DPP3 binders directed to and binding to specific DPP3 epitopes, as well as their use in the prevention or treatment of diseases / acute conditions associated with oxidative stress.

Abstract

The present invention provides a binder directed to and bound to the DPP3 protein or a functional derivative thereof, and its use in a method for preventing or treating a patient's disease or acute condition, where the disease or acute condition is related to oxidative stress. [Solution] In relation to this, specifically, the present invention provides a binder that is directed to and binds to an epitope according to Sequence ID No. 2, wherein the epitope is contained in the DPP3 protein or a functional derivative thereof, and wherein the DPP3 binder recognizes and binds to at least three amino acids of Sequence ID No. 2.

Description

[Technical Field]

[0001] Field of the present invention In a first main embodiment of the present invention, the subject matter of the present invention is a binder directed to and bound to dipeptidyl peptidase 3 (DPP3) protein or a functional derivative thereof.

[0002] In another embodiment of the first main aspect of the present invention, the aforementioned binder is provided for use in the prevention or treatment of a patient's disease or acute condition, where the disease or acute condition is related to oxidative stress. In another embodiment of the first main aspect of the present invention, the aforementioned binder is provided for use in the treatment or prevention of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress, and wherein the disease is selected from the group including neurodegenerative diseases, metabolic syndrome, cardiovascular diseases, autoimmune diseases, inflammatory lung diseases, kidney diseases, liver diseases, gastrointestinal diseases, viral infections, cancer, inflammation, sepsis, septic shock, and SIRS.

[0003] In a second main embodiment of the present invention, a binder is provided which is directed to and binds to an epitope according to SEQ ID NO: 2, wherein the binder recognizes and binds to at least three amino acids of SEQ ID NO: 2, and wherein the epitope is contained in DPP3 as shown in SEQ ID NO: 1.

[0004] The following text refers to the second main aspect of the present invention: An additional aspect of the present invention is the aforementioned binder which is directed to and binds to the epitope defined by SEQ ID NO: 2, wherein the binder is directed to and binds to the epitope defined by SEQ ID NO: 3, and wherein the binder recognizes and binds to at least three amino acids in SEQ ID NO: 3, and wherein the epitope is contained in DPP3 as shown in SEQ ID NO: 1.

[0005] An additional aspect of the present invention is the aforementioned binder which is directed to and binds to the epitope defined by SEQ ID NO: 2, wherein the binder is directed to and binds to the epitope defined by SEQ ID NO: 4, and wherein the DPP3 binder recognizes and binds to at least three amino acids of SEQ ID NO: 4, and wherein the epitope is contained in the DPP3 shown in SEQ ID NO: 1.

[0006] An additional aspect of the present invention is the aforementioned binder directed to and bound to the epitope defined in SEQ ID NO: 2, wherein the binder is selected from the group comprising an antibody, an antibody fragment, or a non-Ig scaffold, and wherein the epitope is contained in DPP3 as shown in SEQ ID NO: 1.

[0007] In a third main embodiment of the present invention, the binder of the second main embodiment of the present invention, which is directed to and binds to the epitope according to SEQ ID NO: 2, is a dipeptidyl peptidase 3 (DPP3) binder directed to and binds to the epitope according to SEQ ID NO: 2, wherein the epitope is contained in the DPP3 protein or a functional derivative thereof, and wherein the DPP3 binder recognizes and binds to at least three amino acids of SEQ ID NO: 2.

[0008] An additional aspect of the present invention is the aforementioned binder directed to and bound to the epitope defined by SEQ ID NO: 2 according to a third aspect of the present invention, wherein the binder is a monoclonal antibody or monoclonal antibody fragment, and wherein the heavy chain complementarity-determining region (CDR) is the following sequence: Sequence ID 7, Sequence ID 8, and / or Sequence ID 9 The light chain complementarity determination region includes the following sequence: Sequence ID 10, KVS, and / or Sequence ID 11 Includes.

[0009] An additional aspect of the present invention is the aforementioned binder directed to and bound to the epitope defined by Sequence ID No. 2 according to a third aspect of the present invention, wherein the binder is a humanized monoclonal antibody or a humanized monoclonal antibody fragment, wherein its heavy chain sequence is the following sequence: Sequence ID 12 It includes, and where the light chain sequence is the following sequence: Sequence ID 13 Includes.

[0010] An additional aspect of the present invention is one of the aforementioned binders directed to and bound to an epitope according to Sequence ID No. 2, for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress, and wherein the epitope is contained in DPP3 as shown in Sequence ID No. 1.

[0011] The specific content of the present invention is a binder directed to and bound to the epitope defined by Sequence ID No. 2, specifically a dipeptidyl peptidase 3 (hereinafter referred to as DPP3) binder, wherein the epitope is contained in the DPP3 protein or a functional derivative thereof, and wherein the DPP3 binder recognizes and binds to at least three amino acids (aa), preferably at least four aa, of Sequence ID No. 2.

[0012] Further aspects of the present invention include a binder directed to and bound to an epitope defined by Sequence ID No. 2, specifically a DPP3 binder, wherein the epitope is contained in the DPP3 protein or a functional derivative thereof, and wherein, for use in the prevention or treatment of a patient's disease or acute condition (due to the disease or acute condition being related to oxidative stress), the DPP3 binder recognizes and binds to at least three amino acids (aa), preferably at least four aa, of Sequence ID No. 2.

[0013] Furthermore, the present invention also includes a binder that binds to the epitope according to Sequence ID No. 2, specifically an anti-DPP3 antibody or an anti-DPP3 antibody fragment, wherein the epitope is contained in a DPP3 protein or its functional derivative or an anti-DPP3 non-Ig scaffold that binds to the epitope according to Sequence ID No. 2, wherein the anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold recognizes and binds to at least three amino acids (aa), preferably at least four aa, of Sequence ID No. 2.

[0014] In relation to the preceding context, and also relating to the present invention, the present invention also relates to a binder according to the present invention that binds to the epitope according to SEQ ID NO: 2, specifically an anti-DPP3 antibody or anti-DPP3 antibody fragment, wherein the epitope is contained in a DPP3 protein or a functional derivative thereof or an anti-DPP3 non-Ig scaffold that binds to the epitope according to SEQ ID NO: 2, wherein the epitope is contained in a DPP3 protein or a functional derivative thereof, and wherein the anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold recognizes and binds to at least three amino acids (aa), preferably at least four aa, of SEQ ID NO: 2.

[0015] Further aspects of the present invention relate to a method for preventing or treating a patient's disease or acute condition (due to the disease or acute condition being related to oxidative stress), characterized in that a binder directed to and bound to DPP3, or a binder directed to and bound to Sequence ID No. 2 as an epitope contained in the DPP3 protein or a functional derivative thereof, or an anti-DPP3 antibody or anti-DPP3 antibody fragment that binds to DPP3, or an anti-DPP3 non-Ig scaffold directed to and bound to Sequence ID No. 2 as an epitope contained in the DPP3 protein or a functional derivative thereof, is administered to the patient in a pharmaceutically effective amount.

[0016] The present invention further relates to a pharmaceutical composition for use in the prevention or treatment of a patient's disease or acute condition (due to the disease or acute condition being related to oxidative stress), comprising a binder directed to and bound to DPP3, or a binder directed to and bound to SEQ ID NO: 2 as an epitope containing the DPP3 protein or a functional derivative thereof, or an anti-DPP3 antibody or anti-DPP3 antibody fragment that binds to DPP3, or an anti-DPP3 non-Ig scaffold directed to and bound to SEQ ID NO: 2 as an epitope contained in the DPP3 protein or a functional derivative thereof.

[0017] Another subject of the present invention is a pharmaceutical composition comprising the binder of the present invention, or a DPP3 binder according to the present invention, specifically an anti-DPP3 antibody or anti-DPP3 antibody fragment bound to DPP3, or an anti-DPP3 non-Ig scaffold bound to DPP3, for use in the prevention or treatment of a patient's disease or acute condition (due to the disease or acute condition being related to the oxidative stress described above), wherein the pharmaceutical composition comprises, for example, at least one additional pharmaceutically active drug that can be used as a primary agent in a method of treating a disease or acute condition, and wherein the treatment induces oxidative stress as a side effect, thereby the binder, the DPP3 binder, the anti-DPP3 antibody or anti-DPP3 antibody fragment bound to DPP3, or the anti-DPP3 non-Ig scaffold bound to DPP3 may play a secondary role, and it reduces or modulates the aforementioned induced oxidative stress.

[0018] Further embodiments of the present invention are a binder, or a kit comprising a DPP3 binder according to the present invention, specifically an anti-DPP3 antibody, an anti-DPP3 antibody fragment bound to DPP3, or an anti-DPP3 non-Ig scaffold bound to DPP3, for use in the prevention or treatment of a patient's disease or acute condition (due to the disease or acute condition being related to the oxidative stress described above), wherein the pharmaceutical composition optionally comprises at least one additional pharmaceutically active drug that can be used as a primary agent in a method of treating a disease or acute condition, and wherein the treatment induces oxidative stress as a side effect, thereby the DPP3 binder, anti-DPP3 antibody, or anti-DPP3 antibody fragment bound to DPP3, or anti-DPP3 non-Ig scaffold bound to DPP3, may play a secondary role, and it may reduce or modulate the aforementioned induced oxidative stress.

[0019] Modified anti-DPP3 antibody, fragment, scaffold Furthermore, the content of the present invention is for use as a medicament, the binder according to the present invention described above, or an anti-DPP3 antibody or anti-DPP3 antibody fragment that binds to DPP3, or an anti-DPP3 non-Ig scaffold that binds to DPP3, wherein the binder, or the antibody or the antibody fragment, or the non-Ig scaffold is a modified binder, antibody or fragment, or scaffold.

[0020] In a preferred embodiment, the modified anti-DPP3 antibody or modified anti-DPP3 fragment, or modified non-Ig scaffold is used for the prevention or treatment of a patient's disease or acute condition (where the disease or acute condition is related to oxidative stress). Furthermore, according to the present invention, the modified anti-DPP3 antibody or anti-DPP3 antibody fragment, or modified non-Ig scaffold according to the present invention modulates the biological activity of DPP3.

[0021] In connection with the content of the present invention, DPP3 biological activity can be defined as the regulation of DPP3 enzyme activity or DPP3 activity in the oxidative stress pathway. According to the present invention, the modified anti-DPP3 antibody or anti-DPP3 antibody fragment, or modified non-Ig scaffold according to the present invention can promote the biological activity of DPP3. In another embodiment of the present invention, the modified anti-DPP3 antibody or anti-DPP3 antibody fragment, or modified non-Ig scaffold according to the present invention can reduce the biological activity of DPP3.

[0022] In another specific embodiment related to the content of the present invention, the "modified" anti-DPP3 antibody or modified anti-DPP3 antibody fragment, or modified non-Ig scaffold described above is an anti-DPP3 antibody or anti-DPP3 antibody fragment, or modified anti-DPP3 non-Ig scaffold that inhibits the biological activity of DPP3 by at least 10%, preferably at least 50%, more preferably >50%, and most preferably 100%.

[0023] In specific embodiments, the modified binder, modified anti-DPP3 antibody or modified anti-DPP3 antibody fragment, or modified anti-DPP3 non-Ig scaffold according to the present invention is used for the prevention or treatment of a patient's disease or acute condition, where the disease or acute condition is related to oxidative stress.

[0024] Another embodiment of the present invention is a kit or assay comprising the previously described DPP3-binding binder, or anti-DPP3 antibody, and / or anti-DPP3 antibody fragment, or an anti-DPP3 non-Ig scaffold-binding anti-DPP3, for use in the treatment or prevention of a patient's disease or acute condition (due to the disease or acute condition being related to oxidative stress). [Background technology]

[0025] background Dipeptidyl peptidase 3 (DPP3) Dipeptidyl peptidase 3, also known as dipeptidylaminopeptidase III, dipeptidylarylamidase III, dipeptidyl peptidase III, enkephalinase B, or erythrocyte angiotensinase, abbreviated as DPP3 or DPPIII-, is a metallopeptidase that removes dipeptides from physiologically active peptides such as enkephalins and angiotensins. Hereafter, the expression "DPP3" will be used throughout the text as an abbreviation for dipeptidyl peptidase 3 mentioned above.

[0026] DPP3 was first identified and its activity measured in 1967 by Ellis and Nuenke in purified bovine anterior pituitary extract. Registered as EC3.4.14.4, this enzyme has a molecular weight of approximately 83 kDa and is highly conserved in prokaryotes and eukaryotes (Prajapati & Chauhan 2011).

[0027] The amino acid sequence of a human variant of DPP3 is shown in Sequence ID No. 1. DPP3 is a ubiquitous, primarily cytoplasmic peptidase. Despite the absence of a signaling sequence, membrane activity has been reported in several studies (Lee & Snyder 1982).

[0028] DPP3 is a zinc-dependent exopeptidase belonging to the M49 peptidase family. It exhibits broad substrate specificity for oligopeptides of various compositions consisting of 3, 4 to 10 amino acids, and can also cleave after the proline. DPP3 is known to hydrolyze dipeptides from the N-terminus of its substrates (angiotensin II, III, IV; angiotensin I-7 (Cruz-Diaz et al., 2016); Leu-enkephalin and Met-enkephalin; endomorphin 1 and 2, etc.). As a metallopeptidase, DPP3 exhibits optimal activity at pH 8.0-9.0 and can be activated by the addition of divalent metal ions such as Co2+ and Mg2+. Structural analysis of DPP3 revealed the catalytic motifs HELLGH (hDPP3, 450-455) and EECRAE (hDPP3, 507-512), along with the following amino acids important for substrate binding and hydrolysis: Glu316, Tyr318, Asp366, Asn391, Asn394, His568, Arg572, Arg577, Lys666, and Arg669 (Prajapati & Chauhan 2011 and Kumar et al. 2016; numbers indicate the sequence of human DPP3 (see SEQ ID NO: 1)). Considering all known amino acids or sequence regions involved in substrate binding and hydrolysis, the active site of human DPP3 can be defined as the region between amino acids 316-669.

[0029] Recent findings on DPP3 indicate that it plays a role not only in part of protein metabolism but also in blood pressure regulation, pain regulation, inflammatory processes, and oxidative stress regulation (Prajapati & Chauhan 2011).

[0030] DPP3 has also been shown to be a promising biomarker in several studies. Elevated DPP3 activity has been demonstrated in homogenates of ovarian and endometrial tumors. DPP3 activity further increases with the severity / malignancy of these tumors (Simaga et al. 1998, 2003). Immunohistochemical and Western blot analyses of glioblastoma cell lines also showed elevated DPP3 levels (Singh et al. 2014).

[0031] DPP3 has also been proposed as a promising arterial risk marker (US2011008805) and a marker for rheumatoid arthropathy (US2006177886). Patent application WO2005106486 describes the ubiquitous expression of DPP3 on or within the cell surface, its activity as a diagnostic marker for all kinds of diseases, and its potential as a therapeutic agent. EP1498480 describes the potential diagnostic and therapeutic applications of hydrolytic enzymes containing DPP3.

[0032] Related prior art can be further summarized as follows: WO2005 / 106486 describes in a general style a screening method for therapeutic agents that may be useful in treating diseases including cardiovascular diseases, infectious diseases, respiratory diseases, cancer, endocrine diseases, metabolic diseases, gastrointestinal diseases, inflammation, hematological diseases, musculoskeletal diseases, neurological and urological diseases. In the screening method, a test compound is brought into contact with a DPP3 polynucleotide, and the binding between the test compound and the DPP3 polynucleotide is detected. Furthermore, the document describes a compound in a general style that can bind to DPP3 and activate and / or inhibit its activation. Furthermore, the present invention describes a pharmaceutical composition that includes such a compound.

[0033] Liu et al. 2007 described the relationship between the activation of antioxidant response elements (AREs) and the overexpression of DPP3 and Sequestome1 in IMR-32 cells. Overexpression of DPP3 and Sequestome1 stimulated the Nrf2 translocation and led to high levels of NAD(P)H:quinone oxireductase 1, a protein transcriptionally regulated by AREs.

[0034] Hast et al. 2013 described a comparative study of genomic profiles and KEAP1-interacting protein spectra of 178 squamous cell lung cancers characterized by the Cancer Genome Atlas, and revealed amplification and mRNA overexpression of the DPP3 gene in tumors with high Nrf2 activity but lacking Nrf2-stabilizing mutations. They further described that tumor-induced mutations in KEAP1 are hypomorphic with respect to Nrf2 inhibition, and that DPP3 overexpression in the presence of these mutants further promotes Nrf2 activation.

[0035] DPP3 overexpression Therefore, conventional techniques have shown that intracellular DPP3 overexpression is closely related to oxidative stress regulation. DPP3 was identified as an activator of antioxidant response elements (AREs) in an unbiased screen of a cDNA library consisting of approximately 15,000 full-length human expression cDNAs (Lu et al. 2007).

[0036] DPP3 disrupts the KEAP1-Nrf2 complex by competing with Nrf2 for the KEAP1 binding site (Hast et al. 2013). This disruption prevents Nrf2 degradation, leading to the migration and localization of Nrf2 into the nucleus and ARE activation. Overexpression of DPP3 in neuroblastoma cells (Lu et al. 2007), HEK293T cells (Hast et al. 2013), or breast cancer cells MCF7 (Lu et al. 2017) activates Nrf2-mediated transcription. Active and inactive mutants of DPP3 were overexpressed in MCF7 cells and showed the same regulatory effects against oxidative stress (Lu et al. 2017). Hast et al. (2013) also demonstrated a loss-of-function effect: silencing DPP3 using specific siRNA reduced Nrf2-mediated transcription to the level of Nrf2 silencing.

[0037] DPP3 is known as an intracellular protein, and DPP3 activity has been detected in several bodily fluids, namely post-placental serum (Shimamori et al. 1986), seminal plasma (Vanha-Perttula et al. 1988), and cerebrospinal fluid (CSF) (Aoyagi et al. 1993). In CSF, DPP3 activity levels were elevated in Alzheimer's disease patients (AD, Aoyagi et al. 1993). DPP3 is known to be expressed as membrane DPP3, intracellular DPP3, or circulating DPP3.

[0038] Because DPP3 can cleave several bioactive peptides, it has been proposed not only as a promising biomarker but also as a promising therapeutic target. Influenza A virus alters host DPP3 levels for replication (cell culture studies, Meliopoulos et al. 2012). Enkephalin and / or angiotensin-degrading enzymes, including DPP3, generally have therapeutic potential as targets for the treatment of pain, cardiovascular disease (CVD), and cancer, and corresponding inhibitors have potential as promising treatments for pain, psychiatric disorders, and CVD (Khaket et al. 2012, Patel et al. 1993, Igic et al. 2007).

[0039] Inhibition of DPP3 DPP3 activity can be nonspecifically inhibited by various common protease inhibitors (e.g., PMSF, TPCK), sulfhydryl reagents (e.g., pHMB, DTNB), and metal chelators (EDTA, o-phenanthroline) (Abramic et al. 2000, EP2949332).

[0040] DPP3 activity can be further specifically inhibited by various types of compounds: the endogenous DPP3 inhibitor is the peptide spinorphin. Several synthetic spinorphin derivatives (e.g., tynorphin) have been produced and shown to inhibit DPP3 activity to varying degrees (Yamamoto et al. 2000). Other publicly available DPP3 peptide inhibitors include propioxatine A and B (US4804676), as well as propioxatine A analogs (Inaoka et al. 1988).

[0041] DPP3 is produced by small molecules such as fluostatins and benzimidazole derivatives. It can also inhibit DPP3 activity. Fluostatin A and B are antibiotics produced by the Streptomyces sp. TA-3391, a species of the genus Streptomyces. They are non-toxic and strongly inhibit DPP3 activity. To date, 20 different benzimidazole derivatives have been synthesized and published (Agic et al. 2007; Rastija et al. 2015), of which two compounds, 1' and 4', show the strongest inhibitory effect (Agic et al. 2007). Several dipeptidylhydroxamic acids have been shown to further inhibit DPP3 activity (Cvitesic et al., 2016).

[0042] Oxidative stress Oxidative stress reflects an imbalance between the overall expression of reactive oxygen species (ROS) / reactive nitrogen species (RNS) and antioxidants, which favors the excessive generation of free radicals. This process leads to the oxidation of biomolecules, accompanied by loss of their biological function and / or homeostatic imbalance, and their expression is a potential source of oxidative damage to cells and tissues. The accumulation of ROS / RNS results in many harmful effects, such as lipid peroxides, protein oxidation, and DNA damage (including base damage and strand disruption). Furthermore, some reactive oxidative species function as cellular messengers in redox signaling. Therefore, oxidative stress can disrupt the normal mechanisms of cellular signaling.

[0043] "ROS" and "RNS" are terms that collectively describe free radicals and other non-radical reactive derivatives, and are also called oxidizing agents. Radicals are less stable than non-radical species, but their reactivity is generally stronger. Molecules that have one or more unpaired electrons in their outer shell are called free radicals. Free radicals are formed from molecules by radical cleavage, where each fragment gives rise to another radical; by the breaking of chemical bonds that hold one electron; and also by redox reactions. Free radicals associated with oxidative stress include hydroxyl (OH·), superoxide (O2·−), nitric oxide (NO·), nitrogen dioxide (NO2·), peroxyl (ROO·), and lipid peroxyl (LOO·). Also, hydrogen peroxide (H2O2), ozone (O3), singlet oxygen ( 1 O2, hypochlorous acid (HOCl), nitrite (HNO2), peroxynitrite (ONOO·), nitrous oxide (N2O3), and lipid peroxides (LOOH) are not free radicals and are generally called oxidizing substances, but they can easily lead to free radical reactions in living organisms.

[0044] The formation of ROS and RNS occurs in cells through two methods: enzymatic and non-enzymatic reactions. Enzymatic reactions that produce free radicals include those associated with the respiratory chain, phagocytosis, prostaglandin synthesis, and the cytochrome P450 system. Free radicals can also be generated from non-enzymatic reactions of organic compounds with oxygen, as well as those initiated by ionizing radiation. Non-enzymatic methods can also occur during oxidative phosphorylation in mitochondria (i.e., aerobic respiration). For a review, see Pham-Huy et al. 2008. Int J Biomed Sci 4 (2): 89-96.

[0045] Diseases related to oxidative stress In light of the preceding description and in relation to the content of the present invention, oxidative stress is related to, and thereby to, many diseases, including, but not limited to, neurodegenerative diseases, metabolic syndrome, cardiovascular diseases, autoimmune diseases, inflammatory lung diseases, kidney diseases, liver diseases, digestive system diseases, viral infections, cancer, and inflammation.

[0046] Conventional techniques have shown that intracellular DPP3 is closely involved in oxidative stress regulation. DPP3 was identified as an activator of antioxidant response elements (AREs) in an unbiased screen of a cDNA library consisting of approximately 15,000 full-length human expression cDNAs (Lu et al. 2007; see also above). AREs regulate the expression of many cytoprotective antioxidant enzymes and scavengers and contribute to endogenous defense against oxidative stress. This antioxidant effect of DPP3 is due to its interference with the KEAP1-Nrf2 signaling pathway. Nrf2 is a transcription factor that regulates the basal and induced expression of a range of antioxidant response element-dependent genes, thereby modulating the physiological and pathophysiological outcomes of oxidative exposure. Under normal or non-stress conditions, Nrf2 binds to Kelch-like ECH-binding protein 1 (KEAP1) via its ETGE motif and its DLG motif. Within this protein cluster, Nrf2 is retained in the cytoplasm, rapidly ubiquitinated, and then degraded by the proteasome. Under oxidative stress, Nrf2 does not decrease; instead, it translocates to the nucleus, binding to the DNA promoter and inducing the expression of a series of antioxidant response element (ARE)-dependent genes. A variety of chemicals, including plant compounds and derivatives (CDDO, sulforaphane), therapeutic agents (ortiplats, auranofin), environmental substances (paraquat, arsenic), and endogenous chemicals [NO, 15d-PGJ2, nitro fatty acids, and 4-hydroxynonenal (4-HNE)], induce ARE genes via Nrf2 (Ma 2013). [Overview of the project]

[0047] Brief Description of the Invention In light of the above, the inventors also surprisingly and unexpectedly found that oxidative stress can be directed towards the DPP3 protein or its functional derivatives, and can be reduced or regulated by binders that bind to it.

[0048] The inventors also found that oxidative stress can be directed to the epitope of SEQ ID NO: 2 and reduced or regulated by a binder that binds to it, where the epitope is located in the DPP3 protein. Furthermore, the inventors have discovered a dipeptidyl peptidase 3 (hereinafter referred to as DPP3) binder that is directed to and binds to the epitope defined by Sequence ID No. 2, wherein the epitope is contained in the DPP3 protein or a functional derivative thereof, and wherein the DPP3 binder recognizes and binds to at least three amino acids (aa), preferably at least four aa, of Sequence ID No. 2.

[0049] In relation to the preceding context, the inventors have specifically found that oxidative stress can be reduced or regulated by an anti-DPP3 antibody or anti-DPP3 antibody fragment that is directed to and binds to an epitope defined by SEQ ID NO: 2, wherein the epitope is contained in a DPP3 protein or a functional derivative thereof, and wherein the DPP3 binder recognizes and binds to at least three amino acids (aa), preferably at least four aa, of SEQ ID NO: 2, or the anti-DPP3 non-Ig scaffold is directed to and binds to an epitope defined by SEQ ID NO: 2, wherein the epitope is contained in a DPP3 protein or a functional derivative thereof, and wherein the DPP3 binder recognizes and binds to at least three amino acids (aa), preferably at least four aa, of SEQ ID NO: 2.

[0050] Therefore, the present invention provides, for use in a method of prophylactic treatment or treatment of a disease or acute condition of a patient, a binder disclosed herein, and an anti-DPP3 antibody or anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to DPP3, wherein the disease or acute condition of the patient is related to oxidative stress.

[0051] Furthermore, with respect to the binders provided herein, specifically DPP3 binders, anti-DPP3 antibodies, or anti-DPP3 antibody fragments that bind to DPP3 or anti-DPP3 non-Ig scaffolds that bind to DPP3, the inventors have found binders for DPP3 that rapidly reduce or modulate oxidative stress in mammalian cells when measured by the respective biomarker measurement methods described further below.

[0052] Another subject of the present invention is a pharmaceutical composition comprising the binder of the present invention, specifically an anti-DPP3 antibody or anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to DPP3, for use in methods of preventing or treating a patient's disease or acute condition (due to the disease or acute condition being related to oxidative stress).

[0053] Accordingly, the pharmaceutical compositions disclosed herein are also provided for use in the prevention or treatment of symptoms, syndromes, or pathologies and acute conditions, as well as disease-related problems, that are mediated by oxidative stress. Diseases related to oxidative stress

[0054] As mentioned earlier, the emergence of oxidative stress is associated with many diseases or disorders, including, according to the present invention, the following: • Neurodegenerative diseases, where the neurodegenerative diseases may be selected from the group including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). • Metabolic syndrome, where said metabolic syndrome may be selected from the group including insulin resistance, obesity, hyperglycemia, dyslipidemia, hypertension, and diabetes. • Cardiovascular disease, where said cardiovascular disease may be selected from the group including atherosclerosis, hypertension, heart failure, cardiovascular ischemia, cerebral ischemic injury, stroke, and myocardial infarction.

[0055] • Autoimmune diseases, where the autoimmune diseases may be selected from the group including rheumatoid arthritis and systemic lupus erythematosus. • Inflammatory lung disease, where the inflammatory lung disease may be selected from the group including COPD and asthma. • Renal disease, where said renal disease may be selected from the group including nephrotoxicity (drug-induced renal disease), acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, and end-stage renal disease (ESRD). • Liver disease, where the liver disease may be selected from the group including hepatotoxicity, viral hepatitis, and cirrhosis.

[0056] • Digestive system disorders, where the digestive system disorders may be selected from the group including inflammatory bowel diseases, such as ulcerative colitis, Crohn's disease, gastritis, pancreatitis, and peptic ulcers. • Viral infections, said viral infections may be selected from the group including blood-derived hepatitis viruses (types B, C, and D), human immunodeficiency virus (HIV), influenza A, Epstein-Barr virus, and respiratory syncytial viruses. Cancer, where said cancer may be selected from the group including prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, skin cancer, stomach cancer, and liver cancer.

[0057] • Inflammation, and • Sepsis, septic shock, and SIRS.

[0058] In light of the above, and in relation to the content of the present invention, a detailed list of diseases and their relationship to oxidative stress is shown in Table 1 below:

[0059] [Table 1-1] [Table 1-2]

[0060] More details are as follows: Oxidative stress is suspected to be important in neurological and neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, depression, multiple sclerosis, late-onset motor disorder (TD), epilepsy, and acute central nervous system disorders, such as spinal cord injury and / or brain injury. The human brain is susceptible to oxidative stress due to several factors, including (i) catecholamine metabolism; (ii) reduced antioxidants; (iii) the presence of transition metals; (iv) the appearance of brain injury; and also (v) the brain being an organ that relatively requires more oxygen and (vi) expressing low levels of antioxidant enzymes, which contribute to the formation of ROS. One of the structures most affected as a result of redox imbalance in the brain is the lipid membrane (Rao and Balachandran 2002. Nutritional Neuroscience 5: 291-309). The common thread in these diseases is oxidative damage to neurons, which likely contributes to the dysfunction or death of nerve cells that influences the disease pathogenesis.

[0061] Alzheimer's disease (AD), the most prevalent neurodegenerative disorder, is characterized by a progressive decline in behavior, cognition, and function, which significantly impairs daily living activities. Numerous studies and clinical trials have demonstrated that oxidative damage plays a major role in neuronal loss and progression to dementia in Alzheimer's disease. The production of β-amyloid (β), a toxic peptide often found in the brains of Alzheimer's patients, is caused by oxidative stress and plays a crucial role in the neurodegenerative process. In addition, the Aβ protein directly initiates free radical generation through the activation of NADPH oxidase. Furthermore, inflammation is involved in increased cytokine, ROS levels, and cytotoxic expression, consequently exacerbating the progression of AD. For reviews, see Liu et al. 2017. Oxidative Medicine and Cellular Longevity 2525967; Manoharan et al. 2016. Oxid Med Cell Longev 8590578.

[0062] Huntington's disease (HD) is a progressive neurodegenerative disease involving the unstable elongation of cytosine, adenine, and guanine (CAG) repeats within the HTT gene. Elongation of the CAG repeat within exon 1 of the HTT gene leads to mutations that result in the extension of the polyglutamine region, and ultimately to HTT protein products that are susceptible to aggregation. mHTT aggregates accumulate throughout the brains of affected individuals, potentially interfering with protein quality control and transcriptional processes. These alterations are potentially involved in the abnormal motor and cognitive problems of HD. While oxidative damage is not frequently reported in the early stages of HD, it is considered one of the major mechanisms of HD as it progresses. High oxidative stress plays a crucial role in the later stages of HD pathogenesis. Electron transport chain dysfunction and mitochondrial dysfunction are key mechanisms involved in the ROS-mediated causative theory of HD. Dysfunction in oxidative phosphorylation components has been recorded in the brain tissue of HD patients. HD patients showed elevated levels of oxidative stress markers, accompanied by reduced antioxidant status, compared to healthy subjects. For review articles, see Liu et al. 2017. Oxidative Medicine and Cellular Longevity 2525967; Manoharan et al. 2016. Oxid Med Cell Longev 8590578.

[0063] Parkinson's disease (PD) is the most common neurodegenerative disease in older adults, characterized by progressive loss of muscle control. PD is most pronounced in the 60s, and men are 1.5 to 2 times more likely to develop the disease than women. Head injury, illness, or exposure to environmental hazards have been identified as risk factors. This neurodegenerative disorder is characterized by tremors, rigidity, bradykinesia, and balance disorders. PD also causes cognitive impairment, psychiatric disorders, autonomic dysfunction, and sensory disturbances. The pathology of PD is characterized by a slow and selective loss of dopaminergic neurons in the substantia nigra pars compacta. An imbalance in dopamine metabolism due to oxidative stress is recognized as a contributing factor to this disease. Key pathological findings include the presence of Levy bodies in the substantia nigra and loss of neurons in part of its anterior layer. Several studies have reported defective respiratory chains and somatic mutations in mitochondrial DNA in the brains of patients with PD, suggesting a large role of oxidative metabolism in PD. High dopamine metabolism in the brains of patients with Parkinson's disease (PD) may contribute to the accumulation of toxic radicals in the brain, such as hydroxyl radicals. Accumulation of iron in redox-active forms in neurons plays a crucial role in the pathogenesis of this disease. Iron accumulation has been reported in the substantia nigra of patients diagnosed with PD, suggesting a key role of iron-induced lipid peroxidation in the pathogenesis of PD. A twofold increase in protein oxidation was shown in the substantia nigra of PD patients compared to healthy subjects. Accumulation of hydroxyl radicals due to reduced glutathione content in the brain has been reported in PD patients. Reduced activity of antioxidant enzymes and non-enzymatic antioxidants may be involved in the progression of PD. For reviews, see Liu et al. 2017. Oxidative Medicine and Cellular Longevity 2525967; Manoharan et al. 2016. Oxid Med Cell Longev 8590578.

[0064] Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of motor neurons in the anterior horn of the spinal cord. It is classified as either familial or idiopathic depending on the presence or absence of a clearly defined, hereditary genetic component. Idiopathic ALS (sALS) typically appears between the ages of 50 and 60. The onset of sALS is unknown, and therefore, the identification of causative genes and environmental factors remains elusive. In familial ALS, approximately 20% of cases arise from mutations in SOD1. The functions of SOD1 are diverse, including the elimination of excessive peroxide radicals, regulation of cellular respiration, energy metabolism, and post-translational modification. SOD dysfunction leads to a loss of antioxidant capacity. In addition, high levels of ROS and ROS-related damage have been widely reported in ALS. Markers of high ROS damage have also been found in the bodily fluids and postmortem tissues of patients with idiopathic ALS. For a review article, see Liu et al. 2017. Oxidative Medicine and Cellular Longevity 2525967.

[0065] Multiple sclerosis (MS) is a multifactorial disease of the central nervous system (CNS) in which both inflammatory and neurodegenerative processes occur simultaneously. During the disease, inflammation decreases, but CNS degeneration progresses. The inflammatory component in MS is important not only due to axonal and neuronal loss, but also because it initiates the degenerative cascade in the early stages of MS. Induction of microglia activation and mitochondrial dysfunction play specific roles in the inflammatory process. Microglia activated by T lymphocytes release proteolytic enzymes, cytokines, oxidation products, and free radicals. It is also important that mitochondrial dysfunction leads to high production of reactive oxygen species (ROS), which are harmful to neurons and glial cells. Oxidative stress damages mitochondria, which disrupts the transport of adenosine triphosphate along axons, and subsequently leads to neurodegeneration. Oxidative stress is associated with dysregulation of bioenergy in axons, cytokine-induced synaptic hyperexcitability, abnormal iron accumulation, and oxidation / antioxidant balance. Markers of oxidative stress assessed in serum, erythrocyte CSF, saliva, and urine may have diagnostic properties, whereas antioxidants may have future clinical applications. For a review, see Adamczyk and Adamczyk-Sowa 2016. Oxidative Medicine and Cellular Longevity 1973-834.

[0066] Oxidative stress is associated with metabolic syndrome and its individual component pathologies, such as obesity, insulin resistance, dyslipidemia, impaired glucose tolerance, and hypertension. Metabolic syndrome is defined by the World Health Organization criteria (Alberti and Zimmet 1998. Diabet Med. 15:539-553; World Health Organization. 1999. Definition, diagnosis and classification of diabetes mellitus and its complications: report of a WHO Consultation. Part 1: diagnosis and classification of diabetes mellitus. Geneva, Switzerland: World Health). (Organization), and the following: (1) Type II diabetes mellitus; (2) Abnormal fasting blood glucose levels; (3) Impaired glucose tolerance; or (4) One of the following conditions of normal fasting glucose levels (<110 mg / dL), with glucose intake lower than the lowest quadrant of the background population in a study under hyperinsulemic conditions, and the following: (1) Blood pressure: ≥140 / 90 mmHg; (2) Dyslipidemia: Triglycerides (TG): ≥1.695 mmol / L and high-density lipoprotein cholesterol (HDL-C) ≤0.9 mmol / L (male), ≤1.0 mmol / L (female); (3) Central obesity: Waist-to-hip ratio >0.90 (male); >0.85 (female), or Body Mass Index >30 kg / m² 2 (4) Microalbuminuria: Requires the presence of insulin resistance as determined by two of the following: urinary albumin excretion ratio ≥ 20 μg / min or albumin:creatinine ratio ≥ 30 mg / g.

[0067] Enhanced oxidative stress appears to play a central role in metabolic syndrome and its elemental pathology, and may be a unifying factor in the progression of this disease. Furthermore, oxidative stress has been identified as a major mechanism of microvascular and macrovascular complications in metabolic syndrome. For a review, see Hutcheson and Rocic 2012. Exp Diabetes Res. 2012:271028.

[0068] Much of the evidence demonstrates that mitochondrial ROS (dominant superoxide anion) overproduction is associated with diabetes and diabetic complications. It has been suggested that glucose may directly stimulate ROS overproduction, and it has also been shown that high glucose (HG) activates various mitochondrial enzyme cascades, including NADPH oxidase activation, NO synthase uncoupling, and xanthine oxidase stimulation. Glycobinding proteins may also be promoters of ROS formation, suggesting that various factors may be involved in ROS overproduction and oxidative stress in diabetes. For a review, see Pitocco et al. 2013. Int. J. Mol. Sci. 2013, 14, 21525-21550.

[0069] Furthermore, oxidative stress plays a crucial role in the pathogenesis and development of cardiovascular diseases, including hypertension, dyslipidemia, atherosclerosis, myocardial infarction, angina pectoris, and heart failure (Elahi et al. 2009. Oxidative Medicine and Cellular Longevity 2(5): 259-269). One key concept regarding the free radical-mediated pathogenesis of cardiovascular diseases is endothelial dysfunction, which impairs the regulation of the vascular wall microenvironment. For example, ROS activity in the vascular wall is thought to contribute to the formation of oxidized LDL, a major cause of atherosclerosis. Oxidative stress also plays a role in the ischemia cascade due to oxygen-reperfusion injury following hypoxia. This cascade includes both stroke (Chen et al. 2011. Antioxidants and Redox Signaling 14(8): 1505-1517) and myocardial infarction (MI) (Hori and Nishida et al. 2009. Cardiovascular Research 81: 457-464). During cerebral ischemia / reperfusion, several detrimental processes occur, including overproduction of oxidants, inactivation of the detoxification system, and consumption of antioxidants. These changes lead to the disruption of the normal antioxidant defense capacity of brain tissue (Chen et al. 2011. Antioxidants and Redox Signaling 14(8): 1505-1517). For further review, see Elahi et al. 2009. Oxidative Medicine and Cellular Longevity 2(5): 259-269.

[0070] Numerous experimental and clinical trials have demonstrated enhanced production of regenerative acids (ROS) in heart failure (HF) and shown that oxidative stress is involved in the pathophysiology of HF in both cardiac and skeletal muscle. The high metabolic activity of mitochondrially rich cardiomyocytes made these findings intuitively apparent. Oxidative stress clearly activates processes in isolated cardiac cells, such as changes in gene expression and cell death, which are now accepted elements of cardiomyocyte regeneration and heart failure. Furthermore, many studies have been conducted in animal models demonstrating the therapeutic effects of antioxidants on the progression of heart failure. For a review, see Tsutsui et al. 2011. Am J Physiol Heart Circ Physiol 301: H2181-H2190.

[0071] Excessive oxidative stress is thought to play a crucial role in the pathogenesis of autoimmune diseases. Numerous studies have shown that T and B lymphocytes are involved in the pathogenesis of autoimmune diseases through the production of autoantibodies and ROS under environmental and genetic influences. Oxidative stress has been associated with autoimmune diseases in which it plays a significant role in the disease course (rheumatoid arthritis, systemic lupus erythematosus, psoriasis, and childhood steatorrhea). Oxidative stress is enhanced in systemic lupus erythematosus (SLE), and it contributes to immune system dysregulation, abnormal activation and processing of cell death signals, autoantibody production mechanisms, and fatal complications. Mitochondrial dysfunction in T cells promotes the release of highly diffusible inflammatory lipid hydroperoxides, which then spread oxidative stress through other intracellular organelles and the bloodstream. Oxidative modification of autoantigens initiates autoimmunity, and the degree of such modification of serum proteins shows a striking correlation with disease activity and organ damage in SLE (Perl 2013. Nat Rev Rheumatol. 9(11): 674-686). Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints and surrounding tissues with macrophage infiltration and activated T cells. The pathogenesis of this disease is due to the production of ROS and RNS at the site of inflammation. RA is one of the conditions that induces oxidative stress. A five-fold increase in mitochondrial ROS production in whole blood and in monocytes in RA patients compared to healthy subjects suggests that oxidative stress is a prominent feature of RA pathogenicity. Free radicals are indirectly involved in joint damage because they also play an important role as secondary mediators in the inflammatory and immunological cellular responses of RA. Increased exposure of T cells to oxidative stress can lead to unresponsiveness to several stimuli, including those related to growth and death, and can perpetuate abnormal immune responses. Meanwhile, free radicals directly degrade articular cartilage, invade its proteoglycans, and inhibit their synthesis (see Quinonez-Flores et al. 2016. Biomed Res Int. 2016:6097417 for a review).

[0072] Currently, there is substantial evidence that inflammatory lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD), are characterized by systemic and localized chronic inflammation and oxidative stress. A key origin of enhanced airway oxidative stress is the recruitment of inflammatory cells into the airways after exposure to trigger factors. These activated cells can produce anionic superoxide through the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase pathway. Mitochondrial dysfunction in airway epithelial cells, resulting from mechanical and environmental stimuli, can also contribute to the formation of anionic superoxide and airway oxidative stress. Subjects with asthma have greater systemic and airway enhanced oxidative stress, which is associated with worse asthma severity. In subjects with COPD, asthma is accompanied by enhanced airway oxidative and nitrosation stress. Patients with COPD have a higher degree of immunohistochemical staining for nitrotyrosine in airway epithelium, and significant imbalances in inflammatory cells in sputum A in airway thiol metabolism have also been described in patients with COPD, which may be related to downstream redox transcriptional changes and pro-inflammatory events. For a review, see Holguin 2013. Ann Am Thorac Soc 10 Supplement: S150-S157.

[0073] Inflammatory bowel disease (IBD) is an incurable chronic inflammatory bowel disorder of the gastrointestinal (GI) tract that dramatically impacts quality of life. Crohn's disease (CD) and ulcerative colitis (UC) are the main types of IBD. CD can occur in any region of the GI tract and affects the ileum and colon in a discontinuous pattern through transmural inflammation, whereas UC continuously affects only the colon and rectum and is limited to the mucus. Accumulated data from both experimental models and clinical trials indicate that oxidative stress signaling is involved in the development of inflammatory bowel disease through multiple levels of function. Oxidative stress leads to damage to the mucosal layer of the GI tract, bacterial invasion sequentially stimulates an immune response, and then initiates inflammatory bowel disease. During inflammation, immune cells, such as leukocytes, monocytes, and neutrophils, increase ROS production during respiration, prostaglandin, and leukotriene metabolism, leading to further tissue damage. For a review, see Tian et al. 2017. Oxid Med Cell Longev 4535194.

[0074] Celiac disease (CD) is an immunotype-mediated chronic inflammatory disorder of the upper small intestine caused by gluten and related prolamins in genetically susceptible individuals. In other autoimmune conditions, environmental, genetic, and immunological factors may be involved in the pathogenesis of CD. In addition, oxidative stress is also involved in the pathogenesis of CD. For example, xanthine oxidase activation is one mechanism of ROS overproduction in the small intestinal mucosa of celiac disease patients. For a review, see Patlevic et al. 2016. Integr Med Res 5: 250-258.

[0075] Gastritis is defined as inflammation of the inner lining of the gastric mucosa and occurs under several conditions, including H. pylori infection, NSAID use, alcohol consumption, and stress. Peptic ulcer disease (PUD) occurs in the proximal GI duct and is often associated with chronic gastritis. Gastric and duodenal ulcers are most commonly referred to as chronic PUD. Gastritis and peptic ulcers are caused by multiple factors, both endogenous and exogenous, and free radicals are closely involved in both conditions. Several factors are involved in the accumulation of ROS in the stomach. Reduced antioxidant enzyme SOD levels and antioxidant vitamin intake are involved in the accumulation of ROS associated with gastric and duodenal inflammatory disease. Ethanol-induced gastritis is associated with enhanced superoxide production. Phagocytes are the primary source of ROS in chronic inflammation, such as that observed in H. pylori-induced gastritis and IBD. A remarkably large number of neutrophils and / or macrophages infiltrate the gastric mucosa during inflammation and produce large amounts of ROS. For a review, see Bhattacharyya et al. 2014. Physiol Rev 94: 329-354.

[0076] Oxidative stress also plays a significant role in liver diseases such as viral hepatitis (types A, B, and C) and cirrhosis. The association of hepatitis C with severe oxidative stress has been clearly established. This was revealed by evaluating the levels of total oxidant / antioxidant status or individual antioxidants in liver tissue and serum / plasma samples from chronic hepatitis C patients using various techniques, including direct measurement of ROS, DNA quantification, and lipid and protein oxidation products. Screening of liver biopsies from chronic hepatitis C virus carriers revealed significant elevations in the levels of oxygen radicals and stress markers malondialdehyde (MDA) and 4-hydroxynonenal-(HNE)-, as well as other protein adducts. In addition, serum / plasma from such patients showed: It is characterized by high levels of a broad array of oxidative stress markers, such as MDA, lipid peroxides, protein carbonyl content, or thioredoxin (see Ivanov et al. 2017. Oncotarget, 2017, Vol. 8, (No. 3), pp: 3895-3932 for a review).

[0077] Patients with chronic hepatitis B show clear signs of oxidative stress. Levels of oxygen radicals in liver specimens from these patients exceed those of healthy individuals. Patients with hepatitis B show signs of oxidative stress not only in the liver but also in plasma / serum. Chronic hepatitis B is accompanied by an increase in the total oxidant state and a corresponding decrease in the total antioxidant state. The plasma / serum of these patients is also characterized by high levels of ROS, including H2O2, and oxidation products of lipids and proteins. Oxidative stress is not only a prominent feature of chronic hepatitis B virus infection and advanced liver disease; it is also observed in acute and latent hepatitis B, as well as asymptomatic HBV infection. Latent hepatitis B infection is characterized by enhanced levels of ROS in lymphocytes and resulting DNA damage. However, the most dramatic changes were described in hepatitis B patients with cirrhosis and acute chronic hepatitis B liver failure (see Ivanov et al. 2017. Oncotarget, 2017, Vol. 8, (No. 3), pp: 3895-3932 for a review). Cirrhosis is a complication of many forms of chronic hepatitis, a late stage of fibrosis, where regenerative nodule formation is surrounded by fibrous bands of the liver. In cirrhosis, oxidative stress is primarily caused by the overproduction of reactive oxygen species, which is the main determinant of endothelial dysfunction due to an imbalance between oxidants and antioxidant enzymes. Enhanced superoxide formation in the presence of equimolar NO leads to the formation of potent ROS and reactive nitrogen species. See Vairappan 2015. World J Hepatol 27; 7(3): 443-459 for a review.

[0078] Hepatotoxicity implies chemically induced liver damage. Drug-induced liver injury is a cause of acute and chronic liver disease. Drug-induced liver injury is involved in 5% of all hospitalizations and 50% of all acute liver failures. The liver is the most frequently targeted organ in relation to drug toxicity. The production of radical species, specifically ROS and RNS, has been shown to be an early event of drug-induced hepatotoxicity and an indicator of the possibility of hepatotoxicity. For example, we have found that many drugs, such as anti-inflammatory drugs, analgesics, anticancer drugs, and antidepressants, can cause oxidative stress, including increased cytooxidants and lipid peroxides, and decreased antioxidants in the liver (Li et al. 2015. Int. J. Mol. Sci. 16: 26087-26124). More than 900 drugs have been linked to the pathogenesis of liver damage, and these are incorporated herein by reference (https: / / livertox.nlm.nih.gov / ; Bjornsson 2016. Int. J. Mol. Sci. 17: 224).

[0079] In the pathogenesis of alcoholic liver disease (ALD), a direct causal relationship with ethanol metabolism also appears to be associated with ROS production, mitochondrial damage, and fatty degeneration, which are common characteristics of acute and chronic alcohol exposure (Li et al. 2015. Int. J. Mol. Sci. 16: 26087-26124).

[0080] Both acute kidney injury (AKI) and chronic kidney disease (CKD), leading to reduced kidney function, are interdependent risk factors for high mortality. End-stage renal disease (ESRD) is an inevitable outcome when left untreated for extended periods. Acute and chronic kidney disease are sometimes caused by imbalances between molecular mechanisms controlling oxidative stress, inflammation, autophagy, and cell death. Numerous studies have suggested that oxidative stress and its systemic effects play a crucial role in the development of AKI. Recent studies have demonstrated enhanced urinary thioredoxin 1 (TRX1) expression as a biomarker of oxidative stress related to kidney injury. Diabetic nephropathy (DN) is a devastating complication of diabetes and a major cause of CKD. In the kidney, the mitochondrial respiratory chain and NADPH oxidase (NOX) are major common sources of regenerative stress (ROS), and NOX has been shown to generate oxidative stress by promoting vascular dysfunction and fibrosis in CKD. For a review, see Sureshbabu et al. 2015. Redox Biology 4: 208-214.

[0081] Drug-induced nephropathy is a serious problem in clinical practice, accounting for 19%–26% of acute kidney injury (AKI) cases among hospitalized patients. Furthermore, AKI creates severe conditions associated with a high probability of developing progressive chronic kidney disease or end-stage renal failure, leading to a high mortality rate. It has been found that most drugs cause nephropathy, exerting toxic effects through one or more common pathogenesis mechanisms. These include altered glomerular hemodynamics, tubulocytotoxicity, inflammation, crystal nephropathy, rhabdomyolysis, and thrombotic microangiopathy. Examples of AKI include acute renal tubular necrosis (ATN) and acute interstitial nephritis (AIN). The basic mechanism of ATN is oxidative stress. Proximal tubular toxicity develops as a result of direct toxic effects on the kidney, such as mitochondrial dysfunction, lysosomal hydrolase inhibition, phospholipid damage, and high intracellular calcium concentrations, leading to the formation of reactive oxygen species (ROS) accompanied by harmful oxidative stress (Hosohata 2016. Int. J. Mol. Sci. 17: 1826).

[0082] Drug therapies that are potentially harmful to the kidneys (nephrotoxic) include antibiotics such as antimicrobial agents (e.g., streptomycin, gentamicin), antiviral agents (e.g., acyclovir, foscarnet), or antifungal agents (e.g., amphoteserin B), analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., ibuprofen, naproxen), diuretics, proton pump inhibitors, chemotherapeutic agents (e.g., cisplatin), contrast agents, cardiovascular agents such as ACE inhibitors or statins, antidepressants, immunosuppressants (e.g., cyclosporine A), and antihistamines (see Naughton 2008. Am Fam Physician. 2008;78 (6):743-750, Table 1; Hosohata 2016. Int. J. Mol. Sci. 17: 1826 for references).

[0083] In carcinogenesis, highly reactive hydroxyl radicals cause oxidative DNA damage, while peroxynitrites cause both oxidative damage and nitration of DNA bases. The majority of mutations caused by ROS appear to be involved in guanine modification, leading to guanine (G) → thymine (T) base transposition. If this is associated with a key gene, such as an oncogene or tumor suppressor gene, cancer initiation or progression can occur. Generally, high metabolism in cancer cells is associated with increased ROS; however, such levels are less harmful in cancer cells than they are in normal cells. For example, ROS levels increase to a modest degree, but oncogenic cells induce a new redox balance, leading to cell adaptation and proliferation. ROS produced by the mitochondrial respiratory chain and by cytoplasmic Nox enzymes are particularly important. In fact, Nox proteins are now considered oncogenic proteins, and mitochondrial dysfunction is associated with tumorigenesis. Oxidative stress is involved in all stages of carcinogenesis, and there is a dose-dependent relationship between the level of persistent or chronic oxidative stress and tumor stage. During the carcinogenic process, normal cells are transformed into abnormal cells due to numerous structural changes and mutations in gene expression. In the subject literature, carcinogenesis is described by three main stages: initiation, promotion, and progression. All of these stages were hypothesized to be linked to the involvement of ROS and RNS. ROS play a crucial role in pathophysiological conditions involved in neoangiogenesis. For example, ROS-producing enzymes, such as NADPH oxidase (e.g., Nox: Nox1-5), activate redox signaling pathways that ultimately lead to angiogenesis. For reviews, see Kruk and Aboul-Einein 2017. Mini-Reviews in Medicinal Chemistry 17: 904-919; Sosa et al. 2013. Ageing Research Reviews 12: 376-390.

[0084] Prostate cancer is the most frequently diagnosed non-cutaneous malignant tumor in men. It is a multifocal, field-type disease, and it forms solid tumors of glandular origin. Prostate cancer is primarily age-related, with most cases occurring in men over 55 years of age. In the last decade, the association between prostate cancer risk and oxidative stress has been recognized, and epidemiological, experimental, and clinical trials have clearly demonstrated the role of oxidative stress in the development and progression of this disease, generally associated with an antioxidant-prosciutative balance shift towards enhanced oxidative stress. Environmental factors such as diet, inflammation, and changes in cellular function related to NAD(P)H oxidase, androgen signaling, mitochondrial DNA mutations, aging, and redox imbalances are putative mechanisms involved in enhancing ROS production. This enhanced ROS can further stimulate cell proliferation, induce somatic DNA mutations, thus promoting genetic instability, cell cycle arrest, and aging, and in cancer cells, it can lead to enhanced angiogenesis and motility. In particular, enhanced Nox expression, which drives ROS production in prostate cancer, may lead to the development of malignant phenotypes by modulating various signaling cascades. For reviews, see Khandrika et al. 2009. Cancer Lett. 282(2): 125-136; Kruk and Aboul-Einein 2017. Mini-Reviews in Medicinal Chemistry 17: 904-919; Sosa et al. 2013. Ageing Research Reviews 12: 376-390.

[0085] Overproduction of ROS in breast cancer cells includes: strong expression of thymine phosphorylase, leading to the degradation of thymidine into thymine and 2-deoxy-D-ribose phosphate; and oxidation of 17-estradiol panoxyl radicals to lactoperoxidase, which is involved in estrogen metabolism and inflammation. In addition to increased free radicals, changes in antioxidants are associated with breast cancer risk; for example, SOD and GPX levels have been found to be higher in the blood of breast cancer patients compared to healthy women, as a response to increased superoxide and hydrogen peroxide production. In breast cancer, the tumor suppressor gene BRCA1 is mutated in 40-50% of hereditary breast cancers and is absent or low-expression in 30-40% of sporadic breast cancers. BRCA1 is a caretaker gene involved in DNA repairing and can upregulate several genes involved in antioxidant responses by controlling the activity of transcription factors Nrf2 and NfκB. Aside from its inhibitory effect on ROS production, BRCA1 also reduces the level of protein nitration due to RNS accumulation in cells, which ultimately promotes DNA repair processes that help cope with oxidative stress. For reviews, see Nourazarian et al. 2014 Asian Pac J Cancer Prev, 15 (12): 4745-4751; Kruk and Aboul-Einein 2017. Mini-Reviews in Medicinal Chemistry 17: 904-919; Sosa et al. 2013. Ageing Research Reviews 12: 376-390.

[0086] Human lungs are continuously exposed to air pollutant oxidants, in addition to endogenously generated ROS and RNS (involved in physiological biochemical mechanisms and normal cellular signaling pathways). It is a commonly accepted hypothesis that tobacco smoking is a major risk factor in lung cancer development. Both clinical and experimental studies have consistently demonstrated the crucial role of OS in lung cancer. Evidence is available supporting the importance of oxidative stress, as well as its correlation with increased incidence of malignant respiratory diseases resulting from inflammation, activation of transcription factors, and DNA damage. During inflammation, increased ROS production triggers DNA damage, inhibition of apoptosis, and activation of proto-oncogenes by initiating signaling pathways. Inflammatory cells are particularly effective in producing ROS and other reactive species, thereby enhancing oxidative damage and promoting carcinogenic mechanisms. The ability of inhaled particles or fibrous dust to enter the respiratory system and reach the alveoli to produce ROS and other oxidants or free radicals has been suggested to be a major factor in their pathogenicity. The synergistic mechanisms of inhalable particulate matter (which penetrates deeply into the alveoli) and other components of air pollution (ozone, nitric oxide, soot, heavy metals, PAHs) with cigarette smoke were tested. The porous surface of wind-borne particles provides a favorable environment for catalyzing the enhanced generation of ROS or other damaging oxidizing agents, which are potential initiators of lung carcinogenesis. For reviews, see Valavanidis et al. 2013. Int. J. Environ. Res. Public Health 10: 3886-3907; Kruk and Aboul-Einein 2017. Mini-Reviews in Medicinal Chemistry 17: 904-919; Sosa et al. 2013. Ageing Research Reviews 12: 376-390.

[0087] Colorectal cancer (CRC) is one of the most common cancers globally, with the highest incidence in Western countries. Colorectal cancer originates from epithelial cells, a lineage of cells in the intestinal tract. These cells divide rapidly and therefore have a high metabolic rate, which has been seen as a potential factor that may be involved in high DNA oxidation. Human colorectal tumors (adenomas and carcinomas) are associated with high levels of various markers of oxidative stress, for example, high levels The study revealed the presence of nitric oxide (NO), 8-oxo dG in DNA, lipid peroxides, glutathione peroxidase (GPx), catalase (CAT), and reduced cytosine methylation in DNA. Beyond lipid modifications, enhanced leukocyte activation was also observed in oncogenic tissues, suggesting the potential involvement of inflammatory cells in response to further oxidative stress. Furthermore, levels of the antioxidants vitamins A, C, and E in the plasma of colorectal cancer patients were shown to be statistically lower compared to healthy individuals. For reviews, see Perse 2013. BioMed Research International 725710; Kruk and Aboul-Einein 2017. Mini-Reviews in Medicinal Chemistry 17: 904-919; Sosa et al. 2013. Ageing Research Reviews 12: 376-390.

[0088] In industrialized countries, bladder cancer is the fourth most frequently occurring malignant tumor. Recent studies have shown the involvement of oxidative and nitrosation stress in the formation and development of this disease. Redox disorders are characteristic of both the onset and progression of bladder cancer. Changes in the activity of transcription factors, such as the nuclear factor NF-κB; transcription factors: AP-1, Nrf2, STAT3, and the hypoxia-inducible factor HIF-1α have been observed. In addition, studies have shown the role of oxidative stress in the regulation of the MAPK cascade and its involvement in the carcinogenic composition of the bladder. Nitric oxide also plays an important role in tumor biology. Numerous studies have shown that bladder cancer is characterized by increased production of NO. High levels of nitric oxide are produced during inflammation, in contrast to ROS, whose overproduction results from exposure to carcinogenic ex vivo substances. Sustained iNOS activity plays a crucial role in carcinogenesis associated with inflammatory responses, which is therefore also characteristic of bladder cancer. For a review, see Sawicka et al. 2015. Poststepy High Med Dosw 69: 744-752.

[0089] Ovarian cancer is the fifth leading cause of cancer death; the most common cause of death from gynecological malignancies; and the second most commonly diagnosed gynecological malignancy. The vast majority of ovarian cancers originate from the surface epithelium of the ovary. Metastatic cancer is achieved by the isolation of single cells or the diffusion of cells from a primary tumor, followed by implantation on the mesothelial wall of the peritoneum. Endometrial cancer, ovarian cancer, and cervical cancer constitute a major problem in oncology due to their diagnosis at advanced stages. Laboratory studies have shown that oxidative stress plays a causal role in the carcinogenesis of two subtypes of ovarian cancer: clear cell carcinoma and endometriotic carcinoma. Evidence suggests that ovarian cancer patients have low levels of This suggests the presence of circulating antioxidants and high levels of oxidative stress. Epithelial ovarian cancer (EOC) tissues and cells have been reported to exhibit a prooxidative state characterized by increased expression of major prooxidative enzymes and decreased expression of antioxidant enzymes. Specifically, EOC cells and tissues showed increased expression of iNOS, MPO, and NAD(P)H oxidase, as well as elevated NO levels. Furthermore, EOC cells exhibited low levels of apoptosis.

[0090] Endometrial cancer has been reported to be associated with endometriotic disease, and high levels of free iron hemosiderin or heme in endometriotic cysts are considered to be major factors involved in the development of oxidative stress and chronic inflammation.

[0091] Cervical cancer is the second most common cancer among women worldwide, and is the subject of extensive research. Several experimental studies suggesting the involvement of oxidative stress in the cervix have shown that the transcription factors AP-1 and NFκB may be inhibited, or that antioxidants may alter the redox balance in cervical cancer cells, or that apoptosis may be induced. For reviews, see Saed et al. 2017. Gynecologic Oncology 145: 595-602; Kruk and Aboul-Einein 2017. Mini-Reviews in Medicinal Chemistry 17: 904-919; Sosa et al. 2013. Ageing Research Reviews 12: 376-390.

[0092] Oxidative damage caused by OS is also associated with leukemia, and low levels of antioxidants, oxidatively modified DNA, and lipids, caused by high ROS production, were observed in the serum of patients with chronic lymphocytic leukemia. Furthermore, it was found that in chronic leukemia cells, intracellular OS can be adapted by upregulating stress-responsive hemoxygenase-1, confirming the involvement of ROS in the pathogenesis of leukemia. A decrease in GSH in lymphocytes of chronic lymphocytic leukemia was also demonstrated in patients with leukemia B. For a review, see Kruk and Aboul-Einein 2017. Mini-Reviews in Medicinal Chemistry 17: 904-919.

[0093] Gastric cancer (GC) is one of the most common diseases in the human population. It is the fourth most common cancer worldwide and the second most common cause of cancer death. The main risk factor for gastric cancer is chronic inflammation caused by bacterial growth. For example, infection by Helicobacter pylori, which enhances the production of reactive oxygen species and nitrogen species in the human stomach, is considered important in the development of gastric cancer. Protein oxidation products were shown to be significantly higher in GC patients. Furthermore, the antioxidant capacity of SOD and catalase was found to be lower in gastric cancer tissue compared to control healthy tissue. For reviews, see Kruk and Aboul-Einein 2017. Mini-Reviews in Medicinal Chemistry 17: 904-919; Ma et al. 2013. Oxidative Medicine and Cellular Longevity 543760.

[0094] Many factors contribute to liver carcinogenesis, including hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, alcohol abuse, non-alcoholic fatty liver disease (NAFLD), aflatoxin B1, obesity, diabetes, dietary habits, and iron accumulation. Generally, oxidative stress can also be triggered by any risk factor or inflammatory signaling and affects multiple cells in the liver. Liver injury can be either an acute or chronic inflammatory process. In a localized inflammatory environment, many types of liver cells are activated, such as hepatic sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs), dendritic cells (DCs), and Kupffer cells (KCs). These cells produce many types of immunotransmitters, cytokines, and chemokines, which can also lead to the production of oxidative stress. In recent years, there has been increased research on the correlation between oxidative stress and hepatic stellate cells. These cells play a central role in the process of liver fibrosis and can induce collagen production after the body is activated by free radicals, which has been shown to be caused by ROS and superoxide anions, and to further induce damage to liver cells.

[0095] It is known that over 80% of HCC cases are associated with chronic HBV or HCV infection. Typically, HBV- and HCV-associated chronic inflammation and fibrosis of the liver are caused by oxidative stress, which contributes to the pathogenesis of hepatocellular carcinogenesis. HBV infection leads to the activation of macrophages that produce various pro-inflammatory cytokines, such as IL-1, IL-6, CXCL-8, and TNF-α. Such persistent abnormal cytokine production and the resulting ROS production influence hepatocellular carcinogenesis. HCV-induced oxidative stress contributes to the development of hepatocellular carcinoma (HCC). Furthermore, levels of oxidative stress markers in chronic hepatitis C patients clearly correlate with the probability of developing HCC and may serve as a prognostic marker for HCC recurrence in chronic hepatitis C patients who have undergone liver transplantation. Carcinogenesis is regulated by multiple ROS-mediated processes. For a review, see Wang et al. 2016. Oxidative Medicine and Cellular Longevity 7891574.

[0096] The skin is the body's primary environmental contact surface and is therefore prone to accidental or occupational exposure to numerous chemical mutagenic and carcinogenic substances. Skin cancer is a major and growing public health problem. It accounts for over 40% of all new cancers diagnosed. 80% of skin cancers originate from basal cell carcinoma (BCC); another 16% from squamous cell carcinoma (SCC); and 4% from melanoma. Much evidence has shown that UV radiation causes DNA damage, resulting in DNA crosslinking. A key process in skin cancer is the production of hydrogen peroxide due to a decrease in catalase activity in melanocytes. Furthermore, there is evidence suggesting that mutations in several genes involved in melanoma result from oxidative stress. For review articles, see Kruk and Aboul-Einein 2017. Mini-Reviews in Medicinal Chemistry 17: 904-919; Narendhirakannan. 2013. Ind J Clin Biochem 28(2):110-115.

[0097] Reactive species generated during infection can have significant consequences for the disease once they are released to any degree. Oxidative stress can initiate harmful effects in various organs. The development of oxidative stress can be accelerated in the process of hypoxia. Hypoxia is a known complication of infection. Hypoxia is not limited to any particular disease. Influenza, viral hepatitis, and tuberculosis are all examples of infections in which hypoxia occurs. Direct reactive oxygen generation can be initiated by metals. For example, iron, copper, and cadmium can catalyze the development of oxidative stress through the Fenton reaction, in which hydrogen peroxide is converted into hydroxyl radicals and hydroxide anions. Heavy metals are involved in physiological processes related to infections, as in other medical conditions. As mentioned, livers damaged by viral hepatitis are more susceptible to heavy metal damage due to incomplete elimination processes. Clinical trials in patients with hepatitis A, B, C, D, and E have demonstrated that copper and iron accumulation contributes to oxidative stress and oxidative damage to the liver tissue of these patients. Like hepatitis, AIDS involves an imbalance in oxidative homeostasis. High markers of oxidative damage to targets in the body and accumulation of reactive oxygen species are common in HIV-infected individuals. Blood antioxidants are reduced over long periods in infected individuals. For a review, see Pohanka 2013. Folia Microbiol 58:503-513.

[0098] Sepsis and septic shock remain the leading cause of death in adult intensive care units. It is widely recognized that sepsis and septic shock are primarily caused by Gram-negative bacteria and their endotoxins. Endotoxins, or lipopolysaccharides (LPS), play a crucial role as triggers for host responses and inflammatory processes caused by Gram-negative bacterial infections. Production of oxygen radicals by neutrophils and macrophages, such as reactive oxygen species (ROS), NO (nitric oxide), and peroxynitrite, promotes the gene expression of pro-inflammatory mediators. ROS and RNS are antimicrobial agents produced by these leukocytes that can directly destroy microbial pathogens. During sepsis, the overproduction of ROS and RNS threatens the preservation of various biomolecules, including proteins, lipids, and lipoproteins, protein oxidation, and DNA, leading to tissue damage through cell membrane peroxidation, protein oxidation, and DNA strand breakage. These mechanisms contribute to multi-organ failure during sepsis, resulting in myocardial depression, hepatic dysfunction, endothelial dysfunction, and decreased vascular catecholamine response. As a major source of ROS production, mitochondria are particularly prone to ROS-mediated damage. Such damage can induce mitochondrial permeability transitions caused by the opening of unspecified high-conductance permeability transition pores in the inner mitochondrial membrane. ROS itself also provides signals that lead to autophagy, apoptosis, and necrosis. Excessive ROS production and a decrease in adenosine triphosphate due to uncoupling of oxidative phosphorylation promote necrotizing cell death. Release of cytochrome c after mitochondrial expansion activates caspases and initiates apoptotic cell death. For a review, see Kaymak et al. 2011. FABAD J. Pharm. Sci. 36: 41-47.

[0099] Therefore, in another specific embodiment of the present invention, the DPP3 binder disclosed herein, specifically the anti-DPP3 antibody provided herein, and / or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to the epitope of SEQ ID NO: 2 (contained in the DPP3 protein or its functional derivative), is provided for use in the prevention or treatment of a patient's disease or acute condition (due to the disease or acute condition being related to oxidative stress), the disease being selected from the group including the previously described neurodegenerative diseases, metabolic syndrome, cardiovascular diseases, autoimmune diseases, inflammatory lung diseases, kidney diseases, liver diseases, gastrointestinal diseases, viral infections, cancer, inflammation, sepsis, septic shock, and SIRS.

[0100] In another specific embodiment, the disease is a neurodegenerative disease (e.g., Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS)), metabolic syndrome (including insulin resistance, obesity, hyperglycemia, dyslipidemia, hypertension, and diabetes), cardiovascular disease (e.g., atherosclerosis, hypertension, heart failure, cardiovascular ischemia, cerebral ischemic injury / stroke, and myocardial infarction), autoimmune disease (e.g., rheumatoid arthritis and systemic lupus erythematosus), inflammatory lung disease (e.g., COPD, asthma), renal disease (nephrotoxicity (drug-induced kidney disease), acute kidney injury, etc.) The group includes AKI, chronic kidney disease (CKD), diabetic nephropathy, end-stage renal disease (ESRD), liver disease (e.g., hepatotoxicity, viral hepatitis, cirrhosis), gastrointestinal disease (inflammatory bowel disease, e.g., ulcerative colitis, Crohn's disease; gastritis, pancreatitis, and peptic ulcer), viral infections (e.g., bloodborne hepatitis viruses (types B, C, and D), human immunodeficiency virus (HIV), influenza A, Epstein-Barr virus, respiratory syncytial virus), cancer (e.g., prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, skin cancer, gastric cancer, liver cancer), as well as inflammation, sepsis, septic shock, and SIRS.

[0101] In another specific embodiment of the present invention, the DPP3 binder disclosed herein, specifically the anti-DPP3 antibody provided herein, and / or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to the epitope of SEQ ID NO: 2 (contained in the DPP3 protein or its functional derivative), is provided for use in the prevention or treatment of acute conditions, where the acute conditions may be selected from the group including hepatotoxicity and nephrotoxicity.

[0102] Toxicity resulting from alcohol consumption, chronic exposure to tobacco smoke, and even as a side effect of various drug therapies. In relation to the present invention, oxidative stress and subsequent toxicity are also induced by chronic alcohol consumption, chronic exposure to tobacco smoke, and as side effects of various drug therapies (see Table 2 below, outlined in Deavall et al. 2012). Acetaminophen—a widely used analgesic and antipyretic—is a typical hepatotoxic agent associated with drug-induced liver injury linked to KEAP-Nrf2 signaling (Ma, 2013). Other treatment-induced oxidative stresses include ortiplats and auranofin (Ma, 2013).

[0103] [Table 2]

[0104] Those skilled in the art will know that the presence and degree of oxidative stress can be measured and quantified by suitable biomarker assays known in the art. Examples of these markers are given below, but these should not be construed as limiting the possibilities of oxidative stress measurement according to the present invention:

[0105] Markers of oxidative stress for assessment: serum, red blood cells, CSF, saliva, urine Free radicals can damage biological molecules, including nucleic acids, proteins, and lipids. The products of these reactions can serve as markers of oxidative stress. Serum is the most common material for assessing the components of oxidative stress. It allows for inferences about most enzymes, substrates, and products of redox reactions. These enzymes include xanthine oxidase, NOS, lipoxygenase, cyclooxygenase, myeloperoxidase, prolyl oligopeptidase, nicotinamide adenine dinucleotide phosphatase 1 (NOX1), and NADPH-dependent oxidases. The following are markers of oxidative lipid damage: e.g., the lipid peroxide products of arachidonic acid, malondialdehyde (MDA), isoprostanes (IsoP-prostaglandin-like substances) that constitute the formation of fluorescent lipid peroxide protein covalent adducts and the increase of conjugated dienes, e.g., 8-iso-prostaglandin (F2α-8-iso-PGF2α). Oxidative stress is involved in the oxidation of proteins and glycosidines. The following are results of this reaction: decreased glycophore content, total levels of advanced protein oxidation (AOPP), protein carbonyl and dityrosine levels, N'-formylkynurenine, and serum protein thiol group levels. Other specific markers of protein oxidation are tyrosine (marker of hydroxyl radicals) and 3-nitrotyrosine (marker of RNS). Furthermore, 3-nitrotyrosine is a specific marker of peroxynitrite-induced cell damage. Other indicators in serum include kynurenine, N'-formylkynurenine, thioredoxin, and 8-hydroxy-2'-deoxyguanosine.

[0106] The measurements of each biomarker related to oxidative stress in humans are summarized in Ilaria Marrocco, Fabio Altieri, and Ilaria Peluso; Review Article: Measurement and Clinical Significance of Biomarkers of Oxidative Stress in Humans; Oxidative Medicine and Cellular Longevity Volume 2017 (2017), Article ID 6501046, p. 32.

[0107] Furthermore, as is well known to those skilled in the art, the physiological activity of DPP3 affected by the DPP3 binders disclosed herein can be measured, for example, by inhibition via suitable assays known in the art. Examples are given below, but these should not be construed as limiting the possibilities for measuring the physiological activity of DPP3:

[0108] Method for detecting and measuring DPP3 inhibition Inhibition of DPP3 activity in a binder-based liquid-phase assay can be measured as follows: Blood samples from patients (e.g., serum, heparin-plasma, Li-plasma, citrate-plasma, whole blood) before and after anti-DPP3 antibody therapy are incubated with a specific DPP3 substrate in a liquid-phase assay. Specific liquid-phase DPP3 activity analysis to measure the inhibitory capacity of inhibitory DPP3 antibodies in blood samples includes the following steps: Add 20 μl of blood sample in 200 μl of 50 mM Tris-HCl, pH 7.5, to a black, unbound microtiter plate (96 wells). Those skilled in the art will recognize that buffering conditions, concentration, pH, etc., can be modified.

[0109] Add the fluorescence-generating substrate Arg-Arg-βNA (20 μl, 2 mM). The sample was incubated at 37°C, and the generation of free βNA in a Twinkle LB970 microplate fluorometer (Berthold Technologies GmbH) was observed for 1 hour. The fluorescence of βNA was detected by exciting it at 340 nm and measuring the emission at 410 nm. • Calculate the increased fluorescence slope (RFU / min) for various samples. • Analyze DPP3 activity levels before and after anti-DPP3 antibody therapy.

[0110] In contrast, solid-phase assays are assays in which each binding event occurs in the solid phase. Inhibition of DPP3 activity in solid-phase assays by binders can be measured as follows: Blood samples (e.g., serum, plasma, whole blood) from patients before and after anti-DPP3 antibody therapy are brought into contact with an immobilized capture binder for a solid-phase enzyme capture activity assay (ECA). It is preferable to select the capture binder with the lowest inhibitory activity for the ECA. The capture binder should inhibit DPP3 activity by less than 50%, preferably less than 40%, and preferably less than 30%. Specific liquid-phase DPP3 activity assays for quantifying the inhibitory activity of possible capture binders are described in detail in Example 1.

[0111] ECA to measure the inhibitory capacity of inhibitory DPP3 antibodies in a blood sample includes the following steps: The sample is brought into contact with a capture binder that binds to full-length DPP3, but preferably inhibits DPP3 activity by less than 50%, more preferably less than 40%, and more preferably 30% in a liquid-phase assay. • Separate DPP3 that binds to the capture binder from the bodily fluid sample. • Add the substrate for DPP3 to the separated DPP3. • DPP3 activity is quantified by measuring the conversion of the DPP3 substrate. • Evaluate the signals measured before and after anti-DPP3 antibody therapy, and analyze DPP3 activity levels.

[0112] The quantitative analysis of active DPP3 may be performed as a liquid-phase assay or a solid-phase assay, but the inhibition of DPP3 activity may be quantified by a liquid assay following the procedure described above.

[0113] In yet another embodiment, a capture or binding assay may be performed to detect and / or quantify the protease activity of DPP3. For example, an antibody that reacts with the DPP3 protein but does not interfere with its peptidase activity may be immobilized on a solid phase. The test sample is passed over the immobilized antibody, and if present, DPP3 is bound to the antibody and immobilized itself 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 description, the term “solid phase” may be used to include, but is not limited to, porous or non-porous materials, test tubes, wells, slides, etc., in or on which the assay may be performed.

[0114] Furthermore, as the present invention demonstrates, those skilled in the art will recognize that the binding affinity of the DPP3 binders disclosed herein to DPP3 can be measured by various suitable assays known in the art. Examples are given below, but these should not be construed as limiting the possibilities for measuring the binding affinity of the DPP3 binders disclosed herein to DPP3:

[0115] A method for measuring the binding affinity of the DPP3 binder of the present invention to an epitope based on the sequence of Sequence ID No. 2. The binding affinity of the DPP3 binder to the epitope according to Sequence ID No. 2 of the present invention may be measured according to Example 1, and further as follows: A binding assay may be performed to detect and / or quantify an antibody that binds to an immunosuppressant peptide (i.e., SEQ ID NO: 2). For example, this immunosuppressant peptide may be immobilized on a solid phase. A test sample (e.g., an antibody solution) is passed over the immobilized immunosuppressant peptide, and the bound antibody is detected. For the purposes of this description, the term “solid phase” may be used to include, but is not limited to, any material or container in which the assay may be performed or on which it may be performed, porous materials, non-porous materials, test tubes, wells, slides, etc.

[0116] Example detection method: - Before contacting the solid phase, the antibody is labeled, and then each label (fluorescence, chemiluminescence, enzyme, etc.) is detected. -Sample- A labeled secondary antibody targeting a specific Fc portion of the antibody is used. The antibody-bound solid phase is incubated with the secondary antibody (e.g., anti-human IgG, anti-mouse IgG), and the respective labels (fluorescence, chemiluminescence, enzyme, etc.) are detected. - A labeled antibody (e.g., labeled AK1967) is used as a competing substance for solid-phase bonding. - The decrease in signal strength is used to quantify the binding affinity. [Brief explanation of the drawing]

[0117] [Figure 1] Characterization of AK1967 (A) Binding and dissociation curves of AK1967-DPP3 binding analysis using Octet. A biosensor loaded with AK1967 was immersed in a dilution series of recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM), and binding and dissociation were observed. (B) Western blot of dilutions of blood cell lysates and detection of DPP3 using AK1967 as the primary antibody. (C) Inhibition curve of native DPP3 from hematopoietic cells using the inhibitory antibody AK1967. Inhibition of DPP3 by the specific antibody is concentration-dependent and has an IC50 at ~15 ng / ml when analyzed against 15 ng / ml of DPP3. [Figure 2]Effects of AK1967 on oxidative stress in rats suffering from septic shock-induced heart failure: (A) Experimental design for a heart failure study in rats in septic shock. (B) Fluorescence images of DHE-labeled myocardium from sham surgery, CLP, and CLP AK1967 animals. (C) Quantification of DHE-stained areas and their representation as percentages of the region of interest (%).

[0118] Further explanation of the present invention Binders for circulating, intracellular, and membrane DPP3 In another embodiment of the present invention, the binder and PP3 binder disclosed herein, specifically an anti-DPP3 antibody, an anti-DPP3 antibody fragment, or an anti-DPP3 non-Ig scaffold, can bind to circulating DPP3, thereby against circulating DPP3.

[0119] In yet another embodiment of the present invention, the binders of the present invention disclosed herein, and DPP3 binders, specifically anti-DPP3 antibodies, anti-DPP3 antibody fragments, or anti-DPP3 non-Ig scaffolds, can bind to intracellular DPP3, thereby against intracellular DPP3. In yet another embodiment of the present invention, the binders of the present invention disclosed herein, and DPP3 binders, specifically anti-DPP3 antibodies, anti-DPP3 antibody fragments, or anti-DPP3 non-Ig scaffolds, can bind to membrane DPP3, thereby against membrane DPP3.

[0120] The scope of the present invention also includes a DPP3 binder, specifically an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold, for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress, and thereby the binder, specifically the DPP3 binder which is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold, is directed to and binds to the epitope of SEQ ID NO: 2, wherein the epitope is contained in the circulating DPP3 protein or a functional derivative thereof.

[0121] The scope of the present invention also includes, for use in the prevention or treatment of a patient's disease or acute condition, a DPP3 binder, specifically an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold, wherein the disease or acute condition is related to oxidative stress, and thereby the DPP3 binder, specifically an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold, is directed to and binds to the epitope of SEQ ID NO: 2, wherein the epitope is contained in an intracellular DPP3 protein or a functional derivative thereof.

[0122] The scope of the present invention also includes a DPP3 binder, specifically an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold, for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress, and thereby the DPP3 binder, specifically the anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold, is directed to and binds to the epitope of SEQ ID NO: 2, wherein the epitope is contained in a membrane DPP3 protein or a functional derivative thereof.

[0123] The present invention further relates to a method for regulating and / or preventing or treating oxidative stress in a patient suffering from a chronic or acute disease or acute condition, characterized by administering to the patient a pharmaceutically effective amount of the binder of the present invention, the DPP3 binder of the present invention, specifically an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold. According to the present invention, the patient is a patient who requires regulation and / or prevention of oxidative stress or requires treatment of oxidative stress.

[0124] Pharmaceutical composition Another subject of the present invention is a pharmaceutical composition comprising the binder of the present invention disclosed herein, or the DPP3 binder, specifically an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold, for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress.

[0125] In another embodiment of the present invention, the pharmaceutical composition is a solution, preferably a prepared solution. In another embodiment of the present invention, the pharmaceutical composition is a prepared solution containing PBS at a pH of 7.4. In another embodiment of the present invention, the pharmaceutical composition exists in a dry state and is reconstituted before use. In another embodiment of the present invention, the pharmaceutical composition exists in a lyophilized state and is reconstituted before use.

[0126] Route of administration In another embodiment of the present invention, The pharmaceutical composition used for the prevention and / or treatment of a patient's disease or acute condition (where the disease or acute condition is related to oxidation) is administered orally, transdermally, subcutaneously, intradermally, sublingually, intramuscularly, intra-arterially, intracerebrally, intraventricularly, intravenously, or via the central nervous system (CNS) or intraperitoneally.

[0127] kit Another embodiment of the present invention is a kit or assay comprising the binder of the present invention disclosed herein, or a DPP3 binder, specifically an anti-DPP3 antibody or anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold, for use in the prevention or treatment of a patient's disease or acute condition (due to the disease or acute condition being related to oxidative stress).

[0128] Specifically, binding antibodies According to the present invention, "anti-DPP3 antibody" is an antibody that specifically binds to DPP3, and "anti-DPP3 antibody fragment" is a fragment of the anti-DPP3 antibody, wherein the fragment specifically binds to DPP3. "Anti-DPP3 non-Ig scaffold" is a non-Ig scaffold that specifically binds to DPP3.

[0129] In relation to the content of the present invention, "specifically, binds to DPP3" also means that it may similarly bind to other antigens. This means that this specificity does not preclude the binder from cross-reacting with other proteins, polypeptides, or peptides containing the epitope according to SEQ ID NO: 2 (for which a binder has been prepared). Specifically, this includes functional variants of DPP3, which also include the epitope according to SEQ ID NO: 2. This also relates to the specificity of the anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold according to the present invention.

[0130] antibody The "antibody" according to the present invention is a protein comprising one or more polypeptides substantially encoded by an immunoglobulin gene that specifically binds to an antigen. Recognized immunoglobulin genes include the κ, λ, α (IgA), γ (IgG1, IgG2, IgG3, IgG4), δ (IgD), ε (IgE), and μ (IgM) constant region genes, as well as various immunoglobulin variable region genes. The full-length immunoglobulin light chain is generally about 25 kD, or 214 amino acids long.

[0131] The full-length immunoglobulin heavy chain is generally about 50 kD, or 446 amino acids long. The light chain is encoded by a variable region gene at the NH2 terminus (about 110 amino acids long) and a constant region gene at the COOH terminus (κ or λ). Similarly, the heavy chain is encoded by a variable region gene (about 116 amino acids long) and one of the other constant region genes.

[0132] The basic structural unit of an antibody generally consists of two identical pairs of immunoglobulin chains, each pair being a tetramer with one light chain and one heavy chain. In each pair, the variable regions of the light and heavy chains bind to the antigen, while the constant region mediates effector function. Immunoglobulins also exist in various other forms (e.g., Fv, Fab, and (Fab')2, as well as bifunctional hybrid antibodies and single-chain antibodies) (e.g., Lanzavecchia et al., Eur. J. Immunol. 17:105,1987; Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883, 1988; Bird et al., Science 242:423-426, 1988; Hood et al., Immunology, Benjamin, NY, 2nd ed., 1984; Hunkapiller and Hood, Nature 323:15-16,1986).

[0133] The variable region of the light or heavy chain of an immunoglobulin contains a framework region that is blocked by three hypervariable regions (also called complementarity-determining regions (CDRs)) (see Sequences of Proteins of Immunological Interest, E. Kabat et al., US Department of Health and Human Services, 1983). As mentioned above, CDRs are primarily involved in the binding of antigens to epitopes. An immune complex is an antibody (such as a monoclonal antibody, chimeric antibody, humanized antibody or human antibody, or functional antibody fragment) that is specifically bound to an antigen.

[0134] A "chimeric antibody" is an antibody in which light and heavy chain genes are generally constructed by genetic engineering from the variable and constant region genes of immunoglobulins belonging to different species. For example, the variable segment of a gene derived from a mouse monoclonal antibody can be linked to a human constant segment such as κ, γ1, or γ3. Therefore, in one example, a therapeutic chimeric antibody is a hybrid protein consisting of a variable domain (antigen-binding domain) derived from a mouse antibody and a constant domain (effector domain) derived from a human antibody (although other mammalian species can also be used), or the variable region can be created by molecular technology. Methods for creating chimeric antibodies are well known in the art (see, for example, U.S. Patent No. 5,807,715). A "humanized" immunoglobulin is an immunoglobulin that contains a human framework region and one or more CDRs derived from non-human (mouse, rat, or synthetic) immunoglobulins. The non-human immunoglobulin that provides the CDRs is called the "donor," and the human immunoglobulin that provides the framework is called the "acceptor."

[0135] In one embodiment of the present invention, all CDRs of the humanized immunoglobulin are derived from donor immunoglobulin. A constant region may be absent, but if present, it must be substantially identical to the constant region of the human immunoglobulin, i.e., at least about 85-90% (e.g., about 95% or more) identical. Thus, all parts of the humanized immunoglobulin (possibly excluding the CDR) are substantially identical to the corresponding parts of the natural human immunoglobulin sequence.

[0136] According to the present invention, a “humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. The humanized antibody binds to the same antigen as the donor antibody that provides the CDR. The humanized immunoglobulin, i.e., the acceptor framework of the antibody, may have a limited number of substitutions with amino acids obtained from the donor framework. Humanized or other monoclonal antibodies may also have further conserved amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Examples of conserved substitutions include, for example, glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Humanized immunoglobulins can be constructed by genetic engineering (see, for example, U.S. Patent No. 5,585,089). Human antibodies are antibodies whose light chain and heavy chain genes are derived from humans. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells that secrete the target antibody. For example, immortalization can be achieved by EBV infection or by fusing human B cells with myeloma cells or hybridoma cells to create trioma cells. Human antibodies may also be produced by phage presentation (see, e.g., Dower et al., PCT Publication No. WO 91 / 17271; McCafferty et al., PCT Publication No. WO 92 / 001047; and Winter, PCT Publication No. WO 92 / 20791), or selected from a human recombinant monoclonal antibody library (see MorphoSys website). Human antibodies can also be produced using genetically modified animals that possess the human immunoglobulin gene (see, e.g., Lonberg et al., PCT Publication No. WO 93 / 12227; and Kucherlapati, PCT Publication No. WO 91 / 10741).

[0137] Therefore, the anti-DPP3 antibody or anti-DPP3 antibody fragment according to the present invention may have a type known in the art. Examples include, but are not limited to, human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-transplanted antibodies, or antibody fragments thereof.

[0138] Monoclonal antibodies In specific embodiments of the present invention, the anti-DPP3 antibody is a monoclonal antibody or a fragment thereof. In one embodiment of the present invention, the anti-DPP3 antibody or anti-DPP3 antibody fragment is a human or humanized antibody or derived therefrom. In one specific embodiment, one or more (mouse) CDRs are implanted within the human antibody or antibody fragment.

[0139] In a preferred embodiment, the antibody according to the present invention is an antibody produced by recombinant DNA, for example, as a typical full-length immunoglobulin such as IgG, or as an antibody fragment (e.g., a chemically bound antibody (antigen-binding fragment)) containing at least the Fv domain of the heavy chain and / or light chain. Examples of such antibody fragments include Fab fragments such as Fab minibody antibodies, single-chain Fab antibodies, and monovalent Fab antibodies with epitope tags (e.g., Fab-V5Sx2); divalent Fab (mini-antibodies) dimerized with a CH3 domain; divalent or polyvalent Fab (e.g., Fab-dHLX-FSx2) produced by multimerization using heterogeneous domains (e.g., dimerization of the dHLX domain); F(ab')2 fragments derived from classes other than G, scFv fragments, multimerized polyvalent and / or multispecific scFv fragments, bivalent and / or bispecific bispecific antibodies, BITE® (bispecific T cell conjugates (engagers)), trifunctional antibodies, and polyvalent antibodies; single-domain antibodies (e.g., nanobodies derived from camel or fish immunoglobulins); and many others.

[0140] Non-Ig scaffolding In addition to anti-DPP3 antibodies or anti-DPP3 antibody fragments, it is well known in this art that other biomolecular scaffolds, so-called non-Ig scaffolds, can be conjugated with target molecules and are used to create biomacromolecules with high specificity to targets. Examples include aptamers, spiegelmers, antikalins, and conotoxins.

[0141] Non-Ig scaffolds may also be protein scaffolds, and since they can bind to ligands or antigens, they may be used as antibody mimetic models. Non-Ig scaffolds include tetranectin-based non-Ig scaffolds (e.g., described in US2010 / 0028995); fibronectin scaffolds (e.g., described in EP1266025); lipocalin-based scaffolds (e.g., described in WO2011 / 154420); ubiquitin scaffolds (e.g., described in WO2011 / 073214); transferrin scaffolds (e.g., described in US2004 / 0023334); protein A scaffolds (e.g., described in EP2231860); ankyrin repeat-based scaffolds (e.g., described in WO2010 / 060748); microprotein (preferably microproteins that form cysteine ​​knots) scaffolds (e.g., described in EP2314308); Fyn The scaffolds may be selected from a group including 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 may be peptide or oligonucleotide aptamers. Aptamers are typically constructed by selecting them from a large random sequence pool and are short chains of oligonucleotides (DNA, RNA, or XNA) (Xu et al. 2010, Deng et al. 2014) or short variable peptide domains bound to protein scaffolds (Li et al. 2011).

[0142] Fragments and fusion proteins In another embodiment, the type of anti-DPP3 antibody is selected from the group comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, F(ab)2 fragment, and scFv-Fc fusion protein. In another preferred embodiment, the type of antibody is selected from the group comprising scFab fragment, Fab fragment, scFv fragment, and bioavailable complexes thereof (PEGylated fragments).

[0143] Monoclonal / Polyclonal Antibodies In relation to the content of this invention, the term "antibody" generally includes monoclonal antibodies and polyclonal antibodies, as well as their binding fragments (particularly Fc fragments and so-called "single-chain antibodies" (Bird et al. 1988)), chimeric antibodies, humanized antibodies (particularly CDR-transplant antibodies), and bi- or quadri-specific antibodies (Holliger et al. 1993). For example, selected by techniques such as phage presentation. This also includes immunoglobulin-like proteins that specifically bind to the target molecule contained in the sample. In this context, the term "specific binding" refers to an antibody against the target molecule or its fragment. An antibody is considered specific if its affinity for the target molecule or its fragment is preferably at least 50 times, more preferably 100 times, and most preferably at least 1000 times, its affinity for other molecules contained in the sample containing the target molecule. Methods for producing antibodies and selecting antibodies with a given specificity are well known in the art.

[0144] In a specific embodiment of the present invention, the anti-DPP3 antibody or anti-DPP3 antibody fragment that binds to the epitope according to SEQ ID NO: 2 (where the epitope is included in the DPP3 protein or a functional derivative thereof) is a monoclonal antibody or a monoclonal antibody fragment thereof. In one embodiment of the present invention, the anti-DPP3 antibody or anti-DPP3 antibody fragment that binds to the epitope according to SEQ ID NO: 2 (where the epitope is included in the DPP3 protein or a functional derivative thereof) is a human or humanized antibody or a derivative thereof, or a humanized antibody fragment or a derivative thereof. In one embodiment, one or more (mouse) CDRs are implanted within a human antibody or antibody fragment.

[0145] Modified anti-DPP3 antibody In specific embodiments, the DPP3 binder of the present invention, specifically the anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold, is a modified DPP3 binder, anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold. The modified DPP3 binder, anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold of the present invention acts inhibitorily and is measured by the previously described method for detecting and measuring DPP3 inhibition; that is, when measuring the effect of the DPP3 binder on the physiological activity of DPP-3, it may block approximately 100%, preferably at least more than 90%, and more preferably at least 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the physiological activity of DPP3.

[0146] In another specific embodiment, the modified DPP3 binder, anti-DPP3 antibody, anti-DPP3, antibody fragment, and anti-DPP3 non-Ig scaffold of the present invention act to upcontrol and, when measured by the previously described methods for detecting and measuring DPP3 inhibition; that is, when measuring the effect of the DPP3 binder on the physiological activity of DPP-3, it can increase the physiological activity of DPP3 by at least 50%, preferably more than 60%, more preferably more than 70%, more preferably more than 80%, even more preferably more than 90%, and even more very preferably more than 95%.

[0147] Synthesis of anti-DPP3 antibodies The anti-DPP3 antibody according to the present invention can be synthesized as follows. DPP3 peptides for immunotherapy were synthesized (see Table 3 below) (JPT Technologies, Berlin, Germany). A cysteine ​​residue was added to the N-terminus of the peptide to bind it to bovine serum albumin (BSA) (if cysteine ​​was not present in the selected DPP3 sequence). The peptide was covalently bonded to BSA using SulfoLink-Coupling Gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to Perbio's instructions. Recombinant GST-hDPP3 was manufactured by USBio.

[0148] Balb / c mice were intraperitoneally (ip) injected with either GST-hDPP3 or DPP3 peptide-BSA conjugate at doses of 84 μg and 100 μg, respectively (emulsified with TiterMax Gold Adjuvant) on day 0, 84 μg and 100 μg, respectively (emulsified with Freund's complete adjuvant) on day 14, and 42 μg and 50 μg, respectively (dissolved in Freund's incomplete adjuvant) on days 21 and 28. On day 49, GST-hDPP3 (42 μg) or DPP3 peptide-BSA conjugate (50 μg) dissolved in physiological saline was intravenously (iv) injected into the animals. Three days later, the mice were sacrificed and immunocellular fusion was performed.

[0149] Splenocytes derived from immunosuppressed mice and cells from the myeloma cell line SP2 / 0 were fused using 1 ml of 50% polyethylene glycol at 37°C for 30 seconds. After washing, the cells were seeded into 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT-Supplement]. After one week, the cells were subcultured three times in HT medium instead of HAT medium, and then returned to standard cell medium.

[0150] Two weeks after fusion, the cell culture supernatant was first screened for recombinant DPP3 that binds to IgG antibodies. Therefore, recombinant GST-tagged DPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng / well) and incubated with 50 μl / well of cell culture supernatant at room temperature for 2 hours. After washing the plates, 50 μl / well of POD-rabbit anti-mouse IgG was added and incubated at room temperature for 1 hour.

[0151] After the next washing step, 50 μl of the dye stock solution (3.7 mM o-phenylenediamine dissolved in [citrate / hydrogen phosphate buffer, 0.012% H2O2]) was added to each well, and the wells were incubated at room temperature for 15 minutes. The color reaction was then stopped by adding 50 μl of 4N sulfuric acid. Absorption was detected at 490 mm.

[0152] The positive test microbial cultures were transferred to 24-well plates for growth. After retesting, the selected cultures were cloned and re-cloned using the limiting dilution method, and the isotype was determined. Ta.

[0153] Antibodies against GST-tagged human DPP3 or DPP3 peptide were prepared using a standard antibody preparation method (Marx et al., 1997) and purified with Protein A. Antibody purity was over 90% based on SDS gel electrophoresis analysis.

[0154] Humanization of mouse antibodies Humanization of mouse antibodies may be carried out using the following procedure: To humanize mouse-derived antibodies, the antibody sequence is analyzed for structural interactions between the framework region (FR) and complementarity-determining region (CDR) and the antigen. Based on structural modeling, an appropriate human-derived FR is selected, and the mouse CDR sequence is transplanted into that human FR. Diversity may be introduced into the amino acid sequence of the CDR or FR to restore structural interactions lost due to species transposition of the FR sequence. This restoration of structural interactions may be achieved by a random method using a phage-presenting library, or by a direct method guided by molecular modeling (Almagro JC, Fransson J., 2008. Humanization of antibodies. Front Biosci. 2008 Jan 1;13:1619-33).

[0155] CDR grafted antibody In another aspect of the present invention, the provided content is a human CDR-transplanted anti-DPP3 antibody or anti-DPP3 antibody fragment directed to and bound to an epitope defined by SEQ ID NO: 2, wherein the epitope is contained in the DPP3 protein or a functional derivative thereof, and wherein the human CDR-transplanted anti-DPP3 antibody or anti-DPP3 antibody fragment is as follows: Sequence ID 5 The antibody heavy chain variable region (H chain) includes and / or the following: Sequence ID 6 It further includes the antibody light chain variable region (L chain) containing the following.

[0156] Further aspects of the present invention in another embodiment include a human CDR-transplanted anti-DPP3 antibody or anti-DPP3 antibody fragment directed to and bound to an epitope defined by SEQ ID NO: 2, wherein the epitope is contained in the DPP3 protein or a functional derivative thereof, and wherein the human CDR-transplanted anti-DPP3 antibody or anti-DPP3 antibody fragment is as follows: Sequence ID 12 The antibody heavy chain variable region (H chain) includes and / or the following: Sequence ID 13 It further includes the antibody light chain variable region (L chain) containing the following.

[0157] In one specific embodiment of the present invention, the present invention relates to a human monoclonal anti-DPP3 antibody or a monoclonal anti-DPP3 antibody fragment directed to and bound to an epitope defined by SEQ ID NO: 2, wherein the epitope is contained in the DPP3 protein or a functional derivative thereof, and wherein its heavy chain is as follows: Sequence ID 7, Sequence ID 8, or Sequence ID 9 It includes at least one CDR, where its light chain is as follows: Sequence ID 8, KVS, or Sequence ID 11 Includes at least one of the following CDRs.

[0158] In relation to the preceding context, the variable region may be attached to any subclass of the constant region (IgG, IgM, IgE, IgA), or only to the scaffold, Fab fragment, Fv, Fab, and F(ab)2. In Example 3 below, a mouse antibody variant having the IgG2a backbone was used. For chimeration and humanization, the human IgG1κ backbone was used.

[0159] Epitope bonding For epitope binding, only the complementarity-determining region (CDR) is important. The heavy and light chain CDRs of the mouse anti-DPP3 antibody (AK1967) of the present invention are shown in SEQ ID NOs. 7, 8, and 9 for the heavy chain, and SEQ ID NOs. 10, sequence KVS, and 11 for the light chain.

[0160] Epitope binding sites According to the present invention, the DPP3 binder provided herein, specifically the anti-DPP3 antibody, anti-DPP3 antibody fragment, and anti-DPP3 non-Ig scaffold provided herein, is directed to and binds to SEQ ID NO: 1, wherein the DPP3 binder, anti-DPP3 antibody, anti-DPP3 antibody fragment, and anti-DPP3 non-Ig scaffold recognize and bind to at least 3 aa, preferably at least 4 aa, more preferably at least 5 aa, and even more preferably at least 6 aa of SEQ ID NO: 1.

[0161] According to the present invention, the DPP3 binder provided herein, specifically the anti-DPP3 antibody, anti-DPP3 antibody fragment, and anti-DPP3 non-Ig scaffold provided herein, is directed to and binds to an epitope defined by SEQ ID NO: 2, where the epitope is contained in the DPP3 protein or a functional derivative thereof, and where the DPP3 binder, anti-DPP3 antibody, anti-DPP3 antibody fragment, and anti-DPP3 non-Ig scaffold recognize and bind to at least three aa, preferably at least four aa, more preferably at least five aa, and even more preferably at least six aa of SEQ ID NO: 2.

[0162] In another aspect of the present invention, the DPP3 binder provided herein, specifically the anti-DPP3 antibody, anti-DPP3 antibody fragment, and anti-DPP3 non-Ig scaffold provided herein, is directed to and binds to an epitope according to SEQ ID NO: 3, the epitope according to SEQ ID NO: 3 is contained in the DPP3 protein or a functional derivative thereof, and thereof the DPP3 binder, anti-DPP3 antibody, anti-DPP3 antibody fragment, and anti-DPP3 non-Ig scaffold recognizes and binds to at least three aa, preferably at least four aa, more preferably at least five aa, and even more preferably six aa of SEQ ID NO: 3.

[0163] In another embodiment of the present invention, the DPP3 binder provided herein, specifically the anti-DPP3 antibody, anti-DPP3 antibody fragment, and anti-DPP3 non-Ig scaffold provided herein, is directed to and binds to an epitope defined by SEQ ID NO: 4, wherein the epitope defined by SEQ ID NO: 4 is contained in the DPP3 protein or a functional derivative thereof, and wherein the DPP3 binder, anti-DPP3 antibody, anti-DPP3 antibody fragment, and anti-DPP3 non-Ig scaffold recognize and bind to at least three, preferably four, aa of SEQ ID NO: 4.

[0164] Inhibitors or effectors of the physiological activity of DPP3 In specific embodiments of the present invention, DPP3 binders provided herein, specifically anti-DPP3 antibodies, anti-DPP3 antibody fragments, and non-Ig anti-DPP3 scaffolds provided herein, which are directed to and bind to an epitope according to SEQ ID NO: 2 (wherein the epitope is contained in the DPP3 protein or a functional derivative thereof), may function as inhibitors or effectors of the physiological activity of DPP3.

[0165] Therefore, the DPP3 binders provided herein, specifically the anti-DPP3 antibody, anti-DPP3 antibody fragment, and non-Ig anti-DPP3 scaffolds provided herein, which are directed to and bind to the epitope defined by Sequence ID No. 2 (where the epitope is contained in the DPP3 protein or its functional derivative), are useful in the prevention or treatment of a patient's disease or acute condition, where the disease or acute condition is related to oxidative stress according to the present invention.

[0166] affinity In a specific embodiment of the present invention, the DPP3 binders provided herein, specifically the anti-DPP3 antibodies, anti-DPP3 antibody fragments, and non-Ig anti-DPP3 scaffolds provided herein, which are directed to and bind to the epitope according to SEQ ID NO: 2 (wherein said epitope is included in the DPP3 protein or a functional derivative thereof), when measured by the method for measuring the binding affinity of the DPP3 binder of the present invention for the epitope according to the sequence of SEQ ID NO: 2 as described above, have an affinity constant of at least 10 7 M ‐1 , preferably at least 10 8 M ‐1 for DPP3, and more preferably the affinity constant is at least 10 9 M ‐1 , and most preferably the affinity constant is at least 10 10 M ‐1 .

[0167] As a result, those skilled in the art know that a lower affinity can be compensated by applying a higher dose of the binder; for example, the anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold of the present invention, and this measurement does not lead outside the scope of the present invention.

[0168] Combination of drugs In another embodiment of the present invention, the DPP3 binders provided herein, specifically the anti-DPP3 antibodies or anti-DPP3 antibody fragments provided herein, or anti-DPP3 non-Ig scaffolds, can be used in combination with at least one additional drug that causes oxidative stress as a side effect.

[0169] Such drugs are administered as primary agents for use in the prevention or treatment of primary diseases and may be selected from a group including antibiotics (e.g., streptomycin, gentamicin), antimicrobial agents such as antivirals (e.g., acyclovir, foscarnet) or antifungals (e.g., amphoteserin B), analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., ibuprofen, naproxen), diuretics, proton pump inhibitors, chemotherapeutic agents (e.g., cisplatin), contrast agents, cardiovascular agents such as ACE inhibitors or statins, antidepressants, immunosuppressants (e.g., cyclosporine A), and antihistamines. As a result, and according to the present invention, DPP3 binders provided herein that bind to DPP3, specifically, anti-DPP3 antibodies or their anti-DPP3 antibody fragments, or DPP3 non-Ig scaffolds provided herein, can be used as secondary agents, either in combination or as standalone drugs, in the prevention or treatment of induced oxidative stress and resulting toxicity as secondary diseases.

[0170] Selective / Specific Binder In preferred embodiments of the present invention, the DPP3 binder provided herein is pharmaceutically acceptable and selective and / or specific to the epitope defined by SEQ ID NO: 2 (which is contained in the DPP3 protein or its functional derivative).

[0171] In a more preferred embodiment of the present invention, the DPP3 binder provided herein is It is pharmaceutically acceptable and is a selective and / or specific inhibitory binder against the epitope regulated by SEQ ID NO: 2 (which is contained in the DPP3 protein or its functional derivatives).

[0172] In one embodiment of the present invention, a selective / specific inhibitor of DPP3 does not bind to other proteins / peptides / enzymes, is not bound to other proteins / peptides / enzymes, and does not inhibit other enzymes / proteases / peptidases other than DPP3. Therefore, in relation to the present invention, preferred inhibitors of DPP3 physiological activity are specific anti-DPP3 antibodies, antibody fragments, or non-Ig scaffolds that bind to DPP3.

[0173] Orange-specific antibody In relation to the content of the present invention, a monospecific anti-DPP3 antibody, a monospecific anti-DPP3 antibody fragment, or a monospecific anti-DPP3 non-Ig scaffold is the antibody, antibody fragment, or non-Ig scaffold that specifically binds to one specific region in target DPP3 that comprises at least three amino acids, preferably at least four aa.

[0174] In relation to the content of the present invention, a monospecific anti-DPP3 antibody, a monospecific anti-DPP3 antibody fragment, or a monospecific anti-DPP3 non-Ig scaffold is an anti-DPP3 antibody, an anti-DPP3 antibody fragment, or an anti-DPP3 non-Ig scaffold that all have affinity for the same antigen as the target, which is an epitope according to Sequence ID No. 2 (contained in the DPP3 protein or its functional derivative) according to the present invention.

[0175] In another specific embodiment, the monospecific anti-DPP3 antibody, monospecific anti-DPP3 antibody fragment, or monospecific anti-DPP3 non-Ig scaffold is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that all have affinity for the same antigen as the target, which is an epitope according to Sequence ID No. 3 (contained in the DPP3 protein or its functional derivative) according to the present invention.

[0176] In another embodiment, a monospecific anti-DPP3 antibody, monospecific anti-DPP3 antibody fragment, or monospecific anti-DPP3 non-Ig scaffold is an anti-DPP3 antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold that all have affinity for the same antigen as the target, which is an epitope according to SEQ ID NO: 4 (included in the DPP3 protein or its functional derivative) according to the present invention. Monospecific antibodies may also be produced by means other than their production from common germ cells.

[0177] In relation to the preceding context, more preferred embodiments within the scope of the present invention are sequentially numbered below: 1. A binder directed to and binding to an epitope defined by SEQ ID NO: 2, wherein the binder recognizes and binds to at least three amino acids in SEQ ID NO: 2, and wherein the epitope is contained in SEQ ID NO: 1, and corresponds to the amino acid sequence of DPP3.

[0178] 2. A binder according to Embodiment 1, which is directed to and binds to the epitope according to SEQ ID NO: 2, wherein the binder is directed to and binds to the epitope according to SEQ ID NO: 3, and wherein the binder recognizes and binds to at least three amino acids in SEQ ID NO: 3, and wherein the epitope is comprised of SEQ ID NO: 1, and it corresponds to the amino acid sequence of DPP3.

[0179] 3. A binder directed to and bound to the epitope according to SEQ ID NO: 2, as described in Embodiment 1 or Embodiment 2, wherein the binder is directed to and bound to the epitope according to SEQ ID NO: 4, and wherein the binder recognizes and binds to at least three amino acids in SEQ ID NO: 4, and wherein the epitope is comprised of SEQ ID NO: 1, and it corresponds to the amino acid sequence of DPP3.

[0180] 4. A binder directed to and bound to the epitope according to SEQ ID NO: 2, as described in any one of Embodiments 1 to 3, wherein the binder is selected from the group comprising an antibody, an antibody fragment, or a non-Ig scaffold, and wherein the epitope is comprised of SEQ ID NO: 1, and it corresponds to the amino acid sequence of DPP3.

[0181] 5. A binder directed to and bound to an epitope according to SEQ ID NO: 2, as described in any one of Embodiments 1 to 4, wherein the binder is a dipeptidyl peptidase 3 (DPP3) binder directed to and bound to an epitope according to SEQ ID NO: 2, wherein the epitope is contained in a DPP3 protein or a functional derivative thereof, and wherein the DPP3 binder recognizes and binds to at least three amino acids of SEQ ID NO: 2.

[0182] 6. A binder directed to and bound to an epitope according to Sequence ID No. 2, as described in any one of Embodiments 1 to 5, wherein the binder is a monoclonal antibody or a monoclonal antibody fragment, and wherein the heavy chain complementarity determining region (CDR) is the following sequence: Sequence ID 7, Sequence ID 8, and / or Sequence ID 9 It includes and the light chain complementarity determination region is the following sequence: Sequence ID 10, KVS and / or Sequence ID 11 Includes.

[0183] 7. A binder directed to and bound to an epitope according to SEQ ID NO: 2, as described in any one of Embodiments 1 to 6, wherein the binder is a human monoclonal antibody or a human monoclonal antibody fragment, wherein the heavy chain has the following sequence: Sequence ID 12 It includes, and where the light chain has the following sequence: Sequence ID 13 Includes.

[0184] 8. A binder directed to and bound to an epitope according to SEQ ID NO: 2, for use in the treatment or prevention of a patient's disease or acute condition, as described in any one of Embodiments 1 to 7, wherein the disease or acute condition is related to oxidative stress.

[0185] 9. A binder directed to and bound to an epitope according to Sequence ID No. 2 for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in Embodiment 8, wherein the disease is selected from the group including neurodegenerative diseases, metabolic syndrome, cardiovascular diseases, autoimmune diseases, inflammatory lung diseases, kidney diseases, liver diseases, gastrointestinal diseases, viral infections, cancer, inflammation, sepsis, septic shock, and SIRS.

[0186] 10. A binder directed to and bound to an epitope according to Sequence ID No. 2 for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in Embodiment 8 or 9, and furthermore:

[0187] The neurodegenerative diseases mentioned above may be selected from the group including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). The aforementioned metabolic syndrome may be selected from a group that includes insulin resistance, obesity, hyperglycemia, dyslipidemia, hypertension, and diabetes. The cardiovascular disease may be selected from the group including atherosclerosis, hypertension, heart failure, cardiovascular ischemia, cerebral ischemic injury, stroke, and myocardial infarction.

[0188] The aforementioned autoimmune diseases may be selected from a group that includes rheumatoid arthritis and systemic lupus erythematosus. The aforementioned inflammatory lung disease may be selected from the group including COPD and asthma. The aforementioned kidney disease may be selected from the group including acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, and end-stage renal disease (ESRD). The aforementioned liver disease may be selected from the group including viral hepatitis and cirrhosis. The aforementioned digestive system disease may be selected from the group including inflammatory bowel disease, such as ulcerative colitis, Crohn's disease, gastritis, pancreatitis, and peptic ulcer.

[0189] The aforementioned viral infection may be selected from the group including blood-derived hepatitis viruses (types B, C, and D), human immunodeficiency virus (HIV), influenza A, Epstein-Barr virus, and respiratory syncytial virus. The aforementioned cancers may be selected from the group including prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, skin cancer, stomach cancer, and liver cancer. • The aforementioned inflammation, and • The sepsis, the septic shock, and the SIRS, That is the case.

[0190] 11. A binder directed to and bound to an epitope according to Sequence ID No. 2 for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in any one of Embodiments 8 to 10, wherein the disease is selected from the group including sepsis, septic shock, and SIRS.

[0191] 12. A binder directed to and bound to an epitope according to Sequence ID No. 2 for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in Embodiment 8, wherein the acute condition is selected from the group including nephrotoxicity and hepatotoxicity.

[0192] 13. A binder directed to and bound to an epitope according to SEQ ID NO: 2 for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in any one of embodiments 8 to 12, wherein the binder is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold bound to the epitope according to SEQ ID NO: 2, wherein the epitope is contained in a DPP3 protein or a functional derivative thereof, wherein the anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold comprises at least 10 ‐7 This shows the binding affinity of M to DPP3.

[0193] 14. A binder directed to and bound to an epitope according to Sequence ID No. 2 for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in any one of embodiments 8 and 12, wherein the acute condition is drug-induced hepatotoxicity or alcohol-induced hepatotoxicity.

[0194] 15. A binder directed to and bound to an epitope according to Sequence ID No. 2 for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in Embodiment 8, wherein the acute condition is nephrotoxicity which is drug-induced nephrotoxicity.

[0195] 16. A binder directed to and bound to an epitope according to Sequence ID No. 2 for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in any one of embodiments 8 to 11, wherein the disease is related to oxidative stress of the myocardium.

[0196] 17. A pharmaceutical composition comprising a binder according to any one of Embodiments 1 to 7 for use in the treatment or prevention of a patient's disease or acute condition (due to the disease or acute condition being related to oxidative stress).

[0197] 18. A kit comprising the binder described in any one of Embodiments 1 to 16.

[0198] 19. A binder directed to and bound to the DPP3 protein or a functional derivative thereof, for use in the treatment or prevention of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress.

[0199] 20. A binder for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in Embodiment 19, wherein the disease is selected from the group including neurodegenerative diseases, metabolic syndrome, cardiovascular diseases, autoimmune diseases, inflammatory lung diseases, kidney diseases, liver diseases, gastrointestinal diseases, viral infections, cancer, inflammation, sepsis, septic shock, and SIRS.

[0200] twenty one. A binder for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in either Embodiment 19 or 20, and furthermore:

[0201] The neurodegenerative diseases mentioned above may be selected from the group including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). The aforementioned metabolic syndrome may be selected from a group that includes insulin resistance, obesity, hyperglycemia, dyslipidemia, hypertension, and diabetes. The cardiovascular disease may be selected from the group including atherosclerosis, hypertension, heart failure, cardiovascular ischemia, cerebral ischemic injury, stroke, and myocardial infarction.

[0202] The aforementioned autoimmune diseases may be selected from a group that includes rheumatoid arthritis and systemic lupus erythematosus. The aforementioned inflammatory lung disease may be selected from the group including COPD and asthma. The aforementioned kidney disease may be selected from the group including acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, and end-stage renal disease (ESRD). The aforementioned liver disease may be selected from the group including viral hepatitis and cirrhosis. The aforementioned digestive system disease may be selected from the group including inflammatory bowel disease, such as ulcerative colitis, Crohn's disease, gastritis, pancreatitis, and peptic ulcer.

[0203] The aforementioned viral infection may be selected from the group including blood-derived hepatitis viruses (types B, C, and D), human immunodeficiency virus (HIV), influenza A, Epstein-Barr virus, and respiratory syncytial virus. The aforementioned cancers may be selected from the group including prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, skin cancer, stomach cancer, and liver cancer. • The aforementioned inflammation, and • The sepsis, the septic shock, and the SIRS, That is the case.

[0204] twenty two. A binder according to any one of Embodiments 19 to 21, wherein the binder is directed to and binds to an epitope by SEQ ID NO: 2, and wherein the binder recognizes and binds to at least three amino acids of SEQ ID NO: 2.

[0205] twenty three. A binder according to any one of Embodiments 19 to 22, wherein the binder is directed to and binds to an epitope defined by SEQ ID NO: 3, and wherein the binder recognizes and binds to at least three amino acids of SEQ ID NO: 3.

[0206] twenty four. A binder according to any one of Embodiments 19 to 23, wherein the binder is directed to and binds to an epitope by SEQ ID NO: 4, and wherein the DPP3 binder recognizes and binds to at least three amino acids of SEQ ID NO: 4.

[0207] twenty five. A binder according to any one of Embodiments 19 to 24, wherein the binder is selected from the group comprising an antibody, an antibody fragment, or a non-Ig scaffold.

[0208] 26. A binder according to any one of Embodiments 19 to 25, wherein the binder is a monoclonal antibody or a monoclonal antibody fragment, and wherein the heavy chain complementarity-determining region (CDR) is the following sequence: Sequence ID 7, Sequence ID 8, and / or Sequence ID 9 It includes and the light chain complementarity determination region is the following sequence: Sequence ID 10, KVS and / or Sequence ID 11 Includes.

[0209] 27. A binder according to any one of Embodiments 19 to 26, wherein the binder is a humanized monoclonal antibody or a humanized monoclonal antibody fragment, wherein the heavy chain has the following sequence: Sequence ID 12 It includes, and where the light chain has the following sequence: Sequence ID 13 Includes.

[0210] 28. A binder according to any one of Embodiments 19 to 27, wherein the binder is a dipeptidyl peptidase 3 (DPP3) binder directed to and bound to an epitope according to Sequence ID No. 2, wherein the epitope is contained in the DPP3 protein or a functional derivative thereof, and wherein the DPP3 binder recognizes and binds to at least three amino acids of Sequence ID No. 2.

[0211] definition According to the present invention, the “DPP3 binder” is directed to and binds to an epitope defined by SEQ ID NO: 2, where the epitope is contained in the DPP3 protein or a functional derivative thereof, and where the DPP3 binder recognizes and binds to at least three aa of SEQ ID NO: 2 or each of its subsequences described in SEQ ID NO: 3 or 4.

[0212] According to the present invention, the DPP3 binder is preferably an anti-DPP3 antibody, an anti-DPP3 antibody fragment, or an anti-DPP3 non-Ig scaffold that is directed to and binds to an epitope according to SEQ ID NO: 2, wherein the epitope is contained in the DPP3 protein or a functional derivative thereof, and wherein the DPP3 binder recognizes and binds to at least three of SEQ ID NOs: aa or each of its subsequences according to SEQ ID NOs: 3 or 4.

[0213] In relation to the present invention, a “functional derivative” of the DPP3 protein means a peptide, polypeptide, or protein that differs from the sequence of SEQ ID NO: 1 by the deletion, addition, or modification of aa, but maintains the physiological activity and function of the native DPP3 protein. As a result, although the physiological activity and function may be affected to some extent due to the modification of SEQ ID NO: 1, the enzymatic protease reaction catalyzed by DPP3 is still maintained when assayed by preferred physiological activity assays described above or commonly known to those skilled in the art.

[0214] Those skilled in the art understand that dipeptidyl peptidase 3 (DPP3) antibody, anti-DPP3 antibody fragment, or anti-DPP3 non-Ig scaffold is synonymous with dipeptidyl peptidase 3 (DPP3) antibody, dipeptidyl peptidase 3 antibody fragment, or DPP3 non-Ig scaffold, and that each means an anti-dipeptidyl peptidase 3 (DPP3) antibody, anti-dipeptidyl peptidase 3 antibody fragment, or anti-DPP3 non-Ig scaffold that binds to DPP3.

[0215] Throughout the text, the term "antibody" generally encompasses monoclonal and polyclonal antibodies, as well as their binding fragments (particularly Fc fragments and so-called "single-chain antibodies" (Bird et al. 1988)), chimeric antibodies, humanized antibodies (particularly CDR-transplant antibodies), and bi- or quadruple-specific antibodies (Holliger et al. 1993). It also includes immunoglobulin-like proteins that specifically bind to target molecules in a sample, selected by techniques such as phage presentation.

[0216] In this context, the term "specific binding" refers to an antibody against a target molecule or fragment thereof. An antibody is considered specific if its affinity for the target molecule or fragment thereof is preferably at least 50 times, more preferably 100 times, and most preferably at least 1000 times, its affinity for other molecules in the sample containing the target molecule. Methods for producing antibodies and selecting antibodies with a given specificity are well known in the art.

[0217] "Diseases related to oxidative stress" include, but are not limited to, neurodegenerative diseases, metabolic syndrome, cardiovascular diseases, autoimmune diseases, inflammatory lung diseases, kidney diseases, liver diseases, digestive system diseases, viral infections, cancer, and inflammation, sepsis, septic shock, and SIRS.

[0218] In relation to the content of the present invention, neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). In relation to the content of the present invention, metabolic syndrome includes insulin resistance, obesity, hyperglycemia, dyslipidemia, hypertension, and diabetes. In relation to the content of the present invention, cardiovascular diseases include atherosclerosis, hypertension, heart failure, cardiovascular ischemia, cerebral ischemic injury / stroke, and myocardial infarction.

[0219] In relation to the content of the present invention, autoimmune diseases include rheumatoid arthritis and systemic lupus erythematosus. In relation to the content of this invention, inflammatory lung diseases include COPD and asthma. In relation to the content of the present invention, kidney disease includes acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, and end-stage renal disease (ESRD). In relation to the content of this invention, liver diseases include viral hepatitis and cirrhosis.

[0220] In relation to the content of the present invention, digestive system diseases include inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease; gastritis, pancreatitis, and peptic ulcers. In relation thereto, viral infections include blood-derived hepatitis viruses (types B, C, and D), human immunodeficiency virus (HIV), influenza A, Epstein-Barr virus, and respiratory syncytial viruses. In relation to the content of the present invention, cancer includes prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, skin cancer, stomach cancer, and liver cancer.

[0221] In relation to the content of this invention, "acute pathological conditions related to oxidative stress" refers to symptoms that develop, change, or rapidly worsen as a result of the occurrence of oxidative stress. Acute pathological conditions related to oxidative stress have a sudden onset. If left untreated, acute pathological conditions related to oxidative stress can lead to chronic syndromes.

[0222] In contrast, "chronic condition" or "chronic syndrome," in relation to the content of this invention, means a condition or symptom that develops and worsens over a long period of time and may persist even after treatment.

[0223] Oxidative stress reflects an imbalance between the overall expression of reactive oxygen species (ROS) / reactive nitrogen species (RNS) and antioxidants, which favors the excessive generation of free radicals. This process leads to the oxidation of biomolecules, accompanied by loss of their biological function and / or homeostatic imbalance, and their expression is a potential source of oxidative damage to cells and tissues. The accumulation of ROS / RNS results in many detrimental effects, such as lipid peroxides, protein oxidation, and DNA damage (including base damage and strand disruption). Furthermore, some reactive oxidative species function as cellular messengers in redox signaling. Therefore, oxidative stress can disrupt the normal mechanisms of cellular signaling.

[0224] In relation to the content of this invention, a "free radical" is a molecule having one or more unpaired electrons in its outer shell. Free radicals are formed from molecules by radical cleavage, where each fragment gives rise to another radical, by the breakdown of chemical bonds that hold one electron, and also by redox reactions. Examples of free radicals associated with oxidative stress include hydroxyl (OH·), superoxide (O2·−), nitric oxide (NO·), nitrogen dioxide (NO2·), peroxyl (ROO·), and lipid peroxyl (LOO·). Also, hydrogen peroxide (H2O2), ozone (O3), singlet oxygen ( 1 O2, hypochlorous acid (HOCl), nitrite (HNO2), peroxynitrite (ONOO·), nitrous oxide (N2O3), and lipid peroxides (LOOH) are not free radicals and are generally called oxidizing substances, but they can easily lead to free radical reactions in living organisms.

[0225] "Primary drug" refers to a drug that acts against the primary cause of the aforementioned disease or condition. "Secondary drug therapy" refers to drug therapy that improves the patient's condition in a symptomatic manner; for example, it reduces or regulates oxidative stress induced by the administration of a primary drug. In connection with the content of the present invention, generally, "biological activity" is defined as the effect that a substance exhibits in vivo or in vitro on a living organism, tissue, organ, or functional unit after its interaction. In this regard, and specifically in connection with the content of the present invention, DPP3 biological activity can be defined as DPP3 enzyme activity or the regulation of the activity of DPP3 in the oxidative stress pathway.

[0226] [Table 3-1] [Table 3-2] [Examples]

[0227] 1. Example 1 Production of antibodies and quantification of DPP3 binding ability: Several mouse antibodies were produced and screened by specific binding assays based on their binding ability to human DPP3 (see Table 3). 1.1. Method: - Peptide / conjugate for immunization: A DPP3 peptide for immunization was synthesized (see Table 3 below) (JPT Technologies, Berlin, Germany). To conjugate the peptide to bovine serum albumin (BSA), a cysteine residue was added to the N-terminus (if cysteine does not exist in the selected DPP3 sequence). The peptide was covalently bound to BSA using SulfoLink-Coupling Gel (Perbio-science, Bonn, Germany). The coupling procedure was carried out according to the instructions of Perbio. Recombinant GST-hDPP3 was from USBio (United States Biological, Salem, MA, USA).

[0228] Mouse immunization, immune cell fusion, and screening Balb / c mice were intraperitoneally (ip) injected with either GST-hDPP3 or DPP3 peptide-BSA conjugate at doses of 84 μg and 100 μg, respectively (emulsified with TiterMax Gold Adjuvant) on day 0, 84 μg and 100 μg, respectively (emulsified with Freund's complete adjuvant) on day 14, and 42 μg and 50 μg, respectively (dissolved in Freund's incomplete adjuvant) on days 21 and 28. On day 49, GST-hDPP3 (42 μg) or DPP3 peptide-BSA conjugate (50 μg) dissolved in physiological saline was intravenously (iv) injected into the animals. Three days later, the mice were sacrificed and immunocellular fusion was performed.

[0229] Splenocytes derived from immunosuppressed mice and cells from the myeloma cell line SP2 / 0 were fused using 1 ml of 50% polyethylene glycol at 37°C for 30 seconds. After washing, the cells were seeded into 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT-Supplement]. After one week, the cells were subcultured three times in HT medium instead of HAT medium, and then returned to standard cell medium.

[0230] Two weeks after fusion, the cell culture supernatant was first screened for recombinant DPP3 that binds to IgG antibodies. Therefore, recombinant GST-tagged DPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng / well) and incubated with 50 μl / well of cell culture supernatant at room temperature for 2 hours. After washing the plates, 50 μl / well of POD-rabbit anti-mouse IgG was added and incubated at room temperature for 1 hour. After the next washing step, 50 μl of the dye stock (3.7 mM o-phenylenediamine dissolved in [citrate / hydrogen phosphate buffer, 0.012% H2O2]) was added to each well and incubated at room temperature for 15 minutes. The color reaction was stopped by adding 50 μl of 4N sulfuric acid. Absorption was detected at 490 mm. Positive test microbial cultures were transferred to 24-well plates for growth. After retesting, the selected culture media were cloned and re-cloned using the limiting dilution method, and the isotype was determined.

[0231] - Production of mouse monoclonal antibodies Antibodies against GST-tagged human DPP3 or DPP3 peptide were prepared using a standard antibody preparation method (Marx et al., 1997) and purified with Protein A. Antibody purity was over 90% based on SDS gel electrophoresis analysis.

[0232] - Antibody Characterization - Binding to hDPP3 and / or Immunotherapy Peptides To analyze the ability of various antibodies and antibody clones to bind DPP3 / immunolytic peptides, binding assays were performed: a) Solid phase Recombinant GST-tagged hDPP3 (SEQ ID NO: 1) or DPP3 peptide (immunized peptide, SEQ ID NO: 2) was immobilized on the surface of a highly binding microtiter plate (96-well polystyrene microplate, Greiner Bio-One international AG, Austria, 1 μg / well in coupling buffer [50 mM Tris, 100 mM NaCl, pH 7.8], RT for 1 hour). After blocking with 5% bovine serum albumin, the microplate was vacuum-dried.

[0233] b) Marking procedure (tracer) 100 μg (100 μl) of various anti-DPP3 antibodies (detection antibody, 1 mg / ml in PBS, pH 7.4) were mixed with 10 μl of acridinium NHS ester (1 mg / ml in acetonitrile, InVent GmbH, Germany; EP 0 353 971) and incubated at room temperature for 30 minutes. Labeled anti-DPP3 antibodies were purified by gel filtration HPLC using Shodex Protein 5 μm KW-803 (Showa Denko, Japan). The purified labeled antibodies were diluted with assay buffer (50 mmol / l potassium phosphate, 100 mmol / l NaCl, 10 mmol / l Na2-EDTA, 5 g / l bovine serum albumin, 1 g / l mouse IgG, 1 g / l bovine IgG, 50 μmol / l astatin, 100 μmol / l leupeptin, pH 7.4). The final concentration is approximately 5-7 per 200 μl. * 10 6 The relative light units (RLU) were labeled from a compound (approximately 20 ng of labeled antibody). Acridinium ester chemiluminescence was measured using a Centro LB960 luminometer (Berthold Technologies GmbH & Co. KG).

[0234] c) hDPP3 binding assay The plate was filled with 200 μl of labeled and diluted detection antibody (tracer) and incubated at 2–8°C for 2–4 hours. Unbound tracers were removed by washing four times with 350 μl of washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Chemiluminescence of firmly bound tracers was measured using a Centro LB960 luminometer (Berthold Technologies GmbH & Co. KG).

[0235] Antibody Characterization - hDPP3 Inhibition Assay To analyze the DPP3 inhibitory activity of various antibodies and antibody clones, a DPP3 activity assay was performed using a known procedure (Jones et al., 1982). Recombinant GST-tagged hDPP3 was diluted in assay buffer (25 ng / ml GST-DPP3 dissolved in [50 mM Tris-HCl (pH 7.5), 100 μM ZnCl2]), and 200 μl of this solution was incubated at room temperature with 10 μg of each antibody. After 1 hour of pre-incubation, the fluorescence-generating substrate Arg-Arg-βNA (20 μl, 2 mM) was added to the solution, and the generation of free βNA over time was monitored at 37°C using a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG). βNA fluorescence was detected by excitation at 340 nm and measurement of emission at 410 nm. The fluorescence increase gradient (RFU / min) for various samples was calculated. The gradient for the buffer (control) and GST-hDPP3 is defined as 100% activity. The inhibitory ability of a potential capture binder is defined by the decrease (%) in GST-hDPP3 activity upon incubation with the capture binder.

[0236] 1.2.Results: The following table shows the selection of the obtained antibodies, their binding rates in relative light units (RLU), and their relative inhibitory activity (%) (Table 3). Monoclonal antibodies obtained against the DPP3 region shown below were selected based on their ability to bind recombinant DPP3 and / or immunized peptides, as well as their inhibitory potential.

[0237] All antibodies obtained against the full-length recombinant hDPP3 with a GST tag showed strong binding to immobilized GST-tagged hDPP3. Antibodies obtained against the peptide of SEQ ID NO: 2 also bind to GST-hDPP3. The SEQ ID NO: 2 antibody also binds strongly to the immunizing peptide. Those antibodies were characterized in more detail (see Example 2). The monoclonal antibody AK1967, which has the ability to inhibit DPP3 activity by 70%, was selected as a prospective therapeutic antibody and was also used as a template for chimerization and humanization.

[0238]

Table 4

[0239] 2. Example 2 The antibodies obtained against SEQ ID NO: 2 were characterized in more detail (epitope mapping, binding affinity, specificity, inhibition potential). Here, the results regarding clone 1967 of SEQ ID NO: 2 (“AK1967”) are shown as an example.

[0240] 2.1. Method: - Determination of the AK1967 epitope on DPP3: For epitope mapping of AK1967, many N- or C-terminal biotinylated peptides were synthesized (peptides & elephants GmbH, Hennigsdorf, Germany). These peptides include the sequences of the complete immunizing peptide (SEQ ID NO: 2) or its fragments with one amino acid removed stepwise from either the C- or N-terminus (see Table 5 for the full list of peptides).

[0241] a) Solid phase High-binding 96-well plates were coated with 2 μg of Avidin (Greiner Bio-One international AG, Austria) per well in coupling buffer (500 mM Tris-HCl, pH 7.8, 100 mM NaCl). The plates were then washed and filled with a specific solution of biotinylated peptides (10 ng / well; 1x PBS containing buffer-0.5% BSA). b) Marking procedure (tracer) The anti-DPP3 antibody AK1967 was labeled by chemiluminescence according to Example 1.

[0242] c) Peptide bonding assay The plate was filled with 200 μl of labeled and diluted detection antibody (tracer) and incubated at room temperature for 4 hours. Unbound tracers were removed by washing four times with 350 μl of washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Chemiluminescence of firmly bound tracers was measured using a Centro LB960 luminometer (Berthold Technologies GmbH & Co. KG). AK1967 binding to each peptide was determined by evaluating relative light units (RLU). Any peptide showing a significantly higher RLU signal than non-specific binding of AK1967 was defined as an AK1967 binder. Combination analysis of bound and unbound peptides revealed a specific DPP3 epitope for AK1967.

[0243] - Measuring binding affinity using Octet: The experiment was conducted using Octet Red96 (ForteBio). AK1967 was captured on a kinetic-grade anti-human Fc (AHC) biosensor. The implanted biosensor was then immersed in a dilution series of recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM). Binding was observed for 120 seconds, followed by dissociation for 180 seconds. The buffers used in the experiment are shown in Table 4. Kinetic analysis was performed using a 1:1 binding model and global fit.

[0244] [Table 5]

[0245] - Western blot analysis of AK1967 binding specificity: Hematopoietic cells from human EDTA blood were washed (3x in PBS), diluted in PBS, and hemolyzed by repeated freeze-thaw cycles. The hematopoietic cell lysates had a total protein concentration of 250 μg / ml and a DPP3 concentration of 10 μg / ml. Dilutions of hematopoietic cell lysates (1:40, 1:80, 1:160, and 1:320) and purified recombinant human His-DPP3 (31.25–500 ng / ml) were subjected to SDS-PAGE and Western blotting. The blots were incubated in 1) inhibitory buffer (1x PBS-T containing 5% skim milk powder), 2) primary antibody solution (AK1967 1:2,000 in inhibitory buffer), and 3) HRP-labeled secondary antibody (goat anti-mouse IgG, 1:1,000 in inhibitory buffer). The bound secondary antibody was detected using Amersham ECL Western Blotting Detection Reagent and Amersham Imager 600 UV (both from GE Healthcare).

[0246] - DPP3 inhibition assay: To analyze the DPP3 inhibitory activity of AK1967, a DPP3 activity assay was performed using a known procedure (Jones et al., 1982). Recombinant GST-tagged hDPP3 was diluted in assay buffer (25 ng / ml GST-DPP3 in 50 mM Tris-HCl, pH 7.5), and then a higher concentration of AK1967 was added. The fluorescence-generating substrate Arg-Arg-βNA was added to the solution, and the generation of free βNA over time was monitored at 37°C using a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG). βNA fluorescence was detected by excitation at 340 nm and emission at 410 nm. The fluorescence increase gradient (RFU / min) was calculated for various samples. The gradient for the buffer (control) and GST-hDPP3 was set to 100% activity. The inhibitory activity of AK1967 is defined by the decrease (%) in GST-hDPP3 activity when incubated with the aforementioned antibody. The obtained decrease in DPP3 activity is shown in the inhibition curve in Figure 1C.

[0247] 2.2.Results: - Epitope mapping: Analysis of peptides to which AK1967 binds and those to which it does not revealed that the DPP3 sequence INPETG (SEQ ID NO: 3) is an epitope required for AK1967 binding (see Table 5).

[0248] [Table 6]

[0249] -Binding affinity: AK1967 is 2.2 * 10 ‐9 It binds to recombinant GST-hDPP3 with M affinity (see Table 6 for further details, and Figure 1A for the dynamic curve).

[0250] [Table 7]

[0251] -Specificity: The only protein detected alongside AK1967 as a primary antibody in the lysate of blood cells was 80 kDa DPP3 (Figure 1B). The total protein concentration of the lysate was 250 μg / ml, while the estimated DPP3 concentration was approximately 10 μg / ml. Despite the presence of more than 25 times the amount of nonspecific proteins in the lysate, AK1967 specifically bound to and detected DPP3, without causing other nonspecific binding.

[0252] - Possible inhibition: AK1967 inhibits 15 ng / ml of DPP3 in a specific DPP3 activity assay using an IC50 of approximately 15 ng / ml (Figure 1C).

[0253] 3. Example 3 We induced heart failure in rats using a septic shock model and characterized the effects of AK1967 on oxidative stress in the myocardium.

[0254] 3.1. Method: -Research design The research procedure is shown in Figure 2A below. After CLP or sham surgery, the animals were allowed to rest for 20 hours with free access to water and food. Then, the rats were anesthetized, tracheostomies were performed, and arterial and venous lines were placed. 24 hours after the CLP surgery, AK1967 or a solvent (physiological saline) was administered at a dose of 2 mg / kg. As a safety measure, circulatory dynamics were continuously monitored invasively (t=0-3h).

[0255] - CLP model of septic shock Male Wistar rats (2-3 months old, 300-400g; see Table 7 for group size) obtained from Centre d'elevage Janvier (France) were randomly assigned to one of three groups. All animals were anesthetized intraperitoneally (ip) with ketamine hydrochloride (90 mg / kg) and xylazine (9 mg / kg). To induce polymicrobial sepsis, cecal ligation puncture (CLP) was performed using a slightly modified Rittirsch protocol. A ventral midline incision (1.5 cm) was made to expose the cecum. The cecum was then ligated just below the ileocecal valve and punctured once with an 18-gauge needle. The abdominal cavity was then closed in two layers, fluid resuscitation was performed (subcutaneous injection of 3 ml / 100 g (body weight) of saline), and the animals were returned to their cages. Animals in the sham surgery group underwent surgery without cecal puncture.

[0256] - Time of the experiment and animal group At t=0 (baseline), all CLP animals were in septic shock and exhibited decreased cardiac function (hypotension, low shortening rate). At this point, AK1967 (2 mg / kg) or a solvent (saline) was injected (iv; 5 minutes after surgery), and saline infusion was initiated. The table below shows a summary of one control group and two CLP groups (Table 7). At the end of the experiment, the animals were euthanized, and organs (e.g., heart) were collected and further analyzed.

[0257] [Table 8]

[0258] - DHE labeling of ROS in myocardium Dihydroethidium (DHE; Sigma-Aldrich) staining was used to evaluate the in situ levels of superoxide anions in myocardium. Ventricular cardiac cryostat sections (7 μm) were incubated with DHE (37 μM) for 30 minutes in a dark, humidified chamber. Fluorescence images of ethidium bromide were acquired using a Leica fluorescence microscope under the same settings for all tissue blocks. The stained area was measured using IPLab software and expressed as a percentage of the region of interest (ROI).

[0259] 3.2.Results: Rats suffering from septic shock-induced heart failure after CLP surgery produce large amounts of reactive oxygen species (ROS) in their myocardium, while sham-surgery-treated animals show almost no oxidative stress (Figures 2B and C). Treatment of diseased (CLP) animals with AK1967 reduces oxidative stress levels in the myocardium to levels found in healthy (sham-surgery-treated) animals. This potent reduction in ROS was achieved within 3 hours of treatment (Figures 2B and C).

[0260] References [Table 9]

[0261] [Table 10]

[0262] [Table 11]

[0263] [Table 12]

[0264] [Table 13]

[0265] [Table 14]

[0266] [Table 15]

[0267] [Table 16]

[0268] Sequence List Sequence ID 1-hDPP3 aa 1~737 [ka]

[0269] SEQ ID NO: 2-hDPP3 aa 474~493(N-Cys)-Immunolytic peptide with additional N-terminal cysteine CETVINPETGEQIQSWYRSGE Epitope of sequence number 3-hDPP3 aa 477~482-AK1967 INPETG Sequence ID 4-hDPP3 aa 480~483 ETGE

[0270] Sequence ID 5 - Variable region of mouse AK1967 in the heavy chain [ka]

[0271] Sequence ID 6 - Variable region of mouse AK1967 in the light chain [ka]

[0272] Sequence ID 7 - CDR1 of mouse AK1967 in the heavy chain GFSLSTSGMS Sequence ID 8 - CDR2 of mouse AK1967 in heavy chain IWWNDNK Sequence ID 9 - CDR3 of mouse AK1967 in the heavy chain ARNYSYDY Sequence ID 10 - CDR1 of mouse AK1967 in the light chain RSLVHSIGSTY CDR2 of mouse AK1967 in light chain KVS Sequence ID 11 - CDR3 of mouse AK1967 in the light chain SQSTHVPWT

[0273] Sequence ID 12 - Humanized AK1967 - Heavy chain sequence (IgG1κ backbone) [ka]

[0274] Sequence ID 13 - Humanized AK1967 - Light chain sequence (IgG1κ backbone) [ka] Some aspects of the present invention are described below. 1. A dipeptidyl peptidase 3 (DPP3) binder directed to and bound to an epitope defined by Sequence ID No. 2, wherein the DPP3 binder recognizes and binds to at least three amino acids, particularly at least four amino acids, more particularly at least five amino acids, in Sequence ID No. 2, and wherein the epitope is contained in Sequence ID No. 1, and wherein the DPP3 binder has an affinity constant of at least 10 7 M ‐1 A dipeptidyl peptidase 3 binder that exhibits affinity for DPP3. 2. A dipeptidyl peptidase 3 binder as described in item 1, which is directed to and binds to the epitope according to SEQ ID NO: 2, wherein the DPP3 binder is directed to and binds to the epitope according to SEQ ID NO: 3, and wherein the DPP3 binder recognizes and binds to at least three amino acids, particularly at least four amino acids, and more particularly at least five amino acids of SEQ ID NO: 3. 3. A dipeptidyl peptidase 3 binder as described in item 1 or 2, which is directed to and binds to an epitope according to SEQ ID NO: 2, wherein the DPP3 binder is directed to and binds to an epitope according to SEQ ID NO: 4, and wherein the DPP3 binder recognizes and binds to at least three amino acids, particularly four amino acids, of SEQ ID NO: 4. 4. A dipeptidyl peptidase 3 binder that is directed to and binds to an epitope according to Sequence ID No. 2, as described in any of items 1 to 3, wherein the DPP3 binder is selected from the group comprising an antibody, an antibody fragment, or a non-Ig scaffold. 5. A dipeptidyl peptidase 3 binder that is directed to and binds to an epitope according to Sequence ID No. 2, as described in any of items 1 to 4, wherein the DPP3 binder is selected from the group comprising a monospecific antibody, a monospecific antibody fragment, or a monospecific non-Ig scaffold. 6. A dipeptidyl peptidase 3 binder that is directed to and binds to an epitope according to Sequence ID No. 2, as described in any of items 1 to 5, wherein the DPP3 binder is a monoclonal antibody or a monoclonal antibody fragment, and the heavy chain complementarity-determining region (CDR) is the following sequence: Sequence ID 7, Sequence ID 8, and / or Sequence ID 9 It includes and the light chain complementarity determination region (CDR) is the following sequence: Sequence ID 10, KVS and / or Sequence ID 11 A dipeptidyl peptidase 3 binder containing this. 7. A dipeptidyl peptidase 3 binder, as described in any of items 1-6, which is directed to and binds to an epitope defined by Sequence ID No. 2, wherein the DPP3 binder is a humanized monoclonal antibody or a humanized monoclonal antibody fragment, and the heavy chain has the following sequence: Sequence ID 12 It includes, and where the light chain has the following sequence: Sequence ID 13 A dipeptidyl peptidase 3 binder containing this. 8. A dipeptidyl peptidase 3 binder, directed to and bound to an epitope according to Sequence ID No. 2, as described in any of items 1 to 7, for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress. 9. A dipeptidyl peptidase 3 binder, directed to and bound to an epitope according to Sequence ID No. 2, as described in any of items 1 to 6, for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in item 7, wherein the disease is selected from the group including neurodegenerative diseases, metabolic syndrome, cardiovascular diseases, autoimmune diseases, inflammatory lung diseases, kidney diseases, liver diseases, gastrointestinal diseases, viral infections, cancer, inflammation, sepsis, septic shock, and SIRS. 10. A dipeptidyl peptidase 3 binder, directed to and bound to an epitope according to Sequence ID No. 2, as described in any of items 1-6, for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in item 7 or 8, and hereby: The neurodegenerative disease may be selected from the group including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). The aforementioned metabolic syndrome may be selected from a group that includes insulin resistance, obesity, hyperglycemia, dyslipidemia, hypertension, and diabetes. The cardiovascular disease may be selected from the group including atherosclerosis, hypertension, heart failure, cardiovascular ischemia, cerebral ischemic injury, stroke, and myocardial infarction. The autoimmune disease may be selected from the group including rheumatoid arthritis and systemic lupus erythematosus. The aforementioned inflammatory lung disease may be selected from the group including COPD and asthma. The aforementioned kidney disease may be selected from a group that includes acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, and end-stage renal disease (ESRD). The aforementioned liver disease may be selected from the group including viral hepatitis and cirrhosis. The aforementioned digestive system disease may be selected from the group including inflammatory bowel disease, such as ulcerative colitis, Crohn's disease, gastritis, pancreatitis, and peptic ulcer. The aforementioned viral infection may be selected from the group including blood-derived hepatitis viruses (types B, C, and D), human immunodeficiency virus (HIV), influenza A, Epstein-Barr virus, and respiratory syncytial virus. The aforementioned cancer may be selected from the group including prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, skin cancer, stomach cancer, and liver cancer. • The aforementioned inflammation, • The aforementioned sepsis, the aforementioned septic shock, the aforementioned SIRS, This is dipeptidyl peptidase 3 binder. 11. A dipeptidyl peptidase 3 binder, directed to and bound to an epitope according to Sequence ID No. 2, as described in any of items 1 to 6, for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in any of items 7 to 9, wherein the disease is selected from the group including sepsis, septic shock, and SIRS. 12. A dipeptidyl peptidase 3 binder, directed to and bound to an epitope according to Sequence ID No. 2, as described in any of items 1 to 6, for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in any of items 7 to 9, and wherein the disease is related to oxidative stress of the myocardium. 13. A dipeptidyl peptidase 3 binder directed to and bound to the DPP3 protein or a functional derivative thereof, for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress. 14. A dipeptidyl peptidase 3 binder for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in item 13, wherein the disease is selected from the group including neurodegenerative diseases, metabolic syndrome, cardiovascular diseases, autoimmune diseases, inflammatory lung diseases, kidney diseases, liver diseases, gastrointestinal diseases, viral infections, cancer, inflammation, sepsis, septic shock, and SIRS. 15. A dipeptidyl peptidase 3 binder for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in item 13 or 14, and furthermore: The neurodegenerative disease may be selected from the group including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). The aforementioned metabolic syndrome may be selected from a group that includes insulin resistance, obesity, hyperglycemia, dyslipidemia, hypertension, and diabetes. The cardiovascular disease may be selected from the group including atherosclerosis, hypertension, heart failure, cardiovascular ischemia, cerebral ischemic injury, stroke, and myocardial infarction. The autoimmune disease may be selected from the group including rheumatoid arthritis and systemic lupus erythematosus. The aforementioned inflammatory lung disease may be selected from the group including COPD and asthma. The aforementioned kidney disease may be selected from a group that includes acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, and end-stage renal disease (ESRD). The aforementioned liver disease may be selected from the group including viral hepatitis and cirrhosis. The aforementioned digestive system disease may be selected from the group including inflammatory bowel disease, such as ulcerative colitis, Crohn's disease, gastritis, pancreatitis, and peptic ulcer. The aforementioned viral infection may be selected from the group including blood-derived hepatitis viruses (types B, C, and D), human immunodeficiency virus (HIV), influenza A, Epstein-Barr virus, and respiratory syncytial virus. The aforementioned cancer may be selected from the group including prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, skin cancer, stomach cancer, and liver cancer. • The aforementioned inflammation, • The aforementioned sepsis, the aforementioned septic shock, the aforementioned SIRS, This is dipeptidyl peptidase 3 binder.

Claims

1. A dipeptidyl peptidase 3 (DPP3) binder directed to and binding to an epitope defined by SEQ ID NO: 2, wherein the DPP3 binder recognizes and binds to at least three amino acids in SEQ ID NO: 2, wherein the epitope is contained in SEQ ID NO: 1, and wherein the DPP3 binder has an affinity constant of at least 10 ７ M ‐１ It exhibits an affinity for DPP3 such that, where the DPP3 binder is a monoclonal antibody or a monoclonal antibody fragment, and where the heavy chain complementarity-determining region (CDR) is the following sequence: Sequence IDs 7, 8, and 9 It includes and the light chain complementarity determination region (CDR) is the following sequence: Sequence ID 10, KVS, and Sequence ID 11 A dipeptidyl peptidase 3 binder containing this.

2. The dipeptidyl peptidase 3 binder according to claim 1, wherein the DPP3 binder recognizes and binds to at least four amino acids of SEQ ID NO:

2.

3. The dipeptidyl peptidase 3 binder according to claim 2, wherein the DPP3 binder recognizes and binds to at least five amino acids of SEQ ID NO:

2.

4. A dipeptidyl peptidase 3 binder according to any one of claims 1 to 3, wherein the DPP3 binder is directed to and binds to the epitope according to SEQ ID NO: 3, and wherein the DPP3 binder recognizes and binds to at least three amino acids of SEQ ID NO:

3.

5. The dipeptidyl peptidase 3 binder according to claim 4, which is directed to and binds to the epitope according to SEQ ID NO: 2, wherein the DPP3 binder recognizes and binds to at least four amino acids of SEQ ID NO:

3.

6. The dipeptidyl peptidase 3 binder according to claim 5, which is directed to and binds to the epitope according to SEQ ID NO: 2, wherein the DPP3 binder recognizes and binds to at least five amino acids of SEQ ID NO:

3.

7. A dipeptidyl peptidase 3 binder according to any one of claims 1 to 6, wherein the DPP3 binder is directed to and binds to the epitope according to SEQ ID NO: 4, and wherein the DPP3 binder recognizes and binds to at least three amino acids of SEQ ID NO:

4.

8. The dipeptidyl peptidase 3 binder according to claim 7, which is directed to and binds to the epitope according to SEQ ID NO: 2, wherein the DPP3 binder recognizes and binds to four amino acids of SEQ ID NO:

4.

9. A dipeptidyl peptidase 3 binder according to any one of claims 1 to 8, which is directed to and binds to an epitope defined in Sequence ID No. 2, wherein the DPP3 binder is selected from the group consisting of an antibody, an antibody fragment, or a non-Ig scaffold.

10. A dipeptidyl peptidase 3 binder according to any one of claims 1 to 9, wherein the DPP3 binder is selected from the group consisting of a monospecific antibody, a monospecific antibody fragment, or a monospecific non-Ig scaffold.

11. A dipeptidyl peptidase 3 binder directed to and bound to an epitope according to SEQ ID NO: 2, as described in any one of claims 1 to 10, wherein the DPP3 binder is a humanized monoclonal antibody or a humanized monoclonal antibody fragment, wherein the heavy chain has the following sequence: Sequence ID 12 It includes, and where the light chain has the following sequence: Sequence ID 13 A dipeptidyl peptidase 3 binder containing this.

12. A dipeptidyl peptidase 3 binder directed to and bound to an epitope according to SEQ ID NO: 2, as described in any one of claims 1 to 11, for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress.

13. A dipeptidyl peptidase 3 binder, directed to and bound to an epitope according to SEQ ID NO: 2, as described in any one of claims 1 to 11, for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in claim 12, wherein the disease is selected from the group consisting of neurodegenerative diseases, metabolic syndrome, cardiovascular diseases, autoimmune diseases, inflammatory lung diseases, kidney diseases, liver diseases, gastrointestinal diseases, viral infections, cancer, inflammation, sepsis, septic shock, and SIRS.

14. A dipeptidyl peptidase 3 binder, directed to and bound to an epitope according to SEQ ID NO: 2, as described in any one of claims 1 to 11, for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in claim 13, and furthermore: The neurodegenerative disease may be selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). The aforementioned metabolic syndrome may be selected from the group consisting of insulin resistance, obesity, hyperglycemia, dyslipidemia, hypertension, and diabetes. The cardiovascular disease may be selected from the group consisting of atherosclerosis, hypertension, heart failure, cardiovascular ischemia, cerebral ischemic injury, stroke, and myocardial infarction. The autoimmune disease may be selected from the group consisting of rheumatoid arthritis and systemic lupus erythematosus. The aforementioned inflammatory lung disease may be selected from the group consisting of COPD and asthma. The aforementioned kidney disease may be selected from the group consisting of acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, and end-stage renal disease (ESRD). The liver disease may be selected from the group consisting of viral hepatitis and cirrhosis. The digestive system disease may be selected from the group consisting of inflammatory bowel diseases, such as ulcerative colitis, Crohn's disease, gastritis, pancreatitis, and peptic ulcers. The aforementioned viral infection may be selected from the group consisting of blood-derived hepatitis viruses (types B, C, and D), human immunodeficiency virus (HIV), influenza A, Epstein-Barr virus, and respiratory syncytial virus. The aforementioned cancer may be selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, skin cancer, stomach cancer, and liver cancer. - The aforementioned inflammation, - Sepsis, septic shock, SIRS, This is dipeptidyl peptidase 3 binder.

15. A dipeptidyl peptidase 3 binder directed to and bound to an epitope according to SEQ ID NO: 2, as described in any one of claims 1 to 11, for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in any one of claims 12 to 14, wherein the disease is selected from the group consisting of sepsis, septic shock, and SIRS.

16. A dipeptidyl peptidase 3 binder directed to and bound to an epitope according to SEQ ID NO: 2, as described in any one of claims 1 to 11, for use in the prevention or treatment of a patient's disease or acute condition, wherein the disease or acute condition is related to oxidative stress as described in any one of claims 12 to 14, wherein the disease is related to oxidative stress of the myocardium.

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