CD200 fusion protein

Abstract

The present invention relates to a fusion protein having an improved pharmacokinetic profile, comprising a mutant CD200 moiety containing K130 and I131 mutations that directly fuse to a non-CD200 moiety and bind to the human CD200 receptor with higher affinity than wild-type CD200. The present invention also relates to a polynucleotide encoding the fusion protein, a pharmaceutical composition containing the same, and the use thereof.

Description

[Technical Field]

[0001] The present invention relates to a fusion protein comprising a mutant CD200 moiety containing K130Y and I131Y mutations that fuse to a non-CD200 moiety and bind to the human CD200 receptor with higher affinity than wild-type CD200, in order to improve the pharmacokinetic profile of the fusion protein. The present invention also relates to polynucleotides encoding the fusion protein, pharmaceutical compositions containing the same, and the use thereof.

[0002] Cross-reference of related applications This application claims the benefit and priority of UK application GB2306711.9, filed on 5 May 2023, which disclosure is incorporated herein by reference in its entirety.

[0003] Explanation of the sequence list This application includes an electronic sequence listing filed via EFS-Web. The said sequence listing, created on April 29, 2024, is named “4549-144PCT-ST26.xml” and has a size of 12,593 bytes. The electronic information of the sequence listing is part of this application and is incorporated herein by reference in its entirety. [Background technology]

[0004] Inflammatory diseases, including autoimmune and allergic diseases, are the second leading cause of chronic illness globally and a major cause of prevalence among women in the United States. A 2008 international survey found that chronic disease patients in the U.S. are more likely to receive inadequate care due to cost concerns compared to patients in other countries (Schoen, C. et al., (2008) Health Affairs Web Exclusive, w1-w16). In addition, these patients tend to have the highest rates of medical errors, experience problems with care coordination, and have higher out-of-pocket medical expenses.

[0005] The American Association for Autoimmune Diseases (AARDA) currently estimates that 50 million Americans have an autoimmune disease. Epidemiological data is lacking to determine the total 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 exceeded $100 billion. While $100 billion is a staggering figure, the true cost of autoimmune diseases is likely a significant underestimate, given that epidemiological studies estimate the annual costs for just 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—to be between $51.8 billion and $70.6 billion annually. Furthermore, these estimates overlook the costs of immunosuppressive therapy during transplantation.

[0006] Autoimmune diseases are chronic conditions for which there is no cure, and they develop when the immune system mistakes healthy cells for foreign substances and attacks them. Depending on the type, autoimmune diseases can affect one or more different types of body tissues, potentially causing abnormal organ growth and altered organ function. The normal regulation of the immune system relies heavily on receptor / ligand pairs, which contain proteins expressed by cells involved in the immune response. However, these receptor / ligand pairs are often involved in signaling cascades that drive the pathogenesis of autoimmune diseases.

[0007] OX-2 membrane glycoprotein, also known as CD200 (differentiation antigen group 200), is a human protein encoded by the CD200 gene that is expressed in various cell types (Barclay, AN (1981) Immunology 44, 727) and has a high degree of homology to molecules of the immunoglobulin gene family. The protein encoded by this gene is a type 1 membrane glycoprotein containing two immunoglobulin domains and binding to the CD200 receptor (CD200R).

[0008] CD200R is expressed in myeloid cells (monocytes, macrophages, dendritic cells, and eosinophils), B cells, ILC2s (type 2 innate lymphoid cells), and T cells (Wright, et al., (2000), Immunity 12, 233-242; Wright, et al., (2003), J. Immunol, 171, 3034-3046).

[0009] The binding of CD200 to CD200R delivers suppressive signals to myelocytes 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 used in 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), and influenza-induced pneumonia (Snelgrove, et al., (2008) Nat. Immunol., 9, It has been shown to alleviate the symptoms in 1074-1083) and HSV-induced inflammatory lesions (Sarangi, et al., (2009) Clin. Immunol. 131, 31-40).

[0011] In addition, CD200 was challenged against the influenza virus. - / - Mice developed more severe disease compared to wild-type controls, associated with increased pulmonary infiltration and pulmonary endothelial damage (Rygiel. TP, et al. (2009) J. Immunol. 183(3), 1990-1996). CD200 - / - The mice induced an immune response that could control the viral load, suggesting that severe disease was caused by a dysregulation of the immune response rather than a beneficial antiviral immune response. As a result, despite a dramatic increase in viral load, disease could be prevented by depleting T cells before viral challenge. Rygiel.TP et al. (2009) concluded that T cells are essential for the manifestation of disease symptoms during influenza infection, and that a lack of downregulation of CD200-CD200R signaling, rather than viral load, exacerbates 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 (alopecia) (Harries, et al., (2013) J. It has been shown to be downcontrolled in diverse patient populations, including patients with Pathol. 231(2), 236-247).

[0013] Agonist CD200 proteins are disclosed, for example, in WO2000 / 061171 and WO2008 / 089022. These documents describe the use of wild-type CD200 molecules to modulate the function of immune cells. The present invention relates to a mutant CD200 protein that binds to the CD200 receptor with higher affinity than wild-type CD200.

[0014] Therefore, therapeutic interventions using molecules that modulate the CD200 pathway provide a means to control excessive or unwanted immune responses and alleviate disease symptoms in patients with chronic or intermittent (recurrent) autoimmune diseases.

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

[0016] A mutant CD200 fusion protein with an improved pharmacokinetic profile is needed. [Overview of the project]

[0017] In one aspect of the present disclosure, a fusion protein is provided comprising (i) a mutant CD200 moiety comprising mutations at amino acid residues 130 and 131, and (ii) a non-CD200 moiety, wherein the mutations are K130Y, I131Y, or a combination thereof, the non-CD200 is a human Fc fragment, and the dimer construct has a) a serum half-life of 5 to 900 hours in serum, b) an AUC of 0.5 to 50,000 μg·day / ml in serum at a dose of 5 mg / kg, or c) both (a) and (b).

[0018] In some embodiments, the present disclosure includes the following dimer constructs. (A) A first fusion polypeptide, (i) A mutant CD200 moiety containing mutations in amino acid residues 130 and 131, (ii) non-CD200 portion; and The mutation is a mutation of K130Y, I131Y, or a combination thereof, The non-CD200 is a human Fc polypeptide, a first fusion polypeptide, (B) a second fusion polypeptide, (i) a mutant CD200 portion containing mutations at amino acid residues 130 and 131, (ii) a non-CD200 portion, comprising, the mutation is a mutation of K130Y, I131Y, or a combination thereof, the non-CD200 is a human Fc polypeptide, a second fusion polypeptide, a dimer construct comprising, the first fusion polypeptide and the second fusion polypeptide dimerize via the human Fc polypeptide, and the dimer construct a) has a serum half-life of 5 to 900 hours in serum, b) has an AUC of 0.5 to 50,000 μg·day / mL in serum at a dose of 5 mg / kg, or c) is (a) and (b), comprising a dimer construct.

[0019] In certain embodiments, the disclosure includes a dimer construct comprising two fusion polypeptides, each polypeptide comprising (i) a mutant CD200 portion containing mutations at amino acid residues 130 and 131, and (ii) a non-CD200 portion, wherein the mutation is a mutation of K130Y, I131Y, or a combination thereof, and the non-CD200 portion is a human Fc polypeptide, the human Fc polypeptides of the two fusion polypeptides are bound to each other via at least one disulfide bond to form one homodimer, the dimer construct a) has a serum half-life of 5 to 900 hours in serum, b) At a dose of 5 mg / kg, the serum AUC is 0.5 to 50,000 μg·day / ml, or c) (a) and (b), Includes dimeric constructs.

[0020] In some embodiments, the non-CD200 portion does not contain mutations. For example, in some embodiments, the non-CD200 portion contains the following mutations: (1) M252Y and S254T and T256E, (2) T256D and T307R and Q311V, (3) T256D and N315D and A378V, (4) T256D and N286D and T307R and Q311V, (5) H285N and T307R and Q311V and A378V, (6) H285D and Q311V and A378V, (7) T256D and H285D and A378V, (8) T (9) T256D and Q311V and A378V, (10) T256D and H285D and N286D and T307R and A378V, (11) M252Y and T256D, (12) T256D and T307Q, (13) T256D and T307W, (14) T307A and E380A and N434A, (15) T250Q and M428L, (16) S228P, M428L and / or N434S, (17) V308P, or (18) M428L and N434S are not included.

[0021] According to another aspect of the present invention, the non-CD200 portion is one of the following mutations: (1) M252Y and S254T and T256E, (2) T256D and T307R and Q311V, (3) T256D and N315D and A378V, (4) T256D and N286D and T307R and Q311V, (5) H285N and T307R and Q311V and A378V, (6) H285D and Q311V and A378V, (7) T256D and H285D and A378V, (8) T256D and Q311V and A378V, (9) T256D and H285D (10) M252Y and T256D, (11) T256D and T307Q, (12) T256D and T307W, (13) T307A and E380A and N434A, (14) T250Q and M428L, (15) S228P, M428L and / or N434S, (16) V308P, (17) S228P, or (18) M428L and N434S, which are Fc fragments of mutant IgG1, IgG2, IgG3, IgG4, or IgA. In a preferred embodiment, the non-CD200 portion is an Fc fragment of mutant IgG1 or IgG4.

[0022] According to another aspect of the present invention, the fusion protein has a desired glycosylation profile compared to the wild-type CD200-Fc fusion protein. The desired glycosylation profile is produced by one or more of the following: mutating one or more glycosylation sites, for example, modifying the glycan profile by selecting a particular clone, or manipulating expression in a bioreactor by changing the type of culture medium and growth conditions. According to another aspect of the present invention, the fusion protein has an increased sialic acid content compared to the wild-type CD200-Fc fusion protein. According to another aspect of the present invention, the fusion protein has a decreased mannose content compared to the wild-type CD200-Fc fusion protein.

[0023] A further aspect of the present invention provides a polynucleotide encoding a fusion protein as defined herein.

[0024] In a further embodiment, a pharmaceutical composition comprising a fusion protein as defined herein is provided.

[0025] In another aspect of the present invention, a fusion protein, polynucleotide, or pharmaceutical composition as defined herein is provided for use in the preparation of pharmaceuticals. In another aspect of the present invention, a fusion protein, polynucleotide, or pharmaceutical composition as defined herein is provided for use in therapeutics. In another aspect of the present invention, a fusion protein, polynucleotide, or pharmaceutical composition as defined herein is provided for use in the treatment of autoimmune diseases, allergic diseases (e.g., rheumatoid arthritis, asthma, or atopic dermatitis), neurodegeneration, neuropathic pain, inflammatory arthralgia, or diabetic neuropathy. In another aspect of the present invention, a fusion protein, polynucleotide, or pharmaceutical composition as defined herein is provided for use in the treatment of rheumatoid arthritis, asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), or Parkinson's disease. In another aspect of the present invention, a fusion protein, polynucleotide, or pharmaceutical composition as defined herein is provided for use in the treatment of autoimmune diseases affecting the neuromuscular system, vascular system, eyes, skin, gastrointestinal tract, lungs, kidneys, liver, peripheral or central nervous system, bones, cartilage, or joints.

[0026] Other features and characteristics of the subject matter of this disclosure, as well as the methods of operation, the functions and combinations of parts of the elements of the related structures, and the economics of manufacture, will become more apparent by examining the following description and the accompanying drawings, sequence listing, and claims, all of which constitute part of this specification. [Brief explanation of the drawing]

[0027] [Figure 1A-1B] Sensorgrams of the BIAcore assay showing the association and dissociation phases of human CD200R binding to captured mutant CD200-Fc fusion protein (DS-118, Figure 1A) or wild-type CD200-Fc (DS-155, Figure 1B). [Figure 2A-2B]Sensorgrams of the BIAcore assay showing the association and dissociation phases of cynomolgus monkey CD200R1 binding to captured mutant CD200-Fc fusion protein (DS-118, Figure 2A) or wild-type CD200-Fc (DS-155, Figure 2B). [Figure 3A-3D] (Figure 3A) Graph showing inhibition of LPS-induced IL-6 release from U937-CD200R cells after treatment with mutant CD200-Fc fusion protein (DS-118, left panel) or wild-type CD200-Fc (DS-155, right panel). (Figure 3B) Bar graph showing inhibition of LPS-induced IL-8 release from U937-CD200R cells after treatment with mutant CD200-Fc fusion protein (DS-118). (Figure 3C) Bar graph showing inhibition of LPS-induced TNFα release from U937-CD200R cells after treatment with mutant CD200-Fc fusion protein (DS-118). (Figure 3D) Bar graph showing LPS-stimulated inhibition of phosphorylated ERK in U937-CD200R cells after treatment with mutant CD200-Fc fusion protein (DS-118, left panel) or wild-type CD200-Fc fusion protein (DS-155, right panel). The inhibition rate (%) is relative to LPS-stimulated cytokine release from U937-CD200R cells not treated with mutant fusion protein / CD200, or to a setting where the phosphorylated ERK level in U937-CD200R cells is 0%. [Figure 4] Binding of mutant CD200-Fc(DS-118) to U937-CD200R cells visualized using a fluorescent anti-human secondary antibody at 1 hour and 4 hours. [Figure 5] Graphs showing IL-6 release in iPSC-derived macrophage cells after 1 hour of treatment with DS-118 and 18 hours of stimulation with LPS+INFγ. The data shown represent the mean ± SEM from three independent replicates. Two-way ANOVA was performed to measure significance compared to the untreated control. *p<0.05, **p<0.01. [Figure 6] This shows the ribbon model protein structure of DS-118, which exhibits mutations in the CD200 domain and Fc region. [Figure 7] Graph showing IL-6 inhibition in response to dose titration of DS-155 (wild-type CD200-Fc), DS-192 (13nM CD200-Fc), and DS-118 (1nM CD200-Fc). [Figure 8] Graphs showing IL-8 inhibition. The upper panel shows IL-8 inhibition by DS-118. The lower panel shows IL-8 inhibition by DS-155. Error bars represent the standard deviation between biological replicates. [Figure 9] A graph showing the inhibition of TNFα by DS-192. Error bars represent the standard deviation between biological replicates. [Figure 10] Graphs showing inhibition of ERK phosphorylation by DS-155, DS-192, and DS-118, measured by flow cytometry in permeabilized cells in the presence of huCD200-Fc fusions using anti-pERK antibodies. [Figure 11] Graph showing clinical scores for ankle arthritis in male DBA / 1J mice measured every other day from days 25 to 36. Data are expressed as mean ± SEM. **p<0.01, ***p<0.001 vs. disease+Dexa, disease+DS-198, and disease+DS-227. Tukey's multiple comparison test was performed following two-way RM ANOVA. [Figure 12] A graph showing the change in ear thickness from day 0 in a humanized mouse model of contact hypersensitivity. The values ​​shown are the combined values ​​for the right and left ears. [Figure 13] Graph showing mean IL-1β cytokine levels in tissue homogenates from four groups (n=8 per treatment group, n=5 in the control group) derived from a humanized mouse model of contact hypersensitivity on day 15. Data are expressed as mean ± SEM. Student's t-tests were performed with †p<0.05, ††p<0.01 vs. isotype control, and *p<0.05, **p<0.01 vs. negative control. [Figure 14]Graph showing mean GM-CSF cytokine levels in tissue homogenates from four groups (n=8 per treatment group, n=5 in the control group) derived from a humanized mouse model of contact hypersensitivity on day 15. Data are expressed as mean ± SEM. Student's t-tests were performed with †p<0.05, ††p<0.01 vs isotype control, and *p<0.05, **p<0.01 vs negative control. [Figure 15] Graph showing mean IL-13 cytokine levels in tissue homogenates from four groups (n=8 per treatment group, n=5 in the control group) derived from 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 16] A schematic diagram showing the timeline of the high-affinity huCD200-Fc study conducted using an NHP pneumonia model. Cynomolgus monkeys were screened for pre-existing susceptibility to the porcine roundworm (Ascaris suum) antigen. On day 0, they were administered 20 mg / kg intravenously with high-affinity huCD200-Fc (DS-118) (n=6), a vehicle control (n=6), and 1 mg / kg intravenously with dexamethasone (n=4). All animals were challenged intrabronchially with 5000 μg / ml of porcine roundworm (Ascaris suum) antigen on day +1. [Figure 17] A graph showing lymphocyte levels in bronchoalveolar lavage fluid (BAL fluid) measured by flow cytometry on day 2 (24 hours after the challenge and 48 hours after drug treatment). [Figure 18] This graph shows the change in airway resistance immediately after the porcine roundworm (A suum) antigen challenge, compared to immediately before the challenge. Pre-administration measurements were taken on day -1 (relative to huCD200-Fc administration), and post-administration measurements were taken on day +1. The same legend as in Figure 17 is applied to the bars in Figure 18 (from left to right: vehicle, human CD200-Fc 20 mg / kg, dexamethasone 1 mg / kg). [Figures 19A-19F](Figure 19A) Graph showing the 100 μg / mL antibody that recognizes CD64(FcγRI)Fc gamma receptor activity when co-incubated with various doses of DS-192(ha CD200-IgG4 Fc) in the U937 cell assay. (Figure 19B) Graph showing the 0 μg / mL antibody that recognizes CD64(FcγRI)Fc gamma receptor activity when co-incubated with various doses of DS-192(ha CD200-IgG4 Fc) in the U937 cell assay. (Figure 19C) Graph showing the 100 μg / mL antibody that recognizes CD16(FcγRIII)Fc gamma receptor activity when co-incubated with various doses of DS-192(ha CD200-IgG4 Fc) in the U937 cell assay. (Figure 19D) Graph showing 0 μg / mL antibodies that recognize CD16(FcγRII)Fc gamma receptor activity when co-incubated with various doses of DS-192(ha CD200-IgG4 Fc) in a U937 cell assay. (Figure 19E) Graph showing 100 μg / mL antibodies that recognize CD32(FcγRII)Fc gamma receptor activity when co-incubated with various doses of DS-192(ha CD200-IgG4 Fc) in a U937 cell assay. (Figure 19F) Graph showing 0 μg / mL antibodies that recognize CD32(FcγRII)Fc gamma receptor activity when co-incubated with various doses of DS-192(ha CD200-IgG4 Fc) in a U937 cell assay. [Figure 20] A graph showing the binding of DS-118 and two different lots of DS-192 to human PBMCs. [Figure 21] Sensorgrams from the BIAcore assay showing the association and dissociation phases of human CD200R binding to captured mutant CD200-Fc fusion protein (ARQ-234). [Figures 22A-22B] Sensorgrams of the BIAcore assay showing the association and dissociation phases of cynomolgus monkey CD200R1 binding to captured mutant CD200-Fc fusion protein (ARQ-234, Figure 22A) or wild-type CD200-Fc (DS-155, Figure 22B). [Figure 23]Bar graphs showing inhibition of LPS-induced IL-6 release from U937-CD200R cells after treatment with the mutant fusion protein (ARQ-234) in either the presence (upper panel) or absence (lower panel) of the Fc block. The shown inhibition rates (%) are relative to 0% when LPS-induced IL-6 release from U937-CD200R cells not treated with the mutant or wild-type fusion protein is set. [Figure 24] Binding of mutant CD200-Fc (ARQ-234) or wild-type CD200-Fc (DS-155) to U937-CD200R cells visualized using fluorescent anti-human secondary at 1 hour, 4 hours, and 24 hours. [Figure 25] Panel A) Schematic diagram of the pharmacokinetic (PK) analysis protocol. Panel B) Graph showing the concentration of mutant CD200 fusion protein (ARQ-234) measured in the serum of cynomolgus monkeys at the indicated time after drug administration (time=0). [Figure 26] Bar graphs showing inhibition of ERK phosphorylation by DS-155 and DS-192, measured by flow cytometry in permeabilized cells in the presence of huCD200-Fc fusions using anti-pERK antibodies. [Figure 27] Schematic diagram of the ARQ-234 CD200-Fc fusion, which exhibits mutations for high-affinity CD200R and FcRn binding. [Figure 28] For clarity, the N-terminus of huCD200 has been omitted, and the ribbon model protein structure of the affinity-enhancing mutation of huCD200 is shown. [Figure 29] Schematic diagram of the mouse CIA model protocol. [Figure 30] Schematic diagram of a humanized mouse model of a contact hypersensitivity protocol. [Modes for carrying out the invention]

[0028] While the subject matter of this disclosure can be embodied in various forms, the following description is intended to disclose only some of these forms as specific examples of the subject matter encompassed in this disclosure.

[0029] Therefore, the subject matter of this disclosure is not intended to be limited to the forms or embodiments described herein.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. While methods and materials for use in the present invention are described herein, other suitable methods and materials known in the art may also be used. Materials, methods, and examples are for illustrative purposes only and are not intended to limit the scope. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including its definitions, shall prevail.

[0031] Other features and advantages of the present invention will become apparent from the following detailed description and drawings, as well as from the claims.

[0032] According to a first aspect of the present invention, (i) A mutant CD200 moiety containing mutations in amino acid residues 130 and 131, (ii) Non-CD200 portions selected from the non-CD200 portions described below, A fusion protein containing, The aforementioned mutations are K130Y and I131Y, The fusion protein provided has a pharmacokinetic profile or a modified profile as described herein.

[0033] According to one aspect of the present invention, (i) A mutant CD200 moiety containing mutations in amino acid residues 130 and 131, (ii) non-CD200 portion; A fusion protein containing, The aforementioned mutations are K130Y and I131Y, The non-CD200 portion is an Fc fragment of IgG4, comprising the S228P mutation and the deletion of the first 5 amino acids of the hinge according to the EU numbering system. The glycine 232 in the mutant CD200 portion is directly fused to the Fc fragment of the non-CD200 IgG4 at the 6th amino acid according to the IMGT numbering system. A fusion protein having the pharmacokinetic profile or modified profile described herein is provided.

[0034] According to a first aspect of the present invention, (i) A mutant CD200 moiety containing mutations in amino acid residues 130 and 131, (ii) non-CD200 portion; A fusion protein containing, The aforementioned mutations are K130Y and I131Y, The aforementioned non-CD200 portion is an Fc fragment of IgG4, and includes mutations S228P, M428L, and N434S according to the EU numbering system, and deletion of the first 5 amino acids of the hinge. The glycine 232 of the mutant CD200 portion is directly fused to the Fc fragment of non-CD200 IgG4 at the 6th amino acid according to the IMGT numbering system. The fusion protein provided herein has a pharmacokinetic profile or a modified profile as described herein.

[0035] According to another aspect of the present invention, (i) A mutant CD200 moiety containing mutations in amino acid residues 130 and 131, (ii) non-CD200 portion; A fusion protein comprising, The aforementioned non-CD200 portion is an Fc fragment of IgG4, and includes mutations M428L and N434S according to the EU numbering system, and deletion of the first 5 amino acids of the hinge. The glycine 232 of the mutant CD200 portion is directly fused to the Fc fragment of non-CD200 IgG4 at the 6th amino acid according to the IMGT numbering system. The fusion protein provided has a pharmacokinetic profile or a modified profile as described herein.

[0036] In some embodiments, the CD200 mutation may be alternatively K130Y, K130F, I131F, I131Y, or a combination thereof. The disclosures in PCT / GB2022 / 052764 are incorporated herein by reference in their entirety.

[0037] In some embodiments, the fusion protein includes a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, or IgG4 as a non-CD200 portion.

[0038] In another embodiment, the fusion protein may exhibit lower affinity for at least one receptor, such as FcγI, FcγIIA, or C1q, compared to a polypeptide containing the Fc region of wild-type human IgG.

[0039] In yet another embodiment, the fusion protein includes the Fc region of human IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM.

[0040] In yet another embodiment, the fusion protein includes the Fc region of human IgG1, IgG2, or IgG4.

[0041] In some embodiments, the fusion protein includes one or more Fc domains, for example, a pair of human Fc domains. In some embodiments, the human Fc domain is the Fc domain of human IgG1, the Fc domain of human IgG2, the Fc domain of human IgG3, or the Fc domain of human IgG4. In some embodiments, the human Fc domain is the Fc domain of human IgG4. In some embodiments, each human Fc domain contains a sequence that is at least 80% identical to human IgG1 (ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8)). In some embodiments, each human Fc domain contains a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 8. In some embodiments, the human Fc domain contains sequence number 8. In some embodiments, the human Fc domain contains a mutation (a “modified profile” having one or more of the mutations disclosed herein or a combination thereof).

[0042] In some embodiments, the human Fc domain includes mutations that eliminate glycosylation and / or mutations that reduce Fc-gamma receptor binding. In some embodiments, the human Fc domain includes mutations of N297Q, N297A, or N297G; in some embodiments, the human Fc domain includes mutations at positions 234 and / or 235, e.g., L235E, or L234A and L235A (in IgG1), or F234A and L235A (in IgG4); and in some embodiments, the human Fc domain is the Fc domain of IgG2, including mutations of V234A, G237A, P238S, H268Q / A, V309L, A330S, or P331S, or combinations thereof (all following Kabat, EU numbering). In some embodiments, each of the human Fc domains includes a mutation in the human IgG1 constant region L234A / L235A ("LALA") or a mutation in the human IgG1 constant region L234A / L235A / P329G ("LALAPG").

[0043] Further examples of manipulated human Fc domains are known to those skilled in the art. Examples of Ig heavy chain constant region amino acids in which a mutation in at least one amino acid results in reduced Fc function include, but are not limited to, mutations at amino acids 228, 233, 234, 235, 236, 237, 239, 252, 254, 256, 265, 270, 297, 318, 320, 322, 327, 329, 330 and 331 (according to EU numbering) in the heavy chain constant region. Examples of mutant amino acid combinations include, but are not limited to, combinations of mutations at amino acids 234, 235, and 331; combinations of mutations at amino acids 234, 235, and 329; combinations of mutations at L234F, L235E, and P331S; or combinations of mutations at amino acids 318, 320, and 322, such as E318A, K320A, and K322A, which are also known in the art.

[0044] Further examples of manipulated Fc domains include F243L / R292P / Y300L / V305I / P396 IgG1, S239D / I332E IgG1, S239D / I332E / A330L IgG1, S298A / E333A / K334A, L234Y / L235Q / G236W / S239M / H268D / D270E / S298A IgG1 in one heavy chain, and D270E / K326D, A330M / K334E IgG, G236A / S239D / I332E IgG1, K326W / E333S IgG1, S267E / H268F / S324T in the other heavy chain. Examples include IgG1, E345R / E430G / S440Y IgG1, N297A or N297Q or N297G IgG1, L235E IgG1, L234A / L235A IgG1, F234A / L235A IgG4, H268Q / V309L / A330S / P331S IgG2, V234A / G237A / P238S / H268A / V309L / A330S / P331S IgG2, M252Y / S254T / T256E IgG1 ("YTE"), M428L / N434S IgG1, S267E / L328F IgG1, N325S / L328F IgG1, etc. In some embodiments, the manipulated Fc domain includes one or more substitutions selected from the group consisting of N297A IgG1, N297Q IgG1, and S228P IgG4. Furthermore, Fc mutations include AAA (T307A / E380A / N434A), QL (T250Q / M428L), V308P, YD (M252Y / T256D), DQ (T256D / T307Q), and DW (T256D / T307W), L234A / L235A ('LALA'), L235E, P329G, L234A / L235A / P329G "LALAPG", E233P, L234V, L235A, and delta G 236, A327G, A330S, P331S, L234A, L235A, G237A, P238S, H268A, A330S, P331S, K322A, L234R, L235A, L234F, L235E, P331 S, L234F / L235Q / K322Q, L234A / L235A / K322A'LALAKA', L234F / L235E / P331S'FES', L234A / G237A, and G236R / L328R.Further mutations are listed below.

[0045] JPEG2026516066000002.jpg114160

[0046] In one embodiment, the fusion protein of the disclosure containing an Fc variant exhibits lower affinity for Fc receptors, such as FcγRI, FcγRIIA, and FcγRIIIA, compared to the unmodified antibody. In one embodiment, the polypeptide containing the Fc variant exhibits affinity for Fc receptors that is at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, at least 5%, or at least 1% lower than the affinity of the wild-type polypeptide.

[0047] In one embodiment, a polypeptide comprising the Fc variant of the present disclosure exhibits a reduction of Fcγ bonds by more than 700 times, or a reduction of Fcγ bonds by more than 3,500 times.

[0048] In some embodiments, the fusion protein fragment includes a variant Fc region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM. In certain embodiments, the fusion protein is an aglycosylate antibody with reduced effector function. In certain embodiments, the variant Fc region of IgG1 includes (a) an amino acid substitution from leucine to alanine at position 234, (b) an amino acid substitution from leucine to alanine at position 235, (c) an amino acid substitution from proline to glycine or arginine at position 329, (d) an amino acid substitution from asparagine to alanine at position 297, (e) an amino acid substitution from asparagine to glutamine at position 297, (f) an amino acid substitution from asparagine to glycine at position 297, or (g) any combination of (a) to (f). In certain embodiments, the variant Fc region of IgG2 includes (g) an amino acid substitution from Pro to glycine or arginine at position 329. In certain embodiments, the variant Fc region of IgG4 includes (h) an amino acid substitution from serine at position 228 to proline, (i) an amino acid substitution from leucine at position 235 to alanine or glutamic acid, (j) an amino acid substitution from proline at position 329 to glycine or arginine, or (k) any combination of (h) to (j).

[0049] This disclosure includes high-affinity CD200 proteins fused to the Fc domain, with a) direct fusion to the Fc hinge, + / - deletion in the hinge region, and b) a linker between the CD200 extracellular domain and Fc.

[0050] This disclosure includes high-affinity CD200 proteins having modifications to CD200 glycosylation to reduce high mannose and increase terminal sialylation, for example, by the use of sialylatesase, galactosesyltransferase, or both.

[0051] This disclosure also relates to expression systems and host cells comprising the expression system. An expression system comprises at least one expression vector, for example, comprising two or more, or three or more, expression vectors. Those skilled in the art will readily understand that one or more expression vectors can be incorporated into a suitable host cell using conventional methods, including, but not limited to, transformation, transfection, or viral infection. An expression system may include one or more nucleic acid sequences encoding the fusion protein of this disclosure.

[0052] The inventors have found that mutations in amino acid residues of the extracellular domain of CD200 result in a mutant CD200 moiety that has a high binding affinity to the CD200 receptor (CD200R). Furthermore, the fusion protein containing the mutant CD200 moiety described herein has significant advantages, particularly in that it provides higher clinical efficacy and treatment at lower doses.

[0053] Therefore, in certain embodiments, the fusion protein contains 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 even further embodiments, the fusion protein is DS-118.

[0054] Sequence ID 1 (also referred to as "DS-118" in this specification) is the following sequence: The image consists of JPEG2026516066000003.jpg37170, where shaded and bold amino acids represent the mutation site relative to wild-type CD200 or IgG4 Fc, and underlined sequences represent non-CD200 Fc fragments.

[0055] In one embodiment, DS-118 may further include an N-terminal signal sequence which is a signal peptide of a human IgG chain. In a further embodiment, the N-terminal signal sequence consists of the amino acid sequence MEFGLSWLFLVAILKGVQC (SEQ ID NO: 3).

[0056] As used herein, the term "CD200 protein" refers to the wild-type CD200 protein.

[0057] As used herein, the term “wild-type” refers to naturally occurring proteins, peptides, amino acids, and nucleotide sequences. For example, as used herein, the term “wild-type CD200 protein” refers to any full-length isoform or any portion thereof of CD200 (UNIPROT P41217 OX2G_HUMAN) that binds to the CD200 receptor (CD200R), including naturally occurring protein polymorphisms. The CD200 protein is also known as the OX-2 membrane glycoprotein.

[0058] Wild-type CD200 is a cell surface protein having an N-terminal extracellular domain, as well as a short transmembrane domain and a cytoplasmic domain. The extracellular domain binds to target receptors such as the CD200 receptor. In one embodiment, the CD200 protein is the extracellular domain of CD200, or any portion thereof, that binds to the CD200 receptor.

[0059] As used herein, the term "~th" refers to the residue number in an amino acid sequence, where the first is the first translated amino acid. Therefore, the numbering of amino acid positions within the CD200 portion as defined herein will be understood to refer to the amino acid sequence containing the N-terminal signal sequence that represents the first 30 amino acids of the CD200 portion (as shown in bold in Sequence ID No. 2).

[0060] As used herein, the terms “mutated” or “mutated” refer to proteins, peptides, amino acids, and nucleotide sequences that have become mutants through changes in their morphology from their wild-type counterparts. For example, a mutant protein or mutant protein may have undergone changes in its amino acid and / or nucleotide sequence compared to its corresponding wild-type sequence, and such changes may also be referred to as mutations.

[0061] In this specification, the terms “mutant CD200 protein” and “mutant CD200 moiety” refer to a full-length CD200 protein or any portion thereof that binds to the CD200 receptor, containing one or more mutant amino acid residues in its 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 present invention as defined herein, the mutant CD200 moiety contains the K130Y and I131Y mutations. Thus, in one embodiment, the mutation is a substitutional mutation.

[0062] In one embodiment, the fusion protein may be prepared synthetically or recombinantly. In a further embodiment, the fusion protein may be prepared synthetically. In an alternative embodiment, the fusion protein may be prepared recombinantly.

[0063] In one embodiment, the mutant CD200 moiety binds to the CD200 receptor with higher affinity than wild-type CD200.

[0064] In one embodiment, the mutant CD200 protein / molecule may comprise the entire extracellular domain of CD200 or a portion thereof. In a further embodiment, the mutant CD200 protein comprises a signal sequence. It will be understood that secreted proteins contain several amino acids at the N-terminus that constitute a signal sequence that can be cleaved before secretion. Therefore, in a particular embodiment, the mutant CD200 molecule comprises an N-terminal signal sequence. In one embodiment, the mutant CD200 molecule 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 molecule. In a further embodiment, the signal sequence is Sequence ID No. 3. Therefore, in a particular embodiment, the fusion protein comprises a sequence defined herein that lacks the amino acids comprising the signal sequence. For example, the mutant CD200 protein lacks amino acids 1-30 of the wild-type CD200 protein and contains a sequence corresponding to amino acids 31-232 of Sequence ID No. 2. Therefore, in further embodiments, the fusion protein includes the amino acid sequence of SEQ ID NO: 2. In further embodiments, the fusion protein consists of the amino acid sequence of SEQ ID NO: 2. In further embodiments, the fusion protein has the amino acid sequence of SEQ ID NO: 3 at the N-terminus of SEQ ID NO: 1.

[0065] Sequence ID 2 is the following sequence: The image consists of JPEG2026516066000004.jpg42170, where bold amino acids represent signal sequences, shaded and bold amino acids represent mutation sites relative to wild-type CD200 or IgG4 Fc, and underlined sequences represent non-CD200 Fc fragments. The disclosure also includes protein sequences that lack C-terminal lysine, such as proteins in which C-terminal lysine (K) is cleaved during secretion from mammalian cells.

[0066] In one embodiment, a fusion protein having the amino acid sequence of SEQ ID NO: 2 is encoded by the polynucleotide of SEQ ID NO: 4. It is important to note that there is degeneracy of the genetic code, that is, most amino acids are identified by more than one codon. Therefore, since many different codons define the same amino acid, more than one polynucleotide sequence can encode the same amino acid sequence. Thus, SEQ ID NO: 4 represents one exemplary permutation of a polynucleotide sequence that can encode a fusion protein having the amino acid sequence of SEQ ID NO: 2. Any permutation and combination of all elements described herein should be considered disclosed by the description herein unless the context indicates otherwise.

[0067] In further embodiments, the fusion protein consists of the amino acid sequence of SEQ ID NO: 5. In yet another embodiment, the fusion protein is ARQ-234.

[0068] Sequence ID 5 (also referred to herein as "ARQ-234") has the following sequence: The image consists of JPEG2026516066000005.jpg37170, where shaded and bold amino acids represent the mutation sites relative to wild-type CD200 or IgG4 Fc, and underlined sequences represent non-CD200 Fc fragments.

[0069] In one embodiment, ARQ-234 may further comprise an N-terminal signal sequence which 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.

[0070] In one embodiment, the mutant CD200 protein / molecule may include the entire extracellular domain of CD200 or a portion thereof. In a further embodiment, the mutant CD200 protein includes a signal sequence. It will be understood that secreted proteins contain several amino acids at the N-terminus that constitute a signal sequence that can be cleaved before secretion. Therefore, in a particular embodiment, the mutant CD200 molecule includes an N-terminal signal sequence. In one embodiment, the mutant CD200 molecule includes a signal sequence at the N-terminus that is cleaved before secretion from the producing cell. In a further embodiment, the signal sequence includes 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 molecule. In a further embodiment, the signal sequence is Sequence ID No. 3. Therefore, in a particular embodiment, the fusion protein includes a sequence defined herein that lacks the amino acids containing the signal sequence. For example, the mutant CD200 protein lacks amino acids 1-30 of the wild-type CD200 protein and includes a sequence corresponding to amino acids 31-232 of Sequence ID No. 6. Therefore, in further embodiments, the fusion protein includes the amino acid sequence of SEQ ID NO: 6. In further embodiments, the fusion protein consists of the amino acid sequence of SEQ ID NO: 6. In further embodiments, the fusion protein has the amino acid sequence of SEQ ID NO: 3 at the N-terminus of SEQ ID NO: 5.

[0071] Sequence ID 6 consists of the following sequence: JPEG2026516066000006.jpg42170 Here, bold amino acids represent signal sequences, shaded and bold amino acids represent the location of mutations relative to wild-type CD200 or IgG4 Fc, and underlined sequences represent non-CD200 Fc fragments. This disclosure also includes protein sequences that are disclosed but lack C-terminal lysine, such as proteins in which C-terminal lysine (K) is cleaved during secretion from mammalian cells.

[0072] In one embodiment, the fusion protein is encoded by the polynucleotide of SEQ ID NO: 7. It is important to note that there is degeneracy in the genetic code, that is, most amino acids are identified by more than one codon. Therefore, since many different codons define the same amino acid, more than one polynucleotide sequence can encode the same amino acid sequence. Thus, SEQ ID NO: 7 represents one exemplary permutation of a polynucleotide sequence that can encode a fusion protein. Any permutation and combination of all elements described herein should be considered disclosed by the description herein unless the context indicates otherwise.

[0073] When the term “part” is used herein in relation to the sequences of proteins, peptides, and amino acids and nucleotides, it refers to fragments and derivatives that are functional, i.e., that bind to their targets.

[0074] As used herein, the term “fragment” refers to a portion of a sequence of a protein, peptide, amino acid, or nucleotide that recognizes and binds to its target, such as a receptor.

[0075] As used herein, the terms “derivative of” and “mutant” refer to a sequence of a protein, peptide, amino acid, or nucleotide that has at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, or 99%) sequence similarity to the wild-type equivalent and functions similarly. Thus, a mutant may be a derivative of the wild-type equivalent.

[0076] 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.

[0077] As shown by the data presented herein, the mutant CD200 protein / molecule of the present invention binds more strongly to the CD200 receptor and has a longer residence time on the receptor than the wild-type CD200 protein.

[0078] ==Fusion protein== According to a first aspect of the present invention as defined herein, a fusion protein is provided which comprises a mutant CD200 protein / moon as defined herein, fused to a non-CD200 moiety.

[0079] As used herein, the term “fusion protein” means one or more amino acid sequences, peptides, and / or proteins linked together using methods well known in the art, for example, the methods described in U.S. Patents 5,434,131 and 5,637,481. The thus linked amino acid sequences, peptides, or proteins form a single fusion protein.

[0080] In some embodiments, the mutant CD200 protein / molecule 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 the N-terminus to the C-terminus is mutant CD200 moiety-non-CD200 Fc fragment. Thus, in further embodiments, the orientation of the fusion protein is mutant CD200 moiety-IgG4 Fc fragment. In another embodiment, the orientation of the fusion protein from the N-terminus to the C-terminus is signal sequence-mutant CD200 moiety-non-CD200 Fc fragment. Thus, in yet further embodiments, the orientation of the fusion protein is signal sequence-mutant CD200 moiety-IgG4 Fc fragment.

[0081] As used herein, the term “non-CD200 moiety” 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, a fusion protein in which the immunoglobulin (Ig) constant region or a portion thereof, or the non-CD200 moiety, is a synthetic molecule such as PEG.

[0082] In one embodiment, the non-CD200 portion is an antibody fragment. In a particular embodiment, the non-CD200 portion is an Fc fragment. Thus, the mutant CD200 fusion protein described herein may also be called mutant CD200-Fc. In further embodiments, the Fc fragment is derived from a mammal, such as human or monkey, and is, for example, human C(gamma)1 including the hinge, CH2, and CH3 regions. In particular, the Fc fragment includes the hinge region. The Fc fragment offers the advantage of increasing the serum half-life of the mutant CD200 protein of the present invention and, in addition, increases binding affinity and enables operative signaling by dimerizing the CD200 protein. It will be understood by those skilled in the art that the Fc region can be mutated to reduce its effector function (see, for example, U.S. Patents 5,637,481 and 6,132,992).

[0083] In one embodiment, the Fc fragment is an Fc fragment of IgG4.

[0084] In further embodiments, the non-CD200 portion is an antibody Fc fragment containing mutations in one or more amino acid residues. Thus, in certain embodiments, the non-CD200 portion is an IgG4 Fc fragment containing an S228P mutation, the location of which follows the EU numbering system. Therefore, in one embodiment, the non-CD200 Fc fragment is an S228P derivative of human IgG4. The S228P mutation inhibits Fab arm exchange in the antibody. Therefore, the presence of the S228P mutation in the Fc fragments described herein is likely to increase the stability of the fusion protein both in vivo and in vitro, resulting in improved therapeutic efficacy and manufacturability. In further embodiments, the non-CD200 IgG4 Fc fragment contains a deletion of the first five amino acids, for example, the first five amino acids of the hinge region of the IgG4 Fc fragment. Thus, in one embodiment, the non-CD200 portion is an IgG4 Fc fragment containing an S228P mutation and a deletion of the first five amino acids of the hinge region, according to the EU numbering system. In further embodiments, the non-CD200 portion is an IgG4 Fc fragment, comprising the deletion of S228P and the first five amino acids of the Fc hinge region.

[0085] In one embodiment, the fusion protein is formed by the direct fusion of the mutant CD200 moiety to the non-CD200 Fc fragment. Therefore, it will be understood that such a fusion does not involve a linker sequence between the mutant CD200 moiety and the non-CD200 Fc fragment. For example, the amino acid glycine 232 of the mutant CD200 moiety may be directly fused to amino acid 1 of the Fc hinge region. In another embodiment, the fusion protein is formed by the direct fusion of the amino acid glycine 232 of the mutant CD200 moiety to amino acid 6 of the IgG4 Fc fragment (in this case, the first 5 amino acids of the Fc hinge region are deleted as previously described herein). In a further embodiment, the direct fusion is the fusion of the amino acid glycine 232 of the mutant CD200 moiety to amino acid 6 of the Fc hinge region of the IgG4 Fc fragment. Therefore, in one embodiment, the glycine 232 of the mutant CD200 moiety is directly fused to the non-CD200 Fc fragment at amino acid 6 of the Fc hinge region. In these embodiments, the position of the fusion in the Fc fragment follows the IMGT numbering system. Direct fusion of the mutant CD200 moiety to amino acid 6 of the hinge region of the IgG4 Fc fragment increases the stability of the resulting fusion protein without affecting strong binding to CD200R, compared to fusion proteins containing a linker sequence. This result is surprising considering previously reported data for Fc fusion proteins containing a linker sequence.

[0086] In some embodiments, the human Fc domain includes mutations that eliminate glycosylation and / or mutations that reduce Fc-gamma receptor binding. In some embodiments, the human Fc domain includes mutations of N297Q, N297A, or N297G; in some embodiments, the human Fc domain includes mutations at positions 234 and / or 235, e.g., L235E, or L234A and L235A (in IgG1), or F234A and L235A (in IgG4); and in some embodiments, the human Fc domain is the Fc domain of IgG2, including mutations of V234A, G237A, P238S, H268Q / A, V309L, A330S, or P331S, or combinations thereof (all following Kabat, EU numbering). In some embodiments, each of the human Fc domains includes a mutation in the human IgG1 constant region L234A / L235A ("LALA") or a mutation in the human IgG1 constant region L234A / L235A / P329G ("LALAPG").

[0087] Further examples of manipulated human Fc domains are known to those skilled in the art. Examples of Ig heavy chain constant region amino acids in which a mutation in at least one amino acid results in reduced Fc function include, but are not limited to, mutations at amino acids 228, 233, 234, 235, 236, 237, 239, 252, 254, 256, 265, 270, 297, 318, 320, 322, 327, 329, 330 and 331 (according to EU numbering) in the heavy chain constant region. Examples of mutant amino acid combinations include, but are not limited to, combinations of mutations at amino acids 234, 235, and 331; combinations of mutations at amino acids 234, 235, and 329; combinations of mutations at L234F, L235E, and P331S; or combinations of mutations at amino acids 318, 320, and 322, such as E318A, K320A, and K322A, which are also known in the art.

[0088] Further examples of manipulated Fc domains include F243L / R292P / Y300L / V305I / P396 IgG1, S239D / I332E IgG1, S239D / I332E / A330L IgG1, S298A / E333A / K334A, L234Y / L235Q / G236W / S239M / H268D / D270E / S298A IgG1 in one heavy chain, and D270E / K326D, A330M / K334E IgG, G236A / S239D / I332E IgG1, K326W / E333S IgG1, S267E / H268F / S324T in the other heavy chain. Examples include IgG1, E345R / E430G / S440Y IgG1, N297A or N297Q or N297G IgG1, L235E IgG1, L234A / L235A IgG1, F234A / L235A IgG4, H268Q / V309L / A330S / P331S IgG2, V234A / G237A / P238S / H268A / V309L / A330S / P331S IgG2, M252Y / S254T / T256E IgG1 ("YTE"), M428L / N434S IgG1, S267E / L328F IgG1, N325S / L328F IgG1, etc. In some embodiments, the manipulated Fc domain includes one or more substitutions selected from the group consisting of N297A IgG1, N297Q IgG1, and S228P IgG4. Furthermore, Fc mutations include AAA (T307A / E380A / N434A), QL (T250Q / M428L), V308P, YD (M252Y / T256D), DQ (T256D / T307Q), and DW (T256D / T307W), L234A / L235A ('LALA'), L235E, P329G, L234A / L235A / P329G "LALAPG", E233P, L234V, L235A, and delta G 236, A327G, A330S, P331S, L234A, L235A, G237A, P238S, H268A, A330S, P331S, K322A, L234R, L235A, L234F, L235E, P331 S, L234F / L235Q / K322Q, L234A / L235A / K322A'LALAKA', L234F / L235E / P331S'FES', L234A / G237A, and G236R / L328R.Further mutations are listed below.

[0089] JPEG2026516066000007.jpg121170

[0090] For the purposes of this explanation, when used herein in relation to mutations within a non-CD200 portion, and the non-CD200 portion is an Fc fragment, the term "~th" refers to the residue number in the amino acid sequence according to the EU numbering system. Therefore, it will be understood that the location of the mutant residue cited herein for the amino acids of the Fc fragment relates to its location according to the EU numbering system. Furthermore, it will be understood that other numbering systems developed for numbering residues in Fc fragment sequences, such as Kabat, AHo, IMGT, Chothia, and Martin (enhanced Chothia), may be used instead. When used herein in relation to the site where a mutant CD200 portion fuses with a non-CD200 Fc fragment, "~th" refers to the residue number in the Fc fragment according to the IMGT numbering system. Therefore, it will be understood that the residue location for the amino acids of the Fc fragment hinge relates to its location according to the IMGT numbering system. Therefore, the numbering of mutations within the Fc fragment as used herein refers to the EU numbering system, and the numbering of hinge amino acids refers to the IMGT numbering system. In some embodiments, glycine 232 of the mutant CD200 portion is directly fused to the non-CD200 Fc fragment at amino acid 224 of the IgG quadruplex of the Fc fragment, according to the EU numbering system.

[0091] The protein of the present invention is preferably produced by a recombinant DNA method, in which a nucleic acid sequence encoding the CD200-Fc fusion protein or any portion thereof is inserted into a recombinant expression vector, and the nucleic acid sequence is expressed in a recombinant expression system under conditions that promote expression. Thus, in one embodiment, the polynucleotide encoding the fusion protein further comprises a vector such as pCDNA3.1. In one embodiment, the fusion protein is flanked by one or more restriction enzyme sites. In another embodiment, the nucleic acid sequence encoding the CD200-Fc fusion protein or any portion thereof is inserted into a recombinant expression vector using infusion cloning. Thus, in a further embodiment, the nucleic acid encoding the CD200-Fc fusion protein or any portion thereof includes at its terminal a nucleic acid sequence complementary to the nucleic acid sequence at the terminal of the linearized vector, such as a duplication between the nucleic acid encoding the CD200-Fc fusion protein and a vector with a base pair / nucleotide between 12 and 21, e.g., a 15-base pair duplication or a 20-base pair duplication.

[0092] Further embodiments of the present invention provide polynucleotides encoding fusion proteins as defined herein. This 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 encoding proteins as defined herein (e.g., mRNA, self-replicating RNA, self-amplifying mRNA, etc.). The nucleic acid sequences encoding proteins provided by the present invention can provide synthetic genes that can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, 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 comprising the amino acid sequence of SEQ ID NO: 1 or 5. In a further embodiment, the polynucleotide encodes DS-118 or ARQ-234. In a particular embodiment, the polynucleotide encodes a fusion protein comprising the amino acid sequence of SEQ ID NO: 2 or 6. In yet another embodiment, the polynucleotide encodes a fusion protein comprising the amino acid sequence of SEQ ID NO: 2 or 6. Exemplary polynucleotide sequences are provided in SEQ ID NO: 4 or 7.

[0093] The recombinant expression vector comprises a synthetic or cDNA-derived nucleic acid fragment encoding a mutant CD200 operably ligated to a suitable transcriptional or translational regulatory element derived from a mammalian, microorganism, virus, or insect gene. Such regulatory elements include a transcription promoter, an optional operator sequence for controlling transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences for controlling transcription and translation termination. Typically, the vector may further incorporate replication capacity in the host, provided by the origin of replication, and select genes to facilitate recognition of the transformant.

[0094] ==Therapeutic Use== The interaction between the CD200 protein and the CD200 receptor is characterized by a rapid dissociation ("off") rate, which results in a low affinity of CD200 to the CD200 receptor; therefore, the present invention has specific applications in therapy. Accordingly, increasing the affinity of fusion proteins containing mutant CD200 proteins and their moieties to the CD200 receptor, as presented herein, can be used in the manufacture of pharmaceutical compositions with more potent properties.

[0095] Furthermore, the manufacturing costs of recombinant proteins are high, and fusion proteins containing mutant CD200 proteins / their moieties with high affinity can be used in pharmaceutical compositions at significantly lower doses than wild-type or non-mutated CD200 proteins to achieve therapeutic effects. Therefore, the use of fusion proteins containing mutant CD200 proteins / their moieties may be more cost-effective in addition to being clinically more effective.

[0096] A further aspect of the present invention provides 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 comprising the amino acid sequence of SEQ ID NO: 1 or 5. In a further embodiment, the pharmaceutical composition comprises DS-118 or ARQ-234.

[0097] In one embodiment, the mutant CD200 protein or fusion protein as defined herein is a modulator of the CD200 receptor. As used herein, the term “modulator” refers to a substance that causes a change, for example, a protein modulator can cause an increase or decrease in the activity of the protein. Given the properties of the mutant CD200 protein and fusion protein of the present invention, they are considered to be agonists of the CD200 receptor and therefore useful in the treatment of autoimmune diseases. Accordingly, in further embodiments, the mutant CD200 protein or fusion protein as defined herein is a CD200 receptor agonist.

[0098] Accordingly, according to further aspects of the present invention, a fusion protein or a pharmaceutical composition as defined herein is provided for use in the treatment of autoimmune diseases.

[0099] As used herein, the terms “autoimmune disease” or “autoimmune disorder” are interchangeable and refer to an undesirable condition resulting from an inappropriate or undesirable immune response to one’s own cells and / or tissues, or to transplanted cells and / or tissues. The terms “autoimmune disease” or “autoimmune disorder” include such conditions, whether mediated by humoral or cellular immune responses.

[0100] In another embodiment, a fusion protein or pharmaceutical composition as defined herein is provided for use in the treatment of allergic diseases. Where used herein, the terms “allergy” or “allergic disease” are used interchangeably and refer primarily to TH2-driven diseases that arise from the activity of type 2 helper T cells (TH2). Examples of allergic diseases include chronic allergic diseases (such as hay fever or allergic rhinitis), allergic contact dermatitis, seasonal allergies, anaphylaxis, and food allergies.

[0101] Fusion proteins containing the mutant CD200 protein / part as defined herein can inactivate activated immune cells with greater efficiency than fusion proteins containing wild-type or non-mutated CD200 protein.

[0102] 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, bones, cartilage, or joints.

[0103] In further embodiments, autoimmune diseases include acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing myelitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), asthma, atopic dermatitis, autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic neuropathy, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), and autoimmune diseases. Immune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and neurological neuropathy, Barlow's disease, Behçet's disease, bullous pemphigoid and related autoimmune blistering disorders, cardiomyopathy, Castleman disease, celiac disease (including refractory celiac disease type II), Chagas disease, idiopathic chronic urticaria, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing polyneuropathy Osteomyelitis (CRMO), chronic idiopathic urticaria, Churg-Strauss syndrome, pemphigoid scarring / benign mucosal pemphigus, Crohn's disease, Cogan's syndrome, cold agglutinin disease, congenital cardiac conduction disorder, coxsackie myocarditis, CREST disease, idiopathic mixed cryoglobulinemia, demyelinating neuropathy, herpetiform dermatitis, dermatomyositis, Devic's disease (neuromyelitis optica), diabetic neuropathy, discoid lupus erythematosus, Dressler syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, fibrotic alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis (GPA) (formerly known as Wegener's granulomatosis), graft-versus-host disease (GvHD), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes zoster of pregnancy, hypogammaglobulinemia, hidradenitis suppurativaStreptococcal purpura (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 mellitus), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosing, lignite keratoconjunctivitis, linear IgA disease (LAD), lupus (SLE), Lyme disease, chronic, macrophage activation syndrome (MAS), mastocytosis, Meniere's disease Romberg disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mohren's ulcer, Mucher-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), neutropenia, ocular scarring pemphigoid, optic neuritis, relapsing rheumatoid arthritis, PANDAS (streptococcal infection-associated pediatric autoimmune neuropsychiatric disorder), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), palmoplantar pustulosis (PPP), Parry-Romberg syndrome, Personne-Turner syndrome, squamous cellulitis (peripheral uveitis), pemphigus, peripheral neuropathy - Perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, and III polyglandular autoimmune syndromes, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcomatoid, It is one or more autoimmune diseases selected from Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, semen-testicular autoimmunity, generalized rigidus syndrome, subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmitis, 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 bullous skin disease, vitiligo, and Wegener's granulomatosis (now called granulomatosis with polyangiitis (GPA)).

[0104] In another embodiment, a protein or fusion protein, or a composition, as defined herein, is provided for use in the treatment of neurodegeneration.

[0105] In yet another embodiment, a protein or fusion protein, or a composition, as defined herein, is provided for use in the treatment of neuropathic pain and inflammatory arthralgia.

[0106] A further aspect of the present invention provides a method for treating an autoimmune disease, an allergic disease (e.g., rheumatoid arthritis, asthma, or atopic dermatitis), neurodegeneration, neuropathic pain, inflammatory arthralgia, diabetic neuropathy, chronic obstructive pulmonary disease, or Parkinson's disease in a subject, comprising administering the fusion protein of the present invention to a subject having at least one autoimmune disease, an allergic disease, neurodegeneration, neuropathic pain, inflammatory arthralgia, diabetic neuropathy, chronic obstructive pulmonary disease, or Parkinson's disease.

[0107] It will be understood that the proteins or fusion proteins of the present invention may be administered as sole therapeutic agents or in combination with one or more other compounds (or therapies) for the treatment of autoimmune diseases, allergic diseases (e.g., rheumatoid arthritis, asthma, or atopic dermatitis), neurodegenerative diseases (e.g., Parkinson's disease), neuropathic pain, inflammatory arthralgia, chronic obstructive pulmonary disease, or diabetic neuropathy.

[0108] Accordingly, according to a further aspect of the present invention, a pharmaceutical composition is provided comprising a fusion protein as defined herein in combination with one or more further therapeutic agents.

[0109] For the treatment of autoimmune diseases, allergic diseases (e.g., rheumatoid arthritis, asthma, or atopic dermatitis), neurodegenerative diseases (e.g., Parkinson's disease), neuropathic pain, inflammatory arthralgia, chronic obstructive pulmonary disease, or diabetic neuropathy, the fusion protein of the present invention can be advantageously used in combination with one or more other pharmaceutical agents, and more specifically, in combination with one or more immunosuppressants or adjuvants in immunosuppressive therapy.

[0110] Other therapeutic agents or treatments that can be administered together with the compounds of the present invention (whether simultaneously or at different time intervals) include, but are not limited to, azathioprine, methotrexate, cyclosporine, monoclonal antibodies (e.g., basiliximab, daclizumab, and muromonab), and corticosteroids.

[0111] Each of the therapeutic agents included in the combination of the present invention can be administered via a different administration schedule and different routes. In addition, the dosage and administration of each of the two or more agents may differ. Each can be administered simultaneously or at different times. Those skilled in the art will be able to understand the administration schedule and combination therapy to be used through general knowledge. For example, the protein or fusion protein of the present invention can be used in combination with one or more other agents administered according to an existing administration schedule.

[0112] Generally, the proteins disclosed herein are used in a purified form with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including physiological saline and / or buffers. Parenteral vehicles include sodium chloride solution, ringer's dextrose, glucose and sodium chloride, and lactated ringer's solution. If it is necessary to retain the polypeptide complex in a suspension, suitable physiologically acceptable adjuvants may be selected from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates.

[0113] The route of administration of the pharmaceutical composition according to the present invention may be any that is generally known to those skilled in the art. For treatments including but not limited to immunotherapy, the protein of the present invention can be administered to any patient according to standard techniques. Administration may be by any suitable mode, including parenteral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, or pulmonary routes, for example, intranasal or inhalation, or, if appropriate, direct injection by catheter, such as intracranial (e.g., ICV into the ventricles of the central nervous system or IT into the spinal cord). The dosage and frequency of administration will depend on the patient's age, sex, and condition, concomitant administration of other drugs, contraindications, and other parameters that the clinician should consider.

[0114] The proteins of the present invention can be freeze-dried for storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective, and freeze-drying and reconstitution techniques known in the art can be used. It will be understood by those skilled in the art that freeze-drying and reconstitution may result in varying degrees of loss of activity, and that upmodulation may be necessary to compensate for such levels.

[0115] It will be understood that all embodiments described herein may be applied to all aspects of the present invention, and vice versa.

[0116] Other features and advantages of the present invention will become apparent from the description provided herein. However, since various changes and modifications will be apparent to those skilled in the art, it should be understood that the description and specific examples herein, while illustrating preferred embodiments of the present invention, are given only as examples. The following studies and protocols illustrate embodiments of the methods described herein.

[0117] ==Numbered aspects== The following numbered aspects are non-limiting aspects of this disclosure. 1. (i) A mutant CD200 moiety containing mutations in amino acid residues 130 and 131, (ii) non-CD200 portion; A fusion protein containing, The aforementioned mutations are K130Y, I131Y, or combinations thereof. The aforementioned non-CD200 is a human Fc fragment, The aforementioned dimer construct is a fusion protein having a serum half-life of 5 to 900 hours. 2. (A) A first fusion polypeptide, (i) A mutant CD200 moiety containing mutations in amino acid residues 130 and 131, (ii) non-CD200 portion; and The aforementioned mutations are K130Y, I131Y, or combinations thereof. The aforementioned non-CD200 is a human Fc polypeptide. The first fusion polypeptide and, (B) A second fusion polypeptide, (i) A mutant CD200 moiety containing mutations in amino acid residues 130 and 131, (ii) non-CD200 portion; and The aforementioned mutations are K130Y, I131Y, or combinations thereof. The aforementioned non-CD200 is a human Fc polypeptide. The second fusion polypeptide, A dimerized construct comprising, A dimer construct comprising the first fusion polypeptide and the second fusion polypeptide, wherein the first fusion polypeptide and the second fusion polypeptide are dimerized via the human Fc polypeptide, and the dimer construct has a serum half-life of 5 to 900 hours. 3. A dimer construct comprising two fusion polypeptides, each polypeptide comprising (i) a mutant CD200 moiety having mutations at amino acid residues 130 and 131, and (ii) a non-CD200 moiety, wherein the mutations are K130Y, I131Y, or combinations thereof, and the non-CD200 moiety is a human Fc polypeptide. The two fusion polypeptides, the human Fc polypeptides, are linked to each other via at least one disulfide bond to form a homodimer. The dimer construct is a dimer construct having a serum half-life of 5 to 900 hours. 4. A fusion protein or dimer construct of any embodiment disclosed herein, having a half-life of 5 to 300 hours. 5. A fusion protein or dimer construct of any embodiment disclosed herein, having a half-life of 444 to 900 hours. 6. A fusion protein or dimer construct of any form disclosed herein, having a half-life of 400 to 650 hours. 7. A fusion protein or dimer construct in any embodiment disclosed herein, having an AUC of 0.5 to 50,000 μg·day / ml in serum at a dose of 5 mg / kg. 8. A fusion protein or dimer construct in any embodiment disclosed herein, having an AUC of 0.5 to 12,926 μg·day / ml in serum at a dose of 5 mg / kg. 9. A fusion protein or dimer construct of any form disclosed herein, having an AUC of 12,928 to 12,960 μg·day / ml in serum at a dose of 5 mg / kg. 10. A fusion protein or dimer construct of any form disclosed herein, having a Cmax of 40-400 μg / ml or 50-300 μg / ml in serum at a dose of 5 mg / kg. 11. A fusion protein or dimer construct of any form disclosed herein, having a Cmax of 100-200 μg / ml in serum at a dose of 5 mg / kg. 12. A fusion protein or dimer construct of any form disclosed herein, having a Cmax of 120-150 μg / ml in serum at a dose of 5 mg / kg. 13. A dimer construct according to any embodiment disclosed herein, wherein the human Fc fragment of the first fusion polypeptide and / or the second fusion polypeptide comprises a hinge region. 14. A dimer construct according to any embodiment disclosed herein, wherein the non-CD200 portion of the first fusion polypeptide and / or the second fusion polypeptide is an Fc fragment of mutant IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM. 15. A dimer construct according to any embodiment disclosed herein, wherein the human Fc fragment of the first fusion polypeptide and / or the second fusion polypeptide comprises at least one Fc domain. 16. A dimer construct according to any embodiment disclosed herein, wherein the at least one Fc domain is selected from a human IgG1 domain, a human IgG2 domain, a human IgG3 domain, a human IgG4 domain, a human IgA domain, a human IgE domain, and a human IgM domain. 17. A dimer construct according to any embodiment disclosed herein, wherein the at least one Fc domain is a human IgG4 Fc domain. 18. A dimer construct of any embodiment disclosed herein, wherein the at least one Fc domain is human IgG1 (ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP A dimer construct containing a sequence that is at least 80% identical to EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 8). 19. A dimer construct according to any embodiment disclosed herein, wherein the at least one Fc domain comprises a sequence that is at least 90% identical to human IgG1 (SEQ ID NO: 8). 20. A dimer construct according to any embodiment disclosed herein, wherein the at least one Fc domain comprises Sequence ID No. 8. 21. A dimer construct according to any embodiment disclosed herein, wherein the first fusion polypeptide has at least one mutation at at least one glycosylation site compared to a fusion polypeptide comprising a wild-type CD200 moiety and a wild-type non-CD200 moiety. 22. A dimer construct according to any embodiment disclosed herein, wherein the second fusion polypeptide has at least one mutation at at least one glycosylation site compared to a fusion polypeptide comprising a wild-type CD200 moiety and a wild-type non-CD200 moiety. 23. A dimer construct according to any embodiment disclosed herein, wherein each of the first fusion polypeptide and the second fusion polypeptide has a lower affinity for at least one Fc-gamma receptor compared to a fusion polypeptide comprising a non-CD200 moiety having a wild-type human IgG Fc fragment. 24. A dimer construct according to any embodiment disclosed herein, wherein the first fusion polypeptide and / or the second fusion polypeptide comprises a deletion of the first five amino acids of the non-CD200 moiety compared to the wild-type non-CD200 moiety, wherein the deletion is located in the hinge region of the human Fc fragment. 25. A dimer construct according to any embodiment disclosed herein, further comprising a linker between the mutant CD200 portion of the first fusion polypeptide and the non-CD200 portion of the first fusion polypeptide. 26. A dimer construct according to any embodiment disclosed herein, further comprising a linker between the mutant CD200 portion of the second fusion polypeptide and the non-CD200 portion of the second fusion polypeptide. 27. A dimer construct according to any embodiment disclosed herein, wherein the glycine 232 of the mutant CD200 portion of the first fusion polypeptide is directly fused to the non-CD200 IgG4 Fc fragment of the first fusion polypeptide at the 6th amino acid according to the IMGT numbering system. 28. A dimer construct according to any embodiment disclosed herein, wherein the glycine 232 of the mutant CD200 portion of the second fusion polypeptide is directly fused at the 6th amino acid according to the IMGT numbering system to the Fc fragment of the non-CD200 IgG4 of the second fusion polypeptide. 29. A dimer construct according to any embodiment disclosed herein, wherein the first fusion polypeptide and / or the second fusion polypeptide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. 30. A dimer construct according to any embodiment disclosed herein, wherein the first fusion polypeptide and / or the second fusion polypeptide further comprises an N-terminal signal sequence, the N-terminal signal sequence comprising the amino acid sequence MEFGLSWLFLVAILKGVQC (SEQ ID NO: 3). 31. A dimer construct according to any embodiment disclosed herein, which has a lower affinity for at least one of FcγRI, FcγRIIA, FcγRIIIA, or C1q receptors compared to a fusion protein comprising a non-CD200 portion having an Fc fragment of wild-type human IgG. 32. A fusion protein or dimer construct in any embodiment disclosed herein, wherein the human Fc polypeptide is wild-type and free of mutations, or the human Fc polypeptide contains one or more mutations disclosed herein. 33. A polynucleotide encoding a fusion protein or fusion polypeptide in any embodiment disclosed herein. 34. A polynucleotide according to embodiment 33, wherein the polynucleotide has at least 80% sequence identity with SEQ ID NO: 4. 35. A polynucleotide encoding a first fusion polypeptide of a dimer construct according to any one of embodiments 2 to 32. 36. A polynucleotide encoding a second fusion polypeptide of a dimer construct according to any one of embodiments 2 to 32. 37. An expression system comprising at least one expression vector containing a polynucleotide as described in any one of embodiments 33 to 36. 38. Host cells containing the expression system described in embodiment 37. 39. A composition comprising a fusion protein or dimer construct according to any one of embodiments 1 to 31, or a polynucleotide according to any one of embodiments 33 to 36, and a pharmaceutically acceptable carrier. 40. A method for treating a subject having an autoimmune disease, an allergic disease, a neurodegenerative disorder, neuropathic pain, an inflammatory disorder, a skin disease, or diabetic neuropathy, comprising administering a fusion protein or dimer construct according to any one of embodiments 1 to 32, or a polynucleotide according to any one of embodiments 33 to 36. 41. A method for treating a subject having rheumatoid arthritis, asthma, atopic dermatitis, inflammatory arthralgia, chronic obstructive pulmonary disease, or Parkinson's disease, comprising administering a fusion protein or dimer construct according to any one of embodiments 1 to 32, or a polynucleotide according to any one of embodiments 33 to 36. 42. A method for treating a subject having an autoimmune disease affecting the neuromuscular system, vascular system, eyes, skin, gastrointestinal tract, lungs, kidneys, liver, peripheral or central nervous system, bones, cartilage, or joints, comprising administering a fusion protein or dimer construct according to any one of embodiments 1 to 32, or a polynucleotide according to any one of embodiments 33 to 36. 43. A method for treating a subject having a skin disease, comprising administering a fusion protein or dimer construct according to any one of embodiments 1 to 32, or a polynucleotide according to any one of embodiments 33 to 36. 44. The method according to embodiment 43, wherein the skin disease is dermatitis. 45. A method according to any one of embodiments 40 to 44, wherein a fusion protein, dimer construct, or polynucleotide is administered as the sole therapeutic agent. 46. ​​A method according to any one of embodiments 40 to 44, wherein a fusion protein, dimer construct, or polynucleotide is administered in combination with one or more other pharmaceuticals indicated for the treatment of autoimmune diseases, allergic diseases, neurodegenerative disorders, neuropathic pain, inflammatory disorders, or diabetic neuropathy. 47. A method according to any one of embodiments 40 to 44, wherein a fusion protein, dimer construct, or polynucleotide is administered in combination with one or more immunosuppressants or adjuvants in immunosuppressive therapy. 48. The method according to embodiment 47, wherein the dimer construct is administered in combination with azathioprine, methotrexate, cyclosporine, a monoclonal antibody, a corticosteroid, or a combination thereof. 49. The method according to embodiment 48, wherein the monoclonal antibody is basiliximab, daclizumab, or muromonab. 50. A fusion protein or dimer construct according to any one of embodiments 1 to 32, a polynucleotide according to any one of embodiments 33 to 36, or a composition according to embodiment 39, for use in the treatment of autoimmune diseases, allergic diseases, neurodegenerative disorders, neuropathic pain, inflammatory disorders, skin diseases, or diabetic neuropathy. [Examples]

[0118] Example 1: Production of mutant and wild-type CD200-Fc molecules Gene synthesis and cloning Gene synthesis of IgG4 S228P Fc (codon-optimized for CHO expression) was performed using GeneArt. This construct was cloned into plasmid pCDNA3.1 using infusion cloning to generate a vector backbone. A codon-optimized DNA sequence encoding residues 1-232 of the mutant or wild-type human CD200 of UniProt P412178(OX2G_Human), including the N-terminal signal sequence, was inserted into the N-terminus of IgG4 S228P Fc to create direct fusion of the amino acid glycine 232 of CD200 to the Fc region. The sequences were confirmed bidirectionally.

[0119] Gigaprep The sequenced plasmids were transformed into E. coli (Escherichia coli) DH5α cells. Single colonies of each target protein were selected and inoculated into 10.0 mL of LB containing ampicillin. Each mutant or wild-type construct was subcultured in 800 mL of Circlegrow medium for gigascale DNA preparation. The DNA was isolated using the Endotoxin Free Quanta Giga Kit.

[0120] Protein expression The CD200-Fc protein was generated by transient transfection of an expression plasmid into CHO-3E7 cells using polyethyleneimine (PEI). Briefly, 4.0 × 10⁶ cells maintained at 37°C in CD-Forti CHO medium were transfected. 6 A 250 mL culture at a cell density of 1 / mL was transfected with a 2 mg / L plasmid using PEI in a 1:5 ratio. 24 hours after transfection, the culture was transferred to 32°C, and protein expression was enhanced by adding 10% feed C, glutamine, glucose, and 0.5 M sodium butyrate to the cells. The batch was monitored, and the supernatant containing overexpressed CD200-Fc was collected on day 7 with approximately 75% viability. The filtered supernatant was subjected to protein purification.

[0121] Protein purification All purification procedures were performed at 4°C. The culture recovery was loaded onto a 5 mL MabSelect SuRe affinity column pre-equilibrated with 50 mM sodium phosphate, 150 mM NaCl, pH 7.4, at a flow rate of 3 mL / min on the AKTA Pure platform. The column was washed with equilibration buffer, and the bound protein was eluted using 20 mM sodium acetate, 150 mM NaCl, pH 3.5. The eluate was neutralized with 10 v / v% 1 M Tris, pH 8.0, and analyzed by SDS-PAGE. The fraction containing CD200-Fc was pooled, concentrated to 5 mL, and subjected to gel filtration chromatography (Hiload 16 / 600 Superdex-200pg column) on the AKTA Pure platform. The protein was treated at 1.2 mL / min in a 50 mM sodium phosphate, 150 mM NaCl, pH 7.4 buffer system, and each fraction was collected. The fraction containing the CD200-Fc dimer was pooled after analysis by SDS-PAGE and concentrated to 1.33 mg / mL (measured at UV280 nm) using Amicon Ultra Centricon (10 kDa molecular weight cutoff). The purified material was subjected to SEC-HPLC, LC-MS, and EndoSafe LAL cartridge testing to evaluate protein purity, molecular weight, and endotoxin content, respectively. The final sample was stored at -80°C.

[0122] Example 2: Binding analysis of wild-type and mutant CD200-Fc proteins The BIAcore experiment was conducted by Syngene International Ltd. (Biocon Park, Plot No. 2 & 3, Bommasandra Industrial Area, Bommasandra-Jigani Link Road, Bangalore-560099, India).

[0123] Assay principle The BIAcore instrument uses surface plasmon resonance (SPR), an optical method, to measure the binding properties of CD200-Fc to the CD200 acceptor (CD200R) of two interacting molecules. In this technique, when the second of two interacting molecules trapped on a chip (sensor) flows over its immobilized partner in solution, the change in the refractive index of the other molecule is measured. In these experiments, CD200-Fc was immobilized on the surface of a chip (sensor), and CD200R was injected onto the trapped CD200-Fc under continuous flow conditions in aqueous buffer. The change in the refractive index of CD200-Fc after CD200R binding was measured in real time, and the results were plotted as response units (RU) versus time to create a sensorgram.

[0124] Apparatus and reagents The experiment was conducted using a GE Healthcare BIAcore T200. Human, cynomolgus monkey, and mouse CD200R proteins were purchased from Creative Biomart (CD200R1-320H, CD200R1-3483C, CD200R1-3280M). All measurements were repeated twice.

[0125] Protocol For the human CD200 construct, anti-human Fc (GE Healthcare) was covalently immobilized on 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. The CD200-Fc protein was diluted to 0.5 μg / mL–4 μg / mL in running buffer (HEPES-buffered saline pH 7.4 containing 1×HBS-EP+pH 7.4 (Cytiva BR100669), 3 mM EDTA, and 0.05 v / v% surfactant P20), and flowed over the immobilized anti-human IgG Fc at a rate of 10 μL / min for 25–100 seconds with a stabilization time of 60 seconds. CD200-Fc between 35 and 250 RU was captured, and higher RU values ​​were used for cynomolgus monkey CD200R binding experiments. Human CD200R or cynomolgus monkey CD200R, along with a buffer blank (0 nM), were serially diluted (3-fold) to five or more concentrations (depending on expected affinity) in running buffer. Association was performed over the captured ligand by flowing at a rate of 30–50 μL / min for 120 seconds, followed by dissociation in running buffer for 120–360 seconds. The analysis temperature was 25°C. Subsequently, the surface was regenerated by flowing 3M MgCl2 at a rate of 30 μL / min for a short period of 30–90 seconds, and then the surface was stabilized by flowing running buffer for 60 seconds.

[0126] For the mouse CD200 construct, muCD200R-Fc (Creative Biomart CD200R1-458M), diluted to 1 μg / mL in running buffer, was flowed onto immobilized anti-human IgG Fc, and CD200 monomers were successively diluted and flowed onto the captured CD200R. Other details were as described above.

[0127] The experimental sensorgrams were analyzed using BIAevaluation software (GE Healthcare). The resulting curves were fitted to a 1:1 Langmuir-binding model by setting Rmax and RI as local parameters. A reaction rate equation using standard parameters (e.g., ligand concentration, time) was used for iterative curve fitting. The closeness of the fit was determined by the algorithm provided by the manufacturer in the BIAevaluation software, and χ² was used. 2 The value is R max Data was accepted if it was less than 10% and the U-value was 15 or less.

[0128] [Table 1]

[0129] Table 2 shows the ID numbers of the CD200-Fc variants included in this application, indicating the CD200 mutation, Fc domain, and equilibrium binding affinity constant (K D This shows the value (up to the nearest integer).

[0130] [Table 2]

[0131] For all IgG4 fusion constructs, affinity to human CD200R was low, but binding to cynomolgus monkey CD200R1 was detected. On the other hand, as shown in Table 2, binding to human CD200R1L or mouse CD200R1 was not detected.

[0132] result The results (Table 3, Figures 1A-1B and 21) show that DS-118 binds to the human CD200 receptor with approximately 137-fold higher affinity than wild-type CD200-Fc (DS-155), and that ARQ-234 binds to the human CD200 receptor with approximately 84-fold higher affinity than wild-type CD200-Fc (DS-155). The off-rates listed in Table 3 and the sensorgrams shown in Figures 1A-1B demonstrate the off-rate and receptor half-life of DS-118, which are consistent with efficient agonism in functional cell assays. Furthermore, the results in Table 4 and Figures 2A-2B show that DS-118 can bind to cynomolgus monkey CD200R (cyno CD200R), and that this fusion protein can be evaluated using standard toxicological protocols.

[0133] [Table 3]

[0134] [Table 4] * In both Tables 3 and 4, ka (1 / Ms), kd (1 / s), and KD (nM) are the average values ​​from two runs, both performed on the same or different days.

[0135] Example 3: Cell Binding and Activation Assay of Wild-Type and Mutant CD200-Fc Proteins Assay principle To demonstrate the agonist activity of DS-118, human monocyte cell line U937 (ATCC, CRL1539) was transduced with human CD200R cDNA. Cytokine production, including IL-6, from these cells could be induced by stimulation with PMA followed by stimulation with LPS.

[0136] Cell line construction The full-length human CD200R gene, including the signal sequence, was cloned downstream of the EF1α promoter in the pCDH-EF1-human CD200R-IRES-Puro lenti vector (System Biosciences). Lentiviral particles containing the expression construct were produced in 293TN-producing cells and concentrated using PEG-it reagent (System Biosciences) according to the manufacturer's instructions.

[0137] Human monocyte immortalized cell line U937 was transduced with lentiviral particles in the MOI range of 5–200 using Transdux 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 establish stable polyclonal CD200R-expressing cells. CD200R expression was confirmed by flow cytometry and Western blotting.

[0138] Cytokine release assay (inhibition of IL-6, IL-8, and TNFα) 50,000 U937-CD200R cells were seeded per well in a 96-well plate and differentiated in 100 nM PMA for 72 hours. After differentiation, the PMA-containing medium was replaced with fresh medium and incubated for 2 hours. CD200-Fc constructs were added to the wells and incubated for 1 hour, then the cells were activated by adding 10 ng / ml LPS and incubated for a further 24 hours. The supernatant was collected and assayed for IL-6, IL-8, or TNFα using a commercially available ELISA kit.

[0139] pERK Inhibition Assay 50,000 U937-CD200R cells per well were seeded in a 24-well plate. Fc block was then added for 30 minutes, followed by CD200-Fc (DS-155 or DS-118), and incubated at 37°C for a further 2 hours. Cells were then induced with PMA (10 nM) for 20 minutes. After incubation, cells were rapidly harvested and centrifuged at 1250 rpm for 5 minutes. The supernatant was discarded, 100 μL of fixation buffer was added to the pellet, and incubated at 4°C for 15 minutes. Cells were then washed once with 1× PBS + 2% FBS, permeabilized with 100 μL of 90% methanol while vortexing for 5 minutes, and then washed again with 1× PBS + 2% FBS. Anti-pERK antibody in a 1:1000 ratio was added for 45 minutes, followed by secondary antibody for a further 30 minutes at 4°C. The cells were washed again, and data was acquired using a flow cytometer.

[0140] [Table 5]

[0141] cell binding U937 cells were resuspended in 50 μL of FACS buffer (1×PBS + 2% FBS) at a cell density of 100,000 cells per test.

[0142] Treatment with the construct (50 μL) was started at 10 μg / mL, diluted 3-fold with FACS buffer to 10 concentrations, and incubated at 37°C for 1 hour, 4 hours, and 24 hours. At the end of each time point, 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, cells were washed and stained at 4°C for 20 minutes to check viability (1 μL dye / 1 million cells / 1 mL 1 × PBS). Cells were washed and fixed in fixation buffer (100 μL per test) at 4°C for 20 minutes. After incubation, cells were washed and the pellet was resuspended in FACS buffer (100 μL per test) for flow cytometry data acquisition.

[0143] At 24 hours, treatment with CD200-Fc protein was performed using cell culture medium. The washing step involved adding 200 μL of FACS buffer and centrifugation at 1400 RPM.

[0144] [Table 6]

[0145] result The data shown in Figures 3A-3D demonstrate that DS-118 can inhibit LPS-induced IL-6 (Figure 3A), IL-8 (Figure 3B), and TNFα (Figure 3C) secretion in a concentration-dependent manner. As can be seen in Figure 3A, DS-118 inhibits LPS-induced IL-6 release more strongly than wild-type CD200-Fc fusion protein (DS-155), and the IC of DS-155's IL-6 release inhibition 50 While the concentration of the IC in the DS-118 was 0.18 μg / ml, the concentration of the IC in the DS-118 was 0.18 μg / ml. 50 The concentration was 0.01 μg / ml. Furthermore, Figure 3D shows that DS-118 has a stronger ability to inhibit LPS-stimulated ERK activation (phosphorylated ERK / pERK) than wild-type CD200-Fc fusion protein (DS-155).

[0146] Figure 4 shows the binding of the mutant DS-118 CD200-Fc protein to CD200R-expressing U937 cells. This data demonstrates good binding of DS-118 to CD200R-expressing cells at all time points.

[0147] The mutant proteins of the present invention showed higher binding affinity than the wild type and many other mutants tested. Human DS-12 and DS-20 are IgG1 fusion constructs, DS-118, DS-155, and DS-192 are IgG4 fusion constructs, and mouse DS-198 and DS-227 are CD200-Fc(IgG2a)Fc fusions. Table 2 shows that the affinity constant (KD) of K130Y was approximately 13 nM, compared to approximately 179 nM for the wild type (IgG4 fusion). As shown in Table 2, the CD200 variant with one mutation (DS-192) increased affinity to human CD200R, and the binding half-life was extended from 21 seconds to approximately 3 minutes. The CD200 variant (DS-118), possessing multiple mutations, surprisingly exhibited a high affinity of approximately 1 nM, representing a more than 130-fold increase in affinity compared to the wild type, and extending the binding half-life from 21 seconds to approximately 38 minutes. Since affinity was measured against monomer binding, this data suggests that the dimer-Fc fusion form confers additional functional affinity.

[0148] As shown in Figure 7, high-affinity (1nM) DS-118 showed stronger inhibition of IL-6 release than wild-type DS-155, while 13nM DS-192 showed intermediate efficacy. As shown in Figure 8, inhibition of IL-8 was observed with DS-118. As shown in Figure 9, inhibition of TNF-α was observed with 13nM DS-192. As shown in Figure 10, inhibition of ERK phosphorylation correlated with CD200 affinity.

[0149] As shown in Figures 19A-19F, antibodies that recognize the Fc gamma receptor did 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 system.

[0150] Example 4: Macrophage activation assay of wild-type and mutant CD200-Fc fusion proteins Macrophage differentiation A single control iPSC line, BIONi010-C, was differentiated into macrophage progenitor cells using a proprietary protocol developed by Censo Biotechnologies. The cells were quality-controlled using flow cytometry (Censo Biotechnologies) according to standard procedures. The macrophage progenitor cells were then matured into macrophages over 7 days before processing, stimulation, and assay.

[0151] Processing and stimulation Mature macrophages were treated with DS-118 at a range of concentrations for an additional 18 hours, one hour before stimulation (Table 7). After stimulation, the cells were used in a cytokine release assay. Six concentrations of DS-118 were obtained by using a 1:3 dilution at the highest concentration of 10 μg / ml.

[0152] [Table 7]

[0153] Cytokine release (IL-6 HTRF) Following the above processing and stimulation, the supernatant was collected and transferred to a new plate. The samples were stored at -80°C until the day of the assay. IL-6 was measured using the Cisbio HTRF kit (62HILo6PEG) according to the manufacturer's instructions and measured using a BMG ClarioSTAR plate reader. Analysis was performed by removing background fluorescence and interpolating the results using a standard curve. All data are presented as mean + / - SEM, and statistical significance was assessed by two-way ANOVA. Controls included wells that were stimulated but not treated with the compound (untreated) and wells that were neither stimulated nor treated to represent baseline cytokine release (unstimulated).

[0154] result Figure 5 shows that DS-118 can inhibit LPS-induced IL-6 release from iPSC-derived macrophages in a concentration-dependent manner.

[0155] Example 5: In vitro proof of concept for high-affinity mouse CD200-Fc Due to the lack of cross-reactivity with mouse CD200R, a mouse CD200-CD200R1 in silico model was created based on the published crystal structure, and a high-affinity surrogate CD200-Fc protein was designed for in vitro proof-of-concept experiments in a mouse model of autoimmunity.

[0156] Protocol The mouse CD200 construct used Uniprot O54901, which contains the signal peptide and extracellular domain. The mutation number refers to the complete Uniprot sequence including the signal peptide.

[0157] The protein was generated by transient transfecting CHO-3E7 cells with a pcDNA3.1-based expression plasmid using polyethyleneimine (PEI). 24 hours after transfection, the culture was transferred to 32°C, and 10% feed C, glutamine, glucose, and 0.5M sodium butyrate were added. The supernatant was collected on day 7 and filtered. Purification was performed at 4°C on an AKTA Pure platform using a pre-equilibriumized MabSelect SuRe 5ml affinity column (Cytiva) with a flow rate of 3 mL / min in 50 mM sodium phosphate, 150 mM NaCl, pH 7.4. The column was washed with equilibration buffer, and the bound protein was eluted using 20 mM sodium acetate, 150 mM NaCl, pH 3.5. The eluate was neutralized with 10 v / v% 1M Tris pH 8.0 and analyzed by SDS-PAGE. The pooled fraction was concentrated to 5 mL and subjected to gel filtration chromatography (Hiload 16 / 600 Superdex-200pg column) on an AKTA Pure platform. The protein was processed at 1.2 mL / min in a buffer system of 50 mM sodium phosphate, 150 mM NaCl, pH 7.4. The protein-containing fraction was pooled and concentrated to 1.33 mg / mL (measured at UV 280 nm) using Amicon Ultra Centricon (10 kDa molecular weight cutoff). The purified product was subjected to SEC-HPLC, LC-MS, and EndoSafe LAL cartridge testing to evaluate 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. Affinity was examined using the same technique as in Example 2 above.

[0158] result As shown in Table 2, the monomer binding affinity of the variant H82Y, T125I combination is 43 nM, which is approximately 14 times higher than that of the wild-type construct containing the mouse IgG2a Fc domain.

[0159] Example 6: In vivo demonstration of the concept of high affinity CD200-Fc Using a mouse model, we demonstrated that higher affinity mouse CD200-Fc protein, when prophylactically administered, reduces clinical scores in a mouse collagen-induced arthritis (CIA) model.

[0160] Mice possess four potential CD200 receptors, CD200R1–CD200R4, of which at least one may be activated, and CD200R1 is a homolog of human CD200R. Knocking out either CD200 or CD200R1 in transgenic mice induces exacerbation or early onset of symptoms in models of many autoimmune conditions, such as alopecia, arthritis, inflammatory bowel disease (IBD)25, and uveoretinitis.

[0161] CD200R agonism in a rodent model using patient samples is known in the art and has been previously achieved using CD200-Fc fusion proteins, suggesting that human CD200-Fc fusion proteins may be used as therapeutic agents for inflammatory diseases. Similar to other cell surface immune receptors, the affinity of CD200 for CD200R is low (in the high nanomolar concentration range), and therefore, an ideal human therapeutic agent would require enhanced affinity for optimal potency. The Fc domain contributes to an antibody-like serum half-life, and the dimer form increases binding avidity and enables receptor crosslinking. Animal model data show that the sequence of the Fc domain is associated with a mouse IgG2a Fc fusion with optimal efficacy, likely due to the promotion of cell-cell interaction formation by binding to the Fc gamma receptor, further increasing avidity. Antibody-dependent cell-mediated cytotoxicity may also contribute by eliminating CD200R1-expressing cells. Therefore, we used an in vivo mouse model to test the efficacy of the mouse high-affinity CD200-Fc protein compared to the wild-type CD200-Fc protein.

[0162] Protocol Wild-type (DS-198) and higher affinity (DS-227) mouse CD200-Fc proteins were tested using the CIA model by initiating dosing immediately prior to symptom onset, as shown in Figure 29. Arthritis was induced in DBA / 1J male mice by intradermal injection of bovine type II collagen in CFA (complete Freund's adjuvant) on day 1, followed by a booster injection of incomplete Freund's adjuvant on day 21. On day 22, the animals were randomized based on body weight and injected three times a week with 3 mg / kg of a mouse IgG2a isotype control antibody, DS-198 (wild-type muCD200-Fc), or DS-227 (high affinity 43 nM muCD200-Fc) until day 36. The positive control group received 0.5 mg / kg of dexamethasone orally daily. The clinical score of ankle arthritis (blinded evaluation) was measured every other day from days 25 to 36. The data shown in Figure 11 are presented as the mean ± standard error of the mean (SEM). ** p < 0.01, *** p < 0.001 vs. disease + Dexa, disease + DS-198, and disease + DS-227. Two-way ANOVA with repeated measures followed by Tukey's multiple comparison test was performed.

[0163] Results As shown in Figure 11, DS-227, a CD200-Fc with higher affinity, significantly and potently decreased the clinical score compared to the wild-type (DS-198) at the selected dose of 3 mg / kg.

[0164] Example 7: Proof-of-Concept Study Using High-Affinity DS-192 Based on the results of the in vivo proof-of-concept of the above high-affinity mouse CD200-Fc study (CIA mouse study), an in vivo proof-of-concept study was conducted to test DS-192, a high-affinity human CD200-Fc fusion protein. A humanized model of oxazolone-induced contact hypersensitivity was designed using NOG-EXL mice that can transplant both human lymphocytes and bone marrow cells (thereby generating huNOG-EXL mice).

[0165] Protocol As shown in Figure 30, human cells were transplanted into female NOG-EXL mice and randomized based on the percentage of CD45+ cells at 20-21 weeks of age (-1 day). On day 0, mice were sensitized by applying oxazolone (100 μL of 3 w / v% oxazolone in an acetone:alcohol ratio of 1:4) to their abdomen. On days 5, 10, and 14, they were challenged by topically administering 20 μL of 2 w / v% oxazolone (acetone:alcohol ratio of 1:4) to each ear (10 μL / side). Oxazolone challenges were repeated by administering DS-192 (huCD200-Fc, 13 nM) or a CD200R agonist antibody (CD200R mAb) to one ear of pre-sensitized huNOG-EXL mice on the same day as each challenge. Isotype control antibodies, CD200R agonist antibodies, and high-affinity huCD200-Fc (DS-192) were administered intravenously at 3 mg / kg four hours prior to days 5, 10, and 14 of the oxazolone challenge. Ear thickness was measured immediately before the challenge and 24 hours after each challenge. On day 15, punch biopsies were taken for cytokine analysis by multiplexing.

[0166] result As shown in Figure 12, changes in ear thickness (surrogate of inflammatory response) were significantly reduced by DS-192 the day after the second and third challenges, in contrast to CD200R mAb, which did not result in a significant reduction, compared to isotype controls. In addition, as shown in Figures 13, 14, and 15, significant reductions in IL-1β, GM-CSF, and IL-13 in ear tissue were observed in DS-192-treated mice at the end of the study. Thus, these results demonstrate that high-affinity CD200-Fc has superior efficacy in a humanized mouse model of contact hypersensitivity. Extrapolating these advantages, since DS-192 has significantly lower CD200 affinity than DS-118, it shows greater efficacy when used with DS-118 for the treatment of allergic diseases and inflammatory skin disorders.

[0167] Example 8: Proof-of-Concept Study Using High-Affinity DS-118 A schematic diagram of the Fc fusion protein DS-118 of the present invention is shown in Figure 6.

[0168] Protocol This study was conducted using cynomolgus monkeys with porcine roundworm (roundworm)-induced pneumonia in NHP, as shown in Figure 16.

[0169] This model is Th2-driven and has been used in the art for some time to evaluate the efficacy of drugs for asthma. Cynomolgus monkeys were screened for pre-existing susceptibility to Ascaris suum antigen and administered 20 mg / kg of high-affinity huCD200-Fc (DS-118) (n=6), a vehicle control (n=6), and 1 mg / kg of dexamethasone (n=4) on day 0. All animals were challenged with 5000 μg / ml of intrabronchial Ascaris suum antigen on day +1, and lymphocyte levels in BAL (bronchial alveolar lavage fluid) were measured by flow cytometry on day +2 (24 hours after challenge and 48 hours after drug treatment). Changes in airway resistance immediately after Ascaris suum antigen challenge were compared to airway resistance immediately before challenge.

[0170] Pre-administration measurements were performed on day -1 (relative to huCD200-Fc administration), and post-administration measurements were performed on day +1. At each time point (pre-administration, 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, and day 28), at least 0.8 mL of blood was collected from each animal via the cephalic vein or saphenous vein. For samples collected within the first hour of administration, a tolerance of ±1 minute was acceptable. For the remaining time points, samples collected within 5% of the scheduled time were acceptable. Tubes containing blood samples and coagulant were stored at room temperature for 30-60 minutes, then centrifuged at 1500 × g for 10 minutes at 4°C. Serum samples were then rapidly frozen on dry ice and stored at -60°C or below until analysis. Protein concentrations were determined by ELISA. 96-well ELISA plates were coated overnight at 4°C with 1 μg / mL goat anti-human IgG in carbonic acid-bicarbonate buffer. After washing and blocking, serially diluted plasma samples were added, and 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 approximately 5–10 minutes. Absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer. Plasma antibody concentrations were obtained by substituting the OD values ​​of the samples into a standard curve. The detection limit of this method is 1 ng / mL. Serum concentrations were subjected to non-compartmental pharmacokinetic analysis using Phoenix WinNonlin® software (version 8.1, Pharsight, Mountain View, CA). The linear / logarithmic trapezoidal law was applied when obtaining PK parameters. The half-life was calculated without using data from less than 1% of Cmax, and the half-life was inaccurate if AUC_%Extrap_obs was greater than 20% or Rsq_adjusted was less than 0.9.

[0171] result As shown in Figure 17, DS-118 administration the day before final sensitization resulted in a significant reduction in the number of infiltrating lymphocytes in the bronchoalveolar lavage (BAL) fluid at 48 hours compared to the vehicle control. As shown in Figure 18, there was a reduction in post-sensitization airway resistance (RL), but this was not statistically significant. Therefore, the data indicate that high-affinity CD200-Fc substantially reduced cellular infiltration in bronchoalveolar lavage (BAL) fluid in a non-human primate (NHP) model of airway inflammation.

[0172] Example 9: In vitro binding study using DS-118, which has the highest affinity. We conducted research to test the binding of the high-affinity CD200-Fc fusion protein DS-118 to human PBMCs.

[0173] Protocol PBMC cells were resuspended in 50 μL of FACS buffer (1×PBS + 2% FBS) at a cell density of 100,000 cells per assay point. Dilutions of huCD200-Fc in 50 μL were prepared using FACS buffer, starting at 10 μg / mL and diluting 3-fold to 10 concentrations, and incubated at 37°C for 1 hour. At the end of each time point, cells were collected and washed (addition of 200 μL of FACS buffer and centrifugation at 1400 RPM). 10 μg / mL of anti-human secondary antibody (Abcam Ab98596) was added along with an excess of Fc block (Innovex Biosciences no. NB309) and incubated at 4°C for 30 minutes. After incubation, cells were washed and stained at 4°C for 20 minutes to check viability (1 μL of dye / 1,000,000 cells / 1 mL of 1×PBS, ThermoFisher C34557A). The cells were washed and fixed in fixation buffer (100 μL per test, BD Sciences 554655) at 4°C for 20 minutes. After incubation, the cells were washed and the pellet was resuspended in FACS buffer (100 μL per test) for flow cytometry data acquisition.

[0174] result As shown in Figure 20, binding to human PBMCs was dose-dependent.

[0175] Example 10: Production of mutant and wild-type CD200-Fc molecules Gene synthesis and cloning Genetic synthesis of IgG4 S228P Fc (codon-optimized for CHO expression) was performed using the mutant M428L+N434S in GeneArt. These constructs were cloned into plasmid pCDNA3.1 using infusion cloning to generate a vector backbone. A codon-optimized DNA sequence encoding residues 1-232 of the mutant or wild-type human CD200 of UniProt P412178(OX2G_Human), including the N-terminal signal sequence, was inserted into the N-terminus of IgG4 S228P Fc to create direct fusion of the amino acid glycine 232 of CD200 to the Fc region. The sequences were confirmed bidirectionally.

[0176] Gigaprep Sequence-confirmed plasmids were used to transform E. coli DH5α cells. Single colonies of each target protein were selected and inoculated into 10.0 mL of LB containing ampicillin. Each mutant or wild-type construct was subcultured in 800 mL of Circlegrow medium for gigascale DNA preparation. DNA was isolated using the Endotoxin Free Quanta Giga Kit.

[0177] Protein expression CD200-Fc protein was generated by transient transfection of an expression plasmid into CHO-3E7 cells using polyethyleneimine (PEI). Briefly, the plasmid was transfected in CD-Forti CHO medium at a density of 4.0 × 10⁶ cells maintained at 37°C. 6A 250 mL culture with a cell / mL concentration was transfected with a 2 mg / L plasmid using PEI in a 1:5 ratio. 24 hours after transfection, the culture was transferred to 32°C, and protein expression was enhanced by supplying cells with 10% feed C, glutamine, glucose, and 0.5 M sodium butyrate. The batch was monitored, and the supernatant containing overexpressed CD200-Fc was collected on day 7 with approximately 75% viability. The filtered supernatant was subjected to protein purification.

[0178] Protein purification All purification procedures were performed at 4°C. The culture recovery was loaded onto a 5 mL MabSelect SuRe affinity column pre-equilibrated with 50 mM sodium phosphate, 150 mM NaCl, pH 7.4 at a flow rate of 3 mL / min on the AKTA Pure platform. The column was washed with equilibration buffer, and the bound protein was eluted using 20 mM sodium acetate, 150 mM NaCl, pH 3.5. The eluate was neutralized with 10 v / v% 1 M Tris pH 8.0 and analyzed by SDS-PAGE. The fraction containing CD200-Fc was pooled, concentrated to 5 mL, and subjected to gel filtration chromatography (Hiload 16 / 600 Superdex-200pg column) on the AKTA Pure platform. The protein was treated at 1.2 mL / min in a 50 mM sodium phosphate, 150 mM NaCl, pH 7.4 buffer system, and each fraction was collected. The fraction containing the CD200-Fc dimer was pooled after analysis by SDS-PAGE and concentrated to 1.33 mg / mL (measured at UV280 nm) using Amicon Ultra Centricon (10 kDa molecular weight cutoff). The purified material was subjected to SEC-HPLC, LC-MS, and EndoSafe LAL cartridge testing to evaluate protein purity, molecular weight, and endotoxin content, respectively. The final sample was stored at -80°C.

[0179] Example 11: Cell Binding and Activation Assay of Wild-Type and Mutant CD200-Fc Proteins Assay principle To demonstrate the agonist activity of ARQ-234, human monocyte cell line U937 (ATCC, CRL1539) was transfected with human CD200R cDNA. Cytokine production, including IL-6, from these cells could be induced by stimulation with PMA followed by stimulation with LPS.

[0180] Protocol Cell line construction, cytokine inhibition testing, and cell binding testing were performed using the same methods as in Example 3 above.

[0181] Cytokine release (IL-6 inhibition) assay 50,000 U937 cells per well were seeded in a 96-well plate and differentiated after 72 hours of incubation in 100 nM PMA. After differentiation, the PMA-containing medium was replaced with fresh assay medium and incubated for a further 2 hours before processing. CD200-Fc constructs were added to the cell cultures with or without Fc blocks and incubated for 1 hour. Cells were then stimulated with 100 ng / ml LPS and incubated for a further 24 hours. After the final incubation, the cell supernatant (1:10 dilution) was collected and IL-6 secretion was quantified by ELISA assay using a commercially available kit.

[0182] [Table 8]

[0183] result The data shown in Figure 23 demonstrate that ARQ-234 can inhibit LPS-induced IL-6 secretion in a concentration-dependent manner. No significant difference in inhibition was observed in vitro in the presence of the Fc blocking reagent (Figure 23, top panel). This is surprising, as the IgG4 Fc domain of ARQ-234 should bind to the Fc gamma receptor, thereby increasing the avidity of its interaction with CD200R, and this mechanism may further enhance the potency of the CD200-Fc protein in vivo.

[0184] Figure 24 shows the binding of wild-type DS-155 and mutant ARQ-234 CD200-Fc proteins to CD200R-expressing U937 cells. This data demonstrates superior binding of ARQ-234 to CD200R-expressing cells compared to DS-155 (wild-type CD200-Fc protein) at all time points.

[0185] Example 12: Pharmacokinetic study of ARQ-234 in the serum of cynomolgus monkeys Protocol Two cynomolgus monkeys per group (one male and one female) were administered an intravenous bolus of protein at time 0, at a dose of 5 mg / kg. Blood samples were collected for PK analysis at the following time points: before administration, 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, and day 28.

[0186] Serum samples for PK analysis At least 0.8 mL of blood samples were collected from the cephalic vein or saphenous vein of two animals at the sampling time. For samples collected within the first hour of administration, a ±1 minute error in sample collection time was acceptable. For remaining time points, samples collected within 5% of the scheduled time were acceptable. All blood samples were collected in commercially available tubes containing a coagulant. The tubes containing the blood samples were kept at room temperature for 30 minutes and then centrifuged. Samples were centrifuged at 1500 × g at 4°C for 10 minutes within one hour of collection. After centrifugation, approximately 400 μL of serum was recovered per time point. The samples were then rapidly frozen on dry ice and kept below -60°C until transferred to dry ice for analysis. All samples were uniquely identified to indicate their origin and collection time.

[0187] Determination of protein concentration in serum The concentration of the analyte in serum was determined using a bioanalytic ELISA method. 96-well ELISA plates were coated overnight at 4°C with 1 μg / ml goat anti-human IgG in carbonic acid-bicarbonate buffer. After washing and blocking, serially diluted plasma samples were added, followed by biotin-labeled goat anti-human IgG (0.0625 μg / mL) as the detection antibody. HRP-streptavidin and TMB substrate were used for color development. The reaction was stopped by adding 2 M HCl after approximately 5–10 minutes. Absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer (SpectraMax® M5e). Plasma concentrations were obtained by substituting the OD values ​​of the samples into a standard curve. The detection limit LLOQ for Fc+Fc using this ELISA method is 1 ng / mL.

[0188] Serum concentrations of ARQ-234 in monkeys were subjected to non-compartmental pharmacokinetic analysis using Phoenix WinNonlin software (version 8.1, Pharsight, Mountain View, CA). The linear / logarithmic trapezoidal law was applied when obtaining PK parameters.

[0189] result The data shown in Table 9 and Figure 25 demonstrate the good serum stability of ARQ-234, with an average half-life of 370.5 hours in the two animals tested.

[0190] As shown in Table 10, serum levels of ARQ-234 decreased more rapidly initially, but the clearance rate decreased more slowly over time, with a mean half-life of 15.5 days for ARQ-234. DS-118 without the LS mutation had a mean half-life of 4.4 days. As shown in Figure 23, ARQ-234 inhibited IL-6 release in an in vitro cell assay of U937-CD200R. The mean volume of distribution was 148 mL / kg.

[0191] [Table 9]

[0192]

Table 10

[0193] The following parameters were tested in Tables 9 and 10. Half-life (T 1 / 2 ), maximum serum concentration (C max ), area under the serum concentration curve 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). The half-life was calculated excluding data less than 1% of Cmax. The half-life was not accurate if AUC_%Extrap_obs exceeded 20% or if Rsq_adjusted was less than 0.9.

[0194] Example 13: Cellular assay binding analysis of a high-affinity CD200-Fc molecule A diagram of ARQ-234, an Fc fusion protein of the present invention, is shown in FIG. 27. Binding of ARQ-234 to U937-CD200R cells was compared with wild-type control CD200-Fc (DS-155) at human physiological temperature (37°C) for 1 hour, 4 hours, and 24 hours, and binding was detected using flow cytometry with an anti-human IgG antibody. As shown in FIG. 24, binding of ARQ-234 to U937-CD200R cells showed stronger target binding compared to the wild-type huCD200-Fc construct DS-155.

[0195] Any of the above protocols or similar variations thereof may be described in various documents related to pharmaceuticals. Such documents may include, but are not limited to, protocols, statistical analysis plans, investigational drug brochures, clinical guidelines, medical guides, risk assessment and mitigation programs, prescribing information, and other documents that may be related to pharmaceuticals. In particular, it is contemplated that such documents may be physically packaged as a kit together with the pharmaceuticals according to the present disclosure, if beneficial or as determined by a regulatory authority.

[0196] The subject matter of this disclosure is described and shown in considerable detail with reference to certain exemplary embodiments, including various combinations and partial combinations of features, but those skilled in the art will readily understand other embodiments, as well as their variations and modifications, that are included within the scope of this disclosure. Furthermore, the description of such embodiments, combinations, and partial combinations is not intended to indicate that the claimed subject matter requires features or combinations of features other than those expressly described in the claims. Accordingly, the scope of this disclosure is intended to include all modifications and modifications that are included within the spirit and scope of the appended claims below.

Claims

1. (i) A mutant CD200 moiety containing mutations in amino acid residues 130 and 131, (ii) non-CD200 portion; A fusion protein containing, The mutations are K130Y, I131Y, or combinations thereof. The non-CD200 is a human Fc fragment, The aforementioned dimer construct is a fusion protein having a serum half-life of 5 to 900 hours.

2. (A) A first fusion polypeptide, (i) A mutant CD200 moiety containing mutations in amino acid residues 130 and 131, (ii) non-CD200 portion; and The mutations are K130Y, I131Y, or combinations thereof. The aforementioned non-CD200 is a human Fc polypeptide. The first fusion polypeptide and, (B) A second fusion polypeptide, (i) A mutant CD200 moiety containing mutations in amino acid residues 130 and 131, (ii) non-CD200 portion; and The mutations are K130Y, I131Y, or combinations thereof. The aforementioned non-CD200 is a human Fc polypeptide. The second fusion polypeptide, A dimerized construct comprising, A dimer construct comprising the first fusion polypeptide and the second fusion polypeptide, wherein the first fusion polypeptide and the second fusion polypeptide are dimerized via the human Fc polypeptide, and the dimer construct has a serum half-life of 5 to 900 hours.

3. A dimer construct comprising two fusion polypeptides, each polypeptide comprising (i) a mutant CD200 moiety having mutations in amino acid residues 130 and 131, and (ii) a non-CD200 moiety, wherein the mutations are K130Y, I131Y, or a combination thereof, and the non-CD200 moiety is a human Fc polypeptide. The two fusion polypeptides, the human Fc polypeptides, are linked to each other via at least one disulfide bond to form a homodimer. The dimer construct is a dimer construct having a serum half-life of 5 to 900 hours.

4. A fusion protein according to claim 1, or a dimer construct according to claim 2 or 3, having a half-life of 5 to 300 hours.

5. A fusion protein according to claim 1, or a dimer construct according to claim 2 or 3, having a half-life of 444 to 900 hours.

6. A fusion protein according to claim 1, or a dimer construct according to claim 2 or 3, having a half-life of 400 to 650 hours.

7. A fusion protein according to claim 1, or a dimer construct according to any one of claims 2 to 6, wherein the fusion protein or dimer construct has an AUC of 0.5 to 50,000 μg / day / ml in serum at a dose of 5 mg / kg.

8. A fusion protein according to claim 1, or a dimer construct according to any one of claims 2 to 6, wherein the fusion protein or dimer construct has an AUC of 0.5 to 12,926 μg / day / ml in serum at a dose of 5 mg / kg.

9. A fusion protein according to claim 1, or a dimer construct according to any one of claims 2 to 6, wherein the fusion protein or dimer construct has an AUC of 12,928 to 12,960 μg / day / ml in serum at a dose of 5 mg / kg.

10. A fusion protein according to claim 1, or a dimer construct according to any one of claims 2 to 9, wherein the fusion protein or dimer construct has a Cmax of 40 to 400 μg / ml or 50 to 300 μg / ml in serum at a dose of 5 mg / kg.

11. A fusion protein according to claim 1, or a dimer construct according to any one of claims 2 to 9, wherein the fusion protein or dimer construct has a Cmax of 100 to 200 μg / ml in serum at a dose of 5 mg / kg.

12. A fusion protein according to claim 1, or a dimer construct according to any one of claims 2 to 9, wherein the fusion protein or dimer construct has a Cmax of 120 to 150 μg / ml in serum at a dose of 5 mg / kg.

13. A dimer construct according to any one of claims 2 to 12, wherein the human Fc fragment of the first fusion polypeptide and / or the second fusion polypeptide comprises a hinge region.

14. A dimer construct according to any one of claims 2 to 13, wherein the non-CD200 portion of the first fusion polypeptide and / or the second fusion polypeptide is an Fc fragment of mutant IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM.

15. A dimer construct according to any one of claims 2 to 14, wherein the human Fc fragment of the first fusion polypeptide and / or the second fusion polypeptide comprises at least one Fc domain.

16. A dimer construct according to claim 15, wherein at least one Fc domain is selected from human IgG1 domain, human IgG2 domain, human IgG3 domain, human IgG4 domain, human IgA domain, human IgE domain, and human IgM domain.

17. A dimer construct according to claim 15 or 16, wherein at least one Fc domain is a human IgG4 Fc domain.

18. A dimer construct according to claim 15 or 16, wherein the at least one Fc domain is human IgG1 (ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK A dimerized construct comprising a sequence that is at least 80% identical to FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 8).

19. A dimer construct according to claim 15 or 16, wherein the at least one Fc domain comprises a sequence that is at least 90% identical to human IgG1 (SEQ ID NO: 8).

20. A dimer construct according to claim 15 or 16, wherein at least one Fc domain comprises Sequence ID No.

8.

21. A dimer construct according to any one of claims 2 to 20, wherein the first fusion polypeptide has at least one mutation at at least one glycosylation site compared to a fusion polypeptide comprising a wild-type CD200 moiety and a wild-type non-CD200 moiety.

22. A dimer construct according to any one of claims 2 to 21, wherein the second fusion polypeptide has at least one mutation at at least one glycosylation site compared to a fusion polypeptide comprising a wild-type CD200 moiety and a wild-type non-CD200 moiety.

23. A dimer construct according to any one of claims 2 to 22, wherein each of the first fusion polypeptide and the second fusion polypeptide has a lower affinity for at least one Fc-gamma receptor compared to a fusion polypeptide comprising a non-CD200 moiety having a wild-type human IgG Fc fragment.

24. A dimer construct according to any one of claims 2 to 23, wherein the first fusion polypeptide and / or the second fusion polypeptide comprises a deletion of the first five amino acids of the non-CD200 portion compared to the wild-type non-CD200 portion, the deletion being located in the hinge region of the human Fc fragment.

25. A dimer construct according to any one of claims 2 to 24, further comprising a linker between the mutant CD200 portion of the first fusion polypeptide and the non-CD200 portion of the first fusion polypeptide.

26. A dimer construct according to any one of claims 2 to 25, further comprising a linker between the mutant CD200 portion of the second fusion polypeptide and the non-CD200 portion of the second fusion polypeptide.

27. A dimer construct according to any one of claims 2 to 26, wherein the glycine 232 of the mutant CD200 portion of the first fusion polypeptide is directly fused to the non-CD200 IgG4 Fc fragment of the first fusion polypeptide at the 6th amino acid according to the IMGT numbering system.

28. A dimer construct according to any one of claims 2 to 27, wherein the glycine 232 of the mutant CD200 portion of the second fusion polypeptide is directly fused to the non-CD200 IgG4 Fc fragment of the second fusion polypeptide at the 6th amino acid according to the IMGT numbering system.

29. A dimer construct according to any one of claims 2 to 28, wherein the first fusion polypeptide and / or the second fusion polypeptide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:

7.

30. A dimer construct according to any one of claims 2 to 29, wherein the first fusion polypeptide and / or the second fusion polypeptide further comprises an N-terminal signal sequence, the N-terminal signal sequence comprising the amino acid sequence MEFGLSWLFLVAILKGVQC (SEQ ID NO: 3).

31. A dimer construct according to any one of claims 2 to 30, which has a lower affinity for at least one of FcγRI, FcγRIIA, FcγRIIIIA, or C1q receptors compared to a fusion protein comprising a non-CD200 portion having an Fc fragment of wild-type human IgG.

32. A fusion protein according to claim 1, or a dimer construct according to claim 2 or 3, wherein the human Fc polypeptide is wild-type and free of mutations.

33. A polynucleotide encoding a fusion protein or fusion polypeptide according to any one of claims 1 to 13.

34. A polynucleotide according to claim 33, wherein the polynucleotide has at least 80% sequence identity with SEQ ID NO:

4.

35. A polynucleotide encoding the first fusion polypeptide of the dimer construct according to any one of claims 2 to 32.

36. A polynucleotide encoding the second fusion polypeptide of the dimer construct according to any one of claims 2 to 32.

37. An expression system comprising at least one expression vector containing a polynucleotide as described in any one of claims 33 to 36.

38. A host cell comprising the expression system described in claim 37.

39. A composition comprising a fusion protein or dimer construct according to any one of claims 1 to 31, or a polynucleotide according to any one of claims 33 to 36, and a pharmaceutically acceptable carrier.

40. A method for treating a subject having an autoimmune disease, an allergic disease, a neurodegenerative disorder, neuropathic pain, an inflammatory disorder, a skin disease, or diabetic neuropathy, comprising administering a fusion protein or dimer construct according to any one of claims 1 to 32, or a polynucleotide according to any one of claims 33 to 36.

41. A method for treating a subject having rheumatoid arthritis, asthma, atopic dermatitis, inflammatory arthralgia, chronic obstructive pulmonary disease, or Parkinson's disease, comprising administering a fusion protein or dimer construct according to any one of claims 1 to 32, or a polynucleotide according to any one of claims 33 to 36.

42. A method for treating a subject having an autoimmune disease affecting the neuromuscular system, vascular system, eyes, skin, gastrointestinal tract, lungs, kidneys, liver, peripheral or central nervous system, bones, cartilage, or joints, comprising administering a fusion protein or dimer construct according to any one of claims 1 to 32, or a polynucleotide according to any one of claims 33 to 36.

43. A method for treating a subject having a skin disease, comprising administering a fusion protein or dimer construct according to any one of claims 1 to 32, or a polynucleotide according to any one of claims 33 to 36.

44. A method according to claim 43, wherein the skin disease is dermatitis.

45. A method according to any one of claims 40 to 44, wherein the fusion protein, the dimer construct, or the polynucleotide is administered as the sole therapeutic agent.

46. A method according to any one of claims 40 to 44, wherein the fusion protein, the dimer construct, or the polynucleotide is administered in combination with one or more other pharmaceuticals indicated for the treatment of autoimmune diseases, allergic diseases, neurodegenerative disorders, neuropathic pain, inflammatory disorders, or diabetic neuropathy.

47. A method according to any one of claims 40 to 44, wherein the fusion protein, the dimer construct, or the polynucleotide is administered in combination with one or more immunosuppressants or adjuvants in immunosuppressive therapy.

48. The method according to claim 47, wherein the dimer construct is administered in combination with azathioprine, methotrexate, cyclosporine, a monoclonal antibody, a corticosteroid, or a combination thereof.

49. The method according to claim 48, wherein the monoclonal antibody is basiliximab, daclizumab, or muromonab.

50. A fusion protein or dimer construct according to any one of claims 1 to 32, a polynucleotide according to any one of claims 33 to 36, or a composition according to claim 39, for use in the treatment of autoimmune diseases, allergic diseases, neurodegenerative disorders, neuropathic pain, inflammatory disorders, skin diseases, or diabetic neuropathy.

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