Therapy guidance and / or therapy monitoring for treatment of shock

Abstract

To provide a method for predicting or diagnosing intractable shock in a subject suffering from shock or having developed shock.SOLUTION: The method comprises the steps of: measuring the concentration of DPP3 in a bodily fluid sample of said subject; and comparing said measured concentration of DPP3 with a predetermined threshhold value, wherein said subject is predicted to suffer from or develop intractable shock if said concentration of DPP3 exceeds said predetermined threshhold value. The subject-matter further relates to vasopressors, angiotensin receptor agonists and / or precursors thereof, inhibitors of DPP3 activity and anti-ADM antibodies for use in the treatment of shock in a subject suffering from or having developed shock.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock, a blood pressure vasopressor, an angiotensin receptor agonist and / or a precursor thereof, a DPP-3 inhibitor, and an anti-ADM antibody for use in treating shock in a subject suffering from or having developed shock, and a method for treating said shock or refractory shock. [Background technology]

[0002] Dipeptidyl peptidase 3, also known as dipeptidyl aminopeptidase III, dipeptidyl arylamidase III, dipeptidyl peptidase III, enkephalinase B, or erythrocyte angiotensinase (abbreviated as DPP3 or DPP III), is a metallopeptidase that removes dipeptides from bioactive peptides such as enkephalins and angiotensin.

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

[0004] DPP3 is a zinc-dependent exopeptidase belonging to the peptidase family M49. It has broad substrate specificity for oligopeptides of 3 or 4 to 10 amino acids of various compositions and can cleave after proline. DPP3 is known to hydrolyze dipeptides from the N-terminus of its substrates, such as angiotensin II, III, and IV; leucine- and methionine-enkephalins; and endomorphin 1 and 2. The metallopeptidase DPP3 has optimal activity at a pH of 8.0 to 9.0 and is highly cleavable by Co. 2+ and Mg 2+ It can be activated by the addition of divalent metal ions such as

[0005] Structural analysis of DPP3 has revealed the catalytic motifs HELLGH (hDPP3 450-455) and EECRAE (hDPP3 507-512), as well as the following amino acids important for substrate binding and hydrolysis: Glu316, Tyr318, Asp366, Asn391, Asn394, His568, Arg572, Arg577, Lys666, and Arg669 (Prajapati & Chauhan 2011; Kumar et al. 2016; numbers refer to the human DPP3 sequence; see SEQ ID NO: 1). Considering all known amino acids or sequence regions involved in substrate binding and hydrolysis, the active site of human DPP3 can be determined to be the region between amino acids 316 and 669.

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

[0007] Although the exact biological function of DPP3 in cellular physiology remains to be elucidated, recent findings indicate its role in pain regulation and inflammatory processes, as well as protein metabolism (Prajapati & Chauhan 2011). Furthermore, DPP3 reduced blood pressure in hypertensive mice infused with Ang II without altering heart rate (PAng et al. 2016). However, normotensive mice showed no change in blood pressure after injection of DPP3.

[0008] The blood pressure reduction caused by administration of DPP3 together with an angiotensin receptor antagonist in Ang II-infused mice was similar to that caused by injection of DPP3 alone or an angiotensin receptor antagonist alone (Pang et al. 2016. Hypertension 68:630-41).

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

[0010] Treatment with Ang II is currently being investigated for its beneficial effects in vasodilatory shock and septic shock (Khanna, A. et al., 2017; Antonucci, E. et al. 2017, Tumlin, JA et al. 2018). Patients with vasodilatory shock (80% of septic shock cases) treated with angiotensin II are more likely to survive for 28 days and show significant improvement in hypotension (Khanna, A. et al., 2017; Tumlin, JA et al. 2018).

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

[0012] Recently, two assays have been developed, evaluated, and validated to specifically detect DPP3 in human body fluids (e.g., blood, plasma, serum): a luminescence immunoassay (LIA) to detect DPP3 protein concentration and an enzyme capture activity assay (ECA) to detect specific DPP3 activity (Rehfeld et al. 2019 JALM 3(6):943-953). The wash steps in both of these methods remove all interfering substances before the actual detection of DPP3 protein or activity occurs. Both methods are highly specific and allow for reproducible detection of DPP3 in blood samples. Summary of the Invention [Problem to be solved by the invention]

[0013] An object of the present invention is to provide a method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock.

[0014] Another object of the present invention is to provide a vasopressor, an angiotensin receptor agonist and / or a precursor thereof, a DPP3 inhibitor, and an anti-ADM antibody for use in the treatment of shock in a subject suffering from or developing shock. [Means for solving the problem]

[0015] The subject of the present invention is a method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock, said method comprising: measuring the concentration of DPP3 in a body fluid sample from said subject; comparing the measured DPP3 concentration with a predetermined threshold, If the measured DPP3 concentration exceeds the predetermined threshold, the subject is predicted to suffer from refractory shock or is diagnosed as suffering from refractory shock.

[0016] The subject of the present invention is a method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock, said method comprising: measuring the concentration of DPP3 in a body fluid sample from said subject; and comparing the measured concentration of DPP3 with a predetermined threshold; and measuring the concentration of proadrenomedullin or a fragment thereof in a body fluid sample from said subject; and comparing the measured concentration of proadrenomedullin or a fragment thereof with a predetermined threshold value, If the measured concentration of proadrenomedullin or a fragment thereof and / or the concentration of DPP3 exceeds the predetermined threshold, the subject is predicted to suffer from refractory shock or is diagnosed as suffering from refractory shock.

[0017] In certain embodiments of the method of predicting or diagnosing refractory shock in a subject suffering from or developing shock, the shock is selected from the group comprising hypovolemic shock, cardiogenic shock, vaso-occlusive shock, and distributive shock.

[0018] In another particular embodiment of the method for predicting or diagnosing refractory shock in a subject suffering from or developing shock, said shock is selected from the group comprising hypovolemic shock, cardiogenic shock, vaso-occlusive shock, and distributive shock, in particular cardiogenic or septic shock.

[0019] In certain embodiments of the method of predicting or diagnosing refractory shock in a subject suffering from or developing shock, the shock is selected from the group comprising cardiogenic shock, hypovolemic shock, vaso-occlusive shock, and distributive shock; In the case of cardiogenic shock, the subject is suffering from acute coronary syndrome (e.g., acute myocardial infarction) or heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism; or In the case of hypovolemic shock, the subject may be suffering from a bleeding disorder, including spontaneous bleeding due to gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor invasion of major blood vessels), and in the setting of anticoagulant use, or a non-hemorrhagic disorder, including vomiting, diarrhea, renal failure, renal loss, skin loss / insensible loss (e.g., burns, heat stroke), or third-space loss in the setting of pancreatitis, cirrhosis, intestinal obstruction, or trauma; or In the case of vaso-occlusive shock, the patient may have cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or In distributive shock, the patient has septic shock, neurogenic shock, anaphylactic shock, or shock due to adrenal crisis.

[0020] In another embodiment of the method for predicting or diagnosing refractory shock in a subject who has or has developed shock, the method is used to initiate and / or terminate and / or stratify and / or guide treatment. [Brief explanation of the drawings]

[0021] [Figure 1]Kaplan-Meier survival rates associated with low DPP-3 concentrations (<40.5 ng / mL) and high DPP-3 concentrations (≥40.5 ng / mL). (A) Seven-day survival rate for patients with sepsis versus DPP-3 plasma concentration; (B) Seven-day survival rate for patients with cardiogenic shock versus DPP-3 plasma concentration; (C) Seven-day survival rate for patients with septic shock versus DPP-3 plasma concentration.

[0022] [Figure 2] SDS-PAGE on a gradient gel (4-20%) of native hDPP3 purified from human erythrocyte lysate. Molecular weight markers are indicated by arrows.

[0023] [Figure 3] 1 shows experimental conditions for the effects of native DPP 3 in animal models.

[0024] [Figure 4] Figure 4(A) shows that DPP 3 injection causes a decrease in contractility, thereby worsening cardiac function, and Figure 4(B) shows that a further decrease in renal function is observed with an increase in the renal resistive index.

[0025] [Figure 5] Association and dissociation curves for AK1967-DPP 3 binding assay using Octet are shown. AK1967-loaded biosensors were immersed in a dilution series of recombinant GST-tagged human DPP 3 (100, 33.3, 11.1, 3.7 nM) to measure association and dissociation.

[0026] [Figure 6] Western blot of dilutions of blood cell lysates and detection of DPP 3 using AK1967 as the primary antibody are shown.

[0027] [Figure 7]Figure 1 shows the inhibition curve of natural DPP-3 from blood cells by the inhibitory antibody AK1967. The inhibition of DPP-3 by the specific antibody is concentration-dependent, with an IC50 of approximately 15 ng / mL when assayed against 15 ng / mL of DPP-3.

[0028] [Figure 8] Experimental conditions for the effect of procizumab on sepsis-induced heart failure.

[0029] [Figure 9] Figure 1 shows that procizumab dramatically improves (A) contractility and (B) mortality in rats with sepsis-induced heart failure.

[0030] [Figure 10] Experimental conditions are shown for mice subjected to isoproterenol-induced cardiac stress followed by treatment with (B) procizumab and (A) control.

[0031] [Figure 11] Figure 1 shows that (A) contractility improved and (B) renal resistive index decreased within 1 and 6 hours after administration of procizumab in mice with isoproterenol-induced heart failure.

[0032] [Figure 12] Figure 12(A) (left panel) shows the number of days requiring vasopressor therapy in patients with septic shock versus DPP-3 plasma concentration. ≥7 indicates vasopressor therapy for ≥7 days or death within 7 days; * indicates p<0.05 in a post-hoc comparison with the 1-4 day group. Figure 12(B) (right panel) shows the need for vasopressor therapy in patients with septic shock versus DPP-3 plasma concentration. Patients who received vasopressor therapy for up to 5 days (≤5) and patients who received vasopressor therapy for more than 5 days or died within 7 days (>5) were grouped together.

[0033] [Figure 13]DPP-3 is associated with refractory shock. High DPP-3 plasma concentrations in patients with cardiogenic shock are associated with a higher risk of developing refractory cardiogenic shock compared with patients with DPP-3 plasma concentrations below a certain threshold.

[0034] [Figure 14] Kaplan-Meier survival (KMS) data show (A) 4-week survival rates for patients with septic shock associated with bio-ADM plasma concentrations; (B) 4-week survival rates for patients with septic shock associated with DPP-3 plasma concentrations; and (C) 4-week survival rates for patients with septic shock associated with bio-ADM and DPP-3 plasma concentrations. Plasma concentrations are grouped into quartiles, and thresholds were determined based on the third quartile of each marker's measured plasma concentration: low DPP-3 < 48.4 ng / mL; low bio-ADM < 213 pg / mL; high bio-ADM ≥ 213 pg / mL; and high DPP-3 ≥ 48.4 ng / mL.

[0035] [Figure 15] Patients requiring vasopressors are shown, grouped according to DPP-3 and bio-ADM plasma concentrations at admission. The proportion of patients receiving vasopressors for 1 to 7 days is shown in grayscale, with lighter colors representing longer treatment durations. Thresholds were assigned based on Q3 (highest 25% of measurements) for both plasma concentrations: low DPP-3 < 48.4 ng / mL; low bio-ADM < 213 pg / mL; high bio-ADM ≥ 213 pg / mL; high DPP-3 ≥ 48.4 ng / mL, and 7 indicating ≥ 7 days of vasopressor administration or death within 7 days.

[0036] [Figure 16]Figure 16(A) shows plasma DPP-3 concentrations in patients with septic refractory shock requiring vasopressor norepinephrine at or below 0.5 μg / kg / min (n=186) and above 0.5 μg / kg / min (n=95) (p<0.001). Figure 16(B) shows Kaplan-Meier survival plots of 4-week survival rates for patients with septic refractory shock versus DPP-3 plasma concentrations, with plasma concentration values ​​grouped into quartiles. DETAILED DESCRIPTION OF THE INVENTION

[0037] As used herein, the term "guiding a therapy" or "guiding a therapy" refers to the application of a particular therapy or medical intervention based on the value of one or more biomarkers and / or clinical parameters and / or clinical scores.

[0038] The term "monitoring therapy" in the context of the present invention means monitoring and / or adjusting the therapy of said patient, for example by obtaining feedback on the effectiveness of the therapy.

[0039] The term "treatment stratification" refers in particular to the grouping or classification of patients into different groups, for example treatment groups that receive or do not receive a treatment measure depending on the classification.

[0040] The term "prediction" refers to associating a likelihood of an outcome with the result obtained from measuring an analyte, such as measuring a particular marker, such as DPP3, in a sample and correlating the measured concentration with the probability that a subject who has or has developed shock will develop refractory shock.

[0041] The present invention also includes a method for short-term prediction of refractory shock in a subject who has fallen into or developed shock, where short-term means within 14 days, preferably within 7 days, more preferably within 3 days, and most preferably within 48 hours.

[0042] In another embodiment of the method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock, the treatment is initiated and / or maintained and / or withheld and / or terminated when the measured concentration of DPP3 exceeds the predetermined threshold.

[0043] In another embodiment of the method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock, the treatment is initiated and / or maintained and / or withheld and / or terminated when the measured concentration of DPP3 and / or proadrenomedullin and / or fragments thereof exceeds the predetermined threshold.

[0044] In a preferred embodiment of the method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock, the treatment is selected from the group of vasopressors, angiotensin receptor agonists or precursors thereof, and / or inhibitors of DPP3 activity.

[0045] In another preferred embodiment of the method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock, the treatment is selected from the group consisting of vasopressors, angiotensin receptor agonists and / or precursors thereof, inhibitors of DPP3 activity, and anti-adrenomedullin antibodies or anti-adrenomedullin antibody fragments.

[0046] In another embodiment of the method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock, either the concentration of DPP3 protein and / or the concentration of active DPP3 is measured and compared to a predetermined threshold value.

[0047] In another embodiment of the method for predicting or diagnosing refractory shock in a subject suffering from or having shock, the concentration of DPP3 is measured by contacting the sample of body fluid with a capture binding agent that specifically binds DPP3.

[0048] In another preferred embodiment of the method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock, the capture binding agent for measuring the concentration of DPP3 may be selected from an antibody, an antibody fragment, or a non-IgG scaffold.

[0049] In certain embodiments of the method for predicting or diagnosing refractory shock in a subject suffering from or having shock, the capture binding agent is an antibody.

[0050] In another particular embodiment of the method for predicting or diagnosing refractory shock in a subject suffering from or having shock, the capture binding agent is a monoclonal antibody.

[0051] In another embodiment of the method for predicting or diagnosing refractory shock in a subject suffering from or having shock, said sample of body fluid is selected from the group of whole blood, plasma, and serum.

[0052] In certain embodiments of the method of prognosing or diagnosing refractory shock in a subject who has or has developed shock, the method of diagnosing or prognosing is performed at least twice.

[0053] Shock is characterized by decreased oxygen delivery and / or increased oxygen consumption or inadequate oxygen utilization, leading to cellular and tissue hypoxia. It is a life-threatening condition of circulatory failure, most commonly manifested as hypotension (systolic blood pressure less than 90 mm Hg or mean arterial pressure (MAP) less than 65 mm Hg). Shock can be divided into four major forms based on the underlying cause: hypovolemic, cardiogenic, vaso-occlusive, and distributive shock (Vincent and De Backer 2014. N. Engl. J. Med. 370(6): 583).

[0054] Hypovolemic shock is characterized by intravascular volume loss and can be broadly divided into two subtypes: hemorrhagic and nonhemorrhagic. Common causes of hemorrhagic hypovolemic shock include gastrointestinal bleeding, trauma, vascular etiologies (e.g., ruptured abdominal aortic aneurysm, tumor invasion of major blood vessels), and spontaneous bleeding in the setting of anticoagulation. Common causes of nonhemorrhagic hypovolemic shock include vomiting, diarrhea, renal failure, renal loss, skin / insensible loss (e.g., burns, heat stroke), or third-space loss in the setting of pancreatitis, cirrhosis, intestinal obstruction, or trauma. For a review, see Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019-2018 Oct 27.

[0055] Cardiogenic shock (CS) is defined as a state of decreased blood flow within critical organs due to reduced cardiac output. CS manifests in a range of conditions, from mild hypoperfusion to severe shock. Established criteria for the diagnosis of CS are: (i) a systolic blood pressure of 90 mmHg or less for more than 30 minutes or the need for vasopressors to achieve a blood pressure of 90 mmHg or more; (ii) pulmonary congestion or elevated left ventricular filling pressure; and (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 (less than 0.5 mL / kg / h or less than 30 mL / h); and (d) elevated serum lactate (Reynolds and Hochman 2008. Circulation 117: 686-697). Acute myocardial infarction (AMI) and subsequent ventricular dysfunction are the most frequent causes of CS, accounting for approximately 80% of cases. Mechanical complications, such as ventricular septal rupture (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 may be caused by decompensated valvular heart disease, acute myocarditis, or arrhythmias due to heterogeneous treatment options. CS affects 40,000-50,000 patients annually in the United States and 60,000-70,000 patients in Europe. Despite advances in treatment, primarily through early revascularization, and subsequent declines in mortality, CS remains the leading cause of death from AMI, with mortality rates approaching 40-50% in recent population-based and randomized studies (Goldberg et al. 2009. Circulation 119: 1211-1219).

[0056] Vaso-occlusive shock is due to physical obstruction of the great vessels or the heart itself. Several conditions can cause this form of shock (e.g., cardiac tamponade, tension pneumothorax, pulmonary embolism, aortic stenosis). For a review, see Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019-2018 Oct 27.

[0057] Based on the cause, there are four types of distributive shock: neurogenic shock (decreased vascular tone due to decreased sympathetic stimulation); anaphylactic shock; septic shock; and shock due to adrenal insufficiency. In addition to sepsis, distributive shock can be caused by systemic inflammatory response syndrome (SIRS) due to conditions other than infection, such as pancreatitis, burns, or trauma. Other causes include toxic shock syndrome (TSS), anaphylaxis (a sudden, severe allergic reaction), adrenal insufficiency (acute worsening of chronic adrenal insufficiency, destruction or removal of the adrenal glands, suppression of adrenal function by exogenous steroids, hypopituitarism, and metabolic disorders of hormone production), reaction to drugs or toxins, heavy metal poisoning, liver failure, and damage to the central nervous system. For a review, see Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019-2018 Oct 27.

[0058] Refractory shock has generally been defined as requiring an infusion of norepinephrine greater than 0.5 μg / kg per minute in addition to adequate resuscitation. Mortality rates for these patients can be as high as 94%, and a much more aggressive approach to their evaluation and management is required to ensure survival. The term "refractory shock" is used when initial corrective measures (e.g., vasopressors) fail to restore tissue perfusion, resulting in the condition sometimes referred to as "hyperpressor-dependent" or "vasopressor-resistant" shock (Udupa and Shetty 2018. Indian J Respir Care 7: 67-72). Patients with refractory shock may have features of inadequate perfusion, such as hypotension (mean arterial blood pressure less than 65 mmHg), tachycardia, cold extremities, delayed capillary refill, and tachypnea due to hypoxia and acidosis. Septic shock may present with fever. Other signs of hypoperfusion, such as paresthesia, hyperlactatemia, and oliguria, may also be present. These well-known signs of shock do not help identify whether the problem is in the pump (heart) or the circuit (vasculature and tissues). Various types of shock can coexist, as evidenced by unresponsiveness to high-dose vasopressors, and all forms of shock can be refractory (Udupa and Shetty 2018. Indian J Respir Care 7: 67-72).

[0059] In certain embodiments of the invention, the refractory shock is vasopressor resistant, which is defined by the patient's unresponsiveness to high doses of vasopressors (e.g., greater than 0.5 μg / kg / min noradrenaline).

[0060] A diagnosis or prognosis that a subject has or will be diagnosed with vasopressor-resistant refractory shock may result in treatment, such as administration of an angiotensin II inhibitor and / or a DPP3 inhibitor. Yet another result may be withholding vasopressors.

[0061] In a preferred embodiment of the method for predicting refractory shock, particularly vasopressor-resistant shock, in a subject who may be suffering from shock, whether or not the subject has suffered from refractory shock, particularly vasopressor-resistant shock, is predicted within 14 days, particularly within 10 days, particularly within 7 days, particularly within 5 days, particularly within 48 hours, particularly within 24 hours, particularly between 24 and 48 hours from the time the sample is collected.

[0062] Another embodiment of the present invention relates to a method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock, wherein treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP3 activity is initiated and / or continued when the concentration of DPP3 in the sample exceeds a certain threshold, and / or treatment with a vasopressor agent is withheld and / or terminated when the measured concentration of DPP3 exceeds said predetermined threshold.

[0063] Another particular embodiment of the present invention relates to a method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock, wherein treatment with a vasopressor is initiated and / or continued when the concentration of DPP3 in the sample is below a certain threshold, and / or treatment with an angiotensin receptor agonist and / or its precursor and / or an inhibitor of DPP3 activity is withheld and / or terminated when the measured concentration of DPP3 is below said predetermined threshold.

[0064] A further embodiment of the present invention relates to a method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock, comprising initiating and / or continuing treatment with a vasopressor when the concentration of DPP3 in the sample is below a certain threshold, and / or suspending and / or terminating treatment with an angiotensin receptor agonist and / or its precursor and / or an inhibitor of DPP3 activity when the measured concentration of DPP3 is below a certain threshold, and measuring the concentration of proadrenomedullin or a fragment thereof, and initiating and / or continuing treatment with an anti-ADM antibody or an anti-ADM antibody fragment when the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with an anti-ADM antibody or an anti-ADM antibody fragment when the measured concentration of proadrenomedullin or a fragment thereof is below a predetermined threshold.

[0065] A further embodiment of the present invention relates to a method for predicting or diagnosing refractory shock in a subject who has suffered from or developed shock according to the present invention, comprising: the subject may be suffering from an acute coronary syndrome (e.g., acute myocardial infarction) (in the case of cardiogenic shock), or the subject is suffering from heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or The subject may have a bleeding disorder, including spontaneous bleeding in the setting of gastrointestinal bleeding (in the case of hypovolemic shock), trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor invasion into major blood vessels) and anticoagulant use, or a non-hemorrhagic disorder, including vomiting, diarrhea, renal failure, renal loss, skin loss / insensitivity loss (e.g., burns, heat stroke), or third-space loss in the setting of pancreatitis, cirrhosis, intestinal obstruction, or trauma; or the subject may be suffering from cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis (in the case of vaso-occlusive shock); or The subject may be suffering from septic shock (in the case of distributive shock), neurogenic shock, anaphylactic shock, or shock due to adrenal insufficiency.

[0066] A further embodiment of the invention relates to a method for predicting or diagnosing refractory shock in a subject who has suffered from or has developed shock according to the invention, which method is used to initiate and / or terminate and / or stratify and / or guide treatment.

[0067] A further embodiment of the present invention relates to a method for predicting or diagnosing refractory shock in a subject who has suffered from or has developed shock according to the present invention, wherein treatment is initiated and / or maintained and / or withheld and / or terminated if the measured DPP3 concentration exceeds the predetermined threshold.

[0068] The subject of the present invention is a vasopressor, an angiotensin receptor agonist and / or a precursor thereof, an inhibitor of DPP3 activity, and / or an anti-adrenomedullin antibody or an anti-adrenomedullin antibody fragment for use in a method for preventing or treating refractory shock in a subject suffering from or having developed shock, wherein the prediction and / or diagnosis of refractory shock is determined by the method of the present invention for predicting or diagnosing refractory shock in a subject suffering from or having developed shock. In one embodiment of the method, either the concentration of DPP3 protein and / or the concentration of active DPP3 is measured and compared with a predetermined threshold, and the concentration of DPP3 is measured by contacting the sample of body fluid with a capture binding agent that specifically binds to DPP3.

[0069] The subject of the present invention is an angiotensin receptor agonist and / or a precursor thereof and an inhibitor of DPP-3 activity for use in a method for preventing or treating refractory shock in a subject suffering from or having developed shock, wherein said prediction and / or diagnosis of refractory shock is determined by a method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock according to the present invention, wherein treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued when the concentration of DPP-3 in the sample exceeds a certain threshold, and / or treatment with a vasopressor is suspended and / or terminated when the measured concentration of DPP-3 exceeds said predetermined threshold.

[0070] The subject of the present invention is an angiotensin receptor agonist and / or a precursor thereof and an inhibitor of DPP-3 activity for use in a method for preventing or treating refractory shock in a subject suffering from or having developed shock, wherein said prediction and / or diagnosis of refractory shock is determined by a method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock according to the present invention, wherein treatment with a vasopressor is initiated and / or continued when the concentration of DPP-3 in the sample is below a certain threshold, and / or treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is suspended and / or terminated when the measured concentration of DPP-3 is below said predetermined threshold.

[0071] The subject of the present invention is an anti-ADM antibody or an anti-ADM antibody fragment for use in a method for preventing or treating refractory shock in a subject suffering from or having developed shock, wherein the prediction and / or diagnosis of refractory shock is determined by a method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock according to the present invention, further comprising measuring the concentration of proadrenomedullin or a fragment thereof, and starting and / or continuing treatment with the anti-ADM antibody or anti-ADM antibody fragment if the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with the anti-ADM antibody or anti-ADM antibody fragment if the measured concentration of proadrenomedullin or a fragment thereof is below the predetermined threshold.

[0072] Another embodiment of the present invention relates to a method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock, wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment and / or an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued when the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold and the measured concentration of DPP-3 exceeds the predetermined DPP-3 threshold.

[0073] The subject of the present invention is an anti-ADM antibody or an anti-ADM antibody fragment and / or an inhibitor of DPP-3 activity for use in a method for preventing or treating refractory shock in a subject suffering from or having developed shock, wherein said prediction and / or diagnosis of refractory shock is determined by the method of the present invention for predicting or diagnosing refractory shock in a subject suffering from or having developed shock, and wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment and / or an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued when proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold and the measured concentration of DPP-3 exceeds the predetermined DPP-3 threshold.

[0074] In certain embodiments of the present invention, the plasma DPP 3 threshold value is within a threshold range of 20-200 ng / mL, preferably 25-150 ng / mL, even more preferably 30-100 ng / mL, even more preferably 35-75 ng / mL, and most preferably a threshold value of 50 ng / mL is used. In particular, the refractory shock in said patient is vasopressor-resistant shock.

[0075] The peptide adrenomedullin (ADM) was isolated from human pheochromocytoma and described as a novel antihypertensive peptide containing 52 amino acids (Kitamura et al. 1993. Biochemical and Biophysical Research Communications 192 (2): 553-560). Cleavage of the 21-amino acid N-terminal signal sequence from preproadrenomedullin (pre-proADM) yields the precursor peptide proadrenomedullin (pro-ADM) (SEQ ID NO: 31). Pro-ADM is further cleaved into PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), ADM-Gly (SEQ ID NO: 35), and CT-proADM (SEQ ID NO: 36). The mature adrenomedullin peptide is an amidated peptide (ADM-NH2), which contains 52 amino acids (SEQ ID NO: 34) and includes amino acids 95-146 of pre-proADM, from which it is generated by proteolytic cleavage. Mature ADM, bio-ADM, and ADM-NH2 are used synonymously throughout this application and refer to the molecule according to SEQ ID NO:34.

[0076] In one embodiment of the present subject matter, the fragment of proadrenomedullin is selected from the group comprising PAMP (SEQ ID NO: 32), MR-proADM (SEQ ID NO: 33), amidated ADM (SEQ ID NO: 34), ADM-Gly (SEQ ID NO: 35), and CT-proADM (SEQ ID NO: 36).

[0077] ADM may be considered a multifunctional regulatory peptide. It is released into the circulation in part in an inactive, glycine-extended form (Kitamura et al. 1998. Biochem. Biophys. Res. Commun. 244(2): 551-555). Binding proteins specific for ADM and likely modulate its effects as well (Pio et al. 2001. The Journal of Biological Chemistry 276(15): 12292-12300) also exist.

[0078] To date, the most important physiological effect of ADM and PAMP has been shown to be their effect on blood pressure. Therefore, ADM is an effective vasodilator. It has been shown that measurable concentrations of ADM in circulatory and other body fluids significantly exceed those found in healthy controls in many pathological conditions. Thus, ADM concentrations are significantly elevated, albeit to varying degrees, in subjects with congestive heart failure, myocardial infarction, kidney disease, hypertensive disorders, diabetes mellitus, acute shock, and sepsis and septic shock. PAMP concentrations are also elevated in several of these pathological conditions, although plasma concentrations are reduced compared to ADM (Eto et al. 2001. Peptides 22: 1693-1711).

[0079] Furthermore, abnormally high concentrations of ADM are known to be observed in sepsis or septic shock (Eto et al. 2001. Peptides 22: 1693-1711; Hirata et al. 1996. Journal of Clinical Endocrinology and Metabolism 81(4): 1449-1453; Ehlenz et al. 1997. Exp Clin Endocrinol Diabetes 105: 156-162; Tomoda et al. 2001. Peptides 22: 1783-1794; Ueda et al. 1999 Am. J. Respir. Crit. Care Med. 160: 132-136; Wang et al. 2001. Peptides 22: 1835-1840). This finding is associated with the typical hemodynamic changes known to be typical events in the course of disease in patients with sepsis and other severe syndromes, such as SIRS. Adrenomedullin plays an important role in the development of sepsis (Wang, Shock 1998, 10(5):383-384; Wang et al. 1998. Archives of surgery 133(12): 1298-1304) and in many acute and chronic diseases (Parlapiano et al. 1999. European Review for Medical and Pharmacological Sciences 3:53-61; Hinson et al. 2000 Endocrine Reviews 21(2):138-167).

[0080] MR-proADM has been identified as a prognostic marker that can stratify the risk of mortality in septic patients with various degrees of organ failure, which may aid in the early detection and individual risk assessment of sepsis, and further facilitate the subsequent clinical management of sepsis and septic shock (for review see Onal et al. 2018. Healthcare 6: 110).

[0081] Several methods have been described to measure circulating levels of ADM, either directly or indirectly by determining the more stable fragments of its cognate precursor peptide. One method has recently been published that describes a method to measure circulating mature ADM (Weber et al. 2017. JALM 2: 222-233).

[0082] Other methods for quantifying fragments derived from ADM precursors have been described, such as by measuring MR-proADM (Morgenthaler et al. 2005. Clin Chem 51(10):1823-9), PAMP (Washimine et al. 1994. Biochem BiopHys Res Commun 202(2):1081-7), and CT-proADM (EP 2 111 552). A commercially available homogeneous time-resolved fluoroimmunoassay for measuring MR-proADM in plasma using a fully automated system is available (BRAHMS MR-proADM KRYPTOR; BRAHMS GmbH, Hennigsdorf, Germany) (Caruhel et al. 2009. Clin Biochem 42(7-8):725-8). Because these peptides are produced in stoichiometric ratios from the same precursor, their plasma concentrations are somewhat correlated.

[0083] The generation of anti-ADM antibodies is known in the art (e.g., WO 2013072512 and WO 2019057992, which are incorporated herein by reference).

[0084] Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment for use in treating shock in a subject suffering from or experiencing shock, wherein the antibody or fragment or scaffold binds to the N-terminal portion (aa 1-21) of mature ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 37).

[0085] In another preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment recognizes and binds to the N-terminus (aa 1) of ADM. By N-terminus, we mean that amino acid 1, "Y" in SEQ ID NOs: 34 and 35, is essential for antibody binding. The antibody or fragment or non-Ig scaffold is not expected to bind to either N-terminally extended ADM, N-terminally modified ADM, or N-terminally degraded ADM.

[0086] Another embodiment of the present invention relates to an anti-ADM antibody or anti-ADM antibody fragment for use in treating shock in a subject suffering from or having shock, wherein the antibody or fragment is a human monoclonal antibody or fragment that binds to the N-terminal region (aa 1-21) of mature ADM (SEQ ID NO: 37) or an antibody fragment thereof.

[0087] Here, the heavy chain comprises the sequences GYTFSRYW (CDR1: SEQ ID NO: 38), ILPGSGST (CDR2: SEQ ID NO: 39), TEGYEYDGFDY (CDR3: SEQ ID NO: 40), and the light chain comprises the sequences QSIVYSNGNTY (CDR1: SEQ ID NO: 41), RVS (CDR2), FQGSHIPYT (CDR3: SEQ ID NO: 42).

[0088] Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment for use in treating shock in a subject suffering from or having shock, wherein the antibody is a monoclonal antibody comprising the following sequence as its heavy chain: [Table 1] (SEQ ID NO: 43) This monoclonal antibody contains the following sequence as the light chain: [Table 2] (SEQ ID NO: 44)

[0089] Endothelial dysfunction, such as that resulting in shock, particularly septic shock, may result from and / or contribute to several disease processes. Preclinical studies of sepsis / septic shock models have shown that administration of anti-ADM antibodies induces an increase in plasma bio-ADM concentrations, which is associated with increased survival rates (Struck et al. 2013. Intensive Care Med Exp 1(1):22). The mechanism underlying this effect was described by Geven et al. in 2018 (Geven et al. 2018. SHOCK 50 (2): 132-140). Briefly, when administered intravenously, this antibody, due to its size, cannot cross the endothelial barrier and enter the interstitium, but remains in the circulation. In contrast, ADM, a small peptide, can freely diffuse across the endothelial barrier. Thus, when administered in large molar excess over endogenous ADM, this antibody binds to virtually all ADM in plasma, simply reaching binding equilibrium and inducing ADM translocation from the interstitium into the circulation. ADM located between cells can bind to vascular smooth muscle cells, inducing relaxation and causing vasodilation, which is reduced by antibody administration. Meanwhile, ADM in plasma binds to endothelial cells, thereby stabilizing or restoring vascular integrity. This function is therefore enhanced by the increase in plasma ADM concentration resulting from the administration of a non-neutralizing antibody. Consequently, antibody binding to ADM reduces the attenuation of ADM proteolysis. In summary, the anti-ADM antibody, adrecizumab, can restore endothelial function during shock, e.g., septic shock.

[0090] If the concentration of proadrenomedullin or a fragment thereof exceeds a certain threshold concentration, an anti-ADM antibody or an anti-ADM antibody fragment that binds to ADM is administered as a therapeutic intervention.

[0091] This means that, in the subject of certain embodiments of the present invention, said anti-ADM antibody or anti-ADM antibody fragment that binds to ADM is therapeutic, where a body fluid sample taken from said patient shows a high concentration of proADM and / or its fragments having at least 5 amino acids above a certain threshold, and a diagnostic method using said proADM and / or fragments therefore serves as a companion diagnostic method.

[0092] In certain embodiments of the invention, the threshold value for plasma ADM-NH2 is within a threshold range of 50-250 pg / mL, preferably 55-200 pg / mL, even more preferably 60-150 pg / mL, even more preferably 65-100 pg / mL, and most preferably a threshold value of 70 pg / mL is used.

[0093] In a particular embodiment of the invention, the threshold value for plasma MR-proADM is within a threshold range of 0.5 to 3 nmol / L, preferably 0.6 to 2 nmol / L, even more preferably 0.7 to 1 nmol / L, most preferably a threshold value of 0.8 nmol / L is used.

[0094] In a particular embodiment of the invention, the threshold value for plasma CT-proADM is in a threshold range of 85-500 pmol / L, preferably 90-350 pmol / L, even more preferably 95-250 pmol / L, even more preferably 100-200 pmol / L, most preferably a threshold value of 150 pmol / L is used.

[0095] The ADM-NH2 concentration or proADM or fragment thereof concentration of the present invention was measured using the described ADM-NH2 measurement method (or the measurement method for proADM or fragment thereof, respectively) as outlined in the Examples (Weber et al. 2017. JALM 2(2):1-4). The DPP-3 concentration of the present invention was measured using the described DPP-3 measurement method as outlined in the Examples. The above-mentioned thresholds may be different for other measurement methods if they are calibrated differently from the measurement system used in the present invention. Therefore, the above-mentioned thresholds should be adapted accordingly to such differently calibrated measurement methods, taking into account the differences in calibration. One possibility for quantifying the differences in calibration is to measure each biomarker (e.g., bio-ADM, DPP-3) in a sample using both methods and then compare (correlate) the measurement method in question with the respective biomarker measurement method used in the present invention. Another possibility, if the test has sufficient analytical sensitivity, is to determine the median biomarker concentration of a representative normal population using the measurement method in question, compare the results with the median biomarker concentration reported in the literature, and recalculate the calibration based on the difference obtained by this comparison. Using the calibration method used in the present invention, samples from normal (healthy) subjects were measured, and the median plasma bio-ADM (mature ADM-NH2) was 24.7 pg / mL, the lowest value was 11 pg / mL, and the 99th percentile was 43 pg / mL (Marino et al. 2014. Critical Care 18:R34). Using the calibration method used in the present invention, samples from 5,400 normal (healthy) subjects (a Swedish single-center prospective population-based study (MPP-RES)) were measured, and the median plasma DPP-3 (interquartile range) was 14.5 ng / mL (11.3 ng / mL to 19 ng / mL).

[0096] The median plasma MR-proADM concentration in normal (healthy) subjects was 0.41 (interquartile range 0.23-0.64) nmol / L (Smith et al. 2009. Clin Chem 55:1593-1595) using the automated sandwich fluorometric assay for the detection of MR-proADM described by Caruhel et al. (Caruhel et al. 2009. Clin Biochem 42:725-8).

[0097] The median plasma concentration of CT-proADM in normal healthy subjects (n=200) was 77.6 pmol / L (minimum: 46.6 pmol / L, maximum: 136.2 pmol / L), with a 95th percentile of 113.8 pmol / L (EP 2 111 552 No. B1).

[0098] In certain embodiments of the invention, the threshold value for plasma ADM-NH2 is 5 times the median concentration of a normal, healthy population, preferably 4 times the median concentration, more preferably 3 times the median concentration, and most preferably 2 times the median concentration.

[0099] In a particular embodiment of the invention, the threshold value for plasma MR-proADM is 5 times the median concentration in a normal, healthy population, preferably 4 times the median concentration, more preferably 3 times the median concentration, and most preferably 2 times the median concentration.

[0100] In a particular embodiment of the invention, the threshold value for plasma CT-proADM is 5 times the median concentration in a normal, healthy population, preferably 4 times the median concentration, more preferably 3 times the median concentration, and most preferably 2 times the median concentration.

[0101] In an embodiment of the present invention, the anti-ADM antibody or anti-ADM antibody fragment used to treat a patient in need thereof may be administered at a dose of at least 0.5 mg / kg body weight, particularly at least 1.0 mg / kg body weight, and more particularly, 1.0 to 20.0 mg / kg body weight, for example, 2.0 to 10 mg / kg body weight, 2.0 to 8.0 mg / kg body weight, or 2.0 to 5.0 mg / kg body weight.

[0102] One embodiment of the present invention relates to a vasopressor agent for use in treating shock in a subject suffering from or having developed shock, wherein said subject has a concentration of DPP3 in a body fluid sample from said subject that is below a predetermined threshold as determined by any of the methods for predicting or diagnosing refractory shock according to the present invention.

[0103] Another embodiment of the present invention relates to a vasopressor agent for use in treating shock in a subject suffering from or experiencing shock, said vasopressor agent being selected from the group comprising dopamine, norepinephrine, norepinephrine equivalents, epinephrine, phenylephrine, and vasopressin.

[0104] Another particular embodiment of the present invention relates to a vasopressor for use in treating shock in a subject suffering from or having developed shock, said vasopressor being administered to said subject as a pharmaceutical formulation.

[0105] Another embodiment of the present invention relates to a vasopressor agent for use in the treatment of shock in a subject suffering from or having developed shock, wherein said subject has a blood pressure of 65 mm Hg or less.

[0106] Another embodiment of the present invention relates to an inhibitor of DPP3 activity for use in the treatment of shock in a subject suffering from or having developed shock, wherein the subject has a DPP3 concentration in a body fluid sample from the subject that exceeds a predetermined threshold as determined by the method described in the previous embodiment.

[0107] Another particular embodiment of the present invention relates to an inhibitor of DPP3 activity for use in the treatment of shock in a subject suffering from or having developed shock, wherein the inhibitor of DPP3 activity is selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

[0108] One embodiment of the present invention relates to an inhibitor of DPP 3 activity for use in the treatment of shock in a subject suffering from or having developed shock, said inhibitor comprising 10 -7 M or less.

[0109] Another embodiment of the present invention relates to an inhibitor of DPP 3 activity for use in the treatment of shock in a subject suffering from or having developed shock, wherein the inhibitor of DPP 3 activity is an antibody.

[0110] Another particular embodiment of the present invention relates to an inhibitor of DPP 3 activity for use in the treatment of shock in a subject suffering from or having developed shock, said inhibitor being a monoclonal antibody.

[0111] Another embodiment of the present invention relates to an inhibitor of DPP-3 activity for use in treating shock in a subject suffering from or having shock, said inhibitor being a monoclonal antibody whose heavy chain complementarity determining regions (CDRs) comprise the sequence of SEQ ID NO:7, SEQ ID NO:8, and / or SEQ ID NO:9 and whose light chain complementarity determining regions (CDRs) comprise the sequence of SEQ ID NO:10, KVS, and / or SEQ ID NO:11.

[0112] Another embodiment of the present invention relates to an inhibitor of DPP-3 activity for use in the treatment of shock in a subject suffering from or having shock, said inhibitor being a humanized monoclonal antibody or humanized monoclonal antibody fragment, the heavy chain of which comprises SEQ ID NO: 12 and the light chain of which comprises SEQ ID NO: 13. The inhibitor of DPP 3 activity may be administered by inhalation.

[0113] One embodiment of the invention relates to an inhibitor of DPP 3 activity for use in the treatment of shock in a subject suffering from or having developed shock, wherein said inhibitor is administered in combination with an angiotensin receptor agonist and / or a precursor thereof.

[0114] Another embodiment of the present invention relates to an inhibitor of DPP 3 activity for use in the treatment of shock in a subject suffering from or having developed shock, wherein the angiotensin receptor agonist and / or precursor thereof is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, angiotensin IV, in particular angiotensin II.

[0115] Another embodiment of the present invention relates to an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity for use in treating shock in a subject suffering from or having developed shock, wherein treatment with said angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued when the concentration of DPP-3 in a sample from said subject exceeds a certain threshold, and / or treatment with a vasopressor agent is suspended and / or terminated when the measured concentration of DPP-3 exceeds said predetermined threshold.

[0116] Another particular embodiment of the present invention relates to a vasopressor agent for use in treating shock in a subject suffering from or having developed shock, wherein treatment with the vasopressor agent is initiated and / or continued when the concentration of DPP 3 in a body fluid sample from the subject is below a certain threshold, and / or treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP 3 activity is suspended and / or terminated when the measured concentration of DPP 3 is below the predetermined threshold.

[0117] Another specific embodiment of the present invention relates to a vasopressor for use in treating shock in a subject suffering from or having developed shock, wherein treatment with the vasopressor is initiated and / or continued when the concentration of DPP 3 in a body fluid sample from the subject is below a certain threshold, and / or treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP 3 activity is suspended and / or terminated when the measured concentration of DPP 3 is below the predetermined threshold, and the concentration of proadrenomedullin or a fragment thereof is measured, and treatment with an anti-ADM antibody or an anti-ADM antibody fragment is initiated and / or continued when the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or treatment with the anti-ADM antibody or an anti-ADM antibody fragment is suspended and / or terminated when the measured concentration of proadrenomedullin or a fragment thereof is below the predetermined threshold.

[0118] Another specific embodiment of the present invention relates to an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity for use in treating shock in a subject suffering from or having developed shock, wherein treatment with the vasopressor is initiated and / or continued when the concentration of DPP-3 in a body fluid sample from the subject is below a certain threshold, and / or treatment with the angiotensin receptor agonist and / or a precursor thereof and / or the inhibitor of DPP-3 activity is suspended and / or terminated when the measured concentration of DPP-3 is below the predetermined threshold, and the concentration of pro-adrenomedullin or a fragment thereof is measured, and treatment with the anti-ADM antibody or anti-ADM antibody fragment is initiated and / or continued when the concentration of pro-adrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or treatment with the anti-ADM antibody or anti-ADM antibody fragment is suspended and / or terminated when the measured concentration of pro-adrenomedullin or a fragment thereof is below the predetermined threshold.

[0119] Another specific embodiment of the present invention relates to an anti-ADM antibody or an anti-ADM antibody fragment for use in treating shock in a subject suffering from or having developed shock, wherein treatment with the anti-ADM antibody or anti-ADM antibody fragment and / or an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued when the concentration of pro-adrenomedullin or a fragment thereof in the sample exceeds a particular threshold and the measured concentration of DPP-3 exceeds the predetermined DPP-3 threshold.

[0120] When the concentration of proadrenomedullin or a fragment thereof in the sample is measured and the concentration of DPP 3 in the sample is measured, the measurements may be performed by a point-of-care device that provides a test for measuring both the concentration of proadrenomedullin or a fragment thereof and the concentration of DPP 3 in the sample.

[0121] Treatment with angiotensin receptor agonists and / or precursors thereof is initiated when a patient has an amount of DPP 3 protein and / or DPP 3 activity in a body fluid sample above a predetermined threshold concentration.

[0122] One embodiment of the present invention relates to a method of treating shock in a subject suffering from or having developed shock, the method comprising administering a vasopressor to the subject, wherein the subject has a concentration of DPP 3 in a body fluid sample from the subject that is below a predetermined threshold, as measured by the method described in the previous embodiment.

[0123] Another embodiment of the present invention relates to a method of treating shock in a subject suffering from or experiencing shock, the method comprising administering to the subject a vasopressor agent, wherein the vasopressor agent is selected from the group consisting of dopamine, norepinephrine, norepinephrine equivalents, epinephrine, phenylephrine, and vasopressin.

[0124] Certain embodiments of the present invention relate to a method of treating shock in a subject suffering from or having developed shock, the method comprising administering to the subject a vasopressor agent, wherein the vasopressor agent is administered to the subject as a pharmaceutical formulation.

[0125] Another embodiment of the present invention relates to a method of treating shock in a subject suffering from or having developed shock, the method comprising administering a vasopressor to the subject, wherein the subject's blood pressure is 65 mm Hg or less.

[0126] One embodiment of the present invention relates to a method of treating shock in a subject suffering from or having developed shock, the method comprising administering to said subject an inhibitor of DPP-3 activity, wherein said subject has a concentration of DPP-3 in a body fluid sample from said subject that exceeds a predetermined threshold, as measured by the method described in the previous embodiment.

[0127] Another embodiment of the present invention relates to a method for treating shock in a subject suffering from or having developed shock, the method comprising administering to said subject an inhibitor of DPP3 activity, wherein said inhibitor of DPP3 activity is selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

[0128] Another particular embodiment of the present invention relates to a method of treating shock in a subject suffering from or having developed shock, the method comprising administering to said subject an inhibitor of DPP 3 activity, said inhibitor comprising 10 -7 M or less.

[0129] Another embodiment of the present invention relates to a method of treating shock in a subject suffering from or having developed shock, the method comprising administering to the subject an inhibitor of DPP 3 activity, wherein the inhibitor of DPP 3 activity is an antibody.

[0130] One embodiment of the present invention relates to a method of treating shock in a subject suffering from or having developed shock, the method comprising administering to the subject an inhibitor of DPP-3 activity, wherein the inhibitor is a monoclonal antibody.

[0131] A particular embodiment of the present invention relates to a method of treating shock in a subject suffering from or having developed shock, the method comprising administering to the subject an inhibitor of DPP-3 activity, wherein the inhibitor is a monoclonal antibody whose heavy chain complementarity determining regions (CDRs) comprise the sequence of SEQ ID NO:7, SEQ ID NO:8, and / or SEQ ID NO:9, and whose light chain complementarity determining regions (CDRs) comprise the sequence of SEQ ID NO:10, KVS, and / or SEQ ID NO:11.

[0132] Another embodiment of the present invention relates to a method of treating shock in a subject suffering from or having developed shock, the method comprising administering to the subject an inhibitor of DPP-3 activity, wherein the inhibitor is a humanized monoclonal antibody or a humanized monoclonal antibody fragment, the heavy chain of which comprises the sequence of SEQ ID NO: 12 and the light chain of which comprises the sequence of SEQ ID NO: 13.

[0133] Another specific embodiment of the present invention relates to a method of treating shock in a subject suffering from or having developed shock, the method comprising administering to the subject an inhibitor of DPP 3 activity, wherein the inhibitor is administered in combination with an angiotensin receptor agonist and / or a precursor thereof.

[0134] Another embodiment of the present invention relates to a method for treating shock in a subject suffering from or having developed shock, the method comprising administering to the subject an inhibitor of DPP 3 activity, wherein the angiotensin receptor agonist and / or precursor thereof is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, angiotensin IV, in particular angiotensin II.

[0135] Another embodiment of the present invention relates to a method of treating shock in a subject suffering from or having developed shock, the method comprising administering to the subject an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity, wherein treatment with the angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued when the concentration of DPP-3 in a sample from the subject exceeds a certain threshold, and / or treatment with a vasopressor agent is suspended and / or terminated when the measured concentration of DPP-3 exceeds the predetermined threshold.

[0136] One embodiment of the present invention relates to a method for treating shock in a subject suffering from or having developed shock, the method comprising administering a vasopressor agent to the subject, wherein treatment with the vasopressor agent is initiated and / or continued when the concentration of DPP 3 in a body fluid sample from the subject is below a certain threshold, and / or treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP 3 activity is withheld and / or terminated when the measured concentration of DPP 3 is below the predetermined threshold.

[0137] One embodiment of the present invention relates to a method for treating shock in a subject suffering from or having developed shock, the method comprising administering to the subject an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity, wherein treatment with the angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued when the concentration of DPP-3 in a sample from the subject exceeds a certain threshold, and / or treatment with a vasopressor is suspended and / or terminated when the measured concentration of DPP-3 exceeds the predetermined threshold, and wherein the concentration of proadrenomedullin or a fragment thereof is measured, and treatment with the anti-ADM antibody or anti-ADM antibody fragment is initiated and / or continued when the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or treatment with the anti-ADM antibody or anti-ADM antibody fragment is suspended and / or terminated when the measured concentration of proadrenomedullin or a fragment thereof is below the predetermined threshold.

[0138] A particular embodiment of the present invention relates to a method for treating shock in a subject suffering from or having developed shock, comprising administering a vasopressor to the subject, wherein treatment with the vasopressor is initiated and / or continued when a concentration of DPP-3 in a body fluid sample from the subject is below a certain threshold, and / or treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is suspended and / or terminated when the measured concentration of DPP-3 is below the predetermined threshold, and wherein the concentration of pro-adrenomedullin or a fragment thereof is measured, and treatment with the anti-ADM antibody or anti-ADM antibody fragment is initiated and / or continued when the concentration of pro-adrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or treatment with the anti-ADM antibody or anti-ADM antibody fragment is suspended and / or terminated when the measured concentration of pro-adrenomedullin or a fragment thereof is below the predetermined threshold.

[0139] Another embodiment of the present invention relates to a method for treating shock in a subject suffering from or having developed shock, comprising administering to the subject an anti-ADM antibody or an anti-ADM antibody fragment; and measuring the concentration of pro-adrenomedullin or a fragment thereof, and initiating and / or continuing treatment with the anti-ADM antibody or anti-ADM antibody fragment when the concentration of pro-adrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with the anti-ADM antibody or anti-ADM antibody fragment when the measured concentration of pro-adrenomedullin or a fragment thereof is below the predetermined threshold, initiating and / or continuing treatment with an angiotensin receptor agonist and / or its precursor and / or an inhibitor of DPP-3 activity when the concentration of DPP-3 in a sample from the subject exceeds a certain threshold, and / or suspending and / or terminating treatment with a vasopressor when the measured concentration of DPP-3 exceeds the predetermined threshold.

[0140] Another specific embodiment of the present invention relates to a method for treating shock in a subject suffering from or having developed shock, the method comprising administering to the subject an anti-ADM antibody or an anti-ADM antibody fragment, wherein treatment with the anti-ADM antibody or anti-ADM antibody fragment and / or an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued when the concentration of pro-adrenomedullin or a fragment thereof in the sample exceeds a particular threshold and the measured concentration of DPP-3 exceeds the predetermined DPP-3 threshold.

[0141] An agonist is a chemical that binds to a receptor and activates it, causing a biological response. Receptors can be activated by either endogenous agonists (such as hormones or neurotransmitters) or exogenous agonists (such as drugs), causing a biological response. A full agonist binds to a receptor and activates it with the maximum response that an agonist can induce at the receptor. A partial agonist is a drug that binds to and activates a specific receptor, but has only partial efficacy at the receptor compared to a full agonist. Potency is the amount of an agonist required to induce a desired response. The potency of an agonist is determined by its EC 50 This EC is inversely proportional to the value. 50 EC can be measured for a particular agonist by determining the concentration of agonist required to elicit half of the agonist's maximal biological response. 50 The EC value is useful for comparing the potency of a drug with similar potencies that produce similar biological effects. 50 The lower the value, the more potent the agonist and the lower the concentration of drug required to elicit the maximal biological response.

[0142] Treatment with angiotensin receptor agonist and / or its precursor may be continued as long as the amount of DPP 3 protein and / or DPP 3 activity in the patient's body fluid sample remains above a predetermined threshold concentration.

[0143] The amount of DPP 3 protein and / or DPP 3 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.

[0144] Treatment with angiotensin receptor agonists and / or precursors thereof may be discontinued if the amount of DPP 3 protein and / or DPP 3 activity in a patient's body fluid sample falls below a predetermined threshold concentration. Angiotensin therapy:

[0145] Angiotensin I, also known as proangiotensin, is produced by the action of renin on angiotensinogen. Renin cleaves the peptide bond between the leucine (Leu) and valine (Val) residues of angiotensinogen to generate the decapeptide (10 amino acids) (des-Asp) angiotensin I. Angiotensin I is converted to angiotensin II by the enzyme angiotensin-converting enzyme (ACE), primarily in the lungs (but also present in endothelial cells, kidney epithelial cells, and the brain), via removal of two C-terminal residues.

[0146] Angiotensin II, angiotensin III, and angiotensin IV are peptide hormones naturally produced in 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, showing significant effects on systemic and renal blood flow (Harrison-Bernard, LM, The renal renin-angiotensin system. Adv PHysiol Educ, 33(4): 270 (2009)).

[0147] Angiotensin II is a hormone produced by the renin-angiotensin-aldosterone system (RAAS) that regulates blood pressure through the regulation of vascular smooth muscle tone and extracellular fluid homeostasis. Angiotensin II mediates its effects on the vasculature by inducing vasoconstriction and sodium retention, thereby making it a target for many treatments for hypertension. In addition to its systemic effects, angiotensin II has a pronounced effect on the renal efferent arteriole, maintaining glomerular filtration when blood flow is reduced. Angiotensin II also mediates the depletion of Na in the proximal tubule. + / H + It regulates sodium reabsorption in the kidney by stimulating the exchanger and inducing the release of aldosterone and vasopressin (Harrison-Bernard 2009. The renal renin-angiotensin system. Adv PHysiol Educ, 33(4):270).

[0148] An angiotensin II therapeutic agent that can be used in the compositions and methods of the present disclosure is 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, such as horses and pigs. The isoleucine can be replaced with valine to generate 5-valine angiotensin II, i.e., Asp-Arg-Val-Tyr-Val-His-Pro-Phe (SEQ ID NO: 14). [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 -Diiodide Tyr 4 -Ile 5 Other angiotensin II analogs, such as ]-angiotensin II (SEQ ID NO: 20), can also be used. Angiotensin II may be synthesized, for example, by solid-phase peptide synthesis, to incorporate modifications such as C-terminal amidation. The term "angiotensin II" is intended to refer, without further specificity, to any of these various forms, as well as combinations thereof.

[0149] In one aspect, the composition containing angiotensin II is selected from 5-valine angiotensin II, 5-valine angiotensin II amide, 5-L-isoleucine angiotensin II, and 5-L-isoleucine angiotensin II amide, or a pharmaceutically acceptable salt thereof, and is preferably manufactured under current Good Manufacturing Practice (cGMP). In one aspect, the composition may contain various forms of angiotensin II in various ratios, such as a mixture of hexapeptide and nonapeptide angiotensin II. The composition containing angiotensin II may be suitable for parenteral administration, such as injection or intravenous infusion.

[0150] The angiotensin II sequences used in the compositions and methods disclosed herein may be homologous to the angiotensin II sequences described above. In certain 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 such variant sequences may be used in place of angiotensin II, as described in the preceding paragraph.

[0151] Sequence identity for all sequences is determined according to the following method: Percentage 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 (% identity = [number of matches x 100] / [length of aligned region, including gaps]).

[0152] Angiotensin III is a metabolite of angiotensin II and has approximately 40% of the activity of angiotensin II. An angiotensin III therapeutic agent that can be used in the compositions and methods of the present disclosure may 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, such as horses and pigs. Isoleucine can be substituted with valine to produce Arg-Val-Tyr-Val-His-Pro-Phe (SEQ ID NO: 22). [Phe3 ]-angiotensin III (SEQ ID NO: 23), [Ile 4 -Ala 7 ]-angiotensin III (SEQ ID NO: 24), and [diiodoTyr 3 -Ile 4 Other angiotensin III analogs, such as ]-angiotensin III (SEQ ID NO: 25), may also be used. Angiotensin III may be synthesized, for example, by solid-phase peptide synthesis, to incorporate modifications such as C-terminal amidation. The term "angiotensin III" is intended to refer to any of these various forms, as well as combinations thereof, without further specificity.

[0153] In one aspect, the composition comprising angiotensin III may 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 pharmaceutically acceptable salt thereof, preferably manufactured under current Good Manufacturing Practice (cGMP). The composition comprising angiotensin III may be suitable for parenteral administration, such as injection or intravenous infusion.

[0154] The angiotensin III sequences used in the compositions and methods disclosed herein may be homologous to the angiotensin III sequences described above. In certain 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 such variant sequences may be used in place of angiotensin II, as described in the preceding paragraph.

[0155] Angiotensin IV is a metabolite of angiotensin III that is less active than angiotensin II. An angiotensin IV therapeutic agent that can be used in the compositions and methods of the present disclosure may 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 substituted with valine to generate Val-Tyr-Val-His-Pro-Phe (SEQ ID NO: 27). [Phe 2 ]-angiotensin III (SEQ ID NO: 28), [Ile 3 -AIa 6 ]-angiotensin IV (SEQ ID NO: 29), and [diiodoTyr 2 -Ile 3 Other angiotensin IV analogs, such as 4-angiotensin IV (SEQ ID NO: 30), may also be used. Angiotensin IV may be synthesized, for example, by solid-phase peptide synthesis, to incorporate modifications such as C-terminal amidation. The term "angiotensin IV" is intended to refer to any of these various forms, as well as combinations thereof, without further specificity.

[0156] In one aspect, the composition comprising angiotensin IV may 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 pharmaceutically acceptable salt thereof, preferably manufactured under current Good Manufacturing Practice (cGMP). The composition comprising angiotensin IV may be suitable for parenteral administration, such as injection or intravenous infusion.

[0157] The angiotensin IV sequences used in the compositions and methods disclosed herein may be homologous to the angiotensin IV sequences described above. In certain 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 such variant sequences may be used in place of angiotensin IV, as described in the preceding paragraph.

[0158] The angiotensin II, angiotensin III, or angiotensin IV therapeutic agent may be used as any suitable salt (e.g., acetate), deprotected form, acetylated form, deacetylated form, and / or prodrug form of the aforementioned peptide, including pegylated forms of the peptides or conjugates disclosed in U.S. Patent Publication No. 2011 / 0081371 (incorporated by reference). The term "prodrug" refers to any precursor compound capable of generating or releasing the aforementioned peptide under biological conditions. Such prodrugs or precursors may be larger peptides that are selectively cleaved to generate the peptides of the invention. For example, in one aspect, the prodrug or precursor may be angiotensinogen, angiotensin I, or a homolog thereof that can generate angiotensin II through the action of specific endogenous or exogenous enzymes. Further prodrugs or precursors include peptides with protected amino acids, e.g., peptides with protecting groups on one or more carboxylic acid and / or amino groups. Suitable protecting groups for amino groups include benzyloxycarbonyl, t-butyloxycarbonyl (BOC), fluorenylmethyloxycarbonyl (FMOC), formyl, and acetyl or acyl groups. Suitable protecting groups for carboxylic acid groups include esters, such as benzyl esters or t-butyl esters. The present invention also contemplates the use of angiotensin II, angiotensin III, angiotensin IV, and / or precursor peptides having amino acid substitutions, deletions, or additions, including standard D and L amino acids, as well as modified amino acids, such as amidated and acetylated amino acids, while maintaining the therapeutic activity of the basic peptide sequence at pharmacologically useful levels.

[0159] 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 counterion acetate is present in a non-stoichiometric ratio. The molecular formula of angiotensin II acetate is C50 H 71 N 13 O 12 (C2H4O2) n (n is the number of acetate molecules; theoretical n is 3) and the average molecular weight (as the free base) is 1046.2.

[0160] One embodiment of the present invention is an angiotensin receptor agonist and / or precursor thereof for use in treating a disease in a subject, said subject having a DPP 3 protein amount and / or DPP 3 activity in a body fluid sample above a predetermined threshold, and wherein measurements of the DPP 3 protein amount and / or DPP 3 activity in the body fluid sample are used to guide and / or monitor therapy for said treatment with said angiotensin receptor agonist and / or precursor thereof.

[0161] In one embodiment of the invention, the amount of DPP3 protein and / or DPP3 activity in a body fluid sample is measured before treating a subject with an angiotensin receptor agonist and / or precursor thereof. In one embodiment of the invention, the amount of DPP3 protein and / or DPP3 activity in a body fluid sample is measured during treatment of a subject with an angiotensin receptor agonist and / or precursor thereof, and is measured at least once during treatment, preferably at least twice during treatment, or preferably at least once daily during treatment. In one embodiment of the invention, the amount of DPP3 protein and / or DPP3 activity in a body fluid sample is measured after treatment of a subject with an angiotensin receptor agonist and / or precursor thereof. In one embodiment of the invention, the amount of DPP3 protein and / or DPP3 activity in a body fluid sample is measured before and / or during and / or after treatment of a subject with an angiotensin receptor agonist and / or precursor thereof.

[0162] One embodiment of the present invention is an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of shock in a subject suffering from or having developed shock, wherein the subject has an amount of DPP-3 protein and / or DPP-3 activity in a body fluid sample above a predetermined threshold, and the body fluid sample from the subject is selected from whole blood, plasma, and serum.

[0163] One embodiment of the present invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of shock in a subject suffering from or having developed shock, wherein the angiotensin receptor agonist and / or precursor thereof is administered to the subject as a pharmaceutical formulation.

[0164] The term "pharmaceutical formulation" means a pharmaceutical (active) ingredient in combination with at least one pharmaceutically acceptable excipient.

[0165] One embodiment of the present invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of shock in a subject suffering from or having shock, wherein the angiotensin receptor agonist and / or precursor thereof is angiotensin II, and wherein said pharmaceutical formulation is 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, wherein said pharmaceutical formulation is in a dry state and is dissolved in aqueous solution before use.

[0166] The pharmaceutical formulations disclosed herein may also contain diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier that can be administered to a patient together with a therapeutically active substance of the present invention (such as angiotensin II), and which does not destroy the pharmacological activity of the therapeutically active substance. The term "pharmaceutically acceptable" refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient.

[0167] The characteristics of the carrier will depend on the route of administration. The term "excipient" refers to an additive in a formulation or composition that is not a pharmaceutically active ingredient.

[0168] The term "pharmaceutical (active) ingredient" means a therapeutic composition that can be combined, if necessary, with pharmaceutically acceptable excipients to provide a pharmaceutical formulation or dosage form.

[0169] Those skilled in the art will understand 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, since the combination of excipients may produce undesirable effects. Excipients of the present invention include, but are not limited to, cosolvents, solubilizers, buffers, pH adjusters, bulking agents, surfactants, encapsulating agents, isotonicity adjusters, stabilizers, protectants, and viscosity adjusters.

[0170] In certain aspects, it may be beneficial to include a pharmaceutically acceptable carrier in the compositions disclosed herein.

[0171] In some aspects, it may be beneficial to include a solubilizing agent in the compositions of the present invention. Solubilizing agents may be useful for increasing the solubility of any component of the formulation or composition, including therapeutically active substances (e.g., angiotensin II, angiotensin III, or angiotensin IV) or excipients. The solubilizing agents described herein are not intended to constitute a complete list, but only represent exemplary solubilizing agents that can be used in the compositions of the present invention. In certain aspects, solubilizing agents include, but are not limited to, ethyl alcohol, tert-butyl alcohol, polyethylene glycol, glycerin, methylparaben, propylparaben, polyethylene glycol, polyvinylpyrrolidone, and any pharmaceutically acceptable salts thereof, and / or combinations thereof.

[0172] In some aspects, it may be beneficial to include a pH adjuster in the compositions of the present invention to adjust the pH of the composition. Altering the pH of a formulation or composition may be useful, for example, to have a beneficial effect on the stability or solubility of a therapeutically active substance, or to make the formulation or composition suitable for parenteral administration. pH adjusters are well known in the art. Therefore, the pH adjusters described herein are not intended to constitute an exhaustive list, but merely represent exemplary pH adjusters that can be used in the compositions of the present invention. pH adjusters may include, for example, acids and bases. In some aspects, pH adjusters may include, but are not limited to, acetic acid, hydrochloric acid, phosphoric acid, sodium hydroxide, sodium carbonate, and combinations thereof.

[0173] The pH of the compositions disclosed herein can be any pH that provides the formulation or composition with desired properties. Desirable properties may include, for example, stability of the therapeutically active agent (e.g., angiotensin II, angiotensin III, or angiotensin IV), increased retention of the therapeutically active agent relative to compositions at a different pH, and improved filtration efficiency. In certain aspects, the pH of the compositions of the present invention may be about 3.0 to about 9.0, e.g., about 5.0 to about 7.0. In certain aspects, the pH of the compositions of the present invention may 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.

[0174] In some aspects, it may be beneficial to include one or more buffering agents in the composition to buffer pH.In certain embodiments, the pKa of the buffering agent may be, for example, about 5.5, about 6.0, or about 6.5.Those skilled in the art will know that they can select a suitable buffering agent to be included in the composition of the present invention based on its pKa and other properties.

[0175] Buffers are well known in the art. Thus, the buffers described herein are not intended to constitute an exhaustive list, but merely exemplary buffers that can be used in the compositions of the present invention. In certain aspects, the buffer may include one or more of Tris, Tris-HCl, potassium phosphate, sodium phosphate, sodium citrate, sodium ascorbate, a combination of sodium and potassium phosphate, Tris / Tris-HCl, sodium bicarbonate, arginine phosphate, arginine hydrochloride, histidine hydrochloride, cacodylate, succinate, 2-(N-morpholino)ethanesulfonate (MES), maleate, bis-Tris, phosphate, carbonate, and any pharmaceutically acceptable salt and / or combination thereof.

[0176] In some aspects, it may be beneficial to include a surfactant in the compositions of the present invention. Surfactants generally reduce the surface tension of a liquid composition, which may provide beneficial properties such as improved filterability. Surfactants can also act as emulsifiers and / or solubilizers. Surfactants are well known in the art. Therefore, the surfactants described herein are not intended to constitute an exhaustive list, but merely exemplary surfactants that may be used in the compositions of the present invention. Surfactants include, but are not limited to, 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 oxide and polyethylene oxide (e.g., Triton X-100), saponins, phospholipids (e.g., lecithin), and combinations thereof.

[0177] In some aspects, it may be beneficial to include an isotonicity adjusting agent in the compositions of the present invention. The isotonicity of a liquid composition is an important consideration, for example, when administering the composition to a patient via parenteral administration. Therefore, an isotonicity adjusting agent can be used to prepare a formulation or composition suitable for administration. Isotonicity adjusting agents are well known in the art. Therefore, the isotonicity adjusting agents described herein are not intended to constitute an exhaustive list, but merely represent exemplary isotonicity adjusting agents that can be used in the compositions of the present invention. Isotonicity adjusting agents may be ionic or nonionic and may include, but are not limited to, inorganic salts, amino acids, carbohydrates, sugars, sugar alcohols, and carbohydrates. Exemplary inorganic salts may include sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate. An exemplary amino acid is glycine. Exemplary sugars may include glycerin, propylene glycol, glucose, sucrose, lactose, and sugar alcohols such as mannitol.

[0178] In some aspects, it may be beneficial to include a stabilizer in the compositions of the present invention. The stabilizer serves to improve the stability of the therapeutically active agent in the compositions of the present invention. This improvement may be caused, for example, by reducing degradation or preventing aggregation of the therapeutically active agent. Without wishing to be bound by theory, mechanisms for improving stability may include protecting the therapeutically active agent from solvents or inhibiting free radical oxidation of the anthracycline compound. Stabilizers are well known in the art. Therefore, the stabilizers described herein are not intended to constitute an exhaustive list, but merely represent exemplary stabilizers that can be used in the compositions of the present invention. Stabilizers may include, but are not limited to, emulsifiers and surfactants.

[0179] The compositions disclosed herein can be administered by a variety of conventional methods. In certain aspects, the compositions of the present invention are suitable for parenteral administration. These compositions may be administered, for example, intraperitoneally, intravenously, intrarenally, intrathecally, or by inhalation. In certain aspects, the compositions of the present invention are injected intravenously. Those skilled in the art will recognize that the method of administering a therapeutically active agent formulation or composition of the present invention depends on factors such as the age, weight, and physical condition of the patient receiving treatment, as well as the disease or condition being treated. Therefore, those skilled in the art will be able to select the most appropriate administration method for each patient, as appropriate.

[0180] Angiotensin receptor agonists and / or their precursors can be administered by any suitable method, but are typically administered by continuous infusion. Therefore, increasing or decreasing the administration rate can be achieved by changing the flow rate of the intravenous infusion, changing the drug concentration during the intravenous infusion, etc. However, the method for changing the administration rate varies depending on the mode of administration of the therapeutic agent. When the therapeutic agent is administered transmucosally or transdermally, the rate may be increased, for example, by changing to a patch or transdermal composition with a high release rate. When the therapeutic agent is administered orally, the rate may be increased, for example, by switching to a higher dose form, administering additional doses, or administering a sustained-release dosage form with a high release rate.

[0181] When a therapeutic agent is administered by inhalation, its rate may be increased, for example, by administering an additional bolus, a concentrated bolus, or a fast-releasing bolus. Other modes of administration (e.g., via subcutaneous injection pump, suppository, etc.) can be adjusted in a similar manner, with a decrease in the rate of administration being achieved by reversing the actions that increase the rate of administration of the therapeutic agent.

[0182] One embodiment of the present invention is an angiotensin receptor agonist and / or a precursor thereof for use in treating shock in a subject suffering from or having developed shock, wherein the angiotensin receptor agonist and / or precursor thereof is angiotensin II and is administered in an amount 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.

[0183] In certain embodiments of the present disclosure, the starting dose (initial amount) 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.

[0184] In another specific embodiment of the present invention, blood pressure responsiveness (e.g., mean arterial pressure; MAP) is monitored for angiotensin II titration. Angiotensin II titration may 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 may be performed in increments of up to 40 ng / kg / min, more preferably up to 20 ng / kg / min, and most preferably 15 ng / kg / min, as needed, to achieve or maintain the maximum target blood pressure. In another preferred embodiment, the dose of angiotensin II should not exceed 80 ng / kg / min during the first 3 hours of treatment. Most preferably, the maintenance dose should not exceed 40 ng / kg / min, and a dose of at least 1.25 ng / kg / min is used.

[0185] In some embodiments, the patient's initial mean arterial pressure (MAP) is less than or equal to about 40 mm Hg, about 45 mm Hg, about 50 mm Hg, 55 mm Hg, about 60 mm Hg, about 65 mm Hg, about 70 mm Hg, or about 75 mm Hg prior to administering the composition. The method may include measuring the patient's mean arterial blood pressure and increasing the dosage of angiotensin II if the mean arterial blood pressure is less than about 40 mm Hg, about 45 mm Hg, about 50 mm Hg, 55 mm Hg, about 60 mm Hg, about 65 mm Hg, about 70 mm Hg, or about 75 mm Hg.

[0186] In one embodiment, the patient may be administered a vasopressor (e.g., norepinephrine, norepinephrine equivalents, epinephrine, dopamine, catecholamines such as phenylephrine), or a combination thereof. In some embodiments, the vasopressor is a vasopressin (e.g., terlipressin, aldipressin, desmopressin, felypressin, lypressin, or ornipressin).

[0187] One embodiment of the present invention is an angiotensin receptor agonist and / or precursor thereof for use in treating shock in a subject suffering from or having developed shock, wherein the angiotensin receptor agonist and / or precursor thereof, in particular angiotensin II, is administered in combination with an inhibitor of DPP 3.

[0188] One embodiment of the present invention is an angiotensin receptor agonist and / or a precursor thereof for use in treating shock in a subject suffering from or having developed shock, in combination with a DPP-3 inhibitor, wherein the DPP-3 inhibitor is selected from the group comprising an anti-DPP-3 antibody or an anti-DPP-3 antibody fragment or an anti-DPP-3 non-Ig scaffold.

[0189] According to the present invention, an "anti-DPP3 antibody" is an antibody that specifically binds to DPP3, and an "anti-DPP3 antibody fragment" is a fragment of said anti-DPP3 antibody, which fragment specifically binds to DPP3. An "anti-DPP3 non-Ig scaffold" is a non-Ig scaffold that specifically binds to DPP3.

[0190] One embodiment of the present invention is an angiotensin receptor agonist and / or a precursor thereof for use in treating shock in a subject suffering from or having developed shock, in combination with a DPP-3 inhibitor, wherein the DPP-3 inhibitor is an anti-DPP-3 antibody or anti-DPP-3 antibody fragment or anti-DPP-3 non-Ig scaffold that binds to SEQ ID NO: 1, in particular SEQ ID NO: 2.

[0191] One embodiment of the present invention is an angiotensin receptor agonist and / or a precursor thereof for use in combination with an inhibitor of DPP 3 for the treatment of shock in a subject suffering from or having developed shock, wherein the inhibitor of DPP 3 is -7 The antibody or fragment or scaffold exhibits a minimum binding affinity to DPP 3 of M or less.

[0192] In accordance with the present invention, those skilled in the art will appreciate that the binding affinity of the DPP 3-binding agents disclosed herein to DPP 3 can be measured by a variety of suitable measurement methods known in the art. Examples of each are provided below, but these should not be construed as limiting the possibilities for measuring the binding affinity of the DPP 3-binding agents disclosed herein to DPP 3.

[0193] For example, the binding affinity of the DPP-3-binding agent to the epitope may be measured. Binding assays may be performed to detect and / or quantify, for example, antibody binding to the immunizing peptide of each binding agent. For example, the immunizing peptide may be immobilized on a solid phase. A test sample (such as an antibody solution) can be passed over the immobilized immunizing peptide to detect bound antibodies. For purposes of this description, the term "solid phase" may be used to include any material or container within or on which an assay may be performed, including, but not limited to, porous materials, non-porous materials, test tubes, wells, slides, magnetic beads, etc.

[0194] Exemplary detection methods are as follows: - Labeling the antibody before contacting it with the solid phase and detecting the respective label (fluorescent, chemiluminescent, enzymatic, etc.); A method using a secondary antibody labeled against a specific Fc portion of the sample antibody, incubating the solid-phase-bound antibody with the secondary antibody (e.g., anti-human IgG, anti-mouse IgG), and detecting the respective label (fluorescent, chemiluminescent, enzyme, etc.); - using a labeled antibody (e.g., labeled AK1967) as a competitor for solid phase binding; and Quantifying binding affinity by signal reduction.

[0195] As an alternative method for measuring the affinity of an antibody for DPP 3, the binding kinetics of DPP 3 to an immobilized antibody may be measured by label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe, Freiburg, Germany). Reversible immobilization of the antibody may be performed using an anti-mouse Fc antibody covalently bound at high density to a CM5 sensor surface (Mouse Antibody Capture Kit; GE Healthcare) (Lorenz et al. 2011. Antimicrob Agents Chemother. 55(1): 165-173).

[0196] One embodiment of the invention is an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of shock in a subject suffering from or having developed shock, in combination with an inhibitor of DPP 3, wherein said inhibitor of DPP 3 is a monospecific antibody or fragment or scaffold, and in one embodiment said inhibitor of DPP 3 is a monoclonal antibody or fragment or scaffold.

[0197] 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. While monoclonal antibodies are monospecific, monospecific antibodies can be generated by means other than producing them from common germ cells.

[0198] In a particular embodiment, the capture binding agent that binds to full-length DPP 3 specifically inhibits DPP 3 activity by less than 50%, preferably less than 40%, and more preferably less than 30% in a solution-phase assay. See above for a definition of a solution-phase assay. In a particular embodiment to prevent inhibition of DPP 3, the capture binding agent should not bind to DPP 3 in the region surrounding the active center and substrate-binding region (amino acids 316-669 of SEQ ID NO: 1).

[0199] One embodiment of the present invention is an angiotensin receptor agonist and / or a precursor thereof for use in treating shock in a subject suffering from or having developed shock in combination with a DPP-3 inhibitor, said DPP-3 inhibitor being an antibody, fragment or scaffold that binds to full-length DPP-3 and inhibits 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% of DPP-3 activity.

[0200] Inhibition of DPP3 activity by a binding agent in a liquid phase assay can be determined as follows: a potential DPP3 capture binding agent is incubated with a recombinant or purified native DPP3 substrate and a specific DPP3 substrate in a liquid phase assay. Preferably, the binding agent with the lowest inhibitory potential is selected as the capture binding agent for the enzyme capture activity assay (ECA). The capture binding agent should inhibit DPP3 activity by less than 50%, preferably less than 40%, and preferably less than 30%. A specific liquid phase DPP3 activity assay to determine the inhibitory potential of a viable capture binding agent comprises the following steps: Incubating 25 ng / mL of native human DPP 3 with 5 μg / mL of each capture binding agent, 50 mM Tris-HCl buffer control (pH 7.5), and 100 μM ZnCl 2 at room temperature for 1 hour; adding the fluorogenic substrate Arg-Arg-βNA (20 μL, 2 mM); Incubating at 37°C and monitoring the production of free βNA over 1 hour in a Twinkle LB 970 microplate fluorometer (Berthold Technologies), where βNA fluorescence is detected by excitation at 340 nm and measuring emission at 410 nm; and Calculating the slope of the fluorescence increment (RFU / min) for the various samples, where the slope of native human DPP 3 with buffer control is designated as 100% activity. The inhibitory potency of a viable capture binding agent is defined as the % decrease in native human DPP 3 activity upon incubation with said capture binding agent.

[0201] In the liquid-phase assay, a biological fluid sample is directly subjected to a fluorogenic substrate (e.g., Arg-Arg-β-NA). Because many different aminopeptidases exist in plasma (Sanderink et al. 1988), the substrate may be cleaved by peptidases other than DPP-3. To avoid this problem, the use of an enzyme-capture activity assay is a preferred method for detecting specific DPP-3 activity.

[0202] In one particular embodiment, measuring active DPP 3 in an enzyme capture assay comprises the following steps: contacting the sample with a capture binding agent that binds to full-length DPP3 but inhibits DPP3 activity by less than 50%, preferably less than 40%, and more preferably less than 30% in a solution-phase assay; wherein, to prevent inhibition of DPP3, the capture binding agent should not bind to DPP3 in the region surrounding the active center and substrate-binding region (amino acids 316-669 of SEQ ID NO: 1). separating the DPP 3 bound to the capture binding agent from the body fluid sample; adding a substrate for DPP3 to the isolated DPP3; and Quantifying DPP 3 activity by measuring the conversion of a DPP 3 substrate.

[0203] An "antibody" according to the present invention is a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes that specifically bind to an antigen. Recognized immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma (IgG1, IgG2, IgG3, IgG4), delta (IgD), epsilon (IgE), and mu (IgM) constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin light chains are generally about 25 kDa or 214 amino acids in length.

[0204] Full-length immunoglobulin heavy chains are generally about 50 kDa or 446 amino acids in length. Light chains are encoded by an NH2-terminal variable region gene (about 110 amino acids in length) and a COOH-terminal kappa or lambda constant region gene. Heavy chains are similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.

[0205] The basic structural unit of an antibody is generally a tetramer of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain, in which the variable regions of the light and heavy chains bind antigen and the constant regions mediate effector functions. Immunoglobulins also exist in a variety of other forms, including, for example, Fv, Fab, and F(ab')2, as well as bifunctional hybrid antibodies and single chains (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, NY, 2nd ed., 1984; Hunkapiller and Hood, Nature 323:15-16,1986).

[0206] The light or heavy chain variable region of an immunoglobulin contains a framework region interrupted by three hypervariable regions, also called complementarity-determining regions (CDRs) (see Sequences of Proteins of Immunological Interest, E. Kabat et al., US Department of Health and Human Services, 1983). As mentioned above, the CDRs are primarily responsible for binding to an epitope of an antigen. The immune complex is an antibody, such as a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody, or a functional antibody fragment that specifically binds to the antigen.

[0207] A "chimeric antibody" is an antibody whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. For example, the variable region of a gene from a mouse monoclonal antibody can be combined with human constant regions such as kappa and gamma 1 or gamma 3. Thus, in one example, a therapeutic chimeric antibody is a hybrid protein composed 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 also be generated by molecular techniques. Methods for producing chimeric antibodies are well known in the art; see, for example, U.S. Pat. No. 5,807,715. A "humanized" immunoglobulin is an immunoglobulin containing a human framework region and one or more CDRs from a non-human immunoglobulin (e.g., mouse, rat, or synthetic). The non-human immunoglobulin providing the CDRs is called the "donor," and the human immunoglobulin providing the framework is called the "acceptor."

[0208] In one embodiment of the invention, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. Constant regions need not be present, but if present, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, e.g., about 95% or more identical. Thus, all portions of the humanized immunoglobulin, excluding possibly the CDRs, are substantially identical to corresponding portions of natural human immunoglobulin sequences.

[0209] A "humanized antibody" according to the present invention is an antibody comprising a humanized light chain immunoglobulin and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provided the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions with amino acids taken from the donor framework. A humanized antibody or other monoclonal antibody may have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Examples of conservative substitutions include gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed by genetic engineering (see, e.g., U.S. Patent No. 5,585,089). A human antibody is an antibody whose light and heavy chain genes are of human origin. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells secreting the desired antibody. Immortalization can be achieved, for example, by EBV infection or by fusing human B cells with myeloma or hybridoma cells to generate trioma cells. Human antibodies can also be produced by phage display methods (see, e.g., PCT Application WO 91 / 17271 to Dower et al.; PCT Application WO 92 / 001047 to McCafferty et al.; and PCT Application WO 92 / 20791 to Winter) or selected from human combinatorial monoclonal antibody libraries (see the MorpHosys website). Human antibodies can also be prepared using transgenic animals carrying human immunoglobulin genes (see, e.g., PCT Application WO 93 / 12227 to Lonberg et al.; and PCT Application WO 91 / 10741 to Kucherlapati).

[0210] Thus, the anti-DPP3 antibody or anti-DPP3 antibody fragment according to the present invention may have a 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.

[0211] In a specific 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 a human antibody or a humanized antibody or is derived therefrom. In a specific embodiment, one or more (murine) CDRs are grafted onto a human antibody or antibody fragment.

[0212] In a preferred embodiment, the antibody according to the invention is a recombinantly produced antibody such as, for example, an IgG, a typical full-length immunoglobulin, or an antibody fragment comprising at least the F variable domain of the heavy and / or light chain; a chemically conjugated antibody (fragment antigen binding) comprising a Fab fragment, including, for example, but not limited to, a Fab minibody, a single-chain Fab antibody, a monovalent Fab antibody bearing an epitope tag, such as Fab-V5Sx2; a bivalent Fab (miniantibody) dimerized at the CH3 domain; a bivalent Fab or multivalent Fab formed via dimerization of dHLX domains, e.g., Fab-dHLX-FSx2, e.g., via multimerization with the aid of heterologous domains; a F(ab')2 fragment, a scFv fragment, a multimerized multivalent and / or multispecific scFv fragment, a bivalent and / or bispecific antibody, a BITE® (bispecific T cell engager), a trifunctional antibody, e.g., from a class different from G, a multivalent antibody; a single domain antibody such as a nanobody derived from, for example, camel or fish immunoglobulins; and many others.

[0213] In addition to anti-DPP3 antibodies or anti-DPP3 antibody fragments, other biopolymer scaffolds, so-called non-Ig scaffolds, are known in the art to conjugate target molecules and have been used to generate highly target-specific biopolymers, such as aptamers, spiegelmers, anticalins, and conotoxins.

[0214] Non-Ig scaffolds in the context of the present invention may be protein scaffolds and may be used as antibody mimics so that they can bind to ligands or antigens. Non-Ig scaffolds include tetranectin-based non-Ig scaffolds (e.g., as disclosed in US 2010 / 0028995); fibronectin scaffolds (e.g., as disclosed in EP 1266 025); lipocalin-based scaffolds (e.g., as disclosed in WO 2011 / 154420); ubiquitin scaffolds (e.g., as disclosed in WO 2011 / 073214); translocation scaffolds (e.g., as disclosed in US 2004 / 0023334); protein A scaffolds (e.g., as disclosed in EP 2231860), ankyrin repeat-based scaffolds (e.g., as disclosed in WO 2010 / 060748); microprotein (preferably, cystine-knot-forming microprotein) scaffolds (e.g., as disclosed in EP 2314308); Fyn SH3 domain-based scaffolds (e.g., as disclosed in WO 2011 / 023685); EGFR-A domain-based scaffolds (e.g., as disclosed in WO 2005 / 040229); and Kunitz domain-based scaffolds (e.g., as disclosed in EP 1941867). The non-Ig scaffold may be a peptide or oligonucleotide aptamer. Aptamers are typically formed by selection from a large collection of random sequences and may be either 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).

[0215] In another embodiment, the anti-DPP3 antibody format 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 antibody format is selected from the group comprising scFab fragments, Fab fragments, scFv fragments, and conjugates thereof that have been optimized for bioavailability, such as PEGylated fragments.

[0216] The term "antibody" in the context of the present invention generally includes monoclonal and polyclonal antibodies and their binding fragments, particularly Fc fragments and so-called "single-chain antibodies" (Bird et al. 1988), chimeric and humanized antibodies, particularly CDR-grafted antibodies, and bispecific or tetraspecific antibodies (Holliger et al. 1993). It also includes immunoglobulin-like proteins selected by techniques including, for example, phage display, for specific binding to a molecule of interest contained in a sample. The term "specific binding" in this context refers to an antibody raised against a molecule of interest or a fragment thereof. An antibody is considered specific if its affinity for the molecule of interest or a fragment thereof is preferably at least 50-fold higher, more preferably 100-fold higher, and most preferably at least 1000-fold higher than its affinity 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.

[0217] In a specific embodiment of the present invention, in said anti-DPP3 antibody or anti-DPP3 antibody fragment binding to an epitope according to SEQ ID NO: 2, said epitope is comprised in the DPP3 protein or a 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 binds to an epitope according to SEQ ID NO: 2, said epitope is comprised in the DPP3 protein or a functional derivative thereof is or is derived from a human antibody or a humanized antibody, or is or is derived from a humanized antibody fragment.

[0218] In certain embodiments, one or more (murine) CDRs are grafted onto a human antibody or antibody fragment.

[0219] In another aspect of the present invention, there is provided a human CDR-grafted anti-DPP3 antibody or anti-DPP3 antibody fragment thereof directed against and binding to an epitope according to SEQ ID NO:2, wherein said epitope is contained within a DPP3 protein or a functional derivative thereof, and wherein said human CDR-grafted anti-DPP3 antibody or anti-DPP3 antibody fragment thereof comprises an antibody heavy chain variable region (H chain) comprising SEQ ID NO:4 and / or further comprises an antibody light chain variable region (L chain) comprising SEQ ID NO:5.

[0220] 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 an epitope according to SEQ ID NO: 2, said epitope being contained within the DPP3 protein or a functional derivative thereof, said human CDR-grafted anti-DPP3 antibody or anti-DPP3 antibody fragment thereof 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.

[0221] In a particular embodiment of the present invention, the subject of the present invention is a human CDR monoclonal anti-DPP3 antibody or monoclonal anti-DPP3 antibody fragment directed against and binding to an epitope according to SEQ ID NO: 2, said epitope being contained within the DPP3 protein or a functional derivative thereof, the heavy chain of which comprises at least one CDR of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, and the light chain of which comprises at least one CDR of SEQ ID NO: 9, KVS or SEQ ID NO: 10.

[0222] The amount of DPP 3 protein and / or DPP 3 activity in a body fluid sample from said subject may be measured by a variety of methods, such as immunoassays, activity assays, mass spectrometry, and the like.

[0223] The amount of DPP 3 protein and / or DPP 3 activity in a sample of body fluid from said subject may be measured, for example, by one of the methods detailed below.

[0224] 1. Luminescence immunoassay (LIA) to quantify DPP-3 protein concentration (Rehfeld et al. 2019 JALM 3(6):943-953). The LIA is a one-step chemiluminescent sandwich immunoassay using white, high-binding polystyrene microtiter plates as the solid phase. These plates are coated with the monoclonal anti-DPP3 antibody AK2555 (capture antibody). The tracer anti-DPP3 antibody AK2553 is conjugated with MA70-acridinium-NHS-ester and used at a concentration of 20 ng per well. 20 μL of sample (e.g., serum, heparinized plasma, citrated plasma, or EDTA plasma from patient blood) and a calibration sample are pipetted into the coated white microtiter plate. After adding the tracer antibody AK2553, the microtiter plate is incubated at room temperature and 600 rpm for 3 hours. Unbound tracer is then removed by four washing steps (350 μL per well). The residual chemiluminescence is measured for 1 second per well using a microtiter plate luminometer. The concentration of DPP 3 is determined using a six-point calibration curve. Calibrators and samples are preferably measured in parallel.

[0225] 2. Enzyme capture activity assay (ECA) to quantify DPP-3 activity (Rehfeld et al. 2019 JALM 3(6):943-953). ECA is a DPP-3 specific activity assay that uses black high-binding polystyrene microtiter plates as the solid phase. These plates are coated with the monoclonal anti-DPP-3 antibody AK2555 (capture antibody). 20 μL of sample (e.g., serum, heparinized plasma, citrated plasma, EDTA plasma, cerebrospinal fluid, and urine) and calibration samples are pipetted into the coated black microtiter plate. After adding assay buffer (200 μL), the microtiter plate is incubated at 22°C and 600 rpm for 2 hours. DPP-3 present in the sample is immobilized by binding to the capture antibody. Unbound sample components are removed by four washing steps (350 μL per well). The specific activity of the immobilized DPP-3 is measured by adding the fluorogenic substrate Arg-Arg-β-naphthylamide (Arg2-βNA) to the reaction buffer and incubating at 37°C for 1 hour. DPP-3 specifically cleaves Arg2-βNA into Arg-Arg dipeptide and fluorescent β-naphthylamine. Fluorescence is measured in a fluorometer using an excitation wavelength of 340 nm and emission detection at 410 nm. DPP-3 activity is determined using a six-point calibration curve. It is preferable to measure the calibration sample and the sample in parallel.

[0226] 3. Liquid-phase assay (LAA) for quantifying DPP 3 activity (modified from Jones et al., Analytical Biochemistry, 1982). The LAA is a liquid-phase assay for measuring DPP-3 activity using a black, non-binding polystyrene microtiter plate. 20 μL of sample (e.g., serum, heparinized plasma, citrated plasma) and a calibration sample are pipetted into a black, non-binding microtiter plate. After adding the fluorescent substrate Arg2-βNA in assay buffer (200 μL), the initial βNA fluorescence (T = 0) is measured in a fluorometer using an excitation wavelength of 340 nm and emission detection at 410 nm. The plate is then incubated at 37°C for 1 hour. The final fluorescence (T = 60) is measured. The difference between the final and initial fluorescence is calculated. DPP-3 activity is determined using a six-point calibration curve. It is preferable to measure the calibration sample and the sample in parallel.

[0227] A variety of immunoassays are known and can be used 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 array assays, protein microarray assays, and rapid testing formats such as instant immunochromatographic strip tests ("dipstick immunoassays") and immunochromatographic assays.

[0228] In one embodiment of the invention, such an assay is a sandwich immunoassay using any type of detection technology, including but not limited to enzyme-labeled, chemiluminescent-labeled, or electrochemiluminescent-labeled, 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 that can be used on any one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, and Alere Triage®.

[0229] In one embodiment of the present invention, the test method may be a so-called POC test (point-of-care test), which is a testing technology that allows the test to be performed near the patient in less than an hour without the need for a fully automated measurement system. An example of this technology is the immunochromatographic testing technology.

[0230] In a particular embodiment of the present invention, the POC test combines the measurement of multiple analytes simultaneously. In another particular embodiment of the present invention, the analytes are selected from the group consisting of DPP 3 and proadrenomedullin or fragments thereof. In a further particular embodiment of the present invention, the POC test combines the measurement of DPP 3 and mature ADM.

[0231] In one embodiment of the invention, at least one of the binding agents is labeled for detection purposes.

[0232] 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.

[0233] Assays may be homogeneous or heterogeneous, competitive, or non-competitive. In one embodiment, the assay is a form of sandwich assay, a non-competitive immunoassay, in which the molecule to be detected and / or quantified is bound to a primary antibody and a secondary antibody. The primary antibody may be bound to a solid phase, such as a bead, the surface of a well or other container, a small piece, or a thin film, and the secondary antibody is an antibody labeled with a dye, a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. Standard compositions and procedures related to "sandwich assays" are well established and known to those skilled in the art (The Immunoassay Handbook, Ed. David Wild, Elsevier LTD, Oxford; 3rd ed. (May 2005), ISBN-13: 978-0080445267; Hultschig C et al., Curr Opin Chem Biol. 2006 Feb;10(1):4-10. PMID: 16376134).

[0234] In another embodiment, the assay comprises two capture molecules, preferably antibodies, both present as dispersion in a liquid reaction mixture, a first label component attached to the first capture molecule, said first label component being part of a labeling system based on fluorescence or chemiluminescence quenching or amplification, and a second label component of said marking system attached to a second capture molecule, such that upon binding of both capture molecules to the analyte, a measurable signal is generated allowing detection of the formed sandwich complex in the sample-containing solution.

[0235] 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 dye of the cyanine type.

[0236] In the context of the present invention, fluorescence-based assays involve the use of dyes, such as, for example, FAM (5 or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes such as CY3, CY5, CY3.5, CY5.5, Cy7, xanthene, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HE), and the like. 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.

[0237] In the context of the present invention, chemiluminescence-based assays involve the use of dyes based on the physical principles described for chemiluminescent materials in Kirk-Othmer, Encyclopedia of Chemical Technology, 4th ed., executive editor, J.I. Kroschwitz; editor, M. Howe-Grant, John Wiley & Sons, 1993, vol. 15, pp. 518-562, including citations to pp. 551-562, which are incorporated herein by reference. Preferred chemiluminescent dyes are acridinium esters.

[0238] As used herein, an "assay" or "diagnostic assay" may be any form of assay suitable for the field of diagnostics. Such an assay may be based on the binding of a detection analyte to one or more capture probes with a specific affinity. For the interaction between the capture molecule and the target molecule, i.e., molecule of interest, the affinity constant is preferably 10 8 M -1 Greater than.

[0239] DPP3 activity can be measured by detecting cleavage products of DPP3-specific substrates.

[0240] Known peptide hormone substrates include leucine enkephalin, methionine 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 these peptide hormones, as well as other untagged oligopeptides (e.g., Ala-Ala-Ala-Ala; Dhanda et al. 2008), can be monitored by detecting the respective cleavage products. Detection methods include, but are not limited to, HPLC analysis (e.g., Lee & Snyder 1982), mass spectrometry (e.g., Abramic et al. 2000), H1-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 reverse-phase chromatography (e.g., Mazocco et al. 2006).

[0241] Detection of fluorescence due to hydrolysis of fluorogenic substrates by DPP3 is a standard procedure for monitoring DPP3 activity. These substrates are specific dipeptides or tripeptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) conjugated to fluorophores. Fluorophores include, but are not limited to, β-naphthylamide (2-naphthylamide, βNA, 2NA), 4-methoxy-β-naphthylamide (4-methoxy-2-naphthylamide), and 7-amido-4-methylcoumarin (AMC, MCA; Abramic et al. 2000, Ohkubo et al. 1999). Cleavage of these fluorogenic substrates leads to the release of fluorescent β-naphthylamine or 7-amino-4-methylcoumarin, respectively. In a solution-phase assay, the ECA substrate and DPP3 are incubated, for example, in a 96-well plate format, and fluorescence is measured using a fluorescence detector (Ellis & Nuenke 1967). Furthermore, samples retaining DPP3 can be immobilized and resolved on electrophoretic gels, gels stained with a fluorescent substrate (e.g., Arg-Arg-βNA) and Fast Garnet GBC, and the fluorescent protein bands are detected by a fluorescence reader (Ohkubo et al. 1999). The same 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 conjugated to a chromophore such as p-nitroanilide diacetate. Detection of a color change upon hydrolysis of the chromogenic substrate can be used to monitor DPP3 activity.

[0242] Another option for detecting DPP3 activity is the Protease-Glo™ assay (commercially available from Promega). In this embodiment of the method, DPP3-specific dipeptides or tripeptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) are conjugated to aminoluciferin. Upon cleavage by DPP3, the aminoluciferin is released and serves as a substrate for the bound luciferase reaction, emitting detectable luminescence.

[0243] In a preferred embodiment, DPP3 activity is measured by adding the fluorogenic substrate Arg-Arg-βna and monitoring the fluorescence in real time.

[0244] In certain embodiments of the method for measuring active DPP 3 in a body fluid sample from a subject, the capture binding agent reactive with DPP 3 is immobilized on a solid phase.

[0245] The test sample is exposed to the immobilized binding agent, whereby DPP 3, if present, binds to the binding agent and is itself immobilized for detection. A substrate may then be added, and the reaction product may be detected to detect the presence or amount of DPP 3 in the test sample. For purposes of this specification, the term "solid phase" may be used to include any material or container within or on which a measurement can be performed, including, but not limited to, porous materials, non-porous materials, test tubes, wells, slides, agarose resins (e.g., Sepharose from GE Healthcare Life Sciences), magnetic particles (e.g., Dynabeads™ or Pierce™ magnetic beads from Thermo Fisher Scientific), etc.

[0246] Binding agents of protein or peptide origin (e.g., antibodies, antibody fragments, non-Ig scaffolds) can be bound to the surface of the target protein by various methods, including physical adsorption (e.g., by electrostatic or hydrophobic interactions), bioaffinity immobilization (e.g., avidin-biotin, protein A / G / L, His-tag, and Ni 2+ Oligonucleotide-based binders (e.g., aptamers) can be immobilized to solid phases by methods including cleavage of nucleotides (e.g., nucleotides containing nucleotides such as α-NTA, GST tags and glutathione, DNA hybridization, aptamers), covalent binding (e.g., amines and N-hydroxysuccinimide), or a combination of the above immobilization methods (Kim & Herr 2013). Oligonucleotide-based binders (e.g., aptamers) can also be immobilized to solid phases using the (strept)avidin-biotin system (Muller et al. 2012, Deng et al. 2014).

[0247] In certain embodiments of the method for measuring DPP 3 activity in a body fluid sample of a subject, the separating step is a washing step that removes components of the sample that are not bound to the capture binding agent from the captured DPP 3. The separating step may also be any other step that separates DPP 3 bound to the capture binding agent from components of the body fluid sample.

[0248] In certain embodiments of the methods for measuring DPP 3 activity in a body fluid sample from a subject, conversion of the DPP 3 substrate by immobilized DPP 3 is measured (detected) by a method selected from the group comprising fluorescence of a fluorogenic substrate (e.g., Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate 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 DPP 3) or Western blot (cleavage products).

[0249] In certain embodiments of the methods for measuring DPP3 activity in a subject's body fluid sample, the substrate may be selected from the group comprising leucine-enkephalin, methionine-enkephalin, endomorphins 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH, and MSH, or dipeptides and tripeptides linked to fluorophores, chromophores, or aminoluciferin (Promega's Protease-Glo™ assay). Dipeptides or tripeptides cleaved by DPP3 include, but are not limited to, Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, and Suc-Ala-Ala-Phe. Fluorophores include, but are not limited to, β-naphthylamide (2-naphthylamide, βNA, 2NA), 4-methoxy-β-naphthylamide (4-methoxy-2-naphthylamide), and 7-amido-4-methylcoumarin (AMC, MCA; Abramic et al. 2000, Ohkubo et al. 1999). Cleavage of these fluorogenic substrates leads to the release of fluorescent β-naphthylamine or 7-amino-4-methylcoumarin, respectively. Chromophores include, but are not limited to, p-nitroanilide diacetate (pNA). Hydrolysis of the peptide-pNA bond within the chromogenic substrate releases pNA and causes a color change. Thus, the change in absorbance (DA / min) is directly proportional to enzyme activity. Using Promega's Protease-Glo™ assay, aminoluciferin is released upon cleavage by DPP3 and serves as a substrate for the bound luciferase reaction, emitting detectable luminescence.

[0250] The subject of the present invention is also a method for prognosing the outcome and / or risk of an adverse event in a subject who has developed refractory shock, said method comprising: measuring the concentration of DPP 3 in a body fluid sample from said subject; comparing the measured concentration of DPP 3 with a predetermined threshold value, correlating the concentration of DPP 3 with the risk of the adverse event in the subject, wherein an elevated concentration above a certain threshold predicts an increased risk of the adverse event; or correlating the concentration of DPP 3 with the success of a treatment or intervention in the subject, wherein a concentration below a certain threshold is predictive of successful treatment or intervention. Includes:

[0251] The term "prognosis" in the context of the present invention refers to predicting how a patient's condition will progress. The term may include estimating the chances of recovery or the chances of adverse events for said patient. Adverse events are defined as organ dysfunction or mortality. Organ dysfunction is defined as renal, cardiac, or hepatic dysfunction.

[0252] Further embodiments of the present invention are as follows. 1. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock, the method comprising: measuring the concentration of DPP 3 in a body fluid sample from said subject; comparing the measured DPP 3 concentration with a predetermined threshold, The subject is predicted to suffer from refractory shock syndrome or diagnosed as suffering from refractory shock syndrome when the measured DPP 3 concentration exceeds the predetermined threshold. 2. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock according to embodiment 1, wherein the shock is selected from the group comprising hypovolemic shock, cardiogenic shock, vaso-occlusive shock, and distributive shock, in particular cardiogenic shock or septic shock. 3. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock according to embodiments 1 and 2, comprising: In the case of cardiogenic shock, the subject may be suffering from an acute coronary syndrome (e.g., acute myocardial infarction), or the subject is suffering from heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or In the case of hypovolemic shock, the subject may have a bleeding disorder, including spontaneous bleeding due to gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor invasion of major blood vessels), and anticoagulant use, or a non-hemorrhagic disorder, including vomiting, diarrhea, renal failure, renal loss, skin loss / unconscious injury (e.g., burns, heat stroke), or third-space loss in the setting of pancreatitis, cirrhosis, intestinal obstruction, or trauma; or In the case of vaso-occlusive shock, the patient may be suffering from cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis; or In the case of distributive shock, the patient may be suffering from septic shock, neurogenic shock, anaphylactic shock, or shock due to adrenal crisis. 4. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock according to embodiments 1 to 3, wherein the method is used to initiate and / or terminate and / or stratify and / or guide treatment. 5. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock according to any of embodiments 1 to 4, wherein treatment is initiated and / or maintained and / or suspended and / or terminated if the measured DPP 3 concentration exceeds the predetermined threshold. 6. A method for predicting or diagnosing refractory shock in a subject who has suffered from or developed shock according to embodiment 5, wherein the treatment is selected from the group consisting of vasopressors, angiotensin receptor agonists and / or precursors thereof, inhibitors of DPP 3 activity, and anti-adrenomedullin antibodies or anti-adrenomedullin antibody fragments. 7. A method for predicting or diagnosing refractory shock in a subject who has suffered from or has developed shock according to any of embodiments 1 to 6, comprising measuring either the concentration of DPP 3 protein and / or the concentration of active DPP 3 and comparing it with a predetermined threshold. 8. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock according to any of embodiments 1 to 7, wherein the concentration of DPP 3 is measured by contacting the body fluid sample with a capture binding agent that specifically binds to DPP 3. 9. The method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock according to embodiment 8, wherein said capture binding agent for measuring the concentration of DPP 3 may be selected from the group of antibodies, antibody fragments, or non-IgG scaffolds. 10. A method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock according to embodiments 8-9, wherein the capture binding agent is an antibody. 11. A method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock according to any of embodiments 8 to 10, wherein the capture binding agent is a monoclonal antibody. 12. A method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock according to any one of embodiments 1 to 11, wherein the body fluid sample is selected from the group consisting of whole blood, plasma, and serum. 13. A method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock according to any one of embodiments 1 to 12, wherein the method for diagnosing or predicting is carried out at least twice. 14. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock according to embodiments 1 to 13, wherein treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued if the concentration of DPP-3 in said sample exceeds a certain threshold, and / or treatment with a vasopressor agent is withheld and / or terminated if the measured concentration of DPP-3 exceeds said predetermined threshold. 15. A method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock according to embodiments 1 to 13, wherein treatment with a vasopressor is initiated and / or continued if the concentration of DPP 3 in said sample is below a certain threshold, and / or treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP 3 activity is withheld and / or terminated if the measured concentration of DPP 3 is below said predetermined threshold. 16. A method for predicting or diagnosing refractory shock in a subject who has fallen into shock or who has developed shock according to embodiments 14 to 15, further comprising measuring the concentration of proadrenomedullin or a fragment thereof, and initiating and / or continuing treatment with an anti-ADM antibody or an anti-ADM antibody fragment if the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with an anti-ADM antibody or an anti-ADM antibody fragment if the measured concentration of proadrenomedullin or a fragment thereof is below a predetermined threshold. 17. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock according to embodiment 14, wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment and / or an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued if the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold and the measured concentration of DPP-3 exceeds the predetermined DPP-3 threshold. 18. A vasopressor for use in treating shock in a subject suffering from or having developed shock, wherein the subject has a concentration of DPP3 in a body fluid sample from the subject that is below a predetermined threshold when measured by a method according to any one of embodiments 1 to 17. 19. A vasopressor for use in treating shock in a subject suffering from or having developed shock according to embodiment 18, wherein the vasopressor is selected from the group comprising dopamine, norepinephrine, norepinephrine equivalents, epinephrine, phenylephrine, and vasopressin. 20. A vasopressor for use in the treatment of shock in a subject suffering from or having developed shock according to embodiment 18 or 19, wherein said vasopressor is administered to said subject as a pharmaceutical formulation. 21. A vasopressor for use in the treatment of shock in a subject suffering from or having developed shock according to any of embodiments 18 to 20, wherein the subject's blood pressure is 65 mm Hg or less. 22. An inhibitor of DPP3 activity for use in the treatment of shock in a subject suffering from or having developed shock, wherein said subject has a DPP3 concentration above a predetermined threshold in a body fluid sample from said subject when measured by a method according to any of embodiments 1 to 17. 23. An inhibitor of DPP3 activity for use in the treatment of shock in a subject suffering from or having developed shock according to embodiment 22, wherein the inhibitor of DPP3 activity is selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold. 24. An inhibitor of DPP 3 activity for use in the treatment of shock in a subject suffering from or having developed shock according to embodiments 22 and 23, wherein the inhibitor is selected from the group consisting of 10 -7 M or less. 25. An inhibitor of DPP 3 activity for use in the treatment of shock in a subject suffering from or having developed shock according to any of embodiments 22 to 24, wherein the inhibitor of DPP 3 activity is an antibody. 26. An inhibitor of DPP 3 activity for use in the treatment of shock in a subject suffering from or having developed shock according to any of embodiments 22 to 25, wherein said inhibitor is a monoclonal antibody. 27. An inhibitor of DPP-3 activity for use in the treatment of shock in a subject suffering from or having developed shock according to any of embodiments 22 to 26, wherein the inhibitor is a monoclonal antibody, the complementarity determining regions (CDRs) in the heavy chain of which comprise the sequences of SEQ ID NO: 7, SEQ ID NO: 8, and / or SEQ ID NO: 9, and the complementarity determining regions (CDRs) in the light chain of which comprise the sequences of SEQ ID NO: 10, KVS, and / or SEQ ID NO: 11. 28. An inhibitor of DPP-3 activity for use in the treatment of shock in a subject suffering from or having developed shock according to any of embodiments 22 to 27, wherein the inhibitor is a humanized monoclonal antibody or a humanized monoclonal antibody fragment, the heavy chain of which comprises SEQ ID NO: 12 and the light chain of which comprises SEQ ID NO: 13. 29. An inhibitor of DPP 3 activity for use in the treatment of shock in a subject suffering from or having developed shock according to any of embodiments 22 to 28, wherein said inhibitor is administered in combination with an angiotensin receptor agonist and / or a precursor thereof. 30. An inhibitor of DPP 3 activity for use in the treatment of shock in a subject suffering from or having developed shock according to embodiment 29, wherein the angiotensin receptor agonist and / or precursor thereof is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, angiotensin IV, in particular angiotensin II. 31. An angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity for use in treating shock in a subject suffering from or having developed shock, wherein treatment with said angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued when the concentration of DPP-3 in a sample from said subject exceeds a certain threshold, and / or treatment with a vasopressor agent is suspended and / or terminated when the measured concentration of DPP-3 exceeds said predetermined threshold. 32. A vasopressor agent for use in treating shock in a subject suffering from or having developed shock, wherein treatment with the vasopressor agent is initiated and / or continued when the concentration of DPP 3 in a body fluid sample from the subject is below a certain threshold, and / or treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP 3 activity is suspended and / or terminated when the measured concentration of DPP 3 is below said predetermined threshold. 33. A vasopressor for use in treating shock in a subject suffering from or having developed shock according to embodiment 32, further comprising measuring the concentration of proadrenomedullin or a fragment thereof, and initiating and / or continuing treatment with an anti-ADM antibody or an anti-ADM antibody fragment if the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with an anti-ADM antibody or an anti-ADM antibody fragment if the measured concentration of proadrenomedullin or a fragment thereof is below the predetermined threshold. 34. An angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity for use in treating shock in a subject suffering from or having developed shock according to embodiment 31, further comprising measuring the concentration of proadrenomedullin or a fragment thereof, and starting and / or continuing treatment with the anti-ADM antibody or anti-ADM antibody fragment if the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with the anti-ADM antibody or anti-ADM antibody fragment if the measured concentration of proadrenomedullin or a fragment thereof is below the predetermined threshold. 35. An anti-ADM antibody or an anti-ADM antibody fragment for use in treating shock in a subject suffering from or having developed shock according to embodiments 33-34, wherein treatment with the anti-ADM antibody or anti-ADM antibody and / or an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued when the concentration of pro-adrenomedullin or a fragment thereof in the sample exceeds a certain threshold and the measured concentration of DPP-3 exceeds the predetermined DPP-3 threshold. 36. A method of treating shock in a subject suffering from or having developed shock, the method comprising administering a vasopressor to the subject, wherein the subject has a concentration of DPP 3 below a predetermined threshold in a body fluid sample from the subject as measured by a method according to any of embodiments 1 to 17. 37. A method for treating shock in a subject suffering from or having developed shock according to embodiment 35, the method comprising administering to the subject a vasopressor agent, wherein the vasopressor agent is selected from the group comprising dopamine, norepinephrine, norepinephrine equivalents, epinephrine, phenylephrine, and vasopressin. 38. A method for treating shock in a subject suffering from or having developed shock according to embodiments 35-36, the method comprising administering a vasopressor to the subject, wherein the vasopressor is administered to the subject as a pharmaceutical formulation. 39. A method for treating shock in a subject suffering from or having developed shock according to embodiments 35-37, the method comprising administering a vasopressor to the subject, wherein the subject's blood pressure is 65 mm Hg or less. 40. A method of treating shock in a subject suffering from or having developed shock, the method comprising administering to the subject an inhibitor of DPP 3 activity, wherein the subject has a concentration of DPP 3 in a body fluid sample from the subject that exceeds a predetermined threshold when measured by a method according to any of embodiments 1 to 17. 41. A method for treating shock in a subject suffering from or having developed shock according to embodiment 39, the method comprising administering to said subject an inhibitor of DPP3 activity, wherein said inhibitor of DPP3 activity is selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold. 42. A method for treating shock in a subject suffering from or experiencing shock according to embodiments 39-40, comprising administering to the subject an inhibitor of DPP-3 activity, wherein the inhibitor is 10 -7 M or less. 43. A method for treating shock in a subject suffering from or having developed shock according to embodiments 39 to 41, the method comprising administering to the subject an inhibitor of DPP-3 activity, wherein the inhibitor of DPP-3 activity is an antibody. 44. A method for treating shock in a subject suffering from or having developed shock according to any one of embodiments 39 to 42, the method comprising administering to the subject an inhibitor of DPP-3 activity, wherein the inhibitor of DPP-3 activity is a monoclonal antibody. 45. A method for treating shock in a subject suffering from or having developed shock according to embodiments 39 to 43, comprising administering to the subject an inhibitor of DPP-3 activity, wherein the inhibitor is a monoclonal antibody whose heavy chain complementarity-determining regions (CDRs) comprise the sequences of SEQ ID NO: 7, SEQ ID NO: 8, and / or SEQ ID NO: 9 and whose light chain complementarity-determining regions (CDRs) comprise the sequences of SEQ ID NO: 10, KVS, and / or SEQ ID NO: 11. 46. ​​A method for treating shock in a subject suffering from or having developed shock according to embodiments 39 to 44, the method comprising administering to the subject an inhibitor of DPP-3 activity, wherein the inhibitor is a humanized monoclonal antibody or a humanized monoclonal antibody fragment, the heavy chain of which comprises the sequence of SEQ ID NO: 12 and the light chain of which comprises the sequence of SEQ ID NO: 13. 47. A method for treating shock in a subject suffering from or having developed shock according to embodiments 39 to 45, the method comprising administering to the subject an inhibitor of DPP 3 activity, wherein the inhibitor is administered in combination with an angiotensin receptor agonist and / or a precursor thereof. 48. A method for treating shock in a subject suffering from or having developed shock according to embodiments 39 to 46, the method comprising administering to the subject an inhibitor of DPP 3 activity, wherein the angiotensin receptor agonist and / or precursor thereof is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, angiotensin IV, in particular angiotensin II. 49. A method for treating shock in a subject suffering from or having developed shock according to embodiments 39 to 47, comprising administering to the subject an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity, wherein treatment with the angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued when a concentration of DPP-3 in a sample from the subject exceeds a certain threshold, and / or treatment with a vasopressor agent is suspended and / or terminated when the measured concentration of DPP-3 exceeds the predetermined threshold. 50. A method for treating shock in a subject suffering from or having developed shock according to embodiments 35 to 38, comprising administering a vasopressor to the subject, wherein treatment with the vasopressor is initiated and / or continued when a concentration of DPP 3 in a body fluid sample from the subject is below a certain threshold, and / or treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP 3 activity is withheld and / or terminated when the measured concentration of DPP 3 is below the predetermined threshold. 51. A method for treating shock in a subject suffering from or having developed shock according to embodiment 49, the method comprising administering to the subject an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP 3 activity, and further measuring the concentration of proadrenomedullin or a fragment thereof, and initiating and / or continuing treatment with the anti-ADM antibody or anti-ADM antibody fragment if the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with the anti-ADM antibody or anti-ADM antibody fragment if the measured concentration of proadrenomedullin or a fragment thereof is below the predetermined threshold. 52. A method for treating shock in a subject suffering from or having developed shock according to embodiment 50, the method comprising administering a vasopressor to the subject, and further measuring the concentration of proadrenomedullin or a fragment thereof, and initiating and / or continuing treatment with the anti-ADM antibody or anti-ADM antibody fragment if the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with the anti-ADM antibody or anti-ADM antibody fragment if the measured concentration of proadrenomedullin or a fragment thereof is below the predetermined threshold. 53. A method for treating shock in a subject suffering from or having developed shock according to embodiments 51 and 52, comprising administering to the subject an anti-ADM antibody or an anti-ADM antibody fragment, and measuring the concentration of proadrenomedullin or a fragment thereof, and starting and / or continuing treatment with the anti-ADM antibody or anti-ADM antibody fragment if the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with the anti-ADM antibody or anti-ADM antibody fragment if the measured concentration of proadrenomedullin or a fragment thereof is below the predetermined threshold. 54. A method for treating shock in a subject suffering from or having developed shock according to embodiments 51 to 53, the method comprising administering to the subject an anti-ADM antibody or an anti-ADM antibody fragment, wherein treatment with the anti-ADM antibody or anti-ADM antibody and / or an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued when the concentration of pro-adrenomedullin or a fragment thereof in the sample exceeds a certain threshold and the measured concentration of DPP-3 exceeds the predetermined DPP-3 threshold.

[0253] The following consecutively numbered embodiments, together with those described above, provide further particular aspects of the present invention. 1. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock, the method comprising: measuring the concentration of DPP 3 in a body fluid sample from said subject; and comparing the measured DPP 3 concentration with a predetermined threshold, The subject is predicted to suffer from refractory shock syndrome or diagnosed as suffering from refractory shock syndrome when the measured DPP 3 concentration exceeds the predetermined threshold. 2. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock according to embodiment 1, wherein the shock is selected from the group comprising hypovolemic shock, cardiogenic shock, vaso-occlusive shock, and distributive shock, in particular cardiogenic shock or septic shock. 3. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock according to embodiments 1 and 2, comprising: In the case of cardiogenic shock, the subject may be suffering from an acute coronary syndrome (e.g., acute myocardial infarction), or the subject is suffering from heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or In the case of hypovolemic shock, the subject may have a bleeding disorder, including spontaneous bleeding due to gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor invasion of major blood vessels), and anticoagulant use, or a non-hemorrhagic disorder, including vomiting, diarrhea, renal failure, renal loss, skin loss / unconscious injury (e.g., burns, heat stroke), or third-space loss in the setting of pancreatitis, cirrhosis, intestinal obstruction, or trauma; or In the case of vaso-occlusive shock, the patient may be suffering from cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis; or In the case of distributive shock, the patient may be suffering from septic shock, neurogenic shock, anaphylactic shock, or shock due to adrenal crisis. 4. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock according to embodiments 1 to 3, wherein the method is used to initiate and / or terminate and / or stratify and / or guide treatment. 5. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock according to any of embodiments 1 to 4, wherein treatment is initiated and / or maintained and / or suspended and / or terminated if the measured DPP 3 concentration exceeds the predetermined threshold. 6. A method for predicting or diagnosing refractory shock in a subject who has suffered from or developed shock according to embodiment 5, wherein the treatment is selected from the group consisting of vasopressors, angiotensin receptor agonists and / or precursors thereof, inhibitors of DPP 3 activity, and anti-adrenomedullin antibodies or anti-adrenomedullin antibody fragments. 7. A method for predicting or diagnosing refractory shock in a subject who has suffered from or developed shock according to any of embodiments 1 to 6, comprising measuring either the concentration of DPP 3 protein and / or the concentration of active DPP 3 and comparing it with a predetermined threshold, and measuring the concentration of DPP 3 by contacting the body fluid sample with a capture binding agent that specifically binds to DPP 3. 8. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock according to embodiments 1 to 7, wherein treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued if the concentration of DPP-3 in said sample exceeds a certain threshold, and / or treatment with a vasopressor agent is suspended and / or terminated if the measured concentration of DPP-3 exceeds said predetermined threshold. 9. A method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock according to embodiments 1 to 7, wherein treatment with a vasopressor is initiated and / or continued if the concentration of DPP 3 in said sample is below a certain threshold, and / or treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP 3 activity is withheld and / or terminated if the measured concentration of DPP 3 is below said predetermined threshold. 10. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock according to embodiment 8, further comprising measuring the concentration of proadrenomedullin or a fragment thereof, and initiating and / or continuing treatment with an anti-ADM antibody or an anti-ADM antibody fragment if the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with an anti-ADM antibody or an anti-ADM antibody fragment if the measured concentration of proadrenomedullin or a fragment thereof is below a predetermined threshold. 11. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock according to embodiment 8, wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment and / or an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued if the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold and the measured concentration of DPP-3 exceeds the predetermined DPP-3 threshold. 12. A vasopressor for use in treating shock in a subject suffering from or having developed shock, wherein the subject has a concentration of DPP3 in a body fluid sample from the subject that is below a predetermined threshold when measured by a method according to any one of embodiments 1 to 10. 13. An inhibitor of DPP-3 activity for use in the treatment of shock in a subject suffering from or having developed shock, wherein said subject has a DPP-3 concentration above a predetermined threshold in a body fluid sample from said subject when measured by a method according to any of embodiments 1 to 10, and wherein the inhibitor of DPP-3 activity is selected from the group comprising an anti-DPP-3 antibody or an anti-DPP-3 antibody fragment or an anti-DPP-3 non-Ig scaffold. 14. An inhibitor of DPP 3 activity for use in the treatment of shock in a subject suffering from or having developed shock according to embodiment 13, wherein said inhibitor is administered in combination with an angiotensin receptor agonist and / or a precursor thereof. 15. An inhibitor of DPP 3 activity for use in the treatment of shock in a subject suffering from or having developed shock according to embodiment 14, wherein the angiotensin receptor agonist and / or precursor thereof is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, angiotensin IV, in particular angiotensin II. 16. A method of treating shock in a subject suffering from or having developed shock, the method comprising administering a vasopressor to the subject, wherein the subject has a concentration of DPP 3 in a body fluid sample from the subject that is below a predetermined threshold when measured by a method according to any of embodiments 1 to 10. 17. A method of treating shock in a subject suffering from or having developed shock, the method comprising administering to said subject an inhibitor of DPP 3 activity, wherein said subject has a concentration of DPP 3 in a body fluid sample from said subject that exceeds a predetermined threshold when measured by a method according to any of embodiments 1 to 10. 18. A method for treating shock in a subject suffering from or having developed shock according to embodiment 17, the method comprising administering to the subject an inhibitor of DPP 3 activity, wherein the inhibitor is administered in combination with an angiotensin receptor agonist and / or a precursor thereof. 19. A method for treating shock in a subject suffering from or having developed shock according to embodiments 16-17, comprising administering to the subject an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity, wherein treatment with the angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued when a concentration of DPP-3 in a sample from the subject exceeds a certain threshold, and / or treatment with a vasopressor agent is suspended and / or terminated when the measured concentration of DPP-3 exceeds the predetermined threshold. 20. A method for treating shock in a subject suffering from or having developed shock according to embodiments 16-17, comprising administering a vasopressor to the subject, wherein treatment with the vasopressor is initiated and / or continued when a concentration of DPP 3 in a body fluid sample from the subject is below a certain threshold, and / or treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP 3 activity is withheld and / or terminated when the measured concentration of DPP 3 is below the predetermined threshold. 21. A method for treating shock in a subject suffering from or having developed shock according to embodiment 18, comprising administering to the subject an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP 3 activity, and further measuring the concentration of proadrenomedullin or a fragment thereof, and initiating and / or continuing treatment with the anti-ADM antibody or anti-ADM antibody fragment if the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with the anti-ADM antibody or anti-ADM antibody fragment if the measured concentration of proadrenomedullin or a fragment thereof is below the predetermined threshold. 22. A method for treating shock in a subject suffering from or having developed shock according to embodiment 19, the method comprising administering a vasopressor to the subject, and further measuring the concentration of proadrenomedullin or a fragment thereof, and initiating and / or continuing treatment with the anti-ADM antibody or anti-ADM antibody fragment if the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with the anti-ADM antibody or anti-ADM antibody fragment if the measured concentration of proadrenomedullin or a fragment thereof is below the predetermined threshold. 23. A method for treating shock in a subject suffering from or having developed shock according to embodiments 20 and 21, comprising administering to the subject an anti-ADM antibody or an anti-ADM antibody fragment, and further measuring the concentration of pro-adrenomedullin or a fragment thereof, and initiating and / or continuing treatment with the anti-ADM antibody or anti-ADM antibody fragment if the concentration of pro-adrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with the anti-ADM antibody or anti-ADM antibody fragment if the measured concentration of pro-adrenomedullin or a fragment thereof is below the predetermined threshold. 24. A method for prognosing the outcome and / or risk of an adverse event in a subject who has developed refractory shock, the method comprising: measuring the concentration of DPP 3 in a body fluid sample from said subject; comparing the measured DPP 3 concentration with a predetermined threshold; and correlating the concentration of DPP 3 with the risk of an adverse event in the subject; or correlating the concentration of DPP 3 with the success of treatment or intervention in the subject; Elevated concentrations above a certain threshold predict an increased risk of said adverse event, while concentrations below a certain threshold predict successful treatment or intervention. [Example]

[0254] Example 1 - Methods for measuring DPP-3 protein and DPP-3 activity For the production of antibodies and measurement of DPP 3 binding ability, several mouse antibodies were produced and screened for their ability to bind to DPP 3 in a specific binding assay (see Table 1).

[0255] Peptides / conjugates for immunization: The DPP 3 peptides for immunization (JPT Technologies, Berlin, Germany) referenced in Table 1 were synthesized with an additional N-terminal cysteine ​​residue (if no cysteine ​​was present in the selected DPP 3 sequence) for conjugation of the peptide to bovine serum albumin (BSA). The peptides were covalently coupled to BSA using Sulfolink binding gel (Perbio-science, Bonn, Germany). The conjugation procedure was performed according to the Perbio manual. Recombinant GST-hDPP 3 was produced by USBio (United States Biological, Salem, MA, USA).

[0256] Mice immunization, immune cell fusion and selection: Balb / c mice were injected intraperitoneally (ip) with 84 μg or 100 μg of GST-hDPP3 or DPP3-peptide-BSA conjugates (emulsified in TiterMax Gold vehicle) on day 0, 84 μg or 100 μg of GST-hDPP3 or DPP3-peptide-BSA conjugates (emulsified in complete Freund's vehicle) on day 14, and 42 μg or 50 μg of GST-hDPP3 or DPP3-peptide-BSA conjugates (emulsified in incomplete Freund's vehicle) on days 21 and 28. On day 49, the animals received an intravenous (iv) injection of 42 μg of GST-hDPP3 or 50 μg of DPP3-peptide-BSA conjugates dissolved in saline. Mice were sacrificed 3 days later, and immune cell fusion was performed.

[0257] Spleen cells from immunized mice and cells of the myeloma cell line SP2 / 0 were fused with 1 mL of 50% polyethylene glycol at 37°C for 30 seconds. After washing, the cells were seeded into 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium (RPMI1640 medium supplemented with 20% fetal bovine serum and HAT supplements). After one week, the HAT medium was replaced with HT medium for three passages, after which the cells were returned to normal cell culture medium.

[0258] Cell culture supernatants were first screened for recombinant DPP3-binding IgG antibodies 2 weeks after fusion. Recombinant GST-tagged hDPP3 (USBiologicals, Salem, USA) was immobilized on a 96-well plate (100 ng / well) and incubated with 50 μL of cell culture supernatant per well at room temperature for 2 hours. After washing the plate, 50 μL / well of POD-rabbit anti-mouse IgG was added and incubated at room temperature for 1 hour. After a washing step, 50 μL of chromogen solution (3.7 mM o-phenylenediamine, 0.012% H2O2 in citrate / phosphate buffer) was added to each well and incubated at room temperature for 15 minutes. The color reaction was stopped by adding 50 μL of 4N sulfuric acid. Absorbance was detected at 490 nm.

[0259] Positively tested microcultures were transferred to 24-well plates for expansion. After retesting, selected cultures were cloned and recloned using limiting dilution techniques and isotyped.

[0260] Production of mouse monoclonal antibodies Antibodies raised against GST-tagged human DPP 3 or DPP 3-peptide were produced by standard antibody production methods (Marx et al. 1997) and purified by protein A. Antibody purity was greater than 90% based on SDS gel electrophoresis analysis.

[0261] Characterization of antibodies-binding to hDPP3 and / or immunizing peptides Binding assays were performed to analyze the ability of various antibodies and antibody clones to bind DPP 3 / immunizing peptide.

[0262] a) Solid phase Recombinant GST-tagged hDPP3 (SEQ ID NO: 1) or DPP3 peptide (immunizing peptide, SEQ ID NO: 2) was immobilized on the surface of a high-binding microtiter plate (96-well polystyrene microplate, Greiner Bio-One International AG, Austria, 1 μg / well in binding buffer [50 mM Tris, 100 mM NaCl, pH 7.8] at room temperature for 1 h). After blocking with 5% bovine serum albumin, the microplate was vacuum dried. b) Labeling procedure (tracer) 100 μg (100 μL) of various anti-DPP-3 antibodies (detection antibody, 1 mg / mL in PBS, pH 7.4) was mixed with 10 μL of acridinium NHS ester (1 mg / mL in acetonitrile, InVent, Germany; EP 0 353 971) and incubated at room temperature for 30 minutes. The labeled anti-DPP-3 antibodies were purified by gel filtration HPLC on Shodex Protein 5 μm KW-803 (Showa Denko, Japan). The purified labeled antibodies were diluted in assay buffer (50 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na2-EDTA, 5 g / L bovine serum albumin, 1 g / L mouse IgG, 1 g / L bovine IgG, 50 μmol / L amastatin, 100 μmol / L leupeptin, pH 7.4). The final concentration is approximately 5-7*10 of labeled compound (approximately 20 ng of labeled antibody) per 200 μL. 6 The chemiluminescence of the acridinium ester was measured using a Centro LB 960 luminance meter (Berthold Technologies).

[0263] c) hDPP 3 binding measurement Plates were filled with 200 μL of labeled and diluted detection antibody (tracer) and incubated for 2–4 h at 2–8°C. Unbound tracer was removed by washing four times with 350 μL of wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Well-bound chemiluminescence was measured using a Centro LB 960 luminometer (Berthold Technologies).

[0264] Antibody characterization - hDPP 3 inhibition analysis To analyze the DPP3 inhibitory potential of various antibodies and antibody clones, DPP3 activity assays were performed using a previously described procedure (Jones et al., 1982). Recombinant GST-tagged hDPP3 was diluted in assay diluent (25 ng / mL GST-DPP3 and 100 μM ZnCl2 in 50 mM Tris-HCl, pH 7.5), and 200 μL of this solution was incubated with 10 μg of each antibody at room temperature. After a 1-hour preincubation, the fluorogenic substrate Arg-Arg-βNA (20 μL, 2 mM) was added to the solution, and the generation of free βNA was monitored over time at 37°C using a Twinkle LB 970 microplate fluorometer (Berthold Technologies). βNA fluorescence was detected by excitation at 340 nm and emission at 410 nm. The slope of the fluorescence increment (RFU / min) for the various samples was calculated. The slope of GST-hDPP3 with the buffer control is designated as 100% activity. The inhibitory capacity of a potential capture binding agent is defined as the reduction in GST-hDPP3 activity by incubation with said capture binding agent, expressed as %.

[0265] The following table shows a selection of the antibodies obtained and their binding rates in relative light units (RLU) and their relative inhibitory potency (%; Table 1). The monoclonal antibodies raised against the DPP 3 regions shown below were selected for their ability to bind to recombinant DPP 3 and / or the immunizing peptide, as well as their inhibitory potency.

[0266] All antibodies raised against the GST-tagged full-length form of recombinant hDPP 3 show strong binding to immobilized GST-tagged hDPP 3. Antibodies raised against the peptide of SEQ ID NO: 2 also bind to GST-hDPP 3. The antibody of SEQ ID NO: 2 also binds strongly to the immunizing peptide. [Table 3]

[0267] Developments in a luminescence immunoassay (DPP-3-LIA) to quantify DPP-3 protein concentration, as well as an enzyme capture activity assay (DPP-3-ECA) to quantify DPP-3 activity, have recently been reported (Rehfeld et al. 2018. JALM. in press), which are incorporated herein by reference in their entireties.

[0268] Example 2 - DPP 3 for short-term mortality prognosis DPP 3 concentrations were measured in the plasma of patients with sepsis / septic shock and cardiogenic shock and were related to the short-term mortality of the patients.

[0269] Study population - Sepsis / septic shock DPP-3 was measured in 574 plasma samples from patients in the Adrenomedullin in Severe Sepsis and Septic Shock and Outcomes (AdrenOSS) study, a prospective, observational, multinational study including 583 patients admitted to the intensive care unit with sepsis or septic shock (Hollinger et al., 2018). 292 patients were diagnosed with septic shock.

[0270] Study population - cardiogenic shock Plasma samples from 108 patients diagnosed with cardiogenic shock were tested for DPP-3. Blood was collected within 6 hours of the onset of cardiogenic shock. Mortality was followed for 7 days.

[0271] hDPP 3 immunoassay: To measure DPP-3 concentrations in human plasma, we used an immunoassay (LIA) or activity assay (ECA) to detect the amount of human DPP-3 (LIA) or activity of human DPP-3 (ECA), respectively. Antibody fixation, labeling, and incubation were performed as described by Rehfeld et al. (Rehfeld et al. 2018).

[0272] result Short-term patient survival in sepsis patients was associated with DPP-3 plasma concentrations at admission. Patients with DPP-3 plasma concentrations above 40.5 ng / mL (third quartile) had an increased risk of mortality compared with patients with DPP-3 plasma concentrations below this threshold (Figure 1A). When this threshold was applied to a subset of patients with septic shock, the short-term mortality risk associated with high DPP-3 plasma concentrations became even more pronounced (Figure 1B). When the same threshold was applied to patients with cardiogenic shock, a similar increased risk of short-term mortality within 7 days was observed in patients with high DPP-3 concentrations (Figure 1C).

[0273] Example 3 - Purification of human native DPP3 Human erythrocyte lysate was applied to a total of 100 mL of Sepharose 4B resin (Sigma-Aldrich), and the flow-through fraction was collected. The resin was washed with a total of 370 mL of PBS buffer, pH 7.4, and the wash fraction was combined with the collected flow-through fraction to a total volume of 2370 mL.

[0274] For the immunoaffinity purification step, 110 mg of the 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%, as determined by quantification of unbound antibody using the Bradford assay. The resin-antibody complex was equilibrated with 10 bed volumes of wash-binding buffer (PBS, 0.1% Triton X-100, pH 7.4), combined with 2370 mL of cleared red blood cell lysate, and incubated at 4°C for 2 hours with continuous agitation. The resulting 100 mL incubation mixture was applied to ten 15 mL polypropylene columns, centrifuged at 1000 x g for 30 seconds, and the flow-through was collected. This process was repeated several times to obtain 2.5 mL of resin loaded with DPP3 per column. Using gravity flow, each column was washed five times with 10 mL of wash-binding buffer. 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 and immediate centrifugation at 1000 x g for 30 seconds. This elution step was repeated three times to obtain a combined eluate of 360 mL. The pH of the neutralized eluate was 8.0.

[0275] 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 Äkta Start system (GE Healthcare). After loading, the column was washed with 5 column volumes of IEX buffer A2 (12 mM NaH2PO4, pH 7.4) to remove unbound proteins. DPP3 elution was achieved with IEX buffer B (12 mM NaH2PO4, 1 M NaCl, pH 7.4) using a sodium chloride gradient ranging from 0 to 1 M NaCl over 10 column volumes (50 mL). The eluate was collected in 2 mL fractions. The buffers used for ion exchange chromatography were sterile filtered using a 0.22 μM bottle-top filter.

[0276] The results of the purification, including the respective yields and activities of each purification step, are shown in Table 2. Figure 2 shows SDS-PAGE on a gradient gel (4-20%) of native hDPP 3 purified from human erythrocyte lysate. [Table 4]

[0277] Example 4 - Effects of native DPP3 in animal models The effects of native hDPP 3 injection in healthy mice were investigated by monitoring the contraction rate and renal resistance index.

[0278] Six wild-type black mice (8–12 weeks old; see Table 3 for group sizes) were acclimated for 2 weeks and then underwent baseline echocardiography. Mice were randomly assigned to one of two groups and subsequently received an intravenous injection of either native DPP-3 protein or PBS via retroorbital injection. DPP-3 protein was administered at a dose of 600 μg / kg.

[0279] After injection of DPP3 or PBS, cardiac function was assessed by echocardiography (Gao et al. 2011), and renal function was assessed by renal resistive index (Lubas et al. 2014, Dewitte et al. 2012) at 15, 60, and 120 minutes (Figure 3). [Table 5]

[0280] result Mice treated with native DPP 3 protein showed a significantly reduced contraction rate compared to the PBS-injected control group (Figure 4A). The WT+DPP 3 group also showed worsening renal function, as observed by an increased renal resistance index (Figure 4B).

[0281] Example 5 - Development of Procizumab Antibodies raised against SEQ ID NO:2 were characterized in more detail (epitope mapping, binding affinity, specificity, inhibitory capacity), and results for clone 1967 of SEQ ID NO:2 (AK1967; "Procizumab") are shown here as an example.

[0282] Determination of AK1967 epitopes on the surface of DPP-3: For epitope mapping of AK1967, several N- or C-terminally biotinylated peptides were synthesized (PE, Hennigsdorf, Germany). These peptides contain the complete immunizing peptide sequence (SEQ ID NO: 2) or fragments thereof, with stepwise removal of one amino acid from either the C- or N-terminus (see Table 5 for a complete list of peptides).

[0283] A high-binding 96-well plate was coated with 2 μg of avidin (Greiner Bio-One International, Austria) per well in binding buffer (500 mM Tris-HCl, pH 7.8, 100 mM NaCl). The plate was then washed and filled with a specific solution of biotinylated peptide (10 ng / well; buffer—1x PBS containing 0.5% BSA). The anti-DPP3 antibody, AK1967, was labeled with a chemiluminescent label according to Example 1.

[0284] The plate was filled with 200 μL of labeled and diluted detection antibody (tracer) and incubated for 4 hours at room temperature. Unbound tracer was removed by washing four times with 350 μL of wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Chemiluminescence bound to the wells was measured using a Centro LB 960 luminometer (Berthold Technologies). AK1967 binding to each peptide was determined by evaluation of relative light units (RLU). Peptides showing RLU signals significantly higher than nonspecific binding of AK1967 were defined as AK1967 binders. Combined analysis of bound and unbound peptides revealed the specific DPP 3 epitope of AK1967.

[0285] Determining Binding Affinity Using Octet: The experiment was performed using Octet Red 96 (ForteBio). AK1967 was captured with a kinetic-grade anti-human Fc (AHC) biosensor. The loaded biosensor was then immersed in a dilution series of recombinant GST-tagged human DPP3 (100, 33.3, 11.1, and 3.7 nM). Association was observed for 120 seconds, followed by dissociation for 180 seconds. The buffers used in the experiment are listed in Table 4. Kinetic analysis was performed using a 1:1 binding model and multiple-response nonlinear regression (global fitting). [Table 6]

[0286] Western blot analysis of AK1967 binding specificity: Blood cells from human EDTA blood were washed (three times with PBS), diluted with PBS, and lysed by repeated freeze-thaw cycles. The total protein concentration of the blood cell lysate was 250 μg / mL, and the DPP3 concentration was 10 μg / mL. 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) were subjected to SDS-PAGE and Western blot analysis. The blots were incubated in 1) blocking buffer (1x PBS-T containing 5% nonfat dry milk), 2) primary antibody solution (AK1967, 1:2,000 in blocking buffer), and 3) HRP-conjugated secondary antibody (goat anti-mouse IgG, 1:1,000 in blocking buffer). Bound secondary antibodies were detected using Amersham ECL Western blot detection reagent and Amersham Imager 600 UV (both from GE Healthcare).

[0287] DPP-3 Inhibition Assay: To analyze the DPP3 inhibitory potency of AK1967, DPP3 activity assays were performed using a known procedure (Jones et al., 1982) as described in Example 1. The inhibitory potency of AK1967 was defined as the percentage reduction in GST-hDPP3 activity upon incubation with the antibody. The resulting reduction in DPP3 activity is shown in the inhibition curve in Figure 1C.

[0288] Epitope mapping: Analysis of AK1967-bound and non-binding peptides revealed the DPP 3 sequence INPETG (SEQ ID NO: 3) as the epitope required for AK1967 binding (see Table 5). [Table 7]

[0289] Binding affinity: AK1967 is 2.2*10 -9It binds to recombinant GST-hDPP3 with an affinity of M (see Figure 5 for kinetic curves).

[0290] Specificity and Inhibitory Potential: The only protein detected by AK1967 as a primary antibody in the blood cell lysate was the 80 kDa DPP-3 (Figure 6). The total protein concentration of the lysate was 250 µg / mL, but the estimated DPP-3 concentration was approximately 10 µg / mL. Although there were more than 25 times as many nonspecific proteins in the lysate, AK1967 specifically bound to DPP-3 and detected DPP-3, without any other nonspecific binding.

[0291] AK1967 inhibited DPP-3 at 15 ng / mL in specific DPP-3 activity assays, with an IC 50 is approximately 15 ng / mL (Figure 7).

[0292] Chimerization / Humanization: The monoclonal antibody AK1967 ("Procizumab"), which has the ability to inhibit DPP 3 activity by up to 70%, was selected as a potential therapeutic antibody and was also used as a template for chimerization and humanization.

[0293] Humanization of mouse antibodies can be achieved by following the procedure below: To humanize an antibody of mouse origin, the antibody sequence is analyzed for the structural interaction between the framework region (FR) and the complementarity-determining region (CDR) and antigen. Based on the structural model, the appropriate FR of human origin is selected, and the mouse CDR sequence is grafted into the human FR. The amino acid sequence of CDR or FR can be changed to restore the structural interaction, which is abolished by the species switching of the FR sequence. This restoration of structural interaction can be achieved by a random method using a phage display library, or by a guided method guided by molecular model (Almagro and Fransson, 2008. Humanization of antibodies. Front Biosci. 13:1619-33).

[0294] In connection with the above, the variable regions can be attached to any subclass of constant regions (IgG, IgM, IgE, IgA) or to a single scaffold, Fab fragment, Fv, Fab, and F(ab)2. In Examples 6 and 7 below, a murine antibody variant with an IgG2a backbone was used. For chimerization and humanization, a human IgG1κ backbone was used.

[0295] For epitope binding, only the CDRs are important. The CDRs of the heavy and light chains of the murine anti-DPP 3 antibody (AK1967) are shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 for the heavy chain, and SEQ ID NO: 9, sequence KVS, and SEQ ID NO: 10 for the light chain, respectively. Sequencing of the anti-DPP 3 antibody (AK1967) revealed the antibody heavy chain variable region (H chain) as shown in SEQ ID NO: 11 and the antibody light chain variable region (L chain) as shown in SEQ ID NO: 12.

[0296] Example 6 - Effect of Procizumab on septic shock-induced heart failure In this experiment, the effect of procizumab injection in rats with sepsis-induced heart failure (Rittirsch et al. 2009) was investigated by monitoring contractility.

[0297] Cecal Ligation and Perforation (CLP) Model of Septic Shock: Male Wistar rats (2–3 months old, 300–400 g; group sizes are shown in Table 6) from the Centre d'élevage Janvier (France) were randomly assigned to one of three groups. All animals were anesthetized with intraperitoneal (ip) administration of ketamine hydrochloride (90 mg / kg) and xylazine (9 mg / kg). To induce polymicrobial sepsis, CLP was performed using the Rittirsch procedure with minor modifications. A midline abdominal incision (1.5 cm) was made to expose the cecum. The cecum was then ligated just below the ileocecal valve and punctured once with an 18-gauge needle. The abdominal cavity was then closed in two layers, followed by fluid resuscitation (3 mL / 100 g body weight of saline subcutaneously) and the animals were returned to their cages. Control animals underwent surgery without puncturing the cecum. CLP animals were randomly treated with control or therapeutic antibodies.

[0298] Considered design: The study flow is shown in Figure 8. After CLP or control surgery, animals were allowed to rest for 20 hours with free access to water and food. They were then anesthetized, tracheotomized, and arterial and venous lines were connected. 24 hours after CLP surgery, either AK1967 or vehicle (saline) was administered as a bolus injection at 5 mg / kg, followed by a 7.5 mg / kg infusion over 3 hours. As a safety measure, hemodynamics was monitored invasively and continuously from the start (t=0) until 3 hours later.

[0299] At t = 0 (baseline), all CLP animals were in a state of septic shock and developed depressed cardiac function (low blood pressure, low contractility). At this point, prolactin or vehicle (PBS) was injected (iv), and saline infusion was initiated. There was one control group and two CLP groups, as summarized in the table below (Table 6). At the end of the experiment, the animals were euthanized, and organs were harvested for subsequent analysis. [Table 8]

[0300] Invasive blood pressure: Hemodynamic changes were acquired using the AcqKnowledge system (BIOPAC Systems, USA), which provides a fully automated blood pressure analysis system. The catheter was connected to the BIOPAC system via a pressure sensor.

[0301] For this procedure, rats were anesthetized (with ketamine and xylazine). The animals were transferred to a heating pad to achieve a desired body temperature of 37–37.5°C. A temperature feedback probe was inserted into the rectum. The rat was placed supine on the operating table. The trachea was opened, and a catheter (16 G) for external ventilation was inserted without damaging the carotid artery or vagus nerve. An arterial catheter was inserted into the right carotid artery. The carotid artery was separated from the vagus nerve before ligation. A central venous catheter was inserted through the left jugular vein to allow for the administration of PCZ or PBS.

[0302] After surgery, animals were allowed to rest in a stable state prior to hemodynamic measurements. Baseline blood pressure (BP) was then recorded. During data collection, saline infusion via the arterial line was stopped.

[0303] Echocardiography: Animals were anesthetized with ketamine hydrochloride. The chest was shaved and the rat was placed in a supine position. Transthoracic echocardiography (TTE) was performed using a commercially available GE Healthcare Vivid 7 ultrasound system equipped with a high-frequency (14 MHz) linear probe and a 10 MHz cardiac probe. All examinations were digitally recorded and stored for subsequent offline analysis.

[0304] Grayscale images were recorded at a depth of 2 cm. Two-dimensional examinations were performed starting with a parasternal longitudinal view to measure the diameter of the aortic annulus and the pulmonary artery. Furthermore, M-mode was used to measure left ventricular (LV) dimensions and assess fractional contractility (FS%). LVFS was calculated as (LV end-diastolic diameter - LV end-systolic diameter) / LV end-diastolic diameter and expressed as a percentage. End-diastole was therefore defined as the maximum LV diameter. End-systole was therefore defined as the minimum diameter during the same cardiac cycle. All parameters were measured manually. Three cardiac cycles were averaged for each measurement.

[0305] Pulmonary artery flow was recorded using pulse wave Doppler from the same parasternal long axis view. The velocity time integral of pulmonary arterial outflow was measured.

[0306] From the apical five-chamber view, flow at the mitral valve was recorded by pulse wave Doppler at the height of the mitral valve tip.

[0307] result: Septic shock-induced heart failure rats treated with PBS (CLP+PBS) showed a decreased contractile rate compared to control animals (Figure 9A). The CLP+PBS group also showed a high mortality rate (Figure 9B). In contrast, administration of prolactin to septic shock-induced heart failure rats improved contractile rate (Figure 9A) and dramatically reduced mortality (Figure 9B).

[0308] Example 7 - Effect of Procizumab on cardiac and renal function The effect of procizumab on isoproterenol-induced heart failure in mice was investigated by monitoring contractility and renal resistance index. Isoproterenol-induced cardiac stress in mice:

[0309] Acute heart failure was induced in 3-month-old male mice by subcutaneous injection of 300 mg / kg isoproterenol, a nonselective β-adrenergic agonist (DL-isoproterenol hydrochloride; ISO, Sigma Chemical Co.), twice daily for 2 days (Vergaro et al., 2016). ISO was diluted in 0.9% NaCl. ISO-treated mice were randomly assigned to two groups (Table 7). After standard echocardiography (Gao et al., 2011), PBS or procizumab (10 mg / kg) was intravenously injected. Renal resistive index measurements (Lubas et al., 2014, Dewitte et al., 2012) were performed on day 3 (Figure 10A and B).

[0310] Cardiac function was assessed by echocardiography (Gao et al., 2011) and by renal resistive index (Lubas et al., 2014, Dewitte et al., 2012) at 1, 6, and 24 hours (Figure 10A and B). A group of mice injected with vehicle (PBS) instead of isoproterenol received no further pharmacological treatment and served as a control group (Table 7). [Table 9]

[0311] result: Administration of procizumab to mice with isoproterenol-induced heart failure restored cardiac function within the first hour after administration (Figure 11A). Renal function in diseased mice showed a significant improvement 6 hours after procizumab injection and was comparable to that of control animals 24 hours later (Figure 11B).

[0312] Example 8 - DPP 3 demonstrates vasopressor requirement and responsiveness to vasopressor therapy Plasma DPP-3 concentrations in patients with septic shock were measured using an hDPP-3 immunoassay and correlated with the need for vasopressor therapy. Study population - septic shock

[0313] Plasma samples from 292 patients diagnosed with septic shock from the AdrenOSS (see Example 2) study were tested for DPP 3. Human DPP 3 was measured as described in Example 1.

[0314] result: Patients who showed an increased need for vasopressors over a 5-day period had higher DPP-3 plasma concentrations (Fig. 12A). In contrast, patients who responded to vasopressor therapy and who appeared to be able to discontinue vasopressors within the first 5 days had significantly lower DPP-3 plasma concentrations (Fig. 12B). This indicates that the development of refractory shock, i.e., increased need for vasopressors due to a decreased response to this therapy, is associated with higher DPP-3 plasma concentrations in patients with septic shock.

[0315] Patients with vasopressor-resistant refractory septic shock (norepinephrine >0.5 μg / kg / min) had significantly higher plasma DPP-3 concentrations compared with patients requiring norepinephrine doses <0.5 μg / kg / min (p<0.001) (Fig. 16A). Furthermore, DPP-3 plasma concentrations were strongly associated with mortality in patients with vasopressor-resistant refractory septic shock (Fig. 16B).

[0316] Example 9 - DPP-3 is associated with refractory shock Plasma DPP-3 concentrations in patients with cardiogenic shock, measured using a human DPP-3 immunoassay, were associated with the development of refractory shock.

[0317] Study population - cardiogenic shock Plasma samples from 57 patients diagnosed with cardiogenic shock after acute myocardial infarction were tested for DPP 3. Human DPP 3 was measured as described in Example 1.

[0318] result Patients with DPP-3 plasma concentrations above a certain threshold (59.1 ng / mL; third quartile) on admission developed refractory shock more frequently (47%) than patients with DPP-3 plasma concentrations below 59.1 ng / mL (12%) (Figure 13).

[0319] Example 10 - DPP 3 and bio-ADM show short-term mortality in shock DPP-3 and bio-ADM concentrations in the plasma of patients with septic shock were measured using hDPP-3 and bio-ADM immunoassays and correlated with the patients' short-term mortality. Study population - septic shock

[0320] Plasma samples from 292 patients diagnosed with septic shock from the AdrenOSS study were tested for DPP-3 and bio-ADM. Human DPP-3 was measured as described in Example 1. Bio-ADM was measured as described in Weber et al. (Weber et al. 2017. JALM 2(2): 1-4).

[0321] result Plasma concentrations of bio-ADM and DPP-3 were measured in patients with septic shock. Patients were divided into groups according to specific thresholds, which were determined as the third quartile of the total measured plasma concentration of each marker (Table 8). A similar number of patients (15.4%) had either elevated DPP-3 alone or elevated bio-ADM alone, but a lower frequency (9.6%) of patients had elevated plasma concentrations of both bio-ADM and DPP-3. [Table 10]

[0322] Mortality within the first 4 weeks after hospitalization was associated with bio-ADM and DPP-3 concentrations at admission. Patients with elevated plasma bio-ADM or DPP-3 concentrations, respectively, had a significantly increased risk of death within the first 4 weeks compared with patients whose plasma concentrations of bio-ADM (Figure 14A) or DPP-3 (Figure 14B) were below a specific threshold (the third quartile). The survival rate of patients with elevated bio-ADM was higher than that of patients with elevated DPP-3.

[0323] When both markers, i.e., bio-ADM and DPP 3, were combined, a higher short-term mortality risk was observed compared to the mortality associated with an increase in either marker alone (Figure 14C).Only 28.6% of patients with high plasma concentrations of bio-ADM and DPP 3 survived the first 4 weeks after hospitalization.

[0324] Example 11 - DPP 3 and bio-ADM DPP 3 demonstrate vasopressor requirement and response to vasopressor therapy Plasma DPP-3 and bio-ADM concentrations were measured in patients with septic shock using hDPP-3 and bio-ADM immunoassays and correlated with the need for vasopressor therapy.

[0325] Study population - septic shock DPP-3 and bio-ADM concentrations were measured in plasma samples from 292 patients diagnosed with septic shock from the AdrenOSS study. Human DPP-3 was measured as described in Example 1. Bio-ADM was measured as described in Weber et al. (Weber et al. 2017. JALM 2(2): 1-4).

[0326] result Plasma concentrations of bio-ADM and DPP-3 were measured in patients with septic shock. Patients were grouped according to specific thresholds determined as the third quartile of the total measured plasma concentration of each marker in the septic shock population (low DPP-3: <48.4 ng / mL, low bio-ADM: <213 pg / mL, high bio-ADM: ≥213 pg / mL, high DPP-3: ≥48.4 ng / mL).

[0327] Patients with high DPP-3 but low bio-ADM plasma concentrations required continuous vasopressor therapy compared with patients with either low DPP-3 and low bio-ADM or low DPP-3 and high bio-ADM (Figure 15; Table 9). In contrast, patients with low DPP-3 and low bio-ADM plasma concentrations or low DPP-3 but high bio-ADM plasma concentrations on admission were able to discontinue vasopressor therapy earlier than patients with high DPP-3 plasma concentrations on admission (Figure 15; Table 9). This indicates that vasopressor therapy can be discontinued earlier in septic shock patients with high bio-ADM due to their superior therapeutic response compared with septic shock patients with high DPP-3. These patients (with high DPP-3) require longer-term vasopressor therapy due to lack of response. [Table 11]

[0328] (Array example) SEQ ID NO: 1: hDPP 3 aa 1-737 [Table 12] SEQ ID NO: 2: hDPP 3 aa 474-493 (N-Cys) - immunizing peptide with an additional N-terminal cysteine CETVINPETGEQIQSWYRSGE SEQ ID NO: 3: hDPP 3 aa 477-482-AK1967 epitope INPETG SEQ ID NO: 4: Variable region of mouse AK1967 in the heavy chain [Table 13] SEQ ID NO: 5: Variable region of mouse AK1967 in the light chain [Table 14] SEQ ID NO: 6: CDR1 of mouse AK1967 in the heavy chain GFSLSTSGMS SEQ ID NO: 7: CDR2 of mouse AK1967 in the heavy chain IWWNDNK SEQ ID NO: 8: CDR3 of mouse AK1967 in the heavy chain ARNYSYDY SEQ ID NO: 9: CDR1 of mouse AK1967 in the light chain RSLVHSIGSTY CDR2 of mouse AK1967 in the light chain KVS SEQ ID NO: 10: CDR3 of mouse AK1967 in the light chain SQSTHVPWT SEQ ID NO: 11: Humanized AK1967-heavy chain sequence (IgG1κ backbone) [Table 15] SEQ ID NO: 12: Humanized AK1967-light chain sequence (IgG1κ backbone) [Table 16] SEQ ID NO: 13: Angiotensin II (also known as 5-isoleucine-angiotensin II) DRVYIHPF SEQ ID NO: 14: Angiotensin II analogue (5-valine-angiotensin II) DRVYVHPF SEQ ID NO: 15: Angiotensin II analogue (Asn 1 -PHe 4 ) NRVFIHPF SEQ ID NO: 16: Angiotensin II hexapeptide VYIHPF SEQ ID NO: 17: Angiotensin II nonapeptide NRVYYVHPF SEQ ID NO: 18: Angiotensin II analog ([Asn 1 -Ile 5 -Ile 8 ]-Angiotensin II) NRVYIHPI SEQ ID NO: 19: Angiotensin II analog ([Asn 1 -Ile 5 -Ala 8 ]-Angiotensin II) NRVYIHPA SEQ ID NO: 20: Angiotensin II analog ([Asn 1 -Diiodide Tyr 4 -Ile 5 ]-Angiotensin II NRVYIHPF SEQ ID NO: 21: Angiotensin III RVYIHPF SEQ ID NO: 22: Angiotensin III analog (Val 4 -Angiotensin III) RVYVHPF SEQ ID NO: 23: Angiotensin III analogue (PHe 3 -Angiotensin III) RVFIHPF SEQ ID NO: 24: Angiotensin III analog ([Ile 4 -Ala 7 ]-Angiotensin III) RVYIHPA SEQ ID NO: 25: Angiotensin III analog (diiodoTyr 3 -Ile 4 ]-Angiotensin III) RVYIHPF SEQ ID NO: 26: Angiotensin IV VYIHPF SEQ ID NO: 27: Angiotensin IV analog (Val 3 -Angiotensin IV) VYVHPF SEQ ID NO: 28: Angiotensin IV analogue (PHe2 -Angiotensin IV) VFIHPF SEQ ID NO: 29: Angiotensin IV analog ([Ile 3 -Ala 6 ]-Angiotensin IV) VYIHPA SEQ ID NO: 30: Angiotensin IV analog ([diiodoTyr 2 -Ile 3 ]-Angiotensin IV) VYIHPF SEQ ID NO: 31 (proADM): 164 amino acids (22 to 185 of preproADM) [Table 17] SEQ ID NO: 32 (proadrenomedullin, N-20 terminal peptide, PAMP): amino acids 22 to 41 of preproADM ARLDVASEF RKKWNKWALS R SEQ ID NO: 33 (mid-region proadrenomedullin, MR-proADM): amino acids 45 to 92 of preproADM [Table 18] SEQ ID NO: 34 (mature adrenomedullin (mature ADM); amidated ADM; bio-ADM; hADM): amino acids 95-146-CONH2 [Table 19] SEQ ID NO: 35 (Adrenomedullin 1-52-Gly (ADM 1-52-Gly)): Amino acids 95 to 147 of preproADM [Table 20] SEQ ID NO: 36 (C-terminal pro-adrenomedullin, CT-proADM): amino acids 148 to 185 of preproADM [Table 21] SEQ ID NO: 37 (N-terminal portion of mature ADM): amino acids 1 to 21 of mature ADM YRQSMNNFQGLRSFGCRFGTC SEQ ID NO: 38 (CDR1 heavy chain anti-ADM antibody) GYTFSRYW SEQ ID NO: 39 (CDR2 heavy chain anti-ADM antibody) ILPGSGST SEQ ID NO: 40 (CDR3 heavy chain anti-ADM antibody) TEGYEYDGFDY SEQ ID NO: 41 (CDR1 light chain anti-ADM antibody) QSIVYSNGNTY SEQ ID NO: 42 (CDR3 light chain anti-ADM antibody) FQGSHIPYT SEQ ID NO: 43 (anti-ADM antibody (adrecizumab) heavy chain) [Table 22] SEQ ID NO: 44 (anti-ADM antibody (adrecizumab) light chain) [Table 23]

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Claims

1. A method for predicting or diagnosing refractory shock in a subject who has fallen into or developed shock, the method comprising: - measuring the concentration of DPP 3 in a body fluid sample from said subject - comparing the measured concentration of DPP 3 with a predetermined threshold value, wherein the subject is predicted to suffer from refractory shock syndrome or diagnosed as suffering from refractory shock syndrome when the measured DPP 3 concentration exceeds the predetermined threshold.

2. 2. The method for predicting or diagnosing refractory shock in a subject suffering from or having developed shock according to claim 1, wherein the shock is selected from the group comprising hypovolemic shock, cardiogenic shock, vaso-occlusive shock, and distributive shock, in particular cardiogenic shock or septic shock.

3. The method for predicting or diagnosing refractory shock syndrome in a subject who has fallen into or developed shock syndrome according to claim 1 or 2, wherein the shock syndrome is vasopressor-resistant shock syndrome.

4. In the case of cardiogenic shock, the subject may be suffering from an acute coronary syndrome (e.g., acute myocardial infarction), or the subject may be suffering from heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism, or In the case of hypovolemic shock, the subject may suffer from a bleeding disorder, including gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor invasion of major blood vessels), and spontaneous bleeding in the setting of anticoagulation, or a non-hemorrhagic disorder, including vomiting, diarrhea, renal loss, skin loss / unconscious injury (e.g., burns, heat stroke), or third space loss in the setting of pancreatitis, liver cirrhosis, intestinal obstruction, or trauma; or In the case of vaso-occlusive shock, the patient may suffer from cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or In the case of distributive shock, the patient may suffer from septic shock, neurogenic shock, anaphylactic shock, or shock due to adrenal crisis; A method for predicting or diagnosing refractory shock syndrome in a subject who has fallen into or developed shock syndrome according to claims 1 to 3.

5. The method for predicting or diagnosing refractory shock syndrome in a subject who has fallen into or developed shock syndrome according to claims 1 to 4, wherein the method is used for initiating and / or terminating and / or stratifying and / or guiding treatment.

6. The method for predicting or diagnosing refractory shock syndrome in a subject who has fallen into or developed shock syndrome according to any one of claims 1 to 5, wherein treatment is initiated and / or maintained and / or suspended and / or terminated when the measured DPP-3 concentration exceeds the predetermined threshold.

7. The method for predicting or diagnosing refractory shock syndrome in a subject who has fallen into or developed shock syndrome according to claim 6, wherein the treatment is selected from the group consisting of a vasopressor, an angiotensin receptor agonist and / or a precursor thereof, an inhibitor of DPP-3 activity, and an anti-adrenomedullin antibody or an anti-adrenomedullin antibody fragment.

8. The method for predicting or diagnosing refractory shock syndrome in a subject who has fallen into or developed shock syndrome according to any one of claims 1 to 7, comprising measuring the concentration of DPP-3 protein and / or the concentration of active DPP-3 and comparing it with a predetermined threshold, and measuring the DPP-3 concentration by contacting the body fluid sample with a capture binding agent that specifically binds to DPP-3.

9. 9. The method for predicting or diagnosing refractory shock syndrome in a subject who has fallen into or developed shock syndrome according to any one of claims 1 to 8, wherein treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued when the concentration of DPP-3 in the sample exceeds a certain threshold, and / or treatment with a vasopressor is suspended and / or terminated when the measured concentration of DPP-3 exceeds the predetermined threshold.

10. 10. The method for predicting or diagnosing refractory shock syndrome in a subject who has fallen into shock syndrome or who has developed shock syndrome according to claims 1 to 9, wherein treatment with a vasopressor is initiated and / or continued when the concentration of DPP-3 in the sample falls below a certain threshold, and / or treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is suspended and / or terminated when the measured concentration of DPP-3 falls below the predetermined threshold.

11. A method for predicting or diagnosing refractory shock syndrome in a subject who has fallen into shock syndrome or who has developed shock syndrome, as described in claim 9, further comprising measuring the concentration of proadrenomedullin or a fragment thereof, and initiating and / or continuing treatment with an anti-ADM antibody or an anti-ADM antibody fragment when the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a specific threshold, and / or suspending and / or terminating treatment with an anti-ADM antibody or an anti-ADM antibody fragment when the measured concentration of proadrenomedullin or a fragment thereof falls below a predetermined threshold.

12. 10. The method for predicting or diagnosing refractory shock syndrome in a subject who has fallen into or developed shock syndrome according to claim 9, wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment and / or an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is initiated and / or continued when the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a specific threshold and the measured concentration of DPP-3 exceeds the predetermined DPP-3 threshold.

13. 12. A vasopressor for use in treating shock in a subject suffering from or having developed shock, wherein the subject has a DPP-3 concentration in a body fluid sample from the subject that is below a predetermined threshold when measured by the method according to any one of claims 1 to 11.

14. 12. An inhibitor of DPP-3 activity for use in treating shock in a subject suffering from or having developed shock, wherein the subject has a DPP-3 concentration in a body fluid sample from the subject that exceeds a predetermined threshold when measured by the method of any one of claims 1 to 11, and wherein the inhibitor of DPP-3 activity is selected from the group comprising an anti-DPP-3 antibody or an anti-DPP-3 antibody fragment or an anti-DPP-3 non-Ig scaffold.

15. 15. The inhibitor of DPP-3 activity for use in the treatment of shock in a subject suffering from or having developed shock according to claim 14, wherein the inhibitor is administered in combination with an angiotensin receptor agonist and / or a precursor thereof.

16. 16. The inhibitor of DPP-3 activity for use in the treatment of shock in a subject suffering from or having developed shock according to claim 15, wherein the angiotensin receptor agonist and / or precursor thereof is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, angiotensin IV, in particular angiotensin II.

17. 12. A method of treating shock in a subject suffering from or having developed shock, the method comprising administering a vasopressor to the subject, wherein the subject has a concentration of DPP 3 in a body fluid sample from the subject that is below a predetermined threshold when measured by the method of any one of claims 1 to 11.

18. 12. A method for treating shock in a subject suffering from or having developed shock, the method comprising administering to the subject an inhibitor of DPP-3 activity, wherein the subject has a concentration of DPP-3 in a body fluid sample from the subject that exceeds a predetermined threshold when measured by the method of any one of claims 1 to 11.

19. 20. The method of treating shock in a subject suffering from or having developed shock according to claim 18, wherein the method comprises administering to the subject an inhibitor of DPP-3 activity, wherein the inhibitor is administered in combination with an angiotensin receptor agonist and / or a precursor thereof.

20. 19. The method for treating shock in a subject suffering from or having developed shock according to claims 17 to 18, wherein the method comprises administering to the subject an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity, wherein treatment with the angiotensin receptor agonist and / or the inhibitor of DPP-3 activity is initiated and / or continued when the concentration of DPP-3 in a sample from the subject exceeds a certain threshold, and / or treatment with a vasopressor agent is suspended and / or terminated when the measured concentration of DPP-3 exceeds the predetermined threshold.

21. 19. The method for treating shock in a subject suffering from or having developed shock according to claims 17 to 18, wherein the method comprises administering a vasopressor to the subject, wherein treatment with the vasopressor is initiated and / or continued when a concentration of DPP-3 in a body fluid sample from the subject is below a certain threshold, and / or treatment with an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity is suspended and / or terminated when the measured concentration of DPP-3 is below the predetermined threshold.

22. 20. The method for treating shock in a subject suffering from or having developed shock according to claim 19, wherein the method comprises administering to the subject an angiotensin receptor agonist and / or a precursor thereof and / or an inhibitor of DPP-3 activity, measuring the concentration of proadrenomedullin or a fragment thereof, and initiating and / or continuing treatment with the anti-ADM antibody or anti-ADM antibody fragment when the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with the anti-ADM antibody or anti-ADM antibody fragment when the measured concentration of proadrenomedullin or a fragment thereof falls below the predetermined threshold.

23. A method for treating shock in a subject suffering from or developing shock as described in claim 20, further comprising administering a vasopressor to the subject, measuring the concentration of proadrenomedullin or a fragment thereof, and initiating and / or continuing treatment with the anti-ADM antibody or anti-ADM antibody fragment when the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with the anti-ADM antibody or anti-ADM antibody fragment when the measured concentration of proadrenomedullin or a fragment thereof falls below the predetermined threshold.

24. A method for treating shock in a subject suffering from or having developed shock, as described in claims 21 and 22, wherein the method comprises administering an anti-ADM antibody or anti-ADM antibody fragment to the subject, further measuring the concentration of proadrenomedullin or a fragment thereof, and initiating and / or continuing treatment with the anti-ADM antibody or anti-ADM antibody fragment when the concentration of proadrenomedullin or a fragment thereof in the sample exceeds a certain threshold, and / or suspending and / or terminating treatment with the anti-ADM antibody or anti-ADM antibody fragment when the measured concentration of proadrenomedullin or a fragment thereof falls below the predetermined threshold.

25. 1. A method for prognosing the outcome and / or risk of an adverse event in a subject who has developed refractory shock, comprising: - measuring the concentration of DPP 3 in a body fluid sample from said subject; - comparing the measured concentration of DPP 3 with a predetermined threshold value; - correlating the concentration of DPP 3 with the risk of said adverse event in said subject, wherein an elevated concentration above a certain threshold predicts an increased risk of said adverse event; - correlating the concentration of DPP 3 with the success of a treatment or intervention in the subject, wherein a concentration below a certain threshold is predictive of successful treatment or intervention; A method comprising:

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