AU2007317755A1 - Polypeptides with enhanced anti-inflammatory and decreased cytotoxic properties and relating methods - Google Patents

Description

WO 2008/057634 PCT/US2007/072771 POLYPEPTIDES WITH ENHANCED ANTI-INFLAMMATORY AND DECREASED CYTOTOXIC PROPERTIES AND RELATING METHODS CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part patent 5 application of PCT Patent Application Number PCT/US 07/08396, filed on April 3, 2007, which claims the benefit of United States Provisional Patent Application Number 60/789,384, filed on April 5, 2006, both of which are incorporated herein by reference. This application also 10 claims the benefit of PCT Patent Application Number PCT/US 06/41791, filed on October 27, 2006, and United States Provisional Patent Application Number 60/734,196, filed on November 7, 2005, both of which are also incorporated herein by reference. 15 STATEMENT REGARDING FEDERALLY FUNDED RESEARCH The Research leading to the present invention was supported in part, by National Institutes of Health Grant No. AI 034662. Accordingly, the U.S. Government may have certain rights in this invention. 20 FIELD OF THE INVENTION The present invention relates to novel method for designing therapeutic polypeptides for treatment of inflammatory diseases. BACKGROUND 25 Although cellular receptors for immunoglobulins were first identified nearly 40 years ago, their central role in the immune response was only discovered in the last decade. They are key players in both the afferent and efferent phase of an immune response, setting thresholds for B cell 30 activation and antibody production, regulating the maturation of dendritic cells and coupling the exquisite specificity of the antibody response to effector pathways, 1 WO 2008/057634 PCT/US2007/072771 such as phagocytosis, antibody dependent cellular cytotoxicity and the recruitment and activation of inflammatory cells. Their central role in linking the humoral immune system to innate effector cells has made them 5 attractive immunotherapeutic targets for either enhancing or restricting the activity of antibodies in vivo. The interaction of antibodies and antibody-antigen complexes with cells of the immune system effects a variety of responses, including antibody dependent cell-mediated 10 cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC), phagocytosis, inflammatory mediator release, clearance of antigen, and antibody half-life (reviewed in Daron, Annu Rev Immunol, 15, 203-234 (1997); Ward and Ghetie, Therapeutic Immunol, 2, 77-94 (1995); Ravetch and 15 Kinet, Annu Rev Immunol, 9, 457-492 (1991)), each of which is incorporated herein by reference). Antibody constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions. Depending on the amino acid sequence of 20 the constant region of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, and 25 IgG4; IgAl and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called a, d, e, ?, and p, respectively. Of the various human immunoglobulin classes, human IgG1 and IgG3 mediate ADCC more effectively than IgG2 and IgG4. 30 Papain digestion of antibodies produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual "Fc" fragment, 2 WO 2008/057634 PCT/US2007/072771 whose name reflects its ability to crystallize readily. The Fc region is central to the effector functions of antibodies. The crystal structure of the human IgG Fc region has been determined (Deisenhofer, Biochemistry, 20, 5 2361-2370 (1981), which is incorporated herein by reference). In human IgG molecules, the Fc region is generated by papain cleavage N-terminal to Cys, 226. IgG has long been appreciated to mediate both pro- and anti-inflammatory activities through interactions mediated 10 by its Fc fragment. Thus, while Fc-FcyR interactions are responsible for the pro-inflammatory 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. The 15 precise mechanism of such paradoxical properties is unclear but it has been proposed that glycosylation of IgG is crucial for regulation of cytotoxicity and inflammatory potential of IgG. IgG contains a single, N-linked glycan at Asn 297 in the 20 CH2 domain on each of its two heavy chains. The covalently linked, complex carbohydrate is composed of a core, biantennary penta-polysaccharide containing N acetylglucosamine (GIcNAc) and mannose (man). Further modification of the core carbohydrate structure is observed 25 in serum antibodies with the presence of fucose, branching GIcNAc, galactose (gal) and terminal sialic acid (sa) moieties variably found. Over 40 different glycoforms have thus been detected to be covalently attached to this single glycosylation site. Fujii et al., J. Biol. Chem 265, 6009 30 (1990). Glycosylation of IgG has been shown to be essential for binding to all FcyRs by maintaining an open conformation of the two heavy chains. Jefferis and Lund, Immune.1 Lett. 3 WO 2008/057634 PCT/US2007/072771 82, 57 (2002), Sondermann et al., J. Mol. Biol. 309, 737 (2001). This absolute requirement of IgG glycosylation for FcyR binding accounts for the inability of deglycosylated IgG antibodies to mediate in vivo triggered inflammatory 5 responses, such as ADCC, phagocytosis and the release of inflammatory mediators. Nimmerjahn and Ravetch, Immunity 24, 19 (2006). Further observations that individual glycoforms of IgG may contribute to modulating inflammatory responses has been suggested by the altered affinities for 10 individual FcyRs reported for IgG antibodies containing or lacking fucose and their consequential affects on cytotoxicity. Shields et al., J. Biol. Chem. 277, 26733 (2002), Nimmerjahn and Ravetch, Science 310, 1510 (2005). A link between autoimmune states and specific glycosylation 15 patterns of IgG antibodies has been observed in patients with rheumatoid arthritis and several autoimmune vasculities in which decreased galactosylation and sialylation of IgG antibodies have been reported. Parekh et al., Nature 316, 452 (1985), Rademacher et al., Proc. Natl. Acad. Sci. USA 20 91, 6123 (1994), Matsumoto et al., 128, 621 (2000), Holland et al., Biochim. Biophys. Acta Dec 27; [Epub ahead of print] 2005. Variations in IgG glycoforms have also been reported to be associated with aging and upon immunization, although the in vivo significance of these alterations have not been 25 determined. Shikata et al., Glycoconj. J. 15, 683 (1998), Lastra, et al., Autoimmunity 28, 25 (1998). Accordingly, there is a need for the development of methods for the generation of polypeptides that would account for the disparate observations of IVIG properties in 30 vivo. 4 WO 2008/057634 PCT/US2007/072771 SUMMARY OF INVENTION The present invention fills the foregoing need by providing such methods and molecules. In one aspect, the invention provides an isolated polypeptide containing at 5 least one IgG Fc region, having altered properties compared to an unpurified antibody preparation, wherein sialylation of the isolated polypeptide is higher than the sialylation of the unpurified antibody preparation. In one embodiment, the isolated polypeptide containing at least one IgG Fc 10 region is glycosylated with at least one galactose moiety connected to a respective terminal sialic acid moiety by a a 2,6 linkage, and wherein said polypeptide having a higher anti-inflammatory activity as compared to an unpurified antibody. In one embodiment the isolated polypeptide 15 containing at least one IgG Fc region is glycosylated with at least one galactose moiety connected to a respective terminal sialic acid moiety by a a 2,6 linkage, and wherein said polypeptide having a reduced binding to an Fc activating receptor as compared to an unpurified antibody 20 preparation. In a further embodiment the Fc activating receptor is selected from the group consisting of Fc?RIIA, Fc?RIIC and Fc?RIIIA. In another aspect, the instant invention provides a pharmaceutical formulation comprising a polypeptide 25 containing at least one Fc region having a higher anti inflammatory activity, in combination with a suitable carrier or diluent. A method of modulating properties of a polypeptide comprising an Fc region comprising altering the sialylation 30 of the polysaccharide chain of the Fc region. In one embodiment the method comprises: providing an unpurified source of the polypeptide containing at least one 5 WO 2008/057634 PCT/US2007/072771 Fc region, said unpurified source of the polypeptide containing at least one Fc region comprising a plurality of the polypeptides containing at least one Fc region having a polysaccharide chain comprising a terminal sialic acid 5 connected to a galactose moiety through a a 2,6 linkage, and a plurality of the polypeptides containing at least one Fc region lacking a polysaccharide chain comprising a terminal sialic acid connected to a galactose moiety through the X 2,6 linkage; and increasing the ratio of the plurality of 10 the polypeptides containing at least one Fc region having the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the X 2,6 linkage to the plurality of the polypeptide containing at least one Fc region lacking the polysaccharide chain comprising the 15 terminal sialic acid connected to the galactose moiety through the X 2,6 linkage. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an illustration of MALDI-Tof analysis of SNA FC linkages. 20 Figure 2 summarizes experiments demonstrating that enrichment of X 2,6 linkages between sialic acid and galactose improves anti-inflammatory properties of IVIG Fc fragments. Figure 3 summarizes experiments demonstrating that 25 removal of a 2,6 linkages between sialic acid and galactose attenuates anti-inflammatory properties of IVIG Fc fragments. DETAILED DESCRIPTION The inventors have surprisingly found that the 30 cytotoxic and anti-inflammatory response of the IgG Fc domain results from the differential sialylation of the Fc linked core polysaccharide. The cytotoxicity of IgG 6 WO 2008/057634 PCT/US2007/072771 antibodies is reduced upon sialylation; conversely, the anti-inflammatory activity of IVIG is enhanced. IgG sialylation is shown to be regulated upon the induction of an antigen-specific immune response, thus providing a novel 5 means of switching IgG from an innate, anti-inflammatory molecule in the steady-state, to a adaptive, pro inflammatory species upon antigenic challenge. The Fc sialylated IgGs bind to a unique receptor on macrophages that in turn upregulates an inhibitory Fcy receptor (FcyR) 10 thereby protecting against autoantibody-mediated pathology. See, generally, Ravetch and Nimmerjahn, J. Experim. Medicine 24(1): 11-15 (2007). Accordingly, the instant disclosure provides an advantageous strategy of creating and selecting IgGs with 15 desired cytotoxic and anti-inflammatory potential. DEFINITIONS Throughout the present specification and claims, the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., Sequences of 20 Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which is expressly incorporated herein by reference. The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. 25 The term "native" or "parent" refers to an unmodified polypeptide comprising an Fc amino acid sequence. The parent polypeptide may comprise a native sequence Fc region or an Fc region with pre-existing amino acid sequence modifications (such as additions, deletions and/or 30 substitutions). The term "polypeptide" refers to any fragment of a protein containing at least one IgG Fc region, including, 7 WO 2008/057634 PCT/US2007/072771 without limitation, fully functional proteins, such as, for example, antibodies, e.g., IgG antibodies. The term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain. The "Fc region" may 5 be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the 10 carboxyl-terminus thereof. The "CH2 domain" of a human IgG Fc region (also referred to as "C?2" domain) usually extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. 15 Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain (Burton, Mol Immunol, 22, 161 20 206 (1985), which is incorporated herein by reference). The "CH3 domain" comprises the stretch of residues C terminal to a CH2 domain in an Fc region (i.e., from about amino acid residue 341 to about amino acid residue 447 of an IgG). 25 The term "hinge region" is generally defined as stretching from Glu216 to Pro230 of human IgG1 (Burton (1985). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S--S bonds in 30 the same positions. The term "binding domain" refers to the region of a polypeptide that binds to another molecule. In the case of 8 WO 2008/057634 PCT/US2007/072771 an FcR, the binding domain can comprise a portion of a polypeptide chain thereof (e.g., the a chain thereof) which is responsible for binding an Fc region. One exemplary binding domain is the extracellular domain of an FcR chain. 5 A "functional Fc region" possesses at least a partial "effector function" of a native sequence Fc region. Exemplary "effector functions" include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); 10 phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays as herein disclosed, for 15 example. A "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 ordinary skill in the art comprises an amino acid 20 sequence which differs from that of a native sequence Fc region 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 sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about 25 one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology with a native sequence 30 Fc region and/or with an Fc region of a parent polypeptide, and more preferably at least about 90% homology therewith, 9 WO 2008/057634 PCT/US2007/072771 more preferably at least about 95% homology therewith, even more preferably, at least about 99% homology therewith. The term "altered glycosylation" refers to a polypeptide, as defined above, be it native or modified, in 5 which the carbohydrate addition to the heavy chain constant region is manipulated to either increase or decrease specific sugar components. For example, polypeptides, such as, for example, antibodies, prepared in specific cell lines, such as, for example, Lec2 or Lec3, may be deficient 10 in the attachment of sugar moieties such as fucose and sialic acid. 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 sequence human 15 FcR. In another embodiment, FcR, including human FcR, binds an IgG antibody (a gamma receptor) and includes receptors of the Fc?RI, Fc?RII, and Fc?RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc?RII receptors include Fc?RIIA (an "activating receptor") 20 and Fc?RIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor Fc?RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor 25 Fc?RIIB 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., 30 J Lab Clin Med, 126, 330-41 (1995), Nimmerjahn and Ravetch 2006, Ravetch Fc Receptors in Fundemental Immunology, ed 10 WO 2008/057634 PCT/US2007/072771 William Paul 5 th Ed. each of which is incorporated herein by reference). "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to an in vitro or in vivo cell-mediated 5 reaction in which cytotoxic cells that express FcRs (e.g., monocytic cells such as natural killer (NK) cells and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. In principle, any effector cell with an activating Fc?R can be triggered 10 to mediate ADCC. One such cell, the NK cell, expresses Fc?RIII only, whereas monocytes, depending on their state of activation, localization, or differentiation, can express Fc?RI, Fc?RII, and Fc?RIII. FcR expression on hematopoietic cells is summarized in Ravetch and Bolland, Annu Rev 15 Immunol, (2001), which is incorporated herein by reference. "Human effector cells" are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least one type of an activating Fc receptor, such as, for example, Fc?RIII and perform ADCC 20 effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, and neutrophils, with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source thereof, 25 e.g., from blood or PBMCs as described herein. The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and 30 antibody fragments so long as they exhibit the desired biological activity. 11 WO 2008/057634 PCT/US2007/072771 The phrase "sialic acid content" of an antibody refers both to the total number of sialic acid residues on an Fc region of a heavy chain of an antibody and to the ratio of sialylated antibodies to asialylated antibodies in an 5 unpurified antibody preparation, unless the phrase is in a context clearly suggesting that another meaning is intended. "Antibody fragments", as defined for the purpose of the present invention, comprise a portion of an intact antibody, generally including the antigen binding or variable region 10 of the intact antibody or the Fc region of an antibody which retains FcR binding capability. Examples of antibody fragments include linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The antibody fragments preferably retain at least 15 part of the hinge and optionally the CH1 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. The term "monoclonal antibody" as used herein refers to 20 an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being 25 directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The 30 modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as 12 WO 2008/057634 PCT/US2007/072771 requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, 5 Nature, 256, 495-497 (1975), which is incorporated herein by reference, or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567, which is incorporated herein by reference) . The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques 10 described in Clackson et al., Nature, 352, 624-628 (1991) and Marks et al., J Mol Biol, 222, 581-597 (1991), for example, each of which is incorporated herein by reference. In other embodiments of the invention, the polypeptide containing at least one IgG Fc region may be fused with 15 other protein fragments, including, without limitation, whole proteins. A person of ordinary skill in the art will undoubtedly appreciate that many proteins may be fused with the polypeptide of the present invention, including, without limitation, other immunoglobulins, especially, 20 immunoglobulins lacking their respective Fc regions. Alternatively, other biologically active proteins or fragments thereof may be fused with the polypeptide of the present invention, as described, for example, in the U.S. Patent No. 6,660,843, which is incorporated herein by 25 reference. This embodiment is especially advantageous for delivery of such biologically active proteins or fragments thereof to cells expressing Fc receptors. Further, different markers, such as, for example, GST tag or green fluorescent protein, or GFP, may be used. 30 The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or 13 WO 2008/057634 PCT/US2007/072771 homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding 5 sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Patent No. 4,816,567; Morrison et al., Proc Natl Acad Sci USA, 81, 6851-6855 10 (1984); Neuberger et al., Nature, 312, 604-608 (1984); Takeda et al., Nature, 314, 452-454 (1985); International Patent Application No. PCT/GB85/00392, each of which is incorporated herein by reference). "Humanized" forms of non-human (e.g., murine) 15 antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a 20 hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human 25 residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, 30 and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially 14 WO 2008/057634 PCT/US2007/072771 all of the FR residues are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For 5 further details, see Jones et al., Nature, 321, 522-525 (1986); Riechmann et al., Nature, 332, 323-329 (1988); Presta, Curr Op Struct Biol, 2, 593-596 (1992); U.S. Patent No. 5,225,539, each of which is incorporated herein by reference. 10 The polypeptides containing at least one IgG Fc region include those in which specific amino acid substitutions, additions or deletions are introduced into a parental sequence through the use of recombinant DNA techniques to modify the genes encoding the heavy chain constant region. 15 The introduction of these modifications follows well established techniques of molecular biology, as described in manuals such as Molecular Cloning (Sambrook and Russel, (2001)). In addition, the polypeptides with at least one Fc region will include those polypeptides which have been 20 selected to contain specific carbohydrate modifications, obtained either by expression in cell lines known for their glycosylation specificity (Stanley P., et al., Glycobiology, 6, 695-9 (1996); Weikert S., et al., Nature Biotechnology, 17, 1116-1121 (1999); Andresen DC and Krummen L., Current 25 Opinion in Biotechnology, 13, 117-123 (2002) ) or by enrichment or depletion on specific lectins or by enzymatic treatment (Hirabayashi et al., J Chromatogr B Analyt Technol Biomed Life Sci, 771, 67-87 (2002); Robertson and Kennedy, Bioseparation, 6, 1-15 (1996)). It is known in the art that 30 quality and extent of antibody glycosylation will differ depending on the cell type and culture condition employed. (For example, Patel et al., Biochem J, 285, 839-845 (1992)) 15 WO 2008/057634 PCT/US2007/072771 have reported that the content of sialic acid in antibody linked sugar side chains differs significantly if antibodies were produced as ascites or in serum-free or serum containing culture media. Moreover, Kunkel et al., 5 Biotechnol Prog, 16, 462-470 (2000) have shown that the use of different bioreactors for cell growth and the amount of dissolved oxygen in the medium influenced the amount of galactose and sialic acid in antibody linked sugar moieties. These studies, however, did not address how varying levels 10 of sialic acid residues influence antibody activity in vivo. Host Expression Systems The polypeptide of the present invention can be expressed in a host expression systems, i.e., host cells, capable of N-linked glycosylation. Typically, such host 15 expression systems may comprise bacterial, fungal, plant, vertebrate or invertebrate expression systems. In one embodiment the host cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell line, (e.g. CHO-K1; ATCC CCL-61), Green Monkey cell line (COS) (e.g. COS 1 (ATCC CRL 20 1650), COS 7 (ATCC CRL-1651)); mouse cell (e.g. NS/0), Baby Hamster Kidney (BHK) cell line (e.g. ATCC CRL-1632 or ATCC CCL-10), or human cell (e.g. HEK 293 (ATCC CRL-1573)), or any other suitable cell line, e.g., available from public depositories such as the American Type Culture Collection, 25 Rockville, Md. Further, an insect cell line, such as a Lepidoptora cell line, e.g. Sf9, a plant cell line, a fungal cell line, e.g., yeast such as, for example, Saccharomyces cerevisiae, Pichia pastoris, Hansenula spp., or a bacterial expression system based on Bacillus, such as B. subtilis, or 30 Eschericiae coli can be used. It will be appreciated by one of ordinary skill in the art that in some cases modifications to host cells may be required to insure that 16 WO 2008/057634 PCT/US2007/072771 N-linked glycosylation and glycan maturation occur to result in a complex, biantennary sugar as typically found on the Fc domain of human IgG. Therapeutic Formulations 5 Therapeutic formulations comprising the polypeptides containing at least one IgG Fc region can be prepared for storage by mixing the polypeptides of the present invention having the desired degree of purity with optional physiologically acceptable carriers, excipients or 10 stabilizers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and 15 include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenyl, butyl or benzyl 20 alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as 25 polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or 30 sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic 17 WO 2008/057634 PCT/US2007/072771 surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). The formulations herein may also contain more than one active compound as necessary for the particular indication 5 being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The active ingredients may also be entrapped in a 10 microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly (methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin 15 microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). In preferred embodiments, the formulations to be used 20 for in vivo administration are sterile. The formulations of the instant invention can be easily sterilized, for example, by filtration through sterile filtration membranes. Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include 25 semipermeable matrices of solid hydrophobic polymers containing the modified antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), 30 or poly(vinylalcohol)), polylactides (see, e.g., U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L glutamate, non-degradable ethylene-vinyl acetate, degradable 18 WO 2008/057634 PCT/US2007/072771 lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid glycolic acid copolymer and leuprolide acetate), and poly-D (-)-3-hydroxybutyric acid. While polymers such as ethylene 5 vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 10 37 0 C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S--S bond formation through 15 thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. 20 Creation of sialylated polypeptides containing at least one IgG Fc region. The polypeptides of the present invention can be further purified or modified so that they have an increased amount of sialic acid compared to unmodified and/or 25 unpurified antibodies. Multiple methods exist to reach this objective. In one method, the source of unpurified polypeptides, such as, for example, IVIG, is passed through the column having lectin, which is known to bind sialic acid. A person of the ordinary skill in the art will 30 appreciate that different lectins display different affinities for X2,6 versus X2,3 linkages between galactose and sialic acid. Thus, selecting a specific lectin will 19 WO 2008/057634 PCT/US2007/072771 allow enrichment of antibodies with the desired type of linkage between the sialic acid and the galactose. In one embodiment, the lectin is isolated from Sambuccus nigra. A person of the ordinary skill in the art will appreciate that 5 the Sambuccus nigra agglutinin (SNA) is specific for sialic acids linked to galactose or N-acetylgalactosamine by a(2-6) linkages. Shibuya et al, J. Biol. Chem., 262: 1596-1601 (1987) . In contrast, the Maakia amurensis ("MAA") lectin binds to sialic acid linked to galactose by a(2-3) linkages. 10 Wang et al, J Biol Chem., 263: 4576-4585 (1988). Thus, a fraction of the polypeptides containing at least one IgG Fc region having a desired linkage between the galactose and the sialic acid will be retained in the column while a fraction lacking such linkage will pass through. 15 The sialylated fraction of the polypeptides containing at least one IgG Fc region can be eluted by another wash with a different stringency conditions. Thus, it is possible to obtain a preparation of the polypeptide of the present invention wherein the content of sialic acid is increased 20 compared to the normal content. Further, one may employ an enzymatic reaction with a sialyltransferase and a donor of sialic acid as described, for example, in the U.S. Pat. No. 20060030521. Suitable non-limiting examples of sialyltransferase 25 enzymes useful in the claimed methods are ST3Gal III, which is also referred to as Q-(2,3)sialyltransferase (EC 2.4.99.6), and Q-(2,6)sialyltransferase (EC 2.4.99.1). Alpha-(2,3)sialyltransferase catalyzes the transfer of sialic acid to the Gal of a Gal-$-1,3GlcNAc or Gal-$ 30 1,4GlcNAc glycoside (see, e.g., Wen et al., J. Biol. Chem. 267: 21011 (1992); Van den Eijnden et al., J. Biol. Chem. 256: 3159 (1991)) and is responsible for sialylation of 20 WO 2008/057634 PCT/US2007/072771 asparagine-linked oligosaccharides in glycopeptides. The sialic acid is linked to a Gal with the formation of an c( linkage between the two saccharides. Bonding (linkage) between the saccharides is between the 2-position of NeuAc 5 and the 3-position of Gal. This particular enzyme can be isolated from rat liver (Weinstein et al., J. Biol. Chem. 257: 13845 (1982)); the human cDNA (Sasaki et al. (1993) J. Biol. Chem. 268: 22782-22787; Kitagawa & Paulson (1994) J. Biol. Chem. 269: 1394-1401) and genomic (Kitagawa et al. 10 (1996) J. Biol. Chem. 271: 931-938) DNA sequences are known, facilitating production of this enzyme by recombinant expression. Activity of Q-(2,6)sialyltransferase results in 6 sialylated oligosaccharides, including 6-sialylated 15 galactose. The name "Q-(2,6)sialyltransferase" refers to the family of sialyltransferases attaching sialic acid to the sixth atom of the acceptor polysaccharide. Different forms of a-(2,6)sialyltransferase can be isolated from different tissues. For example, one specific form of this 20 enzyme, ST6Gal II, can be isolated from brain and fetal tissues. Krzewinski-Recchi et al., Eur. J. Biochem. 270, 950 (2003). In addition, a person of average skill in the art will appreciate that cell culture conditions can be manipulated 25 to change the sialylation rate. For example, to increase the sialic acid content, production rate is decreased and osmolality is generally maintained within a lower margin suitable for the particular host cell being cultured. Osmolality in the range from about 250 mOsm to about 450 30 mOsm is appropriate for increased sialic acid content. This and other suitable cell culture conditions are described in, e.g., U.S. Patent No. 6,656,466. Patel et al., Biochem J, 21 WO 2008/057634 PCT/US2007/072771 285, 839-845 (1992) have reported that the content of sialic acid in antibody linked sugar side chains differs significantly if antibodies were produced as ascites or in serum-free or serum containing culture media. Moreover, 5 Kunkel et al., Biotechnol. Prog., 16, 462-470 (2000) have shown that the use of different bioreactors for cell growth and the amount of dissolved oxygen in the medium influenced the amount of galactose and sialic acid in antibody linked sugar moieties. 10 In another embodiment, host cells, such as, for example, immortalized human embryonic retina cells, may be modified by introducing a nucleic acid encoding a sialyltransferase such as, for example, an Q-2,3 sialyltransferase or an Q-2,6-sialyltransferase, operably 15 linked to a promoter, such as, for example, a CMV promoter. The Q-2,3-sialyltransferase may be the human x-2,3 sialyltransferase, known as SIAT4C or STZ (GenBank accession number L23767), and described, for example, in the U.S. Pat. No. 20050181359. 20 The nucleic acid encoding the sialyltransferase may be introduced into the host cell by any method known to a person of ordinary skill in the art. Suitable methods of introducing exogenous nucleic acid sequences are also described in Sambrook and Russel, Molecular Cloning: A 25 Laboratory Manual ( 3 rd Edition), Cold Spring Harbor Press, NY, 2000. These methods include, without limitation, physical transfer techniques, such as, for example, microinjection or electroporation; transfections, such as, for example, calcium phosphate transfections; membrane 30 fusion transfer, using, for example, liposomes; and viral transfer, such as, for example, the transfer using DNA or retroviral vectors. 22 WO 2008/057634 PCT/US2007/072771 The polypeptide containing at least one IgG Fc region may be recovered from the culture supernatant and can be subjected to one or more purification steps, such as, for example, ion-exchange or affinity chromatography, if 5 desired. Suitable methods of purification will be apparent to a person of ordinary skill in the art. A person of ordinary skill in the art will appreciate that different combinations of sialylation methods, disclosed above, can lead to production of the polypeptides 10 containing at least one IgG Fc region with an extremely high level of sialylation. For example, one can express the polypeptide containing at least one IgG Fc region in the host cells overexpressing sialyltransferase, as described above, and then further enrich the sialylated fraction of 15 these polypeptides by, for example, sialylating these polypeptides in an enzymatic reaction followed by an affinity chromatography using lectin-containing columns. Similarly, an enzymatic reaction followed by affinity chromatography may be used for IVIG source of the 20 polypeptides containing at least one IgG Fc region. To examine the extent of glycosylation on the polypeptides containing at least one IgG Fc region, these polypeptides can be purified and analyzed in SDS-PAGE under reducing conditions. The glycosylzation can be determined 25 by reacting the isolated polypeptides with specific lectins, or, alternatively as would be appreciated by one of ordinary skill in the art, one can use HPLC followed by mass spectrometry to identify the glycoforms. (Wormald, MR et al., Biochem 36:1370 (1997). 30 To describe the instant invention in more details, several non-limiting illustrative examples are given below. 23 WO 2008/057634 PCT/US2007/072771 EXAMPLES EXAMPLE 1. IVIG WITH INCREASED SIALIC ACID CONTENT EXHIBITS DECREASED CYTOTOXICITY To determine if specific glycoforms of IgG are involved 5 in modulating the effector functions of antibodies the role of specific, Asn2 - linked carbohydrates in mediating the cytotoxicity of defined IgG monoclonal antibodies was explored. The anti-platelet antibodies, derived from the 6A6 hybridoma, expressed as either an IgG1, 2a or 2b switch 10 variant in 293 cells as previously described (6), were analyzed by mass spectroscopy to determine their specific carbohydrate composition and structure. These antibodies contain minimal sialic acid residues. Enrichment of the sialic acid containing species by Sambucus nigra lectin 15 affinity chromatography yielded antibodies enriched 60-80 fold in sialic acid content. Comparison of the ability of sialylated and asialylated 6A6-IgGl and 2b antibodies to mediate platelet clearance revealed an inverse correlation between sialylation and in vivo activity. Sialylation of 6A6 20 IgG antibodies resulted in a 40-80% reduction in biological activity. To determine the mechanism of this reduction in activity surface plasmon resonance binding was performed on these antibodies for each of the mouse FcYRs and to its 25 cognate antigen. Surface plasmon resonance analysis was performed as described in Nimmerjahn and Ravetch, Science 310, 1510 (2005) . Briefly, 6A6 antibody variants containing high or low levels of sialic acid residues in their sugar side 30 chains were immobilized on the surface of CM5 sensor chips. Soluble Fcy-receptors were injected at different concentrations through flow cells at room temperature in 24 WO 2008/057634 PCT/US2007/072771 HBS-EP running buffer (10mM Hepes, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20) at a flow rate of 30 uI/min. Soluble Fc-receptors were injected for 3 minutes and dissociation of bound molecules was observed for 7 minutes. 5 Background binding to control flow cells was subtracted automatically. Control experiments were performed to exclude mass transport limitations. Affinity constants were derived from sensorgram data using simultaneous fitting to the association and dissociation phases and global fitting to 10 all curves in the set. A 1:1 Langmuir binding model closely fitted the observed sensorgram data and was used in all experiments. A 5-10 fold reduction in binding affinity was observed for the sialylated forms of these antibodies to their 15 respective activating FcyRs as compared to their asialylated counterparts, while no differences in binding affinity for the antigen were observed. Since IgG2b binds with a higher affinity to its activation receptor, FcyRIV, when compared to IgG1 binding to its activation receptor FcyRIII, the 20 effect of sialylation was to generate a binding affinity for IgG2b for its activation receptor FcyRIV that was comparable to that of asialylated IgG1 binding to its activation receptor FcYRlII. This effect of this quantitative difference in activation receptor binding resulted in 25 sialylated IgG2b displaying an in vivo activity comparable to that of asialylated IgG1. Similarly, sialylation of IgG1 reduces its already low binding affinity for its activation receptor FcyRlll by a factor of 7 thereby generating a physiologically inactive antibody. Thus, sialylation of the 30 Asn linked glycan structure of IgG resulted in reduced binding affinities to the subclass-restricted activation FcyRs and thus reduced their in vivo cytotoxicity. 25 WO 2008/057634 PCT/US2007/072771 To determine the generality of the observation that sialylation of the N-linked glycan of IgG was involved in modulating its in vivo inflammatory activity, we next examined the role of N-linked glycans on the anti 5 inflammatory activity of IVIG. This purified IgG fraction obtained from the pooled serum of 5-10,000 donors, when administered intravenously at high doses (1-2 g/kg), is a widely used therapeutic for the treatment of inflammatory diseases. Dwyer, N. Engl. J. Med. 326, 107 (1992). This 10 anti-inflammatory activity is a property of the Fc fragment and is protective in murine models of ITP, RA and nephrotoxic nephritis. Imbach et al., Lancet 1, 1228 (1981), Samuelsson et al., Science 291, 484 (2001), Bruhns et al., Immunity 18, 573 (2003), Kaneko et al., J. Exp. Med. 15 in press (2006). A common mechanism for this anti-inflammatory activity was proposed involving the induction of surface expression of the inhibitory FcyRIIB molecule on effector macrophages, thereby raising the threshold required for cytotoxic IgG 20 antibodies or immune complexes to induce effector cell responses by activation FcyR triggering. Nimmerjahn and Ravetch, Immunity 24, 19 (2006). EXAMPLE 2. ASIALYLATION OF IVIG DECREASES THE ANTI INFLAMMATORY EFFECT OF IVIG IN MOUSE ARTHRITIS MODEL 25 Mice C57BL/6 and NOD mice were purchased from the Jackson Laboratory (Bar Harbor, ME) . FcyRIIB mice were generated in the inventors' laboratory and backcrossed for 12 generations to the C57BL/6 background. KRN TCR transgenic 30 mice on a C57BU6 background (K/B) were gifts from D. Mathis and C. Benoist (Harvard Medical School, Boston, MA) and were bred to NOD mice to generate K/BxN mice. Female mice at 8-10 26 WO 2008/057634 PCT/US2007/072771 weeks of age were used for all experiments and maintained at the Rockefeller University animal facility. All experiments were done in compliance with federal laws and institutional guidelines and have been approved by the Rockefeller 5 University (New York, NY). Antibodies and soluble Fc receptors 6A6 antibody switch variants were produced by transient transfection of 293T cells followed by purification via protein G as described. Nimmerjahn and Ravetch, Science 10 310, 1510 (2005). Sialic acid rich antibody variants were isolated from these antibody preparations by lectin affinity chromatography with Sambucus nigra agglutinin (SNA) agarose (Vector Laboratories, Burlingame, CA). Enrichment for sialic acid content was verified by lectin blotting (see below). 15 Human intravenous immune globulin (IVIG, 5% in 10% maltose, chromatography purified) was purchased from Octapharma (Hemdon, VA). Digestion of human IVIG was performed as described. Kaneko Y. et al., Exp. Med. in press (2006) . Briefly, IVIG was digested by 0.5 mg/ml papain for 1 hr at 20 37 'C, and stopped by the addition of 2.5 mg/ml iodoasetamide. Fab and Fc resulting fragments were separated from non-digested IVIG on a HiPrep 26/60 S-200HR column (GE Healthcare, Piscataway, NJ), followed by purification of Fc and Fab fragments with a Protein G column (GE Healthcare) 25 and a Protein L column (Pierce, Rockford, IL). Fragment purity was checked by immunoblotting using anti-human IgG Fab or Fc-specific antibodies. (Jackson ImmunoResearch, West Grove, PA). Purity was judged to be greater than 99%. The F4/80 antibody was from Serotec (Oxford, UK). The Ly 17.2 30 antibody was from Caltag (Burlingame, CA). Sheep anti glomerular basement membrane (GBM) antiserum (nephrotoxic serum, NTS) was a gift from M. P. Madaio (University of 27 WO 2008/057634 PCT/US2007/072771 Pennsylvania, Philadelphia, PA). Soluble Fc receptors containing a C-terminal hexa-hisitidine tag were generated by transient transfection of 293T cells and purified from cell culture supernatants with Ni-NTA agarose as suggested 5 by the manufacturer (Qiagen). IVIG was treated with neuraminidase and the composition and structure of the resulting preparation was analyzed by mass spectroscopy. No detectable sialic acid containing glycans remained after neuraminidase treatment. These IgG 10 preparations were then tested for their ability to protect mice from joint inflammation induced by passive transfer of KxN serum, an IgG 1 immune complex-mediated inflammatory disease model. De-sialylation with neuraminidase abrogated the protective effect of the IVIG preparation in the KXN 15 serum induced arthritis model. This loss of activity was not the result of reduced serum half-life of the asialylated IgG preparations or the result of changes to the monomeric composition or structural integrity of the IgG. Removal of all glycans with PNGase had a similar effect and abrogated 20 the protective effect of IVIG in vivo. EXAMPLE 3. IVIG FRACTION WITH ENRICHED SIALIC ACID CONTENT DECREASES INFLAMMATION IN MOUSE ARTHRITIS MODEL Preparation of IVIG with an increased content of sialic acid 25 Since sialic acid appeared to be required for the anti inflammatory activity of IVIG, the basis for the high dose requirement (1 g/kg) for this anti-inflammatory activity could be the limiting concentration of sialylated IgG in the total IVIG preparation. The IVIG was fractionated on an SNA 30 lectin affinity column to obtain IgG molecules enriched for sialic acid modified glycan structures. 28 WO 2008/057634 PCT/US2007/072771 These sialic acid enriched fractions were tested for protective effects in the KxN serum transfer arthritis model as compared to unfractionated IVIG. A 10 fold enhancement in protection was observed for the SNA-binding fraction, such 5 that equivalent protection was obtained at 0.1 g/kg of SNA enriched IVIG as compared to 1 g/kg of unfractionated IVIG. The serum half-life and IgG subclass distribution of the SNA enriched fraction was equivalent to that of unfractionated IVIG. The effect of sialylation was specific to IgG; 10 sialylated N-linked glycoproteins such as fetuin or transferrin with similar bi-antennary, complex carbohydrate structures had no statistically significant anti inflammatory activity at equivalent molar concentrations of IgG. Finally, the mechanism of protection of the sialylated 15 IVIG preparation was similar to unfractionated IVIG in that it was dependent on FcyRIIB expression and resulted in the increased expression of this inhibitory receptor on effector macrophages. EXAMPLE 4. THE INCREASED ANTI-INFLAMMATORY RESPONSE OF IVIG 20 WITH INCREASED SIALIC ACID CONTENT IS MEDIATED BY SIALYLATION OF THE N-LINKED GLYCAN ON THE FC DOMAIN Since the polyclonal IgG in IVIG may also contain 0 and N linked glycans on the light chains or heavy chain variable domains that can be sialylated, we confirmed that the 25 increase in anti-inflammatory activity of the SNA-enriched IgG preparation resulted from increased sialylation of the N-linked glycosylation site on the Fc. Fc fragments were generated from unfractionated and SNA fractionated IVIG and tested for their in vivo activity. As observed for intact 30 IgG, SNA-purified Fc fragments were enhanced for their protective effect in vivo when compared to Fc fragments generated from unfractionated IVIG. In contrast, Fab fragments displayed no anti-inflammatory activity in this in 29 WO 2008/057634 PCT/US2007/072771 vivo assay. Thus, the high dose requirement for the anti inflammatory activity of IVIG can be attributed to the minor contributions of sialylated IgG present in the total preparation. Enrichment of these fractions by sialic acid 5 binding lectin chromatography consequently increased the anti-inflammatory activity. These results using passive immunization of IgG antibodies indicated that the ability of IgG to switch from a pro-inflammatory to an anti-inflammatory species is 10 influenced by the degree of sialylation of the N-linked glycan on the Fc domain. EXAMPLE 5. INCREASE OF ANTI-INFLAMMATORY ACTIVITY, MEDIATED BY SIALYLATION OF IgG, OCCURS DURING AN ACTIVE IMMUNE RESPONSE 15 Murine model for Goodpasture's Disease In this model, mice are first sensitized with sheep IgG together with adjuvant and four days later injected with a sheep anti-mouse glomerular basement membrane preparation (nephrotoxic serum, NTS). Briefly, mice were pre-immunized 20 intraperitoneally with 200 ig of sheep IgG (Serotec) in CFA, followed by intravenous injection of 2.5 jLl of NTS serum per gram of body weight four days later. Blood was collected from non-treated control mice four days after the anti-GBM anti-serum injection, and serum IgG was purified by Protein 25 G (GE Healthcare, Princeton, NJ) and sepharose-bound sheep IgG column, generated by covalently coupling sheep IgG on NHS-activated sepharose column (GE Healthcare, Princeton, NJ), affinity chromatography. Pre-sensitization followed by treatment with NTS 30 induces mouse IgG2b anti-sheep IgG antibodies (NTN immunized). Kaneko Y. et al., Exp. Med., 203:789 (2006). Mouse IgG2b antibodies are deposited in the glomerulus 30 WO 2008/057634 PCT/US2007/072771 together with the NTS antibodies and result in an acute and fulminant inflammatory response by the IgG2b mediated activation of FcyRIV on infiltrating macrophages. In the absence of pre-sensitization inflammation is not observed, 5 indicating that the mouse IgG2b anti-sheep IgG antibodies are the mediators of the inflammatory response. To determine if active immunization resulting in pro inflammatory IgG is associated with a change in sialylation, serum IgG and IgM from preimmune and NTS immunized mice were 10 characterized for sialic acid content by SNA lectin binding. Total IgG sialylation was reduced on average by 40% in immunized mice as compared to the unimmunized controls. The effect was specific for IgG; sialylation of IgM was equivalent pre and post immunization. This difference in 15 sialylation was more pronounced when the sheep specific IgG fraction from mouse serum was analyzed, showing a 50-60% reduction in sialylation compared to preimmune IgG. These results were confirmed by MALDI-TOF-MS analysis. Monosaccharide composition analysis was performed by UCSD 20 Glycotechnology Core Resource (San Diego, CA). Glycoprotein samples were denatured with SDS and 2-mercaptoethanol, and digested with PNGase F. The released mixed N-glycans were purified by reversed-phase HPLC and solid-phase extraction, and then exposed hydroxyl groups of the N-glycans were 25 methylated. The resulting derivatized saccharides were purified again by reversed-phase HPLC and subject to MALDI TOF-MS. The analysis of the pre and post immunization IgGs confirmed that the changes in the N-glycan structure were 30 specific to the terminal sialic acids moieties. The mouse IgG2b anti-sheep antibodies that were deposited in the glomeruli, previously shown to be responsible for engagement 31 WO 2008/057634 PCT/US2007/072771 of the FcyRIV bearing, infiltrating macrophages displayed reduced sialic acid content as compared to the pre-immunized controls. EXAMPLE 6. ANALYSIS OF LINKAGES BETWEEN SIALIC ACID AND 5 GALACTOSE IN IVIG Sequential Maldi-Tof analysis of SNA (Sambuccus Nigra Agglutinin) IVIG Fc linkages was performed to determine the structure of the sialylated IgG Fc fraction that was protective in the ITP, RA and nephrotoxic nephritis models 10 described above. Glycan peaks generated in Maldi-TOF were isolated, further fractionated, and reanalyzed until galactose-sialic acid structures were obtained. The footprint histogram of the enriched galactose-sialic acid structures with in vivo anti-inflammatory activity (Figure 15 1A) were compared to histograms from sialic acid linkage standards, a2-3 sialyllactose (Figure 1B) and a2-6 sialyllactose (Figure 1C). The signature peaks of the standards are identified by arrows, shown by arrows for a2-3 (Figure 1B) or a2-6 (Figures 1A and 1C), respectively, and 20 compared to the peaks obtained from the sample. EXAMPLE 7. ENRICHMENT OF IVIG FC FRAGMENTS IN 0x2,6 LINKAGES BY IN VITRO GLYCOSYLATION IMPROVES ANTI-INFLAMMATORY PROPERTIES OF IVIG. As shown in Figure 2A, glycan Maldi-Tof MS analysis of 25 IVIG Fc fragments showed structures ending in no galactose (peak GO), one galactose (peak G1), two galactose (peak G2), or in sialic acid (indicated by a bracket entitled "Terminal sialic acid"). To determine the in vivo activity of 2,3 or 2,6 sialylated IgG Fc, samples were treated with sialidase, 30 followed by galactose transferase to convert the GO (no galactose) and G1 (single galactose) to G2 (fully galactosylated) to increase potential sialylation sites. As shown in Figure 2B hypergalactosylation was verified by 32 WO 2008/057634 PCT/US2007/072771 comparing relative band intensity ratios of terminal galactose as measured by ECL and coomassie loading controls. In vitro sialylation was performed (Figure 2C) using either a 2-6 sialyltransferase ("ST6Gal") or a 2-3 5 sialyltransferase ("ST3Gal") and confirmed by lectin blotting for a 2-6 linkages with SNA (top) or a2-3 linkages with ECL (middle) and coomassie (bottom). To evaluate the ability of in vitro sialylated Fc to inhibit inflammation (Figure 2D) mice received either 0.66 mg of a 2-6 sialylated 10 Fcs (black triangles) or 0.66 mg a 2-3 sialylated Fcs (red triangles). 1 hour later, 0.2 ml of K/BxN sera was administered, and the swelling of footpads (clinical score) was monitored over the next seven days. Anti-inflammatory activity was observed for the 2,6 sialylated IgG Fc 15 fragments but not for the 2,3 sialylated molecules. These results are consistent with the data shown above and indicate that a preferential linkage of 2,6 sialic acid galactose is involved in the anti-inflammatory activity of sialylated IgG. 20 EXAMPLE 8. REMOVAL OF a 2-6 BUT NOT 2,3 SIALIC ACID LINKAGES ABROGATES THE IMMUNOSUPPRESSIVE PROPERTIES OF IVIG IVIG was treated with linkage specific sialidases (SAs), and the digestion verified by lectin blotting (Figure 3A). The top panel shows positive Sambucus nigra lectin 25 (SNA) staining for a 2-6 linkages in IVIG (left lane), and a 2-3 SA tx IVIG (center lane), but not in a 2-3,6 SA tx IVIG (right lane). The middle panel is a dot blot for a2-3 sialic acid linkages (MAL I), displaying positive staining for the fetuin positive control only; 100 pg protein are loaded per 30 dot. The bottom panel shows coomassie loading control. 10 pg/lane are shown in the blot and gel. To examine the effect of specific removal of sialic acid moieties, mice were given 33 WO 2008/057634 PCT/US2007/072771 lg/kg of IVIG preparations prior to 200 pl of K/BxN sera. As shown in Figure 3B, footpad swelling was observed in mice administered K/BxN sera (white circles) over the course of a week, as measured by clinical scoring. IVIG treated mice 5 showed minimal swelling (black triangles), as did mice treated with a2-3 SA tx IVIG (white triangles), while mice receiving a2-3,6 SA tx IVIG (red squares) were not protected from footpad swelling. All patent and non-patent publications cited in this 10 disclosure are incorporated herein in to the extent as if each of those patent and non-patent publications was incorporated herein by reference in its entirety. Further, even though the invention herein has been described with reference to particular examples and embodiments, it is to 15 be understood that these examples and embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised 20 without departing from the spirit and scope of the present invention as defined by the following claims. 34

Claims (31)

1. An isolated polypeptide containing at least one IgG Fc region, having altered properties compared to an unpurified antibody preparation, wherein sialylation of the 5 isolated polypeptide is higher than the sialylation of the unpurified antibody preparation.

2. The isolated polypeptide of claim 1, wherein said at least one IgG Fc region is glycosylated with at least one 10 galactose moiety connected to a respective terminal sialic acid moiety by a a 2,6 linkage, and wherein said polypeptide having a higher anti-inflammatory activity as compared to an unpurified antibody preparation. 15

3. The isolated polypeptide of claim 1 or 2, wherein said at least one IgG Fc region is glycosylated with at least one galactose moiety connected to a respective terminal sialic acid moiety by a a 2,6 linkage, and wherein said polypeptide having a reduced binding to an Fc 20 activating receptor as compared to an unpurified antibody preparation.

4. The isolated polypeptide of claim 3, wherein the Fc activating receptor is selected from the group consisting 25 of Fc?RIIA, Fc?RIIC and Fc?RIIIA.

5. The polypeptide of any one of claims 1-4 comprising a human IgG1, IgG2, IgG3 or IgG4 Fc region, said polypeptide having a higher content of the at least one 30 galactose moiety connected to the respective terminal sialic 35 WO 2008/057634 PCT/US2007/072771 acid moiety by a a 2,6 linkage as compared to an unpurified antibody.

6. The polypeptide of any one of claims 1-5, having 5 an increased anti-inflammatory activity in vitro.

7. The polypeptide of any one claims 1-6, having an increased anti-inflammatory activity in vivo. 10

8. The polypeptide of any one of claims 1-7, derived from a naturally occurring antibody source.

9. The polypeptide of any one of claims 1-7, derived from a recombinant antibody source. 15

10. The polypeptide of any one of claims 1-7, wherein said unmodified antibody comprises IVIG.

11. The polypeptide of any one of claims 1-7, derived 20 from a cell line having an enhanced activity of creating Q2,6 linkages between at least one galactose moiety and a respective terminal sialic acid in a protein's polysaccharide chain. 25

12. The polypeptide of any one of claims 1-11, modified by treatment with Q2-6 sialyltransferase.

13. The polypeptide of any one of claims 1-12, which is purified. 30 36 WO 2008/057634 PCT/US2007/072771

14. A pharmaceutical formulation comprising the polypeptide of any one of claims 1-13 in combination with a suitable carrier or diluent. 5

15. A method of modulating properties of a polypeptide comprising an Fc region comprising altering the sialylation of the polysaccharide chain of the Fc region.

16. A method of claim 15, wherein said properties 10 comprise a higher anti-inflammatory activity than an unpurified antibody.

17. The method of claim 15 or 16, wherein the step of altering sialylation comprises: 15 providing an unpurified source of the polypeptide containing at least one Fc region, said unpurified source of the polypeptide containing at least one Fc region comprising a plurality of the polypeptides containing at least one Fc region having a polysaccharide chain comprising a terminal 20 sialic acid connected to a galactose moiety through a a 2,6 linkage, and a plurality of the polypeptides containing at least one Fc region lacking a polysaccharide chain comprising a terminal sialic acid connected to a galactose moiety through the a 2,6 linkage; and 25 increasing the ratio of the plurality of the polypeptides containing at least one Fc region having the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the a 2,6 linkage to the plurality of the polypeptide containing at least one 30 Fc region lacking the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the a 2,6 linkage. 37 WO 2008/057634 PCT/US2007/072771

18. The method of any one of claims 15-17, wherein said unpurified source of the polypeptide containing at least one Fc region comprises a human unpurified IgG 5 antibody.

19. The method of any one of claims 15-18, wherein the unpurified source of the polypeptide containing at least one Fc region is provided from expressing a vector comprising a 10 nucleic acid sequence in an expression system, wherein said nucleic acid sequence is translated into an IgG antibody.

20. The method of claim 19, wherein the expression system comprises modified host expression systems capable of 15 N-linked glycosylation selected from the group consisting of bacterial, fungal, plant, vertebrate and invertebrate expression systems, and any combinations thereof.

21. The method of any one of claims 15-20, wherein the 20 step of increasing the ratio of the plurality of the polypeptides containing at least one Fc region having the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the X 2,6 linkage to the plurality of the polypeptide containing at least one 25 Fc region lacking the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the X 2,6 linkage is achieved through a removal of the polypeptides containing at least one Fc region lacking the polysaccharide chain comprising the terminal sialic acid 30 connected to the galactose moiety through the X 2,6 linkage. 38 WO 2008/057634 PCT/US2007/072771

22. The method of claim 21, wherein said removal is achieved by physical or chemical methods.

23. The method of claim 21 wherein said removal is 5 achieved by a method selected from the group consisting of HPLC, lectin affinity chromatography, high pH anion exchange chromatography, and any combination thereof.

24. The method of claim 23, wherein the lectin 10 affinity chromatography is performed using a lectin having a lower affinity to X2,6 linkages than to X2,3 linkages between the galactose moiety and the terminal sialic acid.

25. The method of any one of claims 15-24, wherein 15 the step of increasing the ratio of the plurality of the polypeptides containing at least one Fc region having the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the X 2,6 linkage to the plurality of the polypeptide containing at least one 20 Fc region lacking the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the X 2,6 linkage is achieved through an enrichment of said unpurified source of the polypeptide containing at least one Fc region having the polysaccharide chain 25 comprising the terminal sialic acid connected to the galactose moiety through the X 2,6 linkage.

26. The method of claim 25 wherein said enrichment is achieved by a method selected from the group consisting of 30 HPLC, lectin affinity chromatography, high pH anion exchange chromatography, and any combination thereof. 39 WO 2008/057634 PCT/US2007/072771

27. The method of claim 26, wherein the lectin affinity chromatography is performed using a lectin having a higher affinity to Q2,6 linkages than to x2,3 linkages between the galactose moiety and the terminal sialic acid. 5

28. The method of any one of claims 25-27 wherein said enrichment is achieved by a chemical reaction with an enzyme creating a 2,6 linkages between the carbohydrate attached to the polypeptide containing least one Fc region and a 10 terminal sialic acid.

29. The method of claim 28, wherein the enzyme is c( (2,6)sialyltransferase. 15

30. A method of treating an inflammatory disease comprising administering to a patient a therapeutically effective dose of the polypeptide of any one of claims 1-14.

31. The method of claim 30, wherein the inflammatory 20 disease is selected from the group consisting of arthritis, thrombocytopenia, and nephritis. 40