DPP3 inhibitors for protecting myocardium and preventing myocardial injury in critical patients with blood pressure drop

Abstract

The subject matter of the present invention is a DPP3 activity inhibitor for use in therapy or intervention in critical patients with reduced blood pressure in order to protect the myocardium and / or prevent myocardial damage.

Description

Technical Field

[0001] The subject matter of this invention is a DPP3 activity inhibitor for use in the treatment or intervention of critically ill patients with hypotension, to protect the myocardium and / or prevent myocardial damage. Background Technology

[0002] Dipeptidyl peptidase 3 (also known as dipeptidyl aminopeptidase III, dipeptidyl arylamidoase III, dipeptidyl peptidase III, enkephalinase B, or erythrocyte angiotensinase; abbreviated as DPP3, DPPIII) is a metallopeptidase that removes dipeptides from physiologically active peptides such as enkephalins and angiotensin. DPP3 was identified and its activity measured in 1967 by Ellis and Nuenke in a purified extract of bovine anterior pituitary. The enzyme is listed as EC 3.4.14.4, has a molecular weight of approximately 83 kDa, and is highly conserved in prokaryotes and eukaryotes (Prajapati & Chauhan 2011). The amino acid sequence of the human variant is depicted in SEQ ID NO. 1. DPP3 is a ubiquitously expressed peptidase primarily found in the cytosol. Although a signaling sequence is lacking, several studies have reported membrane activity (Lee & Snyder 1982).

[0003] DPP3 is a zinc-dependent exopeptidase belonging to the M49 family of peptidases. It exhibits broad substrate specificity for oligopeptides of various compositions ranging from 3 / 4 to 10 amino acids and can also cleave proline residues. DPP3 is known to hydrolyze dipeptides from the N-terminus of its substrates, including angiotensin II, III, and IV, Leu- and Met-enkephalins, and endorphins 1 and 2. The metallopeptidase DPP3 exhibits optimal activity at pH 8.0–9.0 and can be further enhanced by the addition of divalent metal ions such as Co. 2+ and Mg 2+ To activate.

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

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

[0006] Recently, two assays were developed, characterized, and validated for the specific detection of DPP3 in human bodily fluids (e.g., blood, plasma, serum): a luminescent immunoassay (LIA) for detecting DPP3 protein concentration and an enzyme capture activity assay (ECA) for detecting specific DPP3 activity (Rehfeld et al., 2019. JALM 3(6): 943-953). A washing step removes all interfering substances before actual detection of DPP3 activity. Both methods are highly specific, allowing for reproducible detection of DPP3 in blood samples.

[0007] Elevated circulating DPP3 levels have been shown in patients with septic, cardiogenic, and vasodilatory shock (Rehfeld et al., 2019. JALM 3(6): 943-953). Furthermore, it is associated with an increased risk of short-term death and severe organ dysfunction in patients with cardiogenic shock (Deaniau et al., 2020. Eur J Heart Fail. 22(2):290-299). Additionally, in patients with severe sepsis or septic shock, higher initial cDPP3 levels have been associated with a greater need for organ support and vasopressors upon admission, and a longer duration of vasopressors, mechanical ventilation, or renal replacement therapy (RRT), leading to a greater need for fluid loading (Blet et al., 2021. Crit Care 25: 61).

[0008] WO2017 / 182561 describes a method for determining the total amount of active DPP3 in a patient sample for the diagnosis of diseases associated with necrosis. It further describes a method for treating necrosis-related diseases with an antibody targeting DPP3.

[0009] WO2019 / 081595 describes DPP3 binders that target and bind to specific DPP3 epitopes and their use in the prevention or treatment of diseases associated with oxidative stress.

[0010] WO2021 / 185786 describes a method for identifying DPP3 in patient samples for the diagnosis, risk prediction, prognosis, and surveillance of patients infected with coronavirus. It further describes a DPP3 activity inhibitor for a therapy or intervention in infected patients.

[0011] Procizumab is a humanized monoclonal IgG1 antibody that specifically binds to circulating DPP3, targeting and modulating DPP3 activity—a crucial regulator of cardiovascular function. Its mechanism of action is associated with acute disease characterized by massive cell death and the uncontrolled release of intracellular DPP3 into the bloodstream. Translocated DPP3 remains active in circulation, where it cleaves bioactive peptides in an uncontrolled manner. Procizumab blocks circulating DPP3, inhibiting the degradation of bioactive peptides in the bloodstream. This blockade leads to stabilization of cardiovascular and renal function and a reduction in short-term mortality. Preclinical studies of Procizumab in animal models of cardiovascular failure have shown impressive and immediate efficacy. In several preclinical models of cardiovascular failure, Procizumab has been shown to normalize ejection fraction and renal function and reduce mortality.

[0012] Examples in this specification demonstrate that injection of Procizumab in pigs suffering from septic shock prevented myocardial injury (given by increases in myocardial IL-6 and troponin, respectively). Furthermore, it has been shown that DPP3 is significantly elevated in patients with hypotension, particularly in shock (e.g., septic shock, cardiogenic shock) and acute coronary syndrome (ACS). Therefore, it is reasonable to infer that DPP3 inhibitors, especially Procizumab, can prevent myocardial injury in patients with hypotension, regardless of indication.

[0013] Therefore, a surprising finding of this invention is that DPP3 activity inhibitors are suitable as a therapy or intervention in critically ill patients with hypotension to protect the myocardium and / or prevent myocardial damage. Summary of the Invention

[0014] The subject matter of this invention is a DPP3 activity inhibitor for use in the treatment or intervention of critically ill patients with hypotension, to protect the myocardium and / or prevent myocardial damage.

[0015] DPP3 level

[0016] The subject matter of this invention is a DPP3 activity inhibitor for use as a therapy or intervention in critically ill patients with hypotension to protect the myocardium and / or prevent myocardial damage, wherein the patient has a DPP3 level above a predetermined threshold.

[0017] In one embodiment of the invention, the level of DPP3 protein and / or the level of active DPP3 are determined and compared with a predetermined threshold level.

[0018] The subject matter of this application is a DPP3 activity inhibitor for a therapy or intervention in critically ill patients with hypotension to protect the myocardium and / or prevent myocardial damage, wherein the patient has a DPP3 level above a predetermined threshold in the subject's bodily fluid sample when measured by different methods (e.g., immunoassay, activity assay, mass spectrometry, etc.).

[0019] DPP3 activity can be measured by detecting the cleavage products of DPP3-specific substrates. Known peptide hormone substrates include Leu-enkephalin, Met-enkephalin, endorphins 1 and 2, valorphin, β-tyrophage, dynorphin, gastreptide, ACTH (adrenocorticotropic hormone), and MSH (melanocyte-stimulating hormone). Abramić et al., 2000; Baršun et al., 2007; Dhanda et al. 2008 The peptide hormone and other unlabeled oligopeptides (e.g., Ala-Ala-Ala-Ala, ...) Dhanda et al., 2008 The cutting of a component can be monitored by detecting the corresponding cutting products. Detection methods include, but are not limited to, HPLC analysis (e.g., HPLC). Lee & Snyder 1982 ), mass spectrometry (e.g.) Abramić et al., 2000 ), H1-NMR analysis (e.g. Vandenberg et al., 1985 ), capillary zone electrophoresis (CE; for example) Baršun et al., 2007 Thin-layer chromatography (e.g.) Dhanda et al., 2008 ) or reversed-phase chromatography (e.g. Mazocco et al., 2006 ).

[0020] Detecting fluorescence generated by the hydrolysis of DPP3 on fluorescent substrates is a standard procedure for monitoring DPP3 activity. These substrates are specific dipeptides or tripeptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) conjugated to fluorophores. Fluoresceins include, but are not limited to, β-naphthamide (2-naphthamide, βNA, 2NA), 4-methoxy-β-naphthamide (4-methoxy-2-naphthamide), and 7-amido-4-methylcoumarin (AMC, MCA). Abramić et al., 2000; Ohkubo et al., 1999 Cleavage of these fluorescent substrates leads to the release of fluorescent β-naphthylamine or 7-amino-4-methylcoumarin, respectively. In the liquid chromatography method, the ECA substrate and DPP3 are incubated in, for example, a 96-well plate, and fluorescence is measured using a fluorescence detector. Ellis & Nuenke 1967In addition, samples carrying DPP3 can be immobilized and separated on a gel by electrophoresis, the gel can be stained with a fluorescent substrate (e.g., Arg-Arg-βNA) and Fast Garnet GBC, and the fluorescent protein bands can be detected by a fluorescence reader. Ohkubo et al. 1999 The same peptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) can be coupled to chromophores such as p-nitroaniline diacetate. Detecting color changes caused by the hydrolysis of the chromophore substrate can be used to monitor DPP3 activity.

[0021] Another option for detecting DPP3 activity is Protease-Glo TM Assay (available commercially from Promega). In this embodiment of the method, a DPP3-specific dipeptide or tripeptide (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) is conjugated with aminoluciferin. Upon cleavage with DPP3, aminoluciferin is released and used as a substrate for a luciferase-coupled reaction, which emits detectable luminescence.

[0022] In a preferred embodiment, DPP3 activity is measured by adding the fluorescent substrate Arg-Arg-βNA and monitoring the fluorescence in real time.

[0023] In another embodiment of the invention, the DPP3 level is determined by contacting the body fluid sample with a capture binder that specifically binds to DPP3.

[0024] In another preferred embodiment of the invention, the capture binder used to determine the DPP3 level may be selected from antibodies, antibody fragments, or non-IgG scaffolds.

[0025] In one particular embodiment of the invention, the capture binder used to determine the DPP3 level is an antibody.

[0026] Another specific embodiment of the invention includes the use of a capture binder that specifically binds to full-length DPP3.

[0027] In another preferred embodiment of the invention, the capturing binder is fixed on a solid phase.

[0028] The test sample is passed through a fixed binder, and DPP3 (if present in the sample) binds to the binder and is immobilized, ready 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 test sample. Alternatively, DPP3 bound to the solid phase can be detected using a second trapping molecule that specifically binds to DPP3.

[0029] For the purposes of this specification, the term "solid phase" may be used to include any material or container in which or on which measurements may be performed, including but not limited to porous materials, non-porous materials, test tubes, pores, glass slides, agarose resins (e.g., Sepharose from GE Healthcare Life Sciences), and magnetic particles (e.g., Dynabeads from Thermo Fisher Scientific). TM Or Pierce TM (Magnetic beads, etc.)

[0030] In one embodiment of the present invention, a method for determining the DPP3 activity in the subject's bodily fluid sample includes the following steps:

[0031] ● Contact the sample with a trapping binder that specifically binds to full-length DPP3.

[0032] ● Separate DPP3 bound to the capturing binder.

[0033] ● Add DPP3 substrate to the separated DPP3.

[0034] ● The DPP3 activity is quantified by measuring and quantifying the conversion of the DPP3 substrate.

[0035] In another embodiment of the invention, the separation step is a washing step, which removes components of the sample that are not bound to the capturing binder from the captured DPP3.

[0036] In another embodiment of the invention, the DPP3 substrate conversion is detected by a method selected from the group consisting of: fluorescence of a fluorescent substrate (e.g., Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate coupled with an aminoluciferin, mass spectrometry, HPLC / FPLC (reversed-phase chromatography, size exclusion chromatography), thin-layer chromatography, capillary zone electrophoresis, gel electrophoresis, and subsequent active staining (immobilization, active DPP3) or Western blot (cleavage product).

[0037] In another embodiment of the invention, the substrate may be selected from the group consisting of angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endorphin 1 and 2, valorphin, β-tyrophin, dynorphin, gastreptide, ACTH and MSH, or a dipeptide coupled to a fluorophore, chromophore or aminofluorescein, wherein the dipeptide is Arg-Arg.

[0038] In another specific embodiment of the invention, the substrate may be selected from the group consisting of dipeptides coupled to a fluorophore, chromophore, or aminofluorescein, wherein the dipeptide is Arg-Arg.

[0039] In one particular embodiment, the binder exhibits at least 10% resistance to DPP3. 7 M -1 Preferably 10 8 M -1 Binding affinity, preferably greater than 10. 9 M -1 The optimal selection is greater than 10. 10 M -1 Those skilled in the art will understand that lower affinity can be compensated for by applying higher doses of the compound, and such measures do not exceed the scope of the invention.

[0040] To determine the antibody affinity for DPP3, the binding kinetics of DPP3 to the immobilized antibody were determined using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany) with label-free surface plasmon resonance. Reversible immobilization of the antibody was performed according to the manufacturer's instructions (Mouse Antibody Capture Kit; GE Healthcare) using anti-mouse Fc antibody covalently coupled to the surface of a CM5 sensor at high density (Lorenz et al., 2011. Antimicrob Agents Chemother. 55(1): 165–173).

[0041] In one embodiment, the assay for determining DPP3 levels is a sandwich immunoassay using any type of detection technology, including but not limited to enzyme labeling, chemiluminescent labeling, and electrochemiluminescent labeling, preferably a fully automated assay. In one embodiment of the diagnostic method, this assay is an enzyme-labeled sandwich assay. Examples of automated or fully automated assays include those usable in systems such as Roche Elecsys®, AbbottArchitect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, and AlereTriage®.

[0042] Various immunoassays are known and can be used in the assays and methods of this invention, including: mass spectrometry (MS), luminescent immunoassay (LIA), radioimmunoassay (“RIA”), homogeneous enzyme-multiplying immunoassay (“EMIT”), enzyme-linked immunosorbent assay (“ELISA”), apoenzyme reactivation immunoassay (“ARIS”), luminescent bead arrays, magnetic bead arrays, protein microarray assays, rapid test formats such as test strip immunoassays, immunochromatographic strip tests, rare earth cavitation compound assays, and automated systems / analyzers.

[0043] In one embodiment of the invention, it could be a so-called POC (point of care) test, a testing technique that allows testing to be performed near the patient in less than one hour without requiring a fully automated assay system. An example of this technique is an immunochromatographic assay, such as a microfluidic device.

[0044] In one particular embodiment, at least one of the two binding agents is labeled in the sandwich immunoassay for detection.

[0045] In another preferred embodiment, the label is selected from the group consisting of chemiluminescent labels, enzyme labels, fluorescent labels, and radioactive iodine labels.

[0046] The assay can be a homogeneous or heterogeneous assay, or a competitive or non-competitive assay. In one embodiment, the assay takes the form of a sandwich assay, a non-competitive immunoassay in which the molecule to be detected and / or quantified is bound to a first antibody and a second antibody. The first antibody may be bound to a solid phase, such as the surface of beads, pores, or other containers, a chip, or a strip, and the second antibody is an antibody labeled, for example, with a dye, a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general composition and procedures of a sandwich assay are well-established and known to those skilled in the art. Immunoassay The Immunoassay Handbook, edited by David Wild, Elsevier LTD, Oxford; 3rd edition, (May) 2005), ISBN-13: 978-0080445267; Hultschig C et al., Curr Opin Chem Biol. 2006 Feb;10 (1):4-10. PMID: 16376134 ).

[0047] In another embodiment, the assay includes two capture molecules, preferably antibodies, both present in dispersion form in a liquid reaction mixture, wherein a first labeling component is attached to the first capture molecule, wherein the first labeling component is part of a labeling system based on fluorescence or chemiluminescence-quenching or amplification, and a second labeling component of the labeling system is attached to the second capture molecule, such that a measurable signal is generated after the two capture molecules bind to the analyte, allowing detection of a sandwich complex formed in a solution containing the sample.

[0048] In another embodiment, the labeling system comprises a rare earth cavitary compound or rare earth chelate in combination with a fluorescent dye or a chemiluminescent dye, particularly anthocyanin-type dyes.

[0049] In the context of this invention, fluorescence-based assays include the use of dyes, which may be selected, for example, 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, xamethonium, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HE) Rhodamine, TET, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes such as BODIPY TMR, Oregon Green, coumarins such as umbelliferone, benzoimides such as Hoechst 33258, phenanthridines such as Texas Red, Imidacloprid Yellow, Alexa Fluor, PET, ethidium bromide, acridine dyes, carbazole dyes, phenazine dyes, porphyrin dyes, polyacetylenes dyes, etc.

[0050] In the context of this invention, chemiluminescence-based assays include the use of dyes based on the physical principles described for chemiluminescent materials in the following literature (Kirk-Othmer, *Encyclopedia of Chemical Technology*, 4th edition, 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). A preferred chemiluminescent dye is an acridine ester.

[0051] The term "assay" or "diagnostic assay" used herein can refer to any type applied in the diagnostic field. Such assays can be based on the binding of the analyte to be detected to one or more capture probes with a certain affinity. Regarding the interaction between the capture molecule and the target molecule or molecule of interest, the affinity constant is preferably greater than 10. 8 M -1 .

[0052] The subject matter of this invention is a DPP3 activity inhibitor used as a therapy or intervention in critically ill patients with hypotension to protect the myocardium and / or prevent myocardial damage, wherein a predetermined threshold for the DPP3 level in the subject's bodily fluid sample is between 20 and 120 ng / mL, more preferably between 30 and 80 ng / mL, even more preferably between 40 and 60 ng / mL, and most preferably the threshold is 50 ng / mL.

[0053] In one particular embodiment, a assay is used to determine DPP3 levels, wherein the assay has a sensitivity capable of quantifying DPP3 in healthy subjects and is < 20 ng / ml, preferably < 30 ng / ml, more preferably < 40 ng / ml.

[0054] In one particular embodiment, the bodily fluid according to the invention is a blood sample. The blood sample may be selected from the group consisting of whole blood, serum, and plasma. In one particular embodiment of the method, the sample is selected from the group consisting of human citrate plasma, heparin plasma, and EDTA plasma.

[0055] In another specific embodiment of the invention, the DPP3 level is determined from different samples obtained from the patient at different time points.

[0056] In another specific embodiment of the invention, the differences in DPP3 levels between different samples obtained from the patient at different time points are determined. These differences can be determined as absolute or relative differences.

[0057] In one particular embodiment of the invention, the DPP3 level is determined at least twice.

[0058] In another specific embodiment of the invention, the therapy is initiated when the relative difference between the DPP3 levels in different samples obtained from the patient at different time points is 100% or more, more preferably 75% or more, even more preferably 50% or more, and most preferably 25% or more.

[0059] In another specific embodiment of the invention, the at least second determination of the DPP3 level is performed within 2 hours, preferably within 4 hours, more preferably within 6 hours, even more preferably within 12 hours, even more preferably within 24 hours, and most preferably within 48 hours.

[0060] The amount of DPP3 protein and / or the level of DPP3 activity in the subject's bodily fluid sample can be determined, for example, by one of the following methods:

[0061] 1. A luminescent immunoassay (LIA) for quantifying DPP3 protein concentration ( Rehfeld et al., 2019 JALM 3 (6): 943-953 ).

[0062] The LIA is a one-step chemiluminescent sandwich immunoassay using white, highly binding polystyrene microtiter plates as the solid phase. These plates are coated with monoclonal anti-DPP3 antibody AK2555 (capture antibody). The tracer anti-DPP3 antibody AK2553 is labeled with MA70-acrididine-NHS-ester and used at a concentration of 20 ng per well. 20 μL of sample (e.g., serum, heparinized plasma, citrate plasma, or EDTA plasma derived from patient blood) and calibrators are aspirated into the coated white microtiter plates. After adding the tracer antibody AK2553, the microtiter plates are incubated at room temperature and 600 rpm for 3 h. Unbound tracer is then removed by four washing steps (350 μL per well). The residual chemiluminescence for 1 s in each well is measured using a microtiter plate chemiluminometer. The concentration of DPP3 is determined using a 6-point calibration curve. It is preferable to run two parallel samples and calibrators.

[0063] 2. Enzyme capture activity assay (ECA) for quantifying DPP3 activity ( Rehfeld et al., 2019 JALM 3(6): 943-953 ).

[0064] The ECA is a DPP3-specific activity assay using black, highly binding polystyrene microtiter plates as the solid phase. These plates are coated with a monoclonal anti-DPP3 antibody AK2555 (capture antibody). 20 μL of sample (e.g., serum, heparinized plasma, citrate plasma, cerebrospinal fluid, and urine) and calibrators are pipetted into the coated black microtiter plates. After adding assay buffer (200 μL), the microtiter plates are incubated at 22 °C and 600 rpm for 2 h. DPP3 present in the sample is immobilized by binding to the capture antibody. Unbound sample components are removed by four washing steps (350 μL per well). The specific activity of the immobilized DPP3 is measured by adding the fluorescent substrate Arg-Arg-β-naphthylamide (Arg2-βNA) to the reaction buffer and then incubating at 37 °C for 1 h. DPP3 specifically cleaves Arg2-βNA into Arg-Arg dipeptide and fluorescent β-naphthylamide. Fluorescence was measured using a fluorometer with an excitation wavelength of 340 nm and emission detected at 410 nm. The activity of DPP3 was determined using a 6-point calibration curve. It is preferable to run two parallel samples and calibrators.

[0065] 3. Liquid chromatography-liquid chromatography (LAA) for quantifying DPP3 activity (from...) Jones et al., Analytical Biochemistry, 1982 Revise).

[0066] The LAA is a liquid chromatography assay that uses a black, non-bound polystyrene microtiter plate to measure DPP3 activity. 20 μL of sample (e.g., serum, heparinized plasma, citrate plasma) and calibrator are pipetted into the non-bound black microtiter plate. After adding the fluorescent substrate Arg2-βNA to the assay buffer (200 μL), the initial βNA fluorescence is measured in a fluorometer using an excitation wavelength of 340 nm (T=0), and emission is detected at 410 nm. The plate is then incubated at 37°C for 1 hour. The final fluorescence is measured (T=60). The difference between the final and initial fluorescence is calculated. DPP3 activity is determined using a 6-point calibration curve. It is preferable to run two parallel runs for both the calibrator and the sample.

[0067] As outlined in the embodiments, the DPP3 level of the present invention has been determined using the described DPP3 assay method. Rehfeld et al., 2019. JALM 3(6): 943-953The values ​​of the above thresholds may differ in other assays if they are calibrated differently from the assay system used in this invention. Therefore, the above cutoff values ​​should accordingly take into account calibration differences and apply to assays with such different calibrations. One possibility for quantifying calibration differences is to perform a methodological comparison analysis (correlation) between the assay in question and the corresponding biomarker assay used in this invention by measuring the corresponding biomarker (e.g., DPP3) in the sample using two methods. Another possibility is to determine the median level of the biomarker in a representative normal population using the assay in question (given the test's sufficient analytical sensitivity), compare the results with the median levels of the biomarker described in the literature, and recalculate the calibration based on the differences obtained through this comparison. Using the calibration used in this invention, samples from 5400 normal (healthy) subjects (Swedish single-center prospective population study (MPP-RES)) were measured: the median (interquartile range) of plasma DPP3 was 14.5 ng / ml (11.3 ng / ml – 19 ng / ml).

[0068] Threshold levels can be obtained, for example, from a Kaplan-Meier analysis, where the occurrence of disease is associated with the quartiles of the biomarkers described in the population. According to this analysis, subjects with biomarker levels above the 75th percentile had a significantly increased risk of developing the disease according to the invention. This result was further supported by Cox regression analysis with adequate adjustment for classical risk factors: the highest quartile was highly significantly associated with an increased risk of developing the disease according to the invention compared to all other subjects.

[0069] Other preferred cutoff values ​​are, for example, the 90th, 95th, or 99th percentile in the normal population. Using a percentile higher than the 75th percentile can reduce the number of false positives identified, but may miss identifying subjects with moderate risk (although still increased). Therefore, one can choose a cutoff value based on whether it is more appropriate to identify the majority of at-risk subjects at the cost of also identifying "false positives," or whether it is more appropriate to primarily identify high-risk subjects at the cost of missing a few moderately at-risk subjects.

[0070] Indications

[0071] As used herein, the term "patient" refers to a living human or non-human organism receiving or deserving medical care due to illness. This includes individuals undergoing pathological examination without a known disease. Therefore, the methods and assays described herein are applicable to both human and veterinary diseases.

[0072] Myocardial injury is defined as an elevation of cardiac troponin levels above the 99th percentile of the upper limit of reference. Specifically, myocardial injury is structural damage to cardiomyocytes and tissues (such as cardiomyocytes, cardiac fibroblasts, smooth muscle cells, or endothelial cells).

[0073] It is considered a prerequisite for diagnosing myocardial infarction, but it is also an entity in itself and may originate from non-ischemic or non-cardiac conditions. Thygesen et al., 2018, The Fourth Universal Definition of Myocardial Infarction Definition of Myocardial Infarction), (2018). Eur Heart J. 40(3): 237-69; Chapman et al., 2016, Assessment and classification of patients with myocardial injury and infarction in clinical practice. Classification of Patients with Myocardial Injury and Infarction in Clinical Practice), Heart 103(1): 10-8 The term "myocardial injury" may be used in the context of direct cardiac injury such as cardiac contusion, but it can also occur in a variety of other clinical situations, such as myocardial infarction, myocarditis, sepsis, and iatrogenic injury.

[0074] Specifically, myocardial injury may have the following causes:

[0075] - Primary myocardial ischemia / myocardial infarction (rupture of atherosclerotic plaques with thrombosis).

[0076] - Myocardial oxygen supply and demand mismatch (coronary artery spasm, microvascular dysfunction, coronary artery embolism / microembolism / dissection, persistent bradycardia / tachyarrhythmia, hypovolemic shock, respiratory failure / severe anemia, left ventricular hypertrophy / hypertrophic cardiomyopathy, severe hypertension).

[0077] - Non-ischemic myocardial injury (heart failure, myocarditis / myocarditis, cardiomyopathy / Tako-tsubo cardiomyopathy, cardiac contusion, iatrogenic (revascularization, cardiac surgery, ablation, pacing, cardioversion, defibrillation), rhabdomyolysis)

[0078] - Multifactorial and systemic causes (sepsis / critical illness, cardiotoxicity (drugs), infiltrative diseases (cardiac amyloidosis, cardiac sarcoidosis), pulmonary embolism / pulmonary hypertension, acute or chronic kidney disease, stroke / subarachnoid hemorrhage).

[0079] The hypothetical mechanisms of myocardial injury include direct cardiac injury with cardiomyocyte damage, myocardial strain due to excessive wall stress, and myocardial ischemia caused by mismatch between myocardial oxygen supply and demand. Myocardial injury may be irreversible and is often accompanied by myocardial necrosis or apoptosis. Park et al., 2017, Cardiac Troponins: From Myocardial Infarction to Chronic Disease From Myocardial Infarction to Chronic Disease), Cardiovasc Res. 113(14): 1708- 18 ).

[0080] In the context of this invention, "blood pressure" refers to mean arterial pressure (MAP), which is the average arterial pressure throughout the entire cardiac cycle, during systole and diastole. MAP is affected by cardiac output and systemic vascular resistance, each of which is influenced by several variables. MAP is a major determinant of perfusion pressure observed in organs throughout the body. Current guidelines recommend a target MAP of 65 mmHg or higher for critically ill patients. Dellinger et al., 2012, Surviving the Battle Against Sepsis: Severe Sepsis and Pseudomonas aeruginosa International guidelines for the management of toxic shock (Surviving sepsis campaign: international guidelines) for management of severe sepsis and septic shock), Crit Care Med. 2013;41(2): 580–637; Peberdy et al., 2010, Post-cardiac arrest care: 2010 American Heart Association Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Post-cardiac arrest care: 2010 American Heart Association Guidelines for cardiopulmonary resuscitation and emergency cardiovascular care), Circulation 122(18 Suppl 3): S768–786 ).

[0081] In one embodiment, the blood pressure drop is MAP < 65 mmHg, more preferably < 60 mmHg, even more preferably < 55 mmHg, and most preferably < 50 mmHg.

[0082] In another embodiment, the decrease in blood pressure is a decrease in MAP of at least 5 mmHg, more preferably at least 10 mmHg, even more preferably at least 15 mmHg, and most preferably at least 20 mmHg.

[0083] Interleukin-6 (IL-6) is an important inflammatory mediator that is secreted into the circulatory system in response to acute infection and tissue damage. IL-6 expression is tightly regulated and is low in healthy individuals. Cardiac cardiomyocytes produce IL-6 under hypoxic and ischemic stress. Fuchs et al., 2003. FASEB J. 17(14): 2118–2120 This activates the JAK / STAT cascade in these cells to exert negative inotropic and cytotoxic effects. The inflammatory response mediates neutrophil infiltration and activation, triggering the release of other cytokines into the bloodstream, which collectively stimulate the vascular endothelium and induce ICAM-1 expression in cardiomyocytes, leading to myocardial fibrosis and ischemia / reperfusion injury. Gwechenberger et al., 1999. Circulation 99(4): 546– 551 This accelerates myocardial damage and dysfunction. Halawa et al., 1999. Pol. Arch. Med. Wewn 101(3): 197–203 ).

[0084] Troponin is a structural protein found in the troponin complex within skeletal and cardiac filaments. The troponin complex consists of three subunits (I, T, and C) and, together with calcium ions, plays a crucial role in the regulation of muscle contraction. Kozinski et al., 2017. Critical Reviews in Clinical Laboratory Sciences 54(3): 143–172 Each molecule plays a specific role in muscle contraction: troponin T attaches the troponin complex to actin filaments, troponin C acts as a calcium-binding site, and troponin I inhibits interaction with the myosin head in the absence of sufficient calcium ions. Garg et al., 2017. Internal and Emergency Medicine 12(2): 147– 155Troponin T and I are primarily located in the cardiac muscle and are therefore known as cardiac tropons (cTnI and cTnT). These biomarkers are generally considered to have the highest specificity in identifying myocardial injury. Chaulin 2021. Vascular Health and Risk Management 17: 299–316 If a detectable cardiac troponin concentration is found to be above the 99th percentile of the upper limit of reference (URL), myocardial injury is determined. Thygesen et al., 2019. Eur. Heart J. 40: 237–269 ).

[0085] Myocardial protection refers to the prevention of myocardial damage.

[0086] Prevention of myocardial injury is defined as the prevention of an increase in circulating cardiac troponin. Specifically, prevention of myocardial injury is defined as the prevention of structural damage to cardiomyocytes and tissues (e.g., cardiomyocytes, cardiac fibroblasts, smooth muscle cells, or endothelial cells), which is defined as an increase in circulating cardiac troponin.

[0087] In one particular implementation, myocardial injury is characterized by blood levels of cardiac troponin above a threshold, increased myocardial expression of pro-inflammatory interleukin-6 (IL-6), and / or the need for vasopressors (to maintain blood pressure and cardiac output).

[0088] The cardiac troponin is selected from the group comprising cardiac troponin T (cTnT) and cardiac troponin I (cTnI). Troponin can be measured using a high-sensitivity troponin (hs-Tn) assay. The threshold for cardiac troponin concentration is, for example, the 99th percentile above the upper limit of reference (URL). Thygesen et al., 2019. Eur. Heart J. 40: 237– 269 ).

[0089] An elevated cardiac troponin level is further defined as an elevation in cardiac high-sensitivity troponin I (hs-cTnI) or cTnT levels, where at least one value is above the 99th percentile of the upper reference limit. The reference limits are sex-dependent (higher values ​​in males than in females). Furthermore, the reference values ​​depend on the assay method used (Sandoval et al., 2022 Circulation 146: 569–581) – see Table 1 below.

[0090]

[0091] Professionals will be able to easily determine the appropriate threshold based on conventional considerations and activities known in the field, and according to the specific measurement conditions.

[0092] Vasopressors increase vasoconstriction, thereby increasing systemic vascular resistance (SVR). Increased SVR leads to increased mean arterial pressure (MAP) and increased organ perfusion. Vasopressors are selected from the group consisting of: isoproterenol, dobutamine, dopamine, phenylephrine, norepinephrine, epinephrine, vasopressin, or terlipressin.

[0093] Vasopressors are required when the mean arterial pressure (MAP) is below 65 mmHg.

[0094] The patients are critically ill patients suffering from a disease selected from severe infectious diseases, sepsis, pulmonary embolism, pulmonary hypertension, acute coronary syndromes (including unstable angina, ST-segment elevation myocardial infarction (STEMI), and non-ST-segment elevation myocardial infarction (NSTEMI)), any type of shock (including cardiogenic shock, septic shock, or anaphylactic shock), cardiac arrest, acute liver failure, and acute respiratory distress syndrome (ARDS).

[0095] "Critically ill" means that the patient has a life-threatening acute illness or condition and may or will die. In one particular implementation, the critically ill patient is an ICU patient.

[0096] The infectious disease may be of bacterial, viral, fungal, or parasitic origin. The viral infection may be caused by influenza virus or coronavirus.

[0097] The coronaviruses mentioned are selected from the group comprising SARS-CoV-1, SARS-CoV-2, and MERS-CoV, particularly SARS-CoV-2. Coronaviruses cause disease in mammals and birds. In humans, these viruses cause respiratory infections, ranging from the usually mild common cold to rare and potentially fatal forms such as SARS, MERS, and COVID-19. SARS-CoV-1 or -2 infection can manifest as mild, moderate, or severe illness; the latter includes severe pneumonia, acute respiratory distress syndrome (ARDS), sepsis, and septic shock.

[0098] Acute respiratory distress syndrome (ARDS) is a respiratory failure characterized by a rapid onset of widespread inflammation in the lungs. Symptoms include shortness of breath, rapid breathing, and bluish skin. A decline in quality of life is common for survivors. Causes may include sepsis, pancreatitis, trauma, pneumonia, and aspiration. Underlying mechanisms include diffuse damage to cells that form the microscopic air sac barrier of the lungs, surfactant dysfunction, immune system activation, and impaired coagulation regulation. Essentially, ARDS impairs the lungs' ability to exchange oxygen and carbon dioxide. Diagnosis is based on a PaO2 / FiO2 ratio (the ratio of arterial oxygen partial pressure to inhaled oxygen fraction) less than 300 mmHg, despite a positive end-expiratory pressure (PEEP) greater than 5 cm H2O. Primary treatment includes mechanical ventilation and treatment targeting the underlying cause. Ventilation strategies include the use of low-volume, low-pressure ventilation. If oxygenation remains inadequate, lung recruitment maneuvers and neuromuscular blocking agents may be used. If this is still insufficient, extracorporeal membrane oxygenation (ECMO) may be an option. The syndrome is associated with a mortality rate between 35% and 50%.

[0099] Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection (see [link to relevant documentation]). Singer et al., 2016. JAMA 315(8): 801-810 Organ dysfunction can be identified as an acute change in total SOFA score ≥2 post-infection. In patients with no known pre-existing organ dysfunction, a baseline SOFA score of zero can be assumed. A SOFA score ≥2 reflects an overall mortality risk of approximately 10% in the general hospital population with suspected infection. Even patients presenting with mild dysfunction may deteriorate further, highlighting the severity of the condition and the need for timely and appropriate intervention (if not already taken). Sepsis is a life-threatening condition that occurs when the body's response to infection damages its own tissues and organs. Suspected infection patients who may require long-term ICU stays or die in the hospital can be promptly identified at the bedside via qSOFA (i.e., altered mental status, systolic blood pressure ≤100 mm Hg, or respiratory rate ≥22 / min).

[0100] Shock is characterized by reduced oxygen delivery and / or increased oxygen consumption or insufficient oxygen utilization, leading to cellular and tissue hypoxia. It is a life-threatening circulatory failure, most commonly manifested as hypotension (systolic blood pressure below 90 mmHg or MAP below 65 mmHg). Based on the underlying cause, shock is classified into four main types: hypovolemic shock, cardiogenic shock, obstructive shock, and distributive shock. Vincent and De Backer 2014. N. Engl. J. Med. 370(6): 583 ).

[0101] Septic shock is a potentially fatal medical condition that occurs when sepsis (organ damage or impairment in response to infection) leads to dangerous hypotension and cellular metabolic abnormalities. The third international consensus definition of sepsis and septic shock (Sepsis-3) defines septic shock as a subclass of sepsis in which particularly severe circulatory, cellular, and metabolic abnormalities compared to sepsis alone are associated with a greater risk of death. Patients with septic shock are clinically identified by the need for vasopressors to maintain a mean arterial pressure of 65 mm Hg or higher in the absence of hypovolemia and serum lactate levels above 2 mmol / L (>18 mg / dL). This combination is associated with over 40% of hospital-acquired mortality. Singer et al., 2016. JAMA. 315(8): 801–10 Primary infections are most commonly caused by bacteria, but can also be caused by fungi, viruses, or parasites. They can occur anywhere in the body, but are most common in the lungs, brain, urinary tract, skin, or abdominal organs. They can cause multiple organ dysfunction syndrome (formerly known as multiple organ failure) and death. People with septic shock are typically cared for in the intensive care unit. It most commonly affects children, immunocompromised individuals, and the elderly, because their immune systems cannot respond to infections as effectively as those of healthy adults. The mortality rate of septic shock is approximately 25-50%.

[0102] Cardiogenic shock (CS) is defined as a state of inadequate perfusion of critical end organs due to reduced cardiac output. Notably, CS forms a spectrum of diseases ranging from mild hypoperfusion to deep shock. The established diagnostic criteria for CS are: (i) systolic blood pressure ≤90 mmHg for >30 minutes, or requiring vasopressors to achieve blood pressure ≥90 mmHg; (ii) pulmonary congestion or elevated left ventricular filling pressure; (iii) signs of impaired organ perfusion with at least one of the following criteria: (a) altered mental status; (b) cold and 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) followed by ventricular dysfunction is the most common cause of CS, accounting for approximately 80% of cases. Mechanical complications such as ventricular septal rupture (4%) or free wall rupture (2%) and acute severe mitral regurgitation (7%) are less common causes of CS after AMI. Hochman et al., 2000. J Am Coll Cardiol 36: 1063–1070 Non-AMI-related CS can be caused by decompensated valvular heart disease, acute myocarditis, arrhythmias, etc., and has different treatment options. This means there are 40,000 to 50,000 patients annually in the United States and 60,000 to 70,000 patients annually in Europe. Although treatment progress has been made mainly through early revascularization and subsequent mortality reduction, according to recent registry and randomized trials, CS remains the leading cause of death from AMI, with a mortality rate still close to 40-50%. Goldberg et al., 2009. Circulation 119: 1211–1219 ).

[0103] Acute coronary syndrome (ACS) refers to a group of diseases that reduce blood flow to the heart and includes ST-segment elevation myocardial infarction (STEMI), non-ST-segment elevation myocardial infarction (NSTEMI), and unstable angina. It is a type of coronary heart disease (CHD) that causes one-third of all deaths in people over 35 years of age. Some forms of CHD may be asymptomatic, but ACS is always symptomatic. According to guidelines, acute myocardial infarction is defined as follows: when there is acute myocardial injury, clinical evidence of acute myocardial ischemia, and an increase and / or decrease in cardiac troponin (cTn) levels, with at least one value above the 99th percentile of the upper limit of reference (URL), and at least one symptom of myocardial ischemia, new ischemic ECG changes, development of pathological Q waves, radiographic evidence of new loss of viable myocardium, or new regional wall motion abnormalities consistent with an ischemic etiology, or when a coronary thrombus is identified by angiography or autopsy, the term acute myocardial infarction should be used. Thygesen et al., 2018, Fourth Universal Definition of Myocardial Infarction Infarction) (2018), Eur Heart J. 40(3): 237-69 In addition, there are myocardial infarctions associated with coronary procedures such as percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG).

[0104] DPP3 inhibitors

[0105] Inhibitors are preferred molecules that significantly inhibit DPP3 activity. These molecules can be peptides and small molecules, antibodies, antibody fragments, or non-Ig scaffolds.

[0106] Significant inhibition means inhibiting DPP3 activity by more than 60%, preferably more than 70%, more preferably more than 80%, more preferably more than 90%, and more preferably almost or practically 100% inhibition.

[0107] DPP3 activity can be nonspecifically inhibited by various universal protease inhibitors (e.g., PMSF, TPCK), thiol reagents (e.g., pHMB, DTNB), and metal chelators (EDTA, o-phenanthroline). Abramić et al., 2000. Biological Chemistry, 381: 1233–1243; EP 2949332 ).

[0108] DPP3 activity can be further specifically inhibited by different types of compounds: endogenous DPP3 inhibitors include the peptide spinorphin. Several synthetic derivatives of spinorphin, such as tynorphin, have been prepared, and they have shown varying degrees of inhibition of DPP3 activity. Yamamoto et al., 2000. Life sciences 62(19): 1767–1773 Other published DPP3 peptide inhibitors are propioxatin A and B (US 4804676) and propioxatin A analogues (…). Inaoka et al. 1988. J. Biochem 104(5): 706–711 ).

[0109] "Derivatives or analogs" are compounds derived from parent compounds through chemical reactions, in which one or a group of atoms is replaced by a functional group. The parent and derived compounds have similar chemical structures.

[0110] DPP3 can also be inhibited by small molecules such as fluostatin and benzimidazole derivatives. Fluostatin A and B are antibiotics produced in Streptomyces sp. TA-3391, which are non-toxic and strongly inhibit DPP3 activity. To date, 20 different benzimidazole derivatives have been synthesized and published. Agić et al., 2007. Bioorganic Chemistry 35(2): 153–169; Rastija et al., 2015. Acta Chimica Slovenica 62: 867–878 Of these, compounds 1' and 4' showed the strongest inhibitory effects. Agić et al., 2007. Bioorganic Chemistry 35 (2): 153–169 Several dipeptidyl isohydroxamic acids have also been shown to inhibit DPP3 activity. Cvitešić et al., 2016. J Enzyme Inhib Med Chem 31(sup2):40-45 ).

[0111] "Small molecules" specifically refer to organic compounds with low molecular weight (more specifically ≤ 1000 Daltons). Specifically, such small molecules can modulate biological processes, for example, by binding to specific biomacromolecules, specifically DPP3 in this invention, and acting as effectors, particularly inhibitors, to alter the activity or function of the biomacromolecules. Small molecules can be of natural origin or synthetic.

[0112] Specific examples of small molecule and peptide inhibitors of DPP3 are shown in Table 2 below. Compounds 1' and 4' exhibited the strongest inhibitory activity (Agic et al., 2007).

[0113] Table 1: Peptides and Small Molecule Inhibitors of DPP3

[0114] The subject matter of this application is a DPP3 activity inhibitor for a therapy or intervention in critically ill patients with hypotension to protect the myocardium and / or prevent myocardial damage, wherein the DPP3 activity inhibitor is selected from the group consisting of: small molecules, anti-DPP3 antibodies, anti-DPP3 antibody fragments, or anti-DPP3 non-Ig stents.

[0115] The subject matter of this application is a DPP3 activity inhibitor for use in a therapy or intervention for critically ill patients with hypotension, to protect the myocardium and / or prevent myocardial damage, wherein the DPP3 activity inhibitor is a small molecule selected from the group consisting of spinorphin, tynorphin, propioxatin A and B, fluostatin A and B, benzimidazole or derivatives or analogs thereof.

[0116] The subject matter of this application is a DPP3 activity inhibitor for use in a therapy or intervention for critically ill patients with hypotension, to protect the myocardium and / or prevent myocardial damage, wherein said inhibitor has a minimum binding affinity to DPP3 equal to or less than 10. -7 M anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold.

[0117] The subject matter of this application is a DPP3 activity inhibitor for use in a therapy or intervention for critically ill patients with hypotension, to protect the myocardium and / or prevent myocardial damage, wherein said antibody is a monoclonal antibody or a fragment of a monoclonal antibody.

[0118] Throughout this specification, the “antibody” or “antibody fragment” or “non-Ig scaffold” according to the invention is capable of binding to DPP3, and is therefore targeted at DPP3, and can thus be referred to as an “anti-DPP3 antibody,” an “anti-DPP3 antibody fragment,” or an “anti-DPP3 non-Ig scaffold.”

[0119] The term "antibody" generally includes monoclonal and polyclonal antibodies and their binding fragments, particularly the Fc-fragment, as well as so-called "single-chain antibodies" ( Bird et al., 1988 ), chimeric, humanized, especially CDR transplantation antibodies and dimer or tetrasomal antibodies ( Holliger et al., 1993 This also includes immunoglobulin-like proteins selected by techniques including, for example, phage display, that specifically bind to molecules of interest contained in a sample. In this context, the term "specific binding" refers to an antibody generated against a molecule of interest or a fragment thereof. An antibody is considered specific if its affinity for the molecule of interest or its aforementioned fragment is at least 50 times, more preferably 100 times, and most preferably at least 1000 times, higher than its affinity for other molecules contained in a sample containing said molecule of interest. How to manufacture antibodies and select antibodies with a given specificity is well known in the art.

[0120] In one embodiment of the present invention, the anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold is single-specific.

[0121] A monospecific anti-DPP3 antibody, a monospecific anti-DPP3 antibody fragment, or a monospecific anti-DPP3 non-Ig scaffold means that the antibody, antibody fragment, or non-Ig scaffold binds to a specific region within the target DPP3 (SEQ ID No. 1) that covers at least 5 amino acids. A monospecific anti-DPP3 antibody, a monospecific anti-DPP3 antibody fragment, or a monospecific anti-DPP3 non-Ig scaffold is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that all have affinity for the same antigen. Monoclonal antibodies are monospecific, but monospecific antibodies can also be produced by means other than those produced from common embryonic cells.

[0122] In one particular embodiment, the anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold is an inhibitory antibody, fragment, or non-Ig scaffold. The anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold inhibits DPP3 activity by more than 50%, preferably more than 60%, preferably more than 70%, more preferably more than 80%, preferably more than 90%, even more preferably more than 95%, and preferably almost or practically 100%.

[0123] The antibodies or fragments according to the invention are proteins comprising one or more polypeptides essentially encoded by immunoglobulin genes that specifically bind to antigens. Recognized immunoglobulin genes include constant region genes for κ, λ, α (IgA), γ (IgG1, IgG2, IgG3, IgG4), δ (IgD), ε (IgE), and μ (IgM), as well as numerous variable region genes for immunoglobulins. Full-length immunoglobulin light chains are typically about 25 kDa or 214 amino acids in length.

[0124] The full-length immunoglobulin heavy chain is typically about 50 kDa or 446 amino acids in length. The light chain is encoded by a variable region gene (approximately 110 amino acids long) located at the NH2 terminus and a κ or λ constant region gene located at the COOH terminus. The heavy chain is also encoded by a variable region gene (approximately 116 amino acids long) and one of the remaining constant region genes.

[0125] The basic structural unit of an antibody is typically a tetramer composed of two identical pairs of immunoglobulin chains, each pair consisting of one light chain and one heavy chain. In each pair, the variable regions of the light and heavy chains bind to the antigen, while the constant regions mediate effector function. Immunoglobulins also exist in a variety of other forms, including, for example, Fv, Fab, and (Fab')2, as well as bifunctional hybrid antibodies and single-chain antibodies (e.g., [missing information]). Lanzavecchia et al., 1987. Eur. J. Immunol. 17:105; Huston et al., 1988. Proc. Natl. Acad. Sci. USA, 85:5879-5883; Bird et al., 1988. Science 242:423-426; Hood et al., 1984, Immunology, Benjamin, NY, 2nd ed.; Hunkapille and Hood 1986. Nature 323:15-16The variable regions of the immunoglobulin light or heavy chains include structural regions interrupted by three hypervariable regions, also known as complementarity-determining regions (CDRs) (see Immunologically Significant Protein Sequences). Sequences of Proteins of Immunological Interest), E. Kabat et al., 1983, US Department of Health and Human Services As noted above, CDRs are primarily responsible for binding to epitopes of antigens. Immune complexes are antibodies, such as monoclonal antibodies, chimeric antibodies, humanized antibodies, or human antibody or functional antibody fragments, that specifically bind to said antigen.

[0126] Chimeric antibodies are antibodies whose light and heavy chain genes are typically constructed by genetic engineering from variable and constant region genes of immunoglobulins from different species. For example, a variable region from a mouse monoclonal antibody gene can be linked to a human constant region such as κ and γ1 or γ3. Thus, in one instance, a therapeutic chimeric antibody is a hybrid protein consisting of a variable or antigen-binding domain from a mouse antibody and a constant or effector domain from a human antibody, although other mammalian species can also be used, or the variable region can be generated by molecular techniques. Methods for manufacturing chimeric antibodies are well known in the art, see, for example, U.S. Patent No. 5,807,715. A “humanized” immunoglobulin is an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDR is referred to as the “donor,” and the human immunoglobulin providing the framework is referred to as the “recipient.” In one embodiment, in a humanized immunoglobulin, all CDRs are derived from the donor immunoglobulin. Constant regions may not necessarily exist, but if they do, they must be substantially identical to the constant regions of human immunoglobulins, i.e., at least about 85-90%, for example, about 95% or higher. Therefore, it is possible that all parts of a humanized immunoglobulin, except for the CDR, are substantially identical to their corresponding parts of the natural human immunoglobulin sequence. A “humanized antibody” is an antibody comprising humanized light chains and humanized heavy chains of immunoglobulin. The humanized antibody binds to the same antigen as the donor antibody that provides the CDR. The receptor framework of the humanized immunoglobulin or antibody may have a limited number of amino acid substitutions derived from the donor framework. Humanized or other monoclonal antibodies may have other conserved amino acid substitutions that substantially do not affect antigen binding or the function of other immunoglobulins. Exemplary conserved substitutions are, for example: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed using genetic engineering (see, for example, U.S. Patent No. 5,585,089). Human antibodies are antibodies 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 human B cells that secrete the target antibody. Immortification can be achieved, for example, through EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce trivalent hybridoma cells. Human antibodies can also be produced using phage display methods (see, for example...). WO91 / 17271; WO92 / 001047; WO92 / 20791 Human antibodies can be selected from human combined monoclonal antibody libraries (see Morphosys website). Human antibodies can also be prepared using transgenic animals carrying human immunoglobulin genes (see, for example, [link to Morphosys website]). WO93 / 12227; WO 91 / 10741 ).

[0127] Therefore, the anti-DPP3 antibody can have a format known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, and CDR transplantation antibodies. In a preferred embodiment, the antibody according to the invention is a recombinant antibody such as IgG, a typical full-length immunoglobulin, or an antibody fragment containing at least a heavy chain and / or a light chain F-variable domain, such as a chemically conjugated antibody (antigen-binding fragment), including but not limited to Fab fragments, including Fab microbodies, single-chain Fab antibodies, monovalent Fab antibodies with epitope tags such as Fab-V5Sx2; divalent Fab (microbodies) dimerized with a CH3 domain; divalent or multivalent Fab, for example formed by polymerization with the help of a heterodomain, such as by dimerization of a dHLX domain, such as Fab-dHLX-FSx2; F(ab')2 fragments, scFv fragments, multivalent and / or multispecific scFv fragments, divalent and / or bispecific dimers, BITE ® (Bispecific T-cell adaptors), trifunctional antibodies, multivalent antibodies, such as those derived from classes other than G; single-domain antibodies, such as nanobodies derived from camel or fish immunoglobulins, etc.

[0128] In one preferred embodiment, the anti-DPP3 antibody format is selected from the group consisting of: Fv fragments, scFv fragments, Fab fragments, scFab fragments, F(ab)2 fragments, and scFv-Fc fusion proteins. In another preferred embodiment, the antibody format is selected from the group consisting of: scFab fragments, Fab fragments, scFv fragments, and bioavailability-optimized conjugates thereof, such as PEGylated fragments. One of the most preferred formats is the scFab format.

[0129] Non-Ig scaffolds can be protein scaffolds and can be used as antibody mimics because they are able to bind to ligands or antigens. In one embodiment, non-Ig scaffolds can be selected from the group consisting of: tetraligin-based non-Ig scaffolds (e.g., in... US 2010 / 0028995 As described in [the text]), fibronectin scaffolds (e.g. in [the text]). EP 1 266 025 (as described in) lipocalcin-based scaffolds (e.g. in WO 2011 / 154420 As described in [the text], pervasive protein scaffolds (e.g., in [the text]). WO 2011 / 073214 As described in [the text]), transferrin scaffolds (e.g., in [the text]). US 2004 / 0023334 As described in [the text]), protein A scaffold (e.g. in [the text]). EP 2 231 860 As described in [the text], scaffolds based on ankyrin repeat sequences (e.g., in [the text]). WO 2010 / 060748 As described in [the text]), microprotein (preferably microproteins that form cysteine ​​knots) scaffolds (e.g. in [the text]). EP 2314308As described in [the text], scaffolds based on the Fyn SH3 domain (e.g., in [the text]). WO 2011 / 023685 As described in [the text], scaffolds based on EGFR-A structural domains (e.g., in [the text]). WO 2005 / 040229 (as described in) and scaffolds based on Kunitz structural domains (e.g. in) EP 1 941 867 (As described in the text).

[0130] In one embodiment of the invention, the anti-DPP3 antibody according to the invention can be produced, as outlined in Example 1, by synthesizing a DPP3 fragment or full-length DPP3 as an antigen. Subsequently, the binder of the fragment is identified using the methods described below or other methods known in the art.

[0131] Humanization of mouse antibodies can be performed according to the following procedure:

[0132] To humanize the murine antibody, the structural interactions between the scaffold region (FR) and complementarity-determining region (CDR) of the antibody sequence and the antigen were analyzed. Based on structural modeling, a suitable human-derived FR was selected, and the murine CDR sequence was transplanted into the human FR. Mutations can be introduced into the amino acid sequences of the CDR or FR to restore structural interactions abolished by species switching in the FR sequence. This restoration of structural interactions can be achieved using phage display libraries via random methods or via a directed method guided by molecular modeling. Almagro and Fransson 2008. Humanization of antibodies. (Humanization of antibodies), Front Biosci. 2008 Jan 1;13:1619-33 ).

[0133] In another preferred embodiment, the anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold is a full-length antibody, antibody fragment, or non-Ig scaffold.

[0134] The subject matter of this application is a DPP3 activity inhibitor for a therapy or intervention in critically ill patients with hypotension to protect the myocardium and / or prevent myocardial injury, wherein the complementarity-determining region (CDR) in the heavy chain comprises the following sequence:

[0135] SEQ ID NO.: 7, SEQ ID NO.: 8 and / or SEQ ID NO.: 9,

[0136] Furthermore, the complementarity determination region (CDR) in the light chain contains the following sequence:

[0137] SEQ ID NO.: 10, KVS and / or SEQ ID NO.: 11.

[0138] The subject matter of this application is a DPP3 activity inhibitor for use in a therapy or intervention for critically ill patients with hypotension to protect the myocardium and / or prevent myocardial damage, wherein the monoclonal antibody or antibody fragment is a humanized monoclonal antibody or a humanized monoclonal antibody fragment.

[0139] The subject matter of this application is a DPP3 activity inhibitor for a therapy or intervention in critically ill patients with hypotension to protect the myocardium and / or prevent myocardial damage, wherein the heavy chain comprises the sequence SEQ ID NO.: 12, and wherein the light chain comprises the sequence SEQ ID NO.: 13.

[0140] The subject matter of this application is a DPP3 activity inhibitor for a therapy or intervention in critically ill patients with hypotension to protect the heart and / or prevent cardiac damage, wherein the inhibitor is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No. 1. In one embodiment, the inhibitor is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 amino acids in length contained in SEQ ID No. 1. In another embodiment, the inhibitor is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 5 amino acids in length contained in SEQ ID No. 1.

[0141] The subject matter of this application is a DPP3 activity inhibitor for a therapy or intervention in critically ill patients with hypotension to protect the myocardium and / or prevent myocardial injury, wherein the inhibitor is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No.: 2, and wherein the epitope is contained in DPP3 as depicted in SEQ ID No.: 1. In one embodiment, the inhibitor is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 amino acids in length contained in SEQ ID No.: 2, and wherein the epitope is contained in DPP3 as depicted in SEQ ID No.: 1. In another embodiment, the inhibitor is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 5 amino acids in length contained in SEQ ID No.: 2, and wherein the epitope is contained in DPP3 as depicted in SEQ ID No.: 1.

[0142] The subject matter of this application is a DPP3 activity inhibitor for a therapy or intervention in critically ill patients with hypotension to protect the myocardium and / or prevent myocardial injury, wherein the inhibitor is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No.: 3, and wherein the epitope is contained in DPP3 as depicted in SEQ ID No.: 1. In one embodiment, the inhibitor binds to an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 amino acids in length contained in SEQ ID No.: 3, and wherein the epitope is contained in DPP3 as depicted in SEQ ID No.: 1. In another embodiment, the inhibitor binds to an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 5 amino acids in length contained in SEQ ID No.: 3, and wherein the epitope is contained in DPP3 as depicted in SEQ ID No.: 1.

[0143] The subject matter of this application is a DPP3 activity inhibitor for a therapy or intervention in critically ill patients with hypotension to protect the myocardium and / or prevent myocardial injury, wherein the inhibitor is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No.: 4, and wherein the epitope is contained in DPP3 as depicted in SEQ ID No.: 1. In one embodiment, the inhibitor is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 amino acids in length contained in SEQ ID No.: 4, and wherein the epitope is contained in DPP3 as depicted in SEQ ID No.: 1. In another embodiment, the inhibitor is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 5 amino acids in length contained in SEQ ID No.: 4, and wherein the epitope is contained in DPP3 as depicted in SEQ ID No.: 1.

[0144] Epitopes, also known as antigenic determinants, are the portions of an antigen that are recognized by the immune system (especially antibodies). For example, an epitope is a specific segment of an antigen that binds to an antibody. The portion of the antigen that binds to the epitope is called the complement. Epitopes of protein antigens are classified into conformational epitopes and linear epitopes based on their structure and interaction with the complement. Conformational and linear epitopes are based on the 3-D conformation of the epitope and its interaction with the complement, the conformation being determined by the surface features of the epitope residues involved and the shape or tertiary structure of other segments of the antigen. Conformational epitopes are formed by the 3-D conformation of interactions between discontinuous amino acid residues. Linear or continuous epitopes are epitopes recognized by antibodies through their linear amino acid sequence or primary structure and are formed by the 3-D conformation of interactions between continuous amino acid residues.

[0145] In one specific embodiment of the invention, the antibody is a monoclonal antibody or a fragment thereof. In one embodiment of the invention, the anti-DPP3 antibody or anti-DPP3 antibody fragment is a human or humanized antibody or derived therefrom. In one specific embodiment, one or more (mouse) CDRs are grafted into a human antibody or antibody fragment.

[0146] In one aspect, the subject matter of the present invention is a human or humanized CDR transplant antibody or antibody fragment thereof that binds to DPP3, wherein the human or humanized CDR transplant antibody or antibody fragment thereof comprises an antibody heavy chain (H chain), the heavy chain comprising:

[0147] GFSLSTSGMS (SEQ ID NO.: 7),

[0148] IWWNDNK (SEQ ID NO.: 8),

[0149] ARNYSYDY (SEQ ID NO.: 9),

[0150] And / or further comprising an antibody light chain (L chain), said light chain comprising:

[0151] RSLVHSIGSTY (SEQ ID NO.: 10),

[0152] KVS (not part of the sequence list)

[0153] SQSTHVPWT (SEQ ID NO.: 11).

[0154] In one particular embodiment of the invention, the subject matter is a human or humanized monoclonal antibody or an antibody fragment thereof that binds to DPP3, wherein the heavy chain comprises at least one CDR selected from the group consisting of:

[0155] GFSLSTSGMS (SEQ ID NO.: 7),

[0156] IWWNDNK (SEQ ID NO.: 8),

[0157] ARNYSYDY (SEQ ID NO.: 9),

[0158] And said light chain contains at least one CDR selected from the group consisting of:

[0159] RSLVHSIGSTY (SEQ ID NO.: 10),

[0160] KVS (not part of the sequence list)

[0161] SQSTHVPWT (SEQ ID NO.: 11).

[0162] The anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold according to the present invention exhibits affinity for human DPP3, such that the affinity constant is greater than 10. -7 M, preferably greater than 10 -8 M, preferably with an affinity greater than 10 -9 M, preferably higher than 10 -10 M. Those skilled in the art will understand that a lower affinity can be compensated for by applying a higher dose of the compound, and such an action will not fall outside the scope of the invention. The affinity constant can be determined according to the method described in Example 1.

[0163] The subject matter of this invention is a monoclonal antibody or fragment binding to DPP3, or an antibody fragment thereof for use in a therapy or intervention for critically ill patients with hypotension, to protect the myocardium and / or prevent myocardial damage, wherein said antibody or fragment comprises the following sequence as a variable heavy chain:

[0164] SEQ ID NO.: 5

[0165] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPSGKGLEWLAHIWWNDNKSYNPALKSRLTISRDTSNNQVFLKIASVVTADTGTYFCARNYSYDYWGQGTTLTVSS

[0166] It contains the following sequence as a variable light chain:

[0167] SEQ ID NO.: 6

[0168] DVVVTQTPLSLSVSLGDPASISCRSSRSLVHSIGSTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

[0169] The subject matter of this invention is a human or humanized monoclonal antibody or fragment that binds to DPP3, or an antibody fragment thereof for use in a therapy or intervention in critically ill patients with hypotension, to protect the myocardium and / or prevent myocardial injury, wherein said antibody or fragment comprises the following sequence as a heavy chain:

[0170] SEQ ID NO.: 12

[0171] MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQVVLTMTNMDPVDTG TYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0172] It also contains the following sequence as a light chain:

[0173] SEQ ID NO.: 13

[0174] METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHV PWTTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0175] In a specific embodiment of the invention, the antibody comprises the following sequence or a sequence with >95%, preferably >98%, preferably >99% identity with it as a heavy chain: SEQ ID NO: 12,

[0176] And contains the following sequence or a sequence with identity > 95%, preferably > 98%, preferably > 99% as a light chain: SEQ ID NO: 13.

[0177] To assess the identity between two amino acid sequences, a pairwise alignment was performed. Identity is defined as the percentage of directly matching amino acids in the alignment.

[0178] In a preferred embodiment, treatment with a DPP3 activity inhibitor is initiated or altered immediately upon receiving sample analysis results indicating the DPP3 level in the sample. In a further embodiment, the treatment may be initiated within 12 hours of receiving the sample analysis results, preferably within 6, 4, 2, 1, 0.5, 0.25 hours, or immediately.

[0179] In some embodiments, the method includes, or comprises, single and / or multiple measurements of DPP3 in samples from a patient in single and / or multiple samples obtained at substantially the same time point, in order to guide and / or monitor and / or stratify therapy, wherein said therapy is the administration of a DPP3 activity inhibitor.

[0180] The term "pharmaceutical formulation" means a pharmaceutical ingredient in combination with at least one pharmaceutically acceptable excipient, in a form that allows the biological activity of the contained pharmaceutical ingredient to be effective and that contains no other components that would have unacceptable toxicity to the subject to whom the formulation will be administered. The term "pharmaceutical ingredient" means a therapeutic composition that may optionally be combined with a pharmaceutically acceptable excipient to provide a pharmaceutical formulation or dosage form.

[0181] The subject matter of this invention is a pharmaceutical preparation for a therapy or intervention in critically ill patients with hypotension to protect the myocardium and / or prevent myocardial damage, comprising an antibody or fragment or stent according to the invention.

[0182] The subject matter of this invention is a pharmaceutical preparation for a therapy or intervention in critically ill patients with hypotension, according to the invention, to protect the myocardium and / or prevent myocardial damage, wherein the pharmaceutical preparation is a solution, preferably a ready-to-use solution.

[0183] The subject matter of this invention is a pharmaceutical preparation according to the invention for a therapy or intervention in critically ill patients with hypotension, to protect the myocardium and / or prevent myocardial damage, wherein the pharmaceutical preparation is in a freeze-dried state.

[0184] The subject matter of this invention is a pharmaceutical preparation according to the invention for a therapy or intervention in critically ill patients with hypotension, to protect the myocardium and / or prevent myocardial damage, wherein the pharmaceutical preparation is administered intramuscularly.

[0185] The subject matter of this invention is a pharmaceutical preparation according to the invention for a therapy or intervention in critically ill patients with hypotension, to protect the myocardium and / or prevent myocardial damage, wherein the pharmaceutical preparation is administered intravascularly.

[0186] The subject matter of this invention is a pharmaceutical preparation according to the invention for a therapy or intervention in critically ill patients with hypotension, to protect the myocardium and / or prevent myocardial damage, wherein the pharmaceutical preparation is administered by infusion.

[0187] The subject matter of this invention is a pharmaceutical preparation according to the invention for a therapy or intervention in critically ill patients with hypotension, to protect the myocardium and / or prevent myocardial damage, wherein the pharmaceutical preparation is to be administered systemically.

[0188] Against the backdrop described above, the following sequentially numbered embodiments provide other specific aspects of the invention:

[0189] 1. A DPP3 activity inhibitor for use in a therapy or intervention for critically ill patients with hypotension to protect the myocardium and / or prevent myocardial damage.

[0190] 2. The DPP3 activity inhibitor for the therapy or intervention in critically ill patients with decreased blood pressure according to Embodiment 1, wherein the decreased blood pressure is a mean arterial pressure (MAP) < 65 mmHg, more preferably < 60 mmHg, even more preferably < 55 mmHg, and most preferably < 50 mmHg.

[0191] 3. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to embodiments 1 and 2, wherein the patient has a DPP3 level above a (predetermined) threshold in the patient's bodily fluid sample.

[0192] 4. The DPP3 activity inhibitor for the therapy or intervention in critically ill patients with decreased blood pressure according to Embodiment 3, wherein the predetermined threshold of DPP3 level in the body fluid sample of the subject is between 20 and 120 ng / mL, more preferably between 30 and 80 ng / mL, even more preferably between 40 and 60 ng / mL, and most preferably the threshold is 50 ng / mL.

[0193] 5. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to any one of embodiments 1 to 4, wherein the sample is a body fluid sample selected from the group consisting of whole blood, plasma, or serum.

[0194] 6. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with hypotension according to any one of embodiments 1 to 5, wherein the myocardial injury is characterized by blood cardiac troponin (cTn) levels above a threshold, increased myocardial expression of pro-inflammatory interleukin-6 (IL-6), and / or the need for vasopressors to maintain blood pressure and cardiac output.

[0195] 7. The DPP3 activity inhibitor for the therapy or intervention in critically ill patients with decreased blood pressure according to Embodiment 6, wherein the cardiac troponin (cTn) is cardiac troponin T (cTnT) or cardiac troponin I (cTnI).

[0196] 8. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with hypotension according to any one of embodiments 1-7, wherein the patient suffers from a serious infectious disease, sepsis, pulmonary embolism, pulmonary hypertension, acute coronary syndrome (including unstable angina, ST-segment elevation myocardial infarction (STEMI), non-ST-segment elevation myocardial infarction (NSTEMI)), any type of shock (including cardiogenic shock, septic shock, or anaphylactic shock), cardiac arrest, acute liver failure, and acute respiratory distress syndrome (ARDS).

[0197] 9. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to any one of embodiments 1-8, wherein the DPP3 activity inhibitor is selected from the group comprising small molecules, anti-DPP3 antibodies or anti-DPP3 antibody fragments or anti-DPP3 non-Ig scaffolds.

[0198] 10. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with reduced lung function according to embodiment 9, wherein the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No. 1.

[0199] 11. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with reduced lung function according to embodiments 9 and 10, wherein the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No. 2.

[0200] 12. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with reduced lung function according to any one of embodiments 9 to 11, wherein the antibody is a monoclonal antibody or a monoclonal antibody fragment.

[0201] 13. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with reduced lung function according to any one of embodiments 9 to 12, wherein the antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the complementarity-determining region (CDR) in the heavy chain comprises the following sequence:

[0202] SEQ ID NO.: 7, SEQ ID NO.: 8 and / or SEQ ID NO.: 9

[0203] Furthermore, the complementarity determination region (CDR) in the light chain contains the following sequence:

[0204] SEQ ID NO.: 10, KVS and / or SEQ ID NO.: 11.

[0205] 14. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with reduced lung function according to any one of embodiments 9 to 13, wherein the monoclonal antibody or antibody fragment is a humanized monoclonal antibody or a humanized monoclonal antibody fragment.

[0206] 15. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with reduced lung function according to any one of embodiments 9 to 14, wherein the antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises sequence SEQ ID NO.: 12, and wherein the light chain comprises sequence SEQ ID NO.: 13.

[0207] 16. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with reduced lung function according to Embodiment 9, wherein the small molecule is selected from the group comprising spinorphin, tynorphin, propioxatin A and B, fluostatin A and B, benzimidazole or derivatives thereof or analogues thereof.

[0208] For the avoidance of doubt, the following embodiments 10a to 16a correspond to embodiments 10 to 16 described above, including the correct reference to embodiment 9, namely, "blood pressure reduction". References to one or more of embodiments 10 to 16 should also refer to one or more of embodiments 10a to 16a.

[0209] 10a. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to Embodiment 9, wherein the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No. 1.

[0210] 11a. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to embodiments 9 and 10, wherein the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No. 2.

[0211] 12a. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to any of embodiments 9 to 11, wherein the antibody is a monoclonal antibody or a fragment of a monoclonal antibody.

[0212] 13a. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to any of embodiments 9 to 12, wherein the antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the complementarity-determining region (CDR) in the heavy chain comprises the following sequence:

[0213] SEQ ID NO.: 7, SEQ ID NO.: 8 and / or SEQ ID NO.: 9

[0214] Furthermore, the complementarity determination region (CDR) in the light chain contains the following sequence:

[0215] SEQ ID NO.: 10, KVS and / or SEQ ID NO.: 11.

[0216] 14a. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to any of embodiments 9 to 13, wherein the monoclonal antibody or antibody fragment is a humanized monoclonal antibody or a humanized monoclonal antibody fragment.

[0217] 15a. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to any of embodiments 9 to 14, wherein the antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises sequence SEQ ID NO.: 12, and wherein the light chain comprises sequence SEQ ID NO.: 13.

[0218] 16a. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to Embodiment 9, wherein the small molecule is selected from the group comprising spinorphin, tynorphin, propioxatin A and B, fluostatin A and B, benzimidazole or derivatives thereof or analogs thereof.

[0219] 17. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to any of the foregoing embodiments, wherein myocardial injury is defined as an increase in cardiac troponin levels above the 99th percentile of the upper limit of reference, and more specifically, myocardial injury is structural damage to cardiomyocytes and tissues (e.g., cardiomyocytes, cardiac fibroblasts, smooth muscle cells, or endothelial cells).

[0220] 18. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to any of the foregoing embodiments, wherein the blood level of cTn and / or myocardial expression of IL-6, as measured in samples obtained from said patients, is elevated, particularly above a threshold, and more specifically, cTn is elevated above the threshold defined in embodiment 17. Attached Figure Description

[0221] Figure 1Kaplan Meyer survival plots and their relationship with low (< 68.6 ng / mL) and high (≥ 68.6 ng / mL) DPP3 plasma concentrations. (A) Relationship between 7-day survival rate and DPP3 plasma concentration (cutoff 68.6 ng / mL) in patients with sepsis / septic shock; (B) Relationship between 7-day survival rate and DPP3 plasma concentration (cutoff 68.6 ng / mL) in patients with cardiogenic shock; (C) Relationship between 7-day survival rate and DPP3 plasma concentration (cutoff 68.6 ng / mL) in patients with acute myocardial infarction; (D) Relationship between 3-month survival rate and DPP3 plasma concentration in patients with dyspnea; (E) Relationship between 4-week survival rate and DPP3 plasma concentration in patients with burns; (F) Relationship between 7-day survival rate and DPP3 plasma concentration in patients with septic shock.

[0222] Figure 2 Natural hDPP3 purified from human erythrocyte lysates was subjected to SDS-PAGE on gradient gels (4–20%). Molecular weight markers are indicated by arrows.

[0223] Figure 3 Experimental Design—The Role of Natural DPP3 in Animal Models

[0224] Figure 4 (A) DPP3 injection leads to a decrease in the fractional shortening, resulting in a deterioration in cardiac function. (B) Decreased renal function was also observed through an increase in the renal resistance index.

[0225] Figure 5 Binding and dissociation curves were obtained using Octet's AK1967-DPP3 binding assay. The biosensor loaded with AK1967 was immersed in serial dilutions (100, 33.3, 11.1, 3.7 nM) of GST-tagged recombinant human DPP3, and binding and dissociation were monitored.

[0226] Figure 6 Western blot and DPP3 detection of blood cell lysate dilutions using AK1967 as the primary antibody.

[0227] Figure 7 : Inhibition curve of the inhibitory antibody AK1967 against native DPP3 derived from blood cells. The inhibition of DPP3 by the specific antibody is concentration-dependent; when analyzed for 15 ng / ml DPP3, the IC50 was [missing value]. 50 It is ~15 ng / ml.

[0228] Figure 8 Experimental setup—the role of Procizumab in sepsis-induced heart failure.

[0229] Figure 9In rats with sepsis-induced heart failure, Procizumab significantly improved the shortening fraction (A) and mortality (B).

[0230] Figure 10 Experimental design: Isoproterenol-induced cardiac stress in mice, followed by Procizumab treatment (B) and control (A).

[0231] Figure 11 In mice with isoproterenol-induced heart failure, Procizumab improved the shortening fraction (A) and reduced the renal resistance index (B) at 1 hour and 6 hours after administration, respectively.

[0232] Figure 12 High DPP3 levels within 24 hours of admission in sepsis patients were associated with the worst SOFA scores.

[0233] Figure 13 In patients with sepsis, elevated cDPP3 plasma levels are associated with organ dysfunction. A bar chart of SOFA scores in the AdrenOSS-1 is presented based on the evolution of DPP3 levels during ICU stays. HH: DPP3 above median at admission and 24 hours; HL: Above median at admission, but below median at 24 hours; LL: Below median at admission and 24 hours; LH: Below median at admission, but above median at 24 hours.

[0234] Figure 14: High cDPP3 levels in sepsis patients 24 hours after admission are associated with the worst SOFA scores by organ. (A) Heart, (B) Kidney, (C) Respiratory system, (D) Liver, (E) Coagulation and (F) Central nervous system SOFA scores, based on dynamic cDPP3 levels from admission to 24 hours (HH: high / high, HL: high / low, LH: low / high, LL: low / low).

[0235] Figure 15 High levels of DPP3 upon ICU admission were associated with subsequent deterioration of renal function within 48 hours. Y-axis: DPP3 measured on day 1 (ICU admission). X-axis: KDIGO stage 0 or 1 or KDIGO stage 2 or 3 (p = 0.002).

[0236] Figure 16: Continuous measurements of DPP3 during ICU stay in COVID-19 patients correlate with disease severity. A, DPP3 levels measured on day 3 of ICU admission (p = 0.02); B, DPP3 levels measured on day 7 of ICU admission (p = 0.013). X-axis: False = P / F ratio > 150; True = P / F ratio < 150.

[0237] Figure 17: High DPP3 levels during ICU admission are associated with adverse outcomes in COVID-19 patients. A, DPP3 levels measured on day 3 of ICU admission; B, DPP3 levels measured on day 7 of ICU admission. X-axis: 0 = alive; 1 = dead.

[0238] Figure 18 High DPP3 levels at ICU admission were associated with the need for vasopressor therapy during ICU stay (Day 3, p=0.05). Y-axis: DPP3 measured on Day 1 (ICU admission). X-axis: None: No vasopressor therapy during ICU stay, or any: Vasopressor therapy was administered.

[0239] Figure 19: Continuous measurements of DPP3 during ICU admission correlate with the need for organ support therapy (especially intravenous-intravenous ECMO). A, DPP3 levels measured on day 3 of ICU admission (p = 0.03); B, DPP3 levels measured on day 7 of ICU admission (p = 0.04). X-axis: 0 = no ECMO; 1 = ECMO.

[0240] Figure 20 Procizumab is used in treatment regimens to protect the heart during septic shock.

[0241] Figure 21 DPP3 activity (U / L) and Procizumab concentration (ng / ml) were measured at different time points during Procizumab infusion in septic pigs (n=16).

[0242] Figure 22 Compared with the Procizumab treatment group, cardiac inflammation was significantly upregulated in the standard care group as assessed by myocardial mRNA expression of the pro-inflammatory cytokine IL-6 (p=0.0024).

[0243] Figure 23 Compared with Procizumab animals, the standard care group showed significantly higher levels of myocardial injury at H12, as assessed by highly sensitive cardiac troponin I release (p=0.0055). No significant difference in troponin levels was observed between the two groups.

[0244] Figure 24 Norepinephrine requirements were assessed based on the titrated norepinephrine infusion dose (µg / kg / min) at each time point from the start of resuscitation (H1) to death (H12) to maintain mean arterial pressure (MAP) between 65 and 75 mmHg. The standard of care group required significantly more norepinephrine to achieve the target MAP compared to the procizumab group (p<0.05 from H4 to H9, p<0.005 from H10 to H12).

[0245] Figure 25 There were significant differences in cardiac output as assessed by pulmonary artery catheter at time points H4, H8, and H12 between the standard care group and the Procizumab group (p<0.05). Example

[0246] Example 1 – Measurement method of DPP3 protein and DPP3 activity

[0247] Antibody generation and DPP3 binding ability determination: Several mouse antibodies were prepared and screened by their ability to bind human DPP3 in a specific binding assay (see Table 3).

[0248] Peptides / conjugates used for immunity:

[0249] A DPP3 peptide for immunization was synthesized (see Table 3, JPT Technologies, Berlin, Germany) containing an additional N-terminal cysteine ​​residue (if cysteine ​​is absent in the selected DPP3 sequence) for conjugation to bovine serum albumin (BSA). The peptide was covalently linked to BSA using a Sulfolink conjugation gel (Perbio-science, Bonn, Germany). The conjugation procedure was performed according to Perbio's manual. Recombinant GST-hDPP3 was produced by USBio (United States Biological, Salem, MA, USA).

[0250] Immunization, immune cell fusion, and screening in mice:

[0251] Balb / c mice were administered 84 µg GST-hDPP3 or 100 µg DPP3 peptide-BSA conjugate (emulsified in TiterMax Gold adjuvant) on day 0, 84 µg or 100 µg (emulsified in complete Freund's adjuvant) on day 14, and 42 µg or 50 µg (incomplete Freund's adjuvant) on days 21 and 28 via intraperitoneal (ip). On day 49, the animals received intravenous (iv) injection of 42 µg GST-hDPP3 or 50 µg DPP3 peptide-BSA conjugate dissolved in saline. Three days later, the mice were sacrificed and immune cell fusion was performed.

[0252] Spleen cells from the immunized mice and cells from the myeloma cell line SP2 / 0 were fused in 1 ml of 50% polyethylene glycol at 37°C for 30 s. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement]. After one week, the HAT medium was replaced with HT medium, passaged three times, and then returned to normal cell culture medium.

[0253] Two weeks after fusion, recombinant DPP3-binding IgG antibodies in the cell culture supernatant were initially screened. Therefore, GST-tagged recombinant hDPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng / well) and incubated at room temperature with 50 µl of cell culture supernatant per well for 2 hours. After washing, 50 µl / well of POD rabbit anti-mouse IgG antibody was added, and the plates were incubated at RT for 1 h. After the next washing step, 50 µl of chromogenic solution (3.7 mM o-phenylenediamine in citrate / hydrogen phosphate buffer, 0.012% H2O2) was added to each well, and the plates were incubated at RT for 15 minutes. The chromogenic reaction was stopped by adding 50 µl of 4N sulfuric acid. Absorption was detected at 490 nm.

[0254] Microcultures that tested positive were transferred to 24-well plates for propagation. After retesting, selected cultures were cloned and re-cloned using limiting dilution techniques to determine isotypes.

[0255] Mouse monoclonal antibody production

[0256] Through standard antibody production methods ( Marx et al., 1997 Antibodies against human DPP3 or DPP3 peptides tagged with GST were produced and purified using protein A. The purity of the antibodies was ≥ 90% according to SDS gel electrophoresis analysis.

[0257] Antibody characterization – binding to hDPP3 and / or immune peptides

[0258] To analyze the ability of different antibodies and antibody clones to bind to DPP3 / immunopeptide, binding assays were performed:

[0259] a) Solid phase

[0260] Recombinant hDPP3 (SEQ ID NO. 1) or DPP3 peptide (immunopeptide, SEQ ID NO. 2) tagged with GST was immobilized on the surface of a highly binding microtiter plate (96-well polystyrene microtiter plate, Greiner Bio-One international AG, Austria, 1 µg / well, in coupling buffer [50 mM Tris, 100 mM NaCl, pH 7.8], RT for 1 h). After blocking with 5% bovine serum albumin, the microtiter plate was vacuum dried.

[0261] b) Labeling procedure (tracer)

[0262] 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-acridone ester (1 mg / ml, in acetonitrile, InVent GmbH, Germany; EP 0 353 971) and incubated at room temperature for 30 min. The labeled anti-DPP3 antibodies were purified by gel filtration HPLC on a Shodex Protein 5 µm KW-803 (Showa Denko, Japan). The purified labeled antibodies were diluted in assay buffer (50 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na2-EDTA, 5 g / L bovine serum albumin, 1 g / L mouse IgG, 1 g / L bovine IgG, 50 µmol / L aprotinin peptide, 100 µmol / L leucopeptide, pH 7.4). The final concentration is approximately 5-7 x 10⁻⁶ per 200 µl. 6 Acridine ester chemiluminescence was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG) of a relative optical units (RLU) of the labeled compound (approximately 20 ng of labeled antibody).

[0263] c) hDPP3 binding assay

[0264] 200 µl of labeled and diluted detection antibody (tracer) was loaded into the plate and incubated at 2–8 °C for 2–4 h. Unbound tracer was removed by washing four times with 350 µl of washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). The chemiluminescence of the bound tracer was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

[0265] Antibody characterization – hDPP3 inhibition analysis

[0266] To analyze the ability of different antibodies and antibody clones to inhibit DPP3, a DPP3 activity assay was performed using a known procedure (Jones et al., 1982). GST-tagged recombinant hDPP3 was diluted in assay buffer (25 ng / ml GST-DPP3 in 50 mM Tris-HCl, pH 7.5, and 100 µM ZnCl2), and 200 µl of this solution was incubated with 10 µg of the corresponding antibody at room temperature. After a 1-hour pre-incubation, the fluorescent substrate Arg-Arg-βNA (20 µl, 2 mM) was added to the solution, and the generation of free βNA over time was monitored 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 measurement at 410 nm. The slope of fluorescence increase for different samples (in RFU / min) was calculated. The slope of GST-hDPP3 using a buffer control was designated as 100% activity. The inhibitory effect of the possible capture binder was defined as the decrease in GST-hDPP3 activity resulting from incubation with the capture binder, expressed as a percentage.

[0267] The following table shows a selected group of antibodies and their binding rates, expressed in relative optical units (RLU), and their relative inhibitory capabilities (%); Table 3. Monoclonal antibodies generated against the DPP3 region described below were selected based on their ability to bind recombinant DPP3 and / or immunopeptides and their inhibitory potential.

[0268] All antibodies generated against the full-length form of recombinant hDPP3 with a GST tag showed strong binding to immobilized GST-tagged hDPP3. Antibodies generated against SEQ ID NO.:2 peptide also bound to GST-hDPP3. The antibody against SEQ ID NO.:2 also strongly bound to the immunopeptide.

[0269]

[0270] The development of a luminescent immunoassay (DPP3-LIA) for quantifying DPP3 protein concentration and an enzyme capture activity assay (DPP3-ECA) for quantifying DPP3 activity has recently been described. Rehfeld et al., 2019. JALM 3(6): 943- 953 (The entire text is incorporated into this paper by reference).

[0271] Example 2 - DPP3 for prognostic effects of short-term mortality

[0272] Using the hDPP3 immunoassay method ( Rehfeld et al., 2019. JALM 3(6): 943-953The concentration of DPP3 in the plasma of various diseased patients was measured and correlated with the short-term mortality of these patients.

[0273] Study cohort – Sepsis and septic shock

[0274] DPP3 screening was performed on plasma samples from 574 patients in the AdrenOSS-1 study of severe sepsis and septic shock. AdrenOSS-1 is a prospective, observational, multinational study that included 583 patients admitted to the intensive care unit for sepsis or septic shock. Hollinger et al., 2018 292 patients were diagnosed with septic shock.

[0275] Study cohort – Cardiogenic shock

[0276] DPP3 screening was performed on plasma samples from 108 patients diagnosed with cardiogenic shock. Blood was drawn within 6 hours of the detection of cardiogenic shock. Mortality was tracked for 7 days.

[0277] Study cohort – Acute coronary syndrome

[0278] DPP3 screening was performed on plasma samples from 720 patients with acute coronary syndrome. Blood was drawn 24 hours after the onset of chest pain. Mortality was tracked for 7 days.

[0279] Study cohort – Dyspnea

[0280] Plasma samples were collected immediately from 1,440 patients presenting with dyspnea (shortness of breath) upon admission to the emergency department of Skåne University Hospital. Patients with dyspnea, among other conditions, may also have acute coronary syndrome or congestive heart failure, and are at high risk of organ failure and short-term death. Mortality was followed for 3 months after emergency department admission.

[0281] Study cohort – Burn patients

[0282] DPP3 screening was performed on plasma samples from 107 patients with severe burns (covering more than 15% of the total body surface area). Blood was drawn upon admission. Mortality was followed up for 4 weeks.

[0283] hDPP3 immunoassay:

[0284] Immunoassays (LIA) or activity assays (ECA) that separately detect the amount (LIA) or activity of human DPP3 are used to determine the DPP3 level in a patient's plasma. Antibody fixation, labeling, and incubation are performed as in Rehfeld et al. ( Rehfeld et al., 2019. JALM 3(6): 943-953 As described in ( ).

[0285] result

[0286] Short-term survival in patients with sepsis / septic shock is associated with plasma DPP3 concentration at admission. Patients with plasma DPP3 concentrations above 68.6 ng / mL (3rd quartile) have an increased risk of death compared to patients with plasma DPP3 concentrations below this threshold. Figure 1 A). The same relationship can be observed when analyzing only the short-term outcomes of septic shock patients in this cohort and their relationship with DPP3 plasma concentrations. Figure 1 F). Patients with elevated DPP3 plasma concentrations had an increased risk of death compared to patients with low DPP3 plasma concentrations. When the same cutoff value was applied to patients with cardiogenic shock, an increased risk of short-term death within 7 days was also observed in patients with high DPP3. Figure 1 B).

[0287] Furthermore, when DPP3 levels were high and a cutoff value of 68.6 ng / mL was applied, the 7-day survival rate of patients with DPP3-related acute coronary syndrome also increased. Figure 1 C).

[0288] When this cutoff value of 68.6 ng / mL was applied to patients with dyspnea, a significantly increased risk of death was detected in patients with high DPP3 levels during a 3-month follow-up period. Figure 1 D).

[0289] Furthermore, in severely burned patients with high DPP3 concentrations above the corresponding cutoff value of 68.6 ng / mL, the risk of 4-week mortality was increased. Figure 1 E).

[0290] Example 3 - Purification of Human Natural DPP3

[0291] Human erythrocyte lysates were applied to a total of 100 mL of Sepharose 4B resin (Sigma-Aldrich), and the flow-through was collected. The resin was washed with a total of 370 mL of PBS buffer at pH 7.4, and the washing fraction was combined with the collected flow-through to obtain a total volume of 2370 mL.

[0292] For the immunoaffinity purification step, 110 mg of monoclonal anti-hDPP3 mAb AK2552 was conjugated to 25.5 mL of UltraLink hydrazide resin (Thermo Fisher Scientific) according to the manufacturer's protocol (GlycoLink immobilization kit). The conjugation efficiency was determined to be 98% by quantification of unconjugated antibody using the Bradford technique. The resin-antibody conjugate was equilibrated with 10 bed volumes of wash-binding buffer (PBS, 0.1% Triton X-100, pH 7.4), combined with 2370 mL of clear erythrocyte lysate, and incubated at 4°C with continuous stirring for 2 h. Subsequently, 100 mL of the incubation mixture was spread onto ten 15 mL polypropylene columns, and the flow-through was collected by centrifugation at 1000 x g for 30 sec. This step was repeated several times, yielding 2.5 mL of DPP3-loaded resin per column. Each column was washed five times with 10 mL of wash-binding buffer using gravity glow discharge. DPP3 was eluted by placing each column in a 15 mL Falcon tube containing 2 mL of neutralization buffer (1 M Tris-HCl, pH 8.0), then adding 10 mL of elution buffer (100 mM glycine-HCl, 0.1% Triton X-100, pH 3.5) to each column and immediately centrifuging at 1000 x g for 30 seconds. This elution step was repeated a total of 3 times, yielding 360 mL of combined eluent. The neutralized eluent was at pH 8.0.

[0293] Using an Äkta Start system (GE Healthcare) sample pump, the combined eluent was loaded onto a 5 mL HiTrap Q-sephare HP column (GE Healthcare) equilibrated with IEX buffer A1 (100 mM glycine, 150 mM Tris, pH 8.0). After sample loading, the column was washed with five column volumes of IEX buffer A2 (12 mM NaH2PO4, pH 7.4) to remove unbound proteins. Eluent for DPP3 was then applied using IEX buffer B (12 mM NaH2PO4, 1 M NaCl, pH 7.4) via a sodium chloride gradient in the range of 0–1 M NaCl over 10 column volumes (50 mL). The eluent was collected in 2 mL fractions. The buffer used for ion exchange chromatography was sterilely filtered using a 0.22 µM top-of-flask filter.

[0294] Table 4 shows the purification table and the corresponding yield and activity for each purification step. Figure 2The image shows SDS-PAGE of natural hDPP3 purified from human erythrocyte lysate on gradient gels (4-20%).

[0295]

[0296] Example 4 – The role of natural DPP3 in animal models

[0297] The effects of injecting natural hDPP3 into healthy mice were investigated by monitoring the shortening fraction and renal resistance index.

[0298] Wild-type Black 6 mice (8–12 weeks old, group sizes refer to Table 5) were acclimatized to the environment within 2 weeks and baseline echocardiography was performed. Mice were randomly assigned to one of two groups and subsequently injected intravenously via retroorbital injection with either natural DPP3 protein or PBS at a dose of 600 µg / kg.

[0299] Cardiac function was assessed by echocardiography at 15, 60, and 120 minutes after injection of DPP3 or PBS. Gao etc., 2011 Renal function was assessed using the renal resistance index. Lubas et al., 2014; Dewitte et al., 2012 () Figure 3 ).

[0300]

[0301] result

[0302] Compared with the control group injected with PBS, mice treated with natural DPP3 protein showed a significant reduction in shortening fraction ( Figure 4 A). The WT+DPP3 group also showed deterioration in renal function, as observed by the increase in the renal resistance index. Figure 4 B).

[0303] Example 5 – Development of Procizumab

[0304] The antibody generated against SEQ ID NO.: 2 was characterized in more detail (epitope mapping, binding affinity, specificity, and inhibitory potential). The results of clone 1967 (AK1967; "Procizumab") of SEQ ID NO.: 2 are shown here as an example.

[0305] Determining the AK1967 position on DPP3:

[0306] To map the epitopes of AK1967, numerous N- or C-terminal biotinylated peptides were synthesized (peptides & elephants GmbH, Hennigsdorf, Germany). These peptides included a fully immune peptide (SEQ ID No. 2) or a sequence of a fragment with one amino acid removed stepwise from its C- or N-terminus (see Table 7 for a complete list of peptides).

[0307] Each well of a high-binding 96-well plate was coated with 2 µg of avidin (Greiner Bio-One international AG, Austria) in conjugation buffer (500 mM Tris-HCl, pH 7.8, 100 mM NaCl). The plate was then washed and loaded with a specific solution of biotinylated peptides (10 ng / well; buffer—1xPBS containing 0.5% BSA).

[0308] According to Example 1, the anti-DPP3 antibody AK1967 was labeled with a chemiluminescent label.

[0309] Plates were loaded with 200 µl of labeled and diluted detection antibody (tracer) and incubated at room temperature for 4 h. Unbound tracer was removed by washing four times with 350 µl of washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Chemiluminescence of well binding was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). Binding of AK1967 to the corresponding peptide was determined by evaluating relative light units (RLU). Any peptide showing a significantly higher RLU signal than nonspecific binding to AK1967 was defined as an AK1967 binder. Combinatorial analysis of bound and unbound peptides revealed the specific DPP3 epitope of AK1967.

[0310] Binding affinity was determined using Octet:

[0311] Experiments were performed using Octet Red96 (ForteBio). AK1967 was captured onto a kinetically grade anti-human Fc antibody (AHC) biosensor. The loaded biosensor was then immersed in serially diluted GST-tagged recombinant human DPP3 buffers (100, 33.3, 11.1, 3.7 nM). Binding was observed for 120 seconds, followed by dissociation for 180 seconds. The buffers used for these experiments are depicted in Table 6. Kinetic analysis was performed using a 1:1 binding model and global fitting.

[0312]

[0313] Western blot analysis of AK1967 binding specificity:

[0314] Blood cells from human EDTA-treated blood were washed (three times in PBS), diluted in PBS, and lysed by repeated freeze-thaw cycles. The total protein concentration of the blood cell lysates was 250 µg / ml, and the DPP3 concentration was 10 µg / ml. SDS-PAGE and Western blotting were performed on the dilutions of the blood cell lysates (1:40, 1:80, 1:160, and 1:320) and the dilutions of purified recombinant human His-DPP3 (31.25–500 ng / ml). The blotting was incubated in 1.) blocking buffer (1xPBS-T containing 5% skim milk), 2.) primary antibody solution (AK1967, 1:2.000 in blocking buffer), and 3.) HRP-labeled secondary antibody (goat anti-mouse IgG antibody, 1:1.000 in blocking buffer). The secondary antibody was detected using Amersham ECL Western blot assay kits and Amersham Imager 600 UV (both from GE Healthcare).

[0315] DPP3 inhibition assay:

[0316] To analyze the ability of AK1967 to suppress DPP3, a known program was used as described in Example 1 ( Jones et al. 1982 DPP3 activity was measured. The inhibitory effect of AK1967 was defined as the percentage decrease in GST-hDPP3 activity due to incubation with the antibody. Figure 7 The inhibition curves show the reduced DPP3 activity obtained.

[0317] Epitope plotting:

[0318] Analysis of AK1967-bound and unbound peptides showed that the DPP3 sequence INPETG (SEQ ID NO.: 3) is an essential epitope for AK1967 binding (see Table 6).

[0319] Combining affinity:

[0320] AK1967 with 2.2*10 -9 M's affinity binds to recombinant GST-hDPP3 (see kinetic curves). Figure 5 ).

[0321]

[0322] Specificity and inhibitory potential:

[0323] The only protein detected in blood cell lysates using AK1967 as the primary antibody was 80 kDa DPP3. Figure 6 The total protein concentration of the lysate was 250 µg / ml, while the estimated DPP3 concentration was approximately 10 µg / ml. Despite the presence of 25-fold more nonspecific proteins in the lysate, AK1967 specifically bound to and detected DPP3 without other nonspecific binding.

[0324] AK1967 inhibited 15 ng / ml DPP3 in the DPP3 specific activity assay, IC50 50 Approximately 15 ng / ml ( Figure 7 ).

[0325] Chimerism / Humanization:

[0326] The monoclonal antibody AK1967 (“Procizumab”) has the ability to inhibit DPP3 activity by 70%, and has been selected as a potential therapeutic antibody. It has also been used as a template for chimeric and humanized conversion.

[0327] Humanization of mouse antibodies can be performed according to the following procedure:

[0328] To humanize the murine antibody, the structural interactions between the scaffold region (FR) and complementarity-determining region (CDR) of the antibody sequence and the antigen were analyzed. Based on structural modeling, a suitable human-derived FR was selected, and the murine CDR sequence was transplanted into the human FR. Mutations can be introduced into the amino acid sequences of the CDR or FR to restore structural interactions abolished by species switching in the FR sequence. This restoration of structural interactions can be achieved using phage display libraries via random methods or via a directed method guided by molecular modeling. Almagro and Fransson, 2008. Humanization of antibodies (Humanization of antibodies), Front Biosci. 13:1619-33 ).

[0329] In the above context, the variable region can be linked to the constant region of any subclass (IgG, IgM, IgE, IgA), or linked to only the scaffold, Fab fragment, Fv, Fab, and F(ab)2. In Examples 6 and 7 below, mouse antibody variants with an IgG2a backbone were used. For chimerism and humanization, a human IgG1κ backbone was used.

[0330] For epitope binding, only the complementarity-determining region (CDR) is important. The CDRs of the heavy and light chains of the mouse anti-DPP3 antibody (AK1967; "Procizumab") are shown in SEQ ID No. 7, SEQ ID No. 8, and SEQ ID No. 9 (for the heavy chain) and SEQ ID No. 10, sequence KVS, and SEQ ID No. 11 (for the light chain), respectively.

[0331] Sequencing of the anti-DPP3 antibody (AK1967; "Procizumab") revealed the antibody heavy chain variable region (H chain) according to SEQ ID NO.: 12 and the antibody light chain variable region (L chain) according to SEQ ID NO.: 13.

[0332] Example 6 – The role of Procizumab in sepsis-induced heart failure

[0333] In this experiment, the effect of Procizumab injection on sepsis-induced heart failure in rats was studied by monitoring the fractional shortening. Rittirsch et al., 2009 The role of ) in.

[0334] CLP model of septic shock:

[0335] Male Wistar rats (2–3 months old, 300–400 g, group sizes refer to Table 8) from the Centre d'élevage Janvier (France) were randomly assigned to one of three groups. All animals were anesthetized intraperitoneally (ip) with ketamine hydrochloride (90 mg / kg) and toluidine (9 mg / kg). To induce multimicrobial sepsis, cecal ligation and puncture (CLP) was performed using a slightly modified Rittirsch protocol. A ventral midline incision (1.5 cm) was made to expose the cecum. The cecum was then ligated directly below the ileocecal valve and punctured once with an 18-gauge needle. The peritoneal cavity was then closed in two layers, followed by fluid resuscitation (subcutaneous injection of 3 ml / 100 g body weight of physiological saline), and the animals were returned to their cages. Animals in the sham-operated group underwent surgery, but the cecum was not punctured. CLP animals were randomly assigned between the placebo group and the therapeutic antibody group.

[0336] Research Design:

[0337] The research process is described in Figure 8Following CLP or sham surgery, animals were allowed 20 hours of rest with free access to water and food. They were then anesthetized, tracheotomized, and arterial and venous sutures were laid. Twenty-four hours after CLP, AK1967 or a carrier (physiological saline) was administered via bolus at a dose of 5 mg / kg, followed by an infusion of 7.5 mg / kg over 3 hours. As a safety precaution, hemodynamic monitoring was performed invasively and continuously from t=0 until 3 hours.

[0338] At t=0 (baseline), all CLP animals were in septic shock and developed decreased cardiac function (hypotension, low shortening fraction). At this time point, they were injected (iv) with Procizumab or a carrier (PBS) and initiated with saline infusion. The table below (Table 8) summarizes one control group and two CLP groups. At the end of the experiment, the animals were euthanized and their organs were harvested for subsequent analysis.

[0339]

[0340] Invasive blood pressure:

[0341] Hemodynamic variables are obtained using the AcqKnowledge system (BIOPAC Systems, Inc., USA). This system provides a fully automated blood pressure analysis system. A catheter is connected to the BIOPAC system via a pressure sensor.

[0342] For the procedure described, the rats were anesthetized (ketamine and toluidine). The animals were moved to a heating pad to achieve the desired body temperature of 37-37.5°C. A temperature feedback probe was inserted into the rectum. The rats were placed in a supine position on the operating table. The trachea was opened, and a 16G catheter was inserted for external ventilator operation without damaging the carotid artery and vagus nerve. An arterial catheter was inserted into the right carotid artery. The carotid artery was separated from the vagus nerve before ligation.

[0343] A central venous catheter is inserted through the left jugular vein to allow administration of PCZ or PBS.

[0344] Postoperatively, the animal was allowed to rest until a stable state before hemodynamic measurements were performed. Baseline blood pressure (BP) was then recorded. During data collection, saline infusion through the arterial line was stopped.

[0345] Echocardiography:

[0346] The animals were anesthetized with ketamine hydrochloride. The hair on the chest was shaved, and the rats were placed in a supine position.

[0347] For transthoracic echocardiography (TTE), a commercial GE Healthcare Vivid 7 ultrasound system equipped with a high-frequency (14-MHz) linear probe and a 10-MHz cardiac probe was used. All examinations were digitally recorded and stored for subsequent offline analysis.

[0348] Grayscale images were recorded at a depth of 2 cm. A two-dimensional examination was initiated in a parasternal long-axis view to measure the aortic annulus diameter and pulmonary artery diameter. M-mode was also used to measure left ventricular (LV) size and assess fractional shortening (FS%). LVFS was calculated as LV end-diastolic diameter - LV end-systolic diameter / LV end-diastolic diameter, expressed as a percentage. Thus, end-diastolic time was defined at the LV at its maximum diameter. Thus, end-systolic time was defined at the LV at its minimum diameter within the same cardiac cycle. All parameters were measured manually. Each measurement was averaged over three cardiac cycles.

[0349] Pulmonary artery blood flow was recorded using pulsed-wave Doppler ultrasound from the same parasternal long-axis view. The velocity-time integral of pulmonary outflow was measured.

[0350] Mitral valve blood flow was recorded at the level of the mitral valve tip using pulsed Doppler in a five-chamber view at the apex of the heart.

[0351] result:

[0352] Compared to sham-operated animals, sepsis-induced heart failure rats treated with PBS (CLP+PBS) showed a reduced fractional shortening ( Figure 9 A). The CLP+PBS group also showed a high mortality rate ( Figure 9 B). In contrast, administration of Procizumab to rats with sepsis-induced heart failure improved the shortening fraction (B). Figure 9 A) and significantly reduced the mortality rate ( Figure 9 B).

[0353] Example 7 – Effects of Procizumab on Cardiac and Renal Function

[0354] The role of Procizumab in isoproterenol-induced heart failure in mice was investigated by monitoring the shortening fraction and renal resistance index.

[0355] Isoproterenol-induced cardiac stress in mice:

[0356] Acute heart failure was induced in 3-month-old male mice by subcutaneous injection of 300 mg / kg isoproterenol (a non-selective β-adrenergic agonist, DL-isoproterenol hydrochloride, Sigma Chemical Co.) (ISO) twice daily for two days. Vergaro et al., 2016ISO dilution was performed in 0.9% NaCl. Mice treated with isoproterenol were randomly assigned to two groups (Table 9), and baseline echocardiography (Gao et al., 2011) and renal resistance index measurement were performed on day 3. Lubas et al., 2014; Dewitte et al., 2012 Following this, administer PBS or Procizumab (10 mg / kg) intravenously. Figure 10 (A and B).

[0357] Echocardiography was performed at 1 hour, 6 hours, and 24 hours. Gao et al., 2011 ) and renal resistance index ( Lubas et al., 2014; Dewitte et al., 2012 Assess cardiac function ( Figure 10 A and B). Mice injected with the carrier (PBS) instead of isoproterenol did not receive further pharmacological treatment and served as the control group (Table 9).

[0358]

[0359] result:

[0360] Administering Procizumab to mice with isoproterenol-induced heart failure restored cardiac function within the first hour after administration. Figure 11 A). Six hours after PCZ injection, the renal function of the affected mice showed significant improvement and was comparable to that of the sham-operated animals at 24 hours. Figure 11 B).

[0361] Example 8 – DPP3 and organ dysfunction in sepsis

[0362] The same study described in Example 2 (AdrenOSS-1) was used to evaluate the association between circulating DPP3 (cDPP3) and organ dysfunction (e.g., cardiovascular and renal) in patients admitted for sepsis and septic shock. AdrenOSS-1 is a prospective, observational, multinational European study (ClinicalTrials.gov NCT02393781) that included 583 patients admitted to the ICU for sepsis or septic shock. The primary outcome (as described in Example 2) was 28-day mortality. Secondary outcomes included organ failure as defined by the SOFA score, organ support with a focus on vasopressor use, and the need for renal replacement therapy. Blood samples were collected for central laboratory use within 24 hours of ICU admission and on day 2.

[0363] To quantify DPP3 protein concentration (DPP3-LIA), a recently described assay was used. Rehfeld et al. 2019. JALM 3(6): 943-953 ).

[0364] The median cDPP3 level at admission was 45.1 ng / mL (interquartile range 27.5–68.6) in all AdrenOSS-1 patients. High cDPP3 levels at admission were associated with poorer metabolic parameters, renal and cardiac function, and SOFA scores: patients with cDPP3 levels below the median had a median SOFA score of 6 (IQR 4–9), compared to a median SOFA score of 8 (IQR 5–11) in patients with cDPP3 levels above the median of 45.1 ng / mL. Figure 12 ).

[0365] Regardless of the cDPP3 level at admission, high cDPP3 levels 24 hours later were associated with the worst SOFA score, both overall ( Figure 13 ) or by organ (Fig. 14 AF).

[0366] In summary, these data demonstrate that high levels of cDPP3 are associated with survival and the degree of organ dysfunction in a large international cohort of patients with sepsis or septic shock. The study found a significant association between cDPP3 < 45.1 ng / ml at admission and short-term survival, with a prognostic cutoff of 45.1 pg / ml in both sepsis and septic shock. Regarding organ dysfunction, a positive correlation was found between cDPP3 and SOFA scores at ICU admission. More importantly, the relationship between cDPP3 levels and the degree of organ dysfunction observed at ICU admission was also evident during the recovery phase. In fact, patients with high cDPP3 levels at admission and showing a decline towards normal cDPP3 values ​​by day 2 were more likely to recover function in all organs, including the cardiovascular, renal, pulmonary, and hepatic systems.

[0367] Example 9 – DPP3 in patients infected with coronavirus (SARS-CoV-2)

[0368] Plasma samples from 12 patients diagnosed with coronavirus (SARS-CoV-2) were screened for DPP3 and other biomarkers. As recently described ( Rehfeld et al., 2019. JALM 3(6): 943-953 The immunoassay (LIA) or activity assay (ECA) that detects the amount (LIA) or activity of human DPP3 (ECA) will be used to determine the level of DPP3 in the patient's plasma.

[0369] Table 10 summarizes the DPP3 concentration in each sample.

[0370]

[0371] DPP3 concentrations ranged from 27 to 975 ng / ml, with a median (IQR) of 156 (59.5–322.3) ng / ml. DPP3 concentrations were significantly elevated compared to healthy subjects. Measurements were taken from samples of 5400 healthy subjects (Swedish single-center prospective population study (MPP-RES)): the median (interquartile range) of plasma DPP3 was 14.5 ng / ml (11.3 ng / ml – 19 ng / ml).

[0372] Example 10 – DPP3 in COVID-19 patients for prognosis, treatment stratification, and follow-up

[0373] Queue description:

[0374] This study included 21 patients who tested positive for SARS-CoV-2 PCR and were admitted to the ICU. Patient characteristics included a median age of 63 years, 76% being male, a median body mass index (BMI) of 28.6, and a Sequential Organ Failure Assessment (SOFA) score of 5 on admission. Exclusion criteria were age <18 years and pregnancy. Analysis was performed using real-time reverse transcription PCR (RT-PCR). Patients were treated according to our ICU standards of care, including mechanical ventilation, intravenous-venous ECMO, and recurrent renal replacement therapy (RRT) if needed.

[0375] Blood was drawn on the day of admission and daily until day 7 for analysis of DPP3 and standard laboratory parameters. As recently described ( Rehfeld et al., 2019. JALM 3(6): 943-953 DPP3 in EDTA plasma was measured using a one-step luminescent sandwich immunoassay (LIA).

[0376] result:

[0377] a) Baseline DPP3 and serial measurements correlate with disease severity

[0378] DPP3 measured at ICU admission was associated with worsening renal function during ICU stay as defined by the KDIGO criteria. Stages 0-1 indicate no to mild renal impairment and low risk, while stages 2-3 indicate renal injury and renal failure. Furthermore, DPP3 values ​​in stages 2-3 were significantly higher than those in stages 0-1. Figure 15 (p = 0.005). High DPP3 values ​​at baseline can be used in conjunction with other clinical parameters to guide the initiation of renal replacement therapy.

[0379] Because COVID-19 positive patients tend to stay in the ICU for an average of 21 days, measurements of DPP3 levels during ICU admission (days 3 and 7) were also associated with a low PaO2 / FiO2 ratio (<150), and therefore with severe acute respiratory distress syndrome (ARDS) (false = P / F ratio >150; true = P / F ratio <150). In COVID-19 patients, serial measurements of DPP3 during ICU admission were associated with disease severity. On day 3 of ICU admission (p = 0.02)... Figure 16A ) and ICU admission day 7 (p = 0.013) Figure 16B DPP3 levels were measured.

[0380] In addition, on the 3rd day ( Figure 17A (p=0.03) and day 7 ( Figure 17B High DPP3 values ​​(p=0.01) were still associated with high mortality rates during ICU admission.

[0381] b) Baseline DPP3 and serial measurements are associated with the need for organ support therapy.

[0382] High DPP3 levels at admission and during ICU stay were significantly associated with the need for organ support therapy, particularly vasopressors (day 3). Figure 18 ) and extracorporeal membrane oxygenation (ECMO) (days 3 and 7; see below for details). Figure 19A And B).

[0383] Example 11 – Procizumab for cardiac protection in an animal model of sepsis

[0384] To assess the cardioprotective effect of anti-DPP3 antibody (Procizumab) during septic shock, we conducted a randomized, open-label, controlled study in 16 anesthetized and mechanically ventilated pigs. Septic shock was induced by fecal peritonitis. Fluid resuscitation, antimicrobial therapy, and abdominal drainage were initiated one hour after the onset of septic shock. Septic pigs were randomly assigned to receive either Procizumab (on top of standard care) or standard care (norepinephrine and fluids) to maintain mean arterial pressure between 65 and 75 mmHg for 12 hours. Figure 20 The experiment used 8 female pigs and 8 male pigs, achieving an appropriate sex balance between the treatment group and the standard care group.

[0385] The results showed that Procizumab could inhibit DPP3 activity in the bloodstream throughout the infusion period. Figure 21 Finally, Procizumab showed a cardioprotective effect compared to the standard care group.

[0386] This effect is primarily observed by comparing cardiac inflammation, assessed through myocardial mRNA expression of the pro-inflammatory cytokine IL-6 between the two groups. Figure 22 In summary, total RNA was extracted from rapidly frozen left ventricular tissue stored at -80°C in RNA later solution (Invitrogen™, RNA later™ stabilization solution, ThermoFisherScientific, MA, USA) using the RNeasy Mini kit (QIAGEN, Germany). Reverse transcription was performed using random hexamer primers and Superscript II reverse transcriptase (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. For RTqPCR, positive and antisense primers for detecting wild boar (sus scrofa) IL-6 were designed using the Primer3 program. For each sample, amplification reactions were performed in triplicate using the iCycler system (BioRad Laboratories) with the SYBRGreen PCR master mix (Quanta Biosciences, Gaithersburg, MD), specific primers, and diluted template complementary DNA. Relative quantification was achieved using the comparative 2-ΔΔCt method after normalization using the housekeeping gene (β-actin). The results were expressed as a relative fold increase in the mean of left ventricular relative mRNA expression, which was arbitrarily fixed to 1 in the sham surgery group.

[0387] Although the standard care group showed high IL-6 myocardial expression levels, the IL-6 expression levels in procizumab-treated animals were normal, comparable to those in sham-operated animals without septic shock (anesthesia, intubation, and ventilation only). Figure 22 In addition, high-sensitivity troponin I (hsTnI) serum levels were assessed at baseline and H12 to evaluate myocardial injury. Figure 23 Baseline hsTnI levels were similar between the treatment and standard care groups, showing no difference between the two groups. However, at time point H12, hsTnI levels in the Procizumab group remained similar to baseline and were significantly lower than in the standard care group. Figure 23 ).

[0388] Through norepinephrine demand ( Figure 24 ) and cardiac output index ( Figure 25 Cardioprotective effects were also observed in both groups. Records were taken hourly starting from hour 1. The norepinephrine dose was measured in g / kg / min. Although the standard care group had a significantly higher norepinephrine requirement, animals treated with Procizumab were infused with the minimum required norepinephrine dose to achieve the same target mean arterial pressure of 65 mmHg. Figure 24 At H4, the difference between the treatment group and the standard care group became significant, and after H10 (10 hours after the start of norepinephrine infusion), the difference between the two groups was even greater. Figure 24 ).

[0389] Finally, we also assessed the cardiac output index via pulmonary artery catheterization. Figure 25 Compared to the standard care group, animals treated with Procizumab consistently had lower central output indices at all time points (H4 to H12) and were comparable to baseline levels. Figure 25 Indexed cardiac indices showed that higher norepinephrine doses were associated with higher cardiac output, reflecting β1 receptor activation and increased myocardial oxygen consumption, leading to increased oxidative stress and myocardial injury. Therefore, Procizumab improves hemodynamic stability, protects the myocardium from inflammation, and mitigates cardiac stress by preventing the use of high doses of norepinephrine. These results suggest that Procizumab has a cardioprotective effect and prevents myocardial injury during the evolution of septic shock.

[0390] sequence

[0391] SEQ ID No. 1 – hDPP3 aa 1-737

[0392] MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSPEAPYIYALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTKFVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLRHLGLGKEGITTYFSGNCTMEDAKLAQDFLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDSEVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESFTQGSIEAHKRGSRFWIQDKGPIVESYIGFIESYRDPFGSRGEFEGFVAVVNKAMSAKFERLVASAEQLLKELPWPPTFEKDKFLTPDFTSLDVLTFAGSGIPAGINIPNYDDLRQTEGFKNVSLGNVLAVAYATQREKLTFLEEDDKDLYILWKGPSFDVQVGLHELLGHGSGKLFVQDEKGAFNFDQETVINPETGEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLYLCLHPQVLEIFGFEGADAEDVIYVNWLNMVRAGLLALEFYTPEAFNWRQAHMQARFVILRVLLEAGEGLVTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTGDVAGGRALYEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEYEASAAGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA

[0393] SEQ ID No. 2 - hDPP3 aa 474-493 (N-Cys) - Immunopeptide with an additional N-terminal cysteine

[0394] CETVINPETGEQIQSWYRSGE

[0395] SEQ ID No. 3 - hDPP3 aa 477-482 - Epitope of AK1967

[0396] INPETG

[0397] SEQ ID No. 4 – hDPP3 aa 480-483

[0398] ETGE

[0399] SEQ ID No. 5 – Variable region in the heavy chain of mouse AK1967

[0400] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPSGKGLEWLAHIWWNDNKSYNPALKSRLTISRDTSNNQVFLKIASVVTADTGTYFCARNYSYDYWGQGTTLTVSS

[0401] SEQ ID No. 6 – Variable region in mouse AK1967 light chain

[0402] DVVVTQTPLSLSVSLGDPASISCRSSRSLVHSIGSTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

[0403] SEQ ID No. 7 – CDR1 in the heavy chain of mouse AK1967

[0404] GFSLSTSGMS

[0405] SEQ ID No. 8 – CDR2 in the heavy chain of mouse AK1967

[0406] IWWNDNK

[0407] SEQ ID No. 9 – CDR3 in the heavy chain of mouse AK1967

[0408] ARNYSYDY

[0409] SEQ ID No. 10 – CDR1 in the mouse AK1967 light chain

[0410] RSLVHSIGSTY

[0411] CDR2 in the AK1967 light chain

[0412] KVS

[0413] SEQ ID No. 11 - CDR3 in the mouse AK1967 light chain

[0414] SQSTHVPWT

[0415] SEQ ID No. 12 - Humanized AK1967 - Heavy Chain Sequence (IgG1κ Framework)

[0416] MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQVVLTMTNMDPVDTGTYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0417] SEQ ID No. 13 - Humanized AK1967 - Light Chain Sequence (IgG1κ Framework)

[0418] METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. A DPP3 activity inhibitor for use in a therapy or intervention for critically ill patients with hypotension, to protect the myocardium and / or prevent myocardial injury.

2. The DPP3 activity inhibitor for the treatment or intervention in critically ill patients with decreased blood pressure according to claim 1, wherein the decreased blood pressure is a mean arterial pressure (MAP) < 65 mmHg, more preferably < 60 mmHg, even more preferably < 55 mmHg, and most preferably < 50 mmHg.

3. The DPP3 activity inhibitor for the therapy or intervention in critically ill patients with decreased blood pressure according to claims 1 and 2, wherein the patient has a DPP3 level above a (predetermined) threshold in the patient's bodily fluid sample.

4. The DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to claim 3, wherein the predetermined threshold for DPP3 level in the subject's body fluid sample is between 20 and 120 ng / mL, more preferably between 30 and 80 ng / mL, even more preferably between 40 and 60 ng / mL, and most preferably the threshold is 50 ng / mL.

5. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to any one of claims 1 to 4, wherein the sample is a body fluid sample selected from the group consisting of whole blood, plasma, or serum.

6. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with hypotension according to any one of claims 1 to 5, wherein the myocardial injury is characterized by blood cardiac troponin (cTn) levels above a threshold, increased myocardial expression of pro-inflammatory interleukin-6 (IL-6), and / or the need for vasopressors to maintain blood pressure and cardiac output.

7. The DPP3 activity inhibitor of the therapy or intervention for critically ill patients with hypotension according to claim 6, wherein the cardiac troponin (cTn) is cardiac troponin T (cTnT) or cardiac troponin I (cTnI).

8. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with hypotension according to any one of claims 1-7, wherein the patient suffers from severe infectious disease, sepsis, pulmonary embolism, pulmonary hypertension, acute coronary syndrome (including unstable angina, ST-segment elevation myocardial infarction (STEMI), non-ST-segment elevation myocardial infarction (NSTEMI)), any type of shock (including cardiogenic shock, septic shock, or anaphylactic shock), cardiac arrest, acute liver failure, and acute respiratory distress syndrome (ARDS).

9. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with hypotension according to any one of claims 1-8, wherein the DPP3 activity inhibitor is selected from the group comprising small molecules, anti-DPP3 antibodies or anti-DPP3 antibody fragments or anti-DPP3 non-Ig scaffolds.

10. The DPP3 activity inhibitor of claim 9 for a therapy or intervention in critically ill patients with hypotension, wherein the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No.

1.

11. The DPP3 activity inhibitor for the therapy or intervention in critically ill patients with hypotension according to claims 9 and 10, wherein the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No.

2.

12. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to any one of claims 9 to 11, wherein said antibody is a monoclonal antibody or a fragment of a monoclonal antibody.

13. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to any one of claims 9 to 12, wherein the antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the complementarity-determining region (CDR) in the heavy chain comprises the following sequence: SEQ ID NO.: 7, SEQ ID NO.: 8 and / or SEQ ID NO.: 9 Furthermore, the complementarity determination region (CDR) in the light chain contains the following sequence: SEQ ID NO.: 10, KVS and / or SEQ ID NO.:

11.

14. The DPP3 activity inhibitor for a therapy or intervention in critically ill patients with decreased blood pressure according to any one of claims 9 to 13, wherein the monoclonal antibody or antibody fragment is a humanized monoclonal antibody or a humanized monoclonal antibody fragment.

15. A DPP3 activity inhibitor for a therapy or intervention in critically ill patients with hypotension according to any one of claims 9 to 14, wherein the antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises sequence SEQ ID NO.: 12, and wherein the light chain comprises sequence SEQ ID NO.:

13.

16. The DPP3 activity inhibitor of claim 9 for a therapy or intervention in critically ill patients with decreased blood pressure, wherein the small molecule is selected from the group comprising spinorphin, tynorphin, propioxatin A and B, fluostatin A and B, benzimidazole or derivatives thereof or analogues thereof.

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