Novel CD200 fusion proteins

Abstract

The present invention relates to a fusion protein comprising a mutated CD200 moiety comprising K130Y and I131Y mutations, which binds to the human CD200 receptor with a higher affinity compared to the wild-type CD200, is fused directly to a non-CD200 IgG4 Fc fragment comprising S28P, M428L and N434S mutations and a deletion of the first 5 amino acids. The invention also relates to polynucleotides encoding the fusion proteins, pharmaceutical compositions comprising the same and uses thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to UK provisional patent application GB2206673.2 filed on May 6, 2022, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing Statement

[0004] This application contains a sequence listing in electronic format submitted via EFS Web. The sequence listing was created on April 27, 2023, named "4549-140ST26.xml", and is 6664 bytes in size. The electronic format information of the sequence listing is part of this application, and its entire contents are incorporated herein by reference. Technical Field

[0005] The present invention relates to a fusion protein comprising a mutated CD200 portion comprising K130Y and I131Y mutations, which binds to a human CD200 receptor with higher affinity than wild-type CD200, directly fused to a non-CD200 IgG4 Fc fragment comprising S228P, M428L and N434S mutations and a deletion of the first 5 amino acids. The present invention also relates to a polynucleotide encoding the fusion protein, a pharmaceutical composition comprising the same and its use. Background Art

[0006] Inflammatory diseases, including autoimmune and allergic diseases, are the second leading cause of chronic disease worldwide and are the leading cause of morbidity among women in the U.S. According to a 2008 international survey, chronic disease patients in the U.S. are more likely to not receive adequate care due to cost burdens compared to other countries (Schoen, C. et al., (2008) Health Affairs Web Exclusive, w1-w16). In addition, these patients are more likely to experience the highest rates of medical errors, care coordination problems, and high out-of-pocket medical expenses.

[0007] Currently, the American Autoimmune Related Diseases Association (AARDA) estimates that 50 million Americans have an autoimmune disease. Epidemiological data are lacking to determine the full direct and indirect costs of autoimmune diseases to the healthcare system as a whole. However, in 2001, Dr. Anthony Fauci, director of the National Institute of Allergy and Infectious Diseases (NIAID), estimated that the annual cost of treating autoimmune diseases was over $100 billion. While $100 billion is a staggering figure, it is likely a significant underestimate of the true cost of autoimmune disease, as epidemiological studies have estimated that only seven of the more than 100 known autoimmune diseases have annual costs between $51.8 billion and $70.6 billion, including Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), rheumatoid arthritis (RA), psoriasis, and scleroderma. In addition, these estimates ignore the cost of immunosuppressive therapy during transplantation.

[0008] Autoimmune diseases are incurable, chronic conditions that develop when the immune system determines that healthy cells are foreign and attacks them. Depending on the type, autoimmune diseases may affect one or more different types of body tissues and may cause abnormal organ growth and changes in organ function. Normal regulation of the immune system is primarily due to receptor / ligand pairs, which include proteins expressed by cells involved in the immune response. However, these receptor / ligand pairs are often involved in signaling cascades that contribute to the pathology of autoimmune diseases.

[0009] OX-2 membrane glycoprotein, also known as CD200 (cluster of differentiation 200), is a human protein encoded by the CD200 gene, expressed in a variety of cell types (Barclay, AN (1981) Immunology 44, 727), and has a high degree of homology with 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).

[0010] 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).

[0011] The binding of CD200 to CD200R delivers inhibitory signals to myeloid cells and T cells, thereby exerting immunosuppressive effects on 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).

[0012] CD200R agonists have been shown to reduce pathology in a variety of mouse disease models, such as arthritis (Gorczynski et al., (2001) Clin. Immunol. 101, 328-34; Gorczynski et al., (2002) Clin. Immunol. 104, 256-264), transplant 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 lung inflammation (Snelgrove et al., (2008) Nat. Immunol., 9, 1074-1083) and HSV-induced inflammatory lesions (Sarangi et al., (2009) Clin. Immunol. 131, 31-40).

[0013] In addition, CD200 - / - Mice developed more severe disease, which was associated with increased lung infiltration and lung endothelial damage (Rygiel. TP et al. (2009) J. Immunol. 183(3), 1990-1996). CD200 - / - The mice did mount an immune response that controlled viral load, suggesting that severe disease was caused by a poorly controlled immune response rather than a beneficial antiviral immune response. Disease was prevented by T cell depletion prior to viral challenge, despite dramatic increases in viral load. Rygiel.TP et al. (2009) concluded that T cells are critical for the manifestation of disease symptoms during influenza infection and that lack of downregulation of CD200-CD200R signaling, rather than viral load, increases immunopathology.

[0014] Analytical studies have shown that hCD200 is downregulated in different patient populations, such as patients with multiple sclerosis (Koning et al., (2007) Ann. Neurol. 62, 504-514), acute asthma attacks (Aoki et al., 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 (hair loss) (Harries et al., (2013) J. Pathol. 231(2), 236-247).

[0015] Agonist CD200 proteins are disclosed in, for example, WO 2000 / 061171 and WO 2008 / 089022. The literature describes the use of wild-type CD200 molecules to modulate immune cell function. The present invention relates to mutant CD200 proteins, which have a greater binding affinity to the CD200 receptor than wild-type CD200.

[0016] Therefore, therapeutic intervention using molecules that modulate the CD200 pathway offers a way to control excessive or undesirable immune responses and reduce pathology in patients with chronic or intermittent (episodic) autoimmune diseases.

[0017] There is a need to provide better clinical efficacy at lower doses and overcome the problems associated with currently available autoimmune disease treatments. Summary of the invention

[0018] According to a first aspect of the present invention, there is provided a fusion protein comprising:

[0019] (i) a mutated CD200 portion comprising mutations at amino acid residue positions 130 and 131, wherein the mutations are K130Y and I131Y; and

[0020] (ii) a non-CD200 portion, wherein the non-CD200 portion is an IgG4 Fc fragment and comprises the S228P, M428L and N434S mutations according to the EU numbering system and a deletion of the first 5 amino acids of the hinge,

[0021] Here, glycine 232 of the mutated CD200 portion was directly fused to the non-CD200 IgG4 Fc fragment at amino acid 6 according to the IMGT numbering system.

[0022] According to another aspect of the present invention, a polynucleotide encoding the fusion protein defined herein is provided.

[0023] According to a further aspect, there is provided a pharmaceutical composition comprising the fusion protein defined herein.

[0024] In another aspect of the present invention, a fusion protein, polynucleotide or pharmaceutical composition as defined herein is provided for the preparation of a drug. In another aspect of the present invention, a fusion protein, polynucleotide or pharmaceutical composition for treatment as defined herein is provided. In another aspect of the present invention, a fusion protein, polynucleotide or pharmaceutical composition as defined herein is provided for the treatment of autoimmune diseases, allergic diseases, neurodegenerative diseases, inflammatory diseases, Th2-induced airway inflammation, diabetic neuropathy, neurodegeneration or neuropathic pain. In another aspect of the present invention, a fusion protein, polynucleotide or pharmaceutical composition as defined herein is provided for the treatment of rheumatoid arthritis, asthma or atopic dermatitis. In another aspect of the present invention, a fusion protein, polynucleotide or pharmaceutical composition as defined herein is provided for the treatment of autoimmune diseases affecting the neuromuscular system, vascular system, eyes, skin, digestive tract, lungs, kidneys, liver, peripheral or central nervous system, bone, cartilage or joints. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1A-1B : Sensorgram of a BIAcore assay showing human CD200R and captured mutant CD200-Fc fusion proteins (ARQ-234; Figure 1A ) or wild-type CD200-Fc (DS-155; Figure 1B ) association and dissociation phases of binding.

[0026] Figure 2A-2B : Sensorgram of a BIAcore assay showing the interaction of cynomolgus monkey CD200R1 with captured mutant CD200-Fc fusion proteins (ARQ-234; Figure 2A ) or wild-type CD200-Fc (DS-155; Figure 2B ) association and dissociation phases of binding.

[0027] Figure 3 : A bar graph showing inhibition of LPS-stimulated IL-6 release after treatment of U937-CD200R cells with mutant fusion protein (ARQ-234) in the presence (upper graph) or absence (lower graph) of Fc blockade. The percentage of inhibition shown is relative to LPS-stimulated IL-6 release in U937-CD200R cells not treated with mutant or wild-type fusion protein (set to 0%).

[0028] Figure 4: After 1, 4 and 24 hours, the binding of mutant CD200-Fc (ARQ-234) or wild-type CD200-Fc (DS-155) to U937-CD200R cells was observed using fluorescent anti-human secondary antibody.

[0029] Figure 5 : A) Schematic diagram of the PK analysis scheme. B) Graph showing the concentrations of mutant CD200 fusion protein (ARQ-234) measured in cynomolgus monkey serum at the indicated times after dosing (time = 0).

[0030] Figure 6 : Bar graph showing the inhibitory effect of IL-6 on dose titration of DS-155 (wt CD200-Fc) and DS-192 (13 nM high affinity CD200-Fc).

[0031] Figure 7 : Bar graph of DS-192 inhibition of TNFα. Error bars represent standard deviation between biological replicates.

[0032] Figure 8 : Bar graph showing the inhibitory effects of DS-155 and DS-192 on ERK phosphorylation measured by flow cytometry in permeabilized cells in the presence of anti-pERK antibody and huCD200-Fc fusion protein.

[0033] Fig. 9 : Line graph showing clinical scores of paw arthritis in male DBA / 1J mice measured every other day from day 25-36. Data are expressed as mean ± SEM. **p<0.01; ***p<0.001 vs. disease + dexamethasone, disease + DS-198, and disease + DS-227, two-way RM ANOVA with Tukey's multiple comparison test.

[0034] Fig.10 : Bar graph showing changes in ear thickness in a humanized mouse model of contact hypersensitivity starting from day 0. Values ​​shown are combined values ​​for the right and left ears.

[0035] Fig.11 : Bar graph showing IL-1β cytokine levels in tissue homogenates of a humanized mouse model of contact hypersensitivity on day 15. Data are presented as mean ± SEM. Unpaired Student's t-test compared with the isotype control group, Compared with the negative control group, *p<0.05, **p<0.01, unpaired Student's t-test.

[0036] Fig.12: Bar graph showing GM-CSF cytokine levels in tissue homogenates of a humanized mouse model of contact hypersensitivity on day 15. Data are presented as mean ± SEM. Unpaired Student's t-test compared with isotype control group, Compared with the negative control group, *p<0.05, **p<0.01, unpaired Student's t-test.

[0037] Fig.13 : Bar graph showing IL-13 cytokine levels in tissue homogenates of a humanized mouse model of contact hypersensitivity on day 15. Data are presented as mean ± SEM. Unpaired Student's t-test compared with the isotype control group, Compared with the negative control group, *p<0.05, **p<0.01, unpaired Student's t-test.

[0038] Fig.14 : Schematic diagram of ARQ-234CD200-Fc fusion showing mutations for high affinity CD200R and FcRn binding.

[0039] Figures 15A-15F :( Fig.15A ) shows the activity of 100 μg / mL antibody recognizing CD64 (FcγRI) Fcγ receptor when co-incubated with different doses of DS-192 (ha CD200-IgG4Fc) in U937 cell assay. ( Fig. 15B ) shows the activity of 0 μg / mL antibody recognizing CD64 (FcγRI) Fcγ receptor when co-incubated with different doses of DS-192 (ha CD200-IgG4 Fc) in U937 cell assay. ( Fig. 15C ) shows the activity of 100 μg / mL antibody recognizing CD16 (FcγRIII) Fcγ receptor when co-incubated with different doses of DS-192 (ha CD200-IgG4 Fc) in U937 cell assay. ( Fig.15D ) shows the activity of 0 μg / mL antibody recognizing CD16 (FcγRII) Fcγ receptor when co-incubated with different doses of DS-192 (ha CD200-IgG4 Fc) in U937 cell assay. ( Fig.15E ) shows the activity of 100 μg / mL antibody recognizing CD32 (FcγRII) Fcγ receptor when co-incubated with different doses of DS-192 (haCD200-IgG4 Fc) in U937 cell assay. ( Fig.15F ) shows the graph of 0 μg / mL antibody recognizing CD32 (FcγRII) Fcγ receptor activity when co-incubated with different doses of DS-192 (ha CD200-IgG4 Fc) in U937 cell assay. DETAILED DESCRIPTION

[0040] According to a first aspect of the present invention, there is provided a fusion protein comprising:

[0041] (i) a mutated CD200 portion comprising mutations at amino acid residue positions 130 and 131, wherein the mutations are K130Y and I131Y; and

[0042] (ii) a non-CD200 portion, wherein the non-CD200 portion is an IgG4 Fc fragment and comprises the S228P, M428L and N434S mutations according to the EU numbering system and a deletion of the first 5 amino acids of the hinge,

[0043] Therein, glycine 232 of the mutated CD200 portion was directly fused to the non-CD200 IgG4-Fc fragment at amino acid 6 according to the IMGT numbering system.

[0044] The inventors have found that mutations in the extracellular domain of CD200 at these amino acid residues produce mutant CD200 portions with increased binding affinity to the CD200 receptor (CD200R). In addition, as described herein, fusion proteins comprising mutant CD200 portions have significant benefits, particularly in providing treatments with higher clinical efficacy and lower dosages.

[0045] Thus, in a specific embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 1. In another embodiment, the fusion protein consists of the amino acid sequence of SEQ ID NO: 1. In another embodiment, the fusion protein is ARQ-234.

[0046] SEQ ID NO: 1 (also referred to herein as "ARQ-234") consists of the following sequence:

[0047]

[0048] The highlighted amino acids indicate the mutation positions relative to wild-type CD200 or IgG4 Fc, and the underlined sequences indicate non-CD200 Fc fragments.

[0049] In one embodiment, ARQ-234 may further include an N-terminal signal sequence, which is a human IgG heavy chain signal peptide. In another embodiment, the N-terminal signal sequence comprises or consists of the amino acid sequence of SEQ ID NO.3.

[0050] SEQ ID NO:3 consists of the following sequence:

[0051] MEFGLSWLFLVAILKGVQC

[0052] As used herein, the term "CD200 protein" refers to the wild-type CD200 protein.

[0053] The term "wild type" as used herein refers to proteins, peptides, amino acids and nucleotide sequences present in nature. For example, the term "wild type CD200 protein" as used herein refers to any full-length isoform (UNIPROT P41217OX2G_HUMAN) or any portion thereof (including naturally occurring protein polymorphisms) of CD200 that binds to the CD200 receptor (CD200R). CD200 protein is also known as OX-2 membrane glycoprotein.

[0054] Wild-type CD200 is a cell surface protein having an N-terminal extracellular domain, a short transmembrane and a cytoplasmic domain. The extracellular domain binds to a target receptor, such as a CD200 receptor. In one embodiment, the CD200 protein is an extracellular domain of CD200 or any portion thereof that binds to a CD200 receptor.

[0055] As used herein, the term "position" refers to the residue number in an amino acid sequence, where 1 is the first translated amino acid. Thus, it is understood that the numbering of amino acid positions within the CD200 portion as defined herein is relative to the amino acid sequence including the N-terminal signal sequence representing the first 30 amino acids of the CD200 portion (as shown in bold in SEQ ID NO: 2).

[0056] As used herein, the term "mutated" or "mutation" refers to proteins, peptides, amino acids and nucleotide sequences that have changed in form from their wild-type equivalents to mutants. For example, a mutant protein or mutant protein may have a change in amino acid and / or nucleotide sequence compared to the corresponding wild-type sequence, and this change may also be referred to as a mutation.

[0057] "Mutated CD200 protein" and "mutated CD200 portion" referred to herein refers to the full-length CD200 protein or any portion thereof that binds to the CD200 receptor, comprising one or more mutant amino acid residues in the amino acid sequence, making it similar to but no longer identical to the wild-type CD200 protein. According to the first aspect of the invention defined herein, the mutated CD200 portion comprises K130Y and I131Y mutations. Thus, in one embodiment, the mutation is a substitution mutation.

[0058] In one embodiment, the fusion protein can be prepared synthetically or recombinantly. In another embodiment, the fusion protein can be prepared synthetically. In another embodiment, the fusion protein can be prepared recombinantly.

[0059] In one embodiment, the mutated CD200 portion binds the CD200 receptor with a higher affinity than wild-type CD200.

[0060] In one embodiment, the mutated CD200 protein / part may include the entire extracellular domain of CD200 or a portion thereof. In a further embodiment, the mutated CD200 protein includes a signal sequence. It should be understood that the secreted protein contains many amino acids at the N-terminus, which constitute a signal sequence that can be cut before secretion. Therefore, in certain embodiments, the mutated CD200 part includes an N-terminal signal sequence. In one embodiment, the mutated CD200 part includes a signal sequence at the N-terminus that is cut before secretion from the production cell. In another embodiment, the signal sequence includes the first 30 amino acids of the wild-type CD200 protein. In another embodiment, the signal sequence represents the first 30 amino acids of the CD200 part. In another embodiment, the signal sequence is SEQ ID NO:3. Therefore, in certain embodiments, the fusion protein includes a sequence as defined herein, wherein there is no amino acid comprising a signal sequence. For example, in the case where amino acids 1-30 of the wild-type CD200 protein do not exist and the mutated CD200 protein includes a sequence corresponding to amino acids 31-232 of SEQ ID NO:2. Therefore, in another embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fusion protein consists of the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fusion protein consists of the amino acid sequence of SEQ ID NO: 3 at the N-terminus of SEQ ID NO: 1.

[0061] SEQ ID NO:2 consists of the following sequence:

[0062]

[0063] in Amino acids represent signal sequences, The amino acids shown indicate the mutation positions relative to wild-type CD200 or IgG4 Fc. Underline The sequence represents a non-CD200 Fc fragment. The present disclosure also includes the disclosed protein sequences, but lacking the C-terminal lysine, such as proteins whose C-terminal lysine (K) is cleaved during secretion from mammalian cells.

[0064] In one embodiment, the fusion protein is encoded by the polynucleotide of SEQ ID NO:4. It is worth noting that there is degeneracy in the genetic code, which means that most amino acids are specified by multiple codons. Therefore, since many different codons define the same amino acid, multiple polynucleotide sequences can encode the same amino acid sequence. Therefore, SEQ ID NO:4 represents an exemplary arrangement of the polynucleotide sequence that can encode the fusion protein. Unless otherwise indicated by the context, any arrangement and combination of all described elements in this application should be considered as disclosed by the description of the application.

[0065]

[0066] As used herein, the term "portion" refers to proteins, peptides, amino acids and nucleotide sequences, and refers to fragments and derivatives that have function, ie, bind to their target.

[0067] As used herein, the term "fragment" refers to a portion of a protein, peptide, amino acid or nucleotide sequence that recognizes and binds to its target (eg, a receptor).

[0068] The terms "derivative" and "mutant" used herein refer to proteins, peptides, amino acids or nucleotide sequences that have at least 70% (such as 75%, 80%, 85%, 90%, 95% or 99%) sequence similarity and similar functions to the wild-type equivalent. Therefore, a mutant may be a derivative of a wild-type equivalent.

[0069] As used herein, the term "amino acid residue" refers to the monomer unit in a polymer chain, ie, a single amino acid in a protein.

[0070] As shown by the data provided herein, the mutant CD200 proteins / portions of the invention bind more tightly to the CD200 receptor and remain on the receptor longer than wild-type CD200 proteins.

[0071] Fusion Protein

[0072] According to a first aspect of the invention as defined herein, there is provided a fusion protein comprising a mutant CD200 protein / part as defined herein fused to a non-CD200 part.

[0073] As used herein, the term "fusion protein" refers to one or more amino acid sequences, peptides and / or proteins linked together using methods well known in the art, such as U.S. Pat. Nos. 5,434,131 and 5,637,481. The linked amino acid sequences, peptides or proteins thus form a fusion protein.

[0074] In some embodiments, the mutant CD200 protein / part as defined herein is fused to a non-CD200 part at the C-terminus. Thus, in one embodiment, the orientation of the fusion protein from N-terminus to C-terminus is: mutant CD200 part-non-CD200 Fc fragment. Thus, in another embodiment, the orientation of the fusion protein is: mutant CD200 part-IgG4 Fc fragment. In another embodiment, the orientation of the fusion protein from N-terminus to C-terminus is: signal sequence-mutated CD200 part-non-CD200 Fc fragment. Thus, in another embodiment, the orientation of the fusion protein is: signal sequence-mutated CD200 part-IgG4 Fc fragment.

[0075] The term "non-CD200 moiety" as used herein may refer to any molecule, peptide or protein that does not bind to the CD200 receptor and does not interfere with the binding of CD200 to its target. Examples include, but are not limited to, an immunoglobulin (Ig) constant region or a portion thereof; or a fusion protein in which the non-CD200 moiety is a synthetic molecule (e.g., PEG), such as PEG.

[0076] In one embodiment, the non-CD200 portion is an antibody fragment. In a specific embodiment, the non-CD200 portion is an Fc fragment. Therefore, the mutant CD200 fusion protein described herein may also be referred to as mutant CD200-Fc. In another embodiment, the Fc fragment is derived from a mammal, such as derived from a human or monkey, such as a human C (γ) 1 including hinge, CH2 and CH3 regions. In particular, the Fc fragment includes a hinge region. The Fc fragment provides the advantage of extending the serum half-life of the mutant CD200 protein of the present invention, and by dimerizing the CD200 protein, the binding affinity is also increased and agonistic signaling is achieved. It will be appreciated by those skilled in the art that the Fc region can be mutated to reduce its effector function (e.g., see US 5,637,481 and US 6,132,992).

[0077] In one embodiment, the Fc fragment is an IgG4 Fc fragment.

[0078] In another embodiment, the non-CD200 portion is an antibody Fc fragment comprising a mutation of one or more amino acid residues. Thus, in a specific embodiment, the non-CD200 portion is an IgG4 Fc fragment comprising S228P, M428L and N434S mutations, wherein the position of the mutation is according to the EU numbering system. Thus, in one embodiment, the non-CD200 Fc fragment is a S228P, M428L and N434S derivative of human IgG4. The S228P mutation prevents the exchange of Fab arms in antibodies. Therefore, the presence of the S228P mutation in the Fc fragment described herein may increase the stability of the fusion protein in vivo and in vitro, thereby improving the therapeutic effect and manufacturability. The presence of the M428L and N434S mutations (also referred to as "LS variants") increases the affinity of the Fc fragment for the human neonatal Fc receptor (hFcRn) at pH 6 and results in an increased serum half-life (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), such as an increased serum half-life of a fusion protein comprising a mutated non-CD200 Fc fragment as defined herein. In another 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 the IgG4 Fc fragment. Thus, in one embodiment, the non-CD200 portion is an IgG4 Fc fragment comprising S228P, M428L and N434S mutations according to the EU numbering system and lacking the first 5 amino acids of the hinge. In another embodiment, the non-CD200 portion is an IgG4 Fc fragment comprising S228P, M428L and N434S and lacking the first 5 amino acids of the Fc hinge region.

[0079] In one embodiment, the fusion protein is formed by direct fusion of the mutated CD200 part with the non-CD200 Fc fragment. Therefore, it is understood that this fusion does not contain a linker sequence between the mutated CD200 part and the non-CD200 Fc fragment. For example, the amino acid glycine 232 of the mutated CD200 part can be directly fused with the 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 part with the amino acid 6 of the IgG4-Fc fragment (in this case, the first 5 amino acids of the Fc hinge region are deleted as described above). In another embodiment, direct fusion is the fusion of the amino acid glycine 232 of the mutated CD200 part with the amino acid 6 of the Fc hinge region of the IgG4-Fc fragment. Therefore, in one embodiment, the glycine 232 of the mutated CD200 part is directly fused to the non-CD200 Fc fragment at the amino acid 6 of the Fc hinge region. According to these embodiments, the position of the fusion in the Fc fragment conforms to the IMGT numbering system. This direct fusion of the mutated CD200 moiety to amino acid 6 of the hinge region of the IgG4-Fc fragment increased the stability of the resulting fusion protein compared to fusion proteins containing a linker sequence without affecting the efficient binding to CD200R. This result was surprising in light of previously reported data on Fc fusion proteins containing a linker sequence.

[0080] For the purpose of this specification, when the non-CD200 portion is an Fc fragment, the term "position" used herein with respect to the mutation of the non-CD200 portion refers to the residue number in the amino acid sequence according to the EU numbering system. Therefore, it is understood that the mutation residue position of the Fc fragment amino acid cited herein is related to its position according to the EU numbering system. It should be further understood that other numbering systems developed for the numbering of residues in the Fc fragment sequence, such as Kabat, AHo, IMGT, Chothia and Martin (enhanced Chothia), can be used alternatively. When used herein for the point where the mutated CD200 portion is fused to the non-CD200 Fc fragment, "position" refers to the residue number within the Fc fragment according to the IMGT numbering system. Therefore, it is understood that the residue position of the amino acid of the hinge of the Fc fragment is related to its position according to the IMGT numbering system. Therefore, the mutation numbering within the Fc fragment herein refers to the EU numbering system, and the numbering of the hinge amino acid refers to the IMGT numbering system. In some embodiments, glycine 232 of the mutated CD200 portion is directly fused to the non-CD200 Fc fragment at amino acid 224 of the IgG4 heavy chain of the Fc fragment according to the EU numbering system.

[0081] The protein of the present invention is preferably produced by a recombinant DNA method 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 that promote expression. Therefore, 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 endonuclease sites. In another embodiment, a nucleic acid sequence encoding a CD200-Fc fusion protein or any portion thereof is inserted into a recombinant expression vector using fusion cloning. Therefore, in another embodiment, the nucleic acid encoding the CD200-Fc fusion protein or any portion thereof comprises a nucleic acid sequence complementary to the end of a linearized vector at its end, such as an overlap of 12 to 21 base pairs / nucleotides between the nucleic acid encoding the CD200Fc fusion protein and the vector, such as an overlap of 15 base pairs or 20 base pairs.

[0082] According to another aspect of the present invention, a polynucleotide encoding a fusion protein as defined herein is provided. The present disclosure includes polynucleotides encoding proteins as defined herein, and the use of such nucleic acids for producing proteins and / or for therapeutic purposes. Such polynucleotides may include DNA and RNA molecules (e.g., mRNA, self-replicating RNA, self-amplifying mRNA, etc.) encoding proteins as defined herein. The nucleic acid sequence encoding the protein provided by the present invention can be assembled from cDNA fragments and short oligonucleotide linkers or a series of oligonucleotides to provide a synthetic gene 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 another embodiment, the polynucleotide encodes a fusion protein consisting of the amino acid sequence of SEQ ID NO: 1. In another embodiment, the polynucleotide encodes ARQ-234. In a specific embodiment, the polynucleotide encodes a fusion protein comprising the amino acid sequence of SEQ ID NO: 2. In another 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.

[0083] The recombinant expression vector includes a synthetic or cDNA-derived nucleic acid fragment encoding a mutant CD200, which is operably linked to a suitable transcriptional or translational regulatory element from a mammalian, microbial, viral or insect gene. Such regulatory elements include a transcriptional promoter, an optional operator sequence that controls transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence that controls transcriptional and translational termination. The ability to replicate in a host, usually conferred by a replication origin, and a selection gene that facilitates recognition of transformants, can also be additionally introduced.

[0084] Therapeutic Uses

[0085] The present invention has particular application in therapy because the interaction between the CD200 protein and the CD200 receptor is characterized by a fast dissociation ("off") rate, which results in a low affinity of CD200 for the CD200 receptor. Therefore, as described herein, increasing the affinity of mutant CD200 proteins and fusion proteins comprising a portion thereof for the CD200 receptor can be used to prepare pharmaceutical compositions with more potent properties.

[0086] In addition, the manufacturing cost of recombinant proteins is high, and mutant CD200 proteins / fusion proteins comprising a portion thereof have higher affinity and can be used in pharmaceutical compositions at much lower doses than wild-type or non-mutated CD200 proteins to achieve therapeutic effects. Therefore, in addition to being more effective clinically, the use of mutant CD200 proteins / fusion proteins comprising a portion thereof may be more cost-effective.

[0087] According to another 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 another embodiment, the pharmaceutical composition comprises a fusion protein consisting of the amino acid sequence of SEQ ID NO: 1. In another embodiment, the pharmaceutical composition comprises ARQ-234.

[0088] In one embodiment, the mutant CD200 protein or fusion protein as defined herein is a modulator of the CD200 receptor. The term "modulator" as used herein refers to a substance that causes a change, such as a modulator of a protein that may cause an increase or decrease in the activity of the protein. In view of the characteristics of the mutant CD200 proteins and fusion proteins of the present invention, they are considered to be agonists of the CD200 receptor and therefore have practicality in treating autoimmune diseases. Therefore, in another embodiment, the mutant CD200 protein or fusion protein as defined herein is an agonist of the CD200 receptor.

[0089] Therefore, according to another aspect of the present invention, there is provided a fusion protein as described herein or a pharmaceutical composition as defined herein for use in the treatment of an autoimmune disease.

[0090] As used herein, the terms "autoimmune disease" or "autoimmune disease" are used interchangeably and refer to adverse symptoms caused by an inappropriate or undesirable immune response to one's own cells and / or tissues or transplanted cells and / or tissues. The terms "autoimmune disease" or "autoimmune disease" are meant to include these symptoms, whether they are mediated by a humoral immune response or a cellular immune response.

[0091] In an alternative embodiment, a fusion protein as defined herein or a pharmaceutical composition as defined herein is provided for the treatment of an allergic disease. As used herein, the terms "allergy" or "allergic disease" are used interchangeably and refer to a disease driven by T helper cell 2 (TH2) primarily caused by TH2 cell activity. Examples of allergic diseases include chronic allergic diseases (such as hay fever or allergic rhinitis), allergic contact dermatitis, seasonal allergies, anaphylaxis, food allergies, asthma, and atopic dermatitis.

[0092] Fusion proteins comprising a mutant CD200 protein / portion as defined herein may more efficiently inactivate activated immune cells compared to fusion proteins comprising wild-type or non-mutated CD200 protein.

[0093] In one embodiment, the autoimmune disease is selected from an autoimmune disease affecting the neuromuscular system, vascular system, eyes, skin, digestive tract, lungs, kidneys, liver, peripheral or central nervous system, bones, cartilage, or joints.

[0094] In another embodiment, the autoimmune disease is selected from one or more of the following: acute disseminated encephalomyelitis (ADEM); acute necrotizing hemorrhagic leukoencephalitis; Addison's disease; aglobulinemia; alopecia areata; amyloidosis; ankylosing spondylitis; anti-GBM / anti-TBM nephritis; antiphospholipid syndrome (APS); asthma, atopic dermatitis; autoimmune angioedema; autoimmune aplastic anemia; autoimmune autonomic dysregulation; autoimmune hepatitis; autoimmune hyperlipidemia; autoimmune immunodeficiency; autoimmune inner ear disease (AIED); autoimmune myocarditis; autoimmune oophoritis; autoimmune pancreatitis; autoimmune retinopathy; autoimmune thrombocytopenia ATP; autoimmune thyroid disease; autoimmune urticaria; axonal and neuronal neuropathy; Barlow disease; Behcet's disease; bullous pemphigoid and related autoimmune blistering diseases; cardiomyopathy; Castleman disease; celiac disease (eg, refractory celiac disease type II); Chagas disease; chronic idiopathic urticaria; chronic inflammatory demyelinating polyneuropathy (CIDP); chronic relapsing multiple osteomyelitis (CRMO); chronic spontaneous urticaria; Churg-Strauss syndrome; cicatricial pemphigoid / benign mucous membrane pemphigoid; Crohn's disease; Cogans syndrome; cold agglutinin disease; congenital heart block; coxsackie myocarditis; CREST disease; primary mixed cryoglobulin Leukemia; demyelinating neuropathy; dermatitis herpetiformis; dermatomyositis; Devic disease (neuromyelitis optica); diabetic neuropathy; discoid 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 Wegener's granulomatosis); graft-versus-host disease (GvHD); Graves' disease; Guillain-Barré syndrome; Hashimoto's encephalitis; Hashimoto's thyroiditis; hemolytic anemia; Henoch-Schonlein purpura; herpes gestationis; hypogammaglobulinemia; suppurative sweating HS; Idiopathic thrombocytopenic purpura (ITP); IgA nephropathy; IgG4-related sclerosing disease; Immunomodulatory lipoprotein-related lesions; Inclusion body myositis; Inflammatory bowel disease (IBD); Inflammatory skin diseases; Interstitial cystitis; Juvenile arthritis; Juvenile diabetes mellitus (type 1 diabetes); Juvenile myositis; Kawasaki syndrome; Lambert-Eaton syndrome; Leukocytoclastic vasculitis; Lichen planus; Lichen sclerosus; Woody conjunctivitis; Linear IgA disease (LAD); Systemic lupus erythematosus (SLE); Lyme disease, chronic; Macrophage activation syndrome (MAS); Mastocytosis; Meniere's disease; Microscopic polyangiitis; Mixed connective tissue disease (MCTD); Mooren's ulcer;Mucha-Habermann disease; multiple sclerosis; myasthenia gravis; myositis; narcolepsy; neuromyelitis optica (Devic's); neutropenia; ocular cicatricial pemphigoid; optic neuritis; pallial rheumatism; PANDAS (autoimmune neuropsychiatric disorders of childhood associated with streptococci); palmoplantar pustulosis (PPP); paraneoplastic cerebellar degeneration (PNH); paroxysmal nocturnal hemoglobinuria; Parry-Lomborg syndrome; Parsonnage-Turner syndrome; pars planitis (peripheral uveitis); pemphigus; peripheral neuropathy; perivenous encephalomyelitis; pernicious anemia; POEMS syndrome; polyarteritis nodosa; autoimmune polyglandular syndromes types I, II, and III; polymyalgia rheumatica; polymyositis; post-myocardial infarction syndrome; postpericardiotomy syndrome; progesterone dermatitis; primary biliary cirrhosis; primary sclerosing cholangitis; psoriasis; psoriatic arthritis; idiopathic pulmonary fibrosis; pyoderma gangrenosum; pure red cell aplasia; Raynaud's syndrome; 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 and testicular autoimmunity; stiff-man syndrome; subacute bacterial endocarditis (SBE); Susac's syndrome; sympathetic ophthalmia; Takayasu's arteritis; temporal arteritis / giant cell arteritis; thrombocytopenic purpura (TTP); Tolosa-Hunt syndrome; transverse myelitis; type 1 diabetes mellitus; ulcerative colitis; undifferentiated connective tissue disease (UCTD); uveitis; vasculitis; vesicular dermatosis; vitiligo; Wegener's granulomatosis (now called granulomatosis with polyangiitis (GPA)).

[0095] In an alternative embodiment, there is provided a protein or fusion protein as defined herein or a composition as defined herein for use in treating a neurodegenerative disease.

[0096] In another 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.

[0097] According to another aspect of the present invention, there is provided a method for treating an autoimmune disease, an allergic disease (e.g., rheumatoid arthritis, asthma, or atopic dermatitis), neuropathy, neuropathic pain, inflammatory joint pain, or diabetic neuropathy in a subject, comprising administering the fusion protein of the present invention to a subject suffering from at least one autoimmune disease, allergic disease, neuropathy, neuropathic pain, inflammatory joint pain, or diabetic neuropathy.

[0098] It should be understood that the protein or fusion protein of the present invention can be administered as the sole therapeutic agent or in combination with one or more other compounds (or therapies) for the treatment of autoimmune diseases, allergic diseases (such as rheumatoid arthritis, asthma or atopic dermatitis), neurodegenerative diseases, neuropathic pain, inflammatory joint pain or diabetic neuropathy.

[0099] Therefore, according to another aspect of the present invention, there is provided a pharmaceutical composition comprising a fusion protein as defined herein in combination with one or more other therapeutic agents.

[0100] In order to treat autoimmune diseases, allergic diseases (such as rheumatoid arthritis, asthma or atopic dermatitis), neurodegenerative diseases, neuropathic pain, inflammatory joint pain or diabetic neuropathy, the fusion protein of the present invention can be advantageously used in combination with one or more other drugs, more specifically, in immunosuppressive therapy with one or more immunosuppressants or adjuvants.

[0101] Examples of other therapeutic agents or treatment methods that can be administered with the compounds of the invention (whether simultaneously or at different time intervals) include, but are not limited to, azathioprine, methotrexate, cyclosporine, monoclonal antibodies (such as basiliximab, daclizumab and muromonab), and corticosteroids.

[0102] Each therapeutic agent present in the combination of the present invention may be administered in a separate different dosage regimen and by different routes. In addition, the posology of each of the two or more drugs may be different: each drug may be administered simultaneously or at different times. A person skilled in the art will know by his or her common sense the dosage regimen and combination therapy to be used. For example, a protein or fusion protein of the present invention may be used in combination with one or more other drugs administered according to their existing combination regimens.

[0103] Typically, the proteins disclosed herein will be used in purified form with a pharmaceutically suitable excipient or carrier. Typically, these excipients or carriers include aqueous or alcohol / aqueous solutions, emulsions or suspensions, including saline and / or buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, and lactated Ringer's solution. If necessary, the polypeptide complex is kept suspended, and suitable physiologically acceptable adjuvants can be selected from thickeners, such as carboxymethyl cellulose, polyvinyl pyrrolidone, gelatin, and alginate.

[0104] The route of administration of the pharmaceutical composition of the present invention can be any one known to those of ordinary skill in the art. For treatment, including but not limited to immunotherapy, the protein of the present invention can be administered to any patient according to standard techniques. It can be administered by any appropriate means, including parenteral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, pulmonary routes, such as intranasal or inhalation, or can also be directly infused by catheter, such as intracranial (e.g., intravenous injection into the central nervous system ventricle or intravenous injection into the spinal cord). The dosage and frequency of administration will depend on the patient's age, sex and condition, simultaneous administration of other drugs, counterindications and other parameters that clinicians need to consider.

[0105] The proteins of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been proven to be effective and freeze drying and reconstitution techniques known in the art can be used. Those skilled in the art will appreciate that freeze drying and reconstitution will result in varying degrees of loss of activity and that levels may have to be adjusted upwards to compensate.

[0106] It should be understood that all embodiments described herein are applicable to all aspects of the invention, and vice versa.

[0107] Other features and advantages of the present invention will become apparent through the description provided herein. However, it should be understood that while indicating preferred embodiments of the present invention, the description and specific examples are intended to be illustrative only, as various changes and modifications will become apparent to those skilled in the art. The following studies and schemes illustrate embodiments of the methods described herein.

[0108] Example 1: Preparation of mutant and wild-type CD200 Fc molecules

[0109] Gene synthesis and cloning

[0110] The gene synthesis of IgG4 S228P-Fc (codon optimized for CHO expression) was performed at GeneArt, and the mutation was M428L+N434S. These constructs were cloned into plasmid pCDNA 3.1 using fusion cloning to produce a vector backbone. The codon optimized DNA sequence (including the N-terminal signal sequence) encoding the mutant or wild-type human CD200 residues 1-232 of UniProt P412178 (OX2G_human) was inserted into the N-terminus of IgG4 S228P-Fc to produce a direct fusion of the amino acid glycine 232 of CD200 with the Fc region. These sequences were confirmed in both directions.

[0111] Gigaprep

[0112] Transform the sequence-confirmed plasmids into E. coli DH5α cells. Select a single colony for each target protein and inoculate into 10.0 mL of LB containing ampicillin. Subculture each mutant or wild-type construct into 800 mL of Circlegrow medium for gigascale DNA preparation. Isolate DNA using the endotoxin-free Quanta Giga kit.

[0113] Protein expression

[0114] CD200 Fc protein was produced by transiently transfecting the expression plasmid into CHO-3E7 cells using polyethyleneimine (PEI). Briefly, 4.0 × 10 6 250mL culture at a density of 10 cells / mL was transfected with 2mg / L of plasmid using PEI at a ratio of 1:5. 24 hours after transfection, the culture was transferred to 32°C and the cells were fed with 10% supplemented medium (feed C), glutamine, glucose and 0.5M sodium butyrate to enhance protein expression. The batch was monitored and the supernatant containing overexpressed CD200 Fc was harvested on day 7 with a viability of approximately 75%. The filtered supernatant was subjected to protein purification.

[0115] Protein purification

[0116] All purification procedures were performed at 4°C. The culture harvest was loaded onto a MabSelect SuRe affinity column (5 mL) and pre-equilibrated with 50 mM sodium phosphate, 150 mM NaCl pH 7.4 at a flow rate of 3 mL / min on the AKTAPure platform. The column was washed with equilibration buffer and the bound protein was eluted with 20 mM sodium acetate, 150 mM NaCl pH 3.5. The eluate was neutralized with 10% v / v 1M Tris pH 8.0 and analyzed by SDS-PAGE. The fractions containing CD200 Fc were combined and concentrated to 5 mL and subjected to gel filtration chromatography analysis on the AKTAPure platform (Hiload 16 / 600 Superdex-200 pg column). The protein was treated at a speed of 1.2 mL / min in a 50 mM sodium phosphate, 150 mM NaCl pH 7.4 buffer system and separated into different fractions for collection. The fractions containing CD200 Fc dimer were pooled and concentrated to 1.33 mg / mL (measured at UV 280 nm) using an Amicon Ultra Centricon (molecular weight cutoff of 10 kDa) by SDS-PAGE analysis. The purified material was tested by SEC-HPLC, LC-MS, and EndoSafe LAL kit to assess protein purity, molecular weight, and endotoxin content, respectively. The final samples were stored at -80°C.

[0117] Example 2: Binding analysis of wild-type and mutant CD200-Fc proteins

[0118] The BIAcore experiments were performed by Syngene International Ltd. (Biocon Park, Plot No 2 & 3, Bommasandra Industrial Area, Bommasadra-Jigani Link Road, Bangalore-560099, India).

[0119] Determination principle

[0120] The BIAcore instrument uses an optical method, surface plasmon resonance (SPR), to measure the binding properties of two interacting molecules; in this case, wild-type CD200Fc or a CD200Fc mutant bound to the CD200 receptor (CD200R). The technology measures the change in the refractive index of one of the two interacting molecules captured on the chip (sensor) as the second molecule flows past the captured partner in solution. In these experiments, CD200Fc is immobilized on the chip (sensor) surface and CD200R is injected in aqueous buffer above the captured CD200Fc under continuous flow conditions. The change in the refractive index of CD200Fc upon CD200R binding is measured in real time and the results are plotted as a graph of response units (RU) versus time to generate the sensorgram.

[0121] Instruments and reagents

[0122] The experiments were performed on GE Healthcare BIAcore T200. Human, cynomolgus monkey and mouse CD200R proteins were purchased from Creative Biomart (CD200R1-320H, CD200R1-3483C, CD200R1-3280M). All measurements were performed in duplicate.

[0123] plan

[0124] For human CD200 constructs: Anti-human Fc (GE Healthcare) was covalently immobilized on a BIAcore CM5 sensor chip (Cytiva, BR100530) by amine coupling using the Cytiva kit (BR100839) according to the manufacturer's instructions, targeting 8000-11000 RU. CD200 Fc protein was diluted to 0.5 μg / mL to 4 μg / mL in electrophoresis buffer (1xHBS EP+ pH7.4 (Cytiva BR100669), HEPES buffered saline pH 7.4, containing 3mM EDTA and 0.05% v / v surfactant P20) and flowed at 10 μL / min over the immobilized anti-human IgG Fc for 25 to 100 seconds with a stability time of 60 seconds. 35 to 250 RU of CD200 Fc were captured, with higher RUs used for cynomolgus CD200R binding experiments. Human CD200R or cynomolgus CD200R were serially diluted (3-fold dilution) to 5 or higher concentrations (depending on the expected affinity) in electrophoresis buffer along with buffer blank (0 nM) and flowed over the captured ligand at a flow rate of 30 to 50 μL / min for 120 seconds of binding followed by 120-360 seconds of dissociation in electrophoresis buffer. The assay temperature was 25°C. Subsequently, the surface was regenerated by a pulse of 3 M MgCl2 at a flow rate of 30 μL / min, followed by stabilization of the surface with 60 seconds of flowing buffer.

[0125] For mouse CD200 constructs, muCD200R-Fc (Creative Biomart CD200R1-458M) diluted to 1 μg / mL in electrophoresis buffer was passed over the immobilized anti-human IgG Fc, and CD200 monomer was serially diluted and passed over the captured CD200R. Other details were as described above.

[0126] Data analysis: Experimental sensorgrams were analyzed in the BIA evaluation software (GE Healthcare). The obtained curves were fitted to a 1:1 Langmuir binding model by setting Rmax and RI as local parameters. Rate equations using standard parameters (e.g., ligand concentration, time) were used for iterative curve fitting. The closeness of fit was determined by the algorithm provided by the manufacturer in the BIA evaluation software, and data were accepted if the Chi2 value was less than 10% of Rmax and the U value was less than or equal to 15.

[0127] Table 1 details the reagents used to develop and perform the assay.

[0128] Table 1: Reagents used in BIAcore experiments

[0129]

[0130] result

[0131] Results (Table 2 and Figure 1A-1B ) showed that the affinity of the mutant CD200-Fc protein (ARQ-234) of the present invention for binding to the human CD200 receptor was about 84 times higher than that of the wild-type CD200-Fc (DS-155). The shedding rates and Figure 1A-1B The sensorgrams shown in show ARQ-234 shedding rates and receptor half-lives that are compatible with potent agonism in functional cell assays. Figure 2A-2B The results in demonstrate that ARQ-234 is able to bind to cynomolgus monkey CD200R, allowing this fusion protein to be evaluated in standard toxicology protocols.

[0132] Table 2: Surface plasmon resonance (SPR) affinity of wild-type and mutant CD200-Fc fusion molecules to human CD200R and force (K D ) and kinetic parameters (k a , K D ,t 1 / 2 )

[0133]

[0134] Table 3: Surface plasmon resonance of wild-type and mutant CD200-Fc fusion molecules on cyno CD200R (SPR) affinity (K D ) and kinetic parameters (k a , K D ,t 1 / 2 )

[0135]

[0136] *ka (1 / Ms), kd (1 / s) and kD (nM) in Tables 2 and 3 are the average values ​​of two rounds.

[0137] Table 4: Mutant CD200 fusion proteins

[0138]

[0139]

[0140] Example 3: Cell Binding and Cell Activation Assays of Wild-Type and Mutant CD200-Fc Proteins

[0141] Determination principle

[0142] To demonstrate the agonist activity of ARQ-234, the human monocytic cell line U937 (ATCC, CRL1539) was transfected with cDNA of human CD200R. Stimulation with PMA and LPS can induce these cells to produce cytokines, including IL-6.

[0143] Cell line construction

[0144] The full-length human CD200R gene (including the signal sequence) was cloned into the pCDH-EF1-human CD200R-IRES-Puro lentiviral vector (System Biosciences) downstream of the EF1α promoter. Lentiviral particles containing the expression construct were produced in 293TN producer cells and concentrated using PEG-it reagent (System Biosciences) according to the manufacturer's instructions.

[0145] According to the manufacturer's instructions, the U937 human monocyte immortalized cell line was transduced with lentiviral particles at an MOI range of 5 to 200 using TransDux and Max Enhancer reagents (System Biosciences). Transduced U937 cells were a) selected using puromycin (with a first optimized puromycin concentration) and b) sorted by flow cytometry to generate a stable polyclonal CD200R expressing line. In addition to flow cytometry, the expression of CD200R was confirmed by Western blot.

[0146] Cytokine release (IL-6 inhibition) assay

[0147] 50,000 U937 cells per well were seeded in a 96-well plate and differentiated after incubation with 100 nM PMA for 72 hours. After differentiation, the medium containing PMA was replaced with fresh assay medium and incubated for another 2 hours before treatment. CD200-Fc constructs with or without Fc blocking were added to the cell culture and incubated for 1 hour, and then the cells were stimulated with 100 ng / ml LPS and incubated for another 24 hours. After the final incubation, the cell supernatant was collected (diluted 1:10) and IL-6 secretion was determined by ELISA using a commercially available kit.

[0148] Cell binding

[0149] U937 cells were resuspended in 50 μL FACS buffer (1XPBS + 2% FBS) at a density of 100,000 cells per assay.

[0150] Treat with CD200 Fc protein (50 μL), starting at 10 μg / mL, diluted 3-fold to 10-fold concentration with FACS buffer, and incubated at 37°C for 1 hour, 4 hours, and 24 hours. At the end of each time point, the cells were collected and washed. 10 μg / mL of anti-human secondary antibody was added and incubated at 4°C for 30 minutes. After incubation, the cells were washed and stained for 20 minutes at 4°C to check the viability (1 μL dye per million cells per mL in 1× PBS). The cells were washed and fixed with fixation buffer (100 μL per test) for 20 minutes at 4°C. After incubation, the cells were washed and the pellet was resuspended in FACS buffer (100 μL per test) to acquire data on the flow cytometer.

[0151] At the 24 hour time point, CD200 Fc protein treatment was performed with cell culture medium. Wash step = add 200 μL FACS buffer, centrifuge at 1400 RPM.

[0152] Table 5: Reagents used in cell binding assays

[0153]

[0154] result

[0155] Figure 3 The data presented show that ARQ-234 is able to inhibit LPS-stimulated IL-6 secretion in a concentration-dependent manner. No significant differences in inhibition were observed in the presence of Fc blockers in vitro ( Figure 3 , top). Although this is surprising because the IgG4 Fc domain of ARQ-234 binds to Fcγ receptors, which should increase the affinity of the interaction with CD200R, it is still possible that this mechanism increases the potency of CD200Fc proteins in vivo.

[0156] Figure 4 Binding of wild-type DS-155 and mutant ARQ-234 CD200 Fc proteins to U937 cells expressing CD200R is shown. These data demonstrate that ARQ-234 binds better to cells expressing CD200R than DS-155 (wild-type CD200 Fc protein) at all time points.

[0157] Example 4: PK study of ARQ-234 in cynomolgus monkey serum

[0158] plan

[0159] Two cynomolgus monkeys (one male and one female) in each group were injected intravenously with protein at a dose of 5 mg / kg at time 0. Blood samples were collected for PK analysis at the following time points: pre-dose, 0.25 hours, 0.5 hours, 1 hour, 4 hours, 8 hours, 24 hours, day 3, day 5, day 7, day 10, day 12, day 14, day 21, day 28.

[0160] Serum samples for PK analysis

[0161] At the sampling time points, at least 0.8 mL of blood samples were collected from the cephalic vein or saphenous vein of both animals. For samples collected within the first hour after dosing, a deviation of ±1 minute in the sample collection time was acceptable. For the remaining time points, samples collected within 5% of the planned time were acceptable. All blood samples were collected into commercially available tubes containing a coagulant. Tubes containing blood samples were kept at room temperature for 30 minutes before centrifugation. Within one hour of collection, samples were centrifuged at 1500 × g for 10 minutes at 4°C. After centrifugation, approximately 400 μL of serum was collected for each time point. The samples were then quickly frozen on dry ice and kept at -60°C or lower until transferred to dry ice for analysis. All samples were uniquely identified to indicate the source and collection time.

[0162] Determination of protein concentration in serum

[0163] The concentration of analytes in serum was determined using a bioanalytical ELISA method. 96-well ELISA plates were coated with 1 μg / ml goat anti-human IgG in carbonate-bicarbonate buffer overnight at 4°C. After washing and blocking, serially diluted plasma samples were added, and biotinylated goat anti-human IgG (0.0625 ug / mL) was used as the detection antibody. HRP-streptavidin and TMB substrates were used for color development. After approximately 5-10 minutes, the reaction was stopped by adding 2M HCl. The plate was analyzed using a microplate spectrophotometer ( M5e) Read absorbance at 450nm and 540nm. Substitute the OD value of the sample into the standard curve to obtain the plasma concentration. The detection limit LLOQ of this ELISA method for Fc+Fc is 1ng / mL.

[0164] Noncompartmental pharmacokinetic analysis of ARQ-234 serum concentrations in monkeys was performed using PhoenixWinNonlin software (version 8.1, Pharsight, Mountain View, CA). The linear / logarithmic trapezoidal rule was applied in deriving PK parameters.

[0165] result

[0166] Table 6 and Figure 5The data presented show that ARQ-234 has good serum stability, with a mean half-life of 370.5 hours in both tested species.

[0167] Table 6: PK parameters

[0168]

[0169]

[0170] ^When AUC_%Extrap_obs is greater than 20% or Rsq_adjusted is less than 0.9, the half-life is inaccurate.

[0171] The following parameters were tested in Table 6: 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 (Cl_obs); mean residence time extrapolated to infinity (MRTINF); distribution volume (Vss_obs). max Half-lives were calculated for 1% of the data.

[0172] Example 5: In vitro proof of concept of high affinity mouse CD200 Fc

[0173] 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 to design a high-affinity surrogate CD200-Fc protein for proof-of-concept experiments in a mouse autoimmunity model.

[0174] plan

[0175] Mouse CD200 constructs used the Uniprot sequence O54901 containing the signal peptide and the extracellular domain. Mutation numbers refer to the complete Uniprot sequence including the signal peptide.

[0176] Protein was produced by transient transfection of pcDNA 3.1-based expression plasmids into CHO-3E7 cells using polyethyleneimine (PEI). 24 hours after transfection, the culture was moved to 32°C and fed with 10% feed C, glutamine, glucose and 0.5M sodium butyrate; the supernatant was harvested and filtered on the 7th day. Purification was performed using a MabSelect SuRe 5ml affinity column (Cytiva) at 4°C, pre-equilibrated with 50mM sodium phosphate, 150mM NaCl pH7.4 at a flow rate of 3mL / min on the AKTAPure platform. The column was washed with equilibration buffer and the bound protein was eluted with 20mM sodium acetate, 150mM NaCl pH 3.5. The eluate was neutralized with 10% v / v 1M Tris pH 8.0 and analyzed by SDS-PAGE. The combined fractions were concentrated to 5 mL and subjected to gel filtration chromatography analysis (Hiload 16 / 600Superdex-200pg column) on the AKTAPure platform. The protein was treated at a speed of 1.2 mL / min in a 50 mM sodium phosphate, 150 mM NaCl pH 7.4 buffer system. The protein-containing components were combined and concentrated to 1.33 mg / mL (measured at UV 280 nm) using an Amicon Ultra Centricon (molecular weight cutoff of 10 kDa). The purified material was tested by SEC-HPLC, LC-MS, and EndoSafe LAL kits to assess protein purity, molecular weight, and endotoxin content, respectively. Mouse CD200-his protein was purified using Ni-NTA agarose resin using standard methods. All proteins were stored at -80 °C.

[0177] result

[0178] As shown in Table 7, the binding affinity of the combined variant H82Y, T125I was 43 nM, which was approximately 14 times higher than that of the wild-type construct containing the mouse IgG2a-Fc domain.

[0179] Table 7: Mouse CD200 variants

[0180] mutation <![CDATA[K D ]]> <![CDATA[T 1 / 2 (s)]]> wt 584.0±56.6 17.3 H82Y,T125I 43.3±4.9 117.6

[0181] Example 6: In vitro proof of concept of high affinity human CD200 Fc

[0182] The identified human CD200-Fc (huCD200Fc) protein was tested for its ability to inhibit cytokine release from LPS-activated pro-monocyte, human myeloid leukemia cells (U937) engineered to express high levels of human CD200R.

[0183] plan

[0184] Cell line construction, cytokine inhibition assays, and cell binding assays were performed using similar techniques as in Example 3 above.

[0185] To test the ability of huCD200 Fc protein to inhibit ERK-phosphorylation, U937-CD200R cells were induced with PMA for 20 min, and the inhibitory effects of DS-155, DS-192, and DS-118 on ERK-phosphorylation were measured by flow cytometry in cells permeabilized with anti-pERK antibody.

[0186] result

[0187] like Figure 6 As shown, 13 nM DS-192 has a higher affinity and a stronger inhibitory effect on IL-6 release than wild-type DS-155. Figure 7 As shown, an inhibitory effect of TNF-α was also observed. Figure 8 As shown, inhibition of ERK phosphorylation correlates with CD200 affinity.

[0188] like Figures 15A-15F As shown, antibodies that recognize Fcγ receptors do not inhibit the activity of DS-192 in vitro, suggesting that the Fc domain does not play an important role in the inhibitory activity in this particular assay.

[0189] Example 7: In vivo proof of concept of high affinity mouse CD200 Fc

[0190] Using a mouse model, we demonstrated that, when administered prophylactically, a high-affinity mouse CD200Fc protein reduced clinical scores in a mouse collagen-induced arthritis (CIA) model.

[0191] Mice have four potential CD200 receptors, CD200R1-CD200R4, at least one of which may be activated; CD200R1 is the homologue of human CD200R. Knocking out CD200 or CD200R1 in transgenic mice exacerbates or induces early onset in many autoimmune disease models, such as alopecia, arthritis, IBD25 and uveoretinitis.

[0192] In vitro CD200R agonism of patient samples in rodent models is known in the art and has been achieved previously with CD200-Fc fusion proteins, suggesting that human CD200Fc fusion proteins could be used as therapeutic approaches for inflammatory diseases. Like other cell surface immune receptors, CD200 has a low affinity for CD200R (in the high nanomolar range), so an ideal human therapeutic would require enhanced affinity for optimal efficacy. The Fc domain confers an antibody-like serum half-life, and the dimeric form increases binding affinity and cross-links the receptor. Animal model data suggest that the sequence of the Fc domain is related to the mouse IgG2a-Fc fusion with the best efficacy, perhaps by binding to Fcγ receptors to promote the formation of cell-cell interactions, thereby further increasing affinity. Antibody-dependent cellular cytotoxicity may also work by removing cells expressing CD200R1. Therefore, an in vivo mouse model was used to test the potency of the mouse version of the high-affinity CD200 Fc protein versus the wild-type CD200 Fc protein.

[0193] plan

[0194] Wild-type (DS-198) and high-affinity (DS-227) mouse CD200-Fc proteins were tested using the CIA model by starting dosing before 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 according to body weight and injected with 3 mg / kg of mouse IgG2a isotype control antibody DS-198 (wild-type muCD200 Fc) or DS-227 (high-affinity 43nM muCD200 Fc) every 3 days until day 36; the positive control group received 0.5 mg / kg dexamethasone orally daily. Clinical scores for paw arthritis were measured every other day from days 25-36 (blind assessment). Fig. 9 Data in are shown as mean ± SEM. **p<0.01, ***p<0.001 compared with disease + dexamethasone, disease + DS-198, and disease + DS-227. Two-way RM ANOVA followed by Tukey's multiple comparison test.

[0195] result

[0196] like Fig. 9 As shown, at the selected dose of 3 mg / kg, the higher affinity CD200 Fc DS-227 was significantly superior to the wild type (DS-198) in reducing clinical scores.

[0197] Example 8: Proof-of-Concept Study Using High-Affinity DS-192

[0198] Based on the in vivo proof-of-concept results from the high-affinity mouse CD200-Fc study described above (CIA mouse study), an in vivo proof-of-concept study was performed to test the high-affinity human CD200-Fc fusion protein DS-192. A humanized model of oxazolone-induced contact hypersensitivity was designed using NOG-EXL mice, which can be engrafted with human lymphocytes and myeloid cells.

[0199] plan

[0200] Female NOG-EXL mice were implanted with human cells and randomized based on the percentage of CD45+ cells at 20-21 weeks (day -1). Mice were sensitized by abdominal application of oxazolone (100 μL of 3% w / v oxazolone in acetone: alcohol 1:4) on day 0 and challenged by topical application of 20 μL of 2% w / v oxazolone (acetone: alcohol 1:4) to each ear on days 5, 10, and 14 (10 μL / side). Pre-sensitized huNOG EXL mice received repeated oxazolone challenges in one ear, with DS-192 (huCD200 Fc, 13 nM) or CD200R agonist antibody (CD200RmAb) given on the same day of each challenge. Isotype control antibody, CD200R agonist antibody, and high affinity huCD200-Fc (DS-192) were injected intravenously at a dose of 3 mg / kg 4 hours before oxazolone challenge on days 5, 10, and 14. Ear thickness was measured before and 24 hours after each challenge, and punch biopsies were performed on day 15 for cytokine analysis by multiplex analysis.

[0201] result

[0202] like Fig.10 As shown, DS-192 significantly reduced changes in ear thickness (a surrogate marker of inflammatory response) on the second and third days after challenge compared to the isotype control group, whereas the CD200R monoclonal antibody did not result in a significant reduction. Fig.11 , 12 As shown in Figures 1 and 13, at the end of the study in DS-192-treated mice, IL-1β, GM-CSF, and IL-13 were significantly reduced in ear tissue. Therefore, the results suggest that high-affinity CD200Fc has superior efficacy compared to CD200R monoclonal antibodies in a humanized mouse model of contact hypersensitivity. Since DS-192 has significantly lower CD200R affinity than ARQ-234, it is inferred that these advantages suggest that ARQ-234 may have greater efficacy when used to treat allergic and inflammatory skin diseases.

[0203] Example 9: Cellular Assay Binding Analysis of High Affinity CD200 Fc Molecules

[0204] The Fc fusion protein ARQ-234 of the present invention is shown in FIG. Fig.14 shown.

[0205] Binding of ARQ-234 to U937-CD200R cells was compared to wild-type control CD200-Fc (DS-155) at human physiological temperature (37°C) for 1, 4, and 24 hours, and binding was detected using flow cytometry and anti-human IgG antibodies. Figure 4 As shown, ARQ-234 binding to U937-CD200R cells showed more potent target binding compared to the wild-type huCD200-Fc construct DS-155.

Claims

1. A fusion protein comprising: (i) a mutated CD200 portion comprising mutations at amino acid residue positions 130 and 131, wherein the mutations are K130Y and I131Y; and (ii) a non-CD200 portion, wherein the non-CD200 portion is an IgG4 Fc fragment and comprises the S228P, M428L and N434S mutations according to the EU numbering system and a deletion of the first 5 amino acids of the hinge, Therein, glycine 232 of the mutated CD200 portion was directly fused to the non-CD200 IgG4-Fc fragment at amino acid 6 according to the IMGT numbering system. 2 . The fusion protein according to claim 1 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:

1.

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

1.

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

3.

7. The fusion protein according to claim 4, wherein the fusion protein comprises 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 a 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, wherein the fusion protein inhibits cytokine secretion.

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

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

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

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

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

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

20. The method of claim 19, wherein the subject has an autoimmune disease affecting the neuromuscular system, vascular system, eyes, skin, digestive tract, lungs, kidneys, liver, peripheral or central nervous system, bones, cartilage, or joints.

21. The method of claim 20, wherein the subject suffers from acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, aglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), asthma, atopic dermatitis, autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic dysregulation, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinal lesions, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and neuronal neuropathy, Barlow disease, Behcet's disease, bullous pemphigoid and related autoimmune blistering diseases, cardiomyopathy, Castleman disease, celiac disease (such as refractory celiac disease type II), Chagas disease, chronic idiopathic urticaria, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multiple osteomyelitis (CRMO), chronic spontaneous urticaria, Churg-Strauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogans syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devic disease (neuromyelitis optica), diabetic neuropathy, discoid 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 Wegener's granulomatosis), graft-versus-host disease (GvHD), Graves' disease, Guillain-Barre syndrome Syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes during pregnancy, hypogammaglobulinemia, hidradenitis suppurativa (HS), idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunomodulatory lipoprotein-related lesions, inclusion body myositis, inflammatory bowel disease (IBD), inflammatory skin diseases, interstitial cystitis, juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, wood-like conjunctivitis, linear IgA disease (LAD), systemic lupus erythematosus (SLE), Lyme disease,chronic, macrophage activation syndrome (MAS), mastocytosis, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's corneal ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis, pallial rheumatic disease, PANDAS (Streptococcal autoimmune neuropsychiatric disorders in children), palmoplantar pustulosis (PPP), paraneoplastic cerebellar degeneration (PNH), paroxysmal nocturnal hemoglobinuria, Parry-Lomborg syndrome, Parsonnage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, autoimmune polyglandular syndromes types I, II, and III, polymyalgia rheumatica , polymyositis, post-myocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's syndrome, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's disease syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-man syndrome, subacute bacterial endocarditis (SBE), Susac's 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, vesicular dermatosis, or vitiligo.

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

23. The method according to any one of claims 18-21, wherein the fusion protein is administered in combination with one or more other drugs for treating autoimmune diseases, allergic diseases, neurodegenerative diseases, neuropathic pain, inflammatory diseases, Th2-induced airway inflammation or diabetic neuropathy.

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

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

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

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

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