DPP3 inhibitors for myocardial protection and prevention of myocardial injury in critically ill patients with hypotension.

Abstract

The subject of this invention is an inhibitor of DPP3 activity for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury.

Description

[Technical Field]

[0001] The subject of this invention is an inhibitor of DPP3 activity for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury. [Background technology]

[0002] Dipeptidyl peptidase 3, also known as dipeptidylaminopeptidase III, dipeptidylarylamidase III, dipeptidylpeptidase III, enkephalinase B, or erythrocyte angiotensinase, abbreviated as DPP3 or DPPIII, is a metallopeptidase that removes dipeptides from physiologically active peptides such as enkephalins and angiotensins. DPP3 was identified and its activity measured in an extract of purified bovine anterior pituitary gland by Ellis & Nuenke 1967. Described as EC 3.4.14.4, this enzyme 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 shown in Sequence ID No. 1. DPP3 is primarily a cytosolic peptidase and is ubiquitously expressed. Despite the lack of a signal sequence, membrane activity has been reported in several studies (Lee & Snyder 1982).

[0003] DPP3 is a zinc-dependent exopeptidase belonging to the peptidase family M49. It has broad substrate specificity for 3 / 4 to 10 amino acid oligopeptides of various compositions and can also cleave after proline. DPP3 is known to hydrolyze dipeptides from the N-terminus of its substrates, including angiotensin II, III, and IV, Leu- and Met-enkephalins, and endomorphin 1 and 2. The metallopeptidase DPP3 has an optimal pH of 8.0 to 9.0 and can be activated by the addition of divalent metal ions such as Co2+ and Mg2+.

[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; numbering refers to the sequence of human DPP3 (see Sequence 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 of amino acids 316-669.

[0005] The most prominent substrate of DPP3 is angiotensin II (Ang II), the 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 (Correa et al. 2015. Crit Care 19:98). In particular, Ang II has been shown to modulate many cardiovascular functions, including blood pressure control and cardiac remodeling.

[0006] Recently, two assays have been developed, characterized, and validated to specifically detect DPP3 in human bodily fluids (e.g., blood, plasma, serum): a luminescence 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 and enable reproducible detection of DPP3 in blood samples.

[0007] In patients with septic shock, cardiogenic shock, and vasodilatory shock, circulating DPP3 levels were shown to be elevated (Rehfeld et al. 2019. JALM 3(6):943-953). Furthermore, in patients with cardiogenic shock, it was associated with an increased risk of short-term mortality and severe organ failure (Deaniau et al. 2020. Eur J Heart Fail. 22(2):290-299). In addition, in patients with severe sepsis or septic shock, higher initial cDPP3 levels were associated with a greater need for organ support and vasopressors at hospitalization, a longer need for vasopressors, mechanical ventilation, or renal replacement therapy (RRT), and a greater need for fluid loading (Blet et al. 2021. Crit Care 25:61).

[0008] International Publication No. 2017 / 182561 describes a method for determining the total amount of active DPP3 in a patient's sample for the diagnosis of diseases associated with the necrotic process. It further describes a method for treating necrotic-related diseases with antibodies against DPP3.

[0009] International Publication No. 2019 / 081595 describes DPP3 conjugates that target specific DPP3 epitopes and their use in the prevention or treatment of diseases associated with oxidative stress.

[0010] International Publication No. 2021 / 185786 describes a method for determining DPP3 in patient samples for diagnosis, risk prediction, prognosis, and monitoring in patients infected with coronavirus. It further describes inhibitors of DPP3 activity for use in the treatment or intervention in infected patients.

[0011] Procizumab, a humanized monoclonal IgG1 antibody that specifically binds to circulating DPP3, targets and modulates the activity of DPP3, an essential regulator of cardiovascular function. Its mechanism of action is associated with acute diseases involving massive cell death and uncontrolled release of intracellular DPP3 into the bloodstream. The migrated DPP3 remains active in the bloodstream, where it uncontrollably cleaves bioactive peptides. Procizumab can block circulating DPP3, thereby inhibiting the degradation of bioactive peptides in the bloodstream. This blockade stabilizes cardiovascular and renal function and reduces short-term mortality. Preclinical studies of procizumab in animal models of cardiovascular failure have shown significant 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] The examples in this description of the present invention demonstrate that procizumab injection prevented myocardial injury (indicated by increases in myocardial IL-6 and troponin, respectively) in pigs with septic shock. Furthermore, DPP3 has been shown to be significantly elevated in patients with hypotension, particularly in patients with shock (e.g., septic shock, cardiogenic shock) and acute coronary syndrome (ACS). Therefore, DPP3 inhibitors, especially procizumab, are likely to be able to prevent myocardial injury in patients with hypotension, regardless of the indication.

[0013] Therefore, it is a remarkable discovery of this invention that inhibitors of DPP3 activity are suitable for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury. [Overview of the Initiative]

[0014] The subject of this invention is an inhibitor of DPP3 activity for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury.

[0015] DPP3 level The subject of the present invention is an inhibitor of DPP3 activity for use as a treatment or intervention in critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury, wherein the patients have DPP3 levels above a predetermined threshold.

[0016] In one embodiment of the present invention, the level of DPP3 protein and / or the level of active DPP3 is determined and compared with a predetermined threshold level.

[0017] The subject matter of this application is an inhibitor of DPP3 activity for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury, wherein the level of DPP3 in a body fluid sample of the subject exceeds a predetermined threshold, as determined by different methods, such as immunoassay, activity assay, mass spectrometry, etc.

[0018] DPP3 activity can be measured by detecting the cleavage products of DPP3-specific substrates. Known peptide hormone substrates include Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctrin, ACTH (adrenocorticotropic hormone), and MSH (melanocyte-stimulating hormone; Abramic et al. 2000, Barsun et al. 2007, Dhanda et al. 2008). Cleavage of the mentioned peptide hormones and other untagged oligopeptides (e.g., Ala-Ala-Ala-Ala, Dhanda et al. 2008) can be monitored by detecting their respective cleavage products. Detection methods are not limited to, but include HPLC analysis (e.g., Lee & Snyder 1982), mass spectrometry (e.g., Abramic et al. 2000), H1-NMR analysis (e.g., Vandenberg et al. 1985), capillary zone electrophoresis (CE; e.g., Barsun et al. 2007), thin-layer chromatography (e.g., Dhanda et al. 2008), or reverse-phase chromatography (e.g., Mazocco et al. 2006).

[0019] Detection of fluorescence by hydrolysis of fluorescence-generating substrates by DPP3 is a standard procedure for monitoring DPP3 activity. These substrates are specific dipeptides or tripeptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) bound to a fluorophore. Examples of fluorophores, though not limited to these, include β-naphthylamide (2-naphthylamide, βNA, 2NA), 4-methoxy-β-naphthylamide (4-methoxy-2-naphthylamide), and 7-amide-4-methylcoumarin (AMC, MCA; Abramic et al. 2000, Ohkubo et al. 1999). The cleavage of these fluorescence-generating substrates results in the release of either fluorescent β-naphthylamine or 7-amino-4-methylcoumarin, respectively. In a liquid-phase assay, the ECA substrate and DPP3 are incubated, for example, in a 96-well plate format, and fluorescence is measured using a fluorescence detector (Ellis & Nuenke 1967). Alternatively, the DPP3-supported sample may be immobilized, separated on a gel by electrophoresis, the gel stained with a fluorescence-generating substrate (e.g., Arg-Arg-βNA) and Fast Garnet GBC, and the fluorescent protein bands detected by a fluorescence reader (Ohkubo et al. 1999). The same peptide (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) may be bound to a chromophore, such as p-nitroanilide diacetate. DPP3 activity may be monitored by detecting the color change due to hydrolysis of the chromogenic substrate.

[0020] Another option for detecting DPP3 activity is the Protease-Glo™ Assay (commercially available from Promega). In this embodiment of the method, a DPP3-specific dipeptide or tripeptide (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) is conjugated to aminoluciferin. When cleaved by DPP3, aminoluciferin is released and acts as a substrate for a coupled luciferase reaction that emits detectable luminescence.

[0021] In a preferred embodiment, DPP3 activity is measured by addition of the fluorogenic substrate Arg-Arg-βNA and monitoring of fluorescence in real time.

[0022] In another embodiment of the invention, the level of DPP3 is determined by contacting the body fluid sample with a capture binder that specifically binds to DPP3.

[0023] In another preferred embodiment of the invention, the capture binder for determining the level of DPP3 can be selected from the group consisting of an antibody, an antibody fragment or a non-IgG scaffold.

[0024] In a specific embodiment of the invention, the capture binder for determining the level of DPP3 is an antibody.

[0025] Another specific embodiment of the invention involves the use of a capture binder that specifically binds to full-length DPP3.

[0026] In another preferred embodiment of the invention, the capture binder is immobilized on a solid phase.

[0027] When a test sample is passed over an immobilized binder, if DPP3 is present in the sample, it binds to the binder and becomes immobilized for detection. The presence or amount of DPP3 in the test sample can then be indicated by the addition of a substrate and the detection of the reaction product. Alternatively, DPP3 bound to the capture molecule on the solid phase can be detected by a second capture molecule that specifically binds to DPP3.

[0028] For the purposes of this specification, the term “solid phase” may be used to include any material or container on which the assay may be performed, and the term includes, but is not limited to, porous materials, non-porous materials, test tubes, wells, slides, agarose resin (e.g., Sepharose from GE Healthcare Life Sciences), and magnetic particles (e.g., Dynabeads® or Pierce® magnetic beads from Thermo Fisher Scientific).

[0029] In one embodiment of the present invention, a method for determining the DPP3 activity in a body fluid sample of the subject is: • A step of contacting the sample with a scavenging agent that specifically binds to full-length DPP3, • A step of separating DPP3 bound to the scavenging agent, • A step of adding a DPP3 substrate to the separated DPP3. The method includes a step of quantifying DPP3 activity by measuring and quantifying the substrate conversion of DPP3.

[0030] In another embodiment of the present invention, the separation step is a washing step that removes sample components not bound to the scavenging agent from the captured DPP3.

[0031] In another embodiment of the present invention, DPP3 substrate conversion is detected by a method selected from the group including fluorescence of a fluorescent substrate (e.g., Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, emission of a substrate bound to aminoluciferin, mass spectrometry, HPLC / FPLC (reverse-phase chromatography, size exclusion chromatography), thin-layer chromatography, capillary zone electrophoresis, gel electrophoresis followed by activation staining (immobilized active DPP3) or Western blotting (cleavage products).

[0032] In another embodiment of the present invention, the substrate may be selected from the group comprising angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctrin, ACTH and MSH, or a dipeptide (the dipeptide being Arg-Arg) conjugated to a fluorophore, chromophore, or aminoluciferin.

[0033] In another specific embodiment of the present invention, the substrate may be selected from the group comprising a fluorophore, a chromophore, or a dipeptide conjugated to aminoluciferin (the dipeptide being Arg-Arg).

[0034] In specific embodiments, the binder exhibits a binding affinity of at least 107M-1, preferably 108M-1, to DPP3, more preferably greater than 109M-1, and most preferably greater than 1010M-1. Those skilled in the art will know that it may be considered to compensate for lower affinity by applying higher doses of the compound, and this measure is not outside the scope of the present invention.

[0035] To determine the affinity of the antibody for DPP3, the binding kinetics of DPP3 to the immobilized antibody were determined by unlabeled surface plasmon resonance using the Biacore2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible antibody immobilization was performed according to the manufacturer's instructions using high-density covalently bound anti-mouse Fc antibody on the surface of a CM5 sensor (Mouse Antibody Capture Kit; GE Healthcare) (Lorenz et al. 2011. Antimicrob Agents Chemother. 55 (1):165-173).

[0036] In one embodiment, such an assay for determining the level of DPP3 is a sandwich immunoassay using any type of detection technique, including but not limited to enzymatic labeling, chemiluminescence labeling, and electrochemiluminescence labeling, and is preferably a fully automated assay. In one embodiment of the diagnostic method, such an assay is an enzymatically labeled sandwich assay. Examples of automated or fully automated assays include assays that can be used with one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, BiomerieuxVidas®, and Alere Triage®.

[0037] Various immunoassays are known and can be used in the assays and methods of the present invention. These include mass spectrometry (MS), luminescence immunoassay (LIA), radioimmunoassay ("RIA"), homogeneous enzyme amplification immunoassay ("EMIT"), enzyme-linked immunosorbent assay ("ELISA"), apoenzyme reactivation immunoassay ("ARIS"), luminescence-based bead arrays, magnetic bead-based arrays, protein microarray assays, rapid test formats such as dipstick immunoassays, immunochromatography strip tests, rare cryptotate assays, and automated systems / analytical instruments.

[0038] In one embodiment of the present invention, this may be a so-called POC test (point-of-care test), which is a testing technique that enables testing to be performed near the patient within one hour without requiring a fully automated assay system. An example of this technique is an immunochromatographic testing technique, such as a microfluidic device.

[0039] In a specific embodiment, at least one of the two binders is labeled for detection in the sandwich immunoassay.

[0040] In another preferred embodiment, the label is selected from the group including chemiluminescent labels, enzyme labels, fluorescent labels, and radioactive iodine labels.

[0041] The assay may be homogeneous or heterogeneous, competitive or non-competitive. In one embodiment, the assay is a sandwich assay, which is a non-competitive immunoassay, in which the molecules to be detected and / or quantified are bound to a first antibody and a second antibody. The first antibody may be bound to a solid phase, e.g., beads, wells or the surface of another container, a tip or strip, and the second antibody may be labeled with, for example, a dye, a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general compositions and procedures involved in the "sandwich assay" are well-established and known to those skilled in the art (The Immunoassay Handbook, Ed. David Wild, Elsevier LTD, Oxford; 3rd ed. (May 2005), ISBN-13:978-0080445267; Hultschig C et al., Curr Opin Chem Biol. 2006 Feb;10(1):4-10. PMID:16376134).

[0042] In another embodiment, the assay comprises two capture molecules, preferably antibodies, both present as dispersions in a liquid reaction mixture, wherein a first labeled component binds to the first capture molecule, the first labeled component being part of a labeling system based on fluorescence, chemiluminescence quenching, or amplification, and a second labeled component of the marking system binds to the second capture molecule, thereby generating a measurable signal that allows detection of a sandwich complex formed in a solution containing the sample when both capture molecules bind to the analyte.

[0043] In another embodiment, the labeling system includes a rare earth cryptotate or rare earth chelate in combination with a fluorescent dye or chemiluminescent dye, particularly a cyanine-type dye.

[0044] In the context of the present invention, fluorescence-based assays include the use of dyes, such as FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes, such as CY3, CY5, CY3.5, CY5.5, Cy7, xanthene, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), TET, 6-carboxy- The following can be selected from the group including 4',5'-dichloro-2',7'-dimethodyfluorescein (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, e.g., BODIPY TMR, Oregon green, coumarin, e.g., umbelliferone, benzimide, e.g., Hoechst 33258; phenanthridine, e.g., Texas red, Yakima yellow, Alexa Fluor, PET, ethidium bromide, acridinium dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, etc.

[0045] In the context of the present invention, a chemiluminescence-based assay includes the use of dyes based on the physical principles described below for chemiluminescent materials (including citations on pages 551-562, incorporated herein by reference: Kirk-Othmer, Encyclopedia of Chemical Technology, 4th ed., executive editor, J. Kroschwitz; editor, M. Howe-Grant, John Wiley & Sons, 1993, vol. 15, pp. 518-562). Preferred chemiluminescent dyes are acridinium esters.

[0046] As used herein, “assay” or “diagnostic assay” can be any type applied in the field of diagnosis. Such assays may be based on the binding of the analyte to be detected to one or more capture probes with a predetermined affinity. With respect to the interaction between the capture molecule and the target molecule or molecule of interest, the affinity constant is preferably greater than 10⁸M⁻¹.

[0047] The subject of the present invention is an inhibitor of DPP3 activity for use as a treatment or intervention in critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury, wherein a predetermined threshold level of DPP3 in a body fluid sample of the subject is 20 to 120 ng / mL, more preferably 30 to 80 ng / mL, even more preferably 40 to 60 ng / mL, and most preferably 50 ng / mL.

[0048] In a specific embodiment, the assay is used to determine the level of DPP3, and the assay sensitivity of the assay is such that it can quantify DPP3 in a healthy subject and is less than 20 ng / ml, preferably less than 30 ng / ml, and more preferably less than 40 ng / ml.

[0049] In a particular embodiment of the present invention, the body fluid is a blood sample. The blood sample may be selected from the group including whole blood, serum, and plasma. In a specific embodiment of the method, the sample is selected from the group including human citrate plasma, heparin plasma, and EDTA plasma.

[0050] In another specific embodiment of the present invention, the level of DPP3 is determined in different samples taken from the patient at different time points.

[0051] In another specific embodiment of the present invention, the difference between the DPP3 levels is determined in different samples taken from the patient at different time points. This difference may be determined as an absolute difference or a relative difference.

[0052] In a specific embodiment of the present invention, the level of the DPP3 is determined at least twice.

[0053] In another specific embodiment of the present invention, treatment is initiated when the relative difference between the DPP3 levels in different samples taken 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.

[0054] In another specific embodiment of the present invention, the level of DPP3 is determined at least a second time 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.

[0055] The level of DPP3 as the amount of DPP3 protein in the body fluid sample of the subject and / or DPP3 activity can be determined, for example, by one of the following methods: 1. Luminescent immunoassay (LIA) for the quantification of DPP3 protein concentration (Rehfeld et al., 2019 JALM 3(6):943-953).

[0056] LIA is a one-step chemiluminescent sandwich immunoassay using white, highly bounded polystyrene microtiter plates as the solid phase. These plates are coated with monoclonal anti-DPP3 antibody AK2555 (capture antibody). Tracer anti-DPP3 antibody AK2553 is labeled with MA70-acridinium-NHS-ester and used at a concentration of 20 ng / well. 20 microliters of sample (e.g., serum, heparinized plasma, citrated plasma, or EDTA plasma derived from patient blood) and calibrator are pipetted into the coated white microtiter plates. After adding tracer antibody AK2553, the microtiter plates are incubated at room temperature and 600 rpm for 3 hours. Unbound tracer is then removed by four washing steps (350 μL / well). The remaining chemiluminescence is measured per well for 1 second using a microtiter plate luminometer. The DPP3 concentration is determined using a 6-point calibration curve. The calibrator and sample are preferably measured in duplicate.

[0057] 2. Enzyme capture activity assay (ECA) for the quantification of DPP3 activity (Rehfeld et al., 2019 JALM 3(6):943-953).

[0058] ECA is a DPP3-specific activity assay that uses black, highly bound polystyrene microtiter plates as the solid phase. These plates are coated with the monoclonal anti-DPP3 antibody AK2555 (capture antibody). 20 microliters of sample (e.g., serum, heparinized plasma, citrated plasma, EDTA plasma, cerebrospinal fluid, and urine) and calibrator are pipetted into the coated black microtiter plate. After adding assay buffer (200 μL), the microtiter plate is incubated at 22°C and 600 rpm for 2 hours. DPP3 present in the sample is immobilized by binding to the capture antibody. Unbound sample components are removed by four washing steps (350 μL / well). The specific activity of the immobilized DPP3 is measured by adding the fluorescence-generating substrate Arg-Arg-β-naphthylamide (Arg2-βNA) to the reaction buffer, followed by incubation at 37°C for 1 hour. DPP3 specifically cleaves Arg2-βNA into Arg-Arg dipeptide and fluorescent β-naphthylamine. Fluorescence is measured using a fluorometer with an excitation wavelength of 340 nm, and emission is detected at 410 nm. The activity of DPP3 is determined using a 6-point calibration curve. Calibrators and samples are preferably measured in duplicate.

[0059] 3. Liquid-phase assay (LAA) for the quantification of DPP3 activity (modified from Jones et al., Analytical Biochemistry, 1982).

[0060] LAA is a liquid-phase assay that measures DPP3 activity using a black unbound polystyrene microtiter plate. 20 μl of sample (e.g., serum, heparinized plasma, citrated plasma) and calibrator are pipetted into an unbound black microtiter plate. After adding the fluorescence-generating substrate Arg2-βNA to the assay buffer (200 μL), the initial βNA fluorescence (T=0) is measured using a fluorometer with an excitation wavelength of 340 nm, and the emission is detected at 410 nm. The plate is then incubated at 37°C for 1 hour. The final fluorescence (T=60) is measured. The difference between the final fluorescence and the initial fluorescence is calculated. DPP3 activity is determined using a 6-point calibration curve. The calibrator and sample are preferably performed in duplicate measurements.

[0061] The DPP3 levels of the present invention were determined using the described DPP3 assay, as outlined in the examples (Rehfeld et al. 2019. JALM 3(6):943-953). The thresholds described above may differ in other assays if they are calibrated in a different way than the assay system used in the present invention. Therefore, the cutoff values ​​described above should be applied to assays calibrated in such different ways, taking into account the differences in calibration. One possibility for quantifying the differences in calibration is a comparative analysis (correlation) method between the assay in question and each biomarker assay used in the present invention, by measuring each biomarker (e.g., DPP3) in the sample using both methods. Another possibility is to use the assay in question, assuming that this test has sufficient analytical sensitivity, to determine the median biomarker level of a representative normal population, compare the result to the median biomarker level as described in the literature, and recalculate the calibration based on the difference obtained by this comparison. Using the calibration employed in this invention, samples from 5,400 normal (healthy) subjects (Swedish single-center prospective population-based study (MPP-RES)) were measured, and the median (interquartile range) of plasma DPP3 was 14.5 ng / ml (11.3 ng / ml to 19 ng / ml).

[0062] Threshold levels can be obtained, for example, from Kaplan-Meier analysis, which correlates disease incidence with the quartile of a biomarker in a population. According to this analysis, subjects with biomarker levels above the 75th percentile have a significantly higher risk of developing the disease according to the present invention. This result is further supported by Cox regression analysis fully adjusted for classical risk factors. The highest quartile relative to all other subjects is very significantly associated with an increased risk of developing the disease according to the present invention.

[0063] Other preferred cutoff values ​​include, for example, the 90th, 95th, or 99th percentile of the normal population. Using a percentile higher than the 75th percentile reduces the number of false positives identified, but may still fail to identify moderately risked subjects who are still at high risk. Therefore, the cutoff value can be adopted depending on whether it is considered more appropriate to identify the majority of at-risk subjects at the expense of also identifying “false positives,” or whether it is considered more appropriate to identify primarily high-risk subjects at the expense of missing some moderately risked subjects.

[0064] Indications As used herein, the term “patient” refers to a living human or non-human organism that is receiving or in need of medical treatment for a disease. This includes humans who do not have a clear disease for which signs of illness are being investigated. Accordingly, the methods and assays described herein are applicable to both human and veterinary diseases.

[0065] Myocardial injury is defined by an increase in cardiac troponin levels above the upper limit of the 99th percentile. In particular, myocardial injury is structural damage to the cells and tissues of the cardiomyocytes (e.g., cardiomyocytes, cardiac fibroblasts, smooth muscle cells, or endothelial cells).

[0066] While this is considered a prerequisite for diagnosing myocardial infarction, it is also a disease entity in itself and can arise from non-ischemic or non-cardiac conditions (Thygesen et al. 2018. Fourth Universal 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. Heart 103(1):10-8). The term "myocardial injury" may be used in situations of direct cardiac injury such as cardiac contusion, but it can also occur in a variety of other clinical scenarios such as myocardial infarction, myocarditis, sepsis, and iatrogenic injury.

[0067] Specifically, myocardial injury can have the following causes: - Primary myocardial ischemia / myocardial infarction (rupture of atherosclerotic plaque accompanied by thrombus formation) - Imbalance between myocardial oxygen supply and demand (coronary spasm, microvascular dysfunction, coronary artery embolism / microembolism / dissection, persistent bradyarrhythmia / tachyarrhythmia, hypovolemic shock, respiratory failure / severe anemia, left ventricular hypertrophy / hypertrophic cardiomyopathy, severe hypertension), - Non-ischemic myocardial injury (heart failure, myocarditis / myocarditis, cardiomyopathy / takotsubo cardiomyopathy, cardiac contusion, iatrogenic causes (revascularization, cardiac surgery, ablation, pacing, cardioversion, defibrillation), rhabdomyolysis) - Multifactorial and systemic causes (sepsis / severe illness, cardiotoxicity (drugs), invasive diseases (cardiac amyloidosis, cardiac sarcoidosis), pulmonary embolism / pulmonary hypertension, acute or chronic kidney disease, stroke / subarachnoid hemorrhage).

[0068] Possible mechanisms of myocardial injury include direct cardiac injury with cardiomyocyte damage, myocardial strain resulting from excessive wall stress, and myocardial ischemia due to an imbalance between myocardial oxygen supply and demand. Myocardial injury can be irreversible and often involves myocardial necrosis or apoptosis (Park et al. 2017. Cardiac Troponins: From Myocardial Infarction to Chronic Disease. Cardiovasc Res. 113 (14): 1708-18).

[0069] In the context of this invention, “blood pressure” means mean arterial pressure (MAP), which is the mean arterial pressure throughout one cardiac cycle, systolic and diastolic. MAP is influenced by cardiac output and systemic vascular resistance, each of which is influenced by several variables. MAP is the primary determinant of perfusion pressure observed in organs within the body. Current guidelines recommend a MAP target of 65 mmHg or higher in critically ill medical patients (Dellinger et al. 2012. 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 Guidelines for cardiopulmonary resuscitation and emergency cardiovascular care. Circulation 122(18 Suppl 3):S768-786).

[0070] In one embodiment, the blood pressure reduction is a MAP of less than 65 mmHg, more preferably less than 60 mmHg, even more preferably less than 55 mmHg, and most preferably less than 50 mmHg.

[0071] In another embodiment, the blood pressure reduction 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.

[0072] Interleukin-6 (IL-6) is an important inflammatory mediator secreted into the circulatory system in response to acute infection and tissue injury. IL-6 expression is strictly regulated, and expression levels are low in healthy individuals. 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, resulting in negative inotropy and cytotoxicity. The inflammatory response mediates neutrophil infiltration and activation, induces further cytokine release into the bloodstream, co-stimulates the vascular endothelium, and induces ICAM-1 expression in cardiomyocytes, leading to myocardial fibrosis and ischemia / reperfusion injury (Gwechenberger et al. 1999. Circulation 99 (4): 546-551). As a result, myocardial damage and dysfunction are accelerated (Halawa et al. 1999. Pol. Arch. Med. Wewn 101 (3):197-203).

[0073] Troponin is a structural protein found in the troponin complex within the thin filaments of skeletal and cardiac muscle. The troponin complex consists of three subunits (I, T, and C) and, together with calcium ions, plays a crucial role in regulating muscle contraction (Kozinski et al. 2017. Critical Reviews in Clinical Laboratory Sciences 54 (3):143-172). Each molecule has a specific role in the muscle contraction process. Troponin T binds the troponin complex to actin filaments, troponin C acts as a calcium binding site, and troponin I inhibits interaction with myosin heads in the absence of sufficient calcium ions (Garg et al. 2017. Internal and Emergency Medicine 12 (2):147-155). Troponin T and I are primarily localized in cardiac muscle and are therefore called cardiac troponins (cTnI and cTnT). These biomarkers are generally recognized as having the highest specificity for identifying myocardial injury (Chaulin 2021. Vascular Health and Risk Management 17:299-316). Myocardial injury is confirmed when detectable cardiac troponin levels exceed the upper limit of the 99th percentile (URL) (Thygesen et al. 2019. Eur. Heart J. 40:237-269).

[0074] Myocardial protection means preventing myocardial injury.

[0075] Prevention of myocardial injury is defined as preventing an increase in cardiac troponin in the bloodstream. In particular, prevention of myocardial injury is defined as preventing structural damage to myocardial cells and tissues (e.g., cardiomyocytes, cardiac fibroblasts, smooth muscle cells, or endothelial cells) which is defined as an increase in cardiac troponin in the bloodstream.

[0076] In specific embodiments, myocardial injury is characterized by blood levels of cardiac troponin exceeding 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).

[0077] The cardiac troponin is selected from the group including cardiac troponin T (cTnT) and cardiac troponin I (cTnI). The troponin can be measured by a high-sensitivity troponin (hs-Tn) assay. The threshold for cardiac troponin concentration is, for example, above the upper limit of the 99th percentile (URL) (Thygesen et al. 2019. Eur. Heart J. 40:237-269).

[0078] Elevated cardiac troponin levels are further defined as elevated cardiac hypersensitivity troponin I (hs-cTnI) or cTnT levels, where at least one value exceeds the upper limit of the 99th percentile. Reference values ​​are sex-dependent (higher values ​​in men compared to women). Furthermore, reference values ​​depend on the assay used (Sandoval et al. 2022 Circulation 146:569-581). See Table 1 below.

[0079] [Table 1] Those skilled in the art can easily determine appropriate thresholds based on conventional considerations and practices that are common knowledge in the art, taking into account specific assay conditions.

[0080] Vasopressors increase vasoconstriction, thereby raising systemic vascular resistance (SVR). An increase in SVR leads to an increase in mean arterial pressure (MAP) and increased perfusion to organs. Vasopressors are selected from a group including isoproterenol, dobutamine, dopamine, phenylephrine, norepinephrine, epinephrine, vasopressin, or terlipressin.

[0081] The need for vasopressors is defined as a mean arterial pressure (MAP) of less than 65 mmHg.

[0082] The patient is a critically ill patient suffering from one of the following conditions: severe infection, sepsis, pulmonary embolism, pulmonary hypertension, acute coronary syndrome (including unstable angina, ST-elevation myocardial infarction (STEMI), and non-ST-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).

[0083] "Critically ill" means that a patient is suffering from an acute illness or condition that is life-threatening, potentially leading to death, or in which death is imminent. In specific embodiments, such critically ill patients are ICU patients.

[0084] The infection may be of bacterial, viral, fungal, or parasitic origin. The viral infection may be selected from infections caused by influenza viruses or coronaviruses.

[0085] The coronavirus in question is selected from a group that includes SARS-CoV-1, SARS-CoV-2, MERS-CoV, and especially SARS-CoV-2. Coronaviruses cause disease in mammals and birds. In humans, the virus typically causes respiratory infections, including the common cold, which are mild, but rarer forms such as SARS, MERS, and COVID-19 can be fatal. SARS-CoV-1 or SARS-CoV-2 infection can result in mild, moderate, or severe illness. The latter includes severe pneumonia, acute respiratory distress syndrome (ARDS), sepsis, and septic shock.

[0086] Acute respiratory distress syndrome (ARDS) is a type of respiratory failure characterized by the rapid onset of widespread inflammation in the lungs. Symptoms include shortness of breath, rapid breathing, and bluish discoloration of the skin. Even patients who survive generally experience a reduced quality of life. Possible causes include sepsis, pancreatitis, trauma, pneumonia, and aspiration. The underlying mechanisms involve diffuse damage to the cells forming the microscopic air sac septa of the lungs, surfactant dysfunction, activation of the immune system, and dysfunction of the body's regulation of blood coagulation. In effect, ARDS impairs the lungs' ability to exchange oxygen and carbon dioxide. Diagnosis is based on a positive end-expiratory pressure (PEEP) greater than 5 cmH2O, but a PaO2 / FiO2 ratio (ratio of arterial oxygen partial pressure to inspired oxygen fraction) less than 300 mmHg. Primary treatment involves mechanical ventilation along with treatment targeting the underlying cause. Ventilation strategies include the use of low volume and low pressure. If oxygenation remains insufficient, lung recruitment procedures and neuromuscular blocking agents may be used. If these are insufficient, extracorporeal membrane oxygenation (ECMO) may be an option. This syndrome is associated with a 35–50% mortality rate.

[0087] Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection (Singer et al. 2016. JAMA 315(8):801-810). Organ dysfunction can be identified as a rapid change in total SOFA score ≥ 2 due to infection. The baseline SOFA score may be considered 0 in patients who are not known to have pre-existing organ dysfunction. An SOFA score ≥ 2 reflects approximately 10% of the overall mortality risk in a general hospital population suspected of infection. Even patients showing mild dysfunction may worsen, highlighting the severity of this condition and the need for rapid and appropriate intervention if not already implemented. Sepsis is a life-threatening condition that arises when the body's response to infection damages its own tissues and organs. Patients suspected of having an infection with a high probability of prolonged ICU stay or in-hospital death can be rapidly identified at the bedside by qSOFA, i.e., altered consciousness, systolic blood pressure ≤ 100 mmHg, or respiratory rate ≥ 22 / min.

[0088] Shock is characterized by decreased oxygen delivery and / or increased oxygen consumption, or insufficient oxygen utilization, resulting in cellular and tissue hypoxia. It is a life-threatening condition of circulatory failure, most commonly presenting as hypotension (systolic blood pressure less than 90 mmHg or MAP less than 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).

[0089] Septic shock is a potentially fatal medical condition that arises when sepsis, which is organ failure or injury in response to infection, leads to dangerously low blood pressure and cellular metabolic abnormalities. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) defines septic shock as a subset of sepsis in which particularly severe circulatory, cellular, and metabolic abnormalities are associated with a higher mortality risk than sepsis alone. Patients with septic shock can be clinically identified by requiring vasopressors to maintain mean arterial pressure above 65 mmHg in the absence of hypovolemia, and by serum lactate levels exceeding 2 mmol / L (greater than 18 mg / dL). This combination is associated with an in-hospital mortality rate exceeding 40% (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. These can be located in any part of the body, but most commonly in the lungs, brain, urinary tract, skin, or abdominal organs. This can lead to multiple organ failure syndrome (formerly known as multiple organ failure) and death. In many cases, patients with septic shock are treated in the intensive care unit. Children, immunocompromised individuals, and the elderly are most susceptible because their immune systems cannot cope with infection as effectively as those of healthy adults. The mortality rate from septic shock is approximately 25–50%.

[0090] Cardiogenic shock (CS) is defined as a state of severe peripheral organ hypoperfusion due to decreased cardiac output. In particular, CS forms a spectrum ranging from mild hypoperfusion to severe shock. Established criteria for diagnosing CS are: (i) systolic blood pressure ≤90 mmHg for more than 30 minutes, or requiring vasopressors to achieve blood pressure ≥90 mmHg; (ii) pulmonary congestion or elevated left ventricular filling pressure; and (iii) signs of organ perfusion impairment by at least one of the following criteria: (a) altered mental state; (b) cold, damp skin; (c) oliguria (<0.5 mL / kg / hour or <30 mL / hour); (d) elevated serum lactate (Reynolds and Hochman 2008. Circulation 117:686-697). Acute myocardial infarction (AMI) and subsequent ventricular dysfunction are the most frequent causes of CS, accounting for approximately 80% of cases. Mechanical complications such as ventricular septal rupture (4%) or free wall rupture (2%), and acute severe mitral regurgitation (7%) are less frequent causes of CS after AMI (Hochman et al. 2000. J Am Coll Cardiol 36:1063-1070). Non-AMI-related CS can be caused by decompensated valvular disease, acute myocarditis, arrhythmias, etc., and the treatment options are diverse. This translates to 40,000-50,000 patients per year in the United States and 60,000-70,000 patients per year in Europe. Despite advances in treatment, mainly through early vascular regeneration, and the subsequent decrease in mortality, CS remains the leading cause of death in AMI, and according to recent registries and randomized trials, the mortality rate is still close to 40-50% (Goldberg et al. 2009. Circulation 119:1211-1219).

[0091] Acute coronary syndrome refers to a group of conditions characterized by reduced blood flow to the heart, including ST-elevation myocardial infarction (STEMI), non-ST-elevation myocardial infarction (NSTEMI), and unstable angina. It is a type of coronary heart disease (CHD) and accounts for one-third of all-cause deaths in people over 35 years of age. While some forms of CHD can be asymptomatic, ACS is always symptomatic. According to guidelines, acute myocardial infarction is defined as follows: The term acute myocardial infarction should be used when there is acute myocardial injury with clinical evidence of acute myocardial ischemia, elevated and / or decreased cardiac troponin (cTn) levels are detected, with at least one of these levels exceeding the upper limit of the 99th percentile (URL), and at least one of the following is present: symptoms of myocardial ischemia, new ischemic ECG changes, appearance of pathological Q waves, new loss of viable myocardium or new focal wall motion abnormalities in a pattern consistent with ischemic etiology, or identification of coronary thrombus by angiography or autopsy (Thygesen et al. 2018. Fourth Universal Definition of Myocardial Infarction (2018). Eur Heart J. 40(3):237-69). Furthermore, there are also coronary intervention-related myocardial infarctions, such as percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG).

[0092] DPP3 inhibitors The inhibitors are preferably molecules that significantly inhibit DPP3 activity. These molecules may be peptides and small molecules, antibodies, antibody fragments, or non-Ig scaffolds.

[0093] Significant inhibition means inhibiting DPP3 activity by more than 60%, preferably more than 70%, more preferably more than 80%, preferably more than 90%, and more preferably nearly or actually 100%.

[0094] DPP3 activity can be nonspecifically inhibited by various common protease inhibitors (e.g., PMSF, TPCK), sulfhydryl reagents (e.g., pHMB, DTNB), and metal chelating agents (EDTA, o-phenanthroline) (Abramic et al. 2000. Biological Chemistry, 381:1233-1243; European Patent No. 2949332).

[0095] DPP3 activity can also be specifically inhibited by different types of compounds: the endogenous DPP3 inhibitor is the peptide spinorphin. Several synthetic derivatives of spinorphin, such as tynorphin, have been produced and shown to inhibit DPP3 activity to varying degrees (Yamamoto et al. 2000. Life Sciences 62(19):1767-1773). Other published peptide inhibitors of DPP3 include propioxatine A and B (US Patent No. 4804676), and propioxatine A analog (Inaoka et al. 1988. J. Biochem 104(5):706-711).

[0096] A "derivative or analogue" is a chemical compound derived from a parent compound through a chemical reaction involving the substitution of one atom or a group of atoms by a functional group. The parent compound and the derivative compound have similar chemical structures.

[0097] DPP3 can also be inhibited by small molecules such as fluostatin and benzimidazole derivatives. Fluostatin A and B are antibiotics produced in a species of Streptomyces (Streptomyces sp.) TA-3391; they are non-toxic and potently inhibit DPP3 activity. To date, 20 different benzimidazole derivatives have been synthesized and published (Agic et al. 2007. Bioorganic Chemistry 35(2):153-169; Rastija et al. 2015. Acta Chimica Slovenica 62:867-878), of which two compounds, 1' and 4', exhibit the strongest inhibitory effects (Agic et al. 2007. Bioorganic Chemistry 35 (2):153-169). Several dipeptidylhydroxamic acids have also been shown to inhibit DPP3 activity (Cvitesic et al., 2016. J Enzyme Inhib Med Chem 31(sup2):40-45).

[0098] "Small molecules" are organic compounds with particularly low molecular weight (more specifically, less than 1000 daltons). Such small molecules can modulate biological processes, for example, by binding to certain biological macromolecules, particularly DPP3 in this invention, and acting as effectors, especially inhibitors, to alter the activity or function of the biological macromolecule. Small molecules may be of natural or artificial origin.

[0099] Specific examples of small molecule and peptide inhibitors of DPP3 are shown in Table 2 below. Compounds 1' and 4' exhibit the strongest inhibitory effects (Agic et al. 2007).

[0100] [Table 2-1] [Table 2-2] [Table 2-3] The subject matter of this application is an inhibitor of DPP3 activity for use in the treatment or intervention in critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury, wherein the inhibitor of DPP3 activity is selected from the group comprising small molecules, anti-DPP3 antibodies, anti-DPP3 antibody fragments, or anti-DPP3 non-Ig scaffolds.

[0101] The subject matter of this application is an inhibitor of DPP3 activity for use in the treatment or intervention in critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury, wherein the inhibitor of DPP3 activity is a small molecule selected from the group including spinorphine, tinorphine, propioxatine A and B, fluostatin A and B, benzimidazole or its derivatives or analogs.

[0102] The subject matter of this application is an inhibitor of DPP3 activity for use in the treatment or intervention in critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury, the inhibitor being 10 -7 This is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that exhibits minimal binding affinity to DPP3 of M or lower.

[0103] The subject matter of this application is an inhibitor of DPP3 activity for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury, wherein the antibody is a monoclonal antibody or a monoclonal antibody fragment.

[0104] Throughout this specification, the “antibody,” “antibody fragment,” or “non-Ig scaffold” according to the present invention can bind to DPP3 and therefore target DPP3, and can therefore be referred to as “anti-DPP3 antibody,” “anti-DPP3 antibody fragment,” or “anti-DPP3 non-Ig scaffold.”

[0105] The term “antibody” generally includes monoclonal and polyclonal antibodies and their conjugated fragments, particularly Fc fragments, as well as so-called “single-chain antibodies” (Bird et al. 1988), chimeric antibodies, humanized antibodies, particularly CDR-transplant antibodies, and diabodies or tetrabodies (Holliger et al. 1993). It also includes, for example, immunoglobulin-like proteins selected via techniques including phage display that specifically bind to the target molecule contained in a sample. In this context, the term “specific binding” refers to an antibody produced against the target molecule or its fragment. An antibody is considered specific if its affinity for the target molecule or its aforementioned fragment is at least preferably 50-fold higher, more preferably 100-fold higher, and most preferably at least 1000-fold higher than its affinity for other molecules contained in the sample containing the target molecule. Methods for producing antibodies and selecting antibodies with a given specificity are well known in the art.

[0106] In one embodiment of the present invention, the anti-DPP3 antibody, the anti-DPP3 antibody fragment, or the anti-DPP3 non-Ig scaffold is monospecific.

[0107] A monospecific anti-DPP3 antibody, monospecific anti-DPP3 antibody fragment, or monospecific anti-DPP3 non-Ig scaffold means that the antibody, antibody fragment, or non-Ig scaffold binds to one specific region encompassing at least five amino acids within the target DPP3 (SEQ ID NO: 1). A monospecific anti-DPP3 antibody, monospecific anti-DPP3 antibody fragment, or monospecific anti-DPP3 non-Ig scaffold is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that all have the same affinity for the same antigen. While monoclonal antibodies are monospecific, monospecific antibodies can also be produced by means other than producing them from common germ cells.

[0108] In specific embodiments, 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 nearly or actually 100%.

[0109] The antibody or fragment according to the present invention is a protein comprising one or more polypeptides substantially encoded by an immunoglobulin gene that specifically binds to an antigen. Recognized immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma (IgG1, IgG2, IgG3, IgG4), delta (IgD), epsilon (IgE), and mu (IgM) constant region genes, as well as numerous immunoglobulin variable region genes. Full-length immunoglobulin light chains are generally about 25 kd or 214 amino acids long.

[0110] Full-length immunoglobulin heavy chains are generally about 50 kd or 446 amino acids long. The light chain is encoded by a variable region gene at the NH2 terminus (about 110 amino acids long) and a kappa or lambda constant region gene at the COOH terminus. The heavy chain is similarly encoded by a variable region gene (about 116 amino acids long) and one of the other constant region genes.

[0111] The basic structural unit of an antibody is generally a tetramer consisting of two identical pairs of immunoglobulin chains, each pair having 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 region mediates effector function. Immunoglobulins also exist in various other forms, including, for example, Fv, Fab, and (Fab')2, as well as bifunctional hybrid antibodies and single-chain antibodies (e.g., Lanzavecchia et al. 1987. Eur. J. Immunol. 1 7:10 5; Huston et al. 1988. Proc. Natl. Acad. Sci. USA, 8 5:5879-588 3; Bird et al. 1988. Science 242:423-426; Hood et al. 1984, Immunology, Benjamin, NY, 2n (d ed.; Hunkapiller and Hood 1986. Nature 323:15-16). The immunoglobulin light chain or heavy chain variable region contains a framework region interrupted by three hypervariable regions also called complementarity determining regions (CDRs) (see Sequences of Proteins of Immunological Interest, E. Kabat et al. 1983, USD Department of Health and Human Services). As described above, CDRs are primarily involved in the binding of antigens to epitopes. An immune complex is an antibody, such as a monoclonal antibody, chimeric antibody, humanized antibody, or human antibody, or a functional antibody fragment, that specifically binds to an antigen.

[0112] Chimeric antibodies are antibodies in which the light chain and heavy chain genes are constructed, typically by genetic engineering, from immunoglobulin variable region genes and constant region genes belonging to different species. For example, the variable segment of a gene derived from a mouse monoclonal antibody can be linked to a human constant segment, such as kappa and gamma 1 or gamma 3. Thus, in one example, a therapeutic chimeric antibody is a hybrid protein composed of a variable domain or antigen-binding domain derived from a mouse antibody and a constant domain or effector domain derived from a human antibody, but other mammalian species may be used, or the variable region may be created by molecular technology. Methods for producing chimeric antibodies are well known in the art; see, for example, U.S. Patent No. 5,807,715. "Humanized" immunoglobulins are immunoglobulins that include a human framework region and one or more CDRs derived from non-human (e.g., mouse, rat, or synthetic) immunoglobulins. The non-human immunoglobulin providing the CDRs is called the "donor," and the human immunoglobulin providing the framework is called the "acceptor." In one embodiment, all CDRs in the humanized immunoglobulin are derived from the donor immunoglobulin. Constant regions are not required, but if present, they must be substantially identical to the human immunoglobulin constant regions, i.e., at least about 85-90%, e.g., about 95% or more identical. Therefore, in some cases, all parts of the humanized immunoglobulin, except for the CDRs, are substantially identical to the corresponding parts of the natural human immunoglobulin sequence. A "humanized antibody" is an antibody containing humanized light chain and humanized heavy chain immunoglobulins. The humanized antibody binds to the same antigen as the donor antibody providing the CDRs. The acceptor framework of the humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies may have further conserved amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Exemplary conservative substitutions include those of gly, ala;val, ile, leu;asp, glu;asn, gln;ser, thr;lys, arg; and phe, tyr.Humanized immunoglobulins can be constructed by genetic engineering (see, for example, U.S. Patent No. 5,585,089). Human antibodies are antibodies whose light and heavy chain genes are of human origin. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells that secrete the antibody of interest. Immortalization can be achieved, for example, by EBV infection, or by fusing human B cells with myeloma or hybridoma cells to produce trioma cells. Human antibodies can also be produced by phage display (see, for example, International Publication No. 91 / 17271; International Publication No. 92 / 001047, International Publication No. 92 / 20791), or selected from a human combinatorial monoclonal antibody library (see Morphosys website). Human antibodies can also be prepared by using transgenic animals that possess human immunoglobulin genes (see, for example, International Publication No. 93 / 12227 and International Publication No. 91 / 10741).

[0113] Therefore, anti-DPP3 antibodies may have formats known in the art. Examples include human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, and CDR-transplanted antibodies. In preferred embodiments, the antibodies according to the present invention include, for example, antibodies recombinantly produced as IgG, a typical full-length immunoglobulin, or antibody fragments containing at least the F variable domain of the heavy chain and / or light chain as, for example, chemically bound antibodies (fragment antigen binding), and include, but are not limited to, Fab minibodies, single-chain Fab antibodies, monovalent Fab antibodies with epitope tags, e.g., Fab-V5Sx2; divalent Fab (mini-antibodies) dimerized with a CH3 domain; divalent or multivalent Fab formed, for example, through multimerization using heterologous domains, e.g., through dimerization of the dHLX domain, e.g., Fab-dHLX-FSx2; F(ab')2 fragments, scFv fragments, multimerized multivalent and / or multispecific scFv fragments, divalent and / or bispecific diabodies, BITE® (bispecific T-cell engager), trifunctional antibodies, multivalent antibodies, e.g., multivalent antibodies derived from a class different from G; single-domain antibodies, e.g., nanobodies derived from camelid or fish immunoglobulins; and many others.

[0114] In a preferred embodiment, the anti-DPP3 antibody format is selected from the group comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, F(ab)2 fragment, and scFv-Fc fusion protein. In another preferred embodiment, the antibody format is selected from the group comprising scFab fragment, Fab fragment, scFv fragment, and their bioavailability-optimized conjugates, such as PEGylated fragments. One of the most preferred formats is the scFab format.

[0115] The non-Ig scaffold may also be a protein scaffold and can be used as an antibody mimetic because it can bind to a ligand or antigen. In one embodiment, the non-Ig scaffold is a tetranectin-based non-Ig scaffold (e.g., described in U.S. Patent Application Publication 2010 / 0028995), a fibronectin scaffold (e.g., described in European Patent No. 1266025); a lipocalin-based scaffold (e.g., described in International Publication 2011 / 154420); a ubiquitin scaffold (e.g., described in International Publication 2011 / 073214); a transferrin scaffold (e.g., described in U.S. Patent Application Publication 2004 / 0023334); a protein A scaffold (e.g., described in European Patent No. 2231860); an ankyrin repeat-based scaffold (e.g., described in International Publication 2010 / 060748); a microprotein, preferably a microprotein forming a cysteine ​​knot, scaffold (e.g., described in European Patent No. 2314308); Fyn The scaffolds may be selected from a group including those based on the SH3 domain (e.g., described in International Publication No. 2011 / 023685), those based on the EGFR-A domain (e.g., described in International Publication No. 2005 / 040229), and those based on the Kunitz domain (e.g., described in European Patent No. 1941867).

[0116] In one embodiment of the present invention, the anti-DPP3 antibody according to the present invention can be prepared by synthesizing a fragment or full-length DPP3 as an antigen, as outlined in Example 1. Subsequently, a binder for the fragment is identified using the method described below or other methods known in the art.

[0117] Humanization of mouse antibodies can be carried out according to the following procedure. For the humanization of mouse-derived antibodies, antibody sequences are analyzed for structural interactions between the antigen and a framework region (FR) containing a complementary determination region (CDR). Based on structural modeling, appropriate human-derived FRs are selected, and mouse CDR sequences are transplanted into human FRs. Mutations in the amino acid sequences of the CDR or FR can be introduced to restore structural interactions lost due to species switching of the FR sequence. This restoration of structural interactions can be achieved by a random approach using a phage display library or by a directed approach guided by molecular modeling (Almagro and Fransson 2008. Humanization of antibodies. Front Biosci. 2008 Jan 1;13:1619-33).

[0118] 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.

[0119] The subject matter of this application is an inhibitor of DPP3 activity for use in the treatment or intervention in critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury, wherein the heavy chain complementarity determining region (CDR) is Includes the sequence of sequence number 7, sequence number 8, and / or sequence number 9, The light chain complementarity-determining region (CDR) includes the sequences of sequence number 10, KVS, and / or sequence number 11.

[0120] The subject matter of this application is an inhibitor of DPP3 activity for use in the treatment or intervention in critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury, wherein the monoclonal antibody or antibody fragment is a humanized monoclonal antibody or humanized monoclonal antibody fragment.

[0121] The subject matter of this application is an inhibitor of DPP3 activity for use in the treatment or intervention in critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury, wherein the heavy chain comprises the sequence of SEQ ID NO: 12 and the light chain comprises the sequence of SEQ ID NO: 13.

[0122] The subject matter of this application is an inhibitor of DPP3 activity for use in the treatment or intervention of critically ill patients with hypotension for cardioprotection and / or prevention of cardiac injury, 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 acid lengths as included in SEQ ID NO: 1. In one embodiment, 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 amino acid lengths as included in SEQ ID NO: 1. In another embodiment, 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 5 amino acid lengths as included in SEQ ID NO: 1.

[0123] The subject matter of this application is an inhibitor of DPP3 activity for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury, 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 acid length included in SEQ ID NO: 2, the epitope being included in DPP3 as shown in SEQ ID NO: 1. In one embodiment, 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 amino acid length included in SEQ ID NO: 2, the epitope being included in DPP3 as shown in SEQ ID NO: 1. In another embodiment, 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 5 amino acid length included in SEQ ID NO: 2, the epitope being included in DPP3 as shown in SEQ ID NO: 1.

[0124] The subject matter of this application is an inhibitor of DPP3 activity for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury, 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 acid length included in SEQ ID NO: 3, the epitope being included in DPP3 as shown in SEQ ID NO: 1. In one embodiment, 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 amino acid length included in SEQ ID NO: 3, the epitope being included in DPP3 as shown in SEQ ID NO: 1. In another embodiment, 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 5 amino acid length included in SEQ ID NO: 3, the epitope being included in DPP3 as shown in SEQ ID NO: 1.

[0125] The subject matter of this application is an inhibitor of DPP3 activity for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury, 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 acid lengths included in SEQ ID NO: 4, the epitope being included in DPP3 as shown in SEQ ID NO: 1. In one embodiment, 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 amino acid lengths included in SEQ ID NO: 4, the epitope being included in DPP3 as shown in SEQ ID NO: 1. In another embodiment, 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 5 amino acid lengths included in SEQ ID NO: 4, the epitope being included in DPP3 as shown in SEQ ID NO: 1.

[0126] Epitopes, also known as antigenic determinants, are parts of an antigen that are recognized by the immune system, particularly by antibodies. For example, an epitope is a specific piece of an antigen to which an antibody binds. The part of the antibody that binds to an epitope is called a paratope. Protein antigen epitopes are divided into two categories, structural epitopes and linear epitopes, based on their structure and interaction with paratopes. Structural and linear epitopes interact with paratopes based on the 3-D stereochemistry adopted by the epitope, which is determined by the surface features of the epitope residues involved and the shape or tertiary structure of other segments of the antigen. Structural epitopes are formed by a 3-D stereochemistry adopted by the interaction of discontinuous amino acid residues. Linear or continuous epitopes are epitopes that are recognized by antibodies by their linear sequence or primary structure of amino acids and are formed by a 3-D stereochemistry adopted by the interaction of adjacent amino acid residues.

[0127] In specific embodiments of the present invention, the antibody is a monoclonal antibody or a fragment thereof. In one embodiment of the present invention, the anti-DPP3 antibody or anti-DPP3 antibody fragment is a human antibody or a humanized antibody, or is derived therefrom. In one specific embodiment, one or more (mouse) CDRs are implanted onto a human antibody or antibody fragment.

[0128] The subject of the present invention, in one embodiment, is a human or humanized CDR-transplantable antibody or antibody fragment thereof that binds to DPP3, wherein the human or humanized CDR-transplantable antibody or antibody fragment thereof comprises the following antibody heavy chain (H chain): GFSLSTSGMS (Sequence ID 7), IWWNDNK (Sequence ID 8), ARNYSYDY (Sequence ID 9) antibody light chains (L chains) including and / or including the following: RSLVHSIGSTY (Sequence ID 10), KVS (not part of the sequence listing), Includes SQSTHVPWT (sequence number 11).

[0129] In one specific embodiment of the present invention, the subject of the present invention is a human or humanized monoclonal antibody or an antibody fragment thereof that binds to DPP3, wherein the heavy chain is GFSLSTSGMS (Sequence ID 7), IWWNDNK (Sequence ID 8), ARNYSYDY (Sequence ID 9) It includes at least one CDR selected from the group including, and the light chain is RSLVHSIGSTY (Sequence ID 10), KVS (not part of the sequence listing), It includes at least one CDR selected from the group including SQSTHVPWT (SEQ ID NO: 11).

[0130] The anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold according to the present invention has an affinity constant of 10 for human DPP3. -7 Larger than M, preferably 10 -8 M, and the preferred affinity is 10 -9 Larger than M, most preferably 10 -10 It exhibits an affinity higher than M. Those skilled in the art will know that it may be considered to compensate for the lower affinity by applying a higher dose of the compound, and this measure is not outside the scope of the present invention. The affinity constant can be determined according to the method described in Example 1.

[0131] The subject of the present invention is a monoclonal antibody or fragment or antibody fragment conjugated to DPP3 for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury, wherein the antibody or fragment has the following sequence as a variable heavy chain: Sequence ID 5 QVTLKESGPGILQPSQTLSLTCSSGFSLSTSGMSVGWIRQPSGKGLEWLAHIWWNDNKSYNPALKSRLTISRDTSNNQVFLKIASVVTADTGTYFCARNYSYDYWGQGTTLTVSS The following sequence is used for the variable light chain: Sequence ID 6 DVVVTQTPLSLSVSLGDPASISCRSSRSLVHSIGSTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK Includes. The subject of the present invention is a human or humanized monoclonal antibody or fragment conjugated to DPP3, or an antibody fragment thereof, for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury, wherein the antibody or fragment has the following sequence as its heavy chain: Sequence ID 12 MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQVVLTMTNMMDPVDTG TYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG The light chain has the following sequence: Sequence ID 13 METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYYCSQSTHV PWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Includes. In a specific embodiment of the present invention, the antibody has the following sequence as its heavy chain: SEQ ID NO: 12 Or it includes a sequence that is more than 95%, preferably more than 98%, preferably more than 99%, identical to it, and the light chain is the following sequence: SEQ ID NO: 13 Or it includes a sequence that is more than 95%, preferably more than 98%, and preferably more than 99% identical to it.

[0132] Pairwise alignment is performed to evaluate the identity between two amino acid sequences. Identity is defined by the percentage of amino acids that directly match in the alignment.

[0133] In a preferred embodiment, treatment with a DPP3 activity inhibitor is initiated or modified as soon as the results of a sample analysis indicating the level of DPP3 in the sample are provided. In a further embodiment, the treatment may be initiated within 12 hours, preferably within 6, 4, 2, 1, 0.5, or 0.25 hours, or immediately after receiving the results of the sample analysis.

[0134] In some embodiments, the method comprises or consists of one and / or multiple measurements of DPP3 in patient-derived samples in single and / or multiple samples obtained at essentially the same point in time, in order to guide and / or monitor and / or stratify treatment, wherein the treatment is the administration of an inhibitor of DPP3 activity.

[0135] The term "pharmaceutical preparation" means a pharmaceutical ingredient combined with at least one pharmaceutically acceptable excipient in a form that enables the biological activity of the contained pharmaceutical ingredient to be effective, and does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is administered. The term "pharmaceutical ingredient" means a therapeutic composition that can be optionally combined with pharmaceutically acceptable excipients to provide a pharmaceutical preparation or dosage form.

[0136] The subject of the present invention is a pharmaceutical formulation comprising an antibody, fragment, or scaffold according to the present invention, for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury.

[0137] The subject of the present invention is a pharmaceutical formulation for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury according to the present invention, the pharmaceutical formulation being a solution, preferably a ready-to-use solution.

[0138] The subject of the present invention is a pharmaceutical formulation for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury according to the present invention, said pharmaceutical formulation is in a lyophilized state.

[0139] The subject of the present invention is a pharmaceutical formulation for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury according to the present invention, the pharmaceutical formulation being administered intramuscularly.

[0140] The subject of the present invention is a pharmaceutical formulation for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury according to the present invention, the pharmaceutical formulation being administered intravascularly.

[0141] The subject of the present invention is a pharmaceutical formulation for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury according to the present invention, the pharmaceutical formulation being administered by infusion.

[0142] The subject of the present invention is a pharmaceutical formulation for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury according to the present invention, the pharmaceutical formulation being administered systemically.

[0143] Based on the above context, the following sequentially numbered embodiments provide further specific aspects of the present invention. 1. An inhibitor of DPP3 activity for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury.

[0144] 2. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having the hypotension described in Embodiment 1, wherein the hypotension is a mean arterial pressure (MAP) of less than 65 mmHg, more preferably less than 60 mmHg, even more preferably less than 55 mmHg, and most preferably less than 50 mmHg.

[0145] 3. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient with hypotension as described in Embodiments 1 and 2, wherein the patient has a level of DPP3 exceeding a (predetermined) threshold in a sample of the patient's body fluids.

[0146] 4. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient with hypotension as described in Embodiment 3, wherein the predetermined threshold level of DPP3 in the body fluid sample of the subject is 20 to 120 ng / mL, more preferably 30 to 80 ng / mL, even more preferably 40 to 60 ng / mL, and most preferably 50 ng / mL.

[0147] 5. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient with hypotension according to any of Embodiments 1 to 4, wherein the sample is a body fluid sample selected from the group including whole blood, plasma, or serum.

[0148] 6. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having hypotension as described in any of Embodiments 1 to 5, characterized by a blood level of cardiac troponin (cTn) exceeding a threshold, increased myocardial expression of pro-inflammatory interleukin-6 (IL-6), and / or the need for a vasopressor to maintain blood pressure and cardiac output.

[0149] 7. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient with hypotension as described in Embodiment 6, wherein the cardiac troponin (cTn) is cardiac troponin T (cTnT) or cardiac troponin I (cTnI).

[0150] 8. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having hypotension as described in any of Embodiments 1 to 7, who is suffering from a severe infection, sepsis, pulmonary embolism, pulmonary hypertension, acute coronary syndrome (including unstable angina, ST-elevation myocardial infarction (STEMI), non-ST-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).

[0151] 9. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient with hypotension according to any of Embodiments 1 to 8, wherein the inhibitor of DPP3 activity is selected from the group comprising a small molecule, an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

[0152] 10. An inhibitor of DPP3 activity for use in the treatment or intervention of a severely ill patient with impaired lung function as described in Embodiment 9, wherein the inhibitor is an anti-DPP3 antibody, an anti-DPP3 antibody fragment, or an anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acid length contained in SEQ ID NO: 1.

[0153] 11. An inhibitor of DPP3 activity for use in the treatment or intervention of severely ill patients with impaired lung function as described in Embodiments 9 and 10, wherein the inhibitor is an anti-DPP3 antibody, an anti-DPP3 antibody fragment, or an anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acid length contained in SEQ ID NO: 2.

[0154] 12. An inhibitor of DPP3 activity for use in the treatment or intervention of a severely ill patient with impaired lung function according to any of Embodiments 9 to 11, wherein the antibody is a monoclonal antibody or a monoclonal antibody fragment.

[0155] 13. The antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, and the complementarity-determining region (CDR) of the heavy chain is Includes the sequence of sequence number 7, sequence number 8, and / or sequence number 9, The complementarity determination region (CDR) of the light chain is An inhibitor of DPP3 activity for use in the treatment or intervention of critically ill patients with impaired lung function as described in any of embodiments 9 to 12, comprising the sequence of SEQ ID NO: 10, KVS, and / or SEQ ID NO: 11.

[0156] 14. An inhibitor of DPP3 activity for use in the treatment or intervention of a severely ill patient with impaired lung function 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.

[0157] 15. An inhibitor of DPP3 activity for use in the treatment or intervention of a severely ill patient with impaired lung function as described in any of Embodiments 9 to 14, wherein the antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, the heavy chain comprising the sequence of SEQ ID NO: 12 and the light chain comprising the sequence of SEQ ID NO: 13.

[0158] 16. An inhibitor of DPP3 activity for use in the treatment or intervention of a severely ill patient with impaired lung function as described in Embodiment 9, wherein the small molecule is selected from the group comprising spinorphine, tynorphine, propioxatine A and B, fluostatin A and B, benzimidazole, or derivatives or analogs thereof.

[0159] To avoid misunderstanding, the following embodiments 10a to 16a correspond to embodiments 10 to 16 above and include a correct reference to embodiment 9, which is described as “blood pressure reduction.” References to one or more embodiments 10 to 16 also mean references to one or more embodiments 10a to 16a.

[0160] 10a. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient with hypotension as described in 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 acid length contained in SEQ ID NO: 1.

[0161] 11a. An inhibitor of DPP3 activity for use in the treatment or intervention of critically ill patients with hypotension as described in 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 acid length contained in SEQ ID NO: 2.

[0162] 12a. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient with hypotension according to any of Embodiments 9 to 11, wherein the antibody is a monoclonal antibody or a monoclonal antibody fragment.

[0163] 13a. The antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, and the complementarity-determining region (CDR) of the heavy chain is Includes the sequence of sequence number 7, sequence number 8, and / or sequence number 9, The complementarity determination region (CDR) of the light chain is An inhibitor of DPP3 activity for use in the treatment or intervention of critically ill patients with hypotension according to any of embodiments 9 to 12, comprising the sequence of SEQ ID NO: 10, KVS, and / or SEQ ID NO: 11.

[0164] 14a. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient with hypotension 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.

[0165] 15a. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient with hypotension according to any of Embodiments 9 to 14, wherein the antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, the heavy chain comprising the sequence of SEQ ID NO: 12 and the light chain comprising the sequence of SEQ ID NO: 13.

[0166] 16a. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient with hypotension as described in Embodiment 9, wherein the small molecule is selected from the group comprising spinorphine, tynorphine, propioxatine A and B, fluostatin A and B, benzimidazole, or derivatives or analogs thereof.

[0167] 17. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having hypotension as described in any of Embodiments 1 to 16a, wherein the myocardial injury is an elevation of cardiac troponin levels above the upper limit of the 99th percentile, and more specifically, the myocardial injury is structural injury of myocardial cells and tissues (e.g., cardiomyocytes, cardiac fibroblasts, smooth muscle cells, or endothelial cells).

[0168] 18. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having hypotension as described in any of Embodiments 1 to 17, wherein the blood cTn level and / or myocardial expression of IL-6 determined in a sample obtained from the patient is elevated, particularly above a threshold, and more specifically, the cTn is elevated above a threshold as defined in Embodiment 17. [Brief explanation of the drawing]

[0169] [Figure 1] Kaplan-Meier survival plots related to low (<68.6 ng / mL) and high (≧68.6 ng / mL) DPP3 plasma concentrations: (A) 7-day survival rate for patients with sepsis / septic shock related to DPP3 plasma concentration (cutoff 68.6 ng / mL); (B) 7-day survival rate for patients with cardiogenic shock related to DPP3 plasma concentration (cutoff 68.6 ng / mL); (C) 7-day survival rate for patients with acute myocardial infarction related to DPP3 plasma concentration (cutoff 68.6 ng / mL); (D) 3-month survival rate for patients with dyspnea related to DPP3 plasma concentration; (E) 4-week survival rate for burn patients related to DPP3 plasma concentration; (F) 7-day survival rate for patients with septic shock related to DPP3 plasma concentration. [Figure 2] SDS-PAGE of natural hDPP3 purified from human erythrocyte lysate on a gradient gel (4-20%). Molecular weight markers are indicated by arrows.

[0170] [Figure 3] Experimental design - Effects of natural DPP3 in animal models.

[0171] [Figure 4] (A) DPP3 injection reduces the shortening rate and leads to a deterioration of cardiac function. (B) A decline in renal function is also observed through an increase in the renal resistance index.

[0172] [Figure 5]Association and dissociation curves for AK1967-DPP3 binding analysis using Octet. Biosensors loaded with AK1967 were immersed in a dilution series of recombinant GST-tagged human DPP3 (100 nM, 33.3 nM, 11.1 nM, 3.7 nM), and association and dissociation were monitored.

[0173] [Figure 6] Western blotting of diluted blood cell lysates and detection of DPP3 using AK1967 as the primary antibody.

[0174] [Figure 7] Inhibition curve of native DPP3 derived from blood cells by the inhibitory antibody AK1967. Inhibition of DPP3 by the specific antibody is concentration-dependent; when analyzed against 15 ng / ml of DPP3, the IC50 is approximately 15 ng / ml.

[0175] [Figure 8] Experimental setup - Efficacy of procyzumab in sepsis-induced heart failure.

[0176] [Figure 9] Procyzumab dramatically improves the shortening rate (A) and mortality rate (B) in rats with sepsis-induced heart failure.

[0177] [Figure 10] Experimental design - Procyzumab treatment (B) and control (A) following isoproterenol-induced cardiac stress in mice.

[0178] [Figure 11] In mice with isoproterenol-induced heart failure, procizumab improved the shortening rate and reduced the renal resistance index within 1 and 6 hours after administration, respectively (A) (B).

[0179] [Figure 12] High DPP-3 levels 24 hours after admission in sepsis patients were associated with the worst SOFA scores.

[0180] [Figure 13] High cDPP3 plasma levels correlate with organ failure in sepsis patients. Bar graph of SOFA score in AdrenOSS-1 according to the progression of DPP3 levels during ICU stay. 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.

[0181] [Figure 14A] High cDPP3 levels 24 hours after admission in patients with sepsis were associated with the worst SOFA score in each organ. SOFA scores for (A) heart, (B) kidney, (C) respiratory, (D) liver, (E) coagulation system, and (F) central nervous system, according to the kinetic level of cDPP3 (HH: high / high, HL: high / low, LH: low / high, LL: low / low) from admission to 24 hours. [Figure 14B] High cDPP3 levels 24 hours after admission in patients with sepsis were associated with the worst SOFA score in each organ. SOFA scores for (A) heart, (B) kidney, (C) respiratory, (D) liver, (E) coagulation system, and (F) central nervous system, according to the kinetic level of cDPP3 (HH: high / high, HL: high / low, LH: low / high, LL: low / low) from admission to 24 hours. [Figure 14C] High cDPP3 levels 24 hours after admission in patients with sepsis were associated with the worst SOFA score in each organ. SOFA scores for (A) heart, (B) kidney, (C) respiratory, (D) liver, (E) coagulation system, and (F) central nervous system, according to the kinetic level of cDPP3 (HH: high / high, HL: high / low, LH: low / high, LL: low / low) from admission to 24 hours. [Figure 14D]High cDPP3 levels 24 hours after admission in patients with sepsis were associated with the worst SOFA score in each organ. SOFA scores for (A) heart, (B) kidney, (C) respiratory, (D) liver, (E) coagulation system, and (F) central nervous system, according to the kinetic level of cDPP3 (HH: high / high, HL: high / low, LH: low / high, LL: low / low) from admission to 24 hours. [Figure 14E] High cDPP3 levels 24 hours after admission in patients with sepsis were associated with the worst SOFA score in each organ. SOFA scores for (A) heart, (B) kidney, (C) respiratory, (D) liver, (E) coagulation system, and (F) central nervous system, according to the kinetic level of cDPP3 (HH: high / high, HL: high / low, LH: low / high, LL: low / low) from admission to 24 hours. [Figure 14F] High cDPP3 levels 24 hours after admission in patients with sepsis were associated with the worst SOFA score in each organ. SOFA scores for (A) heart, (B) kidney, (C) respiratory, (D) liver, (E) coagulation system, and (F) central nervous system, according to the kinetic level of cDPP3 (HH: high / high, HL: high / low, LH: low / high, LL: low / low) from admission to 24 hours.

[0182] [Figure 15] High levels of DPP3 at ICU admission are associated with worsening renal function over the following 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).

[0183] [Figure 16A] Time-series measurements of DPP3 in the ICU are associated with disease severity in COVID-19 patients. For group A, DPP3 levels were measured on day 3 of ICU admission (p=0.02), and for group B, they were measured on day 7 of ICU admission (p=0.013). X-axis: FALSE = P / F ratio > 150; TRUE = P / F ratio < 150. [Figure 16B]Time-series measurements of DPP3 in the ICU are associated with disease severity in COVID-19 patients. For group A, DPP3 levels were measured on day 3 of ICU admission (p=0.02), and for group B, they were measured on day 7 of ICU admission (p=0.013). X-axis: FALSE = P / F ratio > 150; TRUE = P / F ratio < 150.

[0184] [Figure 17A] High DPP3 levels during ICU stay are associated with poor outcomes in COVID-19 patients. For A, DPP3 levels were measured on day 3 of ICU admission, and for B, they were measured on day 7 of ICU admission. X-axis: 0 = survival; 1 = death. [Figure 17B] High DPP3 levels during ICU stay are associated with poor outcomes in COVID-19 patients. For A, DPP3 levels were measured on day 3 of ICU admission, and for B, they were measured on day 7 of ICU admission. X-axis: 0 = survival; 1 = death.

[0185] [Figure 18] High DPP3 levels upon ICU admission are 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, or Yes: Vasopressor therapy during ICU stay.

[0186] [Figure 19A] Time-series measurements of DPP3 in the ICU are associated with the need for organ support therapy, particularly venous ECMO. For group A, DPP3 levels were measured on day 3 of ICU admission (p=0.03), and for group B, they were measured on day 7 of ICU admission (p=0.04). X-axis: 0 = no ECMO; 1 = ECMO. [Figure 19B] Time-series measurements of DPP3 in the ICU are associated with the need for organ support therapy, particularly venous ECMO. For group A, DPP3 levels were measured on day 3 of ICU admission (p=0.03), and for group B, they were measured on day 7 of ICU admission (p=0.04). X-axis: 0 = no ECMO; 1 = ECMO.

[0187] [Figure 20] Procizumab treatment scheme for cardioprotection in septic shock [Figure 21] DPP3 activity (U / L) and procizumab concentration (ng / ml) measured at different time points of procizumab infusion in septic pigs (n=16). [Figure 22] Cardiac inflammation, as assessed by the expression of pro-inflammatory cytokine IL-6 in cardiomyocyte mRNA, was significantly upregulated in the standard treatment group compared to the procizumab-treated group (p=0.0024).

[0188] [Figure 23] Myocardial injury, as assessed by high-sensitivity cardiac troponin I release, was significantly higher at H12 in the standard treatment group compared to animals treated with procizumab (p=0.0055). There was no significant difference in troponin levels between the two groups.

[0189] [Figure 24] The norepinephrine requirement was assessed based on the norepinephrine infusion dose (μg / kg / min) at each time point from the start of resuscitation (H1) to euthanasia (H12), titrated to maintain a mean arterial pressure (MAP) of 65–75 mmHg. The norepinephrine requirement to achieve the target MAP was significantly higher in the standard treatment group compared to the procizumab group (p<0.05 from H4 to H9, p<0.005 from H10 to H12).

[0190] [Figure 25] Cardiac output, as assessed by pulmonary artery catheterization, showed a significant difference between the standard treatment group and the procizumab group at H4, H8, and H12 time points (p<0.05). [Examples]

[0191] Example 1 - Method for measuring DPP3 protein and DPP3 activity Antibody generation and determination of DPP3 binding ability: Several mouse antibodies were generated and screened based on their ability to bind to human DPP3 in specific binding assays (see Table 3).

[0192] Peptides / conjugates for immunization: The DPP3 peptide for immunization was synthesized using an additional N-terminal cysteine ​​residue (if cysteine ​​was not present in the selected DPP3 sequence) for peptide conjugation to bovine serum albumin (BSA) (see Table 3) (JPT Technologies, Berlin, Germany). The peptide was covalently bonded to BSA using a Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the Perbio manual. Recombinant GST-hDPP3 was prepared by USBio (United States Biological, Salem, MA, USA).

[0193] Mouse immunization, immune cell fusion, and screening: Balb / c mice were intraperitoneally (ip) injected with either 84 μg of GST-hDPP3 or 100 μg of DPP3-peptide-BSA conjugate (emulsified in TiterMax Gold Adjuvant) on day 0, 84 μg of GST-hDPP3 or 100 μg of DPP3-peptide-BSA conjugate (emulsified in complete Freund's adjuvant) on day 14, and 42 μg of GST-hDPP3 or 50 μg of DPP3-peptide-BSA conjugate (emulsified in incomplete Freund's adjuvant) on days 21 and 28. On day 49, the animals were intravenously (iv) injected with either 42 μg of GST-hDPP3 or 50 μg of DPP3-peptide-BSA conjugate dissolved in physiological saline. Three days later, the mice were sacrificed and immunocytofusion was performed.

[0194] Splenocytes from immunized mice and cells from the myeloma cell line SP2 / 0 were fused with 1 ml of 50% polyethylene glycol at 37°C for 30 seconds. After washing, the cells were seeded into 96-well cell culture plates. Hybrid clones were selected by growing them in HAT medium (RPMI1640 culture medium supplemented with 20% fetal bovine serum and HAT supplement). After one week, the HAT medium was replaced with HT medium and the cells were passed three times before being returned to standard cell culture medium.

[0195] Cell culture supernatants were screened for recombinant DPP3-conjugated IgG antibodies two weeks after fusion. Recombinant GST-tagged hDPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng / well) and incubated with 50 μl of cell culture supernatant per well at room temperature for 2 hours. After washing the plates, 50 μl / well of POD-rabbit anti-mouse IgG was added and incubated at room temperature for 1 hour. Following the next washing step, 50 μl of dye stock (3.7 mM o-phenylene-diamine, 0.012% H2O2 in citrate / hydrogen phosphate buffer) was added to each well and incubated at room temperature for 15 minutes. The color reaction was stopped by adding 50 μl of 4N sulfuric acid. Absorbance at 490 nm was detected.

[0196] Microcultures that tested positive were transferred to 24-well plates for propagation. After retesting, selected cultures were cloned and re-cloned using the limiting dilution method, and their isotypes were determined.

[0197] Production of mouse monoclonal antibodies Antibodies produced against GST-tagged human DPP3 or DPP3-peptide were prepared using a standard antibody production method (Marx et al. 1997) and purified with Protein A. Antibody purity was 90% or higher based on SDS gel electrophoresis analysis.

[0198] Antibody Characterization - Binding to hDPP3 and / or Immunotherapy Peptides To analyze the ability of different antibodies and antibody clones to bind DPP3 / immunolytic peptides, binding assays were performed. a) Solid phase Recombinant GST-tagged hDPP3 (SEQ ID NO: 1) or DPP3 peptide (immunotherapy peptide, SEQ ID NO: 2) was immobilized on the surface of a highly bound microtiter plate (96-well polystyrene microplate, Greiner Bio-One international AG, Austria, 1 μg / well in coupling buffer [50 mM Tris, 100 mM NaCl, pH 7.8], 1 hour at room temperature). After blocking with 5% bovine serum albumin, the microplate was vacuum-dried.

[0199] b) Marking procedure (tracer) 100 μg (100 μl) of different anti-DPP3 antibodies (detection antibodies, 1 mg / ml in PBS, pH 7.4) were mixed with 10 μl of acridinium NHS ester (1 mg / ml in acetonitrile, InVent GmbH, Germany, European Patent No. 0353971) and incubated at room temperature for 30 minutes. The labeled anti-DPP3 antibodies were purified by gel filtration HPLC using Shodex Protein 5 μm KW-803 (Showa Denko, Japan). The purified labeled antibodies were diluted in assay buffer (50 mmol / l potassium phosphate, 100 mmol / l NaCl, 10 mmol / l Na2-EDTA, 5 g / l bovine serum albumin, 1 g / l mouse IgG, 1 g / l bovine IgG, 50 μmol / l amastatin, 100 μmol / l leupeptin, pH 7.4). The final concentration is approximately 5-7 × 10⁶ per 200 μl. 6 The relative luminescence (RLU) was measured using a labeled compound (approximately 20 ng of labeled antibody). Acridinium ester chemiluminescence was measured using a Centro LB960 luminometer (Berthold Technologies GmbH & Co. KG).

[0200] c) hDPP3 binding assay 200 μl of labeled and diluted detection antibody (tracer) was packed into a plate and incubated at 2–8°C for 2–4 hours. 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 bound to the wells was measured using a Centro LB960 luminometer (Berthold Technologies GmbH & Co. KG).

[0201] Antibody Characterization - Analysis of hDPP3 Inhibition To analyze the DPP3 inhibition ability of different antibodies and antibody clones, a DPP3 activity assay was performed using a known procedure (Jones et al., 1982). Recombinant GST-tagged hDPP3 was diluted in assay buffer (50 mM Tris-HCl, pH 7.5 and 25 ng / ml GST-DPP3 in 100 μM ZnCl2), and 200 μl of this solution was incubated with 10 μg of each antibody at room temperature. After 1 hour of pre-incubation, the fluorescence-generating substrate Arg-Arg-βNA (20 μl, 2 mM) was added to the solution, and the time-dependent generation of free βNA was monitored at 37°C using a Twinkle LB970 microplate fluorometer (Berthold Technologies GmbH & Co. KG). βNA fluorescence was detected by excitation at 340 nm and measurement of emission at 410 nm. The increasing fluorescence slope (RFU / min) for different samples was calculated. The slope of GST-hDPP3 using a buffer control is defined as 100% activity. The inhibitory ability of a possible scavenging agent is defined as the decrease (%) in GST-hDPP3 activity upon incubation with that scavenging agent.

[0202] The following table shows the selected antibodies, their binding rates in terms of relative luminescence (RLU), and their relative inhibitory activity (%) (Table 3). Monoclonal antibodies produced against the DPP3 region shown below were selected based on their binding ability to recombinant DPP3 and / or immunized peptides, and their inhibitory activity.

[0203] All antibodies produced against GST-tagged full-length recombinant hDPP3 show strong binding to immobilized GST-tagged hDPP3. Antibodies produced against SEQ ID NO: 2 peptide also bind to GST-hDPP3. SEQ ID NO: 2 antibodies also bind strongly to the immunized peptide. [Table 3]

[0204] The development of a luminescence immunoassay (DPP3-LIA) for the quantification of DPP3 protein concentration and an enzyme capture activity assay (DPP3-ECA) for the quantification of DPP3 activity have recently been reported (Rehfeld et al. 2019. JALM 3(6):943-953), and the full details are incorporated herein by reference.

[0205] Example 2 - DPP3 for predicting short-term mortality Plasma DPP3 concentrations in various affected patients were determined using an hDPP3 immunoassay (Rehfeld et al. 2019. JALM 3(6):943-953) and associated with short-term mortality in these patients.

[0206] Research cohort - Sepsis and septic shock Plasma samples from 574 patients in the Adrenomedullin and Outcome in Severe Sepsis and Septic Shock (AdrenOSS-1) study were screened for DPP3. AdrenOSS-1 is a prospective observational multinational study (Hollinger et al., 2018) including 583 patients admitted to the intensive care unit with sepsis or septic shock. 292 patients were diagnosed with septic shock.

[0207] Research cohort - Cardiogenic shock Plasma samples from 108 patients diagnosed with cardiogenic shock were screened for DPP3. Blood samples were collected within 6 hours of detection of cardiogenic shock. Mortality was followed over 7 days.

[0208] Research cohort - Acute coronary syndrome Plasma samples from 720 patients with acute coronary syndrome were screened for DPP3. Blood was collected 24 hours after the onset of chest pain. Mortality was followed for 7 days.

[0209] Research cohort - dyspnea: Plasma samples were collected immediately upon arrival at the emergency department of Skane University Hospital from 1440 patients presenting with respiratory distress (shortness of breath). Patients with respiratory distress are at high risk of organ failure and short-term mortality due to conditions such as acute coronary syndrome or congestive heart failure. Mortality rates were followed for three months after emergency department visit.

[0210] Research cohort - burn patients: Plasma samples from 107 patients with severe burns (over 15% of total surface area) were screened for DPP3. Blood samples were collected upon admission. Mortality was followed for 4 weeks.

[0211] hDPP3 immunoassay: To determine the DPP3 levels in patients' plasma, either an immunoassay (LIA) detecting the amount of human DPP3 (LIA) or an activity assay (ECA) detecting the activity of human DPP3 (ECA) was used. Antibody immobilization, labeling, and incubation were performed as described in Rehfeld et al. (Rehfeld et al. 2019. JALM 3(6):943-953).

[0212] result Short-term patient survival in sepsis / septic shock was associated with DPP3 plasma concentration at admission. Patients with DPP3 plasma concentrations above 68.6 ng / mL (third quartile) had an increased risk of death compared to patients with DPP3 plasma concentrations below this threshold (Figure 1A). A similar relationship was observed when analyzing short-term outcomes related to DPP3 plasma concentration only in patients with septic shock from this cohort (Figure 1F). Patients with high DPP3 plasma concentrations had a higher risk of death compared to patients with low DPP3 plasma concentrations. When the same cutoff was applied to patients with cardiogenic shock, an increased risk of short-term death within 7 days was observed in patients with high DPP3 (Figure 1B).

[0213] Furthermore, the 7-day survival rate for patients with acute coronary syndrome associated with DPP3 also increases when DPP3 levels are high and the corresponding cutoff of 68.6 ng / mL is applied (Figure 1C).

[0214] Applying this 68.6 ng / mL cutoff to patients with dyspnea revealed a significantly increased risk of death in patients with high DPP3 levels during a 3-month follow-up period (Figure 1D).

[0215] Furthermore, patients with severe burns who had high DPP3 concentrations exceeding the corresponding cutoff of 68.6 ng / mL had an increased risk of death at 4 weeks (Figure 1E).

[0216] Example 3 - Purification of human natural DPP3 Human red blood cell lysate was applied to a total of 100 ml of Sepharose 4B resin (Sigma-Aldrich), and the flow-through was collected. The resin was washed with a total of 370 mL of PBS buffer at pH 7.4, and the washing fraction was collected and combined with the collected flow-through to obtain a total volume of 2370 mL.

[0217] In the immunoaffinity purification process, 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, Thermo Fisher Scientific). The conjugation efficiency was determined to be 98% by quantifying unbound antibody using the Bradford method. The resin-antibody conjugate was equilibrated with 10 bed volumes of wash-conjugate buffer (PBS, 0.1% Triton X-100, pH 7.4), combined with 2370 mL of clarified erythrocyte lysate, and incubated at 4°C for 2 hours with continuous stirring. As a result, 100 mL of the incubation mixture was divided into 10 15 mL polypropylene columns, and the flow-through was collected by centrifugation at 1000 × g for 30 seconds. This process was repeated several times to obtain 2.5 mL of DPP3-supported resin per column. Each column was washed five times with 10 mL of wash-binding buffer using the gravity flow method. Each column was placed in a 15 mL Falcon tube containing 2 mL of neutralizing buffer (1 M Tris-HCl, pH 8.0), followed by the addition of 10 mL of elution buffer (100 mM glycine-HCl, 0.1% Triton X-100, pH 3.5) per column. DPP3 was immediately eluted by centrifugation at 1000 × g for 30 seconds. The elution process was repeated a total of three times to obtain 360 mL of combined eluate. The pH of the neutralized eluate was 8.0.

[0218] The combined eluate was loaded onto a 5 mL HiTrap Q-sephare HP column (GE Healthcare) equilibrated with IEX buffer A1 (100 mM glycine, 150 mM Tris, pH 8.0) using the sample pump of an Akta Start system (GE Healthcare). After loading the sample, the column was washed with 5 column volumes of IEX buffer A2 (12 mM NaH2PO4, pH 7.4) to remove unbound proteins. Elution of DPP3 was achieved by applying a sodium chloride gradient over 10 column volumes (50 mL) in the range of 0 - 1 M NaCl using IEX buffer B (12 mM NaH2PO4, 1 M NaCl, pH 7.4). The eluate was collected in 2 mL fractions. The buffers used for ion exchange chromatography were sterile filtered using a 0.22 μM bottle top filter.

[0219] A purification table including the yield and activity of each purification step is shown in Table 4. Figure 2 shows SDS-PAGE on a gradient gel (4 - 20%) of native hDPP3 purified from human erythrocyte lysate.

[0220]

Table 4

[0221] b) The total protein amount was determined using the Lowry method modified by Peterson (Peterson 1977. Analytical Biochemistry 356:346 - 356).

[0222] c) The total Arg2-βNA hydrolysis activity in μmol of substrate converted per minute was determined using DPP3-ECA calibrated with β-naphthylamine (0.05 - 100 μM).

[0223] d) The purification yield was calculated from the total Arg2-βNA hydrolysis activity. The Arg2-βNA hydrolysis activity in the starting material was set to 100%.

[0224] e) Specific activity is defined as the amount of converted substrate in μmol per minute and per mg of total protein.

[0225] f) The purification ratio is the quotient of the specific activity before and after each purification step.

[0226] Example 4 - Effects of natural DPP3 in an animal model The effects of natural hDPP3 injection in healthy mice were examined by monitoring the rate of shortening and the renal resistance index.

[0227] Wild-type Black 6 mice (8-12 weeks old, see Table 5 for group size) were acclimatized for 2 weeks and baseline echocardiography was performed. The mice were randomly assigned to one of two groups, and then either natural DPP3 protein or PBS was administered intravenously by retroorbital injection at a dose of 600 μg / kg for DPP3 protein.

[0228] Cardiac function was evaluated by echocardiography at 15, 60, and 120 minutes after DPP3 or PBS injection (Gao et al. 2011), and renal function was evaluated by the renal resistance index (Lubas et al., 2014, Dewitte et al, 2012) (Figure 3).

[0229] [Table 5] result Mice treated with natural DPP3 protein showed a significantly reduced rate of shortening compared to the control group injected with PBS (Figure 4A). The WT+DPP3 group also showed worsening renal function, as observed by an increased renal resistance index (Figure 4B).

[0230] Example 5 - Development of procyzumab The antibodies produced against SEQ ID NO: 2 were characterized in more detail (epitope mapping, binding affinity, specificity, and inhibitory activity). Here, the results for clone 1967 of SEQ ID NO: 2 (AK1967; "procyzumab") are shown as an example.

[0231] Determination of the AK1967 epitope on DPP3: For epitope mapping of AK1967, numerous N- or C-terminally biotinylated peptides were synthesized (peptides&elephants GmbH, Hennigsdorf, Germany). These peptides contain the complete immunotherapy peptide sequence (SEQ ID NO: 2) or a fragment thereof, with one amino acid progressively removed from either the C-terminus or N-terminus (see Table 7 for a complete list of peptides).

[0232] High-binding 96-well plates were coated with 2 μg of avidin (Greiner Bio-One international AG, Austria) per well in coupling buffer (500 mM Tris-HCl, pH 7.8, 100 mM NaCl). The plates were then washed and packed with specific solutions of biotinylated peptides (10 ng / well; buffer-0.5% BSA in 1×PBS). The anti-DPP3 antibody AK1967 was labeled by chemiluminescence according to Example 1.

[0233] The plate was filled with 200 μl of labeled and diluted detection antibody (tracer) and incubated at room temperature for 4 hours. Unbound tracer was removed by washing 4 times with 350 μl of wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Chemiluminescence bound to the wells was measured using a Centro LB960 luminometer (Berthold Technologies GmbH & Co. KG). Binding of AK1967 to each peptide was determined by evaluation of relative light units (RLU). Any peptide showing a significantly higher RLU signal than the non-specific binding of AK1967 was defined as an AK1967 binder. Combinatorial analysis of the binding and non-binding peptides reveals the specific DPP3 epitope of AK1967.

[0234] Determination of binding affinity using Octet: This experiment was carried out using an Octet Red96 (ForteBio). AK1967 was captured on a kinetics grade anti-human Fc (AHC) biosensor. The loaded biosensor was then immersed in a dilution series (100 nM, 33.3 nM, 11.1 nM, 3.7 nM) of recombinant GST-tagged human DPP3. Association was observed for 120 seconds and then dissociation for 180 seconds. The buffer used in the experiment is shown in Table 6. Kinetic analysis was performed using a 1:1 binding model and global fitting.

[0235]

Table 6

[0236] DPP3 inhibition assay: To analyze the DPP3 inhibitory ability by AK1967, a DPP3 activity assay using a known procedure (Jones et al., 1982) was performed as described in Example 1. The inhibitory ability of AK1967 was defined as the decrease (%) in GST-hDPP3 activity by incubation with the antibody. The obtained decrease in DPP3 activity is shown in the inhibition curve of Figure 7.

[0237] Epitope mapping: Analysis of peptides that bind and do not bind to AK1967 revealed the DPP3 sequence INPETG (SEQ ID NO: 3) as the epitope required for AK1967 binding (see Table 6).

[0238] Binding affinity: AK1967 binds to recombinant GST-hDPP3 with an affinity of 2.2*10 -9 M (see the kinetics curve in Figure 5).

[0239] [Table 7] Specificity and inhibitory activity: The only protein detected in blood cell lysates using AK1967 as the primary antibody was 80 kDa DPP3 (Figure 6). The total protein concentration in the lysates was 250 μg / ml, while the estimated DPP3 concentration was approximately 10 μg / ml. Despite the presence of 25 times more nonspecific proteins in the lysates, AK1967 specifically bound to and detected DPP3, with no other nonspecific binding occurring.

[0240] AK1967 inhibits 15 ng / ml of DPP3 with an IC50 of approximately 15 ng / ml in a specific DPP3 activity assay (Figure 7).

[0241] Chimerization / Humanization: The monoclonal antibody AK1967 ("procyzumab"), which can inhibit DPP3 activity by 70%, was selected as a candidate therapeutic antibody and also used as a template for chimerization and humanization.

[0242] Humanization of mouse antibodies can be carried out according to the following procedure. For the humanization of mouse-derived antibodies, antibody sequences are analyzed for structural interactions between the antigen and a framework region (FR) containing a complementary determination region (CDR). Based on structural modeling, appropriate human-derived FRs are selected, and mouse CDR sequences are transplanted into human FRs. Mutations in the amino acid sequences of the CDR or FR can be introduced to restore structural interactions lost due to species switching of the FR sequence. This restoration of structural interactions can be achieved by a random approach using a phage display library or by a directed approach guided by molecular modeling (Almagro and Fransson, 2008. Humanization of antibodies. Front Biosci. 13:1619-33).

[0243] In the context described above, the variable region may be attached to any subclass of the constant region (IgG, IgM, IgE, IgA), or only to the scaffold, Fab fragment, Fv, Fab, and F(ab)2. Examples 6 and 7 below used mouse antibody variants having the IgG2a backbone. For chimerization and humanization, the human IgG1κ backbone was used.

[0244] In epitope binding, only the complementarity-determining region (CDR) is important. The CDRs for the heavy and light chains of the mouse anti-DPP3 antibody (AK1967; "procyzumab") are shown in SEQ ID NOs. 7, 8, and 9 for the heavy chain, and in SEQ ID NOs. 10, sequence KVS, and 11 for the light chain, respectively.

[0245] Sequencing of the anti-DPP3 antibody (AK1967; "procyzumab") revealed the antibody heavy chain variable region (H chain) described in SEQ ID NO: 12 and the antibody light chain variable region (L chain) described in SEQ ID NO: 13.

[0246] Example 6 - Efficacy of procyzumab in sepsis-induced heart failure In this experiment, the effect of procizumab injection in rats with sepsis-induced heart failure (Rittirsch et al. 2009) was tested by monitoring the rate of reduction in heart rate.

[0247] CLP model of septic shock: Male Wistar rats (2-3 months old, 300-400g; see Table 8 for group size) from Centre d'elevage Janvier (France) were randomly assigned to one of three groups. All animals were anesthetized (ip) by intraperitoneal administration of ketamine hydrochloride (90 mg / kg) and xylazine (9 mg / kg). To induce multibacterial sepsis, cecal ligation and puncture (CLP) was performed using a slightly modified Rittirsch protocol. A midline abdominal incision (1.5 cm) was made to expose the cecum. The cecum was then ligated just below the ileocecal valve and punctured once with an 18-gauge needle. The abdominal cavity was then closed in two layers, followed by fluid resuscitation (subcutaneous injection of 3 ml / 100 g of saline), and the animals were returned to their cages. Siamese animals underwent surgery without cecal puncture. CLP animals were randomized to receive either a placebo or a therapeutic antibody.

[0248] Research plan: The study flow is shown in Figure 8. After CLP or Siam surgery, the animals were allowed to rest for 20 hours with free access to water and food. Anesthesia was then administered, a tracheostomy was performed, and arterial and venous lines were placed. 24 hours after CLP surgery, either AK1967 or vehicle (saline) was administered as a bolus injection at 5 mg / kg, followed by a 3-hour infusion at 7.5 mg / kg. As a safety measure, hemodynamics were invasively and continuously monitored from t=0 to 3 hours.

[0249] At t=0 (baseline), all CLP animals were in septic shock and exhibited impaired cardiac function (hypotension, low shortening rate). At this point, procizumab or vehicle (PBS) was injected (iv), and saline infusion was initiated. There was one control group and two CLP groups, which are summarized in the following table (Table 8). At the end of the experiment, the animals were euthanized, and organs were collected for subsequent analysis.

[0250] [Table 8] Invasive blood pressure: Hemodynamic variables were acquired using an AcqKnowledge system (BIOPAC Systems, Inc., USA). This provides a fully automated blood pressure analysis system. The catheter is connected to the BIOPAC system via a pressure sensor.

[0251] For this procedure, rats were anesthetized (ketamine and xylazine). The animals were transferred to a heating pad to achieve a desired body temperature of 37 - 37.5 °C. A temperature feedback probe was inserted into the rectum. The rats were placed in the supine position on the operating table. The trachea was opened and a catheter (16G) was inserted for an external ventilator without damaging the carotid artery and vagus nerve. The arterial catheter was inserted into the right carotid artery. The carotid artery was dissected from the vagus nerve before ligation.

[0252] A central venous catheter was inserted from the left jugular vein to enable administration of PCZ or PBS.

[0253] After surgery, the animals were allowed to rest until they reached a stable state before hemodynamic measurements. Then, baseline blood pressure (BP) was recorded. During data collection, the infusion of physiological saline via the arterial line was stopped.

[0254] Echocardiography: The animals were anesthetized using ketamine hydrochloride. The chest was shaved and the rats were placed in the lateral position.

[0255] For transthoracic echocardiography (TTE) examinations, a commercially available GE Healthcare Vivid 7 Ultra - sound System equipped with a high - frequency (14 MHz) linear probe and a 10 MHz cardiac probe was used. All examinations were digitally recorded and saved for subsequent offline analysis.

[0256] Grayscale images were recorded at a depth of 2 cm. Two-dimensional imaging was initiated in the parasternal long-axis view to measure the aortic valve annulus diameter and pulmonary artery diameter. M-mode was also used to measure left ventricle (LV) dimensions and evaluate the shortening ratio (FS%). LVFS was calculated as LV end-diastolic diameter - LV end-systolic diameter / LV end-diastolic diameter and expressed as a percentage. Therefore, the end-diastolic point was defined as the maximum diameter of the LV. Accordingly, end-systolic was defined as the minimum diameter in the same cardiac cycle. All parameters were measured manually. Three cardiac cycles were averaged for each measurement.

[0257] Pulmonary artery flow was recorded using pulsed Doppler ultrasound from the same parasternal long-axis image. The velocity-time integral of pulmonary artery outflow was measured.

[0258] From the five-chamber view of the apex of the heart, mitral valve blood flow was recorded at the tip level of the mitral valve using pulsed Doppler.

[0259] result: Septic heart failure rats treated with PBS (CLP+PBS) showed a reduced shortening rate compared to Siamese rats (Figure 9A). The CLP+PBS group also showed a higher mortality rate (Figure 9B). In contrast, application of procyzumab to septic heart failure rats improved the shortening rate (Figure 9A) and dramatically reduced mortality (Figure 9B).

[0260] Example 7 - Effects of procyzumab on cardiac and renal function The effects of procyzumab on isoproterenol-induced heart failure in mice were investigated by monitoring the shortening rate and renal resistance index.

[0261] Isoproterenol-induced cardiac stress in mice: Acute heart failure was induced in 3-month-old male mice by subcutaneous injection of isoproterenol (DL-isoproterenol hydrochloride, Sigma Chemical Co) (ISO), a non-selective β-adrenergic agonist, at a dose of 300 mg / kg twice daily for 2 days (Vergaro et al, 2016). ISO was diluted in 0.9% NaCl. Isoproterenol-treated mice were randomly assigned to two groups (Table 9), and baseline echocardiography (Gao et al., 2011) and renal resistance index measurement (Lubas et al., 2014, Dewitte et al, 2012) were performed on day 3, followed by intravenous injection of either PBS or procizumab (10 mg / kg) (Figures 10A and 10B).

[0262] Cardiac function was assessed at 1 hour, 6 hours, and 24 hours by echocardiography (Gao et al., 2011) and renal resistance index (Lubas et al., 2014, Dewitte et al., 2012) (Figures 10A and 10B). The group of mice injected with vehicle (PBS) instead of isoproterenol did not receive further pharmacological treatment and served as a control group (Table 9).

[0263] [Table 9] result: Application of procyzumab to mice with isoproterenol-induced heart failure restored cardiac function within the first hour of administration (Figure 11A). Renal function in diseased mice showed significant improvement 6 hours after PCZ injection, reaching levels comparable to those of Siamese animals at 24 hours (Figure 11B).

[0264] Example 8 - DPP3 and organ dysfunction in sepsis Using the same study (AdrenOSS-1) described in Example 2, we evaluated the association between circulating DPP3 (cDPP3) and organ function (e.g., cardiovascular and renal dysfunction) in patients hospitalized with sepsis and septic shock. AdrenOSS-1 is a European prospective observational multinational study (ClinicalTrials.gov NCT02393781) including 583 patients admitted to the ICU with sepsis or septic shock. The primary outcome (described in Example 2) was 28-day mortality. Secondary outcomes included organ failure as defined by the SOFA score, organ support focused on vasopressor use, and the need for renal replacement therapy. Blood for central laboratory use was collected within 24 hours of ICU admission and on day 2.

[0265] A recently reported assay was used to quantify the DPP3 protein concentration (DPP3-LIA) (Rehfeld et al. 2019. JALM 3(6):943-953).

[0266] In all AdrenOSS-1 patients, the median cDPP3 level measured at admission was 45.1 ng / mL (interquartile range 27.5–68.6). High DPP3 levels measured at admission were associated with worsening metabolic parameters, renal and cardiac function, and SOFA score: patients with DPP3 levels below the median had a median SOFA score of 6 (IQR 4–9), while patients with DPP3 levels above the median of 45.1 ng / mL had a median SOFA score of 8 (IQR 5–11) (Figure 12). Regardless of the cDPP3 level at admission, high cDPP3 levels 24 hours later were associated with the worst SOFA scores, both overall (Figure 13) and by organ (Figures 14A-F).

[0267] In summary, these data showed that high levels of cDPP3 were associated with survival and the degree of organ dysfunction in patients with sepsis or septic shock in a large international cohort. This study found a significant association between cDPP3 <45.1 ng / ml at admission and short-term survival, and that a prognostic cutoff of 45.1 pg / ml was established for both sepsis and septic shock. Regarding organ dysfunction, there was a positive relationship between cDPP3 at ICU admission and the SOFA score. More importantly, the relationship between cDPP3 levels at ICU admission and the degree of organ dysfunction also held during the recovery phase. In fact, patients with high cDPP3 levels at admission who showed a decrease to normal cDPP3 levels by day 2 were more likely to recover all organ function, including cardiovascular, renal, pulmonary, and hepatic organs.

[0268] Example 9 - DPP3 in patients infected with coronavirus (SARS-CoV-2) Plasma samples from 12 patients diagnosed with coronavirus (SARS-CoV-2) infection were screened for DPP3 and other biomarkers. To determine the levels of DPP3 in the patients' plasma, we used either an immunoassay (LIA) to detect the amount of human DPP3 (LIA) or an activity assay (ECA) to detect the activity of human DPP3 (ECA), as recently reported (Rehfeld et al. 2019. JALM 3(6):943-953).

[0269] Table 10 summarizes the DPP3 concentrations in each sample.

[0270] [Table 10] DPP3 concentrations ranged from 27 to 975 ng / ml, with a median (IQR) of 156 (59.5 to 322.3) ng / ml. DPP3 concentrations were significantly elevated compared to healthy subjects. Samples were measured from 5,400 normal (healthy) subjects (Swedish single-center prospective population-based study (MPP-RES)): the median (interquartile range) of plasma DPP3 was 14.5 ng / ml (11.3 ng / ml to 19 ng / ml).

[0271] Example 10 - DPP3 in COVID-19 patients for prognosis, treatment stratification, and follow-up Cohort description: 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% male, a median body mass index (BMI) of 28.6, and a continuous organ failure assessment (SOFA) score of 5 at admission. Exclusion criteria were age under 18 years and pregnancy. Analysis was performed using real-time reverse transcription PCR (RT-PCR). Patient care followed standard ICU care at our hospital, including mechanical ventilation, venous ECMO, and RRT as needed.

[0272] Blood samples were collected daily from the day of admission until day 7 for analysis of DPP3 and standard laboratory parameters. DPP3 was measured in EDTA plasma using a one-step luminescence sandwich immunoassay (LIA), as recently reported (Rehfeld et al. 2019. JALM 3(6):943-953).

[0273] result: a) DPP3 levels at baseline and over time are associated with disease severity. DPP3 levels measured upon ICU admission were associated with deterioration of renal function during ICU stay, as defined by the KDIGO criteria (stages 0-1 indicating no to slight renal impairment and low risk, and stages 2-3 indicating renal injury and renal failure). DPP3 levels in stages 2-3 were significantly higher than those in stages 0-1 (Figure 15; p=0.005). High baseline DPP3 levels, in combination with other clinical parameters, may be used as a guideline for initiating renal replacement therapy.

[0274] Since COVID-19 positive patients tend to remain in the ICU for an average of 21 days, DPP3 levels measured during ICU stay (days 3 and 7) were associated with a low PaO2 / FiO2 ratio (<150) and, consequently, with severe acute respiratory distress syndrome (ARDS) (FALSE = P / F ratio >150; TRUE = P / F ratio <150). Time-series measurements of DPP3 in the ICU were associated with disease severity in COVID-19 patients. DPP3 levels were measured on day 3 of ICU admission (p=0.02) (Figure 16A) and day 7 of ICU admission (p=0.013) (Figure 16B).

[0275] Furthermore, high DPP3 levels measured on day 3 (Figure 17A, p=0.03) and day 7 (Figure 17B, p=0.01) were still associated with a higher mortality rate during ICU stay.

[0276] b) DPP3 levels at baseline and over time are associated with the need for organ support therapy. High DPP3 levels at admission and during ICU stay were significantly associated with the need for organ support therapy, particularly vasopressor therapy (day 3; Figure 18) and extracorporeal membrane oxygenation (ECMO) (days 3 and 7; Figures 19A and 19B, respectively).

[0277] Example 11 - Procyzumab for cardioprotection in an animal model of sepsis To evaluate the effect of an anti-DPP3 antibody (procizumab) on cardioprotection in septic shock, the inventors conducted a randomized, open-label, controlled trial in 16 anesthetized, mechanically ventilated pigs. Septic shock was induced by fecal peritonitis. Resuscitation with fluid therapy, antimicrobial therapy, and peritoneal drainage was initiated one hour after the onset of septic shock. Pigs with sepsis were randomly assigned to receive either procizumab (in addition to standard treatment) or standard treatment (norepinephrine and fluid therapy), and their mean arterial pressure was maintained at 65-75 mmHg for 12 hours (Figure 20). Eight female pigs and eight male pigs were used in the experiment, with sex appropriately balanced between the treatment group and the standard treatment group.

[0278] The results indicate that procizumab can inhibit DPP3 activity in the bloodstream throughout the infusion time (Figure 21). Finally, procizumab had a cardioprotective effect compared to the standard treatment group.

[0279] This effect was observed primarily by comparing cardiac inflammation, assessed by the expression of the pro-inflammatory cytokine IL-6 in cardiac mRNA between the two groups (Figure 22). Briefly, total RNA was extracted from rapidly frozen left ventricular tissue stored at -80°C in RNA later solution (Invitrogen®, RNA later® Stabilization Solution, ThermoFisher Scientific, 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 RTq-PCR, sense and antisense primers for sus scrofa IL-6 were designed using the Primer3 program. For each sample, a triple amplification reaction was performed using the iCycler system (BioRad Laboratories) with 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 by normalizing with the housekeeping gene (beta-actin). Results were expressed as relative increase ratios exceeding the mean of left ventricular relative mRNA expression in the arbitrarily fixed sham group (1).

[0280] The standard treatment group showed high levels of IL-6 myocardial expression, but the procizumab-treated animals had normal levels of IL-6 expression comparable to Siamese animals that did not have septic shock (only anesthesia, catheter insertion, and mechanical ventilation were performed) (Figure 22). Furthermore, to assess myocardial damage, high-sensitivity troponin I (hsTnI) serum levels were evaluated at baseline and H12 (Figure 23). Baseline hsTnI levels were similar between the treatment and standard treatment groups, and no difference was observed between the two groups. However, at H12, hsTnI levels in the procizumab group remained similar to baseline and were significantly lower compared to the standard treatment group (Figure 23).

[0281] Cardiac protective effects were also observed in both groups by norepinephrine requirements (Figure 24) and indexed cardiac output (Figure 25). Norepinephrine dose (μg / kg / min) was recorded hourly from hour 1. The standard treatment group had significantly higher norepinephrine requirements, although procizumab-treated animals received the minimum norepinephrine dose necessary to achieve the same target mean arterial pressure of 65 mmHg (Figure 24). The difference between the treatment and standard treatment groups was significant at H4, and the two groups diverged even more after H10 (10 hours after the start of norepinephrine infusion) (Figure 24).

[0282] Finally, the inventors also evaluated indexed cardiac output, assessed via pulmonary artery catheter (Figure 25). Indexed cardiac output in procizumab-treated animals was consistently lower at all time points (H4-H12) compared to the standard treatment group and was comparable to baseline levels (Figure 25). The indexed cardiac index showed that higher norepinephrine doses were associated with increased cardiac output, reflecting β1 receptor activation and increased oxygen consumption by myocardial tissue, resulting in increased oxidative stress and myocardial injury. Therefore, procizumab reduces cardiac stress by improving hemodynamic stability, protecting the myocardium from inflammation, and preventing the use of high doses of norepinephrine. These results indicate that procizumab has a myocardial protective effect and prevents myocardial injury in the progression of septic shock.

[0283] array SEQ ID NO: 1-hDPP3 Amino Acids 1-737 MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAWYGGLAVLLQTSPEAPYIYALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTKFVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLRHLGLGKEGITTYFSGNCTMEDAKLA QDFLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDSEVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESFTQGSIEAHKRGSRFWIQDKGPIVESYIGFIESYRDPFGSRGEFEFGFVAVVNKAMSAKFERLVASAEQLLKELPWPPTFEKDKF LTPDFTSLDVLTFAGSGIPAGINIPNYDDLRQTEGFKNVSLGNVLAVAYATQREKLTFLEEDDKDLYILWKGPSFDVQVGLHELLGHGSGKLFVQDEKGAFNFDQETVINPETGEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLYLCLHPQVLEIFGFEGADAEDVIYVNWLNMVRAGLL ALEFYTPEAFNWRQAHMQARFVILRVLLEAGEGLVTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTGDVAGGRALYEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEYEASAAGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA SEQ ID NO: 2-hDPP3 Amino acids 474-493 (N-Cys) - Immunotherapy peptide with an additional N-terminal cysteine CETVINPETGEQIQSWYRSGE SEQ ID NO: 3-hDPP3 Epitope of amino acids 477-482-AK1967 INPETG Sequence ID 4-hDPP3 Amino Acids 480-483 ETGE Sequence ID 5 - Variable region of mouse AK1967 in heavy chain QVTLKESGPGILQPSQTLSLTCSSGFSLSTSGMSVGWIRQPSGKGLEWLAHIWWNDNKSYNPALKSRLTISRDTSNNQVFLKIASVVTADTGTYFCARNYSYDYWGQGTTLTVSS Sequence ID 6 - Variable region of mouse AK1967 in the light chain DVVVTQTPLSLSVSLGDPASISCRSSRSLVHSIGSTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK Sequence ID 7 - CDR1 of mouse AK1967 in heavy chain GFSLSTSGMS Sequence ID 8 - CDR2 of mouse AK1967 in heavy chain IWWNDNK Sequence ID 9 - CDR3 of mouse AK1967 in heavy chain ARNYSYDY Sequence ID 10 - CDR1 of mouse AK1967 in light chain RSLVHSIGSTY CDR2 of mouse AK1967 in light chain KVS Sequence ID 11 - CDR3 of mouse AK1967 in light chain SQSTHVPWT Sequence ID 12 - Humanized AK1967 - Heavy chain sequence (IgG1κ backbone) MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQVVLTMTNMMDPVDTG TYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Sequence ID 13 - Humanized AK1967 - Light chain sequence (IgG1κ backbone) METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYYCSQSTHV PWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. An inhibitor of DPP3 activity for use in the treatment or intervention of critically ill patients with hypotension for myocardial protection and / or prevention of myocardial injury.

2. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having the hypotension described in claim 1, wherein the hypotension is a mean arterial pressure (MAP) of less than 65 mmHg, more preferably less than 60 mmHg, even more preferably less than 55 mmHg, and most preferably less than 50 mmHg.

3. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having hypotension according to claims 1 and 2, wherein the patient has a level of DPP3 exceeding a (predetermined) threshold in a sample of the patient's body fluids.

4. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having hypotension, according to claim 3, wherein the predetermined threshold level of DPP3 in the target body fluid sample is 20 to 120 ng / mL, more preferably 30 to 80 ng / mL, even more preferably 40 to 60 ng / mL, and most preferably the threshold is 50 ng / mL.

5. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having hypotension according to any one of claims 1 to 4, wherein the sample is a body fluid sample selected from the group including whole blood, plasma, or serum.

6. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having hypotension as described in any one of claims 1 to 5, wherein the myocardial injury is characterized by a blood level of cardiac troponin (cTn) exceeding a threshold, increased myocardial expression of pro-inflammatory interleukin-6 (IL-6), and / or the need for a vasopressor to maintain blood pressure and cardiac output.

7. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having hypotension, according to claim 6, wherein the cardiac troponin (cTn) is cardiac troponin T (cTnT) or cardiac troponin I (cTnI).

8. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having hypotension as described in any one of claims 1 to 7, the patient suffering from a severe infection, sepsis, pulmonary embolism, pulmonary hypertension, acute coronary syndrome (including unstable angina, ST-elevation myocardial infarction (STEM), non-ST-elevation myocardial infarction (NSTEM)), any type of shock (including cardiogenic shock, septic shock, or anaphylactic shock), cardiac arrest, acute liver failure, and acute respiratory distress syndrome (ARDS).

9. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient with hypotension according to any one of claims 1 to 8, wherein the inhibitor of DPP3 activity is selected from the group comprising a small molecule, an anti-DPP3 antibody, an anti-DPP3 antibody fragment, or an anti-DPP3 non-Ig scaffold.

10. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient with hypotension according to claim 9, wherein the inhibitor is an anti-DPP3 antibody, an anti-DPP3 antibody fragment, or an anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acid length contained in SEQ ID NO:

1.

11. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient with hypotension according to claims 9 and 10, wherein the inhibitor is an anti-DPP3 antibody, an anti-DPP3 antibody fragment, or an anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acid length contained in SEQ ID NO:

2.

12. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having hypotension according to any one of claims 9 to 11, wherein the antibody is a monoclonal antibody or a monoclonal antibody fragment.

13. The antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, and the complementarity determining region (CDR) of the heavy chain is The sequence includes sequence number 7, sequence number 8, and / or sequence number 9, The complementarity determination region (CDR) of the light chain is An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having hypotension according to any one of claims 9 to 12, comprising the sequence of SEQ ID NO: 10, KVS, and / or SEQ ID NO:

11.

14. An inhibitor of DPP3 activity for use in the treatment or intervention of a severely ill patient having hypotension, 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. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having 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, the heavy chain comprising the sequence of SEQ ID NO: 12 and the light chain comprising the sequence of SEQ ID NO:

13.

16. An inhibitor of DPP3 activity for use in the treatment or intervention of a critically ill patient having hypotension, according to claim 9, wherein the small molecule is selected from the group comprising spinorphine, tynorphine, propioxatine A and B, fluostatin A and B, benzimidazole, or derivatives or analogs thereof.

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