DPP3 inhibitors to improve lung function in critically ill patients

Abstract

Subject matter of the present invention is a DPP3 activity inhibitor for use in the treatment or intervention of critical patients with reduced lung function to improve lung function.

Description

Technical Field

[0001] The subject matter of the present invention is a DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function. Background Art

[0002] Dipeptidyl peptidase 3 (also known as dipeptidyl aminopeptidase III, dipeptidyl arylamidase III, dipeptidyl peptidase III, enkephalinase B or erythrocyte angiotensinase; abbreviation: DPP3, DPPIII) is a metallopeptidase that removes dipeptides from bioactive peptides such as enkephalin and angiotensin. Ellis and Nuenke identified DPP3 and measured its activity in an extract of purified bovine anterior pituitary in 1967. The enzyme is numbered EC 3.4.14.4, has a molecular mass of approximately 83 kDa, and is highly conserved in prokaryotes and eukaryotes ( Prajapati & Chauhan 2011 ). The amino acid sequence of the human variant is SEQ ID NO. 1. DPP3 is a mainly expressed peptidase mainly in the cytosol. Despite the lack of a signal sequence, several studies have reported membrane activity ( Lee & Snyder 1982 ).

[0003] DPP3 is a zinc-dependent exopeptidase belonging to the peptidase family M49. It has a broad substrate specificity for variously composed oligopeptides consisting of three / four to ten amino acids and is also capable of cleaving after proline. DPP3 is known to hydrolyze dipeptides from the N-terminus of its substrates, which include angiotensin II, III, and IV; Leu- and Met-enkephalin; endomorphin 1 and 2. The metallopeptidase DPP3 has optimal activity at pH 8.0 - 9.0 and can be activated by adding divalent metal ions such as Co 2+ and Mg 2+ .

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

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

[0006] Recently, two assays have been generated, characterized, and validated for the specific detection of DPP3 in human body fluids (such as 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 ). Before performing the actual detection of DPP3 activity, a washing step removes all interfering substances. Both methods are highly specific and allow for the reproducible detection of DPP3 in blood samples.

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

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

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

[0010] WO2021 / 185786 describes a method for determining DPP3 in a patient sample for the diagnosis, risk prediction, prognosis, and monitoring of patients infected with a coronavirus. It further describes a DPP3 activity inhibitor for treating or intervening in infected patients.

[0011] Procizumab is a humanized monoclonal IgG1 antibody that specifically binds to circulating DPP3, targets and modulates DPP3 activity, and DPP3 is an important regulator of cardiovascular function. Its mode of action is related to acute diseases accompanied by massive cell death and uncontrolled release of intracellular DPP3 into the blood. Translocated DPP3 remains active in the circulation, where it cleaves bioactive peptides in an uncontrolled manner. Procizumab can block circulating DPP3 and inhibit the degradation of bioactive peptides in the bloodstream. This blockade stabilizes cardiovascular and renal function and reduces short-term mortality. As shown in the Examples section, preclinical studies of Procizumab in animal models of cardiovascular failure have shown impressive and immediate efficacy. In several preclinical models of cardiovascular failure, Procizumab has been shown to normalize ejection fraction and renal function and reduce mortality.

[0012] Examples in the description of the present invention show that injection of Procizumab in pigs with septic shock results in a significant improvement in lung function (given by the PaO2 / FiO2 ratio). In addition, it has been demonstrated that DPP3 is significantly elevated in patients in the ICU who are all on ventilation, especially mechanical ventilation. Therefore, it is reasonable to infer that DPP3 inhibitors, especially Procizumab, can improve lung function in patients with reduced ventilated lung function, regardless of the indication.

[0013] The surprising finding of the present invention is that DPP3 activity inhibitors are suitable for treating or intervening in critically ill patients with reduced lung function to improve lung function. Summary of the Invention

[0014] The subject matter of the present invention is a DPP3 activity inhibitor for the treatment or intervention of critically ill patients with reduced lung function to improve lung function.

[0015] DPP3 Level

[0016] The subject matter of the present invention is a DPP3 activity inhibitor for treating or intervening in critically ill patients to improve lung function, wherein the patient has a DPP3 level higher than a predetermined threshold.

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

[0018] The subject matter of the present application is a DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function, wherein when measured by different methods (such as immunoassay, activity assay, mass spectrometry, etc.), the patient has a DPP3 level higher than a predetermined threshold in a body fluid sample of the subject.

[0019] 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, progastrin, ACTH (adrenocorticotropic hormone), and MSH (melanocyte-stimulating hormone; Abramić et al., 2000; Baršun et al., 2007; Dhanda et al., 2008 ). Cleavage of the peptide hormones and other unlabeled oligopeptides (e.g., Ala-Ala-Ala-Ala, Dhanda et al., 2008 ), can be monitored by detecting the corresponding cleavage products. Detection methods include, but are not limited to, HPLC analysis (e.g., Lee & Snyder 1982 ), mass spectrometry (e.g., Abramić et al., 2000 ), H1-NMR analysis (e.g., Vandenberg et al., 1985 ), capillary zone electrophoresis (CE; e.g., Baršun et al., 2007 ), thin layer chromatography (e.g., Dhanda et al., 2008 ), or reverse phase chromatography (e.g., Mazocco et al., 2006 ).

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

[0021] Another option for detecting DPP3 activity is the Protease-Glo TM 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 coupled with aminofluorescein. After cleavage by DPP3, aminofluorescein is released and used as a substrate for a coupled luciferase reaction that emits detectable luminescence.

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

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

[0024] In another preferred embodiment of the invention, the capture binder for determining the DPP3 level can be selected from antibodies, antibody fragments or non-IgG scaffolds.

[0025] In a specific embodiment of the invention, the capture binder for determining the DPP3 level is an antibody.

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

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

[0028] The test sample is passed through the immobilized binder, and DPP3 (if present in the sample) binds to the binder and is itself immobilized for detection.

[0029] Then, a substrate can be added, and the reaction product can be detected to indicate the presence or amount of DPP3 in the test sample. Alternatively, DPP3 bound to the capture molecule on the solid phase can be detected using a second capture molecule that specifically binds to DPP3.

[0030] For the purposes of this specification, the term "solid phase" can be used to include any material or container in or on which an assay can be performed, including but not limited to porous materials, non-porous materials, test tubes, wells, slides, agarose resins (such as Sepharose from GE Healthcare Life Sciences), magnetic particles (such as Dynabeads from Thermo Fisher Scientific TM or Pierce TM magnetic beads), etc.

[0031] In one embodiment of the invention, a method for determining DPP3 activity in a body fluid sample of a subject comprises the following steps: ● Contacting the sample with a capture binder that specifically binds to full-length DPP3, ● Separating the DPP3 bound to the capture binder, ● Adding a DPP3 substrate to the separated DPP3, ● Quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate.

[0032] In another embodiment of the invention, the separation step is a washing step that removes the components of the sample that are not bound to the capture binder from the captured DPP3.

[0033] In another embodiment of the invention, the DPP3 substrate conversion is detected by a method selected from: fluorescence of a fluorogenic substrate (such as Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate conjugated with aminofluorescein, mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis and subsequent activity staining (immobilized, active DPP3), or western blotting (cleavage products).

[0034] In another embodiment of the invention, the substrate can be selected from: angiotensin II, III, and IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, progastrin, ACTH, and MSH, or a dipeptide conjugated with a fluorophore, chromophore, or aminofluorescein, wherein the dipeptide is Arg-Arg.

[0035] In another specific embodiment of the present invention, the substrate can be selected from dipeptides conjugated to a fluorophore, chromophore or aminofluorescein, wherein the dipeptide is Arg-Arg.

[0036] In a specific embodiment, the binder exhibits at least 10 7 M -1 , preferably 10 8 M -1 binding affinity, and more preferably an affinity greater than 10 9 M -1 , most preferably greater than 10 10 M -1 . Those skilled in the art know that it is possible to consider compensating for a lower affinity by applying a higher dose of the compound, and such measures do not exceed the scope of the present invention.

[0037] To determine the affinity of the antibody for DPP3, the Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany) was used to measure the binding kinetics of DPP3 to the immobilized antibody using label-free surface plasmon resonance. The reversible immobilization of the antibody was performed according to the manufacturer's instructions (Mouse Antibody Capture Kit; GE Healthcare) using an anti-mouse Fc antibody covalently coupled to the CM5 sensor surface at high density ( Lorenz et al., 2011. Antimicrob Agents Chemother. 55(1): 165–173 ).

[0038] In one embodiment, this assay for determining DPP3 levels is a sandwich immunoassay using any type of detection technique, including but not limited to enzyme labeling, chemiluminescent labeling, electrochemiluminescent labeling, preferably a fully automated assay. In one embodiment of the diagnostic method, this assay is an enzyme-labeled sandwich assay. Examples of automated or fully automated assays include assays that can be used with one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®.

[0039] A variety of immunoassays are known and can be used in the assays and methods of the present invention, and they include: mass spectrometry (MS), luminescence immunoassay (LIA), radioimmunoassay ("RIA"), homogeneous enzyme multiplied immunoassay ("EMIT"), enzyme-linked immunosorbent assay ("ELISA"), apodezyme reactivation immunoassay ("ARIS"), luminescence-based bead arrays, magnetic bead-based arrays, protein microarray assays, rapid test formats such as strip immunoassays, immunochromatographic strip tests, rare cryptates assays, and automated systems / analyzers.

[0040] In one embodiment of the present invention, it can be a test technique called POC (point-of-care testing), which allows testing to be performed beside the patient in less than 1 hour without the need for a fully automated assay system. An example of this technique is immunochromatographic test technology, such as a microfluidic device.

[0041] In a particular embodiment, at least one of the two binders is labeled in the sandwich immunoassay for detection.

[0042] In another preferred embodiment, the label is selected from chemiluminescent labels, enzyme labels, fluorescent labels, radioiodine labels.

[0043] The assay can be a homogeneous or heterogeneous assay, a competitive and non-competitive assay. In one embodiment, the assay takes the form of a sandwich assay, which is a non-competitive immunoassay in which the molecule to be detected and / or quantified is bound to a first antibody and to a second antibody. The first antibody can be bound to a solid phase, such as the surface of a bead, well or other container, chip or strip, and the second antibody is an antibody labeled, for example, with a dye, radioisotope or reactive or catalytically active moiety. The amount of the labeled antibody bound to the analyte is then measured by an appropriate method. The general composition and procedure involving "sandwich assays" are well established and known to those skilled in the art ( Handbook of Immunoassay (The Immunoassay Handbook), edited by David Wild, Elsevier LTD, Oxford; 3rd edition, (May 2005), ISBN-13: 978-0080445267; Hultschig C et al., Curr Opin Chem Biol. 2006 Feb;10 (1):4-10. PMID: 16376134 )

[0044] In another embodiment, the assay comprises two capture molecules, preferably antibodies, both present in the liquid reaction mixture in the form of a dispersion, wherein a first labeled component is attached to the first capture molecule, wherein the first labeled component is part of a fluorescence- or chemiluminescence-quenching or amplification-based labeling system, and a second labeled component of the labeling system is attached to the second capture molecule such that a measurable signal is generated after the two capture molecules bind to the analyte, thereby allowing detection of the sandwich complex formed in the solution containing the sample.

[0045] In another embodiment, the labeling system comprises a rare earth cryptate or chelate in combination with a fluorescent dye or a chemiluminescent dye, in particular a cyanine type dye.

[0046] In the context of the present invention, fluorescence-based assays comprise the use of dyes which may be selected, for example, from FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes such as CY3, CY5, CY3.5, CY5.5, Cy7, xanthene, 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), TET, 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes such as BODIPY TMR, Oregon green, coumarins such as umbelliferone, benzimidazoles such as Hoechst 33258, phenanthridines such as Texas red, Yakima yellow, Alexa Fluor, PET, ethidium bromide, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, etc.

[0047] In the context of the present invention, chemiluminescence-based assays comprise the use of dyes based on the physical principles described for chemiluminescent materials in the following documents ( Kirk-Othmer, Encyclopedia of chemical technology, 4th edition, executive editor J. I. Kroschwitz; editor M. Howe-Grant, John Wiley & Sons, 1993, vol.15, p. 518-562, incorporated herein by reference, including the citations on pages 551-562 text )). A preferred chemiluminescent dye is acridinium ester.

[0048] As used herein, the term "assay" or "diagnostic assay" may be of any type applied in the field of diagnostics. Such an assay may be based on the binding of an analyte to be detected to one or more capture probes having a certain 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 10 8 M -1 .

[0049] The subject matter of the present invention is a DPP3 activity inhibitor for use in the treatment or intervention in critically ill patients to improve lung function, wherein a predetermined threshold of the DPP3 level in a sample of the subject's body fluid is between 20 and 120 ng / mL, more preferably between 30 and 80 ng / mL, even more preferably between 40 and 60 ng / mL, and most preferably the threshold is 50 ng / mL.

[0050] In a particular embodiment, an assay is used to determine the DPP3 level, wherein the assay sensitivity of the assay is capable of quantifying DPP3 in healthy subjects and is < 20 ng / ml, preferably < 30 ng / ml, more preferably < 40 ng / ml.

[0051] In a particular embodiment of the invention, the body fluid according to the invention is a blood sample. The blood sample may be selected from whole blood, serum and plasma. In a particular embodiment of the method, the sample is selected from human citrate plasma, heparin plasma and EDTA plasma.

[0052] In another particular embodiment of the invention, the DPP3 level is determined in different samples obtained from the patient at different time points.

[0053] In another particular embodiment of the invention, the difference between the DPP3 levels in different samples obtained from the patient at different time points is determined. The difference may be determined as an absolute or relative difference.

[0054] In a particular embodiment of the invention, the DPP3 level is determined at least twice.

[0055] In another particular embodiment of the invention, treatment is initiated when the relative difference between the DPP3 levels in different samples obtained from the patient at different time points is 100% or more, more preferably 75% or more, even more preferably 50% or more, most preferably 25% or more.

[0056] In another particular embodiment of the invention, the at least second determination of the DPP3 level is performed within 2 hours, preferably within 4 hours, more preferably within 6 hours, even more preferably within 12 hours, even more preferably within 24 hours, most preferably within 48 hours.

[0057] The DPP3 level, which is the amount of DPP3 protein and / or DPP3 activity in the subject body fluid sample, can be determined, for example, by one of the following methods:

[0058] 1. Luminescence immunoassay (LIA) for quantifying DPP3 protein concentration ( Rehfeld et al., 2019 JALM 3 (6): 943-953 )

[0059] The LIA is a one-step chemiluminescent sandwich immunoassay using white high-binding polystyrene microtiter plates as the solid phase. These plates are coated with the monoclonal anti-DPP3 antibody AK2555 (capture antibody). The tracer anti-DPP3 antibody AK2553 is labeled with MA70-acridin-NHS-ester and used at a concentration of 20 ng per well. 20 μL of sample (e.g., serum, heparin plasma, citrate plasma, or EDTA plasma derived from a patient's blood) and calibrators are pipetted into the coated white microtiter plates. After adding the tracer antibody AK2553, the microtiter plates are incubated at room temperature and 600 rpm for 3 h. Unbound tracer is then removed by 4 washing steps (350 μL per well). The remaining chemiluminescence of each well is measured using a microtiter plate luminometer for 1 second. The concentration of DPP3 is determined using a 6-point calibration curve. Calibrators and samples are preferably run in duplicate.

[0060] 2. Enzyme Capture Assay (ECA) for quantifying DPP3 activity ( Rehfeld et al., 2019 JALM 3(6): 943-953 )

[0061] The ECA is a DPP3-specific activity assay using black high-binding polystyrene microtiter plates as the solid phase. These plates are coated with the monoclonal anti-DPP3 antibody AK2555 (capture antibody). 20 μL of sample (e.g., serum, heparin plasma, citrate plasma, cerebrospinal fluid, and urine) and calibrators are pipetted into the coated black microtiter plates. After adding the assay buffer (200 μL), the microtiter plates are incubated at 22 °C and 600 rpm for 2 h. DPP3 present in the sample is immobilized by binding to the capture antibody. Unbound sample components are removed by 4 washing steps (350 μL per well). The specific activity of the immobilized DPP3 is measured by adding the fluorogenic substrate Arg-Arg-β-naphthylamide (Arg2-βNA) in the reaction buffer and then incubating at 37 °C for 1 h. 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 an emission wavelength of 410 nm. The activity of DPP3 is determined using a 6-point calibration curve. Calibrators and samples are preferably run in duplicate.

[0062] 3. Liquid Assay (LAA) for quantifying DPP3 activity (modified from Jones et al., Analytical Biochemistry, 1982 )

[0063] The LAA is a liquid-phase assay that uses black non-binding polystyrene microtiter plates to measure DPP3 activity. 20 μL of sample (e.g., serum, heparin plasma, citrate plasma) and calibrator are pipetted into the non-binding black microtiter plates. After adding the fluorogenic substrate Arg2-βNA in the assay buffer (200 μL), the initial βNA fluorescence (T = 0) is measured in a fluorometer using an excitation wavelength of 340 nm, and the emission is detected at 410 nm. The plate is then incubated at 37 °C for 1 hour. The final fluorescence (T = 60) is measured. The difference between the final fluorescence and the initial fluorescence is calculated. The activity of DPP3 is determined using a 6-point calibration curve. The calibrator and sample are preferably run in duplicate in parallel.

[0064] As outlined in the examples, the DPP3 levels of the present invention have been determined using the described DPP3 assay ( Rehfeld Et al., 2019. JALM 3(6): 943-953 ). If the calibration of other assays is different from the assay system used in the present invention, the above thresholds may vary in other assays. Therefore, taking into account the calibration differences, the above critical values should be applied accordingly to such assays with different calibrations. One possibility to quantify the calibration differences is to perform a method comparison analysis (correlation) of the assay under discussion with the corresponding biomarker assay used in the present invention by measuring the corresponding biomarker (e.g., DPP3) in the sample using both methods. Another possibility is to determine the median level of the biomarker in a representative normal population using the assay under discussion (assuming the test has sufficient analytical sensitivity), compare the results with the median level of the biomarker described in the literature, and recalculate the calibration based on the differences obtained from this comparison. Using the calibration method used in the present invention, samples from 5400 normal (healthy) subjects have been measured (Swedish single-center prospective population study (MPP-RES)): the median (interquartile range) of plasma DPP3 was 14.5 ng / ml (11.3 ng / ml – 19 ng / ml).

[0065] The threshold level can be obtained, for example, from a Kaplan-Meier analysis, where the occurrence of the disease is correlated with the quartiles of the biomarker in the population. According to this analysis, the risk of developing the disease of the present invention is significantly increased in subjects with biomarker levels above the 75th percentile. This result is further supported by a Cox regression analysis with full adjustment for classical risk factors: the highest quartile is highly significantly correlated with an increased risk of developing the disease of the present invention compared to all other subjects.

[0066] Other preferred cut-off values are, for example, the 90th, 95th or 99th percentile of the normal population. By using a higher percentile than the 75th percentile, the number of false-positive subjects identified can be reduced, but subjects with a moderate risk (although the risk is still increased) may be missed. Thus, one may adopt a cut-off value based on whether it is considered more appropriate to identify most subjects at risk at the cost of also identifying "false positives", or whether it is considered more appropriate to mainly identify subjects at high risk at the cost of missing a few subjects at moderate risk.

[0067] Indication

[0068] As used herein, the term "patient" refers to a living human or non-human organism that is receiving medical care or is due to receive medical care for a disease. This includes a person without an apparent disease but who is undergoing examination for pathological signs. Thus, the methods and assays described herein are applicable to both human and veterinary diseases.

[0069] The patient is a critically ill patient suffering from a disease selected from severe infectious diseases, sepsis, pneumonia including community-acquired pneumonia (CAP), pulmonary embolism, myocardial infarction, any type of shock (including cardiogenic shock, septic shock or anaphylactic shock) and acute respiratory distress syndrome (ARDS).

[0070] "Critically ill" means that the patient is suffering from a life-threatening acute disease or acute condition and may or will die. In a particular embodiment, the critically ill patient is an ICU patient.

[0071] The infectious disease may be an infectious disease of bacterial, viral, fungal or parasitic origin. The viral infection may be selected from infections caused by influenza virus or coronavirus.

[0072] The coronavirus is selected from SARS-CoV-1, SARS-CoV-2, MERS-CoV, especially SARS-CoV-2. Coronaviruses cause diseases in mammals and birds. In humans, the virus causes respiratory infections, including the usually mild common cold up to rare forms that can be fatal such as SARS, MERS and COVID-19. SARS-CoV-1 or -2 infections may present as mild, moderate or severe disease; the latter includes severe pneumonia, acute respiratory distress syndrome (ARDS), sepsis and septic shock.

[0073] In a particular embodiment, the viral infection is not caused by a coronavirus; in other words, the infection is not a coronavirus infection.

[0074] Acute respiratory distress syndrome (ARDS) is a form of respiratory failure characterized by the rapid onset of widespread inflammation in the lungs. Symptoms include shortness of breath, rapid breathing, and bluish skin. Among those who survive, a decline in quality of life is common. Causes may include sepsis, pancreatitis, trauma, pneumonia, and aspiration. Underlying mechanisms include diffuse damage to the cells that form the microscopic air sac barriers in the lungs, surfactant dysfunction, immune system activation, and dysfunction of the body's blood clotting regulation. In fact, ARDS impairs the lungs' ability to exchange oxygen and carbon dioxide. Diagnosis is based on a PaO2 / FiO2 ratio (ratio of arterial oxygen partial pressure to fraction of inspired oxygen) of less than 300 mmHg despite positive end-expiratory pressure (PEEP) greater than 5 cm H2O. Main treatments include mechanical ventilation and treatment of the underlying cause. Ventilation strategies include the use of low volume and low pressure. If oxygenation remains inadequate, lung recruitment maneuvers and neuromuscular blocking agents may be used. If this is still insufficient, extracorporeal membrane oxygenation (ECMO) may be an option. The syndrome is associated with a mortality rate between 35% and 50%.

[0075] Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection (see Singer et al., 2016. JAMA 315(8): 801-810 ). Organ dysfunction can be identified as an acute change in the total SOFA score ≥2 points after infection. In patients known to have no organ dysfunction, a baseline SOFA score of zero can be assumed. A SOFA score ≥2 reflects an overall mortality risk of approximately 10% in a general hospital population with suspected infection. Even patients showing mild dysfunction can deteriorate further, highlighting the severity of this condition and the need for timely and appropriate intervention (if not already taken). Sepsis is a life-threatening condition that occurs when the body's response to infection damages its own tissues and organs. Patients with suspected infection who may have a prolonged stay in the intensive care unit (ICU) or die in the hospital can be rapidly identified at the bedside by qSOFA (i.e., altered mental status, systolic blood pressure ≤100 mm Hg, or respiratory rate ≥22 / min).

[0076] Shock is characterized by reduced oxygen delivery and / or increased oxygen consumption or inadequate oxygen utilization, leading to cellular and tissue hypoxia. It is a life-threatening circulatory failure disorder, and the most common manifestation is hypotension (systolic blood pressure below 90 mmHg or mean arterial pressure (MAP) below 65 mmHg). Depending on the underlying cause, shock is divided 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 ).

[0077] Septic shock is a potentially life-threatening medical condition that occurs when sepsis, which is organ injury or damage resulting from a response to infection, leads to dangerously low blood pressure and abnormal cellular metabolism. The Third International Consensus Definition for Sepsis and Septic Shock (Sepsis-3) defines septic shock as a subtype of sepsis in which particularly severe circulatory, cellular, and metabolic abnormalities, compared to sepsis alone, are associated with a greater risk of death. Patients with septic shock can be clinically identified by the need for vasopressors to maintain a mean arterial pressure of 65 mm Hg or higher in the absence of hypovolemia and a serum lactate level greater than 2 mmol / L (>18 mg / dL). This combination is associated with a hospital mortality rate of over 40% ( Singer et al., 2016. JAMA. 315(8): 801–10 ). The primary infection is most commonly caused by bacteria but can also be caused by fungi, viruses, or parasites. It can be located anywhere in the body but is most commonly in the lungs, brain, urinary tract, skin, or abdominal organs. It can cause multiple organ dysfunction syndrome (formerly known as multiple organ failure) and death. Typically, people with septic shock are cared for in an intensive care unit. It most commonly affects children, immunocompromised individuals, and the elderly because their immune systems are unable to respond to infection as effectively as those of healthy adults. The mortality rate of septic shock is approximately 25 - 50%.

[0078] Cardiogenic shock (CS) is defined as a state of critical end-organ hypoperfusion due to reduced cardiac output. Notably, CS forms a disease spectrum ranging from mild hypoperfusion to profound shock. The established criteria for diagnosing CS are: (i) systolic blood pressure ≤90 mmHg for >30 minutes, or the need for vasopressors to achieve a blood pressure ≥90 mmHg; (ii) pulmonary congestion or elevated left ventricular filling pressure; (iii) signs of impaired organ perfusion meeting at least one of the following criteria: (a) altered mental status; (b) cold and clammy skin; (c) oliguria (<0.5 mL / kg / h or <30 mL / h); (d) elevated serum lactate ( Reynolds and Hochman 2008. Circulation 117: 686–697 ). Acute myocardial infarction (AMI) secondary to ventricular dysfunction is the most common cause of CS, accounting for approximately 80% of cases. Mechanical complications such as ventricular septal (4%) or free wall rupture (2%) and acute severe mitral regurgitation (7%) are less common causes of CS after AMI ( Hochman et al., 2000. J Am Coll Cardiol 36: 1063–1070 ). Non-AMI-related CS can be caused by decompensated valvular heart disease, acute myocarditis, arrhythmias, etc., and has different treatment options. This means that there are 40,000 to 50,000 patients in the United States each year, and 60,000 to 70,000 patients in Europe. Despite improvements in treatment, mainly through early revascularization to reduce subsequent mortality, CS remains the leading cause of death in AMI, with a mortality rate still approaching 40 - 50% according to recent registries and randomized trials.Goldberg et al., 2009. Circulation 119: 1211–1219 ).

[0079] The Horowitz index (synonyms: post-oxygenation Horowitz, Horowitz coefficient, P / F ratio) is used to evaluate lung function, especially in patients receiving invasive ventilation in intensive care. It can be used to assess the degree of lung injury. The Horowitz index (PaO2 / FiO2 ratio) is a well-known marker of acute lung injury and predicts mortality in patients with acute respiratory failure. The Horowitz index is defined as the ratio of the partial pressure of oxygen in the blood (PaO2) (in mmHg) to the fraction of inspired oxygen (FiO2), i.e., the PaO2 / FiO2 ratio. In a healthy lung, the Horowitz index depends on age and is typically between 350 and 500. Values below 300 are the threshold for mild lung injury, 200 indicates moderately severe lung injury. Values below 100 are used as the criterion for severe injury. The Horowitz index plays an important role in the diagnosis of acute respiratory distress syndrome (ARDS). According to the Berlin definition, the Horowitz index is used to classify ARDS into three severities based on the degree of hypoxemia ( Matthay et al., 2012. J Clin Invest. 122(8): 2731–274 ).

[0080] Lung dysfunction or reduced lung function is defined as a Horowitz index below 350, more preferably below 300, even more preferably below 200, and most preferably below 100.

[0081] In one embodiment of the present invention, the lung function of the patient is reduced, which is defined as a reduction in the Horowitz index of at least 10%, preferably at least 20%, more preferably at least 50%, and most preferably at least 75%.

[0082] Improvement in lung function is defined as an increase in the Horowitz index of more than 5%, more preferably more than 10%, even more preferably more than 20%, even more preferably more than 30%, and most preferably more than 50%.

[0083] Ventilation

[0084] In another specific embodiment, the critically ill patient is under ventilation.

[0085] The term "ventilation" includes non-invasive or invasive ventilation.

[0086] Non-invasive ventilation refers to the use of respiratory support implemented through a face mask, nasal mask, or helmet. Air (usually supplemented with oxygen) is given through the mask under positive pressure.

[0087] Mechanical ventilation or assisted ventilation is a medical term for artificial ventilation, in which mechanical means are used to assist or replace spontaneous breathing.

[0088] This may involve a machine known as a ventilator, or breathing can be manually assisted by a suitably qualified professional such as an anesthesiologist, respiratory therapist (RT), registered nurse, or paramedic, by squeezing a bag-valve-mask device. If mechanical ventilation involves any instrument that enters the trachea through the mouth (such as an endotracheal tube) or any instrument that enters the trachea through the skin (such as a tracheostomy tube), it is called "invasive". Non-invasive ventilation can be used with a face mask or nasal mask in appropriately selected conscious patients.

[0089] Extracorporeal membrane oxygenation (ECMO), also known as extracorporeal life support system (ECLS), is an extracorporeal technique that provides long-term cardiac and respiratory support for people whose heart and lungs are unable to provide sufficient amounts of gas exchange or perfusion to sustain life. The techniques used for ECMO mainly originated from cardiopulmonary bypass, which provides short-term support by suspending the patient's own circulation. The working principle of ECMO is to draw blood from the human body and artificially remove carbon dioxide from the patient's red blood cells and add oxygen. Generally, it is used in the late treatment of patients after cardiopulmonary bypass or with severe heart failure and / or lung failure, although now it has been extended in some medical centers to the treatment of cardiac arrest, allowing treatment of the underlying cause of cardiac arrest while maintaining circulation and oxygenation. ECMO is also used to support patients with acute viral pneumonia related to, for example, COVID-19 in cases where artificial ventilation is insufficient to maintain blood oxygenation levels. In addition, venovenous ECMO can be used to provide respiratory support for patients with septic shock.

[0090] DPP3 inhibitor

[0091] Inhibitors are molecules that preferably significantly inhibit DPP3 activity. These molecules can be peptides and small molecules, antibodies, antibody fragments, or non-Ig scaffolds.

[0092] Significant inhibition means that DPP3 activity is inhibited by more than 60%, preferably more than 70%, more preferably more than 80%, preferably more than 90%, and more preferably almost or actually 100% inhibition.

[0093] DPP3 activity can be non-specifically inhibited by different general protease inhibitors (such as PMSF, TPCK), sulfhydryl reagents (such as pHMB, DTNB), and metal chelators (EDTA, o-phenanthroline) ( Abramić et al., 2000. Biological Chemistry, 381: 1233–1243; EP 2949332 )

[0094] DPP3 activity can be further specifically inhibited by different kinds of compounds: the endogenous DPP3 inhibitor is spinorphin. Several synthetic derivatives of spinorphin, such as tynorphin, have been prepared, and they show inhibitory effects on DPP3 activity to different degrees ( Yamamoto et al., 2000. Life sciences 62(19): 1767–1773 ). Other published peptide inhibitors of DPP3 are propioxatin A and B (US 4804676) and propioxatin A analogues ( Inaoka et al., 1988. J. Biochem 104(5): 706–711 ).

[0095] DPP3 can also be inhibited by small molecules such as fluostatins and benzimidazole derivatives. Fluostatins A and B are antibiotics produced by Streptomyces sp. TA-3391, which are non-toxic and strongly inhibit DPP3 activity. So far, 20 different benzimidazole derivatives ( Agić et al., 2007. Bioorganic Chemistry 35(2): 153–169; Rastija et al., 2015. Acta Chimica Slovenica 62: 867–878 ) have been synthesized and published, and two of the compounds, 1' and 4', show the strongest inhibitory effects ( Agić et al., 2007. Bioorganic Chemistry 35(2): 153–169 ). Several dipeptidyl hydroxamic acids have also been shown to inhibit DPP3 activity ( Cvitešić et al., 2016. J Enzyme Inhib Med Chem 31(sup2):40-45 ).

[0096] The subject matter of the present application is an inhibitor of DPP3 activity, which is used for treatment or intervention in critically ill patients with reduced lung function to improve lung function, wherein the DPP3 activity inhibitor is selected from small molecules, anti-DPP3 antibodies, anti-DPP3 antibody fragments or anti-DPP3 non-Ig scaffolds.

[0097] The subject matter of the present application is an inhibitor of DPP3 activity, which is used for treatment or intervention in critically ill patients with reduced lung function to improve lung function, wherein the DPP3 activity inhibitor is a small molecule selected from the group consisting of spinorphin, tynorphin, propioxatin A and B, fluostatins A and B, benzimidazole or its derivatives or analogues.

[0098] The subject matter of the present application is an inhibitor of DPP3 activity, which is used for treatment or intervention in critically ill patients with reduced lung function to improve lung function, wherein the inhibitor is an anti-DPP3 antibody, anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold with a minimum binding affinity for DPP3 equal to or less than 10 -7 M.

[0099] The subject matter of the present application is an inhibitor of DPP3 activity, which is used for treatment or intervention in critically ill patients with reduced lung function to improve lung function, wherein the antibody is a monoclonal antibody or monoclonal antibody fragment.

[0100] Throughout this specification, an "antibody" or "antibody fragment" or "non-Ig scaffold" according to the invention is capable of binding DPP3, and thus is directed against DPP3, and can thus be referred to as an "anti-DPP3 antibody", "anti-DPP3 antibody fragment" or "anti-DPP3 non-Ig scaffold".

[0101] The term "antibody" generally includes monoclonal and polyclonal antibodies and their binding fragments, in particular Fc-fragments, and so-called "single-chain antibodies" ( Bird et al., 1988 ), chimeric antibodies, humanized antibodies, in particular CDR-grafted antibodies, and diabodies or tetrabodies ( Holliger et al., 1993 ). Also included are immunoglobulin-like proteins selected by techniques including, for example, phage display that specifically bind to a molecule of interest contained in a sample. In such a case, the term "specifically bind" refers to an antibody raised against the molecule of interest or a fragment thereof. An antibody is considered specific if its affinity for the molecule of interest or the above-mentioned fragment thereof is at least preferably 50-fold higher, more preferably 100-fold higher, most preferably at least 1000-fold higher than its affinity for other molecules contained in a sample containing the molecule of interest. How to make antibodies and select antibodies with a given specificity is well known in the art.

[0102] In one embodiment of the invention, the anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold is monospecific.

[0103] A monospecific anti-DPP3 antibody or monospecific anti-DPP3 antibody fragment or monospecific anti-DPP3 non-Ig scaffold means that the antibody or antibody fragment or non-Ig scaffold binds to a specific region within the target DPP3 (SEQ ID No. 1) that encompasses at least 5 amino acids. A monospecific anti-DPP3 antibody or monospecific anti-DPP3 antibody fragment or monospecific anti-DPP3 non-Ig scaffold is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that all have an affinity for the same antigen. Monoclonal antibodies are monospecific, but monospecific antibodies can also be produced by means other than producing them from a common germ cell.

[0104] In a particular embodiment, the anti-DPP3 antibody, anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold is an inhibitory antibody, fragment or non-Ig scaffold. The anti-DPP3 antibody, anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold inhibits DPP3 activity by more than 50%, preferably more than 60%, preferably more than 70%, more preferably more than 80%, preferably more than 90%, even more preferably more than 95%, preferably almost or actually 100%.

[0105] An antibody or fragment according to the invention is a protein that comprises one or more polypeptides encoded substantially by immunoglobulin genes and that specifically binds an antigen. Well-known immunoglobulin genes include the κ, λ, α (IgA), γ (IgG1, IgG2, IgG3, IgG4), δ (IgD), ε (IgE), and μ (IgM) constant region genes, as well as the myriad immunoglobulin variable region genes. A full-length immunoglobulin light chain is typically about 25 Kd or 214 amino acids in length.

[0106] A full-length immunoglobulin heavy chain is typically about 50 Kd or 446 amino acids in length. The light chain is encoded by a variable region gene (about 110 amino acids in length) at the NH2 terminus and a κ or λ constant region gene at the COOH terminus. The heavy chain is likewise encoded by a variable region gene (about 116 amino acids in length) and one of the remaining constant region genes.

[0107] The basic structural unit of an antibody is typically a tetramer composed 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, and the constant regions mediate effector functions. Immunoglobulins also exist in a variety of other forms, including, for example, Fv, Fab, and (Fab')2, as well as bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al., 1987. Eur. J. Immunol. 17:105; Huston et al., 1988. Proc. Natl. Acad. Sci. U.S.A., 85:5879-5883; Bird et al., 1988. Science 242:423-426; Hood et al., 1984, Immunology, Benjamin, N.Y., 2nd ed.; Hunkapille and Hood 1986. Nature 323:15-16 ). The variable region of an immunoglobulin light or heavy chain comprises framework regions interrupted by three hypervariable regions, also known as complementarity-determining regions (CDRs) (see Sequences of Proteins of Immunological Interest, E. Kabat et al., 1983, U.S. Department of Health and Human Services ). As noted above, the CDRs are primarily responsible for binding to the epitope of the antigen. An immune complex is an antibody that specifically binds to the antigen, such as a monoclonal antibody, chimeric antibody, humanized antibody, or human antibody, or a functional antibody fragment.

[0108] Chimeric antibodies are antibodies in which the light and heavy chain genes are typically constructed by genetic engineering from immunoglobulin variable and constant region genes of different species. For example, the variable segments of the genes from a murine monoclonal antibody can be joined to human constant segments such as κ and γ1 or γ3. Thus, in one example, a therapeutic chimeric antibody is a hybrid protein consisting of the variable or antigen-binding domain from a murine antibody and the constant or effector domain from a human antibody, although other mammalian species can also be used, or the variable regions can be generated by molecular techniques. Methods for making chimeric antibodies are well known in the art, see for example U.S. Patent No. 5,807,715. "Humanized" immunoglobulins are immunoglobulins that contain human framework regions and one or more CDRs from a non-human (e.g., murine, rat, or synthetic) immunoglobulin. The non-human immunoglobulin that provides the CDRs is referred to as the "donor", and the human immunoglobulin that provides the framework is referred to as the receptor. In one embodiment, in a humanized immunoglobulin, all of the CDRs are from the donor immunoglobulin. Constant regions are not necessarily present, but if they are present, they must be substantially identical to human immunoglobulin constant regions, i.e., having at least about 85 - 90% identity, such as about 95% or higher. Thus, with the possible exception of the CDRs, all parts of the humanized immunoglobulin are substantially identical to the corresponding parts of the native human immunoglobulin sequence. A "humanized antibody" is an antibody that contains humanized light and heavy chain immunoglobulins. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The receptor framework of the humanized immunoglobulin may have a limited number of amino acid substitutions taken from the donor framework. Humanized or other monoclonal antibodies may have other conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are, for example: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed using genetic engineering (see for example U.S. Patent No. 5,585,089). Human antibodies are antibodies in which the light and heavy chain genes are of human origin. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells that secrete the desired antibody. 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 methods (see for example WO91 / 17271; WO92 / 001047; WO92 / 20791 ), or selected from a human combinatorial monoclonal antibody library (see the Morphosys website). Human antibodies can also be prepared using transgenic animals bearing human immunoglobulin genes (see for example WO93 / 12227; WO 91 / 10741 ).

[0109] Thus, the anti-DPP3 antibody can have a format known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies. In a preferred embodiment, the antibody according to the invention is a recombinantly produced antibody, such as IgG, a typical full-length immunoglobulin, or an antibody fragment containing at least the F-variable domains of the heavy and / or light chains, such as chemically conjugated antibodies (antigen-binding fragments), including but not limited to Fab fragments, including Fab microantibodies, single-chain Fab antibodies, monovalent Fab antibodies with epitope tags such as Fab-V5Sx2; bivalent Fab (microantibodies) dimerized with CH3 domains; bivalent Fab or multivalent Fab, such as formed by multimerization, e.g., dimerization through the dHLX domain, with the help of heterologous domains, such as Fab-dHLX-FSx2; F(ab’)2 fragments, scFv fragments, multimerized multivalent and / or multispecific scFv fragments, bivalent and / or bispecific diabodies, BITE ® (bispecific T-cell engager), trifunctional antibodies, multivalent antibodies, such as from other classes than class G; single-domain antibodies, such as nanobodies derived from camel or fish immunoglobulins, and so on.

[0110] In a preferred embodiment, the anti-DPP3 antibody format is selected from Fv fragments, scFv fragments, Fab fragments, scFab fragments, F(ab)2 fragments, and scFv-Fc fusion proteins. In another preferred embodiment, the antibody format is selected from scFab fragments, Fab fragments, scFv fragments, and their bioavailability-optimized conjugates, such as PEGylated fragments. One of the most preferred formats is the scFab format.

[0111] Non-Ig scaffolds can be protein scaffolds and can be used as antibody mimetics as they are capable of binding to ligands or antigens. Non-Ig scaffolds can be selected from tetranectin-based non-Ig scaffolds (e.g., as described in US 2010 / 0028995 ), fibronectin scaffolds (e.g., as described in EP 1 266 025 ), lipocalin-based scaffolds (e.g., as described in WO 2011 / 154420 ), ubiquitin scaffolds (e.g., as described in WO 2011 / 073214 ), transferrin scaffolds (e.g., as described in US 2004 / 0023334 ), protein A scaffolds (e.g., as described in EP 2 231 860 ), ankyrin-repeat-based scaffolds (e.g., as described in WO 2010 / 060748 ), miniprotein (preferably miniproteins forming a cysteine knot) scaffolds (e.g., as described in EP 2314308 ), Fyn SH3 domain-based scaffolds (e.g., as described in WO 2011 / 023685described), an EGFR-A domain-based scaffold (e.g., as described in WO 2005 / 040229 described) and a Kunitz domain-based scaffold (e.g., as described in EP 1 941 867 described).

[0112] In one embodiment of the invention, the anti-DPP3 antibody according to the invention can be produced by synthesizing a DPP3 fragment or full-length DPP3 as an antigen, as outlined in Example 1. Subsequently, binders for the fragment are identified using the methods described below or other methods known in the art.

[0113] Humanization of murine antibodies can be carried out according to the following procedure:

[0114] To humanize a murine-derived antibody, the antibody sequence is analyzed to understand the structural interactions of the framework regions (FRs) with the complementarity-determining regions (CDRs) and the antigen. Suitable human FRs are selected on the basis of structural modeling, and the murine CDR sequences are transplanted into the human FRs. Variations can be introduced into the amino acid sequences of the CDRs or FRs to regain disrupted structural interactions resulting from the species switch of the FR sequences. Restoration of such structural interactions can be achieved by using random methods with phage display libraries or by directed methods guided by molecular modeling ( Almagro and Fransson 2008. Humanization of antibodies, Front Biosci. 2008 Jan 1;13: 1619-33 ).

[0115] 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.

[0116] The subject matter of the present application is an inhibitor of DPP3 activity for the treatment or intervention of critically ill patients with reduced lung function to improve lung function, wherein the complementarity-determining regions (CDRs) in the heavy chain comprise the following sequences: SEQ ID NO.: 7, SEQ ID NO.: 8 and / or SEQ ID NO.: 9, and the complementarity-determining regions (CDRs) in the light chain comprise the following sequences: SEQ ID NO.: 10, KVS and / or SEQ ID NO.: 11.

[0117] The subject matter of the present application is an inhibitor of DPP3 activity for the treatment or intervention of critically ill patients with reduced lung function to improve lung function, wherein the monoclonal antibody or antibody fragment is a humanized monoclonal antibody or humanized monoclonal antibody fragment.

[0118] The subject matter of this application is a DPP3 activity inhibitor, which is used for the treatment or intervention of critically ill patients with reduced lung function to improve lung function, wherein the heavy chain contains the sequence SEQ ID NO.: 12, and wherein the light chain contains the sequence SEQ ID NO.: 13.

[0119] The subject matter of this application is a DPP3 activity inhibitor, which is used for the treatment or intervention of critically ill patients with reduced lung function to improve lung function, wherein the inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No. 1. In one embodiment, the inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 amino acids in length contained in SEQ ID No. 1. In another embodiment, the inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to an epitope of at least 5 amino acids in length contained in SEQ ID No. 1.

[0120] The subject matter of this application is a DPP3 activity inhibitor, which is used for the treatment or intervention of critically ill patients with reduced lung function to improve lung function, wherein the inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No. 2, and wherein the epitope is contained in DPP3 as depicted in SEQ ID NO.: 1. In one embodiment, the inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 amino acids in length contained in SEQ ID No. 2, and wherein the epitope is contained in DPP3 as depicted in SEQ ID NO.: 1. In another embodiment, the inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to an epitope of at least 5 amino acids in length contained in SEQ ID No. 2, and wherein the epitope is contained in DPP3 as depicted in SEQ ID NO.: 1.

[0121] The subject matter of the present application is a DPP3 activity inhibitor for the treatment or intervention of critically ill patients with reduced lung function to improve lung function, wherein the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No. 3, and wherein the epitope is contained in DPP3 as depicted in SEQ ID NO.: 1. In one embodiment, the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 amino acids in length contained in SEQ ID No. 3, and wherein the epitope is contained in DPP3 as depicted in SEQ ID NO.: 1. In another embodiment, the inhibitor 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 acids in length contained in SEQ ID No. 3, and wherein the epitope is contained in DPP3 as depicted in SEQ ID NO.: 1.

[0122] The subject matter of the present application is a DPP3 activity inhibitor for the treatment or intervention of critically ill patients with reduced lung function to improve lung function, wherein the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No. 4, and wherein the epitope is contained in DPP3 as depicted in SEQ ID NO.: 1. In one embodiment, the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 amino acids in length contained in SEQ ID No. 4, and wherein the epitope is contained in DPP3 as depicted in SEQ ID NO.: 1. In another embodiment, the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 5 amino acids in length contained in SEQ ID No. 4, and wherein the epitope is contained in DPP3 as depicted in SEQ ID NO.: 1.

[0123] An epitope, also known as an antigenic determinant, is the part of an antigen that is recognized by the immune system, particularly by antibodies. For example, an epitope is a specific segment of an antigen that binds to an antibody. The part of the antibody that binds to the epitope is called a paratope. Epitopes of protein antigens are classified into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. Conformational epitopes and linear epitopes interact with the paratope according to the 3-D conformation 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. Conformational epitopes are formed by the 3-D conformation adopted by the interaction of discontinuous amino acid residues. A linear epitope or continuous epitope is an epitope that is recognized by an antibody through its linear sequence of amino acids or primary structure and is formed by the 3-D conformation adopted by the interaction of continuous amino acid residues.

[0124] In one specific embodiment 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 or humanized antibody or is derived therefrom. In a specific embodiment, one or more (murine) CDRs are grafted into a human antibody or antibody fragment.

[0125] In one aspect, the subject matter of the present invention is a human or humanized CDR-grafted antibody or an antibody fragment thereof that binds to DPP3, wherein the human or humanized CDR-grafted antibody or an antibody fragment thereof comprises an antibody heavy chain (H chain), and the heavy chain comprises: GFSLSTSGMS (SEQ ID NO.: 7), IWWNDNK (SEQ ID NO.: 8), ARNYSYDY (SEQ ID NO.: 9), and / or further comprises an antibody light chain (L chain), and the light chain comprises: RSLVHSIGSTY (SEQ ID NO.: 10), KVS (not part of the sequence listing), SQSTHVPWT (SEQ ID NO.: 11).

[0126] In one specific embodiment of the present invention, the subject matter of the present invention is a human or humanized monoclonal antibody that binds to DPP3 or an antibody fragment thereof that binds to DPP3, wherein the heavy chain comprises at least one CDR selected from the following: GFSLSTSGMS (SEQ ID NO.: 7), IWWNDNK (SEQ ID NO.: 8), ARNYSYDY (SEQ ID NO.: 9), and wherein said light chain comprises at least one CDR selected from the following: RSLVHSIGSTY (SEQ ID NO.: 10), KVS (not part of the sequence listing), SQSTHVPWT (SEQ ID NO.: 11).

[0127] The anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold according to the present invention exhibits a certain affinity for human DPP3, such that the affinity constant is greater than 10 -7 M, preferably greater than 10 -8 M, preferably the affinity is greater than 10 -9 M, most preferably higher than 10 -10 M. Those skilled in the art will appreciate that it is possible to consider compensating for a lower affinity by administering a higher dose of the compound, and such a measure will not result in exceeding the scope of the present invention. The affinity constant can be determined according to the method described in Example 1.

[0128] The subject matter of the present invention is a monoclonal antibody or a fragment thereof, or an antibody fragment, that binds DPP3 and is used for treating or intervening in critically ill patients with reduced lung function to improve lung function, wherein said antibody or fragment comprises the following sequence as the variable heavy chain: SEQ ID NO.: 5 QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPSGKGLEWLAHIWWNDNKSYNPALKSRLTISRDTSNNQVFLKIASVVTADTGTYFCARNYSYDYWGQGTTLTVSS and comprises the following sequence as the variable light chain: SEQ ID NO.: 6 DVVVTQTPLSLSVSLGDPASISCRSSRSLVHSIGSTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK.

[0129] The subject matter of the present invention is a human or humanized monoclonal antibody or a fragment thereof, or an antibody fragment, that binds DPP3 and is used for treating or intervening in critically ill patients with reduced lung function to improve lung function, wherein said antibody or fragment comprises the following sequence as the heavy chain: SEQ ID NO.: 12 MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQVVLTMTNMDPVDTGTYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG and comprises the following sequence as the light chain: SEQ ID NO.: 13 METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0130] In a specific embodiment of the present invention, the antibody comprises the following sequence or a sequence having > 95%, preferably > 98%, preferably > 99% identity thereto as the heavy chain: SEQ ID NO: 12, and comprises the following sequence or a sequence having > 95%, preferably > 98%, preferably > 99% identity thereto as the light chain: SEQ ID NO: 13.

[0131] To assess the identity between two amino acid sequences, pairwise alignments were performed. Identity defines the percentage of amino acids that match directly in an alignment.

[0132] In a preferred embodiment, treatment with a DPP3 activity inhibitor is initiated or altered immediately after providing the sample analysis results indicating the DPP3 level in the sample. In a further embodiment, the treatment can be initiated within 12 hours after receiving the sample analysis results, preferably within 6, 4, 2, 1, 0.5, 0.25 hours or immediately.

[0133] In some embodiments, the method comprises, consists of, or consists essentially of making a single and / or multiple measurements of DPP3 in a single sample and / or multiple samples obtained at substantially the same time point from a patient, in order to guide and / or monitor and / or stratify a therapy, wherein the therapy is the administration of a DPP3 activity inhibitor.

[0134] The term "pharmaceutical formulation" means a pharmaceutical ingredient combined with at least one pharmaceutically acceptable excipient, in a form that permits the biological activity of the pharmaceutical ingredient contained therein to be effective and that is free of other components that are unacceptably toxic to the subject to which the formulation is to be administered. The term "pharmaceutical ingredient" means a therapeutic composition that can optionally be combined with a pharmaceutically acceptable excipient to provide a pharmaceutical formulation or dosage form.

[0135] The subject matter of the present invention is a pharmaceutical formulation for treating or intervening in critically ill patients with reduced lung function to improve lung function, which comprises an antibody or fragment or scaffold according to the present invention.

[0136] The subject matter of the present invention is a pharmaceutical formulation for treating or intervening in critically ill patients with reduced lung function to improve lung function, wherein the pharmaceutical formulation is a solution, preferably a ready-to-use solution.

[0137] The subject matter of the present invention is a pharmaceutical formulation for treating or intervening in critically ill patients with reduced lung function to improve lung function, wherein the pharmaceutical formulation is in a lyophilized state.

[0138] The subject matter of the present invention is a pharmaceutical formulation for treating or intervening in critically ill patients with reduced lung function to improve lung function, wherein the pharmaceutical formulation is administered intramuscularly.

[0139] The subject matter of the present invention is a pharmaceutical formulation for treating or intervening in critically ill patients with reduced lung function to improve lung function, wherein the pharmaceutical formulation is administered intravascularly.

[0140] The subject matter of the present invention is a pharmaceutical formulation for treating or intervening in critically ill patients with reduced lung function to improve lung function, wherein the pharmaceutical formulation is administered by infusion.

[0141] The subject matter of the present invention is a pharmaceutical preparation for treating or intervening in critically ill patients with reduced lung function to improve lung function, wherein the pharmaceutical preparation is to be administered systemically.

[0142] In the above context, the following consecutively numbered embodiments provide further specific aspects of the present invention:

[0143] 1. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function.

[0144] 2. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to embodiment 1, wherein the patient has a DPP3 level higher than a (predetermined) threshold in a body fluid sample of the patient.

[0145] 3. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to embodiment 2, wherein the predetermined threshold of the DPP3 level in the body fluid sample of the subject is between 20 and 120 ng / mL, preferably between 30 and 80 ng / mL, more preferably between 35 and 60 ng / mL, and most preferably the threshold is 40 ng / mL.

[0146] 4. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to any one of embodiments 1 to 3, wherein the sample is a body fluid sample selected from whole blood, plasma or serum.

[0147] 5. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to any one of embodiments 1 to 4, wherein the lung function is evaluated using the Horowitz index (PaO2 / FiO2 ratio).

[0148] 6. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to any one of embodiments 1 to 5, wherein the patient has a Horowitz index lower than 350, preferably lower than 300, even more preferably lower than 200, and most preferably lower than 100.

[0149] 7. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to any one of embodiments 1 to 6, wherein the lung function of the patient is reduced by at least 10%, preferably at least 20%, more preferably at least 50%, and most preferably at least 75%.

[0150] 8. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to any one of Embodiments 1 to 6, wherein the lung function is improved by at least 5%, preferably more than 10%, more preferably more than 20%, even more preferably more than 30%, and most preferably more than 50%.

[0151] 9. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to any one of Embodiments 1 to 8, wherein the critically ill patients with reduced lung function are in an invasive or non-invasive ventilation state.

[0152] 10. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to any one of Embodiments 1 to 9, wherein the patient has a severe infectious disease, sepsis, pneumonia including community-acquired pneumonia (CAP), pulmonary embolism, myocardial infarction, any type of shock (including cardiogenic shock, septic shock or anaphylactic shock) and acute respiratory distress syndrome (ARDS).

[0153] 11. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to any one of Embodiments 1 to 10, wherein the DPP3 activity inhibitor is selected from small molecules, anti-DPP3 antibodies, anti-DPP3 antibody fragments or anti-DPP3 non-Ig scaffolds.

[0154] 12. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to Embodiment 11, wherein the small molecule is selected from eporphin, tyrophin, alpiostatin A and B, fluorastatin A and B, benzimidazole or its derivatives or analogs.

[0155] 13. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to Embodiment 11, wherein the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to an epitope comprising at least 4 or 5 amino acids contained in SEQ ID No. 1.

[0156] 14. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to Embodiment 11, wherein the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to an epitope comprising at least 4 or 5 amino acids contained in SEQ ID No. 2.

[0157] 15. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to any one of Embodiments 11, 13 or 14, wherein the antibody is a monoclonal antibody or monoclonal antibody fragment.

[0158] 16. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to any one of Embodiments 11, 13 or 14, wherein the antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, and the complementarity determining regions (CDRs) in the heavy chain comprise the sequences: SEQ ID NO.: 7, SEQ ID NO.: 8 and / or SEQ ID NO.: 9 and the complementarity determining regions (CDRs) in the light chain comprise the sequences: SEQ ID NO.: 10, KVS and / or SEQ ID NO.: 11.

[0159] 17. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to any one of Embodiments 11 or 13 to 16, wherein the monoclonal antibody or antibody fragment is a humanized monoclonal antibody or a humanized monoclonal antibody fragment.

[0160] 18. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function according to any one of Embodiments 11, 13 to 16, wherein the antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, the heavy chain comprises the sequence SEQ ID NO.: 12, and the light chain comprises the sequence SEQ ID NO.: 13. BRIEF DESCRIPTION OF THE DRAWINGS

[0161] Figure 1 : Kaplan Meyer survival plots in relation to low (< 68.6 ng / mL) and high (≥ 68.6 ng / ml) DPP3 plasma concentrations. (A) 7-day survival rate of patients with sepsis / septic shock in relation to DPP3 plasma concentration (cut-off value 68.6 ng / mL); (B) 7-day survival rate of patients with cardiogenic shock in relation to DPP3 plasma concentration (cut-off value 68.6 ng / mL); (C) 7-day survival rate of patients with acute myocardial infarction in relation to DPP3 plasma concentration (cut-off value 68.6 ng / mL); (D) 3-month survival rate of patients with dyspnea in relation to DPP3 plasma concentration; (E) 4-week survival rate of patients with burns in relation to DPP3 plasma concentration; (F) 7-day survival rate of patients with septic shock in relation to DPP3 plasma concentration.

[0162] Figure 2 : SDS-PAGE of native hDPP3 purified from human erythrocyte lysate on a gradient gel (4-20%). Molecular weight markers are indicated by arrows.

[0163] Figure 3: Experimental design - Role of native DPP3 in animal models.

[0164] Figure 4 : (A) DPP3 injection led to a decrease in fractional shortening, resulting in deteriorated cardiac function. (B) A decline in renal function was also observed through an increase in renal resistance index.

[0165] Figure 5 : Association and dissociation curves using the AK1967-DPP3 binding assay of Octet. The biosensor loaded with AK1967 was immersed in serially diluted recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM), and the association and dissociation were monitored.

[0166] Figure 6 : Western blot and DPP3 detection of diluted hemocyte lysates using AK1967 as the primary antibody.

[0167] Figure 7 : Inhibitory curve of inhibitory antibody AK1967 against native DPP3 from hemocytes. The inhibition of DPP3 by the specific antibody is concentration-dependent, and when analyzed against 15 ng / ml DPP3, the IC 50 is ~15 ng / ml.

[0168] Figure 8 : Experimental setup - Role of prorizacumab in sepsis-induced heart failure.

[0169] Figure 9 : In rats with sepsis-induced heart failure, prorizacumab significantly improved fractional shortening (A) and mortality (B).

[0170] Figure 10 : Experimental design - Isoproterenol-induced cardiac stress in mice, followed by prorizacumab treatment (B) and control (A).

[0171] Figure 11 : In mice with isoproterenol-induced heart failure, prorizacumab improved fractional shortening (A) and reduced renal resistance index (B) at 1 hour and 6 hours after administration, respectively.

[0172] Figure 12 : Experimental setup - Role of valsartan in healthy mice injected with DPP3.

[0173] Figure 13 : Valsartan treatment rescued the decrease in fractional shortening caused by DPP3.

[0174] Figure 14:High levels of DPP3 at 24 hours after admission in patients with sepsis were associated with the worst SOFA score.

[0175] Figure 15 :In patients with sepsis, high plasma levels of cDPP3 were associated with organ dysfunction. Bar graph of SOFA scores in AdrenOSS-1 according to the evolution of DPP3 levels during ICU stay. HH: DPP3 above the median at admission and at 24 h; HL: above the median at admission but below the median at 24 h; LL: below the median at admission and at 24 h; LH: below the median at admission but above the median at 24 h.

[0176] Figure 16: High levels of cDPP3 at 24 hours after admission in patients with sepsis were associated with the worst SOFA score for each organ. (A) Heart, (B) Kidney, (C) Respiratory system, (D) Liver, (E) Coagulation, and (F) Central nervous system SOFA score values are shown according to the dynamic levels of cDPP3 between admission and 24 h (HH: high / high, HL: high / low, LH: low / high, LL: low / low).

[0177] Figure 17 :High levels of DPP3 at ICU admission were associated with subsequent deterioration of renal function within 48 h. Y-axis: DPP3 measured on day 1 (at ICU admission). X-axis: KDIGO stage 0 or 1 or KDIGO stage 2 or 3 (p = 0.002).

[0178] Figure 18 :In patients with COVID-19, serial DPP3 measurements during ICU stay were associated with disease severity. A, DPP3 levels were measured on day 3 of ICU admission (p = 0.02); B, DPP3 levels were measured on day 7 of ICU admission (p = 0.013). X-axis: False = P / F ratio > 150; True = P / F ratio < 150.

[0179] Figure 19 :In patients with COVID-19, high DPP3 values during ICU stay were associated with poor prognosis. A, DPP3 levels were measured on day 3 of ICU admission; B, DPP3 levels were measured on day 7 of ICU admission. X-axis: 0 = Alive; 1 = Dead.

[0180] Figure 20 :High DPP3 levels at ICU admission were associated with the need for vasopressor therapy during ICU stay (day 3, p = 0.05). Y-axis: DPP3 measured on day 1 (at ICU admission). X-axis: None: No vasopressor therapy during ICU stay, or Any: Vasopressor therapy.

[0181] Figure 21 : Serial DPP3 measurements during the ICU stay were associated with the need for organ support therapy, particularly veno-venous ECMO. A, DPP3 levels were measured on day 3 of ICU admission (p = 0.03); B, DPP3 levels were measured on day 7 of ICU admission (p = 0.04). X-axis: 0 = no ECMO; 1 = ECMO.

[0182] Figure 22 : Treatment regimen of prozumab for improving lung function in a sepsis animal model.

[0183] Figure 23 : DPP3 activity (U / L) and prozumab concentration (ng / ml) measured at different time points during prozumab infusion in septic pigs (n = 16).

[0184] Figure 24 : PaO2 / FiO2 ratio (A) and median (B) of each individual, and median of thoracic and lung static compliance (C) in animals receiving standard treatment combined with prozumab treatment and those receiving standard treatment alone.

[0185] Figure 25 : Kaplan–Meier plot showing the correlation between DPP3 levels measured at admission and 28-day mortality in patients (n = 390) requiring mechanical ventilation, with DPP3 levels above and below 40.0 ng / mL (HR 2.1, 95% CI 1.5–2.9, p < 0.0001).

[0186] Figure 26 : Kaplan–Meier plot showing the correlation between DPP3 levels measured at admission and 28-day mortality in n = 217 invasively ventilated patients (a) and n = 131 non-invasively ventilated patients (b), with DPP3 levels above and below 40.0 ng / mL. Examples

[0187] Example 1 - Method for Measuring DPP3 Protein and DPP3 Activity

[0188] Production of antibodies and determination of DPP3-binding ability: Several murine antibodies were prepared and screened for their ability to bind human DPP3 in a specific binding assay (see Table 1).

[0189] Peptide / Conjugate for Immunization:

[0190] Synthesize the DPP3 peptide for immunization, see Table 1 (JPT Technologies, Berlin, Germany), which contains an additional N-terminal cysteine residue (if cysteine does not exist in the selected DPP3 sequence) for conjugating the peptide to bovine serum albumin (BSA). Covalently link the peptide to BSA using Sulfolink coupling gel (Perbio-science, Bonn, Germany). Conduct the conjugation procedure according to Perbio's manual. Recombinant GST-hDPP3 is produced by USBio (United States Biological, Salem, MA, USA).

[0191] Immunization of Mice, Immune Cell Fusion, and Screening:

[0192] On day 0, Balb / c mice were intraperitoneally (i.p.) injected with 84 µg of GST-hDPP3 or 100 µg of the DPP3 peptide-BSA conjugate (emulsified in TiterMax Gold adjuvant), on day 14 with 84 µg or 100 µg (emulsified in complete Freund's adjuvant), and on days 21 and 28 with 42 µg or 50 µg (in incomplete Freund's adjuvant). On day 49, the animals received an intravenous (i.v.) injection of 42 µg of GST-hDPP3 or 50 µg of the DPP3 peptide-BSA conjugate dissolved in saline. Three days later, the mice were sacrificed and immunocyte fusion was performed.

[0193] Cells of splenocytes from the immunized mice and 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 in a 96-well cell culture plate. Hybrid clones were selected by growing in HAT medium [RPMI1640 medium supplemented with 20% fetal bovine serum and HAT supplement]. One week later, the HAT medium was replaced with HT medium, passaged three times, and then transferred back to normal cell medium.

[0194] After two weeks of fusion, the recombinant DPP3-binding IgG antibodies in the cell culture supernatant were initially screened. Thus, recombinant GST-tagged hDPP3 (USBiologicals, Salem, USA) was immobilized in a 96-well plate (100 ng / well) and incubated with 50 μl of cell culture supernatant per well for 2 hours at room temperature. After washing the plate, 50 μl / well of POD-rabbit anti-mouse IgG antibody was added and incubated at RT for 1 hour. After the next washing step, 50 μl of chromogenic solution (3.7 mM o-phenylenediamine in citrate / phosphate buffer, 0.012% H2O2) was added to each well and incubated at RT for 15 minutes, and the chromogenic reaction was stopped by adding 50 μl of 4N sulfuric acid. Absorbance was detected at 490 mm.

[0195] The microcultures that tested positive were transferred to a 24-well plate for propagation. After retesting, the selected cultures were cloned and recloned using the limiting dilution technique, and the isotype was determined.

[0196] Production of Mouse Monoclonal Antibodies

[0197] Antibodies against GST-tagged human DPP3 or DPP3 peptides were produced by standard antibody production methods ( Marx et al., 1997 ) and purified by protein A. According to SDS gel electrophoresis analysis, the purity of the antibodies was ≥ 90%.

[0198] Characterization of Antibodies - Binding to hDPP3 and / or Immunopeptides

[0199] To analyze the binding ability of different antibodies and antibody clones to DPP3 / immunopeptides, a binding assay was performed:

[0200] a) Solid phase

[0201] Recombinant GST-tagged hDPP3 (SEQ ID NO. 1) or DPP3 peptide (immunopeptide, SEQ ID NO. 2) was immobilized on the surface of a high-binding microtiter plate (96-well polystyrene microtiter plate, Greiner Bio-One international AG, Austria, 1 μg / well, in coupling buffer [50 mM Tris, 100 mM NaCl, pH 7.8], 1 h at RT). After blocking with 5% bovine serum albumin, the microtiter plate was dried under vacuum.

[0202] b) Labeling procedure (tracer)

[0203] Mix 100 µg (100 µl) of different anti-DPP3 antibodies (detection antibodies, 1 mg / ml, in PBS, pH 7.4) with 10 µl of NHS-acridinium ester (1 mg / ml, in acetonitrile, InVent GmbH, Germany; EP 0 353 971), and incubate for 30 minutes at room temperature. Purify the labeled anti-DPP3 antibodies by gel filtration HPLC on Shodex Protein 5 µm KW-803 (Showa Denko, Japan). Dilute the purified labeled antibodies in assay buffer (50 mmol / l potassium phosphate, 100 mmol / l NaCl, 10 mmol / l Na2-EDTA, 5 g / l bovine serum albumin, 1 g / l mouse IgG, 1 g / l bovine IgG, 50 µmol / l aprotinin peptide, 100 µmol / l leupeptin, pH 7.4). The final concentration is approximately 5 - 7*10 6 relative light units (RLU) of the labeled compound (approximately 20 ng of labeled antibody) per 200 µl. Measure the acridinium ester chemiluminescence using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

[0204] c) hDPP3 binding assay

[0205] Load 200 µl of the labeled and diluted detection antibody (tracer) into the plate and incubate for 2 - 4 hours at 2 - 8°C. Remove the unbound tracer by washing 4 times with 350 µl of wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Measure the chemiluminescence bound to the wells using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

[0206] Antibody Characterization - hDPP3 Inhibition Assay

[0207] To analyze the ability of different antibodies and antibody clones to inhibit DPP3, a DPP3 activity assay of a known procedure was employed (Jones et al., 1982). Recombinant GST-tagged hDPP3 was diluted in assay buffer (25 ng / ml GST-DPP3, in 50 mM Tris-HCl, pH 7.5 and 100 µM ZnCl2), and 200 µl of this solution was incubated with 10 µg of the corresponding antibody at room temperature. After a 1-hour pre-incubation, the fluorescent substrate Arg-Arg-βNA (20 µl, 2 mM) was added to the solution, and the generation of free βNA over time was monitored at 37 °C using a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG). The fluorescence of βNA was detected by an excitation wavelength of 340 nm and an emission wavelength of 410 nm. The slope of the increase in fluorescence over time (in units of RFU / min) was calculated for different samples. The slope of GST-hDPP3 using a buffer control was designated as 100% activity. The inhibitory ability of a potential capture binder was defined as the percentage reduction in GST-hDPP3 activity resulting from incubation with the capture binder.

[0208] The following table represents a selected group of obtained antibodies and their binding rates expressed in relative light units (RLU) and their relative inhibitory abilities (% ; Table 1). Monoclonal antibodies generated against the following DPP3 regions were selected based on their ability to bind recombinant DPP3 and / or immunopeptides and their inhibitory potential.

[0209] All antibodies generated against full-length recombinant hDPP3 with a GST tag showed strong binding to immobilized GST-tagged hDPP3. Antibodies generated against the peptide of SEQ ID NO.: 2 also bound GST-hDPP3. The SEQ ID NO.: 2 antibody also bound strongly to the immunopeptide.

[0210]

[0211] The development of a luminescence immunoassay (DPP3-LIA) for quantifying DPP3 protein concentration and an enzyme capture activity assay (DPP3-ECA) for quantifying DPP3 activity has recently been described ( Rehfeld et al., 2019. JALM 3(6): 943 - 953 ), which is hereby incorporated by reference in its entirety.

[0212] Example 2 - Prognosis of DPP3 for Short - Term Mortality

[0213] The DPP3 concentration in plasma of various diseased patients was measured using an hDPP3 immunoassay ( Rehfeld et al., 2019. JALM 3(6): 943 - 953 ), which was correlated with the short-term mortality of the patients.

[0214] Study Cohort - Sepsis and Septic Shock

[0215] Plasma samples from 574 patients in the Adrenomedullin and Prognosis in Severe Sepsis and Septic Shock (AdrenOSS-1) study were screened for DPP3. AdrenOSS-1 is a prospective, observational, multinational study that included 583 patients admitted to the intensive care unit with sepsis or septic shock ( Hollinger et al., 2018 ). 292 patients were diagnosed with septic shock.

[0216] Study Cohort - Cardiogenic Shock

[0217] Plasma samples from 108 patients diagnosed with cardiogenic shock were screened for DPP3. Blood was drawn within 6 h of the diagnosis of cardiogenic shock. Mortality was followed up for 7 days.

[0218] Study Cohort - Acute Coronary Syndrome

[0219] Plasma samples from 720 patients with acute coronary syndrome were screened for DPP3. Blood was drawn 24 h after the onset of chest pain. Mortality was followed up for 7 days.

[0220] Study Cohort - Dyspnea

[0221] Plasma samples were immediately collected from 1440 patients presenting with dyspnea (tachypnea) upon admission to the emergency department of Skåne University Hospital. In addition to other diseases, patients with dyspnea may have acute coronary syndrome or congestive heart failure and are at high risk of organ failure and short-term death. Mortality was followed up for 3 months after the emergency department visit.

[0222] Study Cohort - Burn Patients

[0223] Plasma samples from 107 patients with severe burns (more than 15% of the total body surface area) were screened for DPP3. Blood was drawn at admission. Mortality was followed up for 4 weeks.

[0224] hDPP3 Immunoassay:

[0225] Immunoassays (LIA) for measuring the amount (LIA) or activity (ECA) of human DPP3 were used to determine DPP3 levels in patient plasma. Antibody immobilization, labeling, and incubation were performed as described by Rehfeld et al. ( Rehfeld et al., 2019. JALM 3(6): 943 - 953 ) were performed.

[0226] Results

[0227] The short-term survival rate of patients with sepsis / septic shock is related to the plasma concentration of DPP3 at admission. Patients with a plasma concentration of DPP3 higher than 68.6 ng / mL (the 3rd quartile) had an increased risk of death compared to patients with a plasma concentration of DPP3 below this threshold ( Figure 1 A). The same relationship was also seen when analyzing only the relationship between the short-term prognosis of septic shock patients in this cohort and the plasma concentration of DPP3 ( Figure 1 F). Patients with elevated DPP3 plasma concentrations had an increased risk of death compared to patients with low DPP3 plasma concentrations. When the same cut-off value was applied to patients with cardiogenic shock, an increased risk of short-term death within 7 days was also observed in patients with high DPP3 ( Figure 1 B).

[0228] In addition, when the DPP3 level was high and the corresponding cut-off value of 68.6 ng / mL was applied, the 7-day survival rate of patients with DPP3-related acute coronary syndrome also increased ( Figure 1 C).

[0229] When this cut-off value of 68.6 ng / mL was applied to patients with dyspnea, a significantly increased risk of death was detected in patients with high DPP3 levels during 3 months of follow-up ( Figure 1 D).

[0230] In addition, in severely burned patients with a high DPP3 concentration above the corresponding cut-off value of 68.6 ng / mL, the risk of 4-week mortality increased ( Figure 1 E).

[0231] Example 3 - Purification of Human Native DPP3

[0232] Human erythrocyte lysate was applied to a total of 100 ml of Sepharose 4B resin (Sigma-Aldrich), and the effluent was collected. The resin was washed with a total of 370 mL of PBS buffer at pH 7.4, and the wash fractions were combined with the collected effluent to obtain a total volume of 2370 mL.

[0233] For the immunoaffinity purification step, 110 mg of monoclonal anti-hDPP3 mAb AK2552 was conjugated to 25.5 mL of UltraLink hydrazide resin (Thermo Fisher Scientific) according to the manufacturer's protocol (GlycoLink Immobilization Kit, Thermo Fisher Scientific). Unconjugated antibody was quantified by Bradford technique and the conjugation efficiency was 98%. The resin-antibody conjugate was equilibrated with 10 bed volumes of wash-binding buffer (PBS, 0.1% TritonX-100, pH 7.4), combined with 2370 mL of clarified erythrocyte lysate, and incubated at 4 °C with continuous stirring for 2 h. Subsequently, 100 mL of the incubation mixture was spread over 10 15 mL polypropylene columns and the effluent was collected by centrifugation at 1000 x g for 30 s. This step was repeated several times to obtain 2.5 mL of DPP3-loaded resin per column. Each column was washed 5 times with 10 mL of wash-binding buffer using gravity flow. DPP3 was eluted by placing each column in a 15 mL falcon tube containing 2 mL of neutralization buffer (1 M Tris-HCl, pH 8.0), then adding 10 mL of elution buffer (100 mM glycine-HCl, 0.1% TritonX-100, pH 3.5) to each column and immediately centrifuging at 1000 x g for 30 s. The elution step was repeated 3 times in total to obtain 360 mL of pooled eluate. The pH of the neutralized eluate was 8.0.

[0234] The pooled eluate was loaded onto a 5 mL HiTrap Q-sephare HP column (GE Healthcare) equilibrated with IEX buffer A1 (100 mM glycine, 150 mM Tris, pH 8.0) using the sample pump of an Äkta Start system (GE Healthcare). After sample loading, the column was washed with five column volumes of IEX buffer A2 (12 mM NaH2PO4, pH 7.4) to remove unbound proteins. Elution of DPP3 was achieved using IEX buffer B (12 mM NaH2PO4, 1 M NaCl, pH 7.4) by applying a sodium chloride gradient in the range of 0 - 1 M NaCl over 10 column volumes (50 mL). The eluate was collected in 2 mL fractions. Buffers used for ion exchange chromatography were sterile filtered using 0.22 µM bottle top filters.

[0235] The purification table and the corresponding yields and activities for each purification step are given in Table 2. Figure 2SDS-PAGE of native hDPP3 purified from human erythrocyte lysate on a gradient gel (4-20%) is shown.

[0236]

[0237] Example 4 - Role of Native DPP3 in Animal Models

[0238] The effect of injecting native hDPP3 in healthy mice was investigated by monitoring the fractional shortening and renal resistance index.

[0239] Wild-type Black 6 mice (8-12 weeks old, number of groups shown in Table 3) were allowed to acclimatize to the environment for 2 weeks before baseline echocardiography. The mice were randomly assigned to one of two groups and subsequently injected intravenously with native DPP3 protein or PBS by retro-orbital injection, at a dose of 600 μg / kg of DPP3 protein.

[0240] Cardiac function was evaluated by echocardiography at 15, 60, and 120 minutes after injection of DPP3 or PBS ( Gao et al., 2011 ), and renal function was evaluated by renal resistance index ( Lubas et al., 2014; Dewitte et al., 2012 ). ( Figure 3 )

[0241]

[0242] Results

[0243] Mice treated with native DPP3 protein showed a significant decrease in fractional shortening compared to the control group injected with PBS ( Figure 4 A). The WT+DPP3 group also showed deterioration of renal function, as observed by an increase in renal resistance index ( Figure 4 B).

[0244] Example 5 - Development of Pronizumab

[0245] Antibodies against SEQ ID NO.: 2 were characterized in more detail (epitope mapping, binding affinity, specificity, inhibitory potential). The results of the study are shown here using clone 1967 of SEQ ID NO.: 2 (AK1967; "prozumab") as an example.

[0246] Epitope Determination of AK1967 on DPP3:

[0247] To localize the epitopes of AK1967, a number of N- or C-terminally biotinylated peptides (peptides & elephants GmbH, Hennigsdorf, Germany) were synthesized. These peptides included the full immunopeptide (SEQ ID No. 2) or the sequences of its fragments with single amino acid stepwise removal from the C- or N-terminus (for the complete list of peptides, see Table 5).

[0248] Using high-binding 96-well plates (Greiner Bio-One international AG, Austria), each well was coated with 2 µg of avidin in coupling buffer (500 mM Tris-HCl, pH 7.8, 100 mM NaCl). Then, the plates were washed and loaded with a specific solution of biotinylated peptide (10 ng / well; buffer - 1xPBS containing 0.5% BSA).

[0249] According to Example 1, the anti-DPP3 antibody AK1967 was labeled with a chemiluminescent label.

[0250] The plates were loaded 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). The chemiluminescence bound to the wells was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). The binding of AK1967 to the corresponding peptide was determined by evaluating the relative light units (RLU). Any peptide showing an RLU signal significantly higher than the non-specific binding of AK1967 was defined as an AK1967 binder. The combined analysis of binding and non-binding peptides revealed the specific epitope of AK1967 on DPP3.

[0251] Measurement of Binding Affinity Using Octet:

[0252] The experiments were performed using an Octet Red96 (ForteBio). AK1967 was captured on a kinetic-grade anti-human Fc antibody (AHC) biosensor. Then, the loaded biosensor was dipped into serially diluted recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM). The binding-phase signal was observed for 120 seconds, followed by an observation of the dissociation phase for 180 seconds. The buffer used in the experiment is shown in Table 4. Kinetic analysis was performed using a 1:1 binding model and global fitting.

[0253]

[0254] Western Blot Analysis of the Binding Specificity of AK1967:

[0255] Wash the blood cells from human EDTA blood (wash 3 times in PBS), dilute in PBS, and lyse by repeated freeze-thaw cycles. The total protein concentration of the cell lysate is 250 µg / ml, and the DPP3 concentration is 10 µg / ml. Dilutions of the cell lysate (1:40, 1:80, 1:160, and 1:320) and dilutions of purified recombinant human His-DPP3 (31.25 - 500 ng / ml) are subjected to SDS-PAGE and Western blotting. Incubate the transfer membrane in 1.) blocking buffer (1xPBS-T containing 5% non-fat dry milk), 2.) primary antibody solution (AK1967, 1:2,000, in blocking buffer), and 3.) HRP-labeled secondary antibody (goat anti-mouse IgG antibody, 1:1,000, in blocking buffer). Detect the bound secondary antibody using Amersham ECL Western blotting detection reagent and Amersham Imager 600 UV (both from GE Healthcare).

[0256] DPP3 Inhibition Assay:

[0257] To analyze the ability of AK1967 to inhibit DPP3, perform DPP3 activity assays using a known procedure as described in Example 1 ( Jones et al., 1982 ). The inhibitory ability of AK1967 is defined as the percentage reduction in GST-hDPP3 activity after incubation with the antibody. The resulting reduced DPP3 activity is presented in the form of an inhibition curve in Figure 7 .

[0258] Epitope Mapping:

[0259] By analyzing peptides with and without AK1967 binding, it was determined that the DPP3 sequence INPETG (SEQ ID NO.: 3) is an essential epitope for AK1967 binding (see Table 5).

[0260] Binding Affinity:

[0261] AK1967 binds to recombinant GST-hDPP3 with an affinity of 2.2*10 -9 M (see kinetic curve in Figure 5 ).

[0262]

[0263] Specificity and Inhibitory Potential:

[0264] The only protein detected in the hemocyte lysate using AK1967 as the first antibody was DPP3 of 80 kDa ( Figure 6 ). The total protein concentration of the lysate was 250 µg / ml, while the estimated DPP3 concentration was approximately 10 µg / ml. Although the non-specific proteins present in the lysate were 25 times that of DPP3, AK1967 still specifically bound and detected DPP3 without any other non-specific binding.

[0265] In the specific DPP3 activity assay, AK1967 showed an inhibitory effect on 15 ng / ml of DPP3, with its IC 50 being approximately 15 ng / ml ( Figure 7 ).

[0266] Chimerization / Humanization:

[0267] The monoclonal antibody AK1967 (“prozumab”), which has the ability to inhibit 70% of DPP3 activity, was selected as a potential therapeutic antibody and was also used as a template for chimerization and humanization.

[0268] Humanization of Mouse Antibodies Can Be Carried Out According to the Following Procedure:

[0269] To humanize the murine antibody, the framework regions (FRs) of the antibody sequence were analyzed for their structural interactions with the complementarity-determining regions (CDRs) and the antigen. Based on structural modeling, appropriate human FRs were selected and the murine CDR sequences were grafted into the human FRs. Variations in the amino acid sequences of the CDRs or FRs could be introduced to regain the disrupted structural interactions caused by the species switch of the FR sequences. The restoration of such structural interactions could be achieved using a phage display library either by a random method or by a directed method guided by molecular modeling ( Almagro and Fransson, 2008. Humanization of antibodies Front Biosci. 13:1619 - 33 ).

[0270] In the above context, the variable regions can be linked to the constant regions of any subclass (IgG, IgM, IgE, IgA), or can be linked to just the scaffold, Fab fragment, Fv, Fab, and F(ab)2. In Examples 6 and 7 below, murine antibody variants with an IgG2a backbone were used. For chimerization and humanization, a human IgG1κ backbone was used.

[0271] For epitope binding, only the complementarity-determining regions (CDRs) are important. The heavy-chain CDRs of the murine anti-DPP3 antibody (AK1967; "Prozumab") are shown in SEQ ID No. 7, SEQ ID No. 8, and SEQ ID No. 9, and the light-chain CDRs are shown in SEQ ID No. 10, Sequence KVS, and SEQ ID No. 11.

[0272] Sequencing of the anti-DPP3 antibody (AK1967; "Prozumab") revealed that the antibody heavy-chain variable region (H chain) corresponded to SEQ ID NO.: 12, and the antibody light-chain variable region (L chain) corresponded to SEQ ID NO.: 13.

[0273] Example 6 - Role of Proxalutamide in Sepsis-Induced Heart Failure

[0274] In this experiment, the effect of Prozumab injection was studied in rats with sepsis-induced heart failure by monitoring the fractional shortening. Rittirsch et al., 2009 ).

[0275] CLP model of septic shock:

[0276] Male Wistar rats (2 - 3 months old, 300 to 400 g, number of groups shown in Table 6) from Centre d'élevage Janvier (France) were randomly assigned to one of three groups. All animals were anesthetized by intraperitoneal (i.p.) administration of ketamine hydrochloride (90 mg / kg) and xylazine (9 mg / kg). To induce polymicrobial sepsis, cecal ligation and puncture (CLP) was performed using a slightly modified Rittirsch protocol. A 1.5 cm incision was made along the midline of the abdomen 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 sutured in two layers, followed by fluid resuscitation (subcutaneous injection of normal saline at 3 ml / 100 g body weight), and the animals were returned to their cages. Sham-operated animals underwent the same surgical procedure but without cecal puncture. CLP animals were randomly assigned to receive either placebo or the therapeutic antibody.

[0277] Study design:

[0278] The study protocol is depicted in Figure 8In the middle. After CLP or sham surgery, the animals were allowed to rest for 20 hours with free access to water and food. Subsequently, they were anesthetized, tracheostomy was performed, and arterial and venous catheters were inserted. At 24 hours after CLP surgery, AK1967 or the control vehicle (saline) was administered by rapid bolus injection (5 mg / kg) followed by a 3-hour infusion (7.5 mg / kg). To ensure safety, invasive and continuous hemodynamic monitoring was performed throughout from t = 0 until 3 hours.

[0279] At t = 0 (baseline), all CLP animals were in septic shock with decreased cardiac function (hypotension, low shortening fraction). At this time, prozumab or the control vehicle (PBS) was injected intravenously (i.v.), and saline infusion was initiated. One control group and two CLP groups are summarized in the following table (Table 6). At the end of the experiment, the animals were euthanized and the organs were harvested for subsequent analysis.

[0280]

[0281] Invasive blood pressure:

[0282] Hemodynamic variables were obtained using the AcqKnowledge system (BIOPAC Systems, Inc., USA). This system provides fully automated blood pressure analysis. The catheter was connected to the BIOPAC system via a pressure sensor.

[0283] For the procedure, the rats were anesthetized (using ketamine and xylazine). The animals were moved 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 a supine position on the operating table. The trachea was incised and a catheter (16G) was inserted and connected to an external ventilator, taking care not to damage the carotid artery and the vagus nerve during the operation. An arterial catheter was inserted into the right carotid artery. The carotid artery was separated from the vagus nerve before ligation.

[0284] A central venous catheter was inserted via the left jugular vein to allow the administration of PCZ or PBS.

[0285] After surgery, the animals were allowed to rest until they reached a stable state before hemodynamic measurements were taken. Then the baseline blood pressure (BP) was recorded. During data collection, the saline infusion via the arterial catheter was stopped.

[0286] Echocardiogram:

[0287] The animals were anesthetized with ketamine hydrochloride. The chest was shaved and the rats were placed in a supine position.

[0288] For transthoracic echocardiography (TTE) examinations, a commercial GE Healthcare Vivid 7 ultrasound system equipped with a high-frequency (14-MHz) linear probe and a 10-MHz cardiac probe was used. All examinations were digitally recorded and stored for subsequent offline analysis.

[0289] Gray-scale images were recorded at a depth of 2 cm. Two-dimensional examinations were initiated in the parasternal long-axis view to measure the aortic annulus diameter and the pulmonary artery diameter. M-mode ultrasound was also used to measure left ventricular (LV) dimensions and to evaluate the fractional shortening (FS%). The calculation of LVFS was: (LV end-diastolic diameter - LV end-systolic diameter) / LV end-diastolic diameter × 100%. Thus, the end-diastolic phase was defined as the phase of the maximum LV diameter. Thus, the end-systolic phase was defined as the phase of the minimum diameter in the same cardiac cycle. All parameters were measured manually. The average of three cardiac cycles was taken for each index.

[0290] From the same parasternal long-axis view, pulmonary artery blood flow was recorded using pulsed-wave Doppler. The velocity-time integral of the pulmonary artery outflow was measured.

[0291] From the apical five-chamber view, mitral valve blood flow was recorded using pulsed Doppler at the level of the mitral valve tip.

[0292] Results:

[0293] Compared with sham-operated animals, sepsis-induced heart failure rats treated with PBS (CLP+PBS) showed a decrease in fractional shortening ( Figure 9 A). The CLP+PBS group also showed a high mortality rate ( Figure 9 B). In contrast, administration of porzulizumab to sepsis-induced heart failure rats not only improved the fractional shortening ( Figure 9 A) and significantly reduced the mortality rate ( Figure 9 B).

[0294] Example 7 - Effect of Proxalutamide on Cardiac and Renal Function

[0295] The effect of porzulizumab in isoproterenol-induced heart failure in mice was investigated by monitoring fractional shortening and renal resistance index.

[0296] Isoproterenol-induced cardiac stress in mice:

[0297] Acute heart failure was induced in 3-month-old male mice by subcutaneous injection of 300 mg / kg of isoproterenol (a non-selective β-adrenergic agonist, DL-isoproterenol hydrochloride, Sigma Chemical Co) twice a day for two days ( Vergaro et al., 2016). ISO was diluted in 0.9% NaCl. Isoproterenol-treated mice were randomly divided into two groups (Table 7), and baseline echocardiography (Gao et al., 2011) and renal resistance index measurement were performed on day 3 ( Lubas et al., 2014; Dewitte et al., 2012 ), followed by intravenous injection of PBS or porzulizumab (10 mg / kg) ( Figure 10 A and B).

[0298] At 1 hour, 6 hours, and 24 hours, cardiac function was evaluated by echocardiography ( Gao et al., 2011 ) and renal resistance index ( Lubas et al., 2014; Dewitte et al., 2012 ) ( Figure 10 A and B). The group of mice injected with the vehicle (PBS) instead of isoproterenol did not receive further pharmacological treatment and served as the control group (Table 8).

[0299]

[0300] Results:

[0301] Application of porzulizumab to isoproterenol-induced heart failure mice restored cardiac function within the first hour after administration ( Figure 11 A). Renal function in diseased mice showed significant improvement 6 hours after PCZ injection and reached renal function comparable to that of sham-operated animals at 24 hours ( Figure 11 B).

[0302] Example 8 - Role of Valsartan

[0303] The effect of an antagonist of angiotensin II type I receptor (ATR1), i.e., valsartan, on healthy mice injected with DPP3 was investigated by monitoring the fractional shortening.

[0304] In this experiment, healthy Black 6 mice (8 - 12 weeks old, number of groups shown in Table 8) drank water containing 50 mg / kg valsartan or water only (Table 8) for two weeks. Subsequently, both groups received intravenous injection of native DPP3 (600 µg / kg), and the fractional shortening was evaluated at 15, 60, and 120 minutes according to the method of Gao et al., 2011 ( Figure 12 ).

[0305]

[0306] Results:

[0307] Injection of DPP3 into healthy mice led to a significant decrease in the fractional shortening ( Figure 13). In contrast, healthy mice treated with the angiotensin II receptor antagonist valsartan and then injected with DPP3 showed no signs of cardiac dysfunction as assessed by fractional shortening. Thus, valsartan treatment restored cardiac function, indicating that DPP3-mediated cardiac dysfunction is angiotensin II-mediated.

[0308] Animals treated with valsartan for two weeks had adapted to the blockade of the type 1 angiotensin II receptor, and subsequent angiotensin II-mediated signal transduction and its regulation of cardiac function were inhibited. Apparently, under valsartan treatment, since the angiotensin signaling system has been inhibited by valsartan, the organism activates cardiac function through other means independent of the type 1 angiotensin II receptor signaling pathway.

[0309] When DPP3 cleaves Ang II and thus inhibits its mediated cardiac function activity, those animals adapted to the downregulation of the angiotensin system (valsartan-treated animals) showed no signs of cardiac dysfunction as evaluated by fractional shortening. In contrast, animals not treated with the angiotensin II receptor antagonist valsartan and not adapted to the inhibition of Ang II-mediated signaling responded to DPP3 injection and subsequent cleavage and inactivation of Ang II, showing a significant decrease in fractional shortening.

[0310] This experiment clearly demonstrated the association between DPP3 and angiotensin II, suggesting that DPP3-induced cardiac dysfunction is angiotensin II-mediated.

[0311] Example 9 - DPP3 and Organ Dysfunction in Sepsis

[0312] The same study as described in Example 2 (AdrenOSS-1) was used to evaluate the association between circulating DPP3 (cDPP3) and organ (e.g., cardiovascular and renal dysfunction) in patients admitted for sepsis and septic shock. AdrenOSS-1 is a European prospective, observational, multinational study (ClinicalTrials.gov identifier NCT02393781) that included 583 patients admitted to the ICU for sepsis or septic shock. The primary outcome (as described in Example 2) was 28-day mortality. Secondary outcomes included organ failure defined by the SOFA score, organ support focused on vasopressor use, and the need for renal replacement therapy. Blood samples were collected in the central laboratory within 24 hours and on day 2 after admission to the ICU.

[0313] To quantify DPP3 protein concentration (DPP3-LIA), a recently reported assay was used ( Rehfeld et al., 2019. JALM 3(6): 943-953 ).

[0314] The median cDPP3 measured at admission in all AdrenOSS-1 patients was 45.1 ng / mL (interquartile range 27.5 - 68.6). High DPP3 levels measured at admission were associated with worse metabolic parameters, renal and cardiac function, and SOFA score: the median SOFA score was 6 (IQR 4 - 9) in patients with DPP3 levels below the median, compared with a median SOFA score of 8 (IQR 5 - 11) in patients with DPP3 levels above the median of 45.1 ng / mL ( Figure 14 ).

[0315] Regardless of the level of cDPP3 at admission, high cDPP3 levels 24 hours later were associated with the worst SOFA scores, both overall ( Figure 15 ) and by organ (Figure 16 A - F).

[0316] In summary, these data indicate that in a large international cohort of patients with sepsis or septic shock, high levels of cDPP3 are associated with survival and the degree of organ dysfunction. The study found a significant correlation between cDPP3 < 45.1 ng / ml at admission and short-term survival, and a prognostic cut-off value of 45.1 pg / ml in both sepsis and septic shock. Regarding organ dysfunction, there was a positive correlation between cDPP3 and the SOFA score at ICU admission. More importantly, the relationship between cPDPP3 levels and the degree of organ dysfunction existed at the time of ICU admission and also during the recovery phase. In fact, patients with high cDPP3 levels at admission but showing a decline towards normal cDPP3 values on day 2 were more likely to recover all organ functions, including cardiovascular, renal, pulmonary, and hepatic.

[0317] Example 10 - DPP3 in Patients Infected with Coronavirus (SARS-CoV-2)

[0318] Plasma samples from 12 patients diagnosed with coronavirus (SARS-CoV-2) infection were screened for DPP3 and other biomarkers. As recently described ( Rehfeld et al., 2019. JALM 3(6): 943-953 ), immunoassays (LIA) or activity assays (ECA) that separately measure the amount (LIA) or activity (ECA) of human DPP3 were used to determine DPP3 levels in patient plasma.

[0319] The DPP3 concentrations in individual samples are summarized in Table 9.

[0320]

[0321] The DPP3 concentration ranged from 27 to 975 ng / ml, with a median (IQR) of 156 (59.5 - 322.3) ng / ml. The DPP3 concentration was significantly elevated compared to healthy subjects. Samples from 5400 normal (healthy) subjects (Swedish single-center prospective cohort study (MPP-RES)) were measured: the median (interquartile range) of plasma DPP3 was 14.5 ng / ml (11.3 ng / ml – 19 ng / ml).

[0322] Example 11 - DPP3 Prognosis, Therapy Stratification, and Follow-Up in COVID-19 Patients

[0323] Cohort description:

[0324] This study included 21 patients with a positive SARS-CoV-2 PCR result who were admitted to the ICU. Patient characteristics included: median age of 63 years, 76% male, median body mass index (BMI) of 28.6, and an admission sequential organ failure assessment (SOFA) score of 5. Exclusion criteria were age < 18 years and pregnancy. Analysis was performed using real-time reverse transcription PCR (RT-PCR). Patient treatment followed the standard of care in the ICU, including mechanical ventilation, veno-venous ECMO, and RRT (if needed).

[0325] Blood samples were taken on the day of admission and daily thereafter (up to day 7) for analysis of DPP3 and standard laboratory parameters. As recently described ( Rehfeld et al., 2019. JALM 3(6): 943-953 ), DPP3 in EDTA plasma was measured using a one-step luminescent sandwich immunoassay (LIA).

[0326] Results:

[0327] a) Baseline and serial DPP3 measurements are associated with disease severity

[0328] DPP3 measured at the time of ICU admission was associated with deterioration of renal function during ICU stay (defined by KDIGO criteria): stages 0 - 1 indicate no renal impairment to mild impairment and low risk, stages 2 - 3 indicate renal injury and renal failure, and DPP3 values in stages 2 - 3 were significantly higher compared to stages 0 - 1 ( Figure 17 ; p = 0.005). High DPP3 values at baseline can be used in combination with other clinical parameters to guide the initiation of renal replacement therapy.

[0329] Since COVID-19 positive patients stay in the ICU for an average of 21 days, DPP3 level measurements during ICU stay (days 3 and 7) were also associated with a low PaO2 / FiO2 ratio (< 150) and thus with severe acute respiratory distress syndrome (ARDS). In COVID-19 patients, continuous monitoring of DPP3 measurements during ICU stay was associated with disease severity (Figure 18 ): The DPP3 level on the 3rd day of ICU admission was shown in Figure 18 A (p = 0.02), and the DPP3 level on the 7th day of ICU admission was shown in Figure 18 B (p = 0.013). (False = P / F ratio > 150; True = P / F ratio < 150).

[0330] In addition, the high DPP3 values measured on the 3rd day ( Figure 19 A, p = 0.03) and the 7th day ( Figure 19 B, p = 0.01) were still associated with a high mortality rate during ICU admission.

[0331] b) Baseline and serial DPP3 measurements are associated with the need for organ support therapy

[0332] High DPP3 values at admission and during ICU stay were significantly associated with the need for organ support therapy, especially vasopressor therapy (on the 3rd day; Figure 20 ) and extracorporeal membrane oxygenation (ECMO) (on the 3rd and 7th days; see Figure 21 A and B respectively).

[0333] Example 12 - Pronizumab for improving lung function in a sepsis animal model

[0334] To evaluate the improvement of pronizumab on lung function during septic shock, a randomized, open-label, controlled study was conducted on 16 anesthetized and mechanically ventilated pigs. Septic shock was induced by fecal peritonitis. One hour after the onset of septic shock, fluid resuscitation, antimicrobial therapy, and abdominal drainage were initiated. Septic pigs were randomly assigned to receive pronizumab (on top of standard care) or standard care alone (norepinephrine and fluid) to maintain the mean arterial pressure between 65 and 75 mmHg for 12 hours ( Figure 22 ). 8 female pigs and 8 male pigs were used in the experiment, and the gender was evenly distributed between the treatment group and the standard care group.

[0335] The results showed that pronizumab could inhibit DPP3 activity in the bloodstream throughout the infusion time ( Figure 23 ). Finally, compared with the standard care group, pronizumab improved respiratory function. From the time point of 4 hours (H4) to 12 hours (H12), the P / F ratio in the pronizumab group was still significantly higher than that in the standard care group. These results indicate that pronizumab has a lung-protective effect during the evolution of septic shock and prevents the decline of respiratory function ( Figure 24A and B). Additionally, at time points H4, H8, and H12, thoracic pulmonary static compliance (i.e., the degree to which the volume of the lungs changes with a given transpulmonary or transmural pressure) was significantly higher in animals treated with poractant alfa compared to placebo ( Figure 24 C). Thoracic pulmonary static compliance affects the ability of the lungs to expand during inspiration. The lower the compliance of the tissue, the more "stiff" or difficult it is to expand. Therefore, the lower the value, the worse the lung condition (as shown in the control group).

[0336] In addition, the percentage improvement in lung function following infusion of poractant alfa at different time points compared to untreated animals was calculated. As shown in Table 10, continuous infusion of poractant alfa after the onset of sepsis resulted in a 43.7% improvement (at time point H4) to a 76.4% improvement (at time point H12) in lung function (measured by the PaO2 / FiO2 ratio) relative to the control group.

[0337]

[0338] Example 13 – DPP3 in the Ventilated ICU General Population

[0339] To evaluate the association between circulating DPP3 (cDPP3) and 28-day mortality in mechanically ventilated patients, n = 390 patients admitted to the ICU and requiring mechanical ventilation on the day of admission were included. The cohort included patients admitted for, among other things, Covid-19 infection, sepsis, septic shock, or cardiogenic shock.

[0340] To quantify DPP3 protein concentration (DPP3-LIA), a recently described assay was used ( Rehfeld et al., 2019. JALM 3(6): 943-953 )

[0341] The median cDPP3 measured at admission for all patients was 35.6 ng / mL (interquartile range 19.9 - 63.4). High DPP3 levels measured at admission were associated with 28-day mortality: patients with DPP3 levels below 40.0 ng / mL had a survival rate of 74.2%, while patients with cDPP3 levels above 40.0 ng / mL had a mortality rate of only 55.5% (HR 2.1, 95% CI 1.5 - 2.9, p < 0.0001, Figure 25 )

[0342] In summary, these data suggest that in a large international cohort of ventilated ICU patients, high levels of cDPP3 are associated with survival. The study found a significant association between cDPP3 < 40.0 ng / ml at admission and short-term survival.

[0343] Example 14 – DPP3 in Septic Shock Patients on Non-Invasive and Invasive Ventilation

[0344] The same study as described in Examples 2 and 9 was used to evaluate the association between circulating DPP3 (cDPP3) and 28-day mortality in patients admitted with invasive and non-invasive ventilation. Among 583 ICU patients admitted with sepsis or septic shock, n = 217 required invasive ventilation and n = 131 required non-invasive ventilation after admission. The primary outcome (as described in Example 2) was 28-day mortality. To quantify DPP3 protein concentration (DPP3-LIA), a recently described assay was used ( Rehfeld et al., 2019. JALM 3(6): 943-953 ).

[0345] Among patients with invasive ventilation (n = 217), the median cDPP3 measured at admission was 31.9 ng / mL (interquartile range 17.5 - 57.1). High DPP3 levels measured at admission were associated with 28-day mortality: the survival rate of patients with DPP3 levels below 40.0 ng / mL was 71.4%, while the mortality rate of patients with cDPP3 levels above 40.0 ng / mL was only 53.6% (HR 2.1, 95% CI 1.3 - 3.3, p = 0.0013, Figure 26 a).

[0346] Among patients with non-invasive ventilation (n = 131), the median cDPP3 measured at admission was 26.7 ng / mL (interquartile range 17.6 - 40.1). High DPP3 levels measured at admission were associated with 28-day mortality: the survival rate of patients with DPP3 levels below 40.0 ng / mL was 86.6%, while the mortality rate of patients with cDPP3 levels above 40.0 ng / mL was only 66.7% (HR 2.8, 95% CI 1.2 - 6.2, p = 0.0097, Figure 26 b).

[0347] In summary, these data suggest that in a large international cohort of ICU patients with sepsis or septic shock on invasive and non-invasive ventilation, high levels of cDPP3 are associated with survival. A significant association was found between cDPP3 < 40.0 ng / ml at admission and short-term survival.

[0348] Sequence

[0349] SEQ ID No. 1 – hDPP3 aa 1-737 MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSPEAPYIYALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTKFVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLRHLGLGKEGITTYFSGNCTMEDAKLAQDFLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDSEVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESFTQGSIEAHKRGSRFWIQDKGPIVESYIGFIESYRDPFGSRGEFEGFVAVVNKAMSAKFERLVASAEQLLKELPWPPTFEKDKFLTPDFTSLDVLTFAGSGIPAGINIPNYDDLRQTEGFKNVSLGNVLAVAYATQREKLTFLEEDDKDLYILWKGPSFDVQVGLHELLGHGSGKLFVQDEKGAFNFDQETVINPETGEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLYLCLHPQVLEIFGFEGADAEDVIYVNWLNMVRAGLLALEFYTPEAFNWRQAHMQARFVILRVLLEAGEGLVTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTGDVAGGRALYEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEYEASAAGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA

[0350] SEQ ID No. 2 – hDPP3 aa 474-493 (N-Cys) – Immunopeptide with an additional N-terminal cysteine CETVINPETGEQIQSWYRSGE

[0351] SEQ ID No. 3 – hDPP3 aa 477-482 – Epitope of AK1967 INPETG

[0352] SEQ ID No. 4 – hDPP3 aa 480-483 ETGE

[0353] SEQ ID No. 5 - Variable region in murine AK1967 heavy chain QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPSGKGLEWLAHIWWNDNKSYNPALKSRLTISRDTSNNQVFLKIASVVTADTGTYFCARNYSYDYWGQGTTLTVSS

[0354] SEQ ID No. 6 - Variable region in murine AK1967 light chain DVVVTQTPLSLSVSLGDPASISCRSSRSLVHSIGSTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

[0355] SEQ ID No. 7 - CDR1 in murine AK1967 heavy chain GFSLSTSGMS

[0356] SEQ ID No. 8 - CDR2 in murine AK1967 heavy chain IWWNDNK

[0357] SEQ ID No. 9 - CDR3 in murine AK1967 heavy chain ARNYSYDY

[0358] SEQ ID No. 10 - CDR1 in murine AK1967 light chain RSLVHSIGSTY

[0359] CDR2 in murine AK1967 light chain KVS

[0360] SEQ ID No. 11 - CDR3 in murine AK1967 light chain SQSTHVPWT

[0361] SEQ ID No. 12 - Humanized AK1967 - Heavy chain sequence (IgG1κ backbone) MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQVVLTMTNMDPVDTGTYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0362] SEQ ID No. 13 - Humanized AK1967 - Light Chain Sequence (IgG1κ Framework) METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0363] SEQ ID No. 14 - Immunopeptide 1 bio-AFNFDQETVINPETGEQIQSWYRSG

[0364] SEQ ID No. 15 - Immunopeptide 2 bio-AFNFDQETVINPETGEQIQ

[0365] SEQ ID No. 16 – Immunopeptide 3 bio-AFNFDQETVINPETGEQI

[0366] SEQ ID No. 17 - Immunopeptide 4 bio-AFNFDQETVINPETGEQ

[0367] SEQ ID No. 18 – Immunopeptide 5 bio-AFNFDQETVINPETGE

[0368] SEQ ID No. 19 - Immunopeptide 6 bio-AFNFDQETVINPETG

[0369] SEQ ID No. 20 – Immunopeptide 7 bio-AFNFDQETVINPET

[0370] SEQ ID No. 21 - Immunopeptide 8 bio-AFNFDQETVINPE

[0371] SEQ ID No. 22 – Immunopeptide 9 bio-AFNFDQETVINP

[0372] SEQ ID No. 23 - Immunopeptide 10 bio-AFNFDQETVIN

[0373] SEQ ID No. 24 - Immunopeptide 11 ETGEQIQSWYK-bio

[0374] SEQ ID No. 25 - Immunopeptide 12 PETGEQIQSWYK-bio

[0375] SEQ ID No. 26 – Immunopeptide 13 NPETGEQIQSWYK-bio

[0376] SEQ ID No. 27 - Immunopeptide 14 INPETGEQIQSWYK-bio

[0377] SEQ ID No. 28 – Immunopeptide 15 VINPETGEQIQSWYK-bio

[0378] SEQ ID No. 29 - Immunopeptide 16 TVINPETGEQIQSWYK-bio

[0379] SEQ ID No. 30 - Immunopeptide 17 ETVINPETGEQIQSWYK-bio。

Claims

1. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function.

2. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function according to claim 1, wherein the patient has a DPP3 level higher than a (predetermined) threshold in a body fluid sample of the patient.

3. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function according to claim 2, wherein the predetermined threshold of the DPP3 level in the body fluid sample of the subject is between 20 ng / mL and 120 ng / mL, preferably between 30 ng / mL and 80 ng / mL, more preferably between 35 ng / mL and 60 ng / mL, and most preferably the threshold is 40 ng / mL.

4. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function according to any one of claims 1 to 3, wherein the sample is a body fluid sample selected from whole blood, plasma or serum.

5. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function according to any one of claims 1 to 4, wherein the lung function is evaluated using the Horowitz index (PaO2 / FiO2 ratio).

6. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function according to any one of claims 1 to 5, wherein the patient has a Horowitz index lower than 350, preferably lower than 300, even more preferably lower than 200, and most preferably lower than 100.

7. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function according to any one of claims 1 to 6, wherein the lung function of the patient is reduced by at least 10%, preferably by at least 20%, more preferably by at least 50%, and most preferably by at least 75%.

8. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function according to any one of claims 1 to 6, wherein the lung function is improved by at least 5%, preferably by more than 10%, more preferably by more than 20%, even more preferably by more than 30%, and most preferably by more than 50%.

9. The DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function according to any one of claims 1 to 8, wherein the critically ill patient with reduced lung function is in an invasive or non-invasive ventilation state.

10. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function, according to any one of claims 1 to 9, wherein the patient has a severe infectious disease, sepsis, pneumonia including community-acquired pneumonia (CAP), pulmonary embolism, myocardial infarction, any type of shock (including cardiogenic shock, septic shock or anaphylactic shock), and acute respiratory distress syndrome (ARDS).

11. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function, according to any one of claims 1 to 10, wherein the DPP3 activity inhibitor is selected from small molecules, anti-DPP3 antibodies, anti-DPP3 antibody fragments or anti-DPP3 non-Ig scaffolds.

12. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function, according to claim 11, wherein the small molecule is selected from epithalon, tyrphostin, epipropisostatins A and B, fluorostatins A and B, benzimidazole or its derivatives or analogs.

13. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function, according to claim 11, wherein the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No.

1.

14. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function, according to claim 11, wherein the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to an epitope of at least 4 or 5 amino acids in length contained in SEQ ID No.

2.

15. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function, according to any one of claims 11, 13 or 14, wherein the antibody is a monoclonal antibody or monoclonal antibody fragment.

16. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function, according to any one of claims 11, 13 or 14, wherein the antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the complementarity-determining regions (CDRs) in the heavy chain comprise the sequences: SEQ ID NO.: 7, SEQ ID NO.: 8 and / or SEQ ID NO.: 9 and the complementarity-determining regions (CDRs) in the light chain comprise the sequences: SEQ ID NO.: 10, KVS and / or SEQ ID NO.:

11.

17. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function, according to any one of claims 11 or 13 to 16, wherein the monoclonal antibody or antibody fragment is a humanized monoclonal antibody or humanized monoclonal antibody fragment.

18. A DPP3 activity inhibitor for treating or intervening in critically ill patients with reduced lung function to improve lung function, according to any one of claims 11, 13 to 16, wherein the antibody or antibody fragment comprises an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises the sequence SEQ ID NO.: 12, and wherein the light chain comprises the sequence SEQ ID NO.: 13.

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