Multiple anti-insulin-degrading enzyme antibodies and multiple uses thereof

Abstract

A variety of isolated anti-insulin-degrading enzyme antibodies are provided. Each of the variety of antibodies comprises an antigen recognition domain comprising the specified complementarity determining region amino acid sequences. Also provided are a variety of methods of making them, a variety of methods of using them, a variety of pharmaceutical compositions and articles of manufacture comprising them.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 964,139, filed January 22, 2020, the contents of which are hereby incorporated by reference in its entirety.

[0003] SEQUENCE LISTING

[0004] An ASCII file named 85636 created on December 10, 2020, containing 45,056 bytes, is submitted herewith, and is hereby incorporated by reference in its entirety.

[0005] TECHNICAL FIELD AND BACKGROUND

[0006] The present invention, in some embodiments thereof, relates to various anti-insulin-degrading enzyme antibodies and various uses thereof.

[0007] Insulin-degrading enzyme (IDE, insulin) is a large zinc-binding protease (about 110 kDa thio zinc-metalloendopeptidase) located in the cytosol, peroxisomes, endosomes and on the cell surface. This enzyme cleaves small proteins of diverse sequences, many of which form amyloid fibrils rich in β-pleated sheets, including amyloid β-protein (Aβ), insulin, glucagon, islet amyloid, atrial natriuretic factor and calcitonin. Thus, IDE is known to cleave short polypeptides and plays an important role in degrading various proteins, such as insulin and IGF-1, which are reported to modulate immune response activity.

[0008] Since its discovery, various IDE inhibitors have been suggested for the treatment of several diseases, including, for example, diabetes, obesity, various autoimmune diseases of the central nervous system, various neurodegenerative diseases, and varicella-zoster virus (VZV) infection (e.g., Maianti et al. (2014) Nature 511, 94-98; Tang, W.-J. (2016) Trends Endocrinol. etab. 27, 24-34; and International Patent Application Publication Nos. WO 2012 / 017439 and WO 2010 / 086867). For example, PCT Publication No. WO 2010 / 086867 discloses, by the same applicant, the use of an isolated peptide comprising an amino acid sequence of no more than 25 amino acids in length, the amino acid sequence including at least one aspartate or a homolog thereof, the peptide having an insulin-degrading enzyme (IDE) inhibitory activity, for the manufacture of a medicament identified for the treatment of a disease selected from the group consisting of diabetes, obesity, hyperglycemia, retinal damage, kidney failure, nerve damage, microvascular damage, and varicella-zoster virus (VZV) infection. PCT Publication No. WO 2012 / 017439, by the same applicant, provides an insulin-degrading enzyme (IDE) inhibitor (e.g., an antibody) for the treatment of a disease selected from the group consisting of an autoimmune disease of the central nervous system and a neurodegenerative disease.

[0009] Several IDE monoclonal and polyclonal antibodies have been described in the art for in vitro detection of rodent and / or human IDE in various applications, including western blotting, immunoprecipitation, immunocytochemistry, immunohistochemistry, and various quantitative sandwich ELISA [see, e.g., Delledonne A. et al. Mol Neurodegener. (2009) 4:39]. SUMMARY

[0010] According to an aspect of some embodiments of the present application there is provided an isolated antibody comprising an antigen recognition region that specifically binds to IDE, wherein said antigen recognition region comprises the complementarity determining region (CDR) amino acid sequences as set forth below:

[0011] (i) SEQ ID NO: 4 (CDR1), SEQ ID NO: 6 (CDR2), and SEQ ID NO: 8 (CDR3), arranged in order from N to C on a heavy chain of the antibody, SEQ ID NO: 12 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 16 (CDR3), arranged in order from N to C on a light chain of the antibody;

[0012] (ii) SEQ ID NO: 20 (CDR1), SEQ ID NO: 22 (CDR2), and SEQ ID NO: 24 (CDR3), arranged in order from N to C on a heavy chain of the antibody, SEQ ID NO: 28 (CDR1), SEQ ID NO: 30 (CDR2), and SEQ ID NO: 32 (CDR3), arranged in order from N to C on a light chain of the antibody; or

[0013] (iii) SEQ ID NO: 36 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 40 (CDR3), arranged in order from N to C on a heavy chain of the antibody, SEQ ID NO: 44 (CDR1), SEQ ID NO: 46 (CDR2), and SEQ ID NO: 48 (CDR3), arranged in order from N to C on a light chain of the antibody.

[0014] According to an aspect of some embodiments of the present application there is provided a pharmaceutical composition comprising the antibody (ii) or (iii) as an active ingredient, and a pharmaceutically acceptable carrier.

[0015] According to an aspect of some embodiments of the present application there is provided a method of treating a disease associated with an IDE activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody (ii) or (iii) or the pharmaceutical composition, thereby preventing or treating the disease associated with the IDE activity.

[0016] According to an aspect of some embodiments of the present application there is provided use of the antibody (ii) or (iii) for treating a disease associated with an IDE activity in a subject in need thereof.

[0017] According to some embodiments of the present application the method further comprises administering to the subject a therapeutic agent for treating the disease.

[0018] According to some embodiments of the present application the use of the antibody further comprises a therapeutic agent for treating the disease.

[0019] According to some embodiments of the present invention, the subject has an IDE level in a biological sample that is higher than a predetermined threshold compared to a control biological sample.

[0020] According to some embodiments of the present invention, the antibody is used to determine the IDE level in the subject's biological sample prior to administration.

[0021] According to one aspect of some embodiments of the present invention, an article of manufacture identified for treating a disease associated with an IDE activity is provided, the article of manufacture comprising the antibody (ii) or (iii) and a therapeutic agent for treating the disease.

[0022] According to some embodiments of the present invention, the antibody and the therapeutic agent are in multiple separate containers.

[0023] According to some embodiments of the present invention, the antibody and the therapeutic agent are in a common formulation.

[0024] According to one aspect of some embodiments of the present invention, a method for diagnosing a disease associated with an IDE activity of a subject is provided, the method comprising using the antibody to determine an IDE level in a biological sample of the subject, wherein the subject is diagnosed with the disease when the IDE level is higher than a predetermined threshold compared to a control biological sample.

[0025] According to one aspect of some embodiments of the present invention, a method is provided for monitoring the efficacy of a therapy in a subject diagnosed with a disease associated with an IDE activity, the method comprising using the antibody to determine an IDE level in a biological sample of the subject receiving the therapy or after receiving the therapy, wherein the therapy is effective when the IDE level decreases from a predetermined threshold during or after receiving the therapy.

[0026] According to one aspect of some embodiments of the present invention, a method is provided for treating a disease associated with IDE activity in a subject in need, the method comprising:

[0027] (a) Diagnose the subject according to the method; and wherein when the level of the IDE is higher than the predetermined threshold.

[0028] (b) Treat the subject with a therapy for the disease, thereby treating the subject's disease.

[0029] According to one aspect of some embodiments of the present invention, a method is provided for treating a disease associated with IDE activity in a subject in need, the method comprising:

[0030] (a) diagnosing the subject according to the method; and wherein when the level of the IDE is higher than the predetermined threshold,

[0031] (b) selecting a therapy based on the level of the IDE,

[0032] thereby treating the disease in the subject.

[0033] According to some embodiments of the application, the therapy comprises the antibody (ii) or (iii).

[0034] According to an aspect of some embodiments of the application, there is provided a composition of matter, comprising a biological sample of a subject diagnosed with a disease associated with an IDE activity, and the antibody.

[0035] According to some embodiments of the application, the disease associated with the IDE activity is selected from the group consisting of: an autoimmune disease of the central nervous system, a neurodegenerative disease, a metabolic syndrome, diabetes, obesity, hyperglycemia, retinal damage, kidney failure, nerve damage, microvascular damage, varicella zoster virus (VZV) infection and a wound.

[0036] According to some embodiments of the application, the disease associated with the IDE activity is selected from the group consisting of: a metabolic syndrome, diabetes and obesity.

[0037] According to some embodiments of the application, the disease is diabetes.

[0038] According to some embodiments of the application, the disease is type 1 diabetes.

[0039] According to some embodiments of the application, the disease is type 2 diabetes.

[0040] According to some embodiments of the application, the disease is a metabolic syndrome.

[0041] According to some embodiments of the application, the neurodegenerative disease is Parkinson’s disease.

[0042] According to some embodiments of the application, the neurodegenerative disease is Alzheimer’s disease.

[0043] According to some embodiments of the application, the autoimmune disease of the central nervous system is selected from the group consisting of: multiple sclerosis, Guillain-Barre syndrome, Lambert-Eaton myasthenic syndrome, Myasthenia gravis, transverse myelitis, progressive multifocal leukoencephalopathy, chronic headache and cerebral palsy.

[0044] According to some embodiments of the application, the autoimmune disease of the central nervous system is multiple sclerosis.

[0045] According to some embodiments of the application, the wound is selected from the group consisting of: a chronic wound, an acute wound, a diabetic wound, an ischemic wound, an ulcer, a burn and a surgical wound.

[0046] According to an aspect of some embodiments of the application, there is provided an isolated polynucleotide encoding the antibody.

[0047] According to some embodiments of the application, the nucleic acid sequences encoding the CDR amino acid sequences are as listed below:

[0048] (i) SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15;

[0049] (ii) SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 31; or

[0050] (iii) SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45 and SEQ ID NO: 47.

[0051] According to an aspect of some embodiments of the present application there is provided a nucleic acid construct comprising the polynucleotide and a cis-acting regulatory element for directing expression of the polynucleotide.

[0052] According to an aspect of some embodiments of the present application there is provided a host cell expressing the antibody, the polynucleotide or the nucleic acid construct.

[0053] According to an aspect of some embodiments of the present application there is provided a method of producing an anti-IDE antibody, the method comprising expressing the polynucleotide or the nucleic acid construct in a host cell.

[0054] According to some embodiments of the present application the method comprises isolating the antibody.

[0055] According to an aspect of some embodiments of the present application there is provided a method of producing an anti-IDE antibody, the method comprising:

[0056] (a) providing a plurality of antibodies;

[0057] (b) screening the plurality of antibodies to select a plurality of antibodies that bind wild type IDE but not mutant IDE, wherein the mutant IDE has a reduced catalytic activity as compared to the wild type IDE.

[0058] According to some embodiments of the present application the wild type IDE is set forth in SEQ ID NO: 50.

[0059] According to some embodiments of the present application the mutant IDE comprises an El l l Q substitution corresponding to SEQ ID NO: 50.

[0060] According to some embodiments of the present application the method further comprises screening the plurality of antibodies to select a plurality of antibodies that inhibit an insulin-degrading activity of the IDE.

[0061] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the application, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. BRIEF DESCRIPTION OF DRAWINGS

[0062] Some embodiments of the application are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the application. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the application can be implemented.

[0063] In the drawings:

[0064] FIGS. 1A-1G Generation of various anti-IDE antibodies is shown. FIGS. 1A-1D Expression and purification of recombinant human wild type (WT) IDE and inactive IDE mutant (E111Q) is shown. FIG. 1A is a graphical map of a pET28a+ plasmid containing the recombinant IDE: the recombinant construct was ordered from Genewiz and inserted into the vector between Ndel and Hindlll. His tag and single nucleotide mutation site for catalytically inactive IDE (E111Q) are also marked. FIG. 1B SDS-PAGE of total cell extracts (15 μg) of Rosetta BL21 E. coli cells carrying pET28a-IDE vector before and after induction with 0.5 mM IPTG (described as "unind" and "IPTG", respectively); purified protein (3 μg) eluted from GE HisTrap column using imidazole (described as "pure"); protein (15 μg) washed off from GE HisTrap column (described as "FT") is shown. FIG. 1C Western blot analysis of total cell extracts (15 μg) from Rosetta BL21 E. coli cells carrying pET28a+ vector: empty vector (described as "mock"); vector with rhIDE wild type (WT) before and after induction (described as "unind" and "IPTG", respectively). IDE was detected using primary anti-IDE polyclonal antibody followed by (IR)-dye secondary antibody. Fluorescent signal was detected by scanning the membrane in an Odyssey scanner (Licor, pixel size 21 μm, 0.5 mm offset, intensity -6 and average quality). FIG. 1Dis a bar graph showing the IDE activity assay. 1.5 μg / L human insulin was incubated with PBS, 12 μg / ml rhIDE WT or E111Q for 2 hours at 37°C. Residual insulin was later analyzed using Mercodia Ultrasensitive Mouse Insulin ELISA. Detection was read at 450 nm by an ELISA plate reader. The results are shown as mean ± SEM (n=3). **p<0.01; ***p<0.001, One way ANOVA with Bonferroni correction. FIG. 1E shows the expression and purification of anti-IDE MBP-scFv. A representative FPLC evaluation of A9 MBP-scFv is shown in the left panel. The soluble fraction of bacterial lysate was applied to a GE HisTrap column. Elution was performed with 0.5 M imidazole at a flow rate of 2 ml / min (peak). An SDS-PAGE analysis of the three selected clones of MBP-scFv (A9, B1 and H3) is shown in the right panel. Lanes 1 and 2: 15 μg total cell extract from Rosetta BL21 E. coli cells carrying pMALc-NHNN-scFv vector before and after induction with 0.5 mM IPTG, respectively; Lane 4: 3 μg purified protein eluted from GE HisTrap column using imidazole; Lane 3: 15 μg protein washed out from GE HisTrap column. FIG. 1F shows the expression and purification of anti-IDE Inclonal IgG. An SDS-PAGE analysis of the light and heavy chains of the insoluble fraction of cell lysate from Rosetta pUBS500 E. coli cells carrying the anti-IDE antibody chains in pHAK vectors is shown in the upper panel. 10 μg of cell lysate was loaded in each lane before and after induction with IPTG (odd and even lanes, respectively). Lanes 1-2: A9 V H ; Lanes 3-4: B1 V H ; Lanes 5-6: H3 V H ; Lanes 7-8: A9 V L ; Lanes 9-10: B1 V L ; Lanes 11-12 H3 V LA representative FPLC assessment of B1 clonal IgG is shown in the intermediate figure. Refolded solutions of the B1 light and heavy chains were applied to a GE MabSelect column. Eluent was obtained with citrate buffer (pH 3.0) at a flow rate of 2 ml / min (peak). The lower figure is a representative SDS-PAGE analysis (described as “VL” and “VH”) of multiple inclusion bodies of the light and heavy chains purified from multiple Rosetta pUBS500 E. coli cells carrying anti-IDE light and heavy chains in a pHAK vector after IPTG induction. L and V H After incubation in refolded solution (described as "refold"); clonal IgG purified using multiple MabSelect columns (described as "IgG"); commercial IgG as a positive control (erbitux), in reduced and non-reduced states. FIG. 1G The purification of the reverse chimeric H3 antibody (rcH3-IgG) is shown. The figure shows an SDS-PAGE (12% gel) analysis of the production and purification of rcH3-IgG as a mouse IgG1. Lane 1: Conditioned medium (loaded 10 μg) 7 days post-transfection; Lane M: Molecular weight marker; Lane 2: Protein-G purified rcH3-IgG (loaded 5 μg).

[0065] FIGS. 2A-2F The binding of multiple anti-IDE phage-displayed antibodies to the corresponding multiple "clonal" IgGs was shown. FIGS. 2A-2C It displays multiple phages identified by phage-ELISA, clone A9 ( FIG. 2A B1 FIG. 2B ) and H3 ( FIG. 2C Multiple graphs of IDE binding of scfv were presented. The analyzed scFvs, showing multiple phages, were added in multiple serial dilutions to multiple ELISA pans coated with 2.5 μg / ml WT rhIDE IDE (described as “IDE”) or multiple unrelated proteins: bovine serum albumin (BSA), a HisTrap-purified recombinant protein (described as “His”), and MBP-LacZ. Multiple binding phages were detected using a mouse anti-M13 antibody, followed by an HRP-conjugated goat anti-mouse secondary antibody. The multiple results are shown as mean ± SEM (n = 3). FIGS. 2D-2F This shows the difference between IDE as determined by ELISA and purified clonal IgG, clonal A9 ( FIG. 2D B1 FIG. 2E ) and H3 ( FIG. 2FFigure 6 shows a series of graphs demonstrating the binding of a plurality of antibodies analyzed. The plurality of antibodies analyzed were added to ELISA plate wells coated with 2 μg / ml WT rhIDE IDE (described as "IDE") or BSA at serial dilutions. Bound antibodies were detected with a HRP conjugated goat anti-human secondary antibody. The results are shown as mean ± SEM (n = 4).

[0066] FIGS. 3A-3B Figure 7 shows IDE inhibition assays using anti-IDE MBP-scFv or clonal IgG. These graphs show the inhibition of IDE activity as determined by residual insulin levels after incubation with the antibodies generated. 1.5 μg / ml IDE was incubated with the indicated concentrations of anti-IDE scFv clone A9, B1 or H3 or a control scFv (described as "AF") (A); or with anti-IDE clonal IgG clone A9 or H3 or an unrelated clonal IgG (described as "AF") (B) and an IDE inhibitor peptide ADT21 (C) for 1 hour at room temperature and then with 2 μg / L human insulin for 2 hours at 37°C. Residual insulin was then analyzed using Mercodia Ultra Sensitive Mouse Insulin ELISA. Detection was by an ELISA plate reader at 450 nm. The results are shown as mean ± SEM (n = 3). *** - P < 0.001, one-way ANOVA with Bonferroni correction. FIG. 3A FIG. 3B Figure 8 shows a representative Western blot demonstrating the conformational specificity of the anti-IDE antibodies generated. WT rhIDE (0.1 - 1 μg) and 20 μg of mouse spleen cell lysate were spotted onto the membrane in native or denatured state (boiled for 5 minutes at 95°C). Anti-IDE MBP-scFv (clone A9, B1 or H3) were then added, followed by a mouse anti-MBP antibody and later a HRP conjugated goat anti-mouse secondary antibody. Detection was by an ECL reaction.

[0067] FIGS. 4A-4C Figure 9 shows a representative Western blot demonstrating the conformational specificity of the anti-IDE antibodies generated. WT rhIDE (0.1 - 1 μg) and 20 μg of mouse spleen cell lysate were spotted onto the membrane in native or denatured state (boiled for 5 minutes at 95°C). Anti-IDE MBP-scFv (clone A9, B1 or H3) were then added, followed by a mouse anti-MBP antibody and later a HRP conjugated goat anti-mouse secondary antibody. Detection was by an ECL reaction. FIG. 4A FIG. 4B ​​Figure 1 is a graph showing the binding of reverse chimeric rcH3-IgG to WT or mutant rhIDE as determined by ELISA. 5 μg / ml of WT rhIDE, inactive IDE mutant (E111Q, described as "mutant IDE") or BSA were incubated with rcH3-IgG at serial dilutions or the negative control antibody rc2E12-IgG (starting at 100 nM). Antibodies were detected using an HRP-conjugated goat anti-mouse secondary antibody. Results are shown as mean ± SEM (n=3). FIG. 4C Figure 2 is a bar graph showing the inhibition of IDE activity as determined by residual insulin levels after incubation with rcH3-IgG or a negative control rc2E12-IgG. 0.1 μg / ml IDE was incubated with rcH3-IgG or rc2E12-IgG for 1 h at room temperature and then with 1.5 μg / L human insulin for 1 h at 37°C. Residual insulin was then analyzed using Mercodia Ultra Sensitive Mouse Insulin ELISA. Detection was read at 450 nm by an ELISA plate reader. Results are shown as mean ± SEM (n=3).

[0068] FIGS. 5A-5C Figure 3 shows the therapeutic effect of the reverse chimeric anti-IDE H3 antibody in a STZ-induced diabetic mouse model. FIGS. 5A-5B Figure 4 shows the insulin levels after intraperitoneal administration of rcH3-IgG or a negative control rc2E12-IgG 2 days after STZ injection as determined by an oGTT assay (n=5 mice per group; intergroup #p<0.01; FIG. 5A ) or an ITT assay (n=5 mice per group; intra-group *p<0.05, ***p<0.001, FIG. 5B ). FIG. 5C Figure 5 is a graph showing the levels of rcH3-IgG at different days after intraperitoneal administration of STZ-treated mice (n=2 to 4 mice per group). Results are shown as mean ± SEM.

[0069] FIGS. 6A-6BThe in vitro effect of multiple Fab2 fragments of the reverse chimeric anti-IDE H3 antibody (herein referred to as "H3 Fab") on the decrease of reactive oxidative species (ROS) in DJ-1 knockdown (KD) microglial cells, which exhibit the neurotoxic phenotype of microglial cells in Parkinson's disease, is shown. In addition, where indicated (labeled "Rot"), the cells were treated with rotenone (Rotenone) to increase the production of ROS. The levels of ROS per methylene blue (MB) ( FIG. 6A ) and the MB values after incubation with the H3 Fab antibody are shown compared to the medium control. DJ-1-KD microglial cells exhibited higher levels of ROS compared to control microglial cells, and the rotenone-induced increase could be attenuated using anti-IDE H3 Fab. The results using the MB test show that insulin or H3 Fab treatment did not significantly affect cell survival after rotenone stimulation. (N=3, repeated 2-4 times in each experiment; total n=6-12). Rot = rotenone; ins = insulin. Results are shown as mean ± SEM. ***p<0.001, N.S. not significant.

[0070] FIGS. 7A-7C An ELISA assay for detecting IDE in human serum is shown. FIG. 7A is a schematic of the ELISA procedure: (I) 96-well ELISA plates were coated with recombinant A9 IgG overnight as the capture antibody; (II) the wells were washed once with PBS and blocked with 3% skim milk; (III) serum was added to the plates, and known concentrations of recombinant human IDE were used as controls; (IV) the wells were incubated with polyclonal rabbit anti-IDE antibody as the detection antibody; (V) the wells were washed and incubated with HRP-conjugated goat anti-rabbit antibody, followed by development as described in the methods. FIG. 7B The sensitivity curve of the ELISA assay is shown. FIG. 7C The IgG A9 recognizes a conformational epitope on rhIDE is shown. A solution of 5 μg / ml rhIDE in PBS was prepared and used directly to coat one half of an ELISA plate or denatured by heating at 80°C for 20 min, then cooled on ice, followed by using it to coat the other half of the ELISA plate. After the coating, blocking, and washing steps, IgG A9 at concentrations of 100, 33.3, 11.1, or 3.7 nM was applied to the wells coated with native or heat-denatured rhIDE in triplicate. The results are shown as mean ± SEM (n=3).

[0071] FIGS. 8A-8B IgG A9 recognizes a conformational epitope on rhIDE. A solution of PBS containing 5 pg / ml rhIDE was prepared and used directly to coat half of an ELISA plate or denatured by heating at 80°C for 20 min, then cooled on ice, before using it to coat the other half of the ELISA plate. The plate was coated overnight at 4°C at 50 mΐ / well. The next day, the plate was washed once with 300 mΐ / well of PBST and blocked for 1 h at 37°C with 300 mΐ / well of a 3% skimmed milk solution in PBS. Then, the plate was washed three times with 300 mΐ / well of PBST and FIG. 8A ) IgG A9 at concentrations of 100, 33.3, 11.1, 3.7 nM was applied to the wells of three columns of wells coated with native or heat denatured rhIDE. These wells were then washed three times with 300 mΐ / well of PBST / well before adding 50 mΐ / well of HRP-conjugated goat anti-human IgG (Jackson ImmunoResearch) diluted 5000-fold in PBST. FIG. 8B ) To quantify the total rhIDE coated on the wells, the other half of the plate was incubated with HRP-conjugated goat anti-His-tag antibody diluted 2000-fold, 4000-fold, 8000-fold, 16000-fold in PBST. The plate was left for 1 h at room temperature before being washed 3 times with 300 mΐ / well of PBST. Finally, 50 mΐ / well of the HRP substrate TMB was added until color appeared. The reaction was stopped after 2 min by adding 50 mΐ / well of 1 M H2SO4 and analyzed using a BioTek Epoch microplate reader. The optical density was measured at 450 and 620 nm wavelengths. Results are shown as mean ± SEM (n = 3).

[0072] FIG. 9 Higher levels of IDE in the serum of MS patients (n = 51) compared to control healthy subjects (n = 24) are shown. Results are shown as mean ± SEM; ***p < 0.001.

[0073] FIGS. 10A-10C Correlation between serum IDE levels and components of MS: triglycerides; r = 0.423, p < 0.01 FIG. 10A ), insulin; r = 0.294, p < 0.05 FIG. 10B ), and HDLc; r = -0.366, p < 0.05 FIG. 10C DETAILED DESCRIPTION

[0074] ​The present application, in some embodiments thereof, relates to anti-IDE antibodies and uses thereof.

[0075] The principles and operation of the present application can be better understood with reference to the drawings and accompanying descriptions.

[0076] Before explaining at least one embodiment of the application in detail, it is to be understood that the application is not limited in its application to the details set forth in the following description or exemplified by the Examples. The application is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting.

[0077] Insulin-degrading enzyme (IDE) is a large zinc-binding protease known to cleave a variety of short peptides and plays an important role in the degradation of insulin. Several IDE monoclonal and polyclonal antibodies have been described for in vitro [see, e.g., Delledonne A. et al. Mol Neurodegener. (2009) 4:39] and in vivo (see PCT Publication No. WO 2012 / 017439) applications. In addition, several IDE inhibitors, including peptidic inhibitors (see PCT Publication No. WO 2010 / 086867), have been previously described for various therapeutic applications.

[0078] In the course of developing specific embodiments of the present application, the present inventors have generated several new monoclonal anti-IDE antibodies that specifically target and inhibit the insulin-degrading activity of IDE. Accordingly, specific embodiments of the present application suggest the use of these anti-IDE antibodies for the diagnosis and treatment of IDE-related diseases and disorders.

[0079] As shown in the section of the Examples below, the present inventors produced plasmids for the expression of human insulin-degrading enzyme (IDE) and a mutant IDE (E111Q) in E. coli (Example 1, FIG. 1A ). The mutant IDE (E111Q) comprises a site-specific mutation that impairs catalytic activity due to the inability to perform a nucleophilic attack on the substrate. Both peptides were therefore used to isolate specific antibody clones that bind to the catalytically active IDE (i.e., wild-type IDE) with high affinity. Accordingly, IDE proteins were expressed and purified from E. coli, and protein purity and identity were verified (see Example 2, below). FIGS. 1B-1D) were isolated using the phage display technology as bait for specific single-chain variable fragment (scFv) clones that recognize and bind to high-affinity epitopes of the recombinant human wild-type IDE. Three clones of phage were selected, namely, A9, H3, and B1 (Example 1, Figure 2-C). The scFvs were then reformatted for production as soluble antibodies and tested in three formats: MBP-scFv, human IgGl produced as "clones" and reverse chimeric IgG produced in mammalian cell culture. The specificity and IDE-inhibitory properties of these antibodies were further validated in vitro (Examples 1-2, FIGS. 1E-4C ) In addition, a reverse chimeric H3 IgG antibody improved glucose levels and insulin activity in a STZ-induced mouse model of diabetes (Example 2, FIGS. 5A-5C ) In addition, a Fab2 fragment of the reverse chimeric H3 IgG antibody reduced the in vitro production of reactive oxygen species in dopaminergic neurons with a phenotype of Parkinson's disease (Example 3, FIGS. 6A-6B ) The present inventors then used the generated antibodies to develop a high-sensitivity ELISA (Example 4, FIGS. 7A-7C ) Using this ELISA, the present inventors were able to demonstrate a strong correlation between human serum IDE levels and the presence and / or severity of the metabolic syndrome (Example 4, FIGS. 9-10C ).

[0080] According to one aspect of the present application, there is provided an isolated antibody comprising an antigen recognition region that specifically binds to IDE, wherein the antigen recognition region comprises the complementarity determining region (CDR) amino acid sequences set forth below:

[0081] (i) SEQ ID NO: 4 (CDR1), SEQ ID NO: 6 (CDR2), and SEQ ID NO: 8 (CDR3), in order from N to C on a heavy chain of the antibody; and SEQ ID NO: 12 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 16 (CDR3), in order from N to C on a light chain of the antibody;

[0082] (ii) SEQ ID NO: 20 (CDR1), SEQ ID NO: 22 (CDR2), and SEQ ID NO: 24 (CDR3), arranged from N to C in a heavy chain of the antibody; SEQ ID NO: 28 (CDR1), SEQ ID NO: 30 (CDR2), and SEQ ID NO: 32 (CDR3), arranged from N to C in a light chain of the antibody; or

[0083] (iii) SEQ ID NO: 36 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 40 (CDR3), arranged from N to C in a heavy chain of the antibody; SEQ ID NO: 44 (CDR1), SEQ ID NO: 46 (CDR2), and SEQ ID NO: 48 (CDR3), arranged from N to C in a light chain of the antibody.

[0084] As used herein, the term "insulin-degrading enzyme (IDE)" refers to the insulysin or insulin protease, a large zinc-binding protease of the M16A metalloprotease subfamily involved in the cellular processing of various short polypeptides, including amyloid beta protein (Αβ), insulin, glucagon, amylin, atrial natriuretic peptide, and calcitonin (e.g., as set forth in GenBank Accession Nos. NM_004969 and NP_004960). According to particular embodiments, IDE is human IDE. An exemplary IDE of the present application is set forth in EC 3.4.24.56.

[0085] As used herein, the term "wild-type human insulin-degrading enzyme," also referred to as WT IDE, refers to the expression product of the IDE gene that is a functional human IDE having an affinity for insulin of 100 nM and degrades insulin at a rate of 36.6 micromoles (pmole) of human recombinant IDE per 1 pmole of human insulin / min. An exemplary WT IDE is set forth in SEQ ID NO: 50.

[0086] As used herein, the term "mutant IDE type E111Q" refers to the altered form of human IDE having reduced catalytic activity, in which at least one catalytic site mutation occurs (glutamic acid 111 to glutamine, corresponding to SEQ ID NO: 50). An exemplary mutant IDE type E111Q is set forth in SEQ ID NO: 52.

[0087] The term "isolated" means at least partially separated from the natural environment, for example, from serum, or as the predominant form of an antibody in a sample containing a plurality of antibodies, or as the only antibody in a biological sample.

[0088] The term "antibody" as used herein includes whole molecules and functional fragments thereof capable of binding to an epitope of an antigen.

[0089] The term "epitope" as used herein refers to any antigenic determinant on an antigen that is bound by a paratope of an antibody. Epitopic determinants are usually composed of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and are usually found on the surface of a molecule. The interaction of an epitope with a paratope is mediated by chemical interactions.

[0090] According to a specific embodiment, the antibody fragments include, but are not limited to, single chains; Fab, Fab' and F(ab')2 fragments; Fd; Fcab; Fv; dsFv; scFv; MBP-scFv; diabodies; minibodies; nanobodies; Fab phage display libraries; or single domain molecules such as VH and VL, which are capable of binding to an epitope of an antigen in an HLA restricted manner.

[0091] According to a specific embodiment, the antibody is a whole or intact antibody.

[0092] According to a specific embodiment, the antibody is an antibody fragment.

[0093] Suitable antibody fragments for use in practicing some embodiments of the present application include a complementarity determining region (CDR) of an immunoglobulin light chain (referred to herein as "light chain"), a complementarity determining region of an immunoglobulin heavy chain (referred to herein as "heavy chain"), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments that essentially comprise the entire variable region of a light chain and a heavy chain, such as a Fv, a single chain Fv (scFv), a disulfide-stabilized Fv (dsFv), a Fab, a Fab', and a F(ab')2, or antibody fragments that comprise the Fc region of an antibody.

[0094] As used herein, the terms "complementarity determining region" or "CDR" are used interchangeably to refer to the antigen binding regions found within the variable region of the heavy and light chain polypeptides. Typically, antibodies include three CDRs in each of the VH (CDR HI or HI; CDR H2 or H2; and CDR H3 or H3) and three CDRs in each of the VL (CDR LI or LI, CDR L2 or L2; and CDR L3 or L3).

[0095] The identity of the amino acid residues that make up a variable region or a CDR in a particular antibody can be determined using methods known in the art and include, for example, sequence variability as defined by Kabat et al. (see, e.g., Kabat et al. 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), the location of the structural loop regions as defined by Chothia et al. (see, e.g., Chothia et al. Nature 342:877-883, 1989.), a compromise between Kabat and Chothia using Oxford Molecular's AbM antibody modeling software (now available via the Worldwide Web at bioinf.org.uk / abs), methods that define the defined available complex crystal structures from the contacts (see, e.g., MacCallum et al. J. Mol. Biol. 262:732-745, 1996) and the "conformational definition" (see, e.g., Makabe et al. Journal of Biological Chemistry, 283:1156-1166, 2008). See Martin et al. 1989, Proc. Natl Acad Sci USA. 86:9268; and Worldwide Web at bioinf-org.uk / abs, methods that define the defined available complex crystal structures from the contacts (see, e.g., MacCallum et al. J. Mol. Biol. 262:732-745, 1996) and the "conformational definition" (see, e.g., Makabe et al. Journal of Biological Chemistry, 283:1156-1166, 2008).

[0096] As used herein, the "variable regions" and "CDRs" can refer to variable regions and CDRs defined by any method known in the art, including combinations of methods.

[0097] According to particular embodiments, the identity of the amino acid residues that make up the variable regions and / or the CDRs in the antibody is determined from the deduced amino acid sequence of the translated coding gene.

[0098] Functional antibody fragments comprising all or substantially all of the variable regions of the light and heavy chains are defined as follows:

[0099] (i) Fv, defined as a genetically engineered fragment consisting of the variable region (VL) of the light chain and the variable region (VH) of the heavy chain, expressed as two chains;

[0100] (ii) single chain Fv ("scFv"), a genetically engineered single chain molecule containing the variable region of the light chain and the variable region of the heavy chain linked together as a gene fusion by a suitable polypeptide linker;

[0101] (iii) disulfide linked Fv ("dsFv"), a genetically engineered antibody containing the variable region of the light chain and the variable region of the heavy chain linked together by a disulfide bond;

[0102] (iv) Fab, a fragment of an antibody molecule that contains a single antigen-binding portion of an antibody molecule, can be obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment consisting of the heavy chain variable domain and the CH1 domain;

[0103] (v) Fab', a fragment of an antibody molecule that contains a single antigen-binding portion of an antibody molecule, can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab' fragments are obtained from each antibody molecule);

[0104] (vi) F(ab')2, a fragment of an antibody molecule that contains a single antigen-binding portion of an antibody molecule, can be obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of two Fab' fragments held together by two disulfide bonds);

[0105] (vii) single domain antibodies or nanobodies consisting of a single VH or VL domain that exhibit sufficient affinity to the antigen; and

[0106] (viii) Fcab, a fragment of an antibody molecule that contains the Fc portion of an antibody, developed by introducing an antigen binding domain into the Fc region of an antibody.

[0107] According to a specific embodiment, the antibody is a scFv.

[0108] According to specific embodiments, the antibody is a scFv stabilized by fusion with the E. coli maltose binding protein, also known as MBP-scFv, as described, for example, by Bach H1 et al. J Mol Biol. (2001) Sep 7;312(1):79-93 and the examples following the section.

[0109] According to various embodiments, the antibody is an IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, or IgE antibody.

[0110] According to various embodiments, the antibody is an IgG antibody.

[0111] According to various embodiments, the antibody isotype is IgGl or IgG4.

[0112] The antibody can be monospecific (capable of recognizing one epitope or protein), bispecific (capable of binding two epitopes or proteins), or multispecific (capable of recognizing multiple epitopes or proteins).

[0113] According to various embodiments, the antibody is a monospecific antibody.

[0114] According to various embodiments, the antibody is multispecific, e.g., bispecific, trispecific, tetraspecific.

[0115] According to various embodiments, the antibody is a bispecific antibody.

[0116] Various bispecific antibodies, also known as bifunctional antibodies, have at least one antigen recognition site for a first antigen and at least one antigen recognition site for a second antigen. Such antibodies can be produced by various recombinant DNA methods, or can be produced chemically by various methods known in the art. Chemically produced bispecific antibodies include, but are not limited to, antibodies that are reduced and reconstituted to retain their bivalent properties, and antibodies that are chemically coupled so that they have at least two antigen recognition sites for each antigen. Bispecific antibodies include conjugates of all antibodies or antibody fragments, or polymeric forms of antibodies that are capable of recognizing two different antigens.

[0117] According to various embodiments, the antibody is a monoclonal antibody.

[0118] Various methods of producing antibodies and fragments thereof are well known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988; incorporated herein by reference).

[0119] According to various embodiments, the antibody is recombinant produced in mammalian cells.

[0120] According to various embodiments, the antibody is recombinantly produced in bacteria.

[0121] According to some embodiments of the application, antibody fragments can be prepared by proteolytic digestion of the antibody or by protein expression in E. coli or mammalian cells (e.g., Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies, by various conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of whole antibodies using pepsin to provide a 5S fragment representing an F(ab')2 fragment. This fragment can be further cleaved using a thiol reducing agent, and optionally a protecting group for the sulfhydryl groups produced by the cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin directly produces two monovalent Fab' fragments and an Fc fragment. For example, these methods are described in Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which are hereby incorporated by reference in their entirety. See also, Porter, R.R. [Biochem. J. 73:119-126 (1959)]. Other methods of cleaving antibodies for their fragments can also be used, such as separating the heavy chains to form monovalent light-heavy chain fragments, further cleaving the fragments, or using other enzymatic, chemical or genetic techniques, as long as the fragments bind to the antigen that is recognized by the whole antibody.

[0122] A number of Fv fragments comprise a combination of VH and VL chains. This combination can be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (1972)]. Alternatively, the variable chains can be joined using intermolecular disulfide bonds or by chemical linkage, such as with glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene in which the VH and VL domains are connected by a short linker peptide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFv are described in, for example, Whitlow and Filpula, Methods 2:97-105 (1991); Bird et al. Science 242:423-426 (1988); Pack et al. Bio / Technology 11 :1271-77 (1993); and U.S. Patent No. 4,946,778, which are incorporated herein by reference in their entireties.

[0123] Another form of an antibody fragment is a peptide which encodes a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding a single CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to amplify the variable region encoding the CDR(s) from mRNA obtained from antibody-producing cells. See, for example, Larrick and Fry [Methods, 2:106-10 (1991)].

[0124] According to particular embodiments, the antibody is a chimeric antibody.

[0125] According to various embodiments, the antibody is a humanized antibody. Various humanized forms of non-human (e.g., murine) antibodies are chimeric molecules, immunoglobulin chains or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also incorporate residues which are not found in the recipient antibody or in the CDR or framework sequences of the donor antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

[0126] Methods for humanizing various non-human antibodies are well known in the art. Typically, a humanized antibody has one or more amino acid residues from a source that is non-human introduced into it. These non-human amino acid residues are often referred to as imported residues, which are typically taken from an imported variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting one or more non-human CDRs or CDR sequences for the corresponding sequence(s) of a human antibody. Thus, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0127] According to specific embodiments, the antibody is a human antibody.

[0128] Human antibodies can also be produced using various techniques known in the art including the use of various phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991); Bitton A, Nahary L, Benhar I. Methods Mol Biol. 1701: 349-363 (2018)]. The techniques described by Cole et al. and Boerner et al. (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147 (1): 86-95 (1991)) can also be used to produce the human monoclonal antibodies. Similarly, the human antibodies can be made through the introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which is largely indistinguishable from that seen in humans in all respects, including gene rearrangement, recombination and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,569,825; 5,625,126; 5,633,425; 5,661,016; and in the following scientific publications: Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

[0129] It will be appreciated that targeting of specific compartments within the cell can be achieved using multiple intracellular antibodies (also known as "intrabodies"). These are essentially SCAs (e.g., ER, mitochondria, nucleus, cytoplasm) to which multiple intracellular localization signals have been added. This technology has been successfully applied in the art (for a review, see Richardson and Marasco, 1995, TIB TECH vol. 13). Multiple intracellular antibodies have been shown to virtually eliminate the expression of other various abundant cell surface receptors and inhibit the function of a protein within a cell (see, e.g., Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92:3137-3141; Deshane et al., 1994, Gene Ther. 1 :332-337; Marasco et al., 1998 Human Gene Ther 9:1627-42; Shaheen et al., 1996 J. Virol. 70:3392-400; Werge, T.M. et al., 1990, FEBS Letters 274:193-198; Carlson, J.R. 1993 Proc. Natl. Acad. Sci. USA 90:7427-7428; Biocca, S. et al., 1994, Bio / Technology 12:396-399; Chen, S-Y. et al., 1994, Human Gene Therapy 5:595-601; Duan, L et al., 1994, Proc. Natl. Acad. Sci. USA 91 :5075-5079; Chen, S-Y. et al., 1994, Proc. Natl. Acad. Sci. USA 91 :5932-5936; Beerli, R.R. et al., 1994, J. Biol. Chem. 269:23931-23936; Mhashilkar, A.M. et al., 1995, EMBO J. 14:1542-1551; Marasco et al., PCT Publication No. WO 94 / 02610; and Duan et al., PCT Publication No. WO 95 / 03832).

[0130] According to specific embodiments, the antibody is an intracellular antibody, since IDE is expressed on the cell surface, but also in the cytosol, peroxisomes and endosomes.

[0131] In this regard, it has been shown that multiple intracellular antibodies can block the translocation of proteins to the appropriate compartment, thereby inhibiting their activity (see, e.g., J. Cell. Mol. Med. Vol 11, No 1, 2007 pp. 54-70; Persic et al. Gene 187 (1997) 1-8; and Shaki-Loewenstein et al. Journal of Immunological Methods 303 (2005) 19-39). Thus, the intrabodies of some embodiments of the application do not necessarily need to inhibit the intrinsic catalytic activity of IDE itself.

[0132] To make an intrabody expression vector, the cDNAs encoding the light and heavy chains of the antibody specific for the target protein of interest are typically isolated from a hybridoma that secretes a monoclonal antibody specific for APLP1. Hybridomas secreting anti-APLP1 monoclonal antibodies or recombinant monoclonal antibodies can be prepared using a variety of methods known in the art. Once a monoclonal antibody (e.g., a hybridoma-derived monoclonal antibody or a recombinant antibody from a combinatorial library) is identified that is specific for the APLP1 protein, the DNA encoding the light and heavy chains of the monoclonal antibody can be isolated by standard molecular biology techniques. For hybridoma-derived antibodies, light and heavy chain cDNAs can be obtained by PCR amplification or cDNA library screening. For recombinant antibodies, e.g., from a phage display library, the cDNAs encoding the light and heavy chains can be recovered from the display package (e.g., phage) isolated in the process of the library screening and the nucleotide sequences of the antibody light and heavy chain genes determined. For example, many such sequences are disclosed in Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. U.S. Department of Health and Human Services, NIH Publication No. 91-3242 and the "Vbase" human germline sequence database. Once obtained, the antibody light and heavy chain sequences are cloned into a recombinant expression vector using standard methods.

[0133] For cytoplasmic expression of the light and heavy chains, the nucleotide sequences encoding the hydrophobic leaders of the light and heavy chains are removed. An intrabody expression vector can encode an intrabody in one of several different formats. For example, in one embodiment, the vector encodes full-length antibody light and heavy chains, such that a full-length antibody is expressed intracellularly. In another embodiment, the vector encodes a full-length light chain but only the VH / CH1 region of the heavy chain, such that a Fab fragment is expressed intracellularly. In another embodiment, the vector encodes a single-chain antibody (scFv), in which the variable regions of the light and heavy chains are linked by a flexible peptide linker [e.g., (Gly4Ser)3] and expressed as a single-chain molecule. To inhibit APLP1 activity in a cell, the expression vector encoding the intrabody is introduced into the cell by standard transfection methods, as previously described.

[0134] In some embodiments, the antibodies provided herein can be functionally conjugated to a cell-penetrating agent. The phrase "cell-penetrating agent" as used herein refers to an agent that enhances translocation of the antibody across a cell membrane.

[0135] An exemplary cell-penetrating agent is a cell-penetrating peptide.

[0136] As used herein, a "cell-penetrating peptide" is a short (about 12 to 30 residues) amino acid sequence or functional motif that confers energy-independent (i.e., non-endocytic) translocation properties associated with transport of the membrane-permeable complex across the plasma membrane and / or nuclear membrane of a cell. The cell-penetrating peptides used in the membrane-permeable complex of some embodiments of the application preferably comprise at least one non-functional cysteine residue, either free or derivatized, to form a disulfide bond with a double-stranded ribonucleic acid that has been modified for such linkage. Representative amino acid motifs that confer such properties are listed in U.S. Patent No. 6,348,185, the contents of which are expressly incorporated herein by reference. The cell-penetrating peptides of some embodiments of the application can include, but are not limited to, penetratin, transportan, pIsl, TAT (48-60), pVEC, MTS, and MAP.

[0137] Additionally or alternatively, lipid particles, e.g., liposomes, can be used as cell-penetrating agents.

[0138] Liposomes comprise any synthetic (i.e., non-naturally occurring) structure composed of lipid bilayers that enclose a volume. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. The liposomes can be prepared by any of a variety of known methods described in the art [Monkkonen, J. et al. 1994, J. Drug Target, 2:299-308; Monkkonen, J. et al. 1993, Calcif. Tissue Int., 53: 139-145; Lasic D D., Liposomes Technology Inc., Elsevier, 1993, 63-105. (chapter 3); Winterhalter M, Lasic D D, Chem Phys Lipids, 1993 September; 64(1-3):35-43]. The liposomes can be positively charged, neutral, or negatively charged.

[0139] Any method known in the art can be used to incorporate an antibody into a liposome, for example, as described by Alfonso et al., [The science and practice of pharmacy, Mack Publishing, Easton Pa 19th ed., (1995)] and Kulkarni et al., [J. Microencapsul. 1995, 12(3) 229-46].

[0140] To determine liposomes particularly suitable according to the present application, a screening assay can be performed, for example, as described in U.S. Patent Application No. 20040266734 and U.S. Patent Application No. 20040266734; and in the assays described in Danenberg et al., Journal of cardiovascular pharmacology 2003, 42:671-9; Circulation 2002, 106:599-605; Circulation 2003, 108:2798-804.

[0141] According to one embodiment, the antibodies of some embodiments of the application are generated as follows. Purified IDE is used to isolate specific single-chain variable fragment (scFv) clones that can recognize epitopes of the human IDE. A human synthetic antibody phage display library is used to exploit phage display technology. Specifically, a human scFv library, such as the Ronit 1 library [described in Azriel-Rosenfeld et al. J Mol Biol. (2004) 335(1): 177-192, incorporated by reference herein in its entirety] is screened. The library is depleted of non-specific proteins and proteins purified from bacteria using affinity columns and selected using the recombinant human wild-type (WT) IDE (SEQ ID NO: 50). In several rounds (e.g., two rounds), the enriched phage is depleted (i.e., negative selection) on mutant IDE (e.g., E111Q) to enrich for positive phage that recognize the WT IDE or better than the mutant IDE according to a particular embodiment. Each of these depletion steps is followed by a selection step (i.e., positive selection) on WT IDE. After the rounds of affinity selection, individual colonies of infected bacteria are picked and grown. Phage from each colony are then tested (e.g., by ELISA) for IDE binding versus non-specific proteins as negative controls. Positive clones are analyzed by PCR amplification, then fingerprinted to detect different clones, and further sequenced to verify integrity and differences between clones.

[0142] An important feature of the isolation of the antibodies of some embodiments of the application is the selection step. Thus, according to one embodiment, the method of producing an antibody comprises (a) providing a plurality of antibodies (e.g., as described above); and (b) screening the plurality of antibodies to select antibodies that bind to wild-type IDE but not to mutant IDE, wherein the mutant IDE has a reduced catalytic activity compared to the wild-type IDE.

[0143] Once antibodies are obtained, they can be tested for activity. Methods of testing antibody activity include, for example, enzyme-linked immunosorbent assay (ELISA) and in vitro insulin degradation assays.

[0144] According to particular embodiments, the antibodies are tested for IDE inhibitory activity. IDE inhibitory activity refers to specific down-regulation of the enzymatic activity of IDE. For example, according to the assay of the IDE inhibitory peptide ADT-21, the IC 50 in the range of 10 to 200 nM (e.g., 1 to 100 nM, 1 to 50 nM, 1 to 20 nM, or 1 to 10 nM).

[0145] According to various exemplary embodiments, the antibody is capable of down-regulating the enzymatic activity of IDE by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%.

[0146] According to various exemplary embodiments, the antibody is capable of down-regulating the insulin-degrading activity of IDE by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%.

[0147] According to a specific embodiment, the antibody is capable of down-regulating the insulin-degrading activity of IDE by at least 50%.

[0148] According to various exemplary embodiments, the antibody is capable of down-regulating the insulin-degrading activity of IDE by 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 10-50%, 50-100%, or 10-100%.

[0149] According to various exemplary embodiments, the antibody is capable of down-regulating the activity of IDE such that IDE degrades insulin at a rate of 1 pmole human insulin / min with 25-50 pmole, 25-75 pmole, 25-100 pmole, 50-75 pmole, 50-100 pmole, or 75-100 pmole of human recombinant IDE.

[0150] As mentioned above, according to various specific embodiments, the plurality of antibodies (e.g. single chain Fv’s) can be produced by recombinant DNA technology, whereby a nucleic acid of the antibody is ligated into an expression vector, which is then introduced into a host cell.

[0151] According to an aspect of the present application, there is thus provided an isolated polynucleotide comprising a nucleic acid sequence encoding the antibody of some embodiments of the application.

[0152] According to an embodiment of the present application, the nucleic acid sequence of the antibody comprises SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15.

[0153] According to another embodiment of the application, the nucleic acid sequence of the antibody comprises SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 31.

[0154] According to yet another embodiment of the application, the nucleic acid sequence of the antibody comprises SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45 and SEQ ID NO: 47.

[0155] Non-limiting examples of nucleic acid sequences encoding the light chain, the heavy chain, the CDRs and the variable regions comprising an antibody that can be used with some embodiments of the application are provided in SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 25, 3SEQ ID NO: 3 and SEQ ID NO: 41.

[0156] According to specific embodiments, therefore, the polynucleotide comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 33 and SEQ ID NO: 41, each possibility representing a separate embodiment of the application.

[0157] As used herein, the term "polynucleotide" or "nucleic acid sequence" refers to a single- or double-stranded nucleic acid sequence, isolated and provided in the form of a RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and / or a composite polynucleotide sequence (e.g. a combination of the above).

[0158] For expression of a foreign antibody in mammalian cells, a polynucleotide sequence encoding the antibody is preferably linked to a nucleic acid construct suitable for expression in mammalian cells.

[0159] According to an aspect of the application, therefore, there is provided a nucleic acid construct comprising the isolated polynucleotide.

[0160] Such a nucleic acid construct or system includes at least one cis-acting regulatory element for directing expression of the nucleic acid sequence. Cis-acting regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as those that direct inducible expression of the nucleotide sequence only under certain conditions. Thus, for example, a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner is included in the nucleic acid construct. Notably, the nucleic acid sequences of the present application can also be used for in vivo use, either as a naked DNA administered to a subject in need (e.g., a diabetic subject) or linked to a nucleic acid construct useful for gene therapy, such as a viral vector.

[0161] Also provided are host cells comprising the polynucleotides / expression vectors described herein.

[0162] Such cells are typically selected for high expression of recombinant proteins (e.g., bacterial, plant, or eukaryotic cells, such as CHO, HEK-293 cells), but can also be an immune cell (e.g., macrophages, dendritic cells, T cells, B cells, or NK cells), for example when the CDRs of the agent are engrafted into a T cell receptor or a CAR transduced into the cells for adoptive cell therapy.

[0163] Thus, according to an aspect of the present application, there is provided a method of producing an anti-IDE antibody, the method comprising expressing the polynucleotide / expression construct in a host cell.

[0164] However, when the antibodies are produced in vitro, according to embodiments, the recovery or isolation of the recombinant antibody is performed after an appropriate incubation time. The phrase "recovering the recombinant antibody" or "isolating the recombinant antibody" refers to the collection of the entire fermentation medium containing the antibody and does not imply additional steps of isolation or purification. Alternatively, "recovering the recombinant antibody" or "isolating the recombinant antibody" refers to the recovery of the product from a lysate of the producing cells. Nonetheless, the antibodies of some embodiments of the application can be purified using standard protein purification methods, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing, and differential solubilization. According to an embodiment, the antibody is at least 80%, 85%, 90%, 95%, 97% pure. As used herein, pure refers to the composition comprising the purified antibody being free of other proteinaceous material that is not the antibody of interest.

[0165] As described in the sections of the examples below, a number of anti-IDE antibodies were generated using the methods described herein.

[0166] Accordingly, according to an aspect of the application, there is provided an isolated antibody comprising an antigen recognition region, wherein the antigen recognition region comprises the complementarity determining region (CDR) amino acid sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16.

[0167] According to one embodiment, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 are arranged in an order (N>C, as CDR 1-3, respectively) on a heavy chain of the antibody, and SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16 are arranged in an order (N>C, as CDR 1-3, respectively) on a light chain of the antibody.

[0168] According to some embodiments of the application, the heavy chain of the antibody comprises an amino acid sequence having at least 80%, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, for example, 100% sequence homology or identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0169] According to one embodiment, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 are arranged in an order (N>C, as CDR 1-3, respectively) on a heavy chain of the antibody, and SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16 are arranged in an order (N>C, as CDR 1-3, respectively) on a light chain of the antibody.

[0170] According to one embodiment, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 are arranged in an order (N>C, as CDR 1-3, respectively) on a heavy chain of the antibody, and SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16 are arranged in an order (N>C, as CDR 1-3, respectively) on a light chain of the antibody.

[0171] According to some embodiments of the application, the heavy chain of the antibody comprises an amino acid sequence having at least 80%, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, for example, 100% sequence homology or identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0172] According to an aspect of the application there is provided an isolated antibody comprising an antigen recognition region, wherein said antigen recognition region comprises the complementarity determining region (CDR) amino acid sequences as set forth in SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 48.

[0173] According to a specific embodiment, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40 are arranged in an order (N>C, as CDR 1-3, respectively) on a heavy chain of the antibody, and SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 48 are arranged in an order (N>C, as CDR 1-3, respectively) on a light chain of the antibody.

[0174] According to some embodiments of the application, the heavy chain of the antibody comprises an amino acid sequence having at least 80%, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, for example, 100% sequence homology or identity to the amino acid sequence set forth in SEQ ID NO: 34.

[0175] When starting from a polypeptide sequence, homology (e.g., percent homology, identity + similarity) can be determined using any homology comparison software, including, for example, the BlastP or TBLASTN software of the National Center of Biotechnology Information (NCBI), such as by using the in-house parameters, to compare the six-frame conceptual translation products of a nucleotide query sequence (double-stranded) to a protein sequence database.

[0176] For example, the in-house parameters for tBLASTX include: Max Target Sequences: 100; Expect threshold: 10; Word Size: 3; Max matches in a query range: 0; Scoring parameters: Matrix - BLOSUM62; Filters and mask: filter software - low complexity regions.

[0177] The antibodies of some embodiments of the application can be used for treating a disease associated with an IDE activity in a subject in need thereof.

[0178] According to one aspect of the application, there is provided a method of treating a disease associated with IDE activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody (ii) or (iii) as described herein, thereby preventing or treating the disease associated with IDE activity.

[0179] According to another or alternative aspect of the application, there is provided use of the antibody (ii) or (iii) as disclosed herein for treating a disease associated with IDE activity in a subject in need thereof.

[0180] In this regard, as mentioned above, since IDE is expressed on the cell surface, but also in the cytosol, peroxisomes and endosomes, an antibody of some embodiments, which is capable of penetrating cells (an intracellular antibody or an antibody functionally associated with a cell-penetrating agent), can inhibit IDE activity by blocking IDE translocation to the appropriate compartment; thus, it is not necessarily required to inhibit the intrinsic catalytic activity of IDE itself to treat a disease associated with IDE activity.

[0181] According to one aspect of the application, there is provided a method of treating a disease associated with IDE activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody, the antibody comprising an antigen recognition region that specifically binds to IDE, wherein the antigen recognition region comprises CDR amino acid sequences as described below:

[0182] (i) SEQ ID NO: 4 (CDR1), SEQ ID NO: 6 (CDR2) and SEQ ID NO: 8 (CDR3), in order from N to C on a heavy chain of the antibody; and SEQ ID NO: 12 (CDR1), SEQ ID NO: 14 (CDR2) and SEQ ID NO: 16 (CDR3), in order from N to C on a light chain of the antibody;

[0183] (ii) SEQ ID NO: 20 (CDR1), SEQ ID NO: 22 (CDR2) and SEQ ID NO: 24 (CDR3), in order from N to C on a heavy chain of the antibody; and SEQ ID NO: 28 (CDR1), SEQ ID NO: 30 (CDR2) and SEQ ID NO: 32 (CDR3), in order from N to C on a light chain of the antibody; or

[0184] (iii) SEQ ID NO: 36 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 40 (CDR3), arranged from N to C terminus on a heavy chain of the antibody; and SEQ ID NO: 44 (CDR1), SEQ ID NO: 46 (CDR2), and SEQ ID NO: 48 (CDR3), arranged from N to C terminus on a light chain of the antibody,

[0185] wherein, when the antibody is the antibody (iii), the antibody is an intracellular antibody or functionally bound to a cell-penetrating agent,

[0186] thereby preventing or treating the disease associated with the IDE activity.

[0187] According to a further or alternative aspect of the present application, there is provided an antibody for use in treating a disease associated with an IDE activity in a subject in need thereof, the antibody comprising an antigen recognition region that specifically binds to IDE, wherein the antigen recognition region comprises CDR amino acid sequences as set out below:

[0188] (i) SEQ ID NO: 4 (CDR1), SEQ ID NO: 6 (CDR2), and SEQ ID NO: 8 (CDR3), arranged from N to C terminus on a heavy chain of the antibody; and SEQ ID NO: 12 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 16 (CDR3), arranged from N to C terminus on a light chain of the antibody;

[0189] (ii) SEQ ID NO: 20 (CDR1), SEQ ID NO: 22 (CDR2), and SEQ ID NO: 24 (CDR3), arranged from N to C terminus on a heavy chain of the antibody; and SEQ ID NO: 28 (CDR1), SEQ ID NO: 30 (CDR2), and SEQ ID NO: 32 (CDR3), arranged from N to C terminus on a light chain of the antibody; or

[0190] (iii) SEQ ID NO: 36 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 40 (CDR3), arranged from N to C terminus on a heavy chain of the antibody; and SEQ ID NO: 44 (CDR1), SEQ ID NO: 46 (CDR2), and SEQ ID NO: 48 (CDR3), arranged from N to C terminus on a light chain of the antibody,

[0191] wherein, when said antibody is said antibody (iii), said antibody is an intracellular antibody or functionally bound to a cell-penetrating agent.

[0192] As used herein, a "disease associated with IDE activity" refers to a medical condition, disease or syndrome in which IDE activity contributes to the onset or progression of the disease.

[0193] As previously mentioned, IDE is an enzyme that cleaves a number of small proteins of different sequences, including insulin, amyloid beta protein (Aβ), glucagon, islet amyloid polypeptide, atrial natriuretic peptide and calcitonin. Thus, in cases where an increase in the levels of these substrates can contribute to the alleviation of symptoms or even cure of the disease, the antibodies of some embodiments of the application can be employed.

[0194] According to a specific embodiment, an increase in the levels of insulin is necessary for the treatment of a disease or condition.

[0195] Such diseases and conditions include, but are not limited to, autoimmune diseases of the central nervous system, neurodegenerative diseases, metabolic syndromes, diabetes, obesity, hyperglycemia, retinal damage, kidney failure, nerve damage, microvascular damage, varicella zoster virus (VZV) infection and wounds. As used herein, an "autoimmune disease of the central nervous system" refers to a disease in which the immune system of the body attacks its own nervous system, preferably the central nervous system (CNS).

[0196] Examples of various autoimmune diseases of the CNS include, but are not limited to, multiple sclerosis, Guillain-Barré syndrome, Lambert-Eaton myasthenia gravis, myasthenia gravis, transverse myelitis, progressive polyleukoencephalopathy, chronic headache, cerebral palsy, lupus, immune dysfunction muscular central nervous system breakdown, primary CNS vasculitis, autoimmune cerebellar degeneration, gait ataxia with late ageonset polyneuropathy (GALOP), neuromyelitis optica, Stiff Person Syndrome, and HTLV-1-associated myelopathy (HAM) / tropical spastic lower limb weakness. paraparesis (TSP).

[0197] According to several specific embodiments, the autoimmune diseases of the central nervous system are selected from the group consisting of: multiple sclerosis, Guillain-Barré syndrome, Lambert-Eaton myasthenia gravis, transverse myelitis, progressive multiple leukoencephalopathy, chronic headache, and cerebral palsy.

[0198] According to one specific embodiment, the autoimmune disease of the central nervous system includes multiple sclerosis (MS).

[0199] As used herein, “a neurodegenerative disease” refers to a condition, disease, or illness of the nervous system (preferably the CNS) characterized by the gradual and progressive loss of nerve tissue, neurotransmitters, or various nerve functions.

[0200] Examples of neurodegenerative disorders include, but are not limited to, amyotrophic lateral sclerosis (ALS or Lou Gehrig's Disease), autoimmune encephalomyelitis, various degenerative neurological diseases, encephalitis (e.g., Rasmussen's encephalitis), Alzheimer's disease, epilepsy, various genetic brain disorders, stroke, Parkinson's disease, and Huntington's disease.

[0201] According to a specific embodiment, the neurodegenerative disease is Parkinson's disease.

[0202] According to a specific embodiment, the neurodegenerative disease is Alzheimer's disease.

[0203] According to specific embodiments, the disease is selected from the group consisting of: a metabolic syndrome, a diabetes, and an obesity.

[0204] According to specific embodiments, the disease is a metabolic syndrome.

[0205] As used herein, the term "metabolic syndrome" refers to a cluster of several metabolic conditions [abdominal obesity, elevated fasting glucose, "dyslipidemia" (i.e., elevated lipid levels), and elevated blood pressure (HBP)] that occur together more often than by chance, that together contribute to the development of type 2 diabetes and cardiovascular disease. Metabolic syndrome is characterized by elevated triglycerides, low high-density lipoprotein cholesterol (HDL-cholesterol), and in some cases moderately elevated low-density lipoprotein cholesterol (LDL-cholesterol) levels, and accelerated progression of atherosclerotic disease due to the stress of the multiple component risk factors.

[0206] According to specific embodiments, the disease is a diabetes.

[0207] As used herein, "diabetes" refers to a disease resulting in an absolute lack of insulin (Type 1 diabetes) or a relative lack of insulin (Type 2 diabetes) in the presence of insulin resistance, i.e., impaired insulin action in an organism, due to a defect in the biosynthesis or production of insulin. Thus, the diabetic has an absolute or relative lack of insulin and can exhibit, among other symptoms and signs, elevated blood glucose concentration, the presence of glucose in the urine, excessive urine output (polyuria), increased thirst (polydipsia), and increased hunger (polyphagia). In Type 1 diabetes, the symptoms can develop quite rapidly (e.g., within weeks or months), particularly in children. In Type 2 diabetes, however, the symptoms can develop more slowly and can be quite mild or completely absent. Diabetes (both types) can also result in a rapid but significant weight loss (despite a normal or even increased diet) and an inability to lose mental fatigue. Diabetes as used herein includes any stage or type of diabetes, including but not limited to overt diabetes, pre-diabetes, and Latent autoimmune diabetes of adults (LADA).

[0208] According to specific embodiments, the diabetes is Type 1 diabetes.

[0209] According to specific embodiments, the diabetes is Type 2 diabetes.

[0210] Examples of diabetes-related diseases include, but are not limited to, Type 1 diabetes, Type 2 diabetes, gestational diabetes, insulin resistance, obesity, hyperglycemia, various eye disorders (e.g., glaucoma, cataract), various skin infections, hypertension, gastroparesis, ketoacidosis (DKA), neuropathy (e.g., diabetic neuropathy), hyperosmolar hyperglycemic nonketotic syndrome (HHNS), kidney disease (nephropathy), and peripheral arterial disease (PAD). According to other specific embodiments, the disease is a wound.

[0211] As used herein, the term "wound" refers to a variety of injuries to the skin and subcutaneous tissues and multiple internal organs caused in any of a variety of ways (e.g., multiple pressure sores caused by prolonged bed rest, multiple wounds caused by trauma, multiple wounds sustained during or after a surgical procedure, etc.) and has a variety of distinct characteristics. Multiple exemplary examples include, but are not limited to, multiple contusions, multiple scratches, multiple burn wounds, multiple sunburn wounds, multiple incision wounds, multiple excision wounds, multiple surgical wounds, necrotizing fasciitis, multiple ulcers, multiple venous stasis ulcers, multiple diabetic ulcers, multiple decubitus ulcers, multiple aphthous ulcers, multiple pressure ulcers, multiple scars, alopecia areata, dermatitis, allergic contact dermatitis, atopic dermatitis, berloque dermatitis, diaper dermatitis, dyshidrotic dermatitis, psoriasis, eczema, erythema, multiple warts, and multiple anal warts. wart, angioma, cherry angioma, athlete's foot, multiple atypical moles, basal cell carcinoma, Bateman's purpura, bullous pemphigoid, candida, chondrodermatitis helicis, Clark's nevus, multiple cold sores, condylomata, multiple cysts, Darier's disease, dermatofibroma, discoid lupus erythematosus, nummular eczema, atopic eczema, dyshidrotic eczemaEczema, hand eczema, erythema nodosum multiforme, Fordyce's condition, folliculitis keloidis nuchae, folliculitis, granuloma annulare, Grover's disease, heat rash, herpes simplex, herpes zoster (shingles), hidradenitis suppurativa, urticaria, hyperhidrosis, ichthyosis, impetigo, keratosis pilaris, multiple scars, keratoacanthoma, lichen planus Lichen planus, lichen-like keratosis, chronic simplex chronicus, lichen sclerosus, lymphomatoid papulosis, lupus of the skin, Lyme disease, lichen striatus, multiple myxoid cysts, mycosis fungooides, molluscum contagiosum, multiple moles, nail fungus, necrobiosis lipoidica dibeticorum, nummular dermatitis, onychoschizia, onychomycosis, pityriasis lichenoides, pityriasis rosea Rosea, Pityriasis rubra Pilaris, Plantar Wart, Poison Ivy, Poison OakOak), Pompholyx, Pseudofolliculitis Barbae, Pruritus Ani, and Pityriasis Alba. Wounds are generally classified into one of four grades depending on the depth of the wound: (i) Grade I: wounds limited to the epithelium; (ii) Grade II: wounds extending into the dermis; (iii) Grade III: wounds extending into the subcutaneous tissue; and (iv) Grade IV (or full-thickness wounds): wounds with bone exposure (e.g., a bony pressure point such as the greater trochanter or the sacrum).

[0212] The term "partial-thickness wound" as used herein refers to wounds comprising Grades I-III; examples of partial-thickness wounds include burn wounds, pressure ulcers, venous stasis ulcers, and diabetic ulcers.

[0213] The term "deep wound" as used herein refers to wounds comprising Grade III and Grade IV wounds.

[0214] The term "chronic wound" as used herein refers to a wound that does not heal within thirty days.

[0215] The term "healing" with respect to a wound refers to the process of repairing a wound, e.g., by scar formation (in exemplary embodiments, healing is without fibrotic tissue formation).

[0216] In a particular embodiment, the compositions of some embodiments of the present application promote, i.e., accelerate, the healing process.

[0217] The phrase "inducing or accelerating a healing process of a skin wound" refers to inducing the formation of granulation tissue that shrinks the wound and / or inducing epithelialization (i.e., producing new cells in the epithelium). Wound healing is conveniently measured by a reduction in wound area.

[0218] Particular embodiments of the present application contemplate treating all wound types, including deep wounds, acute wounds, chronic wounds, diabetes-related wounds, ischemic wounds, ulcers, burns, and surgical wounds.

[0219] According to particular embodiments, the wound is selected from the group consisting of: a chronic wound, an acute wound, a diabetic wound, an ischemic wound, an ulcer, a burn, and a surgical wound.

[0220] Additional diseases and conditions that can be treated in accordance with various embodiments include, but are not limited to, nonketotic hyperosmolar coma; viral infections [e.g., varicella zoster virus (VZV); atherosclerosis; hypertension; various cardiovascular diseases such as congenital heart defects, cardiomyopathy, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), various valve diseases, myocardial infarction; tuberous sclerosis; scleroderma; transplantation; endometriosis; fertility; Von Hippel-Lindau (VHL) syndrome; cirrhosis; transplantation hemophilia; hypercoagulation; idiopathic thrombocytopenic purpura various immunodeficiency; retinitis pigmentosa, autosomal dominant retinitis pigmentosa, autosomal recessive; SEMD, Pakistani type; urofacial syndrome; cholesteryl ester storage disease;Corneal dystrophy, Thiel-Behnke type; Dubin-Johnson syndrome; Leukemia, T-cell acute lymphocytic; Spinocerebellar ataxia, infantile-onset, with sensory neuropathy; Split hand / foot malformation, type 3; Tolbutamide adverse metabolite; Warfarin sensitivity; Wolman disease; Anterior segment mesenchymal dysgenesis and cataract; Cataract, congenital; Neurofibrosarcoma; Retinal damage such as nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR); Chronic renal failure, diabetic nephropathy; Nerve damage such as diabetic neuropathy; Microvascular damage; Multiple diabetic-related foot ulcers and graft versus host disease.

[0221] The term "treatment" refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and / or causing regression or remission of a pathology. Those skilled in the art will understand that various methods and assays can be used to assess the development of a pathology, and similarly, various methods and assays can be used to assess the regression, remission or resolution of a pathology. It will be appreciated that the treatment can be carried out alone or in conjunction with other therapies.

[0222] As used herein, the term "prevention" refers to preventing a disease, disorder or condition from occurring in a subject who can be at risk of the disease but has not yet been diagnosed with the disease.

[0223] As used herein, the term "subject" refers to a mammalian subject (e.g., a human) of either sex and of any age, including neonates, infants, juveniles, adolescents, adults, and the elderly.

[0224] According to embodiments, the subject has an IDE level in a biological sample that is higher than a predetermined threshold compared to a control biological sample, as further described below.

[0225] According to embodiments, therefore, the methods disclosed herein comprise determining the IDE level in a biological sample of the subject using the antibodies disclosed herein prior to the administration.

[0226] The antibodies of some embodiments of the application can be administered to the subject per se or as part of a pharmaceutical composition.

[0227] According to an aspect of the application, there is provided a pharmaceutical composition comprising an antibody (ii) or (iii) disclosed herein as an active ingredient and a pharmaceutically acceptable carrier.

[0228] As used herein, a "pharmaceutical composition" refers to a preparation of one or more active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0229] Accordingly, the term "active ingredient" refers to the antibody responsible for the biological effect.

[0230] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier", which can be used interchangeably, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. Adjuvants are included under these phrases.

[0231] The term "excipient" herein refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0232] Techniques for formulation and administration of drugs can be found in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA latest edition, which is incorporated herein by reference.

[0233] A variety of suitable routes of administration can include, for example, oral, rectal, transmucosal, particularly transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injection.

[0234] Alternatively, the pharmaceutical composition can be administered in a localized rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

[0235] According to a specific embodiment of the application, the antibody is administered by intranasal administration.

[0236] According to a specific embodiment of the application, the antibody is administered by subcutaneous administration.

[0237] The pharmaceutical compositions of the present application can be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0238] The pharmaceutical compositions used in accordance with the present application can, thus, be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent on the chosen route of administration.

[0239] For injection, the active ingredients of the pharmaceutical composition can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0240] For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated in a variety of dosage forms, including tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be prepared using a solid excipient, optionally grinding the result ing mixture, and processing the mixture of granules, after adding suitable agen ts if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropyl methylcellulose, sodium carb o xymethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as a cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0241] Dragee cores are provided with suitable coatings. For this purpose, there can be used lacquer solutions of sugars, including arabic gum, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dragee cores are provided with suitable coatings. For this purpose, there can be used lacquer solutions of sugars, including arabic gum, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to the tablets or dragee coatings, which im prove their appearance.

[0242] Pharmaceutical compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active ingredients can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, liquid polyethylene glycols, or mixtures thereof. In addition, stabilizers can be added. Doses of the pharmaceutical compositions of all formulations should be suited to the chosen route of administration.

[0243] For buccal administration, the compositions can take the form of tablets or lozenges formulated in conventional manner.

[0244] For administration by nasal inhalation, the various active ingredients for use according to the present application are conveniently delivered in the form of an aerosol spray using a nebulizer or a bomb. In the case of a nebulizer, the dosage unit can be determined by providing a device for measuring a metered amount of the compound. Capsules and cartridges of, for example, gelatin for use in a dispenser can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0245] The pharmaceutical compositions described herein can be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, optionally with an added preservative. The compositions can be suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0246] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active agent in water-soluble form. Additionally, suspensions of the active ingredients can be prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for a highly concentrated solution.

[0247] Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

[0248] The pharmaceutical compositions of this application can also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.

[0249] Various pharmaceutical compositions suitable for use in the context of the present application include those wherein the various active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (i.e., antibodies) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., an IDE-related disease) or prolong the survival of the subject being treated.

[0250] According to one embodiment of the application, a therapeutically effective amount results in an increase in blood insulin levels in the subject after administration.

[0251] According to another embodiment of the application, a therapeutically effective amount results in a decrease in pancreatic beta cell destruction in the subject after administration.

[0252] According to another embodiment of the application, a therapeutically effective amount results in an increase in Insulin growth factor 1 (IGF1) in the subject after administration.

[0253] According to another embodiment of the application, a therapeutically effective amount results in a decrease in IL-17 secretion by T lymphocytes in the subject after administration.

[0254] According to another embodiment of the application, a therapeutically effective amount results in a decrease in IFN-g secretion by T lymphocytes in the subject after administration.

[0255] The levels of insulin, IGF1, IL-17 or IFN-g can be assessed using any method known to those skilled in the art, including for example by ELISA.

[0256] The destruction of pancreatic beta cells can be assessed using any method known to those skilled in the art, including for example by measuring beta cell function (e.g., by measuring the levels of various metabolic markers such as C-peptide) or by imaging beta cell mass [e.g., by emission tomography (PET) or by single-photon emission computed tomography (SPECT)], as taught by Lebastchi J. and Herold K.C., Cold Spring Harb Perspect Med. June 2012; 2(6): a007708, which is incorporated herein by reference.

[0257] According to another embodiment of the application, a therapeutically effective amount results in an inhibition or reduction of IDE activity. The reduction or inhibition of IDE activity can include a reduction of IDE activity by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The reduction of IDE activity can be assessed using any method known to those of skill in the art, including, for example, by fluorescent IDE activity assay or by in vitro insulin and IGF-1 degradation assays, as described in detail in the Examples section below.

[0258] The determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0259] For any preparation used in the methods of the application, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0260] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of doses for use in humans. The dosage can vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1).

[0261] Dosage and interval can be adjusted individually to provide plasma levels of the active ingredients that are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0262] Depending on the severity and responsiveness of the condition of the patient to be treated, dosing can be a single or multiple administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0263] Of course, the amount of a composition to be administered will depend on the subject being treated, the severity of the disease, the manner of administration, the judgment of the prescribing physician, etc.

[0264] For example, according to one embodiment, an antibody of the application can be administered intravenously (i.v.) at a dose of between 0.1 mg / kg to 10 mg / kg, 0.1 mg / kg to 5 mg / kg, 0.1 mg / kg to 1 mg / kg, 1 mg / kg to 10 mg / kg, 1 mg / kg to 5 mg / kg, 2.5 mg / kg to 5 mg / kg, 0.5 mg / kg to 5 mg / kg, or 5 mg / kg to 10 mg / kg. According to another embodiment, the antibody of the application can be administered intravenously at a dose of 10 mg / kg to 100 mg / kg, 10 mg / kg to 50 mg / kg, 25 mg / kg to 50 mg / kg, or 50 mg / kg to 100 mg / kg. According to yet another embodiment, the antibody of the application can be administered intravenously at a dose of 100 mg / kg to 1000 mg / kg, 100 mg / kg to 500 mg / kg, 250 mg / kg to 500 mg / kg, or 500 mg / kg to 1000 mg / kg.

[0265] It will be appreciated that there are animal models that can be used to test the various antibodies of the application prior to human treatment. For example, a STZ-induced or a non-obese diabetic (NOD) mouse diabetes model can be used as a diabetes model. Multiple sclerosis animal models include, for example, the murine EAE model (e.g., disease induction in NOD mice by immunization with MOG (35-55) in CFA). Alzheimer’s disease animal models include, for example, the APP / PSI mouse and the Samaritan Alzheimer’s rat model (available from Samaritan Pharmaceuticals). For wound healing, a diabetic mouse wound model can be used, as previously taught by Galeano et al., Diabetes. (2004) 53(9):2509-17, incorporated herein by reference, or a diabetic rat model can be used, as previously taught by Qiu et al., J Surg Res. (2007) 138(1):64-70, incorporated herein by reference. Parkinson’s disease animal models include, for example, the A53T Tg mice expressing human mutant alpha-synuclein in CNS neurons (Giasson et al., 2002) and the C57BL / 6-Tg(Thyl-SNCA*E35K*E46K*E61K)3798Nuber / J (Nuber et al., 2018).

[0266] If desired, the various compositions of the present application can be presented in a pack or dispenser device, such as an FDA-approved kit, which can contain one or more unit dosage forms containing the active ingredient. The pack can, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration. The pack or dispenser device can also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notices, for example, can be labeling approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert. As further detailed above, compositions comprising a preparation of the present application formulated in a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0267] It is to be understood that in addition to the antibodies described, the therapeutic compositions of the present application can comprise other known drugs or therapeutic agents for treating IDE- associated diseases (e.g., diabetes, autoimmune diseases of the CNS, neurodegenerative diseases), such as, but not limited to, steroids, corticosteroids, immunosuppressant drugs, antihistamines, and the like. These drugs can be included in a kit in a single or multiple separate packages.

[0268] According to an aspect of the present application, there is thus provided an article of manufacture identified for use in treating a disease associated with an IDE activity, the article of manufacture comprising an antibody (ii) or (iii) as disclosed herein and a therapeutic agent for treating the disease.

[0269] According to embodiments, the antibody and the therapeutic agent are in separate containers.

[0270] According to other embodiments, the antibody and the therapeutic agent are in a common formulation.

[0271] Further, according to embodiments, the methods and uses disclosed herein further comprise administering to the subject a therapeutic agent (in addition to the antibodies disclosed herein) for treating the disease.

[0272] As shown in the part of the examples below, the present inventors have developed a highly sensitive ELISA using the generated antibodies and used this ELISA to demonstrate a strong correlation between human serum IDE levels and the presence and / or severity of metabolic syndrome, and thus embodiments of the present application further propose analyzing the IDE levels for the purposes of diagnosis, monitoring treatment efficacy and / or determining treatment.

[0273] According to an aspect of the present application, there is thus provided a method of diagnosing a disease associated with an IDE activity in a subject, the method comprising determining an IDE level in a biological sample of the subject using the antibodies disclosed herein, wherein when the level of the IDE is higher than a predetermined threshold as compared to a control biological sample, the subject is diagnosed as having the disease.

[0274] As used herein, the term "diagnosing" refers to determining whether a pathology exists (i.e., cancer belonging to the Ewing tumor family), classifying a pathology or a symptom, determining a severity (e.g., grade or stage) of a pathology, monitoring progression of a pathology, predicting an outcome and / or prognosis of a pathology, and screening a subject for a particular disease.

[0275] According to an aspect of the application, there is provided a method of determining the prognosis of a disease associated with IDE activity in a subject, the method comprising determining the level of IDE in a biological sample of a subject diagnosed with the disease using the disclosed antibody, wherein when the level of IDE is higher than a predetermined threshold as compared to a control biological sample, the prognosis is poor.

[0276] According to embodiments, the method (e.g. the determining, the contacting) is performed in vitro or ex vivo.

[0277] According to embodiments, the method disclosed herein comprises obtaining the biological sample prior to the determining.

[0278] Non-limiting examples of biological samples that can be used in some embodiments of the application include a cell or cells, a tissue, an organ, a blood cell, a bone marrow cell, a body fluid, e.g. blood, serum, plasma, and lavage fluid, that can be in contact with the diseased cells / tissue, obtained from any tissue biopsy.

[0279] The biological sample can be obtained using methods known in the art, e.g. using a syringe with a needle, a scalpel, fine needle biopsy, needle biopsy, coarse needle biopsy, fine needle aspiration (FNA), surgical biopsy, buccal swab, lavage, etc.

[0280] According to embodiments, the protein molecules are extracted from the biological sample of the subject. Thus, according to embodiments, the method further comprises extracting a protein from the biological sample prior to the determining. Methods of extracting protein molecules from biological samples are well known in the art.

[0281] As used herein, the phrase "predetermined threshold" refers to an IDE level determined under the same conditions that characterizes a healthy sample or a sample with a known prognosis of a disease of the same origin. Such a level can be experimentally determined by comparing samples with known IDE levels (e.g. samples obtained from healthy subjects or from subjects with a known prognosis of a disease) to samples from subjects diagnosed with the disease associated with IDE activity. Alternatively, such a level can be obtained from the scientific literature and databases.

[0282] According to embodiments, the predetermined threshold is from a control sample.

[0283] Several control samples can be used with embodiments of the application.

[0284] Since multiple biological characteristics depend on factors such as species and age, the control sample is preferably obtained from a subject of the same species, age, gender and same sub-population (e.g. smokers / non-smokers).

[0285] According to embodiments, the control sample comprises a biological sample of the same type as the biological sample of the subject.

[0286] According to embodiments, the control sample is a healthy control sample.

[0287] According to embodiments, the control sample is a sample of a subject having a mild disease or a disease with a good prognosis.

[0288] According to embodiments, the control sample is taken from the scientific literature or a database.

[0289] According to embodiments, the increase / decrease above or below a predetermined threshold is statistically significant.

[0290] According to embodiments, the predetermined threshold is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold or at least 20-fold compared to the level of IDE in a control sample measured using the same assay, such as Western blot, ELISA, immunoprecipitation (IP).

[0291] According to embodiments, the predetermined threshold is at least 1.5-fold compared to the level of IDE in a control sample.

[0292] According to embodiments, the predetermined threshold is at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, e.g. 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600% compared to the level of IDE in a control sample.

[0293] According to embodiments, the level of IDE in the biological sample can be determined using any method known in the art using the antibodies disclosed herein, such as, but not limited to, ELISA, Western blot, IP.

[0294] According to some embodiments, the detection of the level of IDE is thus performed by contacting the biological sample, the tissue, the cell or fractions or extracts thereof with the antibodies disclosed herein.

[0295] According to several specific embodiments, the contact is performed under several conditions that allow the formation of a complex (i.e., an immune complex) containing an IDE present in the biological sample and the antibody.

[0296] The immune complexes can be formed at a variety of temperatures, salt concentrations, and pH values, which can vary depending on the method used and the biological sample, and those skilled in the art can adjust the various conditions to suit the formation of each immune complex.

[0297] Therefore, according to one aspect of the invention, a composition of a substance is provided comprising a biological sample (or a lysate of a biological sample of a subject diagnosed with a disease associated with IDE activity) and the antibody disclosed herein.

[0298] According to another or alternative aspect of the invention, an article is provided comprising a biological sample (or a lysate of a biological sample of a subject diagnosed with a disease associated with IDE activity) and the antibody disclosed herein in a separate container.

[0299] According to several specific embodiments, the composition or the article further comprises a protease inhibitor.

[0300] According to one specific embodiment, the composition or the article further comprises primary and secondary antibodies capable of binding the antibody.

[0301] According to several specific embodiments, the antibody disclosed herein binds to a detectable portion.

[0302] Examples of multiple detectable portions that can be used in this invention include, but are not limited to, multiple radioisotopes, multiple phosphorescent chemicals, multiple chemiluminescent chemicals, multiple fluorescent chemicals, multiple enzymes, multiple fluorescent peptides, and a radioisotope (e.g.,

[125] Iodine) and multiple epitope tags. The detectable portion may be a member of a binding pair, which can be identified by its interaction with another member of the binding pair, and is a directly visible tag. In one example, the tag is a fluorescent protein or an enzyme that produces a colorimetric reaction.

[0303] Examples of suitable fluorophores include, but are not limited to, phycoerythrin (PE), fluorescein isothiocyanate (FITC), Cy-chrome, rhodamine, green fluorescent protein (GFP), blue fluorescent protein (BFP), Texas red, PE-Cy5, and the like. For additional guidance on fluorophore selection, methods of attaching fluorophores to various types of molecules, see Richard P. Haugland, Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals 1992-1994, 5th ed., Molecular Probes, Inc. (1994); U.S. Patent No. 6,037,137 to Oncoimmunin Inc.; Hermanson, Bioconjugate Techniques, Academic Press New York, N.Y. (1995); Kay M. et al. 1995. Biochemistry 34:293; Stubbs et al. 1996. Biochemistry 35:937; Gakamsky D. et al. "Evaluating Receptor Stoichiometry by Fluorescence Resonance Energy Transfer," in "Receptors: A Practical Approach," 2nd ed., Stanford C. and Horton R. (eds.), Oxford University Press, UK. (2001); U.S. Patent No. 6,350,466 to Targesome, Inc.

[0304] Many types of enzymes can be attached to the antibody, such as horseradish peroxidase (HPR), beta-galactosidase, and alkaline phosphatase (AP).

[0305] Exemplary detectable moieties include, but are not limited to, green fluorescent protein, alkaline phosphatase, peroxidase, histidine tag, biotin, orange fluorescent protein, and streptavidin.

[0306] Further examples of detectable moieties include those detectable by Positron Emission Tomagraphy (PET) and Magnetic Resonance Imaging (MRI), all of which are well known to those skilled in the art.

[0307] According to some embodiments, the detectable moiety is conjugated by translationally fusing the polynucleotide encoding the antibody disclosed herein to the nucleic acid sequence encoding the detectable moiety.

[0308] Additionally or alternatively, the detectable moiety can be chemically conjugated (coupled) to the antibody disclosed herein using any conjugation method known to those skilled in the art.

[0309] According to specific embodiments, the methods disclosed herein comprise confirming the diagnosis using a state-of-the-art technology. Such methods are known in the art and depend on the type of the disease and include, as a non-limiting example, fasting blood glucose blood tests or A1c blood tests for diabetes.

[0310] According to specific embodiments, the method of diagnosis further comprises treating the diagnosed subject with an effective amount of a therapy for the disease.

[0311] According to an aspect of the application, there is thus provided a method of treating a disease associated with IDE activity in a subject in need thereof, the method comprising:

[0312] (a) diagnosing the subject according to the method; and wherein when the level of IDE is higher than the predetermined threshold,

[0313] (b) treating the subject with a therapy for the disease, thereby treating the disease in the subject.

[0314] Since the inventors show IDE as a prognostic marker for metabolic syndrome, the level of IDE can be used to select a treatment regimen (e.g. type, dosage) suitable for the subject. That is, a disease with poor prognosis is treated with a treatment regimen suitable for a poor prognosis; while a disease with good prognosis is treated with a treatment regimen suitable for a good prognosis. According to specific embodiments, the level of IDE can indicate the likelihood that the subject will respond to the given therapy.

[0315] Thus, according to a further or alternative aspect of the application, there is provided a method of treating a disease associated with an IDE activity in a subject in need thereof, the method comprising:

[0316] (a) diagnosing the subject according to the method; and wherein when the level of the IDE is higher than the predetermined threshold,

[0317] (b) selecting a therapy based on the level of the IDE, thereby treating the disease in the subject.

[0318] According to a further or alternative aspect of the application, there is provided a method of treating a disease associated with an IDE activity in a subject in need thereof, the method comprising:

[0319] (a) determining the prognosis of the subject according to the method; and

[0320] (b) treating the subject with a therapy according to the prognosis.

[0321] According to a further or alternative aspect of the application, there is provided a method of treating a disease associated with an IDE activity in a subject in need thereof, the method comprising:

[0322] (a) determining the prognosis of the subject according to the method; and

[0323] (b) selecting a therapy according to the prognosis.

[0324] According to embodiments, the therapy comprises an antibody (ii) or (iii) as disclosed herein.

[0325] According to a further or alternative aspect of the application, there is provided a method of monitoring the efficacy of a therapy for a disease associated with an IDE activity in a subject diagnosed with the disease, the method comprising determining an IDE level in a biological sample of the subject undergoing or after undergoing the therapy using the antibody as disclosed herein, wherein when the level of the IDE is reduced from a predetermined threshold after undergoing the therapy, the therapy is effective.

[0326] Thus, a reduction in the level of the IDE indicates that the therapy is effective.

[0327] On the other hand, if the level of the IDE does not change, or the level of the IDE increases, the therapy is not effective in treating the disease, and additional and / or alternative therapies (e.g., therapeutic regimens) can be used.

[0328] According to specific embodiments of the monitoring aspects disclosed herein, the predetermined threshold is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, or at least 20-fold compared to the level in the subject prior to the therapy.

[0329] According to specific embodiments of the monitoring aspects disclosed herein, the predetermined threshold is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, or at least 20-fold compared to the level in the subject prior to the therapy.

[0330] According to a specific embodiment, the predetermined threshold is at least 1.5-fold compared to the level in a control sample prior to the therapy or in the subject.

[0331] According to specific embodiments, the predetermined threshold is at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, e.g., 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600% compared to the level in a control sample prior to the therapy or in the subject.

[0332] According to other specific embodiments of this aspect of the application, the predetermined threshold can be determined in a subset of subjects having a known outcome of the therapy.

[0333] According to yet another aspect of the application, there is provided a preparation comprising the antibody disclosed herein and a reagent suitable for ELISA, Western blot or IP, a ELISA plate and / or a positive control sample comprising IDE in a separate container. As used herein, the term "about" refers to ± 10%.

[0334] The terms "comprise", "comprising", "include", "including", "having" and their conjugates mean "including but not limited to".

[0335] The term "consisting of means "including and limited by".

[0336] The term "essentially consisting of' means that the composition, method or structure can include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristic(s) of the claimed composition, method or structure.

[0337] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include more than one compound, including mixtures thereof.

[0338] Throughout this application, various embodiments of the application can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and is to be interpreted -in the context of the specification as a whole. Therefore, it should be understood that any reference to a range includes all sub-ranges included in the range, all individual numerical values included in the range, and all sub-ranges and individual numerical values are specifically included in the range. For example, a range of "1 to 6" should be interpreted to include sub-ranges such as "1 to 3", "1 to 4", "1 to 5", "2 to 4", "2 to 6", "3 to 6", etc., as well as individual numbers within the range, such as 1, 2, 3, 4, 5, and 6, This applies equally to ranges with endpoints involving a descriptive term or term. For example, a description of "from 1 to 6 or 8" should be interpreted to include from 1 to 8, 1 to 6, 1 to 8, 2 to 6, 2 to 8, 3 to 8, etc., as well as the individual numbers 1, 2, 3, 4, 5, 6, 7, and 8, and this applies equally to ranges of endpoints involving descriptive terms or terms.

[0339] Whenever a numerical range is indicated herein, it is meant to include any cited endpoint within the specified range. The phrases "range between" and "range from" are interchangeable and mean including the cited first and second endpoints and all the fractions and integers therebetween.

[0340] As used herein, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacologic, biological, biochemical and medical arts.

[0341] It is to be understood that many of the features described herein in the context of separate embodiments also can be provided in combination, or in any other suitable sub-combination, or in any other described embodiment of the application. It is also to be understood that features described herein in the context of separate embodiments can also be provided in the combination of those embodiments, unless the description clearly dictates otherwise.

[0342] The various embodiments and aspects of the application described above and claimed in the claims section below are found to have experimental support in the following examples.

[0343] Examples

[0344] Reference is now made to the following examples, which together with the above description, illustrate the application in a non-limiting fashion.

[0345] Generally, the nomenclature used herein and the laboratory procedures utilized in the present application include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: a laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); "Current Protocols in Molecular Biology", John Wiley & Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook" Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); the series "Methods in Enzymology" Simon and Grossman, eds.(1994); Stites et al. (eds), Basic and Clinical Immunology (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, W. H. Freeman and Co., New York (1980); see also, e.g., U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771; and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-405, Academic Press; "Biochemistry" VoI I & II, Berg T. ed., Mill Valley, CA (1983).1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Additional general references are provided throughout this document. The numerous programs referred to herein are believed to be well understood by those skilled in the art and are provided for ease of understanding the present disclosure. They are incorporated herein by reference in their entirety.

[0346] Materials and methods

[0347] Mice - Groups of C57BL / 6 male mice (Jackson Laboratory) were housed in a specific pathogen-free facility at Tel Aviv University. All experiments were in accordance with Tel Aviv University guidelines and approved by the TAU Animal Care Committee for animal research.

[0348] Generation and expression of IDE vectors - Sequences of wild type (WT) and E111Q recombinant human IDE (rhIDE) optimized for bacterial expression were ordered from Genewiz (South Plainfield, NJ, USA) and received in a pUC57 vector between Ndel and Hindlll restriction sites. The vector encoded rhIDE fused at its C-terminus to the Ni-NTA binding tag His tag. Groups of Rosetta BL21 E. coli cells were transformed with pET28a-rhIDE expression vectors (WT and E111Q) and grown in 0.5 liter LB medium supplemented with 25 pg / ml Kanamycin to OD600= 0.6 at 37°C. Expression of rhIDE was induced by addition of 1 mM IPTG and incubation for 4 hours at 37°C. Cells were harvested by centrifugation at 4000 rpm for 20 minutes at 4°C, and the pellets were stored at -80°C until use. 600nm(600 nm absorbance) = 0.6-0.8. Protein expression was then induced by overnight incubation with 0.5 mM IPTG at 30°C. The cells were harvested by centrifugation at 9000 rpm for 15 min, the pellet was resuspended in 35 ml PBS containing 0.1% Triton X-100 and lysed by 5 repeated cycles of 30 sec sonication and 2 min on ice. The soluble fraction was clarified by centrifugation at 12000 rpm for 30 min at 4°C and then loaded onto a GE HisTrap 5 ml column. The column was washed with PBS containing 5 mM imidazole and eluted with PBS containing 0.5 M imidazole. The purified IDE protein was dialyzed against PBS, diluted to 2 mg / ml in PBS and 5% glycerol and stored at -80°C.

[0349] Bio-panning - Phage display technology was utilized using the "Ronit 1" human synthetic antibody phage display library as previously described in Azriel-Rosenfeld et al. J Mol Biol. (2004) 335(1): 177-192. The library was subjected to four rounds of affinity selection on 10 μg / ml WT rhIDE, which was used to coat the wells of 24-well plates for 2 hours at room temperature, with WT IDE serving as a decoy. In two of the rounds (round 1 and round 3), the mutant E111Q IDE was used as a decoy for depletion, with the aim of isolating antibodies that bind the catalytic site of the WT enzyme with high affinity. The mutant IDE was used at 10 μg / ml to coat the wells of 24-well plates for 2 hours at room temperature. The wells were washed once with 3 ml PBS and blocked with PBS containing sterile 3% non-fat milk for 1 hour at 37°C. The wells were washed once with 3 ml PBS and the phage was applied to the blocked, mutant IDE-coated wells for 1 hour at 25°C. Subsequently, the wells coated with WT rhIDE and similarly blocked with PBS containing 3% non-fat milk were washed once with 3 ml PBS and the phage from the mutant IDE wells (depleted phage) was transferred to the WT rhIDE wells for an additional hour at 25°C. At the end of each round of affinity selection, the bound phage was eluted with 100 mM TEA pH=13 and immediately neutralized with 1 M Tris-HCl pH=7.4. The eluted phage was used to infect XL1-blue E. coli and grown to log phase for clonal amplification. Phage titers for selection input and output allowed determination of the number of copies of each phage clone. 20-100 copies of each phage clone were then tested for binding to WT rhIDE and mutant E111Q IDE by infecting E. coli with the phage and growing to log phase. The cells were harvested by centrifugation at 9000 rpm for 15 min, resuspended in 100 μl PBS and incubated with 100 μl of 10 μg / ml rhIDE or mutant E111Q IDE for 2 hours at room temperature. The cells were then washed once with 3 ml PBS and the bound phage was eluted with 100 mM TEA pH=13 and immediately neutralized with 1 M Tris-HCl pH=7.4. The eluted phage was used to infect fresh E. coli and grown to log phase for clonal amplification.10 CFU / ml M13KO7 helper phage were rescued overnight at 37°C at 250 RPM. Virions from the bacterial growth supernatant were pelleted in 20% PEG / NaCl and resuspended in PBS before being used for the next round of affinity selection (panning). After four rounds of affinity selection, IDE-specific scFv-displaying phage were identified by monoclonal phage ELISA. In the phage ELISA, HRP anti-m13 monoclonal antibody conjugate: GE Healthcare, CAT 27-9421-01 was used to detect bound phage. Adhesins were validated for specificity by testing their binding to several control antigens, all purchased from SIGMA (now Merck) [BSA Merck CAT A7030-500G; Lysozyme (lysozyme from chicken egg white), Merck CAT L6876; Streptavidin (streptavidin from biotin protein Streptomyces avidinii) Merck CAT S0677]. The validated adhesins were reformatted to produce soluble antibodies and tested in three formats: MBP-scFv( FIG. 1E ), as "clonal" produced human IgGl ( FIG. 1F ), and reverse chimeric IgG produced in mammalian cells ( FIG. 1G ).

[0350] Sequencing - The nucleotide sequences of the cloned genes were determined using the ABI 3500xl Genetic Analyzer (Applied Biosystems, USA) according to the supplier's recommendations. The DNA sequences of the DNA fragments of the clones were analyzed using the program ApE - A Plasmid Editor v2.0.47 (Wayne Davis, University of Utah). Antibody variable domain sequences were analyzed and assigned to framework and CDR regions using the IgBlast tool of NCBI (www(dot)ncbi(dot)nlm(dot)nih(dot)gov / igblast).

[0351] Expression and purification of IDE-specific "clonal" IgG - Expression and refolding of full-length "clonal" IgG was achieved using a protocol previously described in [Hakim, R. and Benhar, I. (2009) MAbs 1, 281-287; Buchner, J. et al. (1992) Anal. Biochem. 205, 263-270; and Benhar, I. and Pastan, I. (1994) Protein Eng. Des. Sel. 7, 1509-1515].

[0352] Dot-blot - Dot-blot was performed as previously described [Mazor et al. (2007) J. Immunol. Methods 321, 41-59]. Briefly, using a Slot blot PR648 filter manifold, protein samples containing 0.1-1 μg of purified protein in a total volume of 50 μl were applied to nitrocellulose membranes (Hoefer Scientific Instruments, San Francisco, CA, USA) in native form or after boiling for 5 min at 95°C (denaturing conditions) by a vacuum manifold. The membranes were then blocked with TBS containing 5% BSA for 1 h at room temperature and incubated overnight with 3 μg / ml MBP (maltose binding protein) fused scFv (prepared as described below) diluted in the blocking solution at 4°C. After 3 washes with TBST, the samples were incubated with mouse anti-MBP (1:5,000 in TBST) for 1 h at room temperature, washed and incubated with HRP-conjugated goat anti-mouse antibody (Jackson Immunoresearch Labs 115-035-003; 1:5,000 in TBST) for 1 h at room temperature. After additional 3 washes with TBST, the membranes were developed using an ECL reagent (WBUR0500, Millipore, Milford, MA, USA) according to the manufacturer's instructions, imaged using an Amersham imager 600 (GE Healthcare Biosciences; Pittsburgh, PA) and analyzed using the ImageJ v1.5i.

[0353] Production of MBP-scFv specific for IDE - A expression vector was designed for cytoplasmic expression of MBP-scFv IDE specific fusion proteins in E. coli. The sequences of all scFv were taken from the corresponding pCC16 phagemid vectors that were identified as specific IDE binders at the end of the phage display affinity selection process. The pMALc-NHNN vector [Birnboim-Perach, R. et al. (2019) Production of Stabilized Antibody Fragments in the E. coli Bacterial Cytoplasm and in Transiently Transfected Mammalian Cells. pp. 455-480, Humana Press, New York, NY]. After digestion with Ncol and Notl restriction enzymes, each scFv coding sequence was recovered from the pCC16 phagemid and the sequences were ligated into the pMALc-NHNN vector between the Ncol and Notl restriction sites. The vector encoded IDE specific scFv fused at their N-terminus to the Ni-NTA binding tag His tag and the maltose binding protein (MBP).

[0354] For MBP-scFv production, Rosetta BL21 E. coli cells were transformed with the pMALc-NHNN-MBP-IDE specific scFv expression vector and grown in 0.5 liter LB medium supplemented with 100 pg / ml Ampicillin to OD600nm=0.8. Protein expression was then induced with 0.5 mM IPTG at 30°C overnight. The cells were collected by centrifugation at 9000 rpm for 15 minutes. The pellet was resuspended in 35 ml PBS containing 0.1% Triton X-100 and lysed by 5 repeated cycles of 30 seconds sonication and 2 minutes rest on ice. The soluble fraction was clarified by centrifugation at 12000 RPM for 30 minutes at 4°C and then loaded onto a GE hisTrap 5 ml column. The column was washed with 5 CV of PBS containing 5 mM imidazole, 5 CV of PBS containing 20 mM imidazole and eluted from the column with PBS containing 250 mM imidazole. The purified MBP-scFv fusion proteins were dialyzed against PBS, diluted to 2 mg / ml in PBS and stored at -80°C.

[0355] Multiple splenocyte preparation - multiple spleens harvested from multiple C57BL / 6 mice were homogenized in RIPA lysis buffer x 1 supplemented with 0.1 mM DTT, 0.1 μΜ sodium vanadate, 0.5 mM PMSF and proteinase inhibitor cocktail (PIC) 1 : 100 on ice and incubated for 20 minutes at 25°C. The supernatant was collected by centrifugation at 12000 RPM for 10 minutes at 4°C and stored in multiple 100 μΐ aliquots at -80°C.

[0356] Production of reverse chimeric anti-mouse IDE antibodies - to convert the antibodies into full-size reverse chimeric IgG, polynucleotides encoding the generated antibodies were cloned into pcDNA 3.4 plasmid backbone. These plasmids are pcDNA3.4 vectors controlled by the CMV promoter, which were used as the Expi293 TM The "antibody positive control vector" section of the kit is provided for transient transfection-based expression. The kit also provides the Expi293F TMcells (ThermoFisher, #A14635). A detailed description of cloning multiple antibody variable domains into multiple IgG expression vectors was previously described [Birnboim-Perach, R, et al. (2019) Production of Stabilized Antibody Fragments in the E. coli Bacterial Cytoplasm and in Transiently Transfected Mammalian Cells. pp. 455-480, Humana Press, New York, NY]. Briefly, the vectors were expressed in E. coli Rosetta BL21 cells and purified using a HisTrap column (17-5248-01 GE Healthcare, Uppsala, Sweden). The antibodies were initially produced as fully human IgG (with human gamma 1 heavy chain and human kappa or lambda light chain constant domains), and later they were expressed with mouse gamma 1 heavy chain and mouse kappa light chain mouse constant domains. The heavy and light chain variable domains of the antibodies were amplified by PCR for cloning and then cloned by Gibson assembly into pcDNA 3.4 plasmids already carrying the corresponding constant domains [Gibson, D.G., et al. (2009) Nat. Methods 6, 343-345]. ExpiFectamine TM transfection kit (Gibco, #A14524) was used to transfect Expi293F cells (Life Technologies, USA) with the different pcDNA3.4 vectors. A total amount of 30 pg plasmid DNA for each transfection consisted of the IgL and IgH in a 3:1 molar ratio, respectively. Transfection, cell growth and harvest of the conditioned media were performed according to the supplier's recommendations (Life Technologies Expi293 TM cells. For each transfection, a total amount of 30 pg plasmid DNA consisted of the IgL and IgH in a 3:1 molar ratio, respectively. Transfection, cell growth and harvest of the conditioned media were performed according to the supplier's recommendations (Life Technologies Expi293 TMSuitable for transient transfection-based expression). Conditioned media containing the antibodies were harvested 6 to 7 days post-transfection by centrifugation at 8000 rpm for 10 min at 4°C (Sorvall GSA rotor). The reverse chimeric mouse IgGl mAbs were purified on protein G columns according to the manufacturer's recommendations (GE Healthcare, Chicago, IL, USA). Prior to purification, the antibody-containing media were buffered with 20 mM phosphate buffer, pH 7, and filtered. The mAbs were eluted in 1-ml fractions, which were neutralized with 0.25 ml 1.5 M Tris-HCl pH 8.8. Buffer exchange to sterile DPBS (Sigma) was performed on 10-kDa Ultra centrifugal filters (MilliporeSigma, Burlington, MA, USA; FIG. 1G ) or PD-10 desalting columns (GE Healthcare, Chicago, IL, USA).

[0357] The antibodies were collected by centrifugation and concentrated to a final concentration of 1-2 mg / ml using a centrifugal filter concentrator. Purified antibodies were stored at -80°C in small aliquots. Protein concentration was determined by measuring the absorbance of the protein at O.D. 280 nm in a Thermo Scientific NanoDrop 2000c spectrophotometer and dividing the absorbance value by the extinction coefficient factor of the protein (extinction coefficient was calculated through www(dot)expasy(dot)org / tools / protparam(dot)html). TM 2000c spectrophotometer and dividing the absorbance value by the extinction coefficient factor of the protein (extinction coefficient was calculated through www(dot)expasy(dot)org / tools / protparam(dot)html).

[0358] IDE binding assay - Multiple 96-well ELISA plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with PBS containing 2.5-5 μg / ml of WT rhIDE, mutant IDE, and multiple non-related proteins: bovine serum albumin (BSA), His trap purified proteins (His), and / or MBP-LacZ (in-house recombinantly produced). After 3 washes with PBST, the wells were blocked for 1 hour at room temperature with PBS containing 3% w / v non-fat milk (232100, Difco, Sparks, MD, USA). Subsequently, the wells were incubated with different formats of antibodies specific for the IDE - as phage clones - with serial dilutions, as MBP-scFv (data not shown), and as hlgG or rcIgG at different concentrations, for 2 hours at room temperature. Bound phage was detected with mouse anti-M13 antibody (1 hour at room temperature) followed by a 1 hour incubation at room temperature with HRP conjugated goat anti-mouse secondary antibody (115-035-003, Jackson ImmunoResearch Laboratories, ME; 1 :5,000 in PBS). Human IDE specific IgG antibodies were incubated with HRP conjugated goat anti-human (109-035-088, Jackson ImmunoResearch Laboratories, ME; 1 :5,000 in PBS) or anti-mouse (115-035-062, Jackson ImmunoResearch Laboratories, ME; 1 :5,000 in PBS) for 2 hours at room temperature. After 3 washes with PBST, 3,3',5,5'-tetramethylbenzidine (TMB; eBioscience, San Diego, CA, USA) was added until color developed. The reaction was stopped by the addition of 1 M H2SO4and analyzed at 450 nm using a Spectrafluor plus microplate reader.

[0359] IDE insulin digestion assay - Various concentrations of IDE inhibitors were incubated with 1.5 μg / ml rhIDE for 1 hour at room temperature. Subsequently, recombinant human insulin (41-975-100, Biological Industries, Kibbutz Beit Haemek, Israel) diluted in Mercodia Ultra Sensitive Mouse Insulin ELISA (10-1249-01) calibrator 0 was added to the tubes and incubated for 2 hours at 37°C. 25 μl was removed from each tube and residual insulin concentration was assessed using the Mercodia Ultra Sensitive Mouse Insulin ELISA kit. Absorbance at 450 nm was recorded with a Spectrafluor plus microplate reader (Tecan, Mannedorf, Switzerland).

[0360] Streptozotocin (STZ) diabetic mouse model - 10-week-old C57BL / 6 mice were fasted overnight and then injected intraperitoneally with 150 mg / kg STZ diluted in 100 nM citrate buffer, pH 4.5. All mice developed hyperglycemia within two days of STZ injection. On the third day after STZ injection, each mouse was injected intraperitoneally with 10 mg / kg of the generated inverted chimeric mAbs (rcH3 or the rc2E12 isotype control).

[0361] Oral glucose tolerance test (oGTT) - Mice were fasted for 2 hours, after which they received a single intraperitoneal injection of the generated inverted chimeric mAbs (10 mg / kg), and then fasted for an additional 4 hours (total of 6 hours). At the end of this period, mice were given an oral dose of glucose at 2 g / kg using a gavage needle. Blood glucose was measured using the Contour blood glucose meter (Bayer, Elkhart, IN, USA) before glucose administration (time 0) and 15, 30, 60, 90 and 120 minutes after glucose administration.

[0362] Insulin tolerance test (ITT) - Mice were fasted for 2 hours, after which they received a single intraperitoneal injection of the generated inverted chimeric mAbs (10 mg / kg), and then fasted for an additional 4 hours (total of 6 hours). At the end of this period, mice were injected intraperitoneally with insulin (0.5 U / Kg, dissolved in PBS). Blood glucose was measured using the Contour blood glucose meter (Bayer, Elkhart, IN, USA) before glucose administration (time 0) and 15, 30, 60 and 90 minutes after glucose administration.

[0363] Serum collection - After the ITT and injection of the various reverse chimeric mAbs, mice were bled every 3 days from the facial vein using a 27G needle. Blood samples (no more than 120 μΐ per time) were left at room temperature for 1 hour in 1.5 ml Eppendorf tubes. The clots were removed from the tubes using a needle and the tubes were placed on ice for 40 minutes. Then, the tubes were centrifuged at 4°C at 2300 RPM for 10 minutes; the clear supernatant was then transferred to a new tube for a second cleaning cycle. The supernatant was moved to a new tube and stored at -20°C.

[0364] Detection of anti-IDE reverse chimeric antibodies in serum samples - 96-well ELISA plates were coated overnight at 4°C with PBS containing 5 μg / ml of IDE. After 3 washes in PBST, the wells were blocked with PBS containing 5% (w / v) non-fat milk for 1 hour at room temperature. To detect the reverse chimeric IDE-specific antibodies (rcH3-IgG), serum samples were diluted 1 :2700 in PBS, while samples of control mice injected with rc2E12 antibody were used as a positive control. Known concentrations of the rcH3-IgG antibody diluted in a sample of a control mouse at 1 :2700 were used as a reference for the measurement of IDE concentrations. The samples were incubated for 2 hours at room temperature. After 3 washes, the wells were incubated with 50 μΐ of HRP-conjugated goat anti-mouse antibody (1 :5,000 in PBS) for 1 hour at room temperature. After 3 washes with PBST, TMB was added until color appeared. The reaction was stopped by the addition of 1 M H2SO4 and analyzed at 450 nm using a Spectrafluor plus microplate reader.

[0365] Production of reverse chimeric anti-IDE H3 Fab2 fragments - 10 mg of IgG were digested with 200 μg of IDES (Fabricator) enzyme (www(dot)genovis(dot)com / products / igg-proteases / fabricator / ) for 20 hours at 37°C. The Fab2 fragments were separated from undigested IgG and the Fc fragments on a MabSelect SuRe™ TM affinity column. The Fab2 were stored at -80°C until use.

[0366] Determination of ROS levels in small glial cells - To measure the levels of ROS in cells, N9 small glial cells were seeded at a concentration of 0.5 x 10 5 cells / ml in RPMI medium containing 10% FCS in a clear-bottom black-walled 24-well plate (#4TI-0241, Nunc) and incubated at 37°C in a 5% CO2 atmosphere. Leiden, The Netherlands). After 24 h, the medium was changed and the cells were incubated for 24 h with serum-free RPMI medium with or without 0.1 pg / ml LPS. To detect ROS in DJ-1-KD cells, DJ-1-KD oligodendrocytes were cultured as described previously (Nash et al. 2017). Control and DJ-1-KD oligodendrocytes were seeded at a concentration of 0.8 x 10 5 Control and DJ-1-KD oligodendrocytes were seeded at a concentration of 0.8 x 10

[0367] Multiple subjects - A total of 24 healthy volunteers and 51 Metabolic Syndrome (MS) patients (Table 1 below) from a previously described cohort (Marcus Y et al. J Clin Hypertens (Greenwich). 2016; 18(1): 19-24) were co-enrolled. To qualify for inclusion in a MS patient, subjects aged 18 to 75 must have met the criteria in the Third Report of the Adult Treatment Panel (ATP III) (Circulation. 2002; 106(25): 3143-421). Impaired fasting glucose was considered a glucose level > 100 mg%. None of the subjects were on anti-diabetic medication. The study was approved by Helsinki's committee of the Tel Aviv-Sourasky Medical Center institution. Consent was obtained from each patient after full explanation of the purpose and nature of all procedures used.

[0368] Biochemical analysis of human serum samples - Serum chemistry was measured by routine commercial automated assays (Centaur, Roche, Indianapolis, IN). HbAlc levels were measured using HPLC (Tosoh Bioscience, San Francisco, CA).

[0369] Generation of polyclonal anti-IDE antibodies -

[0370] Animal immunization: NZW female rabbits, 8 weeks old, weighing 2.5 kg were purchased from Harlan (Israel). Polyclonal antisera were raised in each animal by subcutaneous immunization with 100 μg rhIDE emulsified in a 1:1 ratio with 400 μg M. tuberculosis extract H37 in incomplete Freund's adjuvant (CFA) using a 100 μl emulsion. Each animal received booster doses of rhIDE emulsified in the same manner in incomplete Freund's adjuvant (IFA) without the M. tuberculosis extract every week. Rabbit arterial blood was collected before the first vaccination and one week after each boost and as a final (terminal) bleed. A total of three booster immunizations were required to reach a plateau of serum antibody titers (>20,000).

[0371] Analysis of anti-IDE serum polyclonal antibody titers: Blood collected from each animal was incubated for 1 hour at room temperature and for another hour at 4°C to coagulate. Serum supernatant was separated from the coagulated blood by centrifugation at 3000 g for 20 minutes at 4°C. Anti-IDE antibody titers were measured by direct ELISA as follows: a 96-well ELISA plate (Nunc, Roskilde, Denmark) was coated overnight at 4°C with 50 μl / well of rhIDE diluted in PBS to a final concentration of 2.5 μg / ml. The next day, the plate was washed once with 300 μl / well of PBS containing 0.05% Tween 20 (PBST) and blocked with 300 μl / well of PBS containing 3% skimmed milk (w / v; Difco TM skimmed milk, BD) for 1 hour at 37°C. Next, the plate was washed 3 times with 300 μl / well of PBST and serially diluted x3 in PBST, and the serum samples were applied and allowed to bind for 1 hour at room temperature. Subsequently, the plate was washed 3 times with 300 μl / well of PBST. Bound anti-IDE polyclonal antibodies were detected by using HRP-conjugated goat anti-rabbit IgG (#111-035-004, Jackson Immunoresearch Laboratories) diluted 2000-fold in PBS and incubating the plate for 1 hour at room temperature, followed by 3 washes with 300 μl / well of PBST. Finally, 50 μl / well of the HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB; eBioscience, San Diego, CA, USA) was added until color developed. The reaction was stopped by adding 50 μl / well of 1 M H2SO4and analyzed using a Spectrafluor plus microplate reader (Tecan, Maennedorf, Switzerland). The optical density was measured at 450 and 570 nm wavelengths and analyzed using the Magellan software version 2.22 (Tecan). After reaching adequate titer levels, terminal bleeds were performed on multiple animals and serum was isolated by letting the blood clot for 1 hour at 4°C and then centrifuging at 14000 g in an Eppendorf microfuge at 4°C. The serum was divided into 100 μl aliquots and stored at -80°C.

[0372] Determination of IDE Serum Concentration by ELISA – To measure IDE in various body fluids, a highly sensitive sandwich ELISA suitable for quantifying IDE in human and mouse serum was developed. Multiple 96-well ELISA discs (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 50 μl / well of anti-IDE antibody A9, which was diluted in PBS to a final concentration of 0.5 μg / ml. The next day, the discs were washed once with 300 μl / well of PBST and then with 300 μl / well of Difco solution containing 3% skim milk (w / v). TM Skim milk (BD) was blocked in PBS at 37°C for 1 hour. Next, the dish was washed three times with 300 μl / well PBST; and multiple serum samples were diluted ×3 or ×9 in PBS and allowed to bind overnight at 37°C. A concentration profile was prepared by incorporating a control serum (IDE concentration undetectable) with sequential ×2 dilutions of rhIDE in PBS, starting at 500 ng / ml. FIG. 7A The disks were incubated at room temperature (approximately 25°C) for 2 hours, then washed three times with 300 μl / well of PBST. IDE binding was detected by diluting 300 μl / well of rabbit polyclonal anti-IDE serum 2000-fold in PBS and incubating at room temperature for 1 hour, followed by washing three times with 300 μl / well of PBST. Next, 50 μl / well of HRP-conjugated goat anti-rabbit IgG (#111-035-004, Jackson Immunoresearch Laboratories) diluted 2000-fold in PBS was applied, incubated at room temperature for 1 hour, and then washed three times with 300 μl / well of PBST. Finally, 50 μl / well of the HRP matrix TMB was added until color appeared. The reaction was terminated by adding 50 μl / well of 1M H2SO4, and analysis was performed using a Spectrafluorplus microdisk reader (Tecan, Mennedorf, Switzerland). The optical density was measured at wavelengths of 450 and 570 nm and analyzed using Magellan software version 2.22 (Tecan).

[0373] Statistical analysis - GraphPad software (GraphPad Prism v8) was used for all statistical analyses described in Examples 1-3 below. Multiple data comparisons were performed using an unpaired two-tailed Student’s t test when comparing two groups or a one-way ANOVA (with Bonferroni’s post-test) when analyzing three or more groups. Each experiment was repeated at least 3 times. P < 0.05 was considered significant. For Example 4 below, the collected data were analyzed with IBM SPSS version 25.0. The normal distribution of the data was verified by comparing the mean with the median, the mean with the 5% trimmed mean, the values of skewness and kurtosis, and the results of Shapiro-Wilk’s test. The results showed that some of the variables in the MS group did not obey a normal distribution. Differences between the two groups (MS patients and controls) were analyzed by independent samples t-test and Mann-Whitney test for continuous variables and by Chi-square test for categorical variables. Since the results of the t-test and Mann-Whitney test were the same, the results of the t-test are the ones reported. Descriptive statistics are given in mean and standard deviation or frequency and percentage, according to the scale of the variable. Since the difference in age was significant, this factor was controlled and differences were tested by General Linear Models (GLM). A p-value < 0.05 was considered statistically significant. All reported P values are two-tailed.

[0374] Example 1

[0375] Generation of anti-IDE antibodies and evaluation of their in vitro inhibitory effect on IDE activity

[0376] Recombinant human IDE (rhIDE) was purified for use as the antigen for screening and immunization. To this end, the wild type (WT) and the E111Q mutant form of the enzyme were expressed, in which a point mutation in the catalytic site significantly reduces the catalytic activity of the enzyme (35). The genes encoding the two forms of IDE were cloned into a pET28a+ plasmid backbone with a His tag at the C-terminus. FIG. 1A The vector expressing recombinant human IDE (rhIDE) with a His tag at the C-terminus is shown in Figure 1.

[0377] Upon induction of the expression of the vector, a significant increase in the expression of a 110 kDa protein was seen by SDS-PAGE FIG. 1B ), and the identity of the protein was verified by an immunoblot assay using IDE-specific antibodies FIG. 1C ). In the next step, an insulin degradation assay was performed using the purified proteins, and the results showed that the WT IDE protein effectively degraded insulin (about 60% degradation after 2 hours at 37°C). The mutant IDE, under the same conditions, degraded only 15% of the insulin FIG. 1D

[0378] The purified IDE was used to isolate specific scFv clones that recognize epitopes of the recombinant human IDE. Phage display technology was used with a human synthetic antibody phage display library. Specifically, a human scFv library, the Ronit 1 library [described in Azriel-Rosenfeld et al. J Mol Biol. (2004) 335(1): 177-192], was screened. The library was subjected to four rounds of affinity selection on recombinant human wild type (wt) IDE (rhIDE). In two of the rounds, E111Q IDE was depleted, in order to isolate antibodies that bind the catalytic site of the WT enzyme with high affinity. After four rounds of affinity selection, three IDE-specific scFv-displaying phage were identified by monoclonal phage ELISA, herein referred to as A9, B1 and H3. As shown in FIGS. 2A-2F , serial dilutions of phage of A9, B1 and H3 FIGS. 2A-2C ) bound IDE well, and had little to no binding to BSA, MBP or other purified recombinant proteins used as negative controls.

[0379] ​The nucleic acid and amino acid sequences of the heavy and light chains and the CDRs of the three anti-human IDE antibodies were determined. These are set forth in SEQ ID NO: 1 to SEQ ID NO: 2 and SEQ ID NO: 9 to SEQ ID NO: 10 (for A9 heavy and light chains, respectively), SEQ ID NO: 3 to SEQ ID NO: 8 (for A9 heavy chain CDRs), SEQ ID NO: 11 to SEQ ID NO: 16 (for A9 light chain CDRs), SEQ ID NO: 17 to SEQ ID NO: 18 and SEQ ID NO: 25 to SEQ ID NO: 26 (for B1 heavy and light chains, respectively), SEQ ID NO: 19 to SEQ ID NO: 24 (for B1 heavy chain CDRs), SEQ ID NO: 27 to SEQ ID NO: 32 (for B1 light chain CDRs), SEQ ID NO: 33 to SEQ ID NO: 34 and SEQ ID NO: 41 to SEQ ID NO: 42 (for H3 heavy and light chains, respectively), SEQ ID NO: 35 to SEQ ID NO: 40 (for H3 heavy chain CDRs), and SEQ ID NO: 43 to SEQ ID NO: 48 (for H3 light chain CDRs).

[0380] For initial evaluation of full-size IgGs, the antibodies were produced in "clonal" form, i.e., IgGs expressed in an E. coli expression system (29) FIG. 1F The isolated clonal antibodies showed significant binding to IDE, with an EC 50 of about 1 nM FIGS. 2D-2F The clonal antibodies showed little to no binding to BSA, with only a slight increase at saturating concentrations. In summary, these results show that the three "clonal" antibodies bind to IDE with high affinity and specificity.

[0381] The generated anti-IDE antibodies inhibited insulin degradation in vitro

[0382] To test the ability of the generated anti-IDE scFvs and IgGs to inhibit IDE-mediated insulin degradation, rhIDE was incubated with each of the formats of MBP-scFv or a control MBP-scFv that does not bind rhIDE for 1 hour at room temperature. Subsequently, insulin was added for 2 hours at 37°C. Residual insulin activity was measured using Mercodia ultrasensitive mouse insulin ELISA kit. A dose-dependent inhibition of IDE activity was observed in the presence of B1 and H3 scFvs: B1 inhibited 64% (P<0.001) and 35% (P<0.001) of insulin degradation using 100 nM and 10 nM scFv, respectively; H3 inhibited 40% (P<0.001) of insulin degradation using 100 nM, while no significant inhibition was observed using 10 nM (P=0.09). FIG. 3A The A9 and the control MBP-scFvs did not show significant inhibition of IDE activity. After reformattmg the antibodies into full-size human IgGs, their ability to inhibit IDE activity was reevaluated. In the IgG format, the H3 antibody showed significant efficacy in inhibiting IDE activity, while A9 and the negative control antibodies did not (P<0.001, FIG. 3B

[0383] The generated anti-IDE antibodies recognize conformational IDE epitopes

[0384] To assess the binding epitope on IDE bound by the generated antibodies, a dot-blot analysis was performed. Specifically, serial dilutions of rhIDE and a mouse spleen lysate as a control were spotted onto a nitrocellulose membrane under native and denaturing conditions. All antibody clones detected the native rhIDE protein samples, with the B1 MBP-scFv signal being the strongest. All the denatured protein samples produced a signal that was weaker than the native samples or did not give any visible dots FIG. 4A These results show that the selected scFv clones recognize conformational epitopes of rhIDE that are disrupted under denaturing conditions, rather than linear epitopes.

[0385] Example 2

[0386] The therapeutic effect of the generated anti-IDE antibodies in a diabetic animal model converted the anti-IDE H3 antibody into a reverse chimeric IgG

[0387] ​Chimeric antibodies were an important milestone in the development of therapeutic antibodies. Chimeric antibodies are recombinant IgG in which the variable domains are from a mouse antibody and the constant domains are human sequences. Chimeric antibodies are much less immunogenic than mouse monoclonal antibodies (mAbs), limiting the elicitation of human anti-mouse antibodies in human patients upon administration (36). Conversely, reverse chimeras are antibodies with human variable domains and murine constant domains (37). In addition to being useful for finding antibodies in transgenic mice, reverse chimeric antibodies can be used to treat mouse models to avoid eliciting a mouse anti-human immune response. To assess the efficacy of the generated anti-IDE antibodies in a mouse model, the human H3 IgG antibody was converted to a reverse chimeric IgG (rcIgG) of mouse IgG1 isotype and its ability to bind IDE, BSA, and the E111Q IDE mutant was assessed. The antibody rc2E12, which does not bind IDE, was used as an isotype control. As shown in FIG. 76, rcH3-IgG bound IDE with high affinity (EC50= 1.62 nM). In addition, the rcH3-IgG showed specificity for active IDE and did not bind to the mutant IDE or BSA. Furthermore, the ability of the rcH3-IgG antibody to inhibit IDE activity was tested. The rcH3-IgG antibody inhibited IDE activity in a dose-dependent manner (FIG. 77). In summary, these results show that a reverse chimeric H3 IgG retains similar properties as the human H3 IgG version. FIG. 4B 50 FIG. 4C

[0388] rcH3-IgG antibody improves insulin signaling in a diabetic mouse model

[0389] ​​​It was previously suggested that the suppression of IDE can improve insulin activity in a diabetic mouse model (38). Therefore, the ability of rcH3-IgG to lower glucose levels (oGTT) and improve insulin activity (iTT) in multiple STZ-treated mice was evaluated. To this end, rcH3-IgG or an isotype control antibody was administered intraperitoneally to multiple STZ-treated mice 1 hour prior to testing oGTT and iTT. Following a glucose challenge (oGTT), both control and rcH3-IgG-treated mice exhibited an increase in blood glucose levels. However, compared to the control-treated mice, the rcH3-IgG-treated mice exhibited lower glucose levels over time (p<0.05). Post-hoc analysis showed that the rcH3-IgG-treated mice had significantly reduced glucose levels at 90 minutes after glucose administration compared to the isotype control-treated mice (p<0.05; FIG. 5A ). Furthermore, the ability of rcH3-IgG to improve insulin activity (iTT) was evaluated. While the rcH3-IgG-treated mice exhibited a significant reduction in glucose levels starting at 30 minutes after insulin administration (p<0.001), the isotype control-treated mice exhibited a significant reduction in glucose levels only at 90 minutes (p<0.05). Finally, the half-life of the rcH3-IgG antibody in the serum of the administered mice was determined and found to be approximately 11 days. In summary, these results show that treatment with rcH3-IgG improves glucose levels and insulin activity in a diabetic mouse model.

[0390] Example 3

[0391] The effect of the generated anti-IDE antibodies on dopaminergic neurons having a Parkinson's phenotype

[0392] To assess the therapeutic effect of the generated antibodies on Parkinson's disease, DJ-1 -knockdown (KD) microglia cells exhibit the neurotoxic phenotype of microglia cells that occur in Parkinson's disease (Nash et al. J Neurochem. 2017 143(5):584-594; and Trudler et al. J Neurochem. 2014 129(3):434-47). Previous studies have shown that DJ-1 KD microglia cells produce higher levels of reactive oxygen species (ROS) under basal and inflammatory-stimulated conditions (Trudler et al., 2014). To avoid the possibility that the generated anti-IDE antibodies bind to microglia cells through Fc receptors expressed on the microglia cell membrane (Teeling et al., 2012), the Fab2 fragments of the rcH3-IgG antibody (herein referred to as "H3 Fab") were tested in these environments.

[0393] As shown in FIG. 6A , ROS levels in DJ-1 KD microglia cells were increased by 51% compared to control microglia cells, which is consistent with previous reports (Trudler et al., 2014). In addition, rotenone, a known inhibitor of the mitochondrial complex I, was used to transiently stimulate cells as an experimental model of Parkinson's disease (Xiong et al., 2012), which significantly increased the production of ROS in DJ-1 -KD microglia cells (227% compared to control cells at baseline, p < 0.001). While 100 nM insulin stimulation did not reduce ROS production after the rotenone insult, incubation of the cells with 100 nM H3 Fab significantly reduced ROS production in DJ-1 KD cells (p < 0.001) to levels comparable to the basal levels of the cells (151% of control cells at baseline, and 148% of H3 Fab-treated cells after rotenone stimulation). Notably, as shown in FIG. 6B , the effect of the treatments on total cell number, measured using an MB assay, indicated that neither insulin nor H3 treatment significantly affected cell survival after rotenone stimulation. This is an important control to assess the protective and non-toxic effects.

[0394] In summary, these results show that an rcH3 antibody can be used as a therapeutic approach for Parkinson's disease.

[0395] Example 4

[0396] Diagnosis and prognosis of metabolic syndrome using the generated anti-IDE antibodies

[0397] Using the aforementioned generated anti-IDE antibodies, a sandwich ELISA for detecting and quantifying IDE was developed. FIG. 7A ).like FIG. 7B As shown, this ELISA assay can easily detect rhIDE with high sensitivity at multiple concentrations ranging from 5 pg / μl to up to 500 pg / μl. Human and mouse IDE share >95% sequence identity, therefore it is inferred and verified that the resulting polyclonal and monoclonal antibodies will also bind to mouse IDE. To determine whether antibody A9 recognizes the aforementioned conformation of active rhIDE, the differences in binding to the denatured form of rhIDE were evaluated. FIG. 7C As shown, thermal denaturation of rhIDE resulted in a very significant decrease (to an almost negligible signal) in the ELISA signal generated by A9 IgG detection. Notably, thermal denaturation of rhIDE also resulted in a decrease in the ELISA signal of approximately ×2-3, as shown in Figure 8, where rhIDE was detected using a sequentially ×2 diluted primary anti-His-tagged antibody (recognizing a linear epitope), indicating that the binding efficiency of denatured rhIDE to the multiple wells of the ELISA disc may also be low. Taken together, these results demonstrate that A9 IgG specifically recognizes a conformational epitope of the active rhIDE.

[0398] Subsequently, the developed ELISA assay was used to determine multiple IDE levels in multiple serum samples from multiple human patients with metabolic syndrome (MS) and multiple healthy control subjects. Compared with multiple controls, multiple MS subjects had higher BMI, glucose, triglyceride, and insulin levels, and multiple lower HDLc levels (Table 1 below). These multiple results indicate that multiple IDE levels were higher in multiple MS subjects (mean 637.4 + / - 469.5 vs 470.5 + / - 221.8 pg / μL; p < 0.05). FIG. 9 Since the MS group was older than the control group, the IDE of the two groups was also compared after adjusting for age. After this adjustment, the IDE of multiple MS subjects was higher than that of multiple controls (F(1,68) = 6.675, p = 0.012, net eta-squared = 0.089). Furthermore, multiple IDE levels were detected in relation to serum triglycerides (r = 0.423; p < 0.05). FIG. 10A ) and insulin (r = 0.294, p < 0.05, FIG. 10B A positive correlation was observed between IDE and serum C-peptide levels (not shown). IDE was also negatively correlated with multiple HDLc levels (r = -0.366; p < 0.05). FIG. 10CThese findings indicate that several higher IDE levels are quantitatively correlated with several MS components.

[0399] Furthermore, multiple IDE levels in several MS subjects clearly divided into two distinct subgroups: several subjects with low IDE, whose value distribution and mean (n = 25; 272.1 + / - 157.9 pg / μl) were indistinguishable from the normal control group, and several subjects with high IDE (n = 25; 1002.6 + / - 383.7 pg / μl, p < 0.001). The MS group with low IDE was older (age 54 + / - 10 years vs. age 45 + / - 13 years; p < 0.05) and had higher glucose levels in several subjects than the MS group with high IDE (95 + / - 20 vs. 80 + / - 9 mg / dl, p < 0.01). FIG. 9 It is noteworthy that multiple insulin levels were found to be similar between the two groups, as were triglycerides, HDLc, systolic and diastolic blood pressure, and heart rate.

[0400] Table 1: Multiple characteristics of multiple subjects [BP = blood pressure, HR = heart rate, Trig = triglycerides]

[0401]

[0402] While the invention has been described in conjunction with specific embodiments thereof, it will be apparent to those skilled in the art that many alternatives, modifications, and variations will be readily apparent. Therefore, it is intended to include all such alternatives, modifications, and variations falling within the spirit and scope of the appended claims.

[0403] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication, patent, or patent application were specifically and individually identified and incorporated herein by reference. Furthermore, any references cited or indicated should not be construed as an admission that such references are prior art to this invention. The headings in this application should not be construed as necessary limitations. Additionally, any priority documents of this application are incorporated herein by reference in their entirety.

[0404] References

[0405] (Other references are cited throughout the application)

[0406] 1. Alberti, K.G.M.M. and Zimmet, P.Z. (1998) Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1 : diagnosis and classification of diabetes mellitus. Provisional report of a WHO Consultation. Diabet. Med. 15, 539-553

[0407] 2. Kahn, C.R. (1986) Insulin Resistance: A Common Feature of Diabetes Mellitus. N. Engl. J. Med.

[0408] 3. Roglic, G. (2016) WHO Global report on diabetes: A summary. Int. J. Noncommunicable Dis. 1, 3-8

[0409] 4. Vinik, A.I., Fishwick, D.T., and Pittenger, G. (2004) Advances in diabetes for the millennium: toward a cure for diabetes. Medscape Gen. Med. 6

[0410] 5. Mortel, K.F., Meyer, J.S., Sims, P.A., and McClintic, K. (1990) Diabetes mellitus as a risk factor for stroke. South. Med. J. 83, 904-911

[0411] 6. Falanga, V. (2005) Wound healing and its impairment in the diabetic foot. Lancet 366, 1736-1743

[0412] 7. McCrimmon, R. J., Ryan, C. M., and Frier, B. M. (2012) Diabetes and cognitive dysfunction. Lancet 379, 2291-2299

[0413] 8. Kleinridders, A., Ferris, H. A., Cai, W., and Kahn, C. R. (2014) Insulin action in brain regulates systemic metabolism and brain function.

[0414] 9. Cheung, B. M. Y., Ong, K. L., Cherny, S. S., Sham, P.-C, Tso, A. W. K., and Lam, K. S. L. (2009) Diabetes Prevalence and Therapeutic Target Achievement in the United States, 1999 to 2006. Am. J. Med. 122, 443-453

[0415] 10. Rines, A. K., Sharabi, K., Tavares, C. D. J., and Puigserver, P. (2016) Targeting hepatic glucose metabolism in the treatment of type 2 diabetes. Nat. Rev. Drug Discov. 15, 786-804

[0416] 11. Binder, C, Lauritzen, T., Faber, O., and Pramming, S. (1984) Insulin pharmacokinetics. Diabetes Care 7, 188-199

[0417] 12. Williams, G., Pickup, J. C, and Keen, H. (1987) Massive insulin resistance apparently due to rapid clearance of circulating insulin. Am. J. Med. 82, 1247-1252

[0418] 13. Brunton, S. A., Davis, S. N., and Renda, S. M. (2006) Overcoming psychological barriers to insulin use in type 2 diabetes. Clin. Cornerstone 8, S19-S26

[0419] 14. Porter, S. (2001) Human Immune Response to Recombinant Human Proteins. J. Pharm. Sci. 90, 1-11

[0420] 15. Fernandez-Gamba, A., Leal, M., Morelli, L., and Castano, E. (2009) Insulin-Degrading Enzyme: Structure-Function Relationship and its Possible Roles in Health and Disease. Curr. Pharm. Des. 15, 3644-3655

[0421] 16. M16: Pitrilysin: insulin degrading enzyme. IUPHAR / BPS Guid. to Pharmacol.

[0422] 17. Guo, Q., Manolopoulou, M., Bian, Y., Schilling, A. B., and Tang, W.-J. (2010) Molecular Basis for the Recognition and Cleavages of IGF-II, TGF-alpha, and Amylin by Human Insulin-Degrading Enzyme. J. Mol. Biol. 395, 430-443

[0423] 18. Duckworth, W. C. and Kitabchi, A. E. (1974) Insulin and glucagon degradation by the same enzyme. Diabetes 23, 536-543

[0424] 19. Duckworth, W. C, Bennett, R. G., and Hamel, F. G. (1998) Insulin Degradation: Progress and Potential. Endocr. Rev. 19, 608-624

[0425] 20. Hulse, R. E., Ralat, L. A., and Wei-Jen, T. (2009) Structure, Function, and Regulation of Insulin-Degrading Enzyme. Vitam. Horm. 80, 635-648

[0426] 21. Glebov, K., Schiitze, S., and Walter, J. (2011) Functional relevance of a novel SlyX motif in non-conventional secretion of insulin-degrading enzyme. J. Biol. Chem. 286, 22711-22715

[0427] 22. Maianti, J. P., McFedries, A., Foda, Z. H., Kleiner, R. E., Du, X. Q., Leissring, M. A., Tang, W.-J., Charron, M. J., Seeliger, M. A., Saghatelian, A., and Liu, D. R. (2014) Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by multiple hormones. Nature 511, 94-98

[0428] 23. Yang, D., Qin, W., Shi, X., Zhu, B., Xie, M., Zhao, H., Teng, B., Wu, Y., Zhao, R., Yin, F., Ren, P., Liu, L., and Li, Z. (2018) Stabilized β-Hairpin Peptide Inhibits Insulin Degrading Enzyme. J. Med. Chem. 61, 8174-8185

[0429] 24. Tang, W.-J. (2016) Targeting Insulin-Degrading Enzyme to Treat Type 2 Diabetes Mellitus. Trends Endocrinol. Metab. 27, 24-34

[0430] 25. Deprez-Poulain, R., Hennuyer, N., Bosc, D., Liang, W. G., Enee, E., Marechal, X., Charton, J., Totobenazara, J., Berte, G., Jahkiai, J., Verdelet, T., Dumont, J., Dassonneville, S., Woitrain, E., Gauriot, M., Paquet, C, Duplan, I., Hermant, P., Cantrelle, F.-X., Sevin, E., Culot, M., Landry, V., Herledan, A., Piveteau, C, Lippens, G., Leroux, F., Tang, W.-J., van Endert, P., Staels, B., and Deprez, B. (2015) Catalytic site inhibition of insulin-degrading enzyme by a small molecule induces glucose intolerance in mice. Nat. Commun. 6, 8250

[0431] 26. Brekke, O. H. and Sandlie, I. (2003) Therapeutic antibodies for human diseases at the dawn of the twenty-first century. Nat. Rev. Drug Discov. 2, 52-62

[0432] 27. Maynard, J. and Georgiou, G. (2000) Antibody Engineering. Annu. Rev. Biomed. Eng. 2, 339-376

[0433] 28. Azriel-Rosenfeld, R., Valensi, M., and Benhar, I. (2004) A Human Synthetic Combinatorial Library of Arrayable Single-chain Antibodies based on Shuffling in Vivo Formed CDRs into General Framework Regions. J. Mol. Biol. 335, 177-192

[0434] 29. Hakim, R. and Benhar, I. (2009) "Inclonals": IgGs and IgG-enzyme fusion proteins produced in an E. coli expression-refolding system. MAbs 1, 281-287

[0435] 30. Buchner, J., Pastan, I., and Brinkmann, U. (1992) A method for increasing the yield of properly folded recombinant fusion proteins: Single-chain immunotoxins from renaturation of bacterial inclusion bodies. Anal. Biochem. 205, 263-270

[0436] 31. Benhar, I. and Pastan, I. (1994) Cloning, expression and characterization of the Fv fragments of the anti-carbohydrate mAbs B1 and B5 as single-chain immunotoxins. Protein Eng. Des. Sel. 7, 1509-1515

[0437] 32. Mazor, Y., Barnea, I., Keydar, I., and Benhar, I. (2007) Antibody internalization studied using a novel IgG binding toxin fusion. J. Immunol. Methods 321, 41-59

[0438] 33. Birnboim-Perach, R., Grinberg, Y., Vaks, L., Nahary, L., and Benhar, I. (2019) Production of Stabilized Antibody Fragments in the E. coli Bacterial Cytoplasm and in Transiently Transfected Mammalian Cells. pp. 455-480, Humana Press, New York, NY

[0439] 34. Gibson, D.G., Young, L., Chuang, R.-Y., Venter, J.C., Hutchison, C.A., and Smith, H.O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343-345

[0440] 35. Perlman, R. K., Gehm, B. D., Kuo, W.-L., and Rich Rosner, M. (1993) Functional Analysis of Conserved Residues in the Active Site of Insulin-degrading Enzyme. J. Biol. Chem. 268, 21538-21544

[0441] 36. Morrison, S. L. and Schlom, J. (1990) Recombinant chimeric monoclonal antibodies. Important Adv. Oncol. 3-18

[0442] 37. Murphy, A. J., Macdonald, L. E., Stevens, S., Karow, M., Dore, A. T., Pobursky, K., Huang, T. T., Poueymirou, W. T., Esau, L., Meola, M., Mikulka, W., Krueger, P., Fairhurst, J., Valenzuela, D. M., Papadopoulos, N., and Yancopoulos, G. D. Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice.

[0443] 38. Maianti, J. P., McFedries, A., Foda, Z. H., Kleiner, R. E., Du, X. Q., Leissring, M. a, Tang, W.-J., Charron, M. J., Seeliger, M. a, Saghatelian, A., and Liu, D. R. (2014) Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by multiple hormones. Nature 511, 94-98

[0444] 39. Leissring, M. a., Malito, E., Hedouin, S., Reinstatler, L., Sahara, T., Abdul-Hay, S. O., Choudhry, S., Maharvi, G. M., Fauq, A. H., Huzarska, M., May, P. S., Choi, S., Logan, T. P., Turk, B. E., Cantley, L. C., Manolopoulou, M., Tang, W.-J., Stein, R. L., Cuny, G. D., and Selkoe, D. J. (2010) Designed Inhibitors of Insulin-Degrading Enzyme Regulate the Catabolism and Activity of Insulin. PLoS One 5, e10504

[0445] 40. Shen, Y., Joachimiak, A., Rosner, M. R., and Tang, W.-J. (2006) Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism. Nature 443, 870-874

[0446] 41. Shen, Y., Joachimiak, A., Rich Rosner, M., and Tang, W.-J. (2006) Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism. Nature 443, 870-874

[0447] 42. Farris, W., Mansourian, S., Chang, Y., Lindsley, L., Eckman, E. A., Frosch, M. P., Eckman, C. B., Tanzi, R. E., Selkoe, D. J., and Guenette, S. (2003) Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo. Proc Natl Acad Sci USA 100, 4162-4167

[0448] 43. Backer, J. M., Kahn, R. C, and White, M. F. (1990) The Dissociation and Degradation of Internalized Insulin Occur in the Endosomes of Rat Hepatoma Cells. J. Biol. Chem. 265, 14828-14835

[0449] 44. Fakhrai-Rad, H., Nikoshkov, A., Kamel, A., M., Zierath, J. R., Norgren, S., Luthman, H., and Galli, J. (2000) Insulin-degrading enzyme identified as a candidate diabetes susceptibility gene in GK rats. Hum. MoI. Genet. 9, 2149-2158

[0450] 45. Karamohamed, S., Demissie, S., Volcjak, J., Liu, C. Y., Heard-Costa, N., Liu, J., Shoemaker, C. M., Panhuysen, C. I., Meigs, J. B., Wilson, P., Atwood, L. D., Cupples, L. a, and Herbert, a. (2003) Polymorphisms in the insulin-degrading enzyme gene are associated with type 2 diabetes in men from the NHLBI Framingham Heart Study. Diabetes 52, 1562-1567

[0451] 46. Vieira, P. and Rajewsky, K. (1988) The half-lives of serum immunoglobulins in adult mice. Eur. J. Immunol. 18, 313-316

Claims

1. An isolated antibody, characterized in that: The antibody contains an antigen recognition region that specifically binds to insulin-degrading enzymes, wherein the antigen recognition region comprises a plurality of complementarity-determining region amino acid sequences as described below, i.e., CDR amino acid sequences: SEQ ID NO: 36, i.e., CDR1, SEQ ID NO: 38, i.e., CDR2, and SEQ ID NO: 40, i.e., CDR3, are arranged sequentially from N to C on one heavy chain of the antibody; SEQ ID NO: 44, i.e., CDR1, SEQ ID NO: 46, i.e., CDR2, and SEQ ID NO: 48, i.e., CDR3, are arranged sequentially from N to C on one light chain of the antibody.

2. The antibody as described in claim 1, characterized in that: The antibody is a complete IgG antibody.

3. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises, as an active ingredient, the antibody as described in claim 1 and a pharmaceutically acceptable carrier.

4. Use of the antibody as described in claim 1 in the preparation of a medicament for treating a disease associated with insulin-degrading enzyme activity in a subject in need, said disease being selected from diabetes and Parkinson's disease.

5. Use of a therapeutic agent and the antibody as described in claim 1 in the preparation of a medicament for treating a disease associated with the activity of an insulin-degrading enzyme in a subject in need, said disease being selected from diabetes and Parkinson's disease, said therapeutic agent being used for said disease.

6. The use as described in claim 4 or 5, characterized in that: Compared to a control biological sample, the subject had an insulin-degrading enzyme level in a biological sample that was above a predetermined threshold.

7. A pharmaceutical product for treating a disease associated with the activity of an insulin-degrading enzyme, said disease being selected from diabetes and Parkinson's disease, characterized in that: The product comprises the antibody as described in claim 1 and a therapeutic agent for treating the disease.

8. The pharmaceutical product as described in claim 7, characterized in that: The antibody and the therapeutic agent are in multiple separate containers.

9. The pharmaceutical product as described in claim 7, characterized in that: The antibody and the therapeutic agent are in a common formulation.

10. The use as described in claim 4, characterized in that: The disease in question is type 1 diabetes.

11. The pharmaceutical product as described in claim 7, characterized in that: The disease in question is type 1 diabetes.

12. The use as described in claim 4, characterized in that: The disease in question is type 2 diabetes.

13. The pharmaceutical product as described in claim 7, characterized in that: The disease in question is type 2 diabetes.

14. A polynucleotide encoding an isolated antibody as claimed in claim 1.

15. The isolated polynucleotide as described in claim 14, characterized in that: The multiple nucleic acid sequences encoding the multiple CDR amino acid sequences are listed below: SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45 and SEQ ID NO:

47.

16. A nucleic acid construct, characterized in that: The nucleic acid construct comprises the polynucleotide as described in claim 14 or 15 and a cis-regulatory element for guiding the expression of the polynucleotide.

17. A host cell expressing the antibody as claimed in claim 1, the polynucleotide as claimed in claim 14 or 15, or the nucleic acid construct as claimed in claim 16.

18. A method for generating an antibody against insulin-degrading enzymes, characterized in that: The method comprises expressing the polynucleotide as described in claim 14 or 15 or the nucleic acid construct as described in claim 16 in a host cell.

19. The method as described in claim 18, characterized in that: The method includes isolating the antibody.

Images