DPP3 activity inhibitor, pharmaceutical composition comprising same, and use thereof

Abstract

The invention relates to a DPP3 activity inhibitor, a pharmaceutical composition containing the DPP3 activity inhibitor and application of the DPP3 activity inhibitor. The DPP3 activity inhibitor of the present invention prevents or treats a disease or condition associated with or associated with a necrosis process by modulating DPP3 activity in a body fluid of a subject, wherein the disease is selected from the group consisting of heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, hepatic failure, burn, trauma, severe infection (microorganisms, viruses (e.g., AIDS), parasites (e.g., malaria), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g., epilepsy, neurodegenerative diseases), autoimmune diseases, and vascular diseases (e.g., epilepsy, neurodegenerative diseases). Kawasaki syndrome) and hypotension.

Description

Parent case information

[0001] This application is a divisional application of the Chinese patent application with application number "201780039077.1" and title "Methods and treatment methods for determining DPP3". Technical Field

[0002] The present invention relates to a DPP3 activity inhibitor, a pharmaceutical composition containing the same and use thereof. Background Art

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

[0004] DPP3 was first identified by Ellis & Nuenke in 1967 and its activity was measured in purified bovine anterior pituitary extracts. The enzyme is classified as EC 3.4.14.4, has a molecular weight of approximately 83 kDa, and is highly conserved in prokaryotes and eukaryotes (Prajapati et al., 2001). & Chauhan, 2011). The amino acid sequence of the human variant is described in SEQ ID NO 1. Dipeptidyl peptidase III is a ubiquitously expressed major cytoplasmic peptidase. Despite the lack of a signal sequence, some studies have reported membrane activity (Lee & Snyder, 1982).

[0005] DPP3 is a zinc-dependent exopeptidase belonging to the peptidase family M49. It has a broad substrate specificity for oligopeptides of three / four to ten amino acids of various compositions and is also able to cleave after proline. DPP3 is known to hydrolyze peptides from the N-terminus of its substrates, including angiotensin II, angiotensin III, and angiotensin IV; leucine enkephalin and methionine enkephalin; endomorphin 1 and endomorphin 2. The metallopeptidase DPP3 has its optimal activity at pH 8.0 to 9.0 and can be activated by the addition of divalent metal ions such as CO 2+ and Mg 2+To activate. Structural analysis of DPP3 revealed the catalytic motifs HELLGH (hDPP3 450-455) and EECRAE (hDPP3 507-512) and the following amino acids important for substrate binding and hydrolysis: Glu316, Tyr318, Asp366, Asn391, Asn394, His568, Arg572, Arg577, Lys666 and Arg669 (Prajapati & Chauhan, 2011; Kumar et al., 2016; numbering refers to the sequence of human DPP3, see SEQ ID No. 1). Taking into account 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 from amino acid 316 to amino acid 669.

[0006] The activity of DPP3 can be inhibited by different general protease inhibitors (e.g. PMSF, TPCK), sulfhydryl reagents (e.g. pHMB, DTNB), and metal chelators (EDTA, catechol; et al., 2000 ) inhibited nonspecifically.

[0007] DPP3 activity can be specifically inhibited by different classes of compounds: Endogenous DPP3 inhibitors are the peptide spinorphins. Several synthetic spinorphin derivatives have been produced, for example, tynorphin, and have been shown to inhibit DPP3 activity to varying degrees (Yamamoto et al., 2000). Other disclosed DPP3 peptide inhibitors are propioxatin A and propioxatin B (US4804676) and propioxatin A analogs (Inaoka et al., 1988).

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

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

[0010] DPP3 has been shown to be a promising biomarker in several publications, all of which involve intracellular DPP3. DPP3 activity has been shown to be elevated in homogenates of ovarian and endometrial tumors. DPP3 activity even increases with the severity / malignancy of the tumor ( et al., 1998 and 2003). Immunohistological and western blot analysis of glioblastoma cell lines also showed elevated levels of DPP3 (Singh et al., 2014).

[0011] It is also proposed that intracellular or membranous DPP3 is a potential marker of arterial risk (arteriorisk) (US2011008805) and a marker of rheumatoid arthritis (US2006177886). Patent application WO2005106486 claims protection: DPP3 expression and activity as a diagnostic marker and DPP3 as a therapeutic target in all kinds of diseases, due to the widespread expression of DPP3 in cells or on the cell surface. EP1498480 mentions the potential diagnostic and therapeutic uses of hydrolases (including DPP3).

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

[0013] Although DPP3 is known as an intracellular protein, DPP3 activity has been detected in several body fluids: post-placental serum (Shimamori et al., 1986), seminal plasma (Vanha-Perttula et al., 1988) and 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) proposed the release of intracellular DPP3 as a marker for dead and / or dying cells in cell culture systems. It has also been proposed that the release of DPP3 from necrotic cells affects immune responses in mouse models (Gamrekelashvili et al., 2013). Summary of the invention

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

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

[0016] Another object of the present invention is to provide methods for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process and methods for treating the disease.

[0017] The subject of the present invention is a method for determining active DPP3 in a body fluid sample of a subject, comprising the following steps: contacting the sample with a capture binding agent that specifically binds to full-length DPP3, · isolating DPP3 bound to the capture binding agent, adding a substrate of DPP3 to the isolated DPP3, Quantify DPP3 activity by measuring the conversion rate of DPP3 substrates.

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

[0019] All definitions and specific embodiments specified in the specification should apply to all aspects and purposes of the present invention. It should be understood that for one aspect or purpose of the present invention, the definitions and specific embodiments detailed and summarized should also be the definitions and specific embodiments of other aspects and purposes of the present invention. For example, the definition of capture binder or specific capture can be applied to all embodiments of the present invention: for example, methods, assays and kits, diagnostic methods, and therapeutic methods for determining DPP3. Such definitions may not be repeated throughout the specification.

[0020] By specifically binding to full-length DPP3 is meant that the capture binding agent does not bind to any other protein than DPP3.By specifically binding to full-length DPP3 is meant that the capture binding agent does not bind to any other aminopeptidase than DPP.

[0021] A binding agent that binds to full-length DPP3 is a binding agent that binds to the protein of SEQ ID No. 1. A binding agent that binds to full-length DPP3 is a binding agent that binds to SEQ ID No.1.

[0022] In a specific embodiment, the capture binder inhibits DPP3 activity in a liquid phase assay by less than 50%, preferably less than 40%, preferably less than 30%.A liquid phase assay is an assay in which cleavage of a substrate by DPP3 occurs in liquid phase.

[0023] According to the present invention, the inhibition of DPP3 activity by a binding agent in a liquid phase assay can be determined as follows: In a liquid phase assay, a potential DPP3 capture binding agent is incubated with recombinant or purified native DPP3 and a specific DPP3 substrate. Preferably, as a capture binding agent for the ECA, a capture agent with minimal inhibitory capacity is selected. The inhibition of DPP3 activity by the capture binding agent should be less than 50%, preferably less than 40%, preferably less than 30%. The specific liquid phase DPP3 activity assay for determining the inhibitory capacity of a potential capture binding agent is described in detail in Example 1 and comprises the following steps: 25 ng / ml recombinant GST-hDPP3 was mixed with 5 μg / ml of the respective capture binder and buffer control in 50 mM Tris-HCl, pH 7.5 and 100 μM ZnCl 2 Incubate at room temperature for 1 hour. • Add the fluorescent substrate Arg-Arg-βNA (20 μl, 2 mM). • Incubate at 37°C and monitor the production of free βNA in a Twinkle LB 970 microplate fluorimeter (Berthold Technologies GmbH) over 1 hour. The fluorescence of βNA is detected by excitation at 340 nm and measuring emission at 410 nm. • Calculate the slope of the fluorescence increase (in RFU / min) for the different samples. The slope of the buffer control GST-hDPP3 was assigned as 100% activity. The inhibitory capacity of a potential capture binder was defined as the reduction in GST-hDPP3 activity caused by incubation with the capture binder, in percentage.

[0024] In contrast, solid phase assays are assays in which the corresponding binding events take place on a solid phase (see Examples 4 and 5).

[0025] For clarity, the method for determining active DPP3 can be performed as a liquid phase assay and a solid phase assay. However, according to the above process, inhibition of DPP3 activity can be determined in a liquid assay.

[0026] Therefore, solid phase assays are embodiments of the present invention. Contacting the sample with a capture binding agent that specifically binds to full-length DPP3 can occur in liquid phase (liquid phase capture assay), and then the separation step can include fixing the capture binding agent-DPP3-complex. Alternatively, the capture binding agent can be fixed on a surface, and the binding event - capture binding agent and DPP3 - is carried out on a solid phase (solid phase capture assay).

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

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

[0029] Known peptide hormone substrates include angiotensin II, III, and IV, leucine enkephalin, methionine enkephalin, endomorphins 1 and 2, valorphin, β-casomorphin, dynorphin, protoenterin, ACTH (adrenocorticotropic hormone), and MSH (melanocyte stimulating hormone; et al., 2000; et al., 2007; Dhanda et al., 2008). Cleavage of the mentioned peptide hormones as well as other untagged oligopeptides (e.g. Ala-Ala-Ala-Ala, Dhanda et al., 2008) can be monitored by detecting the corresponding cleavage products. Detection methods include, but are not limited to, HPLC analysis (e.g., Lee & Snyder, 1982), mass spectrometry (e.g., et al., 2000), H1-NMR analysis (e.g., Vandenberg et al., 1985), capillary zone electrophoresis (CE; e.g., et al., 2007), thin layer chromatography (e.g., Dhanda et al., 2008), or reverse phase chromatography (e.g., Mazocco et al., 2006).

[0030] Detection of fluorescence caused by the hydrolysis of fluorescent substrates by DPP3 is a standard procedure for monitoring DPP3 activity. Those substrates are specific dipeptides or tripeptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) coupled to fluorophores. 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; et al., 2000; Ohkubo et al., 1999). Cleavage of these fluorescent substrates results in the release of fluorescent β-naphthylamine or 7-amino-4-methylcoumarin, respectively. In a liquid phase assay, alternatively, the ECA substrate and DPP3 are incubated in, for example, a 96-well plate format, and the fluorescence is measured using a fluorescence detector (Ellis & Nuenke, 1967). In addition, the DPP3 carrying sample can be immobilized and separated on a gel by electrophoresis, the gel stained with a fluorescent substrate (e.g., Arg-Arg-βNA) and Fast Garnet GBC, and the fluorescent protein bands detected by a fluorescence reader (Ohkubo et al., 1999).

[0031] The same peptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) can be coupled with a chromophore such as p-nitroaniline diacetate. Detection of a color change due to hydrolysis of the chromogenic substrate can be used to monitor DPP3 activity.

[0032] Another option for detecting DPP3 activity is Protease-Glo TM Determination (commercially available at Promega). In this embodiment of the method, a DPP3-specific dipeptide or tripeptide (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) is coupled to aminoluciferin. After being cleaved by DPP3, aminoluciferin is released and used as a substrate for coupled luciferase reaction, which emits detectable luminescence.

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

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

[0035] The antibodies according to the present invention are proteins comprising one or more polypeptides substantially encoded by immunoglobulin genes that specifically bind to an antigen. Recognized immunoglobulin genes include kappa, lambda, alpha (IgA), gamma (IgG 1 IgG 2 IgG 3 IgG 4 )、δ(IgD)、 The ε (IgE) and μ (IgM) constant region genes, as well as numerous immunoglobulin variable region genes. A full-length immunoglobulin light chain is typically about 25 kD or 214 amino acids in length. A full-length immunoglobulin heavy chain is typically about 50 kD or 446 amino acids in length. The light chain consists of 2 The heavy chain is encoded by a variable region gene (about 110 amino acids in length) at the COOH-terminus and a kappa or lambda constant region gene at the COOH-terminus. The heavy chain is similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.

[0036] The basic structural unit of an antibody is usually a tetramer, which consists of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions bind to the antigen, and the constant regions mediate effector functions. Immunoglobulins also exist in a variety of other forms, including, for example, Fv, Fab, and (Fab') 2 , as well as bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al., 1987; Huston et al., 1988; Bird et al., 1988; Hood et al., 1984; Hunkapiller & Hood, 1986). The immunoglobulin light chain or heavy chain variable region includes a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDRs) (see, Kabat et al., 1983). As described above, the CDRs are primarily responsible for binding to the epitope of the antigen. An immune complex is an antibody that specifically binds to an antigen, such as a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody, or a functional antibody fragment.

[0037] Chimeric antibodies are antibodies that are usually constructed from light chain and heavy chain genes of immunoglobulin variable regions and constant region genes belonging to different species by genetic engineering. For example, the variable segment of the gene from a mouse monoclonal antibody can be connected to a human constant segment such as κ and γ1 or γ3. In one example, therefore, therapeutic chimeric antibodies are hybrid proteins consisting of variable or antigen-binding domains from mouse antibodies and constant or effector domains from human antibodies, but other mammalian species can also be used, or variable regions can be produced by molecular technology. Methods for preparing chimeric antibodies are well known in the art, for example, referring to U.S. Patent No. 5,807,715. "Humanized" immunoglobulins are immunoglobulins including human framework regions and one or more CDRs from non-human (such as mouse, rat or synthetic) immunoglobulins. The non-human immunoglobulins providing CDRs are referred to as "donors", and the human immunoglobulins providing frameworks are referred to as "acceptors". In one embodiment, all CDRs are from donor immunoglobulins in humanized immunoglobulins. The constant region need not be present, but if it is present, it must be substantially identical to the constant region of a human immunoglobulin, i.e., at least about 85% to 90%, such as about 95% or more identical. Thus, except for possible CDRs, all parts of a humanized immunoglobulin are substantially identical to corresponding parts of a natural human immunoglobulin sequence. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody providing the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions made by amino acids taken from the donor framework. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are those such as gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg and phe, tyr. Humanized immunoglobulins can be constructed by genetic engineering (e.g., see U.S. Patent No. 5,585,089). Human antibodies are antibodies in which the light chain and heavy chain genes are of human origin. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells that secrete the antibody of interest. Immortalization can be achieved, for example, by EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce tri-source hybridoma cells. Human antibodies can also be produced by phage display methods (see, e.g., PCT Publication No. WO91 / 17271; PCT Publication No. WO92 / 001047; PCT Publication No. WO92 / 20791, which are incorporated herein by reference), or selected from human combinatorial monoclonal antibody libraries (see Morphosys website).Human antibodies can also be prepared by using transgenic animals carrying human immunoglobulin genes (eg, see PCT Publication No. WO 93 / 12227; PCT Publication No. WO 91 / 10741, which are incorporated herein by reference).

[0038] Thus, the DPP3 antibody may have a form known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies. In a preferred embodiment, the antibody according to the invention is a recombinantly produced antibody, such as an IgG, a typical full-length immunoglobulin, or an antibody fragment containing at least the F-variable domain of a heavy chain and / or a light chain, such as a chemically coupled antibody (fragment antigen binding), including but not limited to Fab fragments, including Fab miniantibodies, single-chain Fab antibodies, monovalent Fab antibodies with epitope tags, for example, Fab-V5SX2; divalent Fab (miniantibodies) dimerized with a CH3 domain; divalent Fab or multivalent Fab, for example formed by multimerization with the aid of a heterologous domain, for example formed by dimerization of a dHLX domain, for example Fab-DHLx-FSX2; F(ab')2-fragments, scFv-fragments, multimeric multivalent or / and multispecific scFv-fragments, divalent and / or bispecific diabodies, (bispecific T-cell engagers), trifunctional antibodies, multivalent antibodies, e.g. from classes other than G; single domain antibodies, e.g. nanobodies derived from camel or fish immunoglobulins, and many others.

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

[0040] The non-Ig scaffold can be a protein scaffold and can be used as an antibody mimetic because it can bind to a ligand or antigen. The non-Ig scaffold can be selected from the group comprising: a tetranectin-based non-Ig scaffold (e.g., described in US2010 / 0028995), a fibronectin scaffold (e.g., described in EP1266025); a lipocalin-based scaffold (e.g., described in WO2011 / 154420); a ubiquitin scaffold (e.g., described in WO2011 / 073214), a transfer scaffold (e.g., described in US2004 / 0023334), a protein A scaffold (e.g., described in EP2231860), an ankyrin repeat-based scaffold (e.g., described in WO2010 / 060748), a microprotein (preferably, a microprotein forms a cystine knot) scaffold (e.g., described in EP2314308), a Fyn-based scaffold (e.g., described in EP2314308), a scaffold based on a lipocalin-based scaffold (e.g., described in WO2011 / 073214), a transfer scaffold (e.g., described in US2004 / 0023334), a protein A scaffold (e.g., described in EP2231860), a scaffold based on ankyrin repeats (e.g., described in WO2010 / 060748), a microprotein (preferably, a microprotein forms a cystine knot) scaffold (e.g., described in EP2314308), a scaffold based SH3 domain-based scaffolds (e.g., described in WO2011 / 023685), EGFR-A domain-based scaffolds (e.g., described in WO2005 / 040229), and Kunitz domain-based scaffolds (e.g., described in EP1941867). Non-Ig scaffolds can be peptide or oligonucleotide aptamers. Aptamers are typically generated by selecting them from large random sequence libraries and are short chains of oligonucleotides (DNA, RNA, or XNA; Xu et al., 2010, Deng et al., 2014) or short variable peptide domains attached to a protein scaffold (Li et al., 2011).

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

[0042] Recombinant DPP3 (e.g., GST-hDPP3 from USBio, Salem, USA), a peptide comprising a portion of the DPP3 amino acid sequence (e.g., conjugated to BSA), or naturally purified DPP3 (e.g., from human erythrocytes, et al., 1988) to immunize mice.

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

[0044] Splenocytes from immunized mice and cells of myeloma cell line SP2 / 0 were fused with 1 ml 50% polyethylene glycol at 37°C for 30 seconds. After washing, cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplement]. After one week, HAT medium was replaced with HT medium for three generations and then returned to normal cell culture medium.

[0045] Two weeks after fusion, the cell culture supernatant was subjected to preliminary screening for recombinant DPP3 binding IgG antibodies. Therefore, recombinant DPP3 (USBiologicals, Salem, USA) with a GST tag was fixed in a 96-well plate (100 ng / well) and incubated with 50 μl of cell culture supernatant / well (i.e., each well) for 2 hours at room temperature. After washing the plate, 50 μl / well of POD-rabbit anti-mouse IgG was added and incubated for 1 hour at room temperature. After the next washing step, 50 μl of chromogen solution (3,7 mM o-phenylenediamine in citrate / hydrogen phosphate buffer, 0.012% H 2 O 2 ), incubate at room temperature for 15 minutes and stop the color reaction by adding 50 μl 4N sulfuric acid. Absorption was detected at 490 mm.

[0046] Microcultures that test positive are transferred to 24-well plates for propagation. After retesting, selected cultures are cloned and recloned using limiting dilution techniques and isotyped.

[0047] Antibodies raised against GST-tagged human DPP3 or DPP3-peptide were produced by standard antibody production methods (Marx et al., 1997) and purified by protein A. Antibody purity was ≥ 90% based on SDS gel electrophoresis analysis.

[0048] Antibodies can be produced with the aid of phage display according to the following procedure:

[0049] The human natural antibody gene library HAL7 / 8 is used for separating recombinant single-chain F-variable domains (scFv) for DPP3 peptide. The antibody gene library is screened with a panning strategy, which includes the use of a peptide containing a biotin tag connected to the DPP3 peptide sequence by two different spacers. A mixed panning round using an antigen bound to non-specific binding and an antigen bound to streptavidin is used to minimize the background of non-specific binding agents. The eluted phage from the third round of panning has been used to produce an Escherichia coli (E.coli) strain expressing a monoclonal scFv. The supernatant from these cloned strains has been directly used for antigen ELISA testing (see also Hust et al., 2011; Schütte et al., 2009).

[0050] Humanization of murine antibodies can be performed according to the following procedure:

[0051] For the humanization of murine antibodies, the antibody sequence is analyzed for the structural interaction between the framework region (FR) and the complementary determining region (CDR) and the antigen. Based on structural modeling, appropriate human FRs are selected and the mouse CDR sequences are transplanted into human FRs. Changes in the amino acid sequence of CDR or FR can be introduced to regain structural interactions that are eliminated by the species switch of the FR sequence. The restoration of this structural interaction can be achieved by a random approach using a phage display library or a directed approach guided by molecular modeling. (Almagro & Fransson, 2008).

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

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

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

[0055] In a specific embodiment of the method for determining active DPP3 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-Ig scaffold or an aptamer.

[0056] In a specific embodiment of the method for determining active DPP3 in a body fluid sample of a subject, the capture binding agent reactive with DPP3 is immobilized on a solid phase.

[0057] The test sample is passed over the immobilized binding agent, and DPP3, if present, binds to the binding agent and itself is immobilized for detection. A substrate may then be added, and the reaction product may be detected to indicate the presence or amount of DPP3 in the test sample. For the purposes of this specification, the term "solid phase" may be used to include any material or vessel in or on 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 from Thermo Fisher Scientific), TM or Pierce TM Magnetic beads) etc.

[0058] The protein or peptide-derived binding agent (e.g., antibody, antibody fragment, non-Ig scaffold) is immobilized on the solid phase by methods including physical adsorption (e.g., by electrostatic or hydrophobic interactions), bioaffinity immobilization (e.g., avidin-biotin, protein A / G / L, His-tag, and Ni 2+-NTA, GST-tag and glutathione, DNA hybridization, aptamers), covalent bonds (e.g., amines and N-hydroxysuccinimide) or a combination of the described immobilization methods (Kim & Herr, 2013). Oligonucleotide-derived binders (e.g., aptamers) can be immobilized on a solid phase by utilizing the (strept)avidin-biotin system (Müller et al., 2012; Deng et al., 2014).

[0059] In a specific embodiment of the method for determining active DPP3 in a body fluid sample of a subject, the separation step is a washing step that removes sample components that are not bound to the capture binding agent from the captured DPP3. The separation step may be any other step that separates the DPP3 bound to the capture binding agent from components of the body fluid sample.

[0060] In a specific embodiment of the method for determining active DPP3 in a body fluid sample of a subject, the DPP3 substrate conversion rate of the immobilized DPP3 is measured (detected) by a method selected from the following: fluorescence of a fluorescent substrate (e.g., Arg-Arg-βNA, Arg-Arg-AMC), color change of a chromogenic substrate, substrate coupled to aminofluorescein (Promega Protease-Glo TM The DPP3 protein was detected by luminescence, 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 blotting (cleavage products).

[0061] In a specific embodiment of the method for determining active DPP3 in a body fluid sample of a subject, the substrate may be selected from: angiotensin II, angiotensin III and angiotensin IV, leucine enkephalin, methionine enkephalin, endomorphin 1 and endomorphin 2, inlofen, β-casomorphin, dynorphin, protoenterin, ACTH and MSH, or dipeptides and tripeptides coupled to fluorophores, chromophores or aminofluorescein (Promega Protease-Glo TM Determination). Di- 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. 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; et al., 2000; Ohkubo et al., 1999). Cleavage of these fluorescent substrates results in the release of fluorescent β-naphthylamine or 7-amino-4-methylcoumarin, respectively. Chromophores include, but are not limited to, p-nitroaniline diacetate (pNA). Hydrolysis of the peptide-pNA bond in the chromogenic substrate results in the release of pNA, which in turn changes color. Thus, the change in absorbance (DA / min) is proportional to the enzyme activity. Protease-Glo from Promega was used. TM The assay states that upon cleavage by DPP3, aminoluciferin is released and used as a substrate for a coupled luciferase reaction that emits detectable luminescence.

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

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

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

[0065] The kit may further comprise a calibrator: The calibrator can be a sample (native, purified or recombinant) with a known DPP3 concentration. • The calibrant can be the cleavage product itself, such as a free fluorophore (eg, 2-naphthylamine), a free chromophore (eg, p-nitroaniline), or free fluorescein.

[0066] The kit may further comprise a washing reagent: • Detergent (can be any aqueous buffer solution with or without detergent. Here we used 8mM Tris-HCl, pH 7,5, 60mM NaCl, 0.02% Tween 20).

[0067] In a specific embodiment, the assay is an enzyme capture assay (ECA, eg, US5612186A, US5601986A).

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

[0069] In a specific embodiment of the assay or kit for determining active DPP3 in a bodily fluid sample of a subject, the binding agent may be selected from an antibody, an antibody fragment or a non-Ig scaffold.

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

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

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

[0073] In a specific embodiment of the assay and kit for determining active DPP3 in a body fluid sample of a subject, the substrate can be selected from: angiotensin II, angiotensin III and angiotensin IV, leucine enkephalin, methionine enkephalin, endomorphin 1 and endomorphin 2, inlofen, β-casomorphin, dynorphin, protoenterin, ACTH and MSH, or dipeptides and tripeptides coupled to fluorophores, chromophores or aminofluorescein (Promega Protease-Glo TMDetermination). Di- 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. 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; et al., 2000; Ohkubo et al., 1999). Cleavage of these fluorescent substrates results in the release of fluorescent β-naphthylamine or 7-amino-4-methylcoumarin, respectively. Chromophores include, but are not limited to, p-nitroaniline diacetate (pNA). Hydrolysis of the peptide-pNA bond in the chromogenic substrate results in the release of pNA, which in turn changes color. Thus, the change in absorbance (DA / min) is proportional to the enzyme activity. Protease-Glo from Promega was used. TM The assay states that upon cleavage by DPP3, aminoluciferin is released and used as a substrate for a coupled luciferase reaction that emits detectable luminescence.

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

[0075] In a specific embodiment of the assay and kit for determining active DPP3 in a body fluid sample of a subject, the calibrator is selected from: A) recombinant DPP3 (e.g., GST-hDPP3 from USBio), purified native DPP3 (e.g., from human erythrocytes, et al., 1988) or DPP3 fragments (natural, synthetic or recombinant); B) the cleavage products themselves: free fluorophores (e.g., 2-naphthylamine), free chromophores (e.g., p-nitroaniline) or free fluorescein for quantification of fluorescence, color change and bioluminescence signals.

[0076] One embodiment of the invention using DPP3 as a diagnostic biomarker includes various formats of immunoassays, such as radioimmunoassays (RIA), chemiluminescent and fluorescent immunoassays, enzyme-linked immunosorbent assays (ELISA), Luminex-based bead arrays, protein microarray assays, and rapid test formats, such as immunochromatographic strip tests.

[0077] The assay can be a homogeneous or heterogeneous assay, a competitive and non-competitive sandwich assay. In a particularly preferred embodiment using two antibodies according to the invention, the assay is in the form of a sandwich assay, which is a non-competitive immunoassay in which the DPP3 or fragment thereof to be detected and / or quantified is bound to a first antibody and a second antibody. The first antibody can be bound to a solid body, for example, a surface, a patch or a strip of a bead, a well or other container, and the second antibody is a labeled antibody, for example, an antibody labeled with a dye, a radioisotope or a reactive or catalytically active portion. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general compositions and procedures involving "sandwich assays" are well-known and known to the skilled person. (The Immunoassay Handbook, Ed. David Wild, 2005; Hultschig et al., 2006).

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

[0079] In the context of the present invention, fluorescence-based assays include the use of dyes, which may be selected, for example, from the group consisting of FAM (5-carboxyfluorescein or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes such as CY3, CY5, CY3.5, CY5.5, Cy7, xanthen, 6-carboxy-2',4',7',4,7-hexa Chlorofluorescein (HEX), TET, 6-carboxy-4',5'-dichloro-2',7'-diformylfluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes such as BODIPY TMR, Oregon Green, coumarins such as umbelliferone, benzimides such as Hoechst 33258; phenanthridines such as Texas Red, Yakima Yellow, Alexa Fluor, PET, phenanthridine bromide, acridinium dyes, carbazole dyes, phenoxazine dyes, porphyrine dyes, polymethyl iodide dyes, etc.

[0080] In the context of the present invention, chemiluminescence-based assays include the use of dyes based on the physical principles described for chemiluminescent materials in Kirk-Othmer, Encyclopedia of chemical technology, Fourth Edition, Executive Editor, JI Kroschwitz; Editor, M. Howe-Grant, John Wiley & Sons, 1993, Volume 15, Pages 518 to 562, which is incorporated herein by reference, including the citations on pages 551 to 562. Preferred chemiluminescent dyes are acridinium esters.

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

[0082] Assays embodying such reagents and reactions can be performed in any suitable reaction vessel, e.g., a test tube or well of a microtiter plate. Alternatively, assay devices can be developed in a disposable format, such as a dipstick or test strip device format, which are well known to those skilled in the art and are easy to manufacture and use. Such disposable assay devices can be packaged in the form of a kit containing all necessary materials, reagents, and instructions for use.

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

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

[0085] In alternative assay embodiments, the rate at which the reaction occurs can be detected as an indication of the amount of DPP3 present in the test sample. For example, the rate at which the substrate reacts can be used to indicate the amount of DPP3 present in the test sample. Alternatively, the rate at which the reaction product is formed can be used to indicate the amount of DPP3 present in the test sample.

[0086] In another embodiment, capture assay or binding assay can be performed to detect and / or quantify proteases. For example, antibodies that react with the DPP3 protein but do not interfere with peptidase activity can be fixed on a solid phase. The test sample is passed through the fixed antibody, and DPP3 (if present) is bound to the antibody and itself is fixed for detection. Substrate can then be added, and the reaction product can be detected to indicate the presence or amount of DPP3 in the test sample. For this specification, the term "solid phase" can be used to include any material or vessel that can be measured therein or thereon, and includes but is not limited to porous materials, non-porous materials, test tubes, holes, slides, etc.

[0087] In another specific embodiment, the DPP3 ECA can be performed as a test strip assay. In an exemplary test strip device, a test sample application pad is optionally attached to one end of a porous strip. The strip contains fixed antibodies that will bind to DPP3 and thus fix DPP3 at a predetermined site for subsequent detection. Optionally, the device may include an assay end indicator that is located at the far end of the test strip, away from the test sample contact site. The assay end indicator generates a detectable signal when in contact with a test sample or assay reagent, thereby indicating that the assay is complete.

[0088] The test sample application pad can be a part of the porous strip itself or a material that is in fluid flow contact with the end (referred to as the proximal end) of the porous strip, so that the test sample can pass through or migrate to the porous strip from the application pad. Fluid flow contact can include physical contact between the application pad and the porous strip and separation of the application pad and the porous strip by intervening space or additional material, which still allows fluid to flow between the application pad and the porous strip. Substantially all application pads can overlap with the porous strip so that the test sample can basically reach the proximal end of the porous strip through any part of the application pad. Alternatively, only a portion of the application pad can be in fluid flow contact with the porous strip. The application pad can be any material that can transfer the test sample to the porous strip.

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

[0090] The particular dimensions of the porous strip will be convenient, depending on the size of the test sample involved, the assay protocol, the means for detecting and measuring the signal, etc. For example, the dimensions may be selected to modulate the rate of fluid migration and the amount of test sample absorbed by the porous strip.

[0091] In a possible test strip device of the present invention, DPP3 substrate and / or DPP3 capture antibody can be fixed on a porous strip to form at least one analyte detection site, that is, a porous strip area with one or more analytical reagents non-diffusively attached thereto. In another device embodiment, the measurement or detection area of ​​the test strip may include multiple sites containing DPP3 substrate and / or fixed anti-DPP3 antibodies. Optionally, different detection sites may contain different amounts of substrate and / or fixed anti-DPP3 antibodies, that is, the amount in the first detection site is higher and the amount in the subsequent sites is less. For example, if 20 nanograms of antibody capture 1nmol / minute / ml of DPP3 equivalents, then the first detection site of the assay device may contain 50 nanograms of anti-DPP3 antibodies, while subsequent sites contain 10 nanograms, 20 nanograms, 30 nanograms, etc. nanograms of antibodies. After adding the test sample, the number of sites that display a detectable signal provides a quantitative indication of the amount of DPP3 present in the sample. The detection site can be configured into any suitable detectable shape, and is typically a bar across the width of the test strip.

[0092] Alternatively, a multi-capture site device can be prepared so that if there is no threshold amount of DPP3 in the test sample, substantially all DPP3 will bind to the antibody in the first capture site and thus be fixed at the site. If there is more than the threshold amount of DPP3 in the test sample, the remaining DPP3 will bind to the subsequent detection zone of the immobilized antibody along the length of the test strip. The larger the amount of DPP3 in the test sample, the larger the number of capture sites that will display a detectable signal due to the presence of DPP3. As will be appreciated by those skilled in the art, a device comprising multiple DPP3 substrate sites can also be produced, wherein the amount of substrate in a single site is designed to produce a quantitative or semi-quantitative measurement result.

[0093] Another important embodiment of the present invention is a method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process, comprising: determining the amount of total DPP3 in a sample of a body fluid of the subject or determining the amount of active DPP3 in a sample of a body fluid of the subject, comparing said determined amount with a predetermined threshold, - wherein if said determined amount is above said predetermined threshold, said subject is diagnosed as having a disease or condition accompanied by or associated with a necrotic process.

[0094] In a specific embodiment of the method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process, the amount of total DPP3 or the amount of active DPP3 is determined in concentration units.

[0095] Methods for determining the amount of total DPP3 or the amount of active DPP3 are known in the art. In the context of the method according to the invention for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process, prior art methods and assays can be used, or the above-described methods and assays for determining DPP3 can be used.

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

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

[0098] The sensitivity and specificity of a diagnostic test depend not only on the analytical "quality" of the test, they also depend on the definition of what constitutes an abnormal result. In practice, a receiver operating characteristic curve (ROC curve) is usually calculated by plotting the values ​​of a variable in a "normal" group (i.e., apparently healthy) and a "disease" group (i.e., patients suffering from an infection) against their relative frequencies. Depending on the specific diagnostic question to be solved, the reference group does not necessarily have to be "normal", but may be a group of patients suffering from another disease or condition, from which the diseased group of interest is distinguished. For any particular marker, the distribution of marker levels for subjects with or without the disease may overlap. In this case, the test cannot absolutely distinguish between normal and disease with 100% accuracy, and the overlapping area indicates that the test cannot distinguish between normal and diseased parts. A threshold 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 below the ROC curve is a measure of the probability that the perceived measurement can correctly identify the condition. ROC curves can be used even when the test results do not necessarily give an exact number. ROC curves can be created as long as the results can be ranked. For example, the test results for "diseased" samples can be ranked by degree (e.g., 1 = low, 2 = normal, and 3 = high). The ranking can be correlated with the results in the "normal" population and a ROC curve created. These methods are well known in the art (see, e.g., Hanley et al., 1982). Preferably, the threshold is selected to provide a ROC curve area greater than about 0.5, more preferably greater than about 0.7. The term "about" in this context refers to + / - 5% of a given measurement.

[0099] Once the threshold is determined by using a previous study cohort and taking all of the above points into consideration, the physician will use the predetermined threshold of the method for diagnosing a disease according to the present invention and will determine whether the subject has a value above or below the predetermined threshold in order to make an appropriate diagnosis.

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

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

[0102] In a specific embodiment of the method for diagnosing a disease or condition of a subject with or associated with a necrotic process, the sample is selected from whole blood, serum and plasma. The body fluid in the context of the method of the present invention may also be selected from blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum and pleural effusion.

[0103] Necrotic processes herein are defined by all processes in vivo that lead to cell death and release of DPP3 from the cytoplasm into the extracellular space and / or body fluids. These processes include, but are not limited to, necrosis, apoptosis, necroptosis, and erythroid cell death.

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

[0105] In another embodiment, the disease is selected from the group consisting of acute heart failure (AHF), myocardial infarction (MI), liver failure, burns, severe infection (microbial, viral (e.g., AIDS), parasitic (e.g., malaria)) or SIRS or sepsis, cancer and acute kidney injury (AKI).

[0106] In one embodiment, the disorder is hypotension.

[0107] Table 1: Diseases associated with or related to necrotic processes.

[0108] Apoptosis is a process of programmed cell death (PCD) that can occur in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. For a review, see Elmore, 2007.

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

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

[0111] Cell death caused by necrosis does not follow the apoptotic signal transduction pathway, but rather various receptors are activated and lead to loss of cell membrane integrity and uncontrolled release of cell death products into the extracellular space. This triggers an inflammatory response in the surrounding tissues, preventing nearby phagocytes from locating and eliminating dead cells by phagocytosis. For this reason, surgery is often required to remove necrotic tissue, a process known as debridement. Untreated necrosis leads to an accumulation of decomposed dead tissue and cell debris at or near the site of cell death.

[0112] A form of programmed necrosis called necroptosis has been recognized as an alternative form of programmed cell death. Necroptosis can serve as a backup to apoptosis when apoptotic signals are blocked by endogenous factors or exogenous factors such as viruses or mutations (Linkermann et al., 2014). Recently, other types of regulated necrosis have been discovered that share multiple signaling events with necroptosis and apoptosis (Vanden Berghe et al., 2014).

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

[0114] Heart failure is a clinical syndrome characterized by a collection 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% to 2%. Heart failure can be divided into chronic HF and acute HF. Patients with chronic HF can be divided into stable chronic HF, worsening signs and symptoms of chronic HF, and acute decompensation of chronic HF. Acute heart failure (AHF) is defined as the rapid onset of signs and symptoms of heart failure that require emergency treatment or hospitalization. AHF can present as acute new HF (new onset of AHF in patients without previously known cardiac insufficiency) or acute decompensation of chronic AHF. AHF is the main cause of hospitalization in adults over 65 years of age. Although the prognosis of patients with chronic heart failure has improved significantly over the past few decades, mainly related to treatment advances, once a patient is hospitalized for decompensated heart failure, the short-term and long-term outcomes are still very poor. Nearly 25% of patients hospitalized for AHF within 30 days of hospitalization require readmission, and the survival rate of patients more than 5 years after hospitalization is <50%. In addition to significantly reducing the survival and quality of life of affected patients, the economic burden of AHF on the healthcare system is enormous. In the United States alone, the total cost of heart failure care was estimated at $31 billion in 2012, with the majority of costs associated with hospitalization. Due to the aging population, this cost is expected to increase to an unprecedented $70 billion by 2030.

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

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

[0117] The term "acute" is used to mean rapid onset and describes worsening or decompensated heart failure, referring to events in which a patient may be characterized by changes in signs and symptoms of heart failure leading to the need for emergency treatment or hospitalization.

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

[0119] Stable chronic HF is characterized by: (i) there is structural or functional failure of the heart that impairs its ability to provide adequate blood flow to meet the body's needs, (ii) There is no volume overload (manifested by pulmonary and / or systemic congestion) and / or a severe decrease in cardiac output (manifested by hypotension, renal insufficiency and / or shock syndrome), and the patient does not require urgent treatment or modification of treatment and does not require hospitalization.

[0120] Chronic HF with worsening signs and symptoms is characterized by: (i) there is structural or functional failure of the heart that impairs its ability to provide adequate blood flow to meet the body's needs, (ii) There is volume overload (manifested by pulmonary and / or systemic congestion) and / or a severe decrease in cardiac output (manifested by hypotension, renal insufficiency, and / or shock syndrome), and the patient does not require urgent treatment and does not require hospitalization, but requires treatment modification.

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

[0122] New-onset acute HF and acute decompensated chronic HF are characterized by: (i) there is structural or functional failure of the heart that impairs its ability to provide adequate blood flow to meet the body's needs, (ii) There is volume overload (manifested by pulmonary and / or systemic congestion) and / or a severe decrease in cardiac output (manifested by hypotension, renal insufficiency, and / or shock syndrome) and the patient requires urgent treatment or modification of treatment and requires hospitalization.

[0123] The above definition of acute heart failure, i.e. new-onset AHF or acute decompensated HF or acute decompensated chronic HF or worsening symptoms / signs of chronic heart failure, is in accordance with that described by Voors et al., 2016.

[0124] In a specific embodiment of the method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process, DPP3 activity may be determined using a solution phase activity assay or using an enzyme capture activity assay.

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

[0126] In a specific embodiment, determination of active DPP3 in an enzyme capture assay comprises the following steps: contacting the sample with a capture binding agent that binds to full-length DPP3 but preferably inhibits DPP3 activity by less than 50%, preferably less than 40%, more preferably less than 30% in a liquid phase assay. To prevent inhibition of DPP3, the capture binding agent should not bind to DPP3 in the region surrounding the active center and substrate binding region (amino acids 316 to 669 of SEQ ID No. 1). separating DPP3 bound to the capture binding agent from the body fluid sample, adding a substrate for DPP3 to the isolated DPP3, Quantify DPP3 activity by measuring the conversion rate of DPP3 substrates, Evaluation of the measured signal compared to a non-diseased control. The threshold value may be predetermined, for example, by measuring the DPP3 concentration and or DPP3 activity in healthy controls and calculating the corresponding 75th percentile. The upper boundary of the 75th percentile defines the threshold value for healthy patients relative to diseased patients.

[0127] In a specific embodiment, the determination of active DPP3 is performed according to the above-described method of the invention.

[0128] In a specific embodiment of the method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process, the binding agent may be selected from an antibody, an antibody fragment or a non-IgG scaffold.

[0129] In a specific embodiment of the method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process, it is an enzyme capture assay (ECA, US5612186A, US5601986A). The DPP3 binding agent in the assay is an antibody.

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

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

[0132] The test sample is passed over the fixed binding agent, and DPP3 (if present) binds to the binding agent and itself is fixed for detection. The substrate can then be added, and the reaction product can be detected to indicate the presence or amount of DPP3 in the test sample. For the purposes of this specification, the term "solid phase" may be used to include any material or vessel in which or on which 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 from Thermo Fisher Scientific TM or Pierce TM Magnetic beads) etc.

[0133] The protein or peptide-derived binding agent (e.g., antibody, antibody fragment, non-Ig scaffold) is immobilized on the solid phase by methods including physical adsorption (e.g., by electrostatic or hydrophobic interactions), bioaffinity immobilization (e.g., avidin-biotin, protein A / G / L, His-tag, and Ni 2+ -NTA, GST-tag and glutathione, DNA hybridization, aptamers), covalent bonds (e.g., amines and N-hydroxysuccinimide) or a combination of the described immobilization methods (Kim & Herr, 2013). Oligonucleotide-derived binders (e.g., aptamers) can be immobilized on a solid phase by utilizing the (strept)avidin-biotin system (Müller et al., 2012; Deng et al., 2014).

[0134] In a specific embodiment of the method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process, the separation step is a washing step that removes sample components that are not bound to the capture binding agent from the captured DPP3.

[0135] In a specific embodiment of the method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process, the DPP3 substrate conversion rate is measured by a method selected from the following: fluorescence of a fluorogenic substrate (e.g., Arg-Arg-2NA, Arg-Arg-AMC), color change of a chromogenic substrate, luminescence of a substrate coupled to aminofluorescein (Promega Protease-Glo TM assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized, active DPP3), or western blotting (cleavage products).

[0136] In a specific embodiment of the method for diagnosing a disease or condition of a subject accompanied by or associated with a necrotic process, the substrate may be selected from: angiotensin II and angiotensin III, leucine enkephalin, methionine enkephalin, endomorphin 1 and endomorphin 2, inlofen, β-casomorphin, dynorphin, protoenterin, ACTH and MSH, or dipeptides and tripeptides coupled to a fluorophore, chromophore or aminofluorescein (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. 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 fluorescent substrates results in the release of fluorescent β-naphthylamine or 7-amino-4-methylcoumarin, respectively. Chromophores include, but are not limited to, p-nitroaniline diacetate (pNA). Hydrolysis of the peptide-pNA bond in the chromogenic substrate results in the release of pNA, which in turn changes color. Thus, the change in absorbance (DA / minute) is proportional to the enzyme activity. Using the Protease-Glo™ assay from Promega, upon cleavage by DPP3, aminoluciferin is released and used as a substrate for a coupled luciferase reaction that emits detectable luminescence.

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

[0138] Another important embodiment of the present invention is an inhibitor of DPP3 activity for use in preventing or treating a disease or disorder in a subject accompanied by or associated with a necrotic process.

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

[0140] Peptide inhibitors of DPP3 include, but are not limited to, spinin, synthetic derivatives of spinin (tynorphin and other peptides, see Table 2; Yamamoto et al., 2000), propioxatin A and propioxatin B (US Pat. No. 4,804,676) and synthetic propioxatin A analogs (Inaoka et al., 1988).

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

[0142] Table 2: Peptide and small molecule inhibitors of DPP3

[0143] In a preferred embodiment of the present invention, the selected inhibitor is pharmaceutically acceptable, selective and / or specific to DPP3, and does not pass through the cell membrane and / or the blood-brain barrier. The selective and specific inhibitors of DPP3 do not bind to other proteins / enzymes or bind to other proteins / enzymes, and do not inhibit any other enzymes / proteases / peptidases except DPP3. Small peptides can be bound and cut by non-specific aminopeptidases, and small molecule inhibitors easily pass through the cell membrane and the 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 pass through the cell membrane or the blood-brain barrier. Therefore, the preferred DPP3 activity inhibitor is a specific anti-DPP3 antibody, antibody fragment or non-Ig scaffold.

[0144] In a specific embodiment of the present invention, an inhibitor of DPP3 activity is used to prevent or treat a disease or condition in a subject accompanied by or associated with a necrotic process, wherein the inhibitor is selected from an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

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

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

[0147] In a specific embodiment of the present invention, an inhibitor of DPP3 activity is used to prevent a disease or condition in a subject, wherein the disease or condition is acute heart failure (AHF), myocardial infarction (MI), liver failure, cancer, acute kidney injury (AKI) and hypotension.

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

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

[0150] In a specific embodiment, the disease or condition is hypotension.

[0151] In a specific embodiment of the present invention, an inhibitor of DPP3 activity is used for preventing or treating a disease or condition in a subject accompanied by or associated with a necrotic process, wherein the inhibitor is a mono-binding or at least bi-binding antibody.

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

[0153] In a specific embodiment of the present invention, an inhibitor or effector of DPP3 activity is used to prevent or treat a disease or condition in a subject accompanied by or associated with a necrotic process, wherein the inhibitor or effector has a minimum binding affinity for DPP3 of less than 10 -7 Antibodies or fragments or scaffolds of M.

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

[0155] In a specific embodiment of the present invention, an inhibitor or effector of DPP3 activity is used to prevent or treat a disease or condition in a subject accompanied by or associated with a necrotic process, wherein the inhibitor or effector is a monospecific antibody or fragment or scaffold.

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

[0157] In another specific and 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, whereby monospecific means that the antibody or antibody fragment or non-Ig scaffold binds to a specific region comprising at least 4 amino acids within the target DPP3. The monospecific antibody or fragment or non-Ig scaffold according to the present invention is an antibody or fragment or non-Ig scaffold that has affinity for the same antigen. Monoclonal antibodies are monospecific, but monospecific antibodies can also be produced by other means besides producing them from common germ cells.

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

[0159] Another embodiment of the present invention is a pharmaceutical composition comprising the above-mentioned DPP3 activity inhibitor for preventing or treating a disease or disorder of a subject accompanied by or associated with a necrotic process.

[0160] Another embodiment of the present invention is a method for preventing or treating a disease or disorder in a subject accompanied by or associated with a necrotic process, wherein an inhibitor of DPP3 activity is administered.

[0161] In a specific embodiment of the method for preventing or treating a disease or condition in a subject accompanied by or associated with a necrotic process, the inhibitor is selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

[0162] In a specific embodiment of the method for preventing or treating a disease or condition in a subject accompanied by or associated with a necrotic process, wherein the disease is selected from heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infection (microbial, viral (e.g., AIDS), parasite (e.g., malaria)) or SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g., epilepsy, neurodegenerative diseases), autoimmune diseases, vascular diseases (e.g., Kawasaki syndrome) and hypotension.

[0163] In a specific embodiment of the method for preventing or treating a disease or condition in a subject accompanied by or associated with a necrotic process, wherein the inhibitor is a mono-binding or at least bi-binding antibody.

[0164] In a specific embodiment of the method for preventing or treating a disease or condition in a subject accompanied by or associated with a necrotic process, the inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to SEQ ID No. 1, and in a specific embodiment, is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to SEQ ID No. 2.

[0165] In a specific embodiment of the method for preventing or treating a disease or condition in a subject accompanied by or associated with a necrotic process, wherein the inhibitor has a minimum binding affinity for DPP3 of less than 10 -7 Antibodies or fragments or scaffolds of M.

[0166] In a specific embodiment of the method for preventing or treating a disease or disorder in a subject accompanied by or associated with a necrotic process, the inhibitor or effector is a monospecific antibody or fragment or scaffold.

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

[0168] In a specific embodiment of the method for preventing or treating a disease or condition in a subject with or associated with a necrotic process, the subject has an elevated level of DPP3. An elevated level is a level above a predetermined threshold. The threshold is as defined above.

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

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

[0171] The pharmaceutical compositions can also be administered via the central nervous system (CNS), e.g., epidural (synonym: peridural) injection or infusion into the epidural space, intracerebral (into the brain) injection directly into the brain, intraventricular (administration into the ventricular system of the brain), or intrathecal (into the spinal canal).

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

[0173] 1. A method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process, comprising: ·Determining the amount of total DPP3 and / or the amount of active DPP3 in a body fluid sample of the subject, ·Comparing the determined amount of total DPP3 or the amount of active DPP3 with a predetermined threshold, ·Wherein if the determined amount is higher than the predetermined threshold, the subject is diagnosed as having a disease or condition accompanied by or associated with a necrotic process.

[0174] 2. The method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process according to claim 1, wherein the amount of total DPP3 or the amount of active DPP3 is determined in concentration units.

[0175] 3. A method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process according to claim 1 or 2, wherein the sample is selected from the group consisting of whole blood, serum and plasma.

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

[0177] 5. A method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process according to any one of claims 1 to 4, wherein the amount of total DPP3 and / or the amount of active DPP3 is determined in a body fluid sample of the subject, and comprising the following steps: contacting the sample with a capture binding agent that specifically binds to full-length DPP3, · isolating DPP3 bound to the capture binding agent, adding a substrate for DPP3 to the isolated DPP3, - Quantify the active DPP3 by measuring and quantifying the conversion rate of DPP3's substrate.

[0178] 6. The method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process according to claim 5, wherein the capture binding agent can be selected from antibodies, antibody fragments or non-IgG scaffolds.

[0179] 7. A method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process according to claim 5 or 6, wherein the capture binding agent is an antibody.

[0180] 8. A method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process according to any one of claims 5 to 7, wherein the capture binding agent is immobilized on a surface.

[0181] 9. A method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process according to any one of claims 5 to 8, wherein the separation step is a washing step which removes sample components not bound to the capture binding agent from the captured DPP3.

[0182] 10. A method for diagnosing a disease or condition of a subject accompanied by or associated with a necrotic process according to any one of claims 5 to 9, wherein the DPP3 substrate conversion rate is detected by a method selected from the group consisting of: 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 aminofluorescein (Promega Protease-Glo TM assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized, active DPP3), or western blotting (cleavage products).

[0183] 11. A method for diagnosing a disease or condition in a subject accompanied by or associated with a necrotic process according to any one of claims 5 to 10, wherein the substrate can be selected from: angiotensin II, angiotensin III and angiotensin IV, leucine enkephalin, methionine enkephalin, endomorphin 1 and endomorphin 2, inlofen, β-casomorphin, dynorphin, protoenterin, ACTH and MSH, or a dipeptide coupled to a fluorophore, a chromophore or aminofluorescein, wherein the dipeptide is Arg-Arg.

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

[0185] 13. Method for monitoring a disease or condition in a subject accompanied by or associated with a necrotic process, wherein the diagnostic method according to any one of claims 1 to 12 is performed at least twice.

[0186] 14. An inhibitor of DPP3 activity for use in preventing or treating a disease or condition in a subject accompanied by or associated with a necrotic process.

[0187] 15. An inhibitor of DPP3 activity for use in preventing or treating a disease or condition in a subject accompanied by or associated with a necrotic process according to claim 14, wherein the inhibitor is selected from an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

[0188] 16. 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 claim 14 or 15, wherein the disease is selected from heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infection (microorganisms, viruses (e.g., AIDS), parasites (e.g., malaria)), or SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g., epilepsy, neurodegenerative diseases), autoimmune diseases, vascular diseases (e.g., Kawasaki syndrome) and hypotension.

[0189] 17. An inhibitor of DPP3 activity for use in preventing or treating a disease or condition in a subject accompanied by or associated with a necrotic process according to any one of claims 14 to 16, wherein the inhibitor is a mono-binding or at least bi-binding antibody.

[0190] 18. An inhibitor of DPP3 activity for use in preventing or treating a disease or condition in a subject accompanied by 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 or an anti-DPP3 non-Ig scaffold that binds to SEQ ID No. 1, in particular to SEQ ID No. 2.

[0191] 19. An inhibitor of DPP3 activity for use in preventing or treating a disease or condition in a subject accompanied by or associated with a necrotic process according to any one of claims 14 to 18, wherein the inhibitor has a minimum binding affinity for DPP3 equal to or less than 10 -7 Antibodies or fragments or scaffolds of M.

[0192] 20. An inhibitor of DPP3 activity for use in preventing or treating a disease or condition in a subject accompanied by 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.

[0193] 21. An inhibitor of DPP3 activity for use in preventing or treating a disease or condition of a subject accompanied by 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 at least 10%, or at least 50%, more preferably at least 60%, even more preferably greater than 70%, even more preferably greater than 80%, even more preferably greater than 90%, even more preferably greater than 95% of DPP3 activity.

[0194] 22. An inhibitor of DPP3 activity for use in preventing or treating a disease or condition in a subject accompanied by 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 cell membranes and / or the blood-brain barrier.

[0195] 23. A DPP3 activity inhibitor for use in preventing or treating a disease or condition in a subject accompanied by or associated with a necrotic process according to any one of claims 14 to 22, wherein the amount of total DPP3 and / or the amount of active DPP3 in a body fluid sample of the subject is above a predetermined threshold.

[0196] 24. A pharmaceutical composition comprising an inhibitor of DPP3 activity according to any one of claims 14 to 23 for use in preventing or treating a disease or condition of a subject accompanied by or associated with a necrotic process.

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

[0198] 26. A method for determining active DPP3 in a body fluid sample of a subject, comprising the steps of: contacting the sample with a capture binding agent that specifically binds to full-length DPP3, · isolating DPP3 bound to the capture binding agent, adding a substrate for DPP3 to the isolated DPP3, - Quantify the active DPP3 by measuring and quantifying the conversion rate of DPP3's substrate.

[0199] 27. The method for determining active DPP3 in a body fluid sample of a subject according to claim 26, wherein the capture binding agent may be selected from an antibody, an antibody fragment or a non-IgG scaffold.

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

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

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

[0203] 31. A method for determining active DPP3 in a body fluid sample of a subject according to any one of claims 26 to 30, wherein the DPP3 substrate conversion rate is detected by a method selected from the group consisting of: 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 aminofluorescein (Promega Protease-Glo TM assay), mass spectrometry, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, activity staining after gel electrophoresis (immobilized, active DPP3), or western blotting (cleavage products).

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

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

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

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

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

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

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

[0211] 39. An assay or kit for determining active DPP3 in a body fluid sample of a subject according to any one of claims 34 to 38, wherein the capture binding agent is immobilized on a surface.

[0212] 40. An assay or kit for determining active DPP3 in a body fluid sample of a subject according to any one of claims 34 to 39, wherein the substrate can be selected from: angiotensin II, angiotensin III and angiotensin IV, leucine enkephalin, methionine enkephalin, endomorphin 1 and endomorphin 2, inlofen, beta-casomorphin, dynorphin, protoenterin, ACTH and MSH, or a dipeptide coupled to a fluorophore, chromophore or aminofluorescein, wherein the preferred dipeptide is Arg-Arg.

[0213] 41. Use of a method for determining active DPP3 in a body fluid sample of a subject according to any one of claims 26 to 33 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 claims 1 to 13, 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 accompanied by or associated with a necrotic process according to any one of claims 1 to 13. DETAILED DESCRIPTION 1. Example 1

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

[0215] Plasma samples were obtained from 388 patients who came directly to the emergency room at their first presentation. According to their final diagnosis, these patients could be divided into 4 subgroups: patients with acute myocardial infarction (AMI), patients with cardiogenic shock, patients with septic shock and patients with liver failure. The control group was a pool of plasma samples from 93 healthy controls. 1.2. hDPP3 capture activity assay:

[0216] Plasma samples (10 μl) were first enriched for DPP3 by an affinity purification step and then their activity was measured by adding the fluorescent substrate Arg-Arg-βNA (for detailed description see Example 4). The slope of the increase in fluorescence of the different samples (in nmol βNA / min / ml sample [nmol βNA min -1 ml -1 ] refers to 10 μl sample size. 1.3 Results:

[0217] For all patients with severe disease or organ failure, patient samples showed significantly higher DPP3 activity values ​​compared to healthy controls ( Fig.10 ). 2. Example 2

[0218] In this experiment, the effects of recombinant hDPP3 injections in healthy rats were studied by monitoring blood pressure. 2.1 Methods:

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

[0220] The animals were first anesthetized with isoflurane for weighing (full g) and intraperitoneal (ip) administration. 1.2 g / kg BW urethane (c = 0.4 g / mL) was injected for long-term anesthesia. The ventral area of ​​the neck is then clipped and wiped with ethanol. The vessel is prepared and the catheter is inserted. Finally, both catheters were flushed with heparinized isotonic sodium chloride solution. Then the pressure transducer (medexlogical, Medex Medical Ltd.) was connected to the patient monitoring system (Datex-Ohmeda, GE). A portable computer connected to the BP monitor recorded the BP data separately through S / 5 collection software.

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

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

[0223] Antibody Generation and Determination of DPP3 Binding Ability: Several murine antibodies were generated and screened for their ability to bind human DPP3 in either sandwich assays or activity assays (see Table 3). 3.1. Methods: - Peptides / conjugates for immunization:

[0224] Synthesize DPP3 peptides for immunization, see Table 3 (JPT Technologies, Berlin, Germany), with an additional N-terminal cysteine ​​(if cysteine ​​is not present in the selected DPP3-sequence) residue for peptide conjugation to bovine serum albumin (BSA). Peptides were covalently linked to BSA using Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling process was performed according to the manual of Perbio. Recombinant GST-hDPP3 was produced by USBio. -Mouse immunization, immune cell fusion and screening:

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

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

[0227] Two weeks after fusion, the cell culture supernatant was screened mainly for recombinant DPP3 binding IgG antibodies. Therefore, recombinant GST-tagged DPP3 (USBiologicals, Salem, USA) was fixed in a 96-well plate (100 ng / well) and incubated with 50 μl of cell culture supernatant per well at room temperature for 2 hours. After washing the plate, 50 μl / well POD-rabbit anti-mouse IgG was added and incubated at room temperature for 1 hour. After the next washing step, 50 μl of chromogen solution (3.7 mM o-phenylenediamine in citrate / hydrogen phosphate buffer, 0.012% H 2 O 2 ), incubate at room temperature for 15 minutes and stop the color reaction by adding 50 μl 4N sulfuric acid. Absorption was detected at 490 mm.

[0228] Positively tested microcultures were transferred to 24-well plates for propagation. After retesting, selected cultures were cloned and recloned using limiting dilution techniques and isotyped. - Mouse monoclonal antibody production

[0229] Antibodies raised against GST-tagged human DPP3 or DPP3-peptide were produced by standard antibody production methods (Marx et al., 1997) and purified by protein A. Antibody purity was ≥ 90% based on SDS gel electrophoresis analysis. - Characterization of antibodies - hDPP3 - Inhibition assays

[0230] To analyze the DPP3 inhibitory capacity of different antibodies and antibody clones, DPP3 activity assay was performed using known procedures (Jones et al., 1982). Recombinant GST-tagged hDPP3 was diluted in assay buffer (50 mM Tris-HCl, pH 7.5 and 100 μM ZnCl 2 25ng / ml GST-DPP3 in , and 200μl of this solution was incubated with 10μg of each antibody at room temperature. After pre-incubation for 1 hour, the fluorescent substrate Arg-Arg-βNA (20μl, 2mM) was added to the solution, and the generation of free βNA over time was monitored using Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co.KG) at 37°C. The fluorescence of βNA was detected by exciting at 340nm and measuring the emission at 410nm. The slope of the fluorescence increase of different samples was calculated (in RFU / minute). The slope of GST-hDPP3 with buffer control was designated as 100% activity. The inhibitory ability of possible capture-binding agents is defined as reducing GST-hDPP3 activity by incubating with the capture-binding agent in percentage. The resulting DPP3 activity reduction is shown in Figure 1a and Table 3. 3.2. Results:

[0231] The following table shows the selection of antibodies obtained and their maximum inhibition rates (Table 3). The monoclonal antibodies generated against the DPP3 region described below were selected by their ability to bind to native DPP3 (mAb-FL-DPP3_2555 as solid phase and_2553 as tracer for immunoassay, see Example 4 for details).

[0232] For the fixed DPP3 activity assay (see Examples 6 and 7), solid phase antibodies that do not inhibit DPP3 activity too strongly must be selected. As a cutoff for antibody screening, solid phase antibodies should not inhibit DPP3 activity by more than 50%, since mAb DPP3_2555 showed the lowest inhibition rate (Table 3, Figure 1A ).

[0233] In order to produce a strong DPP3 inhibitor that can be used therapeutically (see Examples 8 to 13), it is necessary to select a DPP3 binder that shows the highest inhibition rate. The monoclonal antibody mAbDPP3_1967, which has the ability to inhibit DPP3 activity by 70%, was selected as a possible therapeutic antibody (see Figure 1A and Table 3), and all further analyses were performed using this antibody. Figure 1B The inhibition curves of mAbDPP3_1967 are shown, and its IC 50 It is 0.2041μg / ml.

[0234] Table 3: Immunogen sequences, names and characteristics of anti-DPP3 antibodies generated4. Example 4

[0235] Identify antibody combinations that yield high signal-to-noise ratios in hDPP3 immunoassays. 4.1. Methods: -Monoclonal Antibody Production

[0236] Antibodies raised against GST-tagged human DPP3 were produced by standard antibody production methods (Marx et al., 1997) and purified by protein A. Antibody purity was ≥ 90% based on SDS gel electrophoresis analysis. Different clones were analyzed for their ability to bind DPP3. Use the resulting positive clones as solid phase or tracer antibodies. -Solid phase

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

[0238] 100 μg (100 μl) of different anti-DPP3 antibodies (detection antibodies, 1 mg / ml in PBS, pH 7.4) were mixed with 10 μl of acridine NHS-ester (1 mg / ml in acetonitrile, InVent GmbH, Germany; EP 0 353 971) and incubated for 30 minutes at room temperature. The labeled anti-DPP3 antibodies were purified by gel filtration HPLC on ShodexProtein 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 Na 2 -EDTA, 5g / l bovine serum albumin, The final concentration was about 7*10 of the labeled compound. 6Relative light units (RLU) (about 20 ng labeled antibody) / 200 μl. Acridinium ester chemiluminescence was measured by using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). - Calibration object

[0239] Use (50mmol / L potassium phosphate, 100mmol / L NaCl, 10mmol / L Na-EDTA, 5 g / L bovine serum albumin, 1 g / L mouse IgG, 1 g / L bovine IgG, 50 μmol / L aminopeptidase inhibitor peptide, 100 μmol / L leupeptin, pH 7.4) linear dilution of recombinant human GST-DPP3 Stock solution of ELISA (U Biological, USA) in PBS, pH 7.4. The stock solution was stored at -80°C. The calibrators were prepared before use. -hDPP3 immunoassay

[0240] After adding the labeled and diluted detection antibody (200 μl), 10 μl of sample (or calibrator) was pipetted into the coated 96-well microplate and the plate was incubated at 2°C to 8°C. The cells were incubated for 18 to 24 hours at 4 °C. The cells were washed with 350 μl of a washing solution (20 mM PBS, pH Unbound tracer was removed by washing 4 times with 5% 4% Triton X-100 (7.4). Chemiluminescence of the well-bound was measured by using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). 4.2 Results:

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

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

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

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

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

[0246] Plasma samples were obtained from 214 patients who came directly to the emergency department or oncology department at their first presentation. According to their final diagnosis, these patients could be divided into 6 subgroups: patients with acute heart failure (AHF), patients with myocardial infarction (MI), patients with sepsis, patients with cancer, patients with acute kidney injury (AKI), and patients with lower respiratory tract infection (LRTI). The control group was a pool of plasma samples from 93 healthy controls. 5.2 hDPP3 immunoassay:

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

[0248] Together with the corresponding diagnostic details, the resulting data were statistically analyzed ( Figure 2A ). All patients showed significantly elevated DPP3 plasma concentrations compared to healthy controls (standard). Table 6 shows the percentage of patients with DPP3 values ​​above the 75th percentile of the control group and their respective diagnoses. Analysis of plasma DPP3 levels indicates the disease state of the patient. This revelation can be used in the field of diagnosis and is also used to build the basis for therapeutic treatment, for example, by inhibition of DPP3.

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

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

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

[0252] The activity of native human DPP III was determined by hydrolysis of Arg-Arg-β-naphthylamide (Bachem Holdig AG, Switzerland) to form fluorescent β-naphthylamine. 200 μl of buffer (50 mM TRIS / HCl, pH 8.8, 0.04% NaN 3 , 50 μM aminopeptidase, 100 μM leupeptin) and 10 μl of sample (human plasma) were pipetted into a black 96-well microplate (Greiner Bio-One International GmbH, Austria) and preheated at 37°C for 10 minutes. After adding substrate (20 μl, 2 mM), the fluorescence increase was monitored at 37°C for 1 hour using 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 reference calibrant free βNA was expressed as nmol βNA / min / ml sample [nmol βNA min -1 ml -1 ] The slope of the fluorescence increase was calculated for the different samples.

[0253] With the standard soluble DPP3 activity assay, it cannot be determined whether DPP3 activity in plasma or the activity of other aminopeptidases is measured. In order to generate a signal specific for DPP3, an enzyme capture assay was performed, in which 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:

[0254] The solid phase was prepared using black 96-well microplates (Greiner Bio-One International GmbH, Austria) as described in Example 5. 10 μl of sample (plasma or standard) and 200 μl of buffer (50 mmol / l potassium phosphate, 100 mmol / l NaCl, 5 g / l bovine serum albumin, 1 g / l mouse IgG, 1 g / l bovine IgG, 50 μmol / l aminopeptidase inhibitor, 100 μmol / l leupeptin, pH 7.4) was pipetted into the coated microplate and incubated (18 h to 24 h, 2°C to 8°C, 600 rpm). Unbound analyte was removed by washing with wash solution (3×350 μl). After addition of substrate (200 μl, 100 μM in 50 mM Tris / HCl, pH (25°C) 8.8, 0.04%, NaN 3 ) and then the fluorescence increase was monitored for 1 hour at 37°C in a TwinkleLB 970 microplate fluorimeter (Berthold Technologies GmbH & Co. KG) using an excitation wavelength of 340 nm and an emission wavelength of 410 nm. The reference calibrant free βNA was expressed as nmol βNA / min / ml sample [nmol βNAmin -1 ml -1 The slope of the fluorescence increase of different samples was calculated.

[0255] In each activity assay type, free βNA was used as assay calibrant. Therefore, the activity of the βNA was measured in a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG) at 37°C using an excitation wavelength of 340 nm and an emission wavelength of 410 nm to measure the increase in βNA concentration (in 200 μl, in 50 mM Tris / HCl, pH (25°C) 8.8, 0.04% NaN 3 ; 0, 4, 8, 16, 32, 64, 125, 250 μM βNA). All sample measurements were calibrated on this βNA standard. 7. Example 7

[0256] DPP3 activity in the plasma of various diseased patients (patients with acute heart failure (AHF), sepsis, acute kidney injury (AKI), and lower respiratory tract infection (LRTI)) was determined using a specific DPP3 capture activity assay and compared with DPP3 plasma activity of healthy controls. 7.1 Methods:

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

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

[0259] The percentage of patients with DPP3 values ​​above the 75th percentile of the control group and their respective diagnoses are shown in Table 7. The activity showed better discrimination between healthy controls and diseased patients.

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

[0261] To better compare the DPP3 activity assay with the concentration assay, we performed a double-blind double-dip daptomycin assay on AHF ( Figure 4A ) or patients with sepsis ( Figure 4B ) were analyzed by ROC (receiver operating characteristic) analysis. The values ​​of area under the curve (AUC) and confidence interval (CI) are shown in Table 8. Data analysis showed that the activity assay had higher specificity than the sandwich immunoassay.

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

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

[0264] Female BALB / c nude (CAnN.Cg-Foxn1nu / Crl) mice (Charles River GmbH, Sulzfeld, Germany) 4-5 weeks old at delivery and weighing approximately 15 g to 18 g were maintained under optimal hygienic conditions with air conditioning at 10 to 15 air changes per hour, continuously monitored environment, target temperature range of 22 ± 3 °C, relative humidity of 30-70%, 12 h artificial fluorescent light / 12 h darkness. A maximum of 4 animals were housed per ventilated cage (IVC) and fed a diet prepared by M-Zucht (ssniff GmbH) and autoclaved community label water.

[0265] After a 4-day acclimation period, administration of mAbDPP3 was initiated: three different mAbDPP3 concentrations (0.65 mg / kg, 1.9 mg / kg and 5.75 mg / kg) in PBS were injected into each group of 4 mice. MAbDPP3 was administered intraperitoneally (ip) on days 1, 3, 5 and 7, and mice were monitored for 14 days. 8.2 Results:

[0266] All mice survived the 14 days of treatment without side effects, also at the highest dose. MAbDPP3 was safe for use in other animal experiments, which were always performed at a concentration of 1.9 mg / kg. 9. Example 9

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

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

[0269] First, animals were anesthetized with isoflurane for weighing (full g) and injected intraperitoneally (ip) with 1.2 g / kg BW urethane (c=0.4 g / mL) for long-term anesthesia. Then, the ventral area of ​​the neck was cut off and wiped with ethanol. Prepare the container and insert the catheter. Finally, the two catheters were rinsed with heparinized isotonic sodium chloride solution. Then a pressure sensor (medexlogical, Medex Medical Ltd.) was connected to a patient monitoring system (Datex-Ohmeda, GE). BP data were recorded separately by S / 5 collection software through a laptop connected to a BP monitor.

[0270] Rats were treated with PBS, 1.9 mg / kg and 5.75 mg / kg mAb DPP3 in PBS (n=3 per group). Compounds were administered via intravenous catheter. Blood pressure was monitored for 1 hour before compound administration and for more than 6 hours after compound administration. 9.2 Results:

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

[0272] In this study, we analyzed how treatment with mAbDPP3 affects sepsis mortality in the CLP mouse model. 10.1 Methods:

[0273] Male C57Bl / 6 mice (Charles River Laboratories, Germany) aged 12 to 15 weeks were used for the study. Peritonitis was surgically induced under mild isoflurane anesthesia. An incision was made into the left upper quadrant of the peritoneal cavity (normal location of the cecum). The cecum was exposed and a tight ligature was placed around the cecum, sutured distal to the insertion of the small intestine. A puncture wound was placed into the cecum with a 24-gauge needle, and a small amount of cecal contents were expressed through the wound. The cecum was placed back into 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 normal saline was given subcutaneously as a fluid replacement.

[0274] MAbDPP3 (1.9 mg / kg in PBS) was tested against vehicle (PBS). Compounds and vehicle were injected intravenously 5 minutes before CLP (prophylactic treatment) and after sepsis was fully developed, and 2 hours after CLP (therapeutic treatment). Each group contained 10 mice and was followed up for 7 days. 10.2 Results:

[0275] from Figure 6 As can be seen in the Figure 3, mAbDPP3 antibody significantly reduced mortality compared to PBS administration. After 4 days, 75% of mice were alive when treated with mAbDPP3. In contrast, almost all mice died after 4 days when treated with the vehicle. 11. Example 11

[0276] Heart failure was induced in rats using a septic shock model, and the effects of mAbDPP3 on cardiac function were then characterized. 11.1. Methods: - Study design

[0277] The research process is as follows Fig. 7A As shown. After CLP or sham surgery, animals were allowed to rest for 20 hours with free access to water and food. They were then anesthetized, tracheotomy was performed and arterial and venous lines were placed. 24 hours after CLP surgery, 2 mg / kg of mAbDPP3 or vehicle (saline) was administered. Hemodynamics were monitored invasively and continuously from t=0 hours to 3 hours. Echocardiograms of the heart were performed immediately after surgery and at 15 minutes, 1 hour, 2 hours and 3 hours after mAbDPP3 or saline injection. - CLP model of sepsis

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

[0279] Hemodynamic variables were obtained using the AcqKnowledge system (BIOPAC Systems, Inc., USA). It provides a fully automated blood pressure analysis system. The catheter was connected to the BIOPAC system via a pressure transducer.

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

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

[0282] After surgery, animals were allowed to rest to achieve steady state before hemodynamic measurements. Baseline blood pressure (BP) was then recorded. During data collection, saline infusion through the arterial line was stopped. - Echocardiogram

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

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

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

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

[0287] From the apical five-chamber view, mitral flow was recorded at the level of the mitral valve cusp using pulsed Doppler. - Experimental time points and animal groups

[0288] After surgery, baseline BP and echocardiogram were recorded. Then, mAbDPP3 was injected (2 mg / kg) or vehicle (saline) (iv, 5 min after surgery) and saline infusion was started. Hemodynamic points (BP and echocardiogram) were recorded 15 minutes and 1, 2, and 3 hours after mAbDPP3 or vehicle injection. There was 1 control group and 2 CLP groups, summarized in the table below (Table 9). At the end of the experiment, animals were euthanized, blood was drawn for EDTA-plasma generation, and organs were harvested for subsequent analysis. Group Group size CLP deal with 1- False 7 no brine 2-CLP-Saline 7 yes brine 3-CLP-mAbDPP3 10 yes mAbDPP3

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

[0290] Septic rats had very low blood pressure and reduced cardiac fractional shortening compared to sham animals. Administration of mAbDPP3 significantly increased fractional shortening ( Figure 7B ), increased mean blood pressure ( Figure 7C ) and significantly improved the health of rats with sepsis. 12. Example 12

[0291] The aim of the studies described herein was to evaluate the potential antiproliferative effects of mAbDPP3 in an in vitro cell culture system using various cancer cell lines. 12.1. Methods:

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

[0293] A549 cells are adenocarcinoma human alveolar basal epithelial cells. The cell line was first established by removing and culturing cancerous lung tissue in an explanted tumor of a 58-year-old male. In nature, these cells are squamous, and are responsible for some substances (e.g., water and electrolytes) to diffuse into the alveoli. If the A549 cells are cultured in vitro, they grow as monolayer cells and adhere to culture bottles. Another feature is that these cells can synthesize lecithin and contain high levels of desaturated fatty acids. The A549 cell line is widely used as an in vitro model of the II type lung epithelial cell model for drug metabolism and as a transfection host.

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

[0295] The MDA-MB231 cell line represents human breast cancer cells with epithelial morphology. These cells were isolated from the pleural effusion of a Caucasian breast cancer patient.

[0296] For each cell line, prepare a 96-well suspension cell culture plate. Pour 100 μL of a soft agar bottom layer (0.6% final concentration in complete medium) and allow to solidify. Then add 50 μL of a soft agar top layer (0.4% final concentration) containing the corresponding cells and cell number on top, allow to solidify and place the 96-well plate at 37°C, 10% CO. 2 Incubate overnight.

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

[0298] Raw data were converted to percentage soft agar growth relative to high control (solvent 0.1% DMSO) and low control (10 to 5 M staurosporine), which were set as 100% and 0%, respectively. 50 Calculations were performed using GraphPad Prism 5 software with a variable slope sigmoidal response fit model using 0% soft agar growth as bottom constraint, no bottom constraint, and 100% soft agar growth as top constraint. 12.2. Results:

[0299] In the cell culture system, various doses of mAbDPP3 ( Figure 8 ) were used to evaluate the growth of three cancer cell lines (A549, HCT116, MDA-MB231). IC was determined using standard parameters based on the signal of the solvent control as the top constraint (100% soft agar growth) and the signal of the staurosporine control as the bottom constraint (0% soft agar growth). 50 value. Each IC 50 The values ​​are summarized in Table 10.

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

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

[0302] The aim of the studies described herein 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. Methods:

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

[0304] Female BALB / c nude mice (CAnN.Cg-Foxn1) were 4 to 5 weeks old and weighed approximately 15 to 18 g at delivery. nu / Crl) mice (Charles River GmbH, Sulzfeld, Germany) were maintained under optimal hygienic conditions with air conditioning at 10 to 15 air changes per hour, continuously monitored environment, target temperature range of 22 ± 3 °C, relative humidity of 30% to 70%, 12 h artificial fluorescent light / 12 h darkness. A maximum of 4 animals were housed per ventilated cage (IVC) and fed a diet prepared by M-Zucht (ssniff GmbH) and autoclaved community label water.

[0305] Human breast cancer MDA-MB-231 cells and colon cancer HCT-116 cells provided by ATCC will be used in this study. The cells were subcultured for 5 generations before inoculation into mice. 6 cells / 0.1 mL were injected subcutaneously into the right side of the mouse. When the tumor volume reached about 100 mm 3 Up to 200mm 3 At 1: 20, 20 mice with tumors of appropriate size were randomly divided into groups (10 mice per group) according to tumor volume and body weight. Doses were given according to tumor volume and body weight and animals were started to be administered. These groups are shown in the following table (Table 11).

[0306] Table 11: Overview of treatment strategies.

[0307] Animal behavior and health were observed daily, and tumor growth was recorded every two days for 24 days or several days by caliper measurement. Primary tumor size was measured by caliper (manual caliper, OMC Fontana). According to the formula V = W 2× L / 2 (L = length, W = vertical width of the tumor, L>W) to calculate the tumor size. The relative tumor volume (RTV) was calculated as follows: RTV=V t / V 0 , where V t is the daily volume, V 0 is the volume at the start of treatment. 13.2. Results:

[0308] In xenograft cancer models, administration of mAbDPP3 reduced the formation of all tumors studied ( Figures 9A to 9D ). Under mAbDPP3 treatment, breast cell tumors grew for 2 more days, growing to 20 times their size ( Fig. 9B ), the colon cell tumor grew for 2 more days and grew to 10 times its size ( Fig.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

[0309] In order to evaluate the possibility of using DPP3-adsorbents to remove plasma from excess DPP3, it should be analyzed whether DPP3 is fully bound to anti-DPP3 columns. Affinity chromatography columns were prepared by immobilizing DPP3 binding antibodies on GlycoLink columns (Thermo Fisher). The binding of DPP3 to these columns was analyzed by measuring DPP3 concentrations before and after flowing through these columns. 14.1. Methods:

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

[0311] Therefore, 300 μl of 3 mg / ml solution of the respective anti-DPP3 antibody was diluted in 700 μl GlycoLink coupling buffer to a final volume of 1 ml and 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 minutes. The oxidizing agent was removed from the antibody solution using a desalting column. - Preparation of GlycoLink columns

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

[0313] Recombinant GST-hDPP3 or patient plasma was diluted in coupling buffer (final concentration of recombinant DPP3 = 100 ng / ml, 1 ml; 1 ml plasma + 1 ml coupling buffer), applied to the equilibrated mAbDPP3 column and incubated at room temperature for 30 minutes. The entire flow-through was saved to assess binding efficiency and capacity. The column was washed, bound protein was eluted using GlycoLink elution buffer, and the column was equilibrated before storage. - Analysis of DPP3 content

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

[0315] By affinity chromatography, remove almost all DPP3 (referring to Table 12) in plasma and recombinant DPP3 solution from sample.Different mAbDPP3 antibodies show different affinities to DPP3.Because mAbDPP3_2555 shows the highest recDPP3 binding rate, the antibody is selected for the chromatography of plasma sample.Table 12 shows that DPP3 plasma levels can be strongly reduced by affinity chromatography.These results show that DPP3-adsorbents can be used in plasmapheresis to remove excessive DPP3 in patient plasma.

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Zuk et al., Enzyme Immunochromatography--A Quantitative Immunoassay Requiring No Instrumentation, Clinical Chemistry, 31(7): 1144-1150 (1985) sequence SEQ ID NO:1 hDPP3 AS 1-737 SEQ ID NO:2 hDPP3 AS 474-493 (N-Cys) CETVINPETGEQIQSWYRSGE BRIEF DESCRIPTION OF THE DRAWINGS

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

[0318] Figure 2: DPP3 concentration as 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). The median values ​​of the patient groups were significantly different from those of healthy controls (Mann-Whitney test, p<0.005). (B) Comparison of plasma DPP3 concentrations in patients who died shortly after emergency department admission and those who survived. Surviving patients showed significantly lower DPP3 levels (Mann-Whitney test, p<0.05).

[0319] Figure 3 : DPP3 activity as a diagnostic marker

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

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

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

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

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

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

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

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

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

[0329] Fig. 9: Effect of mAbDPP3 on tumor growth in vivo (A) Mice bearing xenografted breast tumor cells were treated with PBS or mAbDPP3 (n=10 per group). Growth curves of relative tumor volume over 24 days showed reduced tumor growth in mAbDPP3-treated mice. (B) Comparison of the time it takes for breast cell tumors to increase their volume 20-fold with and without mAbDPP3 treatment. With mAbDPP3 treatment, growth took significantly longer (Mann-Whitney test, p<0.05). (C) Mice bearing xenografted colon tumor cells were treated with PBS or mAbDPP3 (n=10 per group). Growth curves of relative tumor volume over 30 days showed reduced tumor growth in mAbDPP3-treated mice. (D) Comparison of the time required for colon cell tumors to increase their volume 10-fold with and without mAbDPP3 treatment. When treated with mAbDPP3, growth took longer.

[0330] Fig.10 :DPP3 activity as a diagnostic marker (II)

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

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

Claims

1. A DPP3 activity inhibitor for preventing or treating a disease or condition of a subject accompanied by or associated with a necrotic process by modulating DPP3 activity in a subject's body fluid, wherein the disease is selected from the group consisting of heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infection (microorganisms, viruses (e.g., AIDS), parasites (e.g., malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g., epilepsy, neurodegenerative diseases), autoimmune diseases and vascular diseases (e.g., Kawasaki syndrome) and hypotension.

2. The DPP3 activity inhibitor for preventing or treating a disease or condition of a subject accompanied by or associated with a necrotic process according to claim 1, wherein the inhibitor is selected from the group consisting of an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

3. The DPP3 activity inhibitor for preventing or treating a disease or condition of a subject accompanied by or associated with a necrotic process according to claim 2, wherein the inhibitor is a single-binding or at least two-binding antibody.

4. The DPP3 activity inhibitor for preventing or treating a disease or condition of a subject accompanied by or associated with a necrotic process according to claim 2 or 3, wherein the inhibitor is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to SEQ ID No. 1, in particular to SEQ ID No.

2.

5. The DPP3 activity inhibitor for preventing or treating a disease or condition of a subject accompanied by or associated with a necrotic process according to claim 2 or 3, wherein the inhibitor has a minimum binding affinity for DPP3 equal to or less than 10 -7 Antibodies or fragments or scaffolds of M.

6. The DPP3 activity inhibitor for preventing or treating a disease or condition of a subject accompanied by or associated with a necrotic process according to claim 2 or 3, wherein the inhibitor is a monospecific antibody or fragment or scaffold.

7. A DPP3 activity inhibitor for preventing or treating a disease or condition of a subject accompanied by or associated with a necrotic process according to claim 2 or 3, wherein the inhibitor is an antibody or fragment or scaffold that binds to full-length DPP3 and inhibits at least 10%, or at least 50%, more preferably at least 60%, even more preferably greater than 70%, even more preferably greater than 80%, even more preferably greater than 90%, even more preferably greater than 95% of the DPP3 activity.

8. An inhibitor of DPP3 activity for use in preventing or treating a disease or condition of a subject accompanied by or associated with a necrotic process according to claim 2 or 3, wherein the inhibitor is selective and / or specific for DPP3 and does not cross the cell membrane and / or the blood-brain barrier.

9. A DPP3 activity inhibitor for use in preventing or treating a disease or condition in a subject accompanied by or associated with a necrotic process according to claim 2 or 3, wherein the amount of total DPP3 and / or the amount of active DPP3 in a body fluid sample of the subject is higher than a predetermined threshold.

10. A pharmaceutical composition comprising the DPP3 activity inhibitor according to any one of claims 1 to 9 for preventing or treating a disease or condition of a subject accompanied by or associated with a necrotic process.

11. Use of an inhibitor of DPP3 activity according to any one of claims 1 to 9 in a method for removing DPP3 from plasma in vitro, comprising apheresis and affinity chromatography.

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