molecule and the target molecule or molecule of interest is 10 8 M -1 It is preferable that it is greater than .

[0127] DPP3 activity can be measured by detecting the cleavage products of DPP3-specific substrates. Known peptide hormone substrates include Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH (adrenocorticotropic hormone), and MSH (melanocyte-stimulating hormone; Abramic et al. 2000, Barsun et al. 2007, Dhanda et al. 2008). Cleavage of the mentioned peptide hormones, as well as other oligopeptides without tags (e.g., Ala-Ala-Ala-Ala, Dhanda et al. 2008), can be monitored by detecting the respective cleavage products. Non-limiting examples of detection methods include HPLC analysis (e.g., Lee and Snyder 1982), mass spectrometry (e.g., Abramic et al. 2000), H-lysine hydrochloride (HLA), and glycerol (HLA). 1 -NMR analysis (e.g., Vandenberg et al. 1985), capillary zone electrophoresis (CE; e.g., Barsun et al. 2007), thin-layer chromatography (e.g., Dhanda et al. 2008), or reversed-phase chromatography (e.g., Mazocco et al. 2006).

[0128] Detection of fluorescence resulting from hydrolysis of fluorescent substrates by DPP3 is a standard procedure for monitoring DPP3 activity. These substrates are special di- 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) that couple to fluorophores. Non-limiting examples of fluorophores include β-naphthylamide (2-naphthylamide, βNA, 2NA), 4-methoxy-β-naphthylamide (4-methoxy-2-naphthylamide), and 7-amido-4-methylcoumarin (AMC, MCA; Abramic et al. 2000, Ohkubo et al. 1999). Cleavage of these fluorescent substrates releases fluorescent β-naphthylamide or 7-amido-4-methylcoumarin, respectively. In the liquid-phase assay or ECA, the 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). In addition, samples containing DPP3 can be immobilized on the surface of a gel and resolved by electrophoresis, the gel can be stained with a fluorescent substrate (e.g., Arg-Arg-βNA) and Fast Garnet GBC, and the fluorescent protein bands can be detected by a fluorescence reader (Ohkubo et al. 1999). The same peptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) can be coupled to a chromophore (such as p-nitroanilide). Detection of the color change resulting from hydrolysis of the chromogenic substrate can be used to monitor DPP3 activity.

[0129] Another option for detecting DPP3 activity is the Protease-Glo™ assay (commercially available from Promega). In this embodiment of the method, DPP3-specific di- or tripeptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) are coupled to aminoluciferin. Upon cleavage by DPP3, aminoluciferin is released and serves as a substrate for the reaction of the bound luciferase, releasing detectable luminescence.

[0130] In a preferred embodiment, DPP3 activity is measured by adding the fluorescent substrate Arg-Arg-βNA and monitoring the fluorescence in real time.

[0131] In one particular embodiment of the method for determining active DPP3 in a body fluid sample of a subject, the capture binder that reacts with DPP3 is immobilized on the surface of a solid phase.

[0132] A test sample is passed over the immobile binder, where DPP3, if present, binds to the binder and is itself immobilized and detected. Substrate is then added and a reaction product can be detected, which indicates the presence or amount of DPP3 in the test sample. For purposes herein, the term "solid phase" can include any material or vessel in or on which an assay can be performed, including non-limiting examples of porous or non-porous materials, test tubes, wells, slides, agarose resin (e.g., Sepharose from GE Healthcare Life Sciences), magnetic particles (e.g., Dynabeads™ or Pierce™ magnetic beads from Thermo Fisher Scientific), and the like.

[0133] Proteins or binders of protein origin (e.g. antibodies, antibody fragments, non-Ig scaffolds) can be bound to the nucleosomes by physical adsorption (e.g. by electrostatic or hydrophobic interactions), bioaffinity immobilization (e.g. avidin-biotin, protein A / G / L, His-tag, Ni 2+ The binders can be immobilized on the solid surface by methods including binding to nucleotides (e.g., nucleotides with nucleotides such as α-NTA, GST tags and glutathione, DNA hybridization, aptamers), covalent bonds (e.g., amines and N-hydroxysuccinimide), or a combination of the above immobilization methods (Kim and Herr 2013). Oligonucleotide-based binders (e.g., aptamers) can be immobilized on the solid surface using the (strept)avidin-biotin system (Muller et al. 2012; Deng et al. 2014).

[0134] In a particular embodiment of the method for determining the activity of DPP3 in a body fluid sample of a subject, the separation step is a washing step that removes components of the sample that are not bound to the capture binder from the captured DPP3, although the separation step can be any other step that separates DPP3 bound to the capture binder from components of the body fluid sample.

[0135] In a particular embodiment of the method for determining the activity of DPP3 in a body fluid sample of a subject, the conversion of the DPP3 substrate by immobilized DPP3 is measured by a method selected from the group including fluorescence of a fluorescent substrate (e.g., Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, fluorescence of a substrate bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis followed by activity staining (immobilized active DPP3), or Western blot (cleavage products).

[0136] In a particular embodiment of the method for determining the activity of DPP3 in a body fluid sample of a subject, the substrate can be selected from the group including Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or di- and tripeptides linked to a fluorophore, chromophore or aminoluciferin (Promega's Protease-Glo™ assay). Non-limiting examples of di- or tripeptides cleaved by DPP3 include 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. Non-limiting examples of fluorophores include β-naphthylamide (2-naphthylamide, βNA, 2NA), 4-methoxy-β-naphthylamide (4-methoxy-2-naphthylamide), and 7-amido-4-methylcoumarin (AMC, MCA; Abramic et al. 2000; Ohkubo et al. 1999). Cleavage of these fluorogenic substrates releases fluorescent β-naphthylamide or 7-amido-4-methylcoumarin, respectively. Non-limiting examples of chromophores include p-nitroanilide diacetate (pNA). Hydrolysis of the peptide-pNA bond in the chromogenic substrate releases pNA, which in turn changes color. Thus, the change in absorbance (DA / min) is directly proportional to the enzyme activity. Using the Protease-Glo™ assay from Promega, cleavage by DPP3 releases aminoluciferin, which serves as a substrate for reaction with bound luciferase, releasing detectable luminescence.

[0137] One embodiment of the present invention is an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; Quantifying the DPP3 activity by measuring and quantifying the conversion of a substrate of DPP3.

[0138] One embodiment of the present invention is an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject, the method of determining DPP3 activity in a body fluid sample of said subject comprising the steps of: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; Quantifying the DPP3 activity by measuring and quantifying the conversion of a substrate of DPP3.

[0139] One embodiment of the present invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder may be selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

[0140] One embodiment of the present invention is an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

[0141] One embodiment of the present invention is an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being immobilized on a surface.

[0142] One embodiment of the present invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject using the method described above, said separation step being a washing step in which components of the sample not bound to the capture binder are removed from the captured DPP3.

[0143] One embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof.

[0144] One embodiment of the invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonist and / or precursors thereof, said angiotensin receptor agonist and / or precursors thereof being selected from the group comprising angiotensin I, angiotensin II, angiotensin III, angiotensin IV as outlined above.

[0145] One embodiment of the present invention is the use of a method or assay or a kit for determining the amount of total DPP3 protein and / or the amount of active DPP3 in a body fluid sample of said subject as a treatment guide and / or treatment monitoring for treatment with an angiotensin receptor agonist and / or its precursors, wherein said amount of DPP3 protein and / or said DPP3 activity has been determined at least once in a body fluid sample of said subject before and / or during treatment with said angiotensin receptor agonist and / or its precursors.

[0146] One embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, said body fluid sample of said subject being selected from whole blood, plasma and serum.

[0147] One embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or Quantifying the amount of the DPP3 protein.

[0148] One embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, said method for determining active DPP3 in a body fluid sample of said subject comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3, and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; Quantifying the amount of the DPP3 protein.

[0149] One embodiment of the present invention is the use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds full length DPP3, said capture binder can be selected from the group of antibodies, antibody fragments or non-IgG scaffolds.

[0150] One embodiment of the present invention is the use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or their precursors, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

[0151] One embodiment of the present invention is the use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or their precursors, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being immobilized on a surface.

[0152] One embodiment of the present invention is the use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of the subject as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or their precursors, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step in which components of the sample that are not bound to the capture binder are removed from the captured DPP3.

[0153] A subject of the present invention is also the use of a method or assay or a kit for determining the amount of DPP3 protein and / or the DPP3 activity in a body fluid sample of said subject, intended to predict the risk of an adverse event in said subject, said method comprising: determining the level of DPP3 in a body fluid sample from said subject; comparing the determined level of DPP3 to a predefined threshold; correlating said level of DPP3 with risk of an adverse event in the subject, wherein elevated levels above a certain threshold are predictive of increased risk of the adverse event.

[0154] In the context of the present invention, the term prognosis refers to a prediction of how a patient's condition will progress, which may include a prediction of the likelihood of recovery or of adverse events for said patient.

[0155] Adverse events are defined herein as organ dysfunction, or worsening organ function, or death.

[0156] In the context of the present invention, the subject has a disease selected from the group consisting of: subjects at increased risk of developing sepsis, subjects suspected of having sepsis, SIRS, sepsis, shock (e.g. septic shock), acute and chronic vascular diseases (e.g. acute heart failure, myocardial infarction, stroke, etc.); organ dysfunction of at least one organ, fluid imbalance, and hypotension.

[0157] In a preferred embodiment, the adverse event is organ dysfunction or deterioration of organ function and the subject has a disease selected from the group consisting of a subject at increased risk of developing sepsis, a subject suspected of having sepsis, SIRS, sepsis, and shock (e.g., septic shock).

[0158] As used herein, organ dysfunction refers to a state or health condition in which an organ does not perform the expected function. "Organ failure" refers to an organ dysfunction to the extent that normal homeostasis cannot be maintained without external clinical intervention. The organ failure may involve an organ selected from the group including kidney, liver, heart, lungs, and nervous system. In contrast, organ function refers to the expected function of each organ within physiological ranges. A person skilled in the art is aware of the respective functions of the organs during medical examination.

[0159] Organ dysfunction can be defined by a set of organ failure assessment scores (SOFA scores) or their components. The SOFA score, previously known as the Sepsis-Associated Organ Failure Assessment score (Singer et al., 2016 JAMA 315(8):801-810), is used to track a person's condition during their stay in the intensive care unit (ICU) and to determine the extent of organ function or the rate of failure. This score is based on six different scores, one each related to the respiratory, cardiovascular, hepatic, coagulation, renal, and neurological systems. Each system is given a score from 0 to 4, with higher scores reflecting worsening organ dysfunction. Criteria for assessing the SOFA score are described, for example, by Lambden et al. (for review, see Lambden et al., 2019 Critical Care 23:374). The SOFA score can be calculated traditionally at the time of admission to the ICU and every 24 hours thereafter.

[0160] In particular, said organ dysfunction is selected from the group comprising renal impairment, cardiac dysfunction, hepatic dysfunction or respiratory tract dysfunction.

[0161] In one aspect of the present invention, the death is defined as death after a specified time has elapsed (e.g., after 24 hours to 3 months, preferably after 24 hours to 2 months, more preferably after 24 hours to 30 days, even more preferably after 24 hours to 28 days, even more preferably after 24 hours to 14 days, even more preferably after 24 hours to 7 days, even more preferably after 24 hours to 5 days, even more preferably after 24 hours to 3 days, and most preferably after 24 hours to 48 hours).

[0162] Another particular embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of an adverse event in said subject, said body fluid sample of said subject being selected from whole blood, plasma and serum.

[0163] One embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of an adverse event in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or - Quantifying the amount of DPP3 protein.

[0164] Another embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject for predicting the risk of an adverse event in said subject, the method for determining DPP3 activity in a body fluid sample of a subject comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; Quantifying the DPP3 activity by measuring and quantifying the conversion of a substrate of DPP3.

[0165] One embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for the purpose of predicting the risk of an adverse event in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder can be selected from the group of antibodies, antibody fragments or non-IgG scaffolds.

[0166] Another embodiment of the present invention is the use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for the purpose of predicting the risk of an adverse event in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

[0167] One embodiment of the present invention is the use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for the purpose of predicting the risk of an adverse event in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being immobilized on a surface.

[0168] Another embodiment of the present invention is the use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for the purpose of predicting the risk of an adverse event in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, and said separation step is a washing step in which components of the sample that are not bound to the capture binder are removed from the captured DPP3.

[0169] Another particular embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of an adverse event in said subject, wherein DPP3 activity is determined in a body fluid sample of said subject and conversion of a DPP3 substrate is detected by a method selected from the group comprising: fluorescence of a fluorescent substrate (e.g. Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3) or Western blot (cleavage products).

[0170] Another embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of an adverse event in said subject, wherein DPP3 activity is determined in a body fluid sample of said subject, and said substrate can be selected from the group comprising angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that said dipeptide is Arg-Arg.

[0171] Another embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of an adverse event in said subject, wherein DPP3 activity is determined in a body fluid sample of said subject, and said substrate can be selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, whereby said dipeptide is Arg-Arg.

[0172] Another embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of organ dysfunction or worsening organ function, said method comprising: determining the level of DPP3 in a body fluid sample from said subject; comparing the determined level of DPP3 to a predefined threshold; correlating said level of DPP3 with risk of an adverse event in the subject, wherein elevated levels above a certain threshold are predictive of increased risk of organ dysfunction or deterioration of organ function.

[0173] Another particular embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of organ dysfunction or worsening organ function in said subject, said body fluid sample of said subject being selected from whole blood, plasma and serum.

[0174] One embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of organ dysfunction or worsening organ function in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or Quantifying the amount of the DPP3 protein.

[0175] Another embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of organ dysfunction or organ function deterioration in said subject, the method for determining DPP3 activity in a body fluid sample of said subject comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; Quantifying the DPP3 activity by measuring and quantifying the conversion of a substrate of DPP3.

[0176] Another embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of organ dysfunction or worsening organ function in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder can be selected from the group of antibodies, antibody fragments or non-IgG scaffolds.

[0177] Another embodiment of the present invention is the use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of organ dysfunction or worsening organ function in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

[0178] Another particular embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of organ dysfunction or worsening organ function in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being immobilized on a surface.

[0179] One embodiment of the present invention is the use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or worsening organ function in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, and said separation step is a washing step in which components of the sample that are not bound to the capture binder are removed from the captured DPP3.

[0180] Another embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of organ dysfunction or worsening organ function in said subject, wherein DPP3 activity is determined in a body fluid sample of said subject and conversion of a DPP3 substrate is detected by a method selected from the group comprising: fluorescence of a fluorescent substrate (e.g. Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3) or Western blot (cleavage products).

[0181] Another embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of organ dysfunction or worsening organ function in said subject, wherein DPP3 activity is determined in a body fluid sample of said subject, said substrate may be selected from the group comprising angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that said dipeptide is Arg-Arg.

[0182] One embodiment of the present invention is the use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of organ dysfunction or worsening organ function in said subject, wherein DPP3 activity is determined in a body fluid sample of said subject and the substrate can be selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, whereby said dipeptide is Arg-Arg.

[0183] The subject of the present invention is also a method for predicting the risk of an adverse event in said subject, said method comprising the steps of: determining the level of DPP3 in a body fluid sample from said subject; comparing the determined level of DPP3 to a predefined threshold; correlating said level of DPP3 with risk of an adverse event in the subject, wherein elevated levels above a certain threshold are predictive of increased risk of the adverse event.

[0184] A subject of the present invention is also a method for predicting the risk of an adverse event in said subject.

[0185] Another embodiment of the present invention is a method for predicting risk of an adverse event in said subject, wherein said body fluid sample of said subject is selected from whole blood, plasma, and serum.

[0186] Another particular embodiment of the present invention is a method for predicting the risk of an adverse event in said subject, comprising determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or Quantifying the amount of the DPP3 protein.

[0187] Another embodiment of the invention is a method for predicting the risk of an adverse event in a subject, wherein DPP3 activity is determined in a body fluid sample of the subject, the method comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; Quantifying the DPP3 activity by measuring and quantifying the conversion of a substrate of DPP3.

[0188] Another embodiment of the present invention is a method for predicting the risk of an adverse event in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder can be selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

[0189] Another embodiment of the present invention is a method for predicting the risk of an adverse event in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being an antibody.

[0190] Another embodiment of the present invention is a method for predicting the risk of an adverse event in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being immobilized on a surface.

[0191] Another embodiment of the present invention is a method for predicting the risk of an adverse event in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step in which components of the sample that are not bound to the capture binder are removed from the captured DPP3.

[0192] Another embodiment of the invention is a method for predicting the risk of an adverse event in a subject, wherein DPP3 activity is determined in a body fluid sample of the subject and conversion of a DPP3 substrate is detected by a method selected from the group comprising fluorescence of a fluorescent substrate (e.g. Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3), or Western blot (cleavage products).

[0193] Yet another embodiment of the present invention is a method for predicting the risk of an adverse event in said subject, wherein DPP3 activity is determined in a body fluid sample of said subject, and said substrate can be selected from the group comprising angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that said dipeptide is Arg-Arg.

[0194] One embodiment of the present invention is a method for predicting the risk of an adverse event in a subject, wherein DPP3 activity is determined in a body fluid sample of the subject, and the substrate can be selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, wherein the dipeptide is Arg-Arg.

[0195] One embodiment of the invention is a method for predicting risk of organ dysfunction or worsening organ function in a subject, the method comprising: determining the level of DPP3 in a body fluid sample from said subject; comparing the determined level of DPP3 to a predefined threshold; correlating said level of DPP3 with risk of an adverse event in the subject, wherein elevated levels above a certain threshold are predictive of increased risk of organ dysfunction or deterioration of organ function.

[0196] Another particular embodiment of the present invention is a method for predicting the risk of organ dysfunction or worsening organ function in said subject, wherein said body fluid sample of said subject is selected from whole blood, plasma, and serum.

[0197] One embodiment of the invention is a method for predicting the risk of organ dysfunction or worsening organ function in said subject, comprising determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or Quantifying the amount of the DPP3 protein.

[0198] Another embodiment of the invention is a method for predicting the risk of organ dysfunction or worsening organ function in a subject, wherein DPP3 activity is determined in a body fluid sample of the subject, the method comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; Quantifying the DPP3 activity by measuring and quantifying the conversion of a substrate of DPP3.

[0199] Another particular embodiment of the present invention is a method for predicting the risk of organ dysfunction or worsening organ function in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder can be selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

[0200] Yet another embodiment of the present invention is a method for predicting the risk of organ dysfunction or worsening organ function in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

[0201] One embodiment of the invention is a method for predicting the risk of organ dysfunction or worsening organ function in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being immobilized on a surface.

[0202] Another embodiment of the present invention is a method for predicting the risk of organ dysfunction or worsening organ function in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step in which components of the sample that are not bound to the capture binder are removed from the captured DPP3.

[0203] Another particular embodiment of the present invention is a method for predicting the risk of organ dysfunction or worsening organ function in a subject, wherein DPP3 activity is determined in a body fluid sample of the subject and conversion of a DPP3 substrate is detected by a method selected from the group comprising fluorescence of fluorescent substrates (e.g. Arg-Arg-βNA, Arg-Arg-AMC), color change of chromogenic substrates, luminescence of substrates bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3) or Western blot (cleavage products).

[0204] Another particular embodiment of the present invention is a method for predicting the risk of organ dysfunction or worsening organ function in said subject, wherein DPP3 activity is determined in a body fluid sample of said subject, and said substrate can be selected from the group comprising angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that said dipeptide is Arg-Arg.

[0205] Another embodiment of the present invention is a method for predicting the risk of organ dysfunction or worsening organ function in said subject, wherein DPP3 activity is determined in a body fluid sample of said subject, and said substrate can be selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, wherein said dipeptide is Arg-Arg.

[0206] Yet another embodiment of the present invention is as follows.

[0207] 1. Use of an angiotensin receptor agonist and / or a precursor thereof in the manufacture of a medicament for treating a disease in a subject, comprising: the disease is selected from the group consisting of heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial, viral (e.g. AIDS), parasitic (e.g. malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. stroke, neurodegenerative diseases), autoimmune diseases, vascular diseases, hypotension, and shock; the subject has an amount of DPP3 protein and / or DPP3 activity in a bodily fluid sample that exceeds a predetermined threshold.

[0208] Item 2. Use according to item 1, wherein the angiotensin receptor agonist and / or precursor thereof is selected from the group comprising angiotensin I, angiotensin II, angiotensin III and angiotensin IV, in particular angiotensin II.

[0209] 3. The use according to item 1 or 2, wherein the amount of DPP3 protein and / or the DPP3 activity is determined in a body fluid sample of the subject at least once before and / or during treatment with the angiotensin receptor agonist and / or its precursor.

[0210] The use according to any one of items 1 to 3, wherein the amount of DPP3 protein and / or the DPP3 activity determined in the subject's body fluid sample is used as a therapeutic guide and / or therapeutic monitoring of the treatment with an angiotensin receptor agonist and / or its precursor.

[0211] The use according to any one of items 1 to 4, wherein the subject's body fluid sample is selected from whole blood, plasma, and serum.

[0212] The use according to any one of items 1 to 5, wherein the angiotensin receptor agonist and / or its precursor is administered to the subject in a pharmaceutical formulation.

[0213] Item 7. The use according to item 6, wherein the angiotensin receptor agonist and / or its precursor is angiotensin II acetate and the pharmaceutical formulation is in a dry state to be reconstituted before use.

[0214] The use according to any one of Items 1 to 7, wherein the angiotensin receptor agonist and / or its precursor is angiotensin II administered at a rate of 0.1 to 200 ng / kg / min, preferably 1 to 100 ng / kg / min, more preferably 2 to 80 ng / kg / min, even more preferably 5 to 60 ng / kg / min, even more preferably 10 to 50 ng / kg / min, even more preferably 15 to 40 ng / kg / min, and most preferably 20 ng / kg / min.

[0215] Item 9. The use according to any one of items 1 to 8, wherein the angiotensin receptor agonist and / or its precursor, in particular angiotensin II, is administered in combination with an inhibitor of DPP3.

[0216] 10. The use according to item 9, wherein said inhibitor of DPP3 is selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

[0217] 11. The use according to item 9 or 10, wherein said inhibitor of DPP3 is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to SEQ ID NO: 1, in particular SEQ ID NO: 2.

[0218] The inhibitor of DPP3 has a potency of 10 to 15% against DPP3. -7 12. The use according to any one of items 9 to 11, wherein the antibody or fragment or scaffold exhibits a minimum binding affinity of M or less.

[0219] Item 13. The use according to any one of items 9 to 12, wherein said inhibitor of DPP3 is a monospecific antibody or fragment or scaffold.

[0220] Item 14. The use according to any one of items 9 to 13, wherein the inhibitor of DPP3 is an antibody or fragment or scaffold that binds to full-length DPP3 and inhibits the activity of DPP3 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%.

[0221] The amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample from said subject, contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or The use according to any one of items 1 to 14, further comprising a step of quantifying the amount of the DPP3 protein.

[0222] The method for determining DPP3 activity in a body fluid sample from said subject comprises: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; The use according to any one of items 1 to 15, comprising a step of quantifying the DPP3 activity by measuring and quantifying conversion of a DPP3 substrate.

[0223] 17. The use according to any one of claims 1 to 16, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being selected from the group of an antibody, an antibody fragment, or a non-IgG scaffold.

[0224] Item 18. The use according to any one of items 1 to 17, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and said determining comprises the use of a capture binder that specifically binds to full-length DPP3, and said capture binder is an antibody.

[0225] Item 19. The use according to any one of items 1 to 18, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the determination comprises the use of a capture binder that specifically binds full-length DPP3, and the capture binder is immobilized on a surface.

[0226] 20. The use according to any one of claims 16 to 19, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step in which components of the sample that are not bound to the capture binder are removed from the captured DPP3.

[0227] 21. The use according to any one of claims 16 to 20, wherein DPP3 activity is determined in a body fluid sample of the subject, and conversion of a DPP3 substrate is detected by a method selected from the group comprising fluorescence of a fluorescent substrate (e.g. Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3), or Western blot (cleavage products).

[0228] 22. The use according to any one of claims 16 to 21, wherein DPP3 activity is determined in a body fluid sample of the subject, and the substrate is 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 linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that the dipeptide is Arg-Arg.

[0229] 23. The use according to any one of claims 16 to 22, wherein DPP3 activity is determined in a body fluid sample of the subject, and the substrate is selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, whereby the dipeptide is Arg-Arg.

[0230] 1. A method of treating a disease in a subject, comprising administering to said subject an angiotensin receptor agonist and / or a precursor thereof; the disease is selected from the group consisting of heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial, viral (e.g. AIDS), parasitic (e.g. malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. stroke, neurodegenerative diseases), autoimmune diseases, vascular diseases, hypotension, and shock; said subject having an amount of DPP3 protein and / or DPP3 activity in a bodily fluid sample that exceeds a predetermined threshold.

[0231] Item 2. The method according to item 1, wherein the angiotensin receptor agonist and / or precursor thereof is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, and angiotensin IV, in particular angiotensin II.

[0232] 3. The method according to claim 1 or 2, wherein the amount of DPP3 protein and / or the DPP3 activity is determined in a body fluid sample of the subject at least once before and / or during treatment with the angiotensin receptor agonist and / or its precursor.

[0233] The method according to any one of items 1 to 3, wherein the amount of DPP3 protein and / or DPP3 activity determined in the subject's body fluid sample is used as a therapeutic guide and / or therapeutic monitoring of treatment with an angiotensin receptor agonist and / or its precursor.

[0234] Item 5. The method according to any one of items 1 to 4, wherein the subject's body fluid sample is selected from whole blood, plasma, and serum.

[0235] Item 6. The method according to any one of items 1 to 5, wherein the angiotensin receptor agonist and / or its precursor is administered to the subject in a pharmaceutical formulation.

[0236] Item 7. The method according to item 6, wherein the angiotensin receptor agonist and / or its precursor is angiotensin II acetate and the pharmaceutical formulation is in a dry state to be reconstituted before use.

[0237] The method according to any one of items 1 to 7, wherein the angiotensin receptor agonist and / or its precursor is angiotensin II administered at a rate of 0.1 to 200 ng / kg / min, preferably 1 to 100 ng / kg / min, more preferably 2 to 80 ng / kg / min, even more preferably 5 to 60 ng / kg / min, even more preferably 10 to 50 ng / kg / min, even more preferably 15 to 40 ng / kg / min, and most preferably 20 ng / kg / min.

[0238] Item 9. The method according to any one of items 1 to 8, wherein the angiotensin receptor agonist and / or a precursor thereof, particularly angiotensin II, is administered in combination with an inhibitor of DPP3.

[0239] 10. The method of claim 9, wherein said inhibitor of DPP3 is selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

[0240] 11. The method of claim 9 or 10, wherein said inhibitor of DPP3 is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to SEQ ID NO: 1, in particular SEQ ID NO: 2.

[0241] The inhibitor of DPP3 has a potency of 10 to 15% against DPP3. -7 12. The method of any one of items 9 to 11, wherein the antibody or fragment or scaffold exhibits a minimum binding affinity of M or less.

[0242] Item 13. The method according to any one of items 9 to 12, wherein said inhibitor of DPP3 is a monospecific antibody or fragment or scaffold.

[0243] Item 14. The method according to any one of items 9 to 13, wherein the inhibitor of DPP3 is an antibody or fragment or scaffold that binds to full-length DPP3 and inhibits the activity of DPP3 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%.

[0244] The amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample from said subject, contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or The method according to any one of items 1 to 14, comprising a step of quantifying the amount of the DPP3 protein.

[0245] The method for determining DPP3 activity in a body fluid sample from said subject comprises: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; The method according to any one of items 1 to 15, comprising a step of quantifying the DPP3 activity by measuring and quantifying conversion of a DPP3 substrate.

[0246] 17. The method according to any one of claims 1 to 16, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and said determining comprises the use of a capture binder that specifically binds full-length DPP3, said capture binder being selected from the group of an antibody, an antibody fragment, or a non-IgG scaffold.

[0247] Item 18. The method according to any one of items 1 to 17, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the determination comprises the use of a capture binder that specifically binds to full-length DPP3, and the capture binder is an antibody.

[0248] Item 19. The method according to any one of items 1 to 18, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the determining comprises the use of a capture binder that specifically binds full-length DPP3, and the capture binder is immobilized on a surface.

[0249] 20. The method according to any one of items 16 to 19, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step in which components of the sample that are not bound to the capture binder are removed from the captured DPP3.

[0250] 21. The method according to any one of claims 16 to 20, wherein DPP3 activity is determined in a body fluid sample of the subject, and conversion of a DPP3 substrate 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 bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3), or Western blot (cleavage products).

[0251] 22. The method according to any one of items 16 to 21, wherein DPP3 activity is determined in a body fluid sample of the subject, and the substrate is 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 linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that the dipeptide is Arg-Arg.

[0252] 23. The method according to any one of items 16 to 22, wherein DPP3 activity is determined in a body fluid sample of the subject, and the substrate is selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, whereby the dipeptide is Arg-Arg.

[0253] Yet another embodiment of the present invention is as follows.

[0254] 1. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject, comprising: the disease is selected from the group consisting of heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial, viral (e.g. AIDS), parasitic (e.g. malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. stroke, neurodegenerative diseases), autoimmune diseases, vascular diseases, hypotension, and shock; the subject has an amount of DPP3 protein and / or DPP3 activity in a bodily fluid sample that exceeds a pre-determined threshold, angiotensin receptor agonist and / or precursors thereof.

[0255] 2. The angiotensin receptor agonist and / or its precursor according to paragraph 1 for use in treating a disease in a subject, wherein the angiotensin receptor agonist and / or its precursor is selected from the group including angiotensin I, angiotensin II, angiotensin III, and angiotensin IV, in particular angiotensin II is selected.

[0256] 3. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to paragraph 1 or 2, wherein the amount of DPP3 protein and / or the DPP3 activity has been determined at least once in a body fluid sample of the subject before and / or during treatment with the angiotensin receptor agonist and / or a precursor thereof.

[0257] 4. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of paragraphs 1 to 3, wherein the amount of DPP3 protein and / or the determination of DPP3 activity determined in a body fluid sample of the subject is used as a therapeutic guide and / or therapeutic monitoring of treatment with the angiotensin receptor agonist and / or a precursor thereof.

[0258] 5. An angiotensin receptor agonist and / or a precursor thereof for use in treating a disease in a subject according to any one of items 1 to 4, wherein the subject's body fluid sample is selected from whole blood, plasma, and serum.

[0259] 6. The angiotensin receptor agonist and / or precursor thereof according to any one of items 1 to 5, for use in treating a disease in a subject, wherein the angiotensin receptor agonist and / or precursor thereof is administered to the subject in a pharmaceutical formulation.

[0260] 7. The angiotensin receptor agonist and / or precursor thereof for use in treating a disease in a subject according to item 6, wherein the angiotensin receptor agonist and / or precursor thereof is angiotensin II acetate and the pharmaceutical formulation is in a dry state to be reconstituted before use.

[0261] 8. The angiotensin receptor agonist and / or a precursor thereof for use in treating a disease in a subject according to any one of items 1 to 7, which is angiotensin II administered at a rate of 0.1 to 200 ng / kg / min, preferably 1 to 100 ng / kg / min, more preferably 2 to 80 ng / kg / min, even more preferably 5 to 60 ng / kg / min, even more preferably 10 to 50 ng / kg / min, even more preferably 15 to 40 ng / kg / min, and most preferably 20 ng / kg / min.

[0262] 9. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of items 1 to 8, wherein the angiotensin receptor agonist and / or a precursor thereof, in particular angiotensin II, is administered in combination with an inhibitor of DPP3.

[0263] 10. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to clause 9, wherein said inhibitor of DPP3 is selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

[0264] 11. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to clause 9 or 10, wherein said inhibitor of DPP3 is either an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to SEQ ID NO: 1, in particular SEQ ID NO: 2.

[0265] 12. The inhibitor of DPP3 has an activity against DPP3 of 10 -7 12. The angiotensin receptor agonist and / or precursor thereof for use in treating a disease in a subject according to any one of items 9 to 11, which is an antibody or fragment or scaffold exhibiting a minimum binding affinity of M or less.

[0266] 13. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of clauses 9 to 12, wherein said inhibitor of DPP3 is a monospecific antibody or fragment or a scaffold.

[0267] 14. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of clauses 9 to 13, wherein said inhibitor of DPP3 is an antibody or fragment or scaffold that binds to full-length DPP3 and inhibits the activity of DPP3 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%.

[0268] 15. The amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and is: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or quantitating the amount of the DPP3 protein, Item 15. An angiotensin receptor agonist and / or a precursor thereof for use in treating a disease in a subject according to any one of items 1 to 14.

[0269] 16. A method for determining DPP3 activity in a body fluid sample of the subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; Quantifying the DPP3 activity by measuring and quantifying the conversion of a DPP3 substrate, Item 16. An angiotensin receptor agonist and / or a precursor thereof for use in treating a disease in a subject according to any one of items 1 to 15.

[0270] 17. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 16, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determination comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

[0271] 18. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 17, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

[0272] 19. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 18, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being immobilized on a surface.

[0273] 20. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of claims 16 to 19, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step in which components of the sample that are not bound to the capture binder are removed from the captured DPP3.

[0274] 21. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 16 to 20, wherein DPP3 activity is determined in a body fluid sample of the subject and the conversion of a substrate of DPP3 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 bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3), or Western blot (cleavage products).

[0275] 22. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 16 to 21, wherein DPP3 activity is determined in a body fluid sample of said subject, and said substrate is selected from the group comprising angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, beta-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that said dipeptide is Arg-Arg.

[0276] 23. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of claims 16 to 22, wherein DPP3 activity is determined in a body fluid sample of said subject, and said substrate is selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that said dipeptide is Arg-Arg.

[0277] 24. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof.

[0278] 25. Use of the method or assay or kit according to clause 24 for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, wherein said angiotensin receptor agonists and / or precursors thereof are selected from the group comprising angiotensin I, angiotensin II, angiotensin III and angiotensin IV, in particular angiotensin II is selected.

[0279] 26. Use of the method or assay or kit according to clause 24 or 25 for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonist and / or precursors thereof, wherein said amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject at least once before and / or during treatment with said angiotensin receptor agonist and / or precursors thereof.

[0280] 27. Use of the method or assay or kit according to any one of clauses 24 to 26 for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum.

[0281] 28. The amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and is: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or quantitating the amount of the DPP3 protein, 28. Use of the method or assay or kit according to any one of claims 24 to 27 for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof.

[0282] 29. A method for determining DPP3 activity in a body fluid sample from said subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; - separating the DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3; Quantifying the DPP3 activity by measuring and quantifying the conversion of a DPP3 substrate, 29. Use of the method or assay or kit according to any one of claims 24 to 28 for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof.

[0283] 30. Use of the method or assay or kit according to clause 28 or 29 for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

[0284] 31. Use of the method or assay or kit according to any one of clauses 28 to 30 for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonist and / or precursors thereof, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

[0285] 32. Use of the method or assay or kit according to any one of clauses 28 to 31 for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonist and / or precursors thereof, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being immobilized on a surface.

[0286] 33. Use of the method or assay or kit according to any one of clauses 28 to 32 for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step that removes components of the sample that are not bound to the capture binder from the captured DPP3.

[0287] 34. Use of the method or assay or kit according to any one of clauses 28 to 33 for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, wherein DPP3 activity is determined in a body fluid sample of the subject and conversion of a substrate of DPP3 is detected by a method selected from the group comprising: fluorescence of a fluorescent substrate (e.g. Arg-Arg-βNA, Arg-Arg-AMC), a color change of a chromogenic substrate, luminescence of a substrate bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3), or Western blot (cleavage products).

[0288] 35. Use of the method or assay or kit according to any one of clauses 28 to 34 for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, wherein DPP3 activity is determined in a body fluid sample of said subject and said substrate is selected from the group comprising angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, beta-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that said dipeptide is Arg-Arg.

[0289] 36. Use of the method or assay or kit according to any one of clauses 28 to 35 for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, wherein DPP3 activity is determined in a body fluid sample of said subject and said substrate is selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that said dipeptide is Arg-Arg.

[0290] 37. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 23, wherein said treatment with an angiotensin receptor agonist and / or a precursor thereof is discontinued when the patient has an amount of DPP3 protein and / or DPP3 activity in a body fluid sample that is below a predetermined threshold level.

[0291] 38. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of clauses 1 to 23 and 27, wherein the treatment with the angiotensin receptor agonist and / or a precursor thereof is initiated when the subject has an amount of DPP3 protein and / or DPP3 activity in a body fluid sample that exceeds a predetermined threshold level.

[0292] In the above context, the following sequentially numbered embodiments provide further specific aspects of the invention.

[0293] 1. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject, comprising: the disease is selected from the group consisting of heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial, viral (e.g. AIDS), parasitic (e.g. malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. stroke, neurodegenerative diseases), autoimmune diseases, vascular diseases, hypotension, and shock; the subject has an amount of DPP3 protein and / or DPP3 activity in a bodily fluid sample that exceeds a pre-determined threshold, angiotensin receptor agonist and / or precursors thereof.

[0294] 2. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to item 1, selected from the group comprising angiotensin I, angiotensin II, angiotensin III and angiotensin IV, in particular angiotensin II being selected.

[0295] 3. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to paragraph 1 or 2, wherein the amount of DPP3 protein and / or the DPP3 activity has been determined at least once in a body fluid sample of the subject before and / or during treatment with the angiotensin receptor agonist and / or a precursor thereof.

[0296] 4. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of paragraphs 1 to 3, wherein the amount of DPP3 protein and / or the DPP3 activity determined in a body fluid sample of the subject is used as a treatment guide and / or treatment monitoring for treatment with an angiotensin receptor agonist and / or a precursor thereof.

[0297] 5. An angiotensin receptor agonist and / or a precursor thereof for use in treating a disease in a subject according to any one of items 1 to 4, wherein the subject's body fluid sample is selected from whole blood, plasma, and serum.

[0298] 6. The angiotensin receptor agonist and / or precursor thereof for use in treating a disease in a subject according to any one of items 1 to 5, wherein the angiotensin receptor agonist and / or precursor thereof is administered to the subject in a pharmaceutical formulation.

[0299] 7. The angiotensin receptor agonist and / or precursor thereof for use in treating a disease in a subject according to item 6, wherein the angiotensin receptor agonist and / or precursor thereof is angiotensin II acetate and the pharmaceutical formulation is in a dry state to be reconstituted before use.

[0300] 8. The angiotensin receptor agonist and / or a precursor thereof for use in treating a disease in a subject according to any one of items 1 to 7, wherein the angiotensin receptor agonist and / or a precursor thereof is angiotensin II administered at a rate of 0.1 to 200 ng / kg / min, preferably 1 to 100 ng / kg / min, more preferably 2 to 80 ng / kg / min, even more preferably 5 to 60 ng / kg / min, even more preferably 10 to 50 ng / kg / min, even more preferably 15 to 40 ng / kg / min, and most preferably 20 ng / kg / min.

[0301] 9. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of items 1 to 8, wherein the angiotensin receptor agonist and / or a precursor thereof, in particular angiotensin II, is administered in combination with an inhibitor of DPP3.

[0302] 10. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to clause 9, wherein said inhibitor of DPP3 is selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

[0303] 11. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to clause 9 or 10, wherein said inhibitor of DPP3 is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to SEQ ID NO: 1, in particular SEQ ID NO: 2.

[0304] 12. The inhibitor of DPP3 has an activity against DPP3 of 10 -7 12. The angiotensin receptor agonist and / or precursor thereof for use in treating a disease in a subject according to any one of items 9 to 11, which is an antibody or fragment or scaffold exhibiting a minimum binding affinity of M or less.

[0305] 13. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of clauses 9 to 12, wherein said inhibitor of DPP3 is a monospecific antibody or fragment or a scaffold.

[0306] 14. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of clauses 9 to 13, wherein said inhibitor of DPP3 is an antibody or fragment or scaffold that binds to full-length DPP3 and inhibits the activity of DPP3 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%.

[0307] 15. The amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and is: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or quantitating the amount of the DPP3 protein, Item 15. An angiotensin receptor agonist and / or a precursor thereof for use in treating a disease in a subject according to any one of items 1 to 14.

[0308] 16. A method for determining DPP3 activity in a body fluid sample of the subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; Quantifying the DPP3 activity by measuring and quantifying the conversion of a DPP3 substrate, Item 16. An angiotensin receptor agonist and / or a precursor thereof for use in treating a disease in a subject according to any one of items 1 to 15.

[0309] 17. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 16, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determination comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

[0310] 18. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 17, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

[0311] 19. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 18, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being immobilized on a surface.

[0312] 20. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of claims 16 to 19, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step in which components of the sample that are not bound to the capture binder are removed from the captured DPP3.

[0313] 21. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 16 to 20, wherein DPP3 activity is determined in a body fluid sample of the subject and the conversion of a substrate of DPP3 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 bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3), or Western blot (cleavage products).

[0314] 22. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 16 to 21, wherein DPP3 activity is determined in a body fluid sample of said subject, and said substrate is selected from the group comprising angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, beta-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that said dipeptide is Arg-Arg.

[0315] 23. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of claims 16 to 22, wherein DPP3 activity is determined in a body fluid sample of said subject, and said substrate is selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that said dipeptide is Arg-Arg.

[0316] 24. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof.

[0317] 25. Use of the method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof according to paragraph 24, wherein said angiotensin receptor agonists and / or precursors thereof are selected from the group comprising angiotensin I, angiotensin II, angiotensin III and angiotensin IV, in particular angiotensin II is selected.

[0318] 26. The use of the method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonist and / or precursors thereof according to paragraph 24 or 25, wherein the amount of DPP3 protein and / or DPP3 activity is determined at least once in a body fluid sample of the subject before and / or during treatment with the angiotensin receptor agonist and / or precursors thereof.

[0319] 27. Use of the method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or its precursor according to any one of paragraphs 24 to 26, wherein the subject's body fluid sample is selected from whole blood, plasma, and serum.

[0320] 28. The amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and is: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or quantitating the amount of the DPP3 protein, Use of the method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity according to any one of claims 24 to 27 as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or a precursor thereof.

[0321] 29. A method for determining DPP3 activity in a body fluid sample from said subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; Quantifying the DPP3 activity by measuring and quantifying the conversion of a DPP3 substrate, Use of the method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity according to any one of claims 24 to 28 as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or a precursor thereof.

[0322] 30. Use of the method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof according to clause 28 or 29, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determination comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

[0323] 31. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or precursor thereof according to any one of claims 28 to 30, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determination comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

[0324] 32. Use of the method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or precursor thereof according to any one of paragraphs 28 to 31, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being immobilized on a surface.

[0325] 33. Use of the method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or precursor thereof according to any one of claims 28 to 32, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step in which components of the sample that are not bound to the capture binder are removed from the captured DPP3.

[0326] 34. The use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or its precursor according to any one of claims 28 to 33, wherein DPP3 activity is determined in a body fluid sample of the subject, and conversion of a substrate of DPP3 is detected by a method selected from the group including fluorescence of a fluorescent substrate (e.g. Arg-Arg-βNA, Arg-Arg-AMC), a color change of a chromogenic substrate, luminescence of a substrate bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3), or Western blot (cleavage products).

[0327] 35. Use of the method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or precursor thereof according to any one of claims 28 to 34, wherein DPP3 activity is determined in a body fluid sample of the subject and the substrate is selected from the group comprising angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, beta-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that the dipeptide is Arg-Arg.

[0328] 36. Use of the method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof according to any one of clauses 28 to 35, wherein DPP3 activity is determined in a body fluid sample of the subject and the substrate is selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, whereby the dipeptide is Arg-Arg.

[0329] 37. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject for predicting the risk of an adverse event in said subject, said method comprising: determining the level of DPP3 in a body fluid sample from said subject; comparing the determined level of DPP3 to a predefined threshold; correlating said level of DPP3 with a risk of an adverse event in said subject; wherein elevated levels above a certain threshold are predictive of increased risk of said adverse event.

[0330] 38. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for the purpose of predicting the risk of an adverse event in said subject, wherein said body fluid sample of said subject is selected from whole blood, plasma, and serum.

[0331] 39. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of the subject for predicting the risk of an adverse event in the subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or The use, comprising a step of quantifying the amount of the DPP3 protein.

[0332] 40. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject for predicting the risk of an adverse event in said subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; - Quantifying DPP3 activity by measuring and quantifying the conversion of a substrate of DPP3.

[0333] 41. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of an adverse event in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

[0334] 42. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for the purpose of predicting the risk of an adverse event in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

[0335] 43. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for the purpose of predicting the risk of an adverse event in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being immobilized on a surface.

[0336] 44. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of the subject for the purpose of predicting the risk of an adverse event in the subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step that removes components of the sample that are not bound to the capture binder from the captured DPP3.

[0337] 45. Use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of an adverse event in said subject, wherein DPP3 activity is determined in a body fluid sample of said subject and conversion of a DPP3 substrate is detected by a method selected from the group comprising: fluorescence of a fluorescent substrate (e.g. Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3), or Western blot (cleavage products).

[0338] 46. ​​Use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of an adverse event in said subject, wherein DPP3 activity is determined in a body fluid sample of said subject and said substrate is selected from the group comprising angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, beta-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that said dipeptide is Arg-Arg.

[0339] 47. Use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for the purpose of predicting the risk of an adverse event in said subject, wherein DPP3 activity is determined in a body fluid sample of said subject and said substrate is selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that said dipeptide is Arg-Arg.

[0340] 48. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject for predicting the risk of organ dysfunction or organ dysfunction in said subject, said method comprising: determining the level of DPP3 in a body fluid sample from said subject; comparing the determined level of DPP3 to a predefined threshold; correlating said level of DPP3 with a risk of an adverse event in said subject, A use wherein elevated levels above a certain threshold are predictive of increased risk of said organ dysfunction or deterioration of organ function.

[0341] 49. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or worsening organ function in said subject, wherein said body fluid sample of said subject is selected from whole blood, plasma, and serum.

[0342] 50. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of the subject for predicting the risk of organ dysfunction or organ dysfunction in the subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or The use, comprising a step of quantifying the amount of the DPP3 protein.

[0343] 51. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject for predicting the risk of organ dysfunction or organ dysfunction deterioration in said subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; - Quantifying DPP3 activity by measuring and quantifying the conversion of a substrate of DPP3.

[0344] 52. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of organ dysfunction or worsening organ function in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

[0345] 53. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or worsening organ function in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

[0346] 54. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or worsening organ function in said subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being immobilized on a surface.

[0347] 55. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of the subject for the purpose of predicting the risk of organ dysfunction or worsening organ function in the subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step that removes components of the sample that are not bound to the capture binder from the captured DPP3.

[0348] 56. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of organ dysfunction or deterioration in said subject, wherein DPP3 activity is determined in a body fluid sample of said subject and conversion of a DPP3 substrate 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 bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3), or Western blot (cleavage products).

[0349] 57. Use of a method or assay or a kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of organ dysfunction or worsening organ function in said subject, wherein DPP3 activity is determined in a body fluid sample of said subject and said substrate is selected from the group comprising angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, beta-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that said dipeptide is Arg-Arg.

[0350] 58. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of said subject for predicting the risk of organ dysfunction or worsening organ function in said subject, wherein DPP3 activity is determined in a body fluid sample of said subject and said substrate is selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that said dipeptide is Arg-Arg.

[0351] 59. A method for predicting risk of an adverse event in a subject, the method comprising: determining the level of DPP3 in a body fluid sample from said subject; comparing the determined level of DPP3 to a predefined threshold; correlating said level of DPP3 with a risk of said adverse event in said subject, A method in which elevated levels above a certain threshold are predictive of increased risk of said adverse event.

[0352] 60. A method for predicting the risk of an adverse event in a subject, wherein the subject's body fluid sample is selected from whole blood, plasma, and serum.

[0353] 61. A method for predicting the risk of an adverse event in a subject, comprising determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of the subject, and determining: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or quantifying the amount of DPP3 protein.

[0354] 62. A method for predicting the risk of an adverse event in a subject, comprising determining DPP3 activity in a body fluid sample of the subject, contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; - quantifying said DPP3 activity by measuring and quantifying conversion of a substrate of DPP3.

[0355] 63. A method for predicting the risk of an adverse event in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being selected from the group of an antibody, an antibody fragment, or a non-IgG scaffold.

[0356] 64. A method for predicting the risk of an adverse event in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being an antibody.

[0357] 65. A method for predicting the risk of an adverse event in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being immobilized on a surface.

[0358] 66. A method for predicting the risk of an adverse event in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step that removes components of the sample that are not bound to the capture binder from the captured DPP3.

[0359] 67. A method for predicting the risk of an adverse event in a subject, wherein DPP3 activity is determined in a body fluid sample of the subject, and conversion of a DPP3 substrate 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 bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3), or Western blot (cleavage products).

[0360] 68. A method for predicting the risk of an adverse event in a subject, wherein DPP3 activity is determined in a body fluid sample of the subject, and the substrate is selected from the group comprising angiotensin II, III, and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, with the proviso that the dipeptide is Arg-Arg.

[0361] 69. A method for predicting the risk of an adverse event in a subject, wherein DPP3 activity is determined in a body fluid sample of the subject, and the substrate is selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, wherein the dipeptide is Arg-Arg.

[0362] 70. A method for predicting the risk of organ dysfunction or organ function deterioration in a subject, the method comprising: determining the level of DPP3 in a body fluid sample from said subject; comparing the determined level of DPP3 to a predefined threshold; correlating said level of DPP3 with a risk of an adverse event in said subject, A method, wherein elevated levels above a certain threshold are predictive of increased risk of said organ dysfunction or deterioration of organ function.

[0363] 71. A method for predicting the risk of organ dysfunction in a subject, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum.

[0364] 72. A method for predicting the risk of organ dysfunction or organ function deterioration in a subject, comprising determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of the subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the isolated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or quantifying the amount of DPP3 protein.

[0365] 73. A method for predicting the risk of organ dysfunction or organ function deterioration in a subject, comprising determining DPP3 activity in a body fluid sample of the subject, contacting the sample with a capture binder that specifically binds full-length DPP3; separating DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; - quantifying said DPP3 activity by measuring and quantifying conversion of a substrate of DPP3.

[0366] 74. A method for predicting the risk of organ dysfunction or worsening organ function in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being selected from the group of an antibody, an antibody fragment, or a non-IgG scaffold.

[0367] 75. A method for predicting the risk of organ dysfunction or worsening organ function in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

[0368] 76. A method for predicting the risk of organ dysfunction or worsening organ function in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being immobilized on a surface.

[0369] 77. A method for predicting the risk of organ dysfunction or worsening organ function in a subject, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step that removes components of the sample that are not bound to the capture binder from the captured DPP3.

[0370] 78. A method for predicting the risk of organ dysfunction or organ function deterioration in a subject, wherein DPP3 activity is determined in a body fluid sample of the subject and conversion of a DPP3 substrate 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 bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3), or Western blot (cleavage products).

[0371] 79. A method for predicting the risk of organ dysfunction or worsening organ function in a subject, wherein DPP3 activity is determined in a body fluid sample of the subject, and the substrate is selected from the group comprising angiotensin II, III, and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, chromophore or aminoluciferin, with the proviso that the dipeptide is Arg-Arg.

[0372] 80. A method for predicting the risk of organ dysfunction or worsening organ function in a subject, wherein DPP3 activity is determined in a body fluid sample of the subject, and the substrate is selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, wherein the dipeptide is Arg-Arg.

[0373] 81. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of clauses 1 to 23, wherein said treatment with the angiotensin receptor agonist and / or a precursor thereof is discontinued when the patient has an amount of DPP3 protein and / or DPP3 activity in a body fluid sample that is below a predetermined threshold level.

[0374] 82. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of clauses 1 to 23 and clause 81, wherein said treatment with an angiotensin receptor agonist and / or a precursor thereof is initiated when the patient has an amount of DPP3 protein and / or DPP3 activity in a body fluid sample that exceeds a predetermined threshold level. EXAMPLES

[0375] Example 1: Methods for measuring DPP3 protein and DPP3 activity

[0376] Generation of antibodies and measurement of DPP3-binding ability: Several mouse antibodies were generated and screened for their binding ability to human DPP3 in a specific binding assay (see Table 1).

[0377] Peptides / Conjugates for Immunization

[0378] Immunizing DPP3 peptides were synthesized with an additional N-terminal cysteine ​​residue (if no cysteine ​​was present in the selected DPP3 sequence) for conjugation with bovine serum albumin (BSA) (see Table 1, JPT Technologies, Berlin, Germany). The peptides were covalently coupled to BSA using Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the Perbio manual. Recombinant GST-hDPP3 was produced by USBio (United States Biological, Salem, MA, USA).

[0379] Immunization of mice, immune cell fusion and screening:

[0380] Balb / c mice were injected intraperitoneally (ip) with 84 μg GT-hDDP3 (emulsified in TiterMax Gold adjuvant) or 100 μg DPP3-peptide-BSA-conjugate on day 0, 84 μg or 100 μg (emulsified in complete Freund's adjuvant) on day 14, and 42 μg or 50 μg (emulsified in incomplete Freund's adjuvant) on days 21 and 28. On day 49, mice were injected intravenously (iv) with 42 μg GT-hDDP3 or 50 μg DPP3-peptide-BSA-conjugate in saline. After 3 days, mice were euthanized and immune cells were fused.

[0381] Spleen cells from immunized mice were fused with cells from myeloma cell line SP2 / 0 using 1 ml of 50% polyethylene glycol at 37°C for 30 seconds. After washing, cells were seeded into 96-well cell culture plates. Hybrid clones were selected in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement]. After 1 week, the HAT medium was replaced with HT medium for 3 passages before returning to normal cell culture medium.

[0382] Two weeks after fusion, cell culture supernatants were first screened for IgG antibodies binding to recombinant DPP3. GST-tagged recombinant hDPP3 (USBiologicals, Salem, USA) was therefore immobilized (100 ng / well) in 96-well plates and incubated with 50 μl cell culture supernatant per well for 2 h at room temperature. After washing the plates, 50 μl / well POD-rabbit anti-mouse IgG was added and incubated for 1 h at room temperature. After a subsequent washing step, 50 μl of chromophore solution (citrate / hydrogen phosphate buffer with 3.7 mM o-phenylenediamine, 0.012% H2O2) was added to each well and incubated for 15 min at room temperature before the color reaction was stopped by adding 50 μl of 4N sulfuric acid. Absorbance was detected at 490 nm.

[0383] Positive microcultures were transferred to 24-well plates for expansion, and after retesting, selected cultures were cloned and recloned using limiting dilution techniques and are isotyped.

[0384] Preparation of mouse monoclonal antibodies

[0385] Antibodies against GST-tagged human DPP3 or DPP3 peptides were generated by standard antibody production methods (Marx et al. 1997) and purified through protein A. Purity of the antibodies was greater than 90% based on SDS gel electrophoresis analysis.

[0386] Characterization of antibodies - binding to hDPP3 and / or immunizing peptide

[0387] Binding assays were performed to analyze the ability of different antibodies and antibody clones to bind to the DPP3 / immunizing peptide.

[0388] a) Solid phase

[0389] GST-tagged recombinant hDPP3 (SEQ ID NO: 1) or DPP3 peptide (immunizing peptide, SEQ ID NO: 2) was immobilized on the surface of high-binding microtiter plates (96-well polystyrene microplates, Greiner Bio-One international AG, Austria, 1 μg per well in coupling buffer [50 mM Tris, 100 mM NaCl, pH 7.8] at room temperature for 1 h). After blocking with 5% bovine serum albumin, the microplates were dried under vacuum.

[0390] b) Tracer procedure

[0391] 100 μg (100 μl) of various anti-DPP3 antibodies (1 mg / ml detection antibody in PBS, pH 7.4) were mixed with 10 μl of acridinium NHS ester (1 mg / ml in acetonitrile, InVent GmbH, Germany; EP 0 353 971) and incubated at room temperature for 30 min. Labeled anti-DPP3 antibodies were purified by gel filtration HPLC on Shodex Protein 5 μm KW-803 (Showa Denko, Japan). 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 was approximately 5-7 × 10 labeled compound (approximately 20 ng labeled antibody) per 200 μl. 6 The chemiluminescence of the acridinium ester was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

[0392] c) hDPP3 binding assay

[0393] Plates were filled with 200 μl of labeled and diluted detection antibody (tracer) and incubated for 2–4 h at 2–8°C. Unbound tracer was removed by washing four times with 350 μl of washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Chemiluminescence of well-bound antibodies was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

[0394] Antibody characterization – hDPP3 inhibition assay

[0395] To analyze the DPP3 inhibitory capacity of different antibodies and antibody clones, DPP3 activity assays were performed as previously described (Jones et al., 1982). GST-tagged recombinant hDPP3 was diluted in assay buffer (25 ng / ml GST-DPP3 in 50 mM Tris-HCl, pH 7.5 and 100 μM ZnCl2) and 200 μl of this solution was incubated with 10 μg of each antibody at room temperature. After 1 h of preincubation, the fluorescent substrate Arg-Arg-βNA (20 μl, 2 mM) was added to the solution and the generation of free βNA was monitored at 37°C using a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG). βNA fluorescence was detected by exciting at 340 nm and measuring the emission at 410 nm. The slope of increasing fluorescence (in RFU / min) for the different samples was calculated. The gradient of native human DPP3 with the buffer control is taken as 100% activity. The inhibitory activity of a potential capture binder is defined as the reduction (in %) of GST-hDPP3 activity upon incubation with said capture binder.

[0396] The table below presents the set of antibodies obtained and their binding rates (expressed in relative light units (RLU)) as well as their relative inhibitory potency (%; Table 1). The monoclonal antibodies against the following DPP3 regions were selected for their ability to bind recombinant DPP3 and / or the immunizing peptide and their inhibitory potency.

[0397] All antibodies against the GST-tagged full-length form of recombinant hDPP3 showed strong binding to immobilized GST-tagged hDPP3. Antibodies against peptide of SEQ ID NO:2 also bind to GST-hDPP3. Antibodies of SEQ ID NO:2 also bind strongly to the immunizing peptide.

[0398] [Table 1] Table 1: List of antibodies against full-length hDPP3 or sequences of hDPP3 and their binding capacity (in RLU) to hDPP3 (SEQ ID NO: 1) or the immunizing peptide (SEQ ID NO: 2) as well as the maximum inhibition rate of recombinant GST-hDPP3.

[0399] The development of a fluorescent immunoassay to quantify DPP3 protein concentrations (DPP3-LIA) and an enzyme capture activity assay to quantify DPP3 activity (DPP3-ECA) has recently been reported (Rehfeld et al. 2018 JALM in press), which is incorporated herein by reference in its entirety.

[0400] Example 2: DPP3 for predicting early mortality

[0401] Using hDPP3 immunoassays, we determined plasma DPP3 concentrations in patients with a variety of diseases and correlated them with early mortality in these patients.

[0402] Study cohort – Sepsis and septic shock

[0403] Plasma samples from 574 patients from the Adrenomedullin and Outcomes in Severe Sepsis and Septic Shock (AdrenOSS) study were screened for DPP3. AdrenOSS is a prospective, observational, multi-country study including 583 patients admitted to intensive care units with sepsis or septic shock (Hollinger et al., 2018). 292 patients were diagnosed with septic shock.

[0404] Study cohort – Cardiogenic shock

[0405] Plasma samples from 108 patients diagnosed with cardiogenic shock were screened for DPP3. Blood was collected within 6 hours of detection of cardiogenic shock. Mortality was followed for 7 days.

[0406] Study cohort - acute coronary syndrome

[0407] Plasma samples from 720 patients with acute coronary syndrome were screened for DPP3. Blood was collected 24 hours after the onset of chest pain. Mortality was tracked over a 7-day period.

[0408] Study cohort – dyspnea

[0409] Plasma samples were collected from 1,440 patients with dyspnea (shortness of breath) immediately after admission to the emergency department at Skåne University Hospital. Patients with dyspnea are particularly likely to have acute coronary syndrome or congestive heart failure, and are therefore at high risk of organ failure and premature death. Mortality was tracked over a three-month period following presentation to the emergency department.

[0410] Study cohort – burn patients

[0411] 107 patients with severe burns (>15% of total body surface area) were screened for DPP3. Blood was collected upon admission. Mortality was followed for 4 weeks.

[0412] hDPP3 Immunoassay:

[0413] The levels of DPP3 in the plasma of patients were determined using an immunoassay (LIA) to detect the amount of human DPP3 or an activity assay (ECA) to detect the activity of human DPP3. Antibody immobilization, labeling, and incubation were performed as described by Rehfeld et al. (Rehfeld et al. 2018).

[0414] result

[0415] Short-term patient survival in sepsis / septic shock was associated with DPP3 plasma concentrations at admission. Patients with DPP3 plasma concentrations above 68.6 ng / ml (3 quartiles) had an increased risk of death compared to patients with DPP3 plasma concentrations below this threshold (Figure 1A). The same relationship was found when analyzing the relationship between short-term outcomes and DPP3 plasma concentrations only in this cohort of septic shock patients (Figure 1F). Patients with elevated DPP3 plasma concentrations had an increased risk of death compared to patients with low DPP3 plasma concentrations. When applying the same cut-off values ​​to patients with cardiogenic shock, an increased risk of early death within 7 days is also observed in patients with high DPP3 (Figure 1B).

[0416] In addition, the 28-day survival rate of patients with DPP3-associated acute coronary syndrome was also lower in those with higher DPP3 when applying the respective cutoff value of 68.6 ng / ml ( Figure 1 C).

[0417] When this cutoff value of 68.6 ng / ml was applied to patients suffering from dyspnea, a significantly increased risk of death was detected in patients with high DPP3 over a 3-month follow-up period ( Fig. 1D ).

[0418] Furthermore, severely burned patients with large DPP3 concentrations above the respective cutoff value of 68.6 ng / ml had an increased risk of 4-week mortality ( Fig. 1E ).

[0419] Example 3: Purification of human native DPP3

[0420] Human erythrocyte lysate was applied to a total of 100 ml of Sepharose 4B resin (Sigma-Aldrich) and the flow-through was collected. The resin was washed with a total of 370 ml of PBS buffer, pH 7.4, and the wash fractions were combined with the collected flow-through, giving a total volume of 2370 ml.

[0421] For the immunoaffinity purification step, 110 mg of monoclonal anti-hDPP3 mAb AK2552 was coupled to 25.5 ml of UltraLink hydrazide resin (Thermo Fisher Scientific) according to the manufacturer's protocol (GlycoLink Immobilization Kit, Thermo Fisher Scientific). The coupling efficiency was 98%, determined by quantifying uncoupled antibody by the Bradford technique. The resin-antibody conjugate was equilibrated with 10 bed volumes of wash-binding buffer (PBS, 0.1% Triton X-100, pH 7.4), combined with 2370 ml of clarified red blood cell lysate, and incubated for 2 h at 4° C. with continuous mixing. The resulting 100 ml incubation mixture was spread over the surface of ten 15 ml polypropylene columns, and the flow-through was collected by centrifugation at 1000×g for 30 s. This step was repeated several times, yielding 2.5 ml of DPP3-loaded resin per column. Each column was washed five times with 10 ml of wash-binding buffer using a gravity-glow approach. DPP3 was eluted by placing each column in a 15 ml Falcon tube containing 2 ml of neutralization buffer (1 M Tris-HCl, pH 8.0), followed by the addition of 10 ml of elution buffer (100 mM glycine-HCl, 0.1% Triton X-100, pH 3.5) per column, followed immediately by centrifugation at 1000×g for 30 seconds. This elution step was repeated three times, yielding a total of 360 ml of eluate. The pH of the neutralized eluate was 8.0.

[0422] The combined eluate was loaded onto a 5 ml HiTrap Q-sephare HP column (GE Healthcare) equilibrated with IEX buffer A1 (100 mM glycine, 150 mM Tris, pH 8.0) using the sample pump of an Akta Start system (GE Healthcare). After loading the sample, the column was washed with 5 column volumes of IEX buffer A2 (12 mM NaH2PO4, pH 7.4) to remove unbound proteins. Elution of DPP3 was achieved by applying a sodium chloride gradient ranging from 0 to 1 M NaCl over 10 column volumes (50 ml) using IEX buffer B (12 mM NaH2PO4, 1 M NaCl, pH 7.4). The eluate was collected in 2 ml fractions. Buffers used for ion exchange chromatography were sterile filtered using 0.22 μM bottle-top filters.

[0423] A purification table listing the respective yields and activities of each purification step is shown in Table 2. Figure 2 shows SDS-PAGE on a gradient gel (4-20%) for native hDPP3 purified from human erythrocyte lysate.

[0424] [Table 2] a) The relative amount of DPP3 was determined in all fractions using the DPP3-LIA assay: the amount of DPP3 in the starting material was set to 100% and the amount of residual DPP3 in the purified fractions was correlated to the starting material. b) Total protein amounts were determined using the method of Lowry as modified by Peterson (Peterson 1977 Analytical Biochemistry 356:346-356). c) The total Arg2-βNA hydrolysis activity (unit: micromoles) of substrate converted per minute was determined using Dpp3-ECA and calibrated through β-naphthylamine (0.05-100 μM). d) The purification yield was calculated from the total Arg2-βNA hydrolysis activity, which was set to 100% in the starting material. e) Specific activity is defined as micromoles of substrate converted per minute per mg of total protein. f) The purification factor is the quotient of the specific activity before and after each purification step.

[0425] Example 4: Effects of native DPP3 in animal models

[0426] The effect of injecting native hDPP3 into healthy mice was investigated by monitoring fractional shortening.

[0427] Wild-type Black 6 mice (8–12 weeks old, see Table 3 for group sizes) were allowed to acclimate for 2 weeks before baseline echocardiography. Mice were randomly assigned to one of two groups and then intravenously injected retroorbitally with native DPP3 protein or PBS at a dose of 600 μg / kg DPP3 protein.

[0428] After injection of DPP3 or PBS, cardiac function was assessed by echocardiography (Gao et al. 2011) and renal function was assessed by the renal resistive index (Lubas et al. 2014; Dewitte et al. 2012) at 15, 60, and 120 min (Fig. 3).

[0429] [Table 3] Table 3: List of experimental groups

[0430] result

[0431] Mice treated with native DPP3 protein show a significant decrease in fractional shortening compared to PBS-injected controls (Figure 4A). The wild-type+DPP3 group also shows worsening renal function, as evidenced by an increase in the renal resistive index (Figure 4B).

[0432] Example 5: Development of Procizumab

[0433] Antibodies against SEQ ID NO:2 were characterized in more detail (epitope mapping, binding affinity, specificity, inhibitory capacity). Results for clone 1967 of SEQ ID NO:2 (AK1967; "Procizumab") are shown here as an example.

[0434] Determination of AK1967 epitope on DPP3

[0435] To map the epitope of AK1967, a number of peptides were synthesized that were biotinylated at the N- or C-terminus (peptides&elephants GmbH, Hennigsdorf, Germany). These peptides comprise the sequence of the complete immunizing peptide (SEQ ID NO:2) or fragments thereof with single amino acids removed from the C- or N-terminus (see Table 5 for a complete list of peptides).

[0436] High-binding 96-well plates were coated with coupling buffer (500 mM Tris-HCl, pH 7.8, 100 mM NaCl) containing 2 μg avidin per well (Greiner Bio-One international AG, Austria). Plates were then washed and filled with a specific solution of biotinylated peptides (10 ng / well; buffer - 1x PBS with 0.5% BSA).

[0437] According to Example 1, the anti-DPP3 antibody AK1967 was labeled with a chemiluminescent label.

[0438] 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 four times with 350 μl of washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Chemiluminescence of well-bound antibodies was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). AK1967 binding to each peptide was determined by evaluating the relative light units (RLU). Any peptide with an RLU signal significantly greater than the nonspecific binding of AK1967 is defined as an AK1967 binder. Combinatorial analysis of binding and non-binding peptides reveals the specific DPP3 epitope of AK1967.

[0439] Measurement of binding affinity using Octet:

[0440] 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 dilution series of GSR-tagged recombinant human DPP3 (100, 33.3, 11.1, 3.7 nM). Association was observed for 120 s, followed by dissociation for 180 s. The buffers used for the experiments are listed in Table 4. Kinetic analysis was performed using a 1:1 binding model and global fitting.

[0441] [Table 4] Table 4: Buffers used for Octet measurements

[0442] Western blot analysis of the binding specificity of AK1967

[0443] Blood cells from human EDTA-blood were washed (three times in PBS), diluted in PBS, and lysed by repeated dry-freeze cycles. The blood cell lysate had a total protein concentration of 250 μg / ml and a DPP3 concentration of 10 μg / ml. SDS-PAGE and Western blots were performed with dilutions of blood cell lysate (1:40, 1:80, 1:160, and 1:320) and purified recombinant human His-DPP3 (31.25–500 ng / ml). Blots were incubated 1) in blocking buffer (1×PBS-T containing 5% skim milk powder), 2) in primary antibody solution (AK1967, 1:2,000 in blocking solution), and 3) in HRP-conjugated secondary antibody (goat anti-mouse IgG, 1:1,000 in blocking solution). Bound secondary antibodies were detected using Amersham ECL Western blotting detection reagent and an Amersham Imager 600 UV (both from GE Healthcare).

[0444] DPP3 Inhibition Assay:

[0445] To analyze the inhibitory ability of AK1967 on DPP3, DPP3 activity assay was performed according to known procedures (Jones et al., 1982) as described in Example 1. The inhibitory ability of AK1967 is defined as the reduction in GST-hDPP3 activity by incubation with the antibody (unit: %). The resulting reduction in DPP3 activity is shown in the inhibition curve in Figure 1C.

[0446] Epitope Mapping:

[0447] Analysis of peptides that bind to and do not bind to AK1967 revealed the DPP3 sequence INPETG (SEQ ID NO: 3) as an epitope required for AK1967 binding (see Table 5).

[0448] [Table 5] Table 5: Peptides used for epitope mapping of AK1967

[0449] Binding affinity:

[0450] AK1967 was added to recombinant GST-hDPP3 at 2.2 × 10 -9 It binds with an affinity of M (see kinetic curves in FIG. 5).

[0451] Specificity and Inhibitory Potential:

[0452] The only protein detected in the blood cell lysate using AK1967 as the primary antibody was DPP3 at 80 kDa (Figure 6). The total protein concentration of the lysate was 250 μg / ml, whereas the estimated DPP3 concentration is about 10 μg / ml. Although there were 25-fold more nonspecific proteins in the lysate, AK1967 specifically bound to DPP3 and detected DPP3, but no other nonspecific binding occurred.

[0453] AK1967 inhibited DPP3 at 15 ng / ml in a specific DPP3 activity assay, with an IC 50 is approximately 15 ng / ml (Figure 7).

[0454] Chimerization / Humanization:

[0455] The monoclonal antibody AK1967 ("procizumab"), which has the ability to inhibit DPP3 activity by 70%, was selected as a potential therapeutic antibody and was also used as a template for chimerization and humanization.

[0456] Humanization of a murine antibody can be carried out according to the following procedure:

[0457] To humanize an antibody of murine origin, the antibody sequence is analyzed for structural interactions of the framework regions (FRs) with the complementarity determining regions (CDRs) and the antigen. Based on structural modeling, suitable FRs of human origin are selected and the murine CDR sequences are grafted into the human FRs. Variations can be introduced in the amino acid sequences of the CDRs or FRs to regain the structural interactions that were abolished by a species switch on the FR sequences. This restoration of structural interactions can be achieved by a random approach using phage display libraries or through a directed approach guided by molecular modeling (Almagro and Fransson, 2008. Humanization of Antibodies. Front Biosci. vol. 13: pp. 1619-33).

[0458] In the above context, the variable regions can be connected to any subclass of constant region (IgG, IgM, IgE, IgA) or only to scaffolds, Fab fragments, Fv, Fab and F(ab)2. In examples 6 and 7 below, murine antibody variants with an IgG2a backbone are used. For chimerization and humanization, a human IgG1κ backbone was used.

[0459] For epitope binding, only the complementarity determining regions (CDRs) are important. The CDRs for the heavy and light chains of the murine anti-DPP3 antibody (AK1967; "Procizumab") are shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 for the heavy chain, and in SEQ ID NO:9, SEQ ID NO:KVS, and SEQ ID NO:10 for the light chain, respectively.

[0460] Sequencing of the anti-DPP3 antibody (AK1967; "Procizumab") revealed an antibody heavy chain variable region (H chain) according to SEQ ID NO:11 and an antibody light chain variable region (L chain) according to SEQ ID NO:12.

[0461] Example 6: Effect of Procizumab on Sepsis-Induced Heart Failure

[0462] In this experiment, the effect of injecting procizumab into rats with sepsis-induced heart failure (Rittirsch et al., 2009) was investigated by monitoring fractional shortening.

[0463] CLP model of septic shock:

[0464] Male Wistar rats (2–3 months old, 300–400 g, see Table 6 for group sizes) from Centre d'elevage Janvier (France) were randomly assigned to one of three groups. All rats were anesthetized with ketamine hydrochloride (90 mg / kg) and xylazine (9 mg / kg) intraperitoneally (ip). To induce polymicrobial sepsis, cecal ligation and puncture (CLP) was performed using a slightly modified Rittirsch protocol. A ventral midline incision (1.5 cm) was made to expose the cecum. The cecum was then ligated just below the ileocecal valve and perforated once with an 18-gauge needle. The abdominal cavity was then closed in two layers, after which fluid resuscitation (3 ml saline per 100 g body weight subcutaneously) was performed and the rats were returned to their original cages. Sham-operated rats were operated on but without cecal perforation. CLP rats were randomly divided into a placebo group and a therapeutic antibody group.

[0465] Research design:

[0466] The study flow is shown in Figure 8. After CLP or sham surgery, rats were allowed to rest for 20 h with free access to water and food. Then, rats were anesthetized, a tracheotomy was performed, and arterial and venous lines were secured. 24 h after CLP surgery, AK1967 or vehicle (saline) was administered at 5 mg / kg as a bolus injection, followed by infusion at 7.5 mg / kg for 3 h. As one safety measure, hemodynamics was monitored invasively and continuously from t=0 to 3 h.

[0467] At t=0 (baseline) all CLP rats were in septic shock and had progressively decreased cardiac function (hypotension, small fractional shortening). At this point, procizumab or vehicle (PBS) was injected (iv) and saline infusion was initiated. There was one control group and two CLP groups, which are summarized in the table below (Table 6). At the end of the experiment, rats were euthanized and organs were harvested for later analysis.

[0468] [Table 6] Table 6: List of experimental groups

[0469] Invasive Blood Pressure:

[0470] Hemodynamic variables were acquired using the AcqKnowledge system (BIOPAC Systems, Inc., USA), which provides a fully automated blood pressure analysis system. The catheter was connected to the BIOPAC system via a pressure sensor.

[0471] To proceed with the procedure, rats were anesthetized (ketamine and xylazine). The rats were moved to a heating pad to achieve the desired body temperature of 37-37.5°C. A temperature feedback probe was inserted into the rectum. The rats were placed in a supine position on the workbench. For external ventilation, the trachea was incised and a catheter (16G) was inserted 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.

[0472] A central venous catheter was inserted through the left jugular vein to allow administration of PCZ or PBS.

[0473] After surgery, rats were allowed to rest in a stable state before hemodynamic measurements were performed. Baseline blood pressure (BP) was then recorded. Intra-arterial saline infusion was discontinued during data collection.

[0474] Echocardiography:

[0475] The rats were anesthetized with ketamine hydrochloride. The chest was shaved and the rats were placed in a supine position.

[0476] For transthoracic echocardiography (TTE) examinations, a commercially available 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 later offline analysis.

[0477] Gray-scale images were recorded at a depth of 2 cm. Two-dimensional examination was started in the parasternal axial plane to measure the diameter of the aortic annulus and the diameter of the pulmonary artery. M-mode was also used to measure the left ventricular (LV) size and to evaluate fractional shortening (FS%). LVFS was calculated as LVend diastolic diameter-LVend systolic diameter / LVend diastolic diameter and expressed as a percentage. The end-diastolic time point was therefore defined as the time when the LV was at its maximum diameter. The end-systolic time point was therefore defined as the time when the LV was at its minimum diameter in the same cardiac cycle. All parameters were measured manually. Three cardiac cycles were averaged for each measurement.

[0478] Pulse-wave Doppler was used to record pulmonary artery flow from the same parasternal long axis plane. The velocity time integral of the pulmonary artery outflow was measured.

[0479] Mitral flow was recorded at the level of the mitral valve tip using pulsed Doppler from the apical five-chamber plane.

[0480] result:

[0481] Treatment of rats with sepsis-induced heart failure with PBS (CLP+PBS) reduced fractional shortening compared to sham-operated rats (Figure 9A). The CLP+PBS group also showed high mortality (Figure 9B). In contrast, application of procizumab to rats with sepsis-induced heart failure improved fractional shortening (Figure 9A) and dramatically reduced mortality (Figure 9B).

[0482] Example 7: Effect of Procizumab on Heart and Renal Failure

[0483] The effect of procizumab on isoproterenol-induced heart failure in mice was investigated by monitoring fractional shortening and renal resistive index.

[0484] Cardiac stress induced by isoproterenol in mice:

[0485] Acute heart failure was induced in 3-month-old male mice by subcutaneous injection of the nonselective β-adrenergic agonist isoproterenol (DL-isoproterenol hydrochloride, Sigma Chemical Co.) (ISO) at 300 mg / kg twice daily for 2 days (Vergaro et al., 2016). ISO was diluted in NaCl 0.9%. Mice treated with isoproterenol were randomly assigned to two groups (Table 7) and, after baseline echocardiography (Gao et al., 2011), intravenously injected with PBS or procizumab (10 mg / kg), and renal resistive index measurements (Lubas et al., 2014; Dewitte et al., 2012) were performed on day 3 (Figures 10A and 10B).

[0486] Cardiac function was assessed by echocardiography (Gao et al. 2011) and renal resistive index (Lubas et al. 2014; Dewitte et al. 2012) at 1, 6, and 24 h (Figures S10A and S10B). Mice in the group injected with vehicle (PBS) instead of isoproterenol were not further treated with drugs and served as the control group (Table 7).

[0487] [Table 7] Table 7: List of experimental groups

[0488] result:

[0489] The administration of PCZ to mice with isoproterenol-induced heart failure reversed the heart failure within the first hour of administration (Figure 11A). The renal function of the diseased mice showed a significant improvement at 6 hours after injection of PCZ and was comparable to that of sham-operated rats at 24 hours (Figure 11B).

[0490] Example 8: Effect of Valsartan

[0491] The effects of valsartan, an antagonist of the angiotensin II type 1 receptor (ATR1), were examined by monitoring fractional shortening in healthy mice injected with DPP3.

[0492] In this experiment, healthy Black 6 mice (8-12 weeks old, see Table 8 for group sizes) consumed water containing 50 mg / kg valsartan per day or water alone for a period of 2 weeks (Table 8). Both groups were then intravenously injected with native DPP3 (600 μg / kg) and fractional shortening was assessed according to Gao et al., 2011 at 15, 60 and 120 min (Figure 12).

[0493] [Table 8] Table 8: List of experimental groups

[0494] result:

[0495] Injection of healthy mice with DPP3 significantly reduced fractional shortening (Figure 13). In contrast, healthy mice treated with the angiotensin II receptor agonist valsartan followed by DPP3 injection showed no signs of cardiac dysfunction as assessed by fractional shortening. Thus, DPP3-mediated cardiac dysfunction is angiotensin II-mediated, since cardiac function was restored by treatment with valsartan.

[0496] Mice treated with valsartan for 2 weeks were adapted to the blockade of type 1 angiotensin II receptors and the subsequent inhibition of angiotensin II-mediated signaling and Ang II-mediated cardiac activation. Apparently, under valsartan treatment, the mice switched to a different way of activating the heart that was independent of type 1 angiotensin II receptor signaling, because this angiotensin signaling system was inhibited by valsartan.

[0497] When DPP3 cleaves Ang II and thus inhibits angiotensin II-mediated cardiac activity, mice adapted to a downregulated angiotensin system (treated with valsartan) showed no signs of cardiac dysfunction as assessed by fractional shortening. In contrast, mice not treated with the angiotensin II receptor antagonist valsartan and adapted to inhibited Ang II-mediated signaling showed a significant decrease in fractional shortening in response to injection of DPP3 and subsequent cleavage and inactivation of Ang II.

[0498] This experiment clearly demonstrates a relationship between DPP3 and angiotensin II, implying that DPP3-induced heart failure is angiotensin II-mediated.

[0499] Example 9: DPP3 and organ dysfunction in sepsis

[0500] The same study described in Example 2 (AdrenOSS-1) was utilized to evaluate the relationship between cDPP3 and organs (e.g., cardiovascular and renal dysfunction) in patients hospitalized with sepsis and septic shock. AdrenOSS-1 is a European observational multi-country study that included 583 patients admitted to the ICU with sepsis or septic shock (ClinicalTrials.gov NCT02393781). The primary outcome (as described in Example 2) was 28-day mortality. Secondary outcomes included organ failure as defined by SOFA score and the need for organ support focused on the use of vasopressors and renal replacement therapy. Blood for central laboratory was sampled within 24 hours of admission to the ICU and on day 2.

[0501] The median cDPP3 level measured at admission in all AdrenOSS-1 patients was 45.1 ng / ml (interquartile range 27.5-68.6). High levels of DPP3 measured at admission were associated with worse metabolic parameters, renal and cardiac function, and SOFA scores. Patients with DPP3 levels above the median of 45.1 ng / ml had a median SOFA score of 8 (IQR 5-11), compared with patients with DPP3 levels below the median, who had a median SOFA score of 6 (IQR 4-9) (Figure 14).

[0502] Regardless of cDPP3 levels on admission, high levels of cDPP3 at 24 hours were associated with the worst SOFA scores, both globally (Figure 15) and by organ (Figures 16A-F).

[0503] Taken together, these data indicated that high levels of cDPP3 were associated with survival and the extent of organ dysfunction in patients with sepsis or septic shock in a large international cohort. The study found a significant association between cDPP3 <45.1 ng / ml on admission and short-term survival, as well as a cutoff value of 45.1 ng / ml that was predictive of poor prognosis, in both sepsis and septic shock. With regard to organ dysfunction, a positive association existed between cDPP3 on ICU admission and SOFA score. More importantly, the association between cDPP3 levels and the extent of organ dysfunction seen on ICU admission also held true during the recovery phase. Indeed, patients with high cDPP3 levels on admission that decreased towards normal cDPP3 values ​​on day 2 were more likely to recover the function of all organs (including cardiovascular, renal, pulmonary, and hepatic).

[0504] array

[0505] SEQ ID NO:1 - hDDP3 amino acids 1-737 MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAWYGGLAVLLQTSPEAPYIYALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTKFVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLRHLGLGKEGITTYFSGNCTMEDAKLA QDFLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDSEVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESFTQGSIEAHKRGSRFWIQDKGPIVESYIGFIESYRDPFGSRGEFEFGFVAVVNKAMSAKFERLVASAEQLLKELPWPPTFEKDKF LTPDFTSLDVLTFAGSGIPAGINIPNYDDLRQTEGFKNVSLGNVLAVAYATQREKLTFLEEDDKDLYILWKGPSFDVQVGLHELLGHGSGKLFVQDEKGAFNFDQETVINPETGEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLYLCLHPQVLEIFGFEGADAEDVIYVNWLNMVRAGLL ALEFYTPEAFNWRQAHMQARFVILRVLLEAGEGLVTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTGDVAGGRALYEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEYEASAAGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA

[0506] SEQ ID NO:2 - hDDP3 amino acids 474-493 (N-Cys) - immunizing peptide with an additional N-terminal cysteine CETVINPETGEQIQSWYRSGE

[0507] SEQ ID NO:3 - hDDP3 amino acids 477-482 - epitope of AK1967 INPETG

[0508] SEQ ID NO:4 - Variable region within the heavy chain of mouse AK1967 QVTLKESGPGILQPSQTLSLTCSSGFSLSTSGMSVGWIRQPSGKGLEWLAHIWWNDNKSYNPALKSRLTISRDTSNNQVFLKIASVVTADTGTYFCARNYSYDYWGQGTTLTVSS

[0509] SEQ ID NO:5 - Variable region within the light chain of mouse AK1967 DVVVTQTPLSLSVSLGDPASISCRSSRSLVHSIGSTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

[0510] SEQ ID NO:6 - CDR1 in the heavy chain of mouse AK1967 GFSLSTSGMS

[0511] SEQ ID NO:7 - CDR2 in the heavy chain of mouse AK1967 IWWNDNK

[0512] SEQ ID NO:8 - CDR3 in the heavy chain of mouse AK1967 ARNYSYDY

[0513] SEQ ID NO:9 - CDR1 in the light chain of mouse AK1967 RSLVHSIGSTY

[0514] Mouse AK1967 light chain CDR2 KVS

[0515] SEQ ID NO: 10 - CDR3 in the light chain of mouse AK1967 SQSTHVPWT

[0516] SEQ ID NO:11 - Humanized AK1967 - Heavy chain sequence (IgG1 kappa framework) MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQVVLTMTNMMDPVDTG TYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0517] SEQ ID NO:12 - Humanized AK1967 - Light chain sequence (IgG1 kappa framework) METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYYCSQSTHV PWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0518] SEQ ID NO:13 - Angiotensin II (also known as 5-isoleucine-angiotensin II) DRVYIHPF

[0519] SEQ ID NO:14 - Angiotensin II analogue (5-valine-angiotensin II) DRVYVHPF

[0520] SEQ ID NO:15 - Angiotensin II analogue (Asn 1 -Phe 4 ) NRVFIHPF

[0521] SEQ ID NO:16 - Angiotensin II hexapeptide VYIHPF

[0522] SEQ ID NO:17 - Angiotensin II nonapeptide NRVYYVHPF

[0523] SEQ ID NO:18 - Angiotensin II analogue ([Asn 1 -Ile 5 -Ile 8 ]-Angiotensin II) NRVYIHPI

[0524] SEQ ID NO:19 - Angiotensin II analogue ([Asn 1 -Ile 5 -Ala 8 ]-Angiotensin II) NRVYIHPA

[0525] SEQ ID NO:20 - Angiotensin II analogue ([Asn 1 -Jinde Tyr 4 -Ile 5 ]-Angiotensin II) NRVYIHPF

[0526] SEQ ID NO:21 - Angiotensin III RVYIHPF

[0527] SEQ ID NO:22 - Angiotensin III analogue (Val 4 -Angiotensin III) RVYVHPF

[0528] SEQ ID NO:23 - Angiotensin III analogue (Phe 3-Angiotensin III) RVFIHPF

[0529] SEQ ID NO:24 - Angiotensin III analogue ([Ile 4 -Ala 7 ]-Angiotensin III) RVYIHPA

[0530] SEQ ID NO:25 - Angiotensin III analogue ([DiindoTyr 3 -Ile 4 ]angiotensin III) RVYIHPF

[0531] SEQ ID NO:26 - Angiotensin IV VYIHPF

[0532] SEQ ID NO:27 - Angiotensin IV analogue (Val 3 -Angiotensin IV) VYVHPF

[0533] SEQ ID NO:28 - Angiotensin IV analogue (Phe 2 -Angiotensin IV) VFIHPF

[0534] SEQ ID NO:29 - Angiotensin IV analogue ([Ile 3 -Ala 6 ]-Angiotensin IV) VYIHPA

[0535] SEQ ID NO:30 - Angiotensin IV analog ([DiindoTyr 2 -Ile 3 ]-Angiotensin IV) VYIHPF

Claims

1. 1. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject, comprising: the disease is selected from the group consisting of heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial, viral (e.g. AIDS), parasitic (e.g. malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. stroke, neurodegenerative diseases), autoimmune diseases, vascular diseases, hypotension, and shock; The subject has an amount of DPP3 protein and / or DPP3 activity in a bodily fluid sample that exceeds a predetermined threshold, angiotensin receptor agonist and / or precursor thereof.

2. 2. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to claim 1, selected from the group comprising angiotensin I, angiotensin II, angiotensin III and angiotensin IV, in particular angiotensin II being selected.

3. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to claim 1 or 2, wherein the amount of DPP3 protein and / or PP3 activity has been determined at least once in a body fluid sample of the subject before and / or during treatment with the angiotensin receptor agonist and / or a precursor thereof.

4. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 3, wherein the amount of DPP3 protein and / or the determination of DPP3 activity determined in a body fluid sample of the subject is used as a therapeutic guide and / or therapeutic monitoring for treatment with the angiotensin receptor agonist and / or precursor thereof.

5. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 4, wherein the subject's body fluid sample is selected from whole blood, plasma and serum.

6. 6. The angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 5, wherein the angiotensin receptor agonist and / or precursor thereof is administered to the subject in a pharmaceutical formulation.

7. 7. The angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to claim 6, wherein the angiotensin receptor agonist and / or precursor thereof is angiotensin II acetate and the pharmaceutical formulation is in a dry state to be reconstituted before use.

8. 8. The angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 7, wherein the angiotensin receptor agonist and / or precursor thereof is angiotensin II administered at a rate of 0.1 to 200 ng / kg / min, preferably 1 to 100 ng / kg / min, more preferably 2 to 80 ng / kg / min, even more preferably 5 to 60 ng / kg / min, even more preferably 10 to 50 ng / kg / min, even more preferably 15 to 40 ng / kg / min and most preferably at a rate of 20 ng / kg / min.

9. 9. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 8, wherein said angiotensin receptor agonist and / or precursor thereof, in particular angiotensin II, is administered in combination with an inhibitor of DPP3.

10. 10. The angiotensin receptor agonist and / or its precursor for use in the treatment of a disease in a subject according to claim 9, wherein the inhibitor of DPP3 is selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

11. 11. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to claim 9 or 10, wherein said inhibitor of DPP3 is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to SEQ ID NO: 1, in particular SEQ ID NO:

2.

12. The inhibitor of DPP3 has a potency of 10 to 15% against DPP3. -7 12. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 9 to 11, which is an antibody or fragment or scaffold exhibiting a minimum binding affinity of M or less.

13. 13. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 9 to 12, wherein said inhibitor of DPP3 is a monospecific antibody or fragment or a scaffold.

14. 14. The angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 9 to 13, wherein said inhibitor of DPP3 is an antibody or fragment or scaffold which binds to full-length DPP3 and inhibits the activity of DPP3 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%.

15. The amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample from said subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating the DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or Quantifying the amount of the DPP3 protein 15. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 14, comprising:

16. The method for determining DPP3 activity in a body fluid sample from said subject comprises the steps of: contacting the sample with a capture binder that specifically binds full-length DPP3; separating the DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; Quantifying the DPP3 activity by measuring and quantifying the conversion of a substrate of DPP3.

16. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 15, comprising:

17. 17. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 16, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

18. The angiotensin receptor agonist and / or its precursor for use in the treatment of a disease in a subject according to any one of claims 1 to 17, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

19. 20. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 18, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being immobilised on a surface.

20. 20. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 16 to 19, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step in which components of the sample that are not bound to the capture binder are removed from the captured DPP3.

21. 21. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 16 to 20, wherein DPP3 activity is determined in a body fluid sample of said subject and conversion of a substrate of DPP3 is detected by a method selected from the group comprising: fluorescence of a fluorescent substrate (e.g. Arg-Arg-βNA, Arg-Arg-AMC), colour change of a chromogenic substrate, luminescence of a substrate bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilised active DPP3) or Western blot (cleavage products).

22. 22. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 16 to 21, wherein DPP3 activity is determined in a body fluid sample of said subject and said substrate is selected from the group comprising angiotensin II, III, IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, beta-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, whereby said dipeptide is Arg-Arg.

23. 23. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 16 to 22, wherein DPP3 activity is determined in a body fluid sample of said subject and said substrate is selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, wherein said dipeptide is Arg-Arg.

24. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or their precursors.

25. Use of the method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonist and / or precursors thereof according to claim 24, wherein the angiotensin receptor agonist and / or precursors thereof are selected from the group comprising angiotensin I, angiotensin II, angiotensin III and angiotensin IV, in particular angiotensin II is selected.

26. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or its precursors according to claim 24 or 25, wherein the amount of DPP3 protein and / or the DPP3 activity is determined in a body fluid sample of the subject at least once before and / or during treatment with the angiotensin receptor agonist and / or its precursors.

27. 27. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject as a therapeutic guide and / or therapeutic monitor for treatment with an angiotensin receptor agonist and / or its precursor according to any one of claims 24 to 26, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum.

28. The amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample from said subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating the DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or Quantifying the amount of the DPP3 protein 28. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or precursor thereof according to any one of claims 24 to 27, comprising:

29. The method for determining DPP3 activity in a body fluid sample from said subject comprises the steps of: contacting the sample with a capture binder that specifically binds full-length DPP3; separating the DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; Quantifying the DPP3 activity by measuring and quantifying the conversion of a substrate of DPP3.

29. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or precursor thereof according to any one of claims 24 to 28, comprising:

30. 30. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or its precursors according to claim 28 or 29, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

31. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or precursor thereof according to any one of claims 28 to 30, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

32. 32. Use of a method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or precursor thereof according to any one of claims 28 to 31, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being immobilized on a surface.

33. Use of the method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or its precursors according to any one of claims 28 to 32, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step that removes components of the sample that are not bound to the capture binder from the captured DPP3.

34. 34. Use of the method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof according to any one of claims 28 to 33, wherein DPP3 activity is determined in a body fluid sample of said subject and conversion of a substrate of DPP3 is detected by a method selected from the group comprising: fluorescence of fluorescent substrates (e.g. Arg-Arg-βNA, Arg-Arg-AMC), color change of chromogenic substrates, luminescence of substrates bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3) or Western blot (cleavage products).

35. 35. Use of the method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof according to any one of claims 28 to 34, wherein DPP3 activity is determined in a body fluid sample of said subject and said substrate is selected from the group comprising angiotensin II, III, IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, wherein said dipeptide is Arg-Arg.

36. 36. Use of the method or assay or kit for determining the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of a subject as a therapeutic guide and / or therapeutic monitoring for treatment with an angiotensin receptor agonist and / or precursor thereof according to any one of claims 28 to 35, wherein DPP3 activity is determined in a body fluid sample of said subject and said substrate is selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, wherein said dipeptide is Arg-Arg.

37. 24. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 23, wherein said treatment with an angiotensin receptor agonist and / or precursor thereof is discontinued when the patient has an amount of DPP3 protein and / or DPP3 activity in a body fluid sample that is below a predetermined threshold level.

38. 40. An angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject according to any one of claims 1 to 23 and 37, wherein said treatment with an angiotensin receptor agonist and / or precursor thereof is initiated when the patient has an amount of DPP3 protein and / or DPP3 activity in a body fluid sample above a pre-determined threshold level.

39. 1. A method for predicting risk of an adverse event in a subject, comprising: determining the level of DPP3 in a body fluid sample from said subject; comparing the determined level of DPP3 with a predefined threshold; correlating said level of DPP3 with a risk of an adverse event in said subject; wherein elevated levels above a certain threshold are predictive of increased risk of said adverse event.

40. 40. The method for predicting the risk of an adverse event in a subject according to claim 39, wherein the subject's bodily fluid sample is selected from whole blood, plasma, and serum.

41. The amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample from said subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating the DPP3 bound to the capture binder; A step of quantifying the DPP3 activity by adding a DPP3 substrate to the separated DPP3 and measuring and quantifying the conversion of the DPP3 substrate; or Quantifying the amount of the DPP3 protein 41. A method for predicting the risk of an adverse event in a subject according to claim 39 or 40, comprising:

42. The method for determining DPP3 activity in a body fluid sample of the subject comprises: contacting the sample with a capture binder that specifically binds full-length DPP3; separating the DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; Quantifying the DPP3 activity by measuring and quantifying the conversion of a substrate of DPP3. A method for predicting the risk of an adverse event in a subject according to any one of claims 39 to 41, comprising:

43. 43. A method for predicting the risk of an adverse event in a subject according to any one of claims 39 to 42, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

44. 42. A method for predicting the risk of an adverse event in a subject according to any one of claims 37 to 41, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

45. 45. A method for predicting the risk of an adverse event in a subject according to any one of claims 39 to 44, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being immobilized on a surface.

46. A method for predicting the risk of an adverse event in a subject according to any one of claims 39 to 45, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step that removes components in the sample that are not bound to the capture binder from the captured DPP3.

47. 47. The method for predicting the risk of an adverse event in a subject according to any one of claims 39 to 46, wherein DPP3 activity is determined in a body fluid sample of the subject and conversion of a DPP3 substrate is detected by a method selected from the group comprising: fluorescence of a fluorescent substrate (e.g. Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate bound to aminoluciferin (Promega's Protease-Glo™ assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3), or Western blot (cleavage products).

48. 48. A method for predicting the risk of an adverse event in a subject according to any one of claims 39 to 47, wherein DPP3 activity is determined in a body fluid sample of the subject, and the substrate is selected from the group comprising angiotensin II, III, IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, whereby the dipeptide is Arg-Arg.

49. 49. A method for predicting the risk of an adverse event in a subject according to any one of claims 39 to 48, wherein DPP3 activity is determined in a body fluid sample of the subject, and the substrate is selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, wherein the dipeptide is Arg-Arg.

50. 1. A method for predicting risk of organ dysfunction or organ function deterioration in a subject, comprising: determining the level of DPP3 in a body fluid sample from said subject; comparing the determined level of DPP3 with a predetermined threshold; correlating said level of DPP3 with said risk of organ dysfunction or worsening organ function in said subject. wherein elevated levels above a threshold are predictive of increased risk of organ dysfunction or deterioration of organ function.

51. 51. The method for predicting the risk of organ dysfunction or deterioration in a subject according to claim 50, wherein the subject's bodily fluid sample is selected from whole blood, plasma, and serum.

52. The amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample from said subject, comprising: contacting the sample with a capture binder that specifically binds full-length DPP3; separating the DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3 and then quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate; or Quantifying the amount of the DPP3 protein 52. A method for predicting the risk of organ dysfunction or organ function deterioration in a subject according to claim 50 or 51, comprising:

53. The method for determining DPP3 activity in a body fluid sample from said subject comprises: contacting the sample with a capture binder that specifically binds full-length DPP3; separating the DPP3 bound to the capture binder; adding a DPP3 substrate to the separated DPP3; Quantifying the DPP3 activity by measuring and quantifying the conversion of a substrate of DPP3.

53. A method for predicting the risk of organ dysfunction or organ function deterioration in a subject according to any one of claims 50 to 52, comprising:

54. 54. A method for predicting the risk of organ dysfunction or worsening organ function in a subject according to any one of claims 50 to 53, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

55. 55. A method for predicting the risk of organ dysfunction or worsening organ function in a subject according to any one of claims 50 to 54, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of said subject, said determining comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder being an antibody.

56. 56. A method for predicting the risk of organ dysfunction or worsening organ function in a subject according to claims 50 to 55, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, said determining comprising the use of a capture binder that specifically binds full-length DPP3, said capture binder being immobilized on a surface.

57. 57. A method for predicting the risk of organ dysfunction or organ function deterioration in a subject according to any one of claims 50 to 56, wherein the amount of DPP3 protein and / or DPP3 activity is determined in a body fluid sample of the subject, and the separation step is a washing step in which components of the sample that are not bound to the capture binder are removed from the captured DPP3.

58. 58. The method for predicting the risk of organ dysfunction or deterioration in a subject according to claims 50 to 57, wherein DPP3 activity is determined in a body fluid sample of the subject and conversion of a substrate of DPP3 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 bound to aminoluciferin (Promega's Protease-Glo™ Assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized active DPP3), or Western blot (cleavage products).

59. 59. A method for predicting the risk of organ dysfunction or worsening organ function in a subject according to any one of claims 50 to 58, wherein DPP3 activity is determined in a body fluid sample of said subject, and said substrate is selected from the group comprising angiotensin II, III, IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, whereby said dipeptide is Arg-Arg.

60. 60. A method for predicting the risk of organ dysfunction or worsening organ function in a subject according to any one of claims 50 to 59, wherein DPP3 activity is determined in a body fluid sample of the subject, and the substrate is selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, wherein the dipeptide is Arg-Arg.

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