Novel CD200 fusion proteins

Abstract

The present invention relates to a fusion protein comprising a mutated CD200 moiety comprising the K130Y and I131Y mutations, which binds to the human CD200 receptor with higher affinity than wild-type CD200, directly fused to a non-CD200 IgG4 Fc fragment comprising the S228P, M428L, and N434S mutations and a deletion of the first 5 amino acids. The present invention also relates to polynucleotides encoding the fusion protein, pharmaceutical compositions comprising same, and uses thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to UK Provisional Patent Application No. GB2206673.2, filed May 6, 2022, which is incorporated by reference in its entirety.

[0002] Description of sequence listing This application contains a sequence listing in electronic format submitted via EFS-Web. The sequence listing, created on April 27, 2023, is named "4549-140ST26.xml" and is 6,664 bytes in size. The information in the electronic format of the sequence listing is part of this application and is incorporated herein by reference in its entirety.

[0003] FIELD OF THEINVENTION The present invention relates to a fusion protein comprising a mutated CD200 moiety comprising the K130Y and I131Y mutations, which binds to the human CD200 receptor with higher affinity than wild-type CD200, directly fused to a non-CD200 IgG4 Fc fragment comprising the S228P, M428L, and N434S mutations and a deletion of the first 5 amino acids. The present invention also relates to polynucleotides encoding the fusion protein, pharmaceutical compositions comprising same, and uses thereof. [Background technology]

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

[0005] Currently, the American Autoimmune Disease Association (AARDA) estimates that 50 million Americans are affected by 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 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. Furthermore, these estimates overlook the costs of immunosuppressive therapy during transplantation.

[0006] Autoimmune diseases are chronic conditions with no cure that occur when the immune system attacks healthy cells as foreign. Depending on the type of autoimmune disease, it can affect one or more different types of body tissues, causing abnormal organ growth and altered organ function. Normal control of the immune system relies heavily on receptor / ligand pairs, which involve proteins expressed by cells involved in the immune response. However, these receptor / ligand pairs are involved in signaling cascades that lead to the pathology of autoimmune diseases.

[0007] 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 immunoglobulins and 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, thus exerting immunosuppressive effects on both the innate and adaptive immune systems (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).

[0010] 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), 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] 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. As a result, 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 increases immunopathology.

[0012] Profiling studies have shown that hCD200 expression is associated with multiple sclerosis (Koning et al. (2007) Ann. Neurol. 62, 504-514), 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).

[0013] Agonistic CD200 proteins are disclosed, for example, in WO2000 / 061171 and WO2008 / 089022. The literature describes 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.

[0014] 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.

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

[0016] According to a first aspect of the present invention, (i) a mutated CD200 portion comprising the mutations at amino acid residues 130 and 131, which are K130Y and I131Y; and (ii) a non-CD200 portion which is an IgG4 Fc fragment and contains the S228P, M428L, and N434S mutations according to the EU numbering system and a deletion of the first five amino acids of the hinge; A fusion protein comprising: A fusion protein is provided in which glycine 232 of the mutated CD200 moiety is fused directly to a non-CD200 IgG4 Fc fragment at amino acid 6 according to the IMGT numbering system.

[0017] According to a further aspect of the present invention there is provided a polynucleotide encoding a fusion protein as defined herein. According to yet another aspect, there is provided a pharmaceutical composition comprising a 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, neurodegenerative disorders, inflammatory disorders, Th2-induced airway inflammation, diabetic neuropathy, neurodegenerative, or neuropathic pain. 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 system, vasculature, eyes, skin, gastrointestinal tract, lungs, kidneys, liver, peripheral or central nervous system, bone, cartilage, or joints. [Brief description of the drawings]

[0019] [Figure 1A] Sensorgram of a BIAcore assay showing the association and dissociation phases of human CD200R binding to a captured mutant CD200-Fc fusion protein (ARQ-234). [Figure 1B] Sensorgram of a BIAcore assay showing the association and dissociation phases of human CD200R binding to captured wild-type CD200-Fc (DS-155). [Figure 2A] Sensorgram of a BIAcore assay showing the association and dissociation phases of cynomolgus monkey CD200R1 binding to a captured mutant CD200-Fc fusion protein (ARQ-234). [Figure 2B] Sensorgram of a BIAcore assay showing the association and dissociation phases of cynomolgus CD200R1 binding to captured wild-type CD200-Fc (DS-155). [Diagram 3]Bar graph showing inhibition of LPS-stimulated IL-6 release from U937-CD200R cells following treatment with mutant fusion protein (ARQ-234) either in the presence (top panel) or without Fc block (bottom panel). The % inhibition shown is relative to LPS-stimulated IL-6 release from U937-CD200R cells not treated with mutant or wild-type fusion protein, set at 0%. [Figure 4] Binding of mutant CD200-Fc (ARQ-234) or wild-type CD200-Fc (DS-155) to U937-CD200R cells visualized using a fluorescent anti-human secondary antibody after 1, 4, and 24 hours. [Diagram 5] A) Schematic of the PK analysis protocol. B) Graph showing the concentration of mutant CD200 fusion protein (ARQ-234) measured in serum of cynomolgus monkeys at the indicated times after dosing (time=0). [Figure 6] Bar graph showing inhibition of IL-6 in response to dose titration of DS-155 (wild type CD200-Fc) and DS-192 (13 nM high affinity CD200-Fc). [Figure 7] Bar graph showing inhibition of TNFα by DS-192. Error bars represent standard deviation between biological replicates. [Figure 8] Bar graph showing inhibition of ERK phosphorylation by DS-155 and DS-192 in the presence of huCD200-Fc fusions measured by flow cytometry in permeabilized cells using an anti-pERK antibody. [Figure 9] Line graph showing clinical scores of ankle arthritis in male DBA / 1J mice measured every other day on days 25-36. Data are expressed as mean ± SEM. **p<0.01; ***p<0.001 vs. disease+Dexa, disease+DS-198, and disease+DS-227, 2-way RM ANOVA followed by Tukey's multiple comparison test. [Figure 10] Bar graph showing the change in ear thickness in a humanized mouse model of contact hypersensitivity from day 0. Values ​​shown are the combined values ​​for right and left ears. [Figure 11]Bar graph showing IL-1β cytokine levels in tissue homogenates of a humanized mouse model of contact hypersensitivity on day 15. Data are expressed as mean ± SEM. †p<0.05, ††p<0.01 vs. isotype control, unpaired Student's t-test; *p<0.05, **p<0.01 vs. negative control, unpaired Student's t-test. [Figure 12] Bar graph showing GM-CSF cytokine levels in tissue homogenates of a humanized mouse model of contact hypersensitivity on day 15. Data are expressed as mean ± SEM. †p<0.05, ††p<0.01 vs. isotype control, unpaired Student's t-test; *p<0.05, **p<0.01 vs. negative control, unpaired Student's t-test. [Figure 13] Bar graph showing IL-13 cytokine levels in tissue homogenates of a humanized mouse model of contact hypersensitivity on day 15. Data are expressed as mean ± SEM. †p<0.05, ††p<0.01 vs. isotype control, unpaired Student's t-test; *p<0.05, **p<0.01 vs. negative control, unpaired Student's t-test. [Figure 14] Schematic diagram of the ARQ-234 CD200-Fc fusion showing mutations for high affinity CD200R and FcRn binding. [Figure 15A] Graph showing 100 μg / mL antibody recognizing CD64 (FcγRI) Fcγ receptor activity when co-incubated with various doses of DS-192 (ha CD200-IgG4 Fc) in a U937 cell assay. [Figure 15B] Graph showing 0 μg / mL of antibody recognizing CD64 (FcγRI) Fcγ receptor activity when co-incubated with various doses of DS-192 (ha CD200-IgG4 Fc) in a U937 cell assay. [Figure 15C] Graph showing 100 μg / mL of antibody recognizing CD16 (FcγRIII) Fcγ receptor activity when co-incubated with various doses of DS-192 (ha CD200-IgG4 Fc) in a U937 cell assay. [Figure 15D]Graph showing 0 μg / mL antibody recognizing CD16 (FcγRII) Fcγ receptor activity when co-incubated with various doses of DS-192 (ha CD200-IgG4 Fc) in a U937 cell assay. [Figure 15E] Graph showing 100 μg / mL of antibody recognizing CD32 (FcγRII) Fcγ receptor activity when co-incubated with various doses of DS-192 (ha CD200-IgG4 Fc) in a U937 cell assay. [Figure 15F] Graph showing 0 μg / mL antibody recognizing CD32 (FcγRII) Fcγ receptor activity when co-incubated with various doses of DS-192 (ha CD200-IgG4 Fc) in a U937 cell assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] According to a first aspect of the present invention, (i) a mutated CD200 portion comprising the mutations at amino acid residues 130 and 131, which are K130Y and I131Y; and (ii) a non-CD200 portion which is an IgG4 Fc fragment and contains the S228P, M428L, and N434S mutations according to the EU numbering system and a deletion of the first five amino acids of the hinge; A fusion protein comprising: A fusion protein is provided in which glycine 232 of the mutated CD200 moiety is fused directly to a 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 mutated CD200 portions that have increased binding affinity to the CD200 receptor (CD200R). Moreover, fusion proteins comprising the mutated CD200 portions described herein have significant advantages, particularly with respect 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 ARQ-234.

[0023] SEQ ID NO:1 (also referred to herein as "ARQ-234") consists of the following sequence:

[0024] [ka]

[0025] Highlighted amino acids represent the positions of mutations relative to wild-type CD200 or IgG4 Fc, and underlined sequences represent non-CD200 Fc fragments. In one embodiment, ARQ-234 may further comprise an N-terminal signal sequence that is a human IgG heavy chain signal peptide. In a further embodiment, the N-terminal signal sequence comprises or consists of the amino acid sequence of SEQ ID NO:3.

[0026] SEQ ID NO:3 contains the following sequence: It consists of MEFGLSWLFLVAILKGVQC. As used herein, the term "CD200 protein" refers to a wild-type CD200 protein.

[0027] The term "wild type" as used herein refers to naturally occurring protein, peptide, amino acid, and nucleotide sequences. For example, the term "wild type CD200 protein" as used herein refers to the full length isoform of CD200 (UNIPROT P41217 OX2G_HUMAN) or any portion thereof (including naturally occurring protein polymorphisms) that binds to the CD200 receptor (CD200R). The CD200 protein is also known as OX-2 membrane glycoprotein.

[0028] Wild-type CD200 is a cell surface protein and has an N-terminal extracellular domain, 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.

[0029] The term "position" as used herein 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 moiety defined herein is relative to the amino acid sequence including the N-terminal signal sequence, which represents the first 30 amino acids of the CD200 moiety (shown in bold in SEQ ID NO:2).

[0030] The term "mutated" or "mutant" 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 changes in the amino acid and / or nucleotide sequence when compared to the corresponding wild-type sequence, and such changes may also be referred to as mutations.

[0031] References herein to "mutated CD200 protein" and "mutated CD200 portion" refer to a full length CD200 protein or any portion thereof that binds to the CD200 receptor and comprises 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. According to the first aspect of the invention as defined herein, the mutated CD200 portion comprises a K130Y mutation and an I131Y mutation. Thus, in one embodiment, the mutation is a substitution mutation.

[0032] In one embodiment, the fusion protein may be synthetically or recombinantly produced. In a further embodiment, the fusion protein may be synthetically produced. In an alternative embodiment, the fusion protein may be recombinantly produced.

[0033] In one embodiment, the mutated CD200 portion binds to the CD200 receptor with greater affinity than wild-type CD200. 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 before secretion. Thus, in a particular embodiment, the mutated CD200 portion comprises an N-terminal signal sequence. In one embodiment, the mutated CD200 protein comprises a signal sequence at the N-terminus that is cleaved before 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 portion. In a further embodiment, the signal sequence is SEQ ID NO: 3. Thus, in a particular embodiment, the fusion protein comprises a sequence as defined herein that lacks the amino acids that comprise the signal sequence. For example, amino acids 1-30 of the wild-type CD200 protein are missing and the mutated CD200 protein comprises a 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 SEQ ID NO: 1.

[0034] SEQ ID NO:2 consists of the following sequence:

[0035] [ka]

[0036] The bolded amino acids represent the signal sequence, the highlighted amino acids represent the position of the mutation relative to wild type CD200 or IgG4 Fc, and the underlined sequences represent non-CD200 Fc fragments. The present disclosure also includes the disclosed protein sequences, but lacking the C-terminal lysine, e.g., proteins in which the C-terminal lysine (K) has been cleaved during secretion from mammalian cells.

[0037] In one embodiment, the fusion protein is encoded by the polynucleotide of SEQ ID NO: 4. It is important to note that there is degeneracy of the genetic code, i.e., most amino acids are specified by more than one codon. Thus, multiple polynucleotide sequences can code for the same amino acid sequence, since many different codons specify the same amino acid. Thus, SEQ ID NO: 4 represents one exemplary permutation of polynucleotide sequences that can code for a fusion protein. Any permutation and combination of all elements described in this application should be considered as disclosed by the description of this application, unless the context indicates otherwise. 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.

[0038] 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 has at least 70% (such as 75%, 80%, 85%, 90%, 95%, or 99%) sequence similarity to the wild-type counterpart and functions similarly. Thus, a variant may be a derivative of the wild-type.

[0039] As used herein, the term "amino acid residue" refers to a monomeric unit in a polymer chain, i.e., a single amino acid in a protein. As demonstrated by the data presented herein, the mutant CD200 proteins / portions of the invention bind more tightly to the CD200 receptor and exhibit longer residence times on the receptor than wild-type CD200 protein.

[0040] Fusion proteins According to a first aspect of the invention as defined herein there is provided a fusion protein comprising a mutated CD200 protein / moiety as defined herein fused to a non-CD200 moiety.

[0041] The term "fusion protein" as used herein refers to one or more amino acid sequences, peptides, and / or proteins 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 some embodiments, the mutated CD200 protein / moiety as defined herein is fused to a non-CD200 moiety at the C-terminus. Thus, in one embodiment, the orientation of the fusion protein from N-terminus to C-terminus is mutated CD200 moiety-non-CD200 Fc fragment. Thus, in a further embodiment, the orientation of the fusion protein is mutated CD200 moiety-IgG4 Fc fragment. In another embodiment, the orientation of the fusion protein from N-terminus to C-terminus is signal sequence-mutated CD200 moiety-non-CD200 Fc fragment. Thus, in a further embodiment, the orientation of the fusion protein is signal sequence-mutated CD200 moiety-IgG4 Fc fragment.

[0043] The term "non-CD200 moiety" as used herein may 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, an immunoglobulin (Ig) constant region or portion thereof, or a fusion protein in which the non-CD200 moiety is a synthetic molecule, such as PEG.

[0044] In one embodiment, the non-CD200 moiety is an antibody fragment. In a particular embodiment, the non-CD200 moiety is an Fc fragment. Thus, the mutated CD200 fusion protein described herein can 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, which comprises the hinge, CH2, and CH3 regions. In particular, the Fc fragment comprises the hinge region. The Fc fragment provides the advantage of increasing the serum half-life of the mutated CD200 protein of the invention, and furthermore, it dimerizes the CD200 protein, thereby increasing the binding activity and allowing agonistic signaling. It will be appreciated by those skilled in the art that the Fc region may be mutated to reduce effector functions (see, for example, US 5,637,481 and US 6,132,992).

[0045] In one embodiment, the Fc fragment is an IgG4 Fc fragment. In a further embodiment, the non-CD200 portion is an antibody Fc fragment comprising one or more amino acid residue mutations. Thus, in a particular embodiment, the non-CD200 portion is an IgG4 Fc fragment, comprising S228P, M428L, and N434L mutations, the positions of said mutations being according to the EU numbering system. Thus, in one embodiment, the non-CD200 Fc fragment is an S228P, M428L, and N434L 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 increases the stability of the fusion protein both in vivo and in vitro, likely resulting in improved therapeutic efficacy and manufacturability. The presence of the M428L and N434S mutations (also known as the "LS variant") increases the affinity of the Fc fragment for the human neonatal Fc receptor (hFcRn) at pH 6, resulting in an increased serum half-life, such as an increased serum half-life of a fusion protein as defined herein comprising the mutated non-CD 200 Fc fragment (Zalevsky et al. (2010) Nat. Biotechnol., 28(2):157-159, doi:10.1038 / nbt.1601 and Ko et al. (2014) Nature, 514(7524):642-645, doi:10.1038 / nature13612). In a further embodiment, the non-CD200 IgG4 Fc fragment comprises a deletion of the first 5 amino acids, such as the first 5 amino acids of the hinge region of said IgG4 Fc fragment. Thus, in one embodiment, the non-CD200 portion is an IgG4 Fc fragment and comprises the S228P, M428L, and N434L mutations according to the EU numbering system and a deletion of the first 5 amino acids of the hinge, hi a further embodiment, the non-CD200 portion is an IgG4 Fc fragment and comprises S228P, M428L, and N434L 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 mutated CD200 portion to the non-CD200 Fc fragment. It is therefore understood that such a fusion does not include a linker sequence between the mutated CD200 portion and the non-CD200 Fc fragment. For example, the amino acid glycine 232 of the mutated CD200 portion may be directly fused to amino acid 1 of the Fc hinge region. In another embodiment, the fusion protein is formed by direct fusion of the amino acid glycine 232 of the mutated CD200 portion to amino acid 6 of an IgG4 Fc fragment (in which case the first 5 amino acids of the Fc hinge region are deleted as described hereinbefore). In a further embodiment, the direct fusion is a fusion of the amino acid glycine 232 of the mutated CD200 portion to amino acid 6 of the Fc hinge region of said IgG4 Fc fragment. Thus, in one embodiment, the glycine 232 of the mutated CD200 portion is directly fused to the non-CD200 Fc fragment at amino acid 6 of the Fc hinge region. According to these embodiments, the position in the Fc fragment of the fusion is according to the IMGT numbering system. Such direct fusion of a mutated CD200 moiety to amino acid 6 of the IgG4 Fc fragment hinge region increases the stability of the resulting fusion protein compared to fusion proteins containing linker sequences, without affecting the strong binding to CD200R. This result is surprising in view of the data previously reported for Fc fusion proteins containing linker sequences.

[0047] For the purposes of this description, when the non-CD200 moiety is an Fc fragment, the term "position" as used herein with respect to a mutation in the non-CD200 moiety refers to the residue number in the amino acid sequence according to the EU numbering system. It is therefore understood that the mutated residue positions of amino acids in the Fc fragment referred to herein relate to the position 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. When used herein with respect to a mutated CD200 moiety fused to a non-CD200 Fc fragment, "position" refers to the residue number in the Fc fragment according to the IMGT numbering system. It is therefore understood that the residue position of an amino acid in the Fc fragment hinge relates to its position according to the IMGT numbering system. The numbering herein of the mutations in the Fc fragment therefore 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 moiety is fused directly 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 proteins of the invention are preferably produced by recombinant DNA methods by inserting a nucleic acid sequence encoding a CD200-Fc fusion protein or any portion 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 pCDNA3.1. 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 portion thereof is inserted into a recombinant expression vector using fusion cloning. Thus, in a further embodiment, the nucleic acid encoding the CD200-Fc fusion protein or any portion thereof comprises at its end a nucleic acid sequence complementary to that at the end of the linearized vector, such as an overlap between the nucleic acid encoding the CD200-Fc fusion protein and the vector of 12 to 21 base pairs / nucleotides, e.g., a 15 base pair overlap or a 20 base pair overlap.

[0049] According to a further aspect of the invention, polynucleotides are provided that encode the fusion proteins defined herein. The disclosure includes polynucleotides that encode the proteins defined herein, and the use of such nucleic acids to produce proteins and / or for therapeutic purposes. Such polynucleotides can 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 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 a recombinant expression vector and expressed in a recombinant transcription unit. 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 ARQ-234. 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 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.

[0051] therapeutic use The present invention is particularly applicable in therapy because 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, increasing the affinity of mutant CD200 proteins and fusion proteins comprising portions thereof for the CD200 receptor, as presented herein, can be used in the preparation of pharmaceutical compositions with more potent properties.

[0052] Furthermore, the production costs of recombinant proteins are high, and mutated CD200 proteins / fusion proteins containing portions thereof with high affinity can be used in pharmaceutical compositions at significantly lower doses than wild-type or non-mutated CD200 proteins to achieve therapeutic efficacy. Thus, the use of mutated CD200 proteins / fusion proteins containing portions thereof may be more cost-effective in addition to being more clinically effective.

[0053] According to a further aspect of the present 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 ARQ-234.

[0054] In one embodiment, the mutated CD200 protein or fusion protein defined herein is a modulator 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. Considering 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 useful in the treatment of autoimmune diseases. Thus, in a further embodiment, the mutated CD200 protein or fusion protein 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 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 (e.g., hay fever or allergic rhinitis), allergic contact dermatitis, seasonal allergies, anaphylaxis, food allergies, asthma, and atopic dermatitis.

[0058] Fusion proteins comprising a mutated CD200 protein / portion as defined herein are able to inactivate activated immune cells more efficiently than fusion proteins comprising wild-type or non-mutated CD200 proteins.

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

[0060] 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 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 rheumatoid arthritis; ... Autoimmune myocarditis;Autoimmune oophoritis;Autoimmune pancreatitis;Autoimmune retinopathy;Autoimmune thrombocytopenic purpura (ATP);Autoimmune thyroid disease;Autoimmune urticaria;Axonal and neuropathies;Barrow's disease;Behçet's disease;Bullous pemphigoid and related autoimmune blistering diseases;Cardiomyopathy;Castleman's disease;Celiac disease (including refractory celiac disease type II);Chagas disease;Idiopathic chronic urticaria;Chronic inflammatory demyelinating polyneuropathy (CIDP);Chronic relapsing Multiple myelitis (CRMO); chronic idiopathic urticaria; Churg-Strauss syndrome; cicatricial pemphigoid / benign mucosal 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); diabetic neuropathy; discoid lupus erythematosus; 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 syndrome;Granulomatosis with polyangiitis (GPA) (formerly called Wegener's granulomatosis);Graft-versus-host disease;Graves' disease;Guillain-Barré syndrome;Hashimoto's disease;Hemolytic anemia;Henoch-Schönlein purpura;Herpes gestationis;Hypogammaglobulinemia;Hidradenitis suppurativa (Hidradenitis suppurativa) supporativa) (HS);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;Ligninous keratoconjunctivitis;Linear immunoglobulin A disease (LAD);SLE;Lyme disease, chronic;Macrophage activation syndrome (MAS);Mastocytosis;Meniere's disease;Microscopic polyangiitis;Mixed connective tissue disease (MCTD);Mohren's ulcer;Much-Habermann disease;Multiple sclerosis;Myasthenia gravis;Myositis;Narcolepsy;Neuromyelitis optica (Devic);Neutropenia;Ocular cicatricial pemphigoid;Optic neuritis;Relapsing rheumatoid arthritis;PANDAS (Streptococcal infections) Related pediatric autoimmune neuropsychiatric disorders); Palmoplantar pustulosis (PPP); Paraneoplastic cerebellar degeneration; Paroxysmal nocturnal hemoglobinuria (PNH); Parry-Romberg syndrome; Parsonage-Turner syndrome; Parsplanitis (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; Cardiac Postmembrane incision 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;sarcoidosis;Schmidt's syndrome;scleritis;scleroderma;Sjögren's syndrome;sperm-testicular autoimmunity;stiff body syndrome ; one or more autoimmune diseases selected from subacute 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));

[0061] In an alternative embodiment 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 a further alternative embodiment 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 joint pain.

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

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

[0064] Thus, according to a further aspect of the present invention there is provided a pharmaceutical composition comprising a 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 joint pain, or diabetic neuropathy, the fusion proteins of the invention may be advantageously used in combination with one or more other agents, more particularly with one or more immunosuppressants or adjuvants in immunosuppressive therapy.

[0065] Examples of therapeutic agents or treatments that may be administered together (simultaneously or at different time intervals) with 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 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 or fusion protein of the present invention can be used in combination with one or more agents that are administered according to existing combination dosage regimens.

[0067] 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.When 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.

[0068] 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 by any suitable mode, including parenteral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, via the pulmonary route, e.g., intranasal or inhalation, and also, suitably, by direct injection by catheter, such as intracranial (e.g., icv into the central nervous system ventricles or it into the spinal cord). The dosage and frequency of administration will vary according to the age, sex, and condition of the patient, the co-administration of other drugs, contraindications, and other parameters that should be taken into account by the clinician.

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

[0070] It is understood that any embodiment described herein may apply to any aspect of the 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 the description and specific examples indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications will become apparent to those skilled in the art. The following studies and protocols are illustrative of embodiments of the methods described herein. EXAMPLES

[0071] Example 1: Production of mutant and wild-type CD200-Fc molecules Gene synthesis and cloning Gene synthesis of IgG4 S228P Fc with mutations M428L+N434S (codon optimized for CHO expression) was performed at GeneArt. These constructs were cloned into the plasmid pCDNA3.1 using fusion cloning to generate the vector backbone. Into this backbone, codon-optimized DNA sequences encoding mutant or wild-type human CD200 residues 1-232 of UniProt P412178 (OX2G_Human) including the N-terminal signal sequence were inserted at the N-terminus of IgG4 S228P Fc to create a direct fusion of amino acid glycine 232 of CD200 to the Fc region. Sequences were confirmed in both directions.

[0072] GigaPrep Sequence-verified plasmids were 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 into 800 mL of Circlegrow medium for giga-scale DNA preparation. DNA was isolated using the Endotoxin Free Quanta Giga Kit.

[0073] Protein expression CD200-Fc protein was produced by transient transfection of the expression plasmid into CHO-3E7 cells using polyethylenimine (PEI). Briefly, cells were cultured at a density of 4.0 × 10 cells maintained at 37 °C in CD-Forti CHO medium. 6 A 250 mL culture of cells / mL was transfected with 2 mg / L of plasmid using PEI at a ratio of 1:5. 24 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. Batches were monitored and supernatants containing overexpressed CD200-Fc were harvested on day 7 with a viability of approximately 75%. The filtered supernatant was subjected to protein purification.

[0074] Protein purification All purification steps were performed at 4°C. The culture harvest was loaded onto a MabSelect SuRe affinity column (5mL) pre-equilibrated with 50mM sodium phosphate, 150mM NaCl pH 7.4 on an AKTA Pure platform at a flow rate of 3mL / min. The column was washed with equilibration buffer and bound proteins were eluted using 20mM sodium acetate, 150mM NaCl pH 3.5. The eluate was neutralized with 10v / v% 1M Tris pH 8.0 and analyzed by SDS-PAGE. Fractions containing CD200-Fc were pooled, concentrated to 5mL and subjected to gel filtration chromatography (Hiload 16 / 600 Superdex-200pg column) on an AKTA Pure platform. Proteins were processed in a 50mM sodium phosphate, 150mM NaCl pH 7.4 buffer system at 1.2mL / min and collected in fractions. Fractions containing CD200-Fc dimer as analyzed by SDS-PAGE were pooled and concentrated to 1.33 mg / mL (measured at UV 280 nm) using an Amicon Ultra Centricon (10 kDa molecular weight cutoff). The purified material was subjected to SEC-HPLC, LC-MS, and EndoSafe LAL cartridge testing to assess 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 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 an 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.

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

[0078] protocol For human CD200 constructs, 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 immobilization of 8,000-11,000 RU. 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, with a stabilization time of 60 seconds, and then incubated with the immobilized anti-human IgG. The running buffer was then flowed over the captured ligand at 10 μL / min for 25-100 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 (3-fold dilutions) to 5 or more concentrations (depending on the expected affinity) in running buffer, along with a buffer blank (0 nM), and flowed over the captured ligand at a flow rate of 30-50 μL / min for 120 s, followed by dissociation in running buffer for 120-360 s. The assay temperature was 25°C. The surface was then regenerated by flowing 3M MgCl2 for 30-90 s at a flow rate of 30 μL / min, followed by stabilization of the surface by flowing running buffer for 60 s.

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

[0080] Data Analysis: The experimental sensorgrams were analyzed with BIAevaluation software (GE Healthcare). The obtained curves were analyzed using R maxThe binding profiles were fitted to a 1:1 Langmuir binding model by setting the RI and RI as local parameters. A kinetic 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 expressed as χ 2 Value is R max Data were accepted if the difference was less than 10% and the U value was 15 or less.

[0081] Table 1 details the reagents used in the development and performance of the assay.

[0082] [Table 1]

[0083] result The results (Table 2 and Figures 1A-1B) show that the mutant CD200-Fc protein of the present invention (ARQ-234) binds to the human CD200 receptor with approximately 84-fold higher affinity than wild-type CD200-Fc (DS-155). The off-rates tabulated in Table 2 and the sensorgrams shown in Figures 1A-1B demonstrate that ARQ-234 has an off-rate and half-life on the receptor that is compatible with efficient agonism in a functional cell assay. Furthermore, the results in Table 3 and Figures 2A-2B show that ARQ-234 can bind to cynomolgus monkey CD200R, allowing this fusion protein to be evaluated in standard toxicology protocols.

[0084] [Table 2]

[0085] [Table 3]

[0086] [Table 4]

[0087] Example 3: Cell binding and cell activation assays of wild-type and mutant CD200-Fc proteins Assay Principle To demonstrate the agonist activity of ARQ-234, the human monocytic cell line U937 (ATCC, CRL1539) was transfected with the cDNA for human CD200R. Cytokine production, including IL-6, from these cells was induced by stimulation with PMA followed by LPS.

[0088] Cell line construction The full-length human CD200R gene, including the signal sequence, was cloned into the pCDH-EF1-human CD200R-IRES-Puro lentivector (System Biosciences) downstream of the EF1α promoter. Lentiviral particles containing the expression construct were produced in 293TN producer cells and concentrated using PEG-it reagent (System Biosciences) according to the manufacturer's instructions.

[0089] U937 human monocyte immortalized cell line was transduced with lentiviral particles at MOI ranging from 5 to 200 using Transducx and Max Enhancer reagents (System Biosciences) according to the manufacturer's instructions. Transduced U937 cells were a) selected using puromycin (with a first optimized puromycin concentration) and b) sorted by flow cytometry to generate stable polyclonal CD200R expressing lines. CD200R expression was confirmed by western blot in addition to flow cytometry.

[0090] Cytokine release (IL-6 inhibition) assay 50,000 U937 cells were seeded per well in 96-well plates and incubated with 100 nM PMA for 72 hours before differentiation. After differentiation, the medium containing PMA was replaced with fresh assay medium and incubated for an additional 2 hours before treatment. CD200-Fc constructs were added to the cell cultures with or without Fc block and incubated for 1 hour, then cells were stimulated with 100 ng / ml LPS and incubated for an additional 24 hours. After the final incubation, cell supernatants (1:10 dilution) were collected and assayed for IL-6 secretion by ELISA assay using a commercially available kit.

[0091] cell binding U937 cells were resuspended in 50 μL of FACS buffer (1× PBS+2% FBS) at a density of 100,000 cells per test.

[0092] Treatment with CD200-Fc protein (50 μL) was performed in FACS buffer starting at 10 μg / mL, with 3-fold dilutions up to a maximum of 10 concentrations, and incubated at 37° C. for 1, 4, and 24 hours. At the end of each time point, cells were collected and washed. Anti-human secondary antibody was added at 10 μg / mL and incubated at 4° C. for 30 minutes. After incubation, cells were washed and stained for 20 minutes at 4° C. to check viability (1 μL dye / million cells / 1 mL 1×PBS). Cells were washed and fixed with fixation buffer (100 μL per experiment) for 20 minutes at 4° C. After incubation, cells were washed and pellets were resuspended in FACS buffer (100 μL per experiment) for data acquisition on the flow cytometer.

[0093] At 24 hours, treatment with CD200-Fc protein was performed with cell culture medium. Washing step = addition of 200 μL FACS buffer and centrifugation at 1400 RPM.

[0094] [Table 5]

[0095] result The data shown in Figure 3 demonstrate that ARQ-234 can inhibit LPS-stimulated IL-6 secretion in a concentration-dependent manner. No significant difference in inhibition was observed in the presence of Fc-blocking reagents in vitro (Figure 3, upper panel). This is surprising since the IgG4 Fc domain of ARQ-234 binds to Fcγ receptors, which should increase the avidity of its interaction with CD200R, but this mechanism could further increase the potency of CD200-Fc protein in vivo.

[0096] Figure 4 shows binding of wild-type DS-155 and mutant ARQ-234 CD200-Fc proteins to CD200R-expressing U937 cells. The data demonstrate superior binding of ARQ-234 to CD200R-expressing cells compared to DS-155 (wild-type CD200-Fc protein) at all time points.

[0097] Example 4: PK study of ARQ-234 in serum of cynomolgus monkeys protocol Two cynomolgus monkeys (1 male, 1 female) per group were administered an intravenous bolus of 5 mg / kg protein at time 0. Blood samples were collected for PK analysis at the following time points: pre-dose, 0.25 h, 0.5 h, 1 h, 4 h, 8 h, 24 h, days 3, 5, 7, 10, 12, 14, 21, and 28.

[0098] Serum samples for PK analysis At least 0.8 mL of blood samples were collected from the cephalic or saphenous vein at the sampling time points from two animals. For samples collected within the first hour of dosing, a deviation of ±1 min in sample collection time was allowed. For the remaining time points, samples taken within 5% of the scheduled time were acceptable. All blood samples were collected in commercial tubes containing a coagulant. Tubes containing blood samples were kept at room temperature for 30 min before centrifugation. Samples were centrifuged at 1500×g for 10 min at 4° C. within 1 hour of collection. After centrifugation, approximately 400 μL of serum was collected per time point. Samples were then quickly frozen on dry ice and kept below −60° C. until transferred into dry ice for analysis. All samples were uniquely identified to indicate origin and collection time.

[0099] Determination of protein concentration in serum The concentrations of analytes in serum were determined using a bioanalytical ELISA method. 96-well ELISA plates were 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 then biotin-labeled goat anti-human IgG (0.0625 μg / mL) was used as the detection antibody. HRP-streptavidin and TMB substrate were used for color development. The reaction was stopped by adding 2 M HCl after about 5-10 minutes. Absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer (SpectraMax® M5e). The OD values ​​of the samples were substituted into the standard curve to obtain the plasma concentrations. The detection limit of this ELISA method LLOQ for Fc+Fc is 1 ng / mL.

[0100] Serum concentrations of ARQ-234 in monkeys were subjected to non-compartmental pharmacokinetic analysis by using Phoenix WinNonlin software (version 8.1, Pharsight, Mountain View, Calif.). The linear / logarithmic trapezoidal rule was applied in obtaining PK parameters.

[0101] result The data presented in Table 6 and FIG. 5 demonstrate good serum stability of ARQ-234, with a mean half-life of 370.5 hours in the two animals tested.

[0102] [Table 6]

[0103] Table 6 shows the following parameters: half-life (T 1 / 2 ); maximum serum concentration (C max ); area under serum concentration from time 0 to time t (28 days) (AUC 0-t); measured clearance rate (Cl_obs); mean residence time extrapolated to infinity (MRTINF); volume of distribution (Vss_obs) were tested. Half-life is C max The calculation was performed without using data of less than 1%.

[0104] Example 5: In vitro proof of concept of high affinity murine CD200-Fc Due to the lack of cross-reactivity with mouse CD200R, a mouse CD200-CD200R1 in silico model was generated based on the published crystal structure and a high affinity surrogate CD200-Fc protein was engineered for proof-of-concept experiments in a mouse model of autoimmunity.

[0105] protocol The mouse CD200 construct used the Uniprot sequence O54901, which contains the signal peptide and the extracellular domain. The mutation number refers to the complete Uniprot sequence including the signal peptide.

[0106] Proteins were produced by transient transfection of pcDNA3.1-based expression plasmids into CHO-3E7 cells using polyethylenimine (PEI). 24 hours after transfection, cultures were shifted to 32°C and fed with 10% Feed C, glutamine, glucose, and 0.5 M sodium butyrate, and supernatants were 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 on an AKTA Pure platform at a flow rate of 3 mL / min. The column was washed with equilibration buffer and bound proteins were 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. Pooled fractions were concentrated to 5 mL and subjected to gel filtration chromatography (Hiload 16 / 600 Superdex-200pg column) on an AKTA Pure platform. Proteins were processed at 1.2 mL / min in a 50 mM sodium phosphate, 150 mM NaCl pH 7.4 buffer system. Protein-containing fractions were pooled and concentrated to 1.33 mg / mL (measured at UV 280 nm) using an Amicon Ultra Centricon (10 kDa molecular weight cutoff). Purified material was subjected to SEC-HPLC, LC-MS, and EndoSafe LAL cartridge testing to assess protein purity, molecular weight, and endotoxin content, respectively. Mouse CD200-His protein was purified on Ni-NTA agarose resin using standard methods. All proteins were stored at -80°C.

[0107] result As shown in Table 7, the monomer binding affinity of the variant H82Y, T125I combination is 43 nM, approximately 14-fold higher than the wild-type construct containing the mouse IgG2a Fc domain.

[0108] [Table 7]

[0109] Example 6: In vitro proof of concept of high affinity human CD200-Fc The identified human CD200-Fc (huCD200-Fc) proteins were tested for their ability to inhibit cytokine release from LPS-activated promonocytic human myeloid leukemia cells (U937) engineered to express high levels of human CD200R.

[0110] protocol Cell line construction, cytokine inhibition studies, and cell binding studies were performed using techniques similar to those described in Example 3 above.

[0111] To test the ability of huCD200-Fc protein to inhibit ERK phosphorylation, U937-CD200R cells were induced with PMA for 20 min, and inhibition of ERK phosphorylation by DS-155, DS-192, and DS-118 was measured by flow cytometry in permeabilized cells with an anti-pERK antibody.

[0112] result As shown in Figure 6, higher affinity was observed for 13 nM DS-192, which shows a stronger inhibition of IL-6 release than wild type DS-155. Inhibition of TNFα was also observed, as shown in Figure 7. Inhibition of ERK phosphorylation correlates with CD200 affinity, as shown in Figure 8.

[0113] As shown in Figures 15A-15F, antibodies that recognize Fcγ receptors do not inhibit the activity of DS-192 in vitro, suggesting that the Fc domain does not play a significant role in the inhibitory activity in this particular assay.

[0114] Example 7: In vivo proof of concept of high affinity murine CD200-Fc Using a mouse model, we showed that the higher affinity mouse CD200-Fc protein, with prophylactic dosing, reduced clinical scores in a mouse collagen-induced arthritis (CIA) model.

[0115] Mice have four potential CD200 receptors, CD200R1-CD200R4, at least one of which may be activated, and CD200R1 is the homolog of human CD200R. Knocking out either CD200 or CD200R1 in transgenic mice exacerbates or induces earlier onset in many models of autoimmune conditions, including alopecia, arthritis, IBD25, and uveoretinitis.

[0116] CD200R agonism in rodent models using patient samples is known in the art in vitro and has been previously achieved with CD200-Fc fusion proteins, suggesting that human CD200-Fc fusion proteins could be used as therapy for inflammatory diseases. As with other cell surface immune receptors, the affinity of CD200 for CD200R is low (high nanomolar range), and therefore an ideal human therapeutic would require affinity enhancement for optimal efficacy. The Fc domain provides an antibody-like serum half-life, and the dimeric format increases avidity and allows receptor cross-linking. Animal model data indicates that the sequence of the Fc domain is associated with mouse IgG2a Fc fusions with optimal efficacy, likely by binding to Fcγ receptors to facilitate the formation of cell-cell interactions and further increase avidity. Antibody-dependent cellular cytotoxicity may also contribute by eliminating CD200R1 expressing cells. Therefore, an in vivo mouse model was used to test the efficacy of the murine version of the high affinity CD200-Fc protein compared to the wild type CD200-Fc protein.

[0117] protocol Wild-type (DS-198) and higher affinity (DS-227) murine CD200-Fc proteins were tested using the CIA model by initiating dosing just 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, animals were randomized based on body weight and injected with 3 mg / kg murine IgG2a isotype control antibody, DS-198 (wild-type muCD200-Fc) or DS-227 (high affinity 43 nM muCD200-Fc) once every 3 days until day 36, and the positive control group received oral 0.5 mg / kg dexamethasone daily. Clinical scores (blinded assessment) of ankle arthritis were measured every other day on days 25-36. Data in FIG. 9 are presented as mean±SEM. ** p < 0.01; *** p<0.001 vs. disease+Dexa, disease+DS-198, and disease+DS-227. 2-way RM ANOVA followed by Tukey's multiple comparisons test.

[0118] result As shown in FIG. 9, the higher affinity CD200-Fc, DS-227, was significantly more potent in reducing clinical scores than the wild type (DS-198) at the selected dose of 3 mg / kg.

[0119] Example 8: Proof-of-concept study using high affinity DS-192 Based on the in vivo proof-of-concept results of the high affinity mouse CD200-Fc study (CIA mouse study) described above, an in vivo proof-of-concept study was performed 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 be engrafted with both human lymphocytes and myeloid cells.

[0120] protocol Female NOG-EXL mice were transplanted with human cells and cultured at 20–21 weeks of age with CD45 +Mice were randomized based on the % of cells in the huNOG-EXL mice (day -1). On day 0, mice were sensitized with abdominal application of oxazolone (100 μL of 3 w / v% oxazolone in acetone:alcohol 1:4) and challenged on days 5, 10, and 14 with topical application of 20 μL of 2 w / v% oxazolone (acetone:alcohol 1:4) to each ear (10 μL / side). Oxazolone challenge was repeated in one ear of presensitized huNOG-EXL mice with DS-192 (huCD200-Fc, 13 nM) or a CD200R agonist (CD200R mAb) administered on the same day as each challenge. Isotype control antibody, CD200R agonist antibody, and high affinity huCD200-Fc (DS-192) were administered intravenously at 3 mg / kg 4 hours prior to oxazolone challenge on days 5, 10, and 14. Ear thickness was measured immediately before and 24 hours after each challenge, and on day 15 punch biopsies were taken for multiplex cytokine analysis.

[0121] result As shown in Figure 10, ear thickness change (a proxy for inflammatory response) was significantly reduced by DS-192 on the day after the second and third exposures compared to isotype control, in contrast to CD200R mAb, which did not result in a significant reduction. Furthermore, as shown in Figures 11, 12, and 13, significant reductions 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 superior efficacy compared to CD200R mAb in a humanized mouse model of contact hypersensitivity. Since DS-192 has significantly lower CD200R affinity than ARQ-234, these benefits could be extrapolated to show greater efficacy when using ARQ-234 to treat allergic diseases and skin inflammatory disorders.

[0122] Example 9: Cellular assay binding analysis of high affinity CD200-Fc molecules A diagram of the Fc fusion protein of the present invention, ARQ-234, is shown in FIG. Binding of ARQ-234 to U937-CD200R cells was compared to wild-type control CD200-Fc (DS-155) at 1, 4, and 24 hours at human physiological temperature (37° C.) and binding was detected using flow cytometry with an anti-human IgG antibody. As shown in FIG 4, binding of ARQ-234 to U937-CD200R cells showed stronger target binding compared to DS-155, the wild-type huCD200-Fc construct.

Claims

1. (i) a mutated CD200 portion comprising the mutations at amino acid residues 130 and 131, which are K130Y and I131Y; and (ii) a non-CD200 portion which is an IgG4 Fc fragment and contains the S228P, M428L, and N434S mutations according to the EU numbering system, and a deletion of the first five amino acids of the hinge; A fusion protein comprising: The fusion protein, wherein glycine 232 of the mutated CD200 portion is fused directly to a non-CD200 IgG4 Fc fragment at amino acid 6 according to the IMGT numbering system.

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

1.

3. A fusion protein according to claim 1 or 2, consisting of the amino acid sequence of SEQ ID NO:

1.

4. The fusion protein of any one of claims 1 to 3, wherein the mutated CD200 portion comprises an N-terminal signal sequence representing the first 30 amino acids of the CD200 portion.

5. The fusion protein according to any one of claims 1 to 3, wherein the N-terminal signal sequence is a human IgG heavy chain signal peptide.

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

3.

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

2.

8. The fusion protein according to any one of claims 4 to 7, wherein the N-terminal signal sequence is cleaved prior to secretion from the cell.

9. The fusion protein according to any one of claims 1 to 8, which is a modulator of the CD200 receptor.

10. The fusion protein according to any one of claims 1 to 9, which is an agonist of the CD200 receptor.

11. The fusion protein according to any one of claims 1 to 10, which inhibits cytokine secretion.

12. The fusion protein of claim 11, wherein the cytokine is IL-6.

13. The fusion protein of claim 11, wherein the cytokine is IL-8.

14. The fusion protein of claim 11 , wherein the cytokine is TNFα.

15. The fusion protein of any one of claims 1 to 14, which inhibits ERK activation to a greater extent than wild-type CD200-Fc fusion protein.

16. A polynucleotide encoding the fusion protein according to any one of claims 1 to 15.

17. A composition comprising the fusion protein according to any one of claims 1 to 16 or the polynucleotide according to claim 15, and a pharma- ceutically acceptable carrier.

18. 19. A method of treating a subject having an autoimmune disease, an allergic disease, a neurodegenerative disorder, neuropathic pain, an inflammatory disorder, Th2-induced airway inflammation, or diabetic neuropathy, comprising administering to the subject a fusion protein of claims 1 to 15, a polynucleotide of claim 16, or a composition of claim 17.

19. 20. The method of claim 18, wherein the subject has hay fever, allergic rhinitis, allergic contact dermatitis, seasonal allergies, anaphylaxis, food allergies, asthma, or atopic dermatitis.

20. 20. The method of claim 19, 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, bone, cartilage, or joints.

21. The subjects are 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 Autoimmune myocarditis; Autoimmune oophoritis; Autoimmune pancreatitis; Autoimmune retinopathy; Autoimmune thrombocytopenic purpura (ATP); Autoimmune thyroid disease; Autoimmune urticaria; Axonal and neuropathies; Baro's disease; Behcet's disease; Bullous pemphigoid and related autoimmune blistering diseases; Cardiomyopathy; Castleman's disease; Celiac disease (including refractory celiac disease type II); Chagas disease; Idiopathic chronic urticaria; Chronic inflammatory demyelinating polyneuropathy (C IDP); chronic relapsing multifocal osteomyelitis (CRMO); chronic idiopathic urticaria; Churg-Strauss syndrome; cicatricial pemphigoid / benign mucosal 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); diabetic neuropathy; discoid lupus erythematosus; Dressler's syndrome; Endometriosis; Eosinophilic esophagitis; Eosinophilic fasciitis; Erythema nodosum; Experimental allergic encephalomyelitis; Evans syndrome; Fibrinating 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 disease; Hemolytic anemia; Henoch-Schönlein purpura; Herpes gestationis; hypogammaglobulinemia; hidradenitis suppurativa (HS); 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; Lignin keratoconjunctivitis; Linear immunoglobulin A disease (LAD); Lupus (SLE); Lyme disease, chronic; Macrophage activation syndrome (MAS); Mastocytosis; Meniere's disease; Microscopic polyangiitis; Mixed connective tissue disease (MCTD); Mooren's ulcer; Muh-Ha-Ha Berman's 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 infection); palmoplantar pustulosis (PPP); 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 legs syndrome; Retroperitoneal fibrosis; Rheumatic fever; Rheumatoid arthritis; Sarcoidosis; Schmidt's syndrome; Scleritis; Scleroderma; Sjogren's syndrome; Spermato-testicular autoimmunity; Stiff body syndrome; Subacute bacterial endocarditis (SBE); Susac syndrome; Sympathetic ophthalmia; 21. The method of claim 20, wherein the patient has Takayasu's arteritis; temporal arteritis / giant cell arteritis; thrombocytopenic purpura (TTP); Trosa-Hunt syndrome; transverse myelitis; type 1 diabetes; ulcerative colitis; undifferentiated connective tissue disease (UCTD); uveitis; vasculitis; vesiculobullous skin disease; or vitiligo.

22. The method of any one of claims 18 to 21, wherein the fusion protein or polynucleotide is administered as the sole therapeutic agent.

23. 22. The method of any one of claims 18 to 21, wherein the fusion protein is administered in combination with one or more other pharmaceutical agents indicated for the treatment of an autoimmune disease, an allergic disease, a neurodegenerative disorder, neuropathic pain, an inflammatory disorder, Th2-induced airway inflammation, or diabetic neuropathy.

24. The method of any one of claims 18 to 21, wherein the fusion protein is administered in combination with one or more immunosuppressive agents or adjuvants in an immunosuppressive therapy.

25. 25. The method of claim 24, wherein the fusion protein is administered in combination with azathioprine, methotrexate, cyclosporine, a monoclonal antibody, a corticosteroid, or a combination thereof.

26. 26. The method of claim 25, wherein the monoclonal antibody is basiliximab, daclizumab, or muromonab.

27. The fusion protein of any one of claims 1 to 15, the polynucleotide of claim 16, or the composition of claim 17 for use in the treatment of an autoimmune disease, an allergic disease, a neurodegenerative disorder, neuropathic pain, an inflammatory disorder, Th2-induced airway inflammation, or diabetic neuropathy.

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