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    • 专利摘要:
    ISOLATED NON-SIALILATED POLYPEPTIDES WITH ANTI-INFLAMMATORY ACTIVITY, METHODS TO OBTAIN THEM, NUCLEIC ACIDS THAT CODE THEM, EXPRESSION VECTOR, HOST CELL AND FORMULATIONPHARMACEUTICALS CONTAINING THEM, METHOD AND USE OF THEM IN TREATMENT OF INFLAMMATORY DISEASESThis invention pertains to anti-inflammatory agents, compositions and methods for treating inflammatory disorders.
    公开号:BR112014014549A2
    申请号:R112014014549-0
    申请日:2012-12-10
    公开日:2020-09-24
    发明作者:Jeffrey V. Ravetch；Andrew Pincetic
    申请人:The Rockefeller University；
    IPC主号:
    专利说明:

    [0001] [0001] This application claims priority from US Interim Application No. 61/577,361, filed December 19, 2011. The contents of the application are incorporated herein entirely by reference. INTERESTS OF THE GOVERNMENT
    [0002] [0002] The invention disclosed herein was made, at least in part, with Government support, supported under National Institutes of Health Grant No. NIH AI035875. Accordingly, the US Government has certain rights in this invention. FIELD OF THE INVENTION
    [0003] [0003] This invention relates to inflammatory agents, compositions and methods for treating inflammatory disorders. BACKGROUND OF THE INVENTION
    [0004] [0004] Inflammatory disorders, including autoimmune diseases, are diseases involving abnormal activation and subsequent migration of white blood cells to affect areas of the body. These conditions encompass a wide range of diseases that affect the lives of millions of people around the world. Although several treatments are currently available, many have significant side effects or are not very effective in alleviating all symptoms. Thus, there is a need for anti-inflammatory agents for treating inflammatory disorders and a need for methods of identifying and evaluating such agents.
    [0005] [0005] Immunoglobulin G (IgG) has long been appreciated for mediating both pro- and anti-inflammatory activities through interactions mediated by this Fc fragment. While Fc-FcR interactions are responsible for the proinflammatory properties of immune complexes and cytotoxic antibodies, intravenous gamma globulin (IVIG) and its Fc fragments are anti-inflammatory and are widely used to suppress inflammatory diseases. It has been proposed that glycosylation of IgG is crucial for the regulation of cytotoxicity and inflammatory potential of IgG. For example, it has been suggested that the anti-inflammatory activity of IVIG is a property of the Fc fragment and its linked glycans, requiring α.2,6 terminal sialic acid binding, indicating a combined requirement for the specific polypeptide backbone and glycan structure for immunosuppression. (Anthony, et al., 2008, Science 320: 373-376 and international patent WO 2007/117505 ).
    [0006] [0006] However, only a small population of IgG in IVIG has glycan endings in α.2,6 sialic acid (sFc) and anti-inflammatory activity. As a result, autoantibody suppression triggered a variety of clinical situations, in one of which it was necessary to administer IVIG at high doses (1-2g/kg), to enrich IgG sialylation, or otherwise increase IgG sialylation. (North American Application Nos. 20080206246, and 20090004179, and Nimmerjahn et al. Annu Rev Immunol 26, 513-533 (2008 )).
    [0007] [0007] The present invention addresses and meets the aforementioned needs by identifying sialylation-free anti-inflammatory polypeptides. SUMMARY
    [0008] [0008] This invention relates to agents, such as polypeptides and antibodies, and methods for treating inflammatory disorders, for example, autoimmune diseases.
    [0009] [0009] Accordingly, one aspect of this invention features an isolated polypeptide comprising a modified sequence that is at least 75% (e.g., any number between 75% and 100%, inclusive, e.g., 70%, 80%, 85%, 90% , 95%, 99%, and 100%) identical to an IgG Fc region. The modified sequence is free of sialylation and the polypeptide has an anti-inflammatory activity that is higher than that of a parent polypeptide. The originator polypeptide may comprise the IgG Fc region, such as the sequence of SEQ ID NO: 1 listed below. In some embodiments, the polypeptide has the ability to bind DC-SIGN, and to bind hFcγRIIA or RIIB. In one embodiment, the isolated polypeptide has an ability to bind hFcγRIIA or RIIB at a KD of 2x10 -5 M or less (i.e., KA of 5.0 x 104 M -1 or greater). modified sequence has an FA241 mutation. The modified sequence can be at least 75% (example, any number between 75% and 100%, inclusive, example, 70%, 80%, 85%, 90%, 95%, 99% , and 100%) identical to SEQ ID NO: 2. In some examples, the modified sequence comprises or consists essentially of SEQ ID NO: 2.
    [00010] [00010] In another aspect, the invention provides a method for producing a polypeptide having an anti-inflammatory activity.
    [00011] [00011] In a third aspect, the invention features an isolated nucleic acid comprising a sequence encoding the above-described polypeptide; an expression vector comprising the nucleic acid, and, a host cell comprising the nucleic acid. The invention also features a method of producing a polypeptide. The method includes culturing the host cell in a medium under conditions that permit expression of a polypeptide encoded by the nucleic acid and purifying the polypeptide from the cultured cell or the cell's medium.
    [00012] [00012] In a fourth aspect, the invention features a pharmaceutical formulation comprising (i) the polypeptide or nucleic acid described above, and (ii) a pharmaceutically acceptable carrier.
    [00013] [00013] In a fifth aspect, the invention provides a method of treating inflammatory disease. The method includes administering to a subject in need thereof a therapeutically effective amount of the polypeptide or nucleic acid encoding the aforementioned polypeptide. Also provided is the use of the polypeptide or nucleic acid in the manufacture of a medicament for treating an inflammatory disease. The invention also features an isolated polypeptide, nucleic acid, expression vector, host cell, composition or method for treating an inflammatory disease substantially as shown and described herein.
    [00014] [00014] The details of one or more embodiments of the invention are set out in the description below. Other features, objects, and advantages of the invention will be apparent from the description and claims. DESCRIPTION OF THE FIGURES
    [00015] [00015] FIGURES a-c are diagrams and photographs showing that α.2,6 sialic acid ligand confers DC-SIGN binding activity to recombinant human IgG1 Fc.
    [00016] [00016] FIGURES 2a-b are diagrams and photographs showing that dysregulation of Fc-glycan interactions confers DC-SIGN binding activity to recombinant human IgG1 Fc.
    [00017] [00017] FIGURE 3 is a set of diagrams showing that the FA241 mutation in hlgGl Fc recapitulates anti-inflammatory activity of α.2,6 sFc.
    [00018] [00018] FIGURES 4a-d are diagrams showing characterizing requirements of FA241 anti-inflammatory activity.
    [00019] [00019] FIGURE 5 is a set of diagrams showing that mutation of FA241 enhances Fcγ receptor binding.
    [00020] [00020] FIGURES 6a-b are photographs showing induction of IL-33 mRNA in bone marrow-derived macrophages by FA241. DETAILED DESCRIPTION
    [00021] [00021] This invention is based, at least in part, on an unexpected discovery that non-sialylated IgG Fc variants confer anti-inflammatory activity and mimic the effect of sialylated Fc .2,6 as anti-inflammatory mediators.
    [00022] [00022] IgG is the major serum immunoglobulin. It is a glycoprotein composed of two identical heavy chains and two light chains, which in turn are composed of variable and constant domains. IgG contains a single, N-glycan linkage at Asn297 in the CH2 domain on each of the two heavy chains. The covalently bonded, complex carbohydrate is composed of a core, biantennary pentapolysaccharide containing N-acetylglucosamine (GIcNAc) and mannose (man). Additional modification of the carbohydrate core structure is observed in serum antibodies in the presence of fucose, branching GIcNAc, galactose (gal) and variable terminal sialic acid (sa) radicals found. More than 40 different glycoforms were thus detectable to be covalently linked to this simple glycosylation situation (Fujii et al., J. Biol. Chem. 265, 6009, 1990). IgG glycosylation has been shown to be essential for binding all FcγRs by maintaining an 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 glycosylation of IgG to FcγR binding is believed to explain the inability of deglycosylated IgG antibodies to mediate in vivo triggering of inflammatory responses, such as ADCC, phagocytosis and the release of inflammatory mediators. Nimmerjahn and Ravetch, Immunity 24, 19 (2006). Additional observations that individual IgG glycoforms may contribute to the modulation of inflammatory responses were suggested by the altered affinity for individual FcγRs reported by IgG antibodies containing or lacking fucose and its consequent effects on cytotoxicity. Shields et al, J. Biol. Chem. 277, 26733 (2002 ), Nimmerjahn and Ravetch, Science 310, 1510 (2005 ). A link between autoimmune conditions and specific models of glycosylation of IgG antibodies has been observed in models with rheumatoid arthritis and various autoimmune vasculitides in which reduced galactosylation and sialylation of IgG antibodies has been reported. Parekh et al., Nature 316, 452 (1985), Rademacher et al, Proc. natl. academy Sci. USA 91, 6123 (1994), Matsumoto et al, 128, 621 (2000), Holland et al., Biochim. Biophys. Minutes December 27. Changes in IgG glycoforms have also been reported to be associated with aging and after immunization, although the in vivo significance of these changes has not been determined. Shikata et al., Glycoconj. J. 15, 683 (1998), Lastra, et al., Autoimmunity 28, 25 (1998).
    [00023] [00023] As disclosed herein, certain non-sialylated IgG Fc variants surprisingly also confer anti-inflammatory activity. Such variants include the FA241 variant, which represent species within larger genera of molecules that, by virtue of simulating the structure and biological properties of sialylated Fc, but not requiring sialylation, could be developed as anti-inflammatory therapeutics.
    [00024] [00024] As disclosed herein, this invention provides isolated polypeptides having human IgG Fc variant sequences that lack a polysaccharide chain having a sialic acid terminus connected to a galactose moiety via the α.2,6 linkage in the aforementioned Asn297. Such non-sialylated IgG Fc variants may be derived from a naturally occurring antibody or expressed in a cell line.
    [00025] [00025] In one embodiment, the Fc region includes one or more substitutions from the hlgGl amino acid sequence. Although not limited to these, exemplary IgGl Fc regions are provided as follows: hlgGl Fc (starting from amino acid 210 in the Kabat system): KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
    [00026] [00026] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to describe the combination of amino acid residues in a polymer. A peptide, polypeptide, or protein can be composed of the naturally occurring amino acid pattern, in addition to rare amino acids and analogous synthetic amino acids. They can be any chain of amino acids, regardless of length or post-translational modification (eg, glycosylation or phosphorylation). The peptide, polypeptide, or protein "of this invention" includes recombinantly or synthetically produced versions having the particular domains or portions that bind DC-SIGN, FcγRIIA and FcγRIIB. The term also encompasses polypeptides that have an additional amino terminus methionine (useful for expression in prokaryotic cells).
    [00027] [00027] An "isolated" polypeptide or protein refers to a polypeptide or protein that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide/protein can make up 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%) by dry weight of the purified preparation. Purity can be measured by an appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. An isolated polypeptide/protein described in the invention can be purified from a natural source, produced by recombinant DNA technique, or by chemical methods. A functional equivalent of IgG Fc refers to a polypeptide derived from IgG Fc, for example, a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. Substantially retaining IgG Fc activity, that is, the ability to bind to the respective receptor and trigger the respective cellular response. The isolated polypeptide may contain SEQ ID NO: 2. In general, the functional equivalent is at least 75% (e.g., any number between 75% and 100%, inclusive, e.g., 80%, 85%, 90%, 95%, and 99%) identical to SEQ ID NO: 2.
    [00028] [00028] The "percent identity" of two amino acid sequences or two nucleic acids is determined using the algorithm of Karlin and Altschul Proc. natl. academy Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. natl. academy Sci. USA 90:5873-77, 1993. Thus an algorithm is incorporated into the programs NBLAST and XBLAST (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. Nucleotide BLAST search can be performed as NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. The BLAST protein search can be performed with the XBLAST program, score=50, wordlength=3 to obtain homologous amino acid sequences for the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be used as described in Altschul et al, Acids Nucleic Res. 25(17):3389-3402, 1997. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (example , XBLAST and NBLAST) can be used.
    [00029] [00029] The amino acid composition of the polypeptide described herein may vary without disrupting the polypeptide's ability to bind to the respective receptor and provoke the respective cellular response. For example, it may contain one or more conservative amino acid substitutions. A "conservative amino acid" substitution is one in which the amino acid residue is replaced by a tense amino acid residue on a similar side chain. Families of amino acid residues having similar side chains have been defined 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), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine , cysteine), non-polar 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). Thus, a predicted non-essential amino acid residue, for example, in SEQ ID NO: 2, is preferably substituted with another amino acid residue of the same side chain family. Alternatively, mutations can be introduced randomly along all or part of the sequences, such as by saturation mutagenesis, and the resulting mutants can be selected for their ability to bind to the respective receptor and trigger the respective cellular response to identify mutants that retain the activity as described in the examples below.
    [00030] [00030] A polypeptide as described in this invention can be obtained as a recombinant polypeptide. To prepare a recombinant polypeptide, an encoded nucleic acid encoding it (e.g. FA241, SEQ ID NO: 2) can be linked to another nucleic acid encoding a fusion association, e.g. glutathione-s-transferase (GST), tag 6x-His epitope, or M13 Gene 3 protein. The resulting fusion nucleic acid expresses in a suitable host cell a fusion protein 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 associated fusion and obtain the recombinant polypeptide of this invention.
    [00031] [00031] Another aspect of the invention features an isolated nucleic acid comprising a sequence encoding the above-described polypeptide or protein. A nucleic acid refers to a DNA molecule (eg, a cDNA or genomic DNA), an RNA molecule (eg, an mRNA), or a DNA or RNA analogue. A DNA or RNA analogue can be synthesized from analogue nucleotides. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. An "isolated nucleic acid" refers to a nucleic acid the structure of which is not identical to any naturally occurring nucleic acid or to any fragment of a naturally occurring genomic nucleic acid. The term therefore encompasses, for example, (a) a DNA that has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both coding sequences that flank the part of the molecule in the organism's genome in which it naturally occurs; (b) a nucleic acid incorporated into a vector or the genomic DNA of a prokaryote or eukaryote in such a way that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by Polymerase Chain Reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. The nucleic acid described above can be used to express the fusion protein of this invention. For this purpose, it can be operably linked to nucleic acid with appropriate regulatory sequences to generate an expression vector.
    [00032] [00032] A vector refers to a nucleic acid molecule capable of carrying another nucleic acid to which it has been linked. The vector may be capable of autonomous replication or integrated into a host DNA. Examples of vector include a plasmid, cosmid, or viral vector. The vector includes a nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the vector includes one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed.
    [00033] [00033] A "regulatory sequence" includes promoters, enhancers, and other expression control elements (eg, polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector may depend on such factors as: the choice of host cell to be transformed, the desired level of protein or RNA expression, and the like. The expression vector can be introduced into the host cell to produce a polypeptide of this invention. A promoter is defined as a DNA sequence that directs RNA polymerase to bind DNA and initiate RNA synthesis. A strong promoter is one that causes mRNAs to start at a high frequency.
    [00034] [00034] Any polynucleotide as mentioned above or a biologically equivalent polynucleotide available to those skilled in the art for the same purpose may be inserted into an appropriate expression vector and ligated with other DNA molecules to form "recombinant DNA molecules" expressing this receptor. These vectors can be made up of DNA or RNA; for most cloning purposes DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophages and cosmids, artificial yeast chromosomes, and other forms of episomal or integrated DNA. It is well within the range of the technician to determine an appropriate vector for a particular use.
    [00035] [00035] A variety of mammalian expression vectors can be used to express the above mentioned IgG Fcs in mammalian cells. As noted above, expression vectors can be DNA sequences that are required for the transcription of the cloned DNA and the translation of its mRNAs into an appropriate host. Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, cyanobacteria, plant cells, insect cells and animal cells. Specifically designed vectors allow for DNA reciprocity between hosts such as bacteria-yeast or bacteria-animal cells. A properly constructed expression vector must contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful enzyme restriction situations, a potential for high copy numbers, and active promoters. Expression vectors may include, but are not limited to, specifically designated cloning vectors, modified cloning vectors, plasmids or viruses. Commercially available mammalian expression vectors that may be suitable include, but are not limited to, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCTneo (Promega), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs) , pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and IZD35 (ATCC 37565).
    [00036] [00036] Also within the scope of this invention is a host cell that contains the above-described nucleic acid. Examples include E. coli cells, insect cells (eg, using baculovirus expression vectors), yeast cells, or mammalian cells. See example, Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. To produce a polypeptide of this invention, a host cell can be cultured in a medium under conditions that allow expression of the polypeptide encoded by a nucleic acid of this invention and purify the polypeptide from the culture cell or culture medium. cell. Alternatively, the nucleic acid of this invention can be transcribed and translated in vitro, for example, using the T7 promoter and T7 polymerase regulatory sequences.
    [00037] [00037] All naturally occurring IgG Fcs, genetically engineered IgG Fcs, and chemically synthesized IgG Fcs can be used to practice the invention disclosed herein. IgG Fc obtained by recombinant DNA technology may have the same amino acid sequence as [FA241] SEQ ID
    [00038] [00038] One can verify the effectiveness of such a polypeptide/protein using an animal model, such as a transgenic mouse, as described below. Any statistically significant increase in in vivo expression of basophils or IL-33 expression of the FcγRIIB receptor on macrophage effect indicates that the polypeptide/protein is a candidate for the treatment of the diseases mentioned below. In one embodiment, the assay described above may be based on measuring a binding to DC-SIGN protein or DC-SIGN(+) cells. The article is replete with various techniques available to one of ordinary skill in the art that will be suitable for measuring the ability of a compound to a DC-SIGN or to DC-SIGN(+) cells and related exchanges in the expression of a gene regulated by the DC pathway. -SING, such as IL-33. The skilled person will be able to mix and match these various research tools without undue experimentation. Once purified and tested by standard methods or in accordance with the assays and methods described in the examples below, non-sialylated IgG Fc variants can be included in the pharmaceutical composition for treating inflammatory disorders.
    [00039] [00039] As used herein, "antibody" is used in the broadest sense and specifically involves monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g.,
    [00040] [00040] As used herein, "antibody fragments" may comprise a portion of an intact antibody, generally including the antigen-binding or variable region of the intact antibody or the Fc region of an antibody that retains the ability to bind FcR . Examples of antibody fragments include linear antibodies; single chain antibody molecules; and multispecific antibodies formed from antibody fragments. Antibody fragments preferably retain at least part of the main point and optionally the CHI region of an IgG heavy chain. More preferably, the antibody fragments retain the entire constant region of an IgG heavy chain, and include an IgG light chain.
    [00041] [00041] As used herein, the term "Fc fragment" or "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native Fc region sequence or a variant Fc region. Although the contours of the Fc region of an immunoglobulin heavy chain may vary, the human IgG Fc region heavy chain is usually defined to extend from an amino acid residue at position Cys226, or from Pro230, to the carboxyl termini thereof.
    [00042] [00042] The "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" as appreciated by one of skill in the art comprises an amino acid sequence that differs from a native Fc region sequence by virtue of at least one "amino acid modification." Preferably, the variant Fc region has at least one amino acid substitution compared to a native Fc region sequence or Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, and preferably from about from one to about five amino acid substitutions in a native Fc region sequence or in the Fc region of an originator polypeptide. The Fc variant region herein preferably will have at least about 75 or 80% homology to a native Fc region sequence and/or to an Fc region of an originator polypeptide, and more preferably at least about 90% homology to this region. more preferably at least about 95% homology thereto, even more preferably at least about 99% homology thereto.
    [00043] [00043] The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc region of an antibody. In one embodiment of the invention, FcR is a native human FcR sequence. In another embodiment, the FcR, includes human FcR, linked to an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcΧRII, and FcγRIII subclasses, including allelic variants and alternatively "spliced" forms of these receptors. . FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The FcγRIIA activating receptor contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibition receptor FcΧRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review in Daron, Annu Rev Immunol, 15, 203-234 (1997); FcRs are reviewed in 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), Nimmerjahn and Ravetch 2006, Ravetch Fc Receptors in Fundemental Immunology, ed William Paul 5th Ed. Each of which is incorporated herein by reference).
    [00044] [00044] The term "native" or "originator" refers to an unmodified polypeptide comprising an Fc amino acid sequence. The originator polypeptide may comprise a native Fc region sequence or an Fc region with preexisting amino acid sequence modifications (such as additions, deletions and/or substitutions).
    [00045] [00045] Within the scope of this invention is a composition that contains a suitable carrier and one or more of the agents described above, such as non-sialylated IgG Fc variants. The composition can be a pharmaceutical composition that contains a pharmaceutically acceptable carrier or a cosmetic composition that contains a cosmetically acceptable carrier.
    [00046] [00046] The term "pharmaceutical composition" refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. A "pharmaceutically acceptable carrier", once administered to a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition may be "acceptable" also in the sense that it is compatible with the active ingredient and may be capable of stabilization. One or more solubilizing agents can be used as pharmaceutical vehicles for delivering the active compound. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible carriers, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate.
    [00047] [00047] The above-described composition, in any of the forms described above, can be used for treating diseases characterized by inflammation. An effective amount refers to the amount of an active compound/agent that is required to impart therapeutic effect to a subject being treated. Effective doses will vary, as recognized by those skilled in the art, depending on the types of diseases treated, route of administration, excipient used, and the possibility of co-use with another therapeutic treatment.
    [00048] [00048] A pharmaceutical composition of this invention can be administered parenterally, orally, nasally, rectally, topically, or buccally. The term "parenteral" as used herein refers to subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion.
    [00049] [00049] A sterile injectable composition can be a solution or suspension and a parenterally acceptable non-toxic diluent or solvent. Such solutions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, fixing oils are conventionally employed with a solvent or suspending medium (eg, synthetic mono- or diglycerides). Fatty acids, such as, but not limited to, oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are pharmaceutically acceptable natural oils, such as, but not limited to, olive oil or castor oil, polyoxyethylated versions of these. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as, but not limited to, carboxymethyl cellulose, or similar dispersing agents. Other commonly used surfactants, such as, but not limited to, TWEENS or SPANS or similar emulsifying agents or bioavailability enhancers, commonly used in the manufacture of pharmaceutically acceptable medications whether solid, liquid, or in other dosage forms, may also be used. for the purpose of formulation.
    [00050] [00050] A composition for oral administration may be presented in any orally acceptable dosage form, including capsules, tablets, aqueous emulsions and suspensions, dispersions, and solutions.
    [00051] [00051] In the case of tablets, carriers commonly used include, but are not limited to, lactose and corn starch. Lubricating agents, such as, but not limited to, magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include, but are not limited to, lactose and dry cornstarch. When aqueous suspensions or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oil phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents may be added.
    [00052] [00052] Pharmaceutical compositions for topical administration according to the described invention may be formulated as solutions, ointments, creams, suspensions, lotions, powders, pastes, gels, aerosols or oils. Alternatively, topical formulations may be in the form of dressings or dressings impregnated with active ingredient(s), which may optionally comprise one or more excipients or diluents. In some preferred embodiments, the topical formulations include a material that can enhance absorption or penetration of the active agent(s) through the skin or other affected areas. The topical composition is useful for the treatment of inflammatory skin disorders, including, but not limited to, eczema, acne, rosacea, psoriasis, contact dermatitis, and poison ivy reactions.
    [00053] [00053] A topical composition contains a safe and effective amount of a dermatologically acceptable carrier for proper application to the skin. A "cosmetically acceptable" or "dermatologically acceptable" composition or component refers to a composition or component that is suitable for use in contact with human skin without undue toxicity, incompatibility, instability, allergic response, or similar reactions. The vehicle enables an active agent and optional component to be delivered to the skin at an appropriate concentration. The vehicle in this manner can act as a diluent, dispersant, solvent, or the like to ensure that the active materials are applied and evenly distributed over the selected target at an appropriate concentration. The carrier may be solid, semi-solid, or liquid. The carrier may be in the form of a lotion, a cream, or a gel, in particular one that has a thickness or pour point sufficient to prevent the active materials from settling. The carrier may be inert or have dermatological benefits. It must also be physically and chemically compatible with the active components described in this document, and must not unduly impede the stability, efficacy or other benefits of uses associated with the composition. The topical composition can be a cosmetic or dermatological product in a form known in the art for topical or transdermal applications, including solutions, aerosols, creams, gels, covers, ointments, lotions, or foams.
    [00054] [00054] The described invention provides methods for treating a subject with an inflammatory disorder. The term "inflammatory disorder" refers to a disorder that is characterized by abnormal or unwanted inflammation, such as an autoimmune disease. Autoimmune diseases are diseases characterized by chronic activation of immune cells under conditions of non-activation. Examples include psoriasis, inflammatory bowel diseases (eg, Crohn's disease and ulcerative colitis), rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, lupus, type I diabetes, primary biliary cirrhosis, and transplantation.
    [00055] [00055] Other examples of inflammatory disorders that can be treated by the methods of this invention include asthma, myocardial infarction, stroke, inflammatory dermatoses (e.g. dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria, necrotizing vasculitis, Cutaneous vasculitis, hypersensitivity vasculitis, eosinophilic myositis, polymyositis, deraiatomyositis and eosinophilic fasciitis), acute respiratory distress syndrome, fulminant hepatitis, hypersensitivity lung diseases (eg, hypersensitivity pneumonia, eosinophilic pneumonia, delayed hypersensitivity pneumonia, interstitial lung disease ( interstitial lung disease (ILD), idiopathic pulmonary fibrosis, and ILD associated with rheumatoid arthritis) and allergic rhinitis. Additional examples also include myasthenia gravis, juvenile diabetes, glomerulonephritis, autoimmune thyroiditis, ankylosing spondylitis, systemic sclerosis, acute and chronic inflammatory diseases (e.g., anaphylaxis or hypersensitivity, systemic responses, drug allergies, insect bite allergies, graft rejection, and versus host) and Sjogren's syndrome.
    [00056] [00056] An "individual" refers to a human and non-human animal. Examples of a non-human animal include all vertebrates, for example mammals such as non-human mammals, non-human primates (particularly higher primates), dog, rodent (eg rat or mouse), guinea pig, cat, rabbit, and non-mammals such as birds, amphibians, reptiles, etc. In one embodiment, the individual is a human. In another embodiment, the subject is an experimental model, non-human animal, or animal suitable as a disease model.
    [00057] [00057] A subject to be treated for an inflammatory disorder can be identified by standard techniques for diagnosing the disorder. Optionally, the subject may be examined for the level or percentage of one or more cytokines or cells in a test sample obtained from the subject by methods known in the art. If the level or percentage is at or below a value (which can be obtained from a normal subject), the subject is a candidate for the treatment described herein. To confirm inhibition or treatment, one can assess and/or check the level or percentage of one or more of the aforementioned cytokines, or cells in the subject after treatment.
    [00058] [00058] "Treating" or "treatment" refers to the administration of a compound or agent to a subject who has a disorder for the purpose of curing, alleviating, aiding, repairing, delaying the onset, preventing, or ameliorating the disorder, the symptom of the disorder, the disease state secondary to the disorder, or the predisposition to the disorder.
    [00059] [00059] An "effective amount" or "therapeutically effective amount" refers to an amount of the compound or agent that is capable of producing a desired medical result in a treated subject. The method of treatment may be performed in vivo or ex vivo, alone or in conjunction with other drugs or therapy. A therapeutically effective amount may be administered in one or more administrations, applications or dosages and is not intended to be limited to one route of formulation or administration.
    [00060] [00060] The agent can be co-administered in vivo or ex vivo, alone or administered in conjunction with other drugs or therapy, i.e., a therapeutic cocktail. As used herein, the term "co-administration" or "co-administered" refers to the administration of at least two agents or therapies to an individual. In some embodiments, the co-administration of two or more agents/therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those skilled in the art will understand that the formulations and/or routes of administration of the various agents/therapies used may vary.
    [00061] [00061] In an in vivo approach, a compound or agent is administered to an individual. Generally, the compound or agent is suspended in a pharmaceutically acceptable vehicle (such as, but not limited to, physiological saline) and administered orally or by intravenous infusion, or injected or implanted subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonary.
    [00062] [00062] The dosage required depends on the choice of route of administration; the nature of the formulation; the nature of the patient's illness; the individual's size, weight, surface area, age and sex; of other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.01-100 mg/kg. Variations in dosage requirements are expected in view of the use of a variety of compounds/agents available and the different efficiencies of different routes of administration.
    [00063] [00063] For example, oral administration is expected to require higher dosages than administration via intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routes for optimization as is well understood in the art. Encapsulation of the compound in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) can increase delivery efficiency, particularly by oral delivery.
    [00064] [00064] This example describes general methods and materials used in Examples 2-7.
    [00065] [00065] Wild type C57BL/6 mice were purchased from Jackson Laboratories. SIGNR1 -/- mice were provided by A. McKenzie. CDl lc-DC-SIGN+ transgenic mice were provided by T. Sparwasser. The transgenic hDC-SIGN BAC mice in a SIGNRl-/- practice were generated in the inventors' laboratory as described above. KRN TCR C57BL/6 mice (gift of D. Mathis and C. Benoist) were bred with NOD mice to generate K/BxN mice. Blood from K/BxN mice (6-12 weeks of age) was collected and serum containing arthritogenic antibodies pooled together. Passive transfer of 200 µl K/BxN serum by intravenous injection to primitive rats (8-12 weeks old) induced arthritis. Inflammation was scored from 0-3 points for each paw which together generated an individual total clinical score per mouse. Recombinant Fc Preparation
    [00066] [00066] IDEC-114, a recombinant source of full-length human IgG1 monoclonal antibody, was digested with papain overnight at 37°C to cleave Fab and Fc fragments. After digestion, the reaction was stopped by the addition of 2.5 mg/ml of iodoacetamide. To separate cleaved fragments of undigested antibody, samples were passed through a HiPrep 26/60 S-200HR size exclusion column (GE HEALTHCARE). The Fc fragment was subsequently purified with protein G agarose capsules. The purity of the sample was verified by coomassie brilliant blue stain of SDS-polyacrylamide gel. Alternatively, recombinant Fcs were produced by transient transfection of human IgG1 Fc plasmid expressions into 293T cells, followed by ammonium sulfate precipitation of supernatants fractions and G protein purification. The genetic sequence encoding the Fc region of IgG1 was amplified from 4-4-20 IgG1 by standard PCR protocols and ligated into pSecTag2 (INVITROGEN). Point mutations were introduced into the Fc coding sequence using conventional mutagenesis techniques and verified by DNA sequencing. PCR primers for the Phe to Ala substitution at position 241 (FA241) were: 5'-ggggaccgtcagtcgccctttccccccaa-3' (SEQ ID NO: 4) and 5'-ttgggggggaagagggcgactgacggtcccc-3' (SEQ ID NO: 5).
    [00067] [00067] Protein expression and purification were confirmed by immunoblotting with anti-human Fc antibodies and/or SDS-polyacrylamide gel brilliant blue coomassie stain.
    [00068] [00068] After purification, 10-50 mg/ml Fc fragments were exchanged from the receptacle to the receptacle of a galactosylation reaction (50mM MOPS, pH 7.2; 20mM MnCl2) and incubated overnight at 37°C with 50 mg UDP-galactose and 0.75 U pi,4-galactosyltransferase. Galactosylation was confirmed by lectin stain using ECL to recognize terminal galactose residues. The galactosylated Fcs were then exchanged from the receptacle to the receptacle of a sialylation reaction (100 mM MOPS, 0.2 mg/ml BSA, 0.5% Triton X-100, pH 7.4) and incubated overnight at 37°C. °C with 50 mg CMP-sialic acid and 0.75 U 2,6-sialyltransferase. Siallylation was confirmed by lectin stain using SNA to recognize terminal sialic acid residues with an α.2-6 linkage.
    [00069] [00069] Bone Marrow cells were released from tibias and femurs of DC-SIGNtg or SIGNRl-/- mice, and seeded in untreated tissue culture 10 cm plates in RPMI 1640 growth medium supplemented with 10% FBS, 1 % Pen/Strep, IL-3 (5 ng/ml, PEPROTECH), and M-CSF (5 ng/ml, PEPROTECH). After overnight incubation at 37°C, non-adherent cells recovered and transferred to 10 cm untreated tissue culture plates with RPMI growth medium supplemented with IL-3/M-CSF-, and cultured for 5-7 days. at 37°C. Mature macrophages were trypsinized and plated in 6 culture plates at a density of 2x10 6 cells/culture, and allowed to bind overnight. The following day macrophages were pulsed with the indicated recombinant Fc preparation for 30 minutes at 37°C. Cells were recovered, washed with cold PBS, and 1x106 cells were administered intravenously to wild type C57BL/6 mice. One hour after injection, mice were infected with serum K/BxN. Expression and Purification of Soluble Human DC-SIGN
    [00070] [00070] A plasmid containing the extracellular domain (ECD) cDNA sequence of human DC-SIGN was provided by K. Drickamer. The coding sequence for DC-SIGN ECD was modified to introduce an N-terminus indicative of streptococcus by standard PCR techniques and linked to pET28b(+). pET28b-strepDCSIGN was transformed into E. coli strain BL21/DE3 and grown in 3 liters of TB growth medium at 37°C until the bacteria culture reached OD600 0.7-0.8. Protein expression was induced by adding 100 mg/l of IPTG and the culture incubated at 37°C for 3.5 h. Bacteria were sedimented by centrifugation at 4000xg for 10 minutes at 4°C. The sedimented bacteria were resuspended in 10 mM Tris-HCl, pH 7.8, and lysed by sonication. Inclusion bodies were sedimented by centrifugation at 10,000xg for 15 minutes at 4°C and solubilized 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 applied to a strep-tactin resin (NOVAGEN) to knock down strep labeled DC-SIGN ECD. Bound proteins were eluted from the resin with the elution receptacle provided by the manufacturer (NOVAGEN). Fractions were analyzed by SDS-PAGE and positive fractions pooled and loaded onto a mannose-agarose column to screen for active receptors. DC-SIGN ECD was eluted with 250 mM NaCl; 25 mM Tris-HCl, pH 7.8; 5 mM EDTA. Fractions were analyzed by SDS-PAGE.
    [00071] [00071] To determine the interaction between various recombinant Fc preparations for soluble hDC-SIGN or hFcRs, steady-state affinity measurements were recorded on a Biacore T100 sensor. Receptors, diluted to 20-50 µg/ml in NaOAc pH 5.0, were immobilized on CM5 chip at high density (2000 RU) by standard amine coupling. By hDC-SIGN interactions, injections were performed at a flow rate of 20 μl/minute with the commercially available HBS-P+ receptacle adjusted to pH 9.0 and supplemented with 2 mM CaCl2 and 500 mM NaCl. For hFcRs interactions, injections were performed at a flow rate of 20 μl/minute with the commercially available HBS-EP+ receptacle. The surfaces were regenerated with a short pulse of 50 mM NaOH. Kd values were calculated after subtracting the binding level for a cell control flow using the Biacore Evaluation software. RT-PCR
    [00072] [00072] Total RNA was extracted from bone marrow-derived macrophages using the RNeasy Mini Kit (QIAGEN). One microgram of total RNA was used for analysis of IL-33 mRNA expression by RT-PCR using the OneStep RT-PCR kit (QIAGEN). GAPDH expression served as load control. PCR primers for mIL-33 were 5'-gaagatcccaacagaagacc-3'(SEQ ID NO: 6) and 5'-ttccggaggcgagacgtcac-3'(SEQ ID NO: 7); and mGAPDH were 5'-gccgcctggagaaacctgc-3' (SEQ ID NO: 8) and 5'-tgaggtccaccaccctgttg-3' (SEQ ID NO: 9). PCR conditions were 94°C for 30 s; 55°C for 30 s; 72°C for 60 s x 35 cycles (IL-33) or 25 cycles (GAPDH).
    [00073] [00073] The lower population of antibodies in IVIG preparations suppressed autoantibody-induced inflammation. These antibodies, containing sialic acid ligand terminal 2,6 on the Fc glycan, mediated an anti-inflammatory response by binding to SIGNR1 in marginal zone macrophages or its human ortholog, DC-SIGN, in myeloid cells.
    [00074] [00074] To study the interaction of sFc with DC-SIGN, a soluble form of the extracellular domain of DC-SIGN (DC-SIGN ECD) was purified from bacteria and immobilized on CM5 chips. The sFc was prepared from full-length IDEC-114 antibodies and sialylated in vitro (Figure 1b), and was specifically bound to the conjugated surface of DC-SIGN (Figure la). Steady-state affinity measurements were performed and the KD value for this interaction was calculated to be approximately 1.3x10 -6 M (Figure la). In contrast, an asialylated glycoform of IDEC-114 Fc showed no binding activity for DC-SIGN suggesting that sialylation induces a conformational switch in the Fc backbone to reveal a DC-SIGN binding situation.
    [00075] [00075] As shown in Figure la, recombinant binding of cc2,6-sFc to soluble DC-SIGN as measured by surface plasmon resonance (SPR) was found. Fcs were prepared by papain cleavage of full-length human IgGl monoclonal antibody (IDEC-114) followed by in vitro galactosylation and sialylation reactions. Antibody binding SPR sensorgrams to immobilized DC-SIGN are shown by sialylated and galactosylated glycoforms of hlgG1 Fcs as described above in Example 1. Fc concentrations fluxes in DC-SIGN ECD were found to be in the range of 3-0 .8 μΜ. As shown in Figure 1b, lectin staining with SNA confirmed sialic acid binding with 2,6 binding on Fc
    [00076] [00076] The Asn-linked core oligosaccharide chain makes extensive non-covalent interactions with the Fc amino acid backbone. Conformational changes in Fc induced by different sugar residues attached to the glycan core are measured by these carbohydrate-protein interactions. Alanine substitutions that abrogate key contact point between the Fc backbone and glycan residues appear to confer DC-SIGN binding activity.
    [00077] [00077] As shown in Figure 2A, mutations FA241 and FA243 exhibit DC-SIGN binding activity without enzyme treatment in vitro. Apparent KD values are on the order of 6x10-7 M for FA241 to 3x10-7 M for FA243. Previous reports indicate that these mutations increase antibody sialylation, likely to make the glycan more accessible to glycosol transferase, when expressed in mammalian cells. To verify this, a lectin stain was performed to determine whether binding of FA241 and FA243 to DC-SIGN is due to increased sialylation of transiently expressed proteins. As shown in Figure 2B, SNA stains did not detect terminal sialic acid residues in purified FA241 and FA243 indicating that DC-SIGN interaction was independent of sialic acid modification.
    [00078] [00078] More specifically, residues F241, F243, D265, and
    [00079] [00079] If the FA241 and FA243 mutations mimic the DC-SIGN binding activity of sFc, assays were conducted to examine whether these mutations could replicate the anti-inflammatory activity of sFc in vivo. Matched by age and sex SIGNR1-/- and hDC-SIGN+/SIGNR-/- mice were infected with arthritogenic serum K/BxN and treated with sFc, FA241, or FA243 at an effective dose of 0.033 g/kg. Consistent with previous results, sFc suppressed lower paw swelling in DC-SIGN+ mice, but not in SIGNR1-/- mice. Similarly, FA241 demonstrated comparable anti-inflammatory activity to those with sFc in hDC-SIGN+/SIGNRr-/- mice. Mice dosed with FA243 showed no reduction in joint inflammation. This result suggests that recombinant Fcs having an F241A mutation
    [00080] [00080] As shown in Figure 3, hDC-SIGN+/SIGNRl-/- (open squares) and SIGNR1-/- (closed squares) mice were given 0.7 mg/mouse of sFc, FA241, or FA243 by intravenous injection. . Mice were subsequently infected with serum K/BxN 1 hour later. Lower paw swelling was monitored and recorded over several days. As previously reported, anti-inflammatory activity of sFc is DC-SIGN- dependent (left panel). FA241 also suppressed arthritic inflammation in infected K/BxN mice in a DC-SIGN dependent manner. FA243 did not significantly reduce lower paw swelling at 6 days. Means and standard error of the mean (Standard Error Mean - SEM) of clinical scores from 4-5 rats per group are plotted on Day 6.
    [00081] [00081] To identify the determining factors for the anti-inflammatory activity of FA241, bone marrow-derived macrophages (ΒΜΜΦ) from CDllc.DC-SIGN+ and SIGNR1-/- mice were stimulated with FA241 or other Fc preparations and transferred to mice. WT C57BL/6 K/BxN serum infected recipients.
    [00082] [00082] Briefly, bone marrow-derived macrophages from CDl lc.DC-SIGN+ and SIGNR1-/- mice were cultured in IL-3 (5 ng/ml) and M-CSF (5 ng/ml) for 5-7 days . as shown in Figure 4, DC-SIGN+ ΒΜΜΦ were pulsed with 0.5 mg/ml asialylated (black bars) or sialylated (white bars) glycoforms of indicated Fc preparations. Fc-treated ΒΜΜΦ were transferred to recipient WT C57BL/6 mice followed by -/- K/BxN challenge. as shown in Figure 4b SIGNRl (black bars) and DC-SIGN (white bars) ΒΜΜΦ were pulsed with 0.5 mg/ml of indicated Fc preparation and transferred to WT C57BL/6 recipient mice followed by K/BxN challenge . Similarly, DC-SIGN+ ΒΜΜΦ were pulsed with 0.5 mg/ml deglycosylated FA241 or FA241 (Figure 4c, white bars) or PBS (Figure 4c, black bar) and transferred to WT C57BL/6 recipient mice followed by infection by K/BxN. FA241 was deglycosylated with PNGase F and glycan removal confirmed by lecithin stains. DC-SIGN+ ΒΜΜΦ were pulsed with the indicated asialylated Fc preparation or PBS (Figure 4d, black circle) and transferred into WT C57BL/6 recipient mice followed by K/BxN infection. In all cases, lower paw swelling was monitored and recorded over several days. Means and standard error of the mean (Standard Error Mean - SEM) of clinical scores from 4-5 rats per group are plotted. *P < 0.05, as determined by an analysis of variance (ANOVA) test, followed by Tukey's post hoc test.
    [00083] [00083] As shown in Figure 4A, non-sialylated or sialylated preparations of FA241 were equally effective in suppressing joint inflammation compared to sFc. WT Fc preparations though, required sialic acid bound to .2,6 since DC-SIGN+ ΒΜΜΦ pulsed with asialylated WT Fc did not transfer protection to the deposited mice.
    [00084] [00084] Corresponding to the results shown in Figure 3, both sFc and FA241 required DC-SIGN expression in ΒΜΜΦ to transfer protection (Figure 4B). Although FA241 did not require sialic acid to transfer protection, deglycosylation with
    [00085] [00085] If an alanine substitution at position 241 induces a conformational shift in the Fc, then perhaps the affinity towards human Fc receptors will be altered. It was previously reported that sialylation reduces the affinity of IgGs for FcγRs, consequently attenuating ADCC activity in vivo.
    [00086] [00086] Recombinant binding of IgGl Fc's to soluble FcΧRs was measured by surface plasma resonance (Surface Plasmon Resonance - SPR). Fcs were prepared in the manner as described above. SPR sensorgrams for antibody binding to immobilized hFcγRIIA131R and hFcγRIIB are shown in Figure 5 by asialylated and sialylated glycoforms of WT hlgGl Fc and by asialylated FA241 Fc.
    [00087] [00087] As shown in Figure 5, asialylated glycoforms of WT Fc binding to hFcγRIIA and RIIB with an observed KD value of approximately 2-3xl 0-5 M. The sFc, however, does not become visible to bind hFc RIIA or RIIB. Surprisingly, FA241 becomes visible to bind both hFcγRIIA and RIIB with a higher affinity magnitude degree (KD = ~2x10 -6 M).
    [00088] [00088] SFc induced a TH2-dependent anti-inflammatory pathway, which requires the secretion of IL-4 from a population of FcεRI+, possibly basophilic, leukocytes to upregulate FcεRIIB in regulatory macrophages. Administration of IL-33 in vivo or in vitro stimulates basophils to release reserve IL-4. IL-33 mRNA expression is up-regulated in spleen of WT C57BL/6 mice treated with sFc or IVIG, but not in SIGNR1-/- mice. This suggests that sFc can induce IL-33 expression in SIGNR1-/- or DC-SIGN+ cells. In this example, trials were conducted and showed that stimulation of DC-SIGN+ ΒΜΜΦ with FA241 becomes viable to upregulate IL-33 expression.
    [00089] [00089] More specifically, bone marrow-derived macrophages from CD1 lc.DC-SIGN+ and SIGNR1-/- mice were cultured in the manner described above. ΒΜΜΦ were seeded in 12 culture plates in RPMI-free serum medium and allowed to adhere overnight at 37°C. The next day, cells were pulsed with 0.5 mg/ml of indicated RPMI-free serum Fc medium for 1 hour (Figure 6a.) or 4 hours (Figure 6b.) at 37°C. mRNA was collected from cells at specific time points and 1 μg total RNA used for IL-33 mRNA amplification (upper panels). GAPDH amplification served as a load control (lower panels). Co-expression of plasmid DA265 Fc and human sialyltransferase (ST6Gall) were transfected into 293T cells yielding highly sialylated recombinant Fc (ST6-DA265).
    [00090] [00090] As shown in Figure 6, stimulation of DC-SIGN+ ΒΜΜΦ with FA241 made it feasible to upregulate IL-33 expression. Despite higher basal IL-33 expression levels compared to DC-SIGN+ ΒΜΜΦ, SIGNR1 -/- ΒΜΜΦ down-regulated IL-33 mRNA expression in responses to FA241 treatment.
    [00091] [00091] The preceding example and description of preferred embodiments should be taken as illustrating and not limiting the present invention as defined by the claims. All publications cited in this document are hereby incorporated entirely by reference. The numerous variations and combinations of the above-defined features which may be utilized without departing from the present invention as defined in the claims will be readily appreciated. Such variations are not considered to depart from the scope of the invention, and all such variations are understood to fall within the scope of the following claims.
    权利要求:
    Claims (26)
    [1]
    1. An isolated polypeptide comprising a modified sequence that is at least 75% identical to an IgG Fc region, characterized in that: the modified sequence is free of sialylation and the polypeptide has an anti-inflammatory activity that is greater than that of an original polypeptide.
    [2]
    The isolated polypeptide of claim 1, characterized in that: the original polypeptide comprises the IgG Fc region.
    [3]
    The isolated polypeptide of claim 1 or 2, characterized in that: the IgG Fc region comprises the sequence SEQ ID NO: 1.
    [4]
    The isolated polypeptide of any one of claims 1-3, characterized in that: the isolated polypeptide has an ability to bind DC-SIGN.
    [5]
    The isolated polypeptide of any one of claims 1-4, characterized in that: the isolated polypeptide has an ability to bind hFcγRIIA or RIIB.
    [6]
    The isolated polypeptide of any one of claims 1-5, characterized in that: the isolated polypeptide has an ability to bind hFcγRIIA or RIIB at a KD of 2x10 -5 M or less (i.e., at a KA of 5, 0 x 104 M"1 or greater).
    [7]
    The isolated polypeptide of any one of claims 1-6, characterized in that: the modified sequence has an FA241 mutation.
    [8]
    The isolated polypeptide of any one of claims 1-7, characterized in that: the modified sequence is at least 75% identical to SEQ ID NO: 2.
    [9]
    The isolated polypeptide of claim 8, characterized in that: the modified sequence is at least 80% identical to SEQ ID NO: 2.
    [10]
    The isolated polypeptide of claim 9, characterized in that: the modified sequence is at least 90% identical to SEQ ID NO: 2.
    [11]
    The isolated polypeptide of claim 10, characterized in that: the modified sequence is at least 95% identical to SEQ ID NO: 2.
    [12]
    The isolated polypeptide of claim 11, characterized in that: the modified sequence is at least 99% identical to SEQ ID NO: 2.
    [13]
    The isolated polypeptide of claim 12, characterized in that: the modified sequence comprises SEQ ID NO: 2.
    [14]
    The isolated polypeptide of claim 9, characterized in that: the modified sequence consists essentially of SEQ ID NO: 2.
    [15]
    15. Method for obtaining a polypeptide with an anti-inflammatory activity, characterized by: comprising the steps of:
    providing a parent polypeptide having the sequence of an IgG Fc region or a first nucleic acid sequence encoding the parent polypeptide; and modifying the original polypeptide to obtain a modified polypeptide such that it is free of sialylation and mimics the structure of a sialylated form of the IgG Fc region.
    [16]
    The method of claim 15, characterized in that: the modification step is conducted by modifying the first nucleic acid sequence to obtain a second nucleic acid encoding the modified polypeptide.
    [17]
    17. Polypeptide characterized in that: it is obtained by the method of claims 15 or 16.
    [18]
    An isolated nucleic acid comprising: comprising a sequence encoding the polypeptide of any one of claims 1-14 and 17.
    [19]
    19. An expression vector characterized in that: it comprises a nucleic acid of claim 18.
    [20]
    20. Host cell characterized by: comprising a nucleic acid of claim 18.
    [21]
    21. A method of producing a polypeptide, characterized by: comprising the steps of: culturing the host cell of claim 20 in a medium under conditions that allow the expression of a polypeptide encoded by the nucleic acid, and purifying the polypeptide from the cell culture or the middle of the cell.
    [22]
    22. Pharmaceutical formulation characterized by: comprising:
    (i) a polypeptide of any one of claims 1-14 and 17 or a nucleic acid of claim 18, and (ii) a pharmaceutically acceptable carrier.
    [23]
    A method of treating an inflammatory disease, comprising: comprising administering to a subject in need of treatment a therapeutically effective amount of the polypeptide of any one of claims 1-14 and 17, or a nucleic acid encoding the polypeptide.
    [24]
    24. Isolated polypeptide, nucleic acid, expression vector, host cell, or composition substantially as shown and described herein.
    [25]
    25. A method of treating an inflammatory disease substantially as shown and described herein.
    [26]
    Use of a polypeptide of any one of claims 1-14 and 17 or a nucleic acid of claim 18 in the manufacture of a medicament for treating inflammatory disease.
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    法律状态:
    2018-08-28| B12F| Other appeals [chapter 12.6 patent gazette]|
    2020-09-29| B15I| Others concerning applications: loss of priority|Free format text: PERDA DA PRIORIDADE US 61/577,361 REIVINDICADA NO PPCT/US2012/068718, CONFORME AS DISPOSICOES PREVISTAS NA LEI 9.279 DE 14/05/1996 (LPI) ART. 167O E ART 2O DA RESOLUCAO INPI 179 DE 21/02/2017. ESTA PERDA SE DEU PELO FATO DE O DEPOSITANTE CONSTANTE DA PETICAO DE REQUERIMENTO DO PEDIDO PCT SER DISTINTO DAQUELES QUE DEPOSITARAM A PRIORIDADE REIVINDICADA E NAO APRESENTOU DOCUMENTO COMPROBATORIO DE CESSAO DENTRO DO PRAZO DE 60 DIAS A CONTAR DA DATA DA ENTRADA DA FASE NACIONAL, CONFORME AS DISPOSICOES PREVISTAS NA LEI 9.279 DE 14/05/1996 (LPI) ART. 166O, E NO ART. 28 DA RESOLUCAO INPI-PR 77/2013. |
    2020-10-20| B12F| Other appeals [chapter 12.6 patent gazette]|
    2020-11-17| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |
    2021-05-11| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|
    2021-06-01| B350| Update of information on the portal [chapter 15.35 patent gazette]|
    2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|
    优先权:
    申请号 | 申请日 | 专利标题
    US201161577361P| true| 2011-12-19|2011-12-19|
    US61/577,361|2011-12-19|
    PCT/US2012/068718|WO2013095966A1|2011-12-19|2012-12-10|Non-sialylated anti-inflammatory polypeptides|
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