Novel Proteins

Abstract

The present invention relates generally to mutated CD200 proteins which bind to the CD200 receptor with higher affinity than wild-type CD200, in particular the present invention relates to mutated CD200 proteins comprising specific mutations at amino acid residues 130 and / or 131. The present invention also relates to fusion proteins comprising a protein as defined herein fused directly or via an optional linker moiety to a non-CD200 protein moiety, pharmaceutical compositions comprising a protein as defined herein and uses thereof.

Description

[Technical field]

[0001] The present invention relates generally to mutated CD200 proteins which bind with higher affinity to the CD200 receptor than wild-type CD200, in particular the present invention relates to mutated CD200 proteins comprising specific mutations at amino acid residues 130 and / or 131. The present invention also relates to fusion proteins comprising a protein as defined herein fused directly or via an optional linker moiety to a non-CD200 protein moiety, pharmaceutical compositions comprising a protein as defined herein and uses thereof. [Background technology]

[0002] Autoimmune diseases are the second leading cause of chronic illness worldwide and the leading cause of disease in women in the United States. According to a 2008 international survey, chronic illness patients in the United States are more likely to not receive appropriate care due to cost burden than patients in other countries (Schoen, C. et al. (2008) Health Affairs Web Exclusive, w1-w16). Furthermore, such patients are more likely to experience the highest incidence of medical errors, problems with care coordination, and high out-of-pocket medical expenses.

[0003] Currently, the American Autoimmune Disease Association (AARDA) estimates that 50 million Americans suffer from autoimmune diseases. There is a lack of epidemiologic data to determine the full direct and indirect costs of autoimmune diseases to the entire healthcare system. However, in 2001, Anthony Fauci, director of the National Institute of Allergy and Infectious Diseases (NIAID), estimated that the annual cost of treating autoimmune diseases exceeds $100 billion. Although $100 billion is a staggering figure, the true cost of autoimmune diseases is likely to be greatly underestimated, as epidemiologic studies have estimated that the annual costs of only seven of the more than 100 known autoimmune diseases - Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), rheumatoid arthritis (RA), psoriasis, and scleroderma - total between $51.8 billion and $70.6 billion per year. Furthermore, these estimates overlook the costs of immunosuppressive therapy during transplantation.

[0004] Autoimmune diseases, which occur when the immune system attacks healthy cells as foreign, are chronic conditions with no cure. Depending on the type of autoimmune disease, it can affect one or many different types of body tissues, leading to abnormal organ growth and altered organ function. Normal control of the immune system relies heavily on receptor / ligand pairs that involve proteins expressed by cells involved in the immune response. However, these receptor / ligand pairs are often involved in signaling cascades that cause the pathology of autoimmune diseases.

[0005] OX-2 membrane glycoprotein, also called CD200 (cluster of differentiation 200), is a human protein encoded by the CD200 gene that is expressed in a variety of cell types (Barclay, AN (1981) Immunology 44, 727) and has high homology to molecules of the immunoglobulin gene family. The protein encoded by this gene is a type 1 membrane glycoprotein that contains two immunoglobulin domains and binds to the CD200 receptor (CD200R).

[0006] 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).

[0007] The association of CD200 with CD200R delivers inhibitory signals to myeloid and T cells, thus exerting immunosuppressive effects on both the innate and adaptive arms of the immune system (Rahim SA (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).

[0008] CD200R agonists have been shown to inhibit a wide range of mouse disease models, including 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), and 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).

[0009] In addition, CD200 exposed to influenza virus - / - Mice developed a more severe disease associated with increased pulmonary infiltration and pulmonary endothelial injury compared to wild-type controls (Rygiel. TP et al. (2009) J. Immunol. 183(3), 1990-1996). CD200 - / -Mice induced immune responses capable of controlling viral load, suggesting that severe disease was due to a dysregulated immune response as opposed to a beneficial antiviral immune response. Consequently, depletion of T cells prior to viral exposure prevented disease, despite a dramatic increase in viral load. Rygiel.TP et al. (2009) concluded that T cells are essential for the expression of disease symptoms during influenza infection, and that lack of downregulation of CD200-CD200R signaling rather than viral load augments immunopathology.

[0010] Profiling studies have shown that hCD200 expression is associated with multiple sclerosis (Koning et al. (2007) Ann. Neurol. 62, 504-514) and asthma exacerbations (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 planus pilaris (hair loss) (Harries et al. (2013) J. Pathol. 231(2), 236-247).

[0011] Agonistic CD200 proteins are disclosed, for example, in WO2000 / 061171 and WO2008 / 089022, which describe the use of wild-type CD200 molecules to modulate immune cell function. The present invention relates to mutant CD200 proteins that bind to the CD200 receptor with higher affinity than wild-type CD200.

[0012] Thus, therapeutic intervention with molecules that modulate the CD200 pathway may provide a means to control excessive or unwanted immune responses and alleviate pathology in patients suffering from chronic or intermittent (relapsing) autoimmune diseases.

[0013] Thus, there is a need to provide improved clinical efficacy at lower doses that overcome the problems associated with currently available treatments for autoimmune diseases. Summary of the Invention

[0014] According to a first aspect of the invention, the following mutations are provided: (i) K130F; or (ii) I131F; or (iii) K130F and I131F; or (iv) K130F and I131Y; or (v) K130Y and I131F, The present invention provides a mutated CD200 protein comprising:

[0015] According to a second aspect of the invention there is provided a fusion protein comprising a protein as defined herein fused, either directly or via an optional linker moiety, to a non-CD200 moiety. According to a further aspect of the present invention there is provided a polynucleotide encoding a protein or a fusion protein as defined herein.

[0016] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a protein, polypeptide or fusion protein as defined herein. According to a further aspect of the invention there is provided a protein, fusion protein or pharmaceutical composition as defined herein for use in the treatment of an autoimmune disease, an allergic disease, neurodegenerative or neuropathic pain. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows surface plasmon resonance (SPR) sensorgrams illustrating the binding reaction and off-rates of high affinity CD200-Fc (panels A-F) and wild-type CD200-Fc (panel G) fusion molecules binding to the human CD200 receptor at 25° C. [Diagram 2] FIG. 1 is a bar graph showing inhibition of IL-6 secretion by LPS stimulation of CD200R-expressing U937 cells. [Diagram 3] FIG. 1 is a bar graph showing inhibition of IL-6 secretion by LPS stimulation of CD200R-expressing U937 cells. [Figure 4] FIG. 1 is a bar graph showing inhibition of IL-6 secretion by LPS stimulation of CD200R-expressing U937 cells. [Diagram 5] FIG. 1 is a bar graph showing inhibition of IL-6 secretion by LPS stimulation of CD200R-expressing U937 cells. [Figure 6] FIG. 1 is a bar graph showing inhibition of IL-6 secretion by LPS stimulation of CD200R-expressing U937 cells. [Figure 7] FIG. 1 is a bar graph showing inhibition of IL-6 secretion by LPS stimulation of CD200R-expressing U937 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] According to a first aspect of the invention, the following mutations are provided: (i) K130F; or (ii) I131F; or (iii) K130F and I131F; or (iv) K130F and I131Y; or (v) K130Y and I131F, The present invention provides a mutated CD200 protein comprising:

[0019] According to a further aspect of the present invention, The following mutations at positions 130 and / or 131: (i) K130F; or (ii) I131F; or (iii) K130F and I131F; or (iv) K130F and I131Y; or (v) K130Y and I131F, having QVQVVTQDEREQLYTPASLKCSLQNAQEALIVTWQKKKAVSPENMVTFSENHGVVIQPAYKDKINITQLGLQNSTITFWNITLEDEGCYMCLFNTFGFGKISGTACLTVYVQPIVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLHLGTVTDFKQTVNK (SEQ ID NO: 26) The present invention provides a polypeptide comprising a mutated CD200 protein having at least 90% identity to the following:

[0020] It is understood that the polypeptide sequence of SEQ ID NO: 26 relates to the wild-type polypeptide sequence of the extracellular domain of CD200. In one embodiment, the polypeptide comprises: The following mutations at positions 130 and / or 131: (i) K130F; or (ii) I131F; or (iii) K130F and I131F; or (iv) K130F and I131Y; or (v) K130Y and I131F, having MERLVIRMPFSHLSTYSLVWVMAAVVLCTAQVQVVTQDEREQLYTPASLKCSLQNAQEALIVTWQKKKAVSPENMVTFSENHGVVIQPAYKDKINITQLGLQNSTITFWNITLEDEGCYMCLFNTFGFGKISGTACLTVYVQPIVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLHLGTVTDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILLYWKRHRNQDRGELSQGVQKMT (SEQ ID NO: 27) It contains at least 90% identity with the amino acid sequence of

[0021] It is understood that the polypeptide sequence of SEQ ID NO:27 relates to the full-length wild-type polypeptide sequence of CD200. The inventors have found that mutation of CD200 at specific amino acid residues (i)-(v) produces mutant CD200 with increased binding affinity to the CD200 receptor (CD200R). Furthermore, the inventors have found that optimal efficacy is obtained by combining a molecule that exhibits short residence time on the CD200 receptor, such as not exceeding 3000 seconds, with high affinity binding. Moreover, the mutated CD200 proteins described herein have great advantages, especially in terms of providing higher clinical efficacy and lower dose treatment.

[0022] As used herein, the term "CD200 protein" refers to a wild-type CD200 protein. The term "wild type" as used herein refers to naturally occurring proteins, peptides, amino acids, and nucleotide sequences. For example, the term "wild type CD200 protein" as used herein refers to any full length isoform of CD200 (UNIPROT P41217 OX2G_HUMAN) or any portion thereof (including naturally occurring protein polymorphisms) that binds to the CD200 receptor. The CD200 protein is also known as OX-2 membrane glycoprotein.

[0023] 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 a target receptor, such as the CD200 receptor. In one embodiment, the CD200 protein is the extracellular domain of CD200 or any portion thereof that binds to the CD200 receptor.

[0024] As used herein, the term "position" refers to the residue number in an amino acid sequence, with 1 being the first translated amino acid. The term "mutated" or "mutation" as used herein refers to proteins, peptides, amino acids, and nucleotide sequences that are altered in form from their wild-type counterparts, resulting in a mutant form. For example, a mutated or mutant protein may have an altered amino acid sequence and / or nucleotide sequence when compared to the corresponding wild-type sequence, and such alterations may also be referred to as mutations.

[0025] The term "mutated CD200 protein" as used herein refers to a full-length CD200 protein or any portion thereof that binds to the CD200 receptor and contains a mutated amino acid residue or multiple mutated amino acid residues in the amino acid sequence such that it is similar to, but no longer identical to, the wild-type CD200 protein.

[0026] In one embodiment, the mutated CD200 protein can be made synthetically or recombinantly. In a further embodiment, the mutated CD200 protein can be made synthetically. In an alternative embodiment, the mutated CD200 protein can be made recombinantly.

[0027] In one embodiment, the mutated CD200 protein binds to the CD200 receptor with higher affinity than wild-type CD200. In one embodiment, a mutated CD200 protein may contain a biologically or chemically active non-CD200 component within it or attached to it.

[0028] In one embodiment, the mutated CD200 protein may be soluble (i.e., circulating) or surface bound. In a further embodiment, the mutated CD200 protein is soluble. In an alternative embodiment, the mutated CD200 protein is surface bound.

[0029] In one embodiment, the mutated CD200 protein may comprise the entire extracellular domain of CD200 or a portion thereof. In a further embodiment, the mutated CD200 protein comprises a signal sequence. It is understood that the secreted protein comprises some amino acids at the N-terminus that constitute a signal sequence that may be cleaved prior to secretion. Thus, in a particular embodiment, the mutated CD200 protein comprises a signal sequence at the N-terminus that is cleaved prior to secretion from the producing cell. In a further embodiment, the signal sequence may be cleaved at any position selected from amino acid positions 16-35 of the wild-type CD200 protein. In one embodiment, the signal sequence comprises the first 28 amino acids of the wild-type CD200 protein. In an alternative embodiment, the signal sequence comprises the first 29 amino acids of the wild-type CD200 protein. In a further alternative embodiment, the signal sequence comprises the first 30 amino acids of the wild-type CD200 protein. In a further alternative embodiment, the signal sequence comprises the first 31 amino acids of the wild-type CD200 protein. In a further alternative embodiment, the signal sequence comprises the first 32 amino acids of the wild-type CD200 protein. Thus, in certain embodiments, a mutated CD200 protein comprises a sequence defined herein that lacks amino acids, including the signal sequence, for example, lacking amino acids 1-30 of a wild-type CD200 protein, a mutated CD200 protein comprises a sequence corresponding to amino acids 31-232 of any sequence defined herein.

[0030] The term "portion" as used herein in reference to proteins, peptides, and amino acid and nucleotide sequences refers to functional, i.e., target-binding, fragments and derivatives.

[0031] The term "fragment" as used herein refers to a portion of a protein, peptide, amino acid, or nucleotide sequence that recognizes and binds to its target, such as a receptor. The terms "derivative" and "variant" as used herein refer to a protein, peptide, amino acid, or nucleotide sequence that shares at least 70% (such as 75%, 80%, 85%, 90%, 95%, or 99%) sequence similarity with the wild-type counterpart and functions similarly. Thus, a variant may be a derivative of the wild-type counterpart.

[0032] The term "amino acid residue" as used herein refers to a monomeric unit in a polymer chain, i.e., a single amino acid in a protein. As used herein, the term "at least 90% identity" refers to sequences that share sequence identity or sequence homology. Suitable examples include 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0033] In one embodiment, the protein further comprises one or more mutations, for example 1-15 mutations, present in the amino acid sequence. In one embodiment, the mutated CD200 protein comprises a single substitution mutation of K130F. Specific examples of mutated proteins comprising this single substitution mutation are described herein as DS-175, DS-161, DS-174, and DS-213.

[0034] In an alternative embodiment, the mutated CD200 protein comprises a single substitution mutation of I131F. Specific examples of mutated proteins comprising this single substitution mutation are described herein as DS-215, DS-162, DS-216 and DS-214.

[0035] In an alternative embodiment, the mutated CD200 protein comprises a double substitution mutation of K130F and I131F. Specific examples of mutated proteins comprising this double substitution mutation are described herein as DS-217, DS-150, DS-218 and DS-220.

[0036] In an alternative embodiment, the mutated CD200 protein comprises a double substitution mutation of K130F and I131Y. Specific examples of mutated proteins comprising this double substitution mutation are described herein as DS-164, DS-151, DS-163, DS-219, DS-167 and DS-165.

[0037] In an alternative embodiment, the mutated CD200 protein comprises a double substitution mutation of K130Y and I131F. Specific examples of mutated proteins comprising this double substitution mutation are described herein as DS-221, DS-149, DS-222 and DS-223.

[0038] As shown in FIG. 1 and Table 4, the mutated CD200 proteins of the present invention bind more tightly to the CD200 receptor and exhibit a longer residence time on the receptor than the wild-type CD200 protein.

[0039] Fusion proteins According to a second aspect of the invention there is provided a fusion protein comprising a protein as defined herein fused, either directly or via an optional linker moiety, to a non-CD200 moiety.

[0040] For example, the linker moiety is a peptide comprising 1 to 15 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

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

[0042] In one embodiment, the protein herein is fused at the C-terminus to a non-CD200 moiety directly or via an optional linker moiety. The term "non-CD200 moiety" as used herein refers to any molecule, peptide, or protein that does not specifically bind to the CD200 receptor and does not interfere with the binding of the mutated CD200 protein to its target. Examples include, but are not limited to, immunoglobulin (Ig) constant regions or portions thereof, or fusion proteins in which the non-CD200 moiety is a synthetic molecule, such as PEG.

[0043] In one embodiment, the non-CD200 moiety is an antibody or a fragment thereof. In a further embodiment, the non-CD200 moiety is an Fc fragment. Thus, the mutated CD200 fusion proteins described herein may also be referred to as mutated CD200-Fc. In a further embodiment, the Fc fragment is of mammalian origin, such as human or monkey origin, for example human C(gamma)1, including the hinge, CH2, and CH3 regions. The Fc fragment provides the advantage of increasing the serum half-life of the mutated CD200 proteins of the invention, and further increases the binding activity and allows agonistic signaling by dimerizing the CD200 protein. It will be appreciated by those skilled in the art that the Fc region may be mutated to reduce effector function (see, for example, US 5,637,481 and US 6,132,992).

[0044] In some embodiments, the human Fc domain comprises mutations to remove glycosylation and / or to reduce Fc-gamma receptor binding. In some embodiments, the human Fc domain comprises the mutations N297Q, N297A, or N297G, in some embodiments, the human Fc domain comprises a mutation at position 234 and / or 235, e.g., L235E, or L234A and L235A (in IgG1), or F234A and L235A (in IgG4), in some embodiments, the human Fc domain is an IgG2 Fc domain comprising the mutations V234A, G237A, P238S, H268Q / A, V309L, A330S, or P331S, or a combination thereof (all according to Kabat, EU numbering). In some embodiments, the human Fc domain comprises the human IgG1 constant region mutation L234A / L235A ("LALA") or the human IgG1 constant region mutation L234A / L235A / P329G ("LALAPG"), respectively. Further examples of engineered human Fc domains are known to those of skill in the art. Examples of Ig heavy chain constant region amino acids in which mutation of at least one amino acid results in reduced Fc function include, but are not limited to, mutations of amino acids 228, 233, 234, 235, 236, 237, 239, 252, 254, 256, 265, 270, 297, 318, 320, 322, 327, 329, 330, and 331 in the heavy chain constant region (according to Kabat, EU numbering). Examples of mutated amino acid combinations are also known in the art and include, but are not limited to, combinations of mutations at amino acids 234, 235, and 331, such as combinations of 234, 235, and 329, such as L234F, L235E, and P331S, or combinations of amino acids 318, 320, and 322, such as E318A, K320A, and K322A.

[0045] Further examples of engineered Fc domains include F243L / R292P / Y300L / V305I / P396 IgG1; S239D / I332E IgG1; S239D / I332E / A330L IgG1; S298A / E333A / K334A; in one heavy chain, L234Y / L235Q / G236W / S239M / H268D / D270E / S298A IgG1; and in the opposing heavy chain, D270E / K326D, A330M / K334E IgG1; G236A / S239D / I332E IgG1; K326W / E333S IgG1; S267E / H268F / S324T. Examples of IgG1 include IgG1; E345R / E430G / S440Y IgG1; N297A or N297Q or N297G IgG1; L235E IgG1; L234A / L235A IgG1; F234A / L235A IgG4; H268Q / V309L / A330S / P331S IgG2; V234A / G237A / P238S / H268A / V309L / A330S / P331S IgG2; M252Y / S254T / T256E IgG1; M428L / N434S IgG1; S267E / L328F IgG1; N325S / L328F IgG1, etc. In some embodiments, the engineered Fc domain comprises one or more substitutions selected from the group consisting of N297A IgG1, N297Q IgG1, and S228P IgG4.

[0046] In one embodiment, polypeptides of the disclosure comprising an Fc variant exhibit reduced affinity for an Fc receptor, e.g., FcγRI, FcγRIIA, FcγRIIIA, as compared to an unmodified antibody. In one embodiment, polypeptides comprising an Fc variant exhibit affinity for an Fc receptor that is at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, at least 5%, or at least 1% lower than the affinity of the wild-type polypeptide. In one embodiment, polypeptides comprising an Fc variant of the disclosure exhibit greater than a 700-fold decrease in Fcγ binding, or greater than a 3,500-fold decrease in Fcγ binding.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variant Fc region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM. In certain embodiments, the antibody is an aglycosylated antibody with reduced effector function. In certain embodiments, the variant Fc region of IgG1 comprises (a) an amino acid substitution at position Leu234 with alanine, (b) an amino acid substitution at position Leu235 with alanine, (c) an amino acid substitution at position Pro329 with glycine or arginine, (d) an amino acid substitution at position Asn297 with alanine, (e) an amino acid substitution at position Asn297 with glutamine, (f) an amino acid substitution at position Asn297 with glycine, or (g) any combination of (a)-(f). In certain embodiments, the variant Fc region of IgG2 comprises (g) an amino acid substitution at position Ser228 with proline, (h) an amino acid substitution at position Pro329 with glycine or arginine, or (i) both (g) and (h). In certain embodiments, the variant Fc region of IgG4 comprises (j) an amino acid substitution at position Ser228 with proline, (k) an amino acid substitution at position Leu235 with alanine or glutamic acid, (l) an amino acid substitution at position Pro329 with glycine or arginine, or (m) any combination of (j)-(l).

[0048] In one embodiment, the Fc fragment is or is derived from human IgG2 or human IgG4. In a further embodiment, the non-CD200 moiety is an antibody Fc fragment comprising mutations of one or more amino acid residues.Thus, in a further embodiment, the Fc fragment is an S228P derivative of human IgG4.

[0049] In one embodiment, the fusion protein is an Fc fusion protein formed by direct fusion of amino acid glycine 232 of CD200 to amino acid 1 of the Fc hinge region. In an alternative embodiment, the fusion protein is an Fc fusion protein formed by direct fusion of amino acid glycine 232 of CD200 with amino acid 6 of the Fc hinge region. (In this case, the first five amino acids of the Fc hinge are deleted).

[0050] For purposes of this description, when the non-CD200 moiety is an Fc fragment, the term "position" as used herein with respect to the non-CD200 moiety refers to the residue number in the amino acid sequence according to the EU numbering system. Thus, it is understood that the amino acid residue positions of the Fc fragment cited herein refer to the positions according to the EU numbering system. It is further understood that other numbering systems developed for numbering residues in an Fc fragment sequence, such as Kabat, Aho, IMGT, Chothia, and Martin (enhanced Chothia), may alternatively be utilized. For example, when referring to the Fc hinge region herein, it is intended to refer to the region of the Fc domain beginning at amino acid position 1 as defined by the IMGT numbering (https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html) or amino acid position 216 as defined by the EU numbering system.

[0051] Specific examples of the fusion protein of the present invention are exemplified in Table 1 as SEQ ID NOs: 1 to 22. In one embodiment, the fusion protein is selected from any one of SEQ ID NOs: 1 to 22. In a further embodiment, the fusion protein is selected from any one of SEQ ID NOs: 1 to 16 and 19 to 22.

[0052] [Table 1-1]

[0053] [Table 1-2]

[0054] [Table 1-3]

[0055] [Table 1-4]

[0056] [Table 1-5]

[0057] [Table 1-6]

[0058] [Table 1-7]

[0059] [Table 1-8]

[0060] Specific examples of wild-type CD200-Fc fusion proteins are exemplified in Table 2 as SEQ ID NOs:23-25.

[0061] [Table 2]

[0062] The proteins of the invention are preferably produced by recombinant DNA techniques by inserting a nucleic acid sequence encoding the mutated CD200 protein or any part thereof into a recombinant expression vector and expressing the nucleic acid sequence in a recombinant expression system under conditions promoting expression. Thus, in one embodiment, the polynucleotide encoding the fusion protein further comprises a vector such as pcDNA3.4. In one embodiment, the fusion protein is flanked by one or more restriction enzyme sites such as Hind III and / or Xho I. In a further embodiment, the polynucleotide encoding the fusion protein is flanked by Hind III and Xho I restriction sites.

[0063] In one embodiment, the fusion protein contains one or more restriction enzyme sites, such as Bam HI. According to a further aspect of the invention, there are provided polynucleotides encoding the proteins defined herein, and the use of such nucleic acids to produce proteins and / or for therapeutic purposes. Such polynucleotides may include DNA and RNA molecules (e.g., mRNA, self-replicating RNA, self-amplifying mRNA, etc.) that encode the proteins defined herein. Nucleic acid sequences encoding the proteins provided by the invention may be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide synthetic genes that can be inserted into a recombinant expression vector and expressed in a recombinant transcription unit.

[0064] The recombinant expression vector comprises a synthetic or cDNA-derived nucleic acid fragment encoding a mutated CD200 operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants, can also be incorporated.

[0065] therapeutic use The present invention has particular application in therapy, since the interaction between CD200 protein and CD200 receptor is characterized by a fast dissociation ("off") rate resulting in low affinity of CD200 for the CD200 receptor. Thus, as shown herein, increasing the affinity of mutant CD200 proteins for the CD200 receptor can be used to prepare pharmaceutical compositions with more potent properties.

[0066] Furthermore, the production costs of recombinant proteins are high, and mutant CD200 proteins with high affinity can be used in pharmaceutical compositions at significantly lower doses to achieve therapeutic effect than wild-type or non-mutated CD200 proteins. Thus, the use of mutant CD200 proteins may not only be more clinically effective, but also more cost-effective.

[0067] According to a further aspect of the invention there is provided a pharmaceutical composition comprising a protein, polypeptide or fusion protein as defined herein, or a nucleic acid encoding the protein, polypeptide or fusion protein.

[0068] In one embodiment, the mutated CD200 proteins, polypeptides or fusion proteins defined herein are modulators of the CD200 receptor. The term "modulator" as used herein refers to a substance that brings about a change, for example, a modulator of a protein can bring about an increase or decrease in the activity of said protein. Given the properties of the mutated CD200 proteins and fusion proteins of the present invention, they are believed to be agonists of the CD200 receptor and therefore are believed to be useful in the treatment of autoimmune diseases. Thus, in a further embodiment, the mutated CD200 proteins, polypeptides or fusion proteins defined herein are agonists of the CD200 receptor.

[0069] Thus, according to a further aspect of the invention there is provided a protein, polypeptide or fusion protein as defined herein, or a composition as defined herein, for use in the treatment of an autoimmune disease.

[0070] As used herein, the terms "autoimmune disease" or "autoimmune disorder" are used interchangeably to refer to an undesirable condition resulting from an inappropriate or unnecessary 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 cellular immune response.

[0071] In an alternative embodiment, there is provided a protein, polypeptide, or fusion protein as defined herein, or a composition as defined herein, for use in treating allergic disease. As used herein, the terms "allergy" or "allergic disease" are used interchangeably and refer to T helper 2 (TH2)-driven diseases that develop primarily from the activity of TH2 cells. Examples of allergic diseases include chronic allergic diseases (such as hay fever or allergic rhinitis), allergic contact dermatitis, seasonal allergies, the treatment and prevention of anaphylaxis, and food allergies.

[0072] Fusion proteins comprising a mutated CD200 protein as defined herein are able to inactivate activated immune cells more efficiently than fusion proteins comprising a wild-type or non-mutated CD200 protein.

[0073] In one embodiment, the autoimmune disease is selected from autoimmune diseases affecting the neuromuscular system, vascular system, eyes, gastrointestinal tract, lungs, kidneys, liver, peripheral or central nervous system, bone, cartilage, or joints.

[0074] In further embodiments, the autoimmune disease is selected from the group consisting of acute disseminated encephalomyelitis (ADEM); acute necrotizing hemorrhagic leukoencephalitis; Addison's disease; agammaglobulinemia; alopecia areata; amyloidosis; ankylosing spondylitis; 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; autoimmune pancreatitis; autoimmune retinopathy; autoimmune Thrombocytopenic purpura (ATP);autoimmune thyroid disease;autoimmune urticaria;axonal neuronal neuropathy;Barrow's disease;Behçet's disease;bullous pemphigoid and related autoimmune blistering disorders;cardiomyopathy;Castleman's disease;celiac disease (including refractory type 2 celiac disease);Chagas disease;idiopathic chronic urticaria;chronic inflammatory demyelinating polyneuropathy (CIDP);chronic obstructive pulmonary disease (COPD);chronic relapsing multifocal osteomyelitis (CRMO);chronic idiopathic urticaria;Churg-Strauss syndrome;cicatric pemphigoid / benign mucous membrane pemphigoid;Crohn's disease;Cogan's syndrome ;cold agglutinin disease;congenital cardiac conduction disorders;coxsackie myocarditis;CREST disease;idiopathic mixed cryoglobulinemia;demyelinating neuropathy;dermatitis herpetiformis;dermatomyositis;Devic's disease (neuromyelitis optica);disciform lupus;Dressler 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 called Wegener's granulomatosis);graft-versus-host GvHD;Graves' disease;Guillain-Barre syndrome;Hashimoto's encephalitis;Hashimoto's disease;Hemolytic anemia;Henoch-Schönlein purpura nephritis;Herpes of pregnancy;Hypogammaglobulinemia;Idiopathic thrombocytopenic purpura (ITP);IgA neuropathy;IgG4-related sclerosing disease;Immunomodulatory lipoprotein;Inclusion body myositis;Inflammatory bowel disease (IBD);Inflammatory skin disease;Interstitial cystitis;Juvenile arthritis;Juvenile diabetes mellitus (type 1 diabetes);Juvenile myositis;Kawasaki disease;Lambert-Eaton syndrome;Leukocytoclastic vasculitis;Lichen planus;Lichen sclerosus;Lignin keratoconjunctivitis;Linear IgA disease (LAD);Systemic lupus erythematosus (SLE);Lyme disease, chronic;Macrophage activation syndrome (MAS);Mastocytosis;Meniere's syndrome;Microscopic polyangiitis;Mixed connective tissue disease (MCTD);Mohren's ulcer;Much-Habermann's disease;Multiple sclerosis;Myasthenia gravis;Myositis;Narcolepsy;Neuromyelitis optica (Devic's disease);Neutropenia;Ocular cicatricial pemphigoid;Optic neuritis;Relapsing rheumatoid arthritis;PANDAS (Streptococcus associated childhood autoimmune disease) Autoimmune neuropsychiatric disorders);Paramecular cerebellar degeneration;Paroxysmal nocturnal hemoglobinuria (PNH);Parry-Romberg syndrome;Parsonage-Turner syndrome;Paraplanitis (peripheral uveitis);Pemphigus;Peripheral neuropathy;Perivenous encephalomyelitis;Pernicious anemia;POEMS syndrome;Polyarteritis nodosa;Polyglandular autoimmune syndrome types 1, 2, and 3;Polymyalgia rheumatica;Polymyositis;Post-myocardial infarction syndrome;Post-pericardiotomy syndrome;Progesterone Long's 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;sarcoidosis;Schmidt's syndrome;scleritis;scleroderma;Sjogren's syndrome;sperm and testicular autoimmunity;stiff body syndrome;subacute One or more autoimmune diseases selected from bacterial endocarditis (SBE); Susac syndrome; sympathetic ophthalmia; Takayasu's arteritis; temporal arteritis / giant cell arteritis; thrombocytopenic purpura (TTP); Tolosa-Hunt syndrome; transverse myelitis; type 1 diabetes; ulcerative colitis; undifferentiated connective tissue disease (UCTD); uveitis; vasculitis; vesiculobullous dermatoses; vitiligo; and Wegener's granulomatosis (now called granulomatosis with polyangiitis (GPA));

[0075] In further embodiments, the autoimmune disease is one or more autoimmune diseases selected from atopic dermatitis, alopecia areata, asthma, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), chronic obstructive pulmonary disease (COPD), multiple sclerosis, and rheumatoid arthritis.

[0076] In an alternative embodiment there is provided a protein, polypeptide or fusion protein as defined herein, or a composition as defined herein, for use in the treatment of neurodegeneration.

[0077] In alternative embodiments there is provided a protein, polypeptide or fusion protein as defined herein, or a composition as defined herein, for use in the treatment of neuropathic pain. In further embodiments there is provided a protein, polypeptide or fusion protein as defined herein, or a composition as defined herein, for use in the treatment of neuropathic pain, such as diabetic neuropathy.

[0078] According to a further aspect of the invention there is provided a method of treating an autoimmune disease, an allergic disease, neurodegenerative or neuropathic pain in a subject comprising administering to a subject having at least one autoimmune disease, allergic disease, neurodegenerative or neuropathic pain a protein, polypeptide or fusion protein of the invention.

[0079] It will be understood that the proteins, polypeptides, or fusion proteins of the invention can be administered as the sole therapeutic agent or can be administered in combination therapy with one or more other compounds (or therapies) to treat autoimmune diseases, allergic diseases, neurodegenerative, or neuropathic pain.

[0080] Thus, according to a further aspect of the present invention there is provided a pharmaceutical composition comprising a protein, polypeptide or fusion protein as defined herein in combination with one or more additional therapeutic agents.

[0081] To treat autoimmune diseases, allergic diseases, neurodegenerative or neuropathic pain, the proteins, polypeptides or fusion proteins of the invention may be advantageously used in combination with one or more other agents, more particularly, one or more immunosuppressants or adjuvants in immunosuppressive therapy.

[0082] Examples of therapeutic agents or treatments that may be administered together with the compounds of the invention (simultaneously or at different time intervals) include, but are not limited to, azathioprine, methotrexate, cyclosporine, monoclonal antibodies (basiliximab, daclizumab, and muromonab), and corticosteroids.

[0083] Each therapeutic agent present in the combination of the present invention can be administered individually by different dosage schedules and different routes.Furthermore, the dosage of each of the two or more agents can be different, and each can be administered simultaneously or at different times.Those skilled in the art will understand through general knowledge which dosage regimen and combination therapy to use.For example, the protein, polypeptide, or fusion protein of the present invention can be used in combination with one or more other agents that are administered according to existing combination dosage regimens.

[0084] Generally, the proteins disclosed herein are utilized in purified form together with pharmacologically appropriate excipients or carriers.Typically, these excipients or carriers include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and / or buffered media.Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, and lactated Ringer's solution.If necessary to keep the polypeptide complex in suspension, suitable physiologically acceptable adjuvants can be selected from viscosity enhancing agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates.

[0085] The route of administration of the pharmaceutical composition according to the invention may be any of those generally known to those skilled in the art. For treatment, including but not limited to immunotherapy, the protein of the invention may be administered to any patient according to standard techniques. Administration may be performed in any suitable manner, including parenterally, intravenously, intramuscularly, intraperitoneally, subcutaneously, transdermally, via the pulmonary route, or by direct injection, suitably by catheter. The dosage and frequency of administration will depend on the age, sex, and condition of the patient, the co-administration of other drugs, contraindications, and other parameters that the clinician should take into account.

[0086] 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 lyophilization and reconstitution techniques known in the art can be used.Those skilled in the art will understand that lyophilization and reconstitution may reduce activity to various degrees, and levels may have to be adjusted upward to compensate.

[0087] The following studies and protocols demonstrate embodiments of the methods described herein. EXAMPLES

[0088] Example 1: Production of mutant and wild-type CD200-Fc molecules Gene synthesis DNA sequences encoding residues 1-232 of mutant or wild-type human CD200 of Uniprot P412178 (OX2G_Human), including the N-terminal signal sequence, were fused C-terminally to mutant or wild-type IgG2 or IgG4 Fc, either by fusing amino acid glycine 232 of CD200 to amino acid 1 of the Fc hinge region or directly to amino acid 6 of the Fc hinge region (in the latter case, the first 5 amino acids of the Fc hinge are deleted). Gene synthesis was performed at GeneArt for wild-type and mutant constructs.

[0089] The expression construct was made using the mammalian expression vector pcDNA3.4, with a 5'Hind III and a 3'Xho. An internal Bam HI was introduced to facilitate Fc domain swapping.

[0090] Midi Prep Upon receipt, the lyophilized DNA constructs of CD200-Fc target proteins (both wild-type and mutant CD200-Fc proteins) were suspended in 50 μl MQ and transformed into DH5α cells. A single colony of each target protein was selected and inoculated into 5.0 ml LB containing ampicillin. DNA was then isolated from 2.0 ml cultures for confirmation and separated by agarose gel electrophoresis. The constructs were confirmed by digesting DNA with Hind III and Xho I. Each mutant or wild-type construct was cultured in 100 ml LB and mid-scale DNA was prepared. DNA was isolated using the purelink Hipure plasmid midiprep kit.

[0091] Protein expression CD200-Fc target protein was produced using the ThermoFisher Gibco™ ExpiCHO™ Expression System in a culture volume of 25 ml according to the manufacturer's instructions. Culture supernatants containing expressed CD200-Fc were collected and stored at -80°C until use.

[0092] Protein purification Buffer exchange was performed using a Hiprep desalting column (XK26 / 10) packed with 53 ml of Sephadex G25 to prepare the medium for affinity column purification. The desalting column was equilibrated with buffer A (150 mM NaCl containing 20 mM sodium phosphate pH 7.4) on an AKTA Explorer platform. 30 ml of clarified culture was loaded onto two desalting columns connected in series at a rate of 1 ml / min. Protein was eluted at a rate of 2 ml / min and collected as fractions. Fractions showing maximum absorbance and pH 7.4-7.2 were pooled. Fractions showing lower pH were excluded. After desalting, samples were diluted to approximately 45 ml of wild-type or mutant CD200-Fc supernatant. All purification steps were performed on ice at 4°C.

[0093] The column was washed with 10 column volumes of buffer A (10 ml of 20 mM sodium phosphate pH 7.4, 150 mM NaCl). CD200-Fc protein was eluted with 20 mM sodium phosphate pH 7.4, 150 mM NaCl and 100 mM citrate buffer pH 3.5 in a linear gradient from pH 7.4 to 3.5 over 10 column volumes. The CD200-Fc fraction containing the dimeric form of the protein (calculated to be approximately 103 kDa) was collected. The protein buffer was exchanged using an Amicon ultra centricon with a 10 kDa cutoff and the protein was concentrated to approximately 1 mg / ml.

[0094] Example 2: Binding analysis of wild-type and mutant CD200-Fc molecules Biacore experiments were performed by Syngene International Ltd. (Biocon Park, Plot No2&3, Bommasandra Industrial Area, Bommasadra-Jigani Link Road, Bangalore-560099, India).

[0095] Assay Principle The BIAcore instrument uses an optical technique, surface plasmon resonance (SPR), to measure the binding properties of wild-type CD200-Fc or CD200-Fc mutants that bind to two interacting molecules, in this case the CD200 receptor (CD200R). This technique measures the change in refractive index of one of two interacting molecules captured on a chip (sensor) when the second flows over its captured partner in solution. 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 aqueous buffer. The change in the refractive index of CD200-Fc after CD200R binding was measured in real time and the results plotted in response units (Ru) versus time to generate a sensorgram.

[0096] Equipment and Reagents Experiments were performed on a GE Healthcare BIAcore T200. Table 3 details the reagents used in the development and performance of the assay.

[0097] [Table 3]

[0098] Immobilization of CD200-Fc Anti-human Fc was covalently immobilized on a BIAcore CM5 sensor chip by amine coupling using a kit from GE Healthcare, following the manufacturer's instructions. Maximum immobilization target was set at 10,000-15,000 RU. Flow cell 1 was used as a reference with no immobilized ligand to allow subtraction of non-specific binding to the chip surface. Fc ligand was diluted to 0.5 μg / mL in BIAcore running buffer (HBS-EP+: 10 mM HEPES-buffered saline with 2 mM EDTA and 0.05% surfactant P-20). For the final immobilization step, wild-type CD200-Fc, mutant CD200-Fc and unrelated control proteins (Herceptin / Trastuzumab) at concentrations yielding a minimum of 250 response units (RU) were passed over the chip (using flow cells 2, 3, and 4, respectively) for 120 s, followed by stabilization of the surface in running buffer for 120 s. The CD200-Fc capture procedure was repeated for each concentration of CD200R.

[0099] Crossing of CD200R over a CD200-Fc-bound chip surface After capture of Fc-tagged proteins, CD200R (at different concentrations) was flowed over the captured CD200-Fc and control proteins for 120 seconds (to observe binding), followed by 120 seconds of running buffer (to observe dissociation). The chip surface was then regenerated with 10 mM glycine·HCl (pH 2) for 30 seconds (flow rate 30 μl / min), followed by stabilization of the surface with BIAcore running buffer for 60 seconds before the next cycle. All CD200R concentrations were run in duplicate at the following concentrations: 1 μM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.6 nM, and 0 nM.

[0100] 4. Data Analysis Results were expressed in sensorgrams plotted as response units or resonance units (Ru) versus time. Experimental sensorgrams were analyzed with BIAevaluation software version 1.0 (GE Healthcare). Curve fitting for all CD200-Fc fusion molecules, except for DS-162, was performed using a 1:1 Langmuir binding model. The curve for DS-162 did not fit well with the 1:1 binding model. Both 1:1 and two-state binding models were used to fit the DS-162 results. The dissociation phase of the DS-162 sensorgram was found to fit better with the two-state binding model than with the 1:1 binding model (Figure 1, panels E-F). A kinetic equation using standard parameters (e.g., ligand concentration, time) was used for iterative curve fitting. Closeness of fit was determined by the algorithm provided by the manufacturer in the BIAevaluation software version 1.0.

[0101] result The results (Table 4 and Figure 1) show that the mutated CD200-Fc proteins bind to the human CD200 receptor with 16-70 fold higher affinity than wild-type CD200-Fc. The off-rates tabulated in Table 4 and the sensorgrams shown in Figure 1 indicate the range of receptor off-rates and half-lives. Notably, DS-161 and DS-162 combine high affinity binding to the CD200 receptor with off-rates compatible with efficient agonism in functional cellular assays.

[0102] [Table 4]

[0103] Example 3: Inhibition of IL-6 secretion after LPS stimulation of CD200R-expressing U937 cells To demonstrate the agonistic activity of the CD200-Fc protein, the human monocytic cell line U937 (ATCC, CRL1539) was transfected with the cDNA for human CD200R. Cytokine production, including IL-6, from these cells could be induced by stimulation with PMA followed by LPS.

[0104] 50,000 U937 cells were seeded per well in 96-well plates and incubated with 100 nM PMA for 72 h before differentiation. After differentiation, the medium containing PMA was replaced with fresh assay medium and incubated for an additional 2 h before treatment. CD200-Fc constructs (including DS-226 as a wild-type control) were then added to the cell cultures and incubated for 1 h. Cells were stimulated with 100 ng / ml LPS and incubated for an additional 24 h. After the final incubation, cell supernatants (diluted 1:10) were harvested and IL-6 secretion was quantified using an ELISA assay.

[0105] Figures 2-7 show the concentration-dependent inhibition of IL-6 secretion by the CD200-Fc construct. The data presented demonstrate that the CD200-Fc construct can inhibit LPS-stimulated IL-6 secretion in a concentration-dependent manner.

Claims

1. The following mutations: (i) K130F; or (ii) K130F and I131F; or (iii) K130F and I131Y; or (iv) K130Y and I131F, A mutated CD200 protein comprising:

2. The following mutations at positions 130 and / or 131: (i) K130F; or (ii) I131F; or (iii) K130F and I131F; or (iv) K130F and I131Y; or (v) K130Y and I131F, having QVQVVTQDEREQLYTPASLKCSLQNAQEALIVTWQKKKAVSPENMVTFSENHGVVIQPAYKDKINITQLGLQNSTITFWNITLEDEGCYMCLFNTFGFGKISGTACLTVYVQPIVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLHLGTVTDFKQTVNK (SEQ ID NO: 26) A polypeptide comprising a mutated CD200 protein comprising at least 90% identity to the following, wherein the mutation relates to amino acid residues 130 and / or 131 of SEQ ID NO: 27:

3. The following mutations at positions 130 and / or 131: (i) K130F; or (ii) I131F; or (iii) K130F and I131F; or (iv) K130F and I131Y; or (v) K130Y and I131F, having MERLVIRMPFSHLSTYSLVWVMAAVVLCTAQVQVVTQDEREQLYTPASLKCSLQNAQEALIVTWQKKKAVS PENMVTFSENHGVVIQPAYKDKINITQLGLQNSTITFWNITLEDEGCYMCLFNTFGFGKISGTACLTVYVQP IVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLHLGTVTDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILYWKRHRNQDRGELSQGVQKMT (SEQ ID NO: 27) 3. The polypeptide of claim 2, comprising at least 90% identity with the amino acid sequence of

4. A fusion protein comprising the protein of claim 1 fused directly or via a linker moiety to a non-CD200 moiety.

5. A fusion protein comprising the polypeptide of claim 2 fused directly or via a linker moiety to a non-CD200 moiety.

6. The fusion protein of claim 4 or 5, wherein the non-CD200 moiety is an antibody or a fragment thereof.

7. The fusion protein of claim 6, wherein the non-CD200 portion is an Fc fragment.

8. 8. The fusion protein of claim 7, wherein the Fc fragment is or is derived from human IgG2 or human IgG4, such as the S228P derivative of human IgG4.

9. 8. The fusion protein of claim 7, which is an Fc fusion protein formed by directly fusing the amino acid glycine 232 of CD200 to amino acid 1 of the Fc hinge region, or an Fc fusion protein formed by directly fusing the amino acid glycine 232 of CD200 to amino acid 6 of the Fc hinge region.

10. The fusion protein according to claim 4 or 5, which is selected from any one of SEQ ID NOs: 1 to 22.

11. 3. The protein of claim 1 or the polypeptide of claim 2, which is a modulator of the CD200 receptor.

12. The fusion protein of claim 4 or 5, which is a modulator of the CD200 receptor.

13. 3. The protein of claim 1 or the polypeptide of claim 2, which is an agonist of the CD200 receptor.

14. The fusion protein of claim 4 or 5, which is an agonist of the CD200 receptor.

15. A polynucleotide encoding the protein of claim 1.

16. A polynucleotide encoding the polypeptide of claim 2.

17. A polynucleotide encoding the fusion protein of claim 4.

18. A polynucleotide encoding the fusion protein of claim 5.

19. A pharmaceutical composition comprising the protein of claim 1, the polypeptide of claim 2, the fusion protein of claim 4 or 5, or the polynucleotide of any one of claims 15 to 18, and a carrier.

20. 19. A pharmaceutical composition for use in the treatment of autoimmune diseases, allergic diseases, neurodegenerative or neuropathic pain, comprising the protein of claim 1, the polypeptide of claim 2, the fusion protein of claim 4 or 5, or the polynucleotide of any one of claims 15 to 18.

21. The protein according to claim 1, the polypeptide according to claim 2, the fusion protein according to claim 4 or 5, or the polynucleotide according to any one of claims 15 to 18. Acute disseminated encephalomyelitis (ADEM); acute necrotizing hemorrhagic leukoencephalitis; Addison's disease; agammaglobulinemia; alopecia areata; amyloidosis; ankylosing spondylitis; 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; autoimmune pancreatitis; autoimmune Immune retinopathy; autoimmune thrombocytopenic purpura (ATP); autoimmune thyroid disease; autoimmune urticaria; axonal neuronal neuropathy; Barrow's disease; Behçet's disease; bullous pemphigoid and related autoimmune blistering diseases; cardiomyopathy; Castleman's disease; celiac disease (including refractory type 2 celiac disease); Chagas disease; idiopathic chronic urticaria; chronic inflammatory demyelinating polyneuropathy (CIDP); chronic obstructive pulmonary disease (COPD); chronic relapsing multifocal osteomyelitis (CRMO); chronic idiopathic urticaria; Char Strauss syndrome; cicatricial pemphigoid / benign mucous membrane pemphigoid; Crohn's disease; Cogan's syndrome; cold agglutinin disease; congenital cardiac conduction disorders; Coxsackie myocarditis; CREST disease; idiopathic mixed cryoglobulinemia; demyelinating neuropathy; dermatitis herpetiformis; dermatomyositis; Devic's disease (neuromyelitis optica); discoid lupus; 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 called Wegener's granulomatosis); graft-versus-host disease (GvHD); Graves' disease; Guillain-Barré syndrome; Hashimoto's encephalitis; Hashimoto's disease; hemolytic anemia; Henoch-Schönlein purpura nephritis; herpes gestationis; hypogammaglobulinemia; idiopathic thrombocytopenic purpura (ITP); IgA neuropathy; IgG4-related sclerosing disease; immunoregulatory 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 sclerosus; lignified keratoconjunctivitis; linear immunoglobulin A disease (LAD); systemic lupus erythematosus (SLE); Lyme disease, chronic; macrophage activation syndrome (MAS); mastocytosis; Meniere's syndrome; microscopic polyangiitis; mixed connective tissue disease (MCTD); Morbus myofasciitis Len's ulcer; Much-Habermann's disease; multiple sclerosis; myasthenia gravis; myositis; narcolepsy; neuromyelitis optica (Devic's disease); neutropenia; ocular cicatricial pemphigoid; optic neuritis; relapsing rheumatism; PANDAS (pediatric autoimmune neuropsychiatric disorder associated with streptococcal infection); paraneoplastic cerebellar degeneration; paroxysmal nocturnal hemoglobinuria (PNH); Parry-Romberg syndrome; Parsonage-Turner syndrome; pars planitis (peripheral uveitis); pemphigus; Peripheral neuropathy; perivenous encephalomyelitis; pernicious anemia; POEMS syndrome; polyarteritis nodosa; polyglandular autoimmune syndrome types 1, 2, and 3; 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 leg syndrome; retroperitoneal fibrosis; rheumatic fever; rheumatoid arthritis; sarcoidosis; Schmidt's syndrome; scleritis; scleroderma; Sjogren's syndrome; semen and testicular autoimmunity; stiff body syndrome; subacute bacterial endocarditis (SBE); Susac's syndrome; sympathetic ophthalmia; for use in the treatment of an autoimmune disease selected from Takayasu's arteritis; temporal arteritis / giant cell arteritis; thrombocytopenic purpura (TTP); Tolosa-Hunt syndrome; transverse myelitis; type 1 diabetes; ulcerative colitis; undifferentiated connective tissue disease (UCTD); uveitis; vasculitis; vesiculo-bullous dermatosis; vitiligo; or Wegener's granulomatosis (GPA), or For use in the treatment of inflammatory skin diseases, Pharmaceutical compositions.

22. 19. A pharmaceutical composition for use in treating an autoimmune disease selected from atopic dermatitis, alopecia areata, asthma, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), chronic obstructive pulmonary disease (COPD), multiple sclerosis, and rheumatoid arthritis, comprising the protein of claim 1, the polypeptide of claim 2, the fusion protein of claim 4 or 5, or the polynucleotide of any one of claims 15 to 18.

23. A pharmaceutical composition for use in treating neuropathic pain, which is diabetic neuropathy, comprising the protein of claim 1, the polypeptide of claim 2, the fusion protein of claim 4 or 5, or the polynucleotide of any one of claims 15 to 18.

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