Methods of diagnosing and monitoring disease or condition in subject associated with or associated with necrotic process

Abstract

The present invention relates to a method for diagnosing a disease or condition in a subject associated with or associated with a necrotic process and a method for monitoring said disease or condition. The method comprises: determining the amount of total DPP3 in a body fluid sample of the subject; comparing the determined amount of total DPP3 to a predetermined threshold value; wherein, if the determined amount is above the predetermined threshold, the subject is diagnosed as having a disease or condition associated with or associated with a necrotic process; wherein the sample is selected from whole blood, serum and plasma.

Description

Mother case information

[0001] This application is a divisional application of Chinese patent application number "201780039077.1" entitled "Method and treatment method for determining DPP3". Technical Field

[0002] The present invention relates to a method for diagnosing a disease or condition in an object with or related to a necrosis process, and a method for monitoring said disease or condition. Background Technology

[0003] Dipeptidyl peptidase 3—also known as dipeptidyl aminopeptidase III, dipeptidyl arylamide enzyme III, dipeptidyl peptidase III, enkephalinase B, or erythrocyte angiotensinase; abbreviated as: DPP3 and DPPIII are metallopeptidases that can remove dipeptides from physiologically active peptides, such as enkephalins and angiotensin.

[0004] DPP3 was first identified by Ellis & Nuenke in 1967, and its activity was measured in purified bovine anterior pituitary extract. The enzyme is listed as EC 3.4.14.4, with a molecular weight of approximately 83 kDa, and is highly conserved in prokaryotes and eukaryotes (Prajapati & Chauhan, 2011). The amino acid sequence of the human variant is described in SEQ ID NO 1. Dipeptidyl peptidase III is a widely expressed major cytoplasmic peptidase. Although a signaling sequence is lacking, some studies have reported membrane activity (Lee & Snyder, 1982).

[0005] DPP3 is a zinc-dependent exopeptidase belonging to the M49 peptidase family. It exhibits broad substrate specificity for oligopeptides of three to four to ten amino acids in a variety of compositions and is also capable of post-proline cleavage. DPP3 is known to hydrolyze peptides from the N-terminus of its substrates, including angiotensin II, angiotensin III, and angiotensin IV; leucine enkephalin and methionine enkephalin; and endorphin 1 and endorphin 2. The metallopeptidase DPP3 exhibits optimal activity at pH 8.0 to 9.0 and can be further stimulated by the addition of divalent metal ions such as CO2. 2+ and Mg 2+Activation was performed. Structural analysis of DPP3 revealed the catalytic motifs HELLGH (hDPP3 450-455) and EECRAE (hDPP3 507-512), as well as the following amino acids important for substrate binding and hydrolysis: Glu316, Tyr318, Asp366, Asn391, Asn394, His568, Arg572, Arg577, Lys666, and Arg669 (Prajapati & Chauhan, 2011; Kumar et al., 2016; the numbers refer to the sequence of human DPP3, see SEQ ID No. 1). Considering all known amino acids or sequence regions involved in substrate binding and hydrolysis, the active site of human DPP3 can be defined as the region of amino acids 316 to 669.

[0006] DPP3 activity can be mediated by various universal protease inhibitors (e.g., PMSF, TPCK), thiol reagents (e.g., pHMB, DTNB), and metal chelators (EDTA, catechol). (Etc., 2000) Non-specific inhibition.

[0007] DPP3 activity can be specifically inhibited by different types of compounds: endogenous DPP3 inhibitors are peptides like spinorphins. Several synthetic spinorphin derivatives have been produced, such as tynorphin, and they have shown varying degrees of inhibition of DPP3 activity (Yamamoto et al., 2000). Other publicly disclosed DPP3 peptide inhibitors are propioxatin A and propioxatin B (US4804676) and propioxatin A analogs (Inaoka et al., 1988).

[0008] DPP3 can also be inhibited by small molecules such as fluostatins and benzimidazole derivatives. Fluostatins A and Fluostatins B are antibiotics produced in Streptomyces sp. TA-3391, which are non-toxic and strongly inhibit DPP3 activity. To date, 20 different benzimidazole derivatives have been synthesized and disclosed. (e.g., Rastija et al., 2007; Rastija et al., 2015), in which two compounds, 1' and 4', showed the strongest inhibitory activity ( (Et. et al., 2007). For a complete list of DPP3 inhibitors, see Table 2.

[0009] The exact biological function of DPP3 in cell physiology is unclear, but recent findings suggest that it plays a role not only in protein metabolism but also in blood pressure regulation, pain regulation, and inflammation (Prajapati & Chauhan, 2011).

[0010] Several publications have shown DPP3 to be a promising biomarker, all involving intracellular DPP3. Elevated DPP3 activity has been shown in homogenates of ovarian and endometrial tumors. DPP3 activity even increases with the severity / malignancy of the said tumors. Immunohistochemical and Western blot analyses of glioblastoma cell lines also showed elevated DPP3 levels (Singh et al., 1998 and 2003).

[0011] Intracellular or membranous DPP3 has also been proposed as a potential marker of arterial risk (US2011008805) and a marker of rheumatoid arthritis (US2006177886). Patent application WO2005106486 claims protection for DPP3 expression and activity as diagnostic markers and DPP3 as a therapeutic target in all kinds of diseases due to its widespread expression intracellularly or on the cell surface. EP1498480 mentions the potential diagnostic and therapeutic uses of hydrolases, including DPP3.

[0012] DPP3 has been proposed not only as a potential biomarker but also as a potential therapeutic target due to its ability to cleave several bioactive peptides. Overexpression of DPP3 protects neuroblastoma cells from oxidative stress (Liu et al., 2007). Influenza A virus alters host DPP3 levels for self-replication (cell culture studies, Meliopoulos et al., 2012). General enkephalins and / or angiotensin-degrading enzymes, including DPP3, have therapeutic potential as targets for pain, cardiovascular disease (CVD), and cancer, and corresponding inhibitors have potential therapeutic applications for pain, mental illness, and CVD (Khaket et al., 2012; Patel et al., 1993; Igic et al., 2007).

[0013] Although DPP3 is referred to as an intracellular protein, its activity has been detected in several bodily fluids: postplacental serum (Shimamori et al., 1986), seminal plasma (Vanha-Perttula). (Aoyagi et al., 1988) and CSF (Aoyagi et al., 1993). In CSF, elevated DPP3 activity levels were measured in patients with Alzheimer's disease (AD, Aoyagi et al., 1993). Wattiaux et al. (2007) proposed intracellular DPP3 release as a marker of dead and / or dying cells in cell culture systems. It has also been proposed that DPP3 release from necrotic cells affects immune responses in mouse models (Gamrekelashvili et al., 2013). Summary of the Invention

[0014] The object of the present invention is to provide a method for specifically identifying active DPP3 in a body fluid sample, that is, identifying active DPP3 but not any other aminopeptidases other than DPP3.

[0015] One object of the present invention is to provide corresponding assays and kits.

[0016] Another object of the present invention is to provide a method for diagnosing a disease or condition in an object with or related to a necrosis process, and a method for treating said disease.

[0017] The subject of this invention is a method for determining active DPP3 in a bodily fluid sample of a subject, comprising the following steps: • Contact the sample with a trapping binder that specifically binds to full-length DPP3. • Separate DPP3 bound to the capturing binder. • Add a substrate of DPP3 to the separated DPP3. • DPP3 activity was quantified by measuring the conversion rate of DPP3 substrates.

[0018] In one specific embodiment of the invention, the method is an enzyme capture assay (ECA, see, for example, US5612186A, US5601986A).

[0019] All definitions and specific embodiments specified in this specification shall apply to all aspects and objects of the invention. It should be understood that for one aspect or object of the invention, a detailed and general definition and specific embodiment shall also be a definition and specific embodiment of other aspects and objects of the invention. For example, the definition of a capture binder or specific capture may apply to all embodiments of the invention: for example, methods for determining DPP3, assays and kits, diagnostic methods, and treatment methods. Such definitions may not be repeated throughout the specification.

[0020] Specific binding to full-length DPP3 means that the capture binder does not bind to any other proteins besides DPP3.

[0021] The binder that binds to full-length DPP3 is the same binder that binds to the protein in SEQ ID No. 1.

[0022] In one specific embodiment, the capturing binder inhibits DPP3 activity by less than 50%, preferably less than 40%, and most preferably less than 30% in a liquid phase assay. The liquid phase assay is a determination of the cleavage of the substrate by DPP3 occurring in the liquid phase.

[0023] According to the present invention, the inhibition of DPP3 activity by a binding agent in a liquid phase assay can be determined as follows: in the liquid phase assay, a possible DPP3 capturing binding agent is incubated with recombinant or purified natural DPP3 and a specific DPP3 substrate. Preferably, the capturing binding agent with minimal inhibitory ability is selected as the ECA. The inhibition of DPP3 activity by the capturing binding agent should be less than 50%, preferably less than 40%, and more preferably less than 30%. The specific liquid phase DPP3 activity assay for determining the inhibitory ability of a possible capturing binding agent is described in detail in Example 1 and includes the following steps: • Incubate 25 ng / ml recombinant GST-hDPP3 with 5 μg / ml of each capture binder and buffer control in 50 mM Tris-HCl, pH 7.5 and 100 μM ZnCl2 at room temperature for 1 hour. • Add the fluorescent substrate Arg-Arg-βNA (20 μl, 2 mM). • The generation of free βNA was monitored in a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH) after incubation at 37°C for 1 hour. The fluorescence of βNA was detected by excitation at 340 nm and measurement of emission at 410 nm. • Calculate the slope of fluorescence increase for different samples (in RFU / min). The slope for the buffer control of GST-hDPP3 is designated as 100% activity. The inhibitory effect of the possible capture binder is defined as the percentage decrease in GST-hDPP3 activity resulting from incubation with the capture binder.

[0024] In contrast, solid-phase determination is the determination in which the corresponding binding events occur in the solid phase (see Examples 4 and 5).

[0025] For clarity, the method used to determine the activity of DPP3 can be performed as both liquid-phase and solid-phase assays. However, according to the procedure described above, the inhibition of DPP3 activity can be determined in a liquid-phase assay.

[0026] Therefore, solid-phase assays are an embodiment of the present invention. Contacting the sample with a trapping binder that specifically binds full-length DPP3 can occur in the liquid phase (liquid-phase trapping assay), and the separation step can then include immobilizing the trapping binder-DPP3 complex. Alternatively, the trapping binder can be immobilized on a surface, and the binding event—the trapping binder and DPP3—can occur on the solid phase (solid-phase trapping assay).

[0027] In one specific embodiment, to prevent complete inhibition of DPP3 and to reduce the inhibition of DPP3 activity in the aforementioned liquid chromatography by less than 50%, preferably less than 40%, and more preferably less than 30%, the capture binder should preferably not bind to DPP3 in the region of amino acids 316 to 669 of SEQ ID No. 1 or in the region leading to said region. The region of amino acids 316 to 669 of SEQ ID No. 1 includes the active site and substrate-binding region of DPP3 (Prajapati & Chauhan, 2011; Kumar et al., 2016).

[0028] DPP3 activity can be measured by detecting the cleavage products of DPP3-specific substrates.

[0029] Known peptide hormone substrates include angiotensin II, III and IV, leucine enkephalin, methionine enkephalin, endorphin 1 and 2, valorphin, β-tyrophage, dynorphin, gastreptide, ACTH (adrenocorticotropic hormone), and MSH (melanocyte-stimulating hormone). et al., 2000; (Dhanda et al., 2007; Dhanda et al., 2008). The cleavage of the mentioned 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., ...). (e.g., Vandenberg et al., 2000), 1H-NMR analysis (e.g., Vandenberg et al., 1985), capillary zone electrophoresis (CE; e.g., (e.g., Dhanda et al., 2007), thin-layer chromatography (e.g., Dhanda et al., 2008), or reversed-phase chromatography (e.g., Mazocco et al., 2006).

[0030] Detecting fluorescence induced by the hydrolysis of fluorescent substrates by DPP3 is a standard procedure for monitoring DPP3 activity. These substrates are specific dipeptides or tripeptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) conjugated to fluorophores. Fluoresceins 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). Cleavage of these fluorescent substrates results in the release of fluorescent β-naphthylamine or 7-amino-4-methylcoumarin, respectively. Fluorescence can be measured in liquid chromatography, or, for example, in 96-well plates incubated with ECA substrates and DPP3, and using a fluorescence detector (Ellis & Nuenke, 1967). Alternatively, DPP3 carrying the sample can be immobilized and separated on a gel by electrophoresis, the gel stained with a fluorescent substrate (e.g., Arg-Arg-βNA) and Fast Garnet GBC, and fluorescent protein bands detected by a fluorescence reader (Ohkubo et al., 1999).

[0031] The same peptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) can be coupled to chromophores such as p-nitroaniline diacetate. The detection of color changes caused by hydrolysis of the chromogenic substrate can be used to monitor DPP3 activity.

[0032] Another option for detecting DPP3 activity is the protease-Glo TM Assay (available commercially from Promega). In this embodiment of the method, a DPP3-specific dipeptide or tripeptide (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) is conjugated with luciferin. Upon cleavage by DPP3, luciferin is released and used as a substrate for a luciferase-coupled reaction, which emits detectable luminescence.

[0033] In a preferred embodiment, DPP3 activity is measured by adding the fluorescent substrate Arg-Arg-βNA and monitoring the fluorescence in real time.

[0034] In one embodiment of the method for determining the inhibitory effect of a DPP3 binder and / or the DPP3 activity in a body fluid sample of a subject, the binder may be selected from the group consisting of antibodies, antibody fragments, or non-IgG scaffolds.

[0035] The antibody according to the invention is a protein comprising one or more polypeptides encoded substantially by immunoglobulin genes that specifically bind to antigens. Recognized immunoglobulin genes include constant region genes for κ, λ, α (IgA), γ (IgG1, IgG2, IgG3, IgG4), δ (IgD), ε (IgE), and μ (IgM), as well as numerous variable region genes for immunoglobulins. The full-length immunoglobulin light chain is typically about 25 kDa or 214 amino acids long. The full-length immunoglobulin heavy chain is typically about 50 kDa or 446 amino acids long. The light chain is encoded by a variable region gene (about 110 amino acids long) at the NH2- terminus and a κ or λ constant region gene at the COOH- terminus. The heavy chain is similarly encoded by a variable region gene (about 116 amino acids long) and one of the other constant region genes.

[0036] The basic structural unit of an antibody is typically a tetramer, consisting of two pairs of identical immunoglobulin chains, each pair containing one light chain and one heavy chain. In each pair, the variable regions of the light and heavy chains bind to the antigen, while the constant regions mediate effector function. Immunoglobulins also exist in a variety of other forms, including, for example, Fv, Fab, and (Fab')2, as well as bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al., 1987; Huston et al., 1988; Bird et al., 1988; Hood et al., 1984; Hunkapiller & Hood, 1986). The variable regions of the immunoglobulin light or heavy chains comprise a framework region interrupted by three hypervariable regions (also known as complementarity-determining regions (CDRs)) (see, Kabat et al., 1983). As mentioned above, the CDRs are primarily responsible for binding to epitopes of the antigen. Immune complexes are antibodies that specifically bind to antigens, such as monoclonal antibodies, chimeric antibodies, humanized antibodies, or human antibodies, or functional antibody fragments.

[0037] Chimeric antibodies are antibodies that are typically constructed by genetic engineering from the variable and constant regions of immunoglobulins belonging to different species, creating light and heavy chain genes. For example, a variable region from a mouse monoclonal antibody gene can be linked to a human constant region such as κ and γ1 or γ3. In one instance, therefore, a therapeutic chimeric antibody is a hybrid protein consisting of a variable or antigen-binding domain from a mouse antibody and a constant or effector domain from a human antibody, but other mammalian species may also be used, or the variable region may be produced by molecular techniques. Methods for preparing chimeric antibodies are well known in the art; see, for example, U.S. Patent No. 5,807,715. A “humanized” immunoglobulin is an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDR is referred to as the “donor,” and the human immunoglobulin providing the framework is referred to as the “recipient.” In one embodiment, all CDRs are derived from the donor immunoglobulin within the humanized immunoglobulin. A constant region is not required, but if it exists, it must be substantially identical to the constant region of human immunoglobulins, i.e., at least about 85% to 90%, such as about 95% or more. Therefore, except for possible CDRs, all portions of a humanized immunoglobulin are substantially identical to the corresponding portions of the native human immunoglobulin sequence. A “humanized antibody” is an antibody containing humanized light chains and humanized heavy chains of immunoglobulins. Humanized antibodies bind to the same antigen as the donor antibody that provides the CDR. The receptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions made by amino acids taken from the donor framework. Humanized or other monoclonal antibodies may have additional conserved amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin function. Exemplary conserved substitutions are those such as gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed through genetic engineering (see, for example, U.S. Patent No. 5,585,089). Human antibodies are antibodies in which the light and heavy chain genes are of human origin. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells that secrete the target antibody. Immortification can be achieved, for example, by EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce trivalent hybridoma cells. Human antibodies can also be produced by phage display methods (see, for example, PCT Publication No. WO91 / 17271; PCT Publication No. WO92 / 001047; PCT Publication No. WO92 / 20791, which are incorporated herein by reference) or selected from human combinatorial monoclonal antibody libraries (see Morphosys website).Human antibodies can also be prepared by using transgenic animals carrying human immunoglobulin genes (e.g., see PCT Publication No. WO93 / 12227; PCT Publication No. WO91 / 10741, which are incorporated herein by reference).

[0038] Therefore, DPP3 antibodies can take the forms known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, and CDR-grafted antibodies. In a preferred embodiment, the antibody according to the invention is a recombinant antibody, such as IgG, a typical full-length immunoglobulin, or an antibody fragment containing at least a heavy chain and / or a light chain F-variable domain, such as a chemically conjugated antibody (fragment antigen binding), including but not limited to Fab fragments, including Fab microantibodies, single-chain Fab antibodies, monovalent Fab antibodies with epitope tags, such as Fab-V5SX2; divalent Fab (microantibodies) dimerized with a CH3 domain; divalent or multivalent Fab, for example formed by polymerization via heterodomains, such as by dimerization of the dHLX domain, such as Fab-DHLx-FSX2; F(ab')2-fragments, scFv-fragments, polymerized multivalent or / and multispecific scFv-fragments, divalent and / or bispecific biantibodies, (Bispecific T-cell conjugates), trifunctional antibodies, multivalent antibodies, such as those derived from classes other than G; single-domain antibodies, such as nanobodies derived from camel or fish immunoglobulins and many other immunoglobulins.

[0039] Besides anti-DPP3 antibodies, other biopolymer scaffolds are well known in the art for recombining target molecules and have been used to generate highly target-specific biopolymers. Examples include aptamers, specigelmers, anticalins, and conotoxins.

[0040] Non-Ig scaffolds can be protein scaffolds and can be used as antibody mimics because they are capable of binding to ligands or antigens. Non-Ig scaffolds can be selected from the group consisting of: tetraligin-based non-Ig scaffolds (e.g., described in US2010 / 0028995), fibronectin scaffolds (e.g., described in EP1266025); lipid transport protein-based scaffolds (e.g., described in WO2011 / 154420); ubiquitin scaffolds (e.g., described in WO2011 / 073214), transfer scaffolds (e.g., described in US2004 / 0023334), protein A scaffolds (e.g., described in EP2231860), ankyrin repeat-based scaffolds (e.g., described in WO2010 / 060748), microprotein (preferably, microprotein forming cystine knots) scaffolds (e.g., described in EP2314308), and Fyn-based scaffolds. Scaffolds can be SH3 domain-based (e.g., described in WO2011 / 023685), EGFR-A domain-based (e.g., described in WO2005 / 040229), or Kunitz domain-based (e.g., described in EP1941867). Non-Ig scaffolds can be peptides or oligonucleotide aptamers. Aptamers are typically generated by selecting from large random sequence libraries and are either short chains of oligonucleotides (DNA, RNA, or XNA; Xu et al., 2010; Deng et al., 2014) or short, variable peptide domains attached to a protein scaffold (Li et al., 2011).

[0041] In one embodiment of the present invention, the anti-DPP3 antibody according to the present invention can be produced as follows:

[0042] Recombinant DPP3 (e.g., GST-hDPP3 from USBio, Salem, USA), peptides containing a portion of the DPP3 amino acid sequence (e.g., conjugated with BSA), or naturally purified DPP3 (e.g., from human erythrocytes) can be used. (Etc., 1988) to immunize mice.

[0043] On day 0, Balb / c mice were intraperitoneally (ip) injected with 100 μg of recombinant GST-hDPP3, naturally purified hDPP3, or DPP3-peptide-BSA-conjugate (emulsified in TiterMax Gold adjuvant); on day 14, 100 μg (emulsified in complete Freund's adjuvant); and on days 21 and 28, 50 μg (incomplete Freund's adjuvant). On day 49, animals were intravenously (iv) injected with 50 μg of GST-hDPP3, naturally purified hDPP3, or DPP3-peptide-BSA-conjugate dissolved in saline. Mice were sacrificed three days later for immune cell fusion.

[0044] Spleen cells from immunized mice and 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 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement]. After one week, the HAT medium was replaced with HT medium for three passages, and then the cells were returned to normal cell culture medium.

[0045] Two weeks after fusion, preliminary screening for recombinant DPP3-bound IgG antibodies was performed on the cell culture supernatant. Therefore, GST-tagged recombinant DPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng / well) and incubated at room temperature with 50 μl of cell culture supernatant / well (i.e., per well) for 2 hours. After washing the plates, 50 μl / well of POD-rabbit anti-mouse IgG was added, and the plates were incubated at room temperature for 1 hour. After the next washing step, 50 μl of chromogen solution (3.7 mM o-phenylenediamine in citrate / hydrophosphate buffer, 0.012% H2O2) was added to each well, and the plate was incubated at room temperature for 15 minutes. The colorimetric reaction was terminated by adding 50 μl of 4N sulfuric acid. Absorption was detected at 490 nm.

[0046] Microcultures that tested positive were transferred to 24-well plates for propagation. After retesting, selected cultures were cloned and recloned using restrictive dilution techniques to determine isotypes.

[0047] Antibodies against human DPP3 or DPP3-peptide tagged with GST were produced using a standard antibody production method (Marx et al., 1997) and purified using protein A. Antibody purity was ≥90% based on SDS-PAGE analysis.

[0048] Antibodies can be produced using phage display according to the following procedure:

[0049] The human natural antibody gene library HAL7 / 8 was used to isolate recombinant single-chain F-variable domains (scFv) targeting the DPP3 peptide. The antibody gene library was screened using a panning strategy that included peptides containing biotinylated tags linked to the DPP3 peptide sequence via two distinct spacer regions. A mixed panning wheel using both non-specifically binding antigens and streptavidin-binding antigens was used to minimize the background of non-specific binding agents. Phages eluted from the third round of panning were used to generate *E. coli* strains expressing monoclonal scFv. Supernatants from cultures of these cloned strains were used directly for antigen ELISA assays (see also Hust et al., 2011; Schütte et al., 2009).

[0050] The humanization of mouse antibodies can be performed according to the following procedure:

[0051] For the humanization of murine antibodies, the antibody sequence is analyzed to target the structural interactions between the frame region (FR), complementarity-determining region (CDR), and antigen. Based on structural modeling, an appropriate human FR is selected, and the murine CDR sequence is transplanted into the human FR. Amino acid sequence changes in the CDR or FR can be introduced to restore structural interactions that are eliminated by species switching of the FR sequence. This restoration of structural interactions can be achieved using a randomized method with a phage display library or a directed method guided by molecular modeling. (Almagro & Fransson, 2008).

[0052] In an alternative embodiment, the DPP3 antibody form is selected from the group consisting of Fv fragments, scFv fragments, Fab fragments, scFab fragments, F(ab)2 fragments, and scFv-Fc fusion proteins. In another preferred embodiment, the antibody form is selected from the group consisting of scFab fragments, Fab fragments, scFv fragments, and bioavailability-optimized conjugates thereof, such as PEGylated fragments.

[0053] In one specific embodiment of the method for determining the activity of DPP3 in a body fluid sample of a subject, the binding agent is an antibody.

[0054] In one embodiment, a capture assay or binding assay can be performed to detect and / or quantify DPP3. A binding agent that reacts with the DPP3 protein but interferes with peptidase activity by no more than 50%, preferably less than 40%, and most preferably less than 30% in liquid chromatography can be immobilized on a solid phase. In one embodiment, to prevent inhibition of DPP3, the capture binding agent should preferably not bind to DPP3 in the amino acid region from amino acid positions 316 to 669 of SEQ ID No. 1. The amino acid region from amino acid positions 316 to 669 of SEQ ID No. 1 includes the active site and substrate-binding region of DPP3 (Prajapati & Chauhan, 2011; Kumar et al., 2016).

[0055] In one specific embodiment of the method for determining the activity of DPP3 in a body fluid sample of a subject, the binding agent may be selected from the group consisting of antibodies, antibody fragments, non-Ig scaffolds, or aptamers.

[0056] In one specific embodiment of the method for determining the activity of DPP3 in a body fluid sample of a subject, the capture binder that reacts with DPP3 is immobilized on a solid phase.

[0057] The test sample is passed through a fixed binder, and DPP3 (if present) binds to the binder and is itself immobilized for detection. A substrate can then be added, and the reaction product can be detected to indicate the presence or amount of DPP3 in the test sample. For the purposes of this specification, the term "solid phase" may be used to include any material or vessel in which or on which the determination can be performed, including but not limited to: porous materials, non-porous materials, test tubes, pores, glass slides, agarose resins (e.g., Sepharose from GE Healthcare Life Sciences), magnetic particles (e.g., Dynabeads from Thermo Fisher Scientific). TM Or Pierce TM Magnetic beads, etc.

[0058] Protein or peptide-derived binders (e.g., antibodies, antibody fragments, non-Ig scaffolds) can be immobilized on a solid phase using methods including: physical adsorption (e.g., via electrostatic or hydrophobic interactions), bioaffinity immobilization (e.g., avidin-biotin, protein A / G / L, His-tag, and Ni...). 2+-NTA, GST-tags and glutathione, DNA hybridization, aptamers), covalent bonds (e.g., amines and N-hydroxysuccinimide) or combinations of the aforementioned immobilization methods (Kim & Herr, 2013). Oligonucleotide-derived binders (e.g., aptamers) can be immobilized on a solid phase using the (streptomycin) avidin-biotin system (Müller et al., 2012; Deng et al., 2014).

[0059] In one specific embodiment of the method for identifying active DPP3 in a bodily fluid sample of a subject, the separation step is a washing step that removes sample components not bound to the capture binder from the captured DPP3. This separation step can be any other step that separates the DPP3 bound to the capture binder from the components of the bodily fluid sample.

[0060] In one specific embodiment of the method for determining the activity of DPP3 in a bodily fluid sample of a subject, the DPP3 substrate conversion rate of the immobilized DPP3 is measured (detected) by methods selected from: fluorescence of a fluorescent substrate (e.g., Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, and substrate coupled with aminofluorescein (Promega protease-Glo). TM The determination of the protein can be performed by luminescence, mass spectrometry, HPLC / FPLC (reversed-phase chromatography, size exclusion chromatography), thin-layer chromatography, capillary zone electrophoresis, gel electrophoresis followed by active staining (immobilization, active DPP3) or Western blotting (cleavage products).

[0061] In one specific embodiment of the method for determining the activity of DPP3 in a body fluid sample, the substrate may be selected from: angiotensin II, angiotensin III and angiotensin IV, leucine enkephalin, methionine enkephalin, endorphin 1 and endorphin 2, indomethacin, β-tyrophage, dynorphin, gastreptide, ACTH and MSH, or dipeptides and tripeptides conjugated with a fluorophore, chromophore or aminofluorescein (Promega Protease-Glo). TM (Assay). Dipeptides or tripeptides cleaved by DPP3 include, but are not limited to, Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, and Suc-Ala-Ala-Phe. Fluorophores include, but are not limited to, β-naphthylamide (2-naphthylamide, βNA, 2NA), 4-methoxy-β-naphthylamide (4-methoxy-2-naphthylamide), and 7-amino-4-methylcoumarin (AMC, MCA). Cleavage of these fluorescent substrates leads to the release of fluorescent β-naphthylamine or 7-amino-4-methylcoumarin, respectively. Chromophores include, but are not limited to, p-nitroaniline diacetate (pNA). Hydrolysis of the peptide-pNA bond in the chromogenic substrate results in the release of pNA, which in turn changes color. Therefore, the change in absorbance (DA / min) is directly proportional to enzyme activity. Protease-Glo from Promega was used. TM The assay showed that, upon cleavage by DPP3, aminoluciferin was released and used as a substrate for a luminescent coupled luciferase reaction.

[0062] In a preferred embodiment, DPP3 activity is measured by adding a fluorescent substrate, Arg-Arg-βNA, and monitoring fluorescence in real time. In one specific embodiment of the method for determining the activity of DPP3 in a body fluid sample, the sample is selected from whole blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusion.

[0063] In one specific embodiment of the method for determining active DPP3 in a body fluid sample, the sample is a blood sample selected from whole blood, serum, and plasma.

[0064] Another embodiment of the present invention is a assay or kit for determining the activity of DPP3 in a body fluid sample of a subject, comprising: • A capture binder that specifically binds to full-length DPP3. • Substrate of DPP3.

[0065] The kit may additionally include calibrators: • The calibrator can be a sample (natural, purified or recombinant) with a known DPP3 concentration; • The calibrator can be the lysis product itself, such as a free fluorophore (e.g., 2-naphthylamine), a free chromophore (e.g., p-nitroaniline), or a free fluorescein.

[0066] The kit may additionally include a washing reagent: • Detergent (can be any aqueous buffer solution with or without detergent. Here we use 8 mM Tris-HCl, pH 7.5, 60 mM NaCl, 0.02% Tween 20).

[0067] In one specific embodiment, the assay is an enzyme capture assay (ECA, such as US5612186A, US5601986A).

[0068] In one specific embodiment, the capture binder that binds to full-length DPP3 specifically inhibits less than 50% of DPP3 activity in a liquid chromatography assay, preferably less than 40%, more preferably less than 30%. For the definition of liquid chromatography, see above. In one specific embodiment to prevent DPP3 inhibition, the capture binder should not bind to DPP3 in the region surrounding the active site and substrate-binding region (amino acids 316 to 669 of SEQ ID No. 1).

[0069] In one specific embodiment of the assay or kit for determining the activity of DPP3 in a body fluid sample, the binder may be selected from an antibody, an antibody fragment, or a non-Ig scaffold.

[0070] In one specific embodiment of the assay and kit for determining the activity of DPP3 in a body fluid sample, the binding agent is an antibody.

[0071] The term "antibody" generally includes monoclonal and polyclonal antibodies and their binding fragments, particularly Fc fragments, as well as so-called "single-chain antibodies" (Bird et al., 1988), chimeric antibodies, humanized antibodies, particularly CDR-grafted antibodies, and tri- or tetra-antibodies (Holliger et al., 1993). It also includes immunoglobulin-like proteins selected by techniques including, for example, phage display, to specifically bind to a target molecule contained in a sample. In this document, the term "specific binding" refers to an antibody produced against a target molecule or a fragment thereof. An antibody is considered specific if its affinity for the target molecule or its aforementioned fragment is preferably at least 50 times, more preferably 100 times, and most preferably at least 1000 times, greater than the affinity for other molecules contained in a sample containing the target molecule. It is well known in the art how antibodies are prepared and how antibodies with a given specificity are selected.

[0072] In one specific embodiment of the assay and kit for determining active DPP3 in a target bodily fluid sample, the capture binder is immobilized on a surface.

[0073] In one specific embodiment of the assay and kit for determining the activity of DPP3 in a target bodily fluid sample, the substrate may be selected from: angiotensin II, angiotensin III and angiotensin IV, leucine enkephalin, methionine enkephalin, endorphin 1 and endorphin 2, indomethacin, β-tyrophage, dynorphin, gastreptide, ACTH and MSH, or dipeptides and tripeptides conjugated with a fluorophore, chromophore or aminofluorescein (Promega Protease-Glo). TM(Assay). Dipeptides or tripeptides cleaved by DPP3 include, but are not limited to, Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, and Suc-Ala-Ala-Phe. Fluorophores include, but are not limited to, β-naphthylamide (2-naphthylamide, βNA, 2NA), 4-methoxy-β-naphthylamide (4-methoxy-2-naphthylamide), and 7-amino-4-methylcoumarin (AMC, MCA). (Ohkubo et al., 2000; Ohkubo et al., 1999). Cleavage of these fluorescent substrates leads to the release of fluorescent β-naphthylamine or 7-amino-4-methylcoumarin, respectively. Chromophores include, but are not limited to, p-nitroaniline diacetate (pNA). Hydrolysis of the peptide-pNA bond in the chromogenic substrate leads to the release of pNA, which in turn changes color. Therefore, the change in absorbance (DA / min) is proportional to the enzyme activity. Protease-Glo from Promega was used. TM The assay showed that, upon cleavage by DPP3, aminoluciferin was released and used as a substrate for a luminescent coupled luciferase reaction.

[0074] In a preferred embodiment, DPP3 activity is measured by adding the fluorescent substrate Arg-Arg-βNA and monitoring the fluorescence in real time.

[0075] In one specific embodiment of the assay and kit for determining the activity of DPP3 in a target bodily fluid sample, the calibrator is selected from: A) recombinant DPP3 (e.g., GST-hDPP3 from USBio), purified natural DPP3 (e.g., from human erythrocytes, Abrami...). A) or DPP3 fragments (natural, synthetic or recombinant); B) the cleavage product itself: free fluorophore (e.g., 2-naphthylamine), free chromophore (e.g., p-nitroaniline) or free fluorescein, for the quantification of fluorescence, color changes and bioluminescent signals.

[0076] One embodiment of the present invention using DPP3 as a diagnostic biomarker includes various forms of immunoassay, such as radioimmunoassay (RIA), chemiluminescence and fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), Luminex-based bead arrays, protein microarray assays, and rapid assays such as immunochromatographic strip assays.

[0077] The assay can be homogeneous or heterogeneous, competitive or non-competitive sandwich assay. In a particularly preferred embodiment using two antibodies according to the invention, the assay is in the form of a sandwich assay, which is a non-competitive immunoassay in which the DPP3 to be detected and / or quantified, or a fragment thereof, binds to a first antibody and a second antibody. The first antibody may bind to a solid, such as the surface of a bead, a well, or other container, a piece, or a strip, and the second antibody is a labeled antibody, such as an antibody labeled with a dye, a radioactive isotope, or a reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by a suitable method. General compositions and procedures involving “sandwich assays” are well-known and are known to those skilled in the art. (The Immunoassay Handbook, Ed. David Wild, 2005; Hultschig et al., 2006).

[0078] In a particularly preferred embodiment, the assay comprises a liquid reaction mixture wherein a first labeling component is linked to a first antibody, wherein the first labeling component is part of a labeling system based on fluorescence- or chemiluminescence-quenching or amplification, and a second labeling component of the labeling system is linked to a second antibody, such that after both antibodies bind to the analyte, a measurable signal is generated that allows the detection of the formed sandwich complex in a solution containing the sample.

[0079] In the context of this invention, fluorescence-based determinations include the use of dyes, which may be selected, for example, from the group consisting of: FAM (5-carboxyfluorescein 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-hexanediol. Chlorophyll (HEX), TET, 6-carboxy-4',5'-dichloro-2',7'-dicarboxyfluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes such as BODIPY TMR, Oregon Green, coumarins such as umbelliferone, benzoimides such as Hoechst 33258; phenanthridines such as Texas Red, Imidacloprid Yellow, Alexa Fluor, PET, phenanthridine bromored red, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethyl iodide dyes, etc.

[0080] In the context of this invention, chemiluminescence-based determinations involve the use of dyes, based on the physical principles described in Kirk-Othmer's *Encyclopedia of Chemical Technology*, 4th edition, Executive Editor J.I. Kroschwitz; Editor M. Howe-Grant, John Wiley & Sons, 1993, Vol. 15, pp. 518–562, which is incorporated herein by reference, including the citation on pages 551–562. A preferred chemiluminescent dye is an acridine ester.

[0081] One embodiment of the present invention relates to the chemical determination of DPP3. The determination uses an enzyme substrate that reacts with DPP3 to form a detectable reaction product. Alternatively, the reaction rate of the substrate can be monitored to determine the presence or amount of DPP3 in the test sample. Suitable enzyme substrates include, but are not limited to, dipeptide substrates such as Arg-Arg-β-NA or Arg-Arg-AMC.

[0082] The determination of such reagents and reactions can be performed in any suitable reaction vessel, such as test tubes or wells of a microtiter plate. Alternatively, disposable assay devices, such as dipsticks or strips, can be developed, which are well known to those skilled in the art and are easy to manufacture and use. Such disposable assay devices can be packaged as kits containing all necessary materials, reagents, and instructions for use.

[0083] The measuring device of the present invention can be advantageously designed as a test strip or test piece device. For example, the test strip can be made of a piece of absorbent material containing a chromogenic substrate for DPP3. Alternatively, the test strip can be made of a non-porous material coated thereon with the substrate. After the device is brought into contact with the desired test sample, the substrate and any DPP3 present in the sample will interact to form a detectable reaction on the device.

[0084] In an alternative embodiment, the device may be a test strip in which the substrate is contained in one or more zones along the length of the absorbent material strip. When one end of the absorbent material strip comes into contact with the desired test sample, the liquid sample migrates along the absorbent material. The reaction of the substrate and the generation of a detectable signal indicate the presence of DPP3 in the test sample. In a multi-zone device, the number of discrete or isolated zones along the length of the strip that generate a detectable signal can also indicate the amount of DPP3 present in the test sample. Alternatively, the majority of the test strip may contain the substrate. The length of the coloring reaction formed in a test strip having such a single, elongated substrate zone can be used to indicate the presence or amount of DPP3 in the test sample.

[0085] In alternative assay embodiments, the rate of reaction can be detected as an indicator of the amount of DPP3 present in the test sample. For example, the rate of substrate reaction can be used to indicate the amount of DPP3 present in the test sample. Alternatively, the rate of formation of reaction products can be used to indicate the amount of DPP3 present in the test sample.

[0086] In yet another embodiment, a capture assay or binding assay can be performed to detect and / or quantify the protease. For example, an antibody that reacts with the DPP3 protein but does not interfere with peptidase activity can be immobilized on a solid phase. The test sample is passed through the immobilized antibody, and DPP3 (if present) binds to the antibody and is itself immobilized for detection. A substrate can then be added, and the reaction product can be detected to indicate the presence or amount of DPP3 in the test sample. For the purposes of this specification, the term "solid phase" can be used to include any material or vessel in which or on which the assay can be performed, and includes, but is not limited to, porous materials, non-porous materials, test tubes, pores, glass slides, etc.

[0087] In another specific embodiment, DPP3 ECA can be performed as a test strip assay. In an exemplary test strip device, a test sample application pad is optionally attached to one end of a multi-well strip. The strip contains immobilized antibodies that bind to DPP3 and thus immobilize DPP3 at predetermined sites for subsequent detection. Optionally, the device may include a assay end indicator located at the distal end of the test strip, away from the test sample contact site. The assay end indicator generates a detectable signal upon contact with the test sample or assay reagent, thereby indicating the completion of the assay.

[0088] The test sample application pad can be part of the porous strip itself or a material that makes fluid flow contact with the ends (referred to as the proximal end) of the porous strip, allowing the test sample to pass through or migrate from the application pad to the porous strip. Fluid flow contact can include physical contact between the application pad and the porous strip, as well as separation of the application pad from the porous strip by an intervention space or additional material, which still allows fluid to flow between the application pad and the porous strip. Essentially all of the application pad can overlap the porous strip, allowing the test sample to reach the proximal end of the porous strip substantially through any portion of the application pad. Alternatively, only a portion of the application pad may make fluid flow contact with the porous strip. The application pad can be any material capable of transferring the test sample to the porous strip.

[0089] The porous strip of the measuring device can be any suitable absorbent, porous, water-absorbing, chromatographically processed material or capillary-processed material through which the test sample containing the analyte can be transported via capillary or wicking action. Synthetically modified natural, synthetic, or naturally occurring materials can be used as porous strips, including but not limited to: cellulose materials such as paper, cellulose and cellulose derivatives such as cellulose acetate and cellulose nitrate; glass fibers; fabrics, both naturally occurring (e.g., cotton) and synthetic (e.g., nylon); porous gels such as silica gel, agarose, dextran, and gelatin; porous fiber matrices; starch-based materials such as cross-linked dextran chains; ceramic materials; polyvinyl chloride membranes; and combinations of polyvinyl chloride and silica, etc. The porous strip should not interfere with the generation of a detectable signal. The porous strip should have reasonable inherent strength, or strength can be provided by supplementary support.

[0090] The specific size of the porous strip will be convenient, depending on the size of the test sample involved, the measurement protocol, the tools used to detect and measure the signal, etc. For example, the size can be selected to adjust the rate of fluid migration and the amount of test sample absorbed by the porous strip.

[0091] In one possible test strip device of the present invention, DPP3 substrate and / or DPP3 capturing antibody can be immobilized on a porous strip to form at least one analyte detection site, i.e., a porous strip region having one or more analytical reagents non-diffusively attached thereto. In another device embodiment, the measurement or detection region of the test strip may include multiple sites containing DPP3 substrate and / or immobilized anti-DPP3 antibody. Optionally, different detection sites may contain different amounts of substrate and / or immobilized anti-DPP3 antibody, i.e., a higher amount in the first detection site and a lower amount in subsequent sites. For example, if 20 nanograms of antibody captures 1 nmol / min / ml of DPP3 equivalent, then the first detection site of the assay device may contain 50 nanograms of anti-DPP3 antibody, while subsequent sites may contain 10 nanograms, 20 nanograms, 30 nanograms, etc. of antibody. After the test sample is added, the number of sites displaying a detectable signal provides a quantitative indication of the amount of DPP3 present in the sample. The detection sites can be constructed into any suitable detectable shape and are typically stripes spanning the width of the test strip.

[0092] Optionally, a multi-capture-site device can be fabricated such that if a threshold amount of DPP3 is absent in the test sample, virtually all DPP3 will bind to and be immobilized at the first capture site. If a greater than threshold amount of DPP3 is present in the test sample, the remaining DPP3 will bind to subsequent detection regions of the immobilized antibody along the length of the test strip. The greater the amount of DPP3 in the test sample, the greater the number of capture sites that will display a detectable signal due to the presence of DPP3. As those skilled in the art will understand, devices comprising multiple DPP3 substrate sites can also be fabricated, wherein the amount of substrate in each site is designed to produce quantitative or semi-quantitative assay results.

[0093] Another important embodiment of the present invention is a method for diagnosing a disease or condition in an object accompanied by or related to a necrosis process, comprising: • Determine the total amount of DPP3 or the amount of active DPP3 in the body fluid sample of the subject. • Compare the determined quantity with a predetermined threshold. • If the determined amount is higher than the predetermined threshold, the object is diagnosed as having a disease or condition accompanied by or related to the necrosis process.

[0094] In one specific embodiment of the method for diagnosing a disease or condition in an object with or related to a necrosis process, the amount of total DPP3 or the amount of active DPP3 is determined in units of concentration.

[0095] Methods for determining the amount of total DPP3 or active DPP3 are known in the art. In the context of the method according to the invention for diagnosing a disease or condition of an object with or related to a necrotic process, existing methods and assays may be used, or the methods and assays described above for determining DPP3 may be used.

[0096] A threshold is predetermined by measuring the DPP3 concentration and / or DPP3 activity in healthy controls and calculating, for example, the corresponding 75th percentile, more preferably the 90th percentile, and even more preferably the 95th percentile. The upper boundary of the 75th percentile, more preferably the 90th percentile, and even more preferably the 95th percentile defines the threshold for healthy patients relative to diseased patients. Regarding the percentile, the threshold for dividing healthy and diseased patients using a sandwich anti-DPP3 immunoassay in plasma can be between 5 ng / ml and 25 ng / ml, more preferably 7 ng / ml and 20 ng / ml, more preferably 8 ng / ml and 18 ng / ml, and most preferably between 10 ng / ml and 15 ng / ml (see Example 3). In the DPP3-specific enzyme capture activity assay in plasma, the threshold for dividing healthy and diseased patients can be between 0.5 and 2 nmol βNA min. -1 ml -1 More preferably 0.7 to 1.8 nmol βNAmin -1 ml -1 More preferably 0.8 to 1.5 nmol βNA min -1 ml -1 The optimal value is 1.0 to 1.3 nmol βNA min. -1 ml -1 Between (see Example 5).

[0097] Those skilled in the art know how to determine thresholds from previously conducted studies. Those skilled in the art know that a particular threshold may depend on a group used to calculate a predetermined threshold, which may subsequently be used in routine procedures. Those skilled in the art know that a particular threshold may depend on the calibration used in the determination. Those skilled in the art know that a particular threshold may depend on what appears to be acceptable sensitivity and / or specificity to the practitioner.

[0098] The sensitivity and specificity of diagnostic tests depend not only on the "quality" of the test analysis, but also on the definition of what constitutes an anomalous result. In practice, receiver operating characteristic (ROC) curves are typically calculated by plotting the variable values ​​against their associated frequencies in a "normal" group (i.e., seemingly healthy) and a "diseased" group (i.e., patients experiencing an infection). Depending on the specific diagnostic problem being addressed, the reference group does not necessarily have to be "normal," but may be a group of patients experiencing another disease or condition from which the target disease group is distinguished. For any given marker, the marker level distributions of subjects with or without disease may overlap. In this case, the test cannot absolutely distinguish between normal and disease with 100% accuracy, and the overlapping area indicates that the test cannot distinguish between the normal and diseased portions. A threshold is chosen, above which (or below which, depending on how the marker changes with disease) the test is considered anomalous, and below which the test is considered normal. The area under the ROC curve is a measure of the probability that the perceived measurement correctly identifies the condition. ROC curves can be used even when test results do not necessarily give a precise number. ROC curves can be created as long as the results can be sorted. For example, test results for “illness” samples can be ranked according to severity (e.g., 1 = low, 2 = normal, and 3 = high). This ranking can be correlated with results in the “normal” population, creating an ROC curve. These methods are well known in the art (see, for example, Hanley et al., 1982). Preferably, a threshold is selected to provide an ROC curve area greater than about 0.5, more preferably greater than about 0.7. In this context, the term “about” means + / - 5% for a given measurement.

[0099] Once a threshold is determined by using previous research groups and considering all the above points, the physician will use the predetermined threshold of the method for diagnosing diseases according to the invention and will determine whether the subject has a value higher or lower than the predetermined threshold in order to make an appropriate diagnosis.

[0100] A variety of commercially available DPP3 ELISA kits (e.g., from LifeSpan BioSciences) can be used to measure DPP3 concentrations in tissue homogenates and body fluids. These assays are based on the sandwich assay principle and are for research use only.

[0101] The standard procedure for measuring DPP3 levels is to determine DPP3 activity using a fluorescent substrate (e.g., Arg-Arg-β-naphthamide) in a liquid chromatography assay (Ellis & Nuenke, 1967). Commercially available kits (e.g., from BPS Bioscience) typically contain a low-binding black microtiter plate, recombinant DPP3, the fluorescent substrate, and the corresponding buffer. These kits are frequently used for screening DPP3 substrates and inhibitors.

[0102] In one specific embodiment of the method for diagnosing a disease or condition in an object with or related to a necrotic process, the sample is selected from whole blood, serum, and plasma. In the context of the method of the invention, bodily fluids may also be selected from blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusion.

[0103] The necrosis process in this article is defined by all processes in vivo that lead to cell death and the release of DPP3 from the cytoplasm into the extracellular space and / or body fluids. These processes include, but are not limited to, necrosis, apoptosis, necroptotic apoptosis, and erythrocyte death.

[0104] In one specific embodiment of the method for diagnosing a disease or condition in a subject with or related to a necrosis process, the disease is selected from: heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infection (microorganisms, viruses (e.g., AIDS), parasites (e.g., malaria)) or SIRS or sepsis, cancer, acute kidney injury (AKI), central nervous system (CNS) disorders (e.g., epilepsy, neurodegenerative diseases), autoimmune diseases, vascular diseases (e.g., Kawasaki syndrome), and hypotension. Table 1 lists the clinical symptoms / diseases and corresponding necrosis events with or related to a necrosis process.

[0105] In another embodiment, the disease is selected from: acute heart failure (AHF), myocardial infarction (MI), liver failure, burns, severe infection (microorganisms, viruses (e.g., AIDS), parasites (e.g., malaria)) or SIRS or sepsis, cancer, and acute kidney injury (AKI).

[0106] In one implementation, the disease is low blood pressure.

[0107] Table 1: Diseases with or related to necrosis.

[0108] Apoptosis is a process of programmed cell death (PCD) that can occur in multicellular organisms. Biochemical events lead to characteristic cellular changes (morphological changes) and death. These changes include blistering, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. See Elmore, 2007 for commentary.

[0109] Necrosis is a form of cellular damage that leads to premature death of cells in living tissue through autolysis (Vanlangenakker et al., 2008). Necrosis is caused by factors outside the cells or tissues (e.g., infection, toxins, or trauma) that result in the unregulated digestion of cellular components.

[0110] While apoptosis usually has beneficial effects on the body, necrosis is almost always harmful and can be fatal.

[0111] Cell death caused by necrosis does not follow the apoptosis signaling pathway. Instead, various receptors are activated, leading to loss of cell membrane integrity and the uncontrolled release of cell death products into the extracellular space. This triggers an inflammatory response in the surrounding tissue, preventing nearby phagocytes from locating and eliminating dead cells through phagocytosis. For this reason, surgical removal of necrotic tissue is usually required; this is known as debridement. Untreated necrosis leads to the accumulation of decomposed dead tissue and cellular debris at or near the site of cell death.

[0112] A form of programmed necrosis known as necroptosis has been recognized as another form of programmed cell death. Necrotosis can serve as a backup for apoptosis when apoptotic signals are blocked by endogenous factors or exogenous factors such as viruses or mutations (Linkermann et al., 2014). Recently, other types of regulatory necrosis have also been discovered, which share multiple signaling events with necroptosis and apoptosis (Vanden Berghe et al., 2014).

[0113] Heart failure (HF) is a heart condition that occurs when a structural or functional problem with the heart impairs its ability to provide adequate blood flow to meet the body's needs. It can cause a variety of symptoms, particularly shortness of breath (SOB) during rest or exercise, signs of fluid retention (e.g., lung congestion or ankle swelling), and objective evidence of structural or functional abnormalities of the heart at rest.

[0114] Heart failure is a clinical syndrome characterized by a metabolite of symptoms and signs resulting from cardiac dysfunction. It is a leading cause of morbidity and mortality in developed countries, with a prevalence of 1% to 2%. Heart failure can be classified as chronic HF or acute HF. Patients with chronic HF can be further classified as stable chronic HF, worsening signs and symptoms of chronic HF, and acute decompensation of chronic HF. Acute heart failure (AHF) is defined as a rapid onset of signs and symptoms of heart failure requiring urgent treatment or hospitalization. AHF can present as acute neo-HF (new onset of AHF in patients without prior known heart failure) or acute decompensation of chronic AHF. AHF is a leading cause of hospitalization in adults over 65 years of age. Although the prognosis for patients with chronic heart failure, primarily associated with treatment progression, has improved significantly over the past decades, short-term and long-term outcomes remain very poor once a patient is hospitalized for decompensated heart failure. Nearly 25% of patients hospitalized for AHF within 30 days of initial hospitalization require readmission, and survival rates for patients hospitalized more than 5 years after admission are <50%. In addition to significantly reducing the survival and quality of life of affected patients, heart failure (AHF) places a tremendous economic burden on the healthcare system. In the United States alone, the total cost of heart failure care was estimated at $31 billion in 2012, with the majority of the cost related to hospitalization. Due to an aging population, this cost is projected to rise to an unprecedented $70 billion by 2030.

[0115] Heart failure encompasses a wide range of patients, from those typically considered to have ≥50% of normal left ventricular ejection fraction (LVEF) (also known as HF with preserved EF (HFpEF)) to those typically considered to have <40% reduced LVEF (also known as HF with reduced EF (HFrEF)). Patients with an LVEF range of 40% to 49% represent the “grey zone,” which is defined as HF with moderate EF (HFmrEF) (Ponikowski et al., 2016).

[0116] Heart failure can occur in either acute or chronic heart failure.

[0117] The term "acute" is used to refer to a rapid onset and describes worsening or decompensated heart failure, which refers to an event in which a patient may exhibit changes in the signs and symptoms of heart failure, requiring urgent treatment or hospitalization.

[0118] The term "chronic" refers to a long duration. Chronic heart failure is a long-term condition that is usually kept stable by treating the symptoms (stable chronic HF).

[0119] The characteristics of stable chronic HF are: (i) There is structural or functional failure of the heart to provide adequate blood flow to meet the body's needs. (ii) There is no volume overload (manifested as pulmonary and / or systemic congestion) and / or a severe decrease in cardiac output (manifested as hypotension, renal insufficiency and / or shock syndrome), and the patient does not require emergency treatment or treatment adjustment and does not require hospitalization.

[0120] The characteristics of chronic HF with worsening signs and symptoms are: (i) There is structural or functional failure of the heart to provide adequate blood flow to meet the body's needs. (ii) There is volume overload (manifested as pulmonary and / or systemic congestion) and / or a severe decrease in cardiac output (manifested as hypotension, renal insufficiency and / or shock syndrome), and the patient does not require emergency treatment or hospitalization, but requires treatment adjustment.

[0121] Chronic heart failure can also become decompensated (called acute decompensated heart failure or acute decompensated chronic heart failure), most commonly due to comorbidities (e.g., pneumonia), myocardial infarction, arrhythmias, uncontrolled hypertension, or failure of the patient to maintain fluid restriction, diet, or medication. With treatment, patients with acute decompensated chronic HF can recover to a stable chronic compensated state (stable chronic HF).

[0122] The characteristics of newly diagnosed acute heart disease (HF) and acute decompensated chronic heart disease (HF) are: (i) There is structural or functional failure of the heart to provide adequate blood flow to meet the body's needs. (ii) There is volume overload (manifested as pulmonary and / or systemic congestion) and / or a severe decrease in cardiac output (manifested as hypotension, renal insufficiency and / or shock syndrome), and the patient requires emergency treatment or treatment adjustment and hospitalization.

[0123] The above definition of acute heart failure, namely new-onset AHF, acute decompensated HF, acute decompensated chronic HF, or worsening symptoms / signs of chronic heart failure, is consistent with the definition given by Voors et al. in 2016.

[0124] In one specific embodiment of the method for diagnosing a disease or condition in an object with or related to a necrosis process, DPP3 activity may be determined using a liquid phase activity assay or an enzyme capture activity assay.

[0125] In liquid chromatography, the sample of body fluid is directly subjected to a fluorescent substrate (e.g., Arg-Arg-β-NA). Because many different aminopeptidases exist in plasma (Sanderink et al., 1988), the substrate used may be cleaved by peptidases other than DPP3. To avoid this problem, a preferred method for detecting specific DPP3 activity is to use an enzyme capture activity assay.

[0126] In one specific implementation, the determination of active DPP3 in the enzyme capture assay includes the following steps: • The sample is brought into contact with a capture binder that binds to full-length DPP3 but preferably inhibits DPP3 activity by less than 50%, more preferably less than 40%, and more preferably less than 30% in liquid chromatography. To prevent DPP3 inhibition, the capture binder should not bind to DPP3 in the region surrounding the active site and substrate binding region (amino acids 316 to 669 of SEQ ID No. 1). • Separate DPP3 and body fluid samples that are bound to the said capture binder. • Add the DPP3 substrate to the isolated DPP3. • DPP3 activity is quantified by measuring the conversion rate of DPP3 substrates. • Evaluation of the measured signal compared to non-disease controls. The threshold can be predetermined, for example, by measuring DPP3 concentration and / or DPP3 activity in healthy controls and calculating the corresponding 75th percentile. The upper boundary of the 75th percentile defines the threshold for healthy patients relative to diseased patients.

[0127] In one specific embodiment, the active DPP3 is determined according to the method described above according to the present invention.

[0128] In one specific embodiment of the method for diagnosing a disease or condition in an object with or related to a necrosis process, the binder may be selected from an antibody, an antibody fragment, or a non-IgG scaffold.

[0129] In one specific embodiment of the method for diagnosing a disease or condition in an object with or related to a necrotic process, an enzyme capture assay (ECA, US5612186A, US5601986A) is used. The DPP3 binder in this assay is an antibody.

[0130] In one specific embodiment of the method for diagnosing a disease or condition in an object with or related to a necrotic process, the capture binder is immobilized on a surface. A binder that reacts with the DPP3 protein but does not interfere with peptidase activity exceeding 50%, preferably less than 40%, and more preferably less than 30%, can be immobilized on a solid phase. To prevent inhibition of DPP3, the capture binder should not bind to DPP3 in the region surrounding the active site and substrate-binding region (amino acids 316 to 669 of SEQ ID No. 1).

[0131] In one specific embodiment of the method for determining the activity of DPP3 in a body fluid sample of a subject, the binding agent may be selected from an antibody, an antibody fragment, a non-Ig scaffold, or an aptamer.

[0132] The test sample is passed through a fixed binder, and DPP3 (if present) binds to the binder and is itself fixed for detection. A substrate can then be added, and the reaction product can be detected to indicate the presence or amount of DPP3 in the test sample. For the purposes of this specification, the term "solid phase" may be used to include any material or vessel in which or on which the determination can be performed, including but not limited to: porous materials, non-porous materials, test tubes, pores, glass slides, agarose resins (e.g., Sepharose from GE Healthcare Life Sciences), and magnetic particles (e.g., Dynabeads from Thermo Fisher Scientific). TM Or Pierce TM Magnetic beads, etc.

[0133] Protein or peptide-derived binders (e.g., antibodies, antibody fragments, non-Ig scaffolds) can be immobilized on a solid phase using methods including: physical adsorption (e.g., via electrostatic or hydrophobic interactions), bioaffinity immobilization (e.g., avidin-biotin, protein A / G / L, His-tag, and Ni...). 2+ -NTA, GST-tags and glutathione, DNA hybridization, aptamers), covalent bonds (e.g., amines and N-hydroxysuccinimide) or combinations of the aforementioned immobilization methods (Kim & Herr, 2013). Oligonucleotide-derived binders (e.g., aptamers) can be immobilized on a solid phase using the (streptomycin) avidin-biotin system (Müller et al., 2012; Deng et al., 2014).

[0134] In one specific embodiment of the method for diagnosing a disease or condition in an object with or related to a necrosis process, the separation step is a washing step that removes sample components from the captured DPP3 that are not bound to the capture binder.

[0135] In one specific embodiment of the method for diagnosing a disease or condition in an object with or related to a necrosis process, the DPP3 substrate conversion rate is measured by a method selected from: fluorescence of a fluorescent substrate (e.g., Arg-Arg-2NA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate conjugated with aminofluorescein (Promega protease-Glo). TM Determination methods include mass spectrometry, HPLC / FPLC (reversed-phase chromatography, size exclusion chromatography), thin-layer chromatography, capillary zone electrophoresis, and active staining after gel electrophoresis (immobilization, active DPP3) or Western blotting (cleavage products).

[0136] In one specific embodiment of the method for diagnosing a disease or condition in a subject with or related to a necrotic process, the substrate may be selected from: angiotensin II and angiotensin III, leucine enkephalin, methionine enkephalin, endorphin 1 and endorphin 2, indomethacin, β-tyrofoam, dynorphin, gastreptide, ACTH and MSH, or dipeptides and tripeptides conjugated to a fluorophore, chromophore, or aminofluorescein (as determined by Promega Protease-Glo™). Dipeptides or tripeptides cleaved by DPP3 include, but are not limited to, Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, and Suc-Ala-Ala-Phe. Fluoresceins 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; Abramic et al., 2000; Ohkubo et al., 1999). Cleavage of these fluorescent substrates results in the release of fluorescent β-naphthylamide or 7-amino-4-methylcoumarin, respectively. Chromophores include, but are not limited to, p-nitroaniline diacetate (pNA). Hydrolysis of the peptide-pNA bond in the chromogenic substrate leads to the release of pNA, which in turn changes color. Therefore, the change in absorbance (DA / min) is proportional to enzyme activity. Using Protease-Glo™ from Promega, aminoluciferin is released after cleavage by DPP3 and used as a substrate for a detectable luminescent luciferase-coupled reaction.

[0137] In a preferred embodiment, DPP3 activity is measured by adding the fluorescent substrate Arg-Arg-βNA and monitoring the fluorescence in real time.

[0138] Another important embodiment of the present invention is an inhibitor of DPP3 activity for the prevention or treatment of diseases or conditions in subjects with or related to necrosis.

[0139] Inhibitors are preferably molecules that significantly inhibit DPP3 activity. These molecules can be peptides and small molecules (see Table 2) or antibodies (see Table 3). Significant inhibition means inhibition of more than 80%, preferably more than 90%, and more preferably almost or practically 100% in the liquid chromatography assay described above.

[0140] Peptide inhibitors of DPP3 include, but are not limited to: spin proteins, synthetic derivatives of spin proteins (tynorphin and other peptides, see Table 2; Yamamoto et al., 2000), propioxatin A and propioxatin B (US4804676) and synthetic propioxatin A analogs (Inaoka et al., 1988).

[0141] Small molecule inhibitors of DPP3 include, but are not limited to, fluostatin and benzimidazole derivatives. Fluostatins A and Fluostatins B are antibiotics produced in Streptomyces p. TA-3391, which are non-toxic and strongly inhibit DPP3 activity. To date, 20 different benzimidazole derivatives have been synthesized and disclosed (Agic et al., 2007; Rastija et al., 2015), among which two compounds 1' and 4' show the strongest inhibitory activity (Agic et al., 2007).

[0142] Table 2: Peptides and Small Molecule Inhibitors of DPP3

[0143] In a preferred embodiment of the invention, the selected inhibitor is pharmaceutically acceptable, selective, and / or specific to DPP3, and does not cross the cell membrane and / or blood-brain barrier. The selective and specific inhibitors of DPP3 do not bind to other proteins / enzymes or inhibit any other enzymes / proteases / peptidases besides DPP3. Small peptides can be bound and cleaved by nonspecific aminopeptidases, and small molecule inhibitors readily cross the cell membrane and blood-brain barrier. Anti-DPP3 antibodies, anti-DPP3 antibody fragments, or anti-DPP3 non-Ig scaffolds specifically and selectively bind to DPP3 and do not cross the cell membrane or blood-brain barrier. Therefore, preferred DPP3 activity inhibitors are specific anti-DPP3 antibodies, antibody fragments, or non-Ig scaffolds.

[0144] In one specific embodiment of the present invention, a DPP3 activity inhibitor is used to prevent or treat diseases or conditions in subjects with or related to a necrosis process, wherein the inhibitor is selected from anti-DPP3 antibodies or anti-DPP3 antibody fragments or anti-DPP3 non-Ig scaffolds.

[0145] In one specific embodiment of the invention, a DPP3 activity inhibitor or effector is used to prevent or treat a disease or condition in a subject with or associated with a necrosis process, wherein the disease is selected from heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infection (microorganisms, viruses (e.g., AIDS), parasites (e.g., malaria)) or SIRS or sepsis, cancer, acute kidney injury (AKI), central nervous system (CNS) disorders (e.g., epilepsy, neurodegenerative diseases), autoimmune diseases, vascular diseases (e.g., Kawasaki syndrome), and hypotension.

[0146] In another embodiment, the disease is selected from acute heart failure (AHF), myocardial infarction (MI), liver failure, burns, severe infection (microorganisms, viruses (e.g., AIDS), parasites (e.g., malaria)) or SIRS or sepsis, cancer, and acute kidney injury (AKI).

[0147] In one specific embodiment of the invention, the DPP3 activity inhibitor is used to prevent diseases or conditions of the subject, wherein the diseases or conditions are acute heart failure (AHF), myocardial infarction (MI), liver failure, cancer, acute kidney injury (AKI), and hypotension.

[0148] In one specific embodiment of the invention, the DPP3 activity inhibitor is used to treat a disease or condition of the subject, wherein the disease or condition is acute heart failure (AHF), myocardial infarction (MI), liver failure, cancer, acute kidney injury (AKI), and hypotension.

[0149] In one specific embodiment of all embodiments of the present invention, the disease is not Alzheimer's disease. In one specific embodiment of all embodiments of the present invention, the disease is not cancer. In one specific embodiment of all embodiments of the present invention, the disease is not rheumatoid arthritis.

[0150] In one specific implementation, the disease or condition is low blood pressure.

[0151] In one specific embodiment of the invention, a DPP3 activity inhibitor is used to prevent or treat diseases or conditions in subjects with or related to a necrosis process, wherein the inhibitor is a mono- or at least mono- or mono-binding antibody.

[0152] In one specific embodiment of the present invention, an inhibitor or effector of DPP3 activity is used to prevent or treat a disease or condition in a subject with or related to a necrosis process, wherein the inhibitor or effector is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold bound to SEQ ID No. 1, and in one specific embodiment, it is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold bound to SEQ ID No. 2.

[0153] In one specific embodiment of the invention, an inhibitor or effector of DPP3 activity is used to prevent or treat a disease or condition in a subject with or related to a necrosis process, wherein the inhibitor or effector has a minimum binding affinity for DPP3 of less than 10. -7 M is an antibody, fragment, or scaffold.

[0154] In one specific embodiment of a method for preventing or treating a disease or condition in a subject with or related to a necrosis process, said inhibitor or effector is an antibody, fragment, or scaffold that binds to full-length DPP3 and inhibits at least 10%, or at least 50%, more preferably at least 60%, even more preferably greater than 70%, even more preferably greater than 80%, even more preferably greater than 90%, even more preferably greater than 95% of DPP3 activity. The activity can be determined in a liquid chromatography assay as described above.

[0155] In one specific embodiment of the invention, a DPP3 activity inhibitor or effector is used to prevent or treat a disease or condition in a subject with or associated with a necrosis process, wherein the inhibitor or effector is a single-specific antibody or fragment or scaffold.

[0156] Monospecific anti-DPP3 antibody, monospecific anti-DPP3 antibody fragment, or monospecific anti-DPP3 non-Ig scaffold means that the antibody, antibody fragment, or non-Ig scaffold binds to a specific region containing at least 5 amino acids within the target DPP3. Monospecific anti-DPP3 antibody, monospecific anti-DPP3 antibody fragment, or monospecific anti-DPP3 non-Ig scaffold is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that has affinity for the same antigen.

[0157] In another specific and preferred embodiment, the anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold binding to DPP3 is a monospecific antibody, antibody fragment, or non-Ig scaffold, where monospecificity means that the antibody, antibody fragment, or non-Ig scaffold binds to a specific region of the target DPP3 containing at least four amino acids. The monospecific antibody, fragment, or non-Ig scaffold according to the invention is an antibody, fragment, or non-Ig scaffold with affinity for the same antigen. Monoclonal antibodies are monospecific, but monospecific antibodies can also be produced by means other than from ordinary germ cells.

[0158] In one specific embodiment of the invention, an inhibitor or effector of DPP3 activity is used to prevent or treat a disease or condition in a subject with or associated with a necrosis process, wherein the subject has elevated levels of DPP3. Elevated levels are levels above a predetermined threshold.

[0159] Another embodiment of the present invention is a pharmaceutical composition comprising the above-described DPP3 activity inhibitor for the prevention or treatment of diseases or conditions in subjects with or related to necrosis.

[0160] Another embodiment of the invention is a method for preventing or treating a disease or condition in an object with or related to a necrosis process, wherein a DPP3 activity inhibitor is administered.

[0161] In one specific embodiment of a method for preventing or treating a disease or condition in an object with or related to a necrosis process, the inhibitor is selected from the group comprising anti-DPP3 antibodies or anti-DPP3 antibody fragments or anti-DPP3 non-Ig scaffolds.

[0162] In one specific embodiment of a method for preventing or treating a disease or condition of an object with or related to a necrosis process, said disease is selected from heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infection (microorganisms, viruses (e.g., AIDS), parasites (e.g., malaria)) or SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g., epilepsy, neurodegenerative diseases), autoimmune diseases, vascular diseases (e.g., Kawasaki syndrome), and hypotension.

[0163] In one specific embodiment of a method for preventing or treating a disease or condition of an object with or related to a necrosis process, said inhibitor is a mono- or at least mono- or mono-binding antibody.

[0164] In one embodiment of a method for preventing or treating a disease or condition of an object with or associated with a necrotic process, the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold bound to SEQ ID No. 1, and in another embodiment, it is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold bound to SEQ ID No. 2.

[0165] In one specific embodiment of a method for preventing or treating a disease or condition in an object with or related to a necrotizing process, said inhibitor has a minimum binding affinity of less than 10 for DPP3. -7 M is an antibody, fragment, or scaffold.

[0166] In one embodiment of a method for preventing or treating a disease or condition in an object with or related to a necrosis process, the inhibitor or effector is a single-specific antibody or fragment or scaffold.

[0167] In one specific embodiment of a method for preventing or treating a disease or condition in a subject with or associated with a necrosis process, the inhibitor or effector is an antibody, fragment, or scaffold that binds to full-length DPP3 and inhibits at least 10%, or at least 50%, more preferably at least 60%, even more preferably greater than 70%, even more preferably greater than 80%, even more preferably greater than 90%, even more preferably greater than 95% of DPP3 activity.

[0168] In one embodiment of a method for preventing or treating a disease or condition in an object with or related to a necrosis process, the object has an elevated level of DPP3. The elevated level is above a predetermined threshold, as defined above.

[0169] Another embodiment of the invention is the removal of DPP3 from a patient's blood. Removal can be achieved through various blood component apheresis and / or affinity chromatography steps (Balogun et al., 2010). These methods include, but are not limited to, filtering patient plasma with an adsorbent containing a specific and high-affinity DPP3 antibody conjugated to agarose resin, see Example 12, for analyzing the binding of DPP3 to possible adsorbent materials.

[0170] The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Routes of administration are generally classified according to the site of application of the substance. Common examples include oral, epidermal, subcutaneous, intradermal, sublingual, intramuscular, intra-arterial, intravenous, and intraperitoneal administration.

[0171] The drug composition can also be administered via the central nervous system (CNS), such as epidural injection or infusion into the epidural space, intracerebral injection (into the brain), intraventricular injection (into the ventricular system of the brain), or intrathecal injection (into the spinal canal).

[0172] The specific embodiments of the present invention are summarized below:

[0173] 1. A method for diagnosing a disease or condition in an object with or related to a necrotic process, comprising: • Determine the amount of total DPP3 and / or active DPP3 in the body fluid sample of the subject. • Compare the determined total DPP3 amount or active DPP3 amount with a predetermined threshold. • If the determined amount is higher than the predetermined threshold, the object is diagnosed as having a disease or condition accompanied by or related to the necrosis process.

[0174] 2. The method of claim 1 for diagnosing a disease or condition in an object with or related to a necrosis process, wherein the amount of total DPP3 or the amount of active DPP3 is determined in units of concentration.

[0175] 3. The method for diagnosing a disease or condition of an object with or related to a necrosis process, according to claim 1 or 2, wherein the sample is selected from whole blood, serum, and plasma.

[0176] 4. A method for diagnosing a disease or condition of an object with or related to a necrotic process, according to any one of claims 1 to 3, wherein the disease is selected from heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infection (microorganisms, viruses (e.g., AIDS), parasites (e.g., malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g., epilepsy, neurodegenerative diseases), autoimmune diseases, vascular diseases (e.g., Kawasaki syndrome), and hypotension.

[0177] 5. A method for diagnosing a disease or condition in a subject with or related to a necrotic process, according to any one of claims 1 to 4, wherein the amount of total DPP3 and / or the amount of active DPP3 is determined in a body fluid sample of the subject, and comprising the following steps: • Contact the sample with a trapping binder that specifically binds to full-length DPP3. • Separate DPP3 bound to the capturing binder. • Add the DPP3 substrate to the isolated DPP3. • The active DPP3 is quantified by measuring and quantifying the conversion rate of the DPP3 substrate.

[0178] 6. The method of claim 5 for diagnosing a disease or condition of an object with or related to a necrosis process, wherein the capture binder is selected from an antibody, an antibody fragment, or a non-IgG scaffold.

[0179] 7. The method of claim 5 or 6 for diagnosing a disease or condition of an object with or related to a necrosis process, wherein the capture binder is an antibody.

[0180] 8. A method for diagnosing a disease or condition of an object with or related to a necrosis process, according to any one of claims 5 to 7, wherein the capturing binder is fixed to the surface.

[0181] 9. A method for diagnosing a disease or condition of an object with or related to a necrosis process, according to any one of claims 5 to 8, wherein the separation step is a washing step that removes sample components not bound to the capture binder from the captured DPP3.

[0182] 10. A method for diagnosing a disease or condition in an object with or related to a necrotic process, according to any one of claims 5 to 9, wherein the DPP3 substrate conversion rate is detected by methods selected from: fluorescence of a fluorescent substrate (e.g., Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate conjugated with aminofluorescein (Promega protease-Glucosamine). TM Determination methods include mass spectrometry, HPLC / FPLC (reversed-phase chromatography, size exclusion chromatography), thin-layer chromatography, capillary zone electrophoresis, and active staining after gel electrophoresis (immobilization, active DPP3) or Western blotting (cleavage products).

[0183] 11. A method for diagnosing a disease or condition of a subject with or related to a necrotic process, according to any one of claims 5 to 10, wherein the substrate may be selected from: angiotensin II, angiotensin III and angiotensin IV, leucine enkephalin, methionine enkephalin, endorphin 1 and endorphin 2, indomethacin, β-tyrofoam, dynorphin, gastreptide, ACTH and MSH, or a dipeptide conjugated to a fluorophore, chromophore or aminofluorescein, wherein the dipeptide is Arg-Arg.

[0184] 12. A method for diagnosing a disease or condition of an object with or related to a necrotic process, according to any one of claims 5 to 11, wherein the substrate may be selected from: a dipeptide coupled to a fluorophore, a chromophore, or an aminofluorescein, wherein the dipeptide is Arg-Arg.

[0185] 13. A method for monitoring a disease or condition in an object with or related to a necrosis process, wherein the diagnostic method according to any one of claims 1 to 12 is performed at least twice.

[0186] 14. DPP3 activity inhibitors used for the prevention or treatment of diseases or conditions in subjects with or related to necrosis.

[0187] 15. A DPP3 activity inhibitor of claim 14 for the prevention or treatment of a disease or condition in an object with or related to a necrosis process, wherein the inhibitor is selected from anti-DPP3 antibodies or anti-DPP3 antibody fragments or anti-DPP3 non-Ig scaffolds.

[0188] 16. A DPP3 activity inhibitor of claim 14 or 15 for the prevention or treatment of a disease or condition of a subject with or associated with a necrotic process, wherein the disease is selected from heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infection (microorganisms, viruses (e.g., AIDS), parasites (e.g., malaria)), or SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g., epilepsy, neurodegenerative diseases), autoimmune diseases, vascular diseases (e.g., Kawasaki syndrome), and hypotension.

[0189] 17. A DPP3 activity inhibitor for the prevention or treatment of a disease or condition of a subject with or related to a necrosis process, according to any one of claims 14 to 16, wherein the inhibitor is a mono- or at least mono-binding antibody.

[0190] 18. A DPP3 activity inhibitor for the prevention or treatment of a disease or condition of a subject with or related to a necrosis process, according to any one of claims 14 to 17, wherein the inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to SEQ ID No. 1, particularly to SEQ ID No. 2.

[0191] 19. A DPP3 activity inhibitor according to any one of claims 14 to 18 for the prevention or treatment of a disease or condition in a subject with or related to a necrosis process, wherein the inhibitor has a minimum binding affinity to DPP3 equal to or less than 10. -7M is an antibody, fragment, or scaffold.

[0192] 20. A DPP3 activity inhibitor for the prevention or treatment of a disease or condition of a subject with or related to a necrosis process, according to any one of claims 14 to 19, wherein the inhibitor is a monospecific antibody, fragment, or scaffold.

[0193] 21. A DPP3 activity inhibitor for the prevention or treatment of a disease or condition in a subject with or related to a necrosis process, according to any one of claims 14 to 20, wherein the inhibitor is an antibody, fragment, or scaffold that binds to full-length DPP3 and inhibits at least 10%, or at least 50%, more preferably at least 60%, even more preferably greater than 70%, even more preferably greater than 80%, even more preferably greater than 90%, even more preferably greater than 95% of DPP3 activity.

[0194] 22. A DPP3 activity inhibitor for the prevention or treatment of a disease or condition of a subject with or related to a necrosis process, according to any one of claims 14 to 21, wherein the inhibitor is selective and / or specific to DPP3 and does not cross the cell membrane and / or blood-brain barrier.

[0195] 23. A DPP3 activity inhibitor for the prevention or treatment of a disease or condition of a subject with or related to a necrosis process, according to any one of claims 14 to 22, wherein the subject has a total DPP3 amount and / or an active DPP3 amount in a body fluid sample of the subject that is above a predetermined threshold.

[0196] 24. A pharmaceutical composition comprising a DPP3 activity inhibitor according to any one of claims 14 to 23 for the prevention or treatment of a disease or condition in a subject with or related to a necrosis process.

[0197] 25. Use of the DPP3 activity inhibitor of any one of claims 14 to 23 in a method for in vitro removal of DPP3 from plasma, said method comprising apheresis and affinity chromatography.

[0198] 26. A method for determining active DPP3 in a body fluid sample of a subject, comprising the following steps: • Contact the sample with a trapping binder that specifically binds to full-length DPP3. • Separate DPP3 bound to the capturing binder. • Add the DPP3 substrate to the isolated DPP3. • The active DPP3 is quantified by measuring and quantifying the conversion rate of the DPP3 substrate.

[0199] 27. The method of claim 26 for determining active DPP3 in a body fluid sample of a subject, wherein the capture binder may be selected from antibodies, antibody fragments, or non-IgG scaffolds.

[0200] 28. The method for determining active DPP3 in a body fluid sample of a subject according to claim 26 or 27, wherein the capture binder is an antibody.

[0201] 29. A method for determining active DPP3 in a body fluid sample of a subject according to any one of claims 26 to 28, wherein the capturing binder is fixed on a surface.

[0202] 30. A method for determining active DPP3 in a body fluid sample of a subject according to any one of claims 26 to 29, wherein the separation step is a washing step that removes sample components not bound to the captured DPP3 from the captured DPP3.

[0203] 31. The method for determining the activity of DPP3 in a body fluid sample of a subject according to any one of claims 26 to 30, wherein the DPP3 substrate conversion rate is detected by methods selected from: fluorescence of a fluorescent substrate (e.g., Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate conjugated with aminofluorescein (Promega protease-Glucosamine). TM Determination methods include mass spectrometry, HPLC / FPLC (reversed-phase chromatography, size exclusion chromatography), thin-layer chromatography, capillary zone electrophoresis, and active staining after gel electrophoresis (immobilization, active DPP3) or Western blotting (cleavage products).

[0204] 32. The method for determining active DPP3 in a body fluid sample of a subject according to any one of claims 26 to 31, wherein the substrate may be selected from: a dipeptide coupled to a fluorophore, chromophore, or aminofluorescein, wherein the dipeptide is Arg-Arg.

[0205] 33. The method for determining active DPP3 in a body fluid sample of a subject according to any one of claims 26 to 32, wherein the sample is a blood sample selected from whole blood, serum, and plasma.

[0206] 34. A assay or kit for determining the activity of DPP3 in a body fluid sample of a subject, comprising: • A capture binder that specifically binds to full-length DPP3. • Substrate of DPP3.

[0207] 35. The assay or kit for determining the activity of DPP3 in a body fluid sample of a subject, according to claim 34, wherein the capture binder may be selected from an antibody, an antibody fragment, or a non-IgG scaffold.

[0208] 36. The assay or kit for determining the activity of DPP3 in a body fluid sample of a subject according to claim 34 or 35, wherein the capturing binder inhibits less than 50%, preferably less than 40%, more preferably less than 30% of DPP3 activity in the liquid phase assay.

[0209] 37. The assay or kit for determining the activity of DPP3 in a body fluid sample of a subject according to any one of claims 34 to 36, wherein the binding region of the DPP3 of the capturing binder is not within the region of amino acids 316 to 669 of SEQ Id No. 1.

[0210] 38. A assay or kit for determining the activity of DPP3 in a body fluid sample of a subject, according to any one of claims 34 to 37, wherein the binding agent is an antibody.

[0211] 39. A assay or kit for determining the activity of DPP3 in a body fluid sample of a subject, according to any one of claims 34 to 38, wherein the capturing binder is immobilized on a surface.

[0212] 40. A assay or kit for determining the activity of DPP3 in a body fluid sample of a subject according to any one of claims 34 to 39, wherein the substrate may be selected from: angiotensin II, angiotensin III and angiotensin IV, leucine enkephalin, methionine enkephalin, endorphin 1 and endorphin 2, indomethacin, β-tyrosine, dynorphin, gastreptide, ACTH and MSH, or a dipeptide conjugated with a fluorophore, chromophore or aminofluorescein, wherein the preferred dipeptide is Arg-Arg.

[0213] 41. The use of the method for determining active DPP3 in a body fluid sample of a subject according to any one of claims 26 to 33 in the method for diagnosing or monitoring a disease or condition of a subject with or related to a necrosis process according to any one of claims 1 to 13, or the use of the assay or kit according to any one of claims 34 to 40 in the method for diagnosing or monitoring a disease or condition of a subject with or related to a necrosis process according to any one of claims 1 to 13. Detailed Implementation 1. Example 1

[0214] The specific DPP3 capture activity assay was used to determine the plasma DPP3 activity in patients with various diseases (acute myocardial infarction (AMI), cardiogenic shock, septic shock, and liver failure) and to compare it with the plasma DPP3 activity in healthy controls. 1.1. Study population:

[0215] Plasma samples were obtained from 388 patients who came directly to the emergency room at their first appearance. Based on their final diagnosis, these patients were divided into four subgroups: those who had experienced acute myocardial infarction (AMI), those who had experienced cardiogenic shock, those who had experienced septic shock, and those with liver failure. The control group was a collection of plasma samples from 93 healthy controls. 1.2. hDPP3 capture activity assay:

[0216] First, DPP3 in a 10 μl plasma sample was enriched via affinity purification. Then, its activity was measured by adding the fluorescent substrate Arg-Arg-βNA (see Example 4 for detailed description). The slope of the increase in fluorescence for different samples was calculated (in nmol βNA / min / ml sample [nmol βNA min]). -1 ml -1 The sample size (10 μl) refers to the sample size. 1.3 Results:

[0217] For all patients with severe illness or organ failure, patient samples showed significantly higher DPP3 activity levels compared to healthy controls. Figure 10 ). 2. Example 2

[0218] In this experiment, the effects of recombinant hDPP3 injection in healthy rats were investigated by monitoring blood pressure. 2.1 Method:

[0219] Male Wistar rats aged 2 to 3 months (Charles River Laboratories, Germany) were used in this study. To measure and record blood pressure (BP), a catheter (Introcan-W; 22G / 1'; B. Braun) was inserted into the carotid artery (right common carotid artery). Human recombinant dipeptidyl peptidase 3 (recGST-hDPP3) with an N-terminal GST-tagged interface was injected via the tail vein.

[0220] The animal was first anesthetized with isoflurane for weighing (total g) and then injected intraperitoneally (ip) with 1.2 g / kg BW ethyl carbamate (c = 0.4 g / mL) for prolonged anesthesia. The abdominal area around the neck was then cut off and wiped clean with ethanol. A container was prepared and catheters inserted. Finally, both catheters were flushed with heparinized isotonic sodium chloride solution. A pressure sensor (medexlogical, Medex Medical Ltd.) was then connected to a patient monitoring system (Datex-Ohmeda, GE). A portable computer connected to the BP monitor recorded BP data separately using S / 5 collection software.

[0221] Rats were treated with recGST-hDPP3 0.2 mg / kg in PBS by injection into the tail vein. Blood pressure was monitored continuously before and after injection of DPP3. 2.2 Results:

[0222] Injection of recombinant GST-hDPP3 into healthy rats resulted in an immediate decrease in blood pressure. Figure 11 ). 3. Example 3

[0223] Antibody generation and determination of DPP3 binding ability: Several mouse antibodies were generated and screened by their ability to bind human DPP3 in sandwich assays or activity assays (see Table 3). 3.1. Method: - Peptides / conjugates used for immunity:

[0224] A DPP3 peptide for immunization was synthesized (see Table 3, JPT Technologies, Berlin, Germany), containing an additional N-terminal cysteine ​​residue (if cysteine ​​is absent in the selected DPP3-sequence) to conjugate the peptide to bovine serum albumin (BSA). The peptide was covalently linked to BSA using a Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling process was performed according to Perbio's manual. Recombinant GST-hDPP3 was produced via USBio. - Mouse immunization, immune cell fusion, and screening:

[0225] On day 0, Balb / c mice were intraperitoneally (ip) injected with 84 μg of GST-hDPP3 or 100 μg of DPP3-peptide-BSA-conjugate (emulsified in TiterMax Gold adjuvant); on day 14, they were injected with 84 μg or 100 μg (emulsified in complete Freund's adjuvant); and on days 21 and 28, they were injected with 42 μg or 50 μg (emulsified in incomplete Freund's adjuvant). On day 49, the animals were intravenously (iv) injected with 42 μg of GST-hDPP3 or 50 μg of DPP3-peptide-BSA-conjugate dissolved in saline. Three days later, the mice were sacrificed and immune cell fusion was performed.

[0226] Spleen cells and myeloma cell line SP2 / 0 from immunized mice were fused with 1 ml of 50% polyethylene glycol for 30 seconds at 37°C. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement]. After one week, the HAT medium was replaced with HT medium for three passages, and then the cells were returned to normal cell culture medium.

[0227] Two weeks after fusion, cell culture supernatants were screened primarily for recombinant DPP3-bound IgG antibodies. Therefore, recombinant GST-labeled DPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng / well) and incubated with 50 μl of cell culture supernatant per well at room temperature for 2 hours. After washing the plates, 50 μl / well of POD-rabbit anti-mouse IgG was added and incubated at room temperature for 1 hour. After the next washing step, 50 μl of chromogen solution (3.7 mM o-phenylenediamine in citrate / hydrogen phosphate buffer, 0.012% H2O2) was added to each well, incubated at room temperature for 15 minutes, and the colorimetric reaction was terminated by adding 50 μl of 4N sulfuric acid. Absorption was detected at 490 nm.

[0228] The microcultures from positive tests were transferred to 24-well plates for propagation. After retesting, selected cultures were cloned and re-cloned using restrictive dilution techniques, and isotypes were determined. - Mouse monoclonal antibody production

[0229] Antibodies against GST-labeled human DPP3 or DPP3-peptide were produced using a standard antibody production method (Marx et al., 1997) and purified by protein A. Antibody purity was ≥90% based on SDS-PAGE analysis. Antibody Characterization - hDPP3 Inhibition Analysis

[0230] To analyze the DPP3 inhibitory activity of different antibodies and antibody clones, DPP3 activity was determined using a known procedure (Jones et al., 1982). Recombinant GST-labeled 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 respective antibody at room temperature. After a 1-hour pre-incubation, the fluorescent substrate Arg-Arg-βNA (20 μl, 2 mM) was added to the solution, and the generation of free βNA over time was monitored using a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG) at 37 °C. βNA fluorescence was detected by excitation at 340 nm and measurement of emission at 410 nm. The slope of fluorescence increase for different samples (in RFU / min) was calculated. The slope of GST-hDPP3 with a buffer control was designated as 100% activity. The inhibitory capacity of the potential capture-binding agent was defined as the reduction in GST-hDPP3 activity by percentage incubation with the capture-binding agent. The resulting reduction in DPP3 activity is shown in Figure 1a and Table 3. 3.2. Results:

[0231] The table below shows the selection of antibodies obtained and their maximum inhibition rates (Table 3). Monoclonal antibodies generated targeting the DPP3 region described below were selected based on their ability to bind native DPP3 (mAb-FL-DPP3_2555 was used as the solid phase, and _2553 was used as the tracer for the immunoassay; see Example 4 for details).

[0232] For fixed DPP3 activity assays (see Examples 6 and 7), solid-phase antibodies that do not strongly inhibit DPP3 activity must be selected. As a cutoff for antibody screening, the solid-phase antibody should not inhibit DPP3 activity by more than 50%, as mAbDPP3_2555 showed the lowest inhibition rate (Table 3). Figure 1A ).

[0233] To produce a potent DPP3 inhibitor suitable for therapeutic use (see Examples 8 through 13), a DPP3 binding agent exhibiting the highest inhibition rate must be selected. The monoclonal antibody mAbDPP3_1967, possessing the ability to inhibit 70% of DPP3 activity, was selected as a potential therapeutic antibody (see...). Figure 1A (and Table 3), and used the antibody for all further analyses. Figure 1B The inhibition curve of mAbDPP3_1967 is shown, and its IC50 value is... 50 It was 0.2041 μg / ml.

[0234] Table 3: Immunogen sequences, names, and characteristics of the generated anti-DPP3 antibodies 4. Example 4

[0235] Identify antibody combinations that produce high signal-to-noise ratios in hDPP3 immunoassays. 4.1. Method: Monoclonal antibody production

[0236] Antibodies against the GST-tagged human DPP3 were produced using a standard antibody production method (Marx et al., 1997) and purified via protein A. Antibody purity was ≥90% based on SDS-PAGE analysis. The binding capacity of different clones to DPP3 was analyzed. The obtained positive clones were used as solid-phase or tracer antibodies. -Solid phase

[0237] A 96-well polystyrene microplate (Greiner Bio-One International AG, Austria) was coated with an anti-DPP3 antibody clone (capture antibody; 1.5 μg antibody / 0.25 mL 100 mmol / L NaCl, 50 mmol / L Tris / HCl, pH 7.8) (at room temperature for 1 hour). After blocking with 5% bovine serum albumin, the microplate was vacuum dried. - Labeling process (tracer)

[0238] 100 μg (100 μl) of different anti-DPP3 antibodies (detection antibody, 1 mg / ml in PBS, pH 7.4) were mixed with 10 μl of acridine NHS-ester (1 mg / ml in acetonitrile, InVent GmbH, Germany; EP 0 353 971) and incubated at room temperature for 30 min. The labeled anti-DPP3 antibodies were purified by gel filtration HPLC on a Shodex Protein 5 μm KW-803 (Showa Denko, Japan). The purified labeled antibodies were diluted in assay buffer (50 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na2-EDTA, 5 g / L bovine serum albumin, 1 g / L mouse IgG, 1 g / L bovine IgG, 50 μmol / L aprotinin peptide, 100 μmol / L leucopeptide peptide, pH 7.4). The final concentration was approximately 7 × 10⁻⁶ of the labeled compound. 6Relative optical units (RLU) (approximately 20 ng labeled antibody) / 200 μl. Chemiluminescence of acridine ester was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). - Calibration material

[0239] The stock solution of recombinant human GST-DPP3 (USBiological, USA) was linearly diluted (in PBS, pH 7.4) using (50 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-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 leucopentin peptide, pH 7.4). The stock solution was stored at -80°C. The calibrator was prepared prior to use. -hDPP3 Immunoassay

[0240] After adding labeled and diluted detection antibody (200 μl), pipette 10 μl of sample (or calibrator) into a coated 96-well microplate and incubate the plate at 2 to 8 °C for 18 to 24 hours. Unbound tracer is removed by washing four times with 350 μl of washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Well-bound chemiluminescence is measured using a Centro LB960 chemiluminometer (Berthold Technologies GmbH & Co. KG). 4.2 Results:

[0241] All antibodies were used in sandwich immunoassays, as coated microplates and labeled antibodies, in combinations of the following variations (Tables 4 and 5). Incubation was performed as described for the hDPP3-immunoassay. Results are given as the ratio of specific signal to background signal for recombinant human GST-DPP3 (100 ng / ml, 10 ng / ml, and 1 ng / ml) and native hDPP3 in plasma samples.

[0242] Table 4: Signal-to-noise ratio of anti-DPP3 antibody pairs - Measurement of recombinant GST-hDPP3 (SP-solid phase).

[0243] Table 5: Signal-to-noise ratio in anti-DPP3 antibody pairs - Measurement of human plasma samples (SP - solid phase).

[0244] All combinations showed good signal-to-noise ratios for recombinant GST-hDPP3. Furthermore, all combinations except 2552 and 2554 produced good signal-to-noise ratios relative to the native samples. Therefore, all remaining combinations are suitable for further investigation. Regarding the highest absolute RLU signal, we used 2555 as the solid-phase antibody and 2553 as the labeled antibody. 5. Example 5

[0245] The hDPP3 immunoassay was used to determine the plasma DPP3 concentrations in patients with various diseases (acute heart failure (AHF), myocardial infarction (MI), sepsis, cancer, acute kidney injury (AKI), and lower respiratory tract infection (LRTI)) and to compare them with the plasma DPP3 concentrations in healthy controls. 5.1 Study population:

[0246] Plasma samples were obtained from 214 patients who came directly to the emergency or oncology departments at their first visit. Based on their final diagnoses, these patients were divided into six subgroups: those with acute heart failure (AHF), those with myocardial infarction (MI), those with sepsis, those with cancer, those with acute kidney injury (AKI), and those with lower respiratory tract infection (LRTI). The control group consisted of plasma samples from 93 healthy controls. 5.2hDPP3 Immunoassay:

[0247] mAbDPP3_2555 was used as the solid-phase antibody, and mAbDPP3_2553 was used as the labeled tracer antibody. Antibody fixation, labeling, and incubation were performed as described in Example 2. 5.3. Results:

[0248] Along with the corresponding diagnostic details, the generated data were statistically analyzed. Figure 2A Compared to healthy controls (standard), all patients showed significantly elevated plasma DPP3 concentrations. Table 6 shows the percentage of patients with DPP3 values ​​above the 75th percentile of the control group and their respective diagnoses. Analysis of plasma DPP3 levels indicates a patient's disease state. This insight can be used in the diagnostic field and also as a basis for developing therapeutic treatments, for example, through the inhibition of DPP3.

[0249] Table 6: Comparison of DPP3 values ​​between diseased patients and healthy controls in sandwich immunoassay.

[0250] Analyzing the same study population by their mortality rates, patients who died after being admitted to the emergency room had significantly higher plasma DPP3 levels than emergency room patients who survived in the hospital. Therefore, elevated DPP3 concentrations indicate a poor prognosis in terms of mortality. Figure 2B ). 6. Example 6

[0251] The amount of DPP3 in human plasma can be determined not only by DPP3 concentration but also by activity assay. A standard procedure is a soluble activity assay using Arg-Arg-βNA as a fluorescent substrate:

[0252] The activity of natural human DPP III to form fluorescent β-naphthylamine was determined by hydrolysis of Arg-Arg-β-naphthylamide (Bachem Holdig AG, Switzerland). 200 μl of buffer (50 mM TRIS / HCl, pH 8.8, 0.04% NaN3, 50 μM aprotinin peptide, 100 μM leucopeptide peptide) and 10 μl of sample (human plasma) were pipetted into black 96-well microplates (Greiner Bio-One International GmbH, Austria) and preheated at 37 °C for 10 min. After adding substrate (20 μl, 2 mM), fluorescence increase was monitored for 1 h at 37 °C using a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG) with excitation wavelength of 340 nm and emission wavelength of 410 nm. Reference calibrator was used to measure free βNA at nmol βNA / min / ml sample [nmol βNA min...]. -1 ml -1 ] Calculate the slope of fluorescence increase for different samples.

[0253] Using the standard soluble DPP3 activity assay, it is impossible to determine whether DPP3 activity or the activity of other aminopeptidases in plasma is measured. To generate a DPP3-specific signal, an enzyme capture assay is performed, in which DPP3 is immobilized on a surface by binding with a monoclonal antibody in a first step, and after a washing step, only specific DPP3 activity can be measured.

[0254] Solid-phase preparation was performed using a black 96-well microplate (Greiner Bio-One International GmbH, Austria) as described in Example 5. 10 μl of sample (plasma or standard) and 200 μl of buffer (50 mmol / L potassium phosphate, 100 mmol / L NaCl, 5 g / L bovine serum albumin, 1 g / L mouse IgG, 1 g / L bovine IgG, 50 μmol / L aprotinin peptide, 100 μmol / L leucine peptide, pH 7.4) were pipette into the coated microplate and incubated (18 to 24 hours, 2°C to 8°C, 600 rpm). Unbound analytes were removed by washing with washing solution (3 × 350 μl). After adding the substrate (200 μl, 100 μM, in 50 mM Tris / HCl, pH (25 °C) 8.8, 0.04%, NaN3), fluorescence increase was monitored for 1 hour at 37 °C using a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG) with an excitation wavelength of 340 nm and an emission wavelength of 410 nm. Reference calibrator was used to measure free βNA in nmol βNA / min / ml sample [nmol βNA min]. -1 ml -1 [Calculate] the slope of fluorescence increase for different samples.

[0255] In each activity assay type, free βNA was used as the assay calibrator. Therefore, in the Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH) The concentration increase of βNA was measured at 37 °C using an excitation wavelength of 340 nm and an emission wavelength of 410 nm (in 200 μl, in 50 mM Tris / HCl, pH (25 °C) 8.8, 0.04% NaN3; 0, 4, 8, 16, 32, 64, 125, 250 μM βNA). All sample measurements were calibrated against this βNA standard. 7. Example 7

[0256] The DPP3 capture activity assay was used to determine the plasma DPP3 activity in patients with various diseases (acute heart failure (AHF), sepsis, acute kidney injury (AKI), and lower respiratory tract infection (LRTI)) and to compare it with the DPP3 plasma activity in healthy controls. 7.1 Method:

[0257] DPP3-specific enzyme capture activity assays were performed on some portions of the group analyzed in Example 5. In this assay, DPP3 (10 μl) from plasma samples was first enriched via an affinity purification step, and then its activity was measured by adding the fluorescent substrate Arg-Arg-βNA (see Example 6 for a detailed description). The slope of the fluorescence increase for different samples was calculated (in nmol βNA / min / ml sample [nmol βNA min]). -1 ml -1 The sample size (10 μl) refers to the sample size. 7.2. Results:

[0258] Comparison of patient samples and healthy controls showed that all patients (AHF, sepsis, AKI, and LTRI) had different outcomes. Figure 3 The DPP3 activity value of ) was significantly higher.

[0259] Table 7 shows the percentage of patients with DPP3 values ​​higher than the 75th percentile of the control group and their respective diagnoses. Activity demonstrates better differentiation between healthy controls and diseased patients.

[0260] Table 7: Comparison of DPP3 values ​​between diseased patients and healthy controls in sandwich immunoassay and enzyme capture activity assay.

[0261] To better compare DPP3 activity assays with concentration assays, we analyzed samples from AHF (… Figure 4A or patients with sepsis ( Figure 4B ROC (recipient operating characteristic) analysis was performed to differentiate healthy controls. The area under the curve (AUC) and confidence interval (CI) values ​​are shown in Table 8. Data analysis showed that the activity assay had higher specificity compared to the sandwich immunoassay.

[0262] Table 8: Data from ROC analysis (AUC - area under the curve; CI - confidence interval). 8. Example 8

[0263] In this experiment, the general safety of increased mAbDPP3 doses in healthy mice was monitored. 8.1 Method:

[0264] Female BALB / c nude mice (CAnN.Cg-Foxn1nu / Crl) aged 4-5 weeks at parturition, weighing approximately 15g-18g, were kept under optimal hygienic conditions. Air was conditioned with 10-15 air changes per hour, and the environment was continuously monitored. The target temperature range was 22±3℃, relative humidity was 30-70%, and there was 12 hours of artificial fluorescent lighting / 12 hours of darkness. A maximum of 4 animals were housed in each ventilated cage (IVC), and they were fed with food prepared by M-Zucht (ssniff). The diet consists of community-labeled water (from GmbH) and autoclaved water.

[0265] After a 4-day acclimatization period, mAbDPP3 administration was initiated: three different concentrations of mAbDPP3 in PBS (0.65 mg / kg, 1.9 mg / kg, and 5.75 mg / kg) were injected into four mice in each group. mAbDPP3 was administered intraperitoneally (ip) on days 1, 3, 5, and 7, and the mice were monitored for 14 days. 8.2 Results:

[0266] All mice survived the 14-day treatment period without side effects, even at the highest dose. MAbDPP3 is safe for use in other animal studies, always at a concentration of 1.9 mg / kg. 9. Example 9

[0267] In this experiment, the general safety of mAbDPP3 treatment in healthy rats was investigated by monitoring mean blood pressure. 9.1 Method:

[0268] Male Wistar rats aged 2 to 3 months (Charles River Laboratories, Germany) were used in this study. To measure and record blood pressure (BP), a catheter (Introcan-W; 22G / 1”; B. Braun) was inserted into the carotid artery (right common carotid artery). A drug administration and sampling catheter was inserted into the jugular vein (left jugular vein).

[0269] First, the animal was anesthetized with isoflurane for weighing (total g) and then intraperitoneally (ip) injected with 1.2 g / kg BW ethyl carbamate (c = 0.4 g / mL) for long-term anesthesia. Next, the ventral region of the neck was cut off and wiped clean with ethanol. A container was prepared and catheters inserted. Finally, both catheters were flushed with heparinized isotonic sodium chloride solution. A pressure sensor (medexlogical, Medex Medical Ltd.) was then connected to a patient monitoring system (Datex-Ohmeda, GE). BP data were recorded separately via S / 5 collection software using a laptop connected to the BP monitor.

[0270] Rats were treated with PBS, PBS containing 1.9 mg / kg and PBS containing 5.75 mg / kg mAbDPP3 (n=3 per group). The compound was administered via intravenous catheter. Blood pressure was monitored 1 hour before administration and for more than 6 hours after administration. 9.2 Results:

[0271] Rats responded well to mAbDPP3 treatment. Mice treated with high doses of mAbDPP3 showed a slight increase in mean blood pressure. Figure 5 Generally, mAbDPP3 is safe for use in rat models, even at higher doses. 10. Example 10

[0272] This study analyzed how mAbDPP3 treatment affected sepsis mortality in a CLP mouse model. 10.1 Method:

[0273] Male C57Bl / 6 mice aged 12 to 15 weeks (Charles River Laboratories, Germany) were used for the study. Peritonitis was induced surgically under mild isoflurane anesthesia. An incision was made in the upper left quadrant of the peritoneal cavity (the normal location of the cecum). The cecum was exposed and a tight ligation was placed around it, sutured distal to the small bowel insertion. A puncture wound was made into the cecum using a 24-gauge needle, and a small amount of cecal contents were expressed through the wound. The cecum was returned to the abdominal cavity and the abdominal cavity was closed in the cesarean section position. Finally, the animals were returned to their cages with free access to food and water. 500 μl of physiological saline was administered subcutaneously as a fluid replacement.

[0274] MAbDPP3 (1.9 mg / kg in PBS) was tested relative to the loading agent (PBS). The compound and loading agent were administered intravenously 5 minutes before CLP (prophylactic treatment) and after complete development of sepsis, and 2 hours after CLP (therapeutic treatment). Each group contained 10 mice, and the mice were followed up for 7 days. 10.2 Results:

[0275] from Figure 6 As can be seen, mAbDPP3 antibody significantly reduced mortality compared to PBS administration. Four days later, 75% of mice treated with mAbDPP3 survived. Conversely, almost all mice died four days after treatment with the carrier. 11. Example 11

[0276] We induced heart failure in rats using a septic shock model and then characterized the effect of mAbDPP3 on cardiac function. 11.1. Method: -Research Design

[0277] The research process is as follows: Figure 7A As shown. After CLP or sham surgery, animals were kept at rest for 20 hours with free access to water and food. They were then anesthetized, and a tracheotomy was performed with arterial and venous sutures placed. Twenty-four hours after CLP, 2 mg / kg of mAbDPP3 or a carrier (saline) was administered. Hemodynamics were invasively monitored continuously from t=0 to 3 hours. Echocardiography of the heart was performed immediately after surgery, and at 15 minutes, 1 hour, 2 hours, and 3 hours after mAbDPP3 or saline injection. -CLP model of sepsis

[0278] Male Wistar rats (2 to 3 months old, 300 to 400 g, group sizes see Table 1) from the Centre d'élevage Janvier (France) were randomly assigned to one of three groups. All animals were anesthetized intraperitoneally (ip) with ketamine hydrochloride (90 mg / kg) and toluidine (9 mg / kg). To induce multimicrobial sepsis, cecal ligation and puncture (CLP) was performed using a slightly modified Rittirsch protocol. A ventral midline incision (1.5 cm) was made to laterally ligate the cecum. The cecum was then attached below the ileocecal valve and punctured once with 18 needles. The peritoneal cavity was then sealed in two layers, followed by fluid resuscitation (subcutaneous injection of 3 ml / 100 g body weight saline) and the animals were returned to their cages. Dummy animals underwent surgery without having their cecum punctured. -Invasive Blood Pressure

[0279] Hemodynamic variables are obtained using the AcqKnowledge system (BIOPAC Systems, Inc., USA). It provides a fully automated blood pressure analysis system. A catheter is connected to the BIOPAC system via a pressure sensor.

[0280] For this procedure, the rats were anesthetized (ketamine and toluidine). The animals were moved to a heating pad to achieve the desired body temperature of 37°C to 37.5°C. A temperature feedback probe was inserted into the rectum. The rats were placed on a control table in a supine position. The trachea was opened, and a 16G catheter was inserted as an external ventilator without damaging the carotid artery and vagus nerve. The ductus arteriosus was inserted into the right carotid artery. The carotid artery was separated from the vagus nerve before ligation.

[0281] A central venous catheter was inserted through the left jugular vein to allow for drug administration.

[0282] Following surgery, the animal was allowed to rest to achieve a stable state before hemodynamic measurements. Baseline blood pressure (BP) was then recorded. During data collection, saline infusion through the arterial line was stopped. - Echocardiography

[0283] The animals were anesthetized with ketamine hydrochloride. The incision was shaved and the rats were placed in a recumbent position.

[0284] For transthoracic echocardiography (TTE), a commercial GE Healthcare Vivid 7 ultrasound system equipped with a high-frequency (14-MHz) linear probe and a 10-MHz cardiac probe was used. All examinations were digitally recorded and stored for subsequent offline analysis.

[0285] Grayscale images were recorded at a depth of 2 cm. A two-dimensional examination was initiated in a parasternal long-axis view to measure the diameters of the aortic rings and pulmonary artery. We also measured left ventricular (LV) dimensions and assessed the fractional shortening (FS%) using M-mode. LVFS was calculated as LV end-diastolic diameter – LV end-systolic diameter / LV end-diastolic diameter, expressed as a percentage. Therefore, the duration of end-diastole was defined at the maximum diameter of the LV. Thus, end-systole was defined as the minimum diameter within the same cardiac cycle. All parameters were measured manually. Three cardiac cycles were performed for each measurement.

[0286] Pulmonary artery blood flow was recorded using pulsed-wave Doppler from the same parasternal long-axis view. The velocity-time integral of pulmonary outflow was measured.

[0287] Mitral valve blood flow was recorded at the tip level of the mitral valve using pulsed Doppler in the top five-chamber view. -Experimental time points and animal groups

[0288] Baseline blood pressure and echocardiography were recorded after surgery. Then, mAbDPP3 was injected. (2 mg / kg) or carrier (saline) (iv, 5 minutes after surgery) and begin saline infusion. Hemodynamic points (BP and echocardiography) were recorded at 15 minutes, 1 hour, 2 hours, and 3 hours after mAbDPP3 or the carrier injection. One control group and two CLP groups were included, summarized in Table 9 below. At the end of the experiment, animals were euthanized, blood was collected for EDTA-plasma production, and organs were harvested for subsequent analysis. Group Group size CLP deal with 1-False 7 no brine 2-CLP-Saline 7 yes brine 3-CLP-mAbDPP3 10 yes mAbDPP3

[0289] Table 1: Experimental Groups 11.2. Results:

[0290] Compared to the sham animals, septic rats exhibited very low blood pressure and reduced cardiac shortening fraction. Administration of mAbDPP3 significantly increased the shortening fraction ( Figure 7B ), which increased average blood pressure ( Figure 7C It also greatly improved the health of septic rats. 12. Example 12

[0291] The purpose of the study described in this article is to evaluate the potential antiproliferative effects of mAbDPP3 in in vitro cell culture systems using a variety of cancer cell lines. 12.1. Method:

[0292] The stock solution of mAbDPP3 (1 mg / ml in PBS) was diluted to cover a final concentration range of 0 μg / ml to 100 μg / ml. PBS was used as a reference compound. Cancer cells derived from established cancer cell lines (A549, HCT116, MDA-MB231) were cultured in DMEM containing 10% FCS and penicillin / streptomycin.

[0293] A549 cells are alveolar basal epithelial cells from adenocarcinoma patients. This cell line was first established by removing and culturing cancerous lung tissue from an explanted tumor in a 58-year-old male. In nature, these cells are squamous and responsible for the diffusion of certain substances (e.g., water and electrolytes) into the alveoli. When cultured in vitro, the A549 cells grow as a monolayer, adhering to the culture flask. Another characteristic is that these cells are capable of synthesizing lecithin and contain high levels of desaturated fatty acids. The A549 cell line is widely used as an in vitro model for drug metabolism in type II lung epithelial cells and as a transfection host.

[0294] The HCT116 cell line represents human colon cancer cells. These epithelial cells are adherent and derived from male adults. This cell line is a suitable transfection host. The line contains a mutation at codon 13 of the ras proto-oncogene and can be used as a positive control for PCR assays of mutations in this codon.

[0295] The MDA-MB231 cell line represents human breast cancer cells with an epithelial morphology. These cells were isolated from pleural effusion in Caucasian breast cancer patients.

[0296] For each cell line, prepare a 96-well suspension cell culture plate. Pour in 100 μL of soft agar bottom layer (0.6% final concentration in complete medium) and allow it to solidify. Then add 50 μL of soft agar top layer (0.4% final concentration) containing the corresponding number of cells, solidify, and incubate the 96-well plate overnight at 37°C and 10% CO2.

[0297] The next day, the compound was added to the wells of the plate. The analyte was then incubated in a cell culture incubator. Finally, the assay was developed using Alamar Blue, and fluorescence intensity (excitation: 560 nm; emission: 590 nm) was determined after incubation at 37°C for 3–5 hours. As a low control, cells were treated with 10–5 M astrococcus (6-fold value). As a high control, cells were treated with 0.1% DMSO (solvent control, 6-fold value).

[0298] The raw data were converted into soft agar growth percentages relative to the high control (0.1% DMSO solvent) and low control (10 to 5 M astrococcus), which were set at 100% and 0%, respectively. IC 50 The calculations were performed using GraphPadPrism 5 software with a variable slope S-shaped response fitting model, with 0% soft agar growth as the bottom constraint, no bottom constraint, and 100% soft agar growth as the top constraint. 12.2. Results:

[0299] In cell culture systems, depending on the applied dose of mAbDPP3 ( Figure 8 The growth of three cancer cell lines (A549, HCT116, and MDA-MB231) was evaluated. IC50 was determined using standard parameters based on the signal from the solvent control as the top constraint (100% soft agar growth) and the signal from the astrococcal control as the bottom constraint (0% soft agar growth). 50 Values. Each IC 50 The values ​​are summarized in Table 10.

[0300] MAbDPP3 treatment had an anti-proliferative effect on the three cell lines tested. cell lines Organization source Incubation time <![CDATA[IC 50 ]]> A549 lung 8 days 6.3 μg / ml HCT116 colon 8 days 2.0 μg / ml MDA-MB231 mammary gland 11 days 8.7μg / ml

[0301] Table 10: IC50 values ​​after mAbDPP3 treatment. 13. Example 13

[0302] The purpose of the study described in this article is to evaluate the ability of mAbDPP3 to prevent tumor formation in xenograft models of breast and colon cancer (tumor growth inhibition study). 13.1. Method:

[0303] Monolayers of MDA-MB-231 cells (breast cancer) and HCT-116 cells (colon cancer) were grown in DMEM + 10% FCS. Cells were cultured at 37°C in a humidified atmosphere of 90% air and 10% carbon dioxide. The medium was routinely changed every 3 days. Confluent cultures were separated every 3 to 4 days using trypsin / EDTA at a ratio of 1:3 to 1:3 and at approximately 3 to 4 × 10⁻⁶ cells / day. 6 Cells / 15cm 2 Inoculate at a density of +25 mL of culture medium.

[0304] Female BALB / c nude mice (CAnN.Cg-Foxn1) that were 4 to 5 weeks old at the time of parturition and weighed approximately 15g to 18g were selected. nu / Crl) mice (Charles River GmbH, Sulzfeld, Germany) were kept under optimal sanitary conditions, with air conditioning provided at 10 to 15 ventilations per hour, continuous environmental monitoring, a target temperature range of 22±3°C, a relative humidity of 30% to 70%, and 12 hours of artificial fluorescent lighting / 12 hours of darkness. A maximum of 4 animals were housed in each ventilated cage (IVC) and fed with food prepared by M-Zucht (ssniff) The diet consists of community-labeled water (from GmbH) and autoclaved water.

[0305] Human breast cancer MDA-MB-231 cells and colon cancer HCT-116 cells, provided by ATCC, will be used in this study. The cells were passaged 5 times before being inoculated into mice. 3 × 10⁻⁶ cells were then used. 6 One cell / 0.1 mL was subcutaneously injected into the right side of the mouse. When the tumor volume reached approximately 100 mm²... 3 Up to 200mm 3 At that time, 20 mice with tumors of appropriate size were randomly divided into groups of 10 mice each, based on tumor volume and body weight. Dosage was administered to the animals according to tumor volume and body weight. These groups are shown in Table 11 below.

[0306] Table 11: Overview of treatment strategies.

[0307] Observe the animal's behavior and health status daily, and record tumor growth every two days over 24 days or several days using calipers. Measure the size of the primary tumor using calipers (manual calipers, OMC Fontana). Calculate according to the formula V = W. 2The tumor size is calculated using the formula ×L / 2 (L = length, W = vertical width of the tumor, L > W). The relative tumor volume (RTV) is calculated as follows: RTV=V t / V0, where V t V0 is the daily volume, while V0 is the volume at the start of treatment. 13.2. Results:

[0308] In xenograft cancer models, administration of mAbDPP3 reduced the formation of all tumors studied. Figures 9A to 9D Treatment with mAbDPP3 induced breast cell tumors to grow for an additional 2 days, reaching a size 20 times larger. Figure 9B The colon cell tumor grew to 10 times its original size in just 2 days. Figure 9D In this model, tumors induced by colon cancer cell lines grow much slower than tumors induced by breast cancer cell lines. 14. Example 14

[0309] To assess the feasibility of using DPP3-adsorbents to remove excess DPP3 from plasma, the binding of DPP3 to the anti-DPP3 column should be analyzed. Affinity chromatography columns were prepared by immobilizing DPP3-binding antibodies onto GlycoLink (Thermo Fisher) columns. The binding of DPP3 to these columns was analyzed by measuring the DPP3 concentration before and after passing through them. 14.1. Method:

[0310] In the first step, each of the DPP3-binding antibodies (mAbDPP3_2552, 2553, 2554, 2555) was oxidized and immobilized on a GlycoLink column according to the instructor's manual. Subsequently, recombinant GST-hDPP3 (USBio) or patient plasma samples were loaded onto the column and DPP3 binding was monitored. - Antibody oxidation

[0311] Therefore, 300 μl of each 3 mg / ml anti-DPP3 antibody solution was diluted to a final volume of 1 ml with pH < 6 in 700 μl of GlycoLink coupling buffer. To oxidize the carbohydrate groups of the antibody, 2.1 mg of sodium periodate was added to the solution and incubated at room temperature for 30 minutes. The oxidant was removed from the antibody solution using a desalting column. Preparation of GlycoLink columns

[0312] To catalyze the coupling reaction, 0.1 M aniline was added to the oxidized antibody. The solution was then applied to an equilibrated GlycoLink column and incubated at room temperature for 4 hours. Unbound material was allowed to flow through the column. The column was then washed, equilibrated, and stored until further use. -DPP3 affinity purification

[0313] Dilute recombinant GST-hDPP3 or patient plasma in conjugation buffer (final concentration of recombinant DPP3 = 100 ng / ml, 1 ml; 1 ml plasma + 1 ml conjugation buffer), apply to a equilibrated mAbDPP3 column, and incubate at room temperature for 30 minutes. Maintain the entire flowthrough to assess binding efficiency and capacity. Wash the column, elute bound proteins with GlycoLink elution buffer, and equilibrate the column before storage. -Analysis of DPP3 contents

[0314] The DPP3 concentration of the sample and recombinant GST-hDPP3 prior to affinity purification and flow-through was measured using a sandwich-type luminescent immunoassay (see Example 4 for details). After adding the labeled and diluted detection antibody (200 μl), 20 μl of sample (or calibrator) was pipetted into a coated 96-well microplate and incubated at 2°C to 8°C for 18 to 24 hours. Unbound tracer was removed by washing four times with 350 μl of washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Well-bound chemiluminescence was measured using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). 14.2. Results:

[0315] Almost all DPP3 in the plasma and recombinant DPP3 solution was removed from the samples by affinity chromatography (see Table 12). Different mAbDPP3 antibodies showed varying affinities for DPP3. Since mAbDPP3_2555 exhibited the highest recDPP3 binding rate, this antibody was chosen for the chromatography of plasma samples. Table 12 shows that affinity chromatography significantly reduced plasma DPP3 levels. These results indicate that DPP3-adsorbents can be used in plasma exchange procedures to remove excess DPP3 from patient plasma.

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[0317] Figure 1: Inhibition of DPP3 activity (A) The activity of recombinant GST-hDPP3 was measured in the presence of a variety of different anti-DPP3 antibodies. DPP3-binding antibodies against peptides and / or full-length (FL) native DPP3 showed strong inhibitory effects of up to 70%. (B) Inhibition curve of recombinant GST-hDPP3 with inhibitory mAbDPP3. The inhibition of DPP3 by the specific antibody is concentration-dependent, with IC50 at 100%. 50 It is ~0.2μg / ml.

[0318] Figure 2: DPP3 concentration as a diagnostic marker (A) DPP3 concentrations in EDTA plasma of healthy controls and patients with various diseases (AHF - acute heart failure, MI - myocardial infarction, sepsis, cancer, AKI - acute kidney injury, LRTI - lower respiratory tract infection). The median concentrations in the patient groups were significantly different from those in the healthy controls (Mann-Whitney test, p < 0.005). (B) Comparison of plasma DPP3 concentrations between patients who died shortly after admission to the emergency department and survivors. Survivors showed significantly lower DPP3 levels (Mann-Whitney test, p<0.05).

[0319] Figure 3 DPP3 activity as a diagnostic marker

[0320] DPP3 activity in EDTA plasma was measured in healthy controls and patients with various diseases (AHF - acute heart failure, sepsis, AKI - acute kidney injury, LRTI - lower respiratory tract infection). The median activity in the patient group was significantly different from that in the healthy control group (Mann-Whitney test, p < 0.0001).

[0321] Figure 4: ROC plot analysis of DPP3 activity and concentration determination. (A) ROC analysis of healthy controls and patients with AHF. (B) ROC analysis of healthy controls and patients with sepsis.

[0322] Figure 5 Safety of mAbDPP3 treatment (blood pressure)

[0323] Healthy rats were treated with either PBS or mAbDPP3 (5.75 mg / kg). Blood pressure (BP) was measured and recorded by inserting a catheter into the right common carotid artery. The administration and sampling catheters were inserted into the jugular vein. Treatment slightly increased relative blood pressure compared to PBS-treated rats (n=3 per group).

[0324] Figure 6 Effects of mAbDPP3 on mortality in septic mice

[0325] Septic mice (CLP model) were treated with PBS or mAbDPP3 (1.9 mg / kg) 5 minutes before and 2 hours after CLP. Mortality was monitored over 7 days. Kaplan-Meyer plots showed increased survival in septic mice after mAbDPP3 treatment.

[0326] Figure 7: Effects of mAbDPP3 on heart failure in septic rats (A) Experimental design for a study of heart failure in rats with septic shock. (B) CLP induced heart failure in rats, as indicated by a reduced shortening fraction compared to sham animals. Treatment with mAbDPP3 significantly increased this shortening fraction (2 mg / kg; n ≥ 7 per group; Mann-Whitney test, p < 0.0001). (C) The mean blood pressure of septic rats treated with the carrier decreased over time, while mAbDPP3 treatment led to a significant increase in mBP (2 mg / kg; n≥7 per group; Mann-Whitney test, p<0.005).

[0327] Figure 8 Effects of mAbDPP3 on in vitro tumor growth

[0328] Soft agar assays of tumor cell lines (lung cancer, colon cancer, and breast cancer). The addition of anti-DPP3 antibody reduced tumor cell growth rate.

[0329] Figure 9: Effect of mAbDPP3 on tumor growth in vivo (A) Mice with xenografted breast tumor cells were treated with PBS or mAbDPP3 (n=10 per group). Growth curves of relative tumor volume over 24 days showed reduced tumor growth in mAbDPP3-treated mice. (B) Time comparison: Breast cell tumors required a 20-fold increase in volume with and without mAbDPP3 treatment. With mAbDPP3 treatment, growth required significantly longer (Mann-Whitney test, p<0.05). (C) Mice with xenografted colon tumor cells were treated with PBS or mAbDPP3 (n=10 per group). Growth curves of relative tumor volume over 30 days showed reduced tumor growth in mice treated with mAbDPP3. (D) Time comparison: Colonic cell tumors required a 10-fold increase in volume when treated with and without mAbDPP3. Growth took longer when treated with mAbDPP3.

[0330] Figure 10 DPP3 activity as a diagnostic marker (II)

[0331] DPP3 activity in EDTA plasma was measured in healthy controls and patients with various diseases (acute myocardial infarction (AMI), cardiogenic shock, septic shock, and liver failure). The median activity in the patient group was significantly different from that in the healthy controls (Mann-Whitney test, p<0.05).

[0332] Figure 11 Effects of DPP3 on blood pressure in healthy rats Healthy male Wistar rats were injected with 0.2 mg / kg of recombinant GST-hDPP3. Blood pressure (BP) was measured and recorded by inserting a catheter into the right carotid artery. DPP3 was administered intravenously via the tail vein. DPP3 injection resulted in a decrease in BP.

Claims

1. A method for diagnosing a disease or condition in an object with or related to a necrosis process, comprising: • Determine the total amount of DPP3 in the body fluid sample of the subject. • Compare the determined total DPP3 with a predetermined threshold. • Wherein, if the determined quantity is higher than the predetermined threshold, the object is diagnosed as having a disease or condition accompanied by or related to a necrosis process. • The samples mentioned are selected from whole blood, serum and plasma.

2. The method for diagnosing a disease or condition in an object with or related to a necrosis process, as claimed in claim 1, wherein the amount of total DPP3 is determined in units of concentration.

3. The method for diagnosing a disease or condition of an object with or related to a necrosis process, as described in claim 1 or 2, wherein the necrosis process is a process that leads to cell death and the release of DPP3 from the cytoplasm into the extracellular space and / or the body fluids of the object.

4. The method for diagnosing a disease or condition of an object with or related to a necrotic process, according to any one of claims 1 to 3, wherein the disease or condition is selected from heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infection (microorganisms, viruses (e.g., AIDS), parasites (e.g., malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g., epilepsy, neurodegenerative diseases), autoimmune diseases and vascular diseases (e.g., Kawasaki syndrome), and hypotension.

5. A method for diagnosing a disease or condition in an object with or related to a necrosis process, according to any one of claims 1 to 4, wherein the method comprises contacting the sample in the body fluid of the object with a capture binder that specifically binds full-length DPP3 (SEQ ID NO. 1).

6. The method of claim 5 for diagnosing a disease or condition of an object with or related to a necrosis process, wherein the capture binder may be selected from antibodies, antibody fragments, or non-IgG scaffolds.

7. The method for diagnosing a disease or condition of an object with or related to a necrosis process, as described in claim 5 or 6, wherein the capture binder is an antibody.

8. A method for diagnosing a disease or condition of an object with or related to a necrosis process, according to any one of claims 5 to 7, wherein the capturing binder is fixed to the surface.

9. A method for diagnosing a disease or condition of an object with or related to a necrosis process, according to any one of claims 5 to 8, wherein the method includes a separation step comprising immobilizing a complex formed of a capture binder and DPP3.

10. The method of claim 9 for diagnosing a disease or condition in an object with or related to a necrosis process, wherein the separation step is a washing step in which sample components not bound to the capture binder are removed from the captured DPP3.

11. The method for diagnosing a disease or condition of an object with or related to a necrotic process, according to any one of claims 1 to 10, wherein the amount of DPP3 is determined by an immunoassay.

12. The method of claim 11 for diagnosing a disease or condition in an object with or related to a necrotic process, wherein the immunoassay is a sandwich assay in which the DPP3 to be detected and / or quantified or a fragment thereof binds to a first antibody and a second antibody.

13. A method for monitoring a disease or condition in an object with or related to a necrosis process, wherein the diagnostic method according to any one of claims 1 to 12 is performed at least twice.

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