Non-sialylated anti-inflammatory peptides

Abstract

To provide anti-inflammatory agents, to provide anti-inflammatory compositions, and to provide methods for treating inflammatory diseases.SOLUTION: Provided is a polypeptide having an amino acid sequence having at least 90% identity to a specific sequence, in which amino acid residue 241 (numbered according to the EU index of Kabat) of the amino acid sequence in the peptide is alanine, exhibiting binding to DC-SIGN and hFcγRIIB.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 61 / 577,361, filed on 19 December 2011. The contents of the said application are incorporated herein by reference in their entirety.

[0002] Government rights The inventions disclosed herein were, at least in part, completed with government support under grant number NIH AI035875 from the National Institutes of Health. Accordingly, the U.S. Government has certain rights in these inventions.

[0003] Field of Invention The present invention relates to anti-inflammatory agents, compositions, and methods for treating inflammatory diseases. [Background technology]

[0004] background Inflammatory diseases, including autoimmune diseases, are conditions characterized by abnormal activation of white blood cells and their subsequent migration to affected areas of the body. These conditions encompass a wide range of illnesses that affect the lives of millions of people worldwide. While various treatments are currently available, many have significant side effects or are not sufficiently effective to alleviate all symptoms. Therefore, there is a demand for anti-inflammatory drugs to treat inflammatory diseases, and a demand for methods to identify and evaluate such drugs.

[0005] Immunoglobulin G (IgG) has long been recognized to mediate both pro- and anti-inflammatory activities through interactions mediated by its Fc fragment. The Fc-FcγR interaction is responsible for the pro-inflammatory properties of immune complexes and cytotoxic antibodies, while intravenous gamma globulin (IVIG) and its Fc fragment are anti-inflammatory and are widely used to suppress inflammatory diseases. It has been proposed that the glycosylation of IgG is important for the regulation of the cytotoxic and inflammatory capacity of IgG. For example, the anti-inflammatory activity of IVIG is a property of the Fc fragment and its conjugated glycan, requires terminal α2,6 sialic acid linkage, and indicates that a specific polypeptide backbone and glycan structure are required in combination for immunosuppression (Anthony et al., 20 / 08, Science 320:373-376, and WO2007 / 117505).

[0006] However, only a small population of IgG in IVIG has glycans (sFc) terminated with α2,6 sialic acid and anti-inflammatory activity. As a result, in order to suppress inflammation caused by autoantibodies in various clinical situations, IVIG must be administered at high doses (1-2 g / kg) to enhance sialylated IgG or, if not, the sialylation of IgG must be increased (US Applications Nos. 20080206246, and 20090004179, and Nimmerjahn et al. Annu Rev Immunol 26,513-533 (2008)).

[0007] The present invention addresses and responds to the above requirements by identifying non-sialylated anti-inflammatory peptides. SUMMARY OF THE INVENTION

[0008] Summary The present invention relates to polypeptides and antibodies, and methods for treating inflammatory diseases, such as autoimmune diseases.

[0009] Accordingly, one aspect of the present invention features an isolated polypeptide containing a modified sequence homologous to the IgG Fc region by at least 75% (e.g., any number between 75% and 100%, encompassing 70%, 80%, 85%, 90%, 95%, 99%, and 100%). The modified sequence is not sialylated, and the polypeptide has higher anti-inflammatory activity than that of the parent polypeptide. The parent polypeptide may contain an IgG Fc region such as the sequence of Sequence ID No. 1 shown below. In some embodiments, the polypeptide has the ability to bind to DC-SIGN and to hFcγRIIA or RIIB. In one embodiment, the isolated polypeptide is 2 × 10⁻¹⁶ -5 K below M D (That is, 5.0 × 10 4 M -1 The above K A ) has the ability to bind to hFcγRIIA or RIIB. Preferably, the modified sequence has the FA241 mutation. The modified sequence may be at least 75% homologous to SEQ ID NO: 2 (e.g., any number between 75% and 100%, encompassing 70%, 80%, 85%, 90%, 95%, 99%, and 100%). In some examples, the modified sequence includes SEQ ID NO: 2 or consists essentially of SEQ ID NO: 2.

[0010] In another aspect, the present invention provides a method for producing polypeptides having anti-inflammatory activity. This method includes, among other things, the steps of providing a parent polypeptide having a sequence of IgG Fc regions, or a first nucleic acid sequence encoding the parent polypeptide; and modifying the parent polypeptide to obtain a modified polypeptide such that the modified polypeptide is not sialylated and mimics the structure of a sialylated form of the IgG Fc region. The modification step may be carried out by modifying the first nucleic acid sequence to obtain a second nucleic acid encoding the modified polypeptide. The present invention also provides polypeptides produced by the method described immediately above.

[0011] In a third embodiment, the present invention is characterized by an isolated nucleic acid comprising a sequence encoding the polypeptide; an expression vector comprising the nucleic acid; and a host cell comprising the nucleic acid. The present invention is also characterized by a method for producing the polypeptide. The method comprises culturing host cells in a culture medium under conditions that enable the expression of the polypeptide encoded by the nucleic acid, and purifying the polypeptide from the cultured cells or the culture medium of the cells.

[0012] In a fourth embodiment, the present invention is characterized by a pharmaceutical formulation comprising (i) the polypeptide or nucleic acid described above, and (ii) a pharmaceutically acceptable carrier.

[0013] In a fifth aspect, the present invention provides a method for treating an inflammatory disease, comprising administering a therapeutically effective amount of the polypeptide or nucleic acid encoding the polypeptide to a subject in need. The use of polypeptides or nucleic acids in the manufacture of pharmaceuticals for treating inflammatory diseases is also provided. The present invention also features isolated polypeptides, nucleic acids, expression vectors, host cells, compositions, or methods for treating inflammatory diseases, as substantially shown and described herein.

[0014] Details of one or more embodiments of the present invention are described below. Other features, objects, and advantages of the present invention will be apparent from the specification and claims. [Brief explanation of the drawing]

[0015] [Figure 1A] Figures 1a-c are diagrams and photographs showing that α2,6-linked sialic acid conferred DC-SIGN binding activity to recombinant human IgG1 Fc. [Figure 1B] Figures 1a-c are diagrams and photographs showing that α2,6-linked sialic acid conferred DC-SIGN binding activity to recombinant human IgG1 Fc. [Figure 1C]Figures 1a-c are diagrams and photographs showing that α2,6-linked sialic acid conferred DC-SIGN binding activity to recombinant human IgG1 Fc. [Figure 2A1] Figures 2a-b are diagrams and photographs showing that DC-SIGN binding activity was conferred to recombinant human IgG1 Fc by interfering with Fc-glycan interactions. [Figure 2A2] Figures 2a-b are diagrams and photographs showing that DC-SIGN binding activity was conferred to recombinant human IgG1 Fc by interfering with Fc-glycan interactions. [Figure 2B] Figures 2a-b are diagrams and photographs showing that DC-SIGN binding activity was conferred to recombinant human IgG1 Fc by interfering with Fc-glycan interactions. [Figure 3] Figure 3 is a set diagram showing that the FA241 mutation in hIgG1 Fc replicates the anti-inflammatory activity of α2,6 sFc. [Figure 4] Figures 4a-d show the characteristic requirements for FA241 anti-inflammatory activity. [Figure 5A] Figure 5 is a set of figures showing that the FA241 mutation increased Fcγ receptor binding. [Figure 5B] Figure 5 is a set of figures showing that the FA241 mutation increased Fcγ receptor binding. [Figure 5C] Figure 5 is a set of figures showing that the FA241 mutation increased Fcγ receptor binding. [Figure 6] Figures 6a-b are photographs showing FA241-induced IL-33 mRNA in bone marrow-derived macrophages. [Modes for carrying out the invention]

[0016] Detailed explanation This invention is based, at least in part, on the unexpected discovery that non-sialylated IgG Fc variants confer anti-inflammatory activity and mimic the effects of 2,6-sialylated Fc as anti-inflammatory mediators.

[0017] IgG and Fc sialylation IgG is the major serum immunoglobulin. It is a glycoprotein consisting of two identical heavy chains and two light chains, sequentially composed of variable and constant domains. IgG has an Asn in the CH2 domain on each of its two heavy chains. 297It contains a single N-linked glycan. The covalently linked complex carbohydrate consists of a core containing N-acetylglucosamine (GlcNAc) and mannose (man), and a branched pentapolysaccharide. Further modifications of the core carbohydrate structure are observed in serum antibodies due to the presence of fucose, branched GlcNAc, galactose (gal), and variably found terminal sialic acid (sa) moieties. More than 40 different sugar types have been detected covalently linked to this single glycosylation site (Fujii et al., J. Biol. Chem. 265, 6009, 1990). IgG glycosylation has been shown to be essential for binding to all FcyRs by maintaining the open conformation of the two heavy chains. Jefferis and Lund, Immune.1 Lett. 82,57 (2002); Sondermann et al., J.Mol.Biol. 309,737 (2001). This IgG glycosylation for FcyR binding is considered a major reason why deglycosylated IgG antibodies cannot mediate in vivo induced inflammatory responses such as ADCC, phagocytosis, and the release of inflammatory mediators. Nimmerjahn and Ravetch, Immunity 24,19 (2006). Further findings suggest that individual glycosphagia of IgG may contribute to modulating inflammatory responses, as indicated by the altered affinity for individual FcyRs reported for fucose-containing or fucose-deficient IgG antibodies, and their consequent effects on cytotoxicity. Shields et al., J. Biol. Chem. 277, 26733 (2002); Nimmerjahn and Ravetch, Science 310, 1510 (2005). An association between autoimmune status and specific glycosylation patterns of IgG antibodies has been observed in patients with rheumatoid arthritis and several autoimmune vasculitises in which reduced galactosylation and sialylation of IgG antibodies have been reported.Parekh et al., Nature 316,452 (1985); Rademacher et al., Proc. Natl. Acad. Sci. USA 91,6123 (1994); Matsumoto et al., 128,621 (2000); Holland et al., Biochim. Biophys. Acta, December 27. IgG glycotype diversity has also been reported to be associated with aging and immunization, but the in vivo significance of these changes has not been elucidated. Sikata et al., Glycoconj. J. 15,683 (1998); Lastra et al., Autoimmunity 28,25 (1998).

[0018] As disclosed herein, certain non-sialylated IgG Fc variants also surprisingly confer anti-inflammatory activity. Such variants, including the FA241 variant, represent a species within a larger genus of molecules that can be developed as anti-inflammatory therapeutics without requiring sialylation, by mimicking the structural and biological properties of sialylated Fc.

[0019] polypeptides and nucleic acids Polypeptide As disclosed herein, the present invention relates to the above Asn 297 The present invention provides an isolated polypeptide having a sequence of a human IgG Fc variant lacking a polysaccharide chain with a terminal sialic acid connected to the galactose moiety via an α2,6 linkage. Such non-sialylated IgG Fc variants may be derived from naturally occurring antibodies or expressed in cell lines.

[0020] In one embodiment, the Fc region includes one or more substitutions of the hIgG1 amino acid sequence. A typical IgG1 Fc region is provided below, but is not limited to this:

[0021] [ka]

[0022] [ka]

[0023] [ka]

[0024] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to describe the arrangement of amino acid residues in a polymer. Peptides, polypeptides, or proteins may consist of the standard 20 naturally occurring amino acids, plus rare amino acids and synthetic amino acid analogs. They can be any chain of amino acids, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). “The peptides, polypeptides, or proteins of this invention” include recombinant or synthetically produced versions having specific domains or moieties that bind to DC-SIGN, FcγRIIA, and FcγRIIB. The term also encompasses polypeptides having an added amino-terminal methionine (useful for expression in prokaryotic cells).

[0025] An "isolated" polypeptide or protein refers to a polypeptide or protein that has been isolated from other proteins, lipids, and nucleic acids to which it is naturally bound. Polypeptides / proteins can constitute at least 10% (i.e., any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 99%) of the dry weight of the purified preparation. Purity can be measured by any suitable standard method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. The isolated polypeptides / proteins described in this invention may be purified from natural sources and produced by recombinant DNA technology or chemical methods. A functional equivalent of IgG Fc means an IgG Fc polypeptide derivative, e.g., a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof. It substantially retains the activity of IgG Fc, i.e., its ability to bind to its respective receptor and induce its respective cellular response. The isolated polypeptide may contain SEQ ID NO: 2. Generally, the functional equivalent is at least 75% homologous to SEQ ID NO: 2 (e.g., any number between 75% and 100%, encompassing 70%, 80%, 85%, 90%, 95%, and 99%).

[0026] The "homology percentage" of two amino acid sequences or two nucleic acids is determined using the algorithm of Karlin and Altschul Proc.Natl.Acad.Sci.USA 87:2264-68, 1990, modified in Karlin and Altschul Proc.Natl.Acad.Sci.USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al. J.Mol.Biol. 215:403-10, 1990. BLAST nucleotide searching can be performed using the NBLAST program, score = 100, word length -12, to obtain nucleotide sequences homologous to the nucleic acid molecule of the present invention. BLAST protein search can be performed using the XBLAST program, score=50, and word length=3 to obtain amino acid sequences homologous to the protein molecule of the present invention. If a gap exists between two sequences, Gapped BLAST can be used as described by Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using the BLAST and Gapped BLAST programs, the initial settings parameters of each program (e.g., XBLAST and NBLAST) may be used.

[0027] The amino acid composition of the polypeptides described herein may be altered without disrupting the polypeptide's ability to bind to its respective receptor and induce its respective cellular response. For example, one or more conservative amino acid substitutions may be included. A “conservative amino acid substitution” is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been revealed in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, for example, the non-essential amino acid residue predicted in SEQ ID NO: 2 is preferably replaced with another amino acid residue from the same side-chain family. Alternatively, mutations can be introduced randomly along all or part of the sequence by saturated mutagenesis, and the resulting mutants can be screened for their ability to bind to their respective receptors and induce their respective cellular responses in order to identify mutants that retain the activity described later in the examples.

[0028] Polypeptides as described in the present invention can be obtained as recombinant polypeptides. To prepare a recombinant polypeptide, the nucleic acid encoding it (e.g., FA241, SEQ ID NO: 2) can be ligated to another nucleic acid encoding a fusion partner, such as glutathione-S-transferase (GST), a 6×His epitope tag, or the M13 gene 3 protein. The resulting fusion nucleic acid expresses a fusion protein in a suitable host cell, which can be isolated by methods known in the art. The isolated fusion protein can be further treated, for example, by enzymatic digestion, to remove the fusion partner and obtain the recombinant polypeptide of the present invention.

[0029] nucleic acid Another aspect of the present invention features an isolated nucleic acid comprising a sequence encoding the polypeptide or protein described above. A nucleic acid refers to a DNA molecule (e.g., cDNA or genomic DNA), an RNA molecule (e.g., mRNA), or an analog of DNA or RNA. DNA or RNA analogs may be synthesized from nucleotide analogs. Nucleic acid molecules may be single-stranded or double-stranded, but are preferably double-stranded DNA. “Isolated nucleic acid” means a nucleic acid having a structure that is not homologous to any naturally occurring nucleic acid or any fragment of a naturally occurring genomic nucleic acid. Therefore, this term is, for example, (a) DNA having a sequence of part of a naturally occurring genomic DNA molecule but not flanked by both of the flank coding sequences of that part of the molecule in the genome of a naturally occurring organism; (b) nucleic acids incorporated into a vector or into the genomic DNA of a prokaryotic or eukaryote in such a way that the resulting molecule is not homologous to any naturally occurring vector or genomic DNA; (c) separate molecules such as cDNA, genomic fragments, fragments or restriction fragments produced by polymerase chain reaction (PCR); and (d) recombinant nucleotide sequences that are part of a hybrid gene, i.e., a gene encoding a fusion protein. The above nucleic acids can be used to express the fusion protein of the present invention. For this purpose, nucleic acids can be operatively ligated to appropriate regulatory sequences to create an expression vector.

[0030] A vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. A vector may be self-replicating or integrable into host DNA. Examples of vectors include plasmids, cosmids, or viral vectors. A vector contains nucleic acid in a form suitable for nucleic acid expression in host cells. Preferably, a vector contains one or more regulatory sequences operatively ligated to the nucleic acid sequence to be expressed.

[0031] A “regulatory sequence” includes promoters, enhancers, and other expression regulators (e.g., polyadenylation signals). Regulatory sequences include those that lead to constitutive expression of nucleotide sequences, as well as tissue-specific regulatory and / or inducible sequences. The design of an expression vector may depend on factors such as the selection of host cells to be transformed and the expression level of the desired protein or RNA. The expression vector can be introduced into host cells that produce the polypeptide of the present invention. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one that initiates mRNA at a high frequency.

[0032] Any of the polynucleotides described above, or any bioequivalent polynucleotides available to those skilled in the art for the same intended purpose, may be inserted into a suitable expression vector and ligated with other DNA molecules to form a “recombinant DNA molecule” that expresses this receptor. These vectors may consist of DNA or RNA; most DNA vectors for cloning purposes are preferred. Typical vectors include plasmids, modified viruses, bacteriophages and cosmids, yeast artificial chromosomes, and other forms of episomal or integrated DNA. This is within the scope of knowledge for those skilled in the art to determine a suitable vector for a particular use.

[0033] Various mammalian expression vectors may be used to express the above-mentioned IgG Fc in mammalian cells. As mentioned above, an expression vector may be a DNA sequence required for the transcription of cloned DNA and the translation of its mRNA in a suitable host. Such vectors may be used to express eukaryotic DNA in a variety of hosts such as bacteria, cyanobacteria, plant cells, insect cells, and animal cells. Specially designed vectors allow for the round trip of DNA between hosts, such as bacterial-yeast or bacterial-animal cells. A well-constructed expression vector should include: an origin of replication for self-replication in host cells, a selection marker, a limited number of useful restriction enzyme sites, high copy number capability, and an active promoter. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specially designed plasmids, or viruses. Suitable commercially available mammalian expression vectors include, but are not limited to, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Biolabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), and pRSVneo (ATCC Examples include 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and IZD35 (ATCC 37565).

[0034] Host cells containing the above nucleic acids are also within the scope of the present invention. Examples include E. coli cells, insect cells (e.g., using baculovirus expression vectors), yeast cells, or mammalian cells. See, for example, Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California. To produce the polypeptide of the present invention, the host cells can be cultured in a medium under conditions that allow expression of the polypeptide encoded by the nucleic acid of the present invention, and the polypeptide can be purified from the cultured cells or the cell medium. Alternatively, the nucleic acid of the present invention can be transcribed and translated in vitro, for example, using the T7 promoter regulatory sequence and T7 polymerase.

[0035] All of the naturally occurring IgG Fc, genetically engineered IgG Fc, and chemically synthesized IgG Fc can be used to carry out the invention disclosed herein. The IgG Fc obtained by recombinant DNA technology may have the same amino acid sequence as [FA241] SEQ ID NO: 2 or a functional equivalent thereof. The term "IgG Fc" also includes chemically modified forms. Examples of chemically modified IgG Fc include IgG Fc that has undergone a conformational change, addition or deletion of sugar chains, and IgG Fc to which a compound such as polyethylene glycol is bound.

[0036] The effectiveness of the polypeptide / protein thus produced can be verified using an animal model such as a transgenic mouse, as described below. Any statistically significant increase in the in vivo expression of IL-33 basophils or the expression of the FcγRIIB receptor on effector macrophages indicates that the polypeptide / protein is a candidate for treating the diseases described below. In one embodiment, the above assay may be based on the measurement of the binding of the DC-SIGN protein or DC-SIGN (+) to cells. This technique is based on DC-SIGN or DC-SIGN (+)A wealth of techniques are available to those skilled in the art, suitable for measuring the ability of compounds to reach cells and the associated changes in the expression of genes regulated by the DC-SING pathway, such as IL-33. Those skilled in the art will be able to combine and adapt these various research tools without excessive experimentation. After purification and testing according to standard methods or the assays and methods described in the examples below, non-sialyzed IgG Fc variants can also be incorporated into pharmaceutical compositions for the treatment of inflammatory diseases.

[0037] As used herein, "antibody" is used in its broadest sense and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, insofar as they exhibit the desired biological activity.

[0038] As used herein, “antibody fragment” may include a portion of an intact antibody, and generally includes the antigen-binding or variable region of the intact antibody, or the Fc region of the antibody that retains FcR-binding ability. Examples of antibody fragments include linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The antibody fragment preferably contains at least the hinge portion and optionally the CH1 region of the IgG heavy chain. More preferably, the antibody fragment contains the entire constant region of the IgG heavy chain and includes the IgG light chain.

[0039] As used herein, the terms “Fc fragment” or “Fc region” are used to define the C-terminal region of an immunoglobulin heavy chain. The “Fc region” may be a native sequence Fc region or a mutant Fc region. While the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus.

[0040] The “natural sequence Fc region” includes an amino acid sequence homologous to the amino acid sequence of an Fc region found in nature. As will be understood by those skilled in the art, the “mutant Fc region” includes an amino acid sequence different from that of the natural sequence Fc region by at least one “amino acid modification.” Preferably, the mutant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, compared to the natural sequence Fc region or the Fc region of the parent polypeptide, preferably about 1 to about 5 amino acid substitutions in the natural sequence Fc region or the Fc region of the parent polypeptide. The mutant Fc region as described herein preferably has at least about 75 or 80% homology with the natural sequence Fc region and / or the Fc region of the parent polypeptide, more preferably at least about 90% homology, more preferably at least about 95% homology, and even more preferably at least about 99% homology.

[0041] The term “Fc receptor” or “FcR” is used to describe receptors that bind to the Fc region of an antibody. In one embodiment of the present invention, FcR is the natural sequence human FcR. In another embodiment, FcR, including human FcR, binds to an IgG antibody (gamma receptor) and includes the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), whose cytoplasmic domains have similar amino acid sequences that differ primarily. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activating motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See the general overview by Daron, Annu Rev Immunol, 15, 203-234 (1997); FcR is summarized by Ravetch and Kinet, Annu Rev Immunol, 9, 457-92 (1991); Capel et al., Immunomethods, 4, 25-34 (1994); and de Haas et al., J Lab Clin Med, 126, 330-41 (1995); and Nimmerjahn and Ravetch 2006, Ravetch Fc Receptors in Fundemental Immunology, William Paul ed., 5th edition, each of which is incorporated herein by reference).

[0042] The terms "native" or "parent" refer to an unmodified polypeptide containing an Fc amino acid sequence. The parent polypeptide may contain either a native Fc region or an existing amino acid sequence modified (e.g., addition, deletion, and / or substitution) Fc region.

[0043] composition A composition containing a suitable carrier and one or more of the above-mentioned agents, such as a non-sialylated IgG Fc variant, is within the scope of the present invention. The composition may be a pharmaceutical composition containing a pharmaceutically acceptable carrier, or a cosmetic composition containing a cosmetically acceptable carrier.

[0044] The term “pharmaceutical composition” refers to a combination of an active agent and an inactive or active carrier that makes the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use. A “pharmaceutically acceptable carrier” does not produce undesirable physiological effects after administration to or on a subject. The carrier in a pharmaceutical composition must also be “acceptable” in the sense that it is compatible with the active ingredient and may be able to stabilize it. One or more solubilizers may be used as a pharmaceutically acceptable carrier for the delivery of the active compound. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents for obtaining compositions usable as dosage forms. Other examples of carriers include colloidal silicon dioxide, magnesium stearate, cellulose, and sodium lauryl sulfate.

[0045] The compositions described above may be used in any of the forms described above to treat diseases characterized by inflammation. The effective dose refers to the amount of active compound / agent required to produce a therapeutic effect in the subject being treated. As will be recognized by those skilled in the art, the effective dose will vary depending on the type of disease being treated, the route of administration, the use of excipients, and the possibility of concomitant use with other therapeutic procedures.

[0046] The pharmaceutical compositions of the present invention may be administered parenterally, orally, nasally, rectally, topically, or orally. As used herein, the term “parenteral” refers to subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-sacral, intrasternal, intrathecal, intrafocal, or intracranial injection, as well as any appropriate injection technique.

[0047] Sterile injectable compositions may be solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents. Such solutions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution, and isotonic sodium chloride solutions. Furthermore, fixative oils are conventionally used as solvents or suspension media (e.g., synthetic mono- or di-glycerides). Fatty acids, but not limited to oleic acid and its glyceride derivatives, and naturally pharmaceutically acceptable oils such as olive oil or castor oil, or their polyoxyethylated forms, are useful in the preparation of injectable formulations. These oil solutions or suspensions may also contain long-chain alcohol diluents, or dispersants such as carboxymethylcellulose, or similar dispersing agents, but are not limited to these. Other commonly used surfactants, such as Tweens or Spans, or other similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solids, liquids, or other dosage forms, may also be used for formulation purposes.

[0048] Compositions for oral administration may be any orally acceptable dosage form, including capsules, tablets, emulsions, and aqueous suspensions, dispersions, and solutions. For tablets, commonly used carriers include, but are not limited to, lactose and corn starch. Lubricants such as magnesium stearate are also typically added, though not limited to these. For oral administration in capsule form, useful diluents include, but are not limited to, lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oil phase in combination with an emulsifier or suspending agent. Certain sweeteners, flavorings, or colorants may be added, if desired.

[0049] The pharmaceutical compositions for topical administration according to the described invention may be formulated as solutions, ointments, creams, suspensions, lotions, powders, pastes, gels, sprays, aerosols, or oils. Alternatively, topical formulations may be in the form of patches or bandages impregnated with the active ingredient(s) and may optionally contain one or more excipients or diluents. In some preferred embodiments, topical formulations may contain substances that will enhance the absorption or penetration of the active ingredient(s) through the skin or other affected area. Topical compositions are useful for treating inflammatory skin conditions, including, but are not limited to, eczema, acne, rosacea, psoriasis, contact dermatitis, and poison ivy reactions.

[0050] Topical compositions contain a safe and effective amount of a dermatologically acceptable carrier suitable for application to the skin. A composition or component that is "cosmetically acceptable" or "dermatologically acceptable" means a composition or component that is suitable for use in contact with human skin without excessive toxicity, incompatibility, instability, allergic reactions, etc. The carrier enables the delivery of the active agent and optional components to the skin at an appropriate concentration(s). The carrier can therefore act as a diluent, dispersant, solvent, etc., to ensure that the active substance is applied and spread uniformly at an appropriate concentration on the selected target. The carrier may be solid, semi-solid, or liquid. The carrier may be in the form of a lotion, cream, or gel, in particular, having sufficient thickness or yield point to prevent the active substance from settling. The carrier may be inert or have dermatological advantages. It should also be physically and chemically compatible with the active ingredients described herein and should not excessively impair stability, efficacy, or other advantages of use related to the composition. Topical compositions may include solutions, aerosols, creams, gels, patches, ointments, lotions, or foams, and may be cosmetic or skin products in forms known in the art for topical or transdermal application.

[0051] Treatment method The described invention provides a method for treating inflammatory diseases in a subject. The term “inflammatory disease” refers to a disease characterized by abnormal or undesirable inflammation, such as autoimmune diseases. Autoimmune diseases are diseases characterized by the chronic activation of immune cells under inactivation conditions. Examples include psoriasis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, lupus, type 1 diabetes mellitus, primary biliary cirrhosis, and transplantation.

[0052] Other examples of inflammatory diseases that can be treated by the method of the present invention include asthma, myocardial infarction, stroke, inflammatory skin diseases (e.g., dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria, necrotizing vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, eosinophilic myositis, polymyositis, dermatomyositis, and eosinophilic fasciitis), acute respiratory distress syndrome, fulminant hepatitis, hypersensitivity lung diseases (e.g., hypersensitivity pneumonitis, eosinophilic pneumonia, delayed-type hypersensitivity, interstitial lung disease (ILD), idiopathic pulmonary fibrosis, and ILD associated with rheumatoid arthritis), and allergic rhinitis. Additional examples also include myasthenia gravis, juvenile-onset diabetes, glomerulonephritis, autoimmune thyroiditis, ankylosing spondylitis, systemic sclerosis, acute and chronic inflammatory diseases (e.g., systemic anaphylaxis or hypersensitivity reactions, drug allergies, insect bite allergies, allograft rejection, and graft-versus-host disease), as well as Sjögren's syndrome.

[0053] "Subject" refers to humans and non-human animals. Non-human animals include all vertebrates, such as non-human mammals, non-human primates (especially higher primates), mammals such as dogs, rodents (e.g., mice or rats), guinea pigs, cats and rabbits, as well as non-mammals such as birds, amphibians and reptiles. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental, non-human animal or an animal suitable as a disease model.

[0054] Subjects to be treated for inflammatory diseases can be identified by standard techniques for diagnosing the disease. Optionally, subjects may be examined for the level or percentage of one or more cytokines or cells using methods known in the art in test samples obtained from the subject. If the level or percentage is below a threshold (obtained from a normal subject), the subject is a candidate for the treatment described herein. To confirm inhibition or treatment, the level or percentage of one or more of the above cytokines or cells in the subject after treatment can be evaluated and / or confirmed.

[0055] "To treat" or "to treat" means the administration of a compound or drug to a subject with a disease for the purpose of curing, alleviating, reducing, eliminating, delaying, preventing or improving the onset of, a disease, a condition secondary to a disease, or a predisposition to the disease.

[0056] "Effective dose" or "therapeutic effective dose" refers to the amount of compound or drug that can produce a medically desirable outcome in the subject being treated. Treatment may be carried out in vivo or ex vivo, alone or in combination with other drugs or therapies. A therapeutic effective dose may be administered in one or more doses, topical applications, or medications, and is not intended to be limited to a specific formulation or route of administration.

[0057] Drugs may be administered in vivo or ex vivo, alone, or in combination with other drugs or therapies, i.e., in a cocktail therapy. As used herein, the terms “co-administration” or “co-administered” refer to the administration of at least two drugs or therapies to a subject. In some embodiments, the co-administration of two or more drugs / therapies is simultaneous. In other embodiments, the first drug / therapy is administered before the second drug / therapy. Those skilled in the art will understand that the formulation and / or route of administration of the various drugs / therapies used may differ.

[0058] In an in vivo approach, the compound or drug is administered to the subject. Generally, the compound or drug is suspended in a pharmaceutically acceptable carrier (e.g., physiological saline, but not limited to) and administered orally or by intravenous infusion, or injected or implanted subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastricly, intratracheally, or intrapulmonaryly.

[0059] The required dose depends on the choice of route of administration; the nature of the formulation; the nature of the patient's illness; the size, weight, surface area, age, and sex of the subject; whether other medications are being administered; and the judgment of the attending physician. Appropriate doses range from 0.01 to 100 mg / kg. Variations in the required dose are anticipated in terms of the available compounds / drugs and the different efficiencies of various routes of administration. For example, oral administration is expected to require higher doses than intravenous administration. As is well understood in the art, these dose level variations can be adjusted using standard empirical routines for optimization. Encapsulation of the compound into a suitable delivery vehicle (e.g., polymer microparticles or embedded devices) can increase delivery efficiency, particularly for oral delivery. [Examples]

[0060] Example 1: Method and Materials In this embodiment, the general methods and materials used in Examples 2 to 7 will be described.

[0061] mouse The wild-type C57BL / 6 mice were purchased from Jackson Laboratories. SIGNR1 - / - The mice were provided by A. McKenzie. CD11c-DC-SIGN + Transgenic mice were provided by T. Sparwasser. SIGNR1 - / -hDC-SIGN BAC transgenic mice were created in the inventors' laboratory in the background, as previously described. KRN TCR C57BL / 6 mice (gifts from D. Mathis and C. Benoist) were crossed with NOD mice to produce K / BxN mice. Blood was collected from K / BxN mice (6–12 weeks old), and serum containing arthritis antibodies was pooled together. Passive transport of 200 μL K / BxN serum by intravenous injection into naive mice (8–12 weeks old) induced arthritis. Inflammation was scored from 0–3 for each leg and added together for a total clinical score per individual mouse.

[0062] Preparation of recombinant Fc IDEC-114, the recombinant source of full-length human IgG1 monoclonal antibody, was digested overnight with papain at 37°C to cleave Fab and Fc fragments. After digestion, the reaction was stopped by the addition of 2.5 mg / mL iodoacetamide. To separate the cleaved fragments from the undigested antibody, the sample was passed through a HiPrep26 / 60 S-200HR size exclusion column (GE Healthcare). The Fc fragment was then purified with Protein G agarose beads. Sample purity was confirmed by Coomassie brilliant blue staining of SDS-polyacrylamide gel. Alternatively, recombinant Fc was produced by transient expression of a human IgG1 Fc expression plasmid in 293T cells, followed by ammonium sulfate precipitation and Protein G purification of the supernatant fraction. The gene sequence encoding the Fc region of human IgG1 was amplified from 4-4-20IgG1 by a standard PCR protocol and ligated to pSecTag2 (Invitrogen). A point mutation was introduced into the Fc coding sequence using standard site-directed mutagenesis techniques and confirmed by DNA sequencing. The PCR primer for the Phe-to-Ala substitution at position 241 (FA241) was

[0063] [ka]

[0064] and

[0065] [ka]

[0066] That was the case.

[0067] Protein expression and purity were confirmed by immunoblotting with anti-human Fc antibody and / or Coomassie brilliant blue staining of SDS-polyacrylamide gel.

[0068] Two-step invitrocyanation reaction After purification, 10–50 mg / mL Fc fragments were buffer-exchanged with galactosylation reaction buffer (50 mM MOPS, pH 7.2; 20 mM MnCl2) and incubated overnight at 37°C with 50 mg UDP-galactose and 0.75 U β1,4-galactosyltransferase. Galactosylation was confirmed by lectin blotting using ECL, as it recognizes terminal galactose residues. The galactosylated Fc was then buffer-exchanged with sialylation reaction buffer (100 mM MOPS, 0.2 mg / mL BSA, 0.5% TRITON X-100, pH 7.4) and incubated overnight at 37°C with 50 mg CMP-sialic acid and 0.75 U α2,6-sialylatetransferase. Sialization was confirmed by lectin blotting using SNA, which recognizes terminal sialic acid residues with α2-6 links.

[0069] Matched transplantation of bone marrow-derived macrophages Bone marrow cells are DC-SIGNtg or SIGNR1 - / -Macrophages were extracted from mouse tibia and femur and seeded into non-tissue culture treated 10-cm plates in RPMI1640 growth medium supplemented with 10% FBS, 1% penicillin / streptomycin, IL-3 (5 ng / mL, Peprotec), and M-CSF (5 ng / mL, Peprotec). After overnight incubation at 37°C, non-adherent cells were harvested and transferred to non-tissue culture treated 10-cm plates containing IL-3 / M-CSF supplemented RPMI growth medium, and cultured at 37°C for 5-7 days. Mature macrophages were trypsin-treated and seeded into 6-well plates at a rate of 2 × 10⁶ cells. 6 The cells were seeded at a cell / well density and allowed to adhere overnight. The following day, the macrophages were pulsed at 37°C for 30 minutes with the indicated recombinant Fc preparation. The cells were harvested, washed with cold PBS, and 1 × 10⁶ cells were collected. 6 Cells were administered intravenously to wild-type C57BL / 6 mice. One hour after injection, the recipient mice were challenged with K / BxN serum.

[0070] Expression and purification of soluble human DC-SIGN A plasmid containing the cDNA sequence of the extracellular domain (ECD) of human DC-SIGN was provided by K. Drickamer. The sequence encoding the DC-SIGN ECD was modified by standard PCR techniques to introduce an N-terminal strep tag and ligated to pET28b(+). pET28b-strepDCSIGN was transformed into E. coli strain BL21 / DE3, and bacterial cultures were OD 600The bacteria were grown in 3 L of TB growth medium at 37°C until the pH reached 0.7–0.8. Protein expression was induced by the addition of 100 mg / L of IPTG, and the cultures were incubated at 37°C for 3.5 hours. The bacteria were pelletized by centrifugation at 4000xg for 10 minutes at 4°C. The bacterial pellets were resuspended in 10 mM Tris-HCl, pH 7.8 and dissolved by sonication. The inclusion bodies were pelletized by centrifugation at 10,000xg for 15 minutes at 4°C and dissolved in 100 mL of 6 M guanidine-HCl; 100 mM Tris-HCl, pH 7.8; 0.2% TRITON X-100. Particulate matter was removed by centrifugation at 20,000xg for 30 minutes at 4°C, and the supernatant fraction was dialyzed against 250 mM NaCl; 25 mM Tris-HCl, pH 7.8; 25 mM CaCl2. After dialysis, insoluble precipitates were removed by centrifugation at 20,000xg for 30 minutes at 4°C, and the supernatant fraction was applied to strep-tactin resin (Novagen) to pull down strep-tagged DC-SIGN ECDs. The bound proteins were eluted from the resin with elution buffer supplied by the manufacturer (Novagen). The fractions were analyzed by SDS-PAGE, and the positive fractions were mixed and loaded onto a mannose-agarose column to select the active receptor. The DC-SIGN ECDs were eluted with 250mM NaCl; 25mM Tris-HCl, pH 7.8; 5mM EDTA. The fractions were analyzed by SDS-PAGE.

[0071] Surface plasmon resonance To measure the interaction of various recombinant Fc preparations with soluble hDC-SIGN or hFcγR, steady-state affinity measurements were recorded using a ViaCore T100 sensor. Receptors, diluted to 20–50 μg / mL in NaOAc pH 5.0, were immobilized on CM5 chips at high density (2000 RU) by standard amine coupling. For hDC-SIGN interactions, injection was performed at a flow rate of 20 μL / min using commercially available HBS-P+ buffer adjusted to pH 9.0 and supplemented with 2 mM CaCl2 and 500 mM NaCl. For hFcγR interactions, injection was performed at a flow rate of 20 μL / min using commercially available HBS-EP+ buffer. The surface was regenerated with short pulses of 50 mM NaOH. d The values ​​were calculated after subtracting background binding to the control flow cell using Biacore Evaluation software.

[0072] RT-PCR Total RNA was extracted from bone marrow-derived macrophages using the RNeasy mini-kit (Qiagen). 1 μg of total RNA was used to analyze IL-33 mRNA expression by RT-PCR using the OneStep RT-PCR kit (Qiagen). GAPDH expression served as a loading control. The PCR primers for mIL-33 were 5'-gaagatcccaacagaagacc-3' (SEQ ID NO: 6) and 5'-ttccggaggcgagacgtcac-3' (SEQ ID NO: 7), and the PCR primers for mGAPDH were 5'-gccgcctggagaaacctgc-3' (SEQ ID NO: 8) and 5'-tgaggtccaccaccctgttg-3' (SEQ ID NO: 9). PCR conditions were 94°C for 30 seconds; 55°C for 30 seconds; and 72°C for 60 seconds × 35 cycles (IL-33) or 25 cycles (GAPDH).

[0073] Example 2: A minority population of antibodies in IVIG preparations, in which α2,6-linked sialic acid conferred DC-SIGN binding activity to recombinant human IgG1 Fc, suppresses autoantibody-induced inflammation. These antibodies, containing terminal α2,6-linked sialic acid on Fc glycans, mediate anti-inflammatory responses by binding to SIGNR1 on marginal zone macrophages, its human homologous species, or DC-SIGN on bone marrow cells.

[0074] To study the interaction of sFc with DC-SIGN, the soluble form of the extracellular domain of DC-SIGN (DC-SIGN ECD) was purified from bacteria and immobilized on a CM5 chip. sFc was prepared from full-length IDEC-114 antibody, sialyzed in vitro (Figure 1b), and specifically bound to the conjugated surface of DC-SIGN (Figure 1a). Steady-state affinity measurements were performed to determine the K of this interaction. D The value is ~1.3 × l0 -6 M was calculated (Figure 1c). In contrast, the asialylated glycosylation of IDEC-114 Fc did not show binding activity to DC-SIGN, suggesting that this sialylation induces conformational changes on the Fc skeleton to expose the DC-SIGN binding site.

[0075] As shown in Figure 1a, the binding of recombinant α2,6-sFc to soluble DC-SIGN was found by surface plasmon resonance (SPR). Fc was prepared by papain cleavage of full-length human monoclonal IgG1 antibody (IDEC-114), followed by in vitro galactosylation and sialylation reactions. As described in Example 1, SPR sensorgrams for the antibody binding to immobilized DC-SIGN are shown for the sialylated and galactosylated glycans of hIgG1 Fc. The Fc concentration flowing on the DC-SIGN ECD was found to be in the range of 3–0.8 μM. As shown in Figure 1b, lectin blot by SNA confirmed the sialic acid linkage with a 2,6-bond on Fc (upper panel); a Coomassi-stained loading control is shown in the lower panel. Steady state K of sFc binding to DC-SIGN D The measurements were calculated using via core evaluation software (as shown in a).

[0076] Example 3 Mutations that disrupt the Fc-glycan interaction conferred DC-SIGN binding activity to recombinant human IgG1 Fc. Asn 297 Core oligosaccharides linked to the core oligosaccharide form extensive non-covalent interactions with the amino acid backbone of Fc. Conformational changes of Fc resulting from different sugar residues linked to the coreglycan are mediated by these protein-carbohydrate interactions. Alanine substitutions that invalidate key contact points between the Fc backbone and glycan residues appear to confer DC-SIGN binding activity.

[0077] As shown in Figure 2A, the FA241 and FA243 mutations exhibit DC-SIGN binding activity without in vitro enzyme treatment. Apparent K D The value is 6 × 10 for FA241. -7 From M, 3x10 about FA243 -7This is in the M range. Previous reports have shown that when expressed in mammalian cells, these mutations enhance antibody sialylation, presumably by allowing glycans to approach glycosyltransferases more easily. To verify this, the binding of FA241 and FA243 to DC-SIGN was transiently expressed in proteins. Lectin blotting was performed to determine whether the increase in sialization was due to cereal activity. As shown in Figure 2B, SNA blotting did not detect terminal sialic acid residues in purified FA241 and FA243, indicating that the DC-SIGN interaction was unrelated to sialic acid modification.

[0078] More specifically, along the amino acid backbone of IgG1 Fc, residues F241, F243, D265, and R301 were substituted with alanine to disrupt non-covalent interactions with oligosaccharide residues. Fc was expressed, purified from 293T cells, and analyzed for DC-SIGN binding activity by surface plasmon resonance as described above. Fc with mutant FA241 or FA243 was found to exhibit increased affinity for DC-SIGN compared to affinity measurements for sFc (Figure 1a). Lectin blotting using ECL (Figure 1b, center panel) and SNA (top panel) was performed to measure the terminal sugar moieties on purified Fc from 293T cells. As a positive control for sialylated Fc, FA241 was sialylated in vitro as described in Figure 1a. Coomassi-stained loading controls are shown in the bottom panel of Figure 1b.

[0079] Example 4: The FA241 mutation in hIgG1 Fc recapitulated the anti-inflammatory activity of α2,6sFc. If the FA241 and FA243 mutations mimic the DC-SIGN binding activity of sFc, assays were performed to investigate whether these mutations could reproduce the anti-inflammatory activity of sFc in vivo. Age and sex-matched SIGNR1 - / - and hDC-SIGN + / SIGNR1 - / -Mice were loaded with arthritis K / BxN serum and treated with sFc, FA241, or FA243 at an effective dose of 0.033 g / kg. Consistent with previous findings, sFc was DC-SIGN + Swelling of the mouse's soles was suppressed, but SIGNR1 - / - It was not suppressed. Similarly, FA241 hDC-SIGN + / SIGNR1 - / - It showed anti-inflammatory activity comparable to that of sFc in mice. Mice administered FA243 did not show a reduction in arthritis. These findings suggest that F 241 This suggests that recombinant Fc with the A mutation (FA241) replicates the DC-SIGN binding and anti-inflammatory activity of sFc without sialic acid modification.

[0080] As shown in Figure 3, hDC-SIGN + / SIGNR1 - / - (White square) and SIGNR1 - / - Mice (black squares) were administered 0.7 mg / mouse of sFc, FA241, or FA243 by intravenous injection. Mice were subsequently loaded with K / BxN serum 1 hour later. Plantar swelling was observed and scored over several days. As previously reported, the anti-inflammatory effect of sFc is DC-SIGN-dependent (left panel). FA241 also suppressed arthritis in a DC-SIGN-dependent manner in mice loaded with K / BxN. FA243 did not significantly reduce measured swelling on day 6. The mean clinical scores and SEM of 4-5 mice per group were plotted on day 6.

[0081] Example 5: Characterization of the necessary conditions for the anti-inflammatory activity of FA241 To identify the determinants of the anti-inflammatory activity of FA241, CD11c.DC-SIGN + and SIGNR1 - / - Bone marrow-derived macrophages (BMMΦ) from mice were stimulated with FA241 or other Fc preparations and transplanted into WT C57BL / 6 recipient mice loaded with K / BxN serum.

[0082] In other words, CD11c.DC-SIGN + and SIGNR1 - / - Bone marrow-derived macrophages from mice were cultured in IL-3 (5 ng / mL) and M-CSF (5 ng / mL) for 5–7 days. DC-SIGN is shown in Figure 4. + BMMΦ was pulsed with either non-sialylated (black bar) or sialylated (white bar) glycans of a 0.5 mg / mL indicated Fc preparation. Fc-treated BMMΦ was implanted into WT C57BL / 6 recipient mice, followed by K / BxN loading. As shown in Figure 4b, SIGNR1 - / - (Black bar) and DC-SIGN + (White bar) BMMΦ was pulsed with an Fc preparation labeled at 0.5 mg / mL and implanted into WT C57BL / 6 recipient mice, followed by K / BxN loading. Similarly, DC-SIGN + BMMΦ was pulsed with either 0.5 mg / mL of FA241, deglycosylated FA241 (Figure 4c, white bar), or PBS (Figure 4c, black bar), and transplanted into WT C57BL / 6 recipient mice, followed by K / BxN loading. FA241 was deglycosylated by PNGase F, and glycan removal was confirmed by lectin blotting. DC-SIGN + BMMΦ was pulsed with the indicated Fc preparation or PBS (Figure 4d, black circle) and implanted into WT C57BL / 6 recipient mice, followed by K / BxN loading. In all cases, plantar swelling was observed and scored over several days. The mean clinical scores and SEMs of 4-5 mice per group are plotted. * P<0.05 was determined by analysis of variance (ANOVA), followed by a Tukey post-hoc test.

[0083] As shown in Figure 4A, non-sialylated or sialylated FA241 preparations were equally effective in suppressing arthritis compared to sFc. However, DC-SIGN pulsed with non-sialylated WT Fc was also effective. +Since BMΦ did not transfer protection to recipient mice, the WT Fc preparation required α2,6-linked sialic acid. Consistent with the results shown in Figure 3, both sFc and FA241 required DC-SIGN expression on BMΦ to transfer protection (Figure 4B). Although FA241 did not require sialic acid to transfer protection, deglycosylation by PNGase F neutralized the anti-inflammatory properties of FA241 (Figure 4C), suggesting that Fc glycans are still required. Furthermore, the observed anti-inflammatory activity was F 241 To demonstrate specificity for the A mutation, DC-SIGN + BMΦ was pulsed along with Fc having an alternative mutation that did not confer enhanced DC-SIGN binding. Only FA241-stimulated BMΦ protected K / BxN-loaded recipient mice.

[0084] Example 6: The FA241 mutation enhanced Fcγ receptor binding. If alanine substitution at position 241 induces a conformational change in Fc, then the affinity for the human Fcγ receptor is likely altered. It has been previously reported that sialylation reduces the affinity of IgG for FcγR, resulting in attenuation of ADCC activity in vivo.

[0085] Recombinant IgG1 The binding of Fc to soluble FcγR was measured by surface plasmon resonance (SPR). Fc was prepared by the method described above. Immobilized hFcγRIIA 131R SPR sensorgrams regarding antibody binding to hFcγRIIB are shown in Figure 5 for non-sialylated and sialylated glycosylated forms of WT hIgG1 Fc, as well as for non-sialylated FA241 Fc.

[0086] As shown in Figure 5, the non-sialylated sugar form of WT Fc is ~2-3 × 10 -5 Observed K of M DIt bound to hFcγRIIA and RIIB at a certain value. sFc, however, did not appear to bind to either hFcγRIIA or RIIB. Surprisingly, FA241 had an affinity (K) that was an order of magnitude stronger. D =~2 × 10 -6 M) appears to bind to both hFcγRIIA and RIIB.

[0087] Example 7: IL-33 mRNA induction in bone marrow-derived macrophages by FA241. sFc upregulates FcγRIIB on regulatory macrophages, FcεRI + The leukocyte population, possibly basophils, requires IL-4 secretion. H It induces a 2-dependent anti-inflammatory pathway. In vivo or in vitro, administration of IL-33 stimulates basophils to release IL-4 reserves. IL-33 mRNA expression is upregulated in the spleen of WT C57BL / 6 mice treated with sFc or IVIG, but SIGNR1 - / - This does not happen with a mouse. This is because sFc is SIGNR1 + or DC-SIGN + This suggests the possibility of inducing IL-33 expression in cells. In this example, an assay was performed and DC-SIGN was detected by FA241. + This suggests that stimulating BMMΦ may upregulate IL-33 expression.

[0088] More specifically, CD11c.DC-SIGN + and SIGNR1 - / -Bone marrow-derived macrophages from mice were cultured using the method described above. BMMs were seeded in serum-free RPMI medium in 12-well plates and allowed to adhere overnight at 37°C. The following day, cells were pulsed at 37°C for 1 hour (Figure 6a) or 4 hours (Figure 6b) with 0.5 mg / mL of indicated Fc in serum-free RPMI medium. mRNA was collected from cells at specific time points, and 1 μg of total RNA was used for RT-PCR amplification of IL-33 mRNA (upper panel). GAPDH amplification served as a loading control (lower panel). Plasmids co-expressing DA265 Fc and human sialylate transferase (ST6Gal1) were transformed into 293T cells producing highly sialylated recombinant Fc (ST6-DA265).

[0089] As shown in Figure 6, DC-SIGN is performed by FA241. + Stimulation of BMΦ appears to upregulate IL-33 expression. DC-SIGN + Despite having a higher basal expression level of IL-33 compared to BMMΦ, SIGNR1 - / - BMMΦ downregulated IL-33 mRNA expression in response to FA241 treatment.

[0090] The above examples and descriptions of preferred embodiments should be interpreted as illustrative rather than limiting the invention as defined by the claims. All publications cited herein are incorporated in their entirety by reference. For ease of understanding, numerous variations and combinations of the features described above can be used without departing from the invention as defined in the claims. Such variations are not considered departures from the scope of the invention, and all such variations are intended to be included within the following claims.

Claims

1. A polypeptide fragment consisting solely of amino acid sequences that are at least 90% identical to sequence number 2, In the polypeptide, amino acid residue 241 (numbered according to the EU index of Kabat) is alanine. A polypeptide fragment showing binding to DC-SIGN and hFcγRIIB.

2. An IgG1 Fc variant polypeptide fragment consisting solely of amino acid sequences that are at least 90% identical to SEQ ID NO: 2, In the polypeptide, amino acid residue 241 (numbered according to the EU index of Kabat) is alanine. Shows binding to DC-SIGN and hFcγRIIB. IgG1 Fc variant polypeptide fragment.

3. The polypeptide fragment according to claim 1 or 2, wherein the amino acid sequence is at least 95% identical to that of SEQ ID NO:

2.

4. The polypeptide fragment according to claim 1 or 2, wherein the amino acid sequence is at least 99% identical to that of SEQ ID NO:

2.

5. A pharmaceutical composition for treating an inflammatory disease, comprising (i) a polypeptide fragment according to any one of claims 1 to 4, and (ii) a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, wherein the inflammatory disease is an autoimmune disease.

7. The pharmaceutical composition according to claim 5, wherein the inflammatory disease is inflammatory bowel disease, inflammatory skin disease, hypersensitivity lung disease, or acute or chronic inflammatory disease.

8. Use of a polypeptide fragment according to any one of claims 1 to 4 in the manufacture of a pharmaceutical product for treating inflammatory diseases in patients in need.

9. The use of claim 8, wherein the inflammatory disease is an autoimmune disease.

10. The use according to claim 8, wherein the inflammatory disease is inflammatory bowel disease, inflammatory skin disease, hypersensitivity lung disease, or acute or chronic inflammatory disease.

11. The use according to claim 8, wherein the inflammatory disease is psoriasis, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, lupus, type 1 diabetes, primary biliary cirrhosis, asthma, acute respiratory distress syndrome, fulminant hepatitis, myasthenia gravis, juvenile-onset diabetes, glomerulonephritis, autoimmune thyroiditis, ankylosing spondylitis, systemic sclerosis, or Sjögren's syndrome.

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