Method for determining DPP3 and therapeutic method

Abstract

To provide a method for diagnosing a disease or a condition in a subject accompanied by or related to necrotic processes.SOLUTION: Active DPP3 in a bodily fluid sample is determined.SELECTED DRAWING: Figure 2A

Description

[Technical field]

[0001] The present invention relates to a method for quantifying active DPP3 in a body fluid sample, an assay or kit for quantifying active DPP3 in a body fluid sample, a method for diagnosing a disease or condition in a subject which involves or is associated with a necrotic process, and a method for treating said disease. [Background technology]

[0002] Dipeptidyl peptidase 3 (also known as dipeptidyl aminopeptidase III, dipeptidyl arylamidase III, dipeptidyl peptidase III, enkephalinase B or erythrocyte angiotensinase, abbreviated DPP3 or DPPIII) is a metallopeptidase that removes dipeptides from bioactive peptides such as enkephalins and angiotensin.

[0003] DPP3 was first identified and its activity measured in purified bovine anterior pituitary extracts by Ellis and Nuenke in 1967. The enzyme, listed as EC 3.4.14.4, has a molecular weight of approximately 83 kDa and is highly conserved in prokaryotes and eukaryotes (Prajapati & Chauhan 2011). The amino acid sequence of its human variant is shown in SEQ ID NO:1. Dipeptidyl peptidase III is a ubiquitously expressed, primarily cytoplasmic peptidase. Despite the absence of a signal sequence, activity at membranes has been reported in several studies (Lee & Snyder 1982).

[0004] DPP3 is a zinc-dependent exopeptidase belonging to the M49 peptidase family, with broad substrate specificity for oligopeptides of 3, 4 to 10 amino acids of various compositions, and can also cleave after proline. DPP3 is known to hydrolyze dipeptides from the N-terminus of its substrates (angiotensin II, III, IV; Leu- and Met-enkephalins; endomorphins 1 and 2, etc.). DPP3, a metallopeptidase, has optimal activity at pH 8.0–9.0 and is highly cleavable by Co 2+ and Mg 2+ DPP3 can be activated by the addition of divalent metal ions such as . Structural analysis of DPP3 revealed catalytic motifs HELLGH (hDPP3, 450-455) and EECRAE (hDPP3, 507-512) with the following amino acids Glu316, Tyr318, Asp366, Asn391, Asn394, His568, Arg572, Arg577, Lys666, and Arg669 that are important for substrate binding and hydrolysis (Prajapati & Chauhan 2011 and Kumar et al. 2016; numbers indicate 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 between amino acids 316-669.

[0005] DPP3 activity can be nonspecifically inhibited by a variety of general protease inhibitors (e.g., PMSF, TPCK), sulfhydryl reagents (e.g., pHMB, DTNB), and metal chelators (EDTA, o-phenanthroline) (Abramic et al. 2000).

[0006] DPP3 activity can be specifically inhibited by a variety of compounds: the endogenous DPP3 inhibitor is a peptide called spinorphin. Several synthetic spinorphin derivatives (e.g., tinorphin) have been produced and shown to inhibit DPP3 activity to various degrees (Yamamoto et al. 2000). Other published peptide inhibitors of DPP3 are propioxatin A and B (US4804676), as well as propioxatin A analogues (Inaoka et al. 1988).

[0007] DPP3 can also be inhibited by small molecules such as fluostatins and benzimidazole derivatives. Fluostatins A and B are antibiotics produced by Streptomyces sp. TA-3391 that are non-toxic and strongly inhibit DPP3 activity. Twenty different benzimidazole derivatives have been synthesized and published to date (Agic et al. 2007; Rastija et al. 2015), of which two compounds 1' and 4' show the strongest inhibitory effects (Agic et al. 2007). See Table 2 for a complete list of DPP3 inhibitors.

[0008] Although the exact biological function of DPP3 in cellular physiology is unknown, recent findings indicate that it plays a role not only in protein metabolism but also in blood pressure control, pain regulation and inflammatory processes (Prajapati & Chauhan 2011).

[0009] DPP3 has been shown to be a promising biomarker in several publications (all describing intracellular DPP3). DPP3 activity has been shown to be elevated in homogenates of ovarian and endometrial tumors. DPP3 activity further increases with the severity / grade of these tumors (Simaga et al. 1998, 2003). Immunohistochemical and western blot analysis of glioblastoma cell lines also showed elevated DPP3 levels (Singh et al. 2014).

[0010] Intracellular or membrane DPP3 has also been proposed to be a promising arterial risk marker (US2011008805) and a marker for rheumatoid arthropathy (US2006177886). Patent application WO2005106486 describes the expression of DPP3 and its activity as a diagnostic marker for all kinds of diseases, as well as DPP3 as a therapeutic agent, due to its ubiquitous expression at or within the cell surface. EP1498480 describes potential diagnostic and therapeutic applications of hydrolases, including DPP3.

[0011] DPP3 can cleave several bioactive peptides and has therefore been proposed not only as a promising biomarker but also as a potential therapeutic target. Overexpression of DPP3 protects neuroblastoma cells from oxidative stress (Liu et al. 2007). Influenza A virus alters host DPP3 levels for replication (cell culture studies, Meliopoulos et al. 2012). Enkephalin and / or angiotensin degrading enzymes in general, including DPP3, have therapeutic potential as targets for the treatment of pain, cardiovascular disease (CVD), and cancer, and corresponding inhibitors as promising treatments for pain, psychiatric disorders, and CVD (Khaket et al. 2012, Patel et al. 1993, Igic et al. 2007).

[0012] Although DPP3 is known as an intracellular protein, DPP3 activity has been detected in several body fluids, namely postplacental serum (Shimamori et al. 1986), seminal plasma (Vanha-Perttula et al. 1988), and cerebrospinal fluid (CSF) (Aoyagi et al. 1993). In CSF, elevated levels of DPP3 activity were measured in patients with Alzheimer's disease (AD, Aoyagi et al. 1993). Wattiaux et al. (2007) have proposed the release of intracellular DPP3 as a marker of dead and / or dying cells in cell culture systems. It has also been proposed that the release of DPP3 from necrotic cells affects the immune response in a mouse model (Gamrekelashvili et al. 2013). Summary of the Invention

[0013] An object of the present invention is to provide a method for specifically quantifying active DPP3 in a body fluid sample (ie, the active DPP3 is quantified but aminopeptidases other than DPP3 are not quantified). It is an object of the present invention to provide respective assays and kits.

[0014] Another object of the present invention is to provide methods for diagnosing a disease or condition in a subject involving or associated with a necrotic process, and methods for treating said disease.

[0015] The subject of the present invention is a method for quantifying active DPP3 in a body fluid sample of a subject, comprising the steps of: contacting the sample with a capture binding agent that specifically binds full-length DPP3; Separating the DPP3 bound to the capture binding agent; adding a DPP3 substrate to the separated DPP3; Quantifying DPP3 activity by measuring the conversion rate of the DPP3 substrate; The method includes:

[0016] In certain embodiments of the invention, said method is an enzyme capture assay (ECA; see, for example, US Pat. No. 5,612,186A and US Pat. No. 5,601,986A). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] All definitions and specific aspects specified herein shall apply to all aspects and objects of the present invention. However, definitions and specific aspects detailed for one aspect or object of the present invention shall also be definitions and specific aspects for other aspects and objects of the present invention. For example, the definition of capture binding agent or specific capture shall apply to all aspects of the present invention (e.g., the method, assay and kit for quantifying DPP3, diagnostic method, therapeutic method). Such definitions may not be repeated in this specification.

[0018] The term "specifically binds to full-length DPP3" means that the capture binding agent does not bind to proteins other than DPP3. Also, the term "specifically binds to full-length DPP3" means that the capture binding agent does not bind to aminopeptidases other than DPP3.

[0019] A binding agent that binds to full length DPP3 is one that binds to the protein of SEQ ID NO: 1. A binding agent that binds to full length DPP3 is one that binds to SEQ ID NO:1.

[0020] In certain embodiments, the capture binding agent inhibits DPP3 activity by less than 50%, preferably less than 40%, preferably less than 30% in a solution phase assay, which is an assay in which cleavage of the substrate by DPP3 occurs in the solution phase.

[0021] Inhibition of DPP3 activity by a binding agent in a liquid phase assay may be quantified according to the present invention in the following manner: In a liquid phase assay, a potential DPP3 capture binding agent is incubated with recombinant DPP3 or purified native DPP3, as well as a specific DPP3 substrate. It is preferable to select a capture binding agent for ECA that has the lowest inhibitory potential. The capture binding agent should inhibit DPP3 activity by less than 50%, preferably less than 40%, preferably less than 30%. A specific liquid phase DPP3 activity assay for quantifying the inhibitory potential of a potential capture binding agent is described in detail in Example 1 and includes the following steps: · 25 ng / ml of recombinant GST-hDPP3 was dissolved in 5 μg / ml of each capture binding agent or buffer (control) [50 mM Tris-HCl (pH 7.5), 100 μM ZnCl2] and incubated at room temperature for 1 hour. Add the fluorogenic substrate Arg-Arg-βNA (20 μl, 2 mM). The formation of free βNA was monitored for 1 h at 37°C using a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH), detecting βNA fluorescence by exciting at 340 nm and measuring emission at 410 nm. The slope of the fluorescence increase (relative fluorescence units (RFU) / min) of the various samples is calculated. The slope for GST-hDPP3 and buffer (control) is taken as 100% activity. The inhibitory capacity of a potential capture binding agent is defined as the % reduction in GST-hDPP3 activity upon incubation with said capture binding agent.

[0022] In contrast, a solid phase assay is one in which each binding event occurs in the solid phase (see Examples 4 and 5).

[0023] For clarity, the method for quantifying active DPP3 may be performed as a liquid phase assay or as a solid phase assay, but inhibition of DPP3 activity may be quantified in a liquid phase assay according to the procedure described above.

[0024] Thus, a solid-phase assay is one aspect of the present invention. Contacting the sample with a capture binding agent that specifically binds to full-length DPP3 may occur in liquid phase (liquid-phase capture assay), and then a separation step may include immobilizing the complex of the capture binding agent and DPP3. Alternatively, the capture binding agent may be immobilized on a surface, and the binding event (of the capture binding agent to DPP3) occurs in solid phase (solid-phase capture assay).

[0025] In one particular embodiment, in order to avoid complete inhibition of DPP3 in the solution phase assays described above and inhibit DPP3 activity by less than 50%, preferably less than 40%, preferably less than 30%, it is preferred that the capture binding agent does not bind DPP3 within or to the region of amino acids 316-669 of SEQ ID NO: 1. The region of amino acids 316-669 of SEQ ID NO: 1 contains the active center and substrate binding region of DPP3 (Prajapati & Chauhan 2011; Kumar et al. 2016). DPP3 activity can be measured by detecting cleavage products of a DPP3-specific substrate.

[0026] Known peptide hormone substrates include angiotensin II, III, IV, Leu enkephalin, Met enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH (adrenocorticotropic hormone), and MSH (melanocyte-stimulating hormone) (Abramic et al. 2000, Barsun et al. 2007, Dhanda et al. 2008). Cleavage of the above peptide hormones and other oligopeptides without tags (e.g., Ala-Ala-Ala-Ala (Dhanda et al. 2008)) can be monitored by detection of the respective cleavage products. Detection methods include, but are not limited to, HPLC analysis (e.g., Lee & Snyder 1982), mass spectrometry (e.g., Abramic et al. 2000), H1-NMR analysis (e.g., Vandenberg et al., 1985), capillary zone electrophoresis (CE; e.g., Barsun et al., 2007), thin-layer chromatography (e.g., Dhanda et al. 2008), and reverse-phase chromatography (e.g., Mazocco et al. 2006).

[0027] Detection of fluorescence resulting from hydrolysis of fluorogenic substrates by DPP3 is a standard method for monitoring DPP3 activity. These substrates are specific di- or tripeptides conjugated to fluorophores (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe). Fluorophores include, but are not limited to, β-naphthylamide (2-naphthylamide, βNA, 2NA), 4-methoxy-β-naphthylamide (4-methoxy-2-naphthylamide), and 7-amino-4-methylcoumarin (AMC, MCA; Abramic et al. 2000, Ohkubo et al. 1999). Cleavage of these fluorogenic substrates liberates fluorescent β-naphthylamide or 7-amino-4-methylcoumarin, respectively. In a liquid-phase assay or ECA, the substrate and DPP3 are incubated in, for example, a 96-well plate format, and the fluorescence is measured with a fluorescence detector (Ellis & Nuenke 1967). Alternatively, DPP3-loaded samples can be immobilized and separated by electrophoresis in a gel. The gel is stained with a fluorogenic substrate (e.g., Arg-Arg-βNA) and fast garnet GBC, and the fluorescent protein bands are detected with a fluorescence reader (Ohkubo et al. 1999).

[0028] 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 also be conjugated to a chromophore such as p-nitroanilide diacetate. Detection of a color change upon hydrolysis of the chromogenic substrate can be used to monitor DPP3 activity.

[0029] Another option for detecting DPP3 activity is the Protease-Glo™ Assay (commercially available from Promega). In this embodiment of the method, a dipeptide or tripeptide specific for DPP3 (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) is conjugated to aminoluciferin. Upon cleavage by DPP3, the aminoluciferin is released and serves as a substrate for a reaction with the bound luciferase that produces detectable luminescence.

[0030] In a preferred embodiment, DPP3 activity is measured by adding the fluorogenic substrate Arg-Arg-βNA and monitoring fluorescence in real time.

[0031] In certain embodiments of the method of quantifying the inhibitory effect and / or DPP3 activity of a DPP3-binding agent in a body fluid sample of a subject, the binding agent may be selected from the group of an antibody, an antibody fragment, a non-IgG scaffold.

[0032] An antibody of the present invention is a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes that specifically bind to an antigen. Recognized immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma (IgG1, IgG2, IgG3, IgG4), delta (IgD), epsilon (IgE), and mu (IgM) constant region genes, as well as various immunoglobulin variable region genes. Full-length immunoglobulin light chains are generally about 25 kD or 214 amino acids in length. Full-length immunoglobulin heavy chains are generally about 50 kD or 446 amino acids in length. Light chains are encoded by a variable region gene (about 110 amino acids in length) at the NH2-terminus and a kappa or lambda constant region gene at the COOH-terminus. Heavy chains are similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.

[0033] The basic structural unit of an antibody generally consists of two identical pairs of immunoglobulin chains, each pair being a tetramer with 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 functions. Immunoglobulins also exist in a variety of other forms, such as 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 region of an immunoglobulin light or heavy chain contains a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDRs) (see Kabat et al. 1983). As mentioned above, the CDRs are primarily responsible for binding to an epitope of an antigen. An immune complex is an antibody (such as a monoclonal, chimeric, humanized or human antibody, or a functional antibody fragment) that specifically binds to an antigen.

[0034] Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. For example, the variable segments of genes from a mouse monoclonal antibody can be linked to human constant segments such as kappa, gamma 1 or gamma 3. Thus, in one example, a therapeutic chimeric antibody is a hybrid protein consisting of the variable or antigen binding domain from a mouse antibody and the constant or effector domain from a human antibody (although other mammalian species can be used), or the variable regions can be generated by molecular techniques. Methods for generating chimeric antibodies are well known in the art (see, for example, U.S. Pat. No. 5,807,715). A "humanized" immunoglobulin is an immunoglobulin that contains a human framework region and one or more CDRs from a non-human (mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor". In one embodiment, all CDRs of the humanized immunoglobulin are from the donor immunoglobulin. Constant regions may be absent, but if present, they must be substantially identical to the constant regions of human immunoglobulins, i.e. at least about 85-90% (e.g. about 95% or more) identical. Thus, all parts of a humanized immunoglobulin (except possibly the CDRs) are substantially identical to the corresponding parts of a natural human immunoglobulin sequence. A "humanized antibody" is an antibody that comprises a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin, i.e., an antibody, may have a limited number of substitutions with amino acids taken from the donor framework. Humanized or other monoclonal antibodies may also have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions.Examples of conservative substitutions include, for example, glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Humanized immunoglobulins can be constructed by genetic engineering (see, for example, U.S. Pat. No. 5,585,089). Human antibodies are antibodies whose light and heavy chain genes are of human origin. Human antibodies can be produced using methods known in the art. Human antibodies can be made by immortalizing human B cells that secrete the antibody of interest. For example, immortalization can be achieved by EBV infection or by fusing human B cells with myeloma cells or hybridoma cells to produce trioma cells. Human antibodies may be made by phage display technology (see, e.g., PCT Publication Nos. 91 / 17271, 92 / 001047, 92 / 20791, which are incorporated herein by reference) or selected from human recombinant monoclonal antibody libraries (see the MorphoSys website). Human antibodies can also be made using transgenic animals harboring human immunoglobulin genes (see, e.g., PCT Publication Nos. 93 / 12227 and 91 / 10741, which are incorporated herein by reference).

[0035] Thus, the DPP3 antibody may have any type known in the art, including, for example, human, monoclonal, humanized, chimeric, CDR-grafted, etc. In a preferred embodiment, the antibody according to the invention is a recombinantly produced antibody, e.g., as a typical full-length immunoglobulin IgG, or as an antibody fragment comprising at least the Fv domain of the heavy and / or light chain (e.g., a chemically bonded antibody (antigen-binding fragment)). Such antibody fragments include Fab fragments, such as Fab minibodies, single chain Fab antibodies, monovalent Fab antibodies with an epitope tag (e.g. Fab-V5Sx2); bivalent Fab (miniantibodies) dimerized with a CH3 domain; bivalent Fab or multivalent Fabs, for example generated by multimerization with heterologous domains (e.g. by dimerization of dHLX domains) (e.g. Fab-dHLX-FSx2); F(ab')2 fragments, for example from classes other than G, scFv fragments, multimerized multivalent and / or multispecific scFv fragments, bivalent and / or bispecific bispecific antibodies, BITE® (bispecific T cell engager), trifunctional antibodies, multivalent antibodies; single domain antibodies (e.g. nanobodies derived from camel or fish immunoglobulins); as well as many others.

[0036] In addition to anti-DPP3 antibodies, other biopolymer scaffolds are known in the art to be conjugated with targeting molecules and used to generate highly target-specific biopolymers, including aptamers, spiegelmers, anticalins, and conotoxins.

[0037] The non-Ig scaffolds may be protein scaffolds, which may be used as antibody mimics, since they can bind to ligands or antigens. Non-Ig scaffolds include non-Ig scaffolds based on tetranectin (e.g., as described in US2010 / 0028995); fibronectin scaffolds (e.g., as described in EP1266025); lipocalin-based scaffolds (e.g., as described in WO2011 / 154420); ubiquitin scaffolds (e.g., as described in WO2011 / 073214), transferrin scaffolds (e.g., as described in US2004 / 0023334), protein A scaffolds (e.g., as described in EP2231860), ankyrin repeat-based scaffolds (e.g., as described in WO2010 / 060748), microprotein (preferably microproteins that form cysteine ​​knots) scaffolds (e.g., as described in EP2314308), Fyn scaffolds (e.g., as described in EP2314308), and the like. The scaffold may be selected from the group including SH3 domain-based scaffolds (e.g., as described in WO2011 / 023685), EGFR-A domain-based scaffolds (e.g., as described in WO2005 / 040229), and Kunitz domain-based scaffolds (e.g., as described in EP1941867). The non-Ig scaffold may be a peptide or oligonucleotide aptamer. Aptamers are usually generated by selecting them from a large random sequence pool and are short variable peptide domains attached to short strands of oligonucleotides (DNA, RNA, or XNA) (Xu et al. 2010, Deng et al. 2014) or protein scaffolds (Li et al. 2011).

[0038] In one embodiment of the present invention, the anti-DPP3 antibody of the present invention may be produced as follows.

[0039] Mice are immunized with either recombinant DPP3 (e.g., GST-hDPP3 from USBio, Salem, USA), a peptide containing a portion of the amino acid sequence of DPP3 (e.g., conjugated to BSA), or native purified DPP3 (e.g., from human erythrocytes (Abramic et al. 1988)).

[0040] Balb / c mice were injected intraperitoneally (ip) with 100 μg of recombinant GST-hDPP3, native purified hDPP3, or DPP3 peptide-BSA complex (emulsified in TiterMax Gold Adjuvant) on day 0, 100 μg (emulsified in Freund's complete adjuvant) on day 14, and 50 μg (in Freund's incomplete adjuvant) on days 21 and 28. On day 49, animals were injected intravenously (iv) with 50 μg of GST-hDPP3, native purified hDPP3, or DPP3 peptide-BSA complex (dissolved in saline). Three days later, mice were sacrificed and immunocytofusion was performed.

[0041] Spleen cells from immunized mice were fused with cells of the myeloma cell line SP2 / 0 using 1 ml of 50% polyethylene glycol at 37°C for 30 seconds. After washing, the cells were seeded into 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium (RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT-Supplement). After 1 week, the HAT medium was replaced with HT medium and subcultured three times, after which the cells were returned to normal cell medium.

[0042] Two weeks after fusion, cell culture supernatants were first screened for recombinant DPP3 binding to IgG antibodies. Thus, recombinant GST-tagged DPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng / well) and incubated with 50 μl / well of cell culture supernatant at room temperature for 2 h. After washing the plates, 50 μl / well of POD-rabbit anti-mouse IgG was added and incubated at room temperature for 1 h. After a further 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 min, and the color reaction was stopped by adding 50 μl of 4N sulfuric acid. Absorbance was detected at 490 mm.

[0043] Positive testing microbial cultures were transferred to 24-well plates for growth. After retesting, selected cultures were cloned and recloned by limiting dilution and isotyped.

[0044] Antibodies against GST-tagged human DPP3 or DPP3 peptides were generated by standard antibody production methods (Marx et al., 1997) and purified with protein A. Antibody purity was ≥90% based on SDS gel electrophoresis analysis.

[0045] Antibodies may be generated by phage display according to the following procedure. The human naive antibody gene library HAL7 / 8 was used to isolate recombinant single-chain Fv domains (scFv) against DPP3 peptides. The antibody gene library was screened with a panning strategy that included the use of peptides with a biotin tag linked to the DPP3 peptide sequence via two different spacers. The background of nonspecific binders was minimized by mixing panning rounds with nonspecifically bound antigens and with antigens bound to streptavidin. Phages eluted from the third panning round are used to generate monoclonal scFv expressing E. coli strains. The supernatants from the culture of these clonal strains are used directly for enzyme-linked immunosorbent assay (ELISA) testing of the antigens (Hust et al. 2011; see also Schutte et al. 2009).

[0046] Humanization of a mouse antibody may be carried out by the following procedure. To humanize an antibody of mouse origin, the antibody sequence is analyzed for structural interactions of the framework regions (FRs) and complementarity determining regions (CDRs) with the antigen. Based on structural modeling, appropriate human-derived FRs are selected and mouse CDR sequences are grafted onto the human FRs. Diversity may be introduced into the amino acid sequences of the CDRs or FRs to restore structural interactions lost due to species switching with respect to the FR sequences. This restoration of structural interactions may be achieved in a random manner using phage display libraries or in a directed manner guided by molecular modeling (Almagro & Fransson 2008).

[0047] In another embodiment, the type of DPP3 antibody is selected from the group comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, F(ab)2 fragment, and scFv-Fc fusion protein. In another preferred embodiment, the type of antibody is selected from the group comprising scFab fragment, Fab fragment, scFv fragment, and bioavailability optimized conjugates thereof (PEGylated fragments).

[0048] In certain embodiments of the method for quantifying active DPP3 in a body fluid sample from a subject, the binding agent is an antibody.

[0049] In one embodiment, a capture or binding assay may be performed to detect and / or quantify DPP3. A binding agent may be immobilized on a solid phase that reacts with DPP3 protein but inhibits peptidase activity by no more than 50% (preferably less than 40%, preferably less than 30%) in a liquid phase assay. In one embodiment, to prevent inhibition of DPP3, the capture binding agent preferably does not bind to the region of amino acids 316-669 of SEQ ID NO: 1 of DPP3. The region of amino acids 316-669 of SEQ ID NO: 1 contains the active center and substrate binding region of DPP3 (Prajapati & Chauhan 2011; Kumar et al. 2016).

[0050] In certain embodiments of the method for quantifying active DPP3 in a body fluid sample of a subject, the binding agent may be selected from the group of antibodies, antibody fragments, non-Ig scaffolds, aptamers.

[0051] In certain embodiments of the method for quantifying active DPP3 in a body fluid sample from a subject, the capture binding agent reactive to DPP3 is immobilized on a solid phase.

[0052] When the test sample is passed over the immobilized binding agent, DPP3 (if present) binds to the binding agent and is itself immobilized for detection. Substrate may then be added and a reaction product may be detected that indicates the presence or amount of DPP3 in the test sample. For purposes of this specification, The term "solid phase" may be used to encompass any material or vessel in which an assay may be performed, including, but not limited to, porous materials, non-porous materials, test tubes, wells, slides, agarose resins (e.g., Sepharose from GE Healthcare Life Sciences), magnetic particles (e.g., Dynabeads™ or Pierce™ magnetic beads from Thermo Fisher Scientific), and the like.

[0053] Protein or peptide-based binding agents (e.g., antibodies, antibody fragments, non-Ig scaffolds) can be immobilized by physical adsorption (e.g., electrostatic or hydrophobic interactions), bioaffinity immobilization (e.g., avidin-biotin, protein A / G / L, His-tag, Ni 2+ -NTA, GST tags, and glutathione, DNA hybridization, aptamers), covalent bonds (e.g., amines and N-hydroxysuccinimide), or a combination of the above immobilization methods (Kim & Herr 2013). Oligonucleotide-derived binders (e.g., aptamers) may be immobilized to the solid phase by utilizing the (strept)avidin-biotin system (Muller et al. 2012, Deng et al. 2014).

[0054] In certain embodiments of the method for quantifying active DPP3 in a body fluid sample from a subject, the separation step is a washing step that removes components of the sample that are not bound to the capture binding agent from the captured DPP3, or may be any other step that separates DPP3 bound to the capture binding agent from components of the body fluid sample.

[0055] In certain embodiments of the method for quantifying active DPP3 in a subject's body fluid sample, the conversion of the DPP3 substrate by immobilized DPP3 is measured (detected) by a method selected from the group including fluorescence of a fluorogenic substrate (e.g., Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, light emission of a substrate conjugated to aminoluciferin (Promega Protease-Glo™ Assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis followed by activity staining (immobilized active DPP3) or Western blot (cleavage products).

[0056] In certain embodiments of the method for quantifying active DPP3 in a subject's body fluid sample, the substrate may be selected from the group including angiotensin II, III, IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH, and MSH; and dipeptides or tripeptides conjugated to a fluorophore, chromophore, or aminoluciferin (Promega Protease-Glo™ 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, Suc-Ala-Ala-Phe, and the like. 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; Abramic et al. 2000, Ohkubo et al. 1999). Cleavage of these fluorogenic substrates liberates fluorescent β-naphthylamide or 7-amino-4-methylcoumarin, respectively. Chromophores include, but are not limited to, p-nitroanilide diacetate (pNA). Hydrolysis of the peptide-pNA bond in the chromogenic substrate liberates pNA and causes a color change. Thus, the change in absorbance (DA / min) is directly proportional to the enzyme activity. When using the Protease-Glo™ Assay (Promega), upon cleavage by DPP3, aminoluciferin is liberated and serves as a substrate for a reaction by bound luciferase that produces detectable luminescence.

[0057] In a preferred embodiment, DPP3 activity is measured by adding the fluorogenic substrate Arg-Arg-βNA and monitoring the fluorescence in real time. In a particular embodiment of the method for quantifying active DPP3 in a body fluid sample of a subject, the sample is selected from the group including whole blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusion.

[0058] In a particular embodiment of the method of quantifying active DPP3 in a body fluid sample of a subject, the sample is a blood sample selected from the group comprising whole blood, serum, and plasma.

[0059] Another aspect of the present invention is a method for producing a 1. An assay or kit for quantifying active DPP3 in a body fluid sample from a subject, comprising: a capture binding agent that specifically binds to full-length DPP3; DPP3 Substrates The present invention is an assay or kit comprising: The kit may further include a calibration sample. Calibrator samples may be samples with known concentrations of DPP3 (native, purified or recombinant). The calibrator may be the cleavage product itself, such as free fluorophore (eg 2-naphthylamide), free chromophore (eg p-nitroanilide) or free luciferin. The kit may further comprise a washing reagent. The washing reagent can be any aqueous buffer, with or without detergent. Here, we used 8 mM Tris-HCl (pH 7.5), 60 mM NaCl, 0.02% Tween 20. In a particular embodiment, the assay is an enzyme capture assay (ECA) (eg US5612186A, US5601986A).

[0060] In a particular embodiment, the capture binding agent that binds to full-length DPP3 specifically inhibits DPP3 activity by less than 50%, preferably less than 40%, more preferably less than 30% in a solution-phase assay. See above for a definition of solution-phase assay. In a particular embodiment, to prevent inhibition of DPP3, the capture binding agent should not bind to the region surrounding the active center and substrate binding region of DPP3 (amino acids 316-669 of SEQ ID NO: 1).

[0061] In certain embodiments of said assay or kit for quantifying active DPP3 in a body fluid sample of a subject, said binding agent may be selected from the group of antibodies, antibody fragments, non-IgG scaffolds.

[0062] In a particular embodiment of the assay or kit for quantifying active DPP3 in a body fluid sample of a subject, the binding agent is an antibody.

[0063] The term "antibody" generally includes monoclonal and polyclonal antibodies, as well as binding fragments thereof (especially Fc fragments and so-called "single chain antibodies" (Bird et al. 1988)), chimeric antibodies, humanized antibodies (especially CDR-grafted antibodies), bi- or tetra-specific antibodies (Holliger et al. 1993). Also included are immunoglobulin-like proteins that specifically bind to a molecule of interest contained in a sample, selected, for example, by techniques such as phage display. In this context, the term "specific binding" refers to an antibody directed against a molecule of interest or a fragment thereof. An antibody is considered to be specific if its affinity for the molecule of interest or said fragment is preferably at least 50 times, more preferably 100 times, and most preferably at least 1000 times, higher than its affinity for other molecules contained in the sample containing the molecule of interest. Methods for generating antibodies and for selecting antibodies with a given specificity are well known in the art.

[0064] In certain embodiments of the assay or kit for quantifying active DPP3 in a subject's body fluid sample, the capture binding agent is immobilized on a surface.

[0065] In certain embodiments of the assay or kit for quantifying active DPP3 in a subject's body fluid sample, the substrate may be selected from the group including angiotensin II, III, IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH, and MSH; and dipeptides or tripeptides conjugated to a fluorophore, chromophore, or aminoluciferin (Promega Protease-Glo™ 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, Suc-Ala-Ala-Phe, and the like. 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; Abramic et al. 2000, Ohkubo et al. 1999). Cleavage of these fluorogenic substrates liberates fluorescent β-naphthylamide or 7-amino-4-methylcoumarin, respectively. Chromophores include, but are not limited to, p-nitroanilide diacetate (pNA). Hydrolysis of the peptide-pNA bond in the chromogenic substrate liberates pNA and causes a color change. Thus, the change in absorbance (DA / min) is directly proportional to the enzyme activity. When using the Protease-Glo™ Assay (Promega), upon cleavage by DPP3, aminoluciferin is liberated and serves as a substrate for a reaction by bound luciferase that produces detectable luminescence.

[0066] In a preferred embodiment, DPP3 activity is measured by adding the fluorogenic substrate Arg-Arg-βNA and monitoring fluorescence in real time.

[0067] In a particular embodiment of said assay or kit for quantifying active DPP3 in a subject's body fluid sample, said calibration samples are selected from the group comprising A) recombinant DPP3 (e.g. GST-hDPP3 from USBio), purified native DPP3 (e.g. derived from human erythrocytes (Abramic et al. 1988), and DPP3 fragments (natural, synthetic, or recombinant); and B) the cleavage products themselves, i.e. free fluorophores (e.g. 2-naphthylamide), free chromophores (e.g. p-nitroanilide), or free luciferin for quantifying fluorescence, color change, and bioluminescence signals.

[0068] One embodiment of the invention using DPP3 as a diagnostic biomarker encompasses various formats of immunoassays, e.g., radioimmunoassays (RIA), chemiluminescent and fluorescent immunoassays, enzyme-linked immunoassays (ELISA), Luminex-based bead arrays, protein microarray assays, and rapid test formats, e.g., immunochromatographic strip tests.

[0069] The assay may be a homogeneous or heterogeneous assay, a competitive assay and a non-competitive sandwich assay. In a particularly preferred embodiment, when using two antibodies of the invention, the assay is in the form of a sandwich assay (non-competitive immunoassay), in which the DPP3 or a fragment thereof to be detected and / or quantified is bound to a first antibody and a second antibody. The first antibody may be bound to a solid phase (e.g., a bead, a surface of a well or other container, a chip or a strip), and the second antibody is an antibody labeled with, for example, a dye, a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general composition and procedure of the "sandwich assay" are well established and known to those skilled in the art (The Immunoassay Handbook, Ed. David Wild, 2005;Hultschig et al. 2006).

[0070] In a particularly preferred embodiment, the assay comprises a reaction mixture in which a first label component is conjugated to a first antibody, said first label component being part of a labeling system based on fluorescence quenching or chemiluminescence quenching or amplification, and a second label component of said labeling system being conjugated to a second antibody, such that a measurable signal is generated upon binding of both antibodies to the analyte, thereby allowing detection of sandwich complexes formed in a solution containing a sample.

[0071] In the context of the present invention, a fluorescence-based assay involves the use of a dye. Dyes include, for example, FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes (CY3, CY5, CY3.5, CY5.5, Cy7, etc.), xanthene, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), TET, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes (such as BODIPY TMR), Oregon Green, coumarins (umbelliferone), benzimides (Hoechst 33258); phenanthridines (such as Texas Red), Yakima Yellow, Alexa Fluor, PET, ethidium bromide, acridinium dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, and the like.

[0072] In the context of the present invention, chemiluminescent assays involve the use of dyes that are based on the physical principles described for chemiluminescent materials in Kirk-Othmer, Encyclopedia of chemical technology, 4th ed., executive editor, JI Kroschwitz; editor, M. Howe-Grant, John Wiley & Sons, 1993, vol. 15, p. 518-562, the contents of which are incorporated herein by reference (including citations on pages 551-562). Preferred chemiluminescent dyes are acridinium esters.

[0073] One aspect of the present invention includes a chemical assay for DPP3. The assay uses an enzyme substrate that reacts with DPP3 to produce a detectable reaction product. Alternatively, the reaction rate of the substrate can be monitored to determine the presence or amount of DPP3 in a sample. Suitable enzyme substrates include, but are not limited to, dipeptide substrates such as Arg-Arg-βNA or Arg-Arg-AMC.

[0074] Assays embodying such reagents and reactions can be carried out in any suitable reaction vessel (e.g., a test tube or a well of a microtiter plate). Alternatively, assay devices may be developed in disposable form, such as dipstick or test strip device formats, which are well known to those of skill in the art and easy to manufacture and use. Such disposable assay devices may be packaged in the form of a kit containing all the necessary materials, reagents, and instructions for use.

[0075] The assay device of the present invention may advantageously be in the form of a urine test strip or test strip device. For example, the urine test strip may be made of a small piece of absorbent material containing a chromogenic substrate for DPP3. Alternatively, the urine test strip may be made of a non-porous material to which the substrate is applied. When the device is contacted with the desired test sample, the substrate interacts with any DPP3 present in the sample, producing a detectable reaction on the device.

[0076] In another embodiment, the device may be a test strip containing the substrate in one or more regions along the length of a strip of water-absorbent material. When one end of the strip is contacted with the desired test sample, the liquid sample migrates along the water-absorbent material. Reaction of the substrate and generation of a detectable signal indicates the presence of DPP3 in the test sample. In a device with multiple regions, the number of regions along the length of the strip that generate separate (i.e., independent) detectable signals may indicate the amount of DPP3 present in the test sample. Alternatively, the majority of the test strip may contain the substrate. The length of colored reaction in such a test strip with one long substrate region may be used to indicate the presence or amount of DPP3 in the test sample.

[0077] In another embodiment of the assay, the rate at which the reaction occurs may be detected as an indication of the amount of DPP3 present in the test sample. For example, the rate at which the substrate reacts may be used to indicate the amount of DPP3 present in the test sample. Alternatively, the rate at which the reaction product is produced may be used to indicate the amount of DPP3 present in the test sample.

[0078] In yet another embodiment, a capture or binding assay may be performed to detect and / or quantify the protease. For example, an antibody that reacts with DPP3 protein but does not interfere with peptidase activity may be immobilized on a solid phase. When a test sample is passed over the immobilized antibody, DPP3 (if present) binds to the antibody and is itself immobilized for detection. Substrate may then be added and a reaction product may be detected that indicates the presence or amount of DPP3 in the test sample. For purposes of this specification, the term "solid phase" may be used to encompass any material or container on which an assay may be performed, including, but not limited to, porous materials, non-porous materials, test tubes, wells, slides, and the like.

[0079] In another specific embodiment, the ECA of DPP3 may 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 porous strip. The strip contains immobilized antibodies that bind to DPP3 and thereby immobilize DPP3 at a predetermined site for subsequent detection. Optionally, the device may include an assay indicator end located at the distal end of the strip away from the test sample contact site. The assay indicator end indicates the completion of the assay by generating a detectable signal when contacted with a test sample or assay reagent.

[0080] The test sample application pad may be a part of the porous strip itself, or it may be a material at the interface between the fluid stream and the end (referred to as the proximal end) of the porous strip that allows the test sample to pass or transfer from the application pad to the porous strip. Fluid stream contact may include physical contact between the application pad and the porous strip, as well as cases where the application pad is separated from the porous strip by an intervening space or additional material that allows fluid to flow between the application pad and the porous strip. Substantially the entire application pad may overlap the porous strip, allowing the test sample to pass from substantially any part of the application pad to the proximal end of the porous strip. Alternatively, only a portion of the application pad may be at the interface between the fluid stream and the porous strip. The application pad may be any material that allows the test sample to transfer to the porous strip.

[0081] The porous strip of the assay device may be any suitable absorbent, porous, bibulous, chromatographic, or capillary-containing material capable of transporting the test sample containing the analyte by capillary or wicking action. Natural, synthetic, or synthetically modified naturally occurring materials may be used as the porous strip. Examples of materials include, but are not limited to, cellulosic materials such as paper, cellulose, cellulose derivatives (such as cellulose acetate and nitrocellulose); glass fibers; natural (cotton) and synthetic (nylon) fabrics; porous gels such as silica gel, agarose, dextran, and gelatin; porous fibrous substrates; starch-based materials such as cross-linked dextran chains; ceramic materials; films made of polyvinyl chloride or a combination of polyvinyl chloride and silica. The porous strip should not interfere with the generation of a detectable signal. The porous strip should have reasonable intrinsic strength or strength can be provided by an auxiliary support.

[0082] The particular dimensions of the porous strip may be conveniently selected depending on the size of the test sample involved, the assay protocol, the means of signal detection and measurement, etc. For example, dimensions may be selected to control the rate of fluid movement or the amount of test sample absorbed by the porous strip.

[0083] In one possible test strip device of the invention, a DPP3 substrate and / or a DPP3 capture antibody may be immobilized on a porous strip to form at least one analyte detection site (i.e., that area of ​​the porous strip having one or more assay reagents non-diffusively attached thereto). In another device embodiment, the measurement or detection area of ​​the test strip may include multiple sites containing a DPP3 substrate and / or immobilized anti-DPP3 antibody. If desired, 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 the equivalent of 1 nmol / min / ml of DPP3, the first detection site of the detection device would contain 50 nanograms of anti-DPP3 antibody, and subsequent sites would contain 10 nanograms, 20 nanograms, 30 nanograms, etc. of antibody. The number of sites displaying a detectable signal upon addition of a test sample is a quantitative indication of the amount of DPP3 present in the sample. The detection sites may be configured in any suitably detectable shape, typically in the shape of a bar spanning the width of the test strip.

[0084] If desired, a device with multiple capture sites may be made such that if a threshold amount of DPP3 is not present in the test sample, substantially all of the DPP3 is immobilized at the first capture site by binding to the antibody of that site. If an amount of DPP3 greater than the threshold amount is present in the test sample, the remaining DPP3 will bind to the detection area of ​​the subsequent 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 show a detectable signal due to the presence of DPP3. As will be appreciated by those skilled in the art, devices can also be made that contain multiple DPP3 substrate sites designed such that the amount of substrate at each site provides a quantitative or semi-quantitative assay result.

[0085] Another important aspect of the present invention is a method for diagnosing a disease or condition involving or associated with a necrotic process in a subject, comprising the steps of: Quantifying the amount of total DPP3 or the amount of active DPP3 in the body fluid sample of the subject; comparing said quantified amount to a predetermined threshold value; Including, If the quantified amount exceeds the predetermined threshold, the subject is diagnosed as having a disease or condition involving or associated with a necrotic process.

[0086] In certain embodiments of the methods for diagnosing a disease or condition in a subject involving or associated with a necrotic process, the amount of total or active DPP3 is quantified in concentration units.

[0087] The method of quantifying total or active DPP3 is known to those skilled in the art. For the method of the present invention for diagnosing a disease or condition in a subject that is accompanied by or associated with necrotic process, the current method and assay may be used, or the above-mentioned method and assay for quantifying DPP3 may be used.

[0088] The threshold is preset by measuring the DPP3 concentration and / or DPP3 activity of healthy controls and calculating, for example, the corresponding 75th percentile, more preferably the 90th percentile, and even more preferably the 95th percentile. The higher boundary of the 75th percentile, more preferably the 90th percentile, and even more preferably the 95th percentile, defines the threshold between healthy subjects and disease patients. With respect to said percentiles, the threshold separating healthy subjects and disease patients may be 5-25 ng / ml, more preferably 7-20 ng / ml, more preferably 8-18 ng / ml, and most preferably 10-15 ng / ml in plasma when using a sandwich-type anti-DPP3 immunoassay (see Example 3). In an enzyme capture activity assay specific for DPP3 in plasma, the threshold separating healthy subjects and disease patients is 0.5-2 nmol(βNA)min -1 ml -1 , more preferably 0.7 to 1.8 nmol (βNA) min -1 ml -1 , more preferably 0.8 to 1.5 nmol (βNA) min -1 ml -1 , and most preferably 1.0 to 1.3 nmol (βNA) min-1 ml -1 (See Example 5).

[0089] Those skilled in the art know how to determine the threshold value from previous studies. Those skilled in the art know that the specific threshold value may depend on the cohort used to calculate the predetermined threshold value that can be routinely used later. Those skilled in the art know that the specific threshold value may depend on the calibration used for the test. Those skilled in the art know that the specific threshold value may depend on the sensitivity and / or specificity that the practitioner considers acceptable.

[0090] The sensitivity and specificity of a diagnostic test depend not only on the analytical "nature" of the test, but also on the definition of what constitutes an abnormal result. Indeed, receiver operating characteristic curves (ROC curves) are commonly calculated by plotting the values ​​of a variable against the relative frequency in a "normal" (i.e., supposedly healthy) and a "disease" (i.e., patients suffering from an infectious disease) population. Depending on the particular diagnostic problem to be addressed, the reference group does not necessarily have to be "normal" but can be a group of patients suffering from another disease or condition, which is differentiated from the patient group of interest. For any particular marker, there is likely to be an overlap in the distribution of the marker's levels in subjects with and without disease. Under such conditions, the test will never absolutely distinguish normal from disease with 100% accuracy, and the overlap area indicates the area where the test cannot distinguish normal from disease. A threshold value is selected above which (or below which, depending on how the marker changes with the disease) the test is considered abnormal, and below which the test is considered normal. The area under the ROC curve is a measure of the probability that the recognized measurement correctly identifies a condition. ROC curves can be used even when test results do not necessarily provide an exact numerical value. As long as results can be ranked, a ROC curve can be created. For example, test results on "disease" samples can be ranked according to degree (e.g., 1=low, 2=normal, 3=high). This ranking can be correlated with results on a "normal" population to create a ROC curve. These methods are well known to those skilled in the art (see, for example, Hartley et al, 1982). The threshold value is preferably selected so that the area of ​​the ROC curve is greater than about 0.5, more preferably greater than about 0.7. In this context, the term "about" means ±5% of a given measurement.

[0091] Once the threshold has been determined using past study cohorts and taking into account all the above points, a physician would use the pre-determined threshold in the method of diagnosing disease according to the present invention to determine whether a subject has a value above or below said pre-determined threshold in order to make an appropriate diagnosis.

[0092] DPP3 concentrations in tissue homogenates and body fluids can be measured using several commercially available DPP3 ELISA kits (e.g., from LifeSpan BioSciences), all of which are based on the sandwich-type assay principle and are for research purposes only.

[0093] The standard procedure for measuring DPP3 levels is to quantify DPP3 activity using a fluorogenic substrate (e.g., Arg-Arg-β-naphthylamide) in a liquid-phase assay (Ellis & Nuenke 1967). Commercially available kits (e.g., from BPS Bioscience) usually contain low-binding black microtiter plates, recombinant DPP3, a fluorogenic substrate, and respective buffers. These kits are commonly used as screening assays for DPP3 substrates and inhibitors.

[0094] In a particular embodiment of the method for diagnosing a disease or condition in a subject involving or associated with a necrotic process, the sample is selected from the group comprising whole blood, serum and plasma. For the method of the invention, the body fluid may be selected from the group consisting of blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum and pleural effusion.

[0095] Necrotic processes are defined herein as any process in the body that results in cell death and release of DPP3 from the cytoplasm into the extracellular space and / or body fluids, including, but not limited to, necrosis, apoptosis, necroptosis, and eryptosis.

[0096] In certain embodiments of the method of diagnosing a disease or condition in a subject involving or associated with a necrotic process, the disease is selected from the group including heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial, viral (e.g. AIDS), parasitic (e.g. malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), central nervous system (CNS) disorders (e.g. stroke, neurodegenerative diseases), autoimmune diseases, vascular diseases (e.g. Kawasaki syndrome), and hypotension. Table 1 lists clinical symptoms / diseases involving or associated with a necrotic process and the respective necrotic events.

[0097] In another embodiment, the disease is selected from the group comprising acute heart failure (AHF), myocardial infarction (MI), liver failure, burns, severe infection (microbial, viral (e.g., AIDS, parasitic (e.g., malaria)), SIRS or sepsis, cancer, and acute kidney injury (AKI). In one embodiment, the condition is hypotension. [Table 1]

[0098] Apoptosis is a process of programmed cell death (PCD) that can occur in multicellular organisms. Biochemical events lead to characteristic cellular changes (morphology) and cell death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and fragmentation of chromosomal DNA. For review see Elmore 2007. Toxicol Pathol 35(4): 495-516.

[0099] Necrosis is a form of cell injury that results in the premature death of cells in viable tissues by autolysis (Vanlangenakker et al. 2008. Current Molecular Medicine 8(3): 207-220). Necrosis is caused by extracellular or extracellular factors (such as infection, toxins, or trauma) that result in the unregulated digestion of cellular components.

[0100] While apoptosis often has beneficial effects on the organism, necrosis is almost always harmful and potentially fatal.

[0101] Necrotic cell death does not follow apoptotic signaling pathways and activates various receptors, resulting in the loss of cell membrane integrity and the uncontrolled release of cell death products into the extracellular space. This initiates an inflammatory response in the surrounding tissue, preventing nearby phagocytes from finding and eliminating the dead cells by phagocytosis. This often necessitates surgical removal of necrotic tissue, a procedure known as necrotic debridement. If necrosis is left untreated, dead tissue and cellular debris will decay and accumulate at or near the site of cell death.

[0102] A form of programmed necrosis (called necroptosis) has been recognized as another form of programmed cell death. Necroptosis may act as a complementary cell death to apoptosis when apoptotic signaling is blocked by endogenous or exogenous factors such as viruses or mutations (Linkermann et al. 2014. NEJM 370(5): 455-465). Recently, other types of regulated necrosis have been discovered that share some signaling events with necroptosis and apoptosis (Vanden Berghe et al. 2014. Nature Reviews 15 (135-147)).

[0103] Heart failure (HF) is a cardiac condition that occurs when problems with the heart's structure or function impair its ability to provide adequate blood flow for the body. HF can result in a variety of symptoms, notably shortness of breath (SOB) at rest or during exercise, signs of fluid retention (e.g., pulmonary congestion, ankle swelling), and objective evidence of abnormalities in cardiac structure or function at rest.

[0104] Heart failure is a clinical syndrome characterized by a set of symptoms and signs caused by cardiac dysfunction. It is one of the leading causes of morbidity and mortality in developed countries, with a prevalence of 1–2%. Heart failure can be classified as chronic HF or acute HF. Patients with chronic HF can be classified as having stable chronic HF, chronic HF with worsening signs and symptoms, or acute decompensated chronic HF. Acute heart failure (AHF) is defined as the rapid onset of signs and symptoms of heart failure that requires urgent treatment or hospitalization. AHF can present as de novo acute HF (new onset of AHF in patients with no previous cardiac dysfunction) or acute decompensation of chronic HF. AHF is the leading cause of hospitalization in adults over 65 years of age. Although the prognosis of patients with chronic heart failure has improved significantly, mainly related to advances in treatment over the past decades, once patients are hospitalized with decompensated heart failure, both short-term and long-term outcomes remain very poor. Nearly 25% of hospitalized patients with AHF require readmission within 30 days of discharge, and less than 50% survive longer than 5 years after hospitalization. In addition to significantly reducing survival and quality of life for affected patients, AHF also poses a significant financial burden to the healthcare system: the total cost of treating heart failure was estimated at $31 billion in 2012 in the United States alone, with the majority of this cost associated with hospitalization. Due to the aging population, this cost is projected to increase to an unprecedented $70 billion by 2030.

[0105] Heart failure encompasses a broad range of patients, from those with a normal left ventricular ejection fraction (LVEF) (generally considered to be ≥ 50%), also known as HF with preserved EF (HFpEF), to those with a reduced LVEF (generally considered to be < 40%), also known as HF with reduced EF (HFrEF). Patients with an LVEF in the range of 40-49% are in the "gray area" and are defined as HF with moderate EF (HFmrEF) (Ponikowski et al. 2016. European Heart Journal 18(8): 891-975). Heart failure can occur as acute or chronic heart failure.

[0106] The term "acute" means rapid onset and is used to describe worsening or decompensated heart failure, referring to an episode in which the patient can be characterized by changes in signs and symptoms of heart failure that require urgent treatment or hospitalization.

[0107] The term "chronic" refers to long duration. Chronic heart failure is a long-term condition that is usually kept stable by symptomatic treatment (stable chronic HF). Stable chronic HF is characterized by: (i) There is a defect in the structure or function of the heart that impairs its ability to supply adequate blood flow to the body. (ii) Absence of volume overload (indicated by pulmonary and / or systemic congestion) and / or severe reduction in cardiac output (indicated by hypotension, renal failure, and / or shock syndrome). The patient does not require urgent medical treatment or care coordination and does not require hospitalization.

[0108] Worsening signs and symptoms of chronic HF are characterized by: (i) There is a defect in the structure or function of the heart that impairs its ability to supply adequate blood flow to the body. (ii) volume overload (indicated by pulmonary and / or systemic congestion) and / or severe reduction in cardiac output (indicated by hypotension, renal failure, and / or shock syndrome); The patient does not require urgent medical attention and does not require hospitalization, but care coordination is necessary.

[0109] Chronic heart failure can be decompensated (termed acutely decompensated heart failure or acutely decompensated chronic heart failure). This is most commonly caused by intercurrent illness (such as pneumonia), myocardial infarction, arrhythmias, uncontrolled hypertension, or the patient's failure to maintain fluid, dietary, and drug therapy. After treatment, patients with acutely decompensated chronic HF may revert to a stable compensated chronic state (stable chronic HF).

[0110] New-onset acute HF and acutely decompensated chronic HF are characterized by the following: (i) There is a defect in the structure or function of the heart that impairs its ability to supply adequate blood flow to the body. (ii) volume overload (indicated by pulmonary and / or systemic congestion) and / or severe reduction in cardiac output (indicated by hypotension, renal failure, and / or shock syndrome); The patient requires urgent medical attention and care coordination and requires hospitalization.

[0111] [Table 2]

[0112] The above definitions of acute heart failure, whether new-onset AHF, acutely decompensated HF i.e. acute decompensation of chronic HF, or chronic heart failure with worsening signs / symptoms, are consistent with Voors et al., European Journal of Heart Failure (2016), 18, 716-726.

[0113] In certain embodiments of the above methods for diagnosing a disease or condition in a subject involving or associated with a necrotic process, DPP3 activity can be quantified by a solution phase activity assay or an enzyme capture activity assay.

[0114] In the liquid-phase assay, the body fluid sample is directly exposed to a fluorogenic substrate (e.g., Arg-Arg-βNA). Because many different aminopeptidases are present in plasma (Sanderink et al. 1988), it is possible that the substrate used may be cleaved by peptidases other than DPP3. To circumvent this problem, one preferred method for detecting specific DPP3 activity is to use an enzyme capture activity assay.

[0115] In a particular embodiment, the quantification of active DPP3 in an enzyme capture assay comprises: contacting the sample with a capture binding agent that binds full length DPP3 but preferably inhibits DPP3 activity by less than 50%, preferably less than 40%, more preferably less than 30% in a solution phase assay (to avoid inhibition of DPP3, the capture binding agent should not bind to the region surrounding the active center and substrate binding region of DPP3 (amino acids 316-669 of SEQ ID NO:1)); Separating the DPP3 bound to the capture binding agent from the body fluid sample; adding a DPP3 substrate to the separated DPP3; Quantifying DPP3 activity by measuring the conversion rate of a DPP3 substrate; and evaluating the measured signal in comparison with a non-disease control (the threshold may be pre-determined, for example, by measuring the DPP3 concentration or DPP3 activity in healthy controls and calculating the corresponding 75th percentile, the upper boundary of the 75th percentile defining the threshold between healthy and diseased patients).

[0116] In a particular embodiment, the quantification of active DPP3 is carried out according to the method of the invention described above.

[0117] In certain embodiments of said method of diagnosing a disease or condition in a subject involving or associated with a necrotic process, said binding agent may be selected from the group of antibodies, antibody fragments, non-IgG scaffolds.

[0118] A particular embodiment of the method for diagnosing a disease or condition in a subject involving or associated with a necrotic process is an enzyme capture assay (ECA) (US5612186A, US5601986A), in which the binding agent for DPP3 is an antibody.

[0119] In a particular embodiment of the method for diagnosing a disease or condition in a subject that involves or is associated with a necrotic process, the capture binding agent is immobilized on a surface. A binding agent that reacts with DPP3 protein but inhibits peptidase activity by no more than 50% (preferably less than 40%, preferably less than 30%) may be immobilized on a solid phase. To prevent inhibition of DPP3, the capture binding agent should not bind to the region surrounding the active center and substrate binding region of DPP3 (amino acids 316-669 of SEQ ID NO: 1).

[0120] In certain embodiments of the method for quantifying active DPP3 in a body fluid sample of a subject, the binding agent may be selected from the group of antibodies, antibody fragments, non-Ig scaffolds, aptamers.

[0121] When the test sample is passed over the immobilized binding agent, DPP3 (if present) binds to the binding agent and is itself immobilized for detection. Substrate may then be added, and reaction products may be detected that 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 container in which an assay may be performed, including, but not limited to, porous materials, non-porous materials, test tubes, wells, slides, agarose resins (e.g., Sepharose from GE Healthcare Life Sciences), magnetic particles (e.g., Dynabeads™ or Pierce™ magnetic beads from Thermo Fisher Scientific), and the like.

[0122] Protein or peptide-based binding agents (e.g., antibodies, antibody fragments, non-Ig scaffolds) can be immobilized by physical adsorption (e.g., electrostatic or hydrophobic interactions), bioaffinity immobilization (e.g., avidin-biotin, protein A / G / L, His-tags and Ni 2+-NTA, GST tags and glutathione, DNA hybridization, aptamers), covalent bonds (e.g., amines and N-hydroxysuccinimide), or a combination of the above immobilization methods (Kim & Herr 2013). Oligonucleotide-derived binders (e.g., aptamers) may be immobilized to the solid phase by utilizing the (strept)avidin-biotin system (Muller et al. 2012, Deng et al. 2014).

[0123] In certain embodiments of the methods for diagnosing a disease or condition in a subject involving or associated with a necrotic process, the separating step is a washing step in which components of the sample that are not bound to the capture binding agent are removed from the captured DPP3.

[0124] In certain embodiments of the methods for diagnosing a disease or condition in a subject involving or associated with a necrotic process, the conversion of the DPP3 substrate is measured by a method selected from the group comprising fluorescence of a fluorogenic substrate (e.g., Arg-Arg-2NA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate conjugated to aminoluciferin (Promega Protease-Glo™ Assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis followed by activity staining (fixed active DPP3) or Western blot (cleavage products).

[0125] In certain embodiments of the method for diagnosing a disease or condition in a subject involving or associated with a necrotic process, the substrate may be selected from the group including angiotensin II, III, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH, and MSH; and dipeptides or tripeptides conjugated to a fluorophore, chromophore, or aminoluciferin (Promega Protease-Glo™ 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, Suc-Ala-Ala-Phe, and the like. 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; Abramic et al. 2000, Ohkubo et al. 1999). Cleavage of these fluorogenic substrates liberates fluorescent β-naphthylamide or 7-amino-4-methylcoumarin, respectively. Chromophores include, but are not limited to, p-nitroanilide diacetate (pNA). Hydrolysis of the peptide-pNA bond in the chromogenic substrate liberates pNA and causes a color change. Thus, the change in absorbance (DA / min) is directly proportional to the enzyme activity. When using the Protease-Glo™ Assay (Promega), upon cleavage by DPP3, aminoluciferin is liberated and serves as a substrate for a reaction by bound luciferase that produces detectable luminescence.

[0126] In a preferred embodiment, DPP3 activity is measured by adding the fluorogenic substrate Arg-Arg-βNA and monitoring fluorescence in real time.

[0127] Another important aspect of the present invention is an inhibitor of DPP3 activity for use in the prophylaxis or treatment of a disease or condition in a subject involving or associated with a necrotic process.

[0128] The inhibitor is preferably a molecule that significantly inhibits DPP3 activity. Such molecules may be peptides and small molecules (see Table 2) or antibodies (see Table 3). By significantly inhibiting, we mean more than 80%, preferably more than 90%, more preferably nearly or actually 100% inhibition in the liquid phase assay as described above.

[0129] Peptide inhibitors of DPP3 include, but are not limited to, spinorphin, synthetic spinorphin derivatives (tinorphin and other peptides (see Table 2); Yamamoto et al. 2000), propioxatin A and B (US4804676), and synthetic propioxatin A analogs (Inaoka et al. 1988).

[0130] Small molecule inhibitors of DPP3 include, but are not limited to, fluostatins and benzimidazole derivatives. Fluostatins A and B are antibiotics produced by Streptomyces sp. TA-3391, which are non-toxic and strongly inhibit DPP3 activity. Twenty different benzimidazole derivatives have been synthesized and published so far (Agic et al. 2007; Rastija et al. 2015), of which two compounds 1' and 4' show the strongest inhibitory effects (Agic et al. 2007). [Table 3-1] [Table 3-2] [Table 3-3]

[0131] In a preferred embodiment of the present invention, the selected inhibitor is pharma- ceutically acceptable, selective and / or specific to DPP3, and does not cross the cell membrane and / or blood-brain barrier. A selective and specific DPP3 inhibitor does not bind to or is bound by other proteins / enzymes, and does not inhibit any enzymes / proteases / peptidases other than DPP3. Small peptides can be bound and cleaved by non-specific aminopeptidases, and small molecule inhibitors easily 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 and blood-brain barrier. Thus, preferred DPP3 activity inhibitors are specific anti-DPP3 antibodies, antibody fragments, or non-Ig scaffolds.

[0132] In a particular embodiment of the invention, an inhibitor of DPP3 activity is used for the prevention or treatment of a disease or condition in a subject which involves or is associated with a necrotic process, said inhibitor being selected from the group comprising an anti-DPP3 antibody, an anti-DPP3 antibody fragment, an anti-DPP3 non-Ig scaffold.

[0133] In certain embodiments of the invention, inhibitors or agonists of DPP3 activity are used for the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process, said disease being selected from the group comprising heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial, viral (e.g. AIDS), parasitic (e.g. malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), central nervous system (CNS) disorders (e.g. stroke, neurodegenerative diseases), autoimmune and vascular diseases (e.g. Kawasaki syndrome), and hypotension.

[0134] In another embodiment, the disease is selected from the group comprising acute heart failure (AHF), myocardial infarction (MI), liver failure, burns, severe infections (microbial, viral (e.g. AIDS), parasitic (e.g. malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI).

[0135] In one particular embodiment of the invention, an inhibitor of DPP3 activity is used to prevent a disease or condition in a subject, said disease or condition being acute heart failure (AHF), myocardial infarction (MI), liver failure, cancer, acute kidney injury (AKI), and hypotension.

[0136] In one particular embodiment of the invention, an inhibitor of DPP3 activity is used to treat a disease or condition in a subject, said disease or condition being acute heart failure (AHF), myocardial infarction (MI), liver failure, cancer, acute kidney injury (AKI), and hypotension.

[0137] For all aspects of the invention, in one particular embodiment, the disease is not Alzheimer's disease. For all aspects of the invention, in one particular embodiment, the disease is not cancer. For all aspects of the invention, in one particular embodiment, the disease is not rheumatoid arthritis. In a particular embodiment, the disease or condition is hypotension.

[0138] In a particular embodiment of the invention, an inhibitor of DPP3 activity is used for the prevention or treatment of a disease or condition in a subject which involves or is associated with a necrotic process, said inhibitor being a mono-binding or at least two-binding antibody.

[0139] In a particular embodiment of the invention, an inhibitor or agonist of DPP3 activity is used for the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process, said inhibitor or agonist being an anti-DPP3 antibody or anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to SEQ ID NO: 1, and in a particular embodiment an anti-DPP3 antibody or anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to SEQ ID NO: 2.

[0140] In a particular embodiment of the invention, an inhibitor or agonist of DPP3 activity is used for the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process, said inhibitor or agonist having a 10 -7The antibody or fragment or scaffold exhibits a minimum binding affinity of less than M.

[0141] In certain embodiments of the method for preventing or treating a disease or condition in a subject involving or associated with a necrotic process, the inhibitor or agonist is an antibody or fragment or scaffold that binds to full length DPP3 and inhibits the activity of DPP3 by at least 10% or at least 50%, more preferably at least 60%, even more preferably more than 70%, even more preferably more than 80%, even more preferably more than 90%, even more preferably more than 95%. As described above, activity may be quantified in a liquid phase assay.

[0142] In a particular embodiment of the invention, an inhibitor or agonist of DPP3 activity is used for the prevention or treatment of a disease or condition in a subject which involves or is associated with a necrotic process, said inhibitor or agonist being a monospecific antibody or fragment or scaffold.

[0143] A monospecific anti-DPP3 antibody or a monospecific anti-DPP3 antibody fragment, or a monospecific anti-DPP3 non-Ig scaffold means that said antibody or antibody fragment or non-Ig scaffold binds to one specific region comprising at least 5 amino acids in the target DPP3. A monospecific anti-DPP3 antibody or a monospecific anti-DPP3 antibody fragment, or a monospecific anti-DPP3 non-Ig scaffold is an anti-DPP3 antibody or an anti-DPP3 antibody fragment, or an anti-DPP3 non-Ig scaffold, which all have affinity for the same antigen.

[0144] In another specific preferred embodiment, the anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to DPP3 is a monospecific antibody, antibody fragment, or non-Ig scaffold, respectively, where "monospecific" means that the antibody or antibody fragment or non-Ig scaffold binds to one specific region that contains at least 4 amino acids in the target DPP3. The monospecific antibody or fragment or non-Ig scaffold of the present invention is an antibody or fragment or non-Ig scaffold that has affinity for the same antigen. Although monoclonal antibodies are monospecific, monospecific antibodies may be produced by methods other than producing monoclonal antibodies from common embryonic cells.

[0145] In a particular embodiment of the invention, an inhibitor or agonist of DPP3 activity is used to prevent or treat a disease or condition involving or associated with a necrotic process in a subject, said subject having elevated levels of DPP3, where "elevated levels" refers to levels above a predefined threshold.

[0146] Another aspect of the present invention is a pharmaceutical composition comprising an inhibitor of DPP3 activity as described above for use in the prophylaxis or treatment of a disease or condition in a subject which involves or is associated with a necrotic process.

[0147] Another aspect of the invention is a method for preventing or treating a disease or condition in a subject involving or associated with a necrotic process by administering an inhibitor of DPP3 activity.

[0148] In certain embodiments of the method for preventing or treating a disease or condition in a subject involving or associated with a necrotic process, said inhibitor is selected from the group comprising an anti-DPP3 antibody, an anti-DPP3 antibody fragment, an anti-DPP3 non-Ig scaffold.

[0149] In certain embodiments of the method of preventing or treating a disease or condition in a subject involving or associated with a necrotic process, said disease is selected from the group comprising heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial, viral (e.g. AIDS), parasitic (e.g. malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. stroke, neurodegenerative diseases), autoimmune and vascular diseases (e.g. Kawasaki syndrome), and hypotension.

[0150] In a particular embodiment of the method for preventing or treating a disease or condition in a subject involving or associated with a necrotic process, said inhibitor is an antibody of single binding specificity or at least dual binding specificity.

[0151] In certain embodiments of the method for preventing or treating a disease or condition in a subject involving or associated with a necrotic process, said inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to SEQ ID NO: 1, and in certain embodiments an anti-DPP3 antibody or anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to SEQ ID NO: 2.

[0152] In certain embodiments of the method for preventing or treating a disease or condition in a subject involving or associated with a necrotic process, the inhibitor has an activity of 10 to 15% against DPP3. -7 The antibody or fragment or scaffold exhibits a minimum binding affinity of less than M.

[0153] In certain embodiments of the method of preventing or treating a disease or condition in a subject involving or associated with a necrotic process, said inhibitor or agonist is a monospecific antibody or fragment or scaffold.

[0154] In certain embodiments of the method for preventing or treating a disease or condition in a subject involving or associated with a necrotic process, the inhibitor or agonist is an antibody or fragment or scaffold that binds to full length DPP3 and inhibits the activity of DPP3 by at least 10% or at least 50%, more preferably at least 60%, even more preferably more than 70%, even more preferably more than 80%, even more preferably more than 90%, and even more preferably more than 95%.

[0155] In a particular embodiment of the method for preventing or treating a disease or condition in a subject involving or associated with a necrotic process, said subject has elevated levels of DPP3, where "elevated levels" refers to levels above a predefined threshold, which has been defined above.

[0156] Another aspect of the present invention is the clearance of DPP3 from the patient's blood. Clearance can be achieved by several apheresis techniques and / or affinity chromatography steps (Balogun et al. 2010). Such methods include, but are not limited to, filtering the patient's plasma through an adsorbent containing a specific, high affinity DPP3 antibody bound to an agarose resin (see Example 12 for analysis of DPP3 bound to potential adsorbent materials).

[0157] The pharmaceutical composition of the present invention is formulated to suit the intended route of administration. Routes of administration are generally classified according to the place where the substance is administered. Common examples include oral administration, dermal administration, subcutaneous administration, intradermal administration, sublingual administration, intramuscular administration, intraarterial administration, intravenous administration, and intraperitoneal administration.

[0158] The pharmaceutical composition may be administered via the central nervous system (CNS), including epidural administration by injection or infusion into the epidural space, intracerebral (into the cerebrum) administration by direct injection into the brain, intraventricular (into the ventricular system of the brain) or intrathecal (into the spinal canal) administration. Certain aspects of the invention are outlined below. (Item 1) 1. A method for diagnosing a disease or condition involving or associated with a necrotic process in a subject, comprising: Quantifying the total amount of DPP3 and / or the amount of active DPP3 in the body fluid sample of the subject; and comparing the quantified amount of total DPP3 or active DPP3 with a predetermined threshold value; If the quantified amount exceeds the predetermined threshold, the subject is diagnosed as having a disease or condition involving or associated with a necrotic process. (Item 2) 2. The method for diagnosing a disease or condition in a subject involving or associated with a necrotic process according to item 1, wherein the amount of total DPP3 or active DPP3 is quantified in units of concentration. (Item 3) 3. Method for diagnosing a disease or condition in a subject involving or associated with a necrotic process according to items 1 or 2, wherein said sample is selected from the group comprising whole blood, serum, plasma. (Item 4) 4. The method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process according to any one of items 1 to 3, wherein the disease is selected from the group comprising heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial, viral (e.g. acquired immune deficiency syndrome (AIDS), parasitic (e.g. malaria)), systemic inflammatory response syndrome (SIRS) or sepsis, cancer, acute kidney injury (AKI), central nervous system (CNS) disorders (e.g. stroke, neurodegenerative diseases), autoimmune diseases and vascular diseases (e.g. Kawasaki syndrome), and hypotension. (Item 5) The amount of total DPP3 and / or active DPP3 is quantified in a body fluid sample from the subject; and contacting the sample with a capture binding agent that specifically binds full-length DPP3; Separating the DPP3 bound to the capture binding agent; adding a DPP3 substrate to the separated DPP3; Quantifying the active DPP3 by measuring and quantifying the conversion rate of the DPP3 substrate; 5. A method for diagnosing a disease or condition in a subject involving or associated with a necrotic process according to any one of items 1 to 4, comprising: (Item 6) 6. The method for diagnosing a disease or condition in a subject involving or associated with a necrotic process according to item 5, wherein the capture binding agent may be selected from the group of an antibody, an antibody fragment, or a non-IgG scaffold. (Item 7) 7. The method for diagnosing a disease or condition in a subject involving or associated with a necrotic process according to item 5 or 6, wherein the capture binding agent is an antibody. (Item 8) 8. Method for diagnosing a disease or condition in a subject involving or associated with a necrotic process according to any one of items 5 to 7, wherein the capture binding agent is immobilized on a surface. (Item 9) 9. The method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process according to any one of items 5 to 8, wherein the separating step is a washing step in which components of the sample that are not bound to the capture binding agent are removed from the captured DPP3. (Item 10) 10. The method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process according to any one of items 5 to 9, wherein the conversion of the DPP3 substrate is detected by a method selected from the group comprising: fluorescence of a fluorogenic substrate (e.g. Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate coupled to aminoluciferin (Promega Protease-Glo™ Assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis followed by activity staining (fixed active DPP3) or Western blot (cleavage products). (Item 11) 11. The method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process according to any one of items 5 to 10, wherein the substrate may be selected from the group comprising angiotensin II, III, IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, adrenocorticotropic hormone (ACTH), and melanocyte-stimulating hormone (MSH); and a dipeptide linked to a fluorophore, a chromophore, or an aminoluciferin, wherein the dipeptide is Arg-Arg. (Item 12) 12. The method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process according to any one of items 5 to 11, wherein the substrate may be selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, the dipeptide being Arg-Arg. (Item 13) 13. A method for monitoring a disease or condition in a subject involving or associated with a necrotic process, comprising performing the diagnostic method according to any one of items 1 to 12 at least twice. (Item 14) An inhibitor of DPP3 activity for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process. (Item 15) 15. The DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process according to item 14, wherein said inhibitor is selected from the group comprising anti-DPP3 antibodies and anti-DPP3 antibody fragments, and anti-DPP3 non-Ig scaffolds. (Item 16) 16. The DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject accompanied by or associated with a necrotic process according to item 14 or 15, wherein said disease is selected from the group comprising heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial, viral (e.g. AIDS), parasitic (e.g. malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. stroke, neurodegenerative diseases), autoimmune diseases and vascular diseases (e.g. Kawasaki syndrome), and hypotension. (Item 17) 17. The DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process according to any one of items 14 to 16, wherein the inhibitor is an antibody of single binding specificity (mono-binding) or at least double binding specificity (two-binding). (Item 18) 18. The DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject accompanied by or associated with a necrotic process according to any one of items 14 to 17, wherein the inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment that binds to SEQ ID NO: 1 and in particular binds to SEQ ID NO: 2, or an anti-DPP3 non-Ig scaffold. (Item 19) The inhibitor has a potent potency of 10 -7 19. The DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process according to any one of items 14 to 18, which is an antibody or fragment or scaffold exhibiting a minimum binding affinity of no more than M. (Item 20) 20. The DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process according to any one of items 14 to 19, wherein the inhibitor is a monospecific antibody or fragment or a scaffold. (Item 21) 21. The DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process according to any one of items 14 to 20, wherein the inhibitor is an antibody or fragment or scaffold that binds to full length DPP3 and inhibits the activity of DPP3 by at least 10% or at least 50%, more preferably at least 60%, even more preferably more than 70%, even more preferably more than 80%, even more preferably more than 90%, even more preferably more than 95%. (Item 22) 22. The DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process according to any one of items 14 to 21, wherein the inhibitor is selective and / or specific for DPP3 and does not cross the cell membrane and / or the blood-brain barrier. (Item 23) 23. The DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process according to any one of items 14 to 22, wherein the subject has an amount of total DPP3 and / or active DPP3 in a body fluid sample that exceeds a predetermined threshold. (Item 24) 24. A pharmaceutical composition comprising a DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject accompanied by or associated with a necrotic process according to any one of items 14 to 23. (Item 25) 24. Use of the DPP3 activity inhibitor according to any one of items 14 to 23 in a method for extracorporeal removal of DPP3 from plasma, comprising apheresis and affinity chromatography. (Item 26) 1. A method for quantifying active DPP3 in a body fluid sample from a subject, comprising: contacting the sample with a capture binding agent that specifically binds full-length DPP3; Separating the DPP3 bound to the capture binding agent; adding a DPP3 substrate to the separated DPP3; Quantifying the active DPP3 by measuring and quantifying the conversion rate of the DPP3 substrate; The method includes: (Item 27) 27. The method for quantifying active DPP3 in a body fluid sample of a subject according to item 26, wherein the capture binding agent may be selected from the group of an antibody, an antibody fragment, or a non-IgG scaffold. (Item 28) 28. The method for quantifying active DPP3 in a body fluid sample of a subject according to item 26 or 27, wherein the capture binding agent is an antibody. (Item 29) 29. The method for quantifying active DPP3 in a body fluid sample of a subject according to any one of Items 26 to 28, wherein the capture binding agent is immobilized on a surface. (Item 30) 30. The method for quantifying active DPP3 in a body fluid sample from a subject according to any one of Items 26 to 29, wherein the separation step is a washing step for removing components of the sample that are not bound to the capture binding agent from the captured DPP3. (Item 31) 31. The method for quantifying active DPP3 in a body fluid sample of a subject according to any one of items 26 to 30, wherein the conversion of the DPP3 substrate is detected by a method selected from the group comprising: fluorescence of a fluorogenic substrate (e.g., Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate conjugated to aminoluciferin (Promega Protease-Glo™ Assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis followed by activity staining (fixed active DPP3) or Western blot (cleavage products). (Item 32) 32. The method for quantifying active DPP3 in a body fluid sample of a subject according to any one of items 26 to 31, wherein the substrate may be selected from the group comprising a dipeptide bound to a fluorescent dye molecule, a chromophore, or an aminoluciferin, and the dipeptide is Arg-Arg. (Item 33) 33. The method for quantifying active DPP3 in a body fluid sample from a subject according to any one of Items 26 to 32, wherein the sample is a blood sample selected from the group consisting of whole blood, serum, and plasma. (Item 34) 1. An assay or kit for quantifying active DPP3 in a body fluid sample from a subject, comprising: a capture binding agent that specifically binds to full-length DPP3; DPP3 Substrates 4. An assay or kit comprising: (Item 35) 35. The assay or kit for quantifying active DPP3 in a body fluid sample of a subject according to item 34, wherein the capture binding agent may be selected from the group of an antibody, an antibody fragment, or a non-IgG scaffold. (Item 36) 36. The assay or kit for quantifying active DPP3 in a body fluid sample of a subject according to item 34 or 35, wherein said capture binding agent inhibits DPP3 activity by less than 50%, preferably less than 40%, more preferably less than 30% in a liquid phase assay. (Item 37) 37. The assay or kit for quantifying active DPP3 in a body fluid sample of a subject according to any one of Items 34 to 36, wherein the binding region of DPP3 to the capture binding agent is not within the region of amino acids 316 to 669 of SEQ ID NO: 1. (Item 38) 38. The assay or kit for quantifying active DPP3 in a body fluid sample of a subject according to any one of items 34 to 37, wherein the capture binding agent is an antibody. (Item 39) 39. The assay or kit for quantifying active DPP3 in a body fluid sample of a subject according to any one of items 34 to 38, wherein the capture binding agent is immobilized on a surface. (Item 40) 40. The assay or kit for quantifying active DPP3 in a body fluid sample of a subject according to any one of items 34 to 39, wherein the substrate may be selected from the group comprising angiotensin II, III, IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH, and MSH; and a dipeptide linked to a fluorophore, a chromophore, or an aminoluciferin, wherein the dipeptide is Arg-Arg. (Item 41) Use of the method for quantifying active DPP3 in a body fluid sample of a subject according to any one of items 26 to 33 or the assay or kit according to any one of items 34 to 40 in a method for diagnosing or monitoring a disease or condition in a subject accompanied by or associated with a necrotic process according to any one of items 1 to 13. EXAMPLES

[0159] (1. Example 1) DPP3 activity in plasma from patients with various diseases (patients with acute myocardial infarction (AMI), cardiogenic shock, septic shock, and liver failure) was quantified using a specific DPP3 scavenging activity assay and compared with DPP3 activity in plasma from healthy controls. 1.1. Study cohort

[0160] Plasma samples were obtained from 388 patients who were admitted directly to the emergency department with their first symptoms. The final diagnosis allowed the patients to be divided into four subgroups: patients with acute myocardial infarction (AMI), cardiogenic shock, septic shock, and liver failure. The control group consisted of plasma samples from 93 healthy controls. 1.2. hDPP3 scavenging activity assay

[0161] DPP3 in plasma samples (10 μl) was first concentrated by an affinity purification step, and then its activity was measured by adding the fluorogenic substrate Arg-Arg-βNA (see Example 4 for a detailed description). The slope of the fluorescence increase for the various samples (nmol(βNA) / min / ml(sample) [nmol(βNA)min -1 ml -1 ]) calculations are for a sample size of 10 μl. (1.3.Results)

[0162] When patient samples were compared with healthy controls, all patients with severe disease or organ failure had significantly higher DPP3 activity levels (Figure 10). 2. Example 2

[0163] In this study, the effects of injecting recombinant hDPP3 into healthy rats were studied by monitoring blood pressure. (2.1. Method)

[0164] Male Wistar rats (Charles River Laboratories, Germany) aged 2-3 months were used in this study. A catheter (Introcan-W; 22 G / 1''; B. Braun) was inserted into the right common carotid artery for measurement and recording of blood pressure (BP). N-terminally GST-tagged human recombinant dipeptidyl peptidase 3 (recGST-hDPP3) was injected via the tail vein.

[0165] Animals were first anesthetized with isoflurane, weighed (all in g), and injected intraperitoneally (ip) with 1.2 g / kg (body weight) of urethane (concentration = 0.4 g / mL) for long-term anesthesia. The ventral area of ​​the neck was then clipped and wiped with ethanol. The vessels were prepared and catheterized. Finally, both catheters were flushed with heparinized isotonic saline. A blood pressure measuring device (Medex Logical (Medex Medical Ltd.)) was then connected to a patient monitoring system (Datex-Ohmeda (GE)). A laptop computer was connected to the BP monitor and BP data were recorded separately with S / 5 Collect Software.

[0166] Rats were treated with 0.2 mg / kg of recGST-hDPP3 in PBS via tail vein injection. Blood pressure was constantly monitored before and after injection of DPP3. (2.2.Results)

[0167] Injection of recombinant GST-hDPP3 into healthy rats rapidly reduces blood pressure (Figure 11). 3. Example 3

[0168] Generation of antibodies and quantification of DPP3-binding ability: Several mouse antibodies were generated and screened for their ability to bind to human DPP3 in sandwich or activity assays (see Table 3). (3.1. Method) (Immunization peptides / conjugates)

[0169] DPP3 peptides for immunization were synthesized (see Table 3) (JPT Technologies, Berlin, Germany). To conjugate the peptides to bovine serum albumin (BSA), a cysteine ​​residue was added at the N-terminus (if no cysteine ​​was present in the selected DPP3 sequence). The peptides were covalently coupled to BSA using SulfoLink-Coupling Gel (Perbio-Science, Bonn, Germany). The coupling procedure was performed according to the instructions from Perbio. Recombinant GST-hDPP3 was from USBio. (Mouse immunization, immune cell fusion and screening)

[0170] Balb / c mice were intraperitoneally (ip) injected with 84 μg and 100 μg of GST-hDPP3 or DPP3 peptide-BSA conjugates (emulsified in TiterMax Gold Adjuvant), respectively, on day 0, 84 μg and 100 μg (emulsified in Freund's complete adjuvant), respectively, on day 14, and 42 μg and 50 μg (dissolved in Freund's incomplete adjuvant), respectively, on days 21 and 28. On day 49, animals were intravenously (iv) injected with GST-hDPP3 (42 μg) or DPP3 peptide-BSA conjugates (50 μg) dissolved in saline. Three days later, mice were sacrificed and immunocytofusion was performed.

[0171] Spleen cells from immunized mice were fused with cells of the myeloma cell line SP2 / 0 using 1 ml of 50% polyethylene glycol at 37°C for 30 seconds. After washing, the cells were seeded into 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT-Supplement]. After 1 week, the HAT medium was replaced with HT medium and subcultured three times, after which the cells were returned to normal cell medium.

[0172] Two weeks after fusion, cell culture supernatants were first screened for recombinant DPP3 binding to IgG antibodies. Thus, recombinant GST-tagged DPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng / well) and incubated with 50 μl / well of cell culture supernatant at room temperature for 2 h. After washing the plates, 50 μl / well of POD-rabbit anti-mouse IgG was added and incubated at room temperature for 1 h. After a further 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 min, and the color reaction was stopped by adding 50 μl of 4N sulfuric acid. Absorbance was detected at 490 mm.

[0173] Positive testing microbial cultures were transferred to 24-well plates for growth. After retesting, selected cultures were cloned and recloned by limiting dilution and isotyped. (Preparation of mouse monoclonal antibodies)

[0174] Antibodies against GST-tagged human DPP3 or DPP3 peptides were generated by standard antibody production methods (Marx et al., 1997) and purified with protein A. Antibody purity was ≥90% based on SDS gel electrophoresis analysis. (Antibody characterization-analysis of hDPP3 inhibition)

[0175] To analyze the DPP3 inhibitory potential of various antibodies and antibody clones, DPP3 activity assays were performed as previously described (Jones et al., 1982). Recombinant GST-tagged hDPP3 was diluted in assay buffer (25 ng / ml GST-DPP3 dissolved in [50 mM Tris-HCl (pH 7.5), 100 μM ZnCl2]) and 200 μl of this solution was incubated with 10 μg of each antibody at room temperature. After 1 h preincubation, the fluorogenic substrate Arg-Arg-βNA (20 μl, 2 mM) was added to the solution and the generation of free βNA over time was monitored at 37°C using a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG). Fluorescence of βNA is detected by exciting at 340 nm and measuring the emission at 410 nm. The slope of the fluorescence increase (RFU / min) of the various samples is calculated. The slope between buffer (control) and GST-hDPP3 is taken as 100% activity. The inhibitory capacity of a potential capture binding agent is defined as the % reduction in GST-hDPP3 activity upon incubation with the capture binding agent. The resulting reduced DPP3 activity is shown in Figure 1a and Table 3. (3.2.Results)

[0176] The following table shows a selection of the obtained antibodies and their maximum inhibition rates (Table 3). The monoclonal antibodies against the following DPP3 regions were selected by their binding ability to native DPP3 (mAb-FL-DPP3_2555 was used as the solid phase and _2553 as the tracer for immunoassays. See Example 2 for details).

[0177] For the immobilized DPP3 activity assay (see Examples 4 and 5), it was necessary to select solid-phase antibodies that did not inhibit DPP3 activity too strongly. As a cutoff for antibody screening, solid-phase antibodies should not inhibit DPP3 activity more than 50%. mAbDPP3_2555 showed the lowest inhibition rate (Table 3, Figure 1A).

[0178] To generate a strong DPP3 inhibitor that can be used for treatment (see Examples 6-11), it was necessary to select a DPP3 binder that showed the highest inhibition rate. The monoclonal antibody mAbDPP3_1967, which has the ability to inhibit DPP3 activity by 70%, was selected as a potential therapeutic antibody (see Figure 1A and Table 3), and all subsequent analyses were performed with this antibody. Figure 1B shows the IC 50 The inhibition curve for mAb DPP3_1967 with a value of 0.2041 μg / ml is shown. [Table 4] 4. Example 4

[0179] The antibody combination that produced a high signal / noise (S / N) ratio in the hDPP3 immunoassay is determined. (4.1. Method) (Production of monoclonal antibodies)

[0180] Antibodies against GST-tagged human DPP3 were generated by standard antibody production methods (Marx et al., 1997) and purified with protein A. Antibody purity was more than 90% based on SDS gel electrophoresis analysis. Different clones were analyzed for their DPP3 binding ability. Clones with positive results were used as solid phase or tracer antibodies. (solid phase)

[0181] A 96-well polystyrene microplate (Greiner Bio-One International AG, Austria) was coated (1 h at room temperature) with one anti-DPP3 antibody clone (capture antibody) (1.5 μg / 0.25 mL antibody, 100 mmol / L NaCl, 50 mmol / L Tris / HCl; pH 7.8). After blocking with 5% bovine serum albumin, the microplate was dried under vacuum. (Labeling Procedure (Tracer)) 100 μg (100 μl) of various anti-DPP3 antibodies (detection antibodies, 1 mg / ml dissolved in PBS, pH 7.4) were mixed with 10 μl of acridinium NHS ester (1 mg / ml dissolved in acetonitrile) (InVent GmbH, Germany; EP 0 353 971) and incubated at room temperature for 30 min. Labeled anti-DPP3 antibodies were purified by gel filtration HPLC on Shodex Protein 5 μm KW-803 (Showa Denko, Japan). The purified labeled antibodies were diluted in assay buffer (50 mmol / l potassium phosphate, 100 mmol / l NaCl, 10 mmol / l Na2-EDTA, 5 g / l bovine serum albumin, 1 g / l mouse IgG, 1 g / l bovine IgG, 50 μmol / l amastatin, 100 μmol / l leupeptin; pH 7.4). The final concentration was approximately 7 × 10 relative light units (RLU) of labeled compound per 200 μl. 6 (Approximately 20 ng of labeled antibody) The chemiluminescence of the acridinium ester was measured using a Centro LB 960 fluorometer (Berthold Technologies GmbH & Co. KG). (Calibration sample)

[0182] A stock solution of recombinant human GST-DPP3 (USBiological, USA) in PBS, pH 7.4, was linearly diluted with (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 amastatin, 100 μmol / L leupeptin; pH 7.4). The stock solution was stored at -80°C. Calibration samples were prepared before use. (hDPP3 immunoassay) 10 μl of sample (or calibrator) was pipetted into the coated 96-well microplate, labeled and diluted detection antibody (200 μl) was added, and the plate was incubated at 2–8°C for 18–24 h. Unbound tracer was removed by washing four times with 350 μl wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Fully bound chemiluminescence was measured in a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). (4.2.Results)

[0183] All antibodies were combined in the following variations (Tables 4 and 5) as pre-coated microplates and labeled antibodies and used in sandwich immunoassays. Incubations were performed as described in the hDPP3 immunoassay section. Results for recombinant human GST-DPP3 (100, 10, 1 ng / ml) and native hDPP3 in plasma samples are shown as specific signal / background signal ratios. [Table 5] [Table 6]

[0184] For recombinant GST-hDPP3, all combinations showed good signal / noise ratios. Moreover, for the native samples, all combinations except 2552 and 2554 showed good signal / noise ratios. Therefore, all remaining combinations can be used for further investigation. With regard to the highest absolute value of RLU signal, 2555 was used as the solid-phase antibody and 2553 was used as the labeled antibody. 5. Example 5

[0185] 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)) were quantified by hDPP3 immunoassay and compared with plasma DPP3 concentrations in healthy controls. 5.1 Study cohort

[0186] Plasma samples were obtained from 214 patients who were admitted directly to the emergency department or oncology department with their first symptoms. The final diagnosis allowed the patients to be divided into six subgroups: patients with acute heart failure (AHF), myocardial infarction (MI), sepsis, cancer, acute kidney injury (AKI), and lower respiratory tract infection (LRTI). The control group consisted of plasma samples from 93 healthy controls. 5.2.hDPP3 Immunoassay

[0187] mAbDPP3_2555 was used as a solid-phase antibody, and mAbDPP3_2553 was used as a labeled tracer antibody. Immobilization, labeling, and incubation of the antibodies were performed as described in Example 2. (5.3.Results)

[0188] The data obtained, together with the corresponding diagnostic details, were statistically analyzed (Figure 2A). All patients showed significantly higher plasma DPP3 concentrations compared to healthy controls (normal). Table 6 shows the percentage of patients with DPP3 values ​​above the 75th percentile of the control group and the respective diagnosis. The analysis results of plasma DPP3 levels indicate the disease status of the patients. This finding can also be used in the diagnostic field and also lays the basis for therapeutic treatment (e.g. by inhibition of DPP3). [Table 7]

[0189] The same study cohort was analyzed by mortality. Patients who died after admission to the emergency department had significantly higher plasma DPP3 levels than emergency patients who survived in the hospital. Thus, elevated DPP3 concentrations indicate a poor prognosis with respect to mortality (Figure 2B). 6. Example 6

[0190] The amount of DPP3 in human plasma can be quantified not only by DPP3 concentration but also by activity assays. One standard procedure is a soluble activity assay using Arg-Arg-βNA as a fluorogenic substrate.

[0191] Native human DPPIII activity was quantified by hydrolysis of Arg-Arg-β-naphthylamide (Bachem Holdig AG, Switzerland) to produce fluorescent β-naphthylamide. 200 μl of buffer (50 mM Tris / HCl, pH 8.8, 0.04% NaN3, 50 μM amastatin, 100 μM leupeptin) and 10 μl of sample (human plasma) were pipetted into a 96-well black microplate (Greiner Bio-One International GmbH, Austria) and pre-warmed for 10 min at 37°C. Substrate (20 μl, 2 mM) was added and the increase in fluorescence was monitored for 1 h at 37°C in a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG) at an excitation wavelength of 340 nm and an emission wavelength of 410 nm. The slope of the fluorescence increase of various samples was calculated as nmol(βNA) / min / ml(sample) [nmol(βNA)min -1 ml -1 ] is calculated.

[0192] The standard soluble DPP3 activity assay described does not allow us to determine whether we are measuring DPP3 activity or the activity of other aminopeptidases in plasma. To generate a signal specific for DPP3, we performed an enzyme capture assay. In this assay, in a first step DPP3 is immobilized on a surface by binding to a monoclonal antibody, and after a washing step, only the specific DPP3 activity can be measured.

[0193] Solid phases were prepared as described in Example 3 using 96-well black microplates (Greiner Bio-One International GmbH, Austria). 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 amastatin, 100 μmol / l leupeptin; pH 7.4) were pipetted into the coated microplates and incubated (2-8°C, 600 rpm for 18-24 h). Unbound analyte was removed by washing with washing solution (3 × 350 μl). After addition of the substrate (100 μM in 200 μl [50 mM Tris / HCl (pH 8.8 at 25°C), 0.04% NaN3]), the increase in fluorescence was monitored for 1 h at 37°C with an excitation wavelength of 340 nm and an emission wavelength of 410 nm in a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG). The slope of the fluorescence increase of the various samples was expressed as nmol(βNA) / min / ml(sample) [nmol(βNA)min -1 ml -1 ] is calculated.

[0194] For each activity assay type, free βNA was used as the calibration sample for the assay. Therefore, increasing concentrations of βNA were used (0, 4, 8, 16, 32, 64, 125, 250 μM βNA dissolved in 50 mM Tris / HCl (pH 8.8 at 25 °C), 0.04% NaN3 in 200 μl, respectively) and measured at 37 °C in 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. All sample measurements were calibrated with this βNA standard. 7. Example 7

[0195] DPP3 activity in plasma from patients with various diseases (patients with acute heart failure (AHF), sepsis, acute kidney injury (AKI), and lower respiratory tract infection (LRTI)) was quantified using a specific DPP3 scavenging activity assay and compared with DPP3 activity in plasma from healthy controls. (7.1. Method)

[0196] A portion of the cohort analyzed in Example 3 was subjected to a DPP3-specific enzyme capture activity assay, in which DPP3 from plasma samples (10 μl) was first enriched by an affinity purification step and then its activity was measured by adding the fluorogenic substrate Arg-Arg-βNA (see Example 4 for a detailed description). The slope of the fluorescence increase for the various samples (nmol(βNA) / min / ml(sample) [nmol(βNA)min -1 ml -1 ]) calculations are for a sample size of 10 μl. (7.2.Results)

[0197] When patient samples were compared with healthy controls, DPP3 activity levels were significantly higher in all patients (AHF, sepsis, AKI, and LTRI) (Figure 3).

[0198] Table 7 shows the percentage of patients with DPP3 values ​​above the 75th percentile of the control group and their respective diagnoses. Activity was more adequate in healthy controls and borderline diseased patients. [Table 8]

[0199] To further compare the DPP3 activity assay and concentration assay, receiver operating characteristic (ROC) analysis was performed for the boundary between healthy controls and AHF patients (Figure 4A) or sepsis patients (Figure 4B). 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 than the sandwich immunoassay. [Table 9] 8. Example 8

[0200] In this study, we monitored the overall safety of increasing doses of mAb DPP3 in healthy mice. (8.1. Method)

[0201] Female BALB / c nude mice (CAnN.Cg-Foxn1nu / Crl) (Charles River GmbH, Sulzfeld, Germany), 4–5 weeks old on arrival and weighing approximately 15–18 g, were housed under optimal hygienic conditions in an air-conditioned, continuously monitored environment with 10–15 air changes per hour, a target temperature range of 22 ± 3 °C, a target relative humidity range of 30–70%, and 12 h artificial fluorescent light / 12 h dark. They were housed up to four per individually ventilated cage (IVC) and fed a diet consisting of M-Zucht (ssniff Spezialdiaten GmbH) and autoclaved public tap water.

[0202] After a 4-day acclimation period, administration of mAbDPP3 was initiated. Three different concentrations of mAbDPP3 (0.65 mg / kg, 1.9 mg / kg, and 5.75 mg / kg) were dissolved in PBS and injected into four mice per group. mAbDPP3 was administered intraperitoneally (ip) on days 1, 3, 5, and 7, and the mice were monitored for 14 days. (8.2.Results)

[0203] All mice survived the 14-day treatment without side effects, even at the highest dose. mAbDPP3 is safe for use in other animal studies (always performed at a concentration of 1.9 mg / kg). 9. Example 9

[0204] In this study, the overall safety of mAbDPP3 treatment in healthy rats was studied by monitoring mean blood pressure. (9.1. Method)

[0205] Male Wistar rats (Charles River Laboratories, Germany) aged 2-3 months were used in this study. A catheter (Introcan-W; 22 G / 1''; B. Braun) was inserted into the right common carotid artery for measuring and recording blood pressure (BP). The dosing and sampling catheters were placed in the left jugular vein.

[0206] Animals were first anesthetized with isoflurane, weighed (all in g), and injected intraperitoneally (ip) with 1.2 g / kg (body weight) of urethane (concentration = 0.4 g / mL) for long-term anesthesia. The ventral area of ​​the neck was then clipped and wiped with ethanol. The vessels were prepared and catheterized. Finally, both catheters were flushed with heparinized isotonic saline. A blood pressure measuring device (Medex Logical (Medex Medical Ltd.)) was then connected to a patient monitoring system (Datex-Ohmeda (GE)). A laptop computer was connected to the BP monitor and BP data were recorded separately with S / 5 Collect Software.

[0207] Rats were treated with PBS, 1.9 mg / kg or 5.75 mg / kg of mAbDPP3 in PBS (n=3 per group). Compounds were administered via an intravenous catheter. Blood pressure was monitored 1 hour before and 6 hours after compound administration. (9.2.Results)

[0208] Rats responded well to mAbDPP3 treatment. Rats treated with high doses of mAbDPP3 showed a slight increase in mean blood pressure (Figure 5). In general, mAbDPP3 is also safe for use in the rat model, even at high doses. 10. Example 10

[0209] In this study, we analyzed how treatment with mAbDPP3 might affect sepsis mortality in a CLP mouse model. (10.1. Method)

[0210] Male C57Bl / 6 mice (Charles River Laboratories, Germany) aged 12–15 weeks were used in this study. Peritonitis was surgically induced under mild isoflurane anesthesia. An incision was made in the left upper quadrant of the abdominal cavity (normal location of the cecum). The cecum was exposed and a tight ligation was made around it with sutures distal to the introduction of the small intestine. A single puncture wound was made in the cecum with a 24-gauge needle, through which a small amount of cecal contents was expelled. The cecum was replaced in the abdominal cavity, and the laparotomy site was closed. Finally, the animals were returned to their cages with free access to food and water. 500 μl of saline was administered subcutaneously (sc) as fluid replacement.

[0211] mAbDPP3 (1.9 mg / kg dissolved in PBS) was tested versus vehicle (PBS). Compound or vehicle was injected intravenously (iv) 5 min before CLP (prophylactic treatment) and 2 h after CLP (therapeutic treatment) after full development of sepsis. Each group contained 10 mice and was followed for 7 days. (10.2.Results)

[0212] As can be seen from Figure 6, mAbDPP3 antibody significantly reduced the mortality rate compared to PBS treatment. After 4 days, 75% of mice treated with mAbDPP3 were alive. In contrast, after 4 days, almost all mice treated with the vehicle had died. (11. Example 11)

[0213] A model of septic shock was used to induce heart failure in rats and characterize the effects of mAbDPP3 on cardiac function. (11.1. Method) (Research design)

[0214] The study flow is shown in Figure 7A below. After CLP or sham surgery, animals were allowed to rest for 20 h with free access to water and food. Then, rats were anesthetized, tracheotomized, and arterial and venous lines were placed. 24 h after CLP surgery, mAbDPP3 or vehicle (saline) was administered at 2 mg / kg. Hemodynamics was continuously monitored invasively (t=0–3 h). Echocardiography of the heart was performed immediately after surgery and 15 min, 1 h, 2 h, and 3 h after mAbDPP3 or saline injection. (CLP model of sepsis)

[0215] Male Wistar rats (2–3 months old, 300–400 g, see Table 1 for group sizes) obtained from the Centre d'elevage Janvier (France) were randomly assigned to one of three groups. All animals were anesthetized intraperitoneally (i.p.) with ketamine hydrochloride (90 mg / kg) and xylazine (9 mg / kg). To induce polymicrobial sepsis, a cecal ligation and puncture procedure (CLP) was performed according to a slight modification of the Rittirsch protocol. A ventral midline incision (1.5 cm) was made to expose the cecum. The cecum was then ligated just below the ileocecal valve and punctured once with an 18-gauge needle. The abdominal cavity was then closed in two layers, fluid resuscitation was performed (3 ml / 100 g body weight saline subcutaneously), and the animals were returned to their cages. Animals in the sham group were operated on without cecal puncture. (invasive blood pressure)

[0216] Haemodynamic variables were measured using the AcqKnowledge system (BIOPAC Systems, Inc., USA), which provides a fully automated blood pressure analysis system. The catheter was connected to the BIOPAC system via a pressure sensor.

[0217] For this procedure, rats were anesthetized (ketamine and xylazine). The animals were transferred to a heating pad to the desired body temperature (~37-37.5°C). A temperature feedback probe was inserted into the rectum. The rat was placed on the operating table in a supine position. The trachea was opened and catheters (16G) were inserted into the carotid artery and vagus nerve sparingly for external ventilation. An arterial catheter was inserted into the right carotid artery. The carotid artery was separated from the vagus nerve and then ligated.

[0218] A central venous catheter was inserted through the left jugular vein to allow administration of drugs.

[0219] After surgery, animals were allowed to rest to reach a stable state before hemodynamic measurements were performed. Baseline blood pressure (BP) values ​​were then recorded. Arterial saline infusion was stopped during data collection. (Echocardiogram)

[0220] The animals were anesthetized with ketamine hydrochloride. The hair on the chest was clipped and the rats were placed in a supine position.

[0221] Transthoracic echocardiography (TTE) was performed using a commercially available GE Healthcare Vivid 7 Ultra-sound System equipped with a high-frequency (14 MHz) linear probe and a cardiac probe (10 MHz). All examinations were digitally recorded and stored for subsequent offline analysis.

[0222] Gray-scale images were recorded at a depth of 2 cm. Two-dimensional examination was started in the parasternal long-axis view to measure the aortic annulus and pulmonary artery diameters. In addition, M-mode was used to measure left ventricular (LV) dimensions and evaluate fractional shortening (FS (%)). LVFS was calculated as LV end-diastolic diameter-LV end-systolic diameter / LV end-diastolic diameter and expressed as a percentage. Thus, the time of end-diastole was defined by the maximum diameter of the LV. As a result, end-systole was defined as the minimum diameter in the same cardiac cycle. All parameters were measured manually. Three cardiac cycles were averaged for each measurement.

[0223] Pulmonary artery flow was recorded using pulsed Doppler in the same parasternal long axis view, and the velocity time integral of pulmonary outflow was measured.

[0224] Mitral valve blood flow was recorded at the mitral valve tip using the pulsed Doppler technique from the apical five-chamber view. (Experimental time points and animal groups)

[0225] After surgery, baseline BP and echocardiograms were recorded. mAbDPP3 (2mg / kg) or vehicle (saline) was then injected (iv; 5 min after surgery) and saline infusion was initiated. Hemodynamic parameters (BP and echocardiogram) were recorded 15 min, 1 h, 2 h and 3 h after mAbDPP3 or vehicle injection. The table below provides an overview of one control group and two CLP groups (Table 9). At the end of the experiment, animals were euthanized, blood was collected to prepare EDTA-plasma and organs were harvested for further analysis. [Table 10] (11.2.Results)

[0226] Compared with sham-operated animals, septic rats had very low blood pressure and reduced cardiac shortening fraction. Administration of mAbDPP3 significantly increased the shortening fraction (Fig. 7B), increased the mean blood pressure (Fig. 7C), and greatly improved the health status of septic rats. 12. Example 12

[0227] The aim of the study described here was to evaluate the potential antiproliferative effects of mAb DPP3 in an in vitro cell culture system using several cancer cell lines. (12.1. Method)

[0228] A stock solution of mAbDPP3 (1 mg / ml dissolved in PBS) was diluted to a final concentration ranging from 0 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 / streptavidin.

[0229] A549 cells are human alveolar basal epithelial adenocarcinoma cells. This cell line was first established by culturing explanted lung cancer tissue from a tumor of a 58-year-old male. In nature, these cells are flattened and are involved in the diffusion of several substances, such as water and electrolytes, across the alveoli of the lung. When cultured in vitro, the A549 cells grow as a monolayer of cells and attach to the culture flask. Further characteristics are that these cells can synthesize lecithin and contain high concentrations of unsaturated fatty acids. The A549 cell line has been widely used as an in vitro model of type II lung epithelial cell model for drug metabolism and as a transfection host.

[0230] The HCT116 cell line is a human colon cancer cell line. These epithelial cells have adherent culture properties and are derived from an adult male. This cell line is a suitable transfection host. This line contains a mutation at codon 13 of the ras proto-oncogene and can be used as a positive control for PCR assays for mutations at this codon.

[0231] The MDA-MB231 cell line is a human breast adenocarcinoma with epithelial morphology. These cells were isolated from the pleural effusion of a Caucasian breast cancer patient.

[0232] For each cell line, a 96-well suspension cell culture plate was prepared. A 100 μL bottom layer of soft agar (final concentration 0.6% in complete medium) was poured and allowed to solidify. Then 50 μL of a top layer of soft agar (final concentration 0.4%) containing the corresponding cells and cell number was added on top and allowed to solidify, and the 96-well plate was incubated at 37° C., 10% CO2 overnight.

[0233] The next day, compounds were added to the inner wells of the plate. The test specimens were then incubated in a cell culture incubator. Finally, the test specimens were colored with Alamar Blue and incubated at 37°C for 3 to 5 hours before measuring the fluorescence intensity (excitation: 560 nm, emission: 590 nm). As a low control, cells were cultured at 10 -5As a high control, cells were treated with 0.1% DMSO (solvent control, 6-fold value).

[0234] Raw data were analyzed using high (solvent 0.1% DMSO) and low (10 -5 The IC values ​​were converted to percent soft agar growth in response to M staurosporine. GraphPad Prism 5 software was used to fit a model of a sigmoidal response with a variable slope (IC values ​​with 0% soft agar growth as the lower limit of the constrained range, no lower limit, and 100% soft agar growth as the upper limit). 50 was calculated. (12.2.Results)

[0235] In a cell culture system, the growth of three cancer cell lines (A549, HCT116, MDA-MB231) was evaluated in response to the different doses of mAbDPP3 used (Figure 8). IC was calculated using standard parameters based on the signal of the solvent control at the upper limit (100% soft agar growth) and the signal of the staurosporine control at the lower limit (0% soft agar growth) of the constraint range. 50 The IC values ​​were calculated. 50 The values ​​are summarized in Table 10. mAbDPP3 treatment had an antiproliferative effect on the three cell lines tested. [Table 11] (13. Example 13)

[0236] The aim of the study described here was to evaluate the ability of mAbDPP3 to prevent tumor formation in xenograft models of breast and colon cancer (tumor growth inhibition studies). (13.1. Method)

[0237] Monolayer MDA-MB-231 cells (breast cancer) and HCT-116 cells (colon cancer), respectively, were grown in DMEM + 10% FCS. Cells were cultured at 37 °C in a humidified atmosphere of 90% air and 10% carbon dioxide. Medium was regularly changed every 3 days. Every 3–4 days, confluent cultures were split 1:3 to 1:3 with trypsin / EDTA until the density reached approximately 3–4 × 10 6 Cell / 15cm 2 +25mL medium was inoculated.

[0238] Female BALB / c nude mice (CAnN.Cg-Foxn1) were 4-5 weeks old and weighed approximately 15-18 g upon arrival. nu Mice were housed in an air-conditioned, continuously monitored environment with 10–15 air changes per hour under optimal hygienic conditions, a target temperature range of 22 ± 3°C, a target relative humidity range of 30–70%, and 12 h artificial fluorescent light / 12 h dark. They were housed up to four per individually ventilated cage (IVC) and fed a diet consisting of M-Zucht (ssniff Spezialdiaten GmbH) and autoclaved municipal tap water.

[0239] Human breast cancer cells MDA-MB-231 and colon cancer cells HCT-116 provided by ATCC were used in this study. Cell subcultures of 5 or fewer passages were inoculated into mice. 3 × 10 6 0.1 mL of cells were injected subcutaneously into the right flank of the mice. 3 When the tumor reaches 100%, 20 mice with tumors of appropriate size are randomly divided into groups (10 mice per group) according to tumor volume and body weight. Animals are dosed according to tumor volume and body weight and treatment begins. The groups are shown in the table below (Table 11). [Table 12]

[0240] Animals were observed daily for behavior and health for 24 days, and tumor growth was recorded every 2 days by caliper measurement. Primary tumor size was measured by caliper measurement (OMC Fontana manual caliper). The formula V=W 2 Tumor size was calculated according to × L / 2 (L = length, W = vertical width of the tumor, L > W). Individual relative tumor volumes (RTV) were calculated as follows: RTV = V t / V0(V t is the volume on each day, and V0 is the volume at the start of treatment). (13.2.Results)

[0241] In xenograft cancer models, the formation of all tumors investigated was reduced by administration of mAbDPP3 (Figure 9A-D). When treated with mAbDPP3, breast cell tumors took 2 days longer to grow to 20-fold larger (Figure 9B), and colon cell tumors took 2 days longer to grow to 10-fold larger (Figure 9D). In this model, tumors induced by colon cancer cell lines grew much slower than tumors induced by breast cancer cell lines. (14. Example 14)

[0242] To evaluate the ability of the DPP3 adsorbent to remove excess DPP3 from plasma, we should analyze whether DPP3 binds sufficiently to the anti-DPP3 column. Affinity chromatography columns were prepared by immobilizing DPP3-binding antibodies on GlycoLink columns (Thermo Fisher). The binding of DPP3 to the columns was analyzed by measuring the DPP3 concentration before and after passing the columns. (14.1. Method)

[0243] In the first step, all DPP3-binding antibodies (mAbDPP3_2552, 2553, 2554, 2555) were oxidized and immobilized on one GlycoLink column according to the manufacturer's instructions, and then recombinant GST-hDPP3 (USBio) or patient plasma samples were injected onto the column to monitor DPP3 binding. (Oxidation of Antibodies)

[0244] Therefore, 300 μl of each 3 mg / ml DPP3 antibody solution was diluted into 700 μl of GlycoLink Coupling Buffer to a final volume of 1 ml, pH < 6. To oxidize the carbohydrate groups of the antibody, 2.1 mg of sodium metaperiodate was added to the solution and incubated at room temperature for 30 min. The oxidizing agent was removed from the antibody solution using a desalting column. (Preparation of GlycoLink column)

[0245] To catalyze the coupling reaction, 0.1M aniline was added to the oxidized antibody. The solution was then loaded onto an equilibrated GlycoLink column and incubated at room temperature for 4 hours. Unbound material was flushed from the column, after which the column was washed, equilibrated, and stored until further use. (Affinity purification of DPP3)

[0246] Recombinant GST-hDPP3 or patient plasma was diluted in coupling buffer (final recombinant DPP3 concentration = 100 ng / ml, 1 ml; 1 ml plasma + 1 ml coupling buffer) and injected onto the equilibrated mAbDPP3 column and incubated at room temperature for 30 min. A total flow rate was maintained allowing assessment of binding efficiency and capacity. The column was washed and bound proteins were eluted with GlycoLink Elution Buffer, equilibrated and stored. (Analysis of DPP3 content)

[0247] The DPP3 concentrations of the samples before affinity purification and the recombinant GST-hDPP3 and the input were measured by a sandwich-type luminescence immunoassay (see Example 2 for details). 20 μl of sample (or calibrator) was pipetted into a pre-coated 96-well microplate, diluted labeled detection antibody (200 μl) was added, and the plate was incubated at 2-8°C for 18-24 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). Fully bound chemiluminescence was measured in a Centro LB 960 fluorometer (Berthold Technologies GmbH & Co. KG). (14.2.Results)

[0248] Nearly all of the DPP3 in plasma and recombinant DPP3 solutions is removed from the samples by affinity chromatography (see Table 12). Different mAb DPP3 antibodies show different DPP3 affinities. mAb DPP3_2555 has the highest binding rate to recombinant DPP3, so this antibody was selected for chromatography of plasma samples. Table 12 shows that affinity chromatography can significantly reduce plasma DPP3 concentrations. These results indicate that the DPP3 adsorbent can be used in apheresis to remove excess DPP3 from patients' plasma. [Table 13] (References) [Brief description of the drawings]

[0249] [Figure 1A] Figure 1 shows inhibition of DPP3 activity. (A) The activity of recombinant GST-hDPP3 was measured in the presence of several different anti-DPP3 antibodies. DPP3-binding antibodies raised against peptides and / or full-length (FL) native DPP3 show strong inhibitory effects of up to 70%. [Figure 1B] (B) Inhibition curve of recombinant GST-hDPP3 by inhibitory mAbDPP3. The inhibition of DPP3 by specific antibodies is concentration-dependent, with an IC50 value of approximately 0.2 μg / ml. [Figure 2A] Figure 2 shows the concentration of DPP3, a diagnostic marker. (A) DPP3 concentration 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) is shown. The median value of the patient group is significantly different from that of healthy controls (Mann-Whitney test, p-value <0.005). [Figure 2B] (B) Comparison of plasma DPP3 concentrations between patients who died shortly after admission to the emergency department and those who survived, with survivors having significantly lower DPP3 concentrations (Mann-Whitney test, p-value <0.05). [Diagram 3] Figure 3 shows DPP3 activity as a diagnostic marker. DPP3 activity in EDTA plasma of healthy controls and patients with various diseases (AHF: acute heart failure, sepsis, AKI: acute kidney injury, LRTI: lower respiratory tract infection) is shown. The median values ​​of the patient group are significantly different from the healthy controls (Mann-Whitney test, p-value <0.0001). [Figure 4A] Figure 4 shows ROC plot analysis of DPP3 activity and concentration assays. (A) ROC analysis results for healthy controls and AHF patients are shown. [Figure 4B] (B) ROC analysis results for healthy controls and sepsis patients. [Diagram 5] Figure 5 shows the safety of mAbDPP3 treatment (blood pressure). Healthy rats were treated with PBS or mAbDPP3 (5.75 mg / kg). Blood pressure (BP) was measured and recorded with a catheter inserted into the right common carotid artery. The dosing and sampling catheters were inserted into the left jugular vein. Treatment resulted in a slight increase in relative blood pressure compared to PBS-treated rats (n=3 per group). [Figure 6] Figure 6 shows the effect of mAbDPP3 on the mortality of septic mice. Septic mice (CLP model) were treated with PBS or mAbDPP3 (1.9 mg / kg) 5 min before and 2 h after CLP. Mortality was monitored for 7 days. Kaplan-Meier plot shows that septic mice survived longer after mAbDPP3 treatment. [Figure 7A] 7 shows the effect of mAbDPP3 on heart failure in septic rats. (A) Experimental design of the study on heart failure in rats with septic shock is shown. [Figure 7B] (B) CLP induces cardiac failure in rats, as indicated by a reduced fractional shortening compared to sham-operated rats, which is significantly increased by mAbDPP3 treatment (2 mg / kg; n ≥ 7 per group; Mann-Whitney test, p value < 0.0001). [Figure 7C](C) Mean blood pressure in vehicle-treated septic rats decreases over time, whereas mBP is significantly increased by mAbDPP3 treatment (2 mg / kg; n ≥ 7 per group; Mann-Whitney test, p value < 0.005). [Figure 8] Figure 8 shows the effect of mAbDPP3 on tumor growth in vitro. A soft agar test using tumor cell lines (lung cancer, colon cancer, breast cancer) is shown. Tumor cell growth is reduced by adding anti-DPP3 antibody. [Figure 9A] Figure 9 shows the effect of mAbDPP3 on tumor growth in vivo. (A) Mice bearing mammary tumor xenografts (n=10 per group) were treated with PBS or mAbDPP3. The relative tumor volume growth curves over 24 days show reduced tumor growth in mice treated with mAbDPP3. [Figure 9B] (B) Comparison of the time it takes for breast cell tumors to grow 20-fold in volume with and without mAbDPP3 treatment, with the growth time being significantly longer in the case of mAbDPP3 treatment (Mann-Whitney test, p-value < 0.05). [Figure 9C] (C) Mice bearing colon tumor cell xenografts (n=10 per group) were treated with PBS or mAbDPP3. Relative tumor volume growth curves over 30 days show reduced tumor growth in mice treated with mAbDPP3. [Figure 9D] (D) Comparison of the time it takes for colon cell tumors to increase 10-fold in volume with and without mAbDPP3 treatment, with the growth time being longer in the case of mAbDPP3 treatment. [Figure 10] Figure 10 shows DPP3 activity as a diagnostic marker (II). DPP3 activity in EDTA plasma from healthy controls and patients with various diseases (acute myocardial infarction (AMI), cardiogenic shock, septic shock, and liver failure) is shown. The median values ​​of the patient group are significantly different from those of healthy controls (Mann-Whitney test, p-value < 0.05). [Figure 11]Figure 11 shows the effect 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 with a catheter inserted into the right common carotid artery. DPP3 was injected intravenously through the tail vein. BP is reduced by DPP3 injection.

Claims

1. 1. A method for diagnosing a disease or condition involving or associated with a necrotic process in a subject, comprising: Quantifying the total amount of DPP3 and / or the amount of active DPP3 in the body fluid sample of the subject; and comparing the quantified amount of total DPP3 or active DPP3 to a predetermined threshold value; If the quantified amount exceeds the predetermined threshold, the subject is diagnosed as having a disease or condition involving or associated with a necrotic process.

2. The method for diagnosing a disease or condition in a subject involving or associated with a necrotic process according to claim 1 , wherein the amount of total or active DPP3 is quantified in units of concentration.

3. A method for diagnosing a disease or condition in a subject involving or associated with a necrotic process according to claim 1 or 2, wherein said sample is selected from the group comprising whole blood, serum, plasma.

4. 4. The method for diagnosing a disease or condition in a subject involving or associated with a necrotic process according to any one of claims 1 to 3, wherein said disease is selected from the group comprising heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial, viral (e.g. acquired immune deficiency syndrome (AIDS), parasitic (e.g. malaria)), systemic inflammatory response syndrome (SIRS) or sepsis, cancer, acute kidney injury (AKI), central nervous system (CNS) disorders (e.g. stroke, neurodegenerative diseases), autoimmune diseases and vascular diseases (e.g. Kawasaki syndrome), and hypotension.

5. The amount of total DPP3 and / or active DPP3 is quantified in a body fluid sample from the subject; and contacting the sample with a capture binding agent that specifically binds full-length DPP3; Separating DPP3 bound to the capture binding agent; Adding a DPP3 substrate to the separated DPP3; Quantifying the active DPP3 by measuring and quantifying the conversion rate of the DPP3 substrate; A method for diagnosing a disease or condition in a subject involving or associated with a necrotic process according to any one of claims 1 to 4, comprising:

6. The method for diagnosing a disease or condition in a subject involving or associated with a necrotic process according to claim 5, wherein the capture binding agent may be selected from the group of an antibody, an antibody fragment, or a non-IgG scaffold.

7. A method for diagnosing a disease or condition in a subject involving or associated with a necrotic process according to claim 5 or 6, wherein the capture binding agent is an antibody.

8. A method for diagnosing a disease or condition in a subject involving or associated with necrotic processes according to any one of claims 5 to 7, wherein said capture binding agent is immobilised on a surface.

9. A method for diagnosing a disease or condition in a subject involving or associated with a necrotic process as described in any one of claims 5 to 8, wherein the separation step is a washing step in which components of the sample that are not bound to the capture binding agent are removed from the captured DPP3.

10. 10. Method for diagnosing a disease or condition in a subject involving or associated with a necrotic process according to any one of claims 5 to 9, wherein the conversion of the DPP3 substrate is detected by a method selected from the group comprising: fluorescence of fluorogenic substrates (e.g. Arg-Arg-βNA, Arg-Arg-AMC), color change of chromogenic substrates, light emission of substrates coupled to aminoluciferin (Promega Protease-Glo™ Assay), mass spectrometry, HPLC / FPLC (reversed phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis followed by activity staining (fixed active DPP3) or Western blot (cleavage products).

11. 11. Method for diagnosing a disease or condition in a subject involving or associated with a necrotic process according to any one of claims 5 to 10, wherein the substrate may be selected from the group comprising angiotensin II, III, IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (MSH); and a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, the dipeptide being Arg-Arg.

12. 12. A method for diagnosing a disease or condition in a subject involving or associated with a necrotic process according to any one of claims 5 to 11, wherein the substrate may be selected from the group comprising a dipeptide linked to a fluorophore, a chromophore or an aminoluciferin, the dipeptide being Arg-Arg.

13. A method for monitoring a disease or condition in a subject involving or associated with a necrotic process, comprising carrying out at least two diagnostic methods according to any one of claims 1 to 12.

14. An inhibitor of DPP3 activity for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process.

15. The DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process as described in claim 14, wherein the inhibitor is selected from the group comprising anti-DPP3 antibodies and anti-DPP3 antibody fragments, and anti-DPP3 non-Ig scaffolds.

16. 16. A DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process according to claim 14 or 15, wherein the disease is selected from the group comprising heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial, viral (e.g. AIDS), parasitic (e.g. malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. stroke, neurodegenerative diseases), autoimmune diseases and vascular diseases (e.g. Kawasaki syndrome), and hypotension.

17. A DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process according to any one of claims 14 to 16, wherein the inhibitor is an antibody of single binding specificity (mono-binding) or at least dual binding specificity (two-binding).

18. A DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process according to any one of claims 14 to 17, wherein the inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment that binds to SEQ ID NO: 1 and in particular to SEQ ID NO: 2, or an anti-DPP3 non-Ig scaffold.

19. The inhibitor has a potency of 10 －７ A DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process according to any one of claims 14 to 18, which is an antibody or fragment or scaffold exhibiting a minimum binding affinity of M or less.

20. An inhibitor of DPP3 activity for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process according to any one of claims 14 to 19, wherein the inhibitor is a monospecific antibody or fragment or scaffold.

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

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

23. A DPP3 activity inhibitor for use in the prevention or treatment of a disease or condition in a subject involving or associated with a necrotic process according to any one of claims 14 to 22, wherein the subject has an amount of total DPP3 and / or active DPP3 in a body fluid sample above a predetermined threshold.

24. A pharmaceutical composition comprising a DPP3 activity inhibitor according to any one of claims 14 to 23 for use in the prevention or treatment of a disease or condition in a subject which involves or is associated with a necrotic process.

25. Use of the DPP3 activity inhibitor according to any one of claims 14 to 23 in a method for extracorporeal removal of DPP3 from plasma, comprising apheresis and affinity chromatography.

26. 1. A method for quantifying active DPP3 in a body fluid sample from a subject, comprising: contacting the sample with a capture binding agent that specifically binds full-length DPP3; Separating DPP3 bound to the capture binding agent; Adding a DPP3 substrate to the separated DPP3; Quantifying the active DPP3 by measuring and quantifying the conversion rate of the DPP3 substrate; The method includes:

27. 27. The method of claim 26, wherein the capture binding agent may be selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

28. 28. A method for quantifying active DPP3 in a body fluid sample from a subject according to claim 26 or 27, wherein the capture binding agent is an antibody.

29. The method for quantifying active DPP3 in a body fluid sample of a subject according to any one of claims 26 to 28, wherein the capture binding agent is immobilized on a surface.

30. The method for quantifying active DPP3 in a body fluid sample from a subject described in any one of claims 26 to 29, wherein the separation step is a washing step for removing components of the sample that are not bound to the capture binding agent from the captured DPP3.

31. 31. The method for quantifying active DPP3 in a body fluid sample of a subject according to any one of claims 26 to 30, wherein the conversion of DPP3 substrate is detected by a method selected from the group comprising: fluorescence of fluorogenic substrates (e.g. Arg-Arg-βNA, Arg-Arg-AMC), color change of chromogenic substrates, light emission of substrates conjugated to aminoluciferin (Promega Protease-Glo™ Assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis followed by activity staining (fixed active DPP3) or Western blot (cleavage products).

32. The method for quantifying 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 the group comprising a dipeptide bound to a fluorescent dye molecule, a chromophore, or an aminoluciferin, and the dipeptide is Arg-Arg.

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

34. 1. An assay or kit for quantifying active DPP3 in a body fluid sample from a subject, comprising: a capture binding agent that specifically binds full-length DPP3; Substrates of DPP3 4. An assay or kit comprising:

35. 35. An assay or kit for quantifying active DPP3 in a subject's body fluid sample as described in claim 34, wherein the capture binding agent may be selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

36. 36. An assay or kit for quantifying active DPP3 in a body fluid sample from a subject according to claim 34 or 35, wherein the capture binding agent inhibits DPP3 activity by less than 50%, preferably less than 40%, more preferably less than 30% in a liquid phase assay.

37. 37. An assay or kit for quantifying active DPP3 in a body fluid sample of a subject according to any one of claims 34 to 36, wherein the binding region of DPP3 for the capture binding agent is not within the region of amino acids 316 to 669 of SEQ ID NO:

1.

38. 38. An assay or kit for quantifying active DPP3 in a body fluid sample of a subject according to any one of claims 34 to 37, wherein the capture binding agent is an antibody.

39. 39. An assay or kit for quantifying active DPP3 in a sample of a subject's body fluid according to any one of claims 34 to 38, wherein the capture binding agent is immobilised on a surface.

40. 40. The assay or kit for quantifying active 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 the group comprising angiotensin II, III, IV, Leu enkephalin, Met enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH, and MSH; and a dipeptide linked to a fluorophore, a chromophore, or an aminoluciferin, wherein the dipeptide is Arg-Arg.

41. Use of a method for quantifying active DPP3 in a body fluid sample from a subject according to any one of claims 26 to 33, or use of an assay or kit according to any one of claims 34 to 40, in a method for diagnosing or monitoring a disease or condition in a subject involving or associated with a necrotic process according to any one of claims 1 to 13.

Images