RU2835616C1 - Therapeutic guidance and/or monitoring therapy for treating shock - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely to emergency medicine. Disclosed are methods for predicting and diagnosing refractory shock in a patient, including determining the level of DPP3 in a patient's sample, where, if the DPP3 level exceeds value from 20 to 200 ng/ml, it is predicted or determined that the said patient falls into a state of refractory shock. Methods for predicting the risk of an adverse event and an outcome in a patient developing refractory shock are disclosed. What is presented is the use of a vasopressor in the therapy of shock in a patient. Disclosed is the use of a DPP3 activity inhibitor in the therapy of shock in a patient, where the DPP3 activity inhibitor is an anti-DPP3 antibody, or an anti-DPP3 antibody fragment, or a non-Ig anti-DPP3 framework. Disclosed are methods of treating shock in a patient, involving administering a vasopressor or DPP3 activity inhibitor to said patient.

EFFECT: presented group of inventions provides effective methods of diagnosing, predicting refractory shock, as well as treating shock in a patient by determining the DPP3 level in a biological fluid sample of said patient.

20 cl, 16 dwg, 9 tbl, 11 ex

Description

The present invention relates to a method for predicting or diagnosing refractory shock in a patient who is either falling into shock or has developed shock, and to vasopressors, angiotensin receptor agonists and/or their precursors, DPP3 inhibitors and anti-ADM antibodies for use in the treatment of shock in a patient who is either falling into shock or has developed shock, and to methods for treating said shock or refractory shock.

Dipeptidyl peptidase 3, also known as dipeptidyl aminopeptidase III, dipeptidyl arylamidase III, dipeptidyl peptidase III, enkephalinase B, or erythrocyte angiotensinase; short name: DPP3, DPPIII, is a metallopeptidase that removes dipeptides from physiologically active peptides such as enkephalins and angiotensins.

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

DPP3 is a zinc-dependent exopeptidase belonging to the M49 peptidase family. It has a broad substrate specificity for oligopeptides of three/four to ten amino acids of various compositions and is also capable of cleavage after proline. DPP3 is known to hydrolyze dipeptides from the N-terminus of its substrates, including angiotensin II, III, and IV; Leu- and Met-enkephalin; endomorphin 1 and 2. DPP3 metallopeptidase has an activity optimum at pH 8-9 and can be activated by the addition of divalent metal ions such as Co ²⁺ and Mg ²⁺ .

Structural analysis of DPP3 has revealed the catalytic motifs HELLGH (hDPP3 450–455) and EECRAE (hDPP3 507–512) and the following amino acids important for substrate binding and hydrolysis: Glu316, Tyr, 318, Asp366, Asn391, Asn394, His568, Arg572, Arg577, Lys666, and Arg669 (Prajapati & Chauhan 2011; Kumar et al. 2016; numbering refers to the human DPP3 sequence, see SEQ ID NO. 1). Taking into account all known amino acids or sequence regions that are involved in substrate binding and hydrolysis, the active site of human DPP3 can be defined as the region between amino acids 316 and 669.

The most prominent substrate of DPP3 is angiotensin II (Ang II), the major effector of the renin-angiotensin system (RAS). 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 (Corrêa et al. 2015. Crit Care 2015; 19:98). In particular, Ang II has been shown to modulate many cardiovascular functions, including blood pressure control and cardiac remodeling.

The exact biological function of DPP3 in cellular physiology is not understood, but recent findings suggest a role not only in protein metabolism but also in pain modulation and inflammation (Prajapati & Chauhan 2011). Furthermore, DPP3 reduced blood pressure in AngII-treated hypertensive mice without changing heart rate (Pang et al. 2016). However, there was no change in blood pressure in normotensive mice when injected with DPP3.

The reduction in blood pressure with DPP3 plus angiotensin receptor antagonist in Ang II-treated mice was similar to the reduction in blood pressure with DPP3 alone or angiotensin receptor antagonist alone (Pang et al. 2016. Hypertension 68:630–41).

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

AngII therapy is currently being discussed due to its beneficial effect in vasodilator shock and septic shock (Khanna, A. et al., 2017; Antonucci, E. et al. 2017, Tumlin, JA et al. 2018). Patients with vasodilator shock (80% in septic shock) who received angiotensin II were more likely to survive up to 28 days and demonstrated significant correction of hypotension (Khanna, A. et al., 2017; Tumlin, JA et al. 2018).

It is suggested that angiotensin-converting enzyme (ACE) is highly dysregulated in patients with shock, resulting in an altered angiotensin I/II ratio, and that angiotensin II infusion may compensate for this dysregulation (Tumlin, J.A. et al. 2018).

Recently, two assays have been developed, characterized, and validated for the specific detection of DPP3 in human biological fluids (e.g., blood, plasma, serum): an immunoluminescence assay (LIA) for the determination of DPP3 protein concentration and an enzyme capture assay (ECA) for the specific detection of DPP3 activity (Rehfeld et al. 2019 JALM 3(6):943–953). A wash step in both methods removes any interfering substances before the actual detection of DPP3 protein or activity. Both methods are highly specific and allow reproducible determination of DPP3 in blood samples.

The object of the present invention is to provide a method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock.

Another object of the invention is to provide vasopressors, angiotensin receptor agonists and/or their precursors, DPP3 inhibitors and anti-ADM antibodies for use in the treatment of shock in a patient who is either going into shock or has developed shock.

The object of the present invention is a method for predicting or diagnosing refractory shock in a patient who is either entering a state of shock or has developed shock, wherein said method comprises the steps of:

determination of the level of DPP3 in a sample of biological fluid of the specified patient;

comparison of the specified level of a given DPP3 with a given threshold value,

where the specified patient is predicted to enter into refractory shock, or is diagnosed with refractory shock, if the specified defined DPP3 level exceeds the specified predetermined threshold.

The object of the present invention is a method for predicting or diagnosing refractory shock in a patient who is either entering a state of shock or has developed shock, wherein said method comprises the steps of:

determination of the level of DPP3 in a sample of biological fluid of the specified patient;

comparing the specified level of a given DPP3 with a given threshold value, and

determining the level of proadrenomedullin or its fragments in a sample of biological fluid of the specified patient, and

comparison of the specified level of a certain proadrenomedullin or its fragments with a given threshold value,

wherein said patient is predicted to enter into refractory shock or is diagnosed with refractory shock if said specified level of proadrenomedullin or its fragments and/or said level of DPP3 exceeds said specified threshold value.

In a specific embodiment of said method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, said shock is selected from the group consisting of hypovolemia-induced shock, cardiogenic shock, obstructive shock, and distributive shock.

In another specific embodiment of said method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, said shock is selected from the group consisting of hypovolemia-induced shock, cardiogenic shock, obstructive shock and distributive shock, in particular cardiogenic or septic shock.

In a specific embodiment of said method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, said shock is selected from the group consisting of:

in case of cardiogenic shock, the said patient has suffered an acute coronary syndrome (e.g. acute myocardial infarction) or heart failure (e.g. acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordal rupture, or massive pulmonary embolism, or

in the case of hypovolemic shock, the patient may have suffered a hemorrhagic disorder including gastrointestinal bleeding, trauma, vascular etiologies (e.g. ruptured abdominal aortic aneurysm, tumor invading a major blood vessel), and spontaneous bleeding in the setting of anticoagulation, or a non-hemorrhagic disorder including vomiting, diarrhea, renal failure, cutaneous loss/intangible loss (e.g. burns, heat stroke), or third space loss in the setting of pancreatitis, liver cirrhosis, ileus, trauma, or

in case of obstructive shock, the said patient may have suffered cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or

In case of distributive shock, the said patient suffers from septic shock, neurogenic shock, anaphylactic shock or shock due to adrenal crisis.

In another embodiment of said method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, said method is used to initiate and/or stop and/or stratify treatment and/or guide treatment.

As used herein, the term "therapeutic guidance" or "treatment guidance" refers to the use of certain therapies or medical interventions based on the value of one or more biomarkers and/or a clinical parameter and/or clinical scores.

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

The term "stratification of therapy" specifically refers to the grouping or classification of patients into different groups, such as treatment groups, who receive or do not receive treatments depending on their classification.

The term "prediction" refers to the correlation of the probability of an outcome with the result obtained by measuring an analyte. An example of this is the measurement of a particular marker, such as DPP3, in a sample, the measured level of which correlates with the probability that a patient who either goes into shock or has developed shock will develop refractory shock.

The present invention also includes a method for short-term prediction of refractory shock in a patient who is either going into shock or has developed shock, where short-term means a period equal to or less than 14 days, preferably equal to or less than 7 days, more preferably equal to or less than 3 days, most preferably equal to or less than 48 hours.

In another embodiment of said method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, said treatment is initiated and/or continued and/or delayed and/or stopped if said determined level of DPP3 is above said specified threshold.

In another embodiment of said method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, said treatment is initiated and/or continued and/or delayed and/or stopped if said determined level of DPP3 and/or pro-adrenomedullin and/or fragments thereof is above said specified threshold.

In a preferred embodiment of said method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, said treatment is selected from the group of vasopressors, angiotensin receptor agonists or their precursors and/or DPP3 activity inhibitors.

In another preferred embodiment of said method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, said treatment is selected from the group of vasopressors, angiotensin receptor agonists and/or their precursors, DPP3 activity inhibitors and anti-adrenomedullin antibodies or anti-adrenomedullin antibody fragments.

In another embodiment of the said method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, the DPP3 protein level and/or the active DPP3 level is determined and compared to a predetermined threshold value.

In another embodiment of said method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, the level of DPP3 is determined by contacting said biological fluid sample with a binding capture agent that specifically binds to DPP3.

In another preferred embodiment of said method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, said capture binding agent for determining the level of DPP3 may be selected from the group consisting of an antibody, an antibody fragment, or a non-IgG scaffold.

In a specific embodiment of said method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, said capture binding agent is an antibody.

In another specific embodiment of said method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, said capture binding agent is a monoclonal antibody.

In another embodiment of said method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, said sample of biological fluid is selected from the group of whole blood, plasma and serum.

In a specific embodiment of said method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, said method of diagnosis or prediction is performed at least twice.

Shock is characterized by decreased oxygen delivery and/or increased oxygen consumption or inadequate oxygen utilization, resulting in cellular and tissue hypoxia. It is a life-threatening state of circulatory failure, most often manifested by hypotension (systolic blood pressure less than 90 mmHg or mean arterial pressure less than 65 mmHg). Shock is classified into four main types depending on the underlying cause: hypovolemic, cardiogenic, obstructive, and distributive shock (Vincent and De Backer 2014. N. Engl. J. Med. 370(6): 583).

Hypovolemic shock is characterized by intravascular volume depletion and can be divided into two main subtypes: hemorrhagic and nonhemorrhagic. Common causes of hemorrhagic hypovolemic shock include gastrointestinal bleeding, trauma, vascular etiologies (eg, ruptured abdominal aortic aneurysm, tumor invading a major blood vessel), and spontaneous bleeding secondary to anticoagulant use. Common causes of nonhemorrhagic hypovolemic shock include vomiting, diarrhea, renal failure, cutaneous/non-sensory losses (eg, burns, heatstroke), or third-space loss due to pancreatitis, cirrhosis, ileus, trauma. For review, see Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019-2018 Oct 27.

Cardiogenic shock (CS) is defined as a state of critical endogenous organ hypoperfusion due to decreased cardiac output. Notably, CS has a spectrum ranging from mild hypoperfusion to profound shock. Established diagnostic criteria for CS are: (i) systolic blood pressure ≤90 mmHg for >30 min or vasopressors required to achieve blood pressure ≥90 mmHg; (ii) pulmonary congestion or elevated left ventricular filling pressures; (iii) evidence of impaired organ perfusion with at least one of the following: (a) altered mental status; (b) cold, clammy skin; c) oliguria (<0.5 mL/kg/h or <30 mL/h); (d) elevated serum lactate (Reynolds and Hochman 2008. Circulation 117: 686–697). Acute myocardial infarction (AMI) with subsequent ventricular dysfunction is the most common cause of CS, accounting for approximately 80% of cases. Less common causes of CS after AMI are mechanical complications such as rupture of the interventricular septum (4%) or free wall (2%), and acute severe mitral regurgitation (7%). (Hochman et al. 2000. J Am Coll Cardiol 36: 1063–1070). Non-AMI-related CS may be caused by decompensated valvular disease, acute myocarditis, arrhythmia, etc., with heterogeneous treatment options. This corresponds to 40,000 to 50,000 patients per year in the USA and 60,000 to 70,000 in Europe. Despite advances in treatment, mainly due to early revascularization with subsequent reduction in mortality, CS remains the leading cause of death in AMI, where the mortality rate still approaches 40-50% according to recent data and randomized trials (Goldberg et al. 2009. Circulation 119: 1211-1219.

Obstructive shock results from physical obstruction of large vessels or the heart itself. Several conditions (eg, cardiac tamponade, tension pneumothorax, pulmonary embolism, aortic stenosis) can lead to this form of shock. For review, see Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019-2018 Oct 27.

Depending on the cause, there are four types of distributive shock: neurogenic shock (decreased sympathetic stimulation leading to decreased vascular tone), anaphylactic shock, septic shock, and shock due to adrenal crisis. In addition to sepsis, distributive shock may 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 (sudden severe allergic reaction), adrenal insufficiency (acute worsening of chronic adrenal insufficiency, destruction or removal of the adrenal glands, adrenal suppression due to exogenous steroids, hypopituitarism, and metabolic failure of hormone production), drug or toxin reactions, heavy metal poisoning, liver failure, and CNS disease. For an overview, see Koya and Paul 2018. Shock. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019-2018 Oct 27.

In general, refractory shock is defined as the need for norepinephrine infusion >0.5 mcg/kg/min despite adequate circulating blood volume resuscitation. Mortality in these patients can be as high as 94%, and the evaluation and treatment of these patients requires a much more aggressive approach to survival. The term refractory shock is used when tissue perfusion cannot be restored with initial corrective measures (e.g., vasopressors), and can therefore be referred to as highly vasopressor-dependent or vasopressor-resistant shock (Udupa and Shetty 2018. Indian J Respir Care 7: 67–72). Patients with refractory shock may have signs of inadequate perfusion such as hypotension (mean arterial pressure <65 mmHg), tachycardia, cold peripheral vessels, prolonged capillary refill time, and tachypnea due to hypoxia and acidosis. Fever may be seen in septic shock. Other signs of hypoperfusion such as altered sensorium, hyperlactatemia, and oliguria may also be seen. These well-known signs of shock do not help determine whether the problem is pump (heart) or system (vessels and tissues) related. Different types of shock may coexist, and all forms of shock may become refractory, as demonstrated by unresponsiveness to high doses of vasopressors (Udupa and Shetty 2018. Indian J Respir Care 7: 67–72).

In a specific embodiment of the invention, said refractory shock is resistant to vasopressors, as determined by the patient's lack of response to high doses of vasopressors (e.g., >0.5 mcg/kg/min norepinephrine).

A consequence of diagnosing or predicting that a patient will either develop vasopressor-resistant refractory shock or is diagnosed with vasopressor-resistant refractory shock may be the initiation of treatment such as angiotensin II inhibitors and/or DPP3 inhibitors. In addition, another consequence may be the withdrawal of vasopressors.

In a preferred embodiment of said method for predicting refractory shock, in particular vasopressor-resistant shock, in a patient who may potentially suffer from shock, wherein it is predicted whether said patient will suffer from refractory shock, in particular vasopressor-resistant shock, from the time of taking a sample within 14 days, or in particular within 10 days, or in particular within 7 days, or in particular within 5 days, or in particular within 48 hours, or in particular within 24 hours, or in particular in the range from 24 to 48 hours.

Another embodiment of the present invention relates to a method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, wherein treatment with angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors is started and/or continued when the DPP3 level in said sample is above a certain threshold value, and/or where treatment with vasopressors is delayed and/or stopped if said determined DPP3 level is above said specified threshold value.

Another specific embodiment of the present invention relates to a method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, wherein treatment with vasopressors is started and/or continued when the DPP3 level in said sample is below a certain threshold value, and/or wherein treatment with angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors is postponed and/or stopped if said certain DPP3 level is below said predetermined threshold value.

Another embodiment of the present invention relates to a method for predicting or diagnosing refractory shock in a patient who is either falling into a state of shock or has developed shock, wherein treatment with vasopressors is started and/or continued when the level of DPP3 in said sample is below a certain threshold value and/or wherein treatment with angiotensin receptor agonists and/or their precursors and/or inhibitors of DPP3 activity is postponed and/or stopped if said determined level of DPP3 is below said predetermined threshold value and the level of proadrenomedullin or fragments thereof is determined, and wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment is started and/or continued when the level of proadrenomedullin or fragments thereof in said sample is above a predetermined threshold value and/or wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment is postponed and/or stopped if said determined level of pro-adrenomedullin or fragments thereof is below said predetermined threshold value.

Another embodiment of the present invention relates to a method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, in accordance with the present invention, wherein

the said patient may have suffered from an acute coronary syndrome (e.g. acute myocardial infarction) or where the said patient has heart failure (e.g. acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordal rupture or massive pulmonary embolism (in case of cardiogenic shock), or

the patient in question may have had a hemorrhagic disorder including gastrointestinal bleeding, trauma, vascular etiologies (e.g. ruptured abdominal aortic aneurysm, tumor invading a major blood vessel), and spontaneous bleeding in the setting of anticoagulation, or a non-hemorrhagic disorder including vomiting, diarrhea, renal loss, skin loss/intangible loss (e.g. burns, heat stroke), or third space loss in the setting of pancreatitis, liver cirrhosis, intestinal obstruction, trauma (in the case of hypovolemic shock), or

the patient in question may have had cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis (in the case of obstructive shock), or

The patient in question may be in septic shock, neurogenic shock, anaphylactic shock, or shock due to adrenal crisis (in the case of distributive shock).

Another embodiment of the present invention relates to a method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to the present invention, wherein said method is used to initiate and/or stop and/or stratify treatment and/or guide treatment.

Another embodiment of the present invention relates to a method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock according to the present invention, wherein treatment is started and/or continued and/or delayed and/or stopped if said determined level of DPP3 is above said predetermined threshold value.

The subject of the present invention are vasopressors, angiotensin receptor agonists and/or their precursors, DPP3 activity inhibitors and/or anti-adrenomedullin antibodies or anti-adrenomedullin antibody fragments for use in a method for preventing or treating refractory shock in a patient who is either falling into a state of shock or who has developed shock, wherein said prediction and/or diagnosis of said refractory shock was carried out using a method for predicting or diagnosing refractory shock in a patient who is either falling into a state of shock or who has developed shock according to the present invention. In one embodiment of the above-mentioned methods, the level of DPP3 protein and/or the level of active DPP3 is determined and compared with a predetermined threshold value, wherein the level of DPP3 is determined by contacting said biological fluid sample with a binding capture agent that specifically binds to DPP3.

The subject of the present invention are angiotensin receptor agonists and/or their precursors, DPP3 activity inhibitors for use in a method for preventing or treating refractory shock in a patient who is either falling into a state of shock or who has developed shock, wherein said prediction and/or diagnosis of said refractory shock was carried out using a method for predicting or diagnosing refractory shock in a patient who is either falling into a state of shock or who has developed shock according to the present invention, wherein treatment with angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors is started and/or continued when the DPP3 level in said sample is above a certain threshold value, and/or treatment with vasopressors is postponed and/or stopped if said certain DPP3 level is above said specified threshold value.

The subject of the present invention are angiotensin receptor agonists and/or their precursors, DPP3 activity inhibitors for use in a method for preventing or treating refractory shock in a patient who is either falling into a state of shock or who has developed shock, wherein said prediction and/or diagnosis of said refractory shock was carried out using a method for predicting or diagnosing refractory shock in a patient who is either falling into a state of shock or who has developed shock, according to the present invention, wherein treatment with vasopressors is started and/or continued when the DPP3 level in said sample is below a certain threshold value, and/or wherein treatment with angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors is postponed and/or stopped if said certain DPP3 level is below said specified threshold value.

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 patient who is either falling into a state of shock or who has developed shock, wherein said prediction and/or diagnosis of said refractory shock was carried out using a method for predicting or diagnosing refractory shock in a patient who is either falling into a state of shock or who has developed shock according to the present invention, wherein the level of proadrenomedullin or fragments thereof is further determined, and wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment is started and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment is postponed and/or stopped if said determined level of pro-adrenomedullin or fragments thereof is below said specified threshold value.

Another embodiment of the present invention relates to a method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment and/or angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors is started and/or continued if the level of proadrenomedullin or its fragments in said sample is above a certain threshold value, and said certain level of DPP3 is above said specified threshold value of DPP3.

The subject of the present invention is an anti-ADM antibody or an anti-ADM antibody fragment and/or a DPP3 activity inhibitor for use in a method for preventing or treating refractory shock in a patient who is either falling into a state of shock or has developed shock, wherein said prediction and/or diagnosis of said refractory shock was carried out using a method for predicting or diagnosing refractory shock in a patient who is either falling into a state of shock or has developed shock according to the present invention, wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment and/or angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors is started and/or continued if the level of proadrenomedullin or its fragments in said sample is above a certain threshold value, and said certain level of DPP3 is above said specified threshold value of DPP3.

In a particular embodiment of the invention, the threshold value is in the range of threshold values for DPP3 in plasma, which is from 20 to 200 ng/ml, preferably from 25 to 150 ng/ml, even more preferably from 30 to 100 ng/ml, even more preferably from 35 to 75 ng/ml, most preferably a threshold value of 50 ng/ml is used.

In particular, the refractory shock in this patient is vasopressor-resistant shock.

The peptide adrenomedullin (ADM) was described as a novel 52-amino acid antihypertensive peptide isolated from human pheochromocytoma (Kitamura et al. 1993. Biochemical and Biophysical Research Communications 192 (2): 553-560). Cleavage of the 21-amino acid N-terminal signal sequence from pre-pro-adrenomedullin (pre-pro-ADM) yields pro-adrenomedullin precursor peptide (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). Mature adrenomedullin peptide is an amidated peptide (ADM-NH2) that contains 52 amino acids (SEQ ID No: 34) and contains amino acids 95 to 146 of pre-proADM, from which it is formed by proteolytic cleavage. Mature ADM, bio-ADM and ADM-NH2 are used synonymously in this application and represent the molecule according to SEQ ID No: 34.

In one embodiment of the subject matter of the present invention, said pro-adrenomedullin fragment is selected from the group consisting of 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).

ADM can be considered as a multifunctional regulatory peptide. It is released into the bloodstream partly in an inactive form supplemented with glycine (Kitamura et al. 1998. Biochem. Biophys. Res. Commun. 244(2): 551-555). There is also a binding protein (Pio et al. 2001. The Journal of Biological Chemistry 276(15): 12292-12300) that is specific for ADM and probably similarly modulates the effect of ADM.

The physiological effects of ADM, as well as PAMPs, that have been of primary importance in research to date are those affecting blood pressure. Thus, ADM is an effective vasodilator. Measurable concentrations of ADM in the bloodstream and other biological fluids have been found to be significantly higher than those found in healthy controls in a number of pathological conditions. Thus, ADM levels are significantly elevated, although to varying degrees, in patients with congestive heart failure, myocardial infarction, renal disease, hypertensive disorders, diabetes mellitus, in the acute phase of shock, and in sepsis and septic shock. PAMP concentrations are also increased in some of these pathological conditions, but plasma levels are reduced compared with ADM (Eto et al. 2001. Peptides 22: 1693-1711).

In addition, it is known that unusually high concentrations of ADM are 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). The findings are related to typical hemodynamic changes known to be typical features of the disease course in patients with sepsis and other severe syndromes such as SIRS. Adrenomedullin plays a key 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 numerous 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).

MR-proADM has been identified as a prognostic marker capable of stratifying mortality risk in sepsis patients with different degrees of organ failure. It may be useful for early detection and assessment of individual sepsis risk and may facilitate subsequent clinical management of sepsis and septic shock (for review, see Önal et al. 2018. Healthcare 6: 110).

Several methods have been described to measure circulating ADM levels, either directly by ADM or indirectly by detecting a more stable fragment of its related precursor peptide. Recently, a method describing an assay for measuring circulating mature ADM has been published (Weber et al. 2017. JALM 2: 222-233).

Other methods for quantification of ADM precursor-derived fragments have been described, such as measurement of 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 immunofluorescence assay for measurement of MR-proADM in plasma is available on a fully automated system (BRAHMS MR-proADM KRYPTOR; BRAHMS GmbH, Hennigsdorf, Germany) (Caruhel et al. 2009. Clin Biochem 42(7-8):725-8). Since these peptides are formed in stoichiometric ratio from the same precursor, their plasma levels are correlated to some extent.

The production of antibodies against ADM is known in the art (e.g., WO2013072512 and WO2019057992, incorporated herein by reference).

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment for use in the treatment of shock in a patient who is either entering shock or has developed shock, wherein said antibody or fragment or scaffold binds to the N-terminal portion (amino acids 1-21) of mature ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID No. 37).

In another preferred embodiment, said anti-ADM antibody or anti-ADM antibody fragment recognizes and binds to the N-terminus (aa1) of ADM. N-terminus means that amino acid 1, i.e., "Y" in SEQ ID No. 34 and 35, is essential for antibody binding. Said non-Ig antibody or fragment or framework thereof will not bind to either N-terminally extended, N-terminally modified, or N-terminally cleaved ADM.

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment for use in the treatment of shock in a patient who is either entering shock or has developed shock, wherein said antibody or fragment is a human monoclonal antibody or fragment that binds to the N-terminal region (amino acids 1-21) of mature ADM (SEQ ID No. 37), or an antibody fragment, wherein 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

where the light chain contains the sequences:

QSIVYSNGNTY (CDR1: SEQ ID NO: 41), RVS (CDR2), FQGSHIPYT (CDR3: SEQ ID NO: 42).

Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment for use in the treatment of shock in a patient who is either going into shock or has developed shock, wherein said antibody is a monoclonal antibody comprising the following sequence as a heavy chain:

QVQLVQSGAEVKKPGSSVKVSCKAS GYTFSRYW IEWVRQAPGQGLEWIGE ILPGSGST NYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 43)

and wherein said monoclonal antibody comprises the following sequence as a light chain:

DVVLTQSPLSLPVTLGQPASISCRSS QSIVYSNGNTY LEWYLQRPGQSPRLLIY RVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHIPYT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 44).

Endothelial dysfunction can result from and/or contribute to the development of several disease processes, such as shock, especially septic shock. From preclinical experiments in sepsis/septic shock models, it is known that administration of anti-ADM antibody causes an increase in plasma bio-ADM concentrations, which coincides with an increase in survival (Struck et al. 2013. Intensive Care Med Exp 1(1):22). The mechanism underlying this effect is described by Geven et al. 2018 (Geven et al. 2018. SHOCK 50 (2): 132-140). Briefly, when administered intravenously, the antibody, due to its size, cannot penetrate the endothelial barrier into the interstitium, but remains in the bloodstream. In contrast, ADM as a small peptide can freely diffuse across the endothelial barrier. Thus, the antibody, when administered in significant molar excess over endogenous ADM, binds virtually all of the ADM in plasma and, as a simple consequence of reaching binding equilibrium, results in translocation of ADM from the interstitium into the circulation. Interstitial ADM can bind to vascular smooth muscle cells and induce relaxation leading to vasodilation. This is reduced by administration of the antibody. On the other hand, ADM in plasma binds to endothelial cells and thereby stabilizes or even restores vascular integrity. Thus, this function is enhanced when plasma ADM levels are increased by administration of an antibody that is not a neutralizing antibody. Finally, binding of the antibody to ADM reduces proteolytic degradation of ADM. Taken together, the anti-ADM antibody, adrecizumab, can restore endothelial function in shock, such as septic shock.

If the indicated level of pro-adrenomedullin or its fragments is above a certain threshold level, then an anti-ADM antibody or an anti-ADM antibody fragment that binds to ADM is administered as therapy.

This means that in a particular embodiment of the present invention, said anti-ADM antibody or anti-ADM antibody fragment binding to ADM is intended for therapy, where a biological fluid sample taken from said patients exhibits an increased level of proADM and/or its fragments having at least 5 amino acids above a certain threshold. Thus, a diagnostic method using said proADM and/or its fragments serves as an additional diagnostic method.

In a particular embodiment of the invention, the threshold value is within the threshold range for ADM-NH2 in plasma, which is from 50 to 250 pg/ml, preferably from 55 to 200 pg/ml, even more preferably from 60 to 150 pg/ml, even more preferably from 65 to 100 pg/ml, most preferably a threshold value of 70 pg/ml is used.

In a particular embodiment of the invention, the cut-off value is within the cut-off range for MR-proADM in plasma, which is from 0.5 to 3 nmol/L, preferably from 0.6 to 2 nmol/L, even more preferably from 0.7 to 1 ng/ml, most preferably a cut-off value of 0.8 nmol/L is used.

In a particular embodiment of the invention, the cut-off value is within the cut-off range for CT-proADM in plasma, which is from 85 to 500 pmol/L, preferably from 90 to 350 pmol/L, even more preferably from 95 to 250 pmol/L, even more preferably from 100 to 200 pmol/L, most preferably a cut-off value of 150 pmol/L is used.

The levels of ADM-NH2 of the present invention or the levels of proADM or fragments thereof, respectively, were determined using the described ADM-NH2 assay as set forth in the examples (or the proADM or fragment assays, respectively) (Weber et al. 2017. JALM 2(2):1-4). The levels of DPP3 of the present invention were determined using the described DPP3 assays as set forth in the examples. The above-mentioned cutoff values may differ in other assays if they have been calibrated differently than the assay systems used in the present invention. Therefore, the above-mentioned cutoff values should be applied for such assays with different calibration, respectively, taking into account the differences in calibration. One possibility for quantifying the differences in calibration is a comparative analysis of the methods (correlation) of the assay in question with the corresponding biomarker assay used in the present invention, by measuring the corresponding biomarker (e.g., bio-ADM, DPP3) in samples using both methods. Another possibility is to determine with the assay in question, provided that this test has sufficient analytical sensitivity, the average level of biomarkers of a representative normal population, compare the results with average levels of biomarkers as described in the literature, and recalculate the calibration based on the difference obtained from this comparison. With the calibration used in the present invention, samples from normal (healthy) patients were measured: the median of bio-ADM in plasma (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 used in the present invention, samples from 5400 normal (healthy) patients (Swedish single-center prospective population-based study (MPP-RES) were measured: the median (interquartile range) of DPP3 in plasma was 14.5 ng/mL (11.3 ng/mL - 19 ng/mL).

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 an automated sandwich fluorescence assay for MR-proADM detection as described by Caruhel et al. (Caruhel et al. 2009. Clin Biochem 42(7–8):725–8).

The mean plasma CT-proADM concentration in healthy subjects (n=200) was 77.6 pmol/L (min. 46.6 pmol/L, max. 136.2 pmol/L), and the 95% percentile was 113.8 pmol/L (EP 2111552 B1).

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

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

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

In embodiments of the present invention, an anti-ADM antibody or anti-ADM antibody fragment for use in treating a patient may be administered at a dose of at least 0.5 mg/kg body weight, in particular at least 1 mg/kg body weight, more in particular 1 to 20 mg/kg body weight, such as 2 to 10 mg/kg body weight, 2 to 8 mg/kg body weight, or 2 to 5 mg/kg body weight.

One embodiment of the invention relates to a vasopressor for use in the treatment of shock in a patient who is either entering a state of shock or who has developed shock, wherein said patient has a DPP3 level in a sample of said patient's biological fluid below a predetermined threshold value, as determined by any of the methods for predicting or diagnosing refractory shock according to the present invention.

Another embodiment of the invention relates to a vasopressor for use in the treatment of shock in a patient who is either entering shock or has developed shock, wherein said vasopressor is selected from the group consisting of dopamine, norepinephrine, norepinephrine equivalent, epinephrine, phenylephrine and vasopressin.

Another specific embodiment of the invention relates to a vasopressor for use in the treatment of shock in a patient who is either entering shock or has developed shock, wherein said vasopressor is administered to said patient in the form of a pharmaceutical composition.

Another embodiment of the invention relates to a vasopressor for use in the treatment of shock in a patient who is either going into shock or has developed shock, wherein said patient has a blood pressure equal to or less than 65 mmHg.

Another embodiment of the invention relates to an inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering a state of shock or who has developed shock, wherein said patient has a level of DPP3 in a biological fluid sample that is above a predetermined threshold value when determined by the method described in the above-mentioned embodiments.

Another specific embodiment of the invention relates to an inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering shock or has developed shock, wherein the inhibitor of DPP3 activity is selected from the group consisting of an anti-DPP3 antibody, or an anti-DPP3 antibody fragment, or a non-Ig anti-DPP3 scaffold.

One embodiment of the invention relates to an inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering shock or has developed shock, wherein said inhibitor has a minimum binding affinity for DPP3 equal to or less than 10 ^-7 M.

Another embodiment of the invention relates to an inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering shock or has developed shock, wherein the inhibitor of DPP3 activity is an antibody.

Another specific embodiment of the invention relates to an inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either going into shock or has developed shock, wherein said inhibitor is a monoclonal antibody.

Another embodiment of the invention relates to an inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either going into shock or has developed shock, wherein said inhibitor is a monoclonal antibody in which the complementarity determining regions (CDRs) in the heavy chain comprise the sequences: SEQ ID NO.: 7, SEQ ID NO.: 8 and/or SEQ ID NO.: 9, and the complementarity determining regions (CDRs) in the light chain comprise the sequences: SEQ ID NO.: 10, KVS and/or SEQ ID NO.: 11.

Another embodiment of the invention relates to an inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either going into shock or has developed shock, wherein said inhibitor is a humanized monoclonal antibody or a fragment of a humanized monoclonal antibody, wherein the heavy chain comprises the sequence: SEQ ID NO.: 12, and the light chain comprises the sequence: SEQ ID NO.: 13.

The indicated DPP3 activity inhibitor can be administered by inhalation.

One embodiment of the invention relates to an inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering shock or has developed shock, wherein said inhibitor is administered in combination with an angiotensin receptor agonist and/or its precursors.

Another embodiment of the invention relates to an inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering shock or has developed shock, wherein said angiotensin receptor agonist and/or its precursors are selected from the group consisting of angiotensin I, angiotensin II, angiotensin III, angiotensin IV, in particular angiotensin II.

Another embodiment of the invention relates to angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors for use in the treatment of shock in a patient who is either entering a state of shock or has developed shock, wherein treatment with said angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors is initiated and/or continued when the DPP3 level in a sample of said patient is above a certain threshold value, and/or where treatment with vasopressors is delayed and/or stopped if said certain DPP3 level is above said predetermined threshold value.

Another specific embodiment of the invention relates to a vasopressor for use in the treatment of shock in a patient who is either entering a state of shock or has developed shock, wherein treatment with said vasopressor is started and/or continued when the level of DPP3 in a biological fluid sample of said patient is below a certain threshold value and/or wherein treatment with angiotensin receptor agonists and/or their precursors and/or inhibitors of DPP3 activity is postponed and/or stopped if said certain level of DPP3 is below said specified threshold value.

Another specific embodiment of the invention relates to a vasopressor for use in the treatment of shock in a patient who is either entering a state of shock or has developed shock, wherein treatment with said vasopressor is initiated and/or continued when the level of DPP3 in a biological fluid sample of said patient is below a certain threshold value, and/or wherein treatment with angiotensin receptor agonists and/or their precursors and/or inhibitors of DPP3 activity is delayed and/or stopped if said determined level of DPP3 is below said predetermined threshold value, and wherein a level of pro-adrenomedullin or fragments thereof is determined, and wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment is initiated and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with the anti-ADM antibody or anti-ADM antibody fragment is delayed and/or stopped if said determined level of pro-adrenomedullin or fragments thereof is below the specified threshold value.

Another embodiment of the invention relates to angiotensin receptor agonists and/or precursors thereof and/or DPP3 activity inhibitors for use in the treatment of shock in a patient who is either entering shock or has developed shock, wherein treatment with said vasopressor is initiated and/or continued when the level of DPP3 in a biological fluid sample of said patient is below a certain threshold value, and/or wherein treatment with angiotensin receptor agonists and/or precursors thereof and/or DPP3 activity inhibitors is delayed and/or stopped if said determined level of DPP3 is below said predetermined threshold value, and wherein a level of pro-adrenomedullin or fragments thereof is determined, and wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment is initiated and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with the anti-ADM antibody or anti-ADM antibody fragment is delayed and/or discontinued if the specified defined level of pro-adrenomedullin or its fragments is below the specified predetermined threshold value.

Another specific embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment for use in the treatment of shock in a patient who is either entering a state of shock or has developed shock, wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment and/or angiotensin receptor agonists and/or precursors thereof and/or DPP3 activity inhibitors is initiated and/or continued if the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value and said certain level of DPP3 is above said predetermined threshold value of DPP3.

In case of determination of the level of pro-adrenomedullin or its fragments in the said sample and determination of the level of DPP3 in the said sample, the said determination may be carried out using a device for providing medical care that provides simultaneously: a test for determination of the level of pro-adrenomedullin or its fragments and a test for determination of the level of DPP3 in the said sample.

Initiation of treatment with an angiotensin receptor agonist and/or its precursor is indicated when the patient's DPP3 protein amount and/or DPP3 activity in a biological fluid sample exceeds a predetermined threshold level.

One embodiment of the invention relates to a method for treating shock in a patient who is either entering a state of shock or has developed shock, wherein said method comprises administering a vasopressor to said patient, wherein said patient has a DPP3 level in a sample of said patient's biological fluid below a predetermined threshold value when determined by the method described in the previous embodiments.

Another embodiment of the invention relates to a method of treating shock in a patient who is either entering a state of shock or who has developed shock, wherein the method comprises administering to said patient a vasopressor, wherein said vasopressor is selected from the group consisting of dopamine, norepinephrine, norepinephrine equivalent, epinephrine, phenylephrine and vasopressin.

A particular embodiment of the invention relates to a method of treating shock in a patient who is either entering a state of shock or who has developed shock, wherein the method comprises administering a vasopressor to said patient, wherein said vasopressor is administered to said patient in a pharmaceutical formulation.

Another embodiment of the invention relates to a method of treating shock in a patient who is either going into shock or has developed shock, said method comprising administering a vasopressor to said patient, wherein said patient has a blood pressure equal to or less than 65 mmHg.

One embodiment of the invention relates to a method for treating shock in a patient who is either falling into a state of shock or who has developed shock, wherein said method comprises administering an inhibitor of DPP3 activity to said patient, wherein said patient has a level of DPP3 in a sample of a biological fluid of said patient that exceeds a predetermined threshold value when determined by the method described in the previous embodiments.

Another embodiment of the invention relates to a method of treating shock in a patient who is either entering a state of shock or has developed shock, wherein the method comprises administering an inhibitor of DPP3 activity to said patient, wherein the inhibitor of DPP3 activity is selected from the group consisting of an anti-DPP3 antibody or an anti-DPP3 antibody fragment, or an anti-DPP3 non-Ig scaffold.

Another specific embodiment of the invention relates to a method of treating shock in a patient who is either entering shock or has developed shock, wherein the method comprises administering to said patient an inhibitor of DPP3 activity, wherein said inhibitor has a minimum binding affinity for DPP3 equal to or less than 10 ^-7 M.

Another embodiment of the invention relates to a method of treating shock in a patient who is either entering a state of shock or who has developed shock, wherein the method comprises administering an inhibitor of DPP3 activity to said patient, wherein the inhibitor of DPP3 activity is an antibody.

One embodiment of the invention relates to a method of treating shock in a patient who is either entering shock or has developed shock, wherein the method comprises administering to said patient an inhibitor of DPP3 activity, wherein said inhibitor is a monoclonal antibody.

One specific embodiment of the invention relates to a method of treating shock in a patient who is either going into shock or has developed shock, wherein the method comprises administering an inhibitor of DPP3 activity to said patient, wherein said inhibitor is a monoclonal antibody, wherein the complementarity determining regions (CDRs) in the heavy chain comprise the sequences: SEQ ID NO.: 7, SEQ ID NO.: 8 and/or SEQ ID NO.: 9 and the complementarity determining regions (CDRs) in the light chain comprise the sequences: SEQ ID NO.: 10, KVS and/or SEQ ID NO.: 11.

Another embodiment of the invention relates to a method of treating shock in a patient who is either going into shock or has developed shock, wherein the method comprises administering to said patient an inhibitor of DPP3 activity, wherein said inhibitor is a humanized monoclonal antibody or a fragment of a humanized monoclonal antibody, wherein the heavy chain comprises the sequence: SEQ ID NO.: 12, and the light chain comprises the sequence: SEQ ID NO.: 13.

Another specific embodiment of the invention relates to a method of treating shock in a patient who is either entering shock or has developed shock, wherein the method comprises administering an inhibitor of DPP3 activity to said patient, wherein said inhibitor is administered in combination with an angiotensin receptor agonist and/or its precursors.

Another embodiment of the invention relates to a method for treating shock in a patient who is either entering a state of shock or has developed shock, wherein the method comprises administering an inhibitor of DPP3 activity to said patient, wherein said angiotensin receptor agonist and/or its precursors are selected from the group comprising angiotensin I, angiotensin II, angiotensin III, angiotensin IV, in particular angiotensin II.

Another embodiment of the invention relates to a method of treating shock in a patient who is either falling into a state of shock or who has developed shock, wherein said method comprises administering angiotensin receptor agonists and/or their precursors and/or a DPP3 activity inhibitor to said patient, wherein treatment with said angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors is started and/or continued when the DPP3 level in a sample of said patient is above a certain threshold value and/or wherein treatment with vasopressors is delayed and/or stopped if said certain DPP3 level is above said specified threshold value.

One embodiment of the invention relates to a method for treating shock in a patient who is either falling into a state of shock or who has developed shock, wherein the method comprises administering a vasopressor to said patient, wherein treatment with said vasopressors is started and/or continued when the level of DPP3 in a biological fluid sample of said patient is below a certain threshold value, and/or wherein treatment with angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors is postponed and/or stopped if said certain level of DPP3 is below said specified threshold value.

One embodiment of the invention relates to a method of treating shock in a patient who is either entering a state of shock or has developed shock, said method comprising administering angiotensin receptor agonists and/or precursors thereof and/or a DPP3 activity inhibitor to said patient, wherein treatment with said angiotensin receptor agonists and/or precursors thereof and/or DPP3 activity inhibitors is initiated and/or continued when the DPP3 level in said patient's sample is above a certain threshold value, and/or wherein treatment with vasopressors is delayed and/or stopped if said determined DPP3 level is above said predetermined threshold value, and wherein a level of pro-adrenomedullin or fragments thereof is determined, and wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment is initiated and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with the anti-ADM antibody or anti-ADM antibody fragment is ADM is delayed and/or discontinued if the specified defined level of pro-adrenomedullin or its fragments is below the specified predetermined threshold value.

One particular embodiment of the invention relates to a method of treating shock in a patient who is either going into shock or has developed shock, said method comprising administering a vasopressor to said patient, wherein treatment with said vasopressors is initiated and/or continued when a level of DPP3 in a biological fluid sample of said patient is below a certain threshold value, and/or wherein treatment with angiotensin receptor agonists and/or their precursors and/or inhibitors of DPP3 activity is delayed and/or stopped if said determined level of DPP3 is below said predetermined threshold value, and wherein a level of pro-adrenomedullin or fragments thereof is determined, and wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment is initiated and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with the anti-ADM antibody or anti-ADM antibody fragment is delayed and/or stopped if said determined level pro-adrenomedullin or its fragments below the specified predetermined threshold value.

Another embodiment of the invention relates to a method of treating shock in a patient who is either entering a state of shock or has developed shock, wherein the method comprises administering an anti-ADM antibody or an anti-ADM antibody fragment to said patient, wherein a level of pro-adrenomedullin or fragments thereof is determined, and wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment is started and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with the anti-ADM antibody or anti-ADM antibody fragment is delayed and/or stopped if said determined level of pro-adrenomedullin or fragments thereof is below said predetermined threshold value, and wherein treatment with angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors is started and/or continued when the level of DPP3 in said patient's sample is above a certain threshold value, and/or wherein treatment with vasopressors is delayed and/or stopped, if the specified defined DPP3 level is above the specified defined threshold.

Another specific embodiment of the invention relates to a method of treating shock in a patient who is either entering a state of shock or has developed shock, wherein the method comprises administering an anti-ADM antibody or an anti-ADM antibody fragment to said patient, wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment and/or angiotensin receptor agonists and/or precursors thereof and/or DPP3 activity inhibitors is initiated and/or continued if the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and said certain level of DPP3 is above said predetermined DPP3 threshold value.

An agonist is a chemical that binds to a receptor and activates the receptor, causing a biological response. Receptors can be activated by either endogenous agonists (such as hormones and neurotransmitters) or exogenous agonists (such as drugs), resulting in a biological response. Full agonists bind to a receptor and activate it, with the maximum response that the agonist can elicit at the receptor. Partial agonists are drugs that bind to and activate a given receptor, but are only partially potent at the receptor compared to a full agonist. Potency is the amount of agonist needed to produce the desired response. The potency of an agonist is inversely proportional to its EC50 value. The EC50 can be measured for a given agonist by determining the concentration of agonist needed to produce half the agonist's maximum biological response. The EC50 value is useful for comparing the potency of drugs of similar potency that produce physiologically similar effects. The lower the EC50 value, the greater the potency of the agonist and the lower the concentration of drug required to produce the maximal biological response.

Treatment with an angiotensin receptor agonist and/or its precursor may be continued as long as the patient's DPP3 protein level and/or DPP3 activity in a biological fluid sample exceeds a predetermined threshold level.

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

Treatment with an angiotensin receptor agonist and/or its precursor may be discontinued when the patient's DPP3 protein level and/or DPP3 activity in a biological fluid sample is below a predetermined threshold level.

Angiotensin therapeutic agents:

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

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

Angiotensin II is a hormone produced by the renin-angiotensin-aldosterone system (RAAS) that modulates arterial pressure by regulating vascular smooth muscle tone and extracellular fluid homeostasis. Angiotensin II mediates its effects on the vasculature by inducing vasoconstriction and sodium retention and is therefore the target of many hypertension therapies. In addition to its systemic effects, angiotensin II has profound effects on the efferent arterioles of the kidney, maintaining glomerular filtration when blood flow is reduced. Angiotensin II also regulates sodium reabsorption in the kidneys by stimulating Na+/H+ exchangers in the proximal tubule and causing the release of aldosterone and vasopressin (Harrison-Bernard 2009. The renal renin-angiotensin system. Adv Physiol Educ, 33(4): 270 (2009)).

An angiotensin II therapeutic agent that may be used in the compositions and methods of the present invention may be Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 13), which is also referred to as 5-isoleucine angiotensin II. SEQ ID NO: 13 is an octapeptide naturally present in humans and other species such as horses, pigs, etc. Isoleucine may be replaced by valine to form 5-valine angiotensin II, Asp-Arg-Val-Tyr-Val-His-Pro-Phe (SEQ ID NO: 14). Other angiotensin II analogs such as [Asn ¹ -Phe ⁴ ]-angiotensin II (SEQ ID NO: 15), hexapeptide Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 16), nonapeptide Asn-Arg-Val-Tyr -Tyr-Val-His-Pro-Phe (SEQ ID NO: 17), [Asn ¹ Ile ⁵ Ile ⁸ ]-angiotensin II (SEQ ID NO: 18), [Asn ¹ -lle ⁵ -Ala ⁸ ]-angiotensin II (SEQ ID NO: 19), and [Asn ¹ -diiodo-Tyr ⁴ -Ile ⁵ -angiotensin II (SEQ ID NO: 20) can also be used. Angiotensin II can be synthesized, for example, by solid-phase peptide synthesis to incorporate modifications such as C-terminal amidation. The term "angiotensin II" without further specification is intended to refer to any of these various forms, as well as combinations thereof.

In some aspects, the composition comprising angiotensin II may be selected from 5-valine-angiotensin, 5-valine-angiotensin II amide, 5-L-isoleucine-angiotensin II and 5-L-isoleucine-angiotensin II amide or a pharmaceutically acceptable salt thereof, preferably obtained under current good manufacturing conditions (cGMP). In some aspects, the composition may include different forms of angiotensin II in different percentages, for example, a mixture of hexapeptide and nonapeptide of angiotensin II. The composition comprising angiotensin II may be suitable for parenteral administration, for example, for injection or intravenous infusion.

The angiotensin II sequence used in the compositions and methods disclosed herein may be homologous to the angiotensin II sequences described above. In some aspects, the invention includes isolated, synthetic, or recombinant amino acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 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.

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

Angiotensin III is a metabolite of angiotensin II with approximately 40% of the activity of angiotensin II. An angiotensin III therapeutic agent that may be used in the compositions and methods of the present invention may be Arg-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 21). SEQ ID NO: 21 is a heptapeptide naturally present in humans and other species such as horses, pigs, etc. Isoleucine may be replaced with valine to produce Arg-Val-Tyr-Val-His-Pro-Phe (SEQ ID NO: 22). Other angiotensin III analogues such as [Phe ³ ]-angiotensin III (SEQ ID NO: 23), [Ile ⁴ -Ala ⁷ ]-angiotensin III (SEQ ID NO: 24), and [diiodo-Tyr ³ -Ile ⁴ ]-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" without further specification is intended to refer to any of these various forms, as well as combinations thereof.

In some aspects, 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 obtained under current good manufacturing conditions (cGMP). The composition comprising angiotensin III may be suitable for parenteral administration, such as by injection or intravenous infusion.

The angiotensin III sequence used in the compositions and methods disclosed herein may be homologous to the angiotensin III sequences described above. In some aspects, the invention includes isolated, synthetic, or recombinant amino acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 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.

Angiotensin IV is a metabolite of angiotensin III with less activity than angiotensin II. An angiotensin IV therapeutic agent that may be used in the compositions and methods of the present invention may be Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 26). SEQ ID NO: 26 is a hexapeptide naturally present in humans and other species. Isoleucine may be replaced with valine to yield Val-Tyr-Val-His-Pro-Phe (SEQ ID NO: 27). Other angiotensin IV analogues such as [Phe ² ]-angiotensin III (SEQ ID NO: 28), [Ile ³ -Ala ⁶ ]-angiotensin IV (SEQ ID NO: 29) and [diiodo-Tyr ² -Ile ³ ]-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" without further specification is intended to refer to any of these various forms, as well as combinations thereof.

In some aspects, 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 conditions (cGMP). The composition comprising angiotensin IV may be suitable for parenteral administration, such as by injection or intravenous infusion.

The angiotensin IV sequence used in the compositions and methods disclosed herein may be homologous to the angiotensin IV sequences described above. In some aspects, the invention includes isolated, synthetic, or recombinant amino acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, and/or 20. Any such variant sequences may be used in place of angiotensin IV as described in the preceding paragraph.

The angiotensin II, angiotensin III or angiotensin IV therapeutic agent may be used in the form of any suitable salt (e.g., acetate), deprotected form, acetylated form, deacetylated form and/or prodrug form of the aforementioned peptides, including pegylated forms of the peptides or conjugates as described in U.S. Patent Publication No. 2011/0081371 (incorporated by reference). The term "prodrug" refers to any precursor compound that is capable of generating or releasing the aforementioned peptide under physiological conditions. Such prodrugs or precursors may be larger peptides that are selectively cleaved to form the peptide of the invention. For example, in some aspects, the prodrug or precursor may be angiotensinogen, angiotensin I, or homologues thereof, from which angiotensin II may be derived by the action of certain endogenous or exogenous enzymes. Additional prodrugs or precursors include peptides with protected amino acids, such as those having protecting groups at one or more carboxylic acids and/or amino groups. Suitable protecting groups for amino groups include benzyloxycarbonyl, tert-butyloxycarbonyl (BOC), fluorenylmethyloxycarbonyl (FMOC), formyl, and acetyl or acyl groups. Suitable protecting groups for a carboxylic acid group include esters such as benzyl esters or tert-butyl esters. The present invention also provides for the use of angiotensin II, angiotensin III, angiotensin IV and/or precursor peptides containing amino acid substitutions, deletions, insertions, wherein the substitutions and insertions include standard D- and L-amino acids and modified amino acids, such as, for example, amidated and acetylated amino acids, wherein the therapeutic activity of the basic peptide sequence is maintained at a pharmacologically useful level.

In a preferred embodiment, 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 salt. The acetate counterion is present in a non-stoichiometric ratio. The molecular formula of angiotensin II acetate is C50H71N13O12⋅(C2H4O2)n; (n=number of acetate molecules; theoretical n=3) with an average molecular weight of 1046.2 (as the free base).

One embodiment of the invention is an angiotensin receptor agonist and/or its precursor for use in treating a disease in a patient, wherein said patient has a certain amount of DPP3 protein and/or DPP3 activity in a biological fluid sample above a predetermined threshold value, wherein determining the amount of DPP3 protein and/or DPP3 activity in the biological fluid sample is used as a guide to therapy and/or monitoring therapy for said treatment with an angiotensin receptor agonist and/or its precursor.

In one embodiment of the invention, the amount of DPP3 protein and/or DPP3 activity in a biological fluid sample is determined before treating the patient with an angiotensin receptor agonist and/or a precursor thereof. In one embodiment of the invention, the amount of DPP3 protein and/or DPP3 activity in a biological fluid sample is determined during treatment of the patient with an angiotensin receptor agonist and/or a precursor thereof at least once during the treatment, preferably at least twice or preferably at least once a day during the treatment. In one embodiment of the invention, the amount of DPP3 protein and/or DPP3 activity in a biological fluid sample is determined after treating the patient with an angiotensin receptor agonist and/or a precursor thereof. In one embodiment of the invention, the amount of DPP3 protein and/or DPP3 activity in a biological fluid sample is determined before and/or during and/or after treating the patient with an angiotensin receptor agonist and/or a precursor thereof.

One embodiment of the invention is an angiotensin receptor agonist and/or its precursor for use in the treatment of shock in a patient who is either falling into a state of shock or who has developed shock, where said patient has a certain amount of DPP3 protein and/or DPP3 activity in a biological fluid sample above a specified threshold value, where said biological fluid sample of said patient is selected from whole blood, blood plasma and blood serum.

One embodiment of the invention is an angiotensin receptor agonist and/or its precursor for use in the treatment of shock in a patient who is either falling into shock or has developed shock, wherein said angiotensin receptor agonist and/or its precursor is administered to said patient in a pharmaceutical composition.

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

One embodiment of the invention is an angiotensin receptor agonist and/or its precursor for use in the treatment of shock in a patient who is either falling into shock or has developed shock, wherein said angiotensin receptor agonist and/or its precursor is angiotensin II, and said pharmaceutical composition is a solution, preferably a ready-to-use solution. In another embodiment, the subject of the present invention is also a pharmaceutical composition according to the present invention, wherein said pharmaceutical composition is in a dried state for reconstitution before use.

The pharmaceutical composition described 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 effective substance (such as angiotensin II) of the present invention and which does not interfere with the pharmacological activity of the therapeutically effective substance. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).

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

The term "pharmaceutical (active) ingredient" means a therapeutic composition that may optionally be combined with pharmaceutically acceptable excipients to produce a pharmaceutical formulation or dosage form.

It will be appreciated by one skilled in the art that the choice of any excipient may influence the choice of any other excipient. For example, the choice of a particular excipient may preclude the use of one or more additional excipients because the combination of excipients may cause undesirable effects. Excipients of the invention may include, but are not limited to, co-solvents, solubilizing agents, buffers, pH adjusting agents, fillers, surfactants, encapsulating agents, tonicity adjusting agents, stabilizers, protectants, and viscosity modifiers.

In some aspects, it may be advantageous to include a pharmaceutically acceptable carrier in the compositions disclosed herein.

In some aspects, it may be useful to include a solubilizing agent in the compositions of the invention. Solubilizing agents may be useful to enhance the solubility of any of the components of the formulation or composition, including the therapeutically effective substance (e.g., angiotensin II, angiotensin III, or angiotensin IV) or an excipient. The solubilizing agents described herein are not intended to be an exhaustive list, but are merely provided as illustrative of solubilizing agents that may be used in the compositions of the invention. In some 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 and/or combinations thereof.

In some aspects, it may be useful to adjust the pH of the compositions by including a pH-adjusting agent in the compositions of the invention. Modifying the pH of a formulation or composition may have a beneficial effect, for example, on the stability or solubility of a therapeutically effective substance, or may be useful in creating a formulation or composition suitable for parenteral administration. pH-adjusting agents are well known in the art. Accordingly, the pH-adjusting agents described herein are not intended to be an exhaustive list, but are merely provided as examples of pH-adjusting agents that may be used in the compositions of the invention. pH-adjusting agents may include, for example, acids and bases. In some aspects, the pH-adjusting agent includes, but is not limited to, acetic acid, hydrochloric acid, phosphoric acid, sodium hydroxide, sodium carbonate, and combinations thereof.

The pH of the compositions as disclosed herein can be any pH that provides the desired properties of the formulation or composition. Desirable properties can include, for example, stability of the therapeutically effective substance (e.g., angiotensin II, angiotensin III, or angiotensin IV), increased retention of the therapeutically effective substance compared to compositions at other pHs, and improved filtration efficiency. In some aspects, the pH of the compositions of the invention can be from about 3 to about 9, such as from about 5 to about 7. In particular aspects, the pH of the compositions of the invention can be 5.5±0.1, 5.6±0.1, 5.7±0.1, 5.8±0.1, 5.9±0.1, 6.0±0.1, 6.1±0.1, 6.2±0.1, 6.3±0.1, 6.4±0.1, or 6.5±0.1.

In some aspects, it may be useful to buffer the pH by including one or more buffers in the compositions. In some aspects, the buffer may have a pKa of, for example, about 5.5, about 6, or about 6.5. One skilled in the art will appreciate that a suitable buffer for inclusion in the compositions of the invention may be selected based on its pKa and other properties.

Buffers are well known in the art. Accordingly, the buffers described herein are not intended to be an exhaustive list, but are provided merely as examples of buffers that may be used in the compositions of the invention. In some aspects, a buffer may include one or more of the following: Tris, Tris HCl, potassium phosphate, sodium phosphate, sodium citrate, sodium ascorbate, combinations of sodium and potassium phosphates, Tris/Tris HCl, sodium bicarbonate, arginine phosphate, arginine hydrochloride, histidine hydrochloride, cacodylate, succinate, 2-(N-morpholino)ethanesulfonic acid (MES), maleate, bis-Tris, phosphate, carbonate, and any pharmaceutically acceptable salts and/or combinations thereof.

In some aspects, it may be useful to include a surfactant in the compositions of the invention. Surfactants generally reduce the surface tension of a liquid composition. This can provide useful properties such as increased ease of filtration. Surfactants can also act as emulsifiers and/or solubilizing agents. Surfactants are well known in the art. Accordingly, the surfactants described herein are not intended to be an exhaustive list, but are merely provided as examples of surfactants that can be used in the compositions of the invention. Surfactants that may be included 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 (e.g., Pluronics), ethylene oxides and polyethylene oxides (e.g., Triton X-100), saponins, phospholipids (e.g., lecithin), and combinations thereof.

In some aspects, it may be advantageous to include a tonicity adjusting agent in the compositions of the invention. The tonicity of a liquid composition is an important factor when administering the composition to a patient, for example, by parenteral administration. Accordingly, tonicity adjusting agents can be used to make the formulation or composition suitable for administration. Tonicity adjusting agents are well known in the art. Accordingly, the tonicity adjusting agents described herein are not intended to be an exhaustive list, but are merely provided as examples of tonicity adjusting agents that can be used in the compositions of the invention. Tonicity adjusting agents can be ionic or non-ionic and include, but are not limited to, inorganic salts, amino acids, carbohydrates, sugars, sugar alcohols, and carbohydrates. Examples of inorganic salts can include sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate. An example of an amino acid is glycine. Examples of sugars may include sugar alcohols such as glycerol, propylene glycol, glucose, sucrose, lactose, and mannitol.

In some aspects, it may be useful to include a stabilizing agent in the compositions of the invention. Stabilizing agents help to increase the stability of the therapeutically effective substance in the compositions of the invention. This may occur, for example, by reducing degradation or preventing aggregation of the therapeutically effective substance. Without wishing to be bound by theory, mechanisms for enhancing stability may include sequestering the therapeutically effective substance from the solvent or inhibiting free radical oxidation of the anthracycline compound. Stabilizing agents are well known in the art. Accordingly, the stabilizers described herein are not intended to be an exhaustive list, but are merely provided as examples of stabilizers that may be used in the compositions of the invention. Stabilizing agents may include, but are not limited to, emulsifiers and surfactants.

The compositions disclosed herein can be administered by a variety of conventional routes. In some aspects, the compositions of the invention are suitable for parenteral administration. These compositions can be administered, for example, intraperitoneally, intravenously, intrarenally, intrathecally, or by inhalation. In some aspects, the compositions of the invention are administered intravenously. It will be understood by one skilled in the art that the route of administration of a therapeutically effective substance in a formulation or composition of the invention will depend on factors such as the age, weight, and physical condition of the patient being treated, as well as the disease or condition being treated. Accordingly, a skilled artisan will be able to select the route of administration that is optimal for the patient in each particular case.

The angiotensin receptor agonist and/or its precursor may be administered by any suitable route, but is typically administered by continuous infusion. Accordingly, an increase or decrease in the rate of administration may be achieved by changing the flow rate of the intravenous drip, changing the concentration of the agent in the intravenous drip, etc. However, the method of changing the rate of administration will depend on the route 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 with a higher release rate or a transdermal composition. 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 controlled release dosage forms with a higher release rate.

When the therapeutic agent is administered by inhalation, the rate can be increased, for example, by administering additional boluses, a more concentrated bolus, or a bolus with a faster release. Other routes of administration (via subcutaneous injection pump, suppository, etc.) can be modulated in a similar manner, and a decrease in the rate of administration can be achieved by an action opposite to that which increases the rate of administration of the therapeutic agent.

One embodiment of the invention is an angiotensin receptor agonist and/or a precursor thereof for use in the treatment of shock in a patient who is either entering shock or has developed shock, wherein said angiotensin receptor agonist and/or a precursor thereof is angiotensin II, which is administered at a rate of from 0.1 to 200 ng/kg/min, preferably from 1 to 100 ng/kg/min, more preferably from 2 to 80 ng/kg/min, even more preferably from 5 to 60 ng/kg/min, even more preferably from 10 to 50 ng/kg/min, even more preferably from 15 to 40 ng/kg/min, most preferably at a rate of 20 ng/kg/min.

In a specific embodiment of the present invention, the initial dose (initial rate) of angiotensin II is 80 ng/kg/min, more preferably 40 ng/kg/min, most preferably 20 ng/kg/min by continuous intravenous infusion.

In another specific embodiment, the titration of angiotensin II is monitored by monitoring the blood pressure response (e.g., mean arterial pressure; MAP). The titration of angiotensin II 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, most preferably every 5 minutes. The titration of angiotensin II may be performed in increments of up to 40 ng/kg/min, more preferably up to 20 ng/kg/min, most preferably up to 15 ng/kg/min as needed until the target blood pressure is achieved or maintained. In another preferred embodiment, the dose should not exceed 80 ng/kg/min of angiotensin II during the first 3 hours of treatment. Most preferably, maintenance doses do not exceed 40 ng/kg/min, and doses up to 1.25 ng/kg/min may be used.

In some embodiments, the patient has a baseline mean arterial pressure (MAP) of no more than about 40 mmHg, about 45 mmHg, about 50 mmHg, 55 mmHg, about 60 mmHg, about 65 mmHg, about 70 mmHg, or about 75 mmHg prior to administration of the composition. The method may include measuring the patient's mean atrial arterial pressure and increasing the rate of angiotensin II administration if the mean arterial pressure is less than about 40 mmHg, about 45 mmHg, about 50 mmHg, 55 mmHg, about 60 mmHg, about 65 mmHg, about 70 mmHg, or about 75 mmHg.

In one embodiment, the patient may receive a vasopressor (e.g., a catecholamine such as norepinephrine, norepinephrine equivalent, epinephrine, dopamine, phenylephrine) or a combination thereof. In some embodiments, the vasopressor is vasopressin (e.g., terlipressin, argipressin, desmopressin, felipressin, lypressin, or ornipressin).

One embodiment of the invention is an angiotensin receptor agonist and/or its precursor for use in the treatment of shock in a patient who is either going into shock or has developed shock, wherein said angiotensin receptor agonist and/or its precursor, in particular angiotensin II, is administered in combination with a DPP3 inhibitor.

One embodiment of the invention is an angiotensin receptor agonist and/or a precursor thereof for use in the treatment of shock in a patient who is either entering shock or has developed shock, in combination with a DPP3 inhibitor, wherein said DPP3 inhibitor is selected from the group consisting of an anti-DPP3 antibody or an anti-DPP3 antibody fragment, or a non-Ig anti-DPP3 scaffold.

According to the invention, an "anti-DPP3 antibody" is an antibody that specifically binds to DPP3, an "anti-DPP3 antibody fragment" is a fragment of said anti-DPP3 antibody, wherein said fragment specifically binds to DPP3. An "anti-DPP3 non-Ig scaffold" is a non-Ig scaffold that specifically binds to DPP3.

One embodiment of the invention is an angiotensin receptor agonist and/or a precursor thereof for use in the treatment of shock in a patient who is either going into shock or has developed shock, in combination with a DPP3 inhibitor, wherein said DPP3 inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or a non-Ig anti-DPP3 scaffold that binds to SEQ ID NO. 1, in particular that binds to SEQ ID NO. 2.

One embodiment of the invention is an angiotensin receptor agonist and/or a precursor thereof for use in the treatment of shock in a patient who is either entering shock or has developed shock, in combination with a DPP3 inhibitor, wherein said DPP3 inhibitor is an antibody, fragment or scaffold thereof that exhibits a minimum binding affinity for DPP3 equal to or less than 10 ^-7 M.

In accordance with the present invention, it is well known to those skilled in the art that the binding affinity of the DPP3 binding agent disclosed herein to DPP3 can be measured using various suitable assays known in the art. Relevant examples are provided below, but should not be construed as limiting the possibilities for measuring the binding affinity of the DPP3 binding agent described herein to DPP3.

For example, the binding affinity of a DPP3 binding agent to an epitope can be determined. The binding assay can be performed to detect and/or quantify the binding of an antibody, for example, to an immunizing peptide of the corresponding binding agent. For example, the immunizing peptide can be immobilized on a solid phase. The sample to be tested (e.g., an antibody solution) is passed through the immobilized immunizing peptide, and the bound antibody can be detected. For the purposes of the present description, the term "solid phase" can be used to include any material or vessel in or on which the assay can be performed, and includes, but is not limited to, porous materials, non-porous materials, test tubes, wells, glasses, magnetic beads, etc.

Examples of detection methods:

- Label the antibody before contact with the solid phase and identify the appropriate label (fluorescent, chemiluminescent, enzymatic, etc.).

- Use a labeled secondary antibody against a specific Fc portion of the sample antibody. Incubate the solid-phase bound antibody with the secondary antibody (e.g. anti-human IgG, anti-mouse IgG) and detect the appropriate label (fluorescent, chemiluminescent, enzymatic, etc.).

- Use a labeled antibody as a competitor for solid-phase binding (e.g. labeled AK1967).

- Quantify the binding affinity by signal reduction.

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

One embodiment of the invention is an angiotensin receptor agonist and/or a precursor thereof for use in the treatment of shock in a patient who is either going into shock or has developed shock, in combination with a DPP3 inhibitor, wherein said DPP3 inhibitor is a monospecific antibody or fragment or scaffold, in one embodiment said DPP3 inhibitor is a monoclonal antibody, fragment or scaffold.

Monospecific antibodies or non-Ig fragments or scaffolds according to the invention are antibodies or non-Ig fragments or scaffolds that all have affinity for the same antigen. Monoclonal antibodies are monospecific, but monospecific antibodies can also be obtained by other methods than obtaining them from a normal germ cell.

In a particular embodiment, said capture binding agent that binds to full-length DPP3 specifically inhibits DPP3 activity by less than 50% in a liquid phase assay, preferably less than 40%, more preferably 30%. For the definition of the liquid phase assay, see above. In one particular embodiment, to prevent inhibition of DPP3, the capture binding agent should not bind to DPP3 in the region around the active site and the substrate binding region (amino acids 316-669 of SEQ ID No. 1).

One embodiment of the invention is an angiotensin receptor agonist and/or a precursor thereof for use in the treatment of shock in a patient who is either going into shock or has developed shock, in combination with a DPP3 inhibitor, wherein said DPP3 inhibitor is an antibody or a fragment or scaffold thereof that binds to full-length DPP3 and inhibits DPP3 activity by at least 10% or at least 50%, more preferably by at least 60%, even more preferably by more than 70%, even more preferably by more than 80%, even more preferably by more than 90%, even more preferably by more than 95%.

Inhibition of DPP3 activity in a liquid-phase assay using a binding agent can be determined as follows: candidate binding agents for DPP3 capture are incubated with recombinant or purified native DPP3 and specific DPP3 substrates in a liquid-phase assay. Preferably, the capture binding agent for the ECA is selected to have the lowest inhibitory capacity. The capture binding agent should inhibit DPP3 activity by less than 50%, preferably less than 40%, preferably less than 30%. The specific DPP3 activity assay in liquid phase for determining the inhibitory capacity of candidate capture binding agents comprises the following steps:

Incubation of 25 ng/ml native human DPP3 with 5 μg/ml of the appropriate binding agent and control buffer in 50 mM Tris-HCl, pH 7.5, and 100 μM _ZnCl2 for 1 h at room temperature.

Addition of the fluorogenic substrate Arg-Arg-βNA (20 μl, 2 mM).

Incubation at 37°C and monitoring of free βNA formation on a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH) for 1 h. βNA fluorescence was detected by excitation at 340 nm and emission measurement at 410 nm.

Calculation of slopes (in RFU/min) of fluorescence increase of different samples. Slope of native human DPP3 with buffer control is taken as 100% activity. Inhibitory capacity of candidate capture agent binder is defined as percentage decrease of native human DPP3 activity upon incubation with specified capture agent binder.

In the liquid-phase assay, body fluid samples are directly exposed to fluorogenic substrates (e.g., Arg-Arg-β-NA). Since there are many different aminopeptidases in plasma (Sanderink et al. 1988), it is possible that the substrate used is cleaved by peptidases other than DPP3. To circumvent this problem, one of the preferred methods for detecting specific DPP3 activity is the use of an enzyme capture activity assay.

In one specific embodiment, determining active DPP3 in an enzyme capture assay comprises the following steps:

Contacting said sample with a capture binding agent that binds to full-length DPP3 but preferably inhibits DPP3 activity in a liquid phase assay by less than 50%, preferably less than 40%, more preferably 30%. To prevent inhibition of DPP3, the capture binding agent should not bind to DPP3 in the region around the active site and the substrate binding region (amino acids 316-669 of SEQ ID No. 1),

separating DPP3 bound to said capture binding agent from a biological fluid sample,

adding a DPP3 substrate to said separated DPP3,

quantitative determination of DPP3 activity by measuring the conversion of DPP3 substrate.

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

The heavy chains of full-length immunoglobulins are typically about 50 kDa or 446 amino acids long. The light chains are encoded by a variable region gene at the NH2 terminus (about 110 amino acids long) and a kappa or lambda constant region gene at the COOH terminus. Similarly, the heavy chains are encoded by a variable region gene (about 116 amino acids long) and one of the other constant region genes.

The basic structural unit of an antibody is usually a tetramer consisting of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, 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 chain antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17:105, 1987; Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85:5879-5883, 1988; Bird et al., Science 242:423-426, 1988; Hood et al., Immunology, Benjamin, N.Y., 2nd ed., 1984; Hunkapiller and Hood, Nature 323:15-16, 1986).

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

"Chimeric antibodies" are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes from different species. For example, the variable segments of a mouse monoclonal antibody gene may be fused to human constant segments such as kappa and gamma 1 or gamma 3. Thus, in one example, a therapeutic chimeric antibody is a fusion protein consisting of a variable or antigen-binding domain of a mouse antibody and a constant or effector domain of a human antibody, although other mammalian species may be used, or a variable region obtained by molecular techniques may be used. Methods for producing chimeric antibodies are well known in the art, see, for example, U.S. Patent No. 5916588. A "humanized" immunoglobulin is an immunoglobulin that includes 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."

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

A "humanized antibody" according to the invention is an antibody comprising an immunoglobulin with a humanized light chain and an immunoglobulin with a humanized heavy chain. The humanized antibody binds to the same antigen as the donor antibody that provides the CDR. The acceptor framework of the humanized immunoglobulin or antibody may have a limited number of amino acid substitutions taken from the donor framework. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that do not substantially affect antigen binding or other functions of the immunoglobulin. Examples of conservative substitutions are gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed using genetic engineering (e.g., see U.S. Patent No. 6,159,477). A human antibody is one in which the 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 a human B cell secreting the antibody of interest. Immortalization can be accomplished, for example, by infection with EBV or by fusing a human B cell with a myeloma or hybridoma cell to produce a trioma cell. Human antibodies can also be produced by phage display techniques (see, e.g., Dower et al., PCT Publication No. WO 91/17271; McCafferty et al., PCT Publication No. WO 92/001047; and Winter, PCT Publication No. WO 92/20791), or selected from a library of combinatorial human monoclonal antibodies (see the Morphosys website). Human antibodies can also be produced using transgenic animals carrying a human immunoglobulin gene (see, e.g., Lonberg et al., PCT Publication No. WO 93/12227; and Kucherlapati, PCT Publication No. WO 91/10741).

Thus, the anti-DPP3 antibody or anti-DPP3 antibody fragment according to the invention may have formats known in the art. Examples include, but are not limited to, human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies or antibody fragments thereof.

In a specific embodiment, the anti-DPP3 antibody is a monoclonal antibody or a fragment thereof. In one embodiment, the anti-DPP3 antibody or anti-DPP3 antibody fragment is a human or humanized antibody or a derivative thereof. In one specific embodiment, one or more (murine) CDRs are grafted onto a human antibody or antibody fragment.

In a preferred embodiment, the antibodies of the present invention are recombinantly produced antibodies, such as IgG, a typical full-length immunoglobulin, or antibody fragments comprising at least the F variable domain of the heavy and/or light chain, such as chemically linked antibodies (antigen-binding fragments), including, but not limited to, Fab fragments, including Fab minibodies, single-chain Fab antibodies, monovalent Fab antibodies with epitope tags, such as Fab-V5Sx2; a bivalent Fab (mini-antibody) dimerized with a CH3 domain; a bivalent Fab or a multivalent Fab, such as one formed by multimerization with a heterologous domain, i.e. by dimerization of the dHLX domains, i.e. Fab-dHLX-FSx2; F(ab’)2 fragments, scFv fragments, multimerized polyvalent and/or polyspecific scFv fragments, bivalent and/or bispecific diabodies, BITE® (bispecific T cell recruiter), trifunctional antibodies, polyvalent antibodies, e.g. of a class other than G; single-domain antibodies, i.e. nanoantibodies derived from camelid or fish immunoglobulins, and many others.

In addition to anti-DPP3 antibodies or anti-DPP3 antibody fragments, other biopolymer scaffolds, so-called non-Ig scaffolds, are well known in the art for forming complexes with a target molecule and have been used to prepare biopolymers with high target specificity. Examples include aptamers, spiegelmers, anticalins and conotoxins.

Non-Ig scaffolds in the context of the invention may be protein scaffolds and may be used as antibody mimics since they are capable of binding to ligands or antigens. The non-Ig scaffolds may be selected from the group consisting of tetranectin-based non-Ig scaffolds (e.g. as described in US 2010/0028995), fibronectin scaffolds (e.g. as described in EP 1 266 025); lipocalin-based scaffolds (e.g. as described in WO 2011/154420); ubiquitin scaffolds (e.g. as described in WO 2011/073214), transfer scaffolds (e.g. as described in US 2004/0023334), protein A scaffolds (e.g. as described in EP 2 231 860), ankyrin repeat-based scaffolds (e.g. as described in WO 2010/060748), microprotein scaffolds (preferably cystine knot-forming microproteins) (e.g. as described in in EP 2314308), Fyn SH3 domain-based scaffolds (e.g., described in WO 2011/023685), EGFR-A domain-based scaffolds (e.g., described in WO 2005/040229), and Kunitz domain-based scaffolds (e.g., described in EP 1941867). Non-Ig scaffolds can be peptide or oligonucleotide aptamers. Aptamers are typically designed by selecting them from a large pool of random sequences and are short chains 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).

In an alternative embodiment, the anti-DPP3 antibody format is selected from the group consisting of an Fv fragment, an scFv fragment, a Fab fragment, an scFab fragment, an F(ab)2 fragment, and an scFv-Fc fusion protein. In another preferred embodiment, the antibody format is selected from the group consisting of an scFab fragment, a Fab fragment, an scFv fragment, and bioavailability optimized conjugates thereof, such as pegylated fragments.

In the context of the invention, the term "antibody" generally includes monoclonal and polyclonal antibodies and their binding fragments, in particular Fc fragments, as well as so-called "single chain antibodies" (Bird et al. 1988), chimeric, humanized, in particular CDR-grafted antibodies and di- or tetraantibodies (Holliger et al. 1993). Also included are immunoglobulin-like proteins that are selected by methods including, for example, phage display for specific binding to a molecule of interest contained in a sample. In this context, the term "specific binding" refers to antibodies produced against a molecule of interest or a fragment thereof. An antibody is considered specific if its affinity for the molecule of interest or the above-mentioned fragment thereof is at least preferably 50 times higher, more preferably 100 times higher, most preferably at least 1000 times higher than for other molecules included in a sample containing the molecule of interest. It is well known in the art how to produce antibodies and select antibodies with a given specificity.

In a specific embodiment of the invention, said anti-DPP3 antibody or anti-DPP3 antibody fragment that binds to an epitope of the sequence SEQ ID NO.: 2, wherein said epitope is contained in the DPP3 protein or a functional derivative thereof, is a monoclonal antibody or a fragment of this monoclonal antibody. In one embodiment of the invention, said anti-DPP3 antibody or anti-DPP3 antibody fragment that binds to an epitope of the sequence SEQ ID NO.: 2, wherein said epitope is contained in the DPP3 protein or a functional derivative thereof, is a human or humanized antibody or a derivative thereof, or a fragment of a humanized antibody or a derivative thereof.

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

In another aspect of the invention, the subject of the invention is a human CDR-grafted anti-DPP3 antibody or a fragment of this anti-DPP3 antibody, which is directed to and binds to an epitope of the sequence SEQ ID NO.: 2, wherein said epitope is contained in the DPP3 protein or a functional derivative thereof, and wherein said human CDR-grafted anti-DPP3 antibody or a fragment of this anti-DPP3 antibody comprises an antibody heavy chain variable region (H chain) comprising:

SEQ ID NO.: 4

and/or further comprises a light chain variable region of an antibody (L chain) comprising:

SEQ ID NO.: 5.

Another object of the present invention in another aspect is a human CDR-grafted anti-DPP3 antibody or a fragment of this anti-DPP3 antibody, which is directed to and binds to an epitope of the sequence SEQ ID NO.: 2, wherein said epitope is contained in the DPP3 protein or a functional derivative thereof, and wherein said human CDR-grafted anti-DPP3 antibody or a fragment of this anti-DPP3 antibody comprises an antibody heavy chain variable region (H chain) comprising:

SEQ ID NO.: 11

and/or further comprises a light chain variable region of an antibody (L chain) comprising:

SEQ ID NO.: 12.

In one specific embodiment of the invention, the subject of the present invention is a human monoclonal antibody against DPP3 or a fragment of this monoclonal antibody against DPP3, which is directed to and binds to an epitope of the sequence SEQ ID NO.: 2, wherein said epitope is contained in the DPP3 protein or a functional derivative thereof, and wherein the heavy chain comprises at least one CDR of:

SEQ ID NO.: 6, SEQ ID NO.: 7 or SEQ ID NO.: 8

and wherein the light chain comprises at least one CDR of:

SEQ ID NO.: 9, KVS or SEQ ID NO.: 10.

The amount of DPP3 protein and/or DPP3 activity in a biological fluid sample of a given patient can be determined by various methods, such as immunoassays, activity assays, mass spectrometric methods, etc.

The amount of DPP3 protein and/or DPP3 activity in a biological fluid sample of a specified patient may be determined, for example, by one of the following methods:

1. Luminescent immunoassay for quantitative determination of DPP3 protein concentration (LIA) (Rehfeld et al. 2019 JALM3(6):943-953).

The LIA is a one-step chemiluminescent sandwich immunoassay that uses white high-binding polystyrene microplates as the solid phase. The plates are coated with the anti-DPP3 monoclonal antibody AK2555 (capture antibody). The anti-DPP3 indicator antibody, AK2553, is labeled with MA70-acridinium-NHS ester and used at a concentration of 20 ng per well. Twenty microliters of samples (e.g., serum, heparinized plasma, citrated plasma, or EDTA plasma from patient blood) and calibrators are pipetted onto the coated white microplates. After addition of the indicator antibody AK2553, the microplates are incubated for 3 h at room temperature and 600 rpm. Unbound indicator is then removed by 4 washing steps (350 µl/well). The remaining chemiluminescence is measured for 1 s/well using a microplate luminometer. The DPP3 concentration is determined using a 6-point calibration curve. Calibrators and samples should preferably be run in duplicate.

2. Enzyme capture assay for quantitative determination of DPP3 activity (ECA) (Rehfeld et al. 2019 JALM3(6):943-953).

ECA is a DPP3-specific activity assay that uses high-binding black polystyrene microplates as a solid phase. The plates are coated with the anti-DPP3 monoclonal antibody AK2555 (capture antibody). Twenty microliters of samples (e.g. serum, heparin plasma, citrate plasma, EDTA plasma, cerebrospinal fluid, and urine) and calibrators are pipetted into the coated black microplates. After addition of assay buffer (200 µl), the microplates are incubated for 2 h at 22°C and 600 rpm. DPP3 present in the samples is immobilized by binding to the capture antibody. Unbound sample components are removed by 4 washing steps (350 µl/well). The specific activity of immobilized DPP3 is measured by adding the fluorogenic substrate Arg-Arg-β-naphthylamide (Arg2-βNA) to the reaction buffer followed by incubation at 37°C for 1 hour. DPP3 specifically cleaves Arg2-βNA into the dipeptide Arg-Arg and the fluorescent β-naphthylamine. Fluorescence is measured with a fluorometer using an excitation wavelength of 340 nm and emission is recorded at 410 nm. DPP3 activity is determined using a 6-point calibration curve. Calibrators and samples are preferably run in duplicate.

3. Liquid-phase assay for quantitative determination of DPP3 activity (LAA) (modified from Jones et al., Analytical Biochemistry, 1982).

The LAA is a liquid-phase assay that uses black non-binding polystyrene microplates to measure DPP3 activity. Twenty microliters of samples (e.g., serum, heparin plasma, citrate plasma) and calibrators are pipetted into black microplates. After addition of the fluorogenic substrate Arg2-βNA to the assay buffer (200 μl), the initial fluorescence of βNA (T=0) is measured in a fluorimeter using an excitation wavelength of 340 nm and the emission is detected 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. DPP3 activity is determined using a 6-point calibration curve. Calibrators and samples are preferably run in duplicate.

Various immunoassays are known that can be used for the assays and methods of the present invention, including: radioimmunoassay ("RIA"), homogeneous enzyme-linked immunosorbent assay ("EMIT"), enzyme-linked immunosorbent assay ("ELISA"), apoenzyme reactivation immunoassay ("ARIS"), chemiluminescent and fluorescent immunoassays, Luminex-based microsphere arrays, protein microarray assays, and rapid test formats such as, for example, immunochromatographic strip tests ("strip immunoassays") and immunochromatographic assays.

In one embodiment of the invention, such an assay is a sandwich immunoassay using any detection technology, including but not limited to an enzyme label, a chemiluminescent label, an electrochemiluminescent label, preferably a fully automated assay. In one embodiment of the invention, such an assay is a sandwich assay with a labeled enzyme. Examples of an automated or fully automated assay include assays that can be used for one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®.

In one embodiment of the invention, this may be a so-called POC (point-of-care) test, i.e. a testing technology that allows the test to be performed in less than 1 hour near the patient without the need for a fully automated analysis system. One example of this technology is immunochromatographic testing technology.

In a specific embodiment of the invention, said ROS test combines the measurement of more than one analyte simultaneously. In another specific embodiment of the invention, said analyte is selected from the group consisting of DPP3 and proadrenomedullin or fragments thereof. In a very specific embodiment of the invention, said POC test combines the measurement of DPP3 and mature ADM.

In one embodiment of the invention, at least one of the binding agents is labeled so that it can be detected.

In a preferred embodiment, said label is selected from the group consisting of a chemiluminescent label, an enzyme label, a fluorescent label, and a radioactive iodine label.

The assays may be homogeneous or heterogeneous, competitive or non-competitive. In one embodiment, the assay is a sandwich assay, which is a non-competitive immunoassay in which the molecule to be detected and/or quantified is bound to a first antibody and a second antibody. The first antibody may be bound to a solid phase, such as a microsphere, a well surface or other container, a chip or a test strip, and the second antibody is an antibody that is labeled, such as with a dye, a radioisotope, or a reactive or catalytically active fragment. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general design and procedures associated with 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. 1999 Feb;17(2):176-80).

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

In another embodiment, said labeling system comprises rare earth cryptates or rare earth chelates in combination with a fluorescent dye or a chemiluminescent dye, in particular a cyanine-type dye.

In the context of the present invention, fluorescence-based assays include the use of dyes that can be, for example, selected from the group consisting of 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 (HEX), TET, 6-carboxy-4',5'-dichloro-2',7'-dimethodylfluorescein (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™R, Oregon Green coumarins such as umbelliferone, benzimides such as Hoechst 33258; Phenantridines such as Texas Red, Yakima Yellow, Alexa Fluor, PET, ethidium bromide, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, etc.

In the context of the present invention, chemiluminescence-based assays include 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, incorporated herein by reference, including citations on pages 551-562). Preferred chemiluminescent dyes are acridinium esters.

As mentioned in this document, "assay" or "diagnostic assay" may refer to any type used in the field of diagnostics. Such an assay may be based on the binding of a detectable analyte to one or more capture probes with a certain affinity. With respect to the interaction between the capture molecules and the target molecules or molecules of interest, the affinity constant preferably exceeds 10 ⁸ M ^-1 .

DPP3 activity can be measured by detecting the cleavage products of DPP3-specific substrates.

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

Detection of fluorescence by hydrolysis of fluorogenic substrates by DPP3 is a standard procedure for monitoring DPP3 activity. These substrates are specific di- or tripeptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) linked to a fluorophore. Fluorophores include, but are not limited to, β-naphthylamide (2-naphthylamide, βNA, 2NA), 4-methoxy-β-naphthylamide-(4-methoxy-2-naphthylamide), and 7-amido-4-methylcoumarin (AMC, MCA; Abramić et al. 2000, Ohkubo et al. 1999). Cleavage of these fluorogenic substrates results in the release of fluorescent β-naphthylamine or 7-amino-4-methylcoumarin, respectively. In a liquid-phase assay, the ECA substrate and DPP3 are incubated, for example, in a 96-well plate format, and the fluorescence is measured using a fluorescence detector (Ellis & Nuenke, 1967). In addition, DPP3-bearing samples can be immobilized and separated on gels by electrophoresis, the gels are stained with a fluorogenic substrate (e.g., Arg-Arg-βNA) and Fast Garnet GBC, and the fluorescent protein bands are detected with a fluorescent plate 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 linked to chromophores such as p-nitroanilide diacetate. Detection of color change due to hydrolysis of chromogenic substrates can be used to monitor DPP3 activity.

Another embodiment for detecting DPP3 activity is the Protease-Glo™ assay (commercially available from Promega). In this embodiment of the method, DPP3-specific di- or tripeptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) are coupled to aminoluciferin. Cleavage of DPP3 releases aminoluciferin, which serves as a substrate for a coupled luciferase reaction that emits detectable luminescence.

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

In a specific embodiment of said method for detecting active DPP3 in a patient's biological fluid sample, said binding agent reactive with respect to DPP3 is immobilized on a solid support.

The sample to be tested is passed through the immobilized binding agent and DPP3, if present, binds to the binding agent and is itself immobilized for detection. A substrate can then be added and the reaction product can be detected to indicate the presence or amount of DPP3 in the sample to be tested. For the purposes of this disclosure, the term "solid phase" may be used to refer to any material or vessel in or on which the assay can be performed and includes, but is not limited to: porous materials, non-porous materials, test tubes, wells, glass slides, agarose resins (e.g., Sepharose from GE Healthcare Life Sciences), magnetic particles (e.g., Dynabeads™ or Pierce™ magnetic microspheres from Thermo Fisher Scientific), etc.

Binding agents of protein or peptide origin (e.g. antibody, antibody fragments, non-Ig scaffold) are immobilized on the solid phase by methods including: physical adsorption (e.g. via electrostatic interaction or hydrophobic interaction), bioaffinity immobilization (e.g. avidin-biotin, protein A/G/L, His-tag and Ni2+-NTA, GST-tag and glutathione, DNA hybridization, aptamers), covalent linkage (e.g. amine and N-hydroxysuccinimide) or a combination of these immobilization methods (Kim & Herr 2013). Binding agents of oligonucleotide origin (e.g. aptamers) can be immobilized on the solid phase using the (strept)avidin-biotin system (Müller et al. 2012, Deng et al. 2014).

In a particular embodiment of said method for determining DPP3 activity in a patient's biological fluid sample, said separation step is a washing step in which components of the sample not bound to said capture binding agent are removed from the captured DPP3. This separation step may be any other step that separates DPP3 bound to said capture binding agent from components of said biological fluid sample.

In a specific embodiment of said method for determining DPP3 activity in a patient's biological fluid sample, said conversion of DPP3 substrate by immobilized DPP3 is measured (detected) by a method selected from the group consisting of: fluorescence of fluorogenic substrates (e.g., Arg-Arg-βNA, Arg-Arg-AMC), color change of chromogenic substrates, luminescence of substrates bound to aminoluciferin (Promega Protease-Glo™ assay), mass spectrometry, HPLC/FLC (reversed phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis followed by activity staining (immobilized, active DPP3), or Western blotting (cleavage products).

In a specific embodiment of said method for determining DPP3 activity in a patient's biological fluid sample, said substrate may be selected from the group consisting of: Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or di- and tripeptides linked to a fluorophore, chromophore or aminoluciferin (Promega Protease-Glo™ assay). Di- 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, 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 results in 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 in chromogenic substrates results in the release of pNA, which in turn changes color. Thus, the change in absorbance (DA/min) is directly proportional to the enzymatic activity. Using the Promega Protease-Glo™ assay, cleavage of DPP3 releases aminoluciferin, which serves as a substrate for a coupled luciferase reaction that emits detectable luminescence.

The present invention also provides a method for predicting the outcome and/or risk of an adverse event in a patient who has developed refractory shock, said method comprising the steps of:

determination of the level of DPP3 in a sample of biological fluid of the specified patient;

comparison of the specified level of a given DPP3 with a given threshold value,

correlation of a specified DPP3 level with a specified risk of an adverse event in a specified patient, where an elevated level above a specified threshold is predictive of an increased risk of the specified adverse events or,

correlation of a specified DPP3 level with the success of therapy or intervention in a specified patient, where a level below a specified threshold is predictive of the success of therapy or intervention.

In the context of the present invention, the term "prognosis" means predicting how the patient's health condition will progress. This may include an assessment of the likelihood of recovery or the likelihood of an adverse event for said patient. An adverse event is defined as organ dysfunction or mortality. Organ dysfunction is defined as renal dysfunction, cardiac dysfunction or liver dysfunction.

Other embodiments of the present invention are:

1. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, said method comprising the steps of:

determination of the level of DPP3 in a sample of biological fluid of the specified patient;

comparison of the specified level of a given DPP3 with a given threshold value,

where the specified patient is predicted to enter into refractory shock, or is diagnosed with refractory shock, if the specified defined DPP3 level exceeds the Specified Predetermined Threshold.

2. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to embodiment 1, wherein said shock is selected from the group consisting of hypovolemia-induced shock, cardiogenic shock, obstructive shock and distributive shock, in particular cardiogenic shock or septic shock.

3. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to embodiments 1 and 2, wherein

in case of cardiogenic shock, the said patient may have suffered from an acute coronary syndrome (e.g., acute myocardial infarction) or the said patient has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordal rupture, or massive pulmonary embolism, or

in the case of hypovolemic shock, the patient may have suffered a hemorrhagic disorder including gastrointestinal bleeding, trauma, vascular etiologies (e.g. ruptured abdominal aortic aneurysm, tumor invading a major blood vessel), and spontaneous bleeding in the setting of anticoagulation, or a non-hemorrhagic disorder including vomiting, diarrhea, renal failure, cutaneous loss/intangible loss (e.g. burns, heat stroke), or third space loss in the setting of pancreatitis, liver cirrhosis, ileus, trauma, or

in case of obstructive shock, the said patient may have suffered cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or

In case of distributive shock, the said patient may have septic shock, neurogenic shock, anaphylactic shock, or shock due to adrenal crisis.

4. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to embodiments 1-3, wherein said method is used to initiate and/or stop and/or stratify treatment and/or guide treatment.

5. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to any of embodiments 1-4, wherein treatment is started and/or continued and/or delayed and/or stopped if a specified level of DPP3 is above a specified predetermined threshold.

6. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to embodiment 5, wherein said treatment is selected from the group of vasopressors, angiotensin receptor agonists and/or their precursors, DPP3 activity inhibitors, and anti-adrenomedullin antibodies or anti-adrenomedullin antibody fragments.

7. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to any one of embodiments 1-6, wherein either the DPP3 protein level and/or the active DPP3 level is determined and compared to a predetermined threshold value.

8. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to any one of embodiments 1-7, wherein the DPP3 level is determined by contacting said biological fluid sample with a binding capture agent that specifically binds to DPP3.

9. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to embodiment 8, wherein said DPP3 level detecting capture binding agent may be selected from the group consisting of an antibody, an antibody fragment, or a non-IgG scaffold.

10. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to embodiments 8-9, wherein said capture binding agent is an antibody.

11. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to any one of embodiments 8-10, wherein said capture binding agent is a monoclonal antibody.

12. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to any one of embodiments 1-11, wherein said biological 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 patient who is either going into shock or has developed shock, according to any one of embodiments 1-12, wherein said method of diagnosis or prediction is performed at least twice.

14. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to any of embodiments 1-13, wherein treatment with angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors is initiated and/or continued when the DPP3 level in said sample is above a certain threshold value, and/or wherein treatment with vasopressors is delayed and/or stopped if said certain DPP3 level is above said predetermined threshold value.

15. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to any of embodiments 1-13, wherein treatment with vasopressors is started and/or continued when the DPP3 level in said sample is below a certain threshold value, and/or where treatment with angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors is delayed and/or stopped if said determined DPP3 level is below said predetermined threshold value.

16. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to embodiment 8, wherein the level of pro-adrenomedullin or fragments thereof is further determined, and wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment is started and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment is postponed and/or stopped if said determined level of pro-adrenomedullin or fragments thereof is below said predetermined threshold value.

17. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to embodiment 14, wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment and/or angiotensin receptor agonists and/or its precursors and/or DPP3 activity inhibitors is initiated and/or continued if the level of proadrenomedullin or its fragments in said sample is above a certain threshold value, and said certain level of DPP3 is above said specified threshold value of DPP3.

18. A vasopressor for use in the treatment of shock in a patient who is either entering shock or has developed shock, wherein said patient has a DPP3 level in a biological fluid sample of said patient below a predetermined threshold when determined by a method according to any one of embodiments 1-17.

19. A vasopressor for use in the treatment of shock in a patient who is either entering shock or has developed shock, according to embodiment 18, wherein said vasopressor is selected from the group consisting of dopamine, norepinephrine, norepinephrine equivalent, epinephrine, phenylephrine and vasopressin.

20. A vasopressor for use in the treatment of shock in a patient who is either entering shock or has developed shock, according to embodiment 18 or 19, wherein said vasopressor is administered to said patient in a pharmaceutical formulation.

21. A vasopressor for use in the treatment of shock in a patient who is either entering shock or has developed shock, according to any one of embodiments 18-20, wherein said patient has a blood pressure equal to or less than 65 mmHg.

22. An inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering shock or has developed shock, wherein said patient has a level of DPP3 in a biological fluid sample of said patient above a predetermined threshold, as determined by the method according to any one of embodiments 1-17.

23. An inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering shock or has developed shock, according to embodiment 22, wherein the inhibitor of DPP3 activity is selected from the group consisting of an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

24. An inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering shock or has developed shock, according to embodiments 22 and 23, wherein said inhibitor has a minimum binding affinity for DPP3 equal to or less than 10 ^-7 M.

25. An inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering shock or has developed shock, according to any of embodiments 22-24, wherein the inhibitor of DPP3 activity is an antibody.

26. An inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering shock or has developed shock, according to any of embodiments 22-25, wherein said inhibitor is a monoclonal antibody.

27. An inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering shock or has developed shock, according to any of embodiments 22-26, wherein said inhibitor is a monoclonal antibody, wherein the complementarity determining regions (CDRs) in the heavy chain comprise the sequences: SEQ ID NO.: 7, SEQ ID NO.: 8 and/or SEQ ID NO.: 9, and the complementarity determining regions (CDRs) in the light chain comprise the sequences: SEQ ID NO.: 10, KVS and/or SEQ ID NO.: 11.

28. An inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering shock or has developed shock, according to any of embodiments 22-27, wherein said inhibitor is a humanized monoclonal antibody or a fragment of a humanized monoclonal antibody, wherein the heavy chain comprises the sequence: SEQ ID NO.: 12, and the light chain comprises the sequence: SEQ ID NO.: 13.

29. An inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering shock or has developed shock, according to any of embodiments 22-28, wherein said inhibitor is administered in combination with an angiotensin receptor agonist and/or a precursor thereof.

30. A DPP3 activity inhibitor for use in the treatment of shock in a patient who is either entering shock or has developed shock, according to embodiment 29, wherein said angiotensin receptor agonist and/or its precursor is selected from the group consisting of angiotensin I, angiotensin II, angiotensin III, angiotensin IV, in particular angiotensin II.

31. Angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors for use in the treatment of shock in a patient who is either going into shock or has developed shock, wherein treatment with said angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors is initiated and/or continued when the DPP3 level in said patient's sample is above a specified threshold, and/or treatment with vasopressors is delayed and/or discontinued if a specified DPP3 level is above a specified threshold.

32. A vasopressor for use in the treatment of shock in a patient who is either entering shock or has developed shock, wherein treatment with said vasopressor is initiated and/or continued when the level of DPP3 in a biological fluid sample of said patient is below a specified threshold, and/or wherein treatment with angiotensin receptor agonists and/or their precursors and/or inhibitors of DPP3 activity is delayed and/or stopped if said specified level of DPP3 is below said specified threshold.

33. A vasopressor for use in the treatment of shock in a patient who is either entering shock or has developed shock, according to embodiment 32, wherein a level of pro-adrenomedullin or fragments thereof is further determined, and wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment is started and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment is delayed and/or stopped if said determined level of pro-adrenomedullin or fragments thereof is below said predetermined threshold value.

34. Angiotensin receptor agonists and/or precursors thereof and/or DPP3 activity inhibitors for use in the treatment of shock in a patient who is either entering shock or has developed shock according to embodiment 31, and wherein a level of pro-adrenomedullin or fragments thereof is further determined and wherein treatment with an anti-ADM antibody or anti-ADM antibody fragment is started and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with an anti-ADM antibody or anti-ADM antibody fragment is delayed and/or stopped if said determined level of pro-adrenomedullin or fragments thereof is below said predetermined threshold value.

35. An anti-ADM antibody or anti-ADM antibody fragment for use in the treatment of shock in a patient who is either entering shock or has developed shock according to embodiments 33-34, wherein treatment with said anti-ADM antibody or anti-ADM antibody and/or angiotensin receptor agonists and/or precursors thereof and/or DPP3 activity inhibitors is initiated and/or continued if the level of proadrenomedullin or fragments thereof in said sample is above a certain threshold and said certain level of DPP3 is above said predetermined DPP3 threshold.

36. A method for treating shock in a patient who is either entering shock or has developed shock, said method comprising administering a vasopressor to said patient, wherein said patient has a DPP3 level in a biological fluid sample of said patient below a predetermined threshold when determined by the method according to any one of embodiments 1-17.

37. A method for treating shock in a patient who is either entering shock or has developed shock, according to embodiment 35, comprising administering a vasopressor to said patient,

wherein said vasopressor is selected from the group consisting of dopamine, norepinephrine, norepinephrine equivalent, epinephrine, phenylephrine and vasopressin.

38. A method for treating shock in a patient who is either entering shock or has developed shock, according to embodiments 35-36, comprising administering a vasopressor to said patient, wherein said vasopressor is administered to said patient in a pharmaceutical formulation.

39. A method for treating shock in a patient who is either entering shock or has developed shock, according to embodiments 35-37, comprising administering to said patient a vasopressor,

where said patient has a blood pressure equal to or less than 65 mmHg.

40. A method for treating shock in a patient who is either entering a state of shock or has developed shock, wherein the method comprises administering an inhibitor of DPP3 activity to said patient, wherein said patient has a level of DPP3 in a sample of a biological fluid of said patient that is above a predetermined threshold value when determined by the method according to any of embodiments 1-17.

41. A method for treating shock in a patient who is either entering a state of shock or has developed shock, according to embodiment 39, comprising administering an inhibitor of DPP3 activity to said patient, wherein the inhibitor of DPP3 activity is selected from the group consisting of 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 patient who is either entering shock or has developed shock, according to embodiments 39-40, comprising administering an inhibitor of DPP3 activity to said patient, wherein said inhibitor has a minimum binding affinity for DPP3 equal to or less than 10 ^-7 M.

43. A method for treating shock in a patient who is either entering shock or has developed shock, according to embodiments 39-41, comprising administering an inhibitor of DPP3 activity to said patient, wherein the inhibitor of DPP3 activity is an antibody.

44. A method for treating shock in a patient who is either entering shock or has developed shock, according to embodiments 39-42, comprising administering an inhibitor of DPP3 activity to said patient, wherein said inhibitor is a monoclonal antibody.

45. A method for treating shock in a patient who is either entering shock or has developed shock, according to embodiments 39-43, comprising administering an inhibitor of DPP3 activity to said patient, wherein said inhibitor is a monoclonal antibody, wherein the complementarity determining regions (CDRs) in the heavy chain comprise the sequences: SEQ ID NO.: 7, SEQ ID NO.: 8 and/or SEQ ID NO.: 9, and the complementarity determining regions (CDRs) in the light chain comprise the sequences: SEQ ID NO.: 10, KVS and/or SEQ ID NO.: 11.

46. A method for treating shock in a patient who is either entering shock or has developed shock, according to embodiments 39-44, comprising administering an inhibitor of DPP3 activity to said patient, wherein said inhibitor is a humanized monoclonal antibody or a fragment of a humanized monoclonal antibody, wherein the heavy chain comprises the sequence: SEQ ID NO.: 12, and the light chain comprises the sequence: SEQ ID NO.: 13.

47. A method for treating shock in a patient who is either entering shock or has developed shock, according to embodiments 39-45, comprising administering an inhibitor of DPP3 activity to said patient, wherein said inhibitor is administered in combination with an angiotensin receptor agonist and/or its precursors.

48. A method for treating shock in a patient who is either entering a state of shock or has developed shock, according to embodiments 39-46, comprising administering an inhibitor of DPP3 activity to said patient, wherein said angiotensin receptor agonist and/or its precursors are selected from the group consisting of angiotensin I, angiotensin II, angiotensin III, angiotensin IV, in particular angiotensin II.

49. A method for treating shock in a patient who is either entering shock or has developed shock, according to embodiments 39-47, wherein the method comprises administering to said patient angiotensin receptor agonists and/or precursors thereof and/or a DPP3 activity inhibitor, wherein treatment with said angiotensin receptor agonists and/or DPP3 activity inhibitors is initiated and/or continued when the level of DPP3 in a sample of said patient is above a certain threshold value, and/or wherein treatment with vasopressors is delayed and/or stopped if said certain level of DPP3 is above said predetermined threshold value.

50. A method for treating shock in a patient who is either entering shock or has developed shock, according to embodiments 35-38, comprising administering a vasopressor to said patient, wherein treatment with said vasopressors is initiated and/or continued when the level of DPP3 in a biological fluid sample of said patient is below a certain threshold value and/or wherein treatment with angiotensin receptor agonists and/or their precursors and/or inhibitors of DPP3 activity is delayed and/or stopped if said certain level of DPP3 is below said predetermined threshold value.

51. A method for treating shock in a patient who is either entering a state of shock or has developed shock, according to embodiment 49, comprising administering angiotensin receptor agonists and/or precursors thereof and/or a DPP3 activity inhibitor to said patient, and wherein a level of pro-adrenomedullin or fragments thereof is further determined, and wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment is started and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with the anti-ADM antibody or anti-ADM antibody fragment is postponed and/or stopped if said determined level of pro-adrenomedullin or fragments thereof is below said predetermined threshold value.

52. A method for treating shock in a patient who is either entering a state of shock or has developed shock, according to embodiment 50, comprising administering a vasopressor to said patient, wherein a level of proadrenomedullin or fragments thereof is further determined, and wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment is started and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with the anti-ADM antibody or anti-ADM antibody fragment is delayed and/or stopped if said determined level of pro-adrenomedullin or fragments thereof is below said predetermined threshold value.

53. A method for treating shock in a patient who is either entering a state of shock or has developed shock, according to embodiments 51 and 52, comprising administering an anti-ADM antibody or an anti-ADM antibody fragment to said patient, wherein a level of pro-adrenomedullin or fragments thereof is further determined, and wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment is started and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with the anti-ADM antibody or anti-ADM antibody fragment is delayed and/or stopped if said determined level of pro-adrenomedullin or fragments thereof is below said predetermined threshold value.

54. A method for treating shock in a patient who is either entering a state of shock or has developed shock, according to embodiments 51-53, wherein the method comprises administering an anti-ADM antibody or an anti-ADM antibody fragment to said patient, wherein treatment with said anti-ADM antibody or an anti-ADM antibody fragment and/or angiotensin receptor agonists and/or precursors thereof and/or DPP3 activity inhibitors is initiated and/or continued if the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and said certain level of DPP3 is above said predetermined DPP3 threshold value.

In the above context, the following sequentially numbered embodiments provide further specific aspects of the invention:

1. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, said method comprising the steps of:

determination of the level of DPP3 in a sample of biological fluid of the specified patient;

comparing a specified level of a certain DPP3 with a predetermined threshold value, wherein the specified patient is predicted to enter into a state of refractory shock, or is diagnosed with refractory shock, if the specified level of DPP3 exceeds the specified predetermined threshold value.

2. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock according to embodiment 1, wherein said shock is selected from the group consisting of hypovolemia-induced shock, cardiogenic shock, obstructive shock and distributive shock, in particular cardiogenic shock or septic shock.

3. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to embodiments 1 and 2, wherein

in case of cardiogenic shock, the said patient may have suffered from an acute coronary syndrome (e.g., acute myocardial infarction) or the said patient has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordal rupture, or massive pulmonary embolism, or

in the case of hypovolemic shock, the patient may have suffered a hemorrhagic disorder including gastrointestinal bleeding, trauma, vascular etiologies (e.g. ruptured abdominal aortic aneurysm, tumor invading a major blood vessel), and spontaneous bleeding in the setting of anticoagulation, or a non-hemorrhagic disorder including vomiting, diarrhea, renal failure, cutaneous loss/intangible loss (e.g. burns, heat stroke), or third space loss in the setting of pancreatitis, liver cirrhosis, ileus, trauma, or

in case of obstructive shock, the said patient may have suffered cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or

In case of distributive shock, the said patient may have septic shock, neurogenic shock, anaphylactic shock, or shock due to adrenal crisis.

4. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to embodiments 1-3, wherein said method is used to initiate and/or stop and/or stratify treatment and/or guide treatment.

5. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to any of embodiments 1-4, wherein treatment is started and/or continued and/or delayed and/or stopped if a specified level of DPP3 is above a specified predetermined threshold.

6. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to embodiment 5, wherein said treatment is selected from the group of vasopressors, angiotensin receptor agonists and/or their precursors, DPP3 activity inhibitors, and anti-adrenomedullin antibodies or anti-adrenomedullin antibody fragments.

7. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to any one of embodiments 1-6, wherein either a DPP3 protein level or an active DPP3 level is determined and compared to a predetermined threshold value, wherein the DPP3 level is determined by contacting said biological fluid sample with a binding capture agent that specifically binds to DPP3.

8. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to any of embodiments 1-7, wherein treatment with angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors is initiated and/or continued when the DPP3 level in said sample is above a certain threshold value, and/or wherein treatment with vasopressors is delayed and/or stopped if said certain DPP3 level is above said predetermined threshold value.

9. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to any of embodiments 1-7, wherein treatment with vasopressors is started and/or continued when the DPP3 level in said sample is below a certain threshold value, and/or where treatment with angiotensin receptor agonists and/or their precursors and/or DPP3 activity inhibitors is delayed and/or stopped if said determined DPP3 level is below said predetermined threshold value.

10. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to embodiment 8, wherein the level of pro-adrenomedullin or fragments thereof is further determined, and wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment is started and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment is postponed and/or stopped if said determined level of pro-adrenomedullin or fragments thereof is below said predetermined threshold value.

11. A method for predicting or diagnosing refractory shock in a patient who is either going into shock or has developed shock, according to embodiment 8, wherein treatment with an anti-ADM antibody or an anti-ADM antibody fragment and/or angiotensin receptor agonists and/or its precursors and/or DPP3 activity inhibitors is initiated and/or continued if the level of proadrenomedullin or its fragments in said sample is above a certain threshold value, and said certain level of DPP3 is above said specified threshold value of DPP3.

12. A vasopressor for use in the treatment of shock in a patient who is either entering shock or has developed shock, wherein said patient has a DPP3 level in a biological fluid sample of said patient below a predetermined threshold when determined by the method according to any one of embodiments 1-10.

13. An inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering a state of shock or has developed shock, wherein said patient has a level of DPP3 in a biological fluid sample of said patient above a predetermined threshold when determined by the method according to any one of embodiments 1-10, wherein the inhibitor of DPP3 activity is selected from the group consisting of an anti-DPP3 antibody or an anti-DPP3 antibody fragment, or an anti-DPP3 non-Ig scaffold.

14. An inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering shock or has developed shock, according to embodiment 13, wherein said inhibitor is administered in combination with an angiotensin receptor agonist and/or its precursor.

15. An inhibitor of DPP3 activity for use in the treatment of shock in a patient who is either entering shock or has developed shock, according to embodiment 14, wherein said angiotensin receptor agonist and/or its precursor is selected from the group consisting of angiotensin I, angiotensin II, angiotensin III, angiotensin IV, in particular angiotensin II.

16. A method for treating shock in a patient who is either entering shock or has developed shock, wherein said method comprises administering a vasopressor to said patient, wherein said patient has a DPP3 level in a biological fluid sample of said patient below a predetermined threshold value when determined by the method according to any one of embodiments 1-10.

17. A method for treating shock in a patient who is either entering a state of shock or has developed shock, wherein the method comprises administering an inhibitor of DPP3 activity to said patient, wherein said patient has a level of DPP3 in a sample of a biological fluid of said patient that is above a predetermined threshold value when determined by the method according to any of embodiments 1-10.

18. A method for treating shock in a patient who is either entering a state of shock or has developed shock, according to embodiment 17, wherein the method comprises administering an inhibitor of DPP3 activity to said patient, wherein said inhibitor is administered in combination with an angiotensin receptor agonist and/or its precursor.

19. A method for treating shock in a patient who is either entering a state of shock or has developed shock, according to embodiments 16-17, wherein the method comprises administering to said patient angiotensin receptor agonists and/or precursors thereof, and/or a DPP3 activity inhibitor, wherein treatment with said angiotensin receptor agonists and/or DPP3 activity inhibitors is initiated and/or continued when the DPP3 level in said patient's sample is above a certain threshold value, and/or treatment with vasopressors is delayed and/or stopped if said certain DPP3 level is above said predetermined threshold value.

20. A method for treating shock in a patient who is either entering a state of shock or has developed shock, according to embodiments 16-17, wherein the method comprises administering a vasopressor to said patient, wherein treatment with said vasopressors is initiated and/or continued when the level of DPP3 in a biological fluid sample of said patient is below a certain threshold value and/or wherein treatment with angiotensin receptor agonists and/or their precursors and/or inhibitors of DPP3 activity is delayed and/or stopped if said determined level of DPP3 is below said predetermined threshold value.

21. A method for treating shock in a patient who is either entering a state of shock or has developed shock, according to embodiment 18, wherein the method comprises administering angiotensin receptor agonists and/or precursors thereof and/or a DPP3 activity inhibitor to said patient, and wherein the level of pro-adrenomedullin or fragments thereof is further determined, wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment is started and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with the anti-ADM antibody or anti-ADM antibody fragment is postponed and/or stopped if said determined level of pro-adrenomedullin or fragments thereof is below said predetermined threshold value.

22. A method for treating shock in a patient who is either entering a state of shock or has developed shock, according to embodiment 19, wherein the method comprises administering a vasopressor to said patient, wherein the level of proadrenomedullin or fragments thereof is further determined, and wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment is started and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with the anti-ADM antibody or anti-ADM antibody fragment is postponed and/or stopped if said determined level of pro-adrenomedullin or fragments thereof is below said predetermined threshold value.

23. A method for treating shock in a patient who is either entering a state of shock or has developed shock, according to embodiments 20 and 21, wherein the method comprises administering an anti-ADM antibody or an anti-ADM antibody fragment to said patient, wherein the level of pro-adrenomedullin or fragments thereof is further determined, and wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment is started and/or continued when the level of pro-adrenomedullin or fragments thereof in said sample is above a certain threshold value, and/or wherein treatment with the anti-ADM antibody or anti-ADM antibody fragment is postponed and/or stopped if said determined level of pro-adrenomedullin or fragments thereof is below said predetermined threshold value.

24. A method for predicting the outcome and/or risk of an adverse event in a patient who has developed refractory shock, said method comprising the steps of:

determination of the level of DPP3 in a sample of biological fluid of the specified patient;

comparison of the specified level of a given DPP3 with a given threshold value,

correlation of a specified DPP3 level with a specified risk of an adverse event in a specified patient, where an elevated level above a specified threshold is predictive of an increased risk of the specified adverse events, or

correlation of a specified DPP3 level with the success of therapy or intervention in a specified patient, where a level below a specified threshold is predictive of the success of therapy or intervention.

EXAMPLES

EXAMPLE 1 - METHODS FOR MEASURING DPP 3 PROTEIN AND DPP3 ACTIVITY

Antibody generation and determination of DPP3 binding capacity: Several mouse antibodies were generated and tested for their ability to bind to human DPP3 in a specific binding assay (see Table 1).

Peptides/conjugates for immunization:

DPP3 peptides for immunization, see Table 1, were synthesized (JPT Technologies, Berlin, Germany) with an additional N-terminal cysteine residue (if cysteine is not present in the selected DPP3 sequence) for conjugation of the peptides to bovine serum albumin (BSA). The peptides were covalently linked to BSA using Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the Perbio manual. Recombinant GST-hDPP3 was prepared by USBio (United States Biological, Salem, MA, USA).

Mice immunization, immune cell fusion and screening:

Balb/c mice were injected intraperitoneally (i.p.) with 84 μg GST-hDPP3 or 100 μg DPP3-peptide-BSA conjugates on day 0 (emulsified in TiterMax Gold adjuvant), 84 μg or 100 μg on day 14 (emulsified in complete Freund's adjuvant), and 42 μg or 50 μg on days 21 and 28 (in incomplete Freund's adjuvant). On day 49, the animal was injected intravenously (i.v.) with 42 μg GST-hDPP3 or 50 μg DPP3-peptide-BSA conjugates dissolved in saline. Three days later, mice were sacrificed and immune cell fusion was performed.

Splenocytes from immunized mice and cells of the myeloma cell line SP2/0 were fused with 1 ml of 50% polyethyleneglycol for 30 s at 37°C. After washing, the cells were seeded into 96-well culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 medium supplemented with 20% fetal calf serum and HAT supplement]. After one week, the HAT medium was replaced by HT medium for three passages, followed by a return to normal culture medium.

Cell culture supernatants were first screened for the presence of recombinant DPP3-binding IgG antibodies two weeks after fusion. Therefore, recombinant GST-tagged hDPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng/well) and incubated with 50 μl cell culture supernatant per well for 2 h at room temperature. After washing the plate, 50 μl/well POD rabbit anti-mouse IgG was added and incubated for 1 h at room temperature. After the next washing step, 50 μl of chromogen solution (3.7 mM o-phenylenediamine in citrate-hydrogen phosphate buffer, 0.012% H ₂ O ₂ ) were added to each well, incubated for 15 min at room temperature, and the chromogenic reaction was stopped by adding 50 μl of 4N sulfuric acid. Absorbance was detected at 490 mm.

Positively tested microcultures were transferred to 24-well expansion plates. After retesting, selected cultures were cloned and recloned using the limiting dilution method and isotypes were determined.

Production of mouse monoclonal antibodies

Antibodies raised against GST-tagged human DPP3 or DPP3 peptides were prepared by standard antibody production methods (Marx et al. 1997) and purified with protein A. Antibody purity was ≥ 90% based on SDS gel electrophoresis analysis.

Antibody characterization - binding to hDPP3 and/or immunizing peptide

To analyze the binding ability of the DPP3 peptide/immunizing peptide by different antibodies and antibody clones, a binding assay was performed:

a) Solid phase

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

b) Tagging procedure (Tracer)

100 μg (100 μl) of different anti-DPP3 antibodies (detection antibody, 1 mg/ml in PBS, pH 7.4) were mixed with 10 μl of NHS-acridinium ester (1 mg/ml in acetonitrile, InVent GmbH, Germany; EP 0353.971) and incubated for 30 min at room temperature. The labeled anti-DPP3 antibody was purified by gel filtration HPLC on Shodex Protein 5 μm KW-803 (Showa Denko, Japan). The purified labeled antibody was diluted in assay buffer (50 mmol/L potassium phosphate, 100 mmol/L NaCl, 10 mmol/L _Na2- EDTA, 5 g/L bovine serum albumin, 1 g/L mouse IgG, 1 g/L bovine IgG, 50 μmol/L amastatin, 100 μmol/L leupeptin, pH 7.4). The final concentration was approximately 5-7* ¹⁰⁶ relative light units (RLU) of labeled compound (approximately 20 ng of labeled antibody) per 200 μl. The chemiluminescence of the acridinium ester was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

c) hDPP3 binding assay

The plates were filled with 200 μl of labeled and diluted detection antibody (label) and incubated for 2-4 h at 2-8°C. Unbound indicator was removed by washing 4 times with 350 μl of washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Well-bound chemiluminescence was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

Antibody characterization - hDPP3 inhibition assay

To analyze the DPP3 inhibition ability of different antibodies and antibody clones, a DPP3 activity assay was performed according to a known method (Jones et al., 1982). Recombinant GST-tagged hDPP3 was diluted in assay buffer (25 ng/ml GST-DPP3 in 50 mM Tris-HCl, pH 7.5, and 100 μM ZnCl2) and 200 μl of this solution were incubated with 10 μg of the corresponding antibody at room temperature. After 1 h of preincubation, the fluorogenic substrate Arg-Arg-βNA (20 μl, 2 mM) was added to the solution and the formation of free βNA was monitored over time using a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG) at 37°C. βNA fluorescence was detected by excitation at 340 nm and emission at 410 nm. Slopes (in RFU/min) of the increase in fluorescence of the different samples were calculated. The slope of GST-hDPP3 with buffer control was taken as 100% activity. The inhibitory capacity of a candidate capture agent binder was defined as the percentage decrease in GST-hDPP3 activity upon incubation with the indicated capture agent binder.

The following table shows a selection of antibodies obtained and their binding rate in relative light units (RLU) as well as their relative inhibitory capacity (%; Table 1). Monoclonal antibodies raised against the regions of DPP3 shown below were selected for their ability to bind recombinant DPP3 and/or the immunizing peptide as well as their inhibitory potential.

All antibodies raised against the GST-tagged full-length form of recombinant hDPP3 show strong binding to immobilized GST-tagged hDPP3. Also, antibodies raised against the peptide of SEQ ID NO.: 2 bind to GST-hDPP3. Antibodies of SEQ ID NO.: 2 also bind strongly to the immunizing peptide.

Sequence number Antigen/Immunogen hDPP3 region Clone hDPP3 [RLU] binding Binding of the immunizing peptide [RLU] Max. hDPP3 inhibition SEQ ID NO.: 1 GST-tagged recombinant FL-hDPP3 1-737 2552 3.053.621 0 65% 2553 3,777,985 0 35% 2554 1.733.815 0 30% 2555 3.805.363 0 25% SEQ ID NO.: 2 CETVINPE
TGEQIQSW
YRSGE 474-493 1963 141.822 2.163.038 60% 1964 100.802 2.041.928 60% 1965 99.493 1,986,794 70% 1966 118.097 1.990.702 65% 1967 113.736 1,909,954 70% 1968 105.696 2.017.731 65% 1969 82.558 2.224.025 70% Table 1: List of antibodies raised against full-length hDPP3 or hDPP3 sequences and their ability to bind hDPP3 (SEQ ID NO.: 1) or the immunizing peptide (SEQ ID NO.: 2) in RLU, as well as maximal inhibition of recombinant GST-hDPP3.

Recently, the development of a luminescent immunoassay for the quantitative determination of DPP3 protein concentration (DPP3-LIA) and an enzyme capture assay for the quantitative determination of DPP3 activity (DPP3-ECA) was described (Rehfeld et al. 2018. JALM. in press), which are incorporated herein by reference in their entirety.

EXAMPLE 2 - DPP3 FOR SHORT-TERM MORTALITY PREDICTION

Plasma DPP3 concentrations in patients with sepsis/septic shock and cardiogenic shock were determined and correlated with short-term patient mortality.

Study cohort - sepsis/septic shock

DPP3 was measured in 574 plasma samples from patients participating in the Adrenomedullin and Outcome in Severe Sepsis and Septic Shock (AdrenOSS) study. AdrenOSS is a prospective, observational, multinational study including 583 patients admitted to the intensive care unit with sepsis or septic shock (Hollinger et al., 2018). Septic shock was diagnosed in 292 patients.

Study cohort - Cardiogenic shock

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

hDPP3 immunoassay:

An immunoassay (LIA) or activity assay (ECA) that measures human DPP3 amount (LIA) or human DPP3 activity (ECA), respectively, was used to determine the level of DPP3 in patient plasma. Antibody immobilization, labeling, and incubation were performed as described by Rehfeld et al. (Rehfeld et al. 2018).

Results

Short-term survival of patients with sepsis was associated with plasma DPP3 concentration on admission. Patients with plasma DPP3 concentrations above 40.5 ng/mL (3rd quartile) had an increased risk of mortality compared with patients with plasma DPP3 concentrations below this threshold (Figure 1A). Applying this threshold to the subcohort of patients with septic shock revealed an even more pronounced risk of short-term mortality in association with high plasma DPP3 concentrations (Figure 1B). When the same threshold is applied to patients with cardiogenic shock, patients with high DPP3 also have an increased risk of short-term mortality within 7 days (Figure 1C).

EXAMPLE 3. PURIFICATION OF NATIVE HUMAN DPP3.

Human erythrocyte lysate was loaded onto 100 ml Sepharose 4B resin (Sigma-Aldrich) and the eluate was collected. The resin was washed with a total volume of 370 ml PBS buffer, pH 7.4, and the wash fraction was combined with the collected eluate, resulting in a total volume of 2370 ml.

For the immunoaffinity purification step, 110 mg of 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 conjugate was equilibrated with 10 bed volumes of binding wash buffer (PBS, 0.1% TritonX-100, pH 7.4), combined with 2370 mL of cleared red blood cell lysate, and incubated at 4°C with continuous shaking for 2 h. Therefore, 100 ml of the incubation mixture was distributed over ten 15 ml polypropylene columns and the eluate was collected by centrifugation at 1000 x g for 30 sec. This step was repeated several times, resulting in 2.5 ml of DPP3-containing resin being loaded onto the column. Each column was washed 5 times with 10 ml of wash-binding buffer using the gravity luminescence method. DPP3 was eluted by loading each column into 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% TritonX-100, pH 3.5) onto the column and immediate centrifugation for 30 sec at 1000 x g. The elution step was repeated a total of 3 times, resulting in 360 ml of combined eluates. The pH of the neutralized eluates was 8.

The pooled eluates were applied to a 5 ml HiTrap Q-sephare HP column (GE Healthcare) equilibrated with IEX buffer A1 (100 mM glycine, 150 mM Tris, pH 8) using an Äkta Start system sample pump (GE Healthcare). After sample loading, the column was washed with five column volumes of IEX buffer A2 (12 mM NaH2PO4, pH 7.4) to remove unbound protein. Elution of DPP3 was achieved by applying a 10 column volume (50 ml) sodium chloride gradient ranging from 0-1 M NaCl _using IEX buffer B (12 mM _NaH2PO4 , 1 M NaCl, pH 7.4). The eluates were collected in 2 ml fractions. Buffers used for ion exchange chromatography were sterile filtered using a 0.22 μM bottle filter.

A purification table with corresponding yields and activities of each purification step is given in Table 2. Figure 2 shows a gradient gel SDS-PAGE (4-20%) of native hDPP3 purified from human red blood cell lysate.

Table 2: Purification of DPP 3 from human red blood cells Stage DPP3 Amount in % (LIA) ^a) Total protein in mg ^b) Total activity in µmol/min (ECA) ^c) Output ^d) in % Specific activity in U/mg ^e) Purification factor ^f) Lizat 100 204160 55 100 0.00027 - IAP 80.6 71.2 46.1 84 0.65 2407 IEX 75 6.6 38.7 70 5.9 21852 ^a) The relative amount of DPP3 was determined in all fractions using the DPP3-LIA assay. The amount of DPP3 in the starting material was taken as 100%, and the remaining amount of DPP3 in the purification fractions was correlated with the starting material.
^b) The amount of total protein was determined using the Lowry method modified by Peterson (Peterson, 1977. Analytical Biochemistry 356:346-356).
^c) Total Arg2-βNA hydrolyzing activity in μmol substrate converted per minute was determined using DPP3ECA calibrated with β-naphthylamine (0.05-100 μM).
^d) The yield after purification was calculated from the total hydrolyzing activity of Arg2-βNA. The hydrolyzing activity of Arg2-βNA in the starting material was taken as 100%.
^e) Specific activity was determined as µmol substrate converted per minute and mg total protein.
^f) The purification factor is the ratio of the specific activity after and before each purification stage.

EXAMPLE 4. EFFECT OF NATIVE DPP3 IN AN ANIMAL MODEL.

The effect of native hDPP3 injection in healthy mice was studied by monitoring the fractional shortening and renal resistance index.

Wild-type Black 6 mice (8–12 weeks old, see Table 3 for group size) were acclimated for 2 weeks and baseline echocardiography was performed. Mice were randomly assigned to one of two groups and then administered native DPP3 protein or PBS intravenously via retro-orbital injection at a dose of 600 μg/kg DPP3 protein.

Following DPP3 or PBS injection, cardiac function was assessed by echocardiography (Gao et al. 2011) and renal function was assessed by renal resistance index (Lubas et al., 2014, Dewitte et al, 2012) at 15, 60, and 120 min (Figure 3).

Group Number of animals Processing WT+PBS 3 PBS WT+DPP3 4 Native DPP3 Table 3: List of experimental groups

Results

Mice treated with native DPP3 protein exhibited significantly reduced fractional shortening compared to the PBS-treated control group (Figure 4A). The WT+DPP3 group also exhibited worsening renal function as evidenced by an increase in the renal resistance index (Figure 4B).

EXAMPLE 5 - DEVELOPMENT OF PROCIZUMAB

Antibodies raised against SEQ ID NO. 2 have been characterized in more detail (epitope mapping, binding affinity, specificity, inhibitory potential). The results for clone 1967 SEQ ID NO.: 2 (AK1967; "Procizumab") are shown here as an example.

Determination of the AK1967 epitope on DPP3:

For epitope mapping of AK1967, a series of N- or C-terminal biotinylated peptides were synthesized (PE GmbH, Hennigsdorf, Germany). These peptides comprise the sequence of the complete immunizing peptide (SEQ ID No. 2) or its fragments with stepwise deletion of one amino acid from either the C- or N-terminus (for a complete list of peptides, see Table 5).

High-binding 96-well plates were coated with 2 μg avidin per well (Greiner Bio-One international AG, Austria) in binding buffer (500 mM Tris-HCl, pH 7.8, 100 mM NaCl). The plates were then washed and loaded with specific solutions of biotinylated peptides (10 ng/well; buffer - 1xPBS with 0.5% BSA). The anti-DPP3 antibody AK1967 was labeled with a chemiluminescent label according to Example 1.

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

Determining Binding Affinity Using Octet:

The experiment was performed using Octet Red96 (ForteBio). AK1967 was captured on kinetic grade anti-human Fc (AHC) biosensors. The loaded biosensors were then dipped in a dilution series of recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM). Association was monitored for 120 s, followed by dissociation for 180 s. The buffers used for the experiment are presented in Table 4. Kinetic analysis was performed using a 1:1 binding model and global fitting.

Buffer Composition Analytical buffer PBS with 0.1% BSA, 0.02% Tween-21 Regenerating buffer 10 mM Glycine buffer (pH 1.7) Neutralizing buffer PBS with 0.1% BSA, 0.02% Tween-21 Table 4: Buffers used for Octet measurements

Western blot analysis of AK1967 binding specificity:

Blood cells from human EDTA-treated blood were washed (3 times in PBS), diluted in PBS, and lysed by repeated freeze-thaw cycles. The blood cell lysate contained 250 μg/mL total protein and 10 μg/mL DPP3. 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. Blots were incubated in 1.) blocking buffer (1xPBS-T with 5% dry skim milk), 2.) primary antibody solution (AK1967 1:2,000 in blocking buffer), and 3.) HRP-labeled secondary antibody (goat anti-mouse IgG, 1:1,000 in blocking buffer). Bound secondary antibody was detected using Amersham ECL Western Blot Detection Reagent and Amersham Imager 600 UV (both GE Healthcare).

DPP3 Inhibition Assay:

To analyze the ability of AK1967 to inhibit DPP3, a DPP3 activity assay was performed according to a known procedure (Jones et al., 1982) as described in Example 1. The inhibitory ability of AK1967 was determined as a percentage decrease in GST-hDPP3 activity upon incubation with the indicated antibody. The resulting decreased DPP3 activity is shown in the inhibition curve in Figure 1C.

Epitope mapping:

Analysis of peptides that AK1967 binds and does not bind revealed that the DPP3 INPETG sequence (SEQ ID No.: 3) is a necessary epitope for AK1967 binding (see Table 5).

peptide ID Peptide sequence binding
AK1967 #1 bio a f n f d q e t v i n p E t g e q i q s w y r s g Yes #2 bio a f n f d q e t v i n p E t g e q i q Yes #3 bio a f n f d q e t v i n p e t g e q i Yes #4 bio a f n f d q e t v i n p e t g e q Yes #5 bio a f n f d q e t v i n p e t g e Yes #6 bio a f n f d q e t v i n p e t g Yes #7 bio a f n f d q e t v i n p e t No #8 bio a f n f d q e t v i n p e No #9 bio a f n f d q e t v i n p No #10 bio a f n f d q e t v i n No #11 e t g e q i q s w y k bio No #12 p e t g e q i q s w y k bio No #13 n p e t g e q i q s w y k bio No #14 i n p e t g e q i q s w y k bio Yes #15 v i n p e t g e q i q s w y k bio Yes #16 t v i n p e t g e q i q s w y k bio Yes #17 e t v i n p e t g e q i q s w y k bio Yes Table 5: Peptides used for AK1967 epitope mapping

Binding affinity:

AK1967 binds with an affinity of 2.2*10-9 M to recombinant GST-hDPP3 (see Figure 5 for kinetic curves).

Specificity and inhibitory potential:

The only protein detected with AK1967 as the primary antibody in the blood cell lysate was DPP3 with a molecular weight of 80 kDa (Figure 6). The total protein concentration in the lysate was 250 μg/mL, while the calculated concentration of DPP3 is approximately 10 μg/mL. Despite the fact that there is 25 times more non-specific protein in the lysate, AK1967 specifically binds and detects DPP3, and no other non-specific binding occurs.

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

Chimerization/Humanization:

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

Humanization of mouse antibodies can be carried out according to the following procedure:

To humanize an antibody of murine origin, the antibody sequence is analyzed for structural interactions of the framework regions (FRs) with the complementarity determining regions (CDRs) and the antigen. Based on structural modeling, a suitable FR of human origin is selected and the murine CDR sequences are grafted onto the human FRs. Variations in the amino acid sequence of the CDRs or FRs can be introduced to re-establish structural interactions that were eliminated during species switching for the FR sequence. This re-establishment of structural interactions can be achieved by a random approach using phage display libraries or by a targeted approach based on molecular modeling (Almagro and Fransson, 2008. Humanization of antibodies. Front Biosci. 13:1619–33).

In the above context, the variable region may be linked to any subclass of constant regions (IgG, IgM, IgE, IgA), or only to scaffolds, Fab fragments, Fv, Fab, and F(ab)2. In Examples 6 and 7 below, a murine antibody variant with an IgG2a scaffold was used. For chimerization and humanization, the human IgG1κ scaffold was used.

Only the CDRs are important for epitope binding. The CDRs for the heavy chain and the light chain of the mouse anti-DPP3 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, KVS sequence, and SEQ ID No. 10 for the light chain, respectively. Sequencing of the anti-DPP3 antibody (AK1967) revealed the antibody heavy chain variable region (H chain) according to SEQ ID No.: 11 and the antibody light chain variable region (L chain) according to SEQ ID No.: 12.

EXAMPLE 6. EFFECT OF PROCIZUMAB IN HEART FAILURE CAUSED BY SEPTIC SHOCK

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

Cecal ligation puncture (CLP) model of septic shock:

Male Wistar rats (2–3 months old, 300 to 400 g, for group sizes see Table 6) from the Centre d'élevage Janvier (France) were randomly assigned to one of three groups. All animals were anesthetized with ketamine hydrochloride (90 mg/kg) and xylazine (9 mg/kg) intraperitoneally (i.p.). For the induction of polymicrobial sepsis, CLP was performed according to the Rittirsch protocol with slight modifications. A ventral midline incision (1.5 cm) was made to exteriorize the cecum. The cecum was then ligated just below the ileocecal valve and punctured once with an 18G needle. The abdomen was then closed in two layers, followed by fluid resuscitation (3 ml/100 g body weight saline was injected subcutaneously) and the animal was returned to the cage. Sham-operated animals underwent surgery without cecal puncture. CLP animals were randomized between placebo and therapeutic antibody.

Study design:

The study procedure is shown in Figure 8. Following CLP or sham surgery, animals were allowed to rest for 20 h with free access to food and water. They were then anesthetized, tracheotomized, and catheterized with arterial and venous lines. Twenty-four hours after CLP surgery, either AK1967 or vehicle (saline) was administered at 5 mg/kg as a bolus injection followed by a 3-h infusion of 7.5 mg/kg. As a precaution, hemodynamics were monitored invasively and continuously from t=0 to 3 h.

At t=0 (baseline), all CLP animals were in septic shock and had developed decreased cardiac function (low blood pressure, low shortening fraction). At this point, procizumab or vehicle (PBS) was administered (i.v.) and saline infusion was started. There was 1 control group and 2 CLP groups, which are summarized in the table below (Table 6). At the end of the experiment, animals were euthanized and organs were removed for later analysis.

Group Number of animals CLP Processing Sham 7 No PBS CLP-PBS 6 Yes PBS CLP-PCZ 4 Yes PCZ Table 6: List of experimental groups

Invasive Blood Pressure:

Hemodynamic parameters were obtained using the AcqKnowledge system (BIOPAC Systems, Inc., USA). It provides a fully automated system for analyzing arterial pressure. The catheter is connected to the BIOPAC system via a pressure sensor.

For the procedure, the rats were anesthetized (ketamine and xylazine). The animals were moved onto a heating pad until the desired body temperature of 37-37.5°C was reached. The temperature feedback sensor was inserted into the rectum. The rats were placed on the operating table in a supine position. The trachea was opened and a catheter (16G) was inserted for external mechanical ventilation without damaging the carotid arteries and vagus nerves. The arterial catheter was inserted into the right carotid artery. The carotid artery was separated from the vagus before ligation. A central venous catheter was inserted through the left jugular vein, which allowed the administration of PCZ or PBS.

After surgery, animals were allowed to rest for stabilization before hemodynamic measurements were taken. Baseline arterial pressure (BP) was then recorded. During data collection, saline infusion through the arterial catheter was stopped.

Echocardiography:

Animals were anesthetized with ketamine hydrochloride. The thorax was shaved and the rats were placed in a supine position. Transthoracic echocardiography (TTE) was performed using a commercial GE Healthcare Vivid 7 ultrasound system equipped with a high-frequency (14 MHz) linear transducer and a 10 MHz cardiac transducer. All examinations were digitally recorded and stored for subsequent offline analysis.

Grayscale images were recorded at a depth of 2 cm. Bidimensional studies were initiated in the parasternal long-axis view to measure aortic annulus diameter and pulmonary artery diameter. M-mode was also used to measure left ventricular (LV) dimensions and estimate fractional shortening (FS%). LVFS was calculated as LV end-diastolic diameter - LV end-systolic diameter/LV end-diastolic diameter and expressed as %. Therefore, end-diastolic time was defined as the maximum LV diameter. Accordingly, end-systole was defined as the minimum diameter in the same cardiac cycle. All parameters were measured manually. Three cardiac cycles were averaged for each measurement.

From the same parasternal long-axis view, pulmonary artery blood flow was recorded using pulsed-wave Doppler. The integral of blood flow velocity for pulmonary artery outflow was measured.

From the apical five-chamber projection, mitral blood flow was recorded using pulsed Doppler sonography at the level of the mitral valve tips.

Results:

Rats with septic shock-induced heart failure treated with PBS (CLP+PBS) exhibited reduced fractional shortening compared to sham-operated animals (Fig. 9A). The CLP+PBS group also exhibited high mortality (Fig. 9B). In contrast, procizumab administration to rats with sepsis-induced heart failure improved fractional shortening (Fig. 9A) and dramatically reduced mortality (Fig. 9B).

EXAMPLE 7. EFFECT OF PROCIZUMAB ON CARDIAC AND KIDNEY FUNCTION

The effect of procizumab on isoproterenol-induced heart failure in mice was studied by monitoring fractional shortening and renal resistance index.

Isoproterenol-induced cardiac stress in mice:

Acute heart failure was induced in 3-month-old male mice by two daily subcutaneous injections of 300 mg/kg isoproterenol, a non-selective ß-adrenergic receptor agonist (DL-isoproterenol hydrochloride, Sigma Chemical Co) (ISO) for two days (Vergaro et al, 2016). ISO dilution was performed in NaCl 0.9%. ISO-treated mice were randomly divided into two groups (Table 7) and PBS or procizumab (10 mg/kg) was administered intravenously after baseline echocardiography (Gao et al, 2011) and renal resistance index measurements (Lubas et al, 2014, Dewitte et al, 2012) were performed on day 3 (Figure 10 A and B).

Cardiac function was assessed by echocardiography (Gao et al., 2011) and renal resistance index (Lubas et al., 2014, Dewitte et al, 2012) at 1 hour, 6 hours and 24 hours (Figure 10 A and B). The group of mice that received vehicle (PBS) instead of isoproterenol did not undergo further pharmacological treatment and served as the control group (Table 7).

Group Number of animals Processing Sham+PBS 27 PBS HF+PBS 15 PBS HF+PCZ 20 Procizumab (PCZ) Table 7: List of experimental groups

Results:

Administration of procizumab to mice with isoproterenol-induced heart failure restores cardiac function within the first hour after administration (Fig. 11A). Kidney function of diseased mice shows significant improvement 6 hours after procizumab injection and is comparable to that of sham-operated animals after 24 hours (Fig. 11B).

EXAMPLE 8. DPP3 INDICATES THE NEED FOR VASOPRESSORS AND THE RESPONSE TO VASOPRESSOR THERAPY

Plasma DPP3 concentrations in patients with septic shock were determined using an hDPP3 immunoassay and associated with the need for vasopressor therapy.

Study cohort - septic shock

Plasma samples from 292 patients diagnosed with septic shock in the AdrenOSS study (see Example 2) were screened for the presence of DPP3. Human DPP3 was measured as described in Example 1.

Results

Patients with increased need for vasopressors for more than 5 days had high plasma DPP3 concentrations (Fig. 12A). In contrast, in patients who responded to vasopressors and in whom vasopressors could be discontinued within the first 5 days, plasma DPP3 concentrations were significantly reduced (Fig. 12B). This indicates that the development of refractory shock and, consequently, increased need for vasopressors due to decreased response to this therapy are associated with high plasma DPP3 concentrations in patients with septic shock.

Patients with vasopressor-resistant refractory septic shock (norepinephrine>0.5 μg/kg/min) demonstrated significantly higher plasma DPP3 concentrations compared to patients who required norepinephrine doses<0.5 μg/kg/min (p<0.001) (Figure 16A). Furthermore, plasma DPP3 concentration was strongly associated with mortality in patients with vasopressor-resistant refractory septic shock (Figure 16B).

EXAMPLE 9 - DPP3 IS ASSOCIATED WITH REFRACTORY SHOCK

Plasma DPP3 concentrations in patients with cardiogenic shock were determined using hDPP3 immunoassay and were associated with the development of refractory shock.

Study cohort - Cardiogenic shock

Plasma samples from 57 patients diagnosed with cardiogenic shock following acute myocardial infarction were screened for DPP3. Human DPP3 was measured as described in Example 1.

Results

Patients with plasma DPP3 concentrations above a certain threshold at admission (59.1 ng/mL; 3rd quartile) developed refractory shock to a higher extent (47%) than patients with plasma DPP3 concentrations below 59.1 ng/mL (12%) (Figure 13).

EXAMPLE 10: DPP3 AND BIO-ADM INDICATE SHORT-TERM MORTALITY IN SHOCK.

Plasma DPP3 and bio-ADM concentrations in patients with septic shock were determined using hDPP3 and bio-ADM immunoassay and correlated with short-term patient mortality.

Study cohort - septic shock

Plasma samples from 292 patients diagnosed with septic shock in the AdrenOSS study were tested for DPP3 and bio-ADM. Human DPP3 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).

Results

Plasma bio-ADM and DPP3 concentrations were measured in patients with septic shock. Patients were grouped according to predetermined cutoff values defined as the 3rd quartile of all measured plasma concentrations of the respective marker (Table 8). An equal number of patients had either high DPP3 only or high bio-ADM only (15.4%), while fewer patients had elevated plasma concentrations of both bio-ADM and DPP3 (9.6%).

DPP3 low level (<48.4 ng/ml) DPP3 high level (>48.4 ng/ml) bio-ADM low level (<213 pg/ml) 174 (59.6%) 45 (15.4%) bio-ADM high level (>213 pg/ml) 45 (15.4%) 28 (9.6%) Table 8: Number of patients with low/high DPP3 and low/high bio-ADM. Cutoffs were assigned based on Q3 (highest 25%) for both.

Mortality within the first 4 weeks after admission was associated with bio-ADM and DPP3 concentrations at admission. Patients with only high plasma bio-ADM concentrations or only high plasma DPP3 concentrations had a significantly increased risk of death within the first 4 weeks compared with patients who had plasma concentrations of either bio-ADM (Figure 14A) or DPP3 (Figure 14B) below a certain threshold (3rd quartile). Survival was better in patients with high bio-ADM than in patients with high DPP3.

When both markers, bio-ADM and DPP3, were combined, an even higher risk of short-term mortality was found compared to the mortality that was associated with an increase in only one of the two markers (Figure 14C). Only 28.6% of patients with high bio-ADM and high plasma DPP3 concentrations survived the first 4 weeks after admission.

EXAMPLE 11. DPP3 AND BIO-ADM DPP3 IDENTIFY THE NEED FOR VASOPRESSORS AND THE RESPONSE TO VASOPRESSOR THERAPY

Plasma DPP3 and bio-ADM concentrations in patients with septic shock were determined using hDPP3 and bio-ADM immunoassay and were associated with the need for vasopressor therapy.

Study cohort - septic shock

In 292 plasma samples from patients diagnosed with septic shock in the AdrenOSS study, DPP3 and bio-ADM concentrations were measured. Human DPP3 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).

Results

Plasma bio-ADM and DPP3 concentrations were measured in patients with septic shock. Patients were grouped according to specific cutoffs defined as the 3rd quartile of all measured plasma concentrations of the respective marker in the septic shock cohort (low DPP3<48.4 ng/mL, low bio-ADM<213 pg/mL; high bio-ADM ≥213 pg/mL; high DPP3 ≥48.4 ng/mL).

Patients with high DPP3 but low plasma bio-ADM concentrations had a higher requirement for sequential vasopressors compared with patients who had either low DPP3+low bio-ADM or low DPP3+high bio-ADM (Figure 15; Table 9). In contrast, in patients with low DPP3 and low plasma bio-ADM or in patients with low DPP3 but high plasma bio-ADM concentrations on admission, vasopressors could be discontinued earlier than in patients with high plasma DPP3 concentrations on admission (Figure 15; Table 9). This suggests that vasopressor therapy in septic shock patients with high bio-ADM can be discontinued earlier due to a better therapeutic response compared with septic shock patients with high DPP3 levels. These patients (with high DPP3 levels) require longer term vasopressor treatment due to lack of response.

Days with need for vasopressor DPP3 low
bioADM low DPP3 low
bioADM high DPP3 high
bioADM low DPP3 high
bioADM high 1 42 8 5 2 2 49 11 6 2 3 25 6 8 2 4 16 4 1 2 5 8 3 2 0 6 3 1 6 1 7 31 12 17 19 Table 9: Patients requiring vasopressors are grouped according to their plasma DPP3 and bio-ADM concentrations on admission.

Cutoff thresholds were assigned based on Q3 (highest 25% of determined values) for both; low DPP3<48.4 ng/mL, low bio-ADM<213 pg/mL; high bio-ADM ≥213 pg/mL; high DPP3 ≥48.4 ng/mL; 7: ≥7 days on vasopressor or death within 7 days.

EXAMPLES OF SEQUENCES

SEQ ID No. 1 - hDPP3 aa 1-737

MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSPEAPYIYALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTKFVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLRHLGLGKEGITTYFSGNC™EDAKLAQ DFLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDSEVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESFTQGSIEAHKRGSRFWIQDKGPIVESYIGFIESYRDPFGSRGEFEGFVAVVNKAMSAKFERLVASAEQLLKELPWPPTFEKDKFL TPDFTSLDVLTFAGSGIPAGINIPNYDDLRQTEGFKNVSLGNVLAVAYATQREKLTFLEEDDKDLYILWKGPSFDVQVGLHELLGHGSGKLFVQDEKGAFNFDQETVINPETGEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLYLCLHPQVLEIFGFEGADAEDVIYVNWLNMVRAGLLA LEFYTPEAFNWRQAHMQARFVILRVLLEAGEGLVTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTGDVAGGRALYEGYATTVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEYEASAAGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA

SEQ ID No. 2 - hDPP3 aa 474-493 (N-Cys) - immunizing peptide with additional N-terminal Cysteine

CETVINPETGEQIQSWYRSGE

SEQ ID No. 3 - hDPP3 aa 477-482 - epitope AK1967

INPETG

SEQ ID No. 4 - variable region of mouse AK1967 in the heavy chain

QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPSGKGLEWLAHIWWNDNKSYNPALKSRLTISRDTSNNQVFLKIASVVTADTGTYFCARNYSYDYWGQGTTLTVSS

SEQ ID No. 5 - variable region of mouse AK1967 in the light chain

DVVVTQTPLSLSVSLGDPASISCRSSRSLVHSIGSTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

SEQ ID No. 6 - CDR1 of mouse AK1967 heavy chain

GFSLSTSGMS

SEQ ID No. 7 - CDR2 of mouse AK1967 heavy chain

IWWNDNK

SEQ ID No. 8 - CDR 3 of mouse AK1967 heavy chain

ARNYSYDY

SEQ ID No. 9 - CDR1 of mouse AK1967 light chain

RSLVHSIGSTY

Mouse AK1967 light chain CDR2

KVS

SEQ ID No. 10 - CDR3 of mouse AK1967 light chain

SQSTHVPWT

SEQ ID No. 11 - humanized AK1967 - heavy chain sequence (IgG1κ backbone)

MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQVVL™TNMDPVDTGT YYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID No. 12 - humanized AK1967 - light chain sequence (IgG1κ backbone)

METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHV PWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID No. 13 - Angiotensin II (synonym: 5-isoleucine-angiotensin II)

DRVYIHPF

SEQ ID No. 14 - Angiotensin II analog (5-valine-angiotensin II)

DRVYVHPF

SEQ ID No. 15 - Angiotensin II analog (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 -diiodine-Tyr ⁴ -Ile ⁵ ]-angiotensin II

NRVYIHPF

SEQ ID No. 21 - Angiotensin III

RVYIHPF

SEQ ID No. 22 - Angiotensin III analogue (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 (diiodine-Tyr ³ -Ile ⁴ ]-angiotensin III)

RVYIHPF

SEQ ID No. 26 - Angiotensin IV

VYIHPF

SEQ ID No. 27 - Angiotensin IV analogue (Val ³ -angiotensin IV)

VYVHPF

SEQ ID No. 28 - Angiotensin IV analogue (Phe ² -angiotensin IV)

VFIHPF

SEQ ID No. 29 - Angiotensin IV analogue ([Ile ³ -Ala ⁶ ]-angiotensin IV)

VYIHPA

SEQ ID No. 30 - Angiotensin IV analog ([diiodo-Tyr ² -Ile ³ -angiotensin IV)

VYIHPF

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

ARLDVASEF RKKWNKWALS RGKRELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

SEQ ID No. 32 (Proadrenomedullin N-20 terminal peptide, PAMP): amino acids 22-41 preproADM

ARLDVASEF RKKWNKWALS R

SEQ ID No. 33 (Mid-regional proAdreno-medullin, MR-proADM): amino acids 45-92 preproADM

ELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RV

SEQ ID No. 34 (mature Adrenomedullin (mature ADM); amidated ADM; bio-ADM; hADM): amino acids 95-146 -CONH ₂

YRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGY - CONH ₂

SEQ ID No. 35 (Adrenomedullin 1-52-Gly (ADM 1-52-Gly)): amino acids 95-147 preproADM

YRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYG

SEQ ID No. 36 (C-terminal proAdrenoMedullin, CT-proADM): amino acids 148-185 preproADM

RRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

SEQ ID No. 37 (N-terminal portion of mature ADM): amino acids 1-21 of mature ADM

YRQSMNNFQGLRSFGCRFGTC

SEQ ID No. 38 (anti-ADM antibody heavy chain CDR1)

GYTFSRYW

SEQ ID No. 39 (anti-ADM antibody heavy chain CDR2)

ILPGSGST

SEQ ID No. 40 (CDR3 of anti-ADM antibody heavy chain)

TEGYEYDGFDY

SEQ ID No. 41 (anti-ADM antibody light chain CDR1)

QSIVYSNGNTY

SEQ ID No. 42 (anti-ADM antibody light chain CDR3)

FQGSHIPYT

SEQ ID No. 43 (anti-ADM antibody heavy chain (Adrecizumab))

QVQLVQSGAEVKKPGSSVKVSCKAS GYTFSRYW IEWVRQAPGQGLEWIGE ILPGSGST NYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYC TEGYEYDGFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 44 (anti-ADM antibody light chain (Adrecizumab))

DVVLTQSPLSLPVTLGQPASISCRSS QSIVYSNGNTY LEWYLQRPGQSPRLLIY RVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHIPYT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

DESCRIPTION OF DRAWINGS

Figure 1: Kaplan-Meier survival plots according to low (<40.5 ng/mL) and high (≥ 40.5 ng/mL) DPP3 concentrations. (A) 7-day survival of patients with sepsis according to plasma DPP3 concentration; (B) 7-day survival of patients with cardiogenic shock according to plasma DPP3 concentration; (C) 7-day survival of patients with septic shock according to plasma DPP3 concentration.

Figure 2: SDS-PAGE on a gradient gel (4-20%) of native hDPP3 purified from human red blood cell lysate. The molecular weight marker is indicated by arrows.

Figure 3: Experimental design. Effect of native DPP3 on animal model.

Figure 4: (A) DPP3 injection causes a reduction in the fractional shortening and therefore leads to a deterioration in cardiac function. (B) A decrease in renal function is also observed through an increase in the renal resistance index.

Figure 5: Association and dissociation curve of AK1967-DPP3 binding assay using Octet. AK1967-loaded biosensors were loaded with a dilution series of recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM) and association and dissociation were monitored.

Figure 6: Western blot of blood cell lysate dilutions and detection of DPP3 with AK1967 as primary antibody.

Figure 7: Inhibition curve of native DPP3 from blood cells by the inhibitory antibody AK1967. Inhibition of DPP3 by the specific antibody is concentration dependent, with an IC50 of ~15 ng/mL when assayed against 15 ng/mL DPP3.

Figure 8: Experimental setup - Effect of procizumab in sepsis-induced heart failure.

Figure 9: Procizumab dramatically improves fractional shortening (A) and mortality (B) in rats with sepsis-induced heart failure.

Figure 10: Experimental design. Isoproterenol-induced cardiac stress in mice followed by procizumab treatment (B) and in control (A).

Figure 11. Procizumab improved fractional shortening (A) and reduced renal resistance index (B) at 1 hour and 6 hours post-administration, respectively, in mice with isoproterenol-induced heart failure.

Figure 12: (A) Number of days of vasopressor therapy required in patients with septic shock according to plasma DPP3 concentration. ≥7 days on vasopressor or death within 7 days; *p<0.05 in post hoc comparison with groups 1-4(B). Need for vasopressor therapy in patients with septic shock according to plasma DPP3 concentration. Patients who received vasopressor therapy for a maximum of 5 days (≤5) and patients who received vasopressor therapy for more than 5 days or died within 7 days were grouped together (>5).

Figure 13: DPP3 is associated with refractory shock. High plasma DPP3 concentrations in patients with cardiogenic shock are associated with a higher risk of developing refractory cardiogenic shock compared to patients with plasma DPP3 concentrations below a certain threshold.

Figure 14: Kaplan-Meier survival plots (A) 4-week survival of patients with septic shock according to plasma bio-ADM concentration; values are grouped into quartiles (B) 4-week survival of patients with septic shock according to plasma DPP3 concentration; values are grouped into quartiles (C) 4-week survival of patients with septic shock according to plasma bio-ADM and DPP3 concentrations; Cutoffs were defined based on the 3rd quartile of all measured plasma concentrations of the respective marker: both low, DPP3<48.4 ng/mL, bio-ADM<213 pg/mL; bio-ADM high ≥213 pg/mL; DPP3 high ≥ 48.4 ng/mL.

Figure 15: Patients on vasopressor should be grouped according to their plasma DPP3 and bio-ADM concentrations on admission. The proportion of patients receiving vasopressors for 1-7 days is shown in grey scale - lighter shades indicate longer treatment duration. Cutoff thresholds were assigned based on Q3 (highest 25% of values detected) for both; low DPP3<48.4 ng/mL, low bio-ADM<213 pg/mL; high bio-ADM ≥213 pg/mL; high DPP3 ≥ 48.4 ng/mL; 7: ≥7 days on vasopressor or death within 7 days.

Figure 16: (A) Plasma DPP3 concentration in patients with septic refractory shock requiring vasopressor norepinephrine below (n=186) and above (n=95) 0.5 μg/kg/min (p<0.001). (B) Kaplan-Meier survival plots (A) 4-week survival of patients with septic refractory shock according to plasma DPP3 concentration; values are grouped into quartiles.

LIST OF REFERENCES

1.Hollenberg et al. Ann Intern Med 1999; 131 :47-59.

2.Reynolds HR, Hochman JS. Circulation 2008; 117 :686-697.

3.Tarvasmäki et al. Eur J Heart Fail JohnWiley& Sons, Ltd; 2018; 20 :572-581.

4. Thiele et al. N Engl J Med 2012; 367 :1287-1296.

5.Champion S. Eur J Heart Fail 2018; 20 :197-198.

6.HochmanJS. Circulation 2003; 107 :2998-3002.

7.Shah et al. Clin Res Cardiol 2018; 107 :287-303.

8.Schmidt et al. Eur Heart J 2015; 36 :2246–2256.

9.Muller et al. Intensive Care Med 2016; 42 :370-378.

10.Chen et al. Crit Care 2016; 20 :336.

11.Harjola et al. Eur J Heart Fail John Wiley& Sons, Ltd; 2015; 17 :501-509.

12.Harjola et al. Eur J Heart Fail John Wiley& Sons, Ltd; 2018; 20 :1081-1099.

13.Bakker et al. Am J Surg 1996; 171 :221-226.

14.Attana et al. Acute Card Care 2012; 14 :20-26.

15.ZhangZ, XuX. Crit Care Med 2014; 42 :2118–2125.

16.Allard et al. J Neurochem 1987; 48 :1553–1559.

17.Prajapati SC, Chauhan SS. FEBS J 2011; 278 :3256-3276.

18. Ocaranza MP, Jalil JE. Hypertens (Dallas, Tex1979) 2016; 68 :552-554.

19. Rehfeld L, J Appl Lab Med 2019 JALM 3(6):943-953.

20.Deniau et al. Eur J Heart Fail 2020 Feb;22(2):290-299.

21. Levy J Am Coll Cardiol 2018; 72 :173-182.

22. Kohsaka et al. Arch Intern Med 2005; 165 :1643-1650.

23.Hochman et al. N Engl J Med 1999; 341 :625-634.

24. Thiele et al. N Engl J Med 2012; 367 :1287-1296.

25.Thieleet al. N Engl J Med 2017; 377 :2419-2432.

26.The TRIUMPH Investigators*, Alexander JH, et al. JAMA 2007; 297 :1657.

27.Reyentovichet al. Nat Rev Cardiol 2016; 13 :481-492.

28.Mebazaa et al. Intensive Care Med 2018; 44 :760-773.

29.Bassi et al. Crit Care Res Pract 2013; 2013 :654708.

30.Baran et al. Catheter Cardiovasc Interv 2019; 94 :29-37.

--->

LIST OF SEQUENCES

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Met Ala Asp Thr Gln Tyr Ile Leu Pro Asn Asp Ile Gly Val Ser Ser

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Leu Asp Cys Arg Glu Ala Phe Arg Leu Leu Ser Pro Thr Glu Arg Leu

20 25 30

Tyr Ala Tyr His Leu Ser Arg Ala Ala Trp Tyr Gly Gly Leu Ala Val

35 40 45

Leu Leu Gln Thr Ser Pro Glu Ala Pro Tyr Ile Tyr Ala Leu Leu Ser

50 55 60

Arg Leu Phe Arg Ala Gln Asp Pro Asp Gln Leu Arg Gln His Ala Leu

65 70 75 80

Ala Glu Gly Leu Thr Glu Glu Glu Tyr Gln Ala Phe Leu Val Tyr Ala

85 90 95

Ala Gly Val Tyr Ser Asn Met Gly Asn Tyr Lys Ser Phe Gly Asp Thr

100 105 110

Lys Phe Val Pro Asn Leu Pro Lys Glu Lys Leu Glu Arg Val Ile Leu

115 120 125

Gly Ser Glu Ala Ala Gln Gln His Pro Glu Glu Val Arg Gly Leu Trp

130 135 140

Gln Thr Cys Gly Glu Leu Met Phe Ser Leu Glu Pro Arg Leu Arg His

145 150 155 160

Leu Gly Leu Gly Lys Glu Gly Ile Thr Thr Tyr Phe Ser Gly Asn Cys

165 170 175

Thr Met Glu Asp Ala Lys Leu Ala Gln Asp Phe Leu Asp Ser Gln Asn

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Leu Ser Ala Tyr Asn Thr Arg Leu Phe Lys Glu Val Asp Gly Glu Gly

195 200 205

Lys Pro Tyr Tyr Glu Val Arg Leu Ala Ser Val Leu Gly Ser Glu Pro

210 215 220

Ser Leu Asp Ser Glu Val Thr Ser Lys Leu Lys Ser Tyr Glu Phe Arg

225 230 235 240

Gly Ser Pro Phe Gln Val Thr Arg Gly Asp Tyr Ala Pro Ile Leu Gln

245 250 255

Lys Val Val Glu Gln Leu Glu Lys Ala Lys Ala Tyr Ala Ala Asn Ser

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His Gln Gly Gln Met Leu Ala Gln Tyr Ile Glu Ser Phe Thr Gln Gly

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Ser Ile Glu Ala His Lys Arg Gly Ser Arg Phe Trp Ile Gln Asp Lys

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Gly Pro Ile Val Glu Ser Tyr Ile Gly Phe Ile Glu Ser Tyr Arg Asp

305 310 315 320

Pro Phe Gly Ser Arg Gly Glu Phe Glu Gly Phe Val Ala Val Val Asn

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Lys Ala Met Ser Ala Lys Phe Glu Arg Leu Val Ala Ser Ala Glu Gln

340 345 350

Leu Leu Lys Glu Leu Pro Trp Pro Pro Thr Phe Glu Lys Asp Lys Phe

355 360 365

Leu Thr Pro Asp Phe Thr Ser Leu Asp Val Leu Thr Phe Ala Gly Ser

370 375 380

Gly Ile Pro Ala Gly Ile Asn Ile Pro Asn Tyr Asp Asp Leu Arg Gln

385 390 395 400

Thr Glu Gly Phe Lys Asn Val Ser Leu Gly Asn Val Leu Ala Val Ala

405 410 415

Tyr Ala Thr Gln Arg Glu Lys Leu Thr Phe Leu Glu Glu Asp Asp Lys

420 425 430

Asp Leu Tyr Ile Leu Trp Lys Gly Pro Ser Phe Asp Val Gln Val Gly

435 440 445

Leu His Glu Leu Leu Gly His Gly Ser Gly Lys Leu Phe Val Gln Asp

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Glu Lys Gly Ala Phe Asn Phe Asp Gln Glu Thr Val Ile Asn Pro Glu

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Thr Gly Glu Gln Ile Gln Ser Trp Tyr Arg Ser Gly Glu Thr Trp Asp

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Ser Lys Phe Ser Thr Ile Ala Ser Ser Tyr Glu Glu Cys Arg Ala Glu

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Gly Phe Glu Gly Ala Asp Ala Glu Asp Val Ile Tyr Val Asn Trp Leu

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Asn Met Val Arg Ala Gly Leu Leu Ala Leu Glu Phe Tyr Thr Pro Glu

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Ala Phe Asn Trp Arg Gln Ala His Met Gln Ala Arg Phe Val Ile Leu

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Arg Val Leu Leu Glu Ala Gly Glu Gly Leu Val Thr Ile Thr Pro Thr

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Thr Gly Ser Asp Gly Arg Pro Asp Ala Arg Val Arg Leu Asp Arg Ser

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Lys Ile Arg Ser Val Gly Lys Pro Ala Leu Glu Arg Phe Leu Arg Arg

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Leu Gln Val Leu Lys Ser Thr Gly Asp Val Ala Gly Gly Arg Ala Leu

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Tyr Glu Gly Tyr Ala Thr Val Thr Asp Ala Pro Pro Glu Cys Phe Leu

645 650 655

Thr Leu Arg Asp Thr Val Leu Leu Arg Lys Glu Ser Arg Lys Leu Ile

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Val Gln Pro Asn Thr Arg Leu Glu Gly Ser Asp Val Gln Leu Leu Glu

675 680 685

Tyr Glu Ala Ser Ala Ala Gly Leu Ile Arg Ser Phe Ser Glu Arg Phe

690 695 700

Pro Glu Asp Gly Pro Glu Leu Glu Glu Ile Leu Thr Gln Leu Ala Thr

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Ala Asp Ala Arg Phe Trp Lys Gly Pro Ser Glu Ala Pro Ser Gly Gln

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Ala

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Cys Glu Thr Val Ile Asn Pro Glu Thr Gly Glu Gln Ile Gln Ser Trp

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Ile Asn Pro Glu Thr Gly

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Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

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Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Gly Met Ser Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Ser Tyr Asn Pro Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Asn Asn Gln Val

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Phe Leu Lys Ile Ala Ser Val Val Thr Ala Asp Thr Gly Thr Tyr Phe

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Cys Ala Arg Asn Tyr Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Thr Leu

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Thr Val Ser Ser

115

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Asp Val Val Val Thr Gln Thr Pro Leu Ser Leu Ser Val Ser Leu Gly

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Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Val His Ser

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Ile Gly Ser Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

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Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

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Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

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Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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Gly Phe Ser Leu Ser Thr Ser Gly Met Ser

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Ile Trp Trp Asn Asp Asn Lys

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Arg Ser Leu Val His Ser Ile Gly Ser Thr Tyr

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Ser Gln Ser Thr His Val Pro Trp Thr

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Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser

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Leu Ala Phe Glu Leu Ser Tyr Gly Gln Ile Thr Leu Lys Glu Ser Gly

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Pro Thr Leu Val Lys Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr Phe

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Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg

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Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala His Ile Trp Trp Asn

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Asp Asn Lys Ser Tyr Asn Pro Ala Leu Lys Ser Arg Leu Thr Ile Thr

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Arg Asp Thr Ser Lys Asn Gln Val Val Leu Thr Met Thr Asn Met Asp

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Pro Val Asp Thr Gly Thr Tyr Tyr Cys Ala Arg Asn Tyr Ser Tyr Asp

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Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

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Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

225 230 235 240

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly

465

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Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

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Gly Ser Thr Gly Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser

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Val Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Arg Ser

35 40 45

Leu Val His Ser Ile Gly Ser Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys

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Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr

100 105 110

Cys Ser Gln Ser Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys

115 120 125

Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

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Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

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Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

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Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

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Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

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Asp Arg Val Tyr Ile His Pro Phe

1 5

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Asp Arg Val Tyr Val His Pro Phe

1 5

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Asn Arg Val Phe Ile His Pro Phe

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Val Tyr Ile His Pro Phe

1 5

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Asn Arg Val Tyr Tyr Val His Pro Phe

1 5

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Asn Arg Val Tyr Ile His Pro Ile

1 5

<210>19

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Asn Arg Val Tyr Ile His Pro Ala

1 5

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Asn Arg Val Tyr Ile His Pro Phe

1 5

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Arg Val Tyr Ile His Pro Phe

1 5

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Arg Val Tyr Val His Pro Phe

1 5

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Arg Val Phe Ile His Pro Phe

1 5

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Arg Val Tyr Ile His Pro Ala

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Arg Val Tyr Ile His Pro Phe

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Val Tyr Ile His Pro Phe

1 5

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Val Tyr Val His Pro Phe

1 5

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<223>Angiotensin IV Analogue

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Val Phe Ile His Pro Phe

1 5

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<223>Angiotensin IV Analogue

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Val Tyr Ile His Pro Ala

1 5

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Val Tyr Ile His Pro Phe

1 5

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Ala Arg Leu Asp Val Ala Ser Glu Phe Arg Lys Lys Trp Asn Lys Trp

1 5 10 15

Ala Leu Ser Arg Gly Lys Arg Glu Leu Arg Met Ser Ser Ser Tyr Pro

20 25 30

Thr Gly Leu Ala Asp Val Lys Ala Gly Pro Ala Gln Thr Leu Ile Arg

35 40 45

Pro Gln Asp Met Lys Gly Ala Ser Arg Ser Pro Glu Asp Ser Ser Pro

50 55 60

Asp Ala Ala Arg Ile Arg Val Lys Arg Tyr Arg Gln Ser Met Asn Asn

65 70 75 80

Phe Gln Gly Leu Arg Ser Phe Gly Cys Arg Phe Gly Thr Cys Thr Val

85 90 95

Gln Lys Leu Ala His Gln Ile Tyr Gln Phe Thr Asp Lys Asp Lys Asp

100 105 110

Asn Val Ala Pro Arg Ser Lys Ile Ser Pro Gln Gly Tyr Gly Arg Arg

115 120 125

Arg Arg Arg Ser Leu Pro Glu Ala Gly Pro Gly Arg Thr Leu Val Ser

130 135 140

Ser Lys Pro Gln Ala His Gly Ala Pro Ala Pro Pro Ser Gly Ser Ala

145 150 155 160

About His Phe Leu

<210>32

<211>20

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Ala Arg Leu Asp Val Ala Ser Glu Phe Arg Lys Lys Trp Asn Lys Trp

1 5 10 15

Ala Leu Ser Arg

20

<210>33

<211>48

<212>protein

<213>Homo sapiens

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Glu Leu Arg Met Ser Ser Ser Tyr Pro Thr Gly Leu Ala Asp Val Lys

1 5 10 15

Ala Gly Pro Ala Gln Thr Leu Ile Arg Pro Gln Asp Met Lys Gly Ala

20 25 30

Ser Arg Ser Pro Glu Asp Ser Ser Pro Asp Ala Ala Arg Ile Arg Val

35 40 45

<210>34

<211>52

<212>protein

<213>Artificial sequence

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<223>C-terminal amide (CONH2)

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Tyr Arg Gln Ser Met Asn Asn Phe Gln Gly Leu Arg Ser Phe Gly Cys

1 5 10 15

Arg Phe Gly Thr Cys Thr Val Gln Lys Leu Ala His Gln Ile Tyr Gln

20 25 30

Phe Thr Asp Lys Asp Lys Asp Asn Val Ala Pro Arg Ser Lys Ile Ser

35 40 45

Pro Gln Gly Tyr

50

<210>35

<211>53

<212>protein

<213>Artificial sequence

<220>

<223>Glycine at the C-terminus

<400>35

Tyr Arg Gln Ser Met Asn Asn Phe Gln Gly Leu Arg Ser Phe Gly Cys

1 5 10 15

Arg Phe Gly Thr Cys Thr Val Gln Lys Leu Ala His Gln Ile Tyr Gln

20 25 30

Phe Thr Asp Lys Asp Lys Asp Asn Val Ala Pro Arg Ser Lys Ile Ser

35 40 45

Pro Gln Gly Tyr Gly

50

<210>36

<211>38

<212>protein

<213>Homo sapiens

<400>36

Arg Arg Arg Arg Arg Ser Leu Pro Glu Ala Gly Pro Gly Arg Thr Leu

1 5 10 15

Val Ser Ser Lys Pro Gln Ala His Gly Ala Pro Ala Pro Pro Ser Gly

20 25 30

Ser Ala Pro His Phe Leu

35

<210>37

<211>21

<212>protein

<213>Homo sapiens

<400>37

Tyr Arg Gln Ser Met Asn Asn Phe Gln Gly Leu Arg Ser Phe Gly Cys

1 5 10 15

ArgPheGlyThrCys

20

<210>38

<211>8

<212>protein

<213>Homo sapiens

<400>38

Gly Tyr Thr Phe Ser Arg Tyr Trp

1 5

<210>39

<211>8

<212>protein

<213>Homo sapiens

<400>39

Ile Leu Pro Gly Ser Gly Ser Thr

1 5

<210>40

<211>11

<212>protein

<213>Homo sapiens

<400>40

Thr Glu Gly Tyr Glu Tyr Asp Gly Phe Asp Tyr

1 5 10

<210>41

<211>11

<212>protein

<213>Homo sapiens

<400>41

Gln Ser Ile Val Tyr Ser Asn Gly Asn Thr Tyr

1 5 10

<210>42

<211>9

<212>protein

<213>Homo sapiens

<400>42

Phe Gln Gly Ser His Ile Pro Tyr Thr

1 5

<210>43

<211>448

<212>protein

<213>Homo sapiens

<400>43

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Glu Gly Tyr Glu Tyr Asp Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210>44

<211>219

<212>protein

<213>Homo sapiens

<400>44

Asp Val Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<---

Claims (38)

1. A method for predicting refractory shock in a patient, wherein said method includes the following stages: determination of the level of DPP3 in a sample of biological fluid of the specified patient; comparing the specified level of a given DPP3 with a given threshold value, which is from 20 to 200 ng/ml, where the specified patient is predicted to enter refractory shock if the specified defined DPP3 level exceeds the specified predetermined threshold. 2. The method according to claim 1, wherein said shock is selected from the group including shock caused by hypovolemia, cardiogenic shock, obstructive shock and distributive shock. 3. The method according to claim 1 or 2, wherein said shock is a vasopressor-resistant shock. 4. The method according to item 2, where in case of cardiogenic shock, the said patient may have suffered from an acute coronary syndrome, in particular acute myocardial infarction, or the said patient has heart failure, in particular acute decompensated heart failure, myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordal rupture or massive pulmonary embolism, or in the case of hypovolemic shock, the patient may have suffered a hemorrhagic disorder, including gastrointestinal bleeding, trauma, ruptured abdominal aortic aneurysm, tumor invading a major blood vessel, and spontaneous bleeding in the setting of anticoagulation, or a nonhemorrhagic disorder, including vomiting, diarrhea, renal failure, burns, heat stroke, or third-space loss in the setting of pancreatitis, liver cirrhosis, intestinal obstruction, trauma, or in case of obstructive shock, the said patient may have suffered cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or In case of distributive shock, the said patient may have septic shock, neurogenic shock, anaphylactic shock, or shock due to adrenal crisis. 5. A method for diagnosing refractory shock in a patient, wherein said method includes the following stages: determination of the level of DPP3 in a sample of biological fluid of the specified patient; comparing the specified level of a given DPP3 with a given threshold value, which is from 20 to 200 ng/ml, where a specified patient is diagnosed with refractory shock if a specified defined DPP3 level exceeds a specified predetermined threshold. 6. The method according to claim 5, wherein said shock is selected from the group including shock caused by hypovolemia, cardiogenic shock, obstructive shock and distributive shock. 7. The method according to claim 5 or 6, wherein said shock is a vasopressor-resistant shock. 8. The method according to item 6, where in case of cardiogenic shock, the said patient may have suffered from an acute coronary syndrome, in particular acute myocardial infarction, or the said patient has heart failure, in particular acute decompensated heart failure, myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordal rupture or massive pulmonary embolism, or in the case of hypovolemic shock, the patient may have suffered a hemorrhagic disorder, including gastrointestinal bleeding, trauma, ruptured abdominal aortic aneurysm, tumor invading a major blood vessel, and spontaneous bleeding in the setting of anticoagulation, or a nonhemorrhagic disorder, including vomiting, diarrhea, renal failure, burns, heat stroke, or third-space loss in the setting of pancreatitis, liver cirrhosis, intestinal obstruction, trauma, or in case of obstructive shock, the said patient may have suffered cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or In case of distributive shock, the said patient may have septic shock, neurogenic shock, anaphylactic shock, or shock due to adrenal crisis. 9. A method for predicting the outcome of a patient who has developed refractory shock, wherein said method includes the following stages: determination of the level of DPP3 in a sample of biological fluid of the specified patient; correlation of a specified defined level of DPP3 with the success of therapy or intervention in a specified patient, where a level below a specified threshold, which is between 20 and 200 ng/mL, is predictive of the success of therapy or intervention. 10. A method for predicting the risk of an adverse event in a patient who has developed refractory shock, wherein said method includes the steps of: determination of the level of DPP3 in a sample of biological fluid of the specified patient; correlation of a given defined level of DPP3 with the risk of an adverse event in a given patient, where an elevated level above a given threshold, which is between 20 and 200 ng/mL, is predictive of an increased risk of the given adverse events. 11. The use of a vasopressor in the treatment of shock in a patient, wherein said patient has a DPP3 level in a biological fluid sample of said patient below a predetermined threshold value, which is between 20 and 200 ng/mL. 12. Use of a DPP3 activity inhibitor in the treatment of shock in a patient, wherein said patient has a DPP3 level in a biological fluid sample of said patient above a specified threshold value, which is from 20 to 200 ng/ml, wherein the DPP3 activity inhibitor is selected from the group consisting of an anti-DPP3 antibody, or an anti-DPP3 antibody fragment, or an anti-DPP3 framework that is not related to Ig. 13. Use according to claim 12, wherein said inhibitor is intended for administration in combination with an angiotensin receptor agonist and/or its precursor. 14. The use according to claim 13, wherein said angiotensin receptor agonist and/or its precursor is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, angiotensin IV, in particular angiotensin II. 15. A method for treating shock in a patient, wherein the method comprises administering a vasopressor to said patient, wherein said patient has a DPP3 level in a sample of said patient's biological fluid below a predetermined threshold value, which is from 20 to 200 ng/ml. 16. The method according to claim 15, wherein the level of ADM-NH2 or its fragments is further determined and wherein the treatment with an anti-ADM antibody or an anti-ADM antibody fragment is started and/or continued when the level of ADM-NH2 or its fragments in said sample is above a certain threshold value, which is from 50 to 250 pg/ml, and/or wherein the treatment with an anti-ADM antibody or an anti-ADM antibody fragment is postponed and/or stopped if said determined level of ADM-NH2 or its fragments is below said specified threshold value. 17. A method for treating shock in a patient, wherein the method comprises administering an inhibitor of DPP3 activity to said patient, wherein said patient has a DPP3 level in a sample of said patient's biological fluid above a predetermined threshold value, which is from 20 to 200 ng/ml. 18. The method according to claim 17, wherein said DPP3 activity inhibitor is administered in combination with an angiotensin receptor agonist and/or its precursor. 19. The method according to claim 18, wherein the treatment with said angiotensin receptor agonists and/or DPP3 activity inhibitors is started and/or continued when the DPP3 level in the sample of said patient is above a certain threshold value, which is from 20 to 200 ng/ml. 20. The method according to claim 19, wherein the level of ADM-NH2 or its fragments is further determined, wherein the treatment with said anti-ADM antibody or anti-ADM antibody fragment is started and/or continued when the level of ADM-NH2 or its fragments in said sample is above a certain threshold value, which is from 50 to 250 pg/ml, and/or wherein the treatment with the anti-ADM antibody or anti-ADM antibody fragment is postponed and/or stopped if said determined level of ADM-NH2 or its fragments is below said specified threshold value.

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