Patent Publication Number: US-2018044416-A1

Title: Polymeric Fc proteins and methods of screening to alter their functional characteristics

Description:
The invention relates to polymeric Fc proteins which bind to human Fc-receptors and methods of screening said polymeric Fc proteins to alter their functional characteristics. The invention also relates to therapeutic compositions comprising the polymeric Fc proteins, and their use in the treatment of immune disorders. 
     BACKGROUND 
     Immune disorders encompass a wide variety of diseases with different signs, symptoms, etiologies and pathogenic mechanisms. Many of these diseases are characterised by the active involvement of pathogenic antibodies and/or pathogenic immune complexes. In some diseases such as ITP (variably called immune thrombocytopenia, immune thrombocytic purpura, idiopathic thrombocytopenic purpura) the target antigens for the pathogenic antibodies (Hoemberg, Scand H J Immunol, Vol 74(5), p 489-495, 2011) and disease process are reasonably well understood. Such immune disorders are often treated with a variety of conventional agents, either as monotherapy or in combination. Examples of such agents are corticosteroids, which are associated with numerous side effects, intravenous immunoglobulin (IVIG) and anti-D. 
     Antibodies, often referred to as immunoglobulins, are Y-shaped molecules comprising two identical heavy (H) chains and two identical light (L) chains, held together by interchain disulphide bonds. Each chain consists of one variable domain (V) that varies in sequence and is responsible for antigen binding. Each chain also consists of at least one constant domain (C). In the light chain there is a single constant domain. In the heavy chain there are at least three, sometimes four constant domains, depending on the isotype (IgG, IgA and IgD have three, IgM and IgE have four). 
     In humans there are five different classes or isotypes of immunoglobulins termed IgA, IgD, IgE, IgG and IgM. All these classes have the basic four-chain Y-shaped structure, but they differ in their heavy chains, termed α, δ, ε, γ and μ respectively. IgA can be further subdivided into two subclasses, termed IgA1 and IgA2. There are four sub-classes of IgG, termed IgG1, IgG2, IgG3 and IgG4. 
     The Fc-domain of an antibody typically comprises at least the last two constant domains of each heavy chain which dimerise to form the Fc domain. The Fc domain is responsible for providing antibody effector functions, including determining antibody half-life, principally through binding to FcRn, distribution throughout the body, ability to fix complement, and binding to cell surface Fc receptors. 
     The differences between antibody isotypes are most pronounced in the Fc-domains, and this leads to the triggering of different effector functions on binding to antigen. Structural differences also lead to differences in the polymerisation state of the antibodies. Thus IgG, IgE and IgD are generally monomeric whereas IgM occurs as both a pentamer and a hexamer, IgA occurs predominantly as a monomer in serum and as a dimer in sero-mucous secretions. 
     Intravenous immunoglobulin (IVIG) is the pooled immunoglobulin from thousands of healthy blood donors. IVIG was initially used as an IgG replacement therapy to prevent opportunistic infections in patients with low IgG levels (reviewed in Baerenwaldt, Expert Rev Clin Immunol, Vol 6(3), p 425-434, 2010). After discovery of the anti-inflammatory properties of IVIG in children with ITP (Imbach, Helv Paediatri Acta, Vol 36(1), p 81-86, 1981), IVIG is now licensed for the treatment of ITP, Guillain-Barré syndrome, Kawasaki disease, and chronic inflammatory demyelinating polyneuropathy (Nimmerjahn, Annu Rev Immunol, Vol 26, p 513-533, 2008). 
     In diseases involving pathogenic immune complexes it has been proposed that a minority fraction of the component immunoglobulin fraction is disproportionately effective. It is observed that traces (typically 1-5%) of IgG are present in multimeric forms within IVIG. The majority of this multimeric fraction is thought to be dimer with smaller amounts of trimer and higher forms. It has also been proposed that additional dimers may form after infusion by binding of recipient anti-idiotype antibodies. One theory is that these multimeric forms compete against immune complexes for binding to low affinity Fcγ receptors due to their enhanced avidity (Augener, Blut, Vol 50, p 249-252, 1985; Teeling, Blood Vol 98(4), p 1095-1099, 2001; Machino, Y., Clin Exp Immunol, Vol 162(3), p 415-424, 2010; Machino, Y. et al., BBRC, Vol 418, p 748-753, 2012). Another theory is that sialic acid glycoforms of IgG within IVIG, especially the presence of higher levels of α2-6 sialic acid forms, cause an alteration in Fcγ receptor activation status (Samuelsson, Science, Vol 291, p 484-486, 2001; Kaneko, Science, Vol 313, p 670-673, 2006; Schwab, European J Immunol Vol 42, p 826-830, 2012; Sondermann, PNAS, Vol 110(24), p 9868-9872, 2013). 
     In diseases involving pathogenic antibodies it has been proposed that the very large dose of IVIG administered to humans (1-2 g/kg) effectively overrides the normal IgG homeostasis mechanism performed by FcRn. Effectively a large dilution of recipient IgG by donor IVIG results in enhanced catabolism and a shorter serum half-life of patient pathogenic antibodies. Other proposed mechanisms for the efficacy include anti-idiotypic neutralisation of pathogenic antibodies and transient reductions in complement factors (Mollnes, Mol Immunol, Vol 34, p 719-729, 1997; Crow, Transfusion Medicine Reviews, Vol 22(2), p 103-116, 2008; Schwab, I. and Nimmerjahn, F. Nature Reviews Immunology, Vol 13, p 176-189, 2013). 
     There are significant disadvantages to the clinical use of IVIG. IVIG has variable product quality between manufacturers due to inherent differences in manufacturing methods and donor pools (Siegel, Pharmacotherapy Vol 25(11) p 78S-84S, 2005). IVIG is given in very large doses, typically in the order of 1-2 g/kg. This large dose necessitates a long duration of infusion, (4-8 hours, sometimes spread over multiple days), which can be unpleasant for the patient and can result in infusion related adverse events. Serious adverse events can occur, reactions in IgA deficient individuals being well understood. Cytokine release can also be observed in patients receiving IVIG but this is largely minimised by careful control of dose and infusion rate. As a consequence of the large amounts used per patient and the reliance on human donors, manufacture of IVIG is expensive and global supplies are severely limited. 
     Collectively the disadvantages of IVIG mean that there is need for improvement in terms of clinical supply, administration and efficacy of molecules able to interfere with the disease biology of pathogenic antibodies and pathogenic immune complexes. 
     Several polymeric Fc proteins for use as potential replacement for IVIG therapy have been described in the literature. 
     A polymeric Fc-fusion protein for use as a potential replacement for IVIG therapy has been described in the literature. (Mekhaiel et al; Nature Scientific Reports 1:124, published 19 Oct. 2011). Mekhaiel et al. describe hexameric hIgG1-Fc-LH309/310CL-tailpiece. This protein comprises a double mutation in which leucine at position 309 is substituted with cysteine, and histidine at position 310 is substituted with leucine. 
     Further constructs such as stradomers (Jain et al, 2012 Arthritis Research and Therapy, 14, 1-12) and stradobodies have been described (WO2014031646). 
     Other constructs include hybrid constant region fusion proteins comprising Cmu 3 and Cmu 4 regions as described in U.S. Pat. No. 8,952,134. 
     Other polymeric fusion proteins have been described in the prior art in which the carboxyl-terminal tailpiece from either IgM or IgA was added to the carboxyl-termini of whole IgG molecule constant regions to produce recombinant IgM-like IgGs. (Smith R. I. F. and Morrison, S. L. Biotechnology, Vol 12, p 683-688, 1994; Smith R. I. F. et al, J Immunol, Vol 154, p 2226-2236, 1995; Sorensen V. et al, J Immunol, Vol 156, p 2858-2865, 1996). The IgG molecules studied were intact immunoglobulins comprising variable regions. 
     The present inventors have observed that some of these polymeric Fc proteins may exhibit unwanted side effects, measured for example by cytokine release, C1q binding and platelet activation. The present invention provides methods for identifying suitable amino acid changes in these proteins to reduce unwanted side effects. The methods also extend to methods of improving stability, multimerisation (in particular hexamerization) and efficacy through amino acid substitutions. Efficacy may be measured for example by inhibition of macrophage phagocytosis. The invention therefore also provides improved and/or optimised Fc domain constructs for use in these polymeric Fc proteins in which one or more of features of the protein have been optimised typically for clinical use. 
     DESCRIPTION OF THE INVENTION 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this invention belongs. All publications and patents referred to herein are incorporated by reference. 
     It will be appreciated that any of the embodiments described herein may be combined. 
     In the present specification the EU numbering system is used to refer to the residues in antibody domains, unless otherwise specified. This system was originally devised by Edelman et al, 1969 and is described in detail in Kabat et al, 1987.
     Edelman et al., 1969; “The covalent structure of an entire γG immunoglobulin molecule,” PNAS Biochemistry Vol. 63 pp 78-85.   Kabat et al., 1987; in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA.   

     Where a position number and/or amino acid residue is given for a particular antibody isotype, it is intended to be applicable to the corresponding position and/or amino acid residue in any other antibody isotype, as is known by a person skilled in the art. When referring to an amino acid residue in a tailpiece derived from IgM or IgA, the position number given is the position number of the residue in naturally occurring IgM or IgA, according to conventional practice in the art. 
     As set out above, the present inventors have observed that some of these polymeric Fc proteins may exhibit unwanted side effects, measured for example by cytokine release, C1q binding and platelet activation. The present invention provides methods for identifying suitable amino acid changes in these proteins to reduce unwanted side effects. The methods also extend to methods of improving stability, multimerisation (in particular hexamerization) and efficacy through amino acid substitutions. Efficacy may be measured for example by inhibition of macrophage phagocytosis. The invention therefore also provides improved and/or optimised Fc domain constructs for use in these polymeric Fc proteins in which one or more of features of the protein have been optimised for typically for clinical use. 
     The present invention therefore provides: 
     A method of screening to identify a polymeric Fc protein with one or more desired functional characteristics compared to a parent polymeric Fc protein comprising:
         (a) substituting one or more amino acids in the or each Fc domain of said parent polymeric Fc protein to another amino acid   (b) testing the mutated polymeric Fc protein obtained in step (a) for the desired functional characteristic (s)   (c) selecting the mutated polymeric Fc protein if it has the desired functional characteristic(s) compared to the parent       

     In the method of the present invention the parent polymeric Fc protein may be any starting or reference polymeric Fc protein. 
     Where it is desired to substitute and test one or more amino acid mutations in a polymeric Fc protein it will be appreciated that these may be substituted and tested sequentially or simultaneously. Similarly individual amino acid changes may be tested separately in different polymeric Fc proteins and subsequently combined. 
     Accordingly steps (a)-(c) may be repeated. In one example the method further comprises step (d) in which steps (a)-(c) are repeated with another amino acid. 
     Additionally such mutations may be tested for two or more desired functional characteristics in step (b) simultaneously or sequentially. 
     In one example at least two amino acid substitutions are tested independently in a factorial or fractional factorial design. In certain embodiments Design of Experiment is used to design a factorial or fractional factorial design. Design of Experiment is a structured, organized method that is used to determine the relationship between the different factors affecting a process and the output of that process. This method was first developed in the 1920s and 1930 by Sir Ronald A. Fisher, the renowned mathematician and geneticist. 
     Design of Experiment involves designing a set of experiments in which all relevant factors are varied systematically. When the results of these experiments are analyzed they help to identify amino acid residue changes, the factors that most influence the results and those that do not, as well as details such as the existence of interactions and synergies between factors. Factorial and experimental design, including the principles of Design of Experiment are known by those of skill in the art, and are described in a number of publications, including the following, all of which are incorporated herein by reference in their entirety: Design and Analysis of Experiments, Fifth Edition, by Douglas C. Montgomery (2001, John Wiley and Sons); DOE Simplified: Practical Tools for Effective Experimentation (Quality Management) by Mark Atkinson and Patrick Whitcomb (2000, Productivity Press, Inc.): Design of Experiments for Engineers and Scientists by Jiju Anthony (2003, Butterworth-Heinemann). 
     It will be appreciated that by using the method of the present invention it is possible to generate an optimised polymeric Fc protein which is optimised for one or more desired functional characteristics or which may be a compromise between two or more desired characteristics. In one example the polymeric Fc protein is optimised for clinical use. 
     It will be appreciated that any desired functional characteristic or characteristics may be tested in step (b). 
     In one example a desired functional characteristic is altered cytokine release compared to the parent. This may be reduced or increased. A cytokine release assay, suitable for use with whole blood, is described in Example 8, and other suitable assays are known in the art. The present applicants have found that cytokine release stimulated by polymeric Fc proteins may be amplified in whole blood. Thus, measurement of cytokine release in isolated blood cell populations or cell lines may not give an accurate indication of the true magnitude of cytokine release. Accordingly, in one example, step (b) of the method comprises testing the mutated polymeric Fc protein obtained in step (a) in a whole blood assay and measuring cytokine release. 
     In one example a desired functional characteristic is reduced platelet activation compared to the parent. 
     In one example a desired functional characteristic is reduced C1q binding compared to the parent. 
     In one example a desired functional characteristic is increased potency of inhibition of macrophage phagocytosis of antibody coated target cells compared to the parent. In one example a desired functional characteristic is increased hexamerisation or stability compared to the parent. 
     Typically each polymeric Fc protein contains two or more identical Fc domains, typically derived from IgG. 
     Typically the Fc domain of the polymeric Fc according to the present invention is engineered by site directed mutagenesis to incorporate the one or more amino acid substitutions in step (a). 
     In one example of the method of the present invention residues in one Fc domain isotype known to differ from those in another isotype are preferentially substituted and tested. For example residues in the IgG1 Fc domain that differ from those in IgG4 are substituted for another amino acid, in particular the corresponding IgG4 residues or vice versa. 
     Alternatively the residues in IgG2 Fc domain that differ from those in IgG3 are substituted for another amino acid, in particular the corresponding IgG3 residues or vice versa. 
     It will be appreciated that this can be done for any isotype pair. 
     In one example in step (a) of the method the one or more amino acids substituted with another amino acid are at a position selected from the group consisting of 234, 235, 236, 268, 274, 296, 300, 309, 327, 330, 331, 339, 355, 356, 358, 409, 419 and 445. 
     In one example in step (a) of the method the one or more amino acids substituted with another amino acid are at a position selected from the group consisting of 234, 268, 274, 296, 327, 330, 331, 355, 356, 358, 409, 419 and 445. 
     In one example in step (a) of the method the parent polymeric Fc comprises IgG1 CH2 and CH3 domains and the one or more amino acids that are substituted with another amino acid are selected from the group consisting of L234, H268, K274, Y296, A327, A330, P331, R355, D356, L358, K409, Q419 and P445. 
     In one example in step (a) of the method the parent polymeric Fc comprises IgG1 CH2 and CH3 domains and the one or more amino acid substitutions are selected from the group consisting of L234F, H268Q, K274Q, Y296F, A327G, A330S, P331S, R355Q, D356E, L358M, K409R, Q419E and P445L. The designation of mutations used herein shows the original amino acid, followed by the numerical position of the amino acid, followed by the new amino acid. Thus, L234F indicates that the leucine residue, originally found at position 234, has been replaced by a phenylalanine residue. 
     In one example in step (a) of the method the one or more amino acid substitutions are selected from the group consisting of L235V, G236A, Y300F, L309V and A339T. 
     In one example in step (a) of the method the parent polymeric Fc comprises IgG4 CH2 and CH3 domains and the one or more amino acids that are substituted with another amino acid are selected from the group consisting of F234, Q268, Q274, F296, G327, S330, S331, Q355, E356, M358, R409, E419 and L445. 
     In one example in step (a) of the method the parent polymeric Fc comprises IgG4 CH2 and CH3 domains and the one or amino acid substitutions are selected from the group consisting of F234L, Q268H, Q274K, F296Y, G327A, S330A, S331P, Q355R, E356D, M358L, R409K, E419Q and L445P. 
     It will be appreciated that ‘one or more’ as used herein may be one, two, three, four, five, six, seven, eight, nine, ten, eleven or more, as appropriate. 
     As set out above these amino acid substitutions may be tested using any suitable method and may be varied independently in a factorial or fractional factorial design, optionally using Design of Experiment as described in the Examples herein. 
     The present invention also provides: 
     A polymeric Fc containing protein comprising one or more amino acid substitutions in the Fc domain at a position selected from the group consisting of 234, 235, 236, 268, 274, 296, 300, 309, 327, 330, 331, 339, 355, 356, 358, 409, 419 and 445. 
     A polymeric Fc containing protein comprising one or more amino acid substitutions in the Fc domain selected from the group consisting of L234F, H268Q, K274Q, Y296F, A327G, A330S, P331S, R355Q, D356E, L358M, K409R, Q419E and P445L. 
     A polymeric Fc containing protein comprising one or more amino acid substitutions in the Fc domain selected from the group consisting of F234L, Q268H, Q274K, F296Y, G327A, S330A, S331P, Q355R, E356D, M358L, R409K, E419Q and L445P. 
     A polymeric Fc containing protein in which the CH2 and CH3 domains are derived from IgG1 comprising one or more mutations or pairs of mutations selected from the group consisting of L234F, A327G, and L234F and P331S. 
     A polymeric Fc containing protein in which the CH2 and CH3 domains are derived from IgG4 comprising one or more mutations or pairs of mutations selected from the group consisting of F234L, F234L and F296Y, F234L and F296Y, A327G and S330A, and A327G and S331P. 
     A polymeric Fc containing protein in which the CH2 and CH3 domains are derived from IgG4 comprising one or more mutations or pairs of mutations selected from the group consisting of F234L, F234L and F296Y, F234L and F296Y, A327G and S330A, and A327G and S331P; and one further Q355R mutation. 
     A polymeric Fc containing protein comprising a CH2 domain and a CH3 domain derived from IgG4 in which at least the glutamine residue at position 355 has been substituted with arginine. 
     A polymeric Fc containing protein comprising a CH2 domain from IgG4 and a CH3 domain derived from IgG1. 
     A polymeric Fc containing protein comprising a CH2 domain from IgG4 and a CH3 domain derived from IgG1 comprising one or more mutations or pairs of mutations selected from the group consisting of F234L, F234L and F296Y, F234L and F296Y, A327G and S330A, and A327G and S331P. 
     In one example 
     mutations to decrease macrophage phagocytosis include:
         alanine residue at position 327   leucine residue at position 234   histidine residue at position 268   tyrosine residue at position 296
 
mutations to decrease cytokine release include:
   phenylalanine residue at position 234   glycine residue at position 327   phenylalanine residue at position 296
 
mutations to decrease platelet activation include
   phenylalanine residue at position 234   glutamine residue at position 274
 
mutations to increase macrophage phagocytosis include:
   glycine residue at position 327   phenylalanine residue at position 234   glutamine residue at position 268   phenylalanine residue at position 296
 
mutations to increase cytokine release:
   leucine residue at position 234   alanine residue at position 327   tyrosine residue at position 296
 
mutations to increase platelet activation include:
   leucine residue at position 234   lysine residue at position 274       

     The term ‘polymeric Fc protein’ or ‘polymeric Fc containing protein’ or ‘polymeric Fc fusion protein’ as used herein refers to any protein comprising two or more, such as three, four, five, six, seven, eight, nine, ten, eleven or twelve antibody Fc domains. Typically the polymeric Fc protein is a protein comprising multimers of antibody Fc domain units. 
     Preferably the antibody Fc domains are derived from IgG. 
     Typically the polymeric Fc protein is a recombinantly engineered fusion protein. 
     Generally the antibody Fc domain units of the polymeric Fc protein assemble into multimers such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, dodecamers, preferably hexamers, by virtue of being linked to or by incorporation of a ‘multimerization domain’. Any suitable multimerisation domain known in the art may be used. Examples include an antibody hinge region, an IgG2 hinge region, a isoleucine zipper, an IgM or IgA tailpiece, knobs and holes, a polypeptide linker (such as described in WO2008/012543) solvent exposed cysteines (e.g. L309C) or solvent exposed mutations favouring inter-Fc interactions such as (i) charge-charge interactions, such as E345X (E345R, E340K) E430X (E340G) S440X (S440Y, S440W) and ‘ROY’ (triple E345R E340G S440Y described in WO13004842) and (ii) ‘hot spots’ such as Q386, P247, 1253, S254, Q311, D/E356, T359, E382, Y436, K447 described in WO2013/004842. 
     In one example the polymeric Fc protein is a polymeric antibody comprising IgG Fc domains and a carboxy-terminal tailpiece from IgM or IgA, as described by Smith R, J et al, Immunology 1995 154:2226-2236. Sorensen V. et al, J Immunol, Vol 156, p 2858-2865, 1996. 
     In one example the Fc protein is originally synthesised as a monomeric Fc protein and assembles into a polymeric Fc protein in vivo. Accordingly the method of the invention optionally further comprises step (d) in which one or more mutations identified in step (c) are incorporated into a protein comprising no more than one Fc domain. 
     In one example the polymeric Fc protein is a Stradomer™ comprising a human IgG2 hinge or isoleucine zipper as described in Jain et al, 2012 Arthritis Research and Therapy, 14, 1-12. Typically, the term ‘stradomer’ is used to describe an Fc domain which has both N-terminal and C-terminal antibody hinge domains. The additional number and complexity of hinge cysteines is thought to result in random inter-Fc disulphide bonds. This is especially the case where the C-terminal hinge sequence is that of human IgG2. Human IgG2 hinge contains 4 cysteines and it is well described that these are functionally promiscuous in the context of a full length IgG (Wypych 2008, Martinez 2008, Allen 2009). It is supposed that the presence of these 4 extra and promiscuous cysteines in the un-natural location at the C-terminus of the Fc results in a high possibility of random inter-Fc disulphide bonds. Depending upon the number of disulphide bonds formed, a ‘ladder’ of multivalent Fc-forms from monomer, through dimer to at least hexamer or more are possible. The overall topology of these stradomer forms is likely to be highly complex and variable and a number of ‘branched’, ‘comb-like’ and ‘arrayed’ forms have been contemplated, for example in International patent application numbers PCT/US2008/065428 and PCT/US2011/045768, incorporated herein by reference. The stradomer ‘ladder’ can be broken by use of disulphide reducing agents and hence is substantially formed by covalent interactions. 
     The present inventors have found that wild type stradomers, without the improved Fc-domains of the invention, stimulated very high levels of cytokine release in whole blood cytokine release assays. In marked contrast, stradomers with improved Fc domains, comprising the L234F and/or A327G mutations, produced much lower levels of cytokine release. (Example 17). Thus, in one embodiment, the polymeric Fc containing protein of the invention further comprises a human IgG2 hinge or isoleucine zipper. In one example the polymeric Fc containing protein comprises the amino acid sequence given in SEQ ID NOs: 72, 74 or 76. In one example the polymeric Fc containing protein is encoded by the DNA sequence given in SEQ ID NOs: 73, 75, or 77. 
     In one example, the polymeric Fc protein is a tandem Fc. A tandem Fc comprises two antibody Fc domains, connected by one or more linkers. It is typically formed from two polypeptide chains, each comprising a first heavy chain Fc region and a second heavy chain Fc region, connected by one or more linkers, and optionally possessing one or more native or modified hinges, as shown in Table 20. Typically, the two polypeptide chains then dimerise to form the tandem Fc protein. An example of a suitable linker sequence is GGGGS. (Nagashima H et al., Molecular Immunology 45, (2008) 2752-2763). Diagrams showing example amino acid and DNA sequences for tandem Fc formats with and without the improved Fc domain constructs of the present invention are provided in  FIG. 17 . In one example the polymeric Fc containing protein comprises the amino acid sequence given in SEQ ID NOs: 78, 80, or 82. In one example the polymeric Fc containing protein is encoded by the DNA sequence given in SEQ ID NOs: 79, 81, or 83. 
     In one example the polymeric Fc protein further comprises one or more mutations selected from the group consisting of E345R, E430G, and S440Y. These mutations are believed to enhance the natural propensity of antibody Fc domains to assemble into hexamers. It has recently been found that complement may be activated by IgG hexamers assembled at the cell surface. Specific noncovalent interactions between the Fc segments of IgG antibodies were observed to result in the formation of ordered antibody hexamers after antigen binding on cells. The hexamers recruited and activated C1, triggering the complement cascade. Mutations were identified that significantly enhanced complement-dependent cytotoxicity, including E345R, E430G, and S440Y. A triple mutant, “IgG1-005-RGY”, combining E345R with E430G and S440Y, readily formed hexamers in solution. (Diebolder C. A. et al., Science 343, 1260-1263, (2014). Thus, in one embodiment, the polymeric Fc containing protein of the invention comprises one or more mutations selected from the group consisting of E345R, E430G, and S440Y. In one example the polymeric Fc containing protein comprises the amino acid sequence given in SEQ ID NOs: 66, 68 or 70. In one example the polymeric Fc containing protein is encoded by the DNA sequence given in SEQ ID NOs: 67, 69, or 71. 
     In one embodiment, the polymeric Fc protein of the invention is an X309C ladder. The present inventors have found that antibody Fc domains can be multimerised into multivalent forms by engineering the presence of a cysteine residue at position 309. (Described in detail in our international patent application PCT/EP2015/058338). The cysteine residues have an important role in the spontaneous assembly of the polymeric Fc protein, by forming disulphide bridges between individual pairs of polypeptide monomer units. In one embodiment, the cysteine residue at position 309 is created by a mutation, e.g. for an Fc domain derived from IgG1, the leucine residue at position 309 is substituted with a cysteine residue (L309C); for an Fc-domain derived from IgG2, the valine residue at position 309 is substituted with a cysteine residue (V309C). In one embodiment, the antibody Fc-domain is mutated by substituting the valine residue at position 308 with a cysteine residue (V308C). Thus, in one embodiment, the polymeric Fc containing protein of the invention comprises a cysteine residue at position 309. In one example the polymeric Fc containing protein comprises the amino acid sequence given in SEQ ID NOs: 60, 62 or 64. In one example the polymeric Fc containing protein is encoded by the DNA sequence given in SEQ ID NOs: 61, 63, or 65. 
     In one example the polymeric Fc protein is a Selective Immunomodulator of Fc Receptors (SIF) such as the trimeric Fc protein SIF3™ (Momenta Pharmaceuticals). The Fc domain is a naturally a homodimer formed by two polypeptides. It has long been known that the Fc domain can be engineered to be heterodimeric by means of any one of a number of techniques, including but not limited to CH3 domain interface engineering such as ‘knobs and holes’, ‘charge complementarity’ and ‘Fab arm exchange’ (Carter P, Journal of Immunological Methods 248 (2001) 7-15; Klein C et al., mAbs 4:6 (2012) 653-663; Von Krudenstein T S et al., mAbs 5:5 (2013) 646-654). Introduction of heterodimeric pairing into the Fc domain enables its use as a fusion partner for other domains, including but not limited to other Fc domains. Fc domains have solvent exposed, flexible and non-essential N- and C-terminii. Hence by judicious combination of N-terminal and C-terminal linker peptides and hetero-engineered Fc, multimeric Fc domains can be constructed containing between 1 and 5 Fc domains, an example of such a multimeric Fc domain is SIF3 (Momenta). A detailed description of SIF proteins is provided in WO2015/168643. 
     The present inventors have found that wild type SIF proteins, without the improved Fc-domains of the invention, stimulated very high levels of cytokine release in whole blood cytokine release assays. In marked contrast, SIF proteins with improved Fc domains, comprising the L234F and/or A327G mutations, produced much lower levels of cytokine release. (Example 17). Thus, in one embodiment, the polymeric Fc containing protein of the invention is a Selective Immunomodulator of Fc Receptors (SIF) protein as described in WO2015/168643. In one embodiment, the polymeric Fc containing protein of the invention is a Selective Immunomodulator of Fc Receptors (SIF) protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises (hγ1hinge-hγ1Fc A ) and the 2 nd  polypeptide comprises (hγ1hinge-hγ1Fc A )-(linker)-(hγ1hinge-hγ1Fc B ), wherein two Fc A  regions combine with each other due to interface engineering to form an Fc domain; and two Fc B  regions combine with each other due to alternative interface engineering to form an Fc domain. In one example the polymeric Fc containing protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 87, 89, 91, 93 and 95. In one example the polymeric Fc containing protein is encoded by DNA comprising a DNA sequence selected from the group consisting of SEQ ID Nos: 84, 86, 88, 90, 92 and 94. 
     In one example, the polymeric Fc protein is a HexaBody™, as described by de Jong R N, et al; (2016) PLoS Biol 14(1): e1002344, doi:10.1371/journal.pbio.1002344. As described above, IgG antibodies are known to be able to organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement dependent target cell killing. De Jong et al have used this natural concept to develop HexaBody™ technology for therapeutic antibody potentiation. They have described mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from haematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells. Furthermore, both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. Thus, IgG1 Fc backbones comprising E345K and/or E430G are expected to be useful for generating therapeutics with enhanced effector functions that only become activated upon binding to target cell-expressed antigen. 
     In one example the polymeric Fc protein is a polymeric Fc fusion protein as described by Mekhaiel et al; Nature Scientific Reports 1:124, published 19 Oct. 2011. In one example the polymeric Fc protein is hybrid constant region fusion protein comprising Cmu 3 and Cmu 4 regions as described in U.S. Pat. No. 8,952,134. 
     In one example the polymeric Fc protein is a multimeric fusion protein comprising two or more IgG Fc domains fused at their C-terminus to a tailpiece which causes the Fc domains to assemble into a multimer. In one example this multimeric fusion protein comprises cysteine at position 309 and optionally histidine or leucine at position 310. In one example this multimeric fusion protein does not contain antibody variable domains. 
     In one example the polymeric Fc proteins of the present invention further comprise a fusion partner. In one example the term ‘fusion partner’ specifically excludes one or more antibody variable domains. Typically the term ‘fusion partner’ refers to an antigen, pathogen-associated molecular pattern (PAMP), drug, ligand, receptor, cytokine or chemokine. 
     Said fusion partner, where present, is fused to the N-terminus of the Fc-domain. The fusion partner may be fused directly to the N-terminus of the heavy chain Fc-domain. Alternatively it may be fused indirectly by means of an intervening amino acid sequence, which may include a hinge, where present. For example, a short linker sequence may be provided between the fusion partner and the heavy chain Fc-region. 
     In one example, the polymeric Fc fusion proteins of the present invention do not comprise a fusion partner. 
     In one example the polymeric Fc fusion proteins of the present invention do not comprise one or more antibody variable regions, typically the molecules do not comprise either a VH or a VL antibody variable region. In one example the polymeric Fc fusion proteins of the present invention do not comprise a Fab fragment. 
     In one example the polymeric Fc fusion proteins of the present invention comprise one or more antibody variable regions, such as a VH or a VL antibody variable region. In one example the polymeric Fc proteins of the present invention comprise a Fab fragment. 
     IgM and IgA occur naturally in humans as covalent multimers of the common H 2 L 2  antibody unit. IgM occurs as a pentamer when it has incorporated a J-chain, or as a hexamer when it lacks a J-chain. IgA occurs as monomer and dimer forms. The heavy chains of IgM and IgA possess an 18 amino acid extension to the C-terminal constant domain, known as a tailpiece. This tailpiece includes a cysteine residue that forms a disulphide bond between heavy chains in the polymer, and is believed to have an important role in polymerisation. The tailpiece also contains a glycosylation site. 
     The tailpiece if present in the polymeric Fc fusion proteins of the present invention may comprise any suitable amino acid sequence. It may be a tailpiece found in a naturally occurring antibody, or alternatively, it may be a modified tailpiece which differs in length and/or composition from a natural tailpiece. Other modified tailpieces may be entirely synthetic and may be designed to possess desired properties for multimerisation, such as length, flexibility and cysteine composition. 
     The tailpiece may be derived from any suitable species. Antibody tailpieces are evolutionarily conserved and are found in most species, including primitive species such as teleosts. In one embodiment the tailpiece of the present invention is derived from a human antibody. 
     In one embodiment, the tailpiece comprises all or part of an 18 amino acid tailpiece sequence from human IgM or IgA as shown in Table 1. 
     The tailpiece may be fused directly to the C-terminus of the Fc domain. Alternatively, it may be fused indirectly by means of an intervening amino acid sequence. For example, a short linker sequence may be provided between the tailpiece and the heavy chain Fc-region. 
     The tailpiece may include variants or fragments of the native sequences described above. A variant of an IgM or IgA tailpiece typically has an amino acid sequence which is identical to the native sequence in 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the 18 amino acid positions shown in Table 1. A fragment typically comprises 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acids. The tailpiece may be a hybrid IgM/IgA tailpiece. Fragments of variants are also envisaged. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Tailpiece sequences 
               
            
           
           
               
               
               
            
               
                   
                 Tailpiece 
                 Sequence 
               
               
                   
                   
               
               
                   
                 IgM 
                 PTLYNVSLVMSDTAGTCY SEQ ID NO: 1 
               
               
                   
                   
               
               
                   
                 IgA 
                 PTHVNVSVVMAEVDGTCY SEQ ID NO: 2 
               
               
                   
                   
               
            
           
         
       
     
     The polymeric Fc proteins may optionally possess one or more native or modified hinge region(s). 
     A native hinge region is the hinge region that would normally be found between Fab and Fc domains in a naturally occurring antibody. A modified hinge region is any hinge that differs in length and/or composition from the native hinge region. Such hinges can include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama or goat hinge regions. Other modified hinge regions may comprise a complete hinge region derived from an antibody of a different class or subclass from that of the heavy chain Fc-region. Alternatively, the modified hinge region may comprise part of a natural hinge or a repeating unit in which each unit in the repeat is derived from a natural hinge region. In a further alternative, the natural hinge region may be altered by converting one or more cysteine or other residues into neutral residues, such as serine or alanine, or by converting suitably placed residues into cysteine residues. By such means the number of cysteine residues in the hinge region may be increased or decreased. Other modified hinge regions may be entirely synthetic and may be designed to possess desired properties such as length, cysteine composition and flexibility. 
     A number of modified hinge regions have already been described for example, in U.S. Pat. No. 5,677,425, WO9915549, WO2005003170, WO2005003169, WO2005003170, WO9825971 and WO2005003171 and these are incorporated herein by reference. 
     Examples of suitable hinge sequences are shown in Table 2. 
     In one embodiment the hinge region comprises the mutated sequence CPPC (SEQ ID NO: 11). The core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO: 12) compared to IgG1 which contains the sequence CPPC. The serine residue present in the IgG4 sequence leads to increased flexibility in this region, and therefore a proportion of molecules form disulphide bonds within the same protein chain (an intrachain disulphide) rather than bridging to the other heavy chain in the IgG molecule to form the interchain disulphide. (Angal S. et al, Mol Immunol, Vol 30(1), p 105-108, 1993). Changing the serine residue to a proline to give the same core sequence as IgG1 allows complete formation of inter-chain disulphides in the IgG4 hinge region, thus reducing heterogeneity in the purified product. This altered isotype is termed IgG4P. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Hinge sequences 
               
            
           
           
               
               
            
               
                 Hinge 
                 Sequence 
               
               
                   
               
               
                 Human IgA1 
                 VPSTPPTPSPSTPPTPSPS SEQ ID NO: 3 
               
               
                   
               
               
                 Human IgA2 
                 VPPPPP SEQ ID NO: 4 
               
               
                   
               
               
                 Human IgD 
                 ESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTP SEQ ID NO: 5 
               
               
                   
               
               
                 Human IgG1 
                 EPKSCDKTHTCPPCP SEQ ID NO: 6 
               
               
                   
               
               
                 Human IgG2 
                 ERKCCVECPPCP SEQ ID NO: 7 
               
               
                   
               
               
                 Human IgG3 
                 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP SEQ ID NO: 8 
               
               
                   
               
               
                 Human IgG4 
                 ESKYGPPCPSCP SEQ ID NO: 9 
               
               
                   
               
               
                 Human IgG4(P) 
                 ESKYGPPCPPCP SEQ ID NO: 10 
               
               
                   
               
               
                 Recombinant v1 
                 CPPC SEQ ID NO: 11 
               
               
                   
               
               
                 Recombinant v2 
                 CPSC SEQ ID NO: 12 
               
               
                   
               
               
                 Recombinant v3 
                 CPRC SEQ ID NO: 13 
               
               
                   
               
               
                 Recombinant v4 
                 SPPC SEQ ID NO: 14 
               
               
                   
               
               
                 Recombinant v5 
                 CPPS SEQ ID NO: 15 
               
               
                   
               
               
                 Recombinant v6 
                 SPPS SEQ ID NO: 16 
               
               
                   
               
               
                 Recombinant v7 
                 DKTHTCAA SEQ ID NO: 17 
               
               
                   
               
               
                 Recombinant v8 
                 DKTHTCPPCPA SEQ ID NO: 18 
               
               
                   
               
               
                 Recombinant v9 
                 DKTHTCPPCPATCPPCPA SEQ ID NO: 19 
               
               
                   
               
               
                 Recombinant v10 
                 DKTHTCPPCPATCPPCPATCPPCPA SEQ ID NO: 20 
               
               
                   
               
               
                 Recombinant v11 
                 DKTHTCPPCPAGKPTLYNSLVMSDTAGTCY SEQ ID NO: 21 
               
               
                   
               
               
                 Recombinant v12 
                 DKTHTCPPCPAGKPTHVNVSVVMAEVDGTCY SEQ ID NO: 22 
               
               
                   
               
               
                 Recombinant v13 
                 DKTHTCCVECPPCPA SEQ ID NO: 23 
               
               
                   
               
               
                 Recombinant v14 
                 DKTHTCPRCPEPKSCDTPPPCPRCPA SEQ ID NO: 24 
               
               
                   
               
               
                 Recombinant v15 
                 DKTHTCPSCPA SEQ ID NO: 25 
               
               
                   
               
            
           
         
       
     
     Each polymeric Fc protein of the invention comprises two or more antibody Fc domains, preferably human. Each antibody Fc-domain may be derived from any suitable class of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3 and IgG4), and IgM. In one embodiment, the antibody Fc-domain is derived from IgG1, IgG2, IgG3 or IgG4. In one embodiment the antibody Fc-domain is derived from IgG1. In one embodiment the antibody Fc domain is derived from IgG4. 
     Each antibody Fc-domain comprises two polypeptide chains, each referred to as a heavy chain Fc-region. The two heavy chain Fc regions dimerise to create the antibody Fc-domain. Whilst the two heavy chain Fc regions within the antibody Fc domain may be different from one another it will be appreciated that these will often be the same as one another. Hence where the term ‘the heavy chain Fc-region’ is used herein below this is used to refer to a single heavy chain Fc-region which dimerises with an identical heavy chain Fc-region to create the antibody Fc-domain. 
     Typically each heavy chain Fc-region comprises or consists of two or three heavy chain constant domains. 
     In native antibodies, the heavy chain Fc-region of IgA, IgD and IgG is composed of two heavy chain constant domains (CH2 and CH3) and that of IgE and IgM is composed of three heavy chain constant domains (CH2, CH3 and CH4). These dimerise to create an Fc domain. 
     In the present invention, each Fc domain may comprise heavy chain constant domains from one or more different classes of antibody, for example one, two or three different classes. 
     In one aspect of the present invention the present inventors have unexpectedly found that the CH3 domain plays a significant role in controlling the polymerisation of the monomer units of polymeric Fc proteins of the present invention. Polymerisation was unexpectedly found to vary depending on the IgG subclass from which the Fc-region was derived. Polymeric Fc proteins comprising a CH2 domain and a CH3 domain derived from IgG1 assembled very efficiently into hexamers, with approximately 80% of the molecules being present in hexameric form. In contrast, polymeric Fc proteins comprising a CH2 domain and a CH3 domain derived from IgG4 formed lower levels of hexamers. Investigation of polymeric Fc proteins comprising hybrid Fc-regions in which the CH2 domain was derived from one particular IgG subclass and the CH3 domain was derived from a different IgG subclass revealed that the ability to form hexamers is encoded mainly by the CH3 domain. The presence of a CH3 domain derived from IgG1 significantly increases hexamerisation. Hybrid polymeric Fc proteins in which the CH3 domain is derived from IgG1 and the CH2 domain is derived from IgG4 assembled just as efficiently as IgG1 wild-type, with approximately 80% of the molecules being found as hexamers. (Example 4 and  FIG. 3 ). Thus, replacing the CH3 domain of IgG4 with that of IgG1 improves the levels of hexamerisation compared to wild type IgG4 polymeric Fc proteins. The resulting hybrid has the advantage of high levels of hexamer formation whilst retaining many of the desirable properties of IgG4. 
     In addition, the CH3 domain of IgG1 is known to confer thermal stability. (Garber and Demarest, Biochem and Biophys Res Comm, Vol 355 p 751-757 2007). Thus, hybrid polymeric Fc proteins comprising a CH3 domain derived from IgG1 will also have improved stability. 
     Thus in one embodiment the Fc domain comprises a CH3 domain derived from IgG1. 
     In one embodiment the Fc domain comprises a CH2 domain derived from IgG4 and a CH3 domain derived from IgG1. 
     The inventors have demonstrated that the amino acid at position 355 of the CH3 domain is critical for hexamerisation. The arginine residue normally found at position 355 of the IgG1 CH3 domain was found to promote particularly efficient hexamerisation. See Example 4 described herein below 
     Thus in one embodiment, the heavy chain Fc region comprises an arginine residue at position 355. 
     Substitution of the arginine residue normally found at position 355 of the IgG1 CH3 domain with a cysteine residue (R355C) increased hexamer formation beyond that of wild type IgG1. See Example 4 described herein below. 
     Thus in one embodiment, the heavy chain Fc region comprises a CH3 domain derived from IgG1 in which the arginine residue at position 355 has been substituted with another amino acid. 
     Thus in one embodiment, the heavy chain Fc region comprises a cysteine residue at position 355. 
     In the polymeric Fc proteins of the present invention which comprise a CH3 domain derived from IgG4, substitution of the glutamine residue at position 355 with an arginine residue (Q355R) significantly increases hexamerisation. Thus, the problem of lower hexamerisation of IgG4 containing polymeric Fc proteins can be solved by a single amino acid substitution. This has the advantage that the resulting polymeric Fc protein assembles into hexamers with high efficiency whilst retaining the characteristic properties of IgG4. 
     Thus in one embodiment, the heavy chain Fc region comprises a CH3 domain derived from IgG4 in which the glutamine residue at position 355 has been substituted with another amino acid. 
     Thus in one embodiment, the heavy chain Fc region comprises a CH3 domain derived from IgG4 in which the glutamine residue at position 355 has been substituted with an arginine residue (Q355R) or a cysteine residue (Q355C). 
     In one embodiment the heavy chain Fc-region comprises a CH4 domain from IgM. The IgM CH4 domain is typically located between the CH3 domain and, where present, the tailpiece. 
     In one embodiment the heavy chain Fc-region comprises CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM. 
     It will be appreciated that the heavy chain constant domains for use in producing a heavy chain Fc-region of the present invention may include variants of the naturally occurring constant domains described above. Such variants may comprise one or more amino acid variations compared to wild type constant domains. In one example the heavy chain Fc-region of the present invention comprises at least one constant domain which varies in sequence from the wild type constant domain. It will be appreciated that the variant constant domains may be longer or shorter than the wild type constant domain. Preferably the variant constant domains are at least 50% identical or similar to a wild type constant domain. The term “identity”, as used herein, indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. The term “similarity”, as used herein, indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. For example, leucine may be substituted for isoleucine or valine. Other amino acids which can often be substituted for one another include but are not limited to:
         phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains);   lysine, arginine and histidine (amino acids having basic side chains);   aspartate and glutamate (amino acids having acidic side chains);   asparagine and glutamine (amino acids having amide side chains); and   cysteine and methionine (amino acids having sulphur-containing side chains).       

     Degrees of identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In one example the variant constant domains are at least 60% identical or similar to a wild type constant domain. In another example the variant constant domains are at least 70% identical or similar. In another example the variant constant domains are at least 80% identical or similar. In another example the variant constant domains are at least 90% identical or similar. In another example the variant constant domains are at least 95% identical or similar. 
     The polymeric Fc proteins of the invention may comprise two, three, four, five, six, seven, eight, nine, ten, eleven or twelve or more Fc domains. Such proteins may alternatively be referred to as a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, undecamer, dodecamer, etc., respectively. 
     In one embodiment, the polymeric Fc proteins of the invention comprises six or twelve polypeptide monomer units. 
     In one example, the polymeric Fc protein of the present invention is a hexamer. 
     In one example the polymeric Fc protein of the present invention is a purified hexamer. In one example the term ‘purified’ means greater than 80% hexamer, for example greater than 90% or 95% hexamer. 
     It will be appreciated that the quantity of hexamer in a sample can be determined using any suitable method such as analytical size exclusion chromatography as described herein below. 
     In one example the present invention provides a mixture comprising a polymeric Fc protein of the invention in more than one multimeric form, for example hexamer and docdecamer. In one example the mixture is enriched for the hexameric form of said multimeric fusion protein. 
     In one example such a mixture may comprise greater than 80% hexamer. In one example such a mixture may comprise greater than 85%, 90%, or 95% hexamer. 
     Each Fc domain unit in a polymeric Fc protein of the invention comprises two individual polypeptide chains, or Fc regions as described herein above. The two polypeptide chains within a particular Fc domain unit may be the same as one another, or they may be different from one another. In one embodiment, the two polypeptide chains are the same as one another. 
     Similarly, the polypeptide monomer units or Fc domains within a particular polymeric Fc fusion protein may be the same as one another, or they may be different from one another. In one embodiment, the Fc domains are the same as one another. 
     In one example, a polypeptide chain of an Fc domain of a polymeric Fc protein of the present invention comprises an amino acid sequence as provided in  FIG. 2 , optionally with an alternative hinge or tailpiece sequence. 
     In one example the present invention also provides a polymeric Fc protein which comprises or consists of two identical polypeptide chains each polypeptide chain comprising or consisting of the sequence given in any one of SEQ ID Nos 26 to 47. 
     In one example the present invention provides a polymeric Fc containing protein wherein each Fc domain comprises two heavy chain Fc-regions; wherein each heavy chain Fc-region comprises or consists of the sequence given in amino acids 6 to 222 of any one of SEQ ID NOs 26 to 29 or amino acids 6 to 222 of any one of SEQ ID NOs 32 to 47 or the sequence given in amino acids 6 to 333 of SEQ ID NOs 30 or 31. 
     The polymeric Fc proteins of the present invention comprise one or more mutations as described herein above and in the examples herein that alter the functional properties of the proteins, for example, binding to Fc-receptors such as FcRn or leukocyte receptors, binding to complement, modified disulphide bond architecture or altered glycosylation patterns, as described herein below. It will be appreciated that any of these mutations may be combined in any suitable manner to achieve the desired functional properties, and/or combined with other mutations to alter the functional properties of the proteins. 
     The method of the present invention provides a means of identifying a polymeric Fc protein with one or more desired functional characteristics compared to a parent or reference polymeric Fc protein. 
     In one example the present invention provides polymeric Fc proteins which are particularly suitable for use in the treatment of immune disorders. 
     In one example the method may be used to generate a polymeric Fc protein with one or more of the following desirable functional characteristics. 
     The potency of a polymeric Fc protein for use in the treatment of immune disorders should be as high as possible. Potency may be determined by measuring the inhibition of macrophage phagocytosis of antibody coated target cells as described in Example 6. 
     Unwanted side effects should be as low as possible. Unwanted side effects may be determined by measuring cytokine release, C1q binding and platelet activation as described in Examples 8, 15 and 16 respectively. 
     Wild type IgG1 polymeric Fc proteins comprising a CH2 and CH3 domain derived from IgG1 without any additional mutations may be less suitable for use in the treatment of immune disorders because, although they display high potency of phagocytosis inhibition, they also show high levels of unwanted side effects, measured by cytokine release, C1q binding and platelet activation. 
     Wild type IgG4 polymeric Fc proteins comprising a CH2 and CH3 domain derived from IgG4, produce very low levels of unwanted side effects although its potency is low relative to that of IgG1. Notwithstanding, the potency of wild type IgG4 polymeric Fc proteins may still be significantly higher than that of IVIG, as shown in  FIG. 7 . 
     In one example the present invention provides polymeric Fc fusion proteins that have been engineered to combine the desirable properties of both IgG1 and IgG4 wild type Fc fusion proteins, without the undesirable properties. Examples of these Fc fusion proteins which display effective levels of potency, whilst reducing unwanted side effects to a tolerable level are shown below in Table 3. These polymeric Fc-fusion proteins are expected to be particularly useful for use in the treatment of immune disorders. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                   
                 phagocytosis 
                 IFNγ 
                 C1q 
                 platelet 
               
               
                 Fc-multimer 
                 inhibition 
                 release 
                 binding 
                 activation 
               
               
                   
               
             
            
               
                 wild type IgG1 
                 the standard 
                 the 
                 the 
                 the 
               
               
                 Fc-multimer 
                 for “IgG1- 
                 standard 
                 standard 
                 standard 
               
               
                   
                 like” 
                 for “IgG1- 
                 for 
                 for “IgG1- 
               
               
                   
                   
                 like” 
                 “IgG1- 
                 like” 
               
               
                   
                   
                   
                 like” 
               
               
                 wild type IgG4 
                 the standard 
                 the 
                 the 
                 the 
               
               
                 Fc-multimer 
                 for “IgG4- 
                 standard 
                 standard 
                 standard 
               
               
                   
                 like” 
                 for “IgG4- 
                 for 
                 for “IgG4- 
               
               
                   
                   
                 like” 
                 “IgG4- 
                 like” 
               
               
                   
                   
                   
                 like” 
               
               
                 IgG1 Fc IgM tp L309 
                 high 
                 medium 
                 IgG4-like 
                 IgG4-like 
               
               
                 L234F P331S 
               
               
                 Hybrid Fc IgG4-CH2 
                 medium 
                 IgG4-like 
                 IgG4-like 
                 low 
               
               
                 IgG1-CH3 IgM 
               
               
                 tp L309 
               
               
                 IgG4 Fc IgM tp 
                 medium 
                 IgG4-like 
                 IgG4-like 
                 medium 
               
               
                 L309 F234L 
               
               
                 IgG4 Fc IgM tp L309 
                 medium 
                 IgG4-like 
                 IgG4-like 
                 medium 
               
               
                 F234L F296Y 
               
               
                 IgG4 Fc IgM tp L309 
                 medium 
                 IgG4-like 
                 IgG4-like 
                 IgG4-like 
               
               
                 G327A S330A 
               
               
                 IgG4 Fc IgM tp L309 
                 medium 
                 IgG4-like 
                 medium 
                 IgG4-like 
               
               
                 G327A S331P 
               
               
                 IgG4 Fc IgM tp L309 
                 medium 
                 IgG4-like 
                 medium 
                 IgG4-like 
               
               
                 S330A S331P 
               
               
                   
               
            
           
         
       
     
     In one example the present invention provides polymeric Fc fusion proteins comprising one or more mutations which decrease cytokine release and/or decrease platelet activation and/or decrease C1q binding when compared to the unmodified parent polymeric Fc fusion protein. In one example the unmodified parent is a polymeric Fc fusion protein containing CH2 and CH3 domains derived from IgG1. 
     Desired characteristics such as cytokine release and/or platelet activation and/or C1q binding may be measured by any suitable method known in the art. In one example cytokine release is measured in a whole blood cytokine release assay. In one example platelet activation is measured by flow cytometry using CD62p as an activation marker. In one example C1q binding is measured by ELISA. 
     In one example the present invention provides polymeric Fc proteins comprising one or more mutations which increase the potency of inhibition of macrophage phagocytosis of antibody-coated target cells when compared to the unmodified parent polymeric Fc protein. In one example the unmodified parent is a polymeric Fc protein of the present invention containing a CH2 and CH3 domain derived from IgG4 or a CH2 domain from IgG4 and a CH3 domain from IgG1. 
     Suitable assays for measuring inhibition of macrophage phagocytosis of antibody coated target cells are known in the art and are described in the examples herein. 
     Accordingly in one example each heavy chain Fc-region of a polymeric Fc protein of the present invention comprises CH2 and CH3 domains derived from IgG1 in which the leucine residue at position 234 and/or the proline residue at position 331 and/or the alanine at position 327 and/or the tyrosine at position 296 has been substituted with another amino acid. 
     In one embodiment the heavy chain Fc-region comprises CH2 and CH3 domains derived from IgG1 in which the leucine residue at position 234 has been substituted with a phenylalanine residue and the proline residue at position 331 has been substituted with a serine residue (L234F/P331S). 
     In one embodiment the heavy chain Fc-region is a hybrid comprising a CH2 domain derived from IgG4 and a CH3 domain derived from IgG1. 
     In one example each heavy chain Fc-region of a polymeric Fc protein of the present invention comprises a CH2 domain from IgG4 and a CH3 domain derived from IgG4 or IgG1 in which one or more amino acid residues selected from the group consisting of the phenylalanine residue at position 234, the phenylalanine residue at position 296, the glycine residue at position 327, the serine residue at position 330 and the serine residue at position 331, have been substituted with another amino acid. 
     In one example each heavy chain Fc-region of a polymeric Fc protein of the present invention comprises a CH2 domain from IgG4 and a CH3 domain derived from IgG4 or IgG1 in which one or more amino acid residues or pairs of amino acids selected from the group consisting of F234, F234 and F296, G327, G327 and S331, S330 and S331, and, G327 and S330 have been substituted with another amino acid. 
     In one embodiment the heavy chain Fc-region of a polymeric Fc protein of the present invention comprises CH2 and CH3 domains derived from IgG4 in which the phenylalanine residue at position 234 has been substituted with a leucine residue (F234L). 
     In one embodiment the heavy chain Fc-region comprises CH2 and CH3 domains derived from IgG4 in which the phenylalanine residue at position 234 has been substituted with a leucine residue and the phenylalanine residue at position 296 has been substituted with a tyrosine residue (F234L/F296Y). 
     In one embodiment the heavy chain Fc-region comprises CH2 and CH3 domains derived from IgG4 in which the glycine residue at position 327 has been substituted with an alanine residue and the serine residue at position 330 has been substituted with an alanine residue (G327A/S330A). 
     In one embodiment the heavy chain Fc-region comprises CH2 and CH3 domains derived from IgG4 in which the glycine residue at position 327 has been substituted with an alanine residue and the serine residue at position 331 has been substituted with a proline residue (G327A/S331P). 
     In one embodiment the heavy chain Fc-region comprises CH2 and CH3 domains derived from IgG4 in which the serine residue at position 330 has been substituted with an alanine residue and the serine residue at position 331 has been substituted with a proline residue (S330A/S331P). 
     In one embodiment the heavy chain Fc-region is a hybrid comprising a CH2 domain derived from IgG4 and a CH3 domain derived from IgG1, in which the phenylalanine residue at position 234 has been substituted with a leucine residue (F234L). 
     In one embodiment the heavy chain Fc-region is a hybrid comprising a CH2 domain derived from IgG4 and a CH3 domain derived from IgG1, in which the phenylalanine residue at position 234 has been substituted with a leucine residue and the phenylalanine residue at position 296 has been substituted with a tyrosine residue (F234L/F296Y). 
     In one embodiment the heavy chain Fc-region is a hybrid comprising a CH2 domain derived from IgG4 and a CH3 domain derived from IgG1, in which the glycine residue at position 327 has been substituted with an alanine residue and the serine residue at position 330 has been substituted with an alanine residue (G327A/S330A). 
     In one embodiment the heavy chain Fc-region is a hybrid comprising a CH2 domain derived from IgG4 and a CH3 domain derived from IgG1, in which the glycine residue at position 327 has been substituted with an alanine residue and the serine residue at position 331 has been substituted with a proline residue (G327A/S331P). 
     In one embodiment the heavy chain Fc-region is a hybrid comprising a CH2 domain derived from IgG4 and a CH3 domain derived from IgG1, in which the serine residue at position 330 has been substituted with an alanine residue and the serine residue at position 331 has been substituted with a proline residue (S330A/S331P). 
     The polymeric Fc proteins of the invention may show altered binding to one or more Fc-receptors (FcR&#39;s) in comparison with the parent. The binding to any particular Fc-receptor may be increased or decreased. In one embodiment, the polymeric Fc protein of the invention comprises one or more mutations which alter its Fc-receptor binding profile. 
     The term “mutation” as used herein may include substitution, addition or deletion of one or more amino acids. 
     Human cells can express a number of membrane bound FcR&#39;s selected from FcαR, FcεR, FcγR and FcRn. Some cells are also capable of expressing soluble (ectodomain) FcR (Fridman et al., (1993) J Leukocyte Biology 54: 504-512 for review). FcγR can be further divided by affinity of IgG binding (high/low) and biological effect (activating/inhibiting). Human FcγRI is widely considered to be the sole ‘high affinity’ receptor whilst all of the others are considered as medium to low. FcγRIIb is the sole receptor with ‘inhibitory’ functionality by virtue of its intracellular ITIM motif whilst all of the others are considered as ‘activating’ by virtue of ITAM motifs or pairing with the common FcγR-γchain. FcγRIIIb is also unique in that although activatory it associates with the cell via a GPI anchor. In total, humans express six ‘standard’ FcγR: FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa FcγRIIIb. In addition to these sequences there are a large number of sequence or allotypic variants spread across these families. Some of these have been found to have important functional consequence and so are sometimes considered to be receptor sub-types of their own. Examples include FcγRIIa H134R , FcγRIIb 1190T , FcγRIIIa F158v  and FcγRIIIb NA1 , FcγRIIIb NA2  FcγRIIIb SH . Each receptor sequence has been shown to have different affinities for the 4 sub-classes of IgG: IgG1, IgG2, IgG3 and IgG4 (Bruhns Blood (1993) Vol 113, p 3716-3725). Other species have somewhat different numbers and functionality of FcγR, with the mouse system being the best studied to date and comprising of 4 FcγR; FcγRI FcγRIIb FcγRIII FcγRIV (Bruhns, Blood (2012) Vol 119, p 5640-5649). Human FcγRI on cells is normally considered to be ‘occupied’ by monomeric IgG in normal serum conditions due to its affinity for IgG1/IgG3/IgG4 (˜10 −8 M) and the concentration of these IgG in serum (˜10 mg/ml). Hence cells bearing FcγRI on their surface are considered to be capable for ‘screening’ or ‘sampling’ of their antigenic environment vicariously through the bound polyspecific IgG. The other receptors having lower affinities for IgG sub-classes (in the range of (˜10 −5 -10 −7 M) are normally considered to be ‘unoccupied’. The low affinity receptors are hence inherently sensitive to the detection of and activation by antibody involved immune complexes. The increased Fc density in an antibody immune complex results in increased functional affinity of binding ‘avidity’ to low affinity FcγR. This has been demonstrated in vitro using a number of methods (Shields R. L. et al, J Biol Chem, Vol 276(9), p 6591-6604, 2001; Lux et al., J Immunol (2013) Vol 190, p 4315-4323). It has also been implicated as being one of the primary modes of action in the use of anti-RhD to treat ITP in humans (Crow Transfusion Medicine Reviews (2008) Vol 22, p 103-116). 
     Many cell types express multiple types of FcγR and so binding of IgG or antibody immune complex to cells bearing FcγR can have multiple and complex outcomes depending upon the biological context. Most simply, cells can either receive an activatory, inhibitory or mixed signal. This can result in events such as phagocytosis (e.g. macrophages and neutrophils), antigen processing (e.g. dendritic cells), reduced IgG production (e.g. B-cells) or degranulation (e.g. neutrophils, mast cells). There are data to support that the inhibitory signal from FcγRIIb can dominate that of activatory signals (Proulx Clinical Immunology (2010) 135:422-429. 
     Cytokines are a family of highly potent proteins which modulate cells of the immune system or effect the killing of target cells such as virally infected or pre-cancerous host cells. The high level of potency has been investigated for use as therapeutic proteins on their own or after fusion to targeting moieties. IL-2, TNFα, G-CSF, GM-CSF, IFNα, IFNβ, IFNγ have all been investigated for use in humans. Their extreme potency was evidenced by a broad range of side effects or adverse events which resulted in rather restricted uses in patients with serious or life threatening conditions. 
     Production of cytokines in vivo can be elicited after the systemic administration of therapeutic proteins such as antibodies or immunoglobulins. Cytokine production can be short lived and temporary such as during and immediately after administration by infusion or subcutaneous injection. For example, infusion of intravenous immunoglobulin is known to result in the production of TNFα, IL-6, IL-8 and IFNγ (Aukrust P et al, Blood, Vol 84, p 2136-2143, 1994) which are associated with common infusion related events: fever, chills and nausea. Cytokine production may be longer lived and related to drug mode of action due to activation of effector cells, for example as in so called ‘tumour lysis syndrome’. Extreme examples have been life threatening when administration of a drug causes ‘cytokine storm’ (Suntharalingam G et al, N Engl J Med, Vol 355, p 1018-1028, 2006). 
     There is a clear need to understand and minimise the cytokine release risk of engineered recombinant proteins. Recombinant proteins that contain the Fc domain of antibodies, and that are capable of becoming functionally multivalent, require special attention. 
     In the present study of polymeric Fc domains a whole blood cytokine release assay was deployed to study the effects of mutagenesis with the aim of minimising cytokine release. (Example 8). 
     Platelets (thrombocytes) are small anucleated cells which are very abundant in blood. Platelets are involved, along with clotting factors and others cells in the cessation of bleeding by formation of blood clots. Platelets are involved in a number of maladies. Low platelet count “thrombocytopenia” can be caused by a number of factors and results in increased bruising and bleeding. Inadvertent clotting “thrombosis” includes events such as stroke and deep vein thrombosis. Human and non-human primate platelets, but not mice platelets; express FcγRIIa on their surface. Platelets can respond very quickly to vessel damage by means of pre-formed ‘dense-granules’ and ‘alpha granules’ and release of potent immunokines and other molecules such as histamine, serotonin, thromboxane, PAF, PDGF, TGFγ IL1β and many others (Semple Nature Reviews Immunology 2011 11: 265-274). 
     Platelets have been mechanistically involved in the toxicology of drugs administered to humans. Certain antibodies have been found to be of special interest because both their target antigen and the Fc-domain have been capable of interacting with the platelet, activating them and causing thrombosis (Horsewood 1991 78(4):1019-1026). Direct, dual binding mechanisms have been proposed for anti-CD40 Mabs (Langer Thrombosis and Haemostasis 2005 93:1127-1146). Alternatively, thrombosis can be caused indirectly by antibodies cross-linking with a target molecule which can interact with platelets such as in ‘HIT syndrome’ (heparin induced thrombocytopenia). Unfractionated heparin was associated with venous thrombosis in approximately 12% or recipients (Levine Chest 2006 130: 681-687). In other examples such as Mabs targeting VEGF, the mechanism of action is likely to involve heparin acting as bridge between VEGF, antibody and platelet (Scappaticci 2007 J National Cancer Institute 99:1232-1239; Meyer J. Thrombosis and Haemostasis 2009 7:171-181). Thrombosis can also be caused by aggregated IgG and hence product quality is of importance when manufacturing IgG and perhaps of special importance when manufacturing multimeric Fc-domains (Ginsberg J. Experimental Medicine 1978 147:207-218). 
     Platelet activation is a pre-cursor to, but not necessarily a commitment towards thrombosis. Platelet activation can be followed in vitro by means of serotonin release assays or following activation markers such as CD62p, CD63 or PAC-1. Platelet aggregation can be observed directly in vitro by means of whole blood, platelet rich plasma or washed platelet aggregation assays. Mice transgenic for human FcγRIIa expression on platelets can also be used to study thrombosis or reduced clotting in vivo. The construction of multimeric Fc-domain proteins poses a foreseeable risk with regards to thrombosis, hence in the present invention Fc-engineering and platelet activation assays have been deployed to understand and ensure the safety of the Fc-multimers. 
     FcRn, has a crucial role in maintaining the long half-life of IgG in the serum of adults and children. The receptor binds IgG in acidified vesicles (pH&lt;6.5) protecting the IgG molecule from degradation, and then releasing it at the higher pH of 7.4 in blood. 
     FcRn is unlike leukocyte Fc receptors, and instead, has structural similarity to MHC class I molecules. It is a heterodimer composed of a β 2 -microglobulin chain, non-covalently attached to a membrane-bound chain that includes three extracellular domains. One of these domains, including a carbohydrate chain, together with β 2 -microglobulin interacts with a site between the CH2 and CH3 domains of Fc. The interaction includes salt bridges made to histidine residues on IgG that are positively charged at pH&lt;6.5. At higher pH, the His residues lose their positive charges, the FcRn-IgG interaction is weakened and IgG dissociates. 
     Thus, in one embodiment, the polymeric Fc proteins of the invention bind to human FcRn. 
     In one embodiment, the polymeric Fc protein has a histidine residue at position 310, and preferably also at position 435. These histidine residues are important for human FcRn binding. In one embodiment, the histidine residues at positions 310 and 435 are native residues, i.e. positions 310 and 435 are not mutated. Alternatively, one or both of these histidine residues may be present as a result of a mutation. 
     The polymeric Fc protein of the invention may comprise one or more mutations which alter its binding to FcRn. The altered binding may be increased binding or decreased binding. 
     In one embodiment, the polymeric Fc fusion protein comprises one or more mutations such that it binds to FcRn with greater affinity and avidity than the corresponding native immunoglobulin. 
     In one embodiment, the Fc domain is mutated by substituting the threonine residue at position 250 with a glutamine residue (T250Q). 
     In one embodiment, the Fc domain is mutated by substituting the methionine residue at position 252 with a tyrosine residue (M252Y) 
     In one embodiment, the Fc domain is mutated by substituting the serine residue at position 254 with a threonine residue (S254T). 
     In one embodiment, the Fc domain is mutated by substituting the threonine residue at position 256 with a glutamic acid residue (T256E). 
     In one embodiment, the Fc domain is mutated by substituting the threonine residue at position 307 with an alanine residue (T307A). 
     In one embodiment, the Fc domain is mutated by substituting the threonine residue at position 307 with a proline residue (T307P). 
     In one embodiment, the Fc domain is mutated by substituting the valine residue at position 308 with a cysteine residue (V308C). 
     In one embodiment, the Fc domain is mutated by substituting the valine residue at position 308 with a phenylalanine residue (V308F). 
     In one embodiment, the Fc domain is mutated by substituting the valine residue at position 308 with a proline residue (V308P). 
     In one embodiment, the Fc domain is mutated by substituting the glutamine residue at position 311 with an alanine residue (Q311A). 
     In one embodiment, the Fc domain is mutated by substituting the glutamine residue at position 311 with an arginine residue (Q311R). 
     In one embodiment, the Fc domain is mutated by substituting the methionine residue at position 428 with a leucine residue (M428L). 
     In one embodiment, the Fc domain is mutated by substituting the histidine residue at position 433 with a lysine residue (H433K). 
     In one embodiment, the Fc domain is mutated by substituting the asparagine residue at position 434 with a phenylalanine residue (N434F). 
     In one embodiment, the Fc domain is mutated by substituting the asparagine residue at position 434 with a tyrosine residue (N434Y). 
     In one embodiment, the Fc domain is mutated by substituting the methionine residue at position 252 with a tyrosine residue, the serine residue at position 254 with a threonine residue, and the threonine residue at position 256 with a glutamic acid residue (M252Y/S254T/T256E). 
     In one embodiment, the Fc domain is mutated by substituting the valine residue at position 308 with a proline residue and the asparagine residue at position 434 with a tyrosine residue (V308P/N434Y). 
     In one embodiment, the Fc domain is mutated by substituting the methionine residue at position 252 with a tyrosine residue, the serine residue at position 254 with a threonine residue, the threonine residue at position 256 with a glutamic acid residue, the histidine residue at position 433 with a lysine residue and the asparagine residue at position 434 with a phenylalanine residue (M252Y/S254T/T256E/H433K/N434F). 
     In one embodiment, the polymeric Fc fusion protein comprises one or more mutations such that it binds to FcRn with lower affinity and avidity than the corresponding native immunoglobulin. In one embodiment, a histidine residue at position 310 is mutated to another amino acid residue. In one example, a histidine residue at position 310 is substituted with a leucine residue (H310L). 
     It will be appreciated that any of the mutations listed above may be combined to alter FcRn binding. 
     The polymeric Fc proteins of the invention may comprise one or more mutations which increase its binding to FcγRIIb. FcγRIIb is the only inhibitory receptor in humans and the only Fc receptor found on B cells. B cells and their pathogenic antibodies lie at the heart of many immune diseases, and thus the multimeric fusion proteins may provide improved therapies for these diseases. 
     In one embodiment, the Fc domain is mutated by substituting the proline residue at position 238 with an aspartic acid residue (P238D). 
     In one embodiment, the Fc domain is mutated by substituting the glutamic acid residue at position 258 with an alanine residue (E258A). 
     In one embodiment, the Fc domain is mutated by substituting the serine residue at position 267 with an alanine residue (S267A). 
     In one embodiment, the Fc domain is mutated by substituting the serine residue at position 267 with a glutamic acid residue (S267E). 
     In one embodiment, the Fc domain is mutated by substituting the leucine residue at position 328 with a phenylalanine residue (L328F). 
     In one embodiment, the Fc domain is mutated by substituting the glutamic acid residue at position 258 with an alanine residue and the serine residue at position 267 with an alanine residue (E258A/S267A). 
     In one embodiment, the Fc domain is mutated by substituting the serine residue at position 267 with a glutamic acid residue and the leucine residue at position 328 with a phenylalanine residue (S267E/L328F). 
     It will be appreciated that any of the mutations listed above may be combined to increase FcγRIIb binding. 
     In one embodiment of the invention we provide polymeric Fc proteins which display decreased binding to FcγR. Decreased binding to FcγR may provide improved therapies for use in the treatment of immune diseases involving pathogenic antibodies. 
     In one embodiment the polymeric Fc protein of the present invention comprises one or more mutations that decrease its binding to FcγR. 
     In one embodiment, a mutation that decreases binding to FcγR is used in a multimeric fusion protein of the invention which comprises an Fc-domain derived from IgG1. 
     In one embodiment, the Fc domain is mutated by substituting the leucine residue at position 234 with an alanine residue (L234A). 
     In one embodiment, the Fc domain is mutated by substituting the phenylalanine residue at position 234 with an alanine residue (F234A). 
     In one embodiment, the Fc domain is mutated by substituting the leucine residue at position 235 with an alanine residue (L235A). 
     In one embodiment, the Fc-domain is mutated by substituting the glycine residue at position 236 with an arginine residue (G236R). 
     In one embodiment, the Fc domain is mutated by substituting the asparagine residue at position 297 with an alanine residue (N297A) or a glutamine residue (N297Q). 
     In one embodiment, the Fc domain is mutated by substituting the serine residue at position 298 with an alanine residue (S298A). 
     In one embodiment, the Fc domain is mutated by substituting the leucine residue at position 328 with an arginine residue (L328R). 
     In one embodiment, the Fc-domain is mutated by substituting the leucine residue at position 234 with an alanine residue and the leucine residue at position 235 with an alanine residue (L234A/L235A). 
     In one embodiment, the Fc-domain is mutated by substituting the phenylalanine residue at position 234 with an alanine residue and the leucine residue at position 235 with an alanine residue (F234A/L235A). 
     In one embodiment, the Fc domain is mutated by substituting the glycine residue at position 236 with an arginine residue and the leucine residue at position 328 with an arginine residue (G236R/L328R). 
     It will be appreciated that any of the mutations listed above may be combined to decrease FcγR binding. 
     In one embodiment the polymeric Fc protein of the present invention comprises one or more mutations that decrease its binding to FcγRIIIa without affecting its binding to FcγRII. 
     In one embodiment, the Fc domain is mutated by substituting the serine residue at position 239 with an alanine residue (S239A). 
     In one embodiment, the Fc domain is mutated by substituting the glutamic acid residue at position 269 with an alanine residue (E269A). 
     In one embodiment, the Fc domain is mutated by substituting the glutamic acid residue at position 293 with an alanine residue (E293A). 
     In one embodiment, the Fc domain is mutated by substituting the tyrosine residue at position 296 with a phenylalanine residue (Y296F). 
     In one embodiment, the Fc domain is mutated by substituting the valine residue at position 303 with an alanine residue (V303A). 
     In one embodiment, the Fc domain is mutated by substituting the alanine residue at position 327 with a glycine residue (A327G). 
     In one embodiment, the Fc domain is mutated by substituting the lysine residue at position 338 with an alanine residue (K338A). 
     In one embodiment, the Fc domain is mutated by substituting the aspartic acid residue at position 376 with an alanine residue (D376A). 
     It will be appreciated that any of the mutations listed above may be combined to decrease FcγRIIIa binding. 
     The polymeric Fc proteins of the invention may comprise one or more mutations that alter its binding to complement. Altered complement binding may be increased binding or decreased binding. 
     In one embodiment the polymeric Fc protein comprises one or more mutations which decrease its binding to C1q. Initiation of the classical complement pathway starts with binding of hexameric C1q protein to the CH2 domain of antigen bound IgG and IgM. The multimeric fusion proteins of the invention do not possess antigen binding sites, and so would not be expected to show significant binding to C1q. However, the presence of one or more mutations that decrease C1q binding will ensure that they do not activate complement in the absence of antigen engagement, so providing improved therapies with greater safety. 
     Thus in one embodiment the polymeric Fc proteins of the invention comprises one or more mutations to decrease its binding to C1q. 
     In one embodiment, the Fc domain is mutated by substituting the leucine residue at position 234 with an alanine residue (L234A). 
     In one embodiment, the Fc domain is mutated by substituting the leucine residue at position 235 with an alanine residue (L235A). 
     In one embodiment, the Fc domain is mutated by substituting the leucine residue at position 235 with a glutamic acid residue (L235E). 
     In one embodiment, the Fc domain is mutated by substituting the glycine residue at position 237 with an alanine residue (G237A). 
     In one embodiment, the Fc domain is mutated by substituting the lysine residue at position 322 with an alanine residue (K322A). 
     In one embodiment, the Fc domain is mutated by substituting the proline residue at position 331 with an alanine residue (P331A). 
     In one embodiment, the Fc domain is mutated by substituting the proline residue at position 331 with a serine residue (P331S). 
     In one embodiment, the polymeric Fc protein comprises an Fc domain derived from IgG4. IgG4 has a naturally lower complement activation profile than IgG1, but also weaker binding of FcγR. Thus, in one embodiment, the multimeric fusion protein comprising IgG4 also comprises one or more mutations that increase FcγR binding. 
     It will be appreciated that any of the mutations listed above may be combined to reduce C1q binding. 
     The polymeric Fc fusion protein of the invention may comprise one or more mutations to create or remove a cysteine residue. Cysteine residues have an important role in the spontaneous assembly of the multimeric fusion protein, by forming disulphide bridges between individual pairs of polypeptide monomer units. Alternatively, they may be used for chemical modification of the free SH group. Thus, by altering the number and/or position of cysteine residues, it is possible to modify the structure of the multimeric fusion protein to produce a protein with improved therapeutic properties. 
     In one example the polymeric Fc fusion protein of the present invention does not comprise a cysteine residue at position 309. The amino acid residue at position 309 may be any amino acid residue other than cysteine. In one embodiment, the amino acid residue at position 309 is the wild type residue found in the corresponding naturally occurring antibody. For example, the wild type residue found at position 309 in naturally occurring human IgG1, IgG3 and IgG4 is a leucine residue, that found in naturally occurring IgG2 is a valine residue. 
     In one embodiment, the Fc-domain is mutated by substituting the valine residue at position 308 with a cysteine residue (V308C). 
     In one embodiment of the invention we provide polymeric Fc fusion proteins with improved manufacturability comprising fewer disulphide bonds and/or glycosylation sites. These proteins have less complex disulphide bond architecture and post translational glycosylation patterns and are thus simpler and less expensive to manufacture. 
     In one embodiment, two disulphide bonds in the hinge region are removed by mutating a core hinge sequence CPPC to SPPS. 
     In one embodiment, a disulphide bond in the tailpiece is removed by substituting the cysteine residue at position 575 with a serine, threonine or alanine residue (C575S, C575T, or C575A). 
     In one embodiment a core hinge sequence CPPC is mutated to SPPS, and the tailpiece cysteine residue at position 575 is substituted with a serine, threonine or alanine residue (C575S, C575T, or C575A). 
     In one embodiment the polymeric Fc fusion protein of the invention comprises substantially non-covalent inter-domain interactions. 
     In one embodiment, the polymeric Fc fusion protein of the invention is expressed in a cell such that a practical proportion of the product is hexameric. 
     A practical proportion is preferably greater than or equal to 50%, for example 50-60%, 60-70%, 70-80%, 80-90%, 90-100%. 
     In one embodiment a glycosylation site in the CH2 domain is removed by substituting the asparagine residue at position 297 with an alanine residue (N297A) or a glutamine residue (N297A). In addition to improved manufacturability, these aglycosyl mutants also reduce FcγR binding as described herein above. 
     In one embodiment, a glycosylation site in the tailpiece, where present, is removed by substituting the asparagine residue at position 563 with an alanine residue (N563A) or a glutamine residue (N563Q). 
     In one embodiment a glycosylation site in the CH2 domain and a glycosylation site in the tailpiece are both removed by substituting the asparagine residue at position 297 with an alanine residue or a glutamine residue, and substituting the asparagine residue at position 563 with an alanine residue or a glutamine residue (N297A/N563A or N297A/N563Q or N297Q/N563A or N297Q/N563Q). 
     It will be appreciated that any of the mutations listed above may be combined. 
     The present invention also provides an isolated DNA sequence encoding a polymeric Fc containing protein of the present invention, such as a polypeptide chain of a polypeptide monomer unit of the present invention, such a heavy chain Fc region, or a component part thereof. The DNA sequence may comprise synthetic DNA, for instance produced by chemical processing, cDNA, genomic DNA or any combination thereof. 
     DNA sequences which encode a polypeptide chain of a polymeric Fc containing protein of the present invention can be obtained by methods well known to those skilled in the art. For example, DNA sequences coding for part or all of a polypeptide chain of a polypeptide monomer unit such as a heavy chain Fc region, may be synthesised as desired from the determined DNA sequences or on the basis of the corresponding amino acid sequences. 
     Examples of suitable DNA sequences according to the present invention are provided in SEQ ID NOs 50-59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, and 83. In one embodiment, the present invention provides an isolated DNA which comprises or consists of the sequence given in any one of SEQ ID NOs, 63, 65, 69, 71, 75, 77, 81 and 83. 
     Standard techniques of molecular biology may be used to prepare DNA sequences coding for a polymeric Fc containing protein of the present invention, such as a polypeptide chain of a polypeptide monomer unit of the present invention. Desired DNA sequences may be synthesised completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate. 
     The present invention also relates to a cloning or expression vector comprising one or more DNA sequences of the present invention. Accordingly, provided is a cloning or expression vector comprising one or more DNA sequences encoding a polypeptide chain of a polypeptide monomer unit of the present invention, or a component part thereof. 
     General methods by which the vectors may be constructed, transfection methods and culture methods are well known to those skilled in the art. In this respect, reference is made to “Current Protocols in Molecular Biology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing. 
     Also provided is a host cell comprising one or more cloning or expression vectors comprising one or more DNA sequences encoding a polymeric Fc protein of the present invention. Any suitable host cell/vector system may be used for expression of the DNA sequences encoding the protein of the present invention. Bacterial, for example  E. coli , and other microbial systems such as  Saccharomyces  or  Pichia  may be used or eukaryotic, for example mammalian, host cell expression systems may also be used. Suitable mammalian host cells include CHO cells. Suitable types of chinese hamster ovary (CHO cells) for use in the present invention may include CHO and CHO-K1 cells, including dhfr-CHO cells, such as CHO-DG44 cells and CHO-DXB11 cells, which may be used with a DHFR selectable marker, or CHOK1-SV cells which may be used with a glutamine synthetase selectable marker. Other suitable host cells include NSO cells. 
     The present invention also provides a process for the production of a polymeric Fc proteins according to the present invention, comprising culturing a host cell containing a vector of the present invention under conditions suitable for expression of the fusion protein and assembly into multimers, and isolating and optionally purifying the polymeric Fc proteins. 
     The polymeric Fc proteins of the present invention are expressed at good levels from host cells. Thus the properties of the polymeric Fc fusion protein are conducive to commercial processing. 
     The polymeric Fc proteins of the present invention may be made using any suitable method. In one embodiment, the polymeric Fc fusion protein of the invention may be produced under conditions which minimise aggregation. In one example, aggregation may be minimised by the addition of preservative to the culture media, culture supernatant, or purification media. Examples of suitable preservatives include thiol capping agents such as N-ethyl maleimide, iodoacetic acid, β-mercaptoethanol, β-mercaptoethylamine, glutathione, or cysteine. Other examples include disulphide inhibiting agents such as ethyl enediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), or acidification to below pH 6.0. 
     In one embodiment there is provided a process for purifying a polymeric Fc fusion protein of the present invention comprising the steps: performing anion exchange chromatography in non-binding mode such that the impurities are retained on the column and the antibody is eluted. 
     In one embodiment the purification employs affinity capture on an FcRn, FcγR or C-reactive protein column. 
     In one embodiment the purification employs protein A. 
     Suitable ion exchange resins for use in the process include Q.FF resin (supplied by GE-Healthcare). The step may, for example be performed at a pH about 8. The process may further comprise an initial capture step employing cation exchange chromatography, performed for example at a pH of about 4 to 5, such as 4.5. The cation exchange chromatography may, for example employ a resin such as CaptoS resin or SP sepharose FF (supplied by GE-Healthcare). The antibody or fragment can then be eluted from the resin employing an ionic salt solution such as sodium chloride, for example at a concentration of 200 mM. 
     Thus the chromatograph step or steps may include one or more washing steps, as appropriate. 
     The purification process may also comprise one or more filtration steps, such as a diafiltration step. 
     Polymeric Fc proteins which have the required number of Fc domains can be separated according to molecular size, for example by size exclusion chromatography. 
     Thus in one embodiment there is provided a purified polymeric Fc fusion protein according to the invention, in substantially purified from, in particular free or substantially free of endotoxin and/or host cell protein or DNA. 
     Purified form as used supra is intended to refer to at least 90% purity, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% w/w or more pure. 
     Substantially free of endotoxin is generally intended to refer to an endotoxin content of 1 EU per mg antibody product or less such as 0.5 or 0.1 EU per mg product. 
     Substantially free of host cell protein or DNA is generally intended to refer to host cell protein and/or DNA content 400 μg per mg of antibody product or less such as 100 μg per mg or less, in particular 20 μg per mg, as appropriate. 
     As the polymeric Fc proteins of the present invention are useful in the treatment and/or prophylaxis of a pathological condition, the present invention also provides a pharmaceutical or diagnostic composition comprising a polymeric Fc protein of the present invention in combination with one or more of a pharmaceutically acceptable excipient, diluent or carrier. Accordingly, provided is the use of a polymeric Fc protein of the invention for the manufacture of a medicament. The composition will usually be supplied as part of a sterile, pharmaceutical composition that will normally include a pharmaceutically acceptable carrier. A pharmaceutical composition of the present invention may additionally comprise a pharmaceutically acceptable excipient. 
     The present invention also provides a process for preparation of a pharmaceutical or diagnostic composition comprising adding and mixing the polymeric Fc protein of the present invention together with one or more of a pharmaceutically acceptable excipient, diluent or carrier. 
     The polymeric Fc protein may be the sole active ingredient in the pharmaceutical or diagnostic composition or may be accompanied by other active ingredients including other antibody ingredients or non-antibody ingredients such as steroids or other drug molecules. 
     The pharmaceutical compositions suitably comprise a therapeutically effective amount of the polymeric Fc protein of the invention. The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent needed to treat, ameliorate or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect. For any medicine, the therapeutically effective amount can be estimated initially either in cell culture assays or in animal models, usually in rodents, rabbits, dogs, pigs or primates. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. 
     The precise therapeutically effective amount for a human subject will depend upon the severity of the disease state, the general health of the subject, the age, weight and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, a therapeutically effective amount will be from 0.01 mg/kg to 500 mg/kg, for example 0.1 mg/kg to 200 mg/kg, such as 100 mg/kg. Pharmaceutical compositions may be conveniently presented in unit dose forms containing a predetermined amount of an active agent of the invention per dose. 
     In one embodiment of a polymeric Fc protein according to the invention a single dose may provide up to a 90% reduction in circulating IgG levels. 
     Compositions may be administered individually to a patient or may be administered in combination (e.g. simultaneously, sequentially or separately) with other agents, drugs or hormones. 
     In one embodiment the polymeric Fc proteins according to the present disclosure are employed with an immunosuppressant therapy, such as a steroid, in particular prednisone. 
     In one embodiment the polymeric Fc proteins according to the present disclosure are employed in combination with Rituximab or other B cell therapies. 
     In one embodiment the polymeric Fc proteins according to the present disclosure are employed with any B cell or T cell modulating agent or immunomodulator. Examples include methotrexate, mycophenylate and azathioprine. 
     The dose at which the polymeric Fc protein of the present invention is administered depends on the nature of the condition to be treated, the extent of the disease present and on whether the multimeric fusion protein is being used prophylactically or to treat an existing condition. 
     The frequency of dose will depend on the half-life of the polymeric Fc protein and the duration of its effect. If the polymeric Fc protein has a short half-life (e.g. 2 to 10 hours) it may be necessary to give one or more doses per day. Alternatively, if the polymeric Fc protein has a long half-life (e.g. 2 to 15 days) and/or long lasting pharmacodynamic effects it may only be necessary to give a dosage once per day, once per week or even once every 1 or 2 months. 
     In one embodiment the dose is delivered bi-weekly, i.e. twice a month. 
     Half-life as employed herein is intended to refer to the duration of the molecule in circulation, for example in serum/plasma. 
     Pharmacodynamics as employed herein refers to the profile and in particular duration of the biological action of the polymeric Fc protein according the present disclosure. 
     The pharmaceutically acceptable carrier should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic. Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates and benzoates. 
     Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the patient. 
     Suitable forms for administration include forms suitable for parenteral administration, e.g. by injection or infusion, for example by bolus injection or continuous infusion. Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilising and/or dispersing agents. The protein may be in the form of nanoparticles. Alternatively, the antibody molecule may be in dry form, for reconstitution before use with an appropriate sterile liquid. 
     Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals. However, in one or more embodiments the compositions are adapted for administration to human subjects. 
     Suitably in formulations according to the present disclosure, the pH of the final formulation is not similar to the value of the isoelectric point of the multimeric fusion protein, for example if the pl of the protein is in the range 8-9 or above then a formulation pH of 7 may be appropriate. Whilst not wishing to be bound by theory it is thought that this may ultimately provide a final formulation with improved stability, for example the multimeric fusion protein remains in solution. 
     In one example the pharmaceutical formulation at a pH in the range of 4.0 to 7.0 comprises: 1 to 200 mg/mL of an protein molecule according to the present disclosure, 1 to 100 mM of a buffer, 0.001 to 1% of a surfactant, a) 10 to 500 mM of a stabiliser, b) 10 to 500 mM of a stabiliser and 5 to 500 mM of a tonicity agent, or c) 5 to 500 mM of a tonicity agent. 
     The pharmaceutical compositions of this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes. Hyposprays may also be used to administer the pharmaceutical compositions of the invention. Typically, the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. 
     Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule. 
     It will be appreciated that the active ingredient in the composition will be a protein molecule. As such, it will be susceptible to degradation in the gastrointestinal tract. Thus, if the composition is to be administered by a route using the gastrointestinal tract, the composition will need to contain agents which protect the protein from degradation but which release the protein once it has been absorbed from the gastrointestinal tract. 
     A thorough discussion of pharmaceutically acceptable carriers is available in Remington&#39;s Pharmaceutical Sciences (Mack Publishing Company, N.J. 1991). 
     In one embodiment the formulation is provided as a formulation for topical administrations including inhalation. 
     Suitable inhalable preparations include inhalable powders, metering aerosols containing propellant gases or inhalable solutions free from propellant gases. Inhalable powders according to the disclosure containing the active substance may consist solely of the abovementioned active substances or of a mixture of the abovementioned active substances with physiologically acceptable excipient. These inhalable powders may include monosaccharides (e.g. glucose or arabinose), disaccharides (e.g. lactose, saccharose, maltose), oligo- and polysaccharides (e.g. dextranes), polyalcohols (e.g. sorbitol, mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) or mixtures of these with one another. Mono- or disaccharides are suitably used, the use of lactose or glucose, particularly but not exclusively in the form of their hydrates. 
     Particles for deposition in the lung require a particle size less than 10 microns, such as 1-9 microns for example from 1 to 5 μm. The particle size of the active ingredient (such as the antibody or fragment) is of primary importance. 
     The propellant gases which can be used to prepare the inhalable aerosols are known in the art. Suitable propellant gases are selected from among hydrocarbons such as n-propane, n-butane or isobutane and halohydrocarbons such as chlorinated and/or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane. The abovementioned propellant gases may be used on their own or in mixtures thereof. 
     Particularly suitable propellant gases are halogenated alkane derivatives selected from among TG 11, TG 12, TG 134a and TG227. Of the abovementioned halogenated hydrocarbons, TG134a (1,1,1,2-tetrafluoroethane) and TG227 (1,1,1,2,3,3,3-heptafluoropropane) and mixtures thereof are particularly suitable. 
     The propellant-gas-containing inhalable aerosols may also contain other ingredients such as cosolvents, stabilisers, surface-active agents (surfactants), antioxidants, lubricants and means for adjusting the pH. All these ingredients are known in the art. 
     The propellant-gas-containing inhalable aerosols according to the invention may contain up to 5% by weight of active substance. Aerosols according to the invention contain, for example, 0.002 to 5% by weight, 0.01 to 3% by weight, 0.015 to 2% by weight, 0.1 to 2% by weight, 0.5 to 2% by weight or 0.5 to 1% by weight of active ingredient. 
     Alternatively topical administrations to the lung may also be by administration of a liquid solution or suspension formulation, for example employing a device such as a nebulizer, for example, a nebulizer connected to a compressor (e.g., the Pari LC-Jet Plus® nebulizer connected to a Pari Master® compressor manufactured by Pari Respiratory Equipment, Inc., Richmond, Va.). 
     The multimeric fusion protein of the invention can be delivered dispersed in a solvent, e.g., in the form of a solution or a suspension. It can be suspended in an appropriate physiological solution, e.g., saline or other pharmacologically acceptable solvent or a buffered solution. Buffered solutions known in the art may contain 0.05 mg to 0.15 mg disodium edetate, 8.0 mg to 9.0 mg NaCl, 0.15 mg to 0.25 mg polysorbate, 0.25 mg to 0.30 mg anhydrous citric acid, and 0.45 mg to 0.55 mg sodium citrate per 1 ml of water so as to achieve a pH of about 4.0 to 5.0. A suspension can employ, for example, lyophilised protein. 
     The therapeutic suspensions or solution formulations can also contain one or more excipients. Excipients are well known in the art and include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensions can be encapsulated in liposomes or biodegradable microspheres. The formulation will generally be provided in a substantially sterile form employing sterile manufacture processes. 
     This may include production and sterilization by filtration of the buffered solvent/solution used for the formulation, aseptic suspension of the protein in the sterile buffered solvent solution, and dispensing of the formulation into sterile receptacles by methods familiar to those of ordinary skill in the art. 
     Nebulizable formulation according to the present disclosure may be provided, for example, as single dose units (e.g., sealed plastic containers or vials) packed in foil envelopes. Each vial contains a unit dose in a volume, e.g., 2 mL, of solvent/solutionbuffer. 
     The polymeric Fc fusion proteins disclosed herein may be suitable for delivery via nebulisation. 
     It is also envisaged that the proteins of the present invention may be administered by use of gene therapy. In order to achieve this, DNA sequences encoding the polypeptide chains of the protein molecule under the control of appropriate DNA components are introduced into a patient such that the polypeptide chains are expressed from the DNA sequences and assembled in situ. 
     In one embodiment we provide a polymeric Fc protein of the invention for use in therapy. 
     In one embodiment we provide a polymeric Fc protein of the invention for use in the treatment of immune disorders. 
     In one embodiment we provide the use of polymeric Fc protein of the invention for the preparation of a medicament for the treatment of immune disorders. 
     Examples of immune disorders which may be treated using a polymeric Fc protein of the invention include immune thrombocytopenia (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP), Kawasaki disease and Guillain-Barre syndrome (GBS). 
     The present invention also provides a polymeric Fc protein (or compositions or proteins comprising same) for use in the control of autoimmune diseases, for example Acute Disseminated Encephalomyelitis (ADEM), Acute necrotizing hemorrhagic leukoencephalitis, Addison&#39;s disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, ANCA-associated vasculitis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome (APS), Autoimmune angioedema, Autoimmune aplastic anemia, Autoimmune dysautonomia, Autoimmune hepatitis, Autoimmune hyperlipidemia, Autoimmune immunodeficiency, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune thrombocytopenic purpura (ATP), Autoimmune thyroid disease, Autoimmune urticarial, Axonal &amp; nal neuropathies, Balo disease, Behcet&#39;s disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign mucosal pemphigoid, Crohn&#39;s disease, Cogans syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic&#39;s disease (neuromyelitis optica), Dilated cardiomyopathy, Discoid lupus, Dressler&#39;s syndrome, Endometriosis, Eosinophilic angiocentric fibrosis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Glomerulonephritis, Goodpasture&#39;s syndrome, Granulomatosis with Polyangiitis (GPA) see Wegener&#39;s, Graves&#39; disease, Guillain-Barre syndrome, Hashimoto&#39;s encephalitis, Hashimoto&#39;s thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic hypocomplementemic tubulointestitial nephritis, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related disease, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inflammatory aortic aneurysm, Inflammatory pseudotumour, Inclusion body myositis, Insulin-dependent diabetes (typel), Interstitial cystitis, Juvenile arthritis, Juvenile diabetes, Kawasaki syndrome, Kuttner&#39;s tumour, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (SLE), Lyme disease, chronic, Mediastinal fibrosis, Meniere&#39;s disease, Microscopic polyangiitis, Mikulicz&#39;s syndrome, Mixed connective tissue disease (MCTD), Mooren&#39;s ulcer, Mucha-Habermann disease, Multifocal fibrosclerosis, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic&#39;s), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Ormond&#39;s disease (retroperitoneal fibrosis), Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with  Streptococcus ), Paraneoplastic cerebellar degeneration, Paraproteinemic polyneuropathies, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus vulgaris, Periaortitis, Periarteritis, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, &amp; Ill autoimmune polyglandular syndromes, Polymyalgia rheumatic, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, Reflex sympathetic dystrophy, Reiter&#39;s syndrome, Relapsing polychondritis, Restless legs syndrome, Retroperitoneal fibrosis (Ormond&#39;s disease), Rheumatic fever, Rheumatoid arthritis, Riedel&#39;s thyroiditis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren&#39;s syndrome, Sperm &amp; testicular autoimmunity, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac&#39;s syndrome, Sympathetic ophthalmia, Takayasu&#39;s arteritis, Temporal arteritis/Giant cell arteritis, Thrombotic, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, Transverse myelitis, Ulcerative colitis, Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vesiculobullous dermatosis, Vitiligo, Waldenstrom Macroglobulinaemia, Warm idiopathic haemolytic anaemia and Wegener&#39;s granulomatosis (now termed Granulomatosis with Polyangiitis (GPA). 
     In one embodiment the polymeric Fc proteins and fragments according to the disclosure are employed in the treatment or prophylaxis of epilepsy or seizures. 
     In one embodiment the polymeric Fc proteins and fragments according to the disclosure are employed in the treatment or prophylaxis of multiple sclerosis. 
     In embodiment the polymeric Fc proteins and fragments of the disclosure are employed in the treatment or prophylaxis of alloimmune disease/indications which include:
         Transplantation donor mismatch due to anti-HLA antibodies   Foetal and neonatal alloimmune thrombocytopenia, FNAIT (or neonatal alloimmune thrombocytopenia, NAITP or NAIT or NAT, or foeto-maternal alloimmune thrombocytopenia, FMAITP or FMAIT).       

     Additional indications include: rapid clearance of Fc-containing biopharmaceutical drugs from human patients and combination of multimeric fusion protein therapy with other therapies—IVIg, Rituxan, plasmapheresis. For example multimeric fusion protein therapy may be employed following Rituxan therapy. 
     In one embodiment the polymeric Fc proteins of the disclosure are employed in the treatment or prophylaxis of a neurology disorder such as:
         Chronic inflammatory demyelinating polyneuropathy (CIDP)   Guillain-Barre syndrome   Paraproteinemic polyneuropathies   Neuromyelitis optica (NMO, NMO spectrum disorders or NMO spectrum diseases), and   Myasthenia gravis.       

     In one embodiment the polymeric Fc proteins of the disclosure are employed in a dermatology disorder such as:
         Bullous pemphigoid   Pemphigus vulgaris   ANCA-associated vasculitis   Dilated cardiomyopathy       

     In one embodiment the polymeric Fc proteins of the disclosure are employed in an immunology or haematology disorder such as:
         Idiopathic thrombocytopenic purpura (ITP)   Thrombotic thrombocytopenic purpura (TTP)   Warm idiopathic haemolytic anaemia   Goodpasture&#39;s syndrome   Transplantation donor mismatch due to anti-HLA antibodies       

     In one embodiment the disorder is selected from Myasthenia Gravis, Neuro-myelitis Optica, CIDP, Guillaume-Barre Syndrome, Para-proteinemic Poly neuropathy, Refractory Epilepsy, ITP/TTP, Hemolytic Anemia, Goodpasture&#39;s Syndrome, ABO mismatch, Lupus nephritis, Renal Vasculitis, Sclero-derma, Fibrosing alveolitis, Dilated cardio-myopathy, Grave&#39;s Disease, Type 1 diabetes, Auto-immune diabetes, Pemphigus, Sclero-derma, Lupus, ANCA vasculitis, Dermato-myositis, Sjogren&#39;s Disease and Rheumatoid Arthritis. 
     In one embodiment the disorder is selected from autoimmune polyendocrine syndrome types 1 (APECED or Whitaker&#39;s Syndrome) and 2 (Schmidt&#39;s Syndrome); alopecia universalis; myasthenic crisis; thyroid crisis; thyroid associated eye disease; thyroid ophthalmopathy; autoimmune diabetes; autoantibody associated encephalitis and/or encephalopathy; pemphigus foliaceus; epidermolysis bullosa; dermatitis herpetiformis; Sydenham&#39;s chorea; acute motor axonal neuropathy (AMAN); Miller-Fisher syndrome; multifocal motor neuropathy (MMN); opsoclonus; inflammatory myopathy; Isaac&#39;s syndrome (autoimmune neuromyotonia), Paraneoplastic syndromes and Limbic encephalitis. 
     The polymeric Fc protein according to the present disclosure may be employed in treatment or prophylaxis. 
     The present invention also provides a method of reducing the concentration of undesired antibodies in an individual comprising the steps of administering to an individual a therapeutically effective dose of a polymeric Fc protein described herein. 
     The multimeric fusion protein of the present invention may also be used in diagnosis, for example in the in vivo diagnosis and imaging of disease states involving Fc-receptors, such as B-cell related lymphomas. 
    
    
     
       FIGURE LEGENDS 
         FIG. 1  Example of an expression construct and a multimeric fusion protein according to the invention. SP is signal peptide, CH2 and CH3 are heavy chain constant domains, TP is tailpiece. 
         FIG. 2  Example Sequences 
         2 ( a ) Example amino acid sequences of a polypeptide chain of a polypeptide monomer unit. In each sequence, the tailpiece sequence is underlined, and any mutations are shown in bold and underlined. The hinge is in bold. In constructs comprising a CH4 domain from IgM, this region is shown in italics. 
         2 ( b ) Example amino acid sequences for an Fc-multimer polypeptide chain comprising a CH2 and CH3 domain derived from IgG1 or a CH2 and CH3 domain derived from IgG4. In each sequence, the positions of difference between IgG1 and IgG4 are in bold and highlighted. 
         2 ( c ) Example amino acid sequences for Fc-multimers designed to combine certain selected properties of IgG1 and certain selected properties of IgG4. The mutations are shown in bold and underlined. 
         2 ( d ) Example amino acid sequences for Fc-multimers with hybrid heavy chain Fc-regions engineered by domain exchange. 
         2 ( e ) Example amino acid sequences for Fc-multimers with hybrid heavy chain Fc-regions and additional mutations, that have been engineered to combine certain selected properties of IgG1 and certain selected properties of IgG4. 
         2 ( f ) DNA and amino acid sequences of the B72.3 signal peptide. (i) DNA sequence, (ii) amino acid sequence. 
         2 ( g ) Example DNA sequences for Fc-multimers. 
         FIG. 3  Role of the CH3 domain in Fc-multimer assembly 
         3 ( a ) Size exclusion chromatography traces showing the effect of IgG1/IgG4 CH3 domain exchange on hexamerisation of Fc-multimers. 
         3 ( b ) Effect of point mutations in the CH3 domain on hexamerisation of IgG1 Fc IgM tp. 
         3 ( c ) High levels of hexamerisation of IgG4 Fc IgM tp are achieved by point mutation Q355R. 
         3 ( d ) Effect of mutagenesis of R355 to all other amino acids in IgG1 Fc IgM tp. 
         FIG. 4  Binding of Fc-multimers to FcRn, measured by surface plasmon resonance analysis. The traces demonstrate binding for multimer concentration range: 2.5 μM, 1.25 μM, 0.6250, 0.31250, 0.156250, 0.0781250, 0.03906250. The Fc-multimers shown all comprised histidine at position 310. 
         4 ( a ) hIgG1 Fc-multimer IgM tp L309C binding to low density FcRn. 
         4 ( b ) hIgG1 Fc-multimer IgM tp binding to low density FcRn. 
         4 ( c ) hIgG1 Fc-multimer IgM tp L309C binding to high density FcRn. 
         4 ( d ) hIgG1 Fc-multimer IgM tp binding to high density FcRn. 
         FIG. 5  Fc-multimer inhibition of macrophage phagocytosis. The data show that Fc-multimers derived from human IgG1 or IgG4, polymerised into hexamer or dodecamer forms by IgM tailpiece alone, or IgM tailpiece and L309C, all exhibit potency and maximum levels of inhibition significantly better than human IVIG. 
         FIG. 6  Fc-multimer inhibition of macrophage phagocytosis. The data show the inhibitory effects of Fc-multimers comprising a single cross-over mutation at a selected position of difference between the IgG1 and IgG4 Fc region. 
         FIG. 7  Fc-multimer inhibition of macrophage phagocytosis. The data show the inhibitory effects of Fc-multimers designed for use in the treatment of immune disorders. 
         FIG. 8  Stimulation of cytokine release by Fc-multimers. The data demonstrated that wild type IgG1 Fc-multimers, both with and without L309C, stimulate very high levels of cytokine release. The observed levels of cytokines were higher than those produced by the positive control anti-CD52 antibody, Campath. In marked contrast, IgG4 Fc-multimers, and IgG1 Fc-multimers comprising the FcγR and C1q inert “LALA” mutation (L234A L235A), produced virtually zero cytokine release. 
         FIG. 9  Stimulation of cytokine release by Fc-multimers. The data show the effects of Fc-multimers comprising a single cross-over mutation at a selected position of difference between the IgG1 and IgG4 Fc region. 
         FIG. 10  Stimulation of cytokine release by Fc-multimers. The data show the effects of Fc-multimers engineered with mutations to modulate cytokine release. 
         FIG. 11  Inhibition of FcRn-mediated IgG recycling by Fc-multimers. 
         FIG. 12  Prevention of platelet loss in an in vivo model of acute thrombocytopenia by Fc-multimers. The graph shows the aggregated results for five independent experiments using Balb/c mice. 
         FIG. 13  Prevention of platelet loss in an in vivo model of chronic thrombocytopenia by Fc-multimers. 
         13 ( a ) Single dose of 10 mg/kg Fc-multimer administered on day 3. 
         13 ( b ) Four consecutive daily doses, 10 mg/kg per dose, administered on days 3, 4, 5, and 6. 
       The group size for each point on the graph was n=6. 
         FIG. 14  Binding of Fc-multimers to C1q. 
         FIG. 15  Platelet activation by Fc-multimers. 
       
         FIG. 16 
       
       Analysis of the effects of mutations using Design of Experiments. 
         16 ( a ) Effects of mutations on macrophage phagocytosis 
         16 ( b ) Effects of mutations on cytokine release 
         16 ( c ) Effects of mutations on platelet activation 
       
         FIG. 17 
       
       Example amino acid and DNA sequences for alternative polymeric Fc formats. 
       
         FIG. 18 
       
       Size exclusion chromatography traces for purified polymeric Fc proteins. 
       
         FIG. 19 
       
       Stimulation of cytokine release by polymeric Fc proteins with and without the improved Fc domains of the invention. 
         19 ( a ) stradomer stimulation of IFN-γ release 
         19 ( b ) stradomer stimulation of TNFα release 
         19 ( c ) stradomer stimulation of IL1-β release 
         19 ( d ) stradomer stimulation of IL-6 release 
         19 ( e ) SIF protein stimulation of IFN-γ release 
     
    
    
     EXAMPLES 
     Example 1: Molecular Biology 
     Fc-multimer DNA sequences were assembled using standard molecular biology methods, including PCR, restriction-ligation cloning, point mutagenesis (Quikchange) and Sanger sequencing. Expression constructs were cloned into expression plasmids (pNAFL, pNAFH) suitable for both transient and stable expression in CHO cells. Other examples of suitable expression vectors include pCDNA3 (Invitrogen). 
     A diagram of an expression construct and multimeric fusion protein according to the invention is shown in  FIG. 1 . 
     Diagrams showing example amino acid sequences of a polypeptide chain of a polypeptide monomer unit are provided in  FIG. 2( a )-( e ) . In each sequence, the tailpiece sequence is underlined, and any mutations are shown in bold and underlined. In constructs comprising a CH4 domain from IgM, this region is shown in italics. 
     IgG1/IgG4 Crossover Mutations 
     Various Fc-multimer variants were constructed in which certain key amino acid residues in the Fc-domain were designed to match those found in IgG1, whilst other key amino acid residues were designed to match those found in IgG4. IgG1 and IgG4 differ from one another at seven positions in the CH2 domain and six positions in the CH3 domain as summarised in Table 4. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                   
                 IgG1 
                 IgG4 
                   
                   
               
               
                 position 
                 amino acid 
                 amino acid 
                 mutation of 
                 mutation of 
               
               
                 number 
                 residue 
                 residue 
                 IgG1 to IgG4 
                 IgG4 to IgG1 
               
               
                   
               
             
            
               
                 234 
                 L 
                 F 
                 L234F 
                 F234L 
               
               
                 268 
                 H 
                 Q 
                 H268Q 
                 Q268H 
               
               
                 274 
                 K 
                 Q 
                 K274Q 
                 Q274K 
               
               
                 296 
                 Y 
                 F 
                 Y296F 
                 F296Y 
               
               
                 327 
                 A 
                 G 
                 A327G 
                 G327A 
               
               
                 330 
                 A 
                 S 
                 A330S 
                 S330A 
               
               
                 331 
                 P 
                 S 
                 P331S 
                 S331P 
               
               
                 355 
                 R 
                 Q 
                 R355Q 
                 Q355R 
               
               
                 356 
                 D 
                 E 
                 D356E 
                 E356D 
               
               
                 358 
                 L 
                 M 
                 L358M 
                 M358L 
               
               
                 409 
                 K 
                 R 
                 K409R 
                 R409K 
               
               
                 419 
                 Q 
                 E 
                 Q419E 
                 E419Q 
               
               
                 445 
                 P 
                 L 
                 P445L 
                 L445P 
               
               
                   
               
            
           
         
       
     
     Diagrams showing example amino acid sequences for an Fc-multimer polypeptide chain comprising a CH2 and CH3 domain derived from IgG1 or a CH2 and CH3 domain derived from IgG4 are provided in  FIG. 2( b ) . In each sequence, the positions of difference between IgG1 and IgG4 are in bold and highlighted. 
     Diagrams showing example amino acid sequences for Fc-multimers designed to combine certain selected properties of IgG1 and certain selected properties of IgG4 are provided in  FIG. 2( c ) . The mutations are shown in bold and underlined. 
     It will be appreciated that a particular sequence of interest may be created using either the IgG1 or the IgG4 Fc-domain sequence as a starting point and making the relevant mutations. For example, an IgG4 CH2 domain with mutation F234L is the same as an IgG1 CH2 domain with mutations H268Q, K274Q, Y296F, A327G, A330S, and P331S. 
     Fc-Region Domain Exchange 
     Fc-multimer variants were also constructed comprising hybrid heavy chain Fc-regions in which the CH2 domain was derived from one particular IgG subclass and the CH3 domain was derived from a different IgG subclass. Diagrams showing example amino acid sequences for Fc-multimers with hybrid heavy chain Fc-regions are provided in  FIG. 2( d ) . 
     Diagrams showing example amino acid sequences for Fc-multimers with hybrid heavy chain Fc-regions and additional mutations, that have been designed to combine certain selected properties of IgG1 and certain selected properties of IgG4 are provided in  FIG. 2( e ) . 
     Example 2: Expression 
     Small scale expression was performed using ‘transient’ expression of HEK293 or CHO cells transfected using lipofectamine or electroporation. Cultures were grown in shaking flasks or agitated bags in CD-CHO (Lonza) or ProCHO5 (Life Technologies) media at scales ranging from 50-2000 ml for 5-10 days. Cells were removed by centrifugation and culture supernatants were stored at 4° C. until purified. Preservatives were added to some cultures after removal of cells. 
     The results demonstrated that the multimeric fusion proteins are expressed well. 
     The signal peptide used to express the multimeric fusion proteins was found to have an impact on the level of expression achieved. A signal peptide from antibody B72.3 resulted in higher expression levels than an IL-2 signal peptide sequence described in the prior art. 
     The DNA and amino acid sequences of the B72.3 signal peptide are shown in  FIG. 2   f.    
     Example 3: Purification and Analysis 
     Fc-multimers were purified from culture supernatants after checking/adjusting pH to be ≧6.5, by protein A chromatography with step elution using a pH3.4 buffer. Eluate was immediately neutralised to ˜pH7.0 using 1M Tris pH8.5 before storage at 4° C. Analytical size exclusion chromatography was used to separate various multimeric forms of Fc-domains using S200 columns and fraction collection. Fractions were analysed and pooled after G3000 HPLC and reducing and non-reducing SDS-PAGE analysis. Endotoxin was tested using the limulus amoebocyte lysate (LAL) assay and samples used in assays were &lt;1 EU/mg. 
     The multimeric fusion proteins were found to be expressed and purified predominantly in hexameric form, with some protein in dodecameric and other forms. The results demonstrate that the proteins assemble effectively into multimers in the absence of cysteine at position 309. 
     Purification of the multimeric fusion proteins in the presence of a preservative reduced the tendency to aggregate, producing improved preparations with more uniform structure. Examples of preservatives shown to be effective include thiol capping agents such as N-ethyl maleimide (NEM) and glutathione (GSH); and disulphide inhibiting agents such as ethyl enediaminetetraacetic acid (EDTA). 
     Example 4 Role of the CH3 Domain in Fc-Multimer Assembly 
     The extent of multimerisation was unexpectedly found to vary depending on the IgG subclass from which the Fc-region was derived. Fc-multimers comprising a CH2 domain and a CH3 domain derived from IgG1 assembled very efficiently into hexamers, with approximately 80% of the molecules being present in hexameric form. In contrast, Fc-multimers comprising a CH2 domain and a CH3 domain derived from IgG4 formed lower levels of hexamers. Investigation of Fc-multimers comprising hybrid Fc-regions in which the CH2 domain was derived from one particular IgG subclass and the CH3 domain was derived from a different IgG subclass revealed that the ability to form hexamers is encoded mainly by the CH3 domain. The presence of a CH3 domain derived from IgG1 significantly increases hexamerisation. Hybrid Fc-multimers in which the CH3 domain is derived from IgG1 and the CH2 domain is derived from IgG4 hexamerised just as efficiently as IgG1 wild-type, with approximately 80% of the molecules being found as hexamers. Thus, replacing the CH3 domain of IgG4 with that of IgG1 improves the levels of hexamerisation compared to wild type IgG4 Fc-multimers. The resulting hybrid has the advantage of high levels of hexamer formation whilst retaining many of the desirable properties of IgG4.  FIG. 3( a ) . 
     The CH3 domains of IgG1 and IgG4 differ at six positions as described in Example 1. Starting with an Fc-multimer comprising a CH2 and CH3 domain derived from IgG1, each of these positions was mutated in turn, from the IgG1 residue to the IgG4 residue. The results demonstrated that the amino acid at position 355 is critical for hexamerisation. The amino acid found at position 355 in wild type IgG1, arginine, promotes efficient hexamerisation. That found in wild type IgG4, glutamine, results in lower hexamerisation. The other positions of difference between the IgG1 and IgG4 CH3 domains did not affect hexamerisation.  FIG. 3( b ) . 
     In Fc-multimers comprising a CH3 domain derived from IgG4, substitution of the glutamine residue at position 355 with an arginine residue (Q355R) resulted in high levels of hexamerisation. Thus, the problem of lower hexamerisation of IgG4 Fc-multimers can be solved by a single amino acid substitution. This has the advantage that the resulting Fc-multimer assembles into hexamers with high efficiency whilst retaining the characteristic properties of IgG4.  FIG. 3( c ) . 
     In IgG1 Fc multimers, substitution of arginine at position 355 with cysteine (R355C) increased hexamer formation beyond that of wild type IgG1. Although we do not wish to be bound by theory, this result suggests that a cysteine residue at position 355 may be capable of forming disulphide bonds with the cysteine in the tailpiece. Mutagenesis of R355 to all the other amino acids did not result in further enhancement of hexamer formation in IgG1 Fc-multimers.  FIG. 3( d ) . 
     Example 5: Affinity Measurements for Interactions of Fc-Multimers and FcR 
     Affinity/avidity measurements for the interactions of multimeric fusion proteins and Fc receptors (FcR) including FcγR and FcRn can be performed using well known methods including surface plasmon resonance, competition ELISA and competition binding studies on FcR bearing cell lines. Soluble, extra-cellular domains (ECDs) of FcRs were used in surface plasmon resonance experiments by non-specific immobilisation or tag specific capture onto a BIAcore sensor chip on a Biacore T200. Human FcRn extracellular domain was provided as a non-covalent complex between the human FcRn alpha chain extracellular domain and (32 microglobulin. Multimeric fusion proteins were titrated over the receptors at a variety of concentrations and flow rates in order to best determine the strength of the interaction. Data was analysed using Biacore T200 Evaluation software. 
       FIG. 4  shows data for binding of multimeric fusion proteins to FcRn, measured by surface plasmon resonance analysis. The traces demonstrate binding for multimer concentration range: 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.15625 μM, 0.078125 μM, 0.0390625 μM. The Fc-multimers shown all comprised histidine at position 310. 
     (a) human IgG1 Fc-multimer IgM tp L309C binding to low density FcRn.
 
(b) human IgG1 Fc-multimer IgM tp binding to low density FcRn.
 
(c) human IgG1 Fc-multimer IgM tp L309C binding to high density FcRn.
 
(d) human IgG1 Fc-multimer IgM tp binding to high density FcRn.
 
     Constructs used in (a) and (c) contain a leucine to cysteine substitution at position 309. 
     The results demonstrated that Fc-multimers comprising histidine at position 310 bind to human FcRn. 
     A number of Fc-multimers were also generated which incorporated mutations thought to increase binding to human FcRn. 
     Table 5 shows the dissociation constants for the binding of mutated monomeric human IgG1 Fc fragments to human FcRn at pH6.0. The mutations resulted in increased binding to human FcRn. However, the strength of the interaction of the monomeric fragments is still weak, with dissociation constants in the micromolar range. Multimerisation of the mutated Fc-domains, as described in the present invention, may confer an avidity benefit, so greatly improving the strength of the interaction. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Binding of mutated monomeric IgG1 Fc-fragments to human FcRn at 
               
               
                 pH 6.0 
               
            
           
           
               
               
               
               
            
               
                   
                 Sample mutation 
                 KD (M) 
                 KD (μM) 
               
               
                   
                   
               
               
                   
                 IgG1 Fc, WT 
                 9.78E−07 
                 0.98 
               
               
                   
                 IgG1 Fc, L309S 
                 1.25E−06 
                 1.25 
               
               
                   
                 IgG1 Fc, Q311A 
                 7.69E−07 
                 0.77 
               
               
                   
                 IgG1 Fc, T307A 
                 5.65E−07 
                 0.57 
               
               
                   
                 IgG1 Fc, T307P 
                 6.93E−07 
                 0.69 
               
               
                   
                 IgG1 Fc, V308C 
                 7.00E−07 
                 0.70 
               
               
                   
                 IgG1 Fc, V308F 
                 3.32E−07 
                 0.33 
               
               
                   
                 IgG1 Fc, V308P 
                 1.36E−07 
                 0.14 
               
               
                   
                 IgG1 Fc, WT 
                 1.07E−06 
                 1.07 
               
               
                   
                   
               
            
           
         
       
     
     Example 6: Macrophage Phagocytosis of B Cell Targets 
     An assay was designed to measure antibody-dependent phagocytosis of B cells by human macrophages. To prepare macrophages, human peripheral blood mononuclear cells (PBMC) were first isolated from fresh blood by density-gradient centrifugation. Monocytes were then selected by incubating the PBMCs for 1 hour at 37° C. in 6-well tissue culture coated plates, followed by removal of non-adherent cells. Adherent monocytes were differentiated into macrophages by 5 day culture in macrophage-colony stimulating factor (MCSF). Human B cells were then prepared from a separate (allogeneic) donor by isolation of PBMC followed by purification of B cells by negative selection using MACS (B cell isolation kit II, Miltenyi Biotech). In some assays, B cells were labelled with carboxyfluorescein succinimidyl ester (CFSE) (Molecular Probes). Differentiated macrophages and B cells were co-cultured at a 1:5 ratio in the presence of anti-CD20 mAb (rituximab) to induce antibody-dependent phagocytosis of the B cells. Multimeric fusion proteins or controls were added at the indicated concentrations and the cells incubated at 37° C. 5% CO 2  for 1-24 hrs. At the end of each time-point, cells were centrifuged and resuspended in FACS buffer at 4° C. to stop further phagocytosis and the B cells surface-stained with anti-CD19 allophycocyanin (APC) before analysis by flow cytometry. Macrophages were distinguished by their auto-fluorescence/side-scatter properties and B cells by their CFSE/CD19 labelling. CFSE-positive macrophages negative for CD19 labelling were assumed to contain engulfed B cells. 
     The results demonstrated that the multimeric fusion proteins of the invention inhibit B cell depletion by human macrophages. ( FIG. 5 ). The data show that Fc-multimers derived from human IgG1 or IgG4, polymerised into hexamer or dodecamer forms by IgM tailpiece alone, or IgM tailpiece and L309C, all exhibit potency and maximum levels of inhibition significantly better than human IVIG. 
     The demonstration that the dodecamer form and hexamer form are equally potent is of benefit for product manufacturing and safety, as there is no need for additional purification to remove trace amounts of dodecamer from hexamer. 
     Flow cytometry analysis using CFSE stained B-cells confirmed that the mechanism of action is inhibition of macrophage phagocytosis, and not B-cell killing or apoptosis by other means. 
     In order to assess the ability of any given Fc-multimer construct to inhibit macrophage phagocytosis, its activity was measured in the assay described herein above and compared with the activities of IgG1 and IgG4 wild type Fc-multimers. The activity of each mutant was then summarised as “IgG1-like”, “high”, “medium”, “low”, or “IgG4-like”, based on a visual comparison of its concentration vs. effect curve with those obtained for IgG1 and IgG4 wild type Fc-multimers. 
     Results for Fc-multimers comprising single cross-over mutations at each of the eight positions of difference in the IgG1 and IgG4 Fc region are shown in  FIG. 6  and summarised in Table 6. 
     Wild type IgG1 Fc-multimer, comprising a CH2 and a CH3 domain derived from IgG1, potently inhibits macrophage phagocytosis of antibody-coated target cells. Two of the single mutations in IgG1 Fc multimers (A330S, K409R) have no effect on the potency of inhibition of phagocytosis. Six of the single mutations (L234F, H268Q, K274Q, Y296F, A327G and P331S) result in a modest reduction in the potency of inhibition of phagocytosis. The residues L234F and A327G are of particular interest as mutating these significantly reduces cytokine release (see Example 8), whilst maintaining relatively high potency in inhibition of phagocytosis. This combination of properties will be useful for the treatment of autoimmune disorders. 
     Wild type IgG4 Fc-multimers inhibit macrophage phagocytosis of antibody-coated target cells, but less potently than IgG1 Fc-multimers. Two of the single mutations in IgG4 Fc-multimers (F234L and G327A) modestly increase the potency of inhibition of phagocytosis. Six of the mutations in IgG4 (Q268H, Q274K, F296Y, S330A, S331P, R409K) have no effect on inhibition of phagocytosis when mutated individually. The residues F234L and G327A are of particular interest as, whilst individually mutating either of these positions enhances potency of IgG4 Fc multimers in inhibition of phagocytosis, they have no effect on increasing cytokine production (see Example 8). This combination of properties will be useful for the treatment of autoimmune disorders. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                 Fc-multimer 
                 potency of phagocytosis inhibition 
               
               
                   
                   
               
             
            
               
                   
                 IgG1 Fc IgM tp L309 
                 the standard for “IgG1-like” 
               
               
                   
                 IgG1 Fc IgM tp L309 L234F 
                 high 
               
               
                   
                 IgG1 Fc IgM tp L309 H268Q 
                 high 
               
               
                   
                 IgG1 Fc IgM tp L309 K274Q 
                 high 
               
               
                   
                 IgG1 Fc IgM tp L309 Y296F 
                 high 
               
               
                   
                 IgG1 Fc IgM tp L309 A327G 
                 high 
               
               
                   
                 IgG1 Fc IgM tp L309 A330S 
                 IgG1-like 
               
               
                   
                 IgG1 Fc IgM tp L309 P331S 
                 high 
               
               
                   
                 IgG1 Fc IgM tp L309 K409R 
                 IgG1-like 
               
               
                   
                 IgG4 Fc IgM tp L309 
                 the standard for “IgG4-like” 
               
               
                   
                 IgG4 Fc IgM tp L309 F234L 
                 medium 
               
               
                   
                 IgG4 Fc IgM tp L309 Q268H 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 Q274K 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 F296Y 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 G327A 
                 low 
               
               
                   
                 IgG4 Fc IgM tp L309 S330A 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 S331P 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 R409K 
                 IgG4-like 
               
               
                   
                   
               
            
           
         
       
     
     Results for further Fc-multimer constructs designed for use in the treatment of immune disorders are shown in  FIG. 7  and summarised in Table 7. 
     Wild type IgG1 and IgG4 Fc-multimers inhibit macrophage phagocytosis of antibody-coated target cells more potently than IVIG. 
     IgG1 Fc-multimers comprising L234F (which reduces cytokine production) or L234F and P331S (reduces cytokine production and C1Q binding, see Examples 8 and 15) have modestly reduced potency in inhibition of phagocytosis relative to wild type IgG1 Fc-multimers, but are still highly potent relative to wild type IgG4 Fc-multimers or IVIG. 
     IgG4 Fc-multimers comprising the mutations F234L; F234L and F296Y; G327A and S331P; S330A and S331P; or G327A and S330A; have enhanced potency in inhibition of phagocytosis compared to wild type IgG4 Fc-multimers. 
     
       
         
           
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                   
                 potency of 
               
               
                 Fc-multimer 
                 phagocytosis inhibition 
               
               
                   
               
             
            
               
                 IgG1 Fc IgM tp L309 
                 the standard for 
               
               
                   
                 “IgG1-like” 
               
               
                 IgG4 Fc IgM tp L309 
                 the standard for 
               
               
                   
                 “IgG4-like” 
               
               
                 IgG1 Fc IgM tp L309 L234F 
                 high 
               
               
                 IgG1 Fc IgM tp L309 L234F P331S 
                 high 
               
               
                 Hybrid Fc IgG4-CH2 IgG1-CH3 IgM tp L309 
                 medium 
               
               
                 IgG4 Fc IgM tp L309 F234L 
                 medium 
               
               
                 IgG4 Fc IgM tp L309 F234L F296Y 
                 medium 
               
               
                 IgG4 Fc IgM tp L309 G327A S330A 
                 medium 
               
               
                 IgG4 Fc IgM tp L309 G327A S331P 
                 medium 
               
               
                 IgG4 Fc IgM tp L309 S330A S331P 
                 medium 
               
               
                   
               
            
           
         
       
     
     Example 7: THP1 Cell Phagocytosis of IgG FITC Beads 
     THP1 cells were plated out at passage 7, counted and re-suspended at 5×10 5  cells/ml. 200 μl of cells were added to each well of a 96-well flat bottom plate (1×10 5  cells per well). Beads coated with rabbit IgG (Cambridge bioscience CAY500290-1 ea) were added directly to each well, mixed (1 in 10 dilution, 10 μl/well) and left for the time points: 1 h, 2h, 4h, 8h. Zero time points were effected by adding beads to cells in ice cold buffer on ice. At the end of each time point, cells were centrifuged at 300 g for 3 mins. The cells were resuspended in FACS buffer containing a 1:20 dilution of trypan blue stock solution for 2 minutes. Cells were washed with 150 μl FACS buffer, centrifuged and resuspended in 200 μl FACS buffer and transferred to a round bottom plate ready for FACS. Cells were centrifuged once more and resuspended in 200 μl of FACS buffer before analysis by flow cytometry. THP1 cells were gated on forward and side-scatter and uptake of beads measured as FITC fluorescence. 
     Example 8: Human Whole Blood Cytokine Release Assay 
     Fresh blood was collected from donors in lithium heparin vacutainers. The Fc-multimer constructs of interest or controls were serially diluted in sterile PBS to the indicated concentrations. 12.5 μl of Fc-multimer or control was added to the assay plates, followed by 237.5 μl of whole blood. The assay plates used were typically 96-well U-bottomed tissue culture plates. The plates were incubated at 37° C. without CO 2  supplementation for 24 hrs. Alternatively, the plates may be incubated at 37° C., 5% CO 2  for 24 hrs. Plates were centrifuged at 1800 rpm for 5 minutes and the plasma removed for cytokine analysis. Cytokine analysis was performed by Meso Scale Discovery cytokine multiplex according to the manufacturer&#39;s protocol and read on a Sector Imager 6000. 
     Results are shown in  FIG. 8 . The data demonstrated that wild type IgG1 Fc-multimers, both with and without L309C, stimulate very high levels of cytokine release. The observed levels of cytokines were higher than those produced by the positive control, Campath. In marked contrast, IgG4 Fc-multimers, and IgG1 Fc-multimers comprising the FcγR and C1q inert “LALA” mutation (L234A L235A), produced virtually zero cytokine release. 
     In order to assess the effect of any given Fc-multimer construct on cytokine release its activity was measured in the assay described herein above and compared with the activities of IgG1 and IgG4 wild type Fc-multimers. The activity of the mutant was then summarised as “IgG1-like”, “high”, “medium”, “low”, or “IgG4-like”, based on a visual comparison of its concentration vs. effect curve with those obtained for IgG1 and IgG4 wild type Fc-multimers. 
     Results for Fc-multimers comprising single cross-over mutations at selected positions of difference in the IgG1 and IgG4 Fc region are shown in  FIG. 9  and summarised in Table 8. 
     The results demonstrated that wild type IgG1 Fc-multimer, comprising a CH2 and a CH3 domain derived from IgG1, stimulates the release of very significant levels of cytokines. The results shown are for IFNγ. Similar results were observed for TNFα. 
     Two of the single mutations (L234F, A327G) significantly reduced cytokine release in IgG1 Fc-multimers. One of the single mutations (Y296F) produced a moderate reduction of cytokine release. One of the mutations (P331S) significantly increased cytokine release. Three of the mutations (H268Q, K274Q, A330S) had no effect on cytokine release. 
     In marked contrast, wild type IgG4 Fc-multimer, comprising a CH2 and a CH3 domain derived from IgG4, produced virtually no cytokine release. None of the single cross-over mutations had any effect on cytokine release by IgG4 Fc-multimers, not even those at positions shown to be important for cytokine release in the IgG1 Fc-multimers. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 8 
               
               
                   
                   
               
               
                   
                 Fc-multimer 
                 stimulation of IFNγ release 
               
               
                   
                   
               
             
            
               
                   
                 IgG1 Fc IgM tp L309 
                 the standard for “IgG1-like” 
               
               
                   
                 IgG1 Fc IgM tp L309 L234F 
                 low 
               
               
                   
                 IgG1 Fc IgM tp L309 H268Q 
                 IgG1-like 
               
               
                   
                 IgG1 Fc IgM tp L309 K274Q 
                 IgG1-like 
               
               
                   
                 IgG1 Fc IgM tp L309 Y296F 
                 medium 
               
               
                   
                 IgG1 Fc IgM tp L309 A327G 
                 low 
               
               
                   
                 IgG1 Fc IgM tp L309 A330S 
                 IgG1-like 
               
               
                   
                 IgG1 Fc IgM tp L309 P331S 
                 higher than IgG1 
               
               
                   
                 IgG1 Fc IgM tp L309 K409R 
                 higher than IgG1 
               
               
                   
                 IgG4 Fc IgM tp L309 
                 the standard for “IgG4-like” 
               
               
                   
                 IgG4 Fc IgM tp L309 F234L 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 Q268H 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 Q274K 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 F296Y 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 G327A 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 S330A 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 S331P 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 R409K 
                 IgG4-like 
               
               
                   
                   
               
            
           
         
       
     
     Results for further Fc-multimer constructs designed to modulate cytokine release are shown in  FIG. 10  and summarised in Table 9. 
     The data shows that cytokine release by IgG1 Fc-multimers can be reduced to levels approximately equivalent to IVIG by inclusion of L234F alone or in combination with P331S. Such Fc-multimers may be useful for the treatment of immune disorders. All the IgG4 Fc-multimers containing mutations shown to have other useful properties, for example enhanced potency in phagocytosis (Example 6), retain virtually zero levels of cytokine release, and may thus be useful for the treatment of immune disorders. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Cytokine release by Fc-multimers 
               
            
           
           
               
               
               
            
               
                   
                 Fc-multimer 
                 stimulation of IFNγ release 
               
               
                   
                   
               
               
                   
                 IgG1 Fc IgM tp L309 
                 the standard for “IgG1-like” 
               
               
                   
                 IgG4 Fc IgM tp L309 
                 the standard for “IgG4-like” 
               
               
                   
                 IgG1 Fc IgM tp L309 L234F 
                 low 
               
               
                   
                 IgG1 Fc IgM tp L309 L234F P331S 
                 medium 
               
               
                   
                 Hybrid Fc IgG4-CH2 IgG1-CH3 
                 IgG4-like 
               
               
                   
                 IgM tp L309 
               
               
                   
                 IgG4 Fc IgM tp L309 F234L 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 F234L F296Y 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 G327A S330A 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 G327A S331P 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 S330A S331P 
                 IgG4-like 
               
               
                   
                   
               
            
           
         
       
     
     Example 9: Effect on IgG Recycling in Cells in Culture 
     Cell-based assays were performed using Madin-Darby Canine Kidney (MDCK) II cells which had been stably transfected with a human FcRn and human β2M double gene vector with a Geneticin selection marker. A stable cell clone was selected that was able to recycle and transcytose human IgG and this was used for all subsequent studies. It will be referred to as MDCK II clone 15. Equivalent MDCK cell lines, transfected with either cynomolgus monkey (“clone 40”) or mouse FcRn have been generated in a similar way, for use in assays equivalent to the above. 
     An in vitro assay was established to examine the ability of a multimeric fusion protein of the present invention to inhibit the IgG-recycling capabilities of FcRn. Briefly, MDCK II clone 15 cells were incubated with biotinylated human IgG (1 μg/ml) in the presence or absence of the multimeric fusion protein in an acidic buffer (pH 5.9) to allow binding to FcRn. After a pulse time of 60 mins, all excess protein was removed and the cells incubated in a neutral pH buffer (pH 7.2) which allowed release of surface-exposed, bound IgG into the supernatant. The inhibition of FcRn was followed using an MSD assay to detect the amount of IgG recycled and thus released into the supernatant. 
     MDCK II clone 15 cells were plated at 15,000 cells per well in a 96 well plate and incubated overnight at 37° C., 5% CO 2 . The cells were incubated with 1 μg/ml of biotinylated human IgG (Jackson) in the presence and absence of the multimeric fusion protein in HBSS+(Ca/Mg) pH 5.9+1% BSA for 1 hour at 37° C., 5% CO 2 . The cells were washed with HBSS+pH 5.9 then incubated at 37° C., 5% CO2 for 2 hours in HBSS+pH 7.2. The lysates and supernatant were removed and analysed for total IgG using an MSD assay (using an anti-human IgG capture antibody (Jackson) and a streptavidin-sulpho tag reveal antibody (MSD)). The inhibition curve was analysed by non-linear regression (Graphpad Prism) to determine the EC 50 . 
     The results demonstrated that the multimeric fusion proteins of the invention inhibit FcRn-mediated IgG recycling. ( FIG. 11 ). Human IgG1 Fc/IgM tailpiece multimers which retain the native histidine residue at position 310 block FcRn-mediated IgG transcytosis and recycling in a concentration-dependent manner, both with and without cysteine at position 309. The data clearly shows that the form lacking L309C is more potent than that with L309C. Although we do not wish to be bound by theory, it is likely that this difference is due to steric hindrance by L309C disulphide bonds adjacent to the histidine residue at position 310 which is critically involved in FcRn binding. 
     Table 10 shows the effects of mutations on the blocking activity of Fc-multimers. Three mutations that increase binding to FcRn, V308F, V308P and T307A, were tested in Fc-multimers comprising IgG1 Fc/IgM tailpiece or IgG4 Fc/IgM tailpiece. The results showed significant improvements in the ability of the Fc-multimers to block FcRn-mediated IgG intracellular uptake and IgG recycling. The data demonstrates the utility of these mutations for improving the potency of Fc-multimers comprising either IgG1 or IgG4 Fc-regions. 
     Substitution of histidine at position 310 with leucine (H310L) was shown to destroy the ability of the Fc-multimers to block FcRn-mediated IgG intracellular uptake and IgG recycling. The results demonstrate that Fc-multimers which retain the histidine residue at position 310 have much better binding to FcRn, and are more potent blockers of IgG intracellular uptake and IgG recycling. The data confirms that the Fc-multimers of the invention may provide new improved therapeutic compositions with longer half-life and greater efficacy. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Effects of mutations on Fc-multimer blockade of IgG intracellular 
               
               
                 uptake and IgG recycling 
               
            
           
           
               
               
               
            
               
                   
                 Blockade of IgG 
                 Blockade of IgG 
               
               
                   
                 intracellular uptake 
                 recycling 
               
               
                 Fc-multimer mutants 
                 EC 50  (μg/ml) 
                 EC 50  (μg/ml) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 IgG1 IgM tail-piece, Wild-type 
                 4.4 
                 0.33 
               
               
                 IgG1 IgM tail-piece, V308F 
                 0.12 
                 0.073 
               
               
                 IgG1 IgM tail-piece, V308P 
                 0.34 
                 0.55 
               
               
                 IgG1 IgM tail-piece, T307A 
                 0.54 
                 0.35 
               
               
                 IgG1 IgM tail-piece, H310L 
                 No detectable 
                 Maximal 45% 
               
               
                   
                 inhibition 
                 inhibition 
               
               
                   
                   
                 at 100 μg/ml 
               
               
                 IgG4 IgM tail-piece, Wild-type 
                 5.9 
                 1.0 
               
               
                 IgG4 IgM tail-piece, V308F 
                 0.42 
                 0.56 
               
               
                 IgG4 IgM tail-piece, V308P 
                 0.51 
                 0.42 
               
               
                 IgG4 IgM tail-piece, T307A 
                 0.50 
                 Not tested 
               
               
                   
               
            
           
         
       
     
     Example 10: Efficacy of Fc-Multimers in Acute ITP 
     Efficacy of Fc-multimers was studied in a mouse model of ITP, in which platelet loss is induced by administration of anti-CD41. This antibody binds to glycoprotein IIb on the surface of the platelets, targeting them for destruction. 
     ITP In Vivo Protocol 
     A 5 μl blood sample was taken from the tails of the mice prior to dosing to obtain baseline platelet numbers. 
     Mice were dosed i.v. with 1 mg/kg or 10 mg/kg Fc-multimers. 
     An hour later 1 μg/mouse rat anti-mouse CD41 IgG1 antibody (MWReg30) was dosed i.p. 
     Terminal cardiac puncture was performed 24 hours after anti-CD41 administration. 
     FACs Staining Protocol 
     5 μl of blood was taken from the tail vein. For terminal samples blood was taken by cardiac puncture into a heparin tube and 5 μl was taken for staining. 
     100 μl of antibody cocktail was added to the 5 μl blood sample and was incubated at 4° C. in the dark for 20 minutes. 
     5 mls of FACs buffer was added. 
     Each sample was diluted 1:4 to make a final volume of 200 μl in a ‘vee’ bottom plate and kept on ice until ready to acquire on the Becton Dickinson FACs Canto. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 11 
               
               
                   
               
               
                 Antibody name/ 
                   
                   
                 Supplier/Lot 
               
               
                 Clone 
                 Antibody Colour 
                 Dilution 
                 number 
               
               
                   
               
             
            
               
                 CD45 (30-F11) 
                 PerCPCy5.5 
                  1/400 
                 Ebio E08336-1633 
               
               
                 CD42d (1C2) 
                 PE 
                  1/200 
                 Ebio E14346-104 
               
               
                 Fc block 
                   
                  1/200 
               
               
                   
               
            
           
         
       
     
     FACS Acquisition 
     A set volume of 150 μl of sample was collected at a flow rate of 1.5 μl/sec. The threshold was set at 200. 
     Analysis was performed on FlowJo software. Platelet counts were derived from the CD45-/CD42d+ gated cells. 
     Cell counts were corrected for sample dilution based on the fact that the initial 5 μl blood sample is diluted 1/4000 and 150 μl of this is run through the FACs machine which equates to 0.1875 μl of the original sample being analysed. 5/0.1875=multiplication factor of x 26.7 for platelet/μl. 
     Reagents 
     Rat anti mouse CD41 Functional grade purified (Ebiosciences, MWReg30, lot #E11914-1632) 
     Endotoxin free PBS (Sigma, D8537) 
     FACs buffer: 0.1% FCS, 2 mM EDTA 
     Results 
     Human multimeric fusion proteins (′Fc-multimers) at 1 mg/kg dose were well tolerated. However they were not efficacious at this dose in the model. 
     Positive results were observed using 10 mg/kg human Fc-multimers. 
     Fc-multimers with either an IgM or an IgA tailpiece significantly inhibited platelet decrease caused by the injection of 1 μg/mouse anti-CD41. 
     The results demonstrated that Fc-multimers prevent platelet loss in an in vivo model of acute immune thrombocytopenia. Statistically significant reductions in platelet loss were achieved using human IgG1 Fc/IgM tailpiece multimers, both with and without L309C, and human IgG4 Fc/IgM tailpiece multimers with L309C, at a dose of 10 mg/kg. ( FIG. 12 ) Thus, multimeric fusion proteins hexamerised through the tailpiece alone, or via L309C disulphide and tailpiece, are efficacious in vivo. Human IVIG is active in this model only at a much higher dose of 1000 mg/kg. At the equivalent dose of 10 mg/kg, IVIG was found to be inactive in preventing platelet loss. 
     Example 11: Efficacy of Fc-Multimers in Chronic ITP 
     Efficacy of Fc-multimers was studied in a mouse model of chronic ITP, in which platelet loss is induced by administration of anti-CD41 for a sustained period of time using minipumps. 
     ITP In Vivo Protocol 
     A 5 ul tail bleed was taken prior to dosing to obtain baseline platelet numbers. 
     An alzet mini pump containing rat anti-mouse CD41 at a concentration of 82.5 ug/ml (57.75 ul rat anti-mouse CD41 Ab+642.25 ul PBS/BSA (1.5 mg/ml)) was implanted subcutaneously. The pumps have a flow rate of 0.5 ul/hour, dosing the equivalent of 0.99 ug of anti-CD41 per day. 
     5 ul tail bleeds to obtain platelet counts were done daily. 
     At a time point when a steady state platelet count has been reached mice are dosed intravenously with 1 g/kg IVIg, or Fc-multimers at a range of doses. 
     At 7 days a terminal cardiac puncture was performed. 
     FACs Staining Protocol 
     Take 5 ul of blood via the tail vein. For terminal samples take blood by cardiac puncture into a heparin tube and take 5 ul for staining. 
     Add 100 ul of antibody cocktail and incubate at 4° C. in the dark for 20 minutes. 
     Add 5 mls of FACs buffer 
     Dilute each sample 1:4 to make a final volume of 200 ul in a v-bottom plate and keep on ice until ready to acquire on the BD FACs Canto. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 12 
               
               
                   
               
               
                 Antibody name/ 
                   
                   
                 Supplier/Lot 
               
               
                 Clone 
                 Antibody Colour 
                 Dilution 
                 number 
               
               
                   
               
             
            
               
                 CD45 (30-F11) 
                 PerCPCy5.5 
                  1/400 
                 Ebio E08336-1633 
               
               
                 CD42d (1C2) 
                 PE 
                  1/200 
                 Ebio E14346-104 
               
               
                 Fc block 
                   
                  1/200 
               
               
                   
               
            
           
         
       
     
     FACS Acquisition 
     A set volume of 150 ul of sample will be collected at a flow rate of 1.5 ul/sec. The threshold will be set at 200. 
     Analysis will be performed on FlowJo software. Platelet counts will be derived from the CD45-CD42d+ gated cells. 
     Cell counts will be corrected for sample dilution based on the initial 5 μl blood sample is diluted 1/4000 and 150 ul of this is run through the FACs machine which equates to 0.1875 ul of the original sample being analysed. 5/0.1875=multiplication factor of x26.7 for platelet/ul. 
     Reagents 
     Rat anti mouse CD41 Functional grade purified (Ebiosciences, MWReg30, lot #E11914-1632) 
     Endotoxin free PBS (Sigma, D8537) 
     FACs buffer: 0.1% FCS, 2 mM EDTA 
     10 mg/kg IgG1 IgM tp, (ID:PB0000238), EWBE-017553, 5.69 mg/mg, 
     Endototoxin&lt;0.35 EU/mg 
     IgG1 Fc IgM tp L309C, (ID:PB0000198), EWBE-017400, 6.49 mg/ml, Endotoxin&lt;0.46 
     EU/mg 
     IVIg: Gammunex lot#26NK1N1. 
     The results demonstrated that the multimeric fusion proteins prevent platelet loss in an in vivo model of chronic thrombocytopenia.  FIG. 13  shows the effects achieved using (a) a single dose of 10 mg/kg Fc-multimer, administered on day 3; and (b) four consecutive daily doses, 10 mg/kg per dose, administered on days 3, 4, 5, and 6. Multiple doses of Fc-multimer increased their effectiveness in preventing platelet loss. Human IVIG was effective only at a much higher dose of 1000 mg/kg. 
     Example 12: Disulphide Bond and Glycan Analysis of Hexameric Fc-Multimers by Mass Spectrometry 
     Method 
     Purified samples of hexameric Fc-multimers (100 ug) were denatured in the presence of 8M urea in 55 mM Tris-HCl pH8.0 and free thiols were capped by incubating with 22 mM iodoacetamide (IAM) for 60 minutes at 37° C. The urea concentration was reduced to 6M using ultrafiltration and protein was digested with a LysC/trypsin mix (Promega) for 3 hours at 37° C. The sample was further diluted with 5 volumes of buffer and the digestion continued overnight at 37° C. Peptides were collected, desalted with Waters Oasis HLB cartridges, dried using a centrifugal evaporator and reconstituted in water containing 0.2% formic acid (solvent A). 
     Samples (7.5 uL, ˜7 ug) were loaded at 150 uL/min onto a 2.1×150 mm C18 column (Waters 1.7u PST 300A) equilibrated with solvent A and operated at 40° C. Peptides were eluted by a 60 min gradient to 50% solvent B (4:4:1 acetonitrile:1-propanol:water/0.2% formic acid) into a Waters Xevo mass spectrometer operated in MS E +ve-ion mode. MS E  data, which consists of alternating scans of low and high collision energy, was collected over the range 100-1900m/z. during elution. After running, the digests were reduced by adding 10 mM Tris(hydroxypropyl)phosphine (THP) solution directly to the autosampler vial and incubating for &gt;1 hr at room temperature. Reduced samples were then analysed a second time. 
     MS E  data was searched against the relevant Fc-multimer sequence using Waters BiopharmaLynx™ (BPL). The proportion of free disulphide thiols was calculated from the ratio of IAM-labelled to free peptide in the THP reduced digest. Glycan profiles were determined from the various glycopeptide isoforms detected in the digests. 
     Results 
     The results for glycan analysis of Fc-multimers (IgG1 Fc/IgM tailpiece), with and without L309C, are shown in Table 13. Glycan structures are shown in Table 8. 
     The data demonstrates high occupancy of N297, the glycosylation site in the IgG1 Fc-region, with less than 10% free asparagine residues being found at this position. Glycosylation at N297 was mainly fucosylated biantennary complex, primarily G0F. Occupancy of the IgM tailpiece site, N563, was about 50%, higher than the level of about 20% found in native IgM. Glycosylation at N563 was mainly high mannose. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Glycan analysis 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                 G0F- 
                   
                   
                   
                   
                   
               
               
                   
                 Occupancy 
                 N 
                 G2-F 
                 G1-F 
                 G0-F 
                 M6 
                 M5 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 IgG1 Fc 
                 CH2 
                 93% 
                 14% 
                 5% 
                 20% 
                 38% 
                 1% 
                 15% 
               
               
                 IgM tp 
                 N297 
               
               
                 L309C 
                 Tail 
                 59% 
                 &lt;1% 
                 7% 
                 5% 
                 2% 
                 24% 
                 20% 
               
               
                   
                 piece 
               
               
                   
                 N563 
               
               
                 IgG1 Fc 
                 CH2 
                 91% 
                 14% 
                 &lt;1% 
                 26% 
                 34% 
                 &lt;1% 
                 15% 
               
               
                 IgM tp 
                 N297 
               
               
                   
                 Tail 
                 45% 
                 1% 
                 4% 
                 3% 
                 &lt;1% 
                 14% 
                 22% 
               
               
                   
                 piece 
               
               
                   
                 N563 
               
               
                   
               
            
           
         
       
     
     Results for analysis of interchain disulphide bonds are shown in Table 14. Similar results were also obtained for intrachain disulphide bonds. The data demonstrated that a high proportion of the cysteine residues in the Fc-multimers are disulphide bonded. There was no evidence for significant amounts of scrambled disulphide bonding, and all expected dipeptides were found at high levels before reduction. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Interchain Disulphide bonds 
               
            
           
           
               
               
               
            
               
                   
                 DSB 
                 ‘Free’ 
               
               
                   
                 (%) 
                 thiol (%) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 IgG1 Fc 
                 Hinge 
                 97 
                 3 
               
               
                   
                 IgM tp 
                 CH2 
                 89 
                 10 
               
               
                   
                 L309C 
                 L309C- 
               
               
                   
                   
                 L309C 
               
               
                   
                   
                 Tail 
                 84 
                 14 
               
               
                   
                   
                 piece 
               
               
                   
                   
                 C575- 
               
               
                   
                   
                 C575 
               
               
                   
                 IgG1 
                 Hinge 
                 96 
                 4 
               
               
                   
                 IgM tp 
                 Tail 
                 80 
                 19 
               
               
                   
                   
                 piece 
               
               
                   
                   
                 C575- 
               
               
                   
                   
                 C575 
               
               
                   
                   
               
            
           
         
       
     
     Example 13: Binding of Fc-Multimers to C1q 
     Binding of Fc-multimers to C1q was measured by enzyme-linked immunosorbent assay (ELISA), using a C1q ELISA kit from Abnova Corporation, catalogue number: KA1274, lot number: V14-111723527. The Fc-multimer constructs were titrated in five-fold dilutions from 500 μg/ml through to 4 μg/ml. 100 μl of each Fc-multimer construct was added to the appropriate well, and agitated for one hour to enable binding. The assay was then carried out according to the manufacturer&#39;s protocol and analysed on a plate reader at an absorbance of 450 nm. 
     In order to assess the binding of any given Fc-multimer construct to C1q, its activity was measured and compared with the activities of IgG1 and IgG4 wild type Fc-multimers. The activity of the mutant was then summarised as “IgG1-like”, “high”, “medium”, “low”, or “IgG4-like”, based on a visual comparison of its concentration vs. effect curve with those obtained for IgG1 and IgG4 wild type Fc-multimers. 
     The results are shown in  FIG. 14  and summarised in Table 15. 
     The results demonstrated that wild type IgG1 Fc-multimer, comprising a CH2 and a CH3 domain derived from IgG1, binds strongly to C1q. In contrast, wild type IgG4 Fc-multimer, comprising a CH2 and a CH3 domain derived from IgG4, binds very poorly to C1q. The dominant residue defining C1q binding in the Fc-multimers was found to be proline at position 331 (P331). Substitution of this proline residue with serine (P331S) effectively reduced C1q binding in Fc-multimers with a CH2 domain derived from IgG1. The converse mutant, S331P, increased C1q binding in Fc-multimers with a CH2 domain derived from IgG4. 
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Binding of Fc-multimers to C1q 
               
            
           
           
               
               
               
            
               
                   
                 Fc-multimer 
                 C1q binding 
               
               
                   
                   
               
               
                   
                 IgG1 Fc IgM tp L309 
                 the standard 
               
               
                   
                   
                 for “IgG1-like” 
               
               
                   
                 IgG4 Fc IgM tp L309 
                 the standard 
               
               
                   
                   
                 for “IgG4-like” 
               
               
                   
                 IgG1 Fc IgM tp L309 L234F 
                 IgG1-like 
               
               
                   
                 IgG1 Fc IgM tp L309 P331S 
                 low 
               
               
                   
                 IgG1 Fc IgM tp L309 L234F P331S 
                 IgG4-like 
               
               
                   
                 Hybrid Fc IgG4-CH2 IgG1-CH3 IgM tp L309 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 F234L 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 F234L F296Y 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 G327A S330A 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 G327A S331P 
                 medium 
               
               
                   
                 IgG4 Fc IgM tp L309 S330A S331P 
                 medium 
               
               
                   
                   
               
            
           
         
       
     
     Example 14 Platelet Activation 
     Platelet activation by Fc-multimers was analysed by flow cytometry. Two-fold dilutions of Fc-multimer were prepared in RMPI medium and transferred to FACS tubes. The final concentration was from 100 μg/ml down to 3.12 μg/ml. 5 μl fresh whole blood (from a minimum of 2 human donors) was added per tube. Platelets were gated using anti-CD42b labelled Mab and activation followed with Mabs against markers: CD62p, CD63 and PAC-1. (Becton Dickinson, BD CD42b APC Cat:551061, BD CD62p PE Cat:550561, BD CD63 PE-Cy-7 Cat:561982, BD PAC-1 FITC Cat:340507). Cells were fixed by addition of 500 μl paraformaldehyde 1% before analysis by flow-cytometry. 
     CD62p was found to be the most sensitive of the three markers tested. 
     In order to assess the platelet activation by any given Fc-multimer construct, its activity was measured and compared with the activities of IgG1 and IgG4 wild type Fc-multimers. The activity of the mutant was then summarised as “IgG1-like”, “high”, “medium”, “low”, or “IgG4-like”, based on a visual comparison of its concentration vs. effect curve with those obtained for IgG1 and IgG4 wild type Fc-multimers. 
     Results for induction of CD62p expression are shown in  FIG. 15  and summarised in Table 16. 
     The results demonstrated that wild type IgG1 Fc-multimer, comprising a CH2 and a CH3 domain derived from IgG1, results in significant levels of platelet activation. 
     We have shown in Example 8 that two mutations are useful for reducing cytokine release from IgG1 Fc-multimer, L234F and A327G. Of these two mutations, L234F has much reduced levels of platelet activation compared to IgG1 wild-type Fc-domain, whereas an Fc-multimer with the A327G mutation retains significant levels of platelet activation. Thus L234F is a very useful mutation—it reduces cytokine release and platelet activation with only minor loss of potency in the inhibition of macrophage phagocytosis. 
     Addition of P331S (shown in Example 13 to be a useful C1q reducing mutation) to L234F (L234F P331S) results in low levels of platelet activation. This double mutant is a means of achieving low cytokine, low platelet activation and zero C1q binding. Thus this combination of mutations is particularly useful and is expected to provide new therapies for the treatment of immune disorders. 
     L234F may be dominant over A327G since the triple L234F A327G P331S mutant has low platelet activation. 
     Wild type IgG4 Fc-multimer, comprising a CH2 and a CH3 domain derived from IgG4, results in virtually no platelet activation. 
     Switching the CH3 domain to that of IgG1 retains these reduced levels of platelet activation. 
     The F234L mutation has low levels of platelet activation (and enhanced potency)—but the platelet activation is increased in comparison with wild type IgG4 Fc-multimer. 
     F296Y can be combined with F234L without additionally increasing platelet activation. This observation is important since F234L F296Y has increased potency compared to IgG4 wild type Fc-multimers. Thus this combination of mutations is particularly useful and is expected to provide new therapies for the treatment of immune disorders. 
     G327A mutation does not increase platelet activation. This observation is surprising in view of the results for the reverse mutation A327G in IgG1 Fc-multimers, which retained high levels of platelet activation. 
     Certain double mutants (G327A S330A, G327A S331P and S330A S331P) which also have enhanced potency over IgG4 WT have very low levels of platelet activation (IgG4 like) and are expected to provide new therapies for the treatment of immune disorders. 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Platelet activation by Fc-multimers 
               
            
           
           
               
               
               
            
               
                   
                 Fc-multimer 
                 CD62p 
               
               
                   
                   
               
               
                   
                 IgG1 Fc IgM tp L309 
                 the standard 
               
               
                   
                   
                 for “IgG1-like” 
               
               
                   
                 IgG4 Fc IgM tp L309 
                 the standard 
               
               
                   
                   
                 for “IgG4-like” 
               
               
                   
                 IgG1 Fc IgM tp L309 L234F 
                 low 
               
               
                   
                 IgG1 Fc IgM tp L309 L234F P331S 
                 IgG4-like 
               
               
                   
                 IgG1 Fc IgM tp L309 A327G 
                 IgG1-like 
               
               
                   
                 IgG1 Fc IgM tp L309 A327G A330S P331S 
                 IgG1-like 
               
               
                   
                 IgG1 Fc IgM tp L309 A327G P331S 
                 IgG1-like 
               
               
                   
                 IgG1 Fc IgM tp L309 L234F A327G P331S 
                 medium 
               
               
                   
                 Hybrid Fc IgG4-CH2 IgG1-CH3 IgM tp L309 
                 low 
               
               
                   
                 IgG4 Fc IgM tp L309 F234L 
                 medium 
               
               
                   
                 IgG4 Fc IgM tp L309 F234L F296Y 
                 medium 
               
               
                   
                 IgG4 Fc IgM tp L309 G327A S330A 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 G327A S331P 
                 IgG4-like 
               
               
                   
                 IgG4 Fc IgM tp L309 S330A S331P 
                 low 
               
               
                   
                   
               
            
           
         
       
     
     Example 15 Engineering of Fc-Multimer Variants 
     The previous examples illustrate that Fc-multimers have been created that are particularly suitable for use in the treatment of immune disorders. The Fc-multimers were engineered with the aim of generating Fc multimers which possess the following properties: 
     Inhibition of Macrophage Phagocytosis of Antibody Coated Target Cells. 
     The potency of the Fc-multimer should be as high as possible. 
     Cytokine Release. 
     Stimulation of cytokine release by the Fc-multimer should be as low as possible. 
     C1q Binding. 
     Binding of the Fc-multimer to C1q should be as low as possible. 
     Platelet Activation. 
     Platelet activation by the Fc-multimer should be as low as possible. 
     However, the work in the previous examples has illustrated that it may be necessary to compromise between maximum potency and slightly lower potency in order to achieve reduced side effects. 
     Wild type IgG1 Fc-multimer comprising a CH2 and CH3 domain derived from IgG1 without any additional mutations may be less suitable for use in the treatment of immune disorders because, although it displays high potency of phagocytosis inhibition, it also shows high levels of unwanted side effects, measured by cytokine release, C1q binding and platelet activation. 
     Wild type IgG4 Fc-multimer comprising a CH2 and CH3 domain derived from IgG4, produces very low levels of unwanted side effects although its potency is low relative to that of IgG1. Notwithstanding, the potency of wild type IgG4 Fc-multimer is still significantly higher than that of IVIG, as shown in  FIG. 7 . 
     Fc-multimers have been designed which combine the desirable properties of both IgG1 and IgG4 wild type Fc-multimers, without the undesirable properties. These Fc-multimers display effective levels of potency, whilst reducing unwanted side effects to a tolerable level as shown below in Table 17. These Fc-multimers are expected to be particularly useful for use in the treatment of immune disorders. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 17 
               
               
                   
               
               
                   
                 phagocytosis 
                 IFNγ 
                 C1q 
                 platelet 
               
               
                 Fc-multimer 
                 inhibition 
                 release 
                 binding 
                 activation 
               
               
                   
               
             
            
               
                 wild type IgG1 Fc-multimer 
                 the standard 
                 the 
                 the 
                 the 
               
               
                   
                 for “IgG1- 
                 standard 
                 standard 
                 standard 
               
               
                   
                 like” 
                 for “IgG1- 
                 for 
                 for “IgG1- 
               
               
                   
                   
                 like” 
                 “IgG1- 
                 like” 
               
               
                   
                   
                   
                 like” 
               
               
                 wild type IgG4 Fc-multimer 
                 the standard 
                 the 
                 the 
                 the 
               
               
                   
                 for “IgG4- 
                 standard 
                 standard 
                 standard 
               
               
                   
                 like” 
                 for “IgG4- 
                 for 
                 for “IgG4- 
               
               
                   
                   
                 like” 
                 “IgG4- 
                 like” 
               
               
                   
                   
                   
                 like” 
               
               
                 IgG1 Fc IgM tp L309 L234F P331S 
                 high 
                 medium 
                 IgG4-like 
                 IgG4-like 
               
               
                 Hybrid Fc IgG4-CH2 IgG1-CH3 IgM tp L309 
                 medium 
                 IgG4-like 
                 IgG4-like 
                 low 
               
               
                 IgG4 Fc IgM tp L309 F234L 
                 medium 
                 IgG4-like 
                 IgG4-like 
                 medium 
               
               
                 IgG4 Fc IgM tp L309 F234L F296Y 
                 medium 
                 IgG4-like 
                 IgG4-like 
                 medium 
               
               
                 IgG4 Fc IgM tp L309 G327A S330A 
                 medium 
                 IgG4-like 
                 IgG4-like 
                 IgG4-like 
               
               
                 IgG4 Fc IgM tp L309 G327A S331P 
                 medium 
                 IgG4-like 
                 medium 
                 IgG4-like 
               
               
                 IgG4 Fc IgM tp L309 S330A S331P 
                 medium 
                 IgG4-like 
                 medium 
                 IgG4-like 
               
               
                   
               
            
           
         
       
     
     Example 16 Design of Experiments 
     Design of Experiments (DoE) is a subset of statistical techniques for designing efficient and valid experiments, which is primarily used to study a number of factors at a time in a range of combinations. This is a very efficient way to determine the effect of a number of different factors that have the potential to interact with each other. 
     In the present invention, DoE has been used to investigate the effects of certain key amino acid residues found in the CH2 domain of IgG1, and other key amino acid residues found in the CH2 domain of IgG4. 
     IgG1 and IgG4 differ from one another at seven positions in the CH2 domain as shown below in Table 18. The seven positions of difference give rise to 128 (2 7 ) possible variants. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 18 
               
               
                   
               
               
                   
                 IgG1 
                 IgG4 
                   
                   
               
               
                 position 
                 amino acid 
                 amino acid 
                 mutation of 
                 mutation of 
               
               
                 number 
                 residue 
                 residue 
                 IgG1 to IgG4 
                 IgG4 to IgG1 
               
               
                   
               
             
            
               
                 234 
                 L 
                 F 
                 L234F 
                 F234L 
               
               
                 268 
                 H 
                 Q 
                 H268Q 
                 Q268H 
               
               
                 274 
                 K 
                 Q 
                 K274Q 
                 Q274K 
               
               
                 296 
                 Y 
                 F 
                 Y296F 
                 F296Y 
               
               
                 327 
                 A 
                 G 
                 A327G 
                 G327A 
               
               
                 330 
                 A 
                 S 
                 A330S 
                 S330A 
               
               
                 331 
                 P 
                 S 
                 P331S 
                 S331P 
               
               
                   
               
            
           
         
       
     
     A two-level Resolution IV fractional factorial design was employed to design a balanced n=16 set of Fc-multimers representing all of the potential 128 (2 7 ) variants between IgG1 and IgG4 CH2 sequences. The sixteen Fc-multimers were:
         IgG1 Fc IgM tp L309 (wild-type)   IgG1 Fc IgM tp L309 H268Q K274Q Y296F A330S   IgG1 Fc IgM tp L309 L234F K274Q Y296F   IgG1 Fc IgM tp L309 K274Q Y296F A327G   IgG1 Fc IgM tp L309 L234F H268Q Y296F   IgG1 Fc IgM tp L309 H268Q A327G A330S   IgG1 Fc IgM tp L309 L234F A327G P331S   IgG1 Fc IgM tp L309 Y296F A327G P331S   IgG1 Fc IgM tp L309 L234F Y296F A327G A330S   IgG1 Fc IgM tp L309 Y296F A330S P331S   IgG1 Fc IgM tp L309 L234F H268Q K274Q   IgG1 Fc IgM tp L309 H268Q K274Q P331S   IgG1 Fc IgM tp L309 L234F K274Q A330S   IgG1 Fc IgM tp L309 K274Q A327G A330S P331S   IgG1 Fc IgM tp L309 L234F H268Q A330S P331S   IgG4 Fc IgM tp L309 (wild-type)       

     The Fc-multimers were expressed and purified as hexamers, and their properties were investigated. A separate DoE analysis was run for each of the following properties:
         macrophage phagocytosis of antibody coated target cells.   Cytokine release   Platelet activation   B-Cell Binding       

     The results for each of the assays were analysed in a statistical model, MODDE version 9.1, Umetrics AB. The statistical model is described in detail in “Design of Experiments—Principles and Applications (Third Revised and Enlarged Edition)” L, Eriksson, E. Johansson, N. Kettaneh-Wold, C. Wikstrom, S. Wold; Umetrics Academy. 
     The factors that had a statistically significant effect (p&lt;0.05, either as a main effect or included in an interaction) are noted in Table 19. Those with brackets indicated only a marginal effect on the data. All the other factors showed no statistically significant effects, so were dropped from the model. 
     
       
         
           
               
               
               
               
               
               
             
               
                   
                 TABLE 19 
               
               
                   
                   
               
               
                   
                   
                 Cytokine 
                   
                 B-Cell 
                   
               
               
                   
                 Factor 
                 Release 
                 Platelets 
                 Binding 
                 Phagocytosis 
               
               
                   
                   
               
             
            
               
                   
                 L234F 
                 ✓ 
                 ✓ 
                   
                 ✓ 
               
               
                   
                 H268Q 
                   
                   
                 (✓) 
                 ✓ 
               
               
                   
                 K274Q 
                 (✓) 
                 ✓ 
               
               
                   
                 Y296F 
                 ✓ 
                   
                   
                 ✓ 
               
               
                   
                 A327G 
                 ✓ 
                   
                 (✓) 
                 ✓ 
               
               
                   
                 A330S 
               
               
                   
                 P331S 
                   
                 (✓) 
                 ✓ 
               
               
                   
                   
               
            
           
         
       
     
     (a) Effects of Mutations on Macrophage Phagocytosis 
     The ability of the Fc-multimers to alter macrophage phagocytosis of antibody-coated target cells was measured as described in Example 6. The results were then analysed in the statistical model. 
     The Effects plot showed that position #5 (A327G) had the biggest effect on altering macrophage phagocytosis, with #1 (L234F), #2 (H268Q) and #4 (Y296F) also showing a significant effect. ( FIG. 16( a ) ). 
     It also showed that there were no significant interactions: i.e. that for none of the factors did their effect depend on the levels of other factors. 
     Therefore, for high inhibition of phagocytosis, set #5, #1, #2 and #4 to the amino acid residue found in IgG1. 
     (b) Effects of Mutations on Cytokine Release 
     The ability of the Fc-multimers to stimulate cytokine release, including IFNγ and TNFα, was measured as described in Example 8. The results were then analysed in the statistical model. 
     All the compounded mutants which contained either L234F or A327G had much reduced or very low levels of cytokine vs IgG1 wild-type. The Y296F mutation was also found to be able to reduce cytokine levels in combination with other changes (in the absence of L234F or A327G). Statistical analysis confirmed that only three mutations significantly reduced cytokine release: L234F&gt;A327G&gt;Y296F. ( FIG. 16( b ) ). 
     (c) Effects of Mutations on Platelet Activation 
     The effect of the Fc-multimers on activation of human platelets was determined by measuring expression of CD62p as described in Example 14. 
     Statistical analysis showed that only two positions: L234F and K274Q have a significant influence on reducing the platelet activation risk of IgG1 Fc-multimers. P331S and H268Q have smaller and non-significant influence on reduction of platelet activation. 
     Mutants containing L234F (which also reduced cytokine release) have particular value for inclusion in Fc-multimers and other aggregated Fc constructs. 
     K274Q mutants did not show a strong influence in reducing cytokine release. 
     Mutant Key: 
       
     
       
         
           
               
               
               
             
               
                   
               
               
                 Position 
                 IgG1 
                 IgG4 
               
               
                   
               
             
            
               
                 #1 (234) 
                 L 
                 F 
               
               
                 #2 (268) 
                 H 
                 Q 
               
               
                 #3 (274) 
                 K 
                 Q 
               
               
                 #4 (296) 
                 Y 
                 F 
               
               
                 #5 (327) 
                 A 
                 G 
               
               
                 #6 (330) 
                 A 
                 S 
               
               
                 #7 (331) 
                 P 
                 S 
               
               
                   
               
            
           
         
       
     
     Example 17: Use of the Improved Fc-Domain Constructs in Other Polymeric Fc-Proteins 
     Studies were carried out to examine the effects of the improved Fc-domain constructs in other polymeric Fc-proteins. 
     (1) Stradomer 
     Diagrams showing example amino acid and DNA sequences for stradomer formats with and without the improved Fc-domain constructs are provided in  FIG. 17 . 
     DNA sequences were assembled and expressed in CHO cells as described in Example 1. The expressed proteins were purified and analysed as described in Example 3. Size exclusion chromatography traces for the purified stradomer proteins are shown in  FIG. 18 . 
     The effect of the improved Fc-domain constructs on the stimulation of cytokine release by stradomers was measured as described in Example 8. Cytokines studied included IFNγ, TNFα, IL-1β, and IL-6. 
     Results are shown in  FIGS. 19 ( a )-( d ) . The data demonstrated that stradomers without the improved Fc-domains stimulated very high levels of cytokine release. In marked contrast, stradomers with improved Fc-domains, comprising the L234F or A327G mutations, produced much lower levels of cytokine release. 
     (2) Tandem Fc 
     Diagrams showing example amino acid and DNA sequences for tandem Fc formats with and without the improved Fc-domain constructs are provided in  FIG. 17 . To construct tandem Fc L234F, the leucine residues at positions 9 and 236 were both substituted with phenylalanine, the equivalent positions to position 234 in the naturally occurring IgG1 Fc domain. ( FIG. 17 , SEQ ID NO:80). To construct tandem Fc A327G, the alanine residues at positions 102 and 329 were both substituted with glycine, the equivalent positions to position 327 in the naturally occurring IgG1 Fc domain. ( FIG. 17 , SEQ ID NO:82). 
     DNA sequences were assembled and expressed in CHO cells as described in Example 1. The expressed proteins were purified and analysed as described in Example 3. Size exclusion chromatography traces for the purified tandem Fc proteins are shown in  FIG. 18 . 
     (3) Fc RGY 
     Diagrams showing example amino acid and DNA sequences for Fc RGY formats with and without the improved Fc-domain constructs are provided in  FIG. 17 . 
     DNA sequences were assembled and expressed in CHO cells as described in Example 1. The expressed proteins were purified and analysed as described in Example 3. Size exclusion chromatography traces for the purified Fc RGY proteins are shown in  FIG. 18 . 
     (4) L309C Ladder 
     Diagrams showing example amino acid and DNA sequences for L309C ladder formats with and without the improved Fc-domain constructs are provided in  FIG. 17 . 
     DNA sequences were assembled and expressed in CHO cells as described in Example 1. The expressed proteins were purified and analysed as described in Example 3. Size exclusion chromatography traces for the purified L309C ladder proteins are shown in  FIG. 18 . 
     (5) Selective Immunomodulator of Fc-Receptor (SIF) 
     Diagrams showing example amino acid and DNA sequences for Selective Immunomodulator of Fc-receptor (SIF) formats, with and without the improved Fc-domain constructs are provided in  FIG. 17 . 
     DNA sequences were assembled and expressed in CHO cells as described in Example 1. DNA was transfected into the CHO cells by electroporation at a ratio of 2:1 (Plasmid construct A:Plasmid construct B). 
     The expressed proteins were purified and analysed as described in Example 3. Size exclusion chromatography traces for the purified SIF proteins are shown in  FIG. 18 . 
     The effect of the improved Fc-domain constructs on the stimulation of cytokine release by SIF proteins was measured in a whole blood cytokine release assay, as described in Example 8. Analysis was performed using a Meso Scale Discovery IFN-γ V-flex kit according to the manufacturer&#39;s protocol and read on a Sector Imager 6000. 
     Results are shown in  FIG. 19 ( e ) . The data shown are mean+/−SEM of n=3 donors. The data demonstrated that SIF proteins without the improved Fc-domains stimulated very high levels of IFN-γ production in the whole blood cytokine release assay. In marked contrast, SIF proteins with improved Fc-domains, comprising the L234F or A327G mutations, resulted in greatly reduced IFN-γ production. 
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 summary of polymeric Fc-proteins constructed 
               
            
           
           
               
               
            
               
                 Fc-protein 
                 structure of monomer unit 
               
               
                   
               
               
                 IgG1 Stradomer 
                 hγ1hinge-hγ1Fc-hγ2hinge 
               
               
                 IgG1 Stradomer L234F 
                 hγ1hinge-hγ1Fc-hγ2hinge L234F 
               
               
                 IgG1 Stradomer A327G 
                 hγ1hinge-hγ1Fc-hγ2hinge A327G 
               
               
                 IgG1 Fc RGY 
                 hγ1hinge-hγ1Fc E345R E430G S440Y 
               
               
                 IgG1 Fc RGY L234F 
                 hγ1hinge-hγ1Fc L234F E345R E430G S440Y 
               
               
                 IgG1 Fc RGY A327G 
                 hγ1hinge-hγ1Fc A327G E345R E430G S440Y 
               
               
                 IgG1 Fc L309C ladder 
                 hγ1hinge-hγ1Fc L309C 
               
               
                 IgG1 Fc L309C ladder L234F 
                 hγ1hinge-hγ1Fc L234F L309C 
               
               
                 IgG1 Fc L309C ladder A327G 
                 hγ1hinge-hγ1Fc A327G L309C 
               
               
                 IgG1 Tandem Fc 
                 (hγ1hinge-hγ1Fc)-(GGGGS)-(hγ1hinge-hγ1Fc) 
               
               
                 IgG1 Tandem Fc L234F 
                 (hγ1hinge-hγ1Fc)-(GGGGS)-(hγ1hinge-hγ1Fc) 
               
               
                   
                 with L234F mutation in each Fc domain 
               
               
                 IgG1 Tandem Fc A327G 
                 (hγ1hinge-hγ1Fc)-(GGGGS)-(hγ1hinge-hγ1Fc) 
               
               
                   
                 with A327G mutation in each Fc domain 
               
               
                 SIF 
                 1 st  polypeptide: (hγ1hinge-hγ1Fc A ) with Y349C, 
               
               
                   
                 T366S, L368A, K370E, Y405V 
               
               
                   
                 2 nd  polypeptide: (hγ1hinge-hγ1Fc A )-(5xSGGG 
               
               
                   
                 linker)-(hγ1hinge-hγ1Fc B ) with S354C, E357K, 
               
               
                   
                 T366W in hγ1Fc A  and D399K, K409D in hγ1Fc B   
               
               
                   
                 Fc A  regions of polypeptides dimerise due to interface 
               
               
                   
                 engineering and Fc B  regions of polypeptides dimerise 
               
               
                   
                 due to alternative interface engineering. 
               
               
                 SIF L234F 
                 1 st  polypeptide: (hγ1hinge-hγ1Fc A ) L234F Y349C, 
               
               
                   
                 T366S, L368A, K370E, Y405V 
               
               
                   
                 2 nd  polypeptide: (hγ1hinge-hγ1Fc A )-(5xSGGG 
               
               
                   
                 linker)-(hγ1hinge-hγ1Fc B ) with L234F in each Fc 
               
               
                   
                 domain and S354C, E357K, T366W in hγ1Fc A  and 
               
               
                   
                 D399K, K409D in hγ1Fc B   
               
               
                   
                 Fc A  polypeptides dimerise due to interface engineering 
               
               
                   
                 and Fc B  polypeptides dimerise due to alternative 
               
               
                   
                 interface engineering. 
               
               
                 SIF A327G 
                 (hγ1hinge-hγ1Fc A ) A327G Y349C, T366S, L368A, 
               
               
                   
                 K370E, Y405V 
               
               
                   
                 (hγ1hinge-hγ1Fc A )-(5xSGGG linker)-(hγ1hinge- 
               
               
                   
                 hγ1Fc B ) with A327G in each Fc domain and 
               
               
                   
                 S354C, E357K, T366W in hγ1Fc A  and D399K, 
               
               
                   
                 K409D in hγ1Fc B   
               
               
                   
                 Fc A  polypeptides dimerise due to interface engineering 
               
               
                   
                 and Fc B  polypeptides dimerise due to alternative 
               
               
                   
                 interface engineering.