Novel CD200 fusion protein

Abstract

The present invention relates to a fusion protein comprising a mutant CD200 moiety containing K130Y and I131Y mutations that binds to the human CD200 receptor with higher affinity than wild-type CD200, directly fused to a non-CD200 IgG4 Fc fragment containing the S228P mutation and deletion of the first 5 amino acids. The present invention also relates to a polynucleotide encoding the fusion protein, a pharmaceutical composition containing the same, and its use.

Description

Technical Field

[0001] Cross-reference to Related Applications This application claims the priority of UK Provisional Patent Application No. GB2206672.4 filed on May 6, 2022, the entire content of which is incorporated herein by reference.

[0002] Sequence Listing Description This application includes a sequence listing in electronic format submitted via EFS-Web. The sequence listing created on April 27, 2023, is named "4549-139ST26.xml" and has a size of 6,646 bytes. The electronic format information of the sequence listing is part of this application and is incorporated herein by reference in its entirety.

[0003] Field of the Invention The present invention relates to a fusion protein comprising a mutant CD200 moiety containing K130Y and I131Y mutations that binds to the human CD200 receptor with higher affinity than wild-type CD200, directly fused to a non-CD200 IgG4 Fc fragment containing the S228P mutation and a deletion of the first 5 amino acids. The present invention also relates to a polynucleotide encoding the fusion protein, a pharmaceutical composition containing the same, and its use.

Background Art

[0004] Inflammatory diseases, including autoimmune and allergic diseases, are the second leading cause of chronic diseases worldwide and a major cause of morbidity in women in the United States. According to an international survey in 2008, chronic disease patients in the United States are more likely to not receive appropriate care due to cost burdens compared to patients in other countries (Schoen, C. et al. (2008) Health Affairs Web Exclusive, w1-w16). Furthermore, such patients have the highest incidence of medical accidents, problems with care coordination, and high out-of-pocket medical expenses.

[0005] Currently, the American Autoimmune Related Diseases Association (AARDA) estimates that 50 million Americans are suffering from autoimmune diseases. Epidemiological data for determining the total direct and indirect costs of autoimmune diseases to the entire healthcare system are lacking. However, in 2001, Dr. Anthony Fauci, the Director of the National Institute of Allergy and Infectious Diseases (NIAID) in the United States, estimated that the annual treatment cost of autoimmune diseases exceeded $100 billion. Although $100 billion is an astonishing figure, the true cost of autoimmune diseases is likely to be greatly underestimated, as the annual costs for just seven of the more than 100 known autoimmune diseases, namely Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), rheumatoid arthritis (RA), psoriasis, and scleroderma, are estimated to total between $51.8 billion and $70.6 billion according to epidemiological surveys. Furthermore, these estimates overlook the cost of immunosuppressive therapy during transplantation.

[0006] Autoimmune diseases are chronic conditions without a cure, which occur when the immune system attacks healthy cells, misidentifying them as foreign substances. Depending on the type, autoimmune diseases can affect one or more different types of body tissues, potentially causing abnormal organ growth and changes in organ function. Normal regulation of the immune system depends largely on receptor / ligand pairs, including proteins expressed by cells involved in the immune response. However, these receptor / ligand pairs are involved in the signaling cascades that cause the pathology of autoimmune diseases.

[0007] OX-2 membrane glycoprotein, also known as CD200 (cluster of differentiation 200), is a human protein encoded by the CD200 gene that is expressed in various cell types (Barclay, A.N. (1981) Immunology 44, 727) and has a high homology with molecules of the immunoglobulin gene family. The protein encoded by this gene contains two immunoglobulins and is a type I membrane glycoprotein that binds to the CD200 receptor (CD200R).

[0008] CD200R is expressed on myeloid cells (monocytes, macrophages, dendritic cells, and eosinophils) and T cells (Wright et al. (2000), Immunity 12, 233-242; Wright et al. (2003), J. Immunol, 171, 3034-3046).

[0009] Engagement of CD200 with CD200R delivers inhibitory signals to myeloid and T cells, and thus exerts an immunosuppressive effect on both innate and adaptive immunity of the immune system (Rahim S.A. (2005) AIDS, 19, 1907-1925; Shiratori, I. (2005) J. Immunol, 175, 4441-4449; Misstear, K. et al. (2012), Journal of Virology, 86(11), 6246-6257).

[0010] CD200R agonists have been shown to reduce the pathology in a wide range of mouse disease models, such as arthritis (Gorczynski et al. (2001) Clin. Immunol. 101, 328-34; Gorczynski et al. (2002) Clin. Immunol. 104, 256-264), graft rejection (Gorczynski et al. (2002) Transplantation 73, 1948-1953), pregnancy failure (Gorczynski et al. (2002) Am. J. Reprod. Immunol., 48, 18-26), contact hypersensitivity (Rosenblum et al. (2004) Blood 103, 2691-8), influenza-induced pneumonia (Snelgrove et al. (2008) Nat. Immunol., 9, 1074-1083), and HSV-induced inflammatory lesions (Sarangi et al. (2009) Clin. Immunol. 131, 31-40).

[0011] Furthermore, CD200 mice exposed to influenza virus - / - developed more severe disease associated with increased lung infiltration and lung endothelial damage compared to wild-type controls (Rygiel.T.P. et al. (2009) J. Immunol. 183(3), 1990-1996). CD200 - / -Since the mouse induced an immune response capable of controlling the viral load, it was suggested that severe diseases, in contrast to beneficial antiviral immune responses, were caused by dysregulation of the immune response. As a result, despite a dramatic increase in the viral load, the disease could be prevented by depleting T cells before viral exposure. Rygiel.T.P. et al. (2009) concluded that T cells are essential for the development of disease symptoms during influenza infection, and that the lack of downregulation of CD200-CD200R signaling rather than the viral load increases immunopathology.

[0012] Profiling studies have shown that the expression of hCD200 is downregulated in diverse patient populations such as multiple sclerosis (Koning et al. (2007) Ann. Neurol. 62, 504-514), asthma exacerbation (Aoki et al. (2009) Clin. Exp. Allergy 39, 213-221), Alzheimer's disease (Walker et al. (2009) Exp. Neurol. 215, 5-19), primary hypertrophic osteoarthropathy (Ren et al. (2013) Rheumatol. Int. 33(10), 2509-2512), pregnancy failure (Clark (2009) Am. J. Reprod. Immunol. 61, 75-84), and lichen planopilaris (alopecia) (Harries et al. (2013) J. Pathol. 231(2), 236-247).

[0013] Agonist CD200 proteins are disclosed, for example, in WO2000 / 061171 and WO2008 / 089022. The literature describes the use of wild-type CD200 molecules to regulate the function of immune cells. The present invention relates to mutant CD200 proteins that bind to the CD200 receptor with higher affinity than wild-type CD200.

[0014] Therefore, therapeutic intervention using molecules that regulate the CD200 pathway provides a means to control excessive or unwanted immune responses and alleviate disease conditions in patients suffering from chronic or intermittent (recurrent) autoimmune diseases.

[0015] There is a need to overcome the problems associated with the currently available treatments for autoimmune diseases and improve the clinical efficacy at lower doses.

Summary of the Invention

[0016] According to a first aspect of the present invention, (i) a mutant CD200 moiety comprising mutations K130Y and I131Y at amino acid residues 130 and 131; and (ii) a non-CD200 moiety which is an IgG4 Fc fragment and comprises an S228P mutation and a deletion of the first 5 amino acids of the hinge according to the EU numbering system, a fusion protein comprising wherein glycine 232 of the mutant CD200 moiety is directly fused to amino acid 6 of the non-CD200 IgG4 Fc fragment according to the IMGT numbering system, is provided.

[0017] According to a further aspect of the present invention, there is provided a polynucleotide encoding the fusion protein as defined herein. According to yet another aspect, there is provided a pharmaceutical composition comprising the fusion protein as defined herein.

[0018] In another aspect of the invention, there is provided a fusion protein, polynucleotide, or pharmaceutical composition as defined herein for use in the preparation of a medicament. In another aspect of the invention, there is provided a fusion protein, polynucleotide, or pharmaceutical composition as defined herein for use in therapy. In another aspect of the invention, there is provided a fusion protein, polynucleotide, or pharmaceutical composition as defined herein for use in the treatment of autoimmune diseases, allergic diseases (e.g., rheumatoid arthritis, asthma, or atopic dermatitis), neurodegenerative neuropathic pain, inflammatory joint pain, or diabetic neuropathy. In another aspect of the invention, there is provided a fusion protein, polynucleotide, or pharmaceutical composition as defined herein for use in the treatment of rheumatoid arthritis, asthma, or atopic dermatitis. In another aspect of the invention, there is provided a fusion protein, polynucleotide, or pharmaceutical composition as defined herein for use in the treatment of autoimmune diseases affecting the neuromuscular, vascular, ocular, dermal, gastrointestinal, pulmonary, renal, hepatic, peripheral or central nervous systems, bone, cartilage, or joints.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]

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Mode for Carrying Out the Invention

[0020] According to a first aspect of the present invention, (i) a mutant CD200 moiety comprising mutations that are K130Y and I131Y at amino acid residues 130 and 131; and (ii) a non-CD200 moiety that is an IgG4 Fc fragment and comprises an S228P mutation and a deletion of the first 5 amino acids of the hinge according to the EU numbering system, a fusion protein comprising a fusion protein is provided in which glycine 232 of the mutant CD200 moiety is directly fused to the non-CD200 IgG4 Fc fragment at amino acid 6 according to the IMGT numbering system.

[0021] The inventors have found that mutations in the extracellular domain of CD200 at these amino acid residues result in a mutant CD200 moiety with increased binding affinity for the CD200 receptor (CD200R). Furthermore, fusion proteins comprising the mutant CD200 moieties described herein have significant benefits, particularly with regard to having higher clinical efficacy and providing treatment at lower doses.

[0022] Thus, in certain embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 1. In further embodiments, the fusion protein consists of the amino acid sequence of SEQ ID NO: 1. In further embodiments, the fusion protein is DS-118.

[0023] SEQ ID NO: 1 (also referred to herein as “DS-118”) consists of the following sequence.

[0024]

Chemical Formula

[0025] The bold amino acids represent the positions of mutations relative to wild-type CD200 or IgG4 Fc, and the underlined sequences represent non-CD200 Fc fragments. In one embodiment, DS-118 may further comprise an N-terminal signal sequence that is a human IgG chain signal peptide. In further embodiments, the N-terminal signal sequence consists of the amino acid sequence of MEFGLSWLFLVAILKGVQC (SEQ ID NO: 3).

[0026] As used herein, the term “CD200 protein” refers to the wild-type CD200 protein. As used herein, the term “wild-type” refers to the sequences of proteins, peptides, amino acids, and nucleotides that exist in nature. For example, as used herein, the term “wild-type CD200 protein” refers to the full-length isoform of CD200 (UNIPROT P41217 OX2G_HUMAN) that binds to the CD200 receptor (CD200R) or any portion thereof (including naturally occurring protein polymorphisms). CD200 protein is also known as OX-2 membrane glycoprotein.

[0027] Wild-type CD200 is a cell surface protein and has an N-terminal extracellular domain, as well as a short transmembrane domain and a cytoplasmic domain. The extracellular domain binds to target receptors such as the CD200 receptor. In one embodiment, the CD200 protein is the extracellular domain of CD200 that binds to the CD200 receptor or any portion thereof.

[0028] As used herein, the term "position" refers to the residue number in an amino acid sequence, with 1 being the first translated amino acid. Thus, it is understood that the numbering of amino acid positions within a CD200 portion as defined herein is relative to an amino acid sequence (shown in bold in SEQ ID NO: 2) that includes an N-terminal signal sequence representing the first 30 amino acids of the CD200 portion.

[0029] As used herein, the term "mutated" or "mutant" refers to the sequences of proteins, peptides, amino acids, and nucleotides that have changed in form from their wild-type equivalents to become mutants. For example, a mutated or mutant protein may have changes in its amino acid sequence and / or nucleotide sequence when compared to the corresponding wild-type sequence, and such changes may also be referred to as mutations.

[0030] References herein to "mutant CD200 protein" and "mutant CD200 portion" refer to the full-length CD200 protein that binds to the CD200 receptor or any portion thereof, which is similar to but no longer identical to the wild-type CD200 protein and contains mutant amino acid residue(s) or multiple mutant amino acid residues in the amino acid sequence. According to the first aspect of the invention as defined herein, the mutant CD200 portion includes the K130Y mutation and the I131Y mutation. Thus, in one embodiment, the mutation is a substitution mutation.

[0031] In one embodiment, the fusion protein can be made synthetically or recombinantly. In a further embodiment, the fusion protein can be made synthetically. In an alternative embodiment, the fusion protein can be made recombinantly.

[0032] In one embodiment, the mutant CD200 moiety binds to the CD200 receptor with a higher affinity than wild-type CD200. In one embodiment, the mutant CD200 protein may comprise the entire extracellular domain of CD200 or a portion thereof. In a further embodiment, the mutant CD200 protein comprises a signal sequence. It is understood that secreted proteins contain at the N-terminus several amino acids that constitute a signal sequence that may be cleaved prior to secretion. Thus, in certain embodiments, the mutant CD200 moiety comprises an N-terminal signal sequence. In one embodiment, the mutant CD200 protein comprises at the N-terminus a signal sequence that is cleaved prior to secretion from the producing cell. In a further embodiment, the signal sequence comprises the first 30 amino acids of the wild-type CD200 protein. In a further embodiment, the signal sequence represents the first 30 amino acids of the CD200 moiety. In a further embodiment, the signal sequence is SEQ ID NO: 3. Thus, in certain embodiments, the fusion protein comprises the sequences defined herein, lacking the amino acids comprising the signal sequence. For example, lacking amino acids 1-30 of the wild-type CD200 protein, where the mutant CD200 protein comprises the sequence corresponding to amino acids 31-232 of SEQ ID NO: 2. Thus, in a further embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 2. In a further embodiment, the fusion protein consists of the amino acid sequence of SEQ ID NO: 2. In a further embodiment, the fusion protein consists of the amino acid sequence of SEQ ID NO: 3 at the N-terminus of the sequence of SEQ ID NO: 1.

[0033] SEQ ID NO: 2 consists of the following sequence.

[0034]

Chemical formula

[0035] The amino acids in bold represent the signal sequence, the highlighted amino acids represent the positions of mutations relative to wild-type CD200 or IgG4 Fc, and the underlined sequences represent non-CD200 Fc fragments. The present disclosure also includes protein sequences disclosed herein, but lacking the C-terminal lysine, for example, proteins in which the C-terminal lysine (K) has been cleaved during secretion from mammalian cells.

[0036] In one embodiment, the fusion protein having the amino acid sequence of SEQ ID NO: 2 is encoded by the polynucleotide of SEQ ID NO: 4. It is important to note that there is degeneracy in the genetic code, i.e., most amino acids are specified by more than one codon. Thus, since multiple different codons define the same amino acid, multiple polynucleotide sequences can encode the same amino acid sequence. Thus, SEQ ID NO: 4 represents one exemplary permutation of a polynucleotide sequence that can encode a fusion protein having the amino acid sequence of SEQ ID NO: 2. Any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0037] As used herein, the term "portion" in connection with protein, peptide, and amino acid, and nucleotide sequences refers to fragments and derivatives that are functional, i.e., that bind to a target.

[0038] As used herein, the term "fragment" refers to a part of a protein, peptide, amino acid, or nucleotide sequence that recognizes and binds its target, such as a receptor. As used herein, the terms "derivative" and "variant" refer to sequences of proteins, peptides, amino acids, or nucleotides that have at least 70% (such as 75%, 80%, 85%, 90%, 95%, or 99%) sequence similarity to the wild-type equivalent and that function similarly. Thus, a variant may be a derivative of the wild type.

[0039] As used herein, the term "amino acid residue" refers to a monomer unit in a polymer chain, i.e., a single amino acid in a protein. As shown by the data presented herein, the mutant CD200 protein / portion of the present invention binds more tightly to the CD200 receptor than the wild-type CD200 protein and exhibits a longer residence time on the receptor.

[0040] Fusion Protein According to a first aspect of the present invention as defined herein, there is provided a fusion protein comprising a mutant CD200 protein / portion as defined herein fused to a non-CD200 portion.

[0041] As used herein, the term "fusion protein" is well known in the art and refers to one or more amino acid sequences, peptides, and / or proteins linked using, for example, the methods described in U.S. Patent Nos. 5,434,131 and 5,637,481. The amino acid sequences, peptides, or proteins thus linked form one fusion protein.

[0042] In some embodiments, the mutant CD200 protein / portion as defined herein is fused to a non-CD200 portion at the C-terminus. Thus, in one embodiment, the orientation of the fusion protein from N-terminus to C-terminus is the mutant CD200 portion - non-CD200 Fc fragment. Thus, in a further embodiment, the orientation of the fusion protein is the mutant CD200 portion - IgG4 Fc fragment. In another embodiment, the orientation of the fusion protein from N-terminus to C-terminus is the signal sequence - mutant CD200 portion - non-CD200 Fc fragment. Thus, in a further embodiment, the orientation of the fusion protein is the signal sequence - mutant CD200 portion - IgG4 Fc fragment.

[0043] As used herein, the term "non-CD200 portion" can refer to a molecule, peptide, or protein that does not specifically bind to the CD200 receptor and does not interfere with the binding of the mutant CD200 protein to its target. Examples include, but are not limited to, immunoglobulin (Ig) constant regions or portions thereof, or fusion proteins where the non-CD200 portion is a synthetic molecule, such as PEG.

[0044] In one embodiment, the non-CD200 portion is an antibody fragment. In a particular embodiment, the non-CD200 portion is an Fc fragment. Thus, the mutated CD200 fusion proteins described herein can also be referred to as mutant CD200-Fc. In a further embodiment, the Fc fragment is of mammalian origin, such as human or monkey origin, for example, human C(γ)1 including the hinge, CH2, and CH3 regions. In particular, the Fc fragment includes the hinge region. The Fc fragment provides the advantage of increasing the serum half-life of the mutated CD200 protein of the present invention, and further, by dimerizing the CD200 protein, increases the binding activity and enables operative signal transduction. It will be understood by those skilled in the art that the Fc region may be mutated to reduce effector function (see, for example, US5,637,481 and US6,132,992).

[0045] In one embodiment, the Fc fragment is an IgG4 Fc fragment. In a further embodiment, the non-CD200 moiety is an antibody Fc fragment comprising mutations of one or more amino acid residues. Thus, in certain embodiments, the non-CD200 moiety is an IgG4 Fc fragment, comprising the S228P mutation, the position of said mutation following the EU numbering system. Thus, in one embodiment, the non-CD200 Fc fragment is an S228P derivative of human IgG4. The S228P mutation prevents Fab arm exchange in the antibody. Thus, the presence of the S228P mutation in the Fc fragments described herein is likely to increase the stability of the fusion protein both in vivo and in vitro, resulting in improved therapeutic efficacy and manufacturability. In a further embodiment, the non-CD200 IgG4 Fc fragment comprises a deletion of the first 5 amino acids, for example, the first 5 amino acids of the hinge region of said IgG4 Fc fragment. Thus, in one embodiment, the non-CD200 moiety is an IgG4 Fc fragment, comprising the S228P mutation and a deletion of the first 5 amino acids of the hinge, following the EU numbering system. In a further embodiment, the non-CD200 moiety is an IgG4 Fc fragment, comprising S228P and a deletion of the first 5 amino acids of the Fc hinge region.

[0046] In one embodiment, the fusion protein is formed by direct fusion of the mutant CD200 moiety to the non-CD200 Fc fragment. Thus, it is understood that such a fusion does not include a linker sequence between the mutant CD200 moiety and the non-CD200 Fc fragment. For example, amino acid glycine 232 of the mutant CD200 moiety can be directly fused to amino acid 1 of the Fc hinge region. In another embodiment, the fusion protein is formed by direct fusion of amino acid glycine 232 of the mutant CD200 moiety to amino acid 6 of the IgG4 Fc fragment (in this case, the first 5 amino acids of the Fc hinge region are deleted as previously described herein). In a further embodiment, the direct fusion is the fusion of amino acid glycine 232 of the mutant CD200 moiety to amino acid 6 of the Fc hinge region of the IgG4 Fc fragment. Thus, in one embodiment, glycine 232 of the mutant CD200 moiety is directly fused to the non-CD200 Fc fragment at amino acid 6 of the Fc hinge region. According to these embodiments, the positions in the Fc fragment of the fusion follow the IMGT numbering system. Such direct fusion of the mutant CD200 moiety to amino acid 6 of the IgG4 Fc fragment hinge region increases the stability of the resulting fusion protein without affecting the strong binding to CD200R compared to a fusion protein containing a linker sequence. This result is surprising considering the previously reported data for Fc fusion proteins containing a linker sequence.

[0047] For the purposes of this description, when the non-CD200 portion is an Fc fragment, the term "position" as used herein with respect to a mutation within the non-CD200 portion means the residue number in the amino acid sequence according to the EU numbering system. Thus, it is understood that the positions of the mutated residues of the amino acids of the Fc fragment cited herein relate to positions according to the EU numbering system. Further, it is understood that other numbering systems developed for the numbering of residues in Fc fragment sequences such as Kabat, AHo, IMGT, Chothia, and Martin (enhanced Chothia) may be utilized instead. When used herein with respect to the point at which the mutated CD200 portion is fused to the non-CD200 Fc fragment, "position" refers to the residue number within the Fc fragment according to the IMGT numbering system. Thus, it is understood that the residue positions of the amino acids of the Fc fragment hinge relate to that position according to the IMGT numbering system. Thus, the numbering in this specification of mutations within the Fc fragment refers to the EU numbering system, and the numbering of the hinge amino acids refers to the IMGT numbering system. In some embodiments, glycine 232 of the mutated CD200 portion is directly fused to the non-CD200 Fc fragment at amino acid 224 of the IgG4 heavy chain of the Fc fragment according to the EU numbering system.

[0048] The protein of the present invention is preferably produced by recombinant DNA methods by inserting a nucleic acid sequence encoding a CD200-Fc fusion protein or any part thereof into a recombinant expression vector and expressing the nucleic acid sequence in a recombinant expression system under conditions that promote expression. Thus, in one embodiment, the polynucleotide encoding the fusion protein further comprises a vector such as pCDNA3.1. In one embodiment, the fusion protein is flanked by one or more restriction enzyme sites. In another embodiment, the nucleic acid sequence encoding the CD200-Fc fusion protein or any part thereof is inserted into the recombinant expression vector using fusion cloning. Thus, in a further embodiment, the nucleic acid encoding the CD200-Fc fusion protein or any part thereof comprises, at its ends, an overlap between the nucleic acid encoding the CD200-Fc fusion protein and the vector of 12 to 21 base pairs / nucleotides, for example, an overlap of 15 base pairs or an overlap of 20 base pairs, etc., a nucleic acid sequence complementary to that at the end of the linearized vector.

[0049] According to a further aspect of the invention, there is provided a polynucleotide encoding a fusion protein as defined herein. The disclosure includes polynucleotides encoding a protein as defined herein, and the use of such nucleic acids for producing a protein and / or for therapeutic purposes. Such polynucleotides can include DNA and RNA molecules encoding a protein as defined herein (e.g., mRNA, self-replicating RNA, self-amplifying mRNA, etc.). The nucleic acid sequences encoding the proteins provided by the invention can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide synthetic genes that can be inserted into recombinant expression vectors and expressed in recombinant transcription units. In one embodiment, the polynucleotide encodes a fusion protein comprising the amino acid sequence of SEQ ID NO: 1. In a further embodiment, the polynucleotide encodes a fusion protein consisting of the amino acid sequence of SEQ ID NO: 1. In a further embodiment, the polynucleotide encodes DS-118. In a particular embodiment, the polynucleotide encodes a fusion protein comprising the amino acid sequence of SEQ ID NO: 2. In a further embodiment, the polynucleotide encodes a fusion protein consisting of the amino acid sequence of SEQ ID NO: 2. An exemplary polynucleotide sequence is provided in SEQ ID NO: 4.

[0050] The recombinant expression vector comprises a synthetic or cDNA-derived nucleic acid fragment encoding a mutated CD200, operably linked to appropriate transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. Such regulatory elements include transcriptional promoters, any operator sequences that control transcription, sequences encoding appropriate mRNA ribosome binding sites, and sequences that control the termination of transcription and translation. Usually, the ability to replicate in a host provided by an origin of replication, and a selectable gene for facilitating the recognition of transformants, can also be further incorporated.

[0051] Therapeutic Use The interaction between the CD200 protein and the CD200 receptor is characterized by a fast dissociation ("off") rate that results in a low affinity of CD200 for the CD200 receptor, and thus the present invention is particularly applicable in therapy. Accordingly, as presented herein, increasing the affinity of mutant CD200 proteins and fusion proteins comprising portions thereof for the CD200 receptor can be used in the manufacture of pharmaceutical compositions having more potent properties.

[0052] Furthermore, the production cost of recombinant proteins is high, and mutant CD200 proteins / fusion proteins comprising portions thereof having high affinity can be used in pharmaceutical compositions at significantly lower doses than wild-type or unmutated CD200 proteins to obtain a therapeutic effect. Accordingly, the use of mutant CD200 proteins / fusion proteins comprising portions thereof may be more cost-effective in addition to being clinically more effective.

[0053] According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a fusion protein as defined herein. In one embodiment, the pharmaceutical composition comprises a fusion protein comprising the amino acid sequence of SEQ ID NO: 1. In a further embodiment, the pharmaceutical composition comprises a fusion protein consisting of the amino acid sequence of SEQ ID NO: 1. In a further embodiment, the pharmaceutical composition comprises DS-118.

[0054] In one embodiment, the mutant CD200 protein or fusion protein as defined herein is a modulator of the CD200 receptor. As used herein, the term "modulator" refers to a substance that brings about a change, e.g., a modulator of a protein can bring about an increase or decrease in the activity of said protein. Considering the properties of the mutated CD200 proteins and fusion proteins of the present invention, these are agonists of the CD200 receptor and are thus considered useful in the treatment of autoimmune diseases. Accordingly, in a further embodiment, the mutated CD200 protein or fusion protein as defined herein is an agonist of the CD200 receptor.

[0055] Thus, according to a further aspect of the invention, there is provided a fusion protein as defined herein or a composition as defined herein for use in the treatment of an autoimmune disease.

[0056] As used herein, the terms "autoimmune disease" or "autoimmune disorder" are used interchangeably and refer to an undesirable condition resulting from an inappropriate or unwanted immune response against self-cells and / or tissues, or transplanted cells and / or tissues. The terms "autoimmune disease" or "autoimmune disorder" are meant to include such conditions whether mediated by a humoral or a cellular immune response.

[0057] In an alternative embodiment, there is provided a fusion protein as defined herein or a pharmaceutical composition as defined herein for use in the treatment of an allergic disease. As used herein, the terms "allergy" or "allergic disease" are used interchangeably and refer to T helper 2 (TH2)-driven diseases that primarily develop from the activation of TH2 cells. Examples of allergic diseases include chronic allergic diseases (such as hay fever or allergic rhinitis), allergic contact dermatitis, seasonal allergies, anaphylaxis, and food allergies.

[0058] A fusion protein comprising a mutant CD200 protein / portion as defined herein can inactivate activated immune cells with higher efficiency than a fusion protein comprising a wild-type or non-mutated CD200 protein.

[0059] In one embodiment, the autoimmune disease is selected from autoimmune diseases affecting the neuromuscular system, vascular system, eye, skin, gastrointestinal tract, lung, kidney, liver, peripheral nervous system or central nervous system, bone, cartilage, or joints.

[0060] In further embodiments, the autoimmune disease is acute disseminated encephalomyelitis (ADEM); acute necrotizing hemorrhagic leukoencephalitis; Addison's disease; agammaglobulinemia; alopecia areata; amyloidosis; ankylosing spondylitis; anti-GBM / anti-TBM nephritis; antiphospholipid antibody syndrome (APS); asthma, atopic dermatitis; autoimmune angioedema; autoimmune aplastic anemia; autoimmune autonomic neuropathy; autoimmune hepatitis; autoimmune hyperlipidemia; autoimmune immunodeficiency; autoimmune inner ear disease (AIED); autoimmune myocarditis; autoimmune oophoritis; autoimmune pancreatitis; autoimmune retinopathy; autoimmune thrombocytopenic purpura (ATP); autoimmune thyroid disease; autoimmune urticaria; axonal and neuropathic neuropathy; Baló's disease; Behçet's disease; bullous pemphigoid and related autoimmune blistering diseases; cardiomyopathy; Castleman's disease; celiac disease (such as refractory celiac disease type II); Chagas disease; idiopathic chronic urticaria; chronic inflammatory demyelinating polyneuropathy (CIDP); chronic recurrent multifocal osteomyelitis (CRMO); chronic idiopathic urticaria; Churg-Strauss syndrome; cicatricial pemphigoid / benign mucous membrane pemphigoid; Crohn's disease; Cogan's syndrome; cold agglutinin disease; congenital heart block; coxsackie myocarditis; CREST disease; idiopathic mixed cryoglobulinemia; demyelinating neuropathy; dermatitis herpetiformis; dermatomyositis; Devic's disease (neuromyelitis optica); diabetic neuropathy; discoid lupus erythematosus; Dressler's syndrome; endometriosis; eosinophilic esophagitis; eosinophilic fasciitis; erythema nodosum; experimental allergic encephalomyelitis; Evans syndrome; fibrosing alveolitis; giant cell arteritis (temporal arteritis); giant cell myocarditis; glomerulonephritis; Goodpasture's syndrome; granulomatosis with polyangiitis (GPA) (formerly known as Wegener's granulomatosis); graft-versus-host disease; (GvHD); Graves' disease; Guillain-Barré syndrome; Hashimoto's disease; hemolytic anemia; Henoch-Schönlein purpura; herpes gestationis; hypogammaglobulinemia; hidradenitis suppurativa (HS); idiopathic thrombocytopenic purpura (ITP); IgA neuropathy; IgG4-related sclerosing diseases; immunomodulatory lipoproteins; inclusion body myositis; inflammatory bowel disease (IBD); inflammatory skin diseases; interstitial cystitis; juvenile arthritis; juvenile diabetes (type 1 diabetes); Kawasaki disease; Lambert-Eaton syndrome; leukocytoclastic vasculitis; lichen planus; lichen sclerosus;One or more autoimmune diseases selected from: ligneous keratitis; linear IgA disease (LAD); lupus (SLE); Lyme disease, chronic; macrophage activation syndrome (MAS); mastocytosis; Meniere's disease; microscopic polyangiitis; mixed connective tissue disease (MCTD); Mooren ulcer; Muhlbauer-Herberman disease; multiple sclerosis; myasthenia gravis; myositis; narcolepsy; neuromyelitis optica (Devic); neutropenia; ocular cicatricial pemphigoid; optic neuritis; relapsing rheumatism; PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections); paraneoplastic cerebellar degeneration; paroxysmal nocturnal hemoglobinuria (PNH); palmoplantar pustulosis (PPP); Parry-Romberg syndrome; Personegi-Turner syndrome; pars planitis (peripheral uveitis); pemphigus; peripheral neuropathy; perivenous encephalomyelitis; pernicious anemia; POEMS syndrome; polyarteritis nodosa; type 1, type 2, and type 3 polyglandular autoimmune syndromes; rheumatoid polymyalgia; polymyositis; post-myocardial infarction syndrome; post-pericardiotomy syndrome; progesterone dermatitis; primary biliary cirrhosis; primary sclerosing cholangitis; psoriasis; psoriatic arthritis; idiopathic pulmonary fibrosis; pyoderma gangrenosum; erythroid leukemia; Raynaud's phenomenon; reactive arthritis; reflex sympathetic dystrophy; Reiter's syndrome; relapsing polychondritis; restless legs syndrome; retroperitoneal fibrosis; rheumatic fever; rheumatoid arthritis; sarcoidosis; Schmidt syndrome; scleritis; scleroderma; Sjogren's syndrome; sperm and testicular autoimmunity; stiff-man syndrome; subacute bacterial endocarditis (SBE); Susac syndrome; sympathetic ophthalmia; Takayasu arteritis; temporal arteritis / giant cell arteritis; thrombotic thrombocytopenic purpura (TTP); Troxler-Hunt syndrome; transverse myelitis; type 1 diabetes; ulcerative colitis; undifferentiated connective tissue disease (UCTD); uveitis; vasculitis; vesiculobullous dermatosis; vitiligo; and Wegener's granulomatosis (now called granulomatosis with polyangiitis (GPA)).;

[0061] In alternative embodiments, there is provided a protein or fusion protein as defined herein or a composition as defined herein for use in the treatment of neurodegeneration.; In further alternative embodiments, there is provided a protein or fusion protein as defined herein or a composition as defined herein for use in the treatment of neuropathic pain and inflammatory arthralgia.;

[0062] According to a further aspect of the invention, there is provided a method of treating an autoimmune disease, allergic disease (e.g., rheumatoid arthritis, asthma, or atopic dermatitis), neurodegeneration, neuropathic pain, inflammatory arthralgia, or diabetic neuropathy in a subject, comprising administering the fusion protein of the invention to a subject having at least one of an autoimmune disease, allergic disease, neurodegeneration, neuropathic pain, inflammatory arthralgia, or diabetic neuropathy.

[0063] It is understood that the protein or fusion protein of the invention can be administered as a single therapeutic agent or in combination therapy with one or more other compounds (or therapies) for the treatment of autoimmune diseases, allergic diseases (e.g., rheumatoid arthritis, asthma, or atopic dermatitis), neurodegeneration, neuropathic pain, inflammatory arthralgia, or diabetic neuropathy.

[0064] Thus, according to a further aspect of the invention, there is provided a pharmaceutical composition comprising the fusion protein as defined herein in combination with one or more therapeutic agents. For the treatment of autoimmune diseases, allergic diseases (e.g., rheumatoid arthritis, asthma, or atopic dermatitis), neurodegeneration, neuropathic pain, inflammatory arthralgia, or diabetic neuropathy, the fusion protein of the invention can advantageously be used in combination with one or more other agents, more specifically with one or more immunosuppressants or adjuvants in immunosuppressive therapy.

[0065] Examples of therapeutic agents or therapies that can be administered together with (simultaneously or at different time intervals) the compounds of the invention include, but are not limited to, azathioprine, methotrexate, cyclosporine, monoclonal antibodies (e.g., basiliximab, daclizumab, and muromonab), and corticosteroids.

[0066] Each of the therapeutic agents present in the combination of the present invention can be administered individually at different dosage schedules and by different routes. Further, the dosage and usage of two or more of each may be different, and each can be administered simultaneously or at different times. A person skilled in the art understands the dosage regimen and combination therapy to be used through general knowledge. For example, the protein or fusion protein of the present invention can be used in combination with one or more drugs administered according to an existing combination dosage regimen.

[0067] Generally, the proteins disclosed herein are utilized in a purified form together with pharmaceutically suitable excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including physiological saline and / or buffer media. Examples of parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, and lactated Ringer's. When it is necessary to hold the polypeptide complex in suspension, suitable physiologically acceptable adjuvants can be selected from thickening agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginate.

[0068] The route of administration of the pharmaceutical composition according to the present invention can be any of those generally known to those skilled in the art. For treatments including but not limited to immunotherapy, the protein of the present invention can be administered to any patient according to standard techniques. Administration can be by any suitable mode including parenteral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, via the pulmonary route, for example, intranasal or inhalation, for example, intranasal or inhalation, and, appropriately, direct injection by catheter such as intracranial (e.g., i.c.v. into the central nervous system ventricle or i.t. into the spinal cord). The dosage and frequency of administration vary depending on the age, sex, and condition of the patient, co-administration of other drugs, contraindications, and other parameters that the clinician should consider.

[0069] The protein of the present invention can be lyophilized for storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective, and those known as lyophilization and reconstitution techniques can be used. It is understood by those skilled in the art that the activity can be reduced to various degrees by lyophilization and reconstitution, and there may be a possibility that the level has to be adjusted upward to compensate for it.

[0070] It is understood that all embodiments described herein may be applicable to all aspects of the present invention, and vice versa. Other features and advantages of the present invention will become apparent from the description provided herein. However, it should be understood that various changes and modifications will be apparent to those skilled in the art, so the description showing the preferred embodiments of the present invention and specific examples are given only by way of illustration. The following research and protocols show embodiments of the methods described herein.

Example

[0071] Example 1: Production of Mutant and Wild-Type CD200-Fc Molecules Gene Synthesis and Cloning Gene synthesis of IgG4 S228P Fc (codon-optimized for CHO expression) was performed with GeneArt. This construct was cloned into plasmid pCDNA3.1 using fusion cloning to generate a vector backbone. A codon-optimized DNA sequence encoding a mutant or wild-type human CD200 residues 1-232 of UniProt P412178 (OX2G_Human) containing an N-terminal signal sequence was inserted at the N-terminus of IgG4 S228P Fc to create a direct fusion of the amino acid glycine 232 of CD200 to the Fc region. The sequence was confirmed bidirectionally.

[0072] Gigaprep The plasmid with confirmed array was transformed into E. coli DH5α cells. A single colony of each target protein was selected and inoculated into 10.0 mL of LB containing ampicillin. Each mutant or wild-type construct was subcultured in 800 mL of Circlegrow medium for gigascale DNA preparation. DNA was isolated using the Endotoxin Free Quanta Giga Kit.

[0073] Protein Expression The CD200-Fc protein was generated by transient transfection of the expression plasmid into CHO-3E7 cells using polyethyleneimine (PEI). Briefly, a 250 mL culture maintained at 37 °C at a density of 4.0×10 6 cells / mL was transfected with 2 mg / L of plasmid using PEI at a ratio of 1:5. Twenty-four hours after transfection, the culture was shifted to 32 °C and the cells were fed with 10% Feed C, glutamine, glucose, and 0.5 M sodium butyrate to enhance protein expression. The batch was monitored and the supernatant containing overexpressed CD200-Fc was harvested on day 7 with a viability of approximately 75%. The filtered supernatant was subjected to protein purification.

[0074] Protein Purification All purification procedures were performed at 4°C. The cell culture harvest was loaded onto a MabSelect SuRe affinity column (5 mL) pre-equilibrated with 50 mM sodium phosphate, 150 mM NaCl pH 7.4 at a flow rate of 3 mL / min on an AKTA Pure platform. The column was washed with the equilibration buffer and the bound protein was eluted using 20 mM sodium acetate, 150 mM NaCl pH 3.5. The eluate was neutralized with 10 v / v% 1 M Tris pH 8.0 and analyzed by SDS-PAGE. The fractions containing CD200-Fc were pooled, concentrated to 5 mL, and subjected to gel filtration chromatography (Hiload 16 / 600 Superdex-200pg column) on an AKTA Pure platform. The protein was processed at 1.2 mL / min in a 50 mM sodium phosphate, 150 mM NaCl pH 7.4 buffer system and collected in fractions. The fractions containing the CD200-Fc dimer as analyzed by SDS-PAGE were pooled and concentrated to 1.33 mg / mL (measured at UV280 nm) using an Amicon Ultra Centricon (10 kDa molecular weight cut-off). The purified material was subjected to SEC-HPLC, LC-MS, and EndoSafe LAL cartridge tests to evaluate protein purity, molecular weight, and endotoxin content, respectively. The final sample was stored at -80°C.

[0075] Example 2: Binding Analysis of Wild-Type and Mutant CD200-Fc Proteins BIAcore experiments were performed by Syngene International Ltd. (Biocon Park, Plot No2&3, Bommasandra Industrial Area, Bommasadra-Jigani Link Road, Bangalore-560099, India).

[0076] Assay Principle The BIAcore instrument uses surface plasmon resonance (SPR), an optical technique, to measure the binding characteristics of two interacting molecules, in this case CD200-Fc binding to the CD200 receptor (CD200R). In this technique, when the second of two interacting molecules captured on a chip (sensor) flows over an immobilized partner in solution, the change in refractive index of one molecule is measured. In these experiments, CD200-Fc was immobilized on the chip (sensor) surface, and CD200R was injected over the captured CD200-Fc under continuous flow conditions in an aqueous buffer. The change in refractive index of CD200-Fc after CD200R binding was measured in real time, and the results were plotted as response units (RU) versus time to create a sensorgram.

[0077] Equipment and Reagents Experiments were performed on a GE Healthcare BIAcore T200. Human, cynomolgus monkey, and mouse CD200R proteins were purchased from Creative Biomart (CD200R1-320H, CD200R1-3483C, CD200R1-3280M). All measurements were repeated twice.

[0078] Protocol For the human CD200 construct, anti-human Fc (GE Healthcare) was covalently immobilized onto a BIAcore CM5 sensor chip (Cytiva, BR100530) by amine coupling using a Cytiva kit (BR100839) according to the manufacturer's instructions, targeting an immobilization of 8,000 - 11,000 RU. The CD200-Fc protein was diluted to 0.5 μg / mL - 4 μg / mL in running buffer (1×HBS-EP+ pH 7.4 (Cytiva BR100669), HEPES buffered saline pH 7.4 containing 3 mM EDTA and 0.05 v / v% surfactant P20) and flowed over the immobilized anti-human IgG Fc at 10 μL / min for 25 - 100 s with a stabilization time of 60 s. 35 - 250 RU of CD200-Fc was captured and the higher RU was used for the cynomolgus CD200R binding experiment. Human CD200R or cynomolgus CD200R was serially diluted to five or more concentrations (depending on the expected affinity) in running buffer along with a buffer blank (0 nM) (3-fold dilution) and flowed over the capture ligand at a flow rate of 30 - 50 μL / min for 120 s, followed by dissociation in running buffer for 120 - 360 s. The analysis temperature was 25°C. Subsequently, 3M MgCl 2 was flowed over for 30 - 90 s at a flow rate of 30 μL / min to regenerate the surface, and then running buffer was flowed over for 60 s to stabilize the surface.

[0079] For the mouse CD200 construct, muCD200R-Fc (Creative Biomart CD200R1-458M) diluted to 1 μg / mL in running buffer was flowed over the immobilized anti-human IgG Fc and the CD200 monomer was serially diluted and flowed over the captured CD200R. Other details were as described above.

[0080] Data Analysis: The experimental sensorgrams were analyzed with BIAevaluation software (GE Healthcare). The obtained curves were maxAnd by setting RI as a local parameter, it was fitted to a 1:1 Langmuir binding model. The reaction rate equation using standard parameters (e.g., ligand concentration, time) was used for iterative curve fitting. The closeness of the fit was determined by an algorithm provided by the manufacturer in the BIAevaluation software, and the χ 2 value was less than 10% of R max , and the data was accepted when the U value was 15 or less.

[0081]

Table 1

[0082] Results The results (Table 2 and Figures 1A - 1B) show that the mutant CD200 - Fc protein (DS - 118) of the present invention binds to the human CD200 receptor with an affinity approximately 137 - fold higher than that of wild - type CD200 - Fc (DS - 155). The off - rate listed in Table 2 and the sensorgrams shown in Figures 1A - 1B demonstrate the off - rate and half - life on the receptor of DS - 118, which are rates consistent with efficient agonism in functional cell assays. Furthermore, the results in Table 3 and Figures 2A - 2B show that DS - 118 can bind to cynomolgus CD200R (cyno CD200R), indicating that this fusion protein can be evaluated with standard toxicology protocols.

[0083]

Table 2

[0084]

Table 3

[0085] Example 3: Cell Binding and Cell Activation Assays of Wild-Type and Mutant CD200-Fc Proteins Assay Principle To demonstrate the agonistic activity of DS-118, the cDNA of human CD200R was transduced into the human monocytic cell line U937 (ATCC, CRL1539). Cytokine production, including IL-6, from these cells was induced by stimulation with PMA followed by stimulation with LPS.

[0086] Construction of Cell Lines The full-length human CD200R gene containing the signal sequence was cloned into the pCDH-EF1-human CD200R-IRES-Puro lentiviral vector (System Biosciences) downstream of the EF1α promoter. Lentiviral particles containing the expression construct were produced in 293TN producer cells and concentrated using the PEG-it reagent (System Biosciences) according to the manufacturer's instructions.

[0087] The U937 human monocytic immortalized cell line was transduced with lentiviral particles at an MOI in the range of 5 - 200 using the Transducx and Max Enhancer reagents (System Biosciences) according to the manufacturer's instructions. The transduced U937 cells were selected using a) puromycin (with the first optimized puromycin concentration) and b) sorted by flow cytometry to generate stable polyclonal CD200R-expressing strains. The expression of CD200R was confirmed by Western blot in addition to flow cytometry.

[0088] Cytokine Release (IL-6, IL-8, and TNFα Inhibition) Assay 50,000 U937-CD200R cells per well were seeded in a 96-well plate and differentiated with 100 nM PMA for 72 hours. After differentiation, the PMA-containing medium was replaced with fresh medium and incubated for 2 hours. The CD200-Fc construct was added to the wells and incubated for 1 hour, then the cells were activated by the addition of 10 ng / ml LPS and incubated for an additional 24 hours. The supernatant was collected and assayed for IL-6, IL-8, or TNF by ELISA assay using a commercially available ELISA kit.

[0089] pERK Inhibition Assay 50,000 U937-CD200R cells per well were seeded in a 24-well plate. Next, an Fc block was added for 30 minutes, followed by the addition of CD200-Fc (DS-155 or DS-118), and the mixture was further incubated at 37 °C for 2 hours. Then, the cells were induced with PMA (10 nM) for 20 minutes. After incubation, the cells were quickly collected and centrifuged at 1250 rpm for 5 minutes. The supernatant was discarded, 100 μL of fixation buffer was added to the pellet, and the mixture was incubated at 4 °C for 15 minutes. Next, the cells were washed once with 1×PBS + 2% FBS, permeabilized with 100 μL of 90% methanol while vortexing for 5 minutes, and then further washed with 1×PBS + 2% FBS. An anti-pERK antibody at a ratio of 1:1000 was added for 45 minutes, and then the secondary antibody was added at 4 °C for an additional 30 minutes. The cells were washed again, and data were acquired using a flow cytometer.

[0090]

Table 4

[0091] Cell Binding U937 cells were resuspended at a cell density of 1 million cells per test using 50 μL of FACS buffer (1×PBS + 2% FBS).

[0092] Construct treatment (50 μL) was diluted 3-fold with FACS buffer starting from 10 μg / mL up to 10 concentrations and incubated at 37 °C for 1 hour, 4 hours, and 24 hours. At the end of each time point, the cells were collected and washed. An anti-human secondary antibody at 10 μg / mL was added and incubated at 4 °C for 30 minutes. After incubation, the cells were washed and stained at 4 °C for 20 minutes to confirm viability (1 μL of dye / 1 million cells / 1 mL of 1×PBS). The cells were washed and fixed with fixation buffer (100 μL per test) at 4 °C for 20 minutes. After incubation, the cells were washed, and the pellet was resuspended in FACS buffer (100 μL per test) for data acquisition using a flow cytometer.

[0093] At the 24-hour time point, treatment with the CD200-Fc protein was performed using cell culture medium. Wash step = addition of 200 μL of FACS buffer and centrifugation at 1400 RPM.

[0094]

Table 5

[0095] Results The data shown in FIGS. 3A-3D demonstrate that DS-118 can inhibit LPS-stimulated IL-6 (FIG. 3A), IL-8 (FIG. 3B), and TNFα (FIG. 3C) secretion in a concentration-dependent manner. As seen in FIG. 3A, DS-118 inhibits LPS-stimulated IL-6 release to a greater extent than the wild-type CD200-Fc fusion protein (DS-155), and DS-118 inhibits IL-6 release at an IC 50 compared to 0.18 μg / ml of DS-155, at an IC of 0.01 μg / ml. 50 Furthermore, FIG. 3D shows the ability of DS-118 to inhibit LPS-stimulated ERK activation (phospho-ERK / pERK) to a greater extent than the wild-type CD200-Fc fusion protein (DS-155).

[0096] Figure 4 shows the binding of the mutant DS-118 CD200-Fc protein to CD200R-expressing U937 cells. This data shows good binding of DS-118 to CD200R-expressing cells at all time points.

[0097] Example 4: Macrophage Activation Assays of Wild-Type and Mutant CD200-Fc Fusion Proteins Macrophage Differentiation One control iPSC line, BIONi010-C, was differentiated into macrophage progenitor cells using a proprietary protocol by Censo Biotechnologies. Cells were quality controlled following standard procedures using flow cytometry (Censo Biotechnologies). Macrophage progenitors were then matured into macrophages for 7 days prior to treatment, stimulation, and assay.

[0098] Treatment and Stimulation Mature macrophages were further treated with DS-118 in a range of concentrations for 18 hours, 1 hour prior to addition of the stimulus (Table 6). After stimulation, the cells were used in the cytokine release assay. DS-118 was used at a maximum concentration of 10 μg / ml with 1:3 dilutions to achieve a total of six concentrations.

[0099] [Table 6]

[0100] Cytokine Release (IL-6 HTRF) After treatment and stimulation as described above, the supernatants were collected and transferred to new plates. Samples were stored at -80 °C until the day of the assay. IL-6 was measured using the Cisbio HTRF kit (62HILo6PEG) following the manufacturer's instructions and measured using a BMG ClarioSTAR plate reader. Analysis was performed by removing background fluorescence and interpolating the results using a standard curve. All data are shown as mean ± SEM and two-way ANOVA was performed to assess statistical significance. Controls included wells that were stimulated but not treated with the compound (untreated), and wells that were neither stimulated nor treated to show baseline cytokine release (unstimulated).

[0101] Results Figure 5 shows that DS-118 can inhibit LPS-stimulated IL-6 release from iPSC-derived macrophages in a concentration-dependent manner.

[0102] Example 5: High-Affinity CD200-Fc Protein Protocol The conjugation was tested using the same technique as in Example 2 above.

[0103] The human CD200 construct used amino acids 1 - 232 of Uniprot P41217 - 1 containing the signal peptide and extracellular domain. The mutation numbers refer to the complete Uniprot sequence including the signal peptide. For the human CD200 - Fc fusions, IgG4 Fc (from P01861) was used for DS - 118, DS155, and DS - 192, CD200 was fused to residue 6 of the hinge (IMGT numbering), and CD200 was fused to residue 1 of the hinge via a G3SG4S linker. The IgG4 mutations S228P, M428L, and N434S refer to the EU antibody numbering system.

[0104] Data Analysis This method involved the use of an affinity prediction protocol scripted within MOE software (CCG Inc) and the use of Rosetta (Creative Commons). Fifteen obtained mutations predicted to confer improved binding affinity were individually expressed as monomeric CD200 - Fc fusion proteins for measurement of binding affinity to CD200R by SPR (surface plasmon resonance).

[0105] K for monomeric CD200 protein D and SPR measurements of the binding half - life were performed at 25°C using at least five dilutions of CD200R flowed over immobilized CD200 - Fc (huCD200), or His - tagged CD200 flowed over immobilized CD200R (muCD200) for k a and k d calculated from. The resulting curves were fit with R maxAnd by setting RI as a local parameter, it was fitted to a 1:1 Langmuir binding model. A reaction rate equation using standard parameters (e.g., ligand concentration, time) was used for iterative curve fitting. Human DS-118, DS-155, and DS-192 are IgG4 fusions, mouse DS-131 and DS-169 are mouse monomers used for SPR, and DS-198 and DS-227 are the corresponding CD200-Fc (IgG2a) Fc fusions. The average of two experiments performed on the same day is shown together with the standard deviation. The residue numbering of the CD200 protein refers to the preprotein containing a signal peptide that is cleaved during insertion into the endoplasmic reticulum.

[0106] Results The mutant protein of the present invention showed higher binding affinity than the wild type and many other tested mutants. Table 7 shows the affinity constant (K D ) of about 13 nM for K130Y compared to about 179 nM for the wild type (IgG4 fusion).

[0107] As shown in Table 7, a CD200 variant (DS-192) having one mutation resulted in an increase in affinity for human CD200R, and the binding half-life increased from 21 seconds to about 3 minutes. The CD200 variant (DS-118) of the present invention having multiple mutations surprisingly resulted in a high affinity up to about 1 nM, showing an increase in affinity more than 130-fold from the wild type, and the binding half-life increased from 21 seconds to about 38 minutes. When the affinity was measured for monomer binding, the data suggested that the dimeric Fc fusion format confers additional functional binding activity.

[0108]

Table 7

[0109] Although the affinity for human CD200R was low, binding to cynomolgus CD200R1 was detected in all constructs, but as shown in Table 7, binding to human CD200R1L or mouse CD200R1 was not detected.

[0110] Example 6: In Vitro Proof-of-Concept of High-Affinity Mouse CD200-Fc Due to the lack of cross-reactivity with murine CD200R, a murine CD200-CD200R1 in silico model was generated based on the published crystal structure, and a high-affinity surrogate CD200-Fc protein was engineered for in vitro proof-of-concept experiments in autoimmune mouse models.

[0111] Protocol The murine CD200 construct used Uniprot O54901, which contains a signal peptide and an extracellular domain. Mutation numbers refer to the complete Uniprot sequence including the signal peptide.

[0112] The protein was generated by transiently transfecting a pcDNA3.1-based expression plasmid into CHO-3E7 cells using polyethyleneimine (PEI). Twenty-four hours after transfection, the culture was shifted to 32 °C, supplied with 10% Feed C, glutamine, glucose, and 0.5 M sodium butyrate, and the supernatant was harvested on day 7 and filtered. Purification was performed at 4 °C using a MabSelect SuRe 5 ml affinity column (Cytiva) pre-equilibrated with 50 mM sodium phosphate, 150 mM NaCl pH 7.4 at a flow rate of 3 mL / min on an AKTA Pure platform. The column was washed with the equilibration buffer, and the bound protein was eluted using 20 mM sodium acetate, 150 mM NaCl pH 3.5. The eluate was neutralized with 10 v / v% 1 M Tris pH 8.0 and analyzed by SDS-PAGE. The pooled fractions were concentrated to 5 mL and subjected to gel filtration chromatography (Hiload 16 / 600 Superdex-200pg column) on an AKTA Pure platform. The protein was processed at 1.2 mL / min in a 50 mM sodium phosphate, 150 mM NaCl pH 7.4 buffer system. The fractions containing the protein were pooled and concentrated to 1.33 mg / mL (measured at UV280 nm) using an Amicon Ultra Centricon (10 kDa molecular weight cut-off). The purified material was subjected to SEC-HPLC, LC-MS, and EndoSafe LAL cartridge tests to evaluate protein purity, molecular weight, and endotoxin content, respectively. Mouse CD200-His protein was purified using Ni-NTA agarose resin by standard methods. All proteins were stored at -80 °C. Affinity was studied using a technique similar to that of Example 2 above.

[0113] Results As shown in Table 7, the monomer binding affinity of the combination of variants H82Y, T125I is 43 nM, which is approximately 14-fold higher than that of the wild-type construct containing the mouse IgG2a Fc domain.

[0114] Example 7: In Vitro Proof-of-Concept of High-Affinity Human CD200-Fc The human CD200-Fc (huCD200-Fc) protein identified using the in silico method was tested for its ability to inhibit cytokine release from LPS-activated premonocytic human myeloid leukemia cells (U937) engineered to express high levels of human CD200R.

[0115] Protocol Cell line construction, cytokine inhibition assays, and cell binding assays were performed using techniques similar to those of Example 3 above.

[0116] To test the ability of the huCD200-Fc protein to inhibit ERK phosphorylation, U937-CD200R cells were induced with PMA for 20 minutes, and inhibition of ERK phosphorylation by DS-155, DS-192, and DS-118 was measured by flow cytometry in permeabilized cells using an anti-pERK antibody.

[0117] Results As shown in Figure 7, high-affinity (1 nM) DS-118 showed more potent inhibition of IL-6 release than wild-type DS-155, and intermediate efficacy was observed for 13 nM DS-192. As shown in Figure 8, inhibition of IL-8 was observed for DS-118. As shown in Figure 9, inhibition of TNF-α was observed for 13 nM DS-192. As shown in Figure 10, inhibition of ERK phosphorylation correlates with CD200 affinity.

[0118] As shown in Figures 19A - 19F, antibodies recognizing Fcγ receptors did not inhibit the activity of DS-192 in vitro, suggesting that the Fc domain does not play an important role in the inhibitory activity in this particular assay system.

[0119] Example 8: In Vivo Proof-of-Concept of High-Affinity CD200-Fc In Vivo Proof-of-Concept of High-Affinity Mouse CD200-Fc Using a mouse model, it was shown that a mouse CD200-Fc protein with higher affinity decreased the clinical score in a mouse collagen-induced arthritis (CIA) model using prophylactic dosing.

[0120] Mice have four potential CD200 receptors, CD200R1 - CD200R4, at least one of which may be activated, and CD200R1 is a homolog of human CD200R. Knocking out either CD200 or CD200R1 in transgenic mice worsens symptoms or induces earlier onset in many autoimmune disease models, such as alopecia, arthritis, IBD25, and uveoretinitis.

[0121] CD200R agonism in rodent models using patient samples is known in the art in vitro and has been previously achieved using CD200-Fc fusion proteins, suggesting that human CD200-Fc fusion proteins can be used as a therapy for inflammatory diseases. Similar to other cell surface immune receptors, the affinity of CD200 for CD200R is low (in the high nanomolar range), and thus an ideal human therapeutic requires affinity enhancement for optimal efficacy. The Fc domain provides an antibody-like serum half-life, and the dimer format increases binding activity and enables receptor cross-linking. Animal model data show that the sequence of the Fc domain is associated with a mouse IgG2a Fc fusion with optimal efficacy, perhaps by binding to Fcγ receptors and promoting the formation of cell-cell interactions and further increasing binding activity. Antibody-dependent cell cytotoxicity may also contribute by removing CD200R1-expressing cells. Therefore, an in vivo mouse model was used to test the efficacy of a mouse high-affinity CD200-Fc protein compared to the wild-type CD200-Fc protein.

[0122] Protocol Wild-type (DS-198) and higher affinity (DS-227) mouse CD200-Fc proteins were tested using the CIA model by initiating dosing immediately prior to symptom onset. Arthritis was induced in male DBA / 1J mice by intradermal injection of bovine type II collagen in CFA (complete Freund's adjuvant) on day 1, followed by a booster injection of incomplete Freund's adjuvant on day 21. On day 22, the animals were randomized based on body weight and injected three times a week with 3 mg / kg of mouse IgG2a isotype control antibody, DS-198 (wild-type muCD200-Fc) or DS-227 (high affinity 43 nM muCD200-Fc) until day 36. The positive control group received oral dexamethasone at 0.5 mg / kg daily. Clinical scores of ankle arthritis (blinded assessment) were measured every other day from days 25 to 36. The data shown in Figure 11 are presented as mean ± SEM. ** p < 0.01; *** p < 0.001 vs. disease + Dexa, disease + DS-198, and disease + DS-227. Two-way repeated measures ANOVA, followed by Tukey's multiple comparison test.

[0123] Results As shown in Figure 11, DS-227, the higher affinity CD200-Fc, was significantly more potent in reducing the clinical score than wild-type (DS-198) at the selected dose of 3 mg / kg.

[0124] Example 9: Proof-of-Concept Study Using High-Affinity DS-192 Based on the results of the in vivo proof-of-concept of the above high affinity mouse CD200-Fc study (CIA mouse study), an in vivo proof-of-concept study was conducted to test DS-192, a high affinity human CD200-Fc fusion protein. A humanized model of oxazolone-induced contact hypersensitivity was designed using NOG-EXL mice, which can engraft both human lymphocytes and bone marrow cells (for generating huNOG-EXL mice).

[0125] Protocol Human cells were transplanted into female NOG-EXL mice and randomized based on the percentage of CD45 + cells at 20 - 21 weeks of age (-1 day). On day 0, the mice were sensitized by abdominal application of oxazolone (100 μL of 3 w / v% oxazolone in acetone:alcohol 1:4), and on days 5, 10, and 14, they were exposed by topical application (10 μL / side) of 20 μL of 2 w / v% oxazolone (acetone:alcohol 1:4) to each ear. Oxazolone exposure was repeated on one ear of the pre-sensitized huNOG-EXL mice using DS-192 (huCD200-Fc, 13 nM) or CD200R agonist (CD200R mAb) administered on the same day as each exposure. Isotype control antibody, CD200R agonist antibody, and high-affinity huCD200-Fc (DS-192) were intravenously administered at 3 mg / kg 4 hours before oxazolone exposure on days 5, 10, and 14. Ear thickness was measured immediately before exposure and 24 hours after each exposure, and on day 15, punch biopsies were taken for multiplex cytokine analysis.

[0126] Results As shown in Figure 12, the change in ear thickness (a surrogate for the inflammatory response), in contrast to the CD200R mAb which did not result in a significant decrease, was significantly decreased by DS-192 on the day after the second and third exposures compared to the isotype control. Furthermore, as shown in Figures 13, 14, and 15, significant decreases in IL-1, GM-CSF, and IL-13 in ear tissue were observed at the end of the study in DS-192-treated mice. Thus, the results showed that high-affinity CD200-Fc has excellent efficacy in a humanized mouse model of contact hypersensitivity. Since DS-192 has a significantly lower CD200 affinity than DS-118, extrapolating these advantages, when using DS-118 for the treatment of allergic diseases and skin inflammatory disorders, it shows greater efficacy.

[0127] Example 10: Proof-of-Concept Study Using the Highest-Affinity DS-118 The figure of the Fc fusion protein DS-118 of the present invention is shown in Figure 6. Protocol As shown in FIG. 16, this study was conducted using cynomolgus monkeys with Ascaris suum (roundworm)-induced lung inflammation in NHP.

[0128] This model is Th2-driven and has been previously used in the art to evaluate the efficacy of drugs against asthma. Cynomolgus monkeys were screened for existing sensitivity to Ascaris suum antigen and on day 0, 20 mg / kg of high-affinity huCD200-Fc (DS-118) (n = 6), vehicle control (n = 6), and 1 mg / kg of dexamethasone (n = 4) were administered. All animals were exposed to 5000 μg / ml of intratracheal Ascaris suum antigen on day +1, and lymphocyte levels in the BAL fluid were measured by flow cytometry on day +2 (24 hours after exposure, 48 hours after drug treatment), and changes in airway resistance immediately after Ascaris suum antigen exposure were compared to airway resistance immediately before exposure.

[0129] Pre-dose measurements were taken on day -1 (relative to huCD200-Fc administration), and post-dose measurements were taken on day +1. At each time point (pre-dose, 0.25 hours, 0.5 hours, 1 hour, 4 hours, 8 hours, 24 hours, 3 days, 5 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days), at least 0.8 mL of blood was collected from the lateral cutaneous vein or the saphenous vein of each animal. For samples collected within the first 1 hour of dosing, ±1 minute was acceptable. For the remaining time points, samples collected within 5% of the scheduled time were acceptable. Blood samples + coagulant-containing tubes were stored at room temperature for 30 - 60 minutes and then centrifuged at 1500×g for 10 minutes at 4°C. Serum samples were then rapidly frozen on dry ice and stored at -60°C or below until analysis. Protein concentration was determined by ELISA. A 96-well ELISA plate was coated overnight at 4°C with 1 μg / ml goat anti-human IgG in carbonate-bicarbonate buffer. After washing and blocking, serially diluted plasma samples were added, and biotinylated goat anti-human IgG (0.0625 μg / mL) was used as the detection antibody. HRP-streptavidin and TMB substrate were used for color development. After approximately 5 - 10 minutes, 2M HCl was added to stop the reaction. Absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer. The OD value of the sample was substituted into the standard curve to obtain the plasma antibody concentration. The detection limit of this method is 1 ng / mL. Serum concentration was subjected to non-compartmental pharmacokinetic analysis using Phoenix WinNonlin™ software (version 8.1, Pharsight, Mountain View, CA). The linear / log trapezoidal rule was applied to obtain PK parameters. Half-life was calculated without using data less than 1% of C max and was not accurate if AUC_%Extrap_obs was greater than 20% or Rsq_adjusted was less than 0.9.

[0130] Results As shown in Figure 17, administration of DS-118 on the day before the final sensitization resulted in a significant decrease in the number of infiltrating lymphocytes in the BAL fluid 48 hours later, compared to the vehicle control. As shown in Figure 18, there was a decrease in airway resistance (RL) after sensitization, but this did not reach significance. Thus, the data indicate that high-affinity CD200-Fc substantially reduced cell infiltration in bronchoalveolar lavage (BAL) fluid in a non-human primate (NHP) model of airway inflammation.

[0131] Example 11: In Vitro Binding Study Using the Highest-Affinity DS-118 A study was conducted to test the binding of the high-affinity CD200-Fc fusion protein DS-118 to human or cynomolgus monkey PBMC.

[0132] Protocol PBMC cells were resuspended at a cell density of 1 million cells per assay point using 50 μL of FACS buffer (1×PBS + 2% FBS). Dilutions of huCD200-Fc in 50 μL were added from 10 μg / mL starting with 3-fold dilutions up to 10 concentrations using FACS buffer and incubated at 37°C for 1 hour. At the end of each time point, cells were collected and washed (addition of 200 μL of FACS buffer and centrifugation at 1400 RPM). 10 μg / mL of anti-human secondary antibody (Abcam Ab98596) was added together with excess Fc block (Innovex Biosciences no. NB309) and incubated at 4°C for 30 minutes. After incubation, cells were washed and stained at 4°C for 20 minutes to confirm viability (1 μL of dye / 1 million cells / 1 mL of 1×PBS, ThermoFisher C34557A). Cells were washed and fixed with fixation buffer (100 μL per test, BD Sciences 554655) at 4°C for 20 minutes. After incubation, cells were washed and the pellet was resuspended in FACS buffer (100 μL per test) for data acquisition on a flow cytometer.

[0133] Results As shown in FIGS. 20A-20B, binding to human and cynomolgus monkey PBMCs was dose-dependent.

Claims

1. (i) Mutant CD200 portion containing mutations at amino acid residues 130 and 131, where K130Y and I131Y; and (ii) IgG4 Fc fragment, non-CD200 portion containing the S228P mutation and deletion of the first 5 amino acids of the hinge according to the EU numbering system, A fusion protein containing, The fusion protein is characterized in which glycine 232 of the mutated CD200 portion is directly fused to the non-CD200 IgG4 Fc fragment with amino acid 224 according to the EU numbering system.

2. The fusion protein according to claim 1, comprising the amino acid sequence of SEQ ID NO:

1.

3. The fusion protein according to claim 1, comprising the amino acid sequence of SEQ ID NO:

1.

4. The fusion protein according to claim 1, wherein the mutated CD200 portion includes an N-terminal signal sequence which is the first 30 amino acids of the mutated CD200 portion.

5. The fusion protein according to claim 4, wherein the N-terminal signal sequence is a human IgG chain signal peptide.

6. The fusion protein according to claim 5, wherein the N-terminal signal sequence includes the amino acid sequence of SEQ ID NO:

3.

7. The fusion protein according to claim 4, comprising the amino acid sequence of SEQ ID NO:

2.

8. The fusion protein according to claim 4, wherein the N-terminal signal sequence is cleaved before secretion from the cell.

9. The fusion protein according to claim 1, which is a modulator of the CD200 receptor.

10. The fusion protein according to claim 1, which is an agonist of the CD200 receptor.

11. The fusion protein according to claim 1, which inhibits cytokine secretion.

12. The fusion protein according to claim 11, wherein the cytokine is IL-6, IL-8, or TNFα.

13. The fusion protein according to claim 1, which inhibits ERK activation to a higher degree than the wild-type CD200-Fc fusion protein.

14. A polynucleotide encoding the fusion protein according to any one of claims 1 to 13.

15. A composition comprising a fusion protein according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

16. A composition comprising the polynucleotide described in claim 14 and a pharmaceutically acceptable carrier.

17. A pharmaceutical composition for treating autoimmune diseases, allergic diseases, neurodegenerative disorders, neuropathic pain, inflammatory disorders, Th2-induced airway inflammation, or diabetic neuropathy, comprising a fusion protein according to any one of claims 1 to 13 or a polynucleotide encoding the fusion protein.

18. The pharmaceutical composition according to claim 17, administered to a subject having rheumatoid arthritis, asthma, or atopic dermatitis.

19. The pharmaceutical composition according to claim 18, wherein the subject has an autoimmune disease affecting the neuromuscular system, vascular system, eyes, skin, gastrointestinal tract, lungs, kidneys, liver, peripheral or central nervous system, bones, cartilage, or joints.

20. The target conditions include: acute disseminated encephalomyelitis (ADEM); acute necrotizing hemorrhagic leukoencephalitis; Addison's disease; agammaglobulinemia; alopecia areata; amyloidosis; ankylosing myelitis; anti-GBM / anti-TBM nephritis; antiphospholipid syndrome (APS); asthma, atopic dermatitis; autoimmune angioedema; autoimmune aplastic anemia; autoimmune autonomic neuropathy; autoimmune hepatitis; autoimmune hyperlipidemia; autoimmune immunodeficiency; autoimmune inner ear disease (AIED); autoimmune myocarditis; autoimmune oophoritis; auto Immune pancreatitis; autoimmune retinopathy; autoimmune thrombocytopenic purpura (ATP); autoimmune thyroid disease; autoimmune urticaria; axonal and neurological neuropathy; Barlow's disease; Behçet's disease; bullous pemphigoid and related autoimmune blistering disorders; cardiomyopathy; Castleman disease; celiac disease (including refractory celiac disease type II); Chagas disease; idiopathic chronic urticaria; chronic inflammatory demyelinating polyneuropathy (CIDP); chronic relapsing polymyelitis (CRMO); chronic idiopathic urticaria; Jarg-Strauss syndrome; pemphigoid scarring / benign mucosal pemphigus; Crohn's disease; Cogan's syndrome; cold agglutinin disease; congenital cardiac conduction disorder; coxsackie myocarditis; CREST disease; idiopathic mixed cryoglobulinemia; demyelinating neuropathy; herpetiform dermatitis; dermatomyositis; Devic's disease (neuromyelitis optica); discoid lupus erythematosus; Dressler syndrome; endometriosis; eosinophilic esophagitis; eosinophilic fasciitis; erythema nodosum; experimental allergic encephalomyelitis; Evans syndrome; fibrotic alveolitis; giant cell Temporal arteritis (temporal arteritis); giant cell myocarditis; glomerulonephritis; Goodpasture syndrome; granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis); graft-versus-host disease (GvHD); Graves' disease; Guillain-Barré syndrome; Hashimoto's disease; hemolytic anemia; Henoch-Schönlein purpura; herpes zoster of pregnancy; hypogammaglobulinemia; idiopathic thrombocytopenic purpura (ITP); IgA neuropathy; IgG4-related sclerosing disease; immunomodulatory lipoprotein; inclusion body myositis; inflammatory bowel disease (IBD); Inflammatory skin diseases; interstitial cystitis; juvenile arthritis; juvenile diabetes mellitus (type 1 diabetes); juvenile myositis; Kawasaki disease; Lambert-Eaton syndrome; leukocytoclastic vasculitis; lichen planus; lichen sclerosing; lignite keratoconjunctivitis; linear IgA disease (LAD); lupus (SLE); chronic Lyme disease; macrophage activation syndrome (MAS); mastocytosis; Meniere's disease; microscopic polyangiitis; mixed connective tissue disease (MCTD); Mollen's ulcer; Mucher-Habermann disease; multiple sclerosis; myasthenia gravis; myositis; narcolepsy; neuromyelitis optica (Devic's); neutropenia; ocular pemphigoid; optic neuritis; relapsing rheumatoid arthritis; PANDAS (Streptococcal infection-associated pediatric autoimmune neuropsychiatric disorder); paraneoplastic cerebellar degeneration; paroxysmal nocturnal hemoglobinuria (PNH); Parry-Romberg syndrome; Personegil Turner syndrome; squamous cellulitis (peripheral uveitis); pemphigus; Peripheral neuropathy; perivenous encephalomyelitis; pernicious anemia; POEMS syndrome; polyarteritis nodosa; type 1, 2, and 3 polyglandular autoimmune syndromes; polymyalgia rheumatica; polymyositis; post-myocardial infarction syndrome; post-pericardiotomy syndrome; progesterone dermatitis; primary biliary cirrhosis; primary sclerosing cholangitis; psoriasis; psoriatic arthritis; idiopathic pulmonary fibrosis; pyoderma gangrenosum; pure red cell aplasia; Raynaud's phenomenon; Reactive arthritis; reflex sympathetic dystrophy; Reiter's syndrome; relapsing polychondritis; restless legs syndrome; retroperitoneal fibrosis; rheumatic fever; rheumatoid arthritis; sarcomatoid syndrome; Schmidt syndrome; scleritis; scleroderma; Sjögren's syndrome; seminal testicular autoimmunity; generalized rigidity syndrome; subacute bacterial endocarditis (SBE); Susac syndrome; sympathetic ophthalmitis; The pharmaceutical composition according to claim 19, having Takayasu's arteritis; temporal arteritis / giant cell arteritis; thrombocytopenic purpura (TTP); Toloser-Hunt syndrome; transverse myelitis; type 1 diabetes mellitus; ulcerative colitis; undifferentiated connective tissue disease (UCTD); uveitis; vasculitis; vesicular bullous skin disease; or vitiligo.

21. The pharmaceutical composition according to claim 17, wherein a fusion protein or polynucleotide is administered as a single therapeutic agent.

22. The pharmaceutical composition according to claim 17, wherein the fusion protein is administered in combination with one or more other pharmaceuticals indicated for the treatment of autoimmune diseases, allergic diseases, neurodegenerative disorders, neuropathic pain, inflammatory disorders, Th2-induced airway inflammation, or diabetic neuropathy.

23. The pharmaceutical composition according to claim 17, wherein the fusion protein is administered in combination with one or more immunosuppressants or adjuvants in immunosuppressive therapy.

24. The pharmaceutical composition according to claim 23, wherein the fusion protein is administered in combination with azathioprine, methotrexate, cyclosporine, a monoclonal antibody, a corticosteroid, or a combination thereof.

25. The pharmaceutical composition according to claim 24, wherein the monoclonal antibody is basiliximab, daclizumab, or muromonab.

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