Novel recombinant diamine oxidase and its use for the treatment of diseases characterized by excess histamine - Patent Application 20070122999

Abstract

The present invention relates to a recombinant human diamine oxidase (DAO) with reduced glycosaminoglycan binding affinity, said DAO comprising at least one amino acid modification in the glycosaminoglycan (GAG)-binding domain. The present invention further relates to the use of DAO in the treatment of conditions associated with excess histamine, particularly in the treatment of chronic allergic diseases, more particularly in the treatment of anaphylaxis, anaphylactic shock, chronic urticaria, acute urticaria, asthma, hay fever, allergic rhinitis, allergic conjunctivitis, histamine poisoning, headache, atopic dermatitis, inflammatory diseases, mastocytosis, mast cell activation syndrome (MCAS), pre-eclampsia, hyperemesis gravidarum, preterm labor, peptic ulcer, acid reflux, pruritus, and sepsis.

Description

[Technical Field]

[0001] The present invention relates to recombinant diamine oxidases (DAOs) with reduced glycosaminoglycan binding affinity, which DAOs comprise at least one amino acid modification in the glycosaminoglycan (GAG) binding domain.

[0002] The present invention further relates to the use of DAO in the treatment of conditions associated with excess histamine, in particular the treatment of chronic allergic diseases and / or the treatment of high-risk pregnancies, more particularly the treatment of anaphylaxis, anaphylactic shock, chronic urticaria, acute urticaria, asthma, hay fever, allergic rhinitis, allergic conjunctivitis, histamine poisoning, headache, atopic dermatitis, inflammatory diseases, mastocytosis, mast cell activation syndrome (MCAS), pre-eclampsia, hyperemesis gravidarum, preterm labor, peptic ulcer, acid reflux, pruritus and sepsis. [Background technology]

[0003] Histamine (2-(1H-imidazol-4-yl)ethanamine) is an organic nitrogen compound involved in local immune responses, regulating gastrointestinal physiology, and acting as a neurotransmitter for the brain, spinal cord, and uterus. Histamine is involved in inflammatory responses and plays a central role as an itch mediator. As part of the immune response to foreign pathogens, histamine is produced by basophils and mast cells found in nearby connective tissue. Histamine is stored in an inactive form within the metachromatic granules of mast cells and basophilic leukocytes and is available for immediate release. Histamine can also be synthesized de novo by neutrophils and macrophages during inflammation. After its release, histamine is a potent physiological and pathological mediator that binds to four receptors (H1–H4) expressed on many different cells throughout the body. Histamine binding to its receptors is highly specific, inducing numerous downstream activities. Acute allergic reactions, such as hay fever, runny nose, itchy eyes, or in more severe cases, asthma accompanied by respiratory distress; acute and chronic urticaria, especially those caused by antibiotic or contrast agent administration, peanut or bee allergies, and hypersensitivity reactions, also known as anaphylaxis, are mediated by tissue mast cells and blood basophils that release several mediators, of which histamine is one of the most active. Histamine-induced symptoms include, among others, anaphylaxis, hypersensitivity reactions accompanied by a drop in blood pressure, syncope, and respiratory distress, bronchospasm, flushing (skin redness), pruritus (itching), tachycardia (high pulse rate), syncope (fainting), hypotension (low blood pressure), epigastric pain, abdominal pain, nausea, vomiting, diarrhea, fatigue, memory loss, depression, and headache. Excessive histamine in the blood (>10 ng / ml, symptoms begin at 1-3 ng / ml) is life-threatening. Histamine levels in the blood of about 100 ng / ml can cause cardiac arrest.Especially during pregnancy, hyperhistamineemia can lead to certain pregnancy complications, such as preeclampsia, spontaneous abortion, preterm labor, and hyperemesis gravidarum (Brew O. and Sullivan MHF, J. Reprod. Immunol., 72 (2006), pp. 94-107; Maintz L. et al., Human Reproduction Update, 14, 5, 2008, pp. 485-495).

[0004] Mast cells and basophils are immune system cells with many different functions. Nevertheless, mast cells and histamine play an important role in numerous diseases, such as mast cell activation syndrome (MCAS) (i.e., the inability to tolerate histamine in red wine, cheese, or other foods, often referred to in the literature as histamine intolerance), atopic dermatitis (a skin disease also known as neurodermatitis), mastocytosis (an increase in the number of mast cells in the skin and / or internal organs such as bone marrow, liver, or spleen), peptic ulcer disease (damage to the mucosa of the stomach or duodenum due to acid release following excess histamine), acid reflux, headache, pruritus, and possibly even sepsis, among others. Histamine can also be synthesized de novo under certain disease conditions, for example, by histidine decarboxylase induced in neutrophils and macrophages.

[0005] Furthermore, histamine can enter the body externally by inhalation or orally, for example, by consuming histamine-containing foods such as cheese, wine, canned fish, and sauerkraut. Histamine can also be produced by bacteria in the bacterial flora.

[0006] Antihistamines that counteract the activity of histamine receptors H1 and H2 have been available for decades and are assumed to be effective in treating symptoms such as runny nose and itchy skin and eyes. Antihistamines are among the most frequently prescribed medications worldwide. Nevertheless, thorough data analysis has clearly demonstrated that antihistamines have limited efficacy in some conditions. For example, approximately 25% of patients with chronic urticaria are antihistamine-resistant and suffer from poor quality of life. Treatment of hypersensitivity reactions with antihistamines is widely practiced, but high-quality efficacy data are lacking, and some documents have reported lack of efficacy. Anaphylaxis guidelines do not necessarily recommend the use of histamine H1 receptor blockers for treatment because evidence of benefit is unclear. Histamine H2 receptor antagonists are recommended to be avoided in anaphylaxis because they may worsen symptoms.

[0007] The limited efficacy of antihistamines is not surprising. Several studies have shown that antihistamines can only block the effects of histamine concentrations that are two- to three-fold elevated, and are much less effective at higher histamine concentrations. During anaphylactic or hypersensitivity reactions, circulating histamine concentrations can rise more than 100-fold compared to normal subjects, and even more so in patients with mastocytosis. Patients with mastocytosis have continuously elevated levels of histamine and its metabolites that are five to 15 times higher than in the steady-state, nonanaphylactic state. Because the body cannot break down excess histamine quickly enough, a variety of symptoms inevitably result.

[0008] In mammals, histamine is degraded by two enzymes: diamine oxidase (DAO, EC 1.4.3.6) and histamine-N-methyltransferase (HNMT or NMT for short, EC 2.1.1.8). NMT catalyzes the N-methylation of histamine to N-methylhistamine.

[0009] The structure and inhibition of human DAO are reviewed in McGrath AP et al. (Biochemistry, 2009, 48(41), pp. 9810-9822). DAO catalyzes the oxidative deamination of histamine to imidazole acetaldehyde. DAO was named histaminase because it was first identified as an enzyme that removes exogenous histamine from minced lung and liver samples (Best CH., J. Physiol., 1929, 67, pp. 256-263). Subsequently, a protein identified as diamine oxidase was erroneously named amiloride-binding protein and was found to be involved in the ameloride-sensitive Na+ binding. + It has been linked to the ATP channel. Some databases still refer to AOC1 as ABP1. It was later noted by the original authors (Novotny WF et al., J. Biol. Chem., 1994, 269, pp. 9921-9925) that DAO and ABP1 are in fact the same protein, who also reported the cloning of a human gene corresponding to a 751-amino acid protein. Overexpression of recombinant human DAO (hDAO) was achieved in insect cells (Elmore BO et al., J. Biol. Inorg. Chem., 2002, 7, pp. 565-579). Elmore et al. further reported that DAO contains a heparin-binding consensus sequence (residues 568-575), but the structure did not lead to the inference that the heparin-binding domain actually binds heparin. A 12-mer heparin molecule is approximately 5 nm (50 Å) long, and the diameter of the cyclic heparin-binding domain in DAO is approximately 25 Å. The molecular weight of a 12-mer heparin = 284 x 12 = 3408 Da. LMWHs do not bind strongly to DAO but have an average molecular weight of 5000 Da (18 sugar units). Only HMWHs bind to DAO and have an average molecular weight of 15000 Da, which corresponds to 15000 / 284 = 52 sugar units (in a row).

[0010] The availability of large quantities of recombinant hDAO has enabled in vitro identification of its preferred substrates, demonstrating a clear preference for diamines. Two atypical diamines, histamine and 1-methylhistamine, are particularly good substrates. Each contains an imidazole group in place of one of the primary amines present in typical diamine substrates such as spermidine, putrescine, or cadaverine. hDAO is the first-line enzyme for degrading exogenous histamine, and reduced levels of DAO have been shown to directly correlate with histamine intolerance (Maintz L. et al., Am. J. Clin. Nutr., 2007, 85, pp. 1185-1196). Decreased DAO activity has been found in multiple heterogeneous complications of pregnancy, such as diabetes, threatened abortion, missed abortion, and trophoblastic disease (Maintz L. et al., 2008).

[0011] In the gastrointestinal tract, DAO breaks down dietary histamine to prevent its blood concentration from increasing and protect the body. Nevertheless, except during pregnancy, DAO antigen, and therefore activity, is low or even absent in plasma (Boehm T. et al., Clinical Biochemistry 50, 2017, p. 444-451). During pregnancy, DAO activity increases by more than 100-fold.

[0012] WO 02 / 43745 discloses the systemic use of DAO of plant origin to treat histamine-mediated diseases. However, the administration of enzymes isolated directly from plants presents significant problems, primarily in view of the fact that the legumes disclosed in WO 02 / 43745 have high allergenic potential, as allergens are frequently found in plants.

[0013] WO 2006 / 003213 describes specific dosage forms of animal-derived or recombinantly produced DAO. Expression and purification of recombinant wild-type (wt) human (rh) DAO in CHO cells was reported by Gludovacz E. et al. (J Biotechnol. 2016, 227: pp. 120-130). Nevertheless, the α-phase (distribution phase) half-life of wt rhDAO in rats was less than 10 minutes and contained more than 80% of the injected protein. The β-phase (elimination phase) half-life was approximately 3 hours. Because the scaling factor for biopharmaceuticals extrapolating from rats to humans is approximately 4-8, the PK (pharmacokinetic) profile of recombinant wtDAO is not appropriate for preclinical and clinical development. Mutation of three glycosylation sites in DAO was performed, but glycan mutations did not improve the PK profile. The effect of glycan mutations on the expression and activity of DAO was published by Gludovacz E. et al. (J. Biol. Chem. 2018, 293(3), p. 1070-1087).

[0014] DATABASE UniProt Accession No. G3QJ02, 2011, XP-002792064 discloses a diamine oxidase sequence from the western gorilla (Gorilla gorilla).

[0015] DATABASE UniProt Accession No. Q9SXW5, 2000 discloses a diamine oxidase sequence from pea (Pisum sativum).

[0016] WO 2012 / 028891 reports histaminase derived from vegetables. Because released histamine cannot be effectively blocked or inactivated by antihistamines, new therapeutic and preventive methods for quickly inactivating excess histamine would be of significant benefit to patients suffering from high circulating histamine levels. Many patients suffer from elevated histamine levels for hours. Moreover, the half-life of histamine increases during anaphylaxis and hypotension due to reduced renal filtration and blood flow. Summary of the Invention [Problem to be solved by the invention]

[0017] Thus, there is a high and unmet need for improved treatments for conditions associated with excess histamine. [Means for solving the problem]

[0018] During disease states, excess histamine cannot be effectively counteracted by currently available antihistamines. Elevated histamine levels cause bothersome, severe, debilitating, and life-threatening symptoms, and sometimes death.

[0019] It is an object of the present invention to provide improved regimens for the removal of excess histamine and the treatment and prevention of histamine-induced diseases and conditions. This object is achieved according to the present invention by providing an individual with a recombinant modified DAO that aids or enables the breakdown of histamine by increasing the concentration of active DAO in the individual's body.

[0020] Administration of the recombinant human DAO described herein allows for the decomposition of excess histamine in acute, subacute, subchronic, chronic, and preventative conditions, thereby significantly benefiting patients suffering from or about to suffer from several diseases in which antihistamines are not sufficiently effective in decomposing excess histamine levels, establishing an entirely new treatment option for patients suffering from excess histamine.

[0021] According to the present invention, there is provided a recombinant human diamine oxidase (DAO) having a glycosaminoglycan (GAG) binding affinity reduced compared to the GAG ​​binding affinity of a corresponding wild-type human DAO, wherein the DAO comprises at least one amino acid modification in the glycosaminoglycan (GAG) binding domain, in particular, the GAG ​​binding domain comprises amino acids 568 to 575 in the numbering of SEQ ID NO: 1.

[0022] In particular, the GAG-binding domain is a heparin / heparan sulfate-binding domain. In particular, the amino acid modifications result in a decrease in the GAG-binding affinity of DAO, while maintaining its enzymatic activity towards histamine.

[0023] According to one particular embodiment, the at least one amino acid modification in the GAG-binding domain of the DAO is an amino acid substitution, deletion, insertion or coupling with a chemical moiety. According to a particular embodiment of the present invention, the recombinant DAO of the present invention comprises 2, 3, 4, 5, 6, 7 or 8 amino acid modifications in the GAG-binding domain, in particular, the amino acid residue is replaced by another amino acid residue, in particular, arginine or lysine is replaced by serine or threonine.

[0024] According to an alternative embodiment, the recombinant DAO comprises a GAG-binding domain of the amino acid sequence X1FX2X3X4LPX5, wherein: X1 can be any amino acid, in particular A or S, more in particular S; X2 can be any amino acid, in particular K; X3 may be any amino acid, in particular A or T, more in particular T; X4 can be any amino acid, in particular K; X5 can be any amino acid, in particular K or T, more in particular T.

[0025] In a further specific embodiment, the recombinant DAO comprises an amino acid sequence selected from the group consisting of SFKAKLPK (SEQ ID NO: 33), AFKAKLPT (SEQ ID NO: 34), AFKTKLPK (SEQ ID NO: 35), SFKTKLPK (SEQ ID NO: 36), AFKTKLPT (SEQ ID NO: 37), SFKAKLPK (SEQ ID NO: 38).

[0026] According to one particular embodiment, the recombinant DAO disclosed herein further comprises at least one modification of the solvent-exposed cysteine ​​at amino acid position 123 (cys123) relative to the numbering of SEQ ID NO: 1, in particular the modification of the cysteine ​​is an amino acid substitution, deletion or attachment with a chemical moiety.

[0027] In a preferred embodiment, cys123 according to the numbering of SEQ ID NO: 1 of the DAO is substituted by alanine (cys123ala, C123A). The DAO of the present invention particularly exhibits reduced plasma clearance. The present invention provides a recombinant DAO that has a significantly extended plasma half-life compared to wild-type DAO, particularly a half-life that is at least 1.5 times, particularly at least 2 times longer than wild-type DAO.

[0028] In an alternative or further embodiment, the DAO has an AUC that is increased by at least 10-fold compared to a wild-type DAO. According to one embodiment provided herein, internalization of the recombinant DAO by endothelial cells is reduced by at least 10%, 25%, 50%, 60%, 70%, 80%, particularly 90% compared to wild-type DAO.

[0029] According to a further embodiment, the GAG ​​binding affinity, particularly heparin / heparan sulfate binding affinity, of the DAO described herein is reduced by at least 10%, 25%, 50%, 60%, 70%, 80%, particularly 90% compared to wild-type DAO.

[0030] Further provided herein is a recombinant DAO or a functional derivative or analog thereof comprising the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or having at least 90% sequence identity to any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0031] Human DAO also has multiple N-glycosylation sites involved in secretion and retention in the endoplasmic reticulum. In particular, the glycan at Asn-168 is predominantly biantennary to tetraantennary sialylated.

[0032] According to one embodiment, the recombinant DAO described herein further comprises one modification, particularly an amino acid substitution at position 168 relative to SEQ ID NO: 1. In particular, the modification is a single modification at position 168. More particularly, Asn is substituted with Gln. In particular, the modification increases the PK of the DAO described herein. More particularly, the DAO comprises the amino acid sequence of SEQ ID NO: 106.

[0033] Further provided herein is a recombinant DAO or a functional derivative or analog thereof encoded by or having at least 90% sequence identity to any one of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32.

[0034] Further provided herein is an isolated nucleotide sequence encoding a DAO or functional analog or derivative thereof described herein, particularly comprising the sequence of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32, or a fragment thereof.

[0035] According to one particular embodiment of the present invention, provided herein is a fusion polypeptide comprising the recombinant DAO described herein and the Fc domain of human IgG or human serum albumin (HSA) or a fragment thereof, wherein the fusion polypeptide retains the functional activity of the recombinant DAO.

[0036] Further provided herein is a fusion polypeptide comprising the recombinant DAO described herein and the Fc domain of human IgG, the fusion polypeptide comprising any one of SEQ ID NOs: 56 to 70 or having at least 90% sequence identity to any one of SEQ ID NOs: 56 to 70.

[0037] Further provided herein is a fusion polypeptide encoded by or having at least 90% sequence identity to any one of SEQ ID NOs: 72-102, comprising a recombinant DAO described herein and the Fc domain of human IgG, or a functional derivative or analog thereof.

[0038] Further provided herein are recombinant vectors comprising the nucleotide sequences described herein, particularly where the vectors are bacterial, yeast, baculovirus, plant or mammalian expression vectors.

[0039] According to one embodiment, provided herein is an expression cassette comprising a nucleotide sequence operably linked to a regulatory element. According to one embodiment, provided herein is a recombinant host cell or host cell line of bacterial, yeast, baculovirus, plant or mammalian origin comprising the recombinant DAO described herein, wherein the host cell is selected from the group consisting of CHO cells, Vero cells, MDCK cells, Pichia pastoris cells and SF9 cells, among others.

[0040] Further provided herein is an expression system comprising a vector or expression cassette and a host cell or host cell line as described herein. According to one embodiment, there is further provided a method for producing a recombinant DAO as described herein, comprising: cloning a nucleotide sequence encoding i.DAO into an expression vector; ii. transforming a host cell with the vector; iii. culturing the transformed host cells under conditions in which the DAO is expressed; iv. isolating the DAO from the host cell culture, optionally by disruption of the host cells; and optionally v. DAO purification process Provided herein is a method comprising:

[0041] According to one embodiment, a pharmaceutical composition is provided comprising a recombinant DAO and optionally one or more excipients. In particular, the pharmaceutical compositions may be administered intravenously, intramuscularly, and subcutaneously, or by another parenteral route of administration, such as intraperitoneally or intrathecally.

[0042] In a further embodiment, there is provided a use of the recombinant DAO for preparing a pharmaceutical composition. In particular, there is provided a recombinant DAO for use in the treatment of conditions associated with excess histamine, particularly in the treatment of chronic allergic diseases or diseases associated with elevated histamine or decreased DAO activity, more particularly in the treatment of anaphylaxis, anaphylactic shock, chronic urticaria, acute urticaria, asthma, hay fever, allergic rhinitis, allergic conjunctivitis, histamine poisoning, headache, atopic dermatitis, inflammatory diseases, mastocytosis, mast cell activation syndrome (MCAS), pre-eclampsia, hyperemesis gravidarum, preterm labor, peptic ulcer, acid reflux, pruritus and sepsis.

[0043] In one embodiment of the present invention, the recombinant DAO is used in the manufacture of a medicament for the treatment of conditions associated with excess histamine, in particular for the treatment of chronic allergic diseases or diseases associated with elevated histamine or decreased DAO activity, more particularly for the treatment of anaphylaxis, anaphylactic shock, chronic urticaria, acute urticaria, asthma, hay fever, allergic rhinitis, allergic conjunctivitis, histamine poisoning, headache, atopic dermatitis, inflammatory diseases, mastocytosis, mast cell activation syndrome (MCAS), pre-eclampsia, hyperemesis gravidarum, preterm labor, peptic ulcer, acid reflux, pruritus and sepsis.

[0044] In an alternative embodiment, provided herein is a target-specific ligand that specifically binds to the GAG-binding domain of DAO, in particular to one or more of the amino acids at positions 568 to 575 in the numbering of SEQ ID NO:1.

[0045] Alternatively, provided herein are target-specific ligands that specifically inhibit heparin / heparan sulfate binding to the GAG-binding domain of DAO, particularly to any one or more of the amino acids 568 to 575 in the numbering of SEQ ID NO: 1.

[0046] In particular, the ligand is selected from the group consisting of a nucleic acid, a small molecule inhibitor or an antigen binding protein. In an alternative embodiment, the ligand is, in particular: - an antibody or antibody fragment, such as a Fab, Fd, scFv, diabody, triabody, Fv tetramer, minibody, nanobody, single domain antibody, such as a VH, VHH, IgNAR or V-NAR; antibody mimetics, such as Adnectin™, Affibody®, Affilin®, Affimer®, affitins, alphabodies, aptamers, anticalins, avimers, DARPins®, Fynomer®, Kunitz domain peptides, monobodies or NanoCLAMPs, or - fusion proteins containing one or more immunoglobulin fold domains, antibody domains or antibody mimetics The antigen-binding protein is selected from the group consisting of:

[0047] According to one embodiment, there is provided a method for identifying a compound that modulates heparin binding of a DAO, comprising the steps of: (a) constructing a computer model of the GAG-binding domain defined by the amino acid structural coordinates of the DAO sequence of SEQ ID NO: 1; (b)(i) assembling molecular fragments into said compound; (ii) selection of compounds from a small molecule database; and (iii) de novo ligand design of the compound selecting a potential regulatory compound by a method selected from the group consisting of: (c) to provide an energy-minimized configuration of said compound in the heparin-binding domain. using a computational means to perform a fitting program between a computer model of the compound and a computer model of the GAG ​​binding domain; (d) evaluating the results of the fitting procedure to quantify the association between the compound and the heparin / heparan sulfate binding domain, thereby assessing the ability of the compound to associate with the heparin / heparan sulfate binding domain. Provided herein are methods of identification comprising: [Brief explanation of the drawings]

[0048] [Figure 1-1] The amino acid and nucleotide sequences of wild-type and modified DAOs are shown. For the amino acid sequences, bold refers to substitutions relative to the wt sequence, underlined letters refer to the secretion signal, bold italic letters refer to Fc, and underlined bold italic letters refer to the linker sequence of the IgG1 hinge region. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above. [Figure 1-6] Same as above. [Figure 1-7] Same as above. [Figure 1-8] Same as above. [Figure 1-9] Same as above. [Figure 1-10] Same as above. [Figure 1-11] Same as above. [Figure 1-12] Same as above. [Figure 1-13] Same as above. [Figure 1-14] Same as above. [Figure 1-15] Same as above. [Figure 1-16] Same as above. [Figure 1-17] Same as above. [Figure 1-18] Same as above. [Figure 1-19] Same as above. [Figure 1-20] Same as above. [Figure 1-21] Same as above. [Figure 1-22] Same as above. [Figure 1-23] Same as above. [Figure 1-24] Same as above. [Figure 1-25] Same as above. [Figure 1-26] Same as above. [Figure 1-27] Same as above. [Figure 1-28] Same as above. [Figure 1-29] Same as above. [Figure 1-30] Same as above. [Figure 1-31] Same as above. [Figure 1-32] Same as above. [Figure 1-33] Same as above. [Figure 1-34] Same as above. [Figure 1-35] Same as above. [Figure 1-36] Same as above. [Figure 1-37] Same as above. [Figure 1-38] Same as above. [Figure 1-39] Same as above. [Figure 1-40] Same as above. [Figure 1-41] Same as above. [Figure 1-42] Same as above. [Figure 1-43] Same as above. [Figure 1-44] Same as above. [Figure 1-45] Same as above. [Figure 1-46] Same as above. [Figure 1-47] Same as above. [Figure 1-48] Same as above. [Figure 1-49] Same as above. [Figure 1-50] Same as above. [Figure 2] 1 shows heparin sepharose elution profiles of recombinant human DAO wild-type and heparin / heparan sulfate mutants. [Figure 3] Hepmut 1, 4 and 7 variants elute from heparin sepharose at 50% lower salt concentrations than DAO_WT. [Figure 4] Western blot of SK-Hep1 cell lysates after incubation with DAO_WT and Hepmut variants is shown. [Figure 5] The Hepmut4 variant shows reduced binding to SK-Hep1 cells compared to DAO_WT. [Figure 6] Isothermal titration calorimetry of DAO_WT and Hepmut4 is shown. [Figure 7] (a) Linear y-axis scale and (b) logarithmic y-axis scale are shown. After intravenous injection of 1 mg / kg DAO variant, Hepmut4 increases the AUC (area under the curve) by more than 19-fold compared to wild-type DAO protein. [Figure 8] After intraperitoneal injection, Hepmut4 increases the AUC by more than 16-fold compared to the DAO_WT protein. [Figure 9] The average values ​​measured using 1 mg / kg DAO wild type and different Hepmut variants are shown. [Figure 10] 1 shows the slower clearance of heparin / heparan sulfate binding domain mutants compared to DAO wild-type protein administered at 1 mg / kg. [Figure 11] 1 shows the slower clearance of the heparin-binding domain mutants compared to the DAO wild-type protein. [Figure 12] 1 shows the slower clearance of the heparin-binding domain mutant compared to the DAO wild-type protein administered at 1 mg / kg. [Figure 13] Slow clearance of heparin-binding domain mutants compared to DAO wild-type protein administered at 1 mg / kg. Linear y-axis scale. [Figure 14]Slow clearance of heparin-binding domain mutants compared to DAO wild-type protein administered at 1 mg / kg. First 90 minutes shown. Linear y-axis scale. [Figure 15] Rapid clearance of Fc-DAO wild type compared to Fc-Hepmut4, administered at 1 mg / kg to 6 or 4 rats, respectively. Mean values ​​and standard deviations are shown. Linear y-axis scale. [Figure 16] Rapid clearance of Fc-DAO wild type compared to Fc-Hepmut4, administered at 1 mg / kg to 6 or 4 rats, respectively. Mean values ​​and standard deviations are shown. Logarithmic y-axis scale. [Figure 17] Fc-DAO-Hepmut4 shows a significant increase in AUC after intravenous administration of 1 mg / kg. Logarithmic y-axis scale. [Figure 18] Fc-DAO-Hepmut4 shows a significant increase in AUC after intravenous administration of 1 mg / kg. Linear y-axis scale. [Figure 19] Western blots of DAO mutants are shown. [Figure 20] No significant difference in DAO activity was observed between DAO_WT and the cys123 mutant. DETAILED DESCRIPTION OF THE INVENTION

[0049] Unless otherwise specified or defined, all terms used herein have their ordinary meaning in the art and are clear to those skilled in the art. For example, see standard manuals such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd ed.), vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990) and Janeway et al., "Immunobiology" (5th ed. or later, Garland Science, New York, 2001).

[0050] The claimed subject matter relates, inter alia, to man-made products, which may be variants of naturally occurring (wild-type) products, or methods of using or producing such man-made products. While there may be some sequence identity to naturally occurring structures, it is understood that the materials, methods, and uses of the invention, e.g., with respect to isolated nucleic acid sequences, amino acid sequences, fusion constructs, expression constructs, transformed host cells, and modified proteins, among others, are "man-made" or synthetic and therefore not considered the result of the "laws of nature."

[0051] As used herein, the terms "comprise," "containing," "having," and "include" may be used interchangeably and are therefore understood to be open-ended definitions that allow for additional members, moieties, or elements. "Consisting of" is considered the most restrictive definition, without additional elements related to the characteristics of the "consisting of" definition. Thus, "comprising" is broader and encompasses the "consisting of" definition.

[0052] As used herein, the term "about" relates to the same value or a value that differs by + / - 5% from any value. The human DAO monomer contains approximately 751 amino acids and forms an enzymatically active dimer. Ten of the 14 cysteines in the DAO dimer are involved in disulfide bond formation, while four are not. In particular, cysteines 123 and 633 are not involved in disulfide bond formation.

[0053] As used herein, amino acid refers to the 20 naturally occurring amino acids coded for by 64 triplet codons. These 20 amino acids are divided into those with neutral, positive and negative charges: The "neutral" amino acids are shown below, along with their respective three-letter and one-letter codes and polarities.

[0054] Alanine: (Ala, A) non-polar, neutral; Asparagine: (Asn, N) polar, neutral; Cysteine: (Cys, C) non-polar, neutral; Glutamine: (Gln, Q) polar, neutral; Glycine: (Gly, G) non-polar, neutral; Isoleucine: (Ile, I) non-polar, neutral; Leucine: (Leu, L) non-polar, neutral; Methionine: (Met, M) non-polar, neutral; Phenylalanine: (Phe, F) non-polar, neutral; Proline: (Pro, P) non-polar, neutral; Serine: (Ser, S) polar, neutral; Threonine: (Thr, T) polar, neutral; Tryptophan: (Trp, W) non-polar, neutral; Tyrosine: (Tyr, Y) polar, neutral; Valine (Val, V) non-polar, neutral; and Histidine: (His, H) polar, positive (10%) neutral (90%).

[0055] "Positively" charged amino acids: Arginine: (Arg, R) polar, positive; and Lysine: (Lys, K) Polar, positive.

[0056] "Negatively" charged amino acids: Aspartic acid: (Asp, D) polar, negative; and Glutamic acid: (Glu, E) polar, negative.

[0057] The term "modification" of the DAO of the present invention refers to any amino acid sequence change, including, but not limited to, amino acid substitution, addition, deletion, mutation, and insertion. Modification can also be chemoselective modification. Such modification can be binding or coupling with a chemical moiety, provided that a stable covalent bond is formed between two molecules, at least one of which is a biological molecule. Such bonds can be formed with one or more amino acid residues, for example, but not limited to, maleimide, iodoacetamide, isoacetamide, 2-thiopyridine, 3-arylpropiolonitrile, benzoyl fluoride, isothiocyanate, isocyanate, diazonium salt, PTAD, NaIO4, or PLP.

[0058] Free cysteines rarely occur on protein surfaces, making them excellent choices for chemoselective modification. In particular, the Cys at amino acid positions 123 and 633 of the modified DAOs described herein are solvent-exposed. Specific substitution of the solvent-exposed Cys123, specifically exchanging Cys123 for Ala123, renders the resulting DAO unable to form disulfide bonds or other intermolecular interactions that lead to higher-order aggregates. When recombinant DAO is expressed in CHO cells, a certain percentage (approximately 20%-30%) of DAO molecules form not only dimers but also tetramers, hexamers, and even octamers. These higher-order oligomers have been described and may be considered natural variants with unknown functions. It is unknown whether these higher-order oligomers form during folding and transport from the endoplasmic reticulum to the Golgi apparatus and secretion into the extracellular environment, or whether they form primarily in the extracellular environment. Yet, these tetramers and larger oligomers complicate the expression, purification, characterization, standardization, and selection of optimal formulations of the recombinant DAOs described herein. Due to mutation of relatively solvent-exposed cysteines on the surface of the DAO, particularly Cys123, only DAO dimers are found in the supernatant of host cells, particularly CHO cells.

[0059] Furthermore, according to a further particular embodiment, the cysteine ​​at amino acid position 633 may also be modified as described herein for cys 123. Cys 633 also does not participate in disulfide bond formation.

[0060] Under basic conditions, cysteine ​​residues can be deprotonated to generate thiolate nucleophiles that react with soft electrophiles, such as maleimides and iodoacetamides, resulting in the formation of carbon-sulfur bonds. Another modification of cysteine ​​residues involves the formation of disulfide bonds. Reduced cysteine ​​residues react with exogenous disulfides to generate new disulfide bonds on proteins. Excess disulfides, such as 2-thiopyridone and 3-carboxy-4-nitrothiophenol, are frequently used to facilitate the reaction. Electron-deficient alkynes have been shown to selectively react with protein cysteine ​​residues in the presence of another nucleophilic amino acid residue. Depending on the substitution of the alkyne, this reaction can produce cleavable bioconjugates (when alkynone derivatives are used) or hydrolytically stable bioconjugates (when 3-arylpropiolonitriles are used).

[0061] The term "modification" also includes the substitution of natural amino acids with unnatural amino acids. Unnatural amino acids are not encoded by the universal genetic code. Typically, these unnatural amino acids can be found in nature as metabolic products, particularly in plants and bacteria. Such unnatural amino acids include D-amino acids, homoamino acids, N-methyl amino acids, alpha-methyl amino acids, beta-amino acids, and the like. 2 -amino acid, beta 3 -amino acid, beta 3- may be selected from, but are not limited to, homoamino acids, ACHC, peptides, or heavy amino acids, in particular substituted with 13C and / or 15N atoms, in particular E-acetyl lysine, alanine (3-aminopropionic acid), 6-aminocaproic acid, ?-aminobutyric acid, citrulline, acetamidomethyl-protected cysteine, dimethyl lysine, hydroxyproline, mercaptopropionic acid, methyl lysine, 3-nitrotyrosine, norleucine, pyroglutamic acid, carbobenzoxy.

[0062] The term "GAG-binding domain" as used herein refers to the region in DAO that is involved in binding or interacting with all four glycosaminoglycans, including heparin / heparan sulfate, chondroitin / dermatan sulfate, keratin sulfate, and hyaluronan. Binding with heparin / heparan sulfate is preferred. Heparin is present in mast cells. Heparan sulfate is present on almost all cells, such as endothelial cells.

[0063] The term "GAG binding" refers to the interaction / binding of DAO with glycosaminoglycans, particularly heparin or heparan sulfate. GAGs bind to many different types of proteins, mostly through electrostatic interactions between negatively charged sulfate groups and uronic acids and positively charged amino acids in the protein. Heparin binding affinity can be measured by any method known to those skilled in the art. Many methods are available for analyzing GAG-protein interactions, some of which are based on the K d This provides a direct measure of K values. A common method involves affinity fractionation of proteins on a Sepharose column containing covalently bound GAG chains, usually heparin. Bound proteins are eluted with different concentrations of sodium chloride, and the concentration required for elution is generally determined by the K d High affinity interactions require at least 1M NaCl to displace the bound ligand, which is proportional to 10 -7 ~10 -9 K of M d The binding affinity is converted to a value (measured by equilibrium binding under physiological salt concentrations). -4~10 -6 Proteins of M (M) either do not bind under "normal" conditions (0.15 M NaCl) or require only 0.3–0.5 M NaCl for elution. This method, based on the assumption that GAG-protein interactions are entirely ionic, can be used to assess relative affinity when comparing different GAG-binding proteins. Alternative methods include affinity co-electrophoresis, analytical ultracentrifugation, circular dichroism, competitive ELISA, fluorescence microscopy, ion mobility mass spectrometry, isothermal titration calorimetry, laser light scattering, NMR, surface plasmon resonance, and X-ray, which provide detailed thermodynamic data (e.g., ΔH [change in enthalpy], ΔS [change in entropy], ΔCp [change in molar heat capacity]), kinetic data (association and dissociation rates), and high-resolution data on the atomic connectivity of GAG-protein interactions (Esko JD et al., Essentials of Glycobiology, 3rd ed., Chapter 38, 2017).

[0064] In particular, the DAO variants described herein have reduced or impaired binding to heparin and / or heparan sulfate. In particular, the heparin / heparan sulfate binding affinity of recombinant DAO is reduced by at least 10%, 25%, 50%, 60%, 70%, 80%, and particularly 90% compared to wild-type DAO. According to a specific method, binding affinity is measured using heparin Sepharose chromatography, where the DAO variant is incubated at a low salt concentration and eluted with increasing salt concentration. The salt concentration at which the DAO protein peaks (measured using absorbance at 280 nm) is used as the mM salt concentration at which DAO is eluted.

[0065] The DAO of the present invention further exhibits reduced internalization into endothelial cells compared to the internalized wild-type DAO, particularly reduced internalization by endothelial cells by at least 10%, 25%, 50%, 60%, 70%, 80%, and particularly 90% compared to the wild-type DAO.

[0066] In particular, the GAG-binding domain comprises amino acids 568 to 575 with respect to the numbering of SEQ ID NO: 1. Within said domain, one, two, three, four, five, six, seven or all amino acids may be modified.

[0067] The DAO monomer contains 24 lysines and 44 arginines in its primary amino acid sequence, most of which are located on the surface of the molecule. Additional lysines and arginines appear to be involved in heparin binding. In addition to the GAG-binding domain encompassing amino acids 568–575, additional lysines or arginines on the surface of DAO may be involved in heparin / heparan sulfate binding.

[0068] Modification of one or more of these lysines and / or arginines may further reduce the heparin / heparan sulfate binding of recombinant DAO. The term "enzyme activity" of DAO refers to the ability of the polypeptide to catalyze the oxidative deamination of suitable substrates, such as putrescine or histamine, to aminobutyraldehyde or imidazoleacetaldehyde.Maintaining enzymatic activity means that the DAO of the present invention has the same or similar enzymatic activity as the corresponding wild-type DAO.Enzyme activity that is at least 80%, particularly at least 90% of the wild-type activity is considered to be similar to the enzymatic activity of wild-type DAO.

[0069] Any method known in the art can be used to measure the enzymatic activity of DAO. These methods include assays using horseradish peroxidase (HRP)-mediated luminol oxidation (Bartko J. et al., Alcohol. 2016 Aug;54: pp.51-9), spectrophotometry as described by Holmstedt BO and Tham R. (Acta Physiol. Scand., 1959, 45, pp.152-163) and Bardsley WG et al. (Biochem. J. 1972, 127, pp.875-879), mass spectrometry (Gludovacz E. et al., 2016), liquid scintillation measurement (Okuyama T. and Kobayashi Y., Archives Biochem. Biophys., 19661, 95, pp.242-250), titration assays, manometric assays, fluorometric assays, biological assays or radioactive assays (Zeller EA., The enzymes, (J.B. Sumner and K. Mayback (Ed.), Vol. II, Part A, p. 536, Academic Press, New York 1951; Shore PA et al., J. Pharmacol., Exptl. Therap. 127, 1959, 182; Ahlark A., Acta Physiol. Scand. Suppl., 1944, 28, 9).

[0070] The terms "functional variant" or "functionally active variant" include naturally occurring allelic variants as well as mutants or any other non-naturally occurring variants. As known in the art, allelic variants are alternate forms of nucleic acids or peptides characterized as having one or more nucleotide or one or more amino acid substitutions, deletions, or additions that do not essentially alter the biological function of the nucleic acid or polypeptide.

[0071] Functional variants can be obtained by sequence changes in polypeptide or nucleotide sequences, such as one or more point mutations, which, when used in the combinations of the present invention, maintain or improve the function of the unchanged polypeptide or nucleotide sequence. Such sequence changes can include, but are not limited to, (conservative) substitutions, additions, deletions, mutations, and insertions. Conservative substitutions are substitutions that occur within a family of amino acids that are similar in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, acidic side chains, non-polar aliphatic side chains, non-polar aromatic side chains, uncharged polar side chains, small side chains, large side chains, etc.

[0072] Point mutation is understood to be the engineering of a polynucleotide that results in the expression of an amino acid sequence that differs from the unmanipulated amino acid sequence, in particular in the substitution or exchange, deletion or insertion of one or more single (non-contiguous) or doublet amino acids with different amino acids.

[0073] In an alternative embodiment, a GAG-binding domain may be introduced into any position within the DAO polypeptide by recombinant means. Each domain may consist of the amino acid sequence X1FX2X3X4LPX5, where X1 is any amino acid, particularly A or S, more particularly S; X2 is any amino acid, particularly K; X3 is any amino acid, particularly A or T, more particularly T; X4 is any amino acid, particularly K; and X5 is any amino acid, particularly K or T, more particularly T. In a specific embodiment, one or more of the amino acid sequences SFKAKLPK (SEQ ID NO: 33), AFKAKLPT (SEQ ID NO: 34), AFKTKLPK (SEQ ID NO: 35), SFKTKLPK (SEQ ID NO: 36), AFKTKLPT (SEQ ID NO: 37), and SFKAKLPK (SEQ ID NO: 38) are introduced into the DAO polypeptide described herein.

[0074] The term "sequence identity" as used herein refers to the relationship between two amino acid sequences or two nucleotide sequences, and is described by the degree of sequence identity or sequence complementarity. The sequence identity of a variant, homolog, or ortholog compared to a parent nucleotide sequence or parent amino acid sequence indicates the degree of identity of two or more sequences. Two or more amino acid sequences may have up to 100% identical or conserved amino acid residues at corresponding positions. Two or more nucleotide sequences may have up to 100% identical or conserved base pairs at corresponding positions.

[0075] Sequence similarity searching is an effective and robust strategy for identifying homologs with a significant degree of sequence identity (e.g., at least 50%). Frequently used sequence similarity search tools include BLAST, FASTA, and HMMER.

[0076] Sequence similarity searches can identify such homologous proteins or polynucleotides by detecting excessive similarity and statistically significant similarity that reflects common ancestry. Homologues can include orthologs, which are understood herein as identical proteins in different organisms, e.g., variants of such proteins in different organisms or species.

[0077] To determine the % complementarity of two complementary sequences, one of the two sequences must be converted to its complementary sequence so that the % complementarity can be calculated as the % identity between the first sequence and the second converted sequence using the algorithm described above.

[0078] "Percent identity" with respect to amino acid sequences, homologs, and orthologs described herein is defined as the percentage of amino acid residues in a candidate sequence that are the same as the amino acid residues in a particular polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, not counting any conservative substitutions as part of the sequence identity. Those skilled in the art can identify appropriate parameters for measuring alignment, including any algorithms needed to achieve the best alignment over the full length of the sequences being compared.

[0079] For purposes described herein, the NCBI BLAST program version 2.2.29 (January 6, 2014) is used to determine sequence identity between two amino acid sequences using blastp set to the following exemplary parameters: Program: blastp, String Length: 6, Expectation: 10, Hit List Size: 100, Gap Cost: 11.1, Matrix: BLOSUM62, Filter String: F, Genetic Code: 1, Window Size: 40, Threshold: 21, Composition-based stats: 2.

[0080] For example, "percent identity (%)" for the nucleotide sequence of a nucleic acid molecule or a portion thereof, particularly a coding DNA sequence, is defined as the percentage of nucleotides in a candidate DNA sequence that are the same as the nucleotides in the DNA sequence after aligning the sequences and introducing gaps, if necessary, to achieve the highest percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent nucleotide sequence identity can be achieved in various ways within the skill of the art, for example, using commonly available computer software. Those skilled in the art can identify appropriate parameters for measuring alignment, including any algorithms required to achieve the best alignment across the entire length of the sequences being compared.

[0081] Optimal alignments can be identified using any algorithm suitable for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, available from novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available from soap.genomies.org.cn), and Maq (available from maq.sourceforge.net).

[0082] The DAO described herein may include the amino acid sequences of SEQ ID NOs: 2-16, and any functional variant thereof having 90%, 95%, or 99% sequence identity to any of SEQ ID NOs: 2-16.

[0083] The recombinant DAO has an extended plasma half-life compared to wild-type DAO, in particular the half-life is at least 1.5 times, in particular at least 2 times longer than wild-type DAO. The duration of a drug's action or physical presence is known as its half-life. It is the time required for the concentration or amount of a drug in the body or in whole blood or plasma to decrease by half. A drug's half-life is usually considered in relation to the amount of the drug in plasma or serum. A drug's plasma or serum half-life depends on how quickly the drug is eliminated from the plasma or serum. Drug molecules can be eliminated from the body, transferred to another body fluid compartment, such as intracellular fluid, or destroyed in the blood. The removal of a drug from plasma is known as clearance, and the distribution of a drug in various body tissues is known as the volume of distribution.

[0084] The area under the plasma drug concentration-time curve (AUC) reflects the body's actual exposure to a drug, i.e., the recombinant DAO described herein, after administration of a dose of the drug and is expressed in μg / min / ml. This area under the curve depends on the rate of drug elimination from the body and the administered dose. The total amount of drug eliminated by the body can be evaluated by summing or integrating the amount lost during each period from time zero (the time of drug administration) to infinity. This total amount corresponds to the fraction of the administered dose that reaches the systemic circulation. AUC is directly proportional to the dose when the drug follows linear kinetics. AUC is inversely proportional to the drug's clearance. That is, the higher the clearance, the shorter the time the drug spends in the systemic circulation, and the faster the plasma drug concentration decreases. Therefore, in such a situation, the body's exposure to the drug and the area under the concentration-time curve are smaller.

[0085] The recombinant DAO described herein has an AUC that is increased by at least 2-fold, at least 5-fold, 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold compared to wild-type DAO.

[0086] The term "expression" is understood as follows: A nucleic acid molecule containing the desired coding sequence of an expression product, e.g., a fusion protein described herein, may be used for expression purposes. A host transformed or transfected with the sequence is capable of producing the encoded protein. To achieve transformation, the expression system may be included in a vector, although it is also possible for the relevant DNA to be integrated into the host chromosome. In particular, the term relates to a host cell and a compatible vector under appropriate conditions to express a protein encoded by foreign DNA, e.g., carried in a vector and introduced into the host cell.

[0087] Coding DNA is a DNA sequence that encodes a specific amino acid sequence for a specific polypeptide or protein. Promoter DNA is a DNA sequence that initiates or alternatively regulates, mediates, or controls the expression of coding DNA. Promoter DNA and coding DNA can be from the same gene or different genes, and can be from the same organism or different organisms. Recombinant cloning vectors frequently contain one or more replication systems for cloning or expression, one or more markers for selection in a host, such as antibiotic resistance, one or more nuclear localization signals (NLS), and one or more expression cassettes.

[0088] As used herein, an "expression vector" is defined as a DNA sequence required for the transcription of a cloned recombinant nucleotide sequence, i.e., a recombinant gene, and the translation of its mRNA in a suitable host organism. To achieve expression, a sequence encoding a desired expression product, such as a DAO described herein, is typically cloned into an expression vector containing a promoter that controls transcription. Suitable bacterial and eukaryotic promoters are well known in the art. The promoter used to control nucleic acid expression depends on the specific application. For example, constitutive strong promoters are typically used for the expression and purification of fusion proteins. In contrast, when the expression product is administered in vivo for gene regulation, either a constitutive promoter or an inducible promoter can be used, depending on the specific use of the expression product. Furthermore, a weak promoter may be preferred for administration. The promoter may further include transactivation response elements, such as a hypoxia response element, a Gal4 response element, and a lac repressor response element. An expression vector comprises an expression cassette, and typically also comprises an origin of autonomous replication in a host cell or a genomic integration site, one or more selectable markers (e.g., amino acid synthesis genes or genes that confer resistance to antibiotics, such as zeocin, kanamycin, G418, or hygromycin), multiple restriction enzyme cleavage sites, a suitable promoter sequence, and a transcription terminator whose components are operably linked to each other.

[0089] An "expression cassette" refers to a DNA coding sequence or DNA fragment that encodes an expression product, which can be inserted into defined restriction sites of a vector. The restriction sites of the cassette are designed to ensure insertion of the cassette in the proper reading frame. Generally, foreign DNA is inserted into one or more restriction sites of the vector DNA and is then carried by the vector into a host cell along with the transmissible vector DNA. A DNA fragment or sequence with inserted or added DNA, such as an expression vector, can also be called a "DNA construct."

[0090] Any suitable host cell or cell line that allows proper folding, post-transcriptional modification and enzymatic activity can be used for the expression of recombinant DAO. In particular, recombinant host cells can be selected from CHO cells, COS cells, Vero cells, MDCK cells, Pichia yeast cells, SF9 cells, human cell lines such as HEK and HeLa.

[0091] As used herein, the term "vector" includes both autonomously replicating and genome-integrating nucleotide sequences. A common type of vector is the "plasmid," which is generally a double-stranded DNA self-contained molecule that readily accepts additional (foreign) DNA and can be readily introduced into a suitable host cell. Plasmid vectors frequently contain coding DNA and promoter DNA and have one or more restriction sites suitable for inserting foreign DNA. In particular, the term "vector" or "plasmid" refers to a vehicle by which a DNA or RNA sequence (e.g., a foreign gene) is introduced into a host cell to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. The vector is transfected into a cell, and in the case of stable transfection, the DNA may be integrated into the genome by homologous recombination, or the cell may be transiently transfected. In particular, the vector may be a bacterial, yeast, baculovirus, plant, or mammalian expression vector.

[0092] Any of the known methods for introducing foreign nucleotide sequences into host cells can be used, including calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, episomes as well as integration, and the use of any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into host cells (see, e.g., Sambrook et al.).

[0093] Examples of mammalian expression vectors include adenovirus vectors, pSV and pCMV series plasmid vectors, vaccinia virus vectors, retrovirus vectors, and baculovirus. Cytomegalovirus (CMV) and SV40 promoters are commonly used to drive gene expression in mammalian expression vectors. Non-viral promoters, such as the elongation factor (EF)-1 promoter, are also known.

[0094] Using the vectors and host cells described above, the present invention provides a method for producing a recombinant DAO, comprising the successive steps of cloning a nucleotide sequence encoding a DAO into an expression vector, transforming host cells, particularly mammalian cells, with the vector, culturing the transformed host cells under conditions in which the DAO is expressed, optionally isolating the DAO from the host cell culture by disruption of the host cells or isolating the DAO from the cell culture supernatant, and optionally purifying the DAO.

[0095] Further, pharmaceutical compositions comprising the recombinant DAO provided herein are described. According to a particular embodiment, such pharmaceutical compositions comprising the DAO or functional variants thereof described herein are used to treat any condition associated with excess histamine, particularly excess histamine at a plasma level of >1 ng / ml, particularly chronic allergic diseases, more particularly anaphylaxis, anaphylactic shock, chronic urticaria, acute urticaria, asthma, hay fever, allergic rhinitis, allergic conjunctivitis, histamine poisoning, headache, itch, vomiting, tachycardia, hypotension, cardiac arrest, atopic dermatitis, inflammatory diseases, mastocytosis, mast cell activation syndrome (MCAS), pre-eclampsia, hyperemesis gravidarum, preterm labor, peptic ulcer, acid reflux, pruritus and sepsis.

[0096] In particular, the pharmaceutical compositions described herein further comprise a pharmaceutically acceptable carrier or excipient, e.g., a bulking agent, when used for diagnosis or therapy. These pharmaceutical compositions can be administered in accordance with the present invention as a bolus or infusion, or by continuous infusion. Suitable pharmaceutical carriers for facilitating such administration are well known in the art.

[0097] Pharmaceutically acceptable carriers generally include any suitable solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc. that are physiologically compatible with the DAO provided by the present invention. Further examples of pharmaceutically acceptable carriers include sterile water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and any combination thereof.

[0098] Additional pharmaceutically acceptable carriers are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Gennaro, A.R., ed., Mack Publishing Co, 1985). The liquid preparation may be a solution, emulsion or suspension, and may contain excipients such as suspending agents, solubilizing agents, surfactants, preservatives and chelating agents.

[0099] Exemplary dosage forms to be used for parenteral administration include those suitable for subcutaneous, intramuscular or intravenous injection, for example, as a solution, emulsion or suspension. DAO as described herein is administered in a therapeutically effective amount, which means sufficient amount or activity to achieve beneficial or desired results, including clinical results, particularly when administered to patients, for example, patients suffering from cancer.As such, effective amount or the amount equivalent thereto depends on the situation in which it is administered.Effective amount is intended to mean the amount of compound that is sufficient to treat, prevent or prevent such disease or disorder.

[0100] The amount of the compound, i.e., the recombinant DAO described herein, that corresponds to such an effective amount will depend on various factors, such as the particular drug or compound, pharmaceutical formulation, route of administration, type of disease or disorder, and the individuality of the patient or host being treated, but can nevertheless be routinely determined by one skilled in the art.

[0101] According to one particular embodiment of the present invention, a fusion protein is provided in which a recombinant DAO is linked to a second moiety, which may be, but is not limited to, Fc or human serum albumin (HSA).

[0102] According to a particular embodiment, the DAO bound to the Fc and the Fc comprise any one of SEQ ID NOs: 40 to 70 or a functional fragment thereof having at least 80%, particularly at least 85%, 90%, 95%, 99% sequence identity to any one of SEQ ID NOs: 40 to 70.

[0103] According to a particular embodiment, the DAO linked to the Fc and the Fc are encoded by any one of SEQ ID NOs: 72 to 102 or by a fragment thereof having at least 80%, particularly at least 85%, 90%, 95%, 99% sequence identity to any one of SEQ ID NOs: 72 to 102.

[0104] In the context of the present invention, the term "fusion polypeptide" primarily (although not necessarily) refers to amino acid sequences connected to each other by peptide bonds. The term "fused" according to the fusion polypeptide of the present invention refers to the fact that at least two amino acid sequences of different origins, namely, the modified DAO as defined herein, and a second part, in particular the Fc domain of human IgG or albumin, are covalently linked to each other, either directly or via an amino acid linker or spacer that joins (crosslinks, bonds, covalently binds) the amino acid sequences. Fusion can be performed by chemical conjugation or genetic engineering methods well known in the art.

[0105] In some embodiments, a DAO polypeptide as defined herein is covalently linked via its C-terminus to either the Fc domain of human IgG or HSA, i.e., in some embodiments, from N- to C-terminus, a fusion polypeptide according to the invention comprises a DAO polypeptide and either an Fc domain component or HSA.

[0106] In another embodiment, a DAO polypeptide as defined herein is covalently linked via its N-terminus to either the Fc domain of human IgG or HSA, i.e., in some embodiments, from N- to C-terminus, a fusion polypeptide of the invention comprises an Fc domain component or HSA and a DAO polypeptide.

[0107] The term "polypeptide" as used herein refers to amino acid residues connected by peptide bonds. A polypeptide sequence is generally written from the N-terminus, which contains a free amino group, to the C-terminus, which contains a free carboxyl group. A polypeptide may also be referred to as an amino acid sequence, peptide, or protein, and may be modified, for example, by mannosylation, glycosylation, amidation, carboxylation, or phosphorylation.

[0108] By the term "covalently bound" or "covalently linked" is meant that the specified domains are connected or linked by a covalent bond. In particular, as used herein, the term "Fc fusion polypeptide" includes DAOs of the present disclosure, including proteins containing a full-length Fc domain, as well as Fc domain fragments (e.g., the entire CH2 domain, the entire CH3 domain, a CH2 fragment, a CH3 fragment, or a combination thereof). The Fc fusion protein may also include all or part of the hinge region.

[0109] As used herein, the Fc region includes polypeptides comprising the constant region of an antibody, excluding the first immunoglobulin constant region domain and fragments thereof. Thus, the Fc region relates to the last two immunoglobulin constant region domains of IgA, IgD, and IgG, and the last three immunoglobulin constant region domains of IgE and IgM, and optionally a flexible N-terminal hinge region to those domains. In the case of IgA and IgM, the Fc region may include the J chain. In the case of IgG, the Fc includes the immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and optionally the hinge region between Cgamma1 (Cγ1) and Cgamma2 (Cγ2). Although the boundaries of the Fc region might vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 at its carboxyl terminus, where numbering is according to the EU index as set forth in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Fc may refer to the region alone, or to the region in the context of an antibody, antibody fragment, or Fc fusion protein.

[0110] The HSA sequence fused to the DAO polypeptide described herein can comprise the wild-type sequence (SEQ ID NO: 103) or 90%, particularly at least 95%, more particularly at least 99%, and more particularly at least 99.9% sequence identity to the wild-type sequence. Optionally, a linker sequence, particularly comprising 2 to about 10 amino acid residues, is included between the DAO and HSA.

[0111] In one particular embodiment, the GAG ​​binding of DAO, particularly heparin / heparan sulfate binding, is inhibited, resulting in reduced internalization of DAO and increased amounts of DAO in the systemic circulation, which can be determined by AUC. By modifying DAO as described herein, DAO indeed exhibits significantly reduced heparin / heparan sulfate binding.

[0112] Another option for increasing DAO in the systemic circulation is to provide a target-specific ligand that specifically binds to the heparin / heparan sulfate binding domain. In particular, an antibody, antibody fragment, or any ligand can bind to DAO near the heparin binding domain, thereby blocking access to the heparin binding domain. An alternative is a fusion protein, where a portion of the fusion protein masks the heparin binding domain and blocks its function, thus having the same effect as a mutation or an antibody that directly binds to the heparin binding domain.

[0113] Alternatively, the ligand inhibits the binding of heparin / heparan sulfate to the GAG-binding domain of DAO. The ligand may be, in particular, an antigen-binding protein selected from the group consisting of an antibody or antibody fragment, e.g., Fab, Fd, scFv, diabody, triabody, Fv tetramer, minibody, nanobody, single-domain antibody, e.g., VH, VHH, IgNAR, or V-NAR; antibody mimetics, e.g., Adnectin™ (which shares a beta-sheet sandwich fold with antibody variable domains, including a diversification loop, but differs from antibodies in primary sequence and has a single-domain structure without disulfide bonds), Affibody® (a simple small protein consisting of a bundle of three helices based on one scaffold of the IgG-binding domain of Protein A), Affilin® (structurally derived from human ubiquitin and constructed by modification of the surface-exposed amino acids of the protein, and suitable for display technologies, e.g., phage display and screening). Affilins resemble antibodies in their affinity and specificity for antigens, but not in structure, making them a type of antibody mimetic), Affimers® (small proteins that bind to target molecules with specificity and affinity similar to that of antibodies), affitins (artificial proteins with the ability to selectively bind to antigens), alphabodies, aptamers, anticalins, avimers, DARPins® (genetically engineered antibody mimetic proteins that typically exhibit high-specificity and high-affinity binding to target proteins), Fynomers® (small binding proteins (7 kDa) derived from the human SH3 domain of Fyn kinase that can be engineered to provide specific, high-affinity binding domains that target particular proteins), Kunitz domain peptides, monobodies, or NanoCLAMPs (CLostridal Antibody Mimetic Proteins), or fusion proteins comprising one or more immunoglobulin-fold domains, antibody domains, or antibody mimetics.

[0114] Further provided is a method for identifying a compound that modulates heparin binding of DAO, comprising: (a) constructing a computer model of the GAG-binding domain defined by the amino acid structural coordinates of the DAO sequence of SEQ ID NO: 1; (b) a selecting step, (i) assembling molecular fragments into said compound; (ii) selection of compounds from a small molecule database; and (iii) de novo ligand design of said compound; selecting a potential regulatory compound by a method selected from the group consisting of: (c) to provide an energy-minimized configuration of said compound in the heparin-binding domain. using a computational means to perform a fitting program between a computer model of the compound and a computer model of the GAG ​​binding domain; (d) evaluating the results of the fitting procedure to quantify the association between the compound and the heparin / heparan sulfate binding domain, thereby assessing the ability of the compound to associate with the heparin / heparan sulfate binding domain. An identification method is provided, comprising:

[0115] The term "structural coordinates" refers to a set of values ​​that define the position of one or more amino acid residues relative to an axis system. The term refers to a data set that defines the three-dimensional structure of a molecule (e.g., Cartesian coordinates, temperature factors, and occupancies). Structural coordinates may be slightly modified and still yield approximately the same three-dimensional structure. A measure of the unique set of structural coordinates is the mean square deviation of the resulting structure. Structural coordinates that yield three-dimensional structures (particularly the three-dimensional structures of heparin / heparan sulfate binding domains) that differ from each other by a mean square deviation of less than 3 Å, 2 Å, 1.5 Å, 1.0 Å, or 0.5 Å can be considered highly similar by those skilled in the art.

[0116] As used herein, the term "constructing a computer model" includes quantitative and qualitative analysis of molecular structure and / or function based on atomic structural information and interaction models. The term "modeling" includes traditional numerically based molecular dynamics energy minimization models, interactive computer graphics models, modified molecular mechanics models, distance geometry and other structure-based constraint models.

[0117] The term "fitting program operation" refers to an operation that utilizes the structural coordinates of a chemical moiety, enzyme active center, binding pocket, molecule or molecular complex, or a portion thereof, to associate the chemical moiety with the enzyme active center, binding pocket, molecule or molecular complex, or a portion thereof. This can be achieved by positioning, rotating, or translating the chemical moiety in the enzyme active center to match the shape and electrostatic complementarity of the enzyme active center. Covalent interactions, non-covalent interactions, such as hydrogen bonds, electrostatic interactions, hydrophobic interactions, van der Waals interactions, and non-complementary electrostatic interactions, such as repulsive charge-charge interactions, dipole-dipole interactions, and charge-dipole interactions, can be optimized. Alternatively, the deformation energy of the binding between the chemical moiety and the enzyme active center can be minimized.

[0118] The following items are specific embodiments of the invention provided herein. 1. A recombinant diamine oxidase (DAO) with reduced glycosaminoglycan binding affinity, said DAO comprising at least one amino acid modification in the glycosaminoglycan (GAG) binding domain.

[0119] 2. The recombinant DAO according to item 1, further comprising at least one modification of the solvent-exposed cysteine ​​at amino acid position 123 relative to the numbering of SEQ ID NO: 1, in particular the modification of the cysteine ​​is an amino acid substitution, deletion or coupling with a chemical moiety.

[0120] 3. The recombinant DAO according to item 1 or 2, wherein the cysteine ​​at position 123 according to the numbering of SEQ ID NO: 1 is substituted with alanine. 4. The recombinant DAO according to any one of items 1 to 3, wherein the GAG-binding domain is a heparin / heparan sulfate-binding domain.

[0121] 5. The recombinant DAO according to any one of items 1 to 4, wherein the at least one amino acid modification in the GAG-binding domain is an amino acid substitution, deletion, insertion or coupling with a chemical moiety.

[0122] 6. The recombinant DAO according to any one of items 1 to 5, comprising 2, 3, 4, 5, 6, 7 or 8 amino acid substitutions in the GAG-binding domain. 7. The recombinant DAO according to any one of items 1 to 6, wherein the GAG-binding domain comprises amino acids at positions 568 to 575 in the numbering of SEQ ID NO: 1.

[0123] 8. A GAG-binding domain of the amino acid sequence X1FX2X3X4LPX5, wherein: X1 can be any amino acid, in particular A or S, more in particular S; X2 can be any amino acid, in particular K; X3 may be any amino acid, in particular A or T, more in particular T; X4 can be any amino acid, in particular K; 8. The recombinant DAO according to any one of items 1 to 7, wherein X5 can be any amino acid, in particular K or T, more in particular T.

[0124] 9. The recombinant DAO of item 7 or 8, comprising an amino acid sequence selected from the group consisting of SFKAKLPK (SEQ ID NO: 33), AFKAKLPT (SEQ ID NO: 34), AFKTKLPK (SEQ ID NO: 35), SFKTKLPK (SEQ ID NO: 36), AFKTKLPT (SEQ ID NO: 37), and SFKAKLPK (SEQ ID NO: 38).

[0125] 10. The recombinant DAO according to any one of items 1 to 9, wherein the DAO has an extended plasma half-life compared to wild-type DAO, in particular the half-life is at least 1.5 times, in particular at least 2 times longer than wild-type DAO.

[0126] 11. The recombinant DAO according to any one of items 1 to 10, wherein the DAO has an AUC that is increased by at least 10-fold compared to a wild-type DAO. 12. The recombinant DAO according to any one of items 1 to 11, wherein internalization by endothelial cells is reduced by at least 10%, 25%, 50%, 60%, 70%, 80%, particularly 90% compared to wild-type DAO.

[0127] 13. The recombinant DAO according to any one of items 1 to 12, wherein the GAG ​​binding affinity, in particular the heparin / heparan sulfate binding affinity, is reduced by at least 10%, 25%, 50%, 60%, 70%, 80%, in particular 90% compared to the wild-type DAO.

[0128] 14. The recombinant DAO according to any one of items 1 to 13, comprising an amino acid sequence of SEQ ID NO: 2 to 16. 15. A fusion polypeptide comprising the recombinant DAO according to any one of items 1 to 14 and the Fc domain of human IgG or human serum albumin (HSA), wherein the fusion polypeptide retains the functional activity of the recombinant DAO.

[0129] 16. An isolated nucleotide sequence encoding a DAO according to any one of items 1 to 15, in particular comprising a sequence of SEQ ID NO: 17 to 32. 17. A recombinant vector comprising a nucleotide sequence according to item 16, in particular a bacterial, yeast, baculovirus, plant or mammalian expression vector.

[0130] 18. An expression cassette comprising the nucleotide sequence of item 17, operably linked to a regulatory element. 19. A recombinant host cell or host cell line comprising the recombinant DAO according to any one of items 1 to 15, wherein the host cell is selected from the group consisting of CHO cells, Vero cells, MDCK cells, Pichia yeast cells, and SF9 cells.

[0131] 20. An expression system comprising the vector according to item 17 or the expression cassette according to item 18 and the host cell or host cell line according to item 19. 21. A method for producing a recombinant DAO according to any one of items 1 to 15, comprising: i. cloning a nucleotide sequence encoding the DAO according to any one of items 1 to 15 into an expression vector; ii. transforming a host cell with the vector; iii. culturing the transformed host cells under conditions in which the DAO is expressed; iv. isolating the DAO from the host cell culture, optionally by disruption of the host cells; and optionally v. DAO purification process A production method comprising:

[0132] 22. A pharmaceutical composition comprising the recombinant DAO according to any one of items 1 to 15 and optionally one or more excipients. 23. Use of a recombinant DAO according to any one of items 1 to 15 for preparing a pharmaceutical composition.

[0133] 24. The recombinant DAO according to any one of items 1 to 15 for use in the treatment of conditions associated with excess histamine, in particular in the treatment of chronic allergic diseases, more in particular in the treatment of anaphylaxis, anaphylactic shock, chronic urticaria, acute urticaria, asthma, hay fever, allergic rhinitis, allergic conjunctivitis, histamine poisoning, headache, atopic dermatitis, inflammatory diseases, mastocytosis, mast cell activation syndrome (MCAS), pre-eclampsia, hyperemesis gravidarum, preterm labor, peptic ulcer, acid reflux, pruritus, and sepsis.

[0134] 25. Use of the recombinant DAO according to any one of items 1 to 15 for the manufacture of a medicinal product for the treatment of conditions associated with excess histamine, in particular for the treatment of chronic allergic diseases, more particularly for the treatment of anaphylaxis, anaphylactic shock, chronic urticaria, acute urticaria, asthma, hay fever, allergic rhinitis, allergic conjunctivitis, histamine poisoning, headache, atopic dermatitis, inflammatory diseases, mastocytosis, peptic ulcer, acid reflux, pruritus and sepsis.

[0135] 26. A target-specific ligand that specifically binds to the GAG-binding domain of DAO, in particular to one or more of the amino acids at positions 568 to 575 with respect to the numbering of SEQ ID NO: 1. 27. A target-specific ligand that specifically inhibits the binding of heparin / heparan sulfate to the GAG-binding domain of DAO, in particular to any one or more of the amino acids at positions 568 to 575 in the numbering of SEQ ID NO: 1.

[0136] 28. The target-specific ligand of item 26 or item 27, wherein the ligand is selected from the group consisting of a nucleic acid, a small molecule inhibitor, or an antigen-binding protein. 29. The ligand is, in particular: - an antibody or antibody fragment, such as a Fab, Fd, scFv, diabody, triabody, Fv tetramer, minibody, nanobody, single domain antibody, such as a VH, VHH, IgNAR or V-NAR; antibody mimetics, such as Adnectin™, Affibody®, Affilin®, Affimer®, affitins, alphabodies, aptamers, anticalins, avimers, DARPins®, Fynomer®, Kunitz domain peptides, monobodies or NanoCLAMPs, or - fusion proteins containing one or more immunoglobulin fold domains, antibody domains or antibody mimetics 29. The target-specific ligand of item 28, which is an antigen-binding protein selected from the group consisting of:

[0137] 30. A method for identifying a compound that modulates heparin binding of DAO, comprising: (a) constructing a computer model of the GAG-binding domain defined by the amino acid structural coordinates of the DAO sequence of SEQ ID NO: 1; (b)(i) assembling molecular fragments into said compound; (ii) selection of compounds from a small molecule database; and (iii) de novo ligand design of the compound selecting a potential regulatory compound by a method selected from the group consisting of: (c) to provide an energy-minimized configuration of said compound in the heparin-binding domain. using a computational means to perform a fitting program between a computer model of the compound and a computer model of the GAG ​​binding domain; (d) evaluating the results of the fitting procedure to quantify the association between the compound and the heparin / heparan sulfate binding domain, thereby assessing the ability of the compound to associate with the heparin / heparan sulfate binding domain. An identification method comprising:

[0138] The examples described herein are illustrative of the present invention and are not intended to be limiting thereof. Different embodiments of the present invention have been described by the present invention. Numerous modifications and variations are possible with respect to the techniques described and illustrated herein without departing from the spirit and scope of the present invention. Therefore, it is understood that the examples are illustrative only and do not limit the scope of the present invention. [Example]

[0139] Example 1 DAO with modified GAG-binding domain: Summary: Amino acids in the GAG-binding (heparin / heparan sulfate-binding) domain of DAO were mutated. After mutations in the heparin-binding domain of DAO, animal experiments were conducted using the mutants. In rats, the short alpha-phase half-life was nearly eliminated, and the beta-phase half-life was extended to 6 hours. The alpha-phase half-life relates to the rate at which plasma concentrations decline due to the drug's redistribution from the central to the peripheral compartment, while the beta-phase half-life relates to the rate at which plasma concentrations decline due to the drug's elimination due to metabolism or excretion. The area under the curve (AUC) increased more than 20-fold. The 6-hour half-life in rats was extrapolated to 24-48 hours in humans, which is certainly sufficient for the treatment of acute and subacute conditions caused by histamine excess. For example, anaphylactic or MCAS events last from several hours to 1-2 days, and anaphylaxis can be biphasic in 10-20% of patients, meaning that a second attack can occur within 24 hours. Several mutations were tested, and different double mutants within the heparin-binding domain showed the most significant improvement in pharmacokinetic (PK) parameters. Because DAO is a dimer and the heparin-binding domain forms a ring structure consisting of both monomers, the four mutations are located close to each other. Nevertheless, the expression, stability, and activity of the DAO wild-type and DAO mutants are identical.

[0140] DAO with modified cysteine When DAO is expressed in CHO cells, a certain percentage of DAO molecules (approximately 20% to 30%) form not only dimers but also tetramers, hexamers, and even octamers. These higher-order oligomers may be considered natural variants with unknown functions. It is unknown whether these higher-order oligomers are formed during folding, transport from the endoplasmic reticulum to the Golgi apparatus, and secretion into the extracellular environment, or whether they are formed solely in the extracellular environment. Nevertheless, these tetramers and larger oligomers complicate the expression, purification, characterization, standardization, and selection of optimal formulations of rhDAO. Therefore, relatively solvent-exposed cysteines on the surface of DAO were mutated, and with these mutants, only DAO dimers were found in the supernatant of CHO cells.

[0141] A DAO construct with two point mutations in the heparin / heparan sulfate binding domain and a single mutation in a separate cysteine ​​is a particular embodiment. The following table shows the recombinant DAO. Mutations at threonine and serine, which replace lysine and arginine with polar amino acids, were preferred over alanine and glycine, which helped maintain the native conformation.

[0142] [Table 1]

[0143] The Hepmut4 mutation showed the most significant loss of binding to heparin. Hepmut6 is a triple mutation that substitutes 570 / 571 / 572 with Gly / Gln / Thr, which are present in rodents. The glutamine in the rodent sequence can also bind to negatively charged sulfates and sometimes substitute for arginine or lysine. All animal studies to date have been performed in rodents (mice and rats).

[0144] The following data are from a heparin sepharose experiment using KCl and NaCl to elute purified wild-type and Hepmut variant DAO from heparin sepharose. Heparin is irreversibly coupled to the sepharose, and the purified DAO was incubated in a low salt concentration followed by a linear gradient of increasing KCl or NaCl. The salt concentration at which the DAO protein peaks (measured using absorbance at 280 nm) is used as the mM salt concentration at which DAO elutes.

[0145] Figure 2 shows the heparin Sepharose elution profiles of wild-type (WT) and heparin variants of recombinant human DAO. The hepmut variant elutes earlier than DAO_WT. Purified DAO_WT and hepmut variants were loaded onto a 1 ml HiTrap Heparin HP (High Performance) column using 50 mM HEPES buffer (pH 7.4) at a flow rate of 0.2 ml / min. Elution was performed using a linear gradient of 0% to 70% of 50 mM HEPES (pH 7.4) containing 1 M sodium chloride over a 60-minute gradient period. rh = recombinant human. For KCl elution, purified rhDAO variants were loaded onto a 1 ml HiTrap Heparin HP column using 10 mM potassium phosphate buffer (pH 7.2) at a flow rate of 0.2 ml / min. Elution was performed using a linear gradient of 0% to 100% of 10 mM potassium phosphate buffer (pH 7.2) containing 1 M potassium chloride over a 60-minute gradient period. The elution profile is not shown, but the salt concentrations at the peak elution are shown below.

[0146] Table 2 and Figure 3 show the raw results and normalized results based on data using the DAO wild-type protein. Means and standard deviations (SD) from the two salt experiments were calculated and displayed as bar graphs. The numbers above the bars correspond to the numbers in the table. For DAO_WT, the numbers represent the salt concentration of elution; for the mutants, the numbers represent the difference from the salt concentration of DAO_WT.

[0147] DAO_WT was eluted from heparin-Sepharose at salt concentrations of 372 mM NaCl or 310 mM KCl. Hepmut eluted at significantly lower salt concentrations. The correlation coefficient R between the NaCl and KCl elution profiles was 97%, with a p-value of 0.0056. Therefore, it seemed justified to combine both profiles and calculate the mean and SD. The mean ± SD is represented by the bar height (mean) and error bars (±SD).

[0148] [Table 2]

[0149] Figure 3 shows that Hepmut 1, 4, and 7 variants elute from heparin Sepharose at 50% lower salt concentrations than DAO_WT. This data is from Table 2. Table 3 shows the delta salt concentration of the variants compared to DAO_WT with respect to NaCl and KCl, e.g., 200 mM means that 200 mM less NaCl was required to elute the variants from heparin sepharose compared to DAO_WT.

[0150] [Table 3]

[0151] Based on this data, Hepmut4 may be the weakest heparin-binding mutant, but is not significantly different from Hepmut1 and Hepmut7. Mutation of a single lysine residue in Hepmut2 reduces its affinity for heparin-Sepharose.

[0152] Serum and plasma combined contain approximately 145-150 mM Na + and K. + and 100 mM Cl -Because the elution of Hepmut 1, 4, and 7 variants from heparin Sepharose is near the physiological concentration of ions in serum, these variants should be minimally active in vivo, as shown below in vitro in cells and in vivo in rats and mice.

[0153] These data clearly demonstrate that DAO binds to heparin via the heparin / heparan sulfate-binding domain. To put this result in a larger context, using Hepmut4 with two arginine mutations, mutation of one or two arginine / lysine residues out of 48 lysine and 88 arginine amino acids in the DAO sequence (most of which are surface-exposed) nearly abolishes heparin binding, even though only 2 / 88 = 2.3% of the arginine residues were mutated.

[0154] The clearance of DAO_WT in mice and rats was significantly more rapid than that of the Hepmut variant. The heparin-binding domain was speculated to be responsible for the decreased area under the curve (AUC) values. The heparin-binding variant eluted from heparin-Sepharose at low salt concentrations, suggesting reduced heparin or heparan sulfate binding.

[0155] In the next set of experiments, it was found that DAO not only binds but is actually internalized into endothelial cells, exhibiting intracellular vesicular staining. The Hepmut variant is no longer internalized by endothelial cells. Endothelial cells are the first cells (other than blood cells) to be exposed to DAO after intravenous administration. Endothelial cells are in direct contact with the blood. Endothelial cells are also exposed to DAO after subcutaneous administration because DAO is transported to the blood compartment via the lymphatic system.

[0156] Immunofluorescence microscopy of SK-Hep1 cells, an immortalized endothelial-like cell line derived from a liver cancer patient, after incubation with DAO_WT and Hepmut variants showed that internalization of the Hepmut variants was inhibited. SK-Hep1 cells were incubated with 20 μg / ml of purified recombinant human DAO and Hepmut variants (negative control: no rhDAO added) for 60 minutes at 37°C. Membrane-bound DAO was removed by washing twice with 150 mM glycine buffer (pH 3.0) containing 150 mM sodium chloride and once with PBS. After fixation and permeabilization, cells were incubated with a 1:500 dilution of rabbit-produced anti-ABP1 (ABP1 = amiloride-binding protein 1, another name for DAO) antibody (Sigma-Aldrich). A 1:500 dilution of Alexa Fluor 488-conjugated donkey anti-rabbit (H+L) (Jackson Research, 711-545-152) was used as the secondary antibody. DAPI was used for nuclear counterstaining. Cells were analyzed by fluorescence microscopy using an inverted DMI-6000B microscope (Leica Microsystems) equipped with an HCX PL APO 63x / 1.30 glycerol immersion objective and filter sets A4 (for DAPI) and L5 (Alexa Fluor 488). Similar results were obtained using HUVECs (human umbilical vein endothelial cells), a typical EC used in endothelial cell (EC) studies.

[0157] The Hepmut4 and Hepmut7 variants showed significantly reduced uptake into SK-Hep1 cells, whereas Hepmut1 produced some staining similar to the vesicle-like staining of DAO_WT. These results not only demonstrate that the HepmutDAO variant prevents uptake into ECs, but also demonstrate for the first time that DAO is internalized by cells. While DAO binding to the surface of ECs indicates its intracellular localization, incubation of cells with DAO and the demonstration of DAO_WT internalization suggest the presence of a DAO receptor. It was confirmed that DAO binds to heparan sulfate glycosaminoglycans present on ECs (heparin is only present in human mast cells), and that the Hepmut variant is no longer internalized because it can no longer effectively bind to them. This is also reflected in the in vivo rat and mouse data described herein.

[0158] The immunofluorescence data discussed above were further reflected in the Western blotting data. There is a more than six-fold difference between the Western blotting signals of DAO_WT and Hepmut1 relative to those of Hepmut4 and Hepmut7. These data are consistent with the immunofluorescence data. Hepmut4 and Hepmut7 were not internalized by SK-Hep1 cells.

[0159] Figure 4 shows Western blots of SK-Hep1 cell lysates after incubation with DAO_WT and Hepmut variants. A. Lane 1: negative control, 2: rhDAO_WT, 3: rhDAO-Hepmut1, 4: rhDAO-Hepmut4, 5: rhDAO_Hepmut7. SK-Hep1 cells were incubated with 20 μg / ml of each purified DAO variant (negative control = no rhDAO added) at 37°C for 60 minutes. Membrane-bound DAO was then removed by washing twice with 150 mM glycine buffer (pH 3.0) containing 150 mM sodium chloride and once with PBS. Cells were then lysed by sonication in RIPA buffer. The cell lysates were loaded onto an SDS-PAGE gel and then blotted onto a PVDF membrane. After blocking, the membrane was incubated with a 1:1000 dilution of serum IgG fraction from a rabbit immunized with purified rhDAO. This step was followed by incubation with a 1:5000 dilution of β-actin mABAC-15 (Invitrogen). A 1:5000 dilution of IRDye® 800CW goat anti-rabbit IgG (H+L) secondary antibody and IRDye® 680RD goat anti-mouse IgG (H+L) (Li-Cor) were used as secondary antibodies. Membranes were scanned at 700 nm and 800 nm using an Odyssey Infrared Imaging System (Li-Cor). B. Band intensities were determined relative to purified rhDAO_WT (not shown) and used to calculate the amount of internalized rhDAO.

[0160] In vitro assays showed approximately 5-fold lower binding of DAO_WT and the Hepmut4 variant to SK-Hep1 cells. DAO was labeled with Alexa488 fluorescent dye on glycosaminoglycans and incubated with SK-Hep1 cells. The assay was performed in a microtiter plate. Fluorescence signals were measured after washing and cell lysis.

[0161] FIG. 5 shows that Hepmut4 variants exhibit reduced binding to SK-Hep1 cells compared to DAO_WT. SK-Hep1 cells were grown in 96-well plates and incubated with Alexa488-labeled rhDAO_WT and rhDAO-Hepmut4 at 0–8 μg / ml for 60 minutes at 37°C. Cells were washed twice with 150 mM glycine buffer (pH 3.0) containing 150 mM sodium chloride and once with PBS to remove membrane-bound DAO. After cell lysis with RIPA buffer, fluorescence intensity was measured using a Tecan plate reader. Mean values ​​and standard error of the mean (SEM) of triplicates are shown.

[0162] The binding of DAO_WT and Hepmut4 to high and low molecular weight heparins (HMWH, LMWH) was further examined using isothermal titration calorimetry (ITC), a state-of-the-art tool for measuring label-free binding affinities.

[0163] Figure 6 provides the ITC data for DAO_WT and Hepmut4. 12.5 μM purified rhDAO_WT and 12.0 μM purified rhDAO_Hepmut4 were analyzed by isothermal titration calorimetry (MicroCal PEAQ-ITC, Malvern) using 25×1 μl injections of 160 μM high molecular weight heparin (HMWH, Gilvasan, average molecular weight = 15 kDa) and low molecular weight heparin (LMWH, Lovenox, average molecular weight = 4.5 kDa). The buffer was 50 mM HEPES (pH 7.3) containing 150 mM KCl. Binding was observed only for rhDAO_WT and HMWH, with a K of 423 nM. D was recognized by value.

[0164] Both components, the DAO protein and the two heparin molecules, were unaltered and unimmobilized, allowing for the measurement of true binding affinity. Heparin Sepharose is a multivalent matrix in which molecules glide from one heparin molecule to the next. DAO_WT had a K of 423 nM. d Hepmut4 bound to HMWH at 1000kJ / mL, whereas DAO_WT did not bind to HMWH.

[0165] Intravenous injection of DAO_WT and Hepmut4 into C57BL6 mice C57BL6 mice weighing approximately 20 grams were injected into the tail vein with 1 mg / kg of DAO_WT and Hepmut4 proteins. The protein concentrations and enzymatic activities of the two purified DAO variants were comparable. Protein purification was performed as recently published (Gludovacz 2016). Linear and logarithmic y-axis scales are shown.

[0166] Figure 7 shows a linear y-axis scale (a) and a logarithmic y-axis scale (b). Following intravenous injection of 1 mg / kg DAO variant, Hepmut4 increases the AUC (area under the curve) by more than 19-fold compared to wild-type DAO protein. Values ​​(n = 3-4 mice per time point) ± standard deviation are shown. DAO concentrations were measured using a self-developed human DAO ELISA that does not recognize mouse or rat DAO (Boehm 2017). Half-life and AUC data are summarized in the following table.

[0167] [Table 4]

[0168] To eliminate the short alpha distribution half-life, which is approximately 10 minutes when using DAO_WT, the half-life was calculated from 60 to 1680 minutes. With the Hepmut4 variant, this very rapid disappearance of DAO from the circulation is no longer nearly absent. A high degree of curve fitting was performed using a monoexponential decay function from 60 to 1680 minutes.

[0169] Intraperitoneal injection of DAO_WT and Hepmut4 into C57BL6 mice Mice weighing 21 grams were intraperitoneally injected with DAO_WT and Hepmut4 proteins at 1 mg / kg. As shown in Figure 8, after intraperitoneal injection, Hepmut4 increased the AUC (area under the curve) by more than 16-fold compared to DAO_WT protein. Each time point represents the average of three mice, so a total of 15 DAO_WT mice and 15 Hepmut4 mice were used. Mean values ​​and standard deviations are shown. DAO concentrations were measured using a recently published human DAO ELISA (Boehm 2017). The AUC data are summarized in the following table. Half-life data are not included because we had to assume that DAO_WT and Hepmut4 absorbed the same amount from the intraperitoneal space, which may not be the case.

[0170] [Table 5]

[0171] Intravenous injection of DAO_WT and Hepmut 1, 4, and 7 into rats Figure 9 shows the mean ± standard deviation (SD) of measurements using 1 mg / kg DAO wild-type and different Hepmut variants. Slower clearance of heparin-binding domain mutants compared to DAO wild-type protein administered at 1 mg / kg. DAO_WT, n = 9; Hepmut1, n = 4; Hepmut4, n = 5; Hepmut7, n = 4; linear y-axis scale. Mean and SD are shown.

[0172] Figure 10 shows the slower clearance of heparin-binding domain mutants compared to the DAO wild-type protein administered at 1 mg / kg. DAO_WT, n=9; Hepmut1, n=4; Hepmut4, n=5; Hepmut7, n=4; logarithmic y-axis scale. Mean and SD are shown.

[0173] DAO_WT and Hepmut7 exhibit short alpha-phase half-lives, and two equations are used to obtain the best-fit curves. Nevertheless, the alpha-phase half-life of Hepmut7 is significantly longer than that of DAO_WT. The half-lives are shown below. For Hepmut1 and Hepmut4, a single-exponential decay provides the best fit with the highest adjusted coefficient of determination and the lowest p-value. This is also observed in the figures containing the raw data.

[0174] Figure 11 shows the curves using the different derived exponential equations, which were used to calculate the AUC and half-life shown below. Figure 11: Slow clearance of heparin-binding domain mutants compared to DAO wild-type protein administered at 1 mg / kg. DAO_WT, n=9; Hepmut1, n=4; Hepmut4, n=5; Hepmut7, n=4; logarithmic y-axis scale. These curves were generated using a best-fit exponential equation.

[0175] Figures 12, 13 and 14 show the first 90 minutes to observe the fast clearance when using DAO wild type protein. Figure 12 shows the slow clearance of heparin-binding domain mutants compared to DAO wild-type protein administered at 1 mg / kg. DAO_WT, n=9; Hepmut1, n=4; Hepmut4, n=5; Hepmut7, n=4; logarithmic y-axis scale. Only the first 90 min is shown.

[0176] Below are linear y-axis versions of those figures. Figure 13 shows the slow clearance of heparin-binding domain mutants compared to DAO wild-type protein administered at 1 mg / kg. DAO_WT, n=9; Hepmut1, n=4; Hepmut4, n=5; Hepmut7, n=4. The curves were generated using a best-fit exponential equation. Linear y-axis scale.

[0177] Figure 14 shows the slow clearance of heparin-binding domain mutants compared to DAO wild-type protein administered at 1 mg / kg. DAO_WT, n=9; Hepmut1, n=4; Hepmut4, n=5; Hepmut7, n=4; only the first 90 min is shown; linear y-axis scale.

[0178] Due to the short alpha phase half-life in DAO_WT treated rats, AUC was calculated from 5 min to 240 min and 1440 min as well as from 0 min. Data points within 10 min appear to be quite variable. The data are summarized in the table below.

[0179] [Table 6]

[0180] For the Hepmut variant, the 0-1440 min or 5-1440 min window did not have a significant effect, whereas for the DAO wild-type protein, starting at 5 min significantly decreased the AUC. The DAO concentration calculated using the best-fit exponential equation is not possible because it exceeds the theoretical DAO concentration. A significant increase in AUC is also evident using the 0-1440 min time window.

[0181] In this set of experiments, the Hepmut4 variant also showed the strongest effect, which is also reflected in the half-life data.

[0182] [Table 7]

[0183] [Table 8]

[0184] In conclusion, mutations in the putative heparin-binding domain of DAO significantly increase the AUC after intravenous injection of 1 mg / kg body weight in rats. The best-performing mutant is the double mutant Hepmut4, in which two arginines are removed, which is in good agreement with predictions and heparin-Sepharose elution data.

[0185] Example 2 Fc-DAO with modified GAG binding domain: Additionally, an Fc-DAO fusion variant with heparin-binding mutations was also tested, and PK parameters were further improved with a 9-hour beta-phase half-life and increased AUC. Amino acids involved in high-affinity interactions with Fc-gamma receptors were removed, while amino acids involved in interactions with FcRN were left unchanged.

[0186] Intravenous injection of Fc-DAO_WT and Fc-DAO-Hepmut4 into rats Below are the results using linear and logarithmic scales from six and four rats after intravenous injection of 1 mg / kg of Fc-DAO wild-type and Fc-Hepmut4 proteins. With Fc-DAO wild-type, only 4 hours were measured. Nevertheless, as measured with the Fc-Hepmut4 variant, a derived exponential function can be used to extrapolate up to 1680 minutes (see below). The Fc-DAO fusion protein exhibits a very short alpha-phase half-life, similar to the DAO wild-type protein. The majority of this fusion variant is cleared from plasma within 20 minutes. The half-life is then approximately 120 minutes, calculated using values ​​at 30, 120, and 240 minutes.

[0187] Fc-Hepmut4 is much more stable in plasma. The DAO clearance mechanism clearly dominates the Fc portion. The half-life of human IgG in rats is several days, and this half-life is primarily determined by the binding of the Fc portion to the FcRN receptor.

[0188] Figure 15 shows the rapid clearance of Fc-DAO wild type compared to Fc-Hepmut4 when dosed at 1 mg / kg in 6 or 4 rats, respectively. Mean values ​​and standard deviations are shown; linear y-axis scale.

[0189] Figure 16 shows the rapid clearance of Fc-DAO wild type compared to Fc-Hepmut4 when dosed at 1 mg / kg in 6 or 4 rats, respectively. Mean values ​​and standard deviations are shown; logarithmic y-axis scale.

[0190] Both data sets could be best fitted to a single exponential decay function with a p-value of less than 0.001 and an adjusted coefficient of determination value of >97%. For the Fc-DAO wild-type protein, the half-life after 30 minutes was calculated to be 120 minutes based on data from time points 30, 120, and 240 minutes. A two-factor exponential decay curve fit did not converge. A best-fit equation was used to extrapolate the Fc-DAO data out to 24 hours. The curve is shown below.

[0191] In Figure 17, Fc-DAO-Hepmut4 shows a significant increase in AUC after intravenous administration of 1 mg / kg; logarithmic y-axis scale. In Figure 18, Fc-DAO-Hepmut4 shows a significant increase in AUC after intravenous administration of 1 mg / kg; linear y-axis scale.

[0192] Area under the curve was calculated from 0 and 5 minutes after intravenous injection to 240 or 1440 minutes. Because curve fitting in the first few minutes resulted in very high values ​​at 0 minutes for the Fc-DAO data, AUC starting at 5 minutes was also calculated.

[0193] [Table 9]

[0194] The following table shows the AUC ratios, followed by a table showing the calculated half-lives.

[0195] [Table 10]

[0196] [Table 11]

[0197] The Fc-Hepmut4 variant is more stable in plasma than the Fc-DAO. This is consistent with mouse and rat data using the unfused DAO variant. Nevertheless, the half-life of Fc-Hepmut4 is still rather short compared to IgG antibodies. DAO clearance still appears to dominate over the slower Fc clearance mechanism.

[0198] Example 3 DAO with modified GAG binding domain and modified cys123: Cysteines 123 and 633 are not involved in disulfide bond formation in the DAO dimer.

[0199] The relative exposed surface area or relative solvent accessibility (RSA) of a protein residue is a measure of the solvent exposure of the residue. It can be calculated by the formula: RSA = ASA / MaxASA, where ASA is the solvent accessible surface area and MaxASA is the maximum possible solvent accessible surface area or residue. Both ASA and MaxASA are generally expressed in Å. 2 Measured at.

[0200] RSA Cys123 Cys123=148Å 2 93.84Å of 2 Exposure = 63.4% (Monomer B) Cys123=148Å 2 90.72Å of 2 Exposure = 61.3% (Monomer A) Average = 62.4% RSA Cys633 Cys633 = 34.62 of 148 exposed = 23.4% (Monomer B) Cys633 = 33.49 of 148 exposed = 22.6% (monomer A) average = 23% Cys123 is on the surface, which is unusual. The amino acid cysteine ​​is the rarest and "least and most" conserved amino acid in proteins, likely because it is oxidizable (Marino SM and Gladyshev VN, J Mol Biol. 2010 Dec 17;404(5):pp.902-16). DAO generates hydrogen peroxide, which can cause oxidation of cys123, resulting in the cysteine ​​forming a disulfide bond that can severely impair function. Cysteine ​​is highly conserved as a catalytic amino acid in enzymes, and if it is involved in disulfide bond formation, it is also present in DAO. Cys633 is deeper in the structure and less accessible, so it may not be important for aggregate formation. It may play a role at high DAO concentrations.

[0201] DAO dimers have two exposed Cys123 amino acids, and one dimer can form a disulfide bond with another dimer, resulting in a tetramer, or two dimers can interact to form a hexamer. The molecular weight of DAO lacking the secretory signal, based purely on amino acids (732), would be 166,872 Da for the dimer. The tetramer would be 333,744 Da, the hexamer 500,617 Da, and the octamer 667,489 Da, but the glycans would increase the molecular weight by approximately 25% (Elmore, 2002). The height of a DAO monomer or dimer is approximately 65 Å, but increases to 130 Å for the tetramer, 195 Å for the hexamer, and 260 Å for the octamer, respectively. There is no data on the structure of DAO multimeric stacks. Aggregates may be rigid or flexible. These aggregates are likely more immunogenic. This is because neoepitopes can form between the two dimers and can be repeated two or three times, with potential and possible adverse consequences regarding efficacy and safety (see below).

[0202] [Table 12]

[0203] Higher-order aggregates of DAO have been described. Paolucci et al. (Biochimie. 1971;53(6):pp.735-49) reported that the molecular weight of human placental DAO consists of 125 kDa ± 5 multiples of 1 to 4, i.e., 125, 250, 375, and 500 kDa. Tufvesson (Scand J Clin Lab Invest. 1978 Sep;38(5):pp.463-72) reported that, after purification from human amniotic fluid, DAO molecular weights corresponded to dimers and tetramers, again at 245 kDa and 485 kDa. Wilfingseder et al. (Inflamm Res. 2002 Apr;51 Suppl 1:pp.89-90) demonstrated the "complex formation" of human placental DAO using Western blotting.

[0204] The different DAO expression plasmids were transfected into ExpiCHO cells for transient expression. After 7 days of culture, the supernatants were directly subjected to Western blotting. The results are shown in Figure 19.

[0205] Figure 19: Lane 1: HiMark standard; Lane 2: negative control (empty plasmid); Lane 3: rhDAO_WT; Lane 4: rhDAOΔ123; Lane 5: rhFc-DAO; Lane 6: rhFc-DAOΔ123; Lane 7: rhDAO-Hepmut4; Lane 8: rhDAO-Hepmut4Δ123; Lane 9: rhFc-DAO-Hepmut4; Lane 10: rhFc-DAO-Hepmut4Δ123. rhFc = recombinant human Fc fusion protein containing DAO; 15 μl of each culture supernatant was loaded. SDS-PAGE was performed under non-reducing conditions. Antibody: MUV rabbit serum #408, 1:5000.

[0206] Mutation of Cys123 to Ala123 completely prevented tetramerization and higher-order aggregate formation of recombinant human wild-type DAO and Hepmut4 variants, but had no effect on the rhFc-DAO fusion construct, suggesting that aggregate formation in this Fc fusion variant is not driven by Cys123 but is likely driven by the Fc portion, which also contains a cysteine.

[0207] Under reducing conditions using mercaptoethanol, the disulfide bonds are cleaved, and neither wild-type DAO nor the Hepmuts or fusion variants exhibit tetramer or higher-order aggregates. The molecular weights range from 71 to 117 Da and, based solely on amino acids, would be estimated to be 83,436 Da. These data clearly demonstrate that the mutation of Cys123 to Ala123 completely prevents tetramerization and higher-order n-merization of DAO. Cys633 is not involved.

[0208] To roughly quantify the percentage of higher-order n-mer variants, the signal intensity of each lane was measured using ImageJ software. To compare the WT lane with the corresponding cys123 mutant lane, regions of comparable size were selected (as shown) and signal profiles were generated. The area under the curve (AUC) for each lane was calculated.

[0209] [Table 13]

[0210] The mean (SD) difference between the Cys123 and Ala123 mutations is approximately 19% (5.5%) for the WT and Hepmut4 mutants when averaging the combined data from replicates 1 and 2. For the Fc-DAO variant, the mean (SD) difference is −6% (15.4%); in other words, the cys123 to ala123 mutation is not different.

[0211] Therefore, the ala123 mutation is of great clinical importance, as it has significant advantages for manufacturing and quality control in general, as well as for reduced immunogenicity. Since aggregates are known to be more immunogenic, the use of this mutation will reduce the immunogenicity of rhDAO.

[0212] The supernatants were tested for DAO activity using the standard DAO activity assay described by Bartko (Alcohol. 2016 Aug;54:51-9) or Gludovacz 2016. In this case, it was confirmed that the cys123 to ala123 mutation did not affect DAO activity. Figure 20 shows the DAO activity. WT DAO, Hepmut4, or Fc variants were set at 100% and compared with the cys123 to ala123 mutant. It shows that there is no significant difference in DAO activity between DAO_WT and the cys123 mutation.

[0213] Example 4 Generation of Asn168 glycosylation mutants Site-directed mutagenesis of Asn-168 To replace the N-glycosylation site asparagine Asn-168 (AAT) with glutamine (CAG), the corresponding codon in the DAO expression plasmid (Gludovacz E. et al., 2016, J. Biotechnol., 227, pp. 120-130) was mutated using site-directed mutagenesis. Therefore, PCR was performed using the following 5-phosphorylated primers: Asn-168, CAGaccacaggcttctcattc (forward, SEQ ID NO: 104) and gaggaagaactgatgcag (reverse, SEQ ID NO: 105). Phusion polymerase (Thermo Fisher Scientific) was used: annealing temperature: 56.3°C; extension time: 4 min, 30 cycles. Ligation (T4 DNA ligase, New England Biolabs) and amplification of the final plasmid were performed. Sequence accuracy was verified by DNA sequencing (Eurofins MWG Operon). All cloning techniques were performed according to Green MR and Sambrook, J. (2012), Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and manufacturer's instructions.

[0214] Intravenous administration of rhDAO, competitor proteins, and glycosylation mutants to rats and mice The experimental protocol for handling rats and mice was approved by the local animal welfare committee and the Austrian Ministry of Science, Research, and Economy (GZ 66.009 / 0152-WF / V / 3b / 2014) and was performed in full accordance with the ARRIVE guidelines (Kilkenny, C et al., Br. J. Pharmacol. (2010) 160, pp. 1577-1579). For implantation of a vascular access port (Rodent Vascular Access Port with Detachable Silastic Catheter, Hugo-Sachs Elektronic-Harvard Apparatus) into the jugular vein, rats were anesthetized with an intraperitoneal injection of ketamine (100 mg / kg) and xylazine (5 mg / kg). After intubation, anesthesia was maintained via continuous volume-controlled ventilation (O2-air mixture + 1.5% isoflurane). The surgical area was depilated and then disinfected with iodine solution (Betaisodona, Mundipharma). Metamizole (100 mg / kg) was administered subcutaneously for analgesia. A 1 cm skin incision was made on the dorsum of the rat, and a vascular access port balloon was placed subcutaneously. The port was flushed with saline, and the balloon was then secured between the scapulae with sutures. A second skin incision was made at the site of the left jugular vein, and the jugular vein was opened for catheter insertion. A small incision was made in the exposed left jugular vein, and a catheter was inserted and secured with silk suture (7-0). The subcutaneous tissue and skin were closed with simple interrupted sutures (4-0). The surgical procedure was performed aseptically. The average surgical time was approximately 2 hours. Postoperative analgesia was provided with ad libitum drinking water containing piritramide and glucose (30 mg piritramide and 10 mL 10% glucose in 250 mL drinking water). During the experiment, the vascular access port is filled with a solution containing argatroban (Argatra 100 mg / mL; Mitsubishi Pharma) 10 μg / mL, tissue plasminogen activator (Alteplase, Actilyse, Boehringer Ingelheim) 0.3 μg / mL, and sodium citrate 0.38% in 0.9% NaCl to prevent clotting.

[0215] At designated time points, venous blood samples (0.5 mL) were collected under brief isoflurane anesthesia and placed in tubes containing sodium citrate for anticoagulation. Plasma was prepared within 4 hours and stored at -32°C until analysis. Rats were given a replacement solution (0.5 mL) using saline (0.9% NaCl). After the final blood collection time point, rats were sacrificed by an overdose of pentobarbital (300 mg / kg) under deep anesthesia with ketamine / xylazine (35 mg and 5 mg / kg). Male Sprague-Dawley rats weighing approximately 400 g and female C57BL / 6N mice weighing approximately 20 g were included.

[0216] Male Sprague Dawley rats are purchased from commercial suppliers (Janvier Labs, Le Genest-Saint-Isle, France and the Division Laboratory of Animal Science and Genetics, Medical University of Vienna, Himberg, Austria). Rats are housed under controlled standard conditions (artificial 12:12 light / dark cycle, room temperature 22±2°C, humidity 45±10%). Rats are housed in groups of two (3 cages from Makrolon) and provided with environmental enrichment. Rats have free access to water and complete rat chow (complete rat and mouse chow sniff R / MH, sniff Spezialdiaeten GmbH).

[0217] Female mice (C57BL / 6N) are obtained from commercial suppliers (Charles River Laboratories, Sulzfeld, Germany and the Division Laboratory of Animal Science and Genetics, Medical University of Vienna, Himberg, Austria). Mice are housed under controlled standard conditions (artificial light / dark cycle 12:12, room temperature 22±2°C, humidity 45±10%). Mice are housed in groups of five (two long cages from Makrolon) and have free access to water and complete mouse chow as described for rats.

[0218] Rapid plasma clearance of rhDAO in rats and mice after intravenous administration Purified rhDAO, wild-type rhDAO, and rhDAO containing either one of the HepMut1 to HepMut7 mutations or HepMut1 to HepMut4 with the additional Cys123 and / or Asn168Gln mutations were administered intravenously to rats and mice at 1 mg / kg, and blood samples were collected at the indicated time points. DAO antigen concentrations were measured using a recently published human DAO ELISA (Boehm T. et al., 2017, Clin. Biochem., 50, pp. 444-451). The distribution (alpha) and elimination (beta) half-lives were approximately 3 and 230 minutes, respectively, in rats and approximately 11 and 110 minutes, respectively, in mice. In rats, more than 90% of the injected dose was cleared from the plasma pool within 10 minutes. Rapid clearance in mice was somewhat slower.

Claims

1. 1. A recombinant human diamine oxidase (DAO) having reduced glycosaminoglycan binding affinity compared to a corresponding wild-type human DAO, wherein the DAO has an amino acid substitution at positions 568-575 of a glycosaminoglycan (GAG) binding domain, with reference to the numbering of SEQ ID NO: 1, wherein the amino acid substitution is selected from R568S, K575T, R568S+R571T, R568S+K575T, and R568S+R571T+C123A.

2. A recombinant DAO as described in claim 1, wherein the solvent-exposed cysteine ​​(cys123) at amino acid position 123 with respect to the numbering of SEQ ID NO: 1 is replaced by alanine.

3. The recombinant DAO of claim 1 or 2, wherein the GAG-binding domain is a heparin / heparan sulfate-binding domain.

4. The recombinant DAO of any one of claims 1 to 3, further comprising an amino acid substitution at position 168 relative to SEQ ID NO:

1.

5. 5. The recombinant DAO of any one of claims 1 to 4, having an extended plasma half-life compared to wild-type DAO, said half-life being at least 1.5 times longer than wild-type DAO.

6. 6. The recombinant DAO of any one of claims 1 to 5, having an AUC that is increased by at least 10-fold compared to wild-type DAO.

7. The recombinant DAO of any one of claims 1 to 6, wherein internalization by endothelial cells is reduced by at least 10% compared to wild-type DAO.

8. 8. The recombinant DAO of any one of claims 1 to 7, wherein the GAG ​​binding affinity is reduced by at least 10% compared to wild-type DAO.

9. 9. The recombinant DAO of claim 8, wherein the heparin / heparan sulfate binding affinity is reduced by at least 10% compared to wild-type DAO.

10. The recombinant DAO of any one of claims 1 to 9, comprising any one of the amino acid sequences of SEQ ID NOs: 2, 3, 5, and 8.

11. A fusion polypeptide comprising the recombinant DAO of any one of claims 1 to 9 and the Fc domain of human IgG or human serum albumin (HSA), wherein the fusion polypeptide retains the functional activity of the recombinant DAO.

12. An isolated nucleotide encoding the DAO of any one of claims 1 to 10.

13. 13. The isolated nucleotide of claim 12, comprising any one of SEQ ID NOs: 18, 19, 21, 24, 26, 27, 29, and 32.

14. A recombinant vector comprising the nucleotide sequence of claim 12 or 13.

15. 14. An expression cassette comprising the nucleotide of claim 12 or 13 operably linked to a regulatory element.

16. 11. A recombinant host cell or host cell line comprising the recombinant DAO of any one of claims 1 to 10, wherein the host cell is selected from the group consisting of CHO cells, Vero cells, MDCK cells, Pichia pastoris cells, and SF9 cells.

17. 17. An expression system comprising a vector according to claim 14 or an expression cassette according to claim 15 and a host cell or host cell line according to claim 16.

18. A method for producing the recombinant DAO of any one of claims 1 to 10, comprising: i. cloning a nucleotide sequence encoding the DAO of any one of claims 1 to 10 into an expression vector; ii. Transforming a host cell with the vector; iii. Culturing the transformed host cells under conditions in which the DAO is expressed; iv. isolating the DAO from the host cell culture by disruption of the host cells; and v. Purifying the DAO A production method comprising:

19. A pharmaceutical composition comprising the recombinant DAO of any one of claims 1 to 10 and optionally one or more excipients.

20. 20. The pharmaceutical composition according to claim 19, for the treatment of conditions associated with excess histamine, for the treatment of chronic allergic diseases.

21. 21. The pharmaceutical composition according to claim 19 or 20, which is for the treatment of anaphylaxis, anaphylactic shock, chronic urticaria, acute urticaria, asthma, hay fever, allergic rhinitis, allergic conjunctivitis, histamine poisoning, headache, atopic dermatitis, inflammatory disease, mastocytosis, mast cell activation syndrome (MCAS), preeclampsia, hyperemesis gravidarum, preterm labor, peptic ulcer, acid reflux, pruritus, or sepsis.

22. Use of a recombinant DAO according to any one of claims 1 to 10 for the manufacture of a medicament for the treatment of conditions associated with excess histamine, for the treatment of chronic allergic diseases.

23. 23. The use according to claim 22 for the manufacture of a medicament for the treatment of anaphylaxis, anaphylactic shock, chronic urticaria, acute urticaria, asthma, hay fever, allergic rhinitis, allergic conjunctivitis, histamine poisoning, headache, atopic dermatitis, inflammatory diseases, mastocytosis, peptic ulcer, acid reflux, pruritus, or sepsis.

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