Anti-IDE antibodies and their use

Abstract

Isolated anti-IDE antibodies are provided, each comprising an antigen recognition domain that includes a CDR amino acid sequence. Methods for producing, using, and pharmaceutical compositions and articles of manufacture containing the same are also provided.

Description

Technical Field

[0001] Related Application This application claims the priority of U.S. Patent Application No. 62 / 964,139, filed on January 22, 2020, and the entire content of this patent application is incorporated herein by reference as part of this specification.

[0002] Statement Regarding the Sequence Listing The 45,056-byte ASCII file "85636" created on December 10, 2020, which was filed simultaneously with this application, is incorporated herein by reference as part of this specification.

[0003] The present invention, in some of its embodiments, relates to anti-IDE antibodies and their use.

Background Art

[0004] Insulin-degrading enzyme (IDE, insulin) is a large zinc-binding protease (a total zinc metalloendopeptidase of about 110 kDa) located in the cytosol, peroxisomes, endosomes, and on the cell surface. This enzyme cleaves a variety of small proteins with the common property of forming β-sheet-rich amyloid fibrils, including amyloid β-protein (Aβ), insulin, glucagon, amylin, atrial natriuretic factor, and calcitonin. Thus, IDE is known to cleave a number of short polypeptides and plays an important role in the degradation of various proteins in which the regulation of immune response activities such as insulin and IGF-1 has been reported.

[0005] Since their discovery, IDE inhibitors have been suggested for use in several diseases, including, for example, diabetes, obesity, autoimmune diseases of the central nervous system, neurodegenerative diseases, and varicella-zoster virus (VZV) infections (e.g., Maianti et al. (2014) Nature 511, 94-98; Tang, W.-J. (2016) Trends Endocrinol. Metab. 27, 24-34; and International Publication Nos. 2012 / 017439 and 2010 / 086867). For example, International Publication No. 2010 / 086867 by the same applicant discloses the use of an isolated peptide having insulin-degrading enzyme (IDE) inhibitory activity, which comprises an amino acid sequence of 25 amino acids or less and the amino acid sequence contains at least one aspartic acid or a homolog thereof, in the manufacture of a medicament specified for the treatment of a disease selected from the group consisting of diabetes, obesity, hyperglycemia, retinal damage, renal insufficiency, nerve damage, microvascular damage, and varicella-zoster virus (VZV) infection. International Publication No. 2012 / 017439 by the same applicant provides an insulin-degrading enzyme (IDE) inhibitor (e.g., an antibody) for use in the treatment of a disease selected from the group consisting of autoimmune diseases of the central nervous system and neurodegenerative diseases).

[0006] Several IDE monoclonal and polyclonal antibodies have been described for in vitro detection of rodent and / or human IDE in various applications, including Western blotting, immunoprecipitation, immunocytochemistry, immunohistochemistry, and quantitative sandwich ELISA [see, for example, Delledonne A. et al. Mol Neurodegener. (2009) 4:39]. SUMMARY OF THE INVENTION

[0007] In one aspect of some embodiments of the present invention, there is provided an isolated antibody comprising an antigen recognition region that specifically binds to IDE, wherein the antigen recognition domain comprises the following complementarity-determining region (CDR) amino acid sequences. (i) The sequences SEQ ID NO:4 (CDR1), SEQ ID NO:6 (CDR2), and SEQ ID NO:8 (CDR3) arranged in order from N to C of the heavy chain of the antibody, and the sequences SEQ ID NO:12 (CDR1), SEQ ID NO:14 (CDR2), and SEQ ID NO:16 (CDR3) arranged in order from N to C of the light chain of the antibody, (ii) The sequences SEQ ID NO:20 (CDR1), SEQ ID NO:22 (CDR2), and SEQ ID NO:24 (CDR3) arranged in order from N to C of the heavy chain of the antibody, and the sequences SEQ ID NO:28 (CDR1), SEQ ID NO:30 (CDR2), and SEQ ID NO:32 (CDR3) arranged in order from N to C of the light chain of the antibody, or (iii) The sequences SEQ ID NO:36 (CDR1), SEQ ID NO:38 (CDR2), and SEQ ID NO:40 (CDR3) arranged in order from N to C of the heavy chain of the antibody, and the sequences SEQ ID NO:44 (CDR1), SEQ ID NO:46 (CDR2), and SEQ ID NO:48 (CDR3) arranged in order from N to C of the light chain of the antibody.

[0008] In one aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising antibody (ii) or (iii) as an active ingredient and further comprising a pharmaceutically acceptable carrier.

[0009] In one aspect of some embodiments of the present invention, there is provided a method for treating an IDE activity-related disease in a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of antibody (ii) or (iii) or the pharmaceutical composition, wherein the administration prevents or treats a disease associated with IDE activity.

[0010] In one aspect of some embodiments of the present invention, there is provided antibody (ii) or (iii) for use in treating an IDE activity-related disease in a subject in need of treatment.

[0011] According to some embodiments of the present invention, the method further comprises administering a therapeutic agent for the disease to the subject.

[0012] According to some embodiments of the present invention, the antibody for use further comprises a therapeutic agent for the disease.

[0013] According to some embodiments of the present invention, a biological sample of interest exhibits an IDE level that exceeds a predetermined threshold as compared to a control biological sample.

[0014] According to some embodiments of the present invention, the method includes determining the IDE level in a biological sample of interest using an antibody prior to administration.

[0015] In one aspect of some embodiments of the present invention, there is provided a product defined for the treatment of an IDE activity-related disease, comprising antibody (ii) or (iii) and a therapeutic agent for treating the disease.

[0016] According to some embodiments of the present invention, the antibody and the therapeutic agent are contained in separate containers.

[0017] According to some embodiments of the present invention, the antibody and the therapeutic agent are included in a co-formulation.

[0018] In one aspect of some embodiments of the present invention, there is provided a method for diagnosing an IDE activity-related disease in a subject, the method comprising determining the IDE level in a biological sample of the subject using an antibody, and diagnosing the subject with the disease if the IDE level exceeds a predetermined threshold as compared to a control biological sample.

[0019] In one aspect of some embodiments of the present invention, there is provided a method for monitoring the effectiveness of a therapy for a disease in a subject diagnosed with an IDE activity-related disease, the method comprising determining the IDE level in a biological sample of the subject during or after treatment using an antibody, and determining that the therapy is effective if the IDE level after treatment is lower than a predetermined threshold.

[0020] In one aspect of some embodiments of the present invention, there is provided a method for treating a disease in a subject in need of treatment for an IDE activity-related disease, Diagnose a subject by a method, and when the IDE level exceeds a predetermined threshold, Treat the subject with a therapy for the disease A method for treating a disease of a subject is provided, which includes the above.

[0021] In one aspect of some embodiments of the present invention, a method for treating a disease in a subject in need of treatment for an IDE activity-related disease, comprising: Diagnose a subject by a method, and when the IDE level exceeds a predetermined threshold, Select a therapy based on the IDE level A method for treating a disease of a subject is provided, which includes the above.

[0022] According to some embodiments of the present invention, the therapy includes antibody (ii) or (iii).

[0023] In one aspect of some embodiments of the present invention, a composition is provided, which includes a biological sample of a subject diagnosed with an IDE activity-related disease and an antibody.

[0024] According to some embodiments of the present invention, the IDE activity-related disease is selected from the group consisting of autoimmune diseases of the central nervous system, neurodegenerative diseases, metabolic syndrome, diabetes, obesity, hyperglycemia, retinal damage, renal insufficiency, nerve damage, microvascular damage, varicella-zoster virus (VZV) infection, and wounds.

[0025] According to some embodiments of the present invention, the IDE activity-related disease is selected from the group consisting of autoimmune diseases of the central nervous system, neurodegenerative diseases, diabetes, obesity, hyperglycemia, retinal damage, renal insufficiency, nerve damage, microvascular damage, varicella-zoster virus (VZV) infection, and wounds.

[0026] According to some embodiments of the present invention, the IDE activity-related disease is selected from the group consisting of metabolic syndrome, diabetes, and obesity.

[0027] According to some embodiments of the present invention, the disease is diabetes.

[0028] According to some embodiments of the present invention, the diabetes is type 1 diabetes.

[0029] According to some embodiments of the present invention, the diabetes is type 2 diabetes.

[0030] According to some embodiments of the present invention, the disease is metabolic syndrome.

[0031] According to some embodiments of the present invention, the neurodegenerative disease is Parkinson's disease.

[0032] According to some embodiments of the present invention, the neurodegenerative disease is Alzheimer's disease.

[0033] According to some embodiments of the present invention, the autoimmune disease of the central nervous system is selected from the group consisting of multiple sclerosis, Guillain - Barré syndrome, Lambert - Eaton myasthenic syndrome, myasthenia gravis, transverse myelitis, progressive multifocal leukoencephalopathy, chronic headache, and cerebral palsy.

[0034] According to some embodiments of the present invention, the autoimmune disease of the central nervous system is multiple sclerosis.

[0035] According to some embodiments of the present invention, the wound is selected from the group consisting of chronic wounds, acute wounds, diabetic wounds, ischemic wounds, ulcers, burns, and surgical wounds.

[0036] In one aspect of some embodiments of the present invention, an isolated polynucleotide encoding an antibody is provided.

[0037] According to some embodiments of the present invention, the nucleic acid sequence encoding the CDR amino acid sequence is (i) SEQ ID NO: 3, 5, 7, 11, 13, and 15, (ii) SEQ ID NO: 19, 21, 23, 27, 29, and 31, or (iii) SEQ ID NOs: 35, 37, 39, 43, 45, and 47 are shown in

[0038] In one aspect of some embodiments of the present invention, there is provided a nucleic acid construct comprising a polynucleotide and a cis element that regulates the expression of the polynucleotide.

[0039] In one aspect of some embodiments of the present invention, there is provided a host cell that expresses an antibody, a polynucleotide, or a nucleic acid construct.

[0040] In one aspect of some embodiments of the present invention, there is provided a method for producing an anti-IDE antibody, the method comprising expressing a polynucleotide or a nucleic acid construct in a host cell.

[0041] According to some embodiments of the present invention, the method includes isolating the antibody.

[0042] In one aspect of some embodiments of the present invention, there is provided a method for producing an anti-IDE antibody, the method comprising: (a) providing a plurality of antibodies; and (b) performing screening of the antibodies to select an antibody that binds to wild-type IDE but does not bind to mutant IDE having a reduced catalytic activity compared to wild-type IDE. The method is provided.

[0043] According to some embodiments of the present invention, the sequence of wild-type IDE is the sequence of SEQ ID NO: 50.

[0044] According to some embodiments of the present invention, the mutant IDE includes the conversion of E111Q corresponding to SEQ ID NO: 50.

[0045] According to some embodiments of the present invention, the method further includes screening the antibodies to select an antibody that inhibits the insulin-degrading activity of IDE.

[0046] 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 this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, 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 not intended to be limiting.

[0047] Some embodiments of the present invention will be described herein by reference to the accompanying drawings for purposes of illustration only. The details shown below, with particular reference to the drawings, are for purposes of illustration and to emphasize the detailed description of the embodiments of the present invention. Similarly, by viewing the description together with the drawings, it will be apparent to those skilled in the art how embodiments of the present invention can be practiced.

Brief Description of the Drawings

[0048]

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Best Mode for Carrying Out the Invention

[0049] In some embodiments, the present invention relates to anti-IDE antibodies and their use.

[0050] The principles and procedures of the present invention will be better understood by reference to the drawings and the following description.

[0051] Before detailing at least one embodiment of the present invention, it should be understood that the present invention is not necessarily limited in its application to the details shown in the following description or exemplified in the examples. The present invention is capable of other embodiments and of being practiced or carried out in various ways. Further, the terminology and phrases used herein are for the purpose of description and should not be regarded as limiting.

[0052] Insulin-degrading enzyme (IDE) is a large zinc-binding protease known to cleave multiple small polypeptides and play an important role in insulin degradation. Several IDE monoclonal and polyclonal antibodies have been published in the art for in vitro applications [see Delledonne A. et al. Mol Neurodegener. (2009) 4:39] and in vivo applications (see International Publication No. WO 2012 / 017439). Further, IDE inhibitors, including peptide inhibitors, have already been reported for various therapeutic applications (see International Publication No. WO 2010 / 086867).

[0053] In practicing certain embodiments of the present invention, the inventors have generated novel monoclonal anti-IDE antibodies that specifically target and inhibit the insulin-degrading activity of IDE. As a result, certain embodiments of the present invention suggest the use of anti-IDE antibodies for the diagnosis and treatment of IDE-related diseases and conditions.

[0054] As shown in the examples described below, the inventors prepared plasmids for expressing human insulin-degrading enzyme (IDE) and mutant IDE (E111Q) in E. coli (Example 1, Fig. 1(A)). Mutant IDE (E111Q) contains a site-specific mutation that loses catalytic activity due to its inability to perform a nucleophilic attack on the substrate. Therefore, both peptides were used to isolate specific antibody clones that bind to IDE with high affinity (i.e., wild-type IDE) having catalytic activity. The IDE protein was expressed and purified in E. coli, and protein purification and identification were demonstrated (see Fig. 1(B)-(D)). Using phage display technology, specific single-chain variable fragment (scFv) clones that recognize and bind to the high-affinity epitope of recombinant human wild-type IDE were isolated using purified IDE. Three clones of phage, specifically A9, H3, and B1, were selected (Example 1, Fig. 2(C)). Subsequently, to produce as soluble antibodies, the scFvs were reformatted and tested in the following three formats: MBP-scFv, human IgG1 produced as "Inclonal", and reverse chimeric IgG produced in cultures of mammalian cells. The specificity and IDE inhibitory properties of these antibodies were further demonstrated in vitro (Examples 1-2, Fig. 1(E)-Fig. 4(C)). In addition, the reverse chimeric H3 IgG antibody improved blood glucose levels and insulin activity in a STZ-induced mouse diabetes model (Example 2, Fig. 5(A)-(C)). In addition, the Fab 2 fragment of the reverse chimeric H3 IgG antibody decreased the production of reactive oxygen species in vitro in microglial cells having a Parkinson's disease phenotype (Example 3, Fig. 6(A)-(B)). Subsequently, the inventors developed a highly sensitive ELISA using the prepared antibodies (Example 4, Fig. 7(A)-(C)). Using this ELISA, the inventors were able to show a strong correlation between human serum IDE levels and the presence and / or severity of metabolic syndrome (Example 4, Fig. 9-Fig. 10(C)).

[0055] Thus, according to one aspect of the present invention, there is provided an isolated antibody comprising an antigen recognition region that specifically binds to IDE, wherein the antigen recognition domain comprises the following complementarity determining region (CDR) amino acid sequences. (i) SEQ ID NO: 4 (CDR1), SEQ ID NO: 6 (CDR2), and SEQ ID NO: 8 (CDR3) arranged in order from N to C of the heavy chain of the antibody, and SEQ ID NO: 12 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 16 (CDR3) arranged in order from N to C of the light chain of the antibody, (ii) SEQ ID NO: 20 (CDR1), SEQ ID NO: 22 (CDR2), and SEQ ID NO: 24 (CDR3) arranged in order from N to C of the heavy chain of the antibody, and SEQ ID NO: 28 (CDR1), SEQ ID NO: 30 (CDR2), and SEQ ID NO: 32 (CDR3) arranged in order from N to C of the light chain of the antibody, or (iii) SEQ ID NO: 36 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 40 (CDR3) arranged in order from N to C of the heavy chain of the antibody, and SEQ ID NO: 44 (CDR1), SEQ ID NO: 46 (CDR2), and SEQ ID NO: 48 (CDR3) arranged in order from N to C of the light chain of the antibody.

[0056] As used herein, the term "insulin-degrading enzyme (IDE)" refers to insulysin or insulin protease, which is a large zinc-binding protease of the M16A metalloprotease subfamily involved in the intracellular processing of a plurality of small polypeptides including amyloid β-protein (Aβ), insulin, glucagon, amylin, atrial natriuretic factor, and calcitonin (e.g., GenBank accession numbers NM_004969 and NP_004960). According to a particular embodiment, the IDE is human IDE. An exemplary IDE of the present invention is the one shown in EC3.4.24.56.

[0057] As used herein, "wild-type human insulin-degrading enzyme," also referred to as WT IDE, refers to the expression product of the IDE gene of functional human IDE. It has an affinity of 100 nM for insulin and degrades insulin at a rate of 36.6 micromoles. Human recombinant IDE degrades human insulin at a rate of 1 μmole / min. An exemplary WT IDE is shown in SEQ ID NO: 50.

[0058] As used herein, "mutant IDE E111Q type" refers to a modified type with reduced catalytic activity of human IDE, which has a mutation in at least one catalytic site (from glutamic acid at position 111 corresponding to SEQ ID NO: 50 to glutamine). An exemplary mutant IDE E111Q type is shown in SEQ ID NO: 52.

[0059] As used herein, the term "isolated" means at least partially separated from its natural environment (e.g., serum), or is the main antibody in a sample containing multiple antibodies, or is the only antibody in a biological sample.

[0060] As used in the present invention, the term "antibody" includes intact molecules and their functional fragments (capable of binding to an epitope of an antigen).

[0061] As used herein, the term "epitope" means any antigenic determinant of an antigen to which the paratope of an antibody binds. Epitope determinants are usually composed of chemically active surface groups of molecules such as amino acids or carbohydrate side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics.

[0062] According to certain embodiments, antibody fragments include single-chain, F ab , F ab’ and F (ab’)2 fragments, Fd, Fcab, Fv, dsFv, scFv, MBP-scFv, diabody, minibody, nanobody, F abAn expression library or single domain molecules, such as, but not limited to, VH and VL are included.

[0063] According to certain embodiments, the antibody is a full or intact antibody.

[0064] According to certain embodiments, the antibody is an antibody fragment.

[0065] Suitable antibody fragments for practicing some embodiments of the present invention include complementarity determining regions (CDRs) of immunoglobulin light chains (referred to herein as "light chains"), complementarity determining regions of immunoglobulin heavy chains (referred to herein as "heavy chains"), variable regions of light chains, variable regions of heavy chains, light chains, heavy chains, Fd fragments, and antibody fragments that essentially include the entire variable regions of both light and heavy chains such as Fv, single chain Fv (scFv), disulfide stabilized Fv (dsFv), Fab, Fab’ and F(ab’)2, or antibody fragments that include the Fc region of an antibody.

[0066] As used herein, the terms "complementarity determining region" or "CDR" are used interchangeably to refer to antigen-binding regions found within the variable regions of heavy and light chain polypeptides. Generally, an antibody includes three CDRs each of VH (CDR HI or HI, CDR H2 or H2, and CDR H3 or H3) and three each of VL (CDR LI or LI, CDR L2 or L2, and CDR L3 or L3).

[0067] The identity of amino acid residues within a variable region or a specific antibody that constitutes a CDR can be confirmed using methods well known in the art, including sequence diversity 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 positions of structural loop regions 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 Accelrys®, see Martin et al., 1989, Proc. Natl Acad Sci USA. 86:9268, and the World Wide Web site www.bioinf-org.uk / abs), available complex crystal structures defined by contact definitions (see MacCallum et al., J. Mol. Biol. 262:732-745, 1996), and "steric definition" (see, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008), among other methods.

[0068] As used herein, "variable region" and "CDR" can mean variable regions and CDRs defined by any approach known in the art, including combinations of various approaches.

[0069] According to certain embodiments, the identity of amino acid residues within an antibody that constitutes a variable region and / or CDR is confirmed by the amino acid sequence deduced from the translated coding gene.

[0070] Functional antibody fragments that include all or substantially all of the variable regions of both the light and heavy chains are defined as follows. (i) Fv: A genetically engineered fragment defined as consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH), represented as two chains. (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 by a polypeptide linker suitable as a genetic fusion single-chain molecule. (iii) Disulfide-stabilized Fv ("dsFv"): A genetically engineered antibody containing the variable region of the light chain and the variable region of the heavy chain, linked by a genetically engineered disulfide bond. (iv) Fab: A fragment of an antibody molecule containing the monovalent antigen-binding portion of the antibody molecule, which can be obtained by treating a whole antibody with the enzyme papain to produce an intact light chain and an Fd fragment of the heavy chain composed of the variable domain and the CH1 domain. (v) Fab’: An antibody molecule fragment containing the monovalent antigen-binding portion of the antibody molecule, which can be obtained by treating a whole antibody with the enzyme pepsin and then reducing it (two Fab’ fragments are obtained per antibody molecule). (vi) F(ab’)2: An antibody molecule fragment containing the monovalent antigen-binding portion of the antibody molecule, which can be obtained by treating a whole antibody with the enzyme pepsin (i.e., a dimer of Fab’ fragments linked by two disulfide bonds). (vii) A single-domain antibody or nanobody is composed of a single VH or VL domain that exhibits sufficient affinity for the antigen. (viii) Fcab: A fragment of an antibody molecule containing the Fc portion of the antibody, developed as an antigen-binding domain by introducing antigen-binding ability into the Fc region of the antibody.

[0071] According to a particular embodiment, the antibody is an scFv.

[0072] According to certain embodiments, the antibody is a scFv stabilized by fusion to E. coli maltose-binding protein, also known as MBP-scFv, for example, as described in Bach H1, et al. J Mol Biol. (2001) Sep 7;312(1):79-93 and also described in the examples below.

[0073] According to certain embodiments, the heavy chain constant region of the antibody is IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD or IgE.

[0074] According to certain embodiments, the antibody is an IgG antibody.

[0075] According to certain embodiments, the antibody isotype is IgG1 or IgG4.

[0076] The antibody can be monospecific (able to recognize one epitope or protein), bispecific (able to bind two epitopes or proteins), or multispecific (able to recognize multiple epitopes or proteins).

[0077] According to certain embodiments, the antibody is a monospecific antibody.

[0078] According to certain embodiments, the antibody is multispecific, for example, bispecific, trispecific, tetraspecific.

[0079] According to some embodiments of the present invention, the antibody is a bispecific antibody.

[0080] 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 recombinantly by DNA techniques or chemically by known methods. Chemically produced bispecific antibodies include, but are not limited to, antibodies that are reduced and reconstituted to maintain bivalent properties, and antibodies that are chemically coupled to have at least two antigen recognition sites for each antigen. Bispecific antibodies include all antibodies or antibody complexes capable of recognizing two different antigens, or multimers of antibodies.

[0081] According to certain embodiments, the antibody is a monoclonal antibody.

[0082] Methods for making antibodies and their fragments are well known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, which is incorporated herein by reference).

[0083] According to certain embodiments, the antibody is a recombinant produced in mammalian cells.

[0084] According to certain embodiments, the antibody is a recombinant produced in bacteria.

[0085] Antibody fragments according to some embodiments of the present invention can be produced by proteolytic hydrolysis of the antibody, or by expression in E. coli cells or mammalian cells (e.g., Chinese hamster ovary cell cultures or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin digestion or papain digestion of the complete antibody by conventional methods. For example, an antibody fragment can be generated by obtaining a 5S fragment designated as F(ab’)2 by enzymatic cleavage of the antibody with pepsin. This fragment can be further cleaved using a thiol reducing agent and, optionally, a blocking group for the sulfhydryl groups resulting from cleavage of the disulfide bonds to generate a 3.5S Fab’ monovalent fragment. Alternatively, enzymatic cleavage using pepsin directly generates two monovalent Fab’ fragments and one Fc fragment. These methods are described, for example, in Goldenberg (U.S. Pat. Nos. 4,036,945 and 4,331,647) and the references contained in those patents, the entire contents of which are hereby incorporated herein by reference as part of this specification. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving the antibody (e.g., separation of the heavy chain to form monovalent light and heavy chain fragments, further cleavage of the fragments, or other enzymatic, chemical or genetic techniques) can also be used as long as the fragment binds to the antigen recognized by the intact antibody.

[0086] The Fv fragment contains the association of the VH and VL chains. As described by Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720)], this association may be non-covalent. Alternatively, the variable chains can be linked by intermolecular disulfide bonds or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragment contains a VH chain and a VL chain linked by a peptide linker. The preparation of such single-chain antigen-binding proteins (sFv) is carried out by constructing a structural gene containing DNA sequences encoding VH and VL domains linked by oligonucleotides. This structural gene is inserted into an expression vector and then introduced into a host cell such as E. coli. The recombinant host cell synthesizes a single polypeptide chain with a linker peptide that bridges the two V domains. Methods for generating sFv are described, for example, in 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 (the entire content of which is incorporated herein by reference).

[0087] Another form of the antibody fragment is a peptide encoding a single complementarity-determining region (CDR). The CDR peptide ("minimal recognition unit") can be obtained by constructing a gene encoding the CDR of the antibody of interest. Such genes can be prepared, for example, by synthesizing the variable region from the RNA of antibody-producing cells using the polymerase chain reaction. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].

[0088] According to a particular embodiment, the antibody is a chimeric antibody.

[0089] According to certain embodiments, the antibody is a humanized antibody. A humanized form of a non-human (e.g., murine) antibody is a chimeric molecule of an immunoglobulin, an immunoglobulin chain, or a fragment thereof (e.g., Fv, Fab, Fab’, F(ab’) 2 or other antigen-binding sub-sequence of an antibody) that contains a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies contain residues from complementarity-determining regions (CDRs) of the recipient that have been substituted with residues from CDRs of a non-human species such as a mouse, rat, or rabbit (donor antibody) that have the desired specificity, affinity, and capacity. Optionally, framework residues of the human immunoglobulin are substituted with the corresponding non-human residues. Humanized antibodies may also contain residues that are not found in the transferred CDRs or framework sequences of the recipient antibody. In general, a humanized antibody contains substantially all of at least one (usually two) variable domains, 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. It is also optimal for a humanized antibody to contain at least a portion of the immunoglobulin constant region (Fc), usually 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)].

[0090] Methods for humanizing non-human antibodies are well known in the art. In general, humanized antibodies have one or more amino acid residues introduced from a non-human source. Such non-human amino acid residues are often referred to as grafted residues and are usually obtained from the grafted variable domain. Humanization can essentially be carried out according to the methods of Winter and his colleagues [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 using rodent CDRs or CDR sequences in place of the corresponding sequences of human antibodies. Thus, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which substantially fewer than intact human variable domains are replaced with the corresponding sequences from non-human species. In practice, humanized antibodies are usually human antibodies in which some CDR residues and possibly some FR residues are replaced with residues from the analogous sites of rodent antibodies.

[0091] According to certain embodiments, the antibody is a human antibody.

[0092] The production of human antibodies can also be carried out using various techniques known in the art, including 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 of Cole et al. and Boerner et al. can also be utilized for the preparation of human monoclonal antibodies (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)). Similarly, transgenic animals, for example, mice in which the endogenous immunoglobulin genes are partially or completely inactivated, can be introduced with the human immunoglobulin locus to generate human antibodies. The production of human antibodies is observed upon challenge, which closely resembles that seen in humans in all aspects such as gene rearrangement, construction, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, and the following scientific literature: 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).

[0093] It should be understood that targeting of specific compartments within the cell can be achieved by intracellular antibodies (also known as "intrabodies"). These are essentially SCAs to which intracellular localization signals have been added (e.g., from the ER, mitochondria, nucleus, cytoplasm). This technique has been successfully used in the art (see Richardson and Marasco, 1995, TIBTECH vol. 13 for reports). Intrabodies virtually eliminate the expression of cell surface receptors that would otherwise be present in large amounts and inhibit protein function within the cell (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, see International Publication No. 94 / 02610 of Marasco et al. and International Publication No. 95 / 03832 of Duan et al.).

[0094] According to certain embodiments, since the IDE is expressed not only on the cell surface but also in the cytosol, peroxisomes, and endosomes, the antibody is an intracellular antibody.

[0095] In this regard, it has been shown that intracellular antibodies can inhibit activity by blocking the movement of proteins to the correct compartments (see, for example, Boldicke 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 intracellular antibodies of some embodiments of the present invention do not necessarily need to inhibit the catalytic activity inherent to IDE itself.

[0096] To generate an intracellular antibody expression vector, the light and heavy chains of an antibody specific for a target protein of interest were isolated, typically from a hybridoma that secretes a monoclonal antibody specific for APLP1. Hybridomas that secrete anti-APLP1 monoclonal antibodies or recombinant monoclonal antibodies can be generated by methods known in the art. Once a monoclonal antibody specific for the APLP1 protein (e.g., either a hybridoma-derived monoclonal antibody or a recombinant antibody from a combinatorial library) is isolated, the DNA encoding the light and heavy chains of the monoclonal antibody is isolated using standard molecular biology techniques. In the case of hybridoma-derived antibodies, the cDNA of the light and heavy chains can be obtained, for example, by PCR amplification or screening of a cDNA library. In the case of recombinant antibodies derived from, e.g., a phage display library, the cDNA encoding the light and heavy chains can be recovered from the display package (e.g., phage) isolated in the library screening step, and the nucleotide sequences of the genes for the light and heavy chains of the antibody can be determined. For example, many such sequences are disclosed in Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 and the "Vbase" human germline sequence database. Once obtained, the light and heavy chain sequences of the antibody are cloned into a recombinant expression vector using standard methods.

[0097] For cytoplasmic expression of the light and heavy chains, the nucleotide sequences encoding the hydrophobic leader sequences of the light and heavy chains are removed. An intracellular antibody expression vector can encode one of several forms of an intracellular antibody. For example, in one embodiment, the vector encodes the full-length antibody light and heavy chains and expresses the full-length antibody intracellularly. In another embodiment, the vector encodes the full-length of the light chain but only the VH / CH1 region of the heavy chain and expresses the Fab fragment intracellularly. In another embodiment, the vector encodes a single-chain antibody (scFv), and the variable regions of the light and heavy chains are linked with a flexible peptide linker [e.g., (Gly 4 Ser) 3 and expressed as a single-chain molecule. To inhibit APLP1 activity intracellularly, an expression vector encoding an intracellular antibody is introduced into cells by standard transduction methods described below.

[0098] In some embodiments, the antibodies provided herein can be functionally associated with a cell-penetrating agent. As used herein, the term "cell-penetrating agent" means an agent that enhances the transfer of an antibody across the cell membrane.

[0099] Exemplary cell-penetrating agents are cell-penetrating peptides.

[0100] As used herein, the "cell-penetrating peptide" refers to a peptide containing a short (about 12 to 30 residues) amino acid sequence or its functional motif that confers energy-dependent (i.e., non-endocytotic) translocation properties related to the translocation of a membrane-permeable complex across the cytoplasmic membrane and / or nuclear membrane of a cell. In some embodiments of the present invention, the cell-penetrating peptide used in the membrane-permeable complex preferably contains at least one non-functional cysteine residue that can freely form or be derivatized to form a disulfide bond with a double-stranded ribonucleic acid modified for such a bond. Representative amino acid motifs that confer such properties are listed in U.S. Patent No. 6,348,185, the contents of which are hereby expressly incorporated by reference. Examples of cell-penetrating peptides in some embodiments of the present invention include, but are not limited to, penetratin, transportan, pIsl, TAT(48-60), pVEC, MTS, and MAP.

[0101] In addition or alternatively, lipid particles such as liposomes may be used as cell penetration agents.

[0102] Liposomes include synthetic (i.e., non-natural) structures composed of a lipid bilayer that encloses a volume. Examples of liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes can be prepared by any method known 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]. Liposomes may be positively charged, negatively charged, or neutral.

[0103] For example, any method known in the art can be used to introduce an antibody into a liposome, such as the methods described by Alfonso et al. [The science and practice of pharmacy, Mack Publishing, Easton Pa 19 th ed., (1995)] and Kulkarni et al. [J. Microencapsul. 1995, 12 (3) 229-46].

[0104] To determine the liposomes particularly suitable for the present invention, screening assays can be performed, such assays being described in US Patent Application Publication Nos. 20040266734 and 20040266734, as well as Danenberg et al., Journal of cardiovascular pharmacology 2003, 42:671-9; Circulation 2002, 106:599-605; Circulation 2003, 108:2798-804.

[0105] According to one embodiment, the antibodies in some embodiments of the present invention are prepared as follows. Purified IDE is used to isolate specific single-chain variable fragment (scFv) clones that recognize epitopes of human IDE. Phage display technology using a human synthetic antibody phage display library is used. Specifically, a human scFv library, for example, the Ronit 1 library [described in Azriel-Rosenfeld et al. J Mol Biol. (2004) 335(1):177-192, which is hereby incorporated by reference in its entirety] is screened. The library is subjected to removal of non-specific proteins, the protein is purified from bacteria using an affinity column, and selection is performed using recombinant human wild-type (WT) IDE (SEQ ID NO: 50). Phages enriched by several cycles (e.g., 2 cycles) on mutant IDE (e.g., E111Q) are removed (i.e., negative selection) in order to enrich for positive phages that recognize WT IDE or, according to certain embodiments, recognize more of the mutant IDE than the wild-type IDE. After each of these removal steps, a selection step (i.e., positive selection) on WT IDE follows. After the affinity selection cycles, individual clones of the infected bacteria are taken out and grown. The phages of each clone are then tested (e.g., by ELISA) for binding to non-specific proteins as an IDE-binding pair (as a negative control). The positive clones are analyzed by PCR amplification, followed by fingerprinting to detect different clones, and further sequencing to demonstrate integrity and differences between the clones.

[0106] An important feature in the isolation of the antibodies in some embodiments of the present invention is the selection step. Thus, in one embodiment, a method for producing an antibody comprises (a) providing a plurality of antibodies (such as those described above), and (b) performing screening of the antibodies to select an antibody that binds to wild-type IDE but does not bind to a mutant IDE with reduced catalytic activity compared to the wild-type IDE.

[0107] Once an antibody is obtained, it may be tested for activity. Examples of methods for testing antibody activity include, for example, enzyme-linked immunosorbent assay (ELISA) and in vitro insulin degradation assay.

[0108] According to certain embodiments, the antibody is tested for IDE inhibitory activity. IDE inhibitory activity means specifically downregulating the enzymatic activity of IDE. For example, the IC 50 of the IDE inhibitory peptide ADT-21 was analyzed and was in the range of 10 - 200 nM (e.g., 1 - 100 nM, 1 - 50 nM, 1 - 20 nM or 1 - 10 nM).

[0109] According to an exemplary embodiment, the antibody may downregulate 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% of the catalytic activity of IDE.

[0110] According to an exemplary embodiment, the antibody may downregulate 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% of the insulin degradation activity of IDE.

[0111] According to certain embodiments, the antibody is capable of downregulating at least 50% of the insulin degradation activity of IDE.

[0112] According to an exemplary embodiment, the antibody may downregulate 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% of the insulin degradation activity of IDE.

[0113] According to an exemplary embodiment, the antibody can downregulate the activity of IDE such that the insulin degradation rate of IDE is 25 to 50 micromoles, 25 to 75 micromoles, 25 to 100 micromoles, 50 to 75 micromoles, 50 to 100 micromoles, or 75 to 100 micromoles, and human recombinant IDE degrades at 1 micromole of human insulin per minute.

[0114] As described above, according to certain embodiments, an antibody (e.g., single-chain Fv) may be produced by recombinant DNA techniques in which the nucleic acid of the antibody is ligated to an expression vector and subsequently introduced into a host cell.

[0115] Thus, according to one aspect of the invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding an antibody of some embodiments of the invention.

[0116] According to one embodiment of the invention, the nucleic acid sequence of the antibody comprises SEQ ID NOs: 3, 5, 7, 11, 13, and 15.

[0117] According to another embodiment of the invention, the nucleic acid sequence of the antibody comprises SEQ ID NOs: 19, 21, 23, 27, 29, and 31.

[0118] According to yet another embodiment of the invention, the nucleic acid sequence of the antibody comprises SEQ ID NOs: 35, 37, 39, 43, 45, and 47.

[0119] Non-limiting examples of nucleic acid sequences encoding the light chain, heavy chain, CDR, and variable regions that make up an antibody that can be used with some embodiments of the invention are provided by SEQ ID NOs: 1, 9, 17, 25, 33, and 41.

[0120] Thus, according to certain embodiments, 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 to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, and 41, each possibility representing an individual embodiment of the invention.

[0121] As used herein, the terms "polynucleotide" or "nucleic acid sequence" refer to single-stranded or double-stranded nucleic acid sequences that are isolated and provided in the form of RNA sequences, complementary polynucleotide sequences (cDNA), genomic polynucleotide sequences, and / or complex polynucleotide sequences (e.g., combinations as described above).

[0122] To express an exogenous antibody in mammalian cells, the polynucleotide sequence encoding the antibody is preferably ligated to a nucleic acid construct suitable for mammalian cell expression.

[0123] Thus, according to one aspect of the present invention, a nucleic acid construct comprising an isolated polynucleotide is provided.

[0124] Such nucleic acid constructs or systems contain at least one cis-element that directs the expression of the nucleic acid sequence. Cis-element sequences include not only those that direct the constitutive expression of nucleotide sequences, but also those that direct the inducible expression of nucleotide sequences only under specific conditions. Thus, for example, a promoter sequence for constitutively or inducibly supporting the transcription of a polynucleotide sequence in a cell is included in the nucleic acid construct. It should be noted that the nucleic acid sequences of the present invention can also be used for in vivo applications, which are administered as naked DNA to a subject in need thereof (e.g., a subject with diabetes) or ligated to a nucleic acid construct useful for gene therapy (e.g., a viral vector).

[0125] Host cells containing the polynucleotides / expression vectors described in this application are also provided.

[0126] Such cells are usually selected for high expression of recombinant proteins (e.g., bacterial cells, plant cells, or eukaryotic cells, such as CHO, HEK-293 cells), but can also be immune cells (e.g., macrophages, dendritic cells, T cells, B cells, or NK cells) when, for example, grafting the CDR of a drug into a T cell receptor used in adoptive cell therapy or a CAR transduced into the cell.

[0127] Thus, according to one aspect of the present invention, there is provided a method for producing an anti-IDE antibody, the method comprising expressing a polynucleotide / expression construct in a host cell.

[0128] Further, when the antibody is produced in vitro, according to certain embodiments, the recovery or isolation of the recombinant antibody is carried out over a period of culture. The phrases "recovery of the recombinant antibody" or "isolation of the recombinant antibody" mean the collection of the entire culture medium containing the antibody and do not necessarily mean steps for additional separation or purification. Instead, the phrases "recovery of the recombinant antibody" or "isolation of the recombinant antibody" mean recovering the product from the lysate of acidified cells. Notwithstanding the above, the antibodies in some embodiments of the present invention can also be produced using various standard protein purification techniques, which include, but are 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 certain embodiments, the antibody is purified to at least 80%, 85%, 90%, 95%, 97%. As used herein, "purified" means that the composition containing the antibody is free of other proteinaceous substances that are not the antibody of interest.

[0129] As described in the Examples section below, multiple anti-IDE antibodies were produced using the methodologies described herein.

[0130] Thus, according to one aspect of the present invention, there is provided an isolated antibody comprising an antigen recognition region comprising the complementarity determining region (CDR) amino acid sequences set forth in SEQ ID NOs: 4, 6, 8, 12, 14, and 16.

[0131] In one embodiment, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8 are arranged in order (N→C, as CDR1-3 respectively) on the heavy chain of the antibody, and SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16 are arranged in order (N→C, as CDR1-3 respectively) on the light chain of the antibody.

[0132] According to some embodiments of the present invention, 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%, 98%, 99%, for example, 100% sequence homology or identity to the amino acid sequence of SEQ ID NO: 2.

[0133] Thus, according to one aspect of the present invention, there is provided an isolated antibody comprising an antigen recognition region comprising the complementarity determining region (CDR) amino acid sequences shown in SEQ ID NO: 20, 22, 24, 28, 30, and 32.

[0134] According to a particular embodiment, SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24 are arranged in order (N→C, as CDR1-3 respectively) on the heavy chain of the antibody, and SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32 are arranged in order (N→C, as CDR1-3 respectively) on the light chain of the antibody.

[0135] According to some embodiments of the present invention, 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%, 98%, 99%, for example, 100% sequence homology or identity to the amino acid sequence of SEQ ID NO: 18.

[0136] Thus, according to one aspect of the present invention, there is provided an isolated antibody comprising an antigen recognition region comprising the complementarity determining region (CDR) amino acid sequences shown in SEQ ID NO: 36, 38, 40, 44, 46, and 48.

[0137] According to certain embodiments, SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO: 40 are arranged in sequence (N→C, as CDR1-3 respectively) on the heavy chain of the antibody, and SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48 are arranged in sequence (N→C, as CDR1-3 respectively) on the light chain of the antibody.

[0138] According to some embodiments of the present invention, 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%, 98%, 99%, for example, 100% sequence homology or identity to the amino acid sequence of SEQ ID NO: 34.

[0139] Homology (e.g., percent homology, identity + similarity) can be determined using any homology comparison software, such as, for example, when starting from a polypeptide sequence, BlastP or TBLASTN software of the National Center for Biotechnology Information (NCBI) that uses default parameters. Alternatively, the tBLASTX algorithm (available from NCBI) that uses default parameters for comparing the six-frame conceptual translation products of a nucleotide query sequence (both strands) to a protein sequence database can be mentioned.

[0140] For example, the default parameters of tBLASTX include the following: maximum number of target sequences: 100, expect threshold: 10, word size: 3, maximum number of matches within query range: 0, scoring parameters: Matrix - BLOSUM62, filter and masking: Filter - low complexity regions.

[0141] The antibody in some embodiments of the present invention may be used for the treatment of IDE activity-related diseases in a subject in need of treatment.

[0142] Thus, according to one aspect of the present invention, there is provided a method for treating an IDE activity-related disease in a subject in need of treatment, comprising administering a therapeutically effective amount of the antibody (ii) or (iii) described herein to the subject, wherein said administration prevents or treats a disease associated with IDE activity.

[0143] According to an additional or alternative aspect of the present invention, there is provided the antibody (ii) or (iii) described herein for use in treating an IDE activity-related disease in a subject in need of treatment.

[0144] In this regard, as described above, since IDE is expressed not only on the cell surface but also in the cytosol, peroxisomes, and endosomes, antibodies capable of penetrating into cells (antibodies functionally related to intracellular antibodies or cell-penetrating agents) in some embodiments can block the movement of IDE to the correct compartment and inhibit its activity. Thus, it is not always necessary to inhibit the catalytic activity specific to IDE itself in order to treat IDE activity-related diseases.

[0145] Therefore, according to one aspect of the present invention, there is provided a method for treating an IDE activity-related disease in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of an antibody comprising an antigen recognition region that specifically binds to IDE, wherein said antigen recognition domain comprises the amino acid sequences of the following complementarity-determining regions (CDRs): (i) SEQ ID NO: 4 (CDR1), SEQ ID NO: 6 (CDR2), and SEQ ID NO: 8 (CDR3) arranged in order from N to C of the heavy chain of the antibody, and SEQ ID NO: 12 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 16 (CDR3) arranged in order from N to C of the light chain of the antibody, (ii) SEQ ID NO: 20 (CDR1), SEQ ID NO: 22 (CDR2), and SEQ ID NO: 24 (CDR3) arranged in order from N to C of the heavy chain of the antibody, and SEQ ID NO: 28 (CDR1), SEQ ID NO: 30 (CDR2), and SEQ ID NO: 32 (CDR3) arranged in order from N to C of the light chain of the antibody, or (iii) The sequences SEQ ID NO: 36 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 40 (CDR3) arranged in order from N to C of the heavy chain of the antibody, and the sequences SEQ ID NO: 44 (CDR1), SEQ ID NO: 46 (CDR2), and SEQ ID NO: 48 (CDR3) arranged in order from N to C of the light chain of the antibody, and when the antibody is antibody (iii), the antibody is an intracellular antibody or is functionally associated with a cell permeabilizing agent, A method is provided for preventing or treating a disease associated with IDE activity by such administration.

[0146] According to an additional or alternative aspect of the invention, there is provided an antibody comprising an antigen recognition region that specifically binds to IDE, wherein the antigen recognition domain comprises the following complementarity determining region (CDR) amino acid sequences. (i) The sequences SEQ ID NO: 4 (CDR1), SEQ ID NO: 6 (CDR2), and SEQ ID NO: 8 (CDR3) arranged in order from N to C of the heavy chain of the antibody, and the sequences SEQ ID NO: 12 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 16 (CDR3) arranged in order from N to C of the light chain of the antibody, (ii) The sequences SEQ ID NO: 20 (CDR1), SEQ ID NO: 22 (CDR2), and SEQ ID NO: 24 (CDR3) arranged in order from N to C of the heavy chain of the antibody, and the sequences SEQ ID NO: 28 (CDR1), SEQ ID NO: 30 (CDR2), and SEQ ID NO: 32 (CDR3) arranged in order from N to C of the light chain of the antibody, or (iii) The sequences SEQ ID NO: 36 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 40 (CDR3) arranged in order from N to C of the heavy chain of the antibody, and the sequences SEQ ID NO: 44 (CDR1), SEQ ID NO: 46 (CDR2), and SEQ ID NO: 48 (CDR3) arranged in order from N to C of the light chain of the antibody, and when the antibody is antibody (iii), the antibody is an intracellular antibody or is functionally associated with a cell permeabilizing agent, An antibody is provided for use in the treatment of a disease associated with IDE activity in a subject in need of treatment.

[0147] As used herein, the term "IDE activity-related disease" refers to a pathological condition, disease or syndrome in which IDE activity contributes to the onset or progression.

[0148] As described above, IDE is an enzyme that cleaves a plurality of small proteins consisting of diverse sequences, including insulin, amyloid β-protein (Aβ), glucagon, amylin, atrial natriuretic factor and calcitonin. Thus, in situations where an increase in the levels of these substrates helps to improve symptoms and even achieve a cure for the disease, the antibodies in some embodiments of the present invention may be used.

[0149] According to certain embodiments, an increase in insulin levels forms the basis for the treatment of a disease or disorder.

[0150] Such diseases or abnormalities include, but are not limited to, autoimmune diseases of the central nervous system, neurodegenerative diseases, metabolic syndrome, diabetes, obesity, hyperglycemia, retinal damage, renal failure, nerve damage, microvascular damage, varicella zoster virus (VZV) infection and trauma. As used herein, the term "autoimmune disease of the central nervous system" refers to a disease in which the body's immune system attacks its own nervous system (preferably the CNS).

[0151] Examples of autoimmune diseases of the CNS include, but are not limited to, multiple sclerosis, Guillain-Barré syndrome, Lambert-Eaton myasthenic syndrome, myasthenia gravis, transverse myelitis, progressive multifocal leukoencephalopathy, chronic headache, cerebral palsy, lupus, immune dysfunction muscular central nervous system breakdown, central nervous system vasculitis, autoimmune cerebellar degeneration, gait ataxia with late-onset polyneuropathy (GALOP), neuromyelitis optica, stiff-person syndrome, and HTLV-1 associated myelopathy (HAM) / tropical spastic paraparesis (TSP).

[0152] According to certain embodiments, autoimmune diseases of the central nervous system are selected from the group consisting of multiple sclerosis, Guillain-Barré syndrome, Lambert-Eaton myasthenic syndrome, myasthenia gravis, transverse myelitis, progressive multifocal leukoencephalopathy, chronic headache, and cerebral palsy.

[0153] According to certain embodiments, autoimmune diseases of the central nervous system include multiple sclerosis (MS).

[0154] As used herein, "neurodegenerative disease" means an abnormality, disease, or condition of the nervous system (preferably the CNS) characterized by a progressive and stepwise loss of nerve tissue, neurotransmitters, or nerve function.

[0155] Specific examples of neurodegenerative diseases include, but are not limited to, amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease), autoimmune encephalomyelitis, degenerative nerve diseases, encephalitis (e.g., Rasmussen encephalitis), Alzheimer's disease, epilepsy, hereditary brain disorders, stroke, Parkinson's disease, and Huntington's disease.

[0156] According to certain embodiments, the neurodegenerative disease is Parkinson's disease.

[0157] According to certain embodiments, the neurodegenerative disease is Alzheimer's disease.

[0158] According to certain embodiments, the disease is selected from the group consisting of metabolic syndrome, diabetes, and obesity.

[0159] According to certain embodiments, the disease is metabolic syndrome.

[0160] As used herein, the term "metabolic syndrome" refers to a cluster or set of metabolic symptoms [abdominal obesity, elevated fasting blood glucose, "dyslipidemia" (i.e., elevated lipid levels), and hypertension (HBP)] that occur more often in relation than by chance, and that together promote the development of type 2 diabetes and cardiovascular disorders. Metabolic syndrome is characterized not only by a specific lipid profile of increased triglycerides, decreased high-density lipoprotein cholesterol (HDL-cholesterol), and in some cases, moderately elevated low-density lipoprotein cholesterol (LDL-cholesterol) levels, but also by an accelerated progression of atherosclerosis due to the pressure of the constituent risk factors.

[0161] According to certain embodiments, the disease is diabetes.

[0162] As used herein, "diabetes" means absolute insulin deficiency (type 1 diabetes) due to a defect in biosynthesis or insulin production in an organism, or relative insulin deficiency (type 2 diabetes) in the presence of insulin resistance, i.e., incomplete insulin activity. Diabetic patients thus exhibit absolute or relative insulin deficiency and may show, in addition to other symptoms and signs, elevated blood glucose levels, the presence of sugar in the urine, excessive urination (polyuria), increased thirst in the throat (polydipsia), and increased hunger (polyphagia). In type 1 diabetes, symptoms can occur rather rapidly (e.g., within a few weeks or months). However, the symptoms of type 2 diabetes are more gradual and may be minimal or completely absent. Diabetes (both types) can also cause rapid and significant weight loss and unrelenting mental fatigue (despite normal or increased food intake). As used herein, diabetes encompasses diabetes at any stage or type (including, but not limited to, overt diabetes, borderline diabetes, and latent autoimmune diabetes in adults (LADA)).

[0163] According to certain embodiments, the diabetes is type 1 diabetes.

[0164] According to certain embodiments, the diabetes is type 2 diabetes.

[0165] Examples of diabetes-related diseases include, but are not limited to, type I diabetes, type II diabetes, gestational diabetes, insulin resistance, obesity, hyperglycemia, eye diseases (e.g., glaucoma, cataracts), skin infections, hypertension, gastric paresis, diabetic ketoacidosis (DKA), neuropathy (e.g., diabetic neuropathy), hyperosmolar hyperglycemic nonketotic syndrome (HHNS), kidney diseases (renal impairment), and peripheral arterial disease (PAD).

[0166] According to other specific embodiments, the disease is a wound.

[0167] The term "wound" as used in the present application means damage to internal organs as well as the skin and subcutaneous tissues, which begins by any one of various methods (e.g., displacement, wounds caused by trauma, wounds occurring during or after surgical procedures, etc.) and has various characteristics. Exemplary ones include contusions, scratches, burns, sunburns, incisions, excisions, surgical wounds, necrotizing fasciitis, ulcers, venous stasis ulcers, diabetic ulcers, pressure ulcers, decubitus ulcers, aphthous ulcers, allergic contact dermatitis, atopic dermatitis, Berloque dermatitis, diaper rash, dyshidrotic dermatitis, psoriasis, eczema, warts, anal warts, angiomas, senile angiomas, athlete's foot, atypical moles, basal cell carcinoma, Bateman's purpura, bullous pemphigoid, candidiasis, chondrodermatitis helicis, Clark's nevus, herpes labialis, condyloma, cysts, Darier's disease, dermatofibroma, discoid lupus erythematosus, nummular eczema, atopic eczema, dyshidrotic eczema, hand eczema, erythema nodosum leprosum, Fordyce's disease, folliculitis decalvans, folliculitis, granuloma annulare, Grover's disease, miliaria, herpes simplex, herpes zoster, hidradenitis suppurativa, pityriasis rosea, hyperhidrosis, ichthyosis, impetigo, keratosis pilaris, keloid, keratoacanthoma, lichen planus, lichen planus-like keratosis, lichen simplex chronicus, lichen sclerosus, lymphomatoid papulosis, lupus erythematosus, Lyme disease, lichen striatus, mucinous cyst, pyogenic granuloma, molluscum contagiosum, moles, nail fungus, necrobiosis lipoidica diabeticorum, nummular eczema, onychoschizia, onychomycosis, pityriasis lichenoides, pityriasis rosea, pityriasis rubra pilaris, plantar warts, poison ivy, poison oak, dyshidrosis, pseudofolliculitis barbae, pruritus ani, and pityriasis alba, but is not limited thereto. Wounds are typically classified into one of four grades according to their depth. (i) Grade I: wounds limited to the epithelium, (ii) Grade II: wounds reaching the dermis, (iii) Grade III: wounds reaching the subcutaneous tissue, and (iv) Grade IV (or full-thickness wounds): wounds in which bone is exposed (e.g., bony pressure points such as the greater trochanter or the sacrum).

[0168] The term "partial thickness wound" means, in the present application, wounds including grades I - III, and includes burns, crush injuries, venous stasis ulcers, and diabetic ulcers.

[0169] The term "full - thickness wound" means, in the present application, wounds including both grades III and IV.

[0170] The term "chronic wound" means, in the present application, a wound that has not healed within 30 days.

[0171] The term "healing" for a wound means, for example, the process of wound repair by scar formation (in an exemplary embodiment, healing lacks fibrotic tissue formation).

[0172] In certain embodiments, the compositions in some embodiments of the present invention promote, i.e., accelerate, the healing process.

[0173] The phrase "induce or accelerate the healing process of a skin wound" means either the induction of granulation tissue formation in wound contraction and / or epithelialization (i.e., the production of new cells in the epithelium). Wound healing is easily measured by a decrease in wound area.

[0174] Certain embodiments of the present invention contemplate the treatment of all types of wounds, including full - thickness wounds, acute wounds, chronic wounds, diabetes - related wounds, ischemic wounds, ulcers, burns, and surgical wounds.

[0175] According to certain embodiments, the wound is selected from the group consisting of chronic wounds, acute wounds, diabetic wounds, ischemic wounds, ulcers, burns, and surgical wounds.

[0176] Additional diseases and abnormalities that can be treated in certain embodiments include non-ketotic hyperosmolar coma, viral infections [e.g., varicella-zoster virus (VZV)], atherosclerosis, hypertension, cardiovascular disorders such as congenital heart disease, cardiomyopathy, aortic valve stenosis, atrial septal defect (ASD), common atrioventricular (A-V) valve defect, ductus arteriosus, pulmonary valve stenosis, subaortic stenosis, ventricular septal defect (VSD), valvular disease, myocardial infarction, tuberous sclerosis, scleroderma, transplantation, endometriosis, infertility, von Hippel-Lindau (VHL) syndrome, cirrhosis, transplantation, hemophilia, hypercoagulability, idiopathic thrombocytopenic purpura, immunodeficiency, retinitis pigmentosa (autosomal dominant), retinitis pigmentosa (autosomal recessive), Pakistani type SEMD, urogenital syndrome, cholesterol ester storage disease, Tyrosinase-negative corneal dystrophy, Dubin-Johnson syndrome, T-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, spinocerebellar ataxia with sensory neuropathy, split hand / foot malformation type 3, tolbutamide hypometabolism group, warfarin sensitivity, Wolman disease, anterior segment dysgenesis and cataracts, congenital cataracts, neurofibrosarcoma, retinal disorders such as non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR), chronic renal failure, diabetic nephropathy, nerve damage such as diabetic neuropathy, microvascular damage, diabetes-related foot ulcers, and graft-versus-host disease, but are not limited thereto.

[0177] The term "treating" means causing inhibition, prevention or arrest of the development of a pathology (disease, abnormality or condition), and / or causing a decrease, alleviation or regression of the pathology. One of ordinary skill in the art understands that various methodologies and assays can be used to evaluate the development of a pathology, and similarly, various methodologies and assays can be used to evaluate a decrease, alleviation or regression of the pathology. Treatment can be carried out alone or in combination with other therapies.

[0178] The term "preventing" as used herein means suppressing the development of a disease, abnormality or condition in a subject who may be at risk of a disease but has not been diagnosed as having developed the disease.

[0179] As used herein, the term "subject" refers to mammalian subjects (e.g., humans) of any gender and any age, including neonates, infants, juveniles, adolescents, adults, and the elderly.

[0180] According to certain embodiments, the subject is one diagnosed with, suffering from, or having a predisposition to the above-mentioned condition.

[0181] According to certain embodiments, as described hereinafter, the IDE level in the biological sample of the subject exceeds a predetermined threshold as compared to the control biological sample.

[0182] Thus, according to certain embodiments, the method disclosed herein includes determining the IDE level in the biological sample of the subject using the antibody disclosed herein prior to administration.

[0183] The antibodies in some embodiments of the invention can be administered to a subject either as such or as part of a pharmaceutical composition.

[0184] According to one aspect of the invention, there is provided a pharmaceutical composition comprising the antibody (ii) or (iii) disclosed herein as an active ingredient and further comprising a pharmaceutically acceptable carrier.

[0185] As used herein, the term "pharmaceutical composition" means a preparation comprising one or more active ingredients described herein and other chemical components such as physiologically suitable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to a living being.

[0186] As used herein, the term "active ingredient" means an antibody that effectively participates in a biological effect.

[0187] As used interchangeably herein, the terms “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” mean a carrier or diluent that does not cause significant irritation to an organism and does not inhibit the biological activity and properties of the administered compound. Such terms include adjuvants.

[0188] As used herein, the term “excipient” means an inert substance added to a pharmaceutical composition to facilitate the administration of the active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol.

[0189] Techniques for drug formulation and administration are described in the latest edition of “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA (incorporated herein by reference).

[0190] Suitable routes of administration include, for example, oral delivery, rectal delivery, transmucosal delivery (particularly nasal delivery), enteral delivery, or parenteral delivery, including, for example, intramuscular injection, subcutaneous injection, and intramedullary injection, as well as intrathecal injection, direct intraventricular injection, intravenous injection, intraperitoneal injection, intranasal injection, or intraocular injection.

[0191] Alternatively, for example, the pharmaceutical composition can be administered locally rather than systemically by injecting the pharmaceutical composition directly into a tissue region of the patient.

[0192] According to certain embodiments, the antibodies of the present invention are administered by nasal administration.

[0193] According to certain embodiments, the antibodies of the present invention are administered by subcutaneous administration.

[0194] The pharmaceutical composition of the present invention can be produced by processes well known in the art, for example, by conventional mixing, dissolving, granulating, tablet coating, levigating, emulsifying, encapsulating, entrapping or lyophilization processes.

[0195] Thus, the pharmaceutical compositions used according to some embodiments of the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries that facilitate the processing of the active ingredient into a pharmaceutically usable preparation. The appropriate formulation is determined by the chosen route of administration.

[0196] In the case of injection, the active ingredient of the pharmaceutical composition can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. In the case of transmucosal administration, permeation enhancers suitable for the permeation barrier are used in the formulation. Such permeation enhancers are generally known in the art.

[0197] In the case of oral administration, the pharmaceutical composition can be easily formulated by combining the active compound with pharmaceutically acceptable carriers well known in the art. Such carriers allow the pharmaceutical composition to be formulated as tablets, pills, coated tablets, capsules, solutions, gels, syrups, slurries, suspensions, etc. so that it can be orally ingested by the patient. Pharmacological preparations for oral use are produced using solid excipients, and if necessary, the resulting mixture is comminuted and, if necessary, appropriate auxiliaries are added, and then the mixture of granules is processed to obtain tablet or coated tablet cores. Suitable excipients are, in particular, fillers such as sugars including lactose, sucrose, mannitol or sorbitol, cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If necessary, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salts (for example, sodium alginate) can be added.

[0198] The sugar-coated tablet core is provided with a suitable coating. For this purpose, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solution, and a concentrated sugar solution which may optionally contain a suitable organic solvent or solvent mixture can be used. Dyes or pigments can be added to the tablets or sugar-coated tablet coatings for identification or to characterize various combinations of the active compound dosages.

[0199] Examples of pharmaceutical compositions for oral use include push-fit capsules formed of gelatin and soft-seal capsules formed of gelatin and a plasticizer such as glycerol or sorbitol. In the push-fit capsules, fillers such as lactose, binders such as starch, lubricants such as talc or magnesium stearate, and optionally stabilizers can be blended with the active ingredient. In the soft capsules, the active ingredient can be dissolved or suspended in a suitable liquid (for example, fatty oil, liquid paraffin, or liquid polyethylene glycol). Further stabilizers can be added. All formulations for oral administration should be in a dosage form suitable for the selected route of administration.

[0200] For oral administration, the composition can take the form of tablets or troches formulated by conventional methods.

[0201] For administration by nasal inhalation, the active ingredient for use according to the present invention is conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant (for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide). In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve for delivering a metered amount. Dispensers, for example, gelatin capsules and drug packages, can be formulated to contain a powder mixture of the compound and a suitable powder base (for example, lactose or starch).

[0202] The pharmaceutical compositions described herein can be formulated, for example, for parenteral administration by bolus injection or continuous infusion. Injectable formulations can be supplied in unit dosage forms, for example, in ampoules, or in multiple-dose containers with added preservatives as required. The compositions can be suspensions, solutions or emulsions in oily vehicles or aqueous vehicles, and can contain formulating agents such as suspending agents, stabilizers and / or dispersing agents.

[0203] Pharmaceutical compositions for parenteral administration include aqueous solutions of water-soluble active agents. Additionally, suspensions of the active ingredient 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 acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the active ingredient to enable the preparation of a concentrated solution.

[0204] Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle (e.g., a sterile and pyrogen-free water-based solution) before use.

[0205] The pharmaceutical compositions of the present invention can also be formulated into rectal compositions such as suppositories or retention enemas using conventional suppository bases such as cocoa butter and other glycerides.

[0206] Pharmaceutical compositions suitable for use in the context of the present invention include compositions containing an effective amount of the active ingredient (i.e., the antibody) effective to achieve the original purpose. More specifically, a therapeutically effective amount means an amount of the active ingredient effective to prevent, alleviate or improve the symptoms of a disorder (e.g., an IDE-related disease) or to extend the survival of the subject being treated.

[0207] According to another embodiment of the present invention, a therapeutically effective amount increases the blood insulin level of the subject after administration.

[0208] According to another embodiment of the present invention, a therapeutically effective amount reduces pancreatic beta cell destruction in a subject after administration.

[0209] According to another embodiment of the present invention, a therapeutically effective amount increases blood insulin-like growth factor 1 (IGF1) levels in a subject after administration.

[0210] According to another embodiment of the present invention, a therapeutically effective amount reduces the secretion of IL-17 by T lymphocytes in a subject after administration.

[0211] According to another embodiment of the present invention, a therapeutically effective amount reduces the secretion of IFN-γ by T lymphocytes in a subject after administration.

[0212] The evaluation of the levels of insulin, IGF1, IL-17 or IFN-γ may be carried out using any method known to those skilled in the art (for example, ELISA).

[0213] The evaluation of pancreatic beta cell destruction may be carried out using any method known to those skilled in the art (for example, measurement of beta cell function (such as measurement of metabolic marker levels such as C-peptide), or imaging of beta cell mass [such as emission tomography (PET) or single photon emission computed tomography (SPECT)], which are taught in Lebastchi J. and Herold K.C., Cold Spring Harb Perspect Med. June 2012; 2(6): a007708, which is hereby incorporated by reference).

[0214] According to another embodiment of the present invention, a therapeutically effective amount results in inhibition or reduction of IDE activity. Inhibition or reduction of IDE activity may include a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of IDE activity. Evaluation of IDE activity reduction may be carried out using any method known to those skilled in the art (e.g., fluorescence quantitative IDE activity assay or in vitro insulin and IGF-1 degradation assay), and these methods are described in detail in the Examples section below.

[0215] Determination of a therapeutically effective amount is well within the ability of those skilled in the art, particularly in view of the detailed disclosure provided herein.

[0216] For any preparation used in the methods of the present invention, a therapeutically effective amount or dose can first be estimated from in vitro and cell culture assays. For example, doses can be formulated in animal models to obtain the desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0217] The toxicity and therapeutic efficacy of the active ingredients described herein can be confirmed by standard pharmaceutical procedures in vitro in cell cultures or experimental animals. Using the data obtained from such in vitro and cell culture assays and animal tests, dosages in the range for use in humans can be formulated. The dosage may vary depending on the dosage form used and the administration route utilized. The exact formulation, administration route and dosage can be selected by the individual physician taking into account the patient's condition (see, e.g., Fingl, et al., 1975 in "The Pharmacological Basis of Therapeutics", Ch. 1 p.1).

[0218] The dosage and dosing interval are adjusted individually to obtain a plasma level (minimum effective concentration, MEC) of the active ingredient sufficient to induce or suppress a biological effect. The MEC varies from preparation to preparation but can be estimated from in vitro data. The dosage required to obtain the MEC is determined by the individual characteristics and the route of administration. The concentration in plasma can be confirmed using a detection assay.

[0219] Depending on the severity and responsiveness of the condition being treated, the medication can be administered as a single or multiple doses, and the course of treatment can last from several days to several weeks, or until a cure is effected or the condition is suppressed.

[0220] Naturally, the amount of the composition administered depends on the subject being treated, the severity of the pain, the method of administration, the judgment of the prescribing physician, etc.

[0221] For example, in one embodiment, the antibody of the present invention may be intravenously injected (i.v.) at a dose between 0.1 mg / kg and 10 mg / kg, between 0.1 mg / kg and 5 mg / kg, between 0.1 mg / kg and 1 mg / kg, between 1 mg / kg and 10 mg / kg, between 1 mg / kg and 5 mg / kg, between 2.5 mg / kg and 5 mg / kg, between 0.5 mg / kg and 5 mg / kg, or between 5 mg / kg and 10 mg / kg. In another embodiment, the antibody of the present invention may be administered i.v. at a dose between 10 mg / kg and 100 mg / kg, between 10 mg / kg and 50 mg / kg, between 25 mg / kg and 50 mg / kg, or between 50 mg / kg and 100 mg / kg. In another embodiment, the antibody of the present invention may be administered i.v. at a dose between 100 mg / kg and 1000 mg / kg, between 100 mg / kg and 500 mg / kg, between 250 mg / kg and 500 mg / kg, or between 500 mg / kg and 1000 mg / kg.

[0222] It should be understood that there are animal models in which the antibodies of the present invention can be tested prior to treating humans. For example, STZ-induced or non-obese diabetic (NOD) mouse models of diabetes may be used as models of diabetes. Examples of animal models of multiple sclerosis include, for example, mouse EAE models (e.g., those in which the disease was induced in NOD mice by immunization with MOG(35-55) in CFA). Examples of animal models of Alzheimer's disease include, for example, APP / PSI mice and the Samaritan Alzheimer's rat model (available from Samaritan Pharmaceuticals). For wound healing, examples include the previously described diabetic mouse wound model by Galeano et al., Diabetes. (2004) 53(9):2509-17 (incorporated by reference) or the previously described diabetic rat model by Qiu et al., J Surg Res. (2007) 138(1):64-70 (incorporated by reference). Examples of animal models of Parkinson's disease include, for example, A53T Tg mice that express mutant alpha-synuclein in CNS neurons (Giasson et al. 2002) and C57BL / 6-Tg(Thy1-SNCA*E35K*E46K*E61K)3798Nuber / J (Nuber et al., 2018).

[0223] The composition of the present invention can be provided as a pack or dispenser device such as an FDA-approved kit, if necessary, and can contain one or more unit dosage forms containing the active ingredient. The pack can contain a metal or plastic foil, for example, like a blister pack. Instructions for administration can be attached to the pack or dispenser device. The pack or dispenser may be accompanied by a notice regarding a container in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals, and this notice reflects the approval by the government agency regarding the form of the composition or the administration to humans or animals. Such a notice can be, for example, the indication approved by the US Food and Drug Administration for prescription drugs, or regarding an approved product insert. For a composition containing a preparation of the present invention formulated in a compatible pharmaceutical carrier, as described in more detail above, it can also be prepared, placed in a suitable container, and labeled for the treatment of the indicated condition.

[0224] It should be understood that the therapeutic composition of the present invention may, in addition to the antibody, contain known drugs or therapeutic agents for the treatment of IDE-related diseases (e.g., diabetes, autoimmune diseases of the CNS, neurodegenerative diseases) (e.g., but not limited to, steroids, corticosteroids, immunosuppressants, antihistamines, etc.). The drug can be included in the product as a single package or individual packages.

[0225] Accordingly, according to one aspect of the present invention, there is provided a product defined for the treatment of an IDE activity-related disease, comprising the antibody (ii) or (iii) disclosed in the present application and a therapeutic agent for the treatment of said disease.

[0226] According to a particular embodiment, the antibody and the therapeutic agent are packaged in separate containers.

[0227] According to a particular embodiment, the antibody and the therapeutic agent are packaged as a co-formulation.

[0228] Furthermore, according to certain embodiments, the methods and uses disclosed herein further comprise administering to the subject a therapeutic agent (other than the antibodies disclosed herein) for treating a disease in the subject.

[0229] As shown in the Examples section below, the inventors developed a highly sensitive ELISA using the produced antibodies and used this ELISA to show a strong correlation between human serum IDE levels and the presence and / or severity of metabolic syndrome. Thus, certain embodiments of the present invention further propose a method for analyzing IDE levels for the purpose of diagnosing, monitoring treatment efficacy and / or making treatment decisions.

[0230] Accordingly, according to one aspect of the present invention, there is provided a method for diagnosing an IDE activity-related disease in a subject, comprising determining the IDE level in a biological sample of the subject using an antibody disclosed herein, and diagnosing the subject with the disease when the IDE level exceeds a predetermined threshold as compared to a control biological sample.

[0231] As used herein, the term "diagnosing" means determining the presence or absence of a pathology (i.e., whether cancer is a Ewing's tumor), classifying a pathology or symptom, determining the severity of a pathology (e.g., grade or stage), monitoring the progression of a pathology, predicting the outcome of a pathology and / or the likelihood of recovery, and screening a subject for a particular disease.

[0232] Thus, according to one aspect of the present invention, there is provided a method for predicting the prognosis of an IDE activity-related disease in a subject, comprising determining the IDE level in a biological sample of a subject diagnosed with the disease using an antibody that discloses the IDE level, and determining a poor prognosis when the IDE level exceeds a predetermined threshold as compared to a control biological sample.

[0233] According to certain embodiments, the methods (e.g., determination, contact) are performed in vitro or ex vivo.

[0234] According to certain embodiments, the methods disclosed herein include obtaining a biological sample prior to the determination.

[0235] Non-limiting examples of biological samples that can be used in some embodiments of the invention include cells, tissues, organs, blood cells, bone marrow cells, blood, serum, plasma, and body fluids such as lavage fluids that may have come into contact with diseased cells / tissues obtained from any tissue biopsy.

[0236] Biological samples can be obtained using methods known in the art, such as the use of a syringe with a needle, a scalpel, a fine needle biopsy, a needle biopsy, a core needle biopsy, a fine needle aspiration (FNA), a surgical biopsy, a buccal smear, washing, etc.

[0237] According to certain embodiments, protein molecules are extracted from the biological sample of the subject. Thus, according to certain embodiments, the method further includes extracting proteins from the biological sample prior to the determination. Methods for extracting protein molecules from biological samples are well known in the art.

[0238] As used herein, the phrase "predetermined threshold" means the IDE level that characterizes a healthy sample or a sample showing a known disease prognosis of the same origin analyzed under the same conditions. Such levels can be determined experimentally by comparing samples of known IDE levels (e.g., samples obtained from healthy subjects or subjects with known disease prognoses) with samples from subjects diagnosed with IDE activity-related diseases. Alternatively, such levels can also be obtained from scientific papers or databases.

[0239] According to certain embodiments, the predetermined threshold is derived from a control sample.

[0240] Multiple control samples can be used in certain embodiments.

[0241] Among various factors, biological characteristics depend on species and age. Therefore, it is preferable that the control sample is obtained from subjects of the same species, age, and gender, and belonging to the same subgroup (e.g., smoking group / non-smoking group).

[0242] According to a specific embodiment, the control sample includes the same type of biological sample as the biological sample of the subject.

[0243] According to a specific embodiment, the control sample is a healthy subject sample.

[0244] According to a specific embodiment, the control sample is a sample obtained from a subject having a mild disease or a disease showing a good prognosis.

[0245] According to a specific embodiment, the control sample is obtained from a scientific paper or a database.

[0246] According to a specific embodiment, an increase / decrease exceeding or falling below a predetermined threshold is statistically significant.

[0247] According to a specific embodiment, when measured by the same assay (e.g., ELISA, Western blot, IP), the predetermined threshold is at least 1.5 times, at least 2 times, at least 3 times, at least 5 times, at least 10 times, or at least 20 times compared to the IDE level of the subject sample.

[0248] According to a specific embodiment, the predetermined threshold is at least 1.5 times compared to the IDE level of the subject sample.

[0249] According to certain 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%, for example, 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600% compared to the IDE level of the target sample.

[0250] According to certain embodiments, the IDE level in a biological sample can be determined using any known method that uses the antibodies disclosed in the present application, including, but not limited to, ELISA, Western blot, and IP.

[0251] Thus, according to some embodiments, detection of the IDE level is performed by contacting a biological sample, tissue, cell, or fraction, or an extract thereof, with the antibodies disclosed in the present application.

[0252] According to certain embodiments, the contact is performed under conditions that allow the formation of a complex (i.e., an immune complex) comprising the IDE present in the biological sample and the antibody.

[0253] Immune complexes can be formed at various temperatures, salt concentrations, and pH values, which can vary depending on the method used and the biological sample, but those skilled in the art can prepare the conditions suitable for each immune complex.

[0254] Thus, according to one aspect of the present invention, there is provided a composition comprising a biological sample (or a lysate of the biological sample of a subject) diagnosed with an IDE activity-related disease and the antibodies disclosed in the present application.

[0255] According to additional or alternative aspects of the present invention, there is provided a product comprising a biological sample (or a lysate of the biological sample of a subject) diagnosed with an IDE activity-related disease and the antibodies disclosed in the present application in a separate container.

[0256] According to certain embodiments, the composition or product further comprises a protease inhibitor.

[0257] According to certain embodiments, the composition or product further comprises a secondary antibody capable of binding to the antibody.

[0258] According to certain embodiments, the antibodies disclosed herein are bound to a detectable moiety.

[0259] Examples of detectable moieties that can be used in the present invention include, but are not limited to, radioisotopes, phosphorescent chemicals, chemiluminescent chemicals, fluorescent chemicals, enzymes, fluorescent polypeptides, radioisotopes (

[0125] such as iodine) and epitope tags. The detectable moiety can be a member of a binding pair (which can be identified by interaction with a further member of the binding pair) and a label that is directly visualized. In one example, the member of the binding pair is an antigen that is identified by a corresponding labeled antibody. In one example, the label is a fluorescent protein or an enzyme that produces a colorimetric reaction.

[0260] Examples of suitable fluorophores include, but are not limited to, phycoerythrin (PE), fluorescein isothiocyanate (FITC), CyChrome, rhodamine, green fluorescent protein (GFP), blue fluorescent protein (BFP), Texas Red, PE-Cy5, etc. For further guidance on the selection of fluorophores and methods for conjugating 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 (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 (Targesome, Inc.).

[0261] Multiple enzymes can be conjugated to antibodies [e.g., horseradish peroxidase (HPR), β-galactosidase, and alkaline phosphatase (AP)].

[0262] Examples of identifiable moieties include, but are not limited to, green fluorescent protein, alkaline phosphatase, peroxidase, histidine tag, biotin, orange fluorescent protein, and streptavidin.

[0263] Further examples of detectable moieties include those detectable by positron emission tomography (PET) and magnetic resonance imaging (MRI), all of which are well known to those skilled in the art.

[0264] According to some embodiments, the detectable moiety binds by translationally fusing a polynucleotide encoding an antibody disclosed herein with a nucleic acid sequence encoding a therapeutic moiety or a detectable moiety.

[0265] In addition or alternatively, the therapeutic moiety or detectable moiety can be chemically conjugated (coupled) to the antibody using any conjugation method known to those skilled in the art.

[0266] According to certain embodiments, the methods disclosed herein include enhancing a diagnosis using techniques known in the art. Such methods are known in the art and non-limiting examples include fasting blood glucose tests or A1c blood tests for diabetes.

[0267] According to certain embodiments, the diagnostic method further includes treating the diagnosed subject with an effective amount of therapy for the disease.

[0268] Thus, according to one aspect of the invention, there is provided a method for treating a disease in a subject in need of treatment for an IDE activity-related disease, (a) diagnosing the subject by the method, and when the IDE level exceeds a predetermined threshold, (b) treating the subject with a therapy for the disease thereby providing a method for treating the subject's disease.

[0269] Since the inventors have shown that IDE can be a prognostic marker for metabolic syndrome, IDE levels can be used in the selection of a treatment regimen (e.g., type, dosage) suitable for a subject. That is, a disease with a poor prognosis is treated with a treatment regimen suitable for a poor prognosis, and a disease with a good prognosis is treated with a treatment regimen suitable for a good prognosis. According to certain embodiments, IDE levels can indicate a tendency for a subject to respond to a given therapy.

[0270] Thus, according to an additional or alternative aspect of the invention, a method for treating a disease in a subject in need of treatment for an IDE activity-related disease, comprising: (a) diagnosing the subject by the method and, when the IDE level exceeds a predetermined threshold, (b) selecting a therapy based on the IDE level is provided, thereby treating the disease of the subject.

[0271] According to an additional or alternative aspect of the invention, a method for treating a disease in a subject in need of treatment for an IDE activity-related disease, comprising: (a) performing a prognosis determination of the subject by the method and then (b) treating the subject with a therapy based on the prognosis determination

[0272] According to an additional or alternative aspect of the invention, a method for treating a disease in a subject in need of treatment for an IDE activity-related disease, comprising: (a) performing a prognosis determination of the subject by the method and then (b) selecting a therapy based on the prognosis determination

[0273] According to certain embodiments, the therapy comprises antibody (ii) or (iii) disclosed herein.

[0274] According to an additional or alternative aspect of the present invention, there is provided a method for monitoring the effectiveness of a therapy for an IDE activity-related disease in a subject diagnosed with the disease, the method comprising determining the IDE level in a biological sample of the subject during or after treatment using an antibody disclosed herein, and the therapy being considered effective if the IDE level during or after treatment is reduced below a predetermined threshold.

[0275] Thus, a decrease in the IDE level serves as an indicator that the therapy is effective.

[0276] On the other hand, if there is no change in the IDE level or the IDE level increases, the therapy is not effective for treating the disease, and additional and / or alternative therapies (e.g., treatment regimens) can be used.

[0277] According to a particular embodiment of the monitoring aspect disclosed herein, the predetermined threshold is compared with the IDE level in the subject before treatment.

[0278] According to a particular embodiment of the monitoring aspect 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 lower than the IDE level in a control sample or in the subject before treatment when measured by the same assay (e.g., ELISA, Western blot, IP).

[0279] According to a particular embodiment, the predetermined threshold is at least 1.5-fold lower than the IDE level in a control sample or in the subject before treatment.

[0280] According to certain 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%, for example, 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600% compared to the IDE level in a control sample or a subject prior to treatment.

[0281] According to other specific embodiments of this aspect of the invention, the predetermined threshold can also be determined in a subset of subjects with known treatment outcomes.

[0282] According to a further additional aspect of the invention, there is provided a product comprising the antibody disclosed herein, reagents suitable for ELISA, Western blot, or IP in a separate container, and a positive control sample comprising an ELISA plate and / or IDE.

[0283] As used herein, "about" refers to ±10%.

[0284] The terms "comprises", "comprising", "includes", "including", "having" and their conjugations mean "including but not limited to".

[0285] The term "consisting of" means "including and limited to".

[0286] The term "consisting essentially of" means that a composition, method or structure may include additional components, steps and / or parts. However, this is limited to the case where the additional components, steps and / or parts do not substantially change the basic and novel characteristics of the composition, method or structure recited in the claims.

[0287] As used herein, the singular forms "a", "an", and "the" include the plural reference unless the context clearly dictates otherwise. For example, "a compound" or "at least one compound" includes a plurality of compounds and may also include mixtures thereof.

[0288] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and is not to be construed as a limitation on the flexibility of the scope of the invention. Thus, a range description should be considered to specifically disclose all the sub-ranges possible and the individual numerical values within that range. For example, a range description of 1 to 6 should be considered to specifically disclose not only the sub-ranges of 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., but also the individual numerical values within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the size of the range.

[0289] When a numerical range is indicated herein, it is intended to include any recited number (fractional or integral) within the indicated range. The phrases "range between" a first recited number and a second recited number, and "range from" a first recited number "to" a second recited number are used interchangeably herein and are intended to include the first and second recited numbers, and all the fractional and integral numbers therebetween.

[0290] As used herein, the term "method" means a manner, means, technique, and procedure for achieving a given task and includes, but is not limited to, those known to, or readily developed by, practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine.

[0291] It should be understood that certain features of the invention described in connection with separate embodiments for clarity may also be provided in combination in one embodiment. Conversely, features of the invention described in connection with one embodiment for brevity may also be provided separately or in any suitable partial combination or in relation to any other suitable described embodiment. A given feature described in connection with various embodiments should not be regarded as an essential requirement of that embodiment unless the particular embodiment would be inoperative without that element.

[0292] As described above, the various embodiments and aspects of the invention described herein and claimed in the claims are experimentally supported by the following examples.

Examples

[0293] Reference is now made to the following examples, which illustrate the invention without limiting it in conjunction with the above description.

[0294] Generally, the nomenclature used herein and the experimental procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are well described in the literature. For example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989), "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994), Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and 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), the methods described in U.S. Patent 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), 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); Available immunoassays are widely described in patents and scientific literature (see, for example, U.S. Patent 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. 1-317, 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) can be cited. All of these documents are incorporated herein by reference. Other general references are provided throughout the specification. The procedures described therein are considered well-known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

[0295] Materials and Methods Mice: C57BL / 6 male mice (Jackson Laboratory) were bred in a specific pathogen-free facility within Tel Aviv University. All experiments were conducted in accordance with the guidelines of Tel Aviv University and were performed with the approval of the TAU animal care committee for animal research.

[0296] Construction and expression of the IDE vector: The 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 the form inserted between the NdeI and HindIII restriction sites of the pUC57 vector. The vector encodes rhIDE fused to an Ni-NTA binding tag and a His tag at the C-terminus. Rosetta BL21 E. coli cells were transformed with the pET28a-rhIDE expression vectors (WT and E111Q), and cultured in 0.5 L of LB medium supplemented with 25 μg / ml of kanamycin until OD 600nm = 0.6 - 0.8. Next, protein expression in the culture was induced overnight at 30°C with 0.5 mM IPTG. The cells were harvested by centrifugation at 9000 rpm for 15 minutes. The pellet was resuspended in 35 ml of PBS containing 0.1% Triton X-100 and lysed by repeating 5 times the cycle of 30 seconds of sonication and 2 minutes of incubation on ice. The soluble fraction was 4 It was clarified by centrifugation at 12,000 rpm for 30 minutes at ℃ and loaded onto a 5 ml column of GE HisTrap. The column was washed with 5 mM imidazole in PBS and eluted with 0.5 M imidazole in PBS. The purified IDE protein was dialyzed against PBS, diluted with 2 mg / ml PBS and 5% glycerol, and stored at -80 °C.

[0297] Biopanning: Phage display technology using the "Ronit1" human synthetic antibody phage display library already described in Azriel-Rosenfeld et al. J Mol Biol. (2004) 335(1):177-192 was used. The library was subjected to 4 rounds of affinity selection cycles on 10 μg / ml of WT rhIDE used to coat the wells of a 24-well plate at room temperature for 2 hours, and WT IDE was used as bait. In 2 of those cycles (cycle 1 and cycle 3), depletion was carried out without using the mutated E111Q IDE as bait with the intention of isolating antibodies that bind with high affinity to the catalytic site of the WT enzyme. 10 μg / ml of the mutated IDE was used to coat the wells of a 24-well plate at room temperature for 2 hours. The wells were washed once with 3 ml of PBS and blocked with a 3% sterile PBS solution of skim milk at 37 °C for 1 hour. The wells were washed once with 3 ml of PBS, and phage was applied to the blocked mutated IDE-coated wells at 25 °C for 1 hour. Subsequently, the wells coated with WT rhIDE were similarly blocked with a 3% PBS solution of skim milk, the wells were washed once with 3 ml of PBS, and the phage from the mutated IDE wells (depleted phage) was transferred to the WT rhIDE wells and left to stand at 25 °C for an additional 1 hour. At the end of each affinity selection cycle, 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 for infection of XL1-blue E. coli and cultured to the logarithmic growth phase for clone propagation. The titers of the input and output phage of the selection enabled the determination of the copy number of each phage clone. Later, 10 of the infected E. coli 10Twenty to one hundred copies of each phage clone were rescued by overnight incubation at 37 °C and 250 RPM with M13KO7 helper phage at 2 × 10⁸ CFU / ml. Virus particles from the bacterial growth supernatant were precipitated in 20% PEG / NaCl and resuspended in PBS before use in the next affinity selection (panning) cycle. Following four cycles of affinity selection, IDE-specific scFv-displaying phages were identified by monoclonal phage ELISA. In phage ELISA, HRP anti-m13 monoclonal antibody conjugate (GE Healthcare, CAT 27-9421-01) was used for detection of bound phages. The conjugates were tested for specificity by testing their binding to several control antigens [BSA, Merck CAT A7030-500G; lysozyme (egg white lysozyme), Merck CAT L6876; streptavidin (streptavidin from Streptomyces avidinii) Merck CAT S0677], all purchased from SIGMA (now Merck). The validated conjugates were reformatted for soluble antibody production and tested in the following three formats: MBP-scFv (Figure 1(E)), human IgG1 produced as "inclonal" (Figure 1(F)), and reverse chimeric IgG produced in mammalian cell culture (Figure 1(G)).

[0298] Sequencing: The nucleotide sequence of the cloned gene was determined using an ABI 3500xl genetic analyzer (Applied Biosystems, USA) following the vendor's recommendations. The DNA sequence of the cloned DNA fragment was analyzed with the ApE program - plasmid editor v2.0.47 (University of Utah, Wayne Davis). Analysis of antibody variable domain sequences and assignment to framework and CDR regions were performed with the IgBlast tool of NCBI (www.ncbi.nlm.nih.gov / igblast).

[0299] Expression and purification of IDE-specific “inclonal” IgG: Expression and refolding of full-length “inclonal” IgG were performed according to the protocols described previously 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].

[0300] Dot blot: Dot blot was performed according to the method described previously in [Mazor, Y. et al. (2007) J. Immunol. Methods 321, 41-59]. Briefly, a total volume of 50 μl of protein sample containing 0.1 - 1 μg of purified protein, either in native form or after boiling at 95°C for 5 minutes (denaturing conditions), was applied to a nitrocellulose membrane by a vacuum manifold using a slot blot PR648 filtration manifold (Hoefer Scientific Instruments, San Francisco, CA, USA). Next, the membrane was blocked with 5% BSA in TBS at room temperature for 1 hour and incubated overnight with 3 μg / ml of MBP (maltose-binding protein) - fused scFvs (produced by the method described below) diluted in the blocking solution at 4°C. After washing three times with TBST, the sample was incubated with mouse anti-MBP (1:5000 in TBST) at room temperature for 1 hour, followed by one wash, and then incubated with HRP-conjugated goat anti-mouse antibody (Jackson Immunoresearch Labs, 115-035-003) (1:5,000 in TBST) at room temperature for 1 hour. After further washing three times with TBST, the membrane was developed with ECL reagent (WBLUR0500, Millipore, Milford, MA, USA) according to the manufacturer's recommended method, imaged with an Amersham imager 600 (GE Healthcare Biosciences, Pittsburgh, PA, USA), and analyzed with ImageJ v1.5i.

[0301] Generation of IDE-specific MBP-scFvs: An expression vector was designed for cytoplasmic expression of MBP-scFv IDE-specific fusion proteins in E. coli. The sequences of all scFvs identified as specific IDE binders at the end of the phage display affinity selection process were obtained from the corresponding pCC16 phagemid vectors. 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]. Following digestion with NcoI and NotI restriction enzymes, each scFv coding sequence was recovered from the pCC16 phagemid and ligated into the sequence between the NcoI and NotI restriction sites of the pMALc-NHNN vector. The vector encodes an IDE-specific scFv fused to an Ni-NTA binding tag His tag and an N-terminal maltose binding protein (MBP).

[0302] For the production of MBP-scFv, Rosetta BL21 E. coli cells were transformed with the pMALc-NHNN-MBP-IDE-specific-scFv expression vector and grown in 0.5 L of LB medium supplemented with 100 μg / ml ampicillin until OD600nm = 0.8. Subsequently, protein expression in the culture was induced overnight at 30 °C with 0.5 mM IPTG. The cells were harvested by centrifugation at 9000 rpm for 15 minutes. The pellet was resuspended in 35 ml of PBS containing 0.1% Triton X-100 and lysed by repeating 5 times the cycle of 30 seconds of sonication and 2 minutes of incubation on ice. The soluble fraction was clarified by centrifugation at 12000 rpm for 30 minutes at 4 °C and loaded onto a 5 ml column of GE HisTrap. The column was washed with 5 CV of 5 mM imidazole in PBS and 5 CV of 20 mM imidazole in PBS, and eluted from the column with 250 mM imidazole in PBS. The purified MBP-scFv fusion protein was dialyzed against PBS, diluted with PBS at 2 mg / ml, and stored at -80 °C.

[0303] Preparation of splenocytes: Spleens recovered from C57BL / 6 mice were homogenized on ice in ×1 RIPA lysis buffer supplemented with 0.1 mM DTT, 0.1 μM sodium vanadate, 0.5 mM PMSF and protease inhibitor cocktail (PIC) 1:100 and incubated at 25 °C for 20 minutes. The supernatant was recovered by centrifugation at 12000 RPM for 10 minutes at 4 °C and aliquots of 100 μl were stored at -80 °C.

[0304] Production of anti-mouse IDE inverse chimeric antibody: To convert the antibody into a full-length inverse chimeric IgG, the polynucleotide encoding the produced antibody was cloned into the pcDNA3.4 plasmid backbone. These plasmids are based on the CMV promoter-controlled pcDNA3.4 vector provided as the "antibody expression positive control vector" part of the Expi293™ kit for expression based on transient transfection. The kit also provides Expi293F™ cells (ThermoFisher, #A14635). For a detailed description of the cloning of the antibody variable domain into the IgG expression vector, see the previous report [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 vector was expressed in E. coli RosettaBL21 cells and purified using a HisTrap column (17-5248-01, Uppsala, Sweden, GE Healthcare). The antibody was first produced as a full-length human IgG (having the respective constant regions of the human gamma 1 heavy chain and human kappa or lambda light chain), and subsequently expressed with the mouse constant domain of the mouse gamma 1 heavy chain and the mouse kappa light chain. Cloning was performed by PCR amplification of the respective variable domains of the heavy and light chains of the antibody, followed by cloning into the pcDNA3.4 plasmid already carrying the corresponding constant domain by Gibson Assembly [Gibson, D. G. et al. (2009) Nat. Methods 6, 343-345]. For the transfection of Expi293F™ cells with various pcDNA3.4 vectors, the ExpiFectamine™ transfection kit (Gibco, #A14524) was used according to the manufacturer's recommended method (Life Technologies, USA).In each transfection, 30 μg of plasmid DNA in total contained IgL and IgH at a molar ratio of 3:1. Transfection, cell growth, and collection of conditioned medium were performed according to the method recommended by the vendor (Expi293™ kit for transient transfection based on the expression of Life Technologies). Antibody-containing conditioned medium was collected 6 - 7 days after transfection by centrifugation at 8000 rpm for 10 minutes at 4°C (Sorvall GSA rotor). Reverse chimeric mouse IgG1 mAb was purified on a protein G column according to the method recommended by the manufacturer (GE Healthcare, Chicago, Illinois, USA). Before purification, the antibody-containing medium was buffered with 20 mM phosphate buffer, pH 7, and then filtered. The mAb was eluted in 1 ml fractions and the pH was neutralized with 0.25 ml of 1.5 M Tris-HCl, pH 8.8. Buffer exchange into sterile DPBS (Sigma) was performed using a 10 kDa Amicon® ultrafiltration filter (MilliporeSigma, Burlington, Massachusetts, USA) (Fig. 1(G)) or a PD-10 desalting column (GE Healthcare, Chicago, Illinois, USA).

[0305] The antibody was recovered by centrifugation and concentrated to a final concentration of 1 - 2 mg / ml using a centrifugal filtration filter. The purified antibody was stored in small equal amounts at -80°C. The protein concentration was determined by measuring the absorbance of the protein at O.D. 280 nm using a Thermo Scientific NanoDrop™ 2000c spectrophotometer and dividing the absorbance value by the extinction coefficient of the protein (the extinction coefficient was calculated at www.expasy.org / tools / protparam.html).

[0306] IDE Binding Assay: 96-well ELISA plates (Nunc, Roskilde, Denmark) were coated overnight at 4 °C with 2.5 - 5 μg / ml of WT rhIDE, mutant IDE, and irrelevant proteins BSA, HisTrap purified recombinant protein (His), and / or MBP-LacZ (made in the laboratory by recombination) in PBS. After three washes with PBST, the wells were blocked for 1 hour at room temperature with 3% w / v skim milk (232100, Difco, Sparks, Maryland, USA) in PBS. Subsequently, the wells were incubated for 2 hours at room temperature with IDE-specific antibodies prepared in various formats (serially diluted phage clones, MBP-scFv (data not shown), and various concentrations of hIgG or rcIgG). Bound phage was detected by incubation with mouse anti-M13 antibody (1 hour at room temperature), followed by HRP-conjugated goat anti-mouse secondary antibody (115-035-003, Jackson ImmunoResearch Laboratories, Maine, 1:5,000 in PBS) for 1 hour at room temperature. Human IDE-specific IgG antibodies were incubated for 2 hours at room temperature with HRP-conjugated goat anti-human antibody (109-035-088, Jackson ImmunoResearch Laboratories, Maine, 1:5,000 in PBS) or anti-mouse antibody (115-035-062, Jackson ImmunoResearch Laboratories, Maine, 1:5,000 in PBS). After three washes with PBST, 3,3’,5,5’-tetramethylbenzidine (TMB, eBioscience, California, USA) was added until color development. 2 SO 4 The reaction was stopped by the addition of 1M H

[0307] IDE Insulin Digestion Assay: IDE inhibitors at various concentrations were incubated with 1.5 μg / ml of rhIDE at room temperature for 1 hour. Subsequently, recombinant human insulin (41-975-100, Kibbutz Beit Haemek, Biological Industries, Israel) diluted with Mercodia ultrasensitive mouse insulin ELISA (10-1249-01) calibrator 0 was added to the tubes and incubated at 37°C for 2 hours. 25 μl was taken out from each tube and the residual insulin concentration was evaluated using Mercodia ultrasensitive mouse insulin ELISA. The absorbance at 450 nm was recorded with a Spectrafluor plus microplate reader (Tecan, Männedorf, Switzerland).

[0308] Streptozotocin (STZ) Diabetic Mouse Model: Ten-week-old C57BL / 6 mice were fasted overnight and then intraperitoneally injected with 150 mg / kg of STZ diluted with 100 nM citrate buffer (pH 4.5). All mice developed hyperglycemia within 2 days after STZ injection. On the third day after STZ injection, each mouse was intraperitoneally injected with 10 mg / kg of the prepared reverse chimeric mAb (rcH3 or rc2E12 isotype control).

[0309] Oral Glucose Tolerance Test (oGTT): Mice were fasted for 2 hours, then received a single intraperitoneal injection of the prepared reverse chimeric mAb (10 mg / kg) and were fasted for an additional 4 hours (total 6 hours). At the end of this period, mice were orally administered 2 g / kg of glucose using a gavage needle. Blood glucose levels were measured using a Contour glucometer (Bayer, Elkhart, Indiana, USA) before glucose administration (0 hour) and at 15, 30, 60, 90, and 120 minutes after glucose administration.

[0310] Insulin Tolerance Test (ITT): Mice were fasted for 2 hours, and then received a single intraperitoneal injection of the prepared reverse chimeric mAb (10 mg / kg), followed by an additional 4-hour fast (total 6 hours). At the end of this period, insulin (0.5 U / Kg, dissolved in PBS) was intraperitoneally injected into the mice. Blood glucose levels were measured using a Contour blood glucose meter (Bayer, Elkhart, Indiana, USA) before glucose administration (0 hours) and at 15, 30, 60, and 90 minutes after glucose administration.

[0311] Serum collection: Following ITT and injection of the reverse chimeric mAb, blood was collected from the facial vein of the mice every 3 days using a 27G needle. Blood samples (≤120 μl each time) were added to 1.5 ml Eppendorf tubes and left at room temperature for 1 hour. The blood clot was removed using a needle, and the tubes were placed on ice for 40 minutes. Subsequently, the tubes were subjected to centrifugation at 4°C and 2300 RPM for 10 minutes, and the clear supernatant was transferred to a new tube for the second washing cycle. The supernatant was transferred to a new tube and maintained at -20°C.

[0312] Detection of anti-IDE reverse chimeric antibody in serum samples: A 96-well ELISA plate was coated with 5 μg / ml of IDE in PBS overnight at 4°C. After washing 3 times with PBST, the wells were blocked with 5% (w / v) skim milk in PBS for 1 hour at room temperature. For the detection of the reverse chimeric IDE-specific antibody (rcH3-IgG), serum samples were diluted 1:2700 with PBS, while the control rc2E12 antibody-injected samples were used as positive controls. Known concentrations of rcH3-IgG antibody diluted with a 1:2700 dilution sample from control mice served as references for IDE concentration measurement. The samples were incubated for 2 hours at room temperature. Following 3 washes, 50 μl of HRP-conjugated goat anti-mouse antibody (1:5,000 in PBS) was incubated with the wells for 1 hour at room temperature. Following 3 washes with PBST, TMB was added until color appeared. 2 SO 4 The reaction was stopped by the addition of, and analyzed at 450 nm using a Spectrafluor plus microplate reader.

[0313] Reverse chimeric anti-IDE H3 Fab 2 Generation of segments: 10 mg of IgG was digested with 200 μg of IDES (Fabricator) enzyme (www.genovis.com / products / igg-proteases / fabricator / ) at 37 °C for 20 hours. Fab 2 fragments were separated from undigested IgG and Fc fragments by a MAbSelect™ affinity column. Fab2 was stored at -80 °C until use.

[0314] Determination of ROS levels in microglia: To measure intracellular ROS levels, N9 microglia at a concentration of 0.5×10 5 cells / ml were seeded in a 24-well plate with a clear base and a black frame (#4TI-0241, BIOKE, Leiden, the Netherlands) containing RPMI medium with 10% FCS. After 24 hours, the medium was replaced, and the cells were further incubated for 24 hours with serum-free RPMI with or without 0.1 μg / ml of LPS. To detect ROS in DJ-1-KD cells, DJ-1-KD microglia were grown according to a previously reported method (Nash et al. 2017). Control and DJ-1-KD microglia were seeded at a concentration of 0.8×10 5 cells / ml in RPMI medium containing 10% FCS. The next day, the medium was replaced with serum-free RPMI medium containing 1 μM of rotenone (#R8875, Sigma) for 2 hours. The medium was removed, the wells were washed once with serum-free RPMI medium, and the cells were incubated with the Fab fragment of the anti-IDE antibody (100 nM) for 2 hours. Next, insulin was added to the wells to a final concentration of 100 nM and left standing overnight. At the end of the experiment, the medium was replaced with RPMI medium containing 10 μM of H 2 DCFDA (#D6883, Sigma) and left standing in the dark at 37 °C for 30 minutes according to a previously reported method (Trudler et al. 2014). H 2Following the oxidation of DCFDA to DCF, the fluorescence of the cells was measured using a Synergy HT spectrofluorometer (BioTek, Winooski, Vermont, USA) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Normalization of the ROS level to the number of cells was achieved by subsequent MB staining of the cells (as described above) and dividing the ROS level of each well by the MB signal.

[0315] Subjects: A total of 24 healthy volunteers and 51 patients with metabolic syndrome (MS) were recruited from a previously reported cohort (Marcus Y, et al. J Clin Hypertens (Greenwich). 2016; 18(1): 19-24) (Table 1 below). To participate as an MS patient, subjects aged 18 to 75 years had to meet the criteria of the Third Report of the Adult Treatment Panel (ATP III) (Circulation. 2002; 106(25): 3143-421). The fasting blood glucose value in question was ≥100 mg%. None of the subjects were receiving treatment with antidiabetic drugs. The study had obtained approval from the institutional Helsinki’s committee of the Tel Aviv-Sourasky Medical Center. Informed consent was obtained from each patient after a full explanation of the purpose and nature of all the procedures used.

[0316] Biochemical analysis of human serum samples: Serum chemistry values were measured using a standard commercially available automated assay (Centaur, Roche, Indianapolis, Indiana). HbA1c levels were measured using HPLC (Tosoh Bioscience, San Francisco, California).

[0317] Preparation of polyclonal anti-IDE antibody: Immunization of animals: Female NZW rabbits, 8 weeks old and weighing 2.5 kg, were purchased from Harlan (Israel). Polyclonal antisera were produced by subcutaneous inoculation of each animal with 100 μg of rhIDE emulsified in a 1:1 ratio with 400 μg of M. tuberculosis extract H37 in incomplete Freund's adjuvant (CFA). Each animal received rhIDE emulsified as above with incomplete Freund's adjuvant (IFA) without M. tuberculosis extract once a week as a booster. Rabbit arterial blood was collected before the first inoculation, 1 week after each booster, and for the final (terminal) bleeding. A total of 3 booster immunizations were required for the serum antibody titer (>20000) to reach a plateau.

[0318] Analysis of the titer of anti-IDE serum polyclonal antibody: Blood collected from each animal was incubated at room temperature for 1 hour and further at 4°C for 1 hour for coagulation. The serum supernatant was separated from the coagulated blood by centrifugation at 4°C and 3000 g for 20 minutes. The anti-IDE antibody titer was measured by direct ELISA according to the following procedure. RhIDE diluted with PBS to a final concentration of 2.5 μg / ml was used at 50 μl / well, and a 96-well ELISA plate (Nunc, Roskilde, Denmark) was coated overnight at 4°C. The next day, the plate was washed once with PBS containing 0.05% Tween 20 (PBST) at 300 μl / well, and blocked with PBS containing 3% skim milk (w / v, Difco™ skim milk, BD) at 300 μl / well for 1 hour at 37°C. Next, the plate was washed with PBST at 300 μl / well, and serum samples diluted stepwise ×3 with PBST were applied and allowed to bind at room temperature for 1 hour. Subsequently, the plate was washed 3 times with PBST at 300 μl / well. The bound anti-IDE polyclonal antibody was detected by applying HRP-conjugated goat anti-rabbit IgG (#111-035-004, Jackson Immunoresearch Laboratories) diluted ×2000 with PBS, incubating the plate at room temperature for 1 hour, and then washing the plate 3 times with PBST at 300 μl / well. Finally, 3,3’,5,5’-tetramethylbenzidine (TMB, eBioscience, California, USA), the substrate of HRP, was added until the color developed. 2 SO 4 The reaction was stopped by the addition of, and analyzed using a Spectrafluor plus microplate reader (Tecan, Männedorf, Switzerland). The optical density was measured at wavelengths of 450 and 570 nanometers and analyzed using Magellan software version 2.22 (Tecan). When a sufficient titer was obtained, the animals were sacrificed by exsanguination, the blood was coagulated at 4°C for 1 hour, and the blood clot was separated from the serum by centrifugation at 4°C and 14000 g using an Eppendorf microcentrifuge. The serum was divided into 100 μl aliquots and stored at -80°C.

[0319] Determination of IDE serum concentration by ELISA: To measure IDE in body fluids, a highly sensitive sandwich ELISA suitable for quantifying IDE in human and mouse sera was developed. Anti-IDE antibody A9 diluted with PBS to a final concentration of 0.5 μg / ml was used at 50 μl / well to coat 96-well ELISA plates (Nunc, Roskilde, Denmark) at 4 °C overnight. The next day, the plates were washed once with 300 μl / well of PBST and blocked for 1 hour at 37 °C with 300 μl / well of PBS containing 3% skim milk (w / v, Difco™ Skim Milk, BD). Next, the plates were washed three times with 300 μl / well of PBST, and 50 μl / well of serum samples diluted ×3 or ×9 with PBS were applied and allowed to bind overnight (ON) at 37 °C. Concentration curves were generated by spiking control serum (undetectable IDE concentration) with serial ×2 dilutions of rhIDE starting at 500 ng / ml in PBS (Figure 7(A)). The plates were left standing at room temperature (about 25 °C) for 2 hours and then washed three times with 300 μl / well of PBST. Bound anti-IDE was detected by applying rabbit polyclonal anti-IDE serum diluted ×2000 with PBS, incubating the plates for 1 hour at room temperature, and then washing the plates 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 with PBS was applied, the plates were incubated for 1 hour at room temperature, and then washed three times with 300 μl / well of PBST. Finally, TMB, the substrate for HRP, was added until the color developed. 2 SO 4 The reaction was stopped by the addition of 1M H

[0320]

[0320] SO and analyzed using a Spectrafluor plus microplate reader (Tecan, Männedorf, Switzerland). Optical density was measured at wavelengths of 450 and 570 nanometers and analyzed using Magellan software version 2.22 (Tecan).Statistical analysis: GraphPad software (GraphPad Prism v8) was used for all statistical analyses in Examples 1 to 3 described below. For data comparison, either an independent two-sided Student's t-test for comparing two groups or one-way ANOVA (with Bonferroni correction) for analyzing three or more groups was used. Each experiment was repeated at least three times. P < 0.05 was considered significant. In Example 4 described below, the collected data was analyzed using IBM SPSS, version 25.0. The normal distribution of the data was verified by comparing the mean and median, comparing the mean and 5% trimmed mean, the values of skewness and kurtosis, and the results of the Shapiro-Wilk test. The results showed that some variables in the MS group did not follow a normal distribution. The differences between two groups (MS patients and controls) were analyzed for continuous variables using an independent samples t-test and a Mann-Whitney test, and for categorical variables using a chi-square test. Since the results of the t-test and the Mann-Whitney test were the same, the reported results were those of the t-test. Descriptive statistics were provided as the mean and standard deviation, or frequencies with percentages according to the scale of the variable. Since the difference in age was significant, this factor was controlled for the general linear model (GLM) and the differences were tested. A p-value < 0.05 was considered significant. All reported P-values were two-sided.

[0321] Example 1 Preparation of anti-IDE antibody and evaluation of its inhibitory effect on IDE activity in vitro Preparation of anti-IDE antibody Recombinant human IDE (rhIDE) was purified for use as an antigen for both screening and immunization. For this purpose, both the wild-type (WT) protein and the E111Q mutant enzyme (35) in which the catalytic activity of the enzyme was significantly reduced by a point mutation in the catalytic site were expressed. The genes encoding the two types of IDE were cloned into the pET28a+ plasmid backbone with a His tag at the C-terminus. The vector expressing recombinant human IDE (rhIDE) with a His tag at the C-terminus is shown in (A) of Figure 1.

[0322] After induction of vector expression, a marked increase in 110 kDa protein expression was visualized by SDS-PAGE ((B) in Fig. 1), and the identity of the protein was confirmed by immunoblot assay using an IDE-specific antibody ((C) in Fig. 1). In the next step, an insulin degradation assay with the purified protein was performed. The WT IDE protein effectively degraded insulin (60% degradation after 2 hours at 37°C), while the mutant IDE degraded only 15% of insulin under the same conditions ((D) in Fig. 1).

[0323] Purified IDE was used to isolate specific scFv clones that recognize epitopes of recombinant human IDE. Phage display technology using a human synthetic antibody phage display library was used. Specifically, the Ronit1 library [described in Azriel-Rosenfeld et al. J Mol Biol. (2004) 335(1):177-192], a human scFv library, was screened. The library was subjected to four rounds of affinity selection cycles on recombinant human wild-type (wt) IDE (rhIDE). In two of the cycles, depletion of E111Q IDE was performed with the intention of isolating antibodies that bind with high affinity to the catalytic site of the WT enzyme. Following four cycles of affinity selection, three IDE-specific scFv-displaying phages were identified by monoclonal phage ELISA and are designated herein as A9, B1, and H3. As shown in (A)-(F) of Fig. 2, phages displaying A9, B1, and H3 at serial dilutions ((A)-(C) of Fig. 2, respectively) bound well to IDE and showed 0 to low levels of binding to BSA, MBP, or other purified recombinant proteins used as negative controls in the wells.

[0324] The nucleic acid and amino acid sequences of the CDRs, as well as the heavy and light chains, of three anti-human IDE antibodies were determined. These are shown in SEQ ID NOs: 1-2 and 9-10 (heavy and light chains of A9, respectively), SEQ ID NOs: 3-8 (heavy chain CDRs of A9), SEQ ID NOs: 11-16 (light chain CDRs of A9), SEQ ID NOs: 17-18 and 25-26 (heavy and light chains of B1, respectively), SEQ ID NOs: 19-24 (heavy chain CDRs of B1), SEQ ID NOs: 27-32 (light chain CDRs of B1), SEQ ID NOs: 33-34 and 41-42 (heavy and light chains of H3, respectively), SEQ ID NOs: 35-40 (heavy chain CDRs of H3), and SEQ ID NOs: 43-48 (light chain CDRs of H3).

[0325] For the initial evaluation of full-length IgG, the antibodies were generated as "inclonal", i.e., expressed in an E. coli expression system (29) ((F) in FIG. 1). The isolated inclonal antibodies showed significant binding to IDE, with an EC 50 of approximately 1 nM ((D)-(F) in FIG. 2). The inclonal antibodies showed only a slight, distinct binding to BSA at saturating concentrations. Collectively, these results suggest that the three "inclonal" antibodies bind to IDE with high affinity and specificity.

[0326] Inhibition of Insulin Degradation In Vitro by the Generated Anti-IDE Antibodies To test the ability of the produced anti-IDE scFv and IgG to inhibit IDE-mediated insulin degradation, each antibody in the form of MBP-scFv, or a control MBP-scFv that does not bind to rhIDE, was incubated with rhIDE at room temperature for 1 hour. Subsequently, insulin was added over 2 hours at 37°C. Activity was measured as residual insulin using the Mercodia ultrasensitive mouse insulin ELISA kit. A dose-dependent inhibition of IDE activity was observed in the presence of B1 scFv and H3 scFv. B1 inhibited insulin degradation by 64% (P<0.001) and 35% (P<0.001) using 100 nM and 10 nM scFv, respectively, and H3 inhibited insulin degradation by 40% (P<0.001) using 100 nM, but no significant inhibition was observed using 10 nM (Figure 3(A)). No clear inhibition of IDE activity was observed upon use of A9 and the control MBP-scFv. After reformatting the antibodies to full-length human IgG, the ability to inhibit IDE activity was re-evaluated. In IgG form, the H3 antibody showed significant efficiency for inhibiting IDE activity, while A9 and the negative control antibody did not (p<0.001, Figure 3(B)).

[0327] Recognition of conformational IDE epitopes by the produced anti-IDE antibodies To determine the binding epitopes on IDE to which the produced antibodies bind, dot blot analysis was performed. Specifically, serially diluted rhIDE and mouse spleen lysates as a control were spotted onto nitrocellulose membranes in native and denatured states. Native rhIDE protein samples were detected by all antibody clones, with B1 MBP-scFv showing the highest signal. All denatured protein samples showed weaker signals or did not yield visible dots compared to the native samples (Figure 4(A)). These results suggest that the selected scFv clones recognize conformational epitopes of rhIDE that are disrupted under denaturing conditions rather than linear epitopes.

[0328] Example 2 Therapeutic effect of the prepared anti-IDE antibody in a diabetic animal model Conversion of anti-IDE H3 antibody to reverse chimeric IgG Chimeric antibodies are an important milestone in the development of therapeutic antibodies. A chimeric antibody is a recombinant IgG in which the variable domain is derived from a mouse antibody and the constant domain is a human sequence. Compared with mouse monoclonal antibodies (mAbs), chimeric antibodies are much less immunogenic and limit the production of human anti-mouse antibodies upon administration to human patients (36). On the other hand, a reverse chimeric is an antibody having a human variable domain and a mouse constant domain (37). Reverse chimeric antibodies are not only useful for the discovery of antibodies in transgenic mice but also very useful for the treatment of mouse models in preventing the occurrence of mouse anti-human immune responses. To evaluate the efficacy of the prepared anti-IDE antibody in a mouse model, the human H3 IgG antibody was converted to reverse chimeric IgG (rcIgG) (mouse IgG1 isotype), and its binding ability to IDE, BSA, and the E111Q IDE mutant was evaluated. The antibody rc2E12 that does not bind to IDE served as an isotype control. As shown in (B) of Figure 4, rcH3-IgG bound to IDE with high affinity (EC 50 was 1.62 nM). Furthermore, rcH3-IgG showed specificity for active IDE and did not bind to mutant IDE or BSA. Additionally, the ability of the rcH3-IgG antibody to inhibit IDE activity was tested. The rcH3-IgG antibody inhibited IDE in a dose-dependent manner (Figure 4 (C)). Taken together, these results suggest that reverse chimeric H3 IgG retains the properties of human H3 IgG type.

[0329] Improvement of insulin signaling by rcH3-IgG antibody in a diabetic mouse model Inhibition of IDE has been previously shown to improve insulin activity in a diabetic mouse model (38). Therefore, the ability of rcH3-IgG to lower blood glucose levels (oGTT) and improve insulin activity (iTT) was evaluated in STZ-treated mice. For this purpose, rcH3-IgG or an isotype control antibody was administered intraperitoneally to STZ-treated mice 1 hour before the oGTT and iTT tests. After glucose loading (oGTT), both control and rcH3-IgG-treated mice showed an increase in blood glucose levels. However, rcH3-IgG-treated mice showed lower blood glucose levels during the period compared to control-treated mice (p<0.05). Post hoc analysis revealed that rcH3-IgG-treated mice had significantly lower blood glucose levels compared to isotype control-treated mice 90 minutes after glucose administration (p<0.05, (A) in Figure 5). Furthermore, the ability of rcH3-IgG to improve insulin activity was evaluated (ITT). Although rcH3-IgG-treated mice showed a significant decrease in blood glucose levels 30 minutes after insulin administration, isotype control-treated mice showed a significant decrease in blood glucose levels 90 minutes later (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. Taken together, these results suggest that treatment with rcH3-IgG improves blood glucose levels and insulin activity in a diabetic mouse model.

[0330] Example 3 Effect of the prepared anti-IDE antibody on microglia with Parkinson's disease phenotype To evaluate the therapeutic effect of the produced antibody against Parkinson's disease, DJ-1 knockdown (KD) microglia presenting the neurotoxic phenotype of microglia found 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) were used. Past studies have shown that DJ-1 KD microglia generate high levels of reactive oxygen species (ROS) under basal conditions and inflammatory stimulation conditions (Trudler et al. 2014). To prevent the binding of the produced anti-IDE antibody to microglia that can occur by the Fc receptor expressed on the microglial membrane (Teeling et al. 2012), the Fab 2 segment (referred to as "H3 Fab" in this application) was tested under these conditions.

[0331] As shown in (A) of Figure 6, DJ-1 KD microglia showed a 51% increase in ROS levels compared to control microglia, which was consistent with past reports (Trudler et al. 2014). Furthermore, short-term stimulation of cells with rotenone, known as an inhibitor of mitochondrial complex I, which serves as an experimental model of Parkinson's disease (Xiong et al. 2012), significantly increased the production of ROS in DJ-1-KD microglia (227% compared to baseline control cells, p<0.001). Stimulation with 100 nM insulin following rotenone damage did not reduce ROS production, and incubation of the cells with 100 nM H3 Fab significantly reduced ROS production by DJ-1 KD cells to a level equivalent to the basal value of the cells (151% of the baseline of control cells vs. 148% of H3 Fab-treated cells following rotenone damage) (p<0.001). Notably, as shown in (B) of Figure 6, the MTT assay used to measure the effect of the treatment on the total cell number indicates that insulin or H3 treatment did not have a significant effect on cell viability after rotenone damage. This is an important control for evaluating protective and non-toxic effects.

[0332] In summary, these results suggest that the rcH3 antibody could be a therapeutic approach for Parkinson's disease.

[0333] Example 4 Diagnosis and prognosis determination of metabolic syndrome using the prepared anti-IDE antibody Using the prepared anti-IDE antibody, a sandwich ELISA for the detection and quantification of IDE was developed ((A) in Figure 7). As shown in (B) of Figure 7, this ELISA assay can easily and sensitively detect rhIDE at concentrations from 5 pg / μl to 500 pg / μl. Since human and mouse IDE have >95% sequence identity, it was reasoned and demonstrated that the prepared polyclonal and monoclonal antibodies also bind to mouse IDE. To determine whether antibody A9 recognizes the conformational structure of active rhIDE, the difference in binding to denatured rhIDE was evaluated. As shown in (C) of Figure 7, heat denaturation of rhIDE resulted in a significant decrease (almost negligible) in the ELISA signal generated by detection with A9 IgG. It should be noted that heat denaturation of rhIDE resulted in a decrease of approximately 2 - 3 times the ELISA signal shown in Figure 8, and rhIDE was detected using an anti-His tag antibody (recognizing a linear epitope) diluted in a ×2 stepwise manner, suggesting that denatured rhIDE can also bind to the wells of the ELISA plate with low efficiency. Collectively, these results suggest that A9 IgG specifically recognizes the structural epitope of active rhIDE.

[0334] Subsequently, an ELISA assay developed to determine IDE levels in serum samples from metabolic syndrome (MS) human patients and control healthy individuals was used. Compared to the controls, the MS subjects had higher BMI, glucose, triglyceride, and insulin levels, and lower HDLc levels (Table 1 below). The results indicate that IDE levels are higher in MS controls (mean 637.4 ± 469.5 vs 470.5 ± 221.8 pg / μL, p < 0.05) (Figure 9). Since the MS group was older than the control group, the IDEs of the two groups were compared again after adjusting for age. Even after this adjustment, IDE was higher in MS subjects compared to controls (F(1,68) = 6.675, p = 0.012, partial eta-squared = 0.089). Furthermore, a positive correlation was found between IDE levels and serum triglycerides (r = 0.423, p < 0.05, (A) in Figure 10) and insulin (r = 0.294, p < 0.05, (B) in Figure 10), and a borderline correlation was detected with serum c-peptide (not shown). IDE showed an even more negative correlation with HDLc levels (r = -0.366, p < 0.05, (C) in Figure 10). These findings suggest that higher IDE levels are quantitatively associated with MS components.

[0335] Furthermore, the IDE levels of MS subjects were separated into two different subgroups, namely, subjects with low IDE (n = 25, 272.1 ± 157.9 pg / μl) having a distribution and mean indistinguishable from those of the normal control group, and subjects with high IDE (n = 25, 1002.6 ± 383.7 pg / μl, p < 0.001 for the difference). The low IDE MS group was older (age 54 ± 10 vs 45 ± 13 years, p < 0.05) and had higher blood glucose levels (95 ± 20 vs 80 ± 9 mg / dl, p < 0.01) compared to the high IDE MS group (Figure 9). Notably, the insulin levels of both groups were similar, as were the triglycerides, HDLc, systolic and diastolic blood pressure values, and heart rate.

[0336]

Table 1

[0337] Although the invention has been described in connection with its specific embodiments, numerous alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, all such alternatives, modifications, and variations are intended to be included within the spirit and broad scope of the appended claims.

[0338] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, and patent application were specifically and individually indicated to be incorporated by reference. In addition, any reference or identification of a reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. Also, to the extent that section headings are used, they should not necessarily be construed as limiting. Further, any priority application of this application is hereby incorporated by reference in its entirety with this reference.

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Sequence Listing Free Text

[0340] SEQ ID NO: 1: Anti-human IDE clone A9 VH SEQ ID NO: 2: Anti-human IDE clone A9 VH SEQ ID NO: 3: Anti-human IDE (clone A9 VH)-CDR1 DNA sequence SEQ ID NO: 4: Anti-human IDE (clone A9 VH)-CDR1 SEQ ID NO: 5: Anti-human IDE (clone A9 VH)-CDR2 DNA sequence SEQ ID NO: 6: Anti-human IDE (clone A9 VH)-CDR2 SEQ ID NO: 7: Anti-human IDE (clone A9 VH)-CDR3 DNA sequence SEQ ID NO: 8: Anti-human IDE (clone A9 VH)-CDR3 SEQ ID NO: 9: Anti-human IDE clone A9 VL - DNA sequence SEQ ID NO: 10: Anti-human IDE clone A9 VL Accession No. 11: Anti-human IDE (Clone A9 VL) - CDR1 DNA Sequence Accession No. 12: Anti-human IDE (Clone A9 VL) - CDR1 Accession No. 13: Anti-human IDE (Clone A9 VL) - CDR2 DNA Sequence Accession No. 14: Anti-human IDE (Clone A9 VL) - CDR2 Accession No. 15: Anti-human IDE (Clone A9 VL) - CDR3 DNA Sequence Accession No. 16: Anti-human IDE (Clone A9 VL) - CDR3 Accession No. 17: Anti-human IDE Clone B1 VH - DNA Sequence Accession No. 18: Anti-human IDE Clone B1 VH Accession No. 19: Anti-human IDE (Clone B1 VH) - CDR1 DNA Sequence Accession No. 20: Anti-human IDE (Clone B1 VH) - CDR1 Accession No. 21: Anti-human IDE (Clone B1 VH) - CDR2 DNA Sequence Accession No. 22: Anti-human IDE (Clone B1 VH) - CDR2 Accession No. 23: Anti-human IDE (Clone B1 VH) - CDR3 DNA Sequence Accession No. 24: Anti-human IDE (Clone B1 VH) - CDR3 Accession No. 25: Anti-human IDE Clone B1 VL (kappa) - DNA Sequence Accession No. 26: Anti-human IDE Clone B1 VL (kappa) Accession No. 27: Anti-human IDE (Clone B1 VL) - CDR1 DNA Sequence Accession No. 28: Anti-human IDE (Clone B1 VL) - CDR1 Accession No. 29: Anti-human IDE (Clone B1 VL) - CDR2 DNA Sequence Accession No. 30: Anti-human IDE (Clone B1 VL) - CDR2 Accession No. 31: Anti-human IDE (Clone B1 VL) - CDR3 DNA Sequence Accession No. 32: Anti-human IDE (Clone B1 VL) - CDR3 Accession No. 33: Anti-human IDE Clone H3 VH - DNA sequence Accession No. 34: Anti-human IDE Clone H3 VH Accession No. 35: Anti-human IDE (Clone H3 VH) - CDR1 DNA sequence Accession No. 36: Anti-human IDE (Clone H3 VH) - CDR1 Accession No. 37: Anti-human IDE (Clone H3 VH) - CDR2 DNA sequence Accession No. 38: Anti-human IDE (Clone H3 VH) - CDR2 Accession No. 39: Anti-human IDE (Clone H3 VH) - CDR3 DNA sequence Accession No. 40: Anti-human IDE (Clone H3 VH) - CDR3 Accession No. 41: Anti-human IDE Clone H3 VL (lambda) - DNA sequence Accession No. 42: Anti-human IDE Clone H3 VL (lambda) Accession No. 43: Anti-human IDE (Clone H3 VL) - CDR1 DNA sequence Accession No. 44: Anti-human IDE (Clone H3 VL) - CDR1 Accession No. 45: Anti-human IDE (Clone H3 VL) - CDR2 DNA sequence Accession No. 46: Anti-human IDE (Clone H3 VL) - CDR2 Accession No. 47: Anti-human IDE (Clone H3 VL) - CDR3 DNA sequence Accession No. 48: Anti-human IDE (Clone H3 VL) - CDR3 Accession No. 49: Coding sequence of wild-type IDE Accession No. 50: Polypeptide sequence of wild-type IDE Accession No. 51: Coding sequence with IDE - E111Q mutation Accession No. 52: Polypeptide sequence with IDE - E111Q mutation

Claims

1. An isolated intact IgG antibody comprising an antigen recognition region that specifically binds to IDE, wherein the antigen recognition region comprises the following complementarity determining region (CDR) amino acid sequences. SEQ ID NO: 36 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 40 (CDR3) arranged in order from N to C of the heavy chain of the antibody, and SEQ ID NO: 44 (CDR1), SEQ ID NO: 46 (CDR2), and SEQ ID NO: 48 (CDR3) arranged in order from N to C of the light chain of the antibody.

2. A pharmaceutical composition comprising the antibody according to claim 1 as an active ingredient and further comprising a pharmaceutically acceptable carrier.

3. The antibody according to claim 1 for use in the treatment of IDE activity-related diseases in a subject in need of treatment.

4. The antibody according to claim 3 for use as described above, further comprising a therapeutic agent for the disease.

5. The antibody according to claim 3 or 4 for use as described above, wherein the biological sample of the subject exhibits an IDE level exceeding a predetermined threshold as compared to a control biological sample.

6. A product defined for the treatment of IDE activity-related diseases, comprising the antibody according to claim 1 and a therapeutic agent for treating IDE activity-related diseases.

7. A method for determining the IDE level of a biological sample of a subject using the antibody of claim 1, wherein the IDE level exceeding a predetermined threshold as compared to a control biological sample serves as an indicator of an IDE activity-related disease.

8. A method for determining the IDE level of a biological sample of a subject using the antibody of claim 1, wherein the IDE level decreased from a predetermined threshold serves as an indicator of an effective therapy for an IDE activity-related disease.

9. The antibody according to claim 3 or 4 for use as described above, wherein the IDE activity-related disease is selected from the group consisting of autoimmune diseases of the central nervous system, neurodegenerative diseases, metabolic syndrome, diabetes, obesity, hyperglycemia, retinal damage, renal insufficiency, nerve damage, microvascular damage, varicella zoster virus (VZV) infection, and wounds.

10. The product according to claim 6, wherein the IDE activity-related disease is selected from the group consisting of autoimmune diseases of the central nervous system, neurodegenerative diseases, metabolic syndrome, diabetes, obesity, hyperglycemia, retinal damage, renal insufficiency, nerve damage, microvascular damage, varicella zoster virus (VZV) infection, and wounds.

11. The method according to claim 7 or 8, wherein the IDE activity-related disease is selected from the group consisting of metabolic syndrome, diabetes, and obesity.

12. An isolated polynucleotide encoding the antibody of claim 1.

13. The nucleic acid sequence encoding the CDR amino acid sequence of the antibody is SEQ ID NO: 35, 37, 39, 43, 45 and 47 The isolated polynucleotide according to claim 12, which is shown in.

14. A host cell expressing the antibody of claim 1.

15. A method for producing an anti-IDE antibody, comprising expressing the polynucleotide according to claim 12 or 13 in a host cell.

16. A method for producing an anti-IDE antibody, comprising: (a) providing a plurality of antibodies; and (b) performing screening of the antibodies for selecting an antibody that binds to wild-type IDE but does not bind to mutant IDE with reduced catalytic activity compared to the wild-type IDE. The method comprising.

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