Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P39/00—General protective or antinoxious agents
  • A61P39/06—Free radical scavengers or antioxidants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/515—Complete light chain, i.e. VL + CL
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2318/00—Antibody mimetics or scaffolds
  • C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics

Definitions

• DPP3 binder directed to and binding to specific DPP3-epitopes and its use in the prevention or treatment of diseases / acute conditions that are associated with oxidative
• subject matter of the invention is a binder being directed to and binding to a dipeptidyl peptidase 3 (DPP3) protein or functional derivative thereof.
• DPP3 dipeptidyl peptidase 3
• the aforementioned binder is provided for the use in the prevention or treatment of diseases or acute conditions in a patient, wherein said disease or acute condition is associated with oxidative stress.
• the aforementioned binder is provided for use in the prevention or treatment of diseases or acute conditions in a patient, wherein said disease or acute condition is associated with oxidative stress, and wherein said diseases are selected from a group comprising neurodegenerative diseases, metabolic syndrome, cardiovascular disorders, autoimmune diseases, inflammatory lung diseases, kidney diseases, liver diseases, digestive diseases, viral infectious diseases, cancer, inflammation, sepsis, septic shock and SIRS.
• a binder is provided that is directed to and binding to an epitope according to SEQ ID NO.: 2, and wherein said binder recognizes and binds to at least three amino acids of SEQ ED NO.: 2, and wherein the epitope is comprised in DPP3 as depicted in SEQ ID NO. : 1.
• binder being directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said binder is directed to and binding to an epitope according to SEQ ID NO.: 3, and wherein said binder recognizes and binds to at least three amino acids of SEQ ID NO.: 3, and wherein the epitope is comprised in DPP3 as depicted in SEQ ID NO.: 1.
• Additional subject matter of the invention is the aforementioned binder being directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said binder is directed to and binding to an epitope according to SEQ ID NO.: 4, and wherein said DPP3 binder recognizes and binds to at least three amino acids of SEQ ID NO.: 4, and wherein the epitope is comprised in DPP3 as depicted in SEQ ID NO.: 1..
• binder being directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said binder is selected from a group comprising an antibody or antibody fragment or non-Ig scaffold, and wherein the epitope is comprised in DPP3 as depicted in SEQ ID NO.: 1.
• the above mentioned binder of the second major aspect of the invention that are directed to and binding to an epitope according to SEQ ID NO.: 2 is a dipeptidyl peptidase 3 (DPP3) binder directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein said DPP3 binder recognizes and binds to at least three amino acids of SEQ ID NO.: 2.
• DPP3 dipeptidyl peptidase 3
• binder being directed to and binding to an epitope according to SEQ ID NO.: 2 according to the third aspect of the invention, wherein said binder is a monoclonal antibody or monoclonal antibody fragment, and wherein the complementarity determining regions (CDRs) in the heavy chain comprises the sequences:
• SEQ ID NO.: 7, SEQ ID NO.: 8 and/ or SEQ ID NO.: 9 and the complementarity deteraiining regions in the light chain comprises the sequences: SEQ ID NO.: 10, KVS and/ or SEQ ID NO.: 11.
• binder being directed to and binding to an epitope according to SEQ ID NO.: 2 according to the third aspect of the invention, wherein said binder is a humanized monoclonal antibody or humanized monoclonal antibody fragment, wherein the heavy chain comprises the sequence:
• Additional subject matter of the invention is anyone of the aforementioned binder being directed to and binding to an epitope according to SEQ ID NO.: 2 for use in the prevention or treatment of diseases or acute conditions in a patient, wherein said disease or acute condition is associated with oxidative stress, and wherein the epitope is comprised in DPP3 as depicted in SEQ ID NO.: 1.
• DPP3 binder specifically a dipeptidyl peptidase 3 (hereinafter DPP3) binder, directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein said DPP3 binder recognizes and binds to at least three amino acids (aa), preferably at least 4 aa of SEQ ID NO.: 2.
• DPP3 binder specifically a dipeptidyl peptidase 3 (hereinafter DPP3) binder, directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein said DPP3 binder recognizes and binds to at least three amino acids (aa), preferably at least 4 aa of SEQ ID NO.: 2.
• a binder specifically a DPP3 binder, directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein said DPP3 binder recognizes and binds to at least three amino acids (aa), preferably at least 4 aa of SEQ ID NO.: 2, for use in the prevention or treatment of diseases or acute conditions of a patient, whereby said disease or acute condition is associated with oxidative stress.
• aa amino acids
• subject matter of the present invention is a binder, specifically an anti-DPP3 antibody or an anti-DPP3 antibody fragment, binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof or an anti-DPP3 non-Ig scaffold binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein said 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 4 aa of SEQ ID NO.: 2.
• aa amino acids
• a binder in accordance with the invention specifically an anti-DPP3 antibody or an anti-DPP3 antibody fragment, binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof or an anti-DPP3 non-Ig scaffold binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein said 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 4 aa of SEQ ID NO.: 2, for use in the prevention or treatment of diseases or acute conditions of a patient, whereby said disease or acute condition is associated with oxidative stress.
• aa amino acids
• Further subject matter of the present invention is a method of prevention or treatment of diseases or acute conditions of a patient, whereby said disease or acute condition is associated with oxidative stress, characterized in that a binder directed to and binding to DPP3, or a binder being directed to and binding to SEQ ID.: 2 as epitope that is comprised in DPP3 protein or a functional derivative thereof, or an anti-DPP3 antibody or an anti-DPP3 antibody fragment binding to DPP3 or an anti-DPP3 non-Ig scaffold being directed to and binding to SEQ ID.: 2 as epitope that is comprised in DPP3 protein or a functional derivative thereof is administered to said patient in pharmaceutically effective amounts.
• Subject matter of the present invention is further a pharmaceutical composition
• a pharmaceutical composition comprising a binder directed to and binding to DPP3, or a binder being directed to and binding to SEQ ID.: 2 as epitope that is comprised in DPP3 protein or a functional derivative thereof, or an anti- DPP3 antibody or an anti-DPP3 antibody fragment binding to DPP3 or an anti-DPP3 non-Ig scaffold being directed to and binding to SEQ ID.: 2 as epitope that is comprised in DPP3 protein or a functional derivative thereof for the use in the prevention or treatment of diseases or acute conditions of a patient, whereby said disease or acute condition is associated with oxidative stress.
• Another subject of the present invention is a pharmaceutical composition
• a pharmaceutical composition comprising a binder of the invention, or a DPP3 binder in accordance with the invention, specifically an anti-DPP3 antibody or an anti-DPP3 antibody fragment binding to DPP3 or an anti-DPP3 non-Ig scaffold binding to DPP3 for use in the prevention or treatment of diseases or acute conditions of a patient, whereby said disease or acute condition is associated with oxidative stress as described above, and wherein said pharmaceutical composition comprises at least one additional pharmaceutically active drug which e.g.
• the said binder, or DPP3 binder, anti-DPP3 antibody or the anti-DPP3 antibody fragment binding to DPP3 or the anti-DPP3 non-Ig scaffold binding to DPP3 may act as secondary medicament, which reduces or regulates the said induced oxidative stress.
• a further embodiment of the invention is a kit comprising a binder, or a DPP3 binder in accordance with the invention, specifically an anti-DPP3 antibody or an anti-DPP3 antibody fragment binding to DPP3 or an anti-DPP3 non-Ig scaffold binding to DPP3 for use in the prevention or treatment of diseases or acute conditions of a patient, whereby said disease or acute condition is associated with oxidative stress as described above, and wherein said pharmaceutical composition optionally comprises at least one additional pharmaceutically active drug which e.g.
• DPP3 binder, anti-DPP3 antibody or the anti-DPP3 antibody fragment binding to DPP3 or the anti-DPP3 non-Ig scaffold binding to DPP3 may act as secondary medicament, which reduces or regulates the said induced oxidative stress.
• subject matter of the present invention is a binder in accordance with the invention, or an anti-DPP3 antibody or an anti-DPP3 antibody fragment binding to DPP3 or an anti- DPP3 non-Ig scaffold binding to DPP3 as described above for the use as a medicament, wherein said binder, or antibody or said antibody fragment or said non-Ig scaffold is a modulating binder, antibody or fragment or scaffold.
• said modulating anti-DPP3 antibody or a modulating anti-DPP3 fragment or a modulating non-Ig scaffold is used in the prevention or treatment of diseases or an acute condition in a patient, whereby said disease or acute condition is associated with oxidative stress.
• said modulating anti-DPP3 antibody or an anti- DPP3 antibody fragment or a modulating non-Ig scaffold of the invention regulates the bioactivity ofDPP3.
• DPP3 bioactivity may be defined as the DPP3 enzyme activity or the regulating activity of DPP3 in the oxidative stress pathway.
• said modulating anti-DPP3 antibody or an anti-DPP3 antibody fragment or modulating non-Ig scaffold of the invention may enhance the bioactivity ofDPP3.
• said modulating anti-DPP3 antibody or an anti-DPP3 antibody fragment or modulating non-Ig scaffold of the invention may reduce the bioactivity ofDPP3.
• a "modulating" anti-DPP3 antibody or a modulating anti-DPP3 antibody fragment or a modulating non-Ig scaffold as described above is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or a modulating anti-DPP3 non-Ig scaffold blocks the bioactivity of DPP3 at least 10 %, preferably at least 50 %, more preferably > 50 %, most preferably 100%.
• a modulating binder, or modulating anti-DPP3 antibody or a modulating anti-DPP3 antibody fragment or a modulating anti-DPP3 non-Ig scaffold according to the present invention is used for the prevention or treatment of diseases or an acute condition of a patient, wherein said disease or acute condition is associated with oxidative stress.
• Another embodiment of the present invention is a kit or an assay comprising the above described binder, or anti-DDP3 antibody, and/or an anti-DPP3 antibody ragment binding to DPP3 or an anti-DPP3 non-Ig scaffold binding to DPP3 for use in the prevention or treatment of a disease or acute condition of a patient, whereby said disease or acute condition is associated with oxidative stress.
• DPP3 Dipeptidyl peptidase 3
• Dipeptidyl peptidase 3 also known as Dipeptidyl aminopeptidase III, Dipeptidyl arylamidase III, Dipeptidyl peptidase ⁇ , Enkephalinase B or red cell angiotensinase; short name: DPP3, or DPPIII - is a metallopeptidase that removes dipeptides from physiologically active peptides, such as enkephalins and angiotensins.
• DPP3 a metallopeptidase that removes dipeptides from physiologically active peptides, such as enkephalins and angiotensins.
• DPP3 was first identified and its activity measured in extracts of purified bovine anterior pituitary by Ellis & Nuenke, 1967.
• the enzyme which is listed as EC 3.4.14.4, has a molecular mass of about 83 kDa and is highly conserved in procaryotes and eucaryotes (Prajapati & Chauhan, 2011).
• DPP3 is a mainly cytosolic peptidase, which is ubiquitously expressed. Despite lacking a signal sequence, a few studies reported membranous activity (Lee & Snyder, 1982).
• DPP3 is a zinc-depending exo-peptidase belonging to the peptidase family M49. It has broad substrate specificity for oligopeptides from three or four to ten amino acids of various compositions and is also capable of cleaving after proline. DPP3 is known to hydrolyze dipeptides from the N-terminus of its substrates, including angiotensin II, III and IV; angiotensin 1-7 (Cruz-Diaz et al., 2016); Leu- and Met-enkephalin; endomorphin 1 and 2.
• the metallopeptidase DPP3 has its activity optimum at pH 8.0 - 9.0 and can be activated by addition of divalent metal ions, such as Co 2+ and Mg 2+ .
• Structural analysis of DPP3 revealed the catalytic motifs HELLGH (hDPP3 450 - 455) and EECRAE (hDPP3 507 - 512), as well as the following amino acids, that are important for substrate binding and hydrolysis: Glu 316, Tyr 318, Asp 366, Asn 391, Asn 394, His 568, Arg 572, Arg 577, Lys 666 and Arg 669 (Prajapati & Chauhan, 2011; Kumar et al., 2016; numbering refers to the sequence of human DPP3, see SEQ ID No.: 1). Considering all known amino acids or sequence regions that are involved in substrate binding and hydrolysis, the active site of human DPP3 can be defined as the region between amino acids 316 and 669.
• DPP3 has been also shown to be a promising biomarker in several publications. It has been shown that DPP3 activity is elevated in homogenates of ovarian and endometrial tumors. DPP3 activity even increases with the severity/malignancy of said tumors (Simaga et al., 1998 and 2003). Immune histology and western blot analysis of glioblastoma cell lines also revealed elevated DPP3 levels (Singh et al., 2014).
• DPP3 was also proposed to be a potential arterio-risk marker (US 2011008805) and as a marker for rheumatoid arthritis (US 2006177886).
• the patent application WO 2005/106486 describes DPP3-expression and activity as diagnostic marker and DPP3 as therapeutic target in all kinds of diseases, due to ubiquitous expression of DPP3 in or at surface of cell.
• EP 1498480 mentions the potential diagnostic and therapeutic use of hydrolytic enzymes, including DPP3.
• the relevant prior art can be further summarized as follows:
• WO 2005/106486 describes in a general manner a method of screening for therapeutic agents which may be useful in the treatment of diseases, comprising cardiovascular diseases, infections, respiratory diseases, cancer, endocrinological diseases, metabolic diseases, gastroenterological diseases, inflammation, haematological diseases, muscle skeleton diseases, neurological and urological diseases.
• a test compound is contacted with a DPP3 polynucleotide and the binding between said test compound and said DPP3 polynucleotide is detected.
• the document describes in a general manner compounds, which may bind to and / or activate or inhibit the activity of DPP3.
• the invention describes pharmaceutical compositions, which comprise such compounds.
• Hast et al. 2013 describe a comparison of the spectrum of KEAP1 interacting proteins with the genomic profile of 178 squamous cell lung carcinomas characterized by The Cancer Genome Atlas and reveal amplification and mRNA over-expression of the DPP3 gene in tumors with high Nrf2activity but lacking Nrf2 stabilizing mutations. They further describe that tumor-derived mutations in KEAPl are hypomorphic with respect to Nrf2 inhibition and that DPP3 over-expression in the presence of these mutants further promotes Nrf2 activation.
• DPP3 intracellular DPP3 was identified as an activator of the antioxidant response element (ARE) in an unbiased screen of a cDNA library consisting of approximately 15,000 full-length human expression cDNAs (Liu et al. 2007).
• ARE antioxidant response element
• DPP3 disrupt the KEAPl -Nrf2 complex by competing with Nrf2 about the KEAPl binding site (Hast et al. 2013). This disruption prevents NRF2 degradation and subsequently leads to translocalization of Nrf2 into the nucleus and ARE activation.
• Overexpression of DPP3 in neuroblastoma cells in neuroblastoma cells (Liu et al. 2007), in HEK293T cells (Hast et al. 2013) or in MCF7 breast cancer cells (Lu et al. 2017) activates Nrf2-mediated transcription. Active and inactive variants of DPP3 were overexpressed in MCF7 cells and showed the same regulatory effect on oxidative stress (Lu et al. 2017). Hast et al.
• AD Alzheimer's disease
• DPP3 is known for being expressed as membranous, intracellular or circulating DPP3.
• DPP3 has been not only proposed as potential biomarker but also as potential therapeutic target due to its ability to cleave several bioactive peptides. Influence A virus changes host DPP3 levels for own replication (cell culture studies, Meliopoulos et al. 2012). Enkephalin and/or angiotensin degrading enzymes in general, including DPP3, have a therapeutic potential as targets for treatment of pain, cardiovascular diseases (CVD) and cancer and the corresponding inhibitors as potential treatments of pain, mental illnesses and CVD (Khaket et al; 2012, Patel et al. 1993, Igic et al. 2007).
• CVD cardiovascular diseases
• DPP3 can be inhibited unspecifically by different general protease inhibitors (e.g. PMSF, TPCK), sulfhydryl reagents (e.g. pHMB, DTNB) and metal chelators (EDTA, o-phenantroline) (Abramid et al. 2000, EP 2949332).
• general protease inhibitors e.g. PMSF, TPCK
• sulfhydryl reagents e.g. pHMB, DTNB
• EDTA metal chelators
• DPP3 activity can be further inhibited specifically by different kinds of compounds: an endogenous DPP3-inhibitor is the peptide spinorphin.
• peptide spinorphin Several synthetic derivatives of spinorphin, e.g. ⁇ , have been produced and shown to inhibit DPP3 activity to varying extents (Yamamoto et al. 2000).
• Other published peptide inhibitors of DPP3 are propioxatin A and B (US 4804676) and propioxatin A analogues (Inaoka et al. 1988).
• DPP3 can also be inhibited by small molecules such as fluostatins and benzimidazol derivatives. Fluostatins A and B are antibiotics produced in Streptomyces sp.
• TA-3391 that are non-toxic and strongly inhibit DPP3 activity.
• 20 different derivatives of benzimidazol have been synthesized and published (Agic et al., 2007; Rastija et al., 2015), of which the two compounds ⁇ and 4' show the strongest inhibitory effect (Agid et al., 2007).
• Several dipeptidyl hydroxamic acids have been shown to inhibit DPP3 activity as well (Cvitesic et al., 2016).
• Oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species (hereinafter ROS) / reactive nitrogen species (hereinafter RNS) and antioxidants in favour of excessive generation of free radicals. This process leads to the oxidation of biomolecules with consequent loss of its biological functions and/or homeostatic imbalances, whose manifestation is the potential oxidative damage to cells and tissues. Accumulation of ROS / RNS can result in a number of deleterious effects such as lipid peroxidation, protein oxidation and DNA damage (including base damage and strand breaks). Further, some reactive oxidative species act as cellular messengers in redox signalling. Thus, oxidative stress can cause disruptions in normal mechanisms of cellular signalling.
• ROS reactive oxygen species
• RNS reactive nitrogen species
• ROS and RNS are the terms collectively describing free radicals and other non-radical reactive derivatives, which are also called oxidants. Radicals are less stable than non-radical species, although their reactivity is generally stronger. A molecule with one or more unpaired electron in its outer shell is called a free radical. Free radicals are formed from molecules via the breakage of a chemical bond such that each fragment keeps one electron, by cleavage of a radical to give another radical and, also via redox reactions. Free radicals related to oxidative stress include hydroxyl ( ⁇ ), superoxide ( ⁇ 2 ⁇ -), nitric oxide ( ⁇ ), nitrogen dioxide ( ⁇ 2 ⁇ ), peroxyl (ROO ⁇ ) and lipid peroxyl (LOO «).
• H2O2 hydrogen peroxide
• ozone O 3
• singlet oxygen ⁇ 2
• hypochlorous acid HOC1
• nitrous acid HNO 2
• peroxynitrite ONOO ⁇
• dinitrogen trioxide N2O 3
• lipid peroxide LOOH
• ROS and RNS can occur in the cells by two ways: enzymatic and non- enzymatic reactions.
• Enzymatic reactions generating free radicals include those involved in the respiratory chain, the phagocytosis, the prostaglandin synthesis and the cytochrome P4S0 system.
• Free radicals can be produced from non-enzymatic reactions of oxygen with organic compounds as well as those initiated by ionizing radiations.
• the non-enzymatic process can also occur during oxidative phosphorylation (i.e. aerobic respiration) in the mitochondria.
• oxidative stress is linked to a number of diseases, including but not limited to neurodegenerative diseases, metabolic syndrome, cardiovascular disorders, autoimmune diseases, inflammatory lung diseases, kidney diseases, liver diseases, digestive diseases, viral infectious diseases, cancer and inflammation, and thus associated therewith.
• DPP3 intracellular DPP3 is known to be closely linked to oxidative stress regulation.
• DPP3 was identified as an activator of the antioxidant response element (ARE) in an unbiased screen of a cDNA library consisting of approximately 15,000 full-length human expression cDNAs (Liu et al. 2007; see also above).
• ARE antioxidant response element
• Nrf2 is a transcription factor that controls the basal and induced expression of an array of antioxidant response element-dependent genes to regulate the physiological and pathophysiological outcomes of oxidant exposure. Under normal or unstressed conditions, Nrf2 is bound to Kelch like-ECH-associated protein 1 (KEAP1) via its ETGE and its DLG motif. Within this protein cluster Nrf2 is kept in the cytoplasm, quickly ubiquinated and degraded by proteasome.
• KEAP1 Kelch like-ECH-associated protein 1
• Nrf2 Under oxidative stress, Nrf2 is not degraded, but instead translocates to the nucleus where it binds to a DNA promoter and induces expression of an array of antioxidant response element (ARE)-dependent genes.
• ARE antioxidant response element
• oxidative stress can be also reduced or regulated by a binder directed to and binding to a DPP3 protein or functional derivative thereof.
• oxidative stress can be also reduced or regulated by a binder directed to and binding to an epitope of SEQ ID NO.: 2, wherein the epitope is comprised in the DPP3 protein.
• the present inventors found a dipeptidyl peptidase 3 (hereinafter DPP3) binder directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein said DPP3 binder recognizes and binds to at least three amino acids (aa), preferably at least 4 aa of SEQ ID NO.: 2.
• DPP3 binder recognizes and binds to at least three amino acids (aa), preferably at least 4 aa of SEQ ID NO.: 2.
• oxidative stress may be reduced or regulated by an anti-DPP3 antibody or an anti DPP3-antibody fragment directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein said DPP3 binder recognizes and binds to at least three amino acids (aa), preferably at least 4 aa of SEQ ID NO.: 2 or an anti- DPP3 non-Ig scaffold directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein said DPP3 binder recognizes and binds to at least three amino acids (aa), preferably at least 4 aa of SEQ ID NO.: 2.
• the present invention provides the herein disclosed binder, and anti-DPP3 antibody or an anti-DPP3 antibody fragment binding to DPP3 or an anti-DPP3 non-Ig scaffold binding to DPP3 for use in methods of the preventive treatment or treatment of diseases or acute conditions of a patient, whereby said disease or acute condition is associated with oxidative stress.
• binder DPP3 binder, and specifically the anti-DPP3 antibody, or anti DPP3 -antibody fragment binding to DPP3 or an anti-DPP3 non-Ig scaffold binding to DPP3, the inventors have found binder to DPP3 which rapidly reduce or regulate oxidative stress in cells of a mammal when determined by the methods of respective biomarker measurements as further set out below.
• Another subject of the present invention is a pharmaceutical composition
• a pharmaceutical composition comprising the binder of the invention, DPP3 binder of the invention, and specifically comprising the anti- DPP3 antibody or an anti-DPP3 antibody fragment binding to DPP3 or an anti-DPP3 non-Ig scaffold binding to DPP3 of the invention for use in methods of the prevention or treatment of diseases or acute conditions of a patient, whereby said disease or acute condition is associated with oxidative stress.
• compositions are also provided for use in the prevention or treatment of symptoms, or syndromes, or pathological and acute conditions and disease associated problems, which are mediated by oxidative stress.
• oxidative stress is linked to a number of diseases or disorders, which in accordance of the invention include:
• neurodegenerative diseases wherein said neurodegenerative diseases may be selected from a group comprising Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS)),
• AD Alzheimer's disease
• PD Parkinson's disease
• HD Huntington's disease
• ALS amyotrophic lateral sclerosis
• MS multiple sclerosis
• metabolic syndrome wherein said metabolic syndrome may be selected from a group comprising insulin resistance, obesity, hyperglycemia, dyslipidemia, hypertension and diabetes,
• cardiovascular disorders wherein said cardiovascular disorders may be selected from a group comprising atherosclerosis, hypertension, heart failure, cardiovascular ischemia, cerebral ischemic injury, stroke and myocardial infarction,
• autoimmune diseases wherein said autoimmune diseases may be selected from a group comprising rheumatoid arthritis, systemic lupus erythematosus,
• inflammatory lung diseases wherein said inflammatory lung diseases may be selected from a group comprising COPD, asthma,
• kidney diseases wherein said kidney diseases may be selected from a group comprising renal toxicity (drug-induced kidney disease), acute kidney injury (AKJ), chronic kidney disease (CKD), diabetic nephropathy, end-stage renal disease (ESRD),
• AKJ acute kidney injury
• CKD chronic kidney disease
• ESRD end-stage renal disease
• liver diseases wherein said liver diseases may be selected from a group comprising hepatotoxicity, viral hepatitis, cirrhosis,
• digestive diseases wherein said digestive diseases may be selected from a group comprising inflammatory bowel disease e.g. Ulcerative colitis, Crohn's disease, gastritis, pancreatitis and peptic ulcer, • viral infectious diseases wherein said viral infectious diseases may be selected from a group comprising blood-borne hepatitis viruses (B, C, and D), human immunodeficiency virus (HIV), influenza A, Epstein-Barr virus, respiratory syncytial virus,
• inflammatory bowel disease e.g. Ulcerative colitis, Crohn's disease, gastritis, pancreatitis and peptic ulcer
• viral infectious diseases wherein said viral infectious diseases may be selected from a group comprising blood-borne hepatitis viruses (B, C, and D), human immunodeficiency virus (HIV), influenza A, Epstein-Barr virus, respiratory syncytial virus,
• cancer wherein said cancer may be selected from a group comprising prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, skin cancer, stomach cancer, liver cancer,
• Table 1 The association of oxidative stress with diseases in accordance with the invention
• 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, tardive dyskinesia (TD), epilepsy and acute diseases of the central nervous system, such as spinal cord injuries and/or brain traumatic.
• the human brain is vulnerable to oxidative stress due to many facts such as (i) metabolism of catecholamines; (ii) decrease in antioxidants; (iii) presence of transition metals; (iv) occurrence of brain trauma/injury; and also (v) the brain is an organ that proportionally requires more oxygen and (vi) expresses low levels of antioxidant enzymes, which contribute to formation of ROS.
• lipid membrane As a consequence of redox unbalance in brain, one of the most affected structures is the lipid membrane (Rao and Balachandran 2002. Nutritional Neuroscience 5: 291-309). A common feature of these diseases is oxidative damage of neurons, which might be responsible for the dysfunction or death of neuronal cells that contributes to disease pathogenesis.
• AD Alzheimer's disease
• ⁇ B-amyloid
• ⁇ proteins can directly initiate free radical formation via the activation of NADPH oxidase.
• inflammation is responsible for increased expression of cytokines, ROS levels, and cellular toxicity, thereby exacerbating AD progression.
• HD Huntington's disease
• CAG cytosine, adenine, guanine
• the expansion of CAG repeats within the exonl of the HIT gene gives rise to a mutation that leads to the elongation of polyglutamine tract, resulting in an HIT protein product that is susceptible to aggregation.
• the mHTT aggregates are accumulated throughout the brain of the affected individuals, which can interrupt protein quality control and transcription process. Those alterations are potentially responsible for the aberrant motor and cognitive problems in HD.
• oxidative damage is not much reported in the early stages of HD, it is proposed as one of the major mechanisms in HD as it progresses.
• Elevated oxidative stress plays a critical role in the late stage of HD pathogenesis. Impairment in the electron transport chain and mitochondrial dysfunction are the major mechanisms involved in the ROS mediated etiopathogenesis of HD. Dysfunction in the oxidative phosphorylation components has been documented in the brain tissues of HD patients. HD patients showed an increased level of oxidative stress markers accompanied by a decrease in antioxidant status compared to healthy subjects. For review see Liu et al. 2017. Oxidative Medicine and Cellular Longevity 2525967: Manoharan et al. 2016. Oxid Med Cell Loneev 8590578.
• Parkinson's disease the most common neurodegenerative disease of the elderly, is characterized by progressive loss of muscle control. PD is predominant at the 6 th decade of life and men are 1.5 to 2 times more likely to contract the disease than women. Head trauma, illness, or exposure to environmental toxins is identified as a risk factor. This neurodegenerative disorder is characterized by tremor, rigidity, bradykinesia, and impairment in balance. PD also causes cognitive, psychiatric, autonomic, and sensory disturbances. The pathology of PD is characterized by the gradual and selective loss of dopaminergic neurons in the substantia nigra pars compacta. Imbalance in dopamine metabolism due to oxidative stress has been recognised as a contributor to this disease.
• the major pathological findings include the presence of Lewy bodies in the substantia nigra and loss of nerve cells in the portions of its ventral tier.
• Several studies have reported impaired respiratory chain and somatic mitochondrial DNA mutations in the brain of patients with PD, which suggests the extensive role of oxidative metabolism in PD.
• Enhanced dopamine metabolism in the brain of patients with PD could account for the accumulation of toxic radicals such as hydroxyl in the brain.
• Iron accumulation in the neurons in the redox active form plays a crucial role in pathogenesis of this disease.
• Amyotrophic lateral sclerosis is characterized by progressive loss of motor neurons in the anterior horn of the spinal cord. It is classified as either familial or sporadic depending on whether there is a clearly defined, inherited genetic element. Sporadic ALS (sALS) typically emerges between SO and 60 years old. The onset of sALS is unknown, and thus the identification of causal genes and environmental factors remains elusive. In familial ALS, about 20% of the cases resulted from mutations in SOD1. The functions of SOD1 are diverse and include scavenging excessive superoxide radical, modulating cellular respiration, energy metabolism, and posttranslational modification. SOD dysfunction leads to a loss of antioxidant capability.
• MS Multiple sclerosis
• CNS central nervous system
• ROS reactive oxygen species
• Oxidative stress damages the mitochondria, which disrupts the transport of adenosine triphosphate along the axon, and consequently leads to neurodegeneration.
• Oxidative stress is associated with the dysregulation of axonal bioenergetics, cytokine-induced synaptic hyperexcitability, abnormal iron accumulation, and the oxidant/antioxidant balance. Markers of oxidative stress assessed in the serum, erythrocytes CSF, saliva, and urine may have diagnostic properties whereas antioxidants may have clinical application in the future. For review see Adamczvk and Adamczvk-Sowa 2016. Oxidative Medicine and Cellular Longevity 1973834.
• Oxidative stress is related to metabolic syndrome and its individual component pathologies, e.g. obesity, insulin resistance, dyslipidemia, impaired glucose tolerance and high blood pressure.
• the metabolic syndrome was defined by the World Health Organization criteria (Albert and Zimmet 1998. Diabet Med 15:539-553: World Health Organization. 1999. Definition, diagnosis and classification of diabetes mellitiis and its complications: report of a WHO Consultation. Part 1: diagnosis and classification of diabetes mellitus. Geneva.
• oxidative stress has emerged as playing a central role in metabolic syndrome and its component pathologies and may be a unifying factor in the progression of this disease. Moreover, oxidative stress has been identified as a major mechanism of micro- and macrovascular complications in the metabolic syndrome. For review see Hutcheson and Rocic 2012. Exp Diabetes Res. 2012:271028. There is a bulk of evidence demonstrating that mitochondrial ROS (predominantly superoxide anion) overproduction is involved in diabetes and diabetic complications.
• oxidative stress plays an important role in the pathogenesis and development of cardiovascular diseases, including hypertension, dyslipidernia, atherosclerosis, myocardial infarction, angina pectoris, and heart failure (Elahi et al. 2009. Oxidative Medicine and Cellular Longevity 2(5): 259-269).
• One of the key concepts of free radical mediated pathogenesis of cardiovascular disease is endothelial dysfunction, whereby the regulation of vascular wall microenvironment is disrupted.
• ROS activity in the vessel wall for example, is thought to contribute to the formation of oxidized LDL, a major contributor to the pathogenesis of atherosclerosis.
• Oxidative stress also plays a role in the ischemic 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).
• MI myocardial infarction
• brain ischemia/reperfusion multiple detrimental processes take place, including overproduction of oxidants, inactivation of detoxification systems, and consumption of antioxidants. These changes cause the disruption of the normal antioxidative defense ability of brain tissue (Chen et al. 2011. Antioxidants and Redox Signaling 14(8): 1505-1517).
• Oxidative Medicine and Cellular Longevity 2(5): 259-269 For further review see Elahi et al. 2009. Oxidative Medicine and Cellular Longevity 2(5): 259-269.
• Oxidative stress is thought to have an important role in the pathogenesis of autoimmune diseases. Many studies have shown that T and B lymphocytes contribute to the pathogenesis of autoimmune diseases by the production of autoantibodies and ROS under environmental and genetic influence. Oxidative stress has been implicated in autoimmune disorders (rheumatoid arthritis, systemic lupus erythematosus, psoriasis, and celiac disease) where it plays an important role in the disease process. Oxidative stress is increased in systemic lupus erythematosus (SLE), and it contributes to immune system deregulation, abnormal activation and processing of cell-death signals, autoantibody production and fatal comorbidities.
• SLE systemic lupus erythematosus
• RA Rheumatoid arthritis
• RA is an autoimmune disease characterized by chronic inflammation of the joints and tissue around the joints with infiltration of macrophages and activated T cells. The pathogenesis of this disease is due to the generation of ROS and RNS at the site of inflammation. RA is one of the conditions that induce oxidative stress.
• oxidative stress is a pathogenic hallmark in RA.
• Free radicals are indirectly implicated in joint damage because they also play an important role as secondary messengers in inflammatory and immunological cellular response in RA. T-cell exposure to increased oxidative stress becomes refractory to several stimuli including those for growth and death and may perpetuate the abnormal immune response.
• free radicals can degrade directly the joint cartilage, attacking its proteoglycan and inhibiting its synthesis (for review see Ouinonez-Flores et al. 2016. Biomed Res Int. 2016:6097417).
• inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) are characterized by systemic and local chronic inflammation and oxidative stress.
• An important source for increased airway oxidative stress is the recruitment of inflammatory cells into the airway after exposure to trigger factors. These activated cells can generate anion superoxide through reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase pathway.
• NADPH nicotinamide adenine dinucleotide phosphate
• Mitochondrial dysfunction in airway epithelial cells which occurs in response to mechanical and environmental stimuli, can also contribute to the formation of anion superoxide and airway oxidative stress.
• Subjects with asthma have greater systemic and airway increased oxidative stress, which is associated with worse asthma severity.
• IBD Inflammatory bowel disease
• GI gastrointestinal
• Crohn's disease (CD) and ulcerative colitis (UC) are the principal types of IBD.
• CD may occur in any region of the GI tract involving the ileum and colon in a discontinuous pattern by transmural inflammation, while UC affects only the colon and rectum continuously and is restricted to the mucosa.
• Oxidative stress signalling is involved in and contributes to the development of IBD through multiple levels of function. Oxidative stress leads to damages of the mucosal layer in the GI tract and bacterial invasion, which in turn stimulates the immune response and initiates IBD.
• Celiac disease is an immune-mediated chronic inflammatory disorder of the upper small intestine induced by gluten and related prolamines in genetically susceptible individuals.
• environmental, genetic, and immunological factors may be involved in the pathogenesis of CD.
• oxidative stress is also implicated in the pathogenesis of CD.
• activation of xanthine oxidase is one of the mechanisms of ROS overproduction in small intestinal mucosa of celiac patients.
• Gastritis is defined as inflammation of the stomach mucosal lining and occurs in several conditions including H. pylori infection, NSAID use, alcohol consumption, and stress.
• Peptic ulcer disease occurs in the proximal GI tract and is often associated with chronic gastritis. Gastric and duodenal ulcers represent the most common and chronic PUDs. Gastritis and peptic ulcer are caused by multiple factors, both endogenous and exogenous, and free radicals are closely linked to both conditions.
• ROS ROS
• Reduced antioxidant enzyme SOD levels and antioxidant vitamin intake contribute to the accumulation of ROS associated with gastroduodenal inflammatory diseases. Ethanol-induced gastric inflammation is associated with increased superoxide generation.
• Phagocytic leukocytes are the main source of ROS in chronic inflammation such as one observes in H. pylori induced gastritis and IBD. Significant numbers of neutrophils and/or macrophages infiltrate the gastric mucosa during inflammation, generating large amounts of ROS. For review see Bhattacharwa et al. 2014. Phvsiol Rev 94: 329-354. Oxidative stress also plays a critical role in liver diseases like viral Hepatitis (Type A, B and C) and liver cirrhosis. It has been clearly established that hepatitis C is associated with strong oxidative stress.
• liver tissues and in blood serum/plasma samples of Cchronic hepatitis C patients using a variety of techniques, including direct measurement of ROS, quantification of DNA, lipid and protein oxidation products, as well as by assessing the total oxidant/antioxidant status or the levels of individual antioxidants.
• Screening of the liver biopsies of chronic hepatitis C virus carriers revealed significant elevation of the levels of oxygen radicals and stress markers malondialdehyde (MDA) and 4-hydroxynonenal- (HNE)- and other protein adducts.
• MDA malondialdehyde
• HNE 4-hydroxynonenal-
• serum/plasma of such patients is characterized by increased levels of a wide array of oxidative stress markers such as MDA, lipid peroxides, protein carbonyl content or thioredoxin (for review see Ivanov et al. 2017. Oncotarget. 2017. Vol. 8. (No. 3). pp: 3895-3932).
• Patients with chronic hepatitis B exhibit signs of pronounced oxidative stress. Levels of oxygen radicals in liver specimens from these patients exceed the levels in healthy people. Patients with hepatitis B exhibit signs of oxidative stress not only in the liver but also in plasma/sera. Chronic hepatitis B is accompanied by an increase in total oxidant status and a concomitant reduction of total antioxidant status. Plasma/serum of these patients was also characterized by the elevated levels of ROS, including H 2 0 2 , and oxidation products of lipids and proteins. Oxidative stress is not just a hallmark of chronic HBV infection and advanced liver disease; it is also observed in acute and occult hepatitis B, as well as in asymptomatic HBV infections.
• Occult hepatitis B infection is characterized by increased levels of ROS in lymphocytes and consequent DNA damage.
• Cirrhosis is a complication of many forms of chronic liver diseases and is a late stage of fibrosis, in which regenerative nodular formation surrounded by fibrous bands of the liver.
• Hepatotoxicity implies chemical-driven liver damage. Drug-induced liver injury is a cause of acute and chronic liver disease. Drug-induced liver injury is responsible for 5% of all hospital admissions and 50% of all acute liver failures. The liver is the most frequently targeted organ in terms of drug toxicity.
• the production of radical species, specifically ROS and RNS, has been proposed as an early event of drugs hepatotoxicity and as an indicator of hepatotoxic potential. It has been discovered that a lot of drugs could induce oxidative stress including increase of cellular oxidants and lipid peroxidation, depletion of antioxidants in the liver, such as anti-inflammation drugs, anti-analgesic drugs, anti-cancer drugs and antidepressants (Li et al. 2015. Int. J. Mol. Sci.
• CKD chronic kidney disease
• ESRD end stage renal disease
• TRX1 urinary thioredoxin 1
• AKI acute kidney injury
• AKI drugs-induced kidney injury
• AKI causes a severe condition associated with high probabilities of developing progressive chronic kidney disease or end-stage renal disease, thus leading to high mortality rates.
• Most drugs found to cause nephrotoxicity exert toxic effects by one or more common pathogenic mechanisms. These include altered intraglomerular hemodynamics, tubular cell toxicity, inflammation, crystal nephropathy, rhabdomyolysis, and thrombotic microangiopathy.
• AKI includes acute tubular necrosis (ATN) and acute interstitial nephritis (AIN).
• a mechanism underlying ATN is oxidative stress.
• Proximal tubular toxicity develops due to direct nephrotoxic effects such as mitochondrial dysfunction, lysosomal hydrolase inhibition, phospholipid damage, and increased intracellular calcium concentration, leading to formation of reactive oxygen species (ROS) with injurious oxidative stress (Hosohata 2016. Int. J. Mol. Sci. 17: 1826).
• ROS reactive oxygen species
• Medications that are potentially harmful to the kidneys are, e.g. antimicrobials like antibiotics (for example streptomycin, gentamicin) or antivirals (for example acyclovir, foscarnet) or antifungal (for example amphotecerin B), analgesics, non- steroidal anti-inflammatory drugs (NSAID) (for example ibuprofen, naproxen), diuretics, proton pump inhibitors, chemotherapeutics (for example cisplatin), contrast dyes, cardiovascular agents like ACE-inhibitors or statins, anti-depressants, immune suppressants (for example cyclosporine A) and antihistamines (for reference see Naughton 2008. Am Fam Physician. 2008:78(6):743-750. Table 1: Hosohata 2016. Int. J. Mol. Sci. 17: 1826).
• NSAID non- steroidal anti-inflammatory drugs
• chemotherapeutics for example cisplatin
• contrast dyes for example cardiovascular
• ROS generated by the respiratory chain in the mitochondria and by the Nox enzymes in the cytoplasm are particularly important.
• Nox proteins are now considered to be oncogenic proteins, and mitochondrial dysfunction is associated with tumorigenesis.
• Oxidative stress is involved in all stages of carcinogenesis and there is a dose-dependent association between level of the persistent or chronic oxidative stress and the tumor stage.
• the carcinogenesis process the normal cells are transformed into abnormal cells owing to a number of structural changes and mutations in genes expression.
• carcinogenesis is described by three main stages: initiation, promotion and progression. All these stages have been postulated to be linked with contribution of ROS and RNS.
• ROS have an important role in the pathophysiological states involved in neovascularization.
• ROS-generating enzymes such as NADPH oxidases (e.g., Nox: Nox 1-5), activate redox signaling pathways that ultimately lead to angiogenesis.
• NADPH oxidases e.g., Nox: Nox 1-5
• redox signaling pathways that ultimately lead to angiogenesis.
• Prostate cancer is the most frequently diagnosed non-cutaneous malignancy in males. This is a multi-focal, filed-type disease, which forms solid tumors of glandular origin. Prostate cancer is mainly a disease of aging, with most cases occurring in men over the age of 55. Over the last decade association between prostate cancer risk and oxidative stress has been recognized, and epidemiological, experimental and clinical studies have unequivocally proven a role for oxidative stress in the development and progression of this disease, commonly associated with a shift in the antioxidant-prooxidant balance towards increased oxidative stress.
• the excessive production of ROS in breast cancer cells include: a strong expression of thymine phosphorylase leading to degradation of thymidine to thymine and 2-deoxy-D-ribose phosphate; oxidation of 17-estradiol panoxyl radicals to lactoperoxidase participating in metabolism of estrogens and inflammation.
• antioxidant changes are related to breast cancer risk, for example the levels of SOD and GPX were found to be higher in the blood of breast cancer cases compared to that of healthy women as a reply on the increased production of superoxide and hydrogen peroxide.
• the tumor suppressor gene breast cancer gene 1 (BRCA1) is mutated in 40-50% of hereditary breast cancers and absent or expressed at low levels in 30-40% of sporadic breast cancers.
• BRCA1 is a caretaker gene that is responsible for repairing DNA, and it is able to upregulate several genes involved in the antioxidant response by controlling the activity of the transcription factors Nrf2 and NfkB.
• Nrf2 and NfkB Apart from the inhibitory action of BRCA1 on ROS generation, BRCA1 also reduces the levels of protein nitration due to RNS accumulation in cells, and it enhances DNA repair processes that ultimately help to cope with oxidative stress.
• respirable particles or fibrous dusts to penetrate the respiratory system and reach the lung alveoli in order to generate ROS and other oxidants or free radicals is suggested to be the main factor involved in their pathogenic potential.
• Synergistic mechanisms of inhalable particulate matter (penetrating deep into the lung's alveoli) and other components of air pollution (ozone, nitric oxide, soot, heavy metals, PAHs) and tobacco smoke have been studied.
• the porous surfaces of airborne particles provide a fertile ground for catalyzing the increased generation of ROS or other damaging oxidants, which are potential initiators of pulmonary carcinogenesis.
• Colorectal cancer is one of the most common cancers worldwide, with the highest incidence rates in western countries. Colon cancer originates from the epithelial cells that line the bowel. These cells divide rapidly and have a high metabolic rate, which has been found as a potential factor that may be responsible for increased oxidation of DNA. It was found that the human colorectal tumors (adenomas and carcinomas) have increased levels of different markers of oxidative stress, such as increased levels nitric oxide (NO), 8-oxodG in DNA, lipid peroxides, glutathione peroxidase (GPx), catalase (CAT), and decreased methylation of cytosine in DNA.
• NO nitric oxide
• 8-oxodG lipid peroxides
• GPx glutathione peroxidase
• CAT catalase
• bladder cancer is the fourth most frequently occurring malignant tumors. Recent studies indicate the involvement of oxidative and nitrosative stress in the formation and development of this disease. Red-ox disorders are characteristic for both, the initiation and progression of bladder cancer. There are observed changes in the activity of transcription factors, such as nuclear factor NF-kB; transcription factors: AP-1, Nrf2 and STAT3 and hypoxia-inducible factor HIF- ⁇ . In addition, studies indicate a role for oxidative stress in the regulation of MAPK cascade and its involvement in carcinogenesis consisting bladder. Nitric oxide also plays an important role in tumor biology. Numerous studies show that the bladder cancer is characterized by an intensified production of NO.
• Ovarian cancer is the fifth leading cause of cancer death; the leading cause of death from gynecologic malignancies, and the second most commonly diagnosed gynecologic malignancy.
• the overwhelming majority of ovarian cancers are derived from ovarian surface epithelium. Metastasis is achieved through detachment of single cells or clusters of cells from the primary tumor followed by implantation on peritoneal mesothelial lining.
• Ovarian, endometrial, and cervical cancer consist a great problem in oncology due to their diagnosis in advanced stage. Research finding have shown that oxidative stress plays a causal role in the carcinogenesis of two subtypes of ovarian cancer: clear cell carcinoma and endometriosis carcinoma.
• EOC epithelial ovarian cancer
• Endometrial cancer has been reported to be associated with endometriosis disease, and the high levels of free iron hemosiderin or heme in endometrial cysts are considered as a main factor responsible for the oxidative stress development and chronic inflammation.
• Cervical cancer is the second most common cancer in women worldwide being a subject of intensive research.
• oxidative stress in cervical, indicating that antioxidants can alter the redox balance in cervical cancer cells, inhibit transcription factors AP-1 and NF- ⁇ or induce cell apoptosis.
• For review 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.
• Oxidative damage induced by OS has been also implicated in leukemia and the decreased levels of antioxidants and oxidatively modified DNA and lipids caused by high ROS production were found in serum of chronic lymphocytic leukemia patients. Moreover, it was found that the chronic leukemia cells were able to adapt to intracellular OS through upregulation the stress-responsive hemeoxygenase- 1 confirming involvement of ROS in the pathogenesis of leukemia cancer. Also, GSH depletion in lymphocytes of the chronic lymphocytic has been demonstrated in leukemia B patients. For review see Kruk and Aboul- Kunststoff- Kunststoff- Kunststoff- Kunststoff- Kunststoff- Kunststoff- Kunststoff- Kunststoffe.
• GC Gastric cancer
• liver carcinogenesis There are many factors involved in liver carcinogenesis, including hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, alcohol abuse, and nonalcoholic fatty liver disease (NAFLD), aflatoxin Bl, obesity, diabetes, dietary habits, and iron accumulation.
• HBV hepatitis B virus
• HCV hepatitis C virus
• NAFLD nonalcoholic fatty liver disease
• oxidative stress can be triggered by any dangerous or inflammatory signal and affects multiple cells in the liver.
• Liver injury can be either an acute or a chronic inflammatory process.
• liver sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs), dendritic cells (DCs), and Kupffer cells (KCs) are activated.
• HBV- and HCV-related chronic inflammation and fibrosis of the liver are usually induced by oxidative stress, which contributes to the pathogenesis of hepatocarcinogenesis.
• HBV infection results in activation of macrophages to produce a variety of proinflammatory cytokines, such as IL- ⁇ , IL-6, CXCL-8, and TNF-a.
• cytokines such as IL- ⁇ , IL-6, CXCL-8, and TNF-a.
• HCV-induced oxidative stress contributes to the development of hepatocellular carcinoma (HCC).
• oxidative stress markers in chronic hepatitis C patients correlate positively with the probability of development of HCC and can serve as prognostic markers for HCC recurrence in chronic hepatitis C patients who underwent liver transplantation.
• Carcinogenesis is orchestrated by multiple ROS-mediated processes. For review see Wang et al. 2016. Oxidative Medicine and Cellular Longevity 7891574. Skin is a major environmental interface for the body, which accidentally or occupationally gets exposed to a number of chemical mutagens and carcinogens. Skin cancer represents a major and growing public health problem. It accounts for more than 40 % of all new cancer diagnosed.
• Reactive species generated during infection may have serious consequences for the disease once they are released to any degree.
• the oxidative stress can initiate adverse effects in different organs. Development of oxidative stress can be accelerated in the course of hypoxia. Hypoxia is a known complication of infectious diseases. Hypoxia is not peculiar to one disease. Influenza, viral hepatitis and tuberculosis are all examples of infectious diseases in which hypoxia takes place.
• Direct generation of reactive oxygen species can be initiated by metals.
• iron, cooper, and cadmium can catalyze the development of oxidative stress by Fenton reaction in which hydrogen peroxide is converted into hydroxyl radical and hydroxide anion. Heavy metals are involved in pathological processes linked to infections like other pathologies.
• livers damaged by viral hepatitis are vulnerable to heavy metals due to faulty elimination processes.
• Clinical studies on patients with viral hepatitis A, B, C, D, and E demonstrated that accumulation of copper and iron caused oxidative stress and oxidative damage to patients' liver tissue.
• AIDS is accompanied by imbalance in oxidative homeostasis. Elevated markers of oxidative damage of targets in the body and accumulation of reactive oxygen species are common in HIV-infected patients. Blood antioxidants are reduced over the long term in the infected individuals. For review see Pohanka 2013. Folia Microbiol 58:503-513.
• ROS reactive oxygen species
• NO nitric oxide
• peroxynitrite peroxynitrite
• ROS and RNS During sepsis, excess production of ROS and RNS threatens the integrity of various biomolecules including proteins, lipids as well as lipoproteins, protein oxidation and DNA resulting in tissue damage, by lipid peroxidation of cell membranes, protein oxidation and DNA strand breaks. These mechanisms contribute to multi organ failure during sepsis resulting in myocardial depression, hepatocellular dysfunction, endothelial dysfunction, and vascular catecholamine hypo- responsiveness. As a major source of ROS production, mitochondria are especially prone to ROS -mediated damage. Such damage can induce the mitochondrial permeability transition caused by opening of nonspecific high conductance permeability transition pores in the mitochondrial inner membrane.
• ROS themselves also provide a signal leading to the induction of autophagy, apoptosis, and necrosis.
• Excessive ROS production and adenosine triphosphate depletion from uncoupling of oxidative phosphorylation promote necrotic cell death.
• Release of cytochrome-c after mitochondrial swelling activates caspases and initiates apoptotic cell death.
• the herein disclosed DPP3 binder specifically the herein provided anti-DDP3 antibody, and/or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold which are binding to an epitope according to SEQ ID NO.: 2, which is comprised in a DPP3 protein or a functional derivative thereof are provided for the use in the prevention or treatment of a disease or acute condition of a patient, whereby said disease or acute condition is associated with oxidative stress, said disease is selected from the group comprising the above described neurodegenerative diseases, metabolic syndrome, cardiovascular disorders, autoimmune diseases, inflammatory lung diseases, kidney diseases, liver diseases, digestive diseases, viral infectious diseases, cancer, inflammation, sepsis, septic shock and SIRS.
• said disease is selected from the group comprising neurodegenerative diseases (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 disorders (e.g. atherosclerosis, hypertension, heart failure, cardiovascular ischemia, cerebral ischemic injury/ stroke and myocardial infarction), autoimmune diseases (e.g. rheumatoid arthritis and systemic lupus erythematosus), inflammatory lung diseases (e.g.
• neurodegenerative diseases 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 disorders e.g. at
• COPD chronic kidney disease
• AKJ acute kidney injury
• CKD chronic kidney disease
• ESRD end-stage renal disease
• liver diseases e.g. hepatotoxicity, viral hepatitis, cirrhosis
• digestive diseases including inflammatory bowel disease e.g. Ulcerative colitis, Crohn's disease; gastritis, pancreatitis and peptic ulcer
• viral infectious diseases e.g. blood-borne hepatitis viruses (B, C, and D), human immunodeficiency virus (HIV), influenza A, Epstein-Barr virus, respiratory syncytial virus)
• cancer e.g.
• the herein disclosed DPP3 binder specifically the herein provided anti-DDP3 antibody, and/or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold which are binding to an epitope according to SEQ ID NO.: 2, which is comprised in a DPP3 protein or a functional derivative thereof are provided for the use in the prevention or treatment of an acute condition, wherein said acute condition may be selected from a group comprising hepatotoxicity and kidney toxicity.
• Toxicities resultant from alcohol consumption, chronic exposure to cigarette smoke and as a side-effect of different drug treatments
• oxidative stress and subsequent toxicities can also be induced by chronic alcohol consumption, chronic exposure to cigarette smoke and as a side- effect of different drug treatments (reviewed in Deavall et al. 2012, see table 2 below).
• Acetaminophen - a widely used analgesic and antipyretic drug - is a prototypical hepatotoxicant for drug-induced liver injury that is connected to KEAP-Nrf2 signaling (Ma, 2013).
• Other therapeutics inducing oxidative stress include oltipraz and auranofin (Ma, 2013).
• Table 2 Examples of toxicities associated with drug-induced oxidative stress (from Deavall etoL 2012)
• oxidative stress may be determined and quantified by suitable biomarker assays known in the art. Respective examples for these markers are given below, but these shall be not construed as limiting possibilities to measure oxidative stress in accordance with the invention: Markers of oxidative stress for assessment: Serum, Erythrocytes, CSF, Saliva, Urine
• Free radicals can damage biological molecules including nucleic acids, proteins, and lipids.
• the products of these reactions can become markers of oxidative stress.
• Serum is the most common material for the evaluation of the components of oxidative stress. It allows the estimation of most enzymes, substrates, and products of redox reactions. These enzymes include xanthine oxidase, NOS, lipoxygenase, cyclooxygenase, myeloperoxidase, prolyl- oligopeptidase, nicotinamide adenine dinucleotide phosphate-oxidase 1 (NOX1), and NADPH-dependent oxidase.
• isoprostanes IsoP-prostaglandin like substances
• 8-iso-prostaglandin F2a-8- iso-PGF2a
• MDA malondialdehyde
• Oxidative stress involves the oxidation of proteins and glycoxidation.
• the glycophore content the total level of advanced protein oxidation (AOPP), protein carbonyls, dityrosine level, N' -formylkynurenine, and a decreased level of serum protein thiol groups.
• Other specific markers of protein oxidation are tyrosine (a marker for hydroxyl radical) and 3- nitrotyrosine (a marker for RNS).
• 3-nitrotyrosine is a specific marker of peroxynitrite-induced cellular damage.
• Other indicators in the serum include kynurenine, N'- formylkynurenine, thioredoxin, and 8-hydroxy-2'-deoxyguanosine.
• Inhibition of DPP3 activity in a liquid phase assay by a binder may be determined as followed: Blood samples (e.g. serum, heparin-plasma, Li-plasma, citrate-plasma, whole blood) of patients before and after anti-DPP3 antibody treatment is incubated with specific DPP3 substrates in a liquid phase assay.
• the specific liquid phase DPP3 activity assay to determine the inhibitory ability of inhibitory DPP3 antibodies in blood samples comprises the following steps:
• a solid phase assay is an assay where the respective binding events take place at the solid phase.
• Inhibition of DPP3 activity in a solid phase assay by a binder may be determined as followed according: Blood samples (e.g. serum, plasma, whole blood) of patients before and after anti-DPP3 antibody treatment are contacted with an immobilized capture-binder for enzyme capture activity assay (ECA) on a solid phase.
• ECA enzyme capture activity assay
• the capture- binder should inhibit DPP3 activity less than 50 %, preferably less than 40 %, preferably less than 30 %.
• the specific liquid phase DPP3 activity assay to determine the inhibitory ability of possible capture-binders is described in detail in Example 1 below.
• the ECA to determine the inhibitory ability of inhibitory DPP3 antibodies in blood samples comprises the following steps:
• the method for determining active DPP3 may be conducted as liquid phase assay and as solid phase assay. Inhibition of DPP3 activity may be determined in a liquid assay nevertheless according to the above-described procedure.
• a capture or binding assay may be performed to detect and/or quantitate the protease activity of DPP3.
• an antibody reactive with DPP3 protein, but which does not interfere with peptidase activity may be immobilized upon a solid phase.
• the test sample is passed over the immobile antibody, and DPP3, if present, binds to the antibody and is itself immobilized for detection.
• a substrate may then be added, and the reaction product may be detected to indicate the presence or amount of DPP3 in the test sample.
• solid phase may used to include any material or vessel in which or on which the assay may be performed and includes, but is not limited to, porous materials, nonporous materials, test tubes, wells, slides, etc.
• the binding affinity of the herein disclosed DPP3 binder to DPP3 may be measured by various suitable assays known in the art. Respective examples are given below, but these shall be not construed as limiting possibilities to measure binding affinity of the herein disclosed DPP3 binder to DPP3: Method for measuring the binding affinity of the DPP3 binder of the invention to the epitope according to sequence SEQ ID NO.: 2
• the binding affinity of the DPP3 binder to the epitope according to SEQ ID NO.: 2 in accordance with the invention may be determined in accordance with Example 1 and as further set out below:
• a binding assay may be performed to detect and/or quantitate antibody binding to the immunization peptide (i.e. SEQ ID NO.: 2).
• this immunization peptide may be immobilized upon a solid phase.
• the test sample e.g. antibody solution
• the term "solid phase" may be used to include any material or vessel in which or on which the assay may be performed and includes, but is not limited to, porous materials, nonporous materials, test tubes, wells, slides, etc.
• the herein disclosed binder of the invention, and DPP3 binder, specifically the anti-DPP3 antibodies, anti-DPP3 antibody fragments, or anti- DPP3 non-Ig scaffolds are capable to bind circulating DPP3, and thus are directed against circulating DPP3.
• the herein disclosed binder of the invention, and DPP3 binder, specifically the anti-DPP3 antibodies, anti-DPP3 antibody fragments, or anti- DPP3 non-Ig scaffolds are capable to bind intracellular DPP3, and thus are directed against intracellular DPP3.
• the herein disclosed binder of the invention DPP3 binder, specifically the anti-DPP3 antibodies, anti-DPP3 antibody fragments, or anti- DPP3 non-Ig scaffolds are capable to bind membranous DPP3, and thus are directed against membranous DPP3.
• DPP3 binder specifically is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold for use in the prevention or treatment of diseases or acute conditions in a patient, wherein said diseases or acute conditions are associated with oxidative stress
• said binder, DPP3 binder specifically is an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold are directed to and binding to an epitope of SEQ ID NO.: 2, wherein said epitope is comprised in a circulating DPP3 protein or functional derivative thereof.
• Also subject matter of the present invention are the herein disclosed binder of the invention , DPP3 binder, specifically an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold for use in the prevention or treatment of diseases or acute conditions in a patient, wherein said diseases or acute conditions are associated with oxidative stress, and whereby said binder, DPP3 binder, specifically an anti-DPP3 antibody or an anti- DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold are directed to and binding to an epitope of SEQ ID NO.: 2, wherein said epitope is comprised in an intracellular DPP3 protein or functional derivative thereof.
• DPP3 binder specifically an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold for use in the prevention or treatment of diseases or acute conditions in a patient, wherein said diseases or acute conditions are associated with oxidative stress
• said DPP3 binder, specifically an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold are directed to and binding to an epitope of SEQ ID NO.: 2, wherein said epitope is comprised in a membranous DPP3 protein or functional derivative thereof.
• Subject matter of the present invention is further a method for regulating and/or preventing or treatment of oxidative stress in a patient having a chronic or acute disease or acute condition, characterized in that to said patient a binder of the invention, or a DPP3 binder of the invention, specifically an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti- DPP3 non-Ig scaffold is administered in pharmaceutically effective amounts.
• said patient is a patient in need of regulating and/or preventing or in need of treatment of oxidative stress.
• Another subject of the present invention is a pharmaceutical composition
• a pharmaceutical composition comprising the herein disclosed binder of the invention, or DPP3 binder, specifically comprising an anti- DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold for use in the prevention or treatment of diseases or acute conditions of a patient, wherein said disease or acute condition is associated with oxidative stress.
• said pharmaceutical composition is a solution, preferably a ready-to-use solution.
• said pharmaceutical composition is a solution, preferably a ready-to-use solution comprising PBS at a pH of 7.4.
• said pharmaceutical composition is in a dried state that is to be reconstituted before use.
• said pharmaceutical composition is in a freeze -dried state that is to be reconstituted before use.
• said pharmaceutical composition that is to be used in the prevention and/or treatment of a disease or an acute condition of a patient, wherein said disease or acute condition is associated with oxidative stress is administered orally, epicutaneously, subcutaneously, intradermally, sublingually, intramuscularly, intraarterially, intracerebrally, intracerebroventricularly, intravenously, or via the central nervous system (CNS) or via intraperitoneal administration.
• CNS central nervous system
• kits or an assay comprising the herein disclosed binder of the invention, or DPP3 binder, specifically comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold for use in the prevention or treatment of a disease or acute condition of a patient, whereby said disease or acute condition is associated with oxidative stress.
• DPP3 binder specifically comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold for use in the prevention or treatment of a disease or acute condition of a patient, whereby said disease or acute condition is associated with oxidative stress.
• an “anti-DPP3 antibody” is an antibody that binds specifically to DPP3
• an “anti-DPP3 antibody fragment” is a fragment of said anti-DPP3 antibody, wherein said fragment binds specifically to DPP3.
• An “anti-DPP3 non-Ig scaffold” is a non-Ig scaffold that binds specifically to DPP3.
• DPP3 specifically binding to DPP3 may also allow binding to other antigens as well. This means, this specificity would not exclude that the binder may cross-react with other proteins or polypeptides or peptides that contain the epitope according to SEQ ID NO.: 2 against which the binder has been raised. This specifically includes functional variants of DPP3, which also comprise an epitope according to SEQ ID NO.: 2. This also pertains to the specificity of the anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold in accordance with the invention.
• an “antibody” is a protein including one or more polypeptides substantially encoded by immunoglobulin genes that specifically binds an antigen.
• the recognized immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma (IgGi, IgG 2 , IgG 3 , IgG 4 ), delta (IgD), epsilon (IgE) and mu (IgM) constant region genes, as well as the myriad immunoglobulin variable region genes.
• Full-length immunoglobulin light chains are generally about 25 kDa or 214 amino acids in length.
• Full-length immunoglobulin heavy chains are generally about SO kDa or 446 amino acids in length.
• Light chains are encoded by a variable region gene at the NH2-terminus (about 110 amino acids in length) and a kappa or lambda constant region gene at the COOH-terminus.
• Heavy chains are similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.
• the basic structural unit of an antibody is generally a tetramer that consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions bind to an antigen, and the constant regions mediate effector functions.
• Immunoglobulins also exist in a variety of other forms including, for example, Fv, Fab, and F(ab') 2 , as well as Afunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al., Eur. J. Immunol. 17:105,1987; Huston et al, Proc. Natl. Acad. Sci. U.S.A., 85:5879-5883, 1988; Bird et al., Science 242:423-426, 1988; Hood et al, Immunology, Benjamin, N.Y., 2nd ed., 1984; Hunkapiller and Hood, Nature 323:15-16,1986).
• An immunoglobulin light or heavy chain variable region includes a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDR's) (see, Sequences of Proteins of Immunological Interest, E. Kabat et al, U.S. Department of Health and Human Services, 1983). As noted above, the CDRs are primarily responsible for binding to an epitope of an antigen.
• An immune complex is an antibody, such as a monoclonal antibody, chimeric antibody, humanized antibody or human antibody, or functional antibody fragment, specifically bound to the antigen.
• Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species.
• the variable segments of the genes from a mouse monoclonal antibody can be joined to human constant segments, such as kappa and gamma 1 or gamma 3.
• a therapeutic chimeric antibody is thus a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species can be used, or the variable region can be produced by molecular techniques. Methods of making chimeric antibodies are well known in the art, e.g., see U.S. Patent No. 5,807,715.
• a “humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) immunoglobulin.
• the non-human immunoglobulin providing the CDRs is termed a "donor” and the human immunoglobulin providing the framework is termed an "acceptor.”
• all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85- 90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences.
• a "humanized antibody” in accordance with the invention is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs.
• the acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework.
• Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are those such as gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr.
• Humanized immunoglobulins can be constructed by means of genetic engineering (e.g., see U.S. Patent No. 5,585,089).
• a human antibody is an antibody wherein the light and heavy chain genes are of human origin.
• Human antibodies can be generated using methods known in the art. Human antibodies can be produced by immortalizing a human B cell secreting the antibody of interest. Immortalization can be accomplished, for example, by EBV infection or by fusing a human B cell with a myeloma or hybridoma cell to produce a trioma cell. Human antibodies can also be produced by phage display methods (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 combinatorial monoclonal antibody library (see the Morphosys website).
• phage display methods see, e.g., Dower et al, PCT Publication No. WO 91/17271; McCafferty et al, PCT Publication No. WO 92/001047; and Winter
• Human antibodies can also be prepared by using transgenic animals carrying a human immunoglobulin gene (for example, see Lonberg et al, PCT Publication No. WO 93/12227; and Kucherlapati, PCT Publication No. WO 91/10741).
• the anti-DPP3 antibody or anti-DPP3 antibody fragment in accordance with the invention may have the formats known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies or antibody fragments thereof, but not limited to.
• the anti-DPP3 antibody is a monoclonal antibody or a fragment thereof.
• the anti-DPP3 antibody or the anti- DPP3 antibody fragment is a human or humanized antibody or derived therefrom.
• one or more (murine) CDR's are grafted into a human antibody or antibody fragment.
• antibodies according to the present invention are recombinantly produced antibodies as e.g. IgG, a typical full-length immunoglobulin, or antibody fragments containing at least the F-variable domain of heavy and/or light chain as e.g. chemically coupled antibodies (fragment antigen binding) including but not limited to Fab-fragments including Fab minibodies, single chain Fab antibody, monovalent Fab antibody with epitope tags, e.g. Fab-V5Sx2; bivalent Fab (mini-antibody) dimerized with the CH3 domain; bivalent Fab or multivalent Fab, e.g. formed via multimerization with the aid of a heterologous domain, e.g.
• chemically coupled antibodies fragment antigen binding
• fragment antigen binding including but not limited to Fab-fragments including Fab minibodies, single chain Fab antibody, monovalent Fab antibody with epitope tags, e.g. Fab-V5Sx2; bivalent Fab (mini-antibody) dimerized
• dHLX domains e.g. Fab-dHLX-FSx2; F(ab')2-fragments, scFv-fragments, multimerized multivalent and/or multispecific scFv-fragments, bivalent and/or bispecific diabodies, BITE ® (bispecific T-cell engager), trifunctional antibodies, polyvalent antibodies, e.g. from a different class than G; single-domain antibodies, e.g. nanobodies derived from camelid or fish immunoglobulines and numerous others.
• Non-Ig scaffolds In addition to anti-DPP3 antibodies or anti-DPP3 antibody fragments, other biopolymer scaffolds, so called non-Ig scaffolds, are well known in the art to complex a target molecule and have been used for the generation of highly target specific biopolymers. Examples are aptamers, spiegelmers, anticalins and conotoxins.
• Non-Ig scaffolds with the context of the invention may be protein scaffolds and may be used as antibody mimics as they are capable to bind to ligands or antigens.
• Non-Ig scaffolds may be selected from the group comprising tetranectin-based non-Ig scaffolds (e.g. described in US 2010/0028995), fibronectin scaffolds (e.g.
• Non-Ig scaffolds may be peptide or oligonucleotide aptamers. Aptamers are usually created by selecting them from a large random sequence pool and are either short strands of oligonucleotides (DNA, RNA or XNA; Xu et al. 2010, Deng et al. 2014) or short variable peptide domains attached to a protein scaffold (Li et al. 2011).
• the anti-DPP3 antibody format is selected from the group comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, F(ab)2 fragment and scFv-Fc Fusion protein.
• the antibody format is selected from the group comprising scFab fragment, Fab fragment, scFv fragment and bioavailability optimized conjugates thereof, such as PEGylated fragments.
• antibody generally comprises monoclonal and polyclonal antibodies and binding fragments thereof, in particular Fc-fragments as well as so called “single-chain-antibodies” (Bird et al. 1988), chimeric, humanized, in particular CDR- grafted antibodies, and di- or tetrabodies (Holliger et al. 1993). Also comprised are immunoglobulin-like proteins that are selected through techniques including, for example, phage display to specifically bind to the molecule of interest contained in a sample.
• specific binding refers to antibodies raised against the molecule of interest or a fragment thereof.
• An antibody is considered to be specific, if its affinity towards the molecule of interest or the aforementioned fragment thereof is at least preferably 50-fold higher, more preferably 100-fold higher, most preferably at least 1000-fold higher than towards other molecules comprised in a sample containing the molecule of interest. It is well known in the art how to make antibodies and to select antibodies with a given specificity.
• said anti-DPP3 antibody or anti-DPP3 antibody fragment binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or functional derivative thereof is a monoclonal antibody or a monoclonal antibody fragment thereof.
• the anti-DPP3 antibody or the anti-DPP3 antibody fragment binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or functional derivative thereof is a human or humanized antibody or derived therefrom or humanized antibody fragment or derived therefrom.
• one or more (murine) CDR's are grafted into a human antibody or antibody fragment.
• said DPP3 binder of the invention specifically said anti-DPP3 antibody, anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold is a modulating DPP3 binder, anti-DPP3 antibody, anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold.
• a modulating DPP3 binder, anti-DPP3 antibody, anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold of the invention may act inhibitory and may block the bioactivity of DPP3 to nearly 100%, preferably to at least more than 90%, more preferably to at least 80, or 70, or 60, or 50, or 40, or 30, or 20, or 10 % when determined by means of the above described method for detecting and measuring the inhibition of DPP3; i.e. measuring the DPP3 binder influence on DPP-3 bioactivity.
• a modulating DPP3 binder, anti-DPP3 antibody, anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold of the invention may act upregulating and thus may enhance the bioactivity of DPP3 to at least 50 %, preferably to at least more than 60 %, more preferably to at least more than 70 %, more preferably to at least more than 80 %, even more preferably to at least more than 90 %, even more so preferably to at least 95 % when determined by means of the above described method for detecting and measuring the inhibition of DPP3; i.e. measuring the DPP3 binder influence on DPP-3 bioactivity.
• Anti-DPP3 antibodies according to the present invention may be synthesised as follows:
• DPP3 peptides for immunization were synthesized, see table 3 below, (JPT Technologies, Berlin, Germany) with an additional N-terminal cystein (if no cystein is present within the selected DPP3-sequence) residue for conjugation of the peptides to Bovine Serum Albumin (BSA).
• BSA Bovine Serum Albumin
• the peptides were covalently linked to BSA by using Sulfolink-coupling gel (Perbio- science, Bonn, Germany). The coupling procedure was performed according to the manual of Perbio. Recombinant GST-hDPP3 was produced by USBio.
• mice were intraperitoneally (i.p.) injected with 84 ⁇ g GST-hDPP3 or 100 ⁇ g DPP3-peptide-BSA-conjugates at day 0 (emulsified in TiterMax Gold Adjuvant), 84 ⁇ g or 100 ⁇ £ at day 14 (emulsified in complete Freund's adjuvant) and 42 ⁇ g or 50 ⁇ g at day 21 and 28 (in incomplete Freund's adjuvant).
• the animal received an intravenous (i.v.) injection of 42 ⁇ g GST-hDPP3 or 50 ⁇ g DPP3-peptide-BSA-conjugates dissolved in saline. Three days later the mice were sacrificed and the immune cell fusion was performed.
• Splenocytes from the immunized mice and cells of the myeloma cell line SP2/0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37°C. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT- Supplement] . After one week, the HAT medium was replaced with HT Medium for three passages followed by returning to the normal cell culture medium.
• HAT medium RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT- Supplement
• the cell culture supernatants were primarily screened for recombinant DPP3 binding IgG antibodies two weeks after fusion. Therefore, recombinant GST-tagged DPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng/ well) and incubated with 50 ⁇ cell culture supernatant per well for 2 hours at room temperature. After washing of the plate, 50 ⁇ / well POD-rabbit anti mouse IgG was added and incubated for 1 h at RT.
• the positive tested microcultures were transferred into 24-well plates for propagation. After retesting the selected cultures were cloned and recloned using the limiting-dilution technique and the isotypes were determined.
• Antibodies raised against GST-tagged human DPP3 or DPP3 -peptides were produced via standard antibody production methods (Marx et al. 1997) and purified via Protein A. The antibody purities were > 90 % based on SDS gel electrophoresis analysis. Humanization of murine antibodies
• Humanization of murine antibodies may be conducted according to the following procedure:
• the antibody sequence is analyzed for the structural interaction of framework regions (FR) with the complementary determining regions (CDR) and the antigen. Based on structural modelling an appropriate FR of human origin is selected and the murine CDR sequences are transplanted into the human FR. Variations in the amino acid sequence of the CDRs or FRs may be introduced to regain structural interactions, which were abolished by the species switch for the FR sequences. This recovery of structural interactions may be achieved by random approach using phage display libraries or via directed approach guided by molecular modeling (Almagro JC, Fransson J., 2008. Humanization of antibodies. Front Biosci. 2008 Jan l;13:1619-33).
• the provided subject matter is a human CDR-grafted anti- DPP3 antibody or anti-DPP3 antibody fragment thereof that is directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein said human CDR-grafted anti-DPP3 antibody or anti-DPP3 antibody fragment thereof comprises an antibody heavy chain variable region (H chain) comprising
• L chain antibody light chain variable region
• Further subject matter of the present invention in another aspect is a human CDR-grafted anti- DPP3 antibody or anti-DPP3 antibody fragment thereof that is directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein the said human CDR-grafted anti-DPP3 antibody or anti-DPP3 antibody fragment thereof comprises an antibody heavy chain variable region (H chain) comprising:
• SEQ ID NO.: 12 and/or further comprises an antibody light chain variable region (L chain) comprising:
• subject matter of the present invention is a human monoclonal anti-DPP3 antibody or monoclonal anti-DPP3 antibody fragment thereof that is directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein the heavy chain comprises at least one CDR of:
• variable region can be connected to any subclass of constant regions (IgG, IgM, IgE. IgA), or only scaffolds, Fab fragments, Fv, Fab and F(ab)2.
• IgG constant regions
• IgM constant regions
• IgE. IgA scaffolds
• Fab fragments fragments
• Fv fragments
• Fab fragments
• F(ab)2 the murine antibody variant with an IgG2a backbone was used.
• chimerization and humanization a human IgG IK backbone was used.
• CDRs Complementarity Determining Regions
• AK1967 The CDRs for the heavy chain and the light chain of the murine anti-DPP3 antibody of the present invention (AK1967) are shown in SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9 for the heavy chain and SEQ ID NO. 10, sequence KVS and SEQ ID NO. 11 for the light chain, respectively.
• the herein provided DPP3 binder specifically the herein provided anti-DPP3 antibodies, anti-DPP3 antibody fragments and anti-DPP3 non Ig- scaffolds are directed to and binding to SEQ ID NO.: 1, and wherein said 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 4 aa, more preferably at least 5 aa, even more preferably at least 6 aa of said SEQ ID NO.: 1.
• the herein provided DPP3 binder specifically the herein provided anti-DPP3 antibodies, anti-DPP3 antibody fragments and anti-DPP3 non Ig- scaffolds are directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein said 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 4 aa, more preferably at least 5 aa, even more preferably at least 6 aa of SEQ ID NO.: 2.
• the herein provided DPP3 binder specifically the herein provided anti-DPP3 antibodies, anti-DPP3 antibody fragments and anti-DPP3 non Ig- scaffolds are directed to and binding to an epitope according to SEQ ID NO.: 3, and wherein said epitope according to SEQ ID NO.: 3 is comprised in a DPP3 protein or a functional derivative thereof, and wherein said 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 4 aa, more preferably at least 5 aa, even more preferably to 6 aa of SEQ ID NO.: 3.
• the herein provided DPP3 binder specifically the herein provided anti-DPP3 antibodies, anti-DPP3 antibody fragments and anti-DPP3 non Ig- scaffolds are directed to and binding to an epitope according to SEQ ID NO.: 4, and wherein said epitope according to SEQ ID NO.: 4 is comprised in a DPP3 protein or a functional derivative thereof, and wherein said DPP3 binder, anti-DPP3 antibody, anti-DPP3 antibody fragment and anti-DPP3 non Ig-scaffold recognizes and binds to at least three aa, preferably to four aa of SEQ ID NO.: 4.
• the herein provided DPP3 binder specifically the herein provided anti-DPP3 antibodies, anti-DPP3 antibody fragments and anti-DPP3 non Ig- scaffolds which are directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, may act as inhibitor or effector of the bioactivity of DPP3.
• the herein provided DPP3 binder specifically the herein provided anti-DPP3 antibodies, anti-DPP3 antibody fragments and anti-DPP3 non Ig-scaffolds which are directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof are useful in the prevention or treatment of a disease or acute condition in a patient, wherein said disease or acute condition is associated with oxidative stress in accordance with the invention.
• the herein provided DPP3 binder specifically the herein provided anti-DPP3 antibodies, anti-DPP3 antibody fragments and anti-DPP3 non Ig- scaffolds which are directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, exhibit an affinity towards DPP3 in such that the affinity constant is at least 10 "7 M "1 , preferably at least 10- 8 M “1 , more preferably the affinity constant is at least 10 "9 M "1 , most preferred the affinity constant is at least 10 "10 M '1 when determined by means of the methods for measuring the binding affinity of the DPP3 binder of the invention to the epitope according to sequence SEQ ID NO.: 2 as described above.
• the herein provided DPP3 binder specifically the herein provided anti-DPP3 antibody or anti-DPP3 antibody fragment thereof or anti-DPP3 non-Ig scaffold may be used in combination with at least one additional drug that induces oxidative stress as side effect.
• Such drugs are administered as primary medicament for use in the prevention or treatment of a primary disease and may be selected from a group comprising antimicrobials like antibiotics (for example streptomycin, gentarnicin) or antivirals (for example acyclovir, foscarnet) or antifungal (for example amphotecerin B), analgesics, non-steroidal anti-inflammatory drugs (NSAID) (for example ibuprofen, naproxen), diuretics, proton pump inhibitors, chemotherapeutics (for example cisplatin), contrast dyes, cardiovascular agents like ACE- inhibitors or statins, anti-depressants, immune suppressants (for example cyclosporine A) and antihistamines.
• antibiotics for example streptomycin, gentarnicin
• antivirals for example acyclovir, foscarnet
• antifungal for example amphotecerin B
• analgesics non-steroidal anti-inflammatory drugs (NSAID) (for example ibuprofen
• the herein provided DPP3 binder specifically the herein provided anti-DPP3 antibody or anti-DPP3 antibody fragment thereof or a DPP3 non-Ig scaffold binding to DPP3 may be used as secondary medicament either in combination or as stand-alone drug in the prevention or treatment of the induced oxidative stress and resultant toxicities as secondary diseases.
• the herein provided DPP3 binder are pharmaceutically acceptable, selective and/or specific for an epitope according to SEQ ID NO.: 2, which is comprised in a DPP3 protein or a functional derivative thereof.
• the herein provided DPP3 binder is an inhibitory binder that is pharmaceutically acceptable, selective and/or specific for an epitope according to SEQ ID NO.: 2, which is comprised in a DPP3 protein or a functional derivative thereof.
• selective and specific inhibitors of DPP3 do not bind to other proteins/peptides/enzymes or are bound by other proteins/peptides/enzymes, and do not inhibit any other enzyme/protease/peptidase other than DPP3. Therefore, the preferred inhibitors of DPP3 bioactivity with the context of the invention are specific anti-DPP3 antibodies, antibody fragments or non-Ig scaffolds binding to DPP3.
• Monospecific anti-DPP3 antibody or monospecific anti-DPP3 antibody fragment or monospecific anti-DPP3 non-Ig scaffold with the context of the invention means that said antibody or antibody fragment or non-Ig scaffold binds specifically to one specific region encompassing at least 3 amino acids, preferably at least 4 aa within the target DPP3.
• monospecific anti-DPP3 antibody or monospecific anti- DPP3 antibody fragment or monospecific anti-DPP3 non-Ig scaffold are anti-DPP3 antibodies or anti-DPP3 antibody fragments or anti-DPP3 non-Ig scaffolds all have affinity for the same antigen as a target which is in accordance with the invention an epitope according to SEQ ID NO.: 2, which is comprised in a DPP3 protein or a functional derivative thereof.
• monospecific anti-DPP3 antibody or monospecific anti- DPP3 antibody fragment or monospecific anti-DPP3 non-Ig scaffold are anti-DPP3 antibodies or anti-DPP3 antibody fragments or anti-DPP3 non-Ig scaffolds all have affinity for the same antigen as a target which is in accordance with the invention an epitope according to SEQ ID NO.: 3, which is comprised in a DPP3 protein or a functional derivative thereof.
• monospecific anti-DPP3 antibody or monospecific anti-DPP3 antibody fragment or monospecific anti-DPP3 non-Ig scaffold are anti-DPP3 antibodies or anti-DPP3 antibody fragments or anti-DPP3 non-Ig scaffolds all have affinity for the same antigen as a target which is in accordance with the invention an epitope according to SEQ ID NO: 1
• binder is selected from a group comprising an antibody or antibody fragment or non-Ig scaffold, and wherein said epitope is comprised in SEQ ID NO.: 1, which corresponds to the amino acid sequence of DPP3.
• DPP3 dipeptidyl peptidase 3
• binder directed to and binding to an epitope according to SEQ ID NO.: 2 of any of the preceding embodiments, wherein said binder is a monoclonal antibody or monoclonal antibody fragment, and wherein the complementarity determining regions (CDR's) in the heavy chain comprises the sequences:
• SEQ ID NO.: 7, SEQ ID NO.: 8 and/ or SEQ ID NO.: 9 and the complementarity determining regions in the light chain comprises the sequences:
• SEQ ID NO.: 10 KVS and/ or SEQ ID NO.: 11.
• binder directed to and binding to an epitope according to SEQ ID NO.: 2 of any of the preceding embodiments, wherein said binder is a human monoclonal antibody or human monoclonal antibody fragment, wherein the heavy chain comprises the sequence:
• SEQ ID NO.: 12 and wherein the light chain comprises the sequence: SEQ ID NO.: 13.
• AD Alzheimer's disease
• PD Parkinson's disease
• HD amyotrophic lateral sclerosis
• MS multiple sclerosis
• o metabolic syndrome may be selected from a group comprising insulin resistance, obesity, hyperglycemia, dyslipidemia, hypertension and diabetes,
• o cardiovascular disorder may be selected from a group comprising aterosclerosis, hypertension, heart failure, cardiovascular ischemia, cerebral ischemic injury, stroke and myocardial infarction,
• o autoimmune disease may be selected from a group comprising rheumatoid arthritis, systemic lupus erythematosus,
• o inflammatory lung disease may be selected from a group comprising COPD, asthma,
• o kidney disease may be selected from a group comprising acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, end-stage renal disease (ESRD),
• AKI acute kidney injury
• CKD chronic kidney disease
• ESRD end-stage renal disease
• o liver disease may be selected from a group comprising viral hepatitis, and cirrhosis,
• o digestive disease may be selected from a group comprising inflammatory bowel disease e.g. Ulcerative colitis, Crohn's disease, gastritis, pancreatitis and peptic ulcer,
• o viral infectious disease may be selected from a group comprising blood-borne hepatitis viruses (B, C, and D), human immunodeficiency virus (HIV), influenza A, Epstein-Barr virus, respiratory syncytial virus, cancer may be selected from a group comprising prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, skin cancer, stomach cancer, liver cancer,
• B, C, and D blood-borne hepatitis viruses
• HAV human immunodeficiency virus
• influenza A Epstein-Barr virus
• cancer may be selected from a group comprising prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, skin cancer, stomach cancer, liver cancer,
• the binder directed to and binding to an epitope according to SEQ ID NO.: 2 for use in the prevention or treatment of diseases or acute conditions in a patient, wherein said disease or acute condition is associated with oxidative stress according to any of the embodiments 8 to 10, wherein said disease is selected from a group comprising sepsis, septic shock, and SIRS.
• the binder directed to and binding to an epitope according to SEQ ID NO.: 2 for use in the prevention or treatment of diseases or acute conditions in a patient, wherein said disease or acute condition is associated with oxidative stress according to embodiment 8, wherein said acute condition is selected from a group comprising renal toxicity and hepatotoxicity.
• the binder directed to and binding to an epitope according to SEQ ID NO.: 2 for use in the prevention or treatment of diseases or acute conditions in a patient, wherein said disease or acute condition is associated with oxidative stress according to any of the embodiments 8 to 11, wherein said disease is associated with oxidative stress in the myocard.
• composition comprising a binder according to any of the embodiments 1 to 7 for use in the prevention or treatment of a disease or acute condition of a patient, whereby said disease or acute condition is associated with oxidative stress.
• a kit comprising a binder according to any of the embodiments 1 to 16.
• the binder for use in the prevention or treatment of diseases or acute conditions in a patient, wherein said disease or acute condition is associated with oxidative stress according to any of the embodiments 19 or 20, and wherein said: o neurodegenerative disease may be selected from a group comprising
• AD Alzheimer's disease
• PD Parkinson's disease
• o metabolic syndrome may be selected from a group comprising insulin resistance, obesity, hyperglycemia, dyslipidemia, hypertension and diabetes,
• o cardiovascular disorder may be selected from a group comprising aterosclerosis, hypertension, heart failure, cardiovascular ischemia, cerebral ischemic injury, stroke and myocardial infarction,
• o autoimmune disease may be selected from a group comprising rheumatoid arthritis, systemic lupus erythematosus,
• o inflammatory lung disease may be selected from a group comprising COPD, asthma,
• o kidney disease may be selected from a group comprising acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, end-stage renal disease (ESRD),
• AKI acute kidney injury
• CKD chronic kidney disease
• ESRD end-stage renal disease
• o liver disease may be selected from a group comprising viral hepatitis, and cirrhosis,
• o digestive disease may be selected from a group comprising inflammatory bowel disease e.g. Ulcerative colitis, Crohn's disease, gastritis, pancreatitis and peptic ulcer,
• o viral infectious disease may be selected from a group comprising blood-borne hepatitis viruses (B, C, and D), human immunodeficiency virus (HIV), influenza
• o cancer may be selected from a group comprising prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, skin cancer, stomach cancer, liver cancer,
• binder according to any of the embodiments 19 to 24, wherein said binder is selected from a group comprising an antibody or antibody fragment or non-Ig scaffold.
• binder according to any of the embodiments 19 to 25, wherein said binder is a monoclonal antibody or monoclonal antibody fragment, and wherein the complementarity determining regions (CDR's) in the heavy chain comprises the sequences:
• SEQ ID NO.: 7, SEQ ID NO.: 8 and/ or SEQ ID NO.: 9 and the complementarity determining regions in the light chain comprises the sequences:
• SEQ ID NO.: 10 KVS and/ or SEQ ID NO.: 11.
• binder according any of the embodiments 19 to 26, wherein said binder is a humanized monoclonal antibody or humanized monoclonal antibody fragment, wherein the heavy chain comprises the sequence:
• SEQ ID NO.: 13 28.
• said binder is a dipeptidyl peptidase 3 (DPP3) binder directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein said DPP3 binder recognizes and binds to at least three amino acids of SEQ ID NO.: 2.
• DPP3 dipeptidyl peptidase 3
• an "DPP3 binder'' is directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein said DPP3 binder recognizes and binds to at least three aa of SEQ ID NO.: 2 or a respective subsequence thereof according to the SEQ ID NO'S.: 3 or 4.
• a DPP3 binder is preferably an anti-DPP3 antibody, or an anti-DPP3 antibody fragment, or an anti-DPP3 non-Ig scaffold directed to and binding to an epitope according to SEQ ID NO.: 2, wherein said epitope is comprised in a DPP3 protein or a functional derivative thereof, and wherein said DPP3 binder recognizes and binds to at least three aa of SEQ ID NO.: 2 or a respective subsequence thereof according to the SEQ ID NO'S.: 3 or 4.
• a “functional derivative" of a DPP3 protein denotes a peptide, polypeptide or protein that differs from the sequence of SEQ ID NO.: 1 by means of deletion of aa, addition of aa or changes of specific aa, but remains the bioactivity and function of a native DPP3 protein.
• the bioactivity and function may be influenced to a certain extent, but the enzymatic protease reaction catalysed by DDP 3 is still maintained when assay by a suitable bioactivity assay as described above or commonly known by the skilled person.
• a dipeptidyl peptidase 3 (DPP3) antibody or an anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold is synonymous to dipeptidyl peptidase 3 (DPP3) antibody or a dipeptidyl peptidase 3 antibody fragment or DPP3 non-Ig scaffold and means ami- dipeptidyl peptidase 3 (DPP3) antibody or an anti- dipeptidyl peptidase 3 antibody fragment or anti-DPP3 non-Ig scaffold binding to DPP3, respectively.
• antibody generally comprises monoclonal and polyclonal antibodies and binding fragments thereof, in particular Fc-fragments as well as so called “single-chain-antibodies” (Bird et al. 1988), chimeric, humanized, in particular CDR-grafted antibodies, and di- or tetrabodies (Holliger et al. 1993). Also comprised are immunoglobulin- like proteins that are selected through techniques including, for example, phage display to specifically bind to the molecule of interest contained in a sample.
• the term "specific binding” refers to antibodies raised against the molecule of interest or a fragment thereof.
• An antibody is considered to be specific, if its affinity towards the molecule of interest or the aforementioned fragment thereof is at least preferably 50-fold higher, more preferably 100-fold higher, most preferably at least 1000-fold higher than towards other molecules comprised in a sample containing the molecule of interest. It is well known in the art how to make antibodies and to select antibodies with a given specificity.
• Diseases associated with oxidative stress include, but are not limited to, neurodegenerative diseases, metabolic syndrome, cardiovascular disorders, autoimmune diseases, inflammatory lung diseases, kidney diseases, liver diseases, digestive diseases, viral infectious diseases, cancer, and inflammation, sepsis, septic shock, SIRS.
• neurodegenerative diseases comprise Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS).
• AD Alzheimer's disease
• PD Parkinson's disease
• HD Huntington's disease
• ALS amyotrophic lateral sclerosis
• MS multiple sclerosis
• metabolic syndrome comprises insulin resistance, obesity, hyperglycemia, dyslipidemia, hypertension and diabetes.
• cardiovascular disorders comprise atero sclerosis, hypertension, heart failure, cardiovascular ischemia, cerebral ischemic injury/ stroke and myocardial infarction.
• autoimmune diseases comprise rheumatoid arthritis and systemic lupus erythematosus.
• inflammatory lung diseases comprise COPD and asthma.
• kidney diseases comprise renal toxicity (drug-induced kidney disease), acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy and end-stage renal disease (ESRD).
• AKI acute kidney injury
• CKD chronic kidney disease
• ESRD end-stage renal disease
• liver diseases comprise hepatotoxicity, viral hepatitis, cirrhosis.
• digestive diseases comprise inflammatory bowel disease e.g. Ulcerative colitis, Crohn's disease; gastritis, pancreatitis and peptic ulcer.
• viral infectious diseases comprise blood-borne hepatitis viruses (B, C, and D), human immunodeficiency virus (HIV), influenza A, Epstein-Barr virus and respiratory syncytial virus.
• cancer comprises prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, skin cancer, stomach cancer and liver cancer.
• Acute condition associated with oxidative stress with the context of the present invention denote symptoms that appear and change or worsen rapidly due to the occurrence of oxidative stress.
• An acute condition associated with oxidative stress is sudden in onset.
• An acute condition associated with oxidative stress may lead to a chronic syndrome, if untreated.
• a “chronic condition” or a “chronic syndrome”, respectively, with the context of the present invention denote a condition or symptom that develops and worsens over an extended period of time, and may be persistent, even if treated.
• "Oxidative stress” reflects an imbalance between the systemic manifestation of reactive oxygen species (ROS)/ reactive nitrogen species (RNS) and antioxidants in favour of excessive generation of free radicals. This process leads to the oxidation of biomolecules with consequent loss of its biological functions and/or homeostatic imbalances, whose manifestation is the potential oxidative damage to cells and tissues. Accumulation of ROS/RNS can result in a number of deleterious effects such as lipid peroxidation, protein oxidation and DNA damage (including base damage and strand breaks). Further, some reactive oxidative species act as cellular messengers in redox signalling. Thus, oxidative stress can cause disruptions in normal mechanisms of cellular signalling.
• a "free radical in the context of the present invention is a molecule with one or more unpaired electron in its outer shell. Free radicals are formed from molecules via the breakage of a chemical bond such that each fragment keeps one electron, by cleavage of a radical to give another radical and, also via redox reactions. Free radicals related to oxidative stress include hydroxyl ( ⁇ ), superoxide ( ⁇ 2 ⁇ "" ) » nitric oxide (NO), nitrogen dioxide ( ⁇ 2 ⁇ ). peroxyl (ROO») and lipid peroxyl (LOO).
• H2O2 hydrogen peroxide
• ozone O 3
• singlet oxygen 102
• hypochlorous acid HOC1
• nitrous acid HNO2
• peroxynitrite ONOO ⁇
• dinitrogen trioxide N 2 O 3
• lipid peroxide LOOH
• Primary medicament means a medicament that acts against the primary cause of said disease or condition.
• “Secondary medication” is a medication that improves the condition of the patient in a supportive way; e.g. reduces or regulates oxidative stress which is induced by the administration of a primary medicament.
• DPP3 bioactivity is defined as the effect that a substance takes on a living organism or tissue or organ or functional unit in vivo or in vitro (e.g. in an assay) after its interaction.
• DPP3 bioactivity may be defined as the DPP3 enzyme activity or the regulating activity of DPP3 in the oxidative stress pathway.
• pHMB polyhexanide polyhexamethylene biguanide
• DPP3 peptides for immunization were synthesized, see table 3, (JPT Technologies, Berlin, Germany) with an additional N-terminal cystein (if no cystein is present within the selected DPP3 -sequence) residue for conjugation of the peptides to Bovine Serum Albumin (BSA).
• BSA Bovine Serum Albumin
• the peptides were covalently linked to BSA by using Sulfolink- coupling gel (Perbio- science, Bonn, Germany). The coupling procedure was performed according to the manual of Perbio. Recombinant GST-hDPP3 was produced by USBio (United States Biological, Salem, MA, USA).
• mice were intraperitoneally (i.p.) injected with 84 ⁇ g GST-hDPP3 or 100 ⁇ g DPP3-peptide-BSA-conjugates at day 0 (emulsified in TiterMax Gold Adjuvant), 84 ⁇ g or 100 ⁇ g at day 14 (emulsified in complete Freund's adjuvant) and 42 ⁇ g or 50 ⁇ g at day 21 and 28 (in incomplete Freund's adjuvant).
• the animal received an intravenous (i.v.) injection of 42 ⁇ g GST-hDPP3 or 50 ⁇ g DPP3- peptide-BSA-conjugates dissolved in saline. Three days later the mice were sacrificed and the immune cell fusion was performed.
• Splenocytes from the immunized mice and cells of the myeloma cell line SP2/0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37°C. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT- Supplement] . After one week, the HAT medium was replaced with HT Medium for three passages followed by returning to the normal cell culture medium. The cell culture supernatants were primarily screened for recombinant DPP3 binding IgG antibodies two weeks after fusion.
• recombinant GST-tagged hDPP3 (USBiologicals, Salem, USA) was immobiliz»d in 96-well plates (100 ng/ well) and incubated with 50 ⁇ cell culture supernatant per well for 2 hours at room temperature. After washing of the plate, 50 ⁇ / well POD-rabbit anti mouse IgG was added and incubated for 1 h at RT.
• a chromogen solution (3,7 mM o-phenylendiamin in citrate/ hydrogen phosphate buffer, 0.012% 3 ⁇ 4(1 ⁇ 4) were added to each well, incubated for 15 minutes at RT and the chromogenic reaction stopped by the addition of 50 ⁇ 4N sulfuric acid. Absorption was detected at 490 mm. The positive tested microcultures were transferred into 24-well plates for propagation.
• Antibodies raised against GST-tagged human DPP3 or DPP3 -peptides were produced via standard antibody production methods (Marx et al. 1997) and purified via Protein A. The antibody purities were > 90% based on SDS gel electrophoresis analysis.
• Recombinant GST-tagged hDPP3 (SEQ ID No. 1) or a DPP3 peptide (immunization peptide, SEQ ID No. 2) was immobilized onto a high binding microtiter plate surface (96- Well polystyrene microplates, Greiner Bio-One international AG, Austria, 1 ⁇ g/well in coupling buffer [50 mM Tris, 100 mM NaCl, pH7,8], lh at RT). After blocking with 5% bovine serum albumin, the microplates were vacuum dried. b) Labelling procedure (Tracer)
• the purified labeled antibody was diluted in assay buffer (SO mmol/1 potassium phosphate, 100 mmol/1 NaCl, 10 mmol/1 Na 2 -EDTA, S g/1 bovine serum albumin, 1 g/1 murine IgG, 1 g/1 bovine IgG, 50 umol/1 amastatin, 100 umol/l leupeptin, pH 7.4).
• the final concentration was approx. 5-7* 10 6 relative light units (RLU) of labelled compound (approx. 20 ng labeled antibody) per 200 ⁇ .
• RLU relative light units
• acridinium ester chemiluminescence was measured by using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).
• the plates were filled with 200 ⁇ of labeled and diluted detection antibody (tracer) and incubated for 2-4 h at 2-8 °C. Unbound tracer was removed by washing 4 times with 350 ⁇ washing solution (20 mM PBS, pH 7.4, 0.1 % Triton X-100). Well- bound chemiluminescence was measured by using the Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).
• the following table represents a selection of obtained antibodies and their binding rate in Relative Light Units (RLU) as well as their relative inhibitory ability (%; table 3).
• RLU Relative Light Unit
• % relative inhibitory ability
• Antibodies raised against SEQ ID NO. 2 were characterized in more detail (epitope mapping, binding affinities, specificity, inhibitory potential). Here the results for clone
• AntiDPP3 antibody AK1967 was labelled with a chemiluminescence label according to Example 1. c) Peptide binding assay
• the experiment was performed using Octet Red96 (ForteBio). AK1967 was captured on kinetic grade anti-humanFc (AHC) biosensors. The loaded biosensors were then dipped into a dilution series of recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM). Association was observed for 120 seconds followed by 180 seconds of dissociation. The buffers used for the experiment are depicted in table 4. Kinetic analysis was performed using a 1 :1 binding model and global fitting.
• Blood cells from human EDTA-blood were washed (3x in PBS), diluted in PBS and lysed by repeated freeze-thaw-cycles.
• the blood cell lysate had a total protein concentration of 250 ⁇ g/ml, and a DPP3 concentration of 10 ⁇ g/ml.
• Dilutions of blood cell lysate (1:40, 1:80, 1:160 and 1:320) and of purified recombinant human His-DPP3 (31.25-500 ng/ml) were subjected to SDS-PAGE and Western Blot.
• the blots were incubated in 1.) blocking buffer (lxPBS-T with 5% skim milk powder), 2.) primary antibody solution (AK1967 1:2.000 in blocking buffer) and 3.) HRP labelled secondary antibody (goat anti mouse IgG, 1:1.000 in blocking buffer). Bound secondary antibody was detected using the Amersham ECL Western Blotting Detection Reagent and the Amersham Imager 600 UV (both from GE Healthcare).
• a DPP3 activity assay with known procedure (Jones et al., 1982) was performed. Recombinant GST-tagged hDPP3 was diluted in assay buffer (25 ng/ ml GST-DPP3 in 50 mM Tris-HCl, pH7,5) and increasing concentrations of AK1967 were added. Fluorogenic substrate Arg-Arg- ⁇ was added to the solution and the generation of free ⁇ over time was monitored using the Twinkle LB 970 microplate fiuorometer (Berthold Technologies GmbH & Co.
• AK1967 binds with an affinity of 2.2* 10 "9 M to recombinant GST-hDPP3 (for details see table 6 and for kinetic curves see Figure 1 A).
• AK1967 inhibits 15 ng/ ml DPP3 in a specific DPP3 activity assay with an IC50 of about 15 ng/ml ( Figure 1C).
• a septic shock model was used to induce heart failure in rats and then to characterize AK1967's influence on oxidative stress in myocardium.
• mice Male Wistar rats (2-3 months, 300 to 400 g, group size refer to table 7) from the Centre d'elevage Janvier (France) were allocated randomly to one of three groups. All the animals were anesthetized using ketamine hydrochloride (90 mg/ kg) and xylazine (9 mg/ kg) intraperitoneally (i.p.). For induction of polymicrobial sepsis, cecal ligation and puncture (CLP) was performed using Rittirsch's protocol with minor modification. A ventral midline incision (1.5 cm) was made to allow exteriorization of the cecum. The cecum is then ligated just below the ileocecal valve and punctured once with an 18-gauge needle.
• CLP cecal ligation and puncture
• Dihydroethidium (DHE; Sigma-Aldrich) staining was used to evaluate the in situ levels of superoxide anion in the myocardium. Cardiac cryostat sections (7 um) of the ventricles were incubated with DHE (37 uM) for 30 min in a dark humidified chamber. Acquisition of fluorescent images of ethidium bromide with Leica fluorescence microscope was performed under identical setting whatever the block tissue. The stained area was measured with IPLab software and expressed as a percentage of area of interest (% of ROI).
• ROS reactive oxygen species
• Human dipeptidyl peptidase III acts as a post-proline-cleaving enzyme on endomorphins. Biological Chemistry, 388(3), pp.343-348.
• Oxidative stress An essential factor in pathogenesis of gastrointestinal mucosal diseases. Physio.l Rev. 94, pp. 329-354. Bird et al., 1988. Single-chain antigen-binding proteins. Science 242:423-426.
• Oxidative stress as a mediator of cardiovascular disease. Oxidative
• Dipeptidyl peptidase III a multifaceted oligopeptide
• Fig. 1 AK1967 characterization (A) Association- and dissociation curve of the AK1967-DPP3 binding analysis using Octet.
• AK1967 loaded biosensors were dipped into a dilution series of recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM) and association and dissociation monitored.
• Inhibition of DPP3 by a specific antibody is concentration dependent, with an IC50 at ⁇ 15 ng/ml when analyzed against IS ng/ml DPP3.
• Fig. 2 Influence of AK1967 on oxidative stress in rats with septic shock induced heart failure

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