USE OF AN EFFECTIVE AMOUNT OF A POLYMERIC PROTEIN UNDERSTANDING SIX POLYMEPTIDE DEMONOMER UNITS

专利摘要:
IMMUNOMODULATORY PROTEINS. The present invention relates to a method for treating a mammalian subject for an autoimmune or inflammatory disease, the method comprising: administering to a mammalian subject an effective amount of a polymeric protein comprising five, six or seven units of polypeptide monomer ; wherein each polypeptide monomer unit comprises an Fc receptor binding portion comprising two immunoglobulin G heavy chain constant regions; wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue that is linked via a disulfide bond to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomer unit; wherein the polymeric protein does not comprise an additional immunomodulatory portion; or a portion of the antigen that causes antigen-specific immunosuppression when administered to the mammalian subject
公开号:BR112015008663B1
申请号:R112015008663-2
申请日:2012-10-17
公开日:2021-01-12
发明作者:Richard John Pleass
申请人:CSL Behring Lengnau AG；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[0001] The invention relates to proteins designed to have an immunomodulatory function, and their medical uses for the treatment of autoimmune or inflammatory diseases. In particular, the designed proteins can be used as a substitute for intravenous immunoglobulin (IVIG). BACKGROUND OF THE INVENTION
[0002] Autoimmune and inflammatory diseases are responsible for substantial morbidity and mortality. In 2003, autoimmune diseases were the sixth most frequent cause of death in all age groups below 75 years. An important treatment modality is intravenous immunoglobulin (IVIG) or IgG. Human plasma immunoglobulin products were initially developed for the treatment of immune deficiencies. However, seventy percent of the prescribed IVIG is now used to treat autoimmune or inflammatory conditions. Worldwide consumption of IVIG increased from 300 kilograms per year in 1980 to 100 tons per year in 2010. IVIG is derived from plasma collected from ~ 3,000 anonymous donors, according to a time-consuming and expensive manufacturing process. The need for extensive donor screening and donated plasma for viruses contributes to the high cost. Given the growing demand, and the strict regulation of IVIG production, a shortage of IVIG may occur. IVIG preparations may be subject to inadequate sterility, presence of impurities and variation from batch to batch. They can vary greatly in their immunoglobulin A content, and IgA can cause allergic or anaphylactic reactions in IgA-deficient receptors, making them unsuitable for some patients. Very high doses of IVIG have to be given to patients, typically 2 g per kg of body weight, and this can cause adverse reactions in some patients. In view of these restrictions, defined substitutions for IVIG are required.
[0003] Although the mechanisms of action for IVIG remain unclear, derivatives of Fc-fragments of IVIG can cure children suffering from idiopathic purple thrombotic (ITP) (Debre M et al, 1993). The interactions between the Fc portion of IgG with both FcyRs inhibitors and activators found in monocytes and macrophages are considered to be important, although the exact receptors involved are much debated (Samuelsson A. et al, 2001; Bazin R. et al, 2006 ; Corvo. AR er ai, 2003; Leontyev D. et al, 2012 ;. Siragam V. et al, 2006) and may vary for the disease for which IVIG is used (Araújo LM et al, 1 201; Anthony RM et al, 201 1). The Fc inhibitor FcyRllb receptor has been shown to be necessary for the protective effect of IVIG in a mouse model of ITP (Samuelsson et al, 2001). A role of activation of the Fc FcyRllla receptor has also been demonstrated in several diseases (reviewed by Mekhaiel et al, 2011 b). The FcRn responsible for maintaining the long plasma Fc half-life has also been postulated to be involved (Roopenian & Akilesh, 2007), although recent work has shown that it is not involved in improving ITP in the mouse model (Corvo et al, 2011 ). Fc portions not only interact with Fc receptors, but also certain lectins. It is known to bind CD22 IVIG lectin (Siglec-2) in B lymphocytes via a terminal sialic acid in Fc-glycan (Seite JF et al, 2010), and a recent study has shown that sialylated Fes are responsible for anti-inflammatory effects of IVIG in a mouse arthritis model as a result of its interaction with human lectin receptor, DC-SIGN, on DCs (Anthony RM et al, 201 1).
[0004] IVIG can also work through a multi-stage model, where the first IVIg forms injected into a type of immuno-complex in the patient (Clynes et al, 2005; Siragam et al, 2005, 2006; Machino et al, 2012). Once these immune complexes are formed, they interact with these receptors and can mediate anti-inflammatory effects helping to reduce the severity of the autoimmune disease or the inflammatory state (Siragam et al, 2006). Fc multimers are formed in IVIG by anti-idiotype interactions (Machino et al, 2010; Machino et al, 2012; Roux & Tankersley, 1990; Teeling et al, 2001) or by covalent Fc interactions (Yoo et al, 2003 ). IgG multimers are postulated to show greater binding avidity for the above receptors and by the nature of the cross-linking of said receptors to induce protective signals that are not induced by Fc monomers. This is supported by the reversion of ITP in mice by immunocomplexes (ICs) (Bazin et al, 2006), and by the observation that CIs improves the tolerogenicity of immature dendritic cells through FcyRllb to promote attenuation of lupus (Zhang et al, 2011 ). The multimeric proportion of IgG and / or sialylated to commercially available IVIG IgG is very low (<1% and 5%, respectively), which may contribute to the need to administer large quantities (Nimmerjahn & Ravetch, 2007).
[0005] Other proposed mechanisms of action of IVIG are restoration of the idiotypic anti-idiotypic network; suppression or neutralization of cytokines by means of specific antibodies in the IVIG; blocking the binding of adhesion molecules in leukocytes to the vascular endothelium; inhibition of complement absorption in target tissues; neutralization of microbial toxins; blockade of Fas-mediated apoptosis binding by anti-Fas antibodies in IGIV; induction of apoptosis with anti-Fas antibodies at high concentrations of IVIG; apoptosis of neutrophils by anti-Siglec-9 antibodies in IGIV; saturation of FcRn receptors to improve the elimination of autoantibodies; induction of FcyRllb inhibitor receptors in effector macrophages; neutralization of growth factors for B cells, such as the activating B-factor cell; inhibition of proliferative T cell responses; expansion, activation, or both of a population of regulatory T cells; inhibition of differentiation and maturation of dendritic cells; improvement of differentiation and maturation of “activated” dendritic cells (reviewed in Ballow, 1 201; Mekhaiel et al, 2011 b).
[0006] Recombinant proteins for use in the treatment of autoimmune diseases and / or as IVIG replacement compounds have been proposed. US 2011/0081345 (Moore) describes Fc (SCFC) single-stranded proteins, having one Fc unit per molecule, composed of two linked Fc domain amino acid chains, which may be useful for the treatment of autoimmune disease. US 2004/0062763 (Temple University; Mosser) describes the use of multivalent abs or portions thereof to bind FcyRI to upregulate IL-10 production, for the treatment of autoimmune disorders. The agent can be two or more Fc fragments coupled together or delivered in a single recombinant peptide. US 2008/0206246 (The Rockefeller University; Ravetch) discloses a polypeptide containing at least one IgG Fc region, which is sialylated, and its use as an IVIG replacement compound. Although the compounds described in these documents may target Fc receptors, compounds containing monomeric or dimeric Fc cannot bind to Fc receptors with sufficient avidity to be effective or fully effective as IVIG replacement compounds. They would therefore not be suitable for biomimicry of the IVIG multimeric fraction. Higher order multimers are not revealed in these documents. Nor is it any engineering means of higher order multimers that operate as IVIG replacement compounds.
[0007] US 2010/0239633 (University of Maryland, Baltimore; Strome) discloses IVIG replacement compounds that comprise multiples attached to Fc moieties. Star-shaped arrangements are planned using the 0μ4 IgM domain and the J chain to carry out the polymerization. However, the working examples have not been described, and the computer simulations reported here suggest that the exemplary molecules do not polymerize effectively and / or that the Fc moieties would not be provided for the effective binding of Fc or other receptors. In addition, complex biological agents containing more than one different polypeptide chain can be more difficult to manufacture uniformity, because not all polypeptide subunits can interact in a stable and predictable manner. For example, cell lines that express heavy and light chain antibodies that produce active intact antibody containing the correct proportion of light and heavy chains can also therapeutically produce inactive heavy chain dimers, without light chain fixation, or halved molecules.
[0008] Linear structures were also proposed in US 2010/0239633, in which the individual chains of amino acids dimerize by pairing between identical chains of amino acids from the Fc domain, generating Fc regions. For example, the Hinge regions can form inter-chain disulfide bonds between the two individual amino acid chains. However, where several chains of Fe amino acids are linked in series, there may be several different ways in which these chains can be paired, as illustrated in Figures 11 and 12 of US 2010/0239633. Thus, a uniform product with reliable and predictable properties would not be expected. It is proposed to include a terminal IgE heavy chain domain to avoid this "zigzag" effect. However, IgE receptor binding molecules can have undesirable consequences, including the risk of anaphylactic shock or other allergic responses.
[0009] US 2010/0239633 also suggests producing cluster molecules in which multimerization regions, such as lgG2a hinge or isoleucine zippers, cause amino acid chains to dimerize or multimerize, thus bringing together Fc pairs of amino acid chains amino domain to form functional Fc units. In a follow-up study, Jain et al (2012) described the production of recombinant proteins in which the human IgG2 Hinge sequence or the isoleucine zipper were fused with murine IgG2a. The authors report that any region of multimerization caused the formation of homodimers (that is, individual functional Fc units referred to herein as monomers) and multimers. Ultimeric fractions of these proteins had some efficacy in the treatment of a mouse model of ITP or in the model of collagen-induced rat arthritis. However, most of the protein produced that contains the IgG2 hinge was monomeric, or existed as dimers or trimers. Higher-order oligomers were responsible for only a small proportion, with unknown quaternary structure. In proteins containing the isoleucine zipper, the proportion of higher order multimers was lower. Higher-order multimers appeared heterogeneous in size, and their structures, including the nature of the N297-linked glycans, are unknown. Isolating some or all of these multimers for therapeutic use, considerable waste would be necessary given their low proportion in the protein produced, and it seems unlikely to be commercially viable.
[0010] There remains a need for compounds of defined structure that effectively as adequate target mechanisms underlying the biological activity of IVIG for the treatment of autoimmune and inflammatory diseases.
[0011] The listing or discussion of a state of the art document published in this specification should not be taken as an acknowledgment that the document is part of the state of the art or of general common knowledge. SUMMARY OF THE INVENTION
[0012] A first aspect of the invention provides a method for treating a mammalian subject for an autoimmune or inflammatory disease, the method comprising:
[0013] administering to the mammal an effective amount of a polymeric protein comprising five, six or seven monomeric units of polypeptide;
[0014] wherein each polypeptide monomer unit comprises an Fc receptor binding portion comprising two immunoglobulin G heavy chain constant regions;
[0015] wherein each immunoglobulin L heavy region constant region comprises a cysteine residue that is linked via a disulfide bond to a cysteine residue of an immunoglobulin L heavy chain constant region of a polypeptide monomer unit adjacent;
[0016] wherein the polymeric protein does not comprise an additional immunomodulatory portion; or a portion of antigen that causes specific immunosuppression for the antigen, when administered to the mammalian subject.
[0017] A second aspect of the invention provides a method for treating a mammalian subject for an autoimmune or inflammatory disease, the method comprising:
[0018] administering to the mammal an effective amount of a polymeric protein consisting of five, six or seven units of polypeptide monomer;
[0019] wherein each polypeptide monomeric unit consists of an Fc receptor-binding portion consisting of two constant regions of immunoglobulin G heavy chains; and, optionally, a polypeptide linker that links the two constant regions of the immunoglobulin G heavy chain as a single Fc chain; and
[0020] wherein each immunoglobulin L heavy chain constant region comprises a cysteine residue that is linked via a disulfide bond to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomer unit .
[0021] A third aspect of the invention provides a method for treating a mammalian subject for an autoimmune or inflammatory disease, the method comprising:
[0022] administering to the mammal an effective amount of a polymeric protein consisting of five, six or seven units of polypeptide monomer;
[0023] wherein each polypeptide monomeric unit consists of a Fc receptor binding portion and a cap region;
[0024] wherein the Fc receptor binding portion consists of two immunoglobulin G heavy chain constant regions; and, optionally, a polypeptide linker that links the two constant regions of the immunoglobulin G heavy chain as a single Fc chain;
[0025] wherein each modified human immunoglobulin L heavy chain constant region chain comprises a cysteine residue that is linked via a disulfide bond to a human immunoglobulin cysteine residue of modified unit L heavy chain constant regions of adjacent polypeptide monomer; and
[0026] where the capping region is fused to each of the two L modified heavy immunoglobulin constant regions of the polypeptide monomer unit heavy chain, and facilitates the assembly of the monomer units in a polymer.
[0027] A fourth aspect of the invention provides a polymeric protein comprising five, six or seven units of polypeptide monomer;
[0028] wherein each polypeptide monomer unit comprises an Fc receptor binding portion comprising two immunoglobulin G heavy chains constant regions;
[0029] wherein each immunoglobulin constant L heavy chain comprises a cysteine residue that is linked via a disulfide bond to an immunoglobulin constant L heavy chain cysteine residue of an adjacent polypeptide monomer unit;
[0030] wherein the polymeric protein does not comprise an additional immunomodulatory moiety; or a portion of antigen that causes specific immunosuppression for the antigen, when administered to a mammalian subject;
[0031] wherein each unit of polypeptide monomer does not include a cap region fused to each of the two immunoglobulin G heavy chains constant regions.
[0032] A fifth aspect of the invention provides a polymeric protein consisting of five, six or seven units of polypeptide monomer;
[0033] wherein each polypeptide monomeric unit consists of an Fc receptor binding portion consisting of two constant regions of immunoglobulin G heavy chains; and, optionally, a polypeptide linker that links the two constant regions of the immunoglobulin G heavy chain as a single Fc chain; and
[0034] wherein each immunoglobulin constant region L heavy chain comprises a cysteine residue that is linked via a disulfide bond to a cysteine residue of an immunoglobulin G heavy chain of the constant region of an adjacent polypeptide monomer unit .
[0035] A sixth aspect of the invention provides a nucleic acid molecule comprising a coding portion that encodes a polypeptide monomer unit of a polymeric protein, as defined according to the fourth or fifth aspect of the invention.
[0036] Other aspects of the invention are an expression vector comprising the nucleic acid molecule of the sixth aspect of the invention; a host cell comprising the expression vector; and a therapeutic composition comprising the polymeric protein of the fourth or fifth aspect of the invention.
[0037] Medical uses corresponding to the treatment methods of the first to third aspects of the invention are also considered. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 shows hexamerica-Fc and structural characterization. A Fc hexameric model showing Cys309 and Cys360 disulfide bonds (cap) formed between a monomer and its adjacent monomers (left panel). Beating mode atomic force microscopy images (panels on the right) reveal bar-shaped, six times symmetric complexes consistent with hexamers. In smaller check sizes (bottom of the two panels on the right), these complexes are shown to be ~ 20 nm in diameter (scale bar on the bottom panel is 50 nm). B Hexamerica-Fc produced from mammalian cell lines can be detected as molecules of approximately 312 kDa by size exclusion chromatography (trace highlighted as hexamer). A small proportion of dimers is also detected.
[0039] Figure 2 shows the binding of hexamoxylin-Fc to human and mouse FCY-receptors (FCYRS). Titrated amounts (50-0.3 nM of Fc proteins were coated on ELISA wells. Coating by human glutathione-S-transferase or mouse (GST) -fused FcyRs as indicated were visualized using horseradish peroxidase (HRP) conjugated to anti- GST Values represent triplicate determinations Error bars are standard errors (EP) around the mean Fused antigen-Fc proteins weakly bound to Fc receptors, as described in Mekhaiel et al, 2011a.
[0040] Figure 3 shows the hexameric-Fc binding to human CD19 + from human B lymphocytes. Characteristic flow cytometry plot, showing the different populations of human leukocytes represented by their front and lateral dispersion profiles (left panel). B CD19 + individual lymphocytes stained with human anti-CD19-FITC (boxed, middle panel) were investigated closed and for hexameric-Fc binding (right panel). The attachment of 50 g of hexa-Fc to CD19 + B cells is indicated by the dash farthest to the right (with hexa-Fc arrows). Before incubation of cells with monoclonal antibody specific for FcRL5 509F6 (with hexa-Fc + FcRL5 arrows blocking mAb 509F6) ablated Fc-hexameric binding indicating that FcRL5 was responsible for some of the hexameric-Fc binding to B cells.
[0041] Fig 4 shows complementary analysis of binding to Fc proteins. C1q (left panel) and the deposition of C5-9 (right panel) of Fc-fusions determined by ELISA. Each point represents the average optical density (+/- SD) of duplicate wells for each mouse within a given group. Data from one of three replicate experiments is shown.
[0042] Figure 5 shows the hexameric protein Fc protects against platelet loss in an ITP mouse model. Balb / c mice were injected ip with Hexa-Fc, IVIG (Gammagard), or PBS. One hour later, ITP was induced in all rats. Platelets were moistened and indicated by the post-treatment time points. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
[0043] According to a first aspect of the invention, a method for treating a mammalian subject for an autoimmune or inflammatory disease is provided. The individual is treated by administering a polymeric protein comprising five, six or seven units of polypeptide monomer; wherein each polypeptide monomer unit comprises an Fc receptor binding portion comprising two immunoglobulin G heavy chain constant regions.
[0044] The term "immunoglobulin G heavy chain constant regions" means a native immunoglobulin G heavy chain region, or variant or fragment. The Fc receptor binding portion typically comprises the Fe portion of an immunoglobulin G, or fragment or variant thereof. The term "Fc pore" includes a fragment of an IgG molecule that is obtained by limited proteolysis with the enzyme papain, which acts on the IgG Hinge region. An Fc portion obtained in this manner contains two peptides with identical disulfide bonds containing the IgG heavy chain CH2 and CH3 domains, also referred to as Cy2 and Cy3 domains, respectively. The two peptides are linked by two disulfide bonds between cysteine residues in the N-terminal parts of the peptides. The arrangement of the disulfide bonds for IgG described refer to natural human antibodies. There may be some variation between antibodies to other species of mammals, although such antibodies may be suitable in the context of the present invention. Antibodies are also found in birds, reptiles and amphibians, and may also be suitable. The nucleotide and amino acid sequences of human IgG Fc are disclosed, for example, in Ellison et al. (1982) Nucleic Acids Res. 10: 4071-4079. The nucleotide and amino acid sequences of murine IgG2a Fc are disclosed, for example, in Bourgois et al. (1974) of euros. J. Biochem. 43: 423-435. Immunoglobulin G from heavy chain constant regions can typically be produced by recombinant expression techniques, and associated as monomer units by disulfide bonds, as occurs in native antibodies. Alternatively, the two constant regions can be produced as a single chain of amino acids with an intervening linking region, i.e., as a single Fc chain (SCFC) typically also by recombinant expression techniques. SCFC molecules are described in US 2011/0081345, including examples that have the following general structure from N to the C-terminus: Hinge-CH2-CH3-ligand-Hinge-CH2-CH3.
[0045] Typically, each of the immunoglobulin G heavy chain constant regions comprises an amino acid sequence of a mammal heavy chain constant region, preferably a human heavy chain constant region; or a variant thereof. A suitable human IgG subtype is IgGl
The Fc receptor binding portion may comprise more than the Fe portion of an immunoglobulin. For example, it can include the immunoglobulin Hinge region, which occurs between CH1 and CH2 of a native immunoglobulin. For certain immunoglobulins, the Hinge region is necessary for binding to Fc receptors. Preferably, the Fc receptor binding portion lacks a CH1 domain and heavy chain variable region (VH) domain. The Fc receptor-binding portion can be truncated at the C- and / or N-terminal compared to the Fc portion of the corresponding immunoglobulin. This binding portion is an Fc receptor, thus a "fragment" of the Fc portion.
[0047] The polymeric protein is formed by virtue of each immunoglobulin heavy chain G comprising the constant region of a cysteine residue that is linked via a disulfide bond to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomer unit. Since IgM and IgA are naturally polymeric, whereas IgG is naturally monomeric, the ability of IgG-based monomer units from heavy chain constant regions to form polymers can be improved by modifying the IgG parts of heavy chain constant regions to be more like the corresponding parts of IgM or IgA. Suitably, each of the immunoglobulin heavy chain constant regions or variants thereof is an IgG heavy chain constant region comprising an amino acid sequence comprising a cysteine residue at position 309, according to the UE, and preferably also a leucine residue at position 310. The EU numeraling system for IgG is described in Kabat EA et al, 1983, Protein sequences of immunological interest. Department of Health and Human Services, National Institutes of Health, Washington DC. Sorensen et al (1996) J Immunol 156: 2858-2865 describes the Leu 309 to Cys 309 mutation of a human IgG3 molecule, comprising an IgM terminal piece to promote polymer formation. Leu 309 corresponds, by sequence homology to Cys 414 in the C3 IgM domain of Cys 309 and in the Ca2 IgA domain. (Note that the amino acid residue humeration differs between IgM and IgA or IgG classes). Other mutations can also be advantageous.
[0048] Suitably, each polypeptide monomer unit comprises a cap region fused to each of the two regions contained in the immunoglobulin G heavy chain; where the cap region of each polypeptide monomer unit facilitates the assembly of the monomer units in a polymer. Typically, the cap region is fused at the C-terminus for each of the two immunoglobulin constant heavy chain regions. Suitably, the ending region is an IgM or IgA ending, or fragment or variant thereof. These caps are the final portions of the IgM C 4 domain or the IgA Ca3 domain, respectively.
[0049] Whenever a region is described as being fused at the C-terminus of another region, the first region can be fused directly with the C-terminus of the last region, or it can be fused with an intermediate amino acid sequence that is fused in itself with the C-terminal of the latter region. N-terminal fusion can be understood in an analogous way.
[0050] A sequence of intervening amino acids can be provided between the constant region of the heavy chain and the cap, or the cap can be fused directly with the C terminal of the constant region of the heavy chain. For example, a short linker sequence can be provided between the immunoglobulin heavy chain cap region and the constant region. Typical linker sequences are between 1 and 20 amino acids in length, typically 2, 3, 4, 5, 6 or even 8, 10, 12, or 16 amino acids in length. A suitable linker to include between the heavy chain region and cap region encodes for Leu-Val-Leu-Gly.
[0051] A preferred cap region is the human IgM cap region, which is PTL Y VS LVM S DTAGTC Y (Rabbitts TH et al, 1981. Nucleic Acids Res 9 (18), 4509-4524 ;. Smith et al (1995) J. Immunol 154: 2226-2236). Suitably, this end piece can be modified at the N-terminal by substituting for the initial Pro Thr, thus generating the sequence of PPL YN VS LVM S DTAGTC Y. This does not affect the ability of the cap to promote polymerization of the monomer. Other suitable variants of the human IgM cap are described in Sorensen et al (1996) J Immunol 156: 2858-2865. Another IgM capping sequence is rodent GKFTLYNVSLIMSDTGGTCY (Abbas and Lichtman, cellular and Molecular Immunology, Elsevier Saunders, 5 th Edn, 2005). A preferred alternative tailing region is the human IgA tailing region, which is PTHVNVSWMAQVDGTCY (Putnam, FW et al, 1979, J. Biol Chem 254: 2865-2874)] Other suitable tailings from IgM or IgA from other species , or even synthetic sequences that facilitate the assembly of the monomer units in a polymer, can be used. It is not necessary to use an immunoglobulin terminal piece from the same species from which the heavy chains of immunoglobulin constant regions are derived, although it is preferred to do so.
[0052] "Variants" and "fragments" are as defined in relation to the regions contained in the heavy chain. A variant of an IgM tailpiece typically has an amino acid sequence that is identical to PPLYNVSLVMSDTAGTCY at 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the amino acid positions 18. A variant of an end piece of IgA typically has an amino acid sequence that is identical to PTHVNVSWMAQVDGTCY at 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the amino acid positions 18. Fragments of these IgM or IgA caps typically comprise 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids. Fragments of variants are also provided for. Typically, fragments and variants of the IgM or IgA cap retain the penultimate cysteine residue, as it is believed to form a disulfide bond between the two monomer units in a polymeric protein.
[0053] The ability of a given cap region to facilitate the assembly of the monomer units in a polymer can be tested by comparing the proportion of protein having a high molecular weight, when formed from monomer units a terminal piece with units is missing of monomer comprising an end piece. The latter can form a greater proportion of large molecule polymers under native conditions. Native molecular weights can be determined by size exclusion chromatography, for example, on Sephadex-200 columns on an AKTA FPLC (Amersham). Alternatively, non-reducing gel electrophoresis can be used, as described in Smith et al (supra) or Sorensen et al (supra).
[0054] When the monomeric units have mounted on a polymer, the receptor of Fc binding portions are arranged in a flat polymeric structure that is spatially oriented to allow each portion of binding to the Fc receptor to bind to an Fc receptor. IgM is naturally pentameric or hexametic and IgA naturally forms dimers, trimers or tetramers. These properties seem to be determined, at least in part, by the ability to cause the cap to associate the monomers to polymers in. Pentameric IgM is formed when the IgM associated with the J chain, although it is typically hexameric in the absence of the J chain. The J chain may or may not be included as an additional component of the polymeric fusion protein of the invention. Production is simplified by omitting the J chain polypeptide, which in any case is not necessary for the polymerization of IgG (Ghumra et al, 2008). IgM or secretory IgA on mucous surfaces found contains secretory component (CS), a part of the polymeric Ig receptor used to translocate them from blood to secretions. CS may or may not be included as an additional component of the polymeric protein of the invention. Production is simplified by the omission of SC, which, in any case, is not necessary for polymerization.
[0055] The polymeric protein comprising five, six or seven units of polypeptide monomer is used according to the first aspect of the invention. The exemplary Fc protein comprising human IgG1 from heavy chain constant regions and a cap of human IgM described herein, forms hexamers and dimers. Variants based on other regions of the IgG heavy chain, including different caps or non-caps, can form polymers having different numbers of monomers. In particular, they can be pentamers or heptamers instead of hexamers. Where Fc proteins naturally associate in polymers with different numbers of monomer units, polymers having the required number of monomer units can be separated according to the molecular size, for example, by gel filtration. Mixtures, in which at least part of the protein is in the form of a pentamer, hexamer and / or heptamer can also be used. Pentamers, hexamers and suitably / or heptamers are used in the absence of proteins that have other numbers of monomer units.
[0056] In the polymer proteins to be used, according to the first aspect of the invention, the Fc receptor binding portion of each monomer unit is capable of binding to an Fc receptor. It will be appreciated that portions comprising the Fe portion of a particular immunoglobulin binding to the Fc receptor, will bind to different Fc receptors, depending on the specificity of binding of the particular immunoglobulin. Typically, the Fc receptor binding portion will have an affinity for a particular Fc receptor that is at least comparable with the affinity of a native monomeric immunoglobulin molecule. However, the lower affinities can be tolerated, as the polymeric protein comprises multiple binding portions of such Fc receptors, and will therefore bind to Fc receptors with greater avidity. Therefore, the Fc receptor binding portion will typically have an affinity that is at least a tenth, suitably at least a fifth, and more suitably at least a half of the affinity of the corresponding monomeric native immunoglobulin molecule that binds to the given receptor. Fc.
[0057] Affinity constants can be easily determined by Plasmon Resonance surface analysis (Biacore). The receiver of Fc binding portions can be passed over the flow cells from CM5 amine sensor chips coupled to the Fc receptors. Equimolar concentrations of the binding portion to the Fc receptor or intact monomeric antibody can be injected over each Fc receptor, association and dissociation observed in real time. The data from a BIAcore X 3000 or machines can be analyzed using BIAevaluation 3.0 software to determine accurate affinity constants.
[0058] There are three classes of human receptor (Gessner Fey et al (1998) Ann Hematol 76: 231-48; Raghavan and Bjorkman (1996) Ann Rev cell Dev Biol 12: 181-220). FcyRI (CD64) binds to monomeric IgG with high affinity. I FcyRI (CD32) and 11 FcyRI (CD16) are the low affinity receptors for Fc and can only interact with high affinity with antibodies that are presented to the immune system in the form of immune complexes (ICs). Although greater (> 350 kDa) multimeric IgG and circulating ICs are removed mostly in the preparation of IVIG, these are common in healthy individuals in which they can contribute as much as 10% to the total plasma Ab concentration, indicating a physiological role in immune maintenance homeostasis in these healthy individuals (Nezlin R (2009) Immunol Lett 122,141-4).
[0059] FcyRII and FcyRIII are closely related in the structure of their ligand-binding domains. In humans, three separate genes, FcyRIIA, FcyRIIB, and FcyRIIC, two of which give rise to alternative splicing variants, code for FcyRII. FcyRlla provides signs of activation of FcyRllb whereas it provides inhibitory signals. The functional basis for the divergent signaling signals comes from motifs located on the cytoplasmic tails of receptors. A tyrosine-based immunoreceptor inhibitor (ITIM) motif located in the cytoplasmic tail of FcyRllb is involved in signaling the negative receptor. The ITIM motif is a unique feature of the FcyRllb receptor since it is apparently not present in any other class of Fey receptor. In contrast, an activation motif based on tyrosine immunoreceptor activators or ITAM is located on the cytoplasmic tail of FcYRIIa. ITAM reasons transduce activation signals. They are also found in the FcRy chains, which are identical to the high affinity IgE receptor (FceRI) y chains. While FcYRIIa and FcyRI ID are widely expressed in myeloid cells and some subsets of T cells that are notably absent from N cells. There are two alleles for the FcYRIIa receptor in humans, referred to as His131 (H131) and Arg131 (R131). The FcYRIIa-Arg131 allele is associated with an increased susceptibility to infection by encapsulated bacteria, such as Haemophilus influenzae, Streptococcus pneumoniae and Neiserria meningitidis, which trigger IgG2 responses (Pleass RJ & Woof JM, 2001). The Fc receptor encoded by this allele cannot bind IgG2 and is therefore unable to cause clearance of IgG2-coated bacteria. Both variants bind to human IgG1, however.
[0060] Human FcyRI II is also present in several isoforms derived from two distinct genes (FCYRII IA and IB) FCYRII. FcYRIIIb is unique in its attachment to the cell membrane by means of a glycosylphosphatidyl anchor. FcYRIIIb expression is restricted to neutrophils while FcyRIIIa is expressed by macrophages, and NK cells. FcYRIIIa is also expressed by certain subsets of determined T cells and monocytes. FcyRI I la requires the presence of the FcRY chain or 0Q3Ç-O 3 n for expression on the cell surface and in signal transduction. The FcRY chain and 003Z — on8ín are dimeric and have ITAM motifs. FcYRIIIa forms a multimeric complex with these subunits and signaling is transduced through them. Thus, there is considerable heterogeneity and diversified FcyR receptor expression
[0061] The binding sites for FcyRI I and FcyRIII map to the hinge and proximal region of the Ig2 CH2 domain, the same region originally identified for FcyRI (Duncan et al (1988) Nature 332: 563-4; Morgan et al (1995) Immunol 86: 319-324; Lund et al (1991) J Immunol 147: 2657 2662).
[0062] Receptors FCY activatorio (FCYRS) trigger and / or inhibitory signaling pathways that set limits for cell activation and culminate in a balanced immune response (Nimmerjahn F & Ravetch JV (2008) Nat Immunol Rev. 8: 34-47). FcR activation and inhibitors are widely expressed throughout the hematopoietic system, but particularly in cells presenting professional antigens (APCs) (Ravetch Nimmerjahn F & JV (2008) supra). For example, in humans, FcyRI is constitutively expressed by blood myeloid dendritic cells (DC) and FCYRII has been detected in each DC subset examined to date, while the expression of FcyRI, FCYRIIB and FcyRI II dominate in murine DC (Ravetch JV (2003) in Fundamental Immunology (ed Paul WE.) 685-700 (Lippincott- Raven, Philidelphia); Bajtay Z et al (2006) Immunol Lett 104: 46-52) ... FCYRS also play a prominent role in the presentation of antigens and immuno-complex Mediated maturation of dendritic cells (DC), and in the regulation of B cell activation and survival of plasma cells (Ravetch JV (2003) supra; Bajtay Z et al, (2006) supra). In addition, by regulating DC activity, FcyRs control whether an immunogenic or tolerogenic response is initiated after the recognition of antigenic peptides displayed on the surface of DCs for cytotoxic T cells, helper T cells and regulatory T cells. FcyRs also cooperate with Toll-like receptors (TLRs) in controlling the levels of important regulatory cytokines, IL-12 and IL-10 (Polumuri SK, 2007, J. Immunol 179: 236-246). Thus, FcyRs are involved in the regulation of innate and adaptive immune responses, which makes them attractive to the targets of novel developmental immunotherapeutic approaches (Nimmerjahn F & Ravetch JV (2008) supra).
[0063] It is known that the FcyRIIB inhibitor controls the magnitude of the immune response, as DCs derived from FcyRI IB-knockout mice generate stronger and longer-lasting immune responses in vitro and in vivo (Bergtold A, Desai DD, et al (2005 ) Immunity 23: 503-514; Kalergis AM & Ravetch JV (2002) J. Exp Med 195: 1653-1659) Most importantly, IB-deficient FcyRI DCs or DC incubated with an mAb that blocks binding to immune complex FcyRIIB showed spontaneous maturation (Boruchov AM, et al (2005) J. Clin Invest 115: 2914-2923; Dhodapkar KM, et al (2005) Proc Natl Acad Sci USA 102: 2910-2915). This suggests that the FcyR inhibitor not only regulates the magnitude of cell activation, but also actively prevents spontaneous DC maturation under non-inflammatory steady-state conditions. Indeed, low levels of immune complexes can be seen in the serum of healthy donors, with an emphasis on the importance of regulatory mechanisms that prevent unwanted DC activation (Dhodapkar KM, et al (2005) Proc Natl Acad Sci USA 102: 2910-2915 ). The loss of FcyRIIB also results in the initiation of more antigen-specific T cells (Kalergis AM & Ravetch JV (2002) J. Exp Med 195: 1653-1659). Therefore, the binding of the polymeric Fc protein of multiple copies of FcyRIIB can induce negative responses from cells expressing this receptor.
[0064] FcRL5 has recently been described as an Fc receptor capable of inhibitory signaling, which is expressed in B cells and which binds to aggregated IgG, but not monomeric IgG (Wilson et al, 2012). These authors propose that IgG can cooperatively bind to FcRL5 and FcyRllb co-expressed in B cells. As described in Example 3, an exemplary hexameric protein can bind independently of FcyRL11 FcRL5.
[0065] When the Fc receptor binding portion comprises immunoglobulin heavy chain constant regions of a human IgG isotype or its variants, which will typically bind to human Fcy receptors (FcyRI, FcyRII and FcyRIII) and / or Human FcRL5. Plasmon Surface
[0066] Resonance analysis as described above can be used to determine affinity constants. Typical affinity constants for binding human IgG1 or IgG3 to FcyRI are about 10 "9 M; for FcyRII it is about 0.6-2.5x10" 6 M; for FcyRIIIA it is about 5x10 "5 M; for FcyRIIIB it is about 0.6-2.5x10" 6 M. Monomeric IgG does not visibly bind to FcRL5, although FcRL5 binds with high avidity to aggregated IgG, suggesting FcRL5 is a low affinity for medium receptor for monomeric IgG with an affinity constant of about 10 '5 -10 "6 M (taken from Wilson et al, 2012). Alternatively, ELISA can be used, essentially as described in Example 3 to obtain a semi-quantitative indication binding properties.
[0067] Fc receptor binding moieties also typically bind to lectins, also referred to as glycan receptors, in particular lectins that bind to sialic acid. Exemplary lectins are human DC-SIGN (or mouse SIGN-R1) and CD22, which contribute to the therapeutic properties of IVIG (reviewed in Mekhaiel, 2011 b). Polymeric proteins are predicted to interact with these receptors, as explained in Example 3. The binding can be determined by surface plasma resonance, as described in Jain et al SINAL-R1, 2012. Analogous methods can be used to determine the CD22 or DC-SIGN connection. Human 2.6 sialylated Fc links SINAL-R1 and human DC-SIGN, with affinity constants 2.7x10-6 M and 3.6x10-6 M, respectively (Anthony et al, 2008, PNAS 105: 19571-19578). non-sialylated Fc do not bind appreciably. Multimeric hemagglutinin sialoproteins for example, are known to bind to sialic acid receptors with affinities in the order of 10 "8 M compared to monomers that bind 10" 2 M (Mammen H, Choi SK, Whitesides, GM (1998) Angew Chem Int Edit 37: 2755 -2794). Thus, as an increase in avidity can improve an inherently weak substrate affinity, having terminal sialic acid residues from polymeric proteins would also have to be expected to significantly improve binding to sialic acid receptors.
[0068] The appropriate limits for determining and affinity constants for the portions comprising the heavy chain variants of native immunoglobulin constant regions, or their fragments of Fc portions, as described above is binding to the Fc receptor.
[0069] A "variant" refers to a protein in which at one or more positions there were amino acid insertions, deletions or substitutions, conservative or non-conservative.
[0070] A "variant" can have modified amino acids. Modifications include acetylation, glycosylation, hydroxylation, methylation, nucleotidylation, phosphorylation, ADP-ribosylation, and other suitable modifications known in the art. Such modifications can occur postranslationally where the peptide is made using recombinant techniques. Otherwise, modifications of synthetic peptides can be made using techniques known in the art.
[0071] Modifications can be included before incorporating an amino acid into a peptide. Carboxylic acid groups can be esterified or can be converted to an amide, an amino group can be alkylated, for example, methylated. A variant can also be modified post-translation, for example, to remove the carbohydrate side chains or individual sugar moieties, for example sialic acid groups or to add sialic acid groups.
[0072] By "conservative substitutions" are intended combinations such as Vai, lie, Leu, Ala, Met; Asp, Glu; Asn, Gin; Ser, Thr, Gly, Ala; Lys, Arg, His; and Phe, Tyr, Trp. Preferred conservative substitutions include Gly, Ala; Val, lie, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
[0073] Typical immunoglobulin G variants of heavy chain constant regions will have an amino acid sequence that is at least 70%, typically at least 80%, at least 90%, at least 95%, at least 99% or at least 99 , 5% identical to immunoglobulin G of the heavy chain constant region of a corresponding native immunoglobulin. Suitably, the variant is a variant of human heavy chain immunoglobulin G1 and the constant region has an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to the latter .
[0074] A "fragment" refers to a protein in which one or more positions have been deleted. Typically, a fragment of an Fc portion comprises at least 60%, more typically at least 70%, 80%, 90%, 95% or up to 99% of the complete sequence of the Fc part. Variant fragments are also included.
[0075] The percentage of sequence identity between two polypeptides can be determined using suitable computer programs, for example, the University of Wisconsin Genetic Computing Group's GAP program and it is noted that the percentage identity is calculated in relation to polypeptides , whose sequence was optimally aligned.
[0076] The alignment can alternatively be carried out using the program Clustal W (Thompson et a /., (1994) Nucleic Acids Res., 22 (22), 4673-80). The parameters used can be as follows: • Quick pair alignment parameters: K-tuple (word) size; 1, the window size; 5, interval penalty; 3, diagonal upper number; 5. Classification method: x percent. • Various alignment parameters: gap open penalty; 10, penalty extension gap; 0.05. • scoring matrix: BLOSUM.
[0077] The variants can be natural or made using the methods of protein engineering and site-directed mutagenesis as are well known in the prior art.
[0078] "Peptides" generally contain up to 10, 20, 50 or 100 amino acids. The peptides and polypeptides can be conveniently blocked at the N- or C-terminus in order to help reduce the susceptibility to exoproteolytic digestion. Peptides and polypeptides can be produced by recombinant protein expression or in in vitro translation systems (Sambrook, et al, "Molecular Cloning: A Laboratory Manual", 2001, 3 rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). The peptides can be synthesized by the solid phase peptide synthesis Fmoc-polyamide method as disclosed by Lu et al (1981) J. Org. Chem. 46, 3433 and references therein.
[0079] Suitably, in the Fc protein for use, according to the first aspect of the invention, each of the heavy chain immunoglobulin G constant regions comprises an amino acid sequence that is modified in comparison to the amino acid sequence of a native immunoglobulin G of the heavy chain constant region, to modify the affinity of the Fc receptor binding portion for at least one Fc receptor. Typically, the affinity of the Fc receptor binding portion for a low inhibitor affinity and / or activating the Fey receptor can be increased.
[0080] The interactions between the Fc and IgG receptors were analyzed in biochemical and structural studies using wild type and Fc mutant. A consensus indicates that some Fc receptor binding regions are located in the part of the Hinge region closest to the CH2 domain and the amino terminal of the CH2 domain that is adjacent to the Hinge, including, for example, residues 233-239 (Glu-Leu -Leu-Gli- Gly-Pro-Ser). Mutations within this region can result in altered binding to Fc receptors. This region appears to be responsible for some of the direct interactions with Fc receptors (Woof JM & Burton D, Nature Reviews Immunology 2004, 4: 89-99). Further on, in the CH2 domain, and away from the hinge, there are other residues that can, at least in some contexts, contribute to binding to the F receptor, including, for example, human IgG1 Pro-329 (in an EU humeration) that appears be involved in direct contact with the Fc and Asn-297 receptor, which appears to be the only site for N-linked glycosylation in the human IgG1 Fc region. The presence of carbohydrates to this residue can contribute to the binding to Fc receptors.
[0081] Activating Fey Receptors are as described above; in humans, the low affinity Fey receptor activators are FcyRIIA / C and FcyRIIIA. FcyRI is the receptor for high affinity activators. FcyRIIB is a human inhibitory Fc receptor. Since the ligand-binding properties of FcyRIIB and FcyRIIA / C are the same, it may not be possible to increase the affinity for FcyRIIA / C, while simultaneously decreasing affinity for FcyRIIB. Lazar et al (2006) PNAS 103: 4005-10 describes mutations in the Fe portion of a human IgG that affect the binding affinity for different Fc receptors. A wild-type IgG bound to FcyRllla with a K D of 252 nM; K D of an I332E mutant was 30 nM and the K D of an S239D / I332E mutant was 2 nM. Combination of an A330L mutation with S239D / I332E increased FcyRllla affinity and reduced FcyRllb affinity. Shields RL et al (2001) J. Biol. Chem. 276: 6591-6604 describes mutations in the Fc portion of human IgG1 that affect binding affinity for different Fc receptors. The S298A mutation increased affinity for FcyRllla and decreased affinity for FcyRIIA; the E333A mutation increased affinity for FcyRllla and decreased affinity for FcyRIIA; the K334A mutation has the highest affinity for FcyRllla. Any or all of the above mutations can be used individually or in combination. Other suitable mutations can be identified by routine methods.
[0082] The affinity of the Fc portion receptor for CD22 or DC-SIGN / SINAL-R1 binding can be increased by increasing the amount of sialic acid bound to the Fc receptor binding portion. Typically, this is achieved by increasing the amount or proportion of terminal sialic acid residues in the N297 glycan, which can be added translational powders to the Fc peptide in the endoplasmic reticulum.
[0083] Other mutations can be adequately done to improve the effectiveness of the Fc receptor binding moieties. Suitably, each of the heavy chain immunoglobulin G constant regions comprises an amino acid sequence that is modified compared to the amino acid sequence of an L of the native immunoglobulin heavy chain constant region, to increase the in vivo half-life of the protein polymeric, appropriately, increasing the affinity of the Fc receptor binding portion to the neonatal Fc receptor for. Increasing serum persistence allows for higher circulating levels, less frequent administration and reduced doses. This can be achieved by increasing the binding of the Fe region to neonatal FcR (FcRn). FcRn, which is expressed on the surface of endothelial cells, binds to Fc in a pH-dependent manner and protects it from degradation. Although hexameric-Fc described in the Examples was unable to bind to human FcRn which binds very well to rat FcRn. The M252Y amino acid substitutions / S254T / T256E and / or H433K / N434F can be introduced into the Fc receptor binding portion to increase the in vivo IgG half-life without unduly affecting FcyR interactions (Vaccaro C, et al (2005) Nat. Biotech. 23: 1,283-1,288). In addition, or alternatively, H310 can be restored.
[0084] The binding of multiple Fc receptors by a polymeric Fc protein can cause different intracellular signaling phenomena than the binding of a single Fc receptor by a monomeric Fc or IgG protein. The binding of various Fc receptors by the polymeric protein may be more effective in blocking the binding of Fc receptors to other ligands, particularly in the case of low affinity Fe receptors. The effectiveness of the polymeric protein can be compared with the effectiveness of a monomeric unit that does not make polymers in many ways. In such tests, it is typical for the polymeric Fc protein monomeric units to individually have the same affinity for a given Fc receptor as the monomeric unit that does not form polymers.
[0085] Polymer proteins will have greater avidity for low affinity receptors, including Fc receptors than those of monomeric control units. They may also have a greater avidity for FcRL, FcRn, CD22, DC-SIGN or other Fc receptors. Greed is the global connecting force of a multi-purpose interaction. The interaction between an Fc binding region and an affinity Fc receptor has a characteristic, while the avidity of the interaction increases almost geometrically for each interaction. For low Fc affinity receptors, the increase in binding strength may allow a biologically relevant interaction with a polymeric protein, which could not be achieved by a monomeric or dimeric unit. Multivalent binding by polymeric proteins results in a considerable increase in stability, as measured by the constant (LJmol) balance, compared to the binding of a control monomeric protein. For example, a typical monovalent interaction between an Fc moiety and an Fc receptor can have an equilibrium constant of about 104 L / mol. The hexavalent interaction can predict an equilibrium constant of about 1011 L / mol. The equilibrium constant can vary, depending on the Fe portion and the Fc receptor. However, a hexameric protein will typically show an increase in binding energy compared to a control monomeric protein of up to about 10 4, 105 or 106 times, or even greater than 10 6 times. Similar increases in energy constants and binding balance can be expected for multivalent interactions with FcRL5, FcRn, CD22 or DC-SIGN. For a description of greed and affinity see book Immunology by Roitt, Brostoff and Male, 2 nd edition of 1989.
[0086] The avidity for Fe receptors of the polymer protein can be compared with that of the monomeric unit that do not form polymers by the Plasmon Resonance Analysis surface (Biacore), as described above.
[0087] It has been found that the fusion of a bulky antigen to the polymeric protein may decrease the ability of the polymeric protein to bind to the Fc receptors (Mekhaiel et al, 2011a). Suitably, the polymeric protein for use according to the first aspect of the invention does not comprise (for example, to be fused or conjugated to) a radical that reduces the binding avidity for FcyRII. The effect on avidity can be determined by comparing binding avidity for a given protein receptor comprising the polymeric portion to be tested with the avidity achieved by the protein lacking the polymeric radical. Typically, if the avidity is reduced by more than 10 times, or more than 5 times or more than twice, the protein comprising the polymeric portion may not be suitable. Reduced binding avidity for FcyRI It would also be indicative of reduced binding avidity for other Fc receptors, including FcyRI or FcyRI II. Alternatively, linkage for each or both of these other receivers could be tested.
[0088] Increased avidity of polymeric proteins for low affinity receptors, as described in Example 3, may involve the biological mechanisms underlying IVIG therapy, particularly those mediated by low affinity receptors FcyRIIB, FcyRIIIA, CD22 and DC-SIGN . Increased avidity for the high affinity IgG FcyRI receptor has also been observed for the exemplary hexameric protein. FcyRI is implicated in autoimmune inflammatory disease (OA Hussein et al, Immunol Invest 2010, 39: 699-712) and FcyRI immunotoxins aimed at inhibiting arthritis (Van Vuuren AJ et al, 2006, J. Immunol 176: 5833-8) . MicroRNA-127 also inhibits lung inflammation, targeting FcyRI (Xie T et al, 2012, J. Immunol 188, 2437-44). In addition, FcyRI binding to macrophages has been proposed to induce the production of IL-10 anti-inflammatory cytokines in US 2004/0062763 (Temple Univeristy; Mosser). Thus, the binding of FcyRI can also have a beneficial effect in the context of the present invention, although FcyRI is not thought to be the basis of the effects of IVIG.
[0089] The immunomodulatory properties of polymeric protein arise from interactions between the F-binding portions and Fc receptors and / or other immune system receptors and components, which interact with Fc portions. The polymeric protein for use according to the first aspect of the invention does not comprise an additional immunomodulatory moiety; or a portion of antigen that causes specific immunosuppression for the antigen, when administered to the mammalian subject. Contrary to therapeutic approaches that provide immunosuppressive agents, such as the TNF- receptor, the polymeric proteins described here are based on a different therapeutic mechanism, which can make them more widely useful in therapy. Nor do polymeric proteins depend on an antigen-specific immunosuppressive effect, which limits the application of other therapeutic approaches to specific diseases.
[0090] An "immunomodulatory moiety" is an agent that has an immunomodulatory activity in a healthy or diseased mammalian subject, when covalently linked to a polymeric protein, as described herein, or, when present, in the absence of said polymeric protein .
[0091] "Immunomodulatory activity" refers to the alteration of an immune response in an individual, to increase or decrease components of the immune system, such as cytokines or antibodies; or to increase or decrease immune functions, such as antigen presentation. An "immunomodulator" portion can be a chemokine or chemokine receptor, cytokine or cytokine receptor, such as Toll receptor (TLR), acute phase protein, complement component, immune receptor, CD molecule, or signal transduction molecule, for example. Such agents are known to have immunomodulatory activities in a healthy or sick mammal subject in the absence of the polymeric protein. Examples of such agents are described in textbooks such as Immunology (Abbas and Lichtman, Cellular and Molecular Immunology, Elsevier Saunders, 5 th Edn, 2005), and the skilled worker can find other examples of relevant literature. Thus, conjugates or fusion proteins that comprise any of these agents and the polymeric protein are excluded from the scope of the invention. Immunomodulatory portions found in the Fc monomeric fusion proteins known as etanercept, alefacept, abatacept, belatacept, Atacicept, briobacept, rilonacept or afilbercept are particularly excluded. Antibody fragments, such as Fab or scFv can also be considered as "immunomodulatory portions". Such fragments can bind to components of the immune system, thus having an immunomodulatory activity. When fused or conjugated to monomeric units of the polymer protein, antibody fragments can recapitulate the structure of the complete antibody, i.e., Fc and antigen combining regions. Such agents may have a more pronounced immunomodulation activity when covalently linked to the polymeric protein, than when present alone. Immunomodulatory portions found in therapeutic antibodies, such as the antibody known as Remicade that binds to TNFa, are particularly excluded. Suitably, the polymeric protein does not comprise any other than immunoglobulin G heavy chain regions with constant antibody portions and, optionally, the Hinge region, whether immunomodulatory or not.
[0092] As noted above, the immunomodulatory properties of the polymeric protein arise from interactions between the F-receptor binding portions and Fc receptors and / or other immune system receptors and components, which interact with Fc portions. Modifications of the Fc receptor binding moieties, such as modified glycosylation, which modify the binding to Fc receptors, lectins or other components of the immune system are not considered as new immunomodulatory moieties, but as components of the Fc receptor binding moieties. Therefore, they are not excluded from the scope of the invention.
A suitable experimental test for an immunomodulatory portion is to provide a polymeric protein without the putative immunomodulatory portion and a polymeric protein comprising the immunomodulatory and putative test protein, either for effectiveness in the ITP mouse model described in the Example 4. If the putative immunomodulatory portion has immunomodulatory activity, it can alter the parameters of the response. For example, it can change the rate or extent of platelet recovery.
[0094] An "antigen" is a molecule that specifically binds to an antibody or a TCR. Antigens that bind to antibodies include all classes of molecules, and are called B cell antigens. Examples of molecule types include peptides, polypeptides, glycoproteins, polysaccharides, gangliosides, lipids, phospholipids, DNA, RNA, fragments, parts of same and combinations thereof. TCR binds only to peptide fragments of proteins complexed with MHC molecules; both the peptide ligand and the native protein from which it is derived are called T cell antigens. "Epitope" refers to an antigenic determinant of a B cell or T cell to the antigen. In the case of a B cell epitope it is a peptide or polypeptide, which typically comprises three or more amino acids, generally at least 5 and more usually at least 8 to 10 amino acids. Amino acids can be adjacent amino acid residues in the primary structure of the polypeptide, or they can become spatially juxtaposed in the folded protein. T cell epitopes can bind to MHC class I or MHC Class II molecules. Typically MHC Class T epitopes of 1-binding cells are 8 to 11 amino acids in length. Class II molecules bind to peptides that can be 10 to 30 residues in length or more, the ideal length being 12 to 16 residues. Peptides that bind to a specific allelic form of an MHC molecule contain amino acid residues that allow complementary interactions between the peptide and the MHC allele molecule. The ability of a putative T cell epitope to bind to an MHC molecule can be predicted and confirmed experimentally (I Dimitrov et al, Bioinformatics 2010 Aug 15; 26 (16): 2066-8).
[0095] The polymeric protein for use, according to the first aspect of the invention does not comprise a portion of antigen that causes specific immunosuppression for the antigen, when administered to the mammalian subject. By "comprise", which include antigens that are covalently linked to at least one of the peptide monomer units, such as by chemical conjugation or recombinant fusion. Typically, the polymeric protein does not comprise a portion that acts as an antigen, either by causing immunosuppression or immunostimulation, when administered to the mammalian subject.
[0096] By "antigen-specific immunosuppression", suppression of responses and / or suppression of antibody T cell responses has been included. T cell responses can be suppressed by clonal deletion of reactive antigen T cells, anergy or suppression, such as through the induction of regulatory T cells. T cell responses can also be shifted more aggressively to less aggressive forms, for example, from Th1 type to Th2 type responses.
[0097] A suitable test for a portion of antigen that causes specific immunosuppression for the antigen is to provide a polymeric protein missing a portion of polymeric protein antigen that comprises the portion of the antigen and to test any protein in the mammal subject to be treated for autoimmune or inflammatory disease. Polymeric proteins lacking the antigenic potion containing the antigen can be administered sequentially to the same mammalian individual, and the subject monitored for an immune response to the antigen after treatment with any of the proteins. Alternatively, groups of subjects, as can be treated with the polymeric protein lacking the antigen comprising the protein of the antigen. Conventional techniques can be used to identify and monitor the immune response to the antigen, whether a B cell response or a T cell response. If there is no difference in the degree or type of immune response to the antigen in subjects following administration of the protein polymeric that lacks the antigen, compared to the polymeric protein comprising the antigen, then the antigen does not cause specific immunosuppression for the antigen when administered to the mammalian subject. In this way, specific means for the resulting immunosuppression antigen from a portion of a polymeric protein antigen are excluded. This does not mean that the immune response to a particular antigen may not decrease in response to therapy. The immune response to an autoantigen implicated in an autoimmune disease may decrease in response to therapy, but this can be achieved by the protein lacking the polymeric antigen as well, as well as by a polymeric protein comprising the antigen. Similar tests can be used to identify whether the polymeric protein comprises a portion that acts as an antigen when administered to the mammalian subject. In these tests, dose, formulation and administration characteristics of polymeric proteins are the same to allow a meaningful comparison.
[0098] Alternatively, polymeric proteins can be tested in healthy mammalian individuals to determine whether a portion that acts as an antigen is present. Such assays are particularly useful in identifying antigen-specific immunostimulation. Suitably, antigen-specific immunostimulation does not occur. Such assays can be useful for confirming the non-immunogenicity of the polymeric protein. The components of the polymeric protein that are not naturally found in the mammalian subject to which the protein is being administered, such as peptide ligands, can be tested for immunogenicity and the selected non-immunogenic components. In such tests, the polymeric protein is administered and, after an appropriate period of time to allow an immune response to develop against a putative antigen, for example, two weeks, a blood sample is tested to determine the level of antibodies against the putative antigen using ELISA.
[0099] Suitable animal models can also be used to test for the presence of a portion of antigen that causes specific immunosuppression for the antigen or a portion that acts as an antigen, when administered to the mammalian subject. For example, the ITP mouse model described in Example 4 can be used. The extent and type of immune response to the antigen is then tested following administration of the polymeric protein lacking the putative antigen or antigen, or polymeric protein comprising the putative antigen or antigen.
[00100] Suitably, the polymeric protein for use, according to the first aspect of the invention, does not activate the classical complement pathway, although it may be able to bind to C1q (Mekhaiel et al, 2011a). Complement binding and activation can be assessed by ELISA in wells coated with polymeric or monomeric IgG or Fc control protein, as described in Lewis M et al, Mol. Immunol 2008, 45: 818-827, and as performed in Example 3. 96 well plate wells were coated overnight with 100μI protein in carbonate buffer, pH 9. After washing, the plates were incubated with human serum diluted 1/100 in 100μI buffered saline containing veronal containing 0 , 5 mM MgCl 2, 2 mM cacl2, 0.05% Tween-20, 0.1% gelatin and 0.5% BSA, for 1 h at room temperature. After washing, the plates are incubated with 100μl of either a 1/800 dilution of sheep anti-C1q-HRP (Serotec) or a dilution of conjugate with biotin anti-C5b-9 (Quidel, Santa Clara, CA), followed by 1 / 500 100μl per streptavidin-HRP (Dako) diluted 1/1000 in PBS-T, 0.5% BSA for 1 hour at room temperature. Absorbance values below 0.4 are considered negative. In a typical C1q binding assay, absorbance values above 0.4 are obtained when the plates are coated with polymeric protein above 2 ng / ml, or above 4 ng / ml or greater than 10 ug / ml. In a typical assay for C5b-9 deposition, indicative of complement activation, absorbance values above 0.4 are not achieved when the plates are coated with polymeric protein of up to 2 µg / ml, up to 4 µg / ml, up to 10 ug / ml, up to 20 ug / ml or up to 50 ug / ml.
[00101] Suitably, the polymeric protein for use, according to the first aspect of the invention is capable of binding Protein G or Protein A with sufficient avidity to allow any substrate to be used as a capture agent for the purification of the polymeric protein . Protein G binds in the region between CY2-Cy3 domains and was used to purify the hexameric Fc monomer proteins and in the Examples. Other receptors that bind to the same region can also bind to polymeric protein, such as TRIM21 (Mallery DL et al, 2010, Proc Natl Acad Sci USA 107 (46): 19.985-19.990; McEwan et al WA 201, BioEssays 33 : 803-809). TRIM21 is thought to be important in removing immunity to the virus.
[00102] Suitably, the polymeric protein for use, according to the first aspect of the invention has a molecular weight of about 230 to 400 kD. For example, it can have a molecular weight of about 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400 kD. Polymers within this size range are typically easier for mammalian cells to synthesize and assemble than molecules that have higher molecular weights, such as ~ 750kDa IgM, or complex proteins, such as antibodies that require the expression and assembly of two chains different polypeptides (light and heavy chains). Proteins with several different polypeptide chains, including antibodies, are more difficult to produce homogeneity during manufacture.
[00103] Suitably, the polymeric protein for use, according to the first aspect of the invention, has a diameter of about 20 nm, such as from 15 to 25 nm or 30 nm up to. 20 nm particles have been shown to be more easily delivered to lymphatics of 45 and 100 nm nanoparticles, which may be important for tissue function and penetration (Reddy et al, 2007). Smaller particles are also more easily manufactured by recombinant expression technology.
[00104] As a consequence of the molecular size and diameter, the polymeric protein typically has a good degree of penetration into the tissue, which can aid its therapeutic effect (Vollmers & Brandlein, 2006). Compared to small molecules or monomers, polymers will naturally show slower penetration over time, but IgM molecules still intact (750 kDa) target tumors in mice and the primary tumors and metastases in patients after implantation. v. or ip administration (Vollmers et al, 1998a, b, Oncol Rep 5: 549552; Oncol Rep 5: 35-40). It can be demonstrated by standard immunohistochemistry techniques that pentameric IgM, when injected ip can reach and shrink tumors transplanted subcutaneously on the animals' backs (all Vollmers refs, supra). This shows that molecules as large as 750 kDa are able to leave the peritoneal cavity, enter the circulation and reach implanted tumors. In their path, the molecules have to pass several endothelial barriers of the lymphatic and blood vessels before they reach their targets, Jain et al, 2001 J. Controle. Release 74: 7-25. Taken together, it is predicted that polymeric proteins for use according to the invention, including the exemplified hexameric Fc protein, will show intermediate penetrance times (hours) between the highest IgM (days) and lowest IgG (minutes). For conditions such as ITP that require biological activity, intermediate penetration and accumulation can be an advantage over treatments using antibodies as carriers where rapid penetration is preferred.
[00105] Suitably, the polymeric protein for use, according to the first aspect of the invention has a spatial orientation in relation to the cell surface which is cis (each Fc is in the same plane parallel to the cell surface and the longitudinal axis of each Fc is perpendicular to the cell surface), rather than trans. The cis orientation allows the connection of several receptors in the same cell, since the receptor binding portions are closely juxtaposed to the same cell. In contrast, a trans orientation in which the Fes are in a plane perpendicular to the cell surface can result in cross-linking of two cells by the same protein. In the cis orientation, the biological effects of receptor binding are centered on a particular cell, and therefore are more likely to be productive.
[00106] The polymeric proteins are intended for the treatment of autoimmune or inflammatory diseases, according to the first aspect of the invention. "Autoimmune disease" includes any disease in which the immune system attacks the body's own tissues. "Inflammatory disease" includes any disease characterized by destructive inflammation, which can be recurrent or chronic and is not associated with the repair of normal tissues. Such diseases particularly include "autoinflammatory" diseases, in which the innate immune system that causes inflammation may be, for unknown reasons. Autoinflammatory diseases are characterized by intense episodes of inflammation that results in symptoms such as fever, rash or swelling of the joints. These diseases they also carry the risk of amyloidosis, a potentially fatal build-up of a protein in the blood in vital organs.
[00107] Autoimmune or inflammatory diseases suitable for treatment include those that are treatable with intravenous immunoglobulin (IVIG). These can be diseases that are currently routinely treated with IVIG or where IVIG has been found to be clinically useful, such as autoimmune cytopenias, Guillain-Barre syndrome, myasthenia gravis, anti-autoimmune factor VIII disease, dermatomyositis, vasculitis and uveitis (See, van der Meche FG et al, Lancet, i, 406 (1984); Sultan Y et al, Lancet II, 765 (1984) ;. Dalakas MC et al, N. Engl J. Med 329, 1993 (1993) .; Jayne DR et al, Lancet 337, 1137 (1991); LeHoang P et al, Ocul Immunol Inflamm 8, 49 (2000) IVIG is typically used to treat idiopathic thrombocytopenic purpura (ITP), Kawasaki disease, Guillain syndrome Barre, syndrome and chronic inflammatory demyelinating polyneuropathy (Laranja et al, 2006, J Allergy Clin Immunol 117: S525-53). IVIG is also increasingly used to treat a diverse range of other autoimmune diseases that are non-responsive to mainstay therapies, including arthritis, diabetes, myositis, Crohn's colitis and lupus erythemat systemic bone.
[00108] Autoimmune or inflammatory diseases suitable for treatment include autoimmune cytopenias, idiopathic purple thrombocytopenia, rheumatoid arthritis, erythematous systemic lupus, asthma, Kawasaki disease, Guillain-Barre syndrome, Stevens-Johnson syndrome, Crohn's colitis, diabetes, poline chronic inflammatory demyelinating myasthenia gravis, anti-Factor VIII autoimmune disease, dermatomyositis, vasculitis, uveitis or Alzheimer's disease.
[00109] Conditions to be treated may include an inflammatory disease with an imbalance in cytokine networks, an autoimmune disorder mediated by pathogenic autoantibodies or auto-aggressive T cells, or an acute or chronic phase of an autoimmune chronic relapse , or inflammatory disease or infectious process. In addition, other medical conditions that have an inflammatory component are included, such as Amyotrophic Lateral Sclerosis, Huntington's disease, Alzheimer's disease, Parkinson's disease, myocardial infarction, stroke, Hepatitis B, Hepatitis C, virus inflammation associated with Human immunodeficiency, adrenoleukodystrophy, and epileptic disorders, especially those believed to be associated with post-viral encephalitis, including Rasmussen's syndrome, West's syndrome, and Lennox-Gastaut syndrome.
[00110] Conditions to be treated can be hematoimmunological diseases, for example, idiopathic purple thrombocytopenia, alloimmune / autoimmune thrombocytopenia, acquired immune thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, B19-associated red cell aplasia, Acquired antifactor VIII autoimmunity, acquired von Willebrand disease, Multiple myeloma and Monoclonal Gammopathy of Unknown Significance, aplastic anemia, pure red series aplasia, Diamond-Blackfan anemia neutropenia, hemolytic disease of the newborn, Immunomediated, refractory to platelet transfusion, post-transfusion neonatal purpura, hemolytic-uremic syndrome, systemic vasculitis, purple thrombotic thrombocytopenic or Evan's syndrome.
[00111] As an alternative, a neuroimmunological disease can be treated, for example, Guillain-Barre syndrome, chronic inflammatory demyelinating Polyradiculoneuropathy Paraproteinemic IgM demyelinating polyneuropathy, mystenic Lambert-Eaton syndrome, myasthenia gravis, Motor neuropathy Multifocal, neuron syndrome Multifocal associated with anti-GM1 antibodies, demyelination, multiple sclerosis and optic neuritis, Man Stiff syndrome, Paraneoplastic cerebellar degeneration with anti-Yo antibodies, paraneoplastic encephalomyelitis, sensory neuropathy with anti-Hu antibodies, epilepsy, encephalitis, myelitis, myelopathy especially associated with a Human lymphotropic T-cell virus-1 Autoimmune Diabetic Neuropathy, or acute idiopathic dysautonomy neuropathy or Alzheimer's disease.
[00112] Rheumatic disease can be treated, for example, Kawasaki disease, rheumatoid arthritis, Felty syndrome, ANCA-positive vasculitis, Spontaneous Polymyositis, dermatomyositis, antiphospholipid syndromes, recurrent spontaneous abortions, systemic lupus erythematosus, juvenile arthritis, idiopathic arthritis CREST Raynaud's syndrome or uveitis.
[00113] Dermatoimmunological disease can be treated, for example, Epidermal Gangrene Necrolysis, Granuloma, skin autoimmune bullous diseases, including pemphigus vulgaris, Pemphigoid, and pemphigus foliaceus, Vitiligo, streptococcal toxic shock syndrome, scleroderma, sclerosis and sclerosis limited systemic skin, atopic dermatitis or steroid-dependent atopic dermatitis.
[00114] Musculoskeletal immune disease can be treated, for example, Myositis Body Inclusion, necrotizing fasciitis, inflammatory myopathies, myositis, Anti-Decorin (BJ antigen) Myopathy, Paraneoplastic Necrotic Myopathy, linked to X vacuolados Myopathy, induced by Penacillamine polymyositis, Atherosclerosis, Coronary Artery disease, or cardiomyopathy.
[00115] A gastrointestinal immune disease can be treated, for example, pernicious anemia, active chronic autoimmune hepatitis, primary biliary cirrhosis, celiac disease, herpetiform dermatitis, cryptogenic cirrhosis, reactive arthritis, Crohn's disease, Whipple's disease, ulcerative colitis or cholangitis sclerosing.
[00116] The disease can be, for example, inflammation of the disease, asthma, type 1 diabetes mellitus with anti-beta cellular antibodies, Sjogren's syndrome, connective tissue disease, mixed Addison's disease, Vogt Koyanagi-Harada Syndrome, membranoproliferative glomerulonephritis , post-infectious Goodpasture syndrome, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, micropoliarteritis, Churg-Strauss syndrome, nodular polyarthritis or multiple organ failure.
[00117] An exemplary disease for the treatment of idiopathic purple thrombocytopenic (ITP).
[00118] Polymer proteins for use in a given species typically include Fc portions of IgG of that species, and may also include tailpiece portions of IgA or IgM of that species. Typically, the mammal subject to be treated is a human being, although other mammals and birds are actually amphibian reptiles and can be treated.
[00119] Polymeric proteins are generally supplied as properly formulated therapeutic compositions. Suitably, they are supplied as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. In addition, if desired, minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents can be included.
[00120] The carrier may preferably be a liquid formulation, and is preferably an isotonic, buffered aqueous solution. Suitably, the therapeutic composition has a pH that is physiological, or close to physiological. It is suitably physiological or close to physiological osmolarity and salinity and / or is sterile and free from endotoxin. It may contain sodium chloride and / or sodium acetate. Pharmaceutically acceptable carriers can also include excipients, such as diluents and the like, and additives, such as stabilizing agents, preservatives, solubilizing agents, and the like. As used herein, the term "pharmaceutically acceptable" means approved by a US or EU regulatory agency or other government or listed in the US Pharmacopoeia or other pharmacopoeia generally recognized for use in humans.
[00121] Pharmaceutical compositions can be formulated for any suitable mode of administration, including, for example, topical (e.g., transdermal or ocular), buccal, nasal, vaginal, rectal or oral parenteral. The term parenteral as used herein includes subcutaneous, intradermal, intravenous (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial injection and intraperitoneal injection, as well as any similar technical injection or infusion. Suitable forms for oral use include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. The compositions provided herein can be formulated as a lyophilizate. Typically, the composition is formulated for intravenous administration.
[00122] Aqueous suspensions contain the active ingredient (s) in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents (for example, sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and acacia gum); and dispersing or wetting agents (for example, naturally occurring phosphatides, such as lecithin, alkylene oxide condensation products with fatty acids, such as polyoxyethylene stearate, ethylene oxide condensation products with long chain aliphatic alcohols, such as heptadecaethylenoxyethanol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as monooleate polyethylene sorbitan). The aqueous suspensions may also comprise one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
[00123] The formulations can be for local or topical administration, such as for topical application to the skin, wounds or mucous membranes, as in the eye. Formulations for topical administration typically comprise a topical vehicle combined with the active agent (s), with or without optional additional components. Suitable topical vehicles and additional components are well known in the art, and it will be apparent that the choice of a vehicle will depend on the particular physical form and mode of administration. Topical vehicles include water; organic solvents, such as alcohols (for example, ethanol or isopropyl alcohol) or glycerin; glycols (for example, butylene, isoprene or propylene glycol); aliphatic alcohols (for example, lanolin); mixtures of water and organic solvents and mixtures of organic solvents such as alcohol and glycerin; lipid-based materials such as fatty acids, acylglycerols (including oils, such as mineral oil, and fats of natural or synthetic origin), phosphoglycerides, sphingolipids and waxes; protein-based materials such as collagen and gelatin; silicone-based materials (both non-volatile and volatile); and materials, such as micro spheres and hydrocarbon polymer based matrices.
[00124] A pharmaceutical composition can be formulated as inhalable formulations, including sprays, mists or aerosols. For formulations for inhalation, the compounds provided herein can be administered by any inhalation methods known to those skilled in the art. Such methods and devices for inhalation include, but are not limited to, dosing inhalers with CFC propellants, such as either HFA or propellants that are physiologically and environmentally acceptable. Other suitable devices are multi-dose inhalers operated for breathing, dry powder inhalers and aerosol nebulizers. Aerosol formulations for use in the method of the present invention typically include propellants, surfactants and cosolvents and can be placed in conventional aerosol containers that are closed by a suitable metering valve.
[00125] Inhalant compositions may comprise liquid or powder compositions containing the active ingredient that are suitable for nebulization and intrabronchial use, or aerosol compositions administered through an aerosol unit that dispenses metered doses. Suitable liquid compositions comprise the active ingredient in a pharmaceutically acceptable inhalant aqueous solvent, for example, isotonic saline or bacteriostatic water. The solutions are administered by means of a pump or by pressing the nebulized spray dispenser, or by any other conventional means to cause or enable the required dosage amount of the liquid composition to be inhaled into the patient's lungs. Suitable formulations, in which the carrier is a liquid, for administration, such as, for example, a nasal spray or nasal drops such as, include aqueous or oily solutions of the active ingredient.
[00126] Formulations or compositions suitable for nasal administration, in which the carrier is a solid, include a coarse powder having a particle size, for example, in the range of 20 to 500 microns, into which the powder is administered (i.e. , by rapid inhalation through the nasal passage from a powder container kept close to the nose). Suitable powder compositions include, by way of illustration, powder preparations of the active ingredient carefully intermixed with lactose or other acceptable inert powders for intrabronchial administration. The powder compositions can be administered through an aerosol dispenser or contained in a breakable capsule that can be inserted by the patient in a device that pierces the capsule and blows the powder out in a stable stream suitable for inhalation.
[00127] The effective dosage amount of a composition administered to a mammalian subject can be determined by physical and physiological factors such as body weight, severity of the condition, type of disease to be treated, therapeutic interventions previously or simultaneously , patient's idiopathic and route of administration. The administering physician will, in any event, determine the concentration of active ingredient (s) in an appropriate composition and dosage (s) for the individual subject.
[00128] Pharmaceutical compositions can comprise, for example, at least about 0.1% of an active compound. In other embodiments, an active compound can comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any derivable range therein. In other non-limiting examples, a dose can also comprise between about 1 milligram / kg / body weight, about 5 mg / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / weight body weight, about 100 milligrams / kg / body weight per administration, and any derivable range in it. The effective dose of polymeric protein is generally about 1% to about 20% of the effective dose of IVIG. The effective dose of IVIG can generally be in the range of about 100 mg / kg to about 2 grams / kg, depending on the condition being treated.
[00129] Convenience of protein polymers and therapeutic compositions can be tested in animal models, prior to administration to human patients. For an animal model to be suitable, it is important that the Fc receptors at which the animal is able to bind to the Fc receptor portions of the polymeric binding protein. It is known that human immunoglobulins can bind to mouse Fc receptors. For example, human IgM binds to the mouse-FCM receptor. Pleass RJ, 2009 Parasite Immunology 31: 529-538 reports that the Fc receptors can bind to the antibodies from which the species. However, where the polymeric binding protein comprises Fc portions of human immunoglobulin derived from heavy chain sequences, the use of transgenic mice that express human Fc receptors may be advantageous. A suitable transgenic mouse expresses the human FcyRI (CD64) receptor that binds to human IgG1 and IgG3 (Heijnen IA et al, J Clin Invest 15 Jan 1996; 97 (2): 331-8). Transgenic mice expressing low affinity FcRs are also available, such as FcyRI IA (CD32) (McKenzie SE 2002, Blood Ap 16: 35).
[00130] Suitable mouse models are the ITP mouse model described in Example 4. Other suitable models include collagen-induced arthritis in mice (Jain et al, 2012) or non-obese diabetes (NOD) mouse model (Inoue, Y. et al. (2007) J. Immunol. 179, 764-774).
[00131] According to a second and third aspect of the invention, a method for treating a mammalian subject for an autoimmune or inflammatory disease is provided.
[00132] According to the second aspect, the subject is treated by administering a polymeric protein comprising five, six or seven units of polypeptide monomer; wherein each polypeptide monomeric unit consists of an Fc receptor-binding portion consisting of two immunoglobulin G heavy chains constant regions; and, optionally, a polypeptide linker that links the two constant regions of the immunoglobulin G heavy chain as a single Fc chain; wherein each immunoglobulin constant L heavy chain comprises a cysteine residue that is linked via a disulfide bond to a cysteine residue of an immunoglobulin G heavy chain of the constant region of an adjacent polypeptide monomer unit.
[00133] According to the third aspect, the subject is treated by administering a polymeric protein consisting of five, six or seven units of polypeptide monomer; wherein each polypeptide monomeric unit consists of an Fc receptor binding portion and a cap region; wherein the Fc receptor binding portion consists of two heavy chains of immunoglobulin G constant regions; and, optionally, a polypeptide linker that links the two constant regions of the immunoglobulin G heavy chain as a single Fc chain; wherein each heavy chain L modified human immunoglobulin constant region comprises a cysteine residue that is linked via a disulfide bond to a cysteine residue of a modified human heavy chain immunoglobulin L constant region of an adjacent polypeptide monomer unit; and where the capping region is fused to each of the two regions contained in the modified human immunoglobulin heavy chain of the polypeptide monomer unit, and facilitates the assembly of the monomer units in a polymer.
[00134] For aspects of either the second or third term "immunoglobulin G heavy chain constant region" is as described in connection with the first aspect of the invention. The immunoglobulin G heavy chain constant region typically associates as monomeric units by disulfide bonds, such as occurs in native antibodies. Alternatively, the two constant regions can be produced as a chain of amino acids with a single intervening link region, that is, as a single chain of Fc (SCFC).
[00135] The polymeric protein for use according to the second aspect of the invention is formed by virtue of each immunoglobulin G heavy chain constant region comprising a cysteine residue that is linked via a disulfide bond to a cysteine residue of an L of the immunoglobulin heavy chain constant region of an adjacent polypeptide monomer unit. A cap is not included. In contrast, the polymeric protein for use according to the third aspect of the invention includes a cap, which facilitates the assembly of the monomer units in a polymer. The end piece is as described in connection with the first aspect of the invention. A short linker sequence can be provided between the immunoglobulin heavy chain cap region and the constant region.
[00136] Regarding the second and third aspects, when the monomer units were assembled in a polymer, the Fc receptor binding portions are arranged in a polymeric structure that is spatially oriented to allow each Fc receptor binding portion to connect to an Fc receiver. The Fc receptor portion of each of the binding monomer units is as described in connection with the first aspect of the invention. When the Fc receptor binding portion comprises immunoglobulin heavy chain constant regions of a human IgG isotype or its variants, which will typically bind to human FCY- receptors (FcyRI, FcyRII and FcyRIII) and / or human FcRL5 and / or CD22 and / or DC-PLATE. Polymer proteins will be more avid for Fc receptors than monomeric control units (which do not polymerize because they lack the necessary cysteine residue).
[00137] The immunomodulatory properties of polymeric protein for use according to the second or third aspects of the invention arise from interactions between the F-receptor binding portions and Fc receptors and / or other immune system receptors and components, which interact with portions Fc. In particular, no more immunomodulatory portion is needed; and no antigen portion that causes specific immunosuppression for the antigen, when administered to the mammalian subject is required. Typically, there is no portion that acts as an antigen when administered to the mammalian subject.
[00138] Other characteristics of the polymeric protein for use, according to the second or third aspects are described in relation to the first aspect of the invention. Typically, the polymeric protein activates the classical complement pathway, although it can bind to C1q; Typically, it can bind protein G or protein A; typically has a molecular weight of about 230 to 400 kDa; typically has a diameter of about 20 nm; it typically has a good degree of tissue penetration; and, typically, it has a spatial orientation in relation to the cell surface which is cis.
[00139] Polymeric proteins for use, according to the second or third aspects of the invention are intended for the treatment of autoimmune or inflammatory diseases, as described in connection with the first aspect of the invention.
[00140] A fourth aspect of the invention provides a polymeric protein comprising five, six or seven units of polypeptide monomer; wherein each polypeptide monomer unit comprises an Fc receptor binding portion comprising two immunoglobulin G heavy chain constant regions; wherein each heavy chain L immunoglobulin constant region comprises a cysteine residue which is linked via a disulfide bond to a cysteine residue of an heavy chain L immunoglobulin constant region of an adjacent polypeptide monomer unit; wherein the polymeric protein does not comprise an additional immunomodulatory moiety; or a portion of antigen that causes specific immunosuppression for the antigen, when administered to a mammalian subject; wherein each polypeptide monomer unit does not include a cap region fused to each of the two immunoglobulin G heavy chain constant regions.
[00141] The polymeric protein of the fourth aspect is, as described in relation to the polymeric protein for use according to the first aspect, except that it is expressly excluded that each unit of polypeptide monomer comprises a cap region fused to each of the two heavy chain immunoglobulin G constant regions. Typically, none of the monomer units comprise a cap region. The polymeric protein does not comprise a cap region.
[00142] A fifth aspect of the invention provides a polymeric protein consisting of five, six or seven units of polypeptide monomer; wherein each monomeric unit of a polypeptide consists of the Fc-binding portion of the receptor that consists of two immunoglobulin G heavy chains constant regions; and, optionally, a polypeptide linker that links the two constant regions of the immunoglobulin G heavy chain as a single Fc chain; and wherein each L of the immunoglobulin heavy chain comprises the constant region of a cysteine residue that is linked via a disulfide bond to a cysteine residue of an L heavy chain constant region of an immunoglobulin of an adjacent polypeptide monomer unit .
[00143] The polymeric protein of the fifth aspect is, as described in relation to the polymeric protein for use according to the third aspect of the invention.
[00144] A sixth aspect of the invention provides a nucleic acid molecule comprising a coding portion that encodes a polypeptide monomer unit of a polymeric protein, as defined according to the fourth or fifth aspect of the invention. Nucleic acid molecules encoding the polypeptide monomeric units of a polymeric protein as described in connection with the first, second or third aspects of the invention.
[00145] Conventional recombinant DNA methodologies can be explored to generate polymeric proteins, as described, for example in (Sambrook et al, "Molecular Cloning: A Laboratory Manual", 2001, 3 rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). The monomer unit constructs are preferably generated at the DNA level, and the resulting DNAs integrated into expression vectors, and expressed to produce the monomeric units that come together to form the polymeric protein.
The nucleic acid molecule of the sixth aspect of the invention comprises a coding portion, which comprises, in the 5 'to 3' direction, a sequence encoding an immunoglobulin L heavy chain constant region or a single Fc chain (SCFC). In suitable embodiments, the latter is merged into a frame with a coding sequence for a cap region. DNA encoding the coding sequences can be in your genomic configuration or cDNA your configuration. It will be appreciated that additional coding sequences between the heavy chain and cap coding sequences can be provided, to allow these components to be separated from each other in the protein expressed by linker sequences. Nucleic acids encoding linker sequences can be included, for example, to allow insertion of useful restriction sites and / or to allow the heavy chain region and cap coding regions to be transcribed in the frame. Suitable coding regions can be amplified by PCR and manipulated using standard techniques (Sambrook et al, supra). Mutations compared to native nucleic acid sequences can be done by PCR SOEing or site-directed mutagenesis.
[00147] Suitably, the coding portion of the nucleic acid molecule that encodes a signal peptide, which is contiguous with the polypeptide monomer unit. This facilitates the isolation of the expressed monomer units from a host cell. The nucleic acid molecule, therefore, will comprise a coding portion which comprises, in the 5 'to 3' direction, a signal sequence fused in frame with the coding sequence of the monomer unit. The portion of the DNA encoding the signal sequence preferably encodes a peptide segment, which directs the secretion of the monomer unit and is subsequently cleaved away from the remainder of the monomer unit. The signal sequence is a polynucleotide that encodes an amino acid sequence that initiates the transport of a protein across the membrane of the endoplasmic reticulum. Signal sequences that are useful include antibody light chain signal sequences, for example, antibody 14.18 (et al (1989) J. of Immunol Meth 125 Gillies: 191), heavy chain antibody signal sequences, for example example, the MOPC141 antibody heavy chain signal sequence (Sakano et al (1980) Nature 286: 5774), and any other signal sequences that are known in the art (see, for example, Watson (1984) Nucleic Acids Research 12 : 5145).
[00148] Other aspects of the invention are an expression vector comprising the nucleic acid molecule of the sixth aspect of the invention; a host cell comprising the expression vector; and a therapeutic composition comprising the polymeric protein of the fourth or fifth aspect of the invention. Therapeutic compositions are as described with respect to polymeric proteins for use, according to the first aspect of the invention.
[00149] Nucleic acid molecules, as described herein, can generally be part of expression vectors. As used herein, the term "vector" is understood to mean any nucleic acid comprising a nucleotide sequence competent to be incorporated into a host cell and to be recombined with and integrated into the genome of the host cell, or to replicate autonomously as an episome . Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and the like. Non-limiting examples of a viral vector include a retrovirus, an adenovirus and an adeno-associated virus. As used herein, the term "gene expression" or "expression" monomer unit means the transcription of a DNA sequence, translation of the mRNA, and optionally also the secretion of a monomer unit.
[00150] Typically, host cells are provided for expression of the expression vector. The cell can be one of mammals, birds, insects, reptiles, bacterial, vegetable or fungal cell. Examples of mammalian cells include, but are not limited to, human, rabbit, chicken, rodent cells (e.g., mouse, rat). Typical mammalian cells include a myeloma cell, an Sp2 / 0 cell, a CHO cell, L cells, COS cells, fibroblasts, MDCK cells, HT29 cells, HEK cells, or a T84 cell. A preferred host cell is CHO-K1. Expression vectors can be introduced into host cells using conventional techniques, including transfection with calcium phosphate, nuclear microinjection, transfection with DEAE-dextran, fusion of bacterial protoplasts and electroporation.
[00151] Polymeric proteins can be prepared by methods that include (1) the preparation of a vector comprising the nucleic acid molecule that codes for the monomer unit; (2) transfection of a host cell with the vector; (3) culturing the host cell to provide expression; and (4) recovering the polymeric protein.
[00152] Polymeric protein can be recovered in the form of products of different molecular sizes. Typically, the desired polymeric protein comprising five, six or seven monomer units is responsible for at least 40% of the total protein composed of monomer units, by weight. Suitably, it accounts for at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the total protein composed of monomer units, by weight. The high proportion of monomer units that can be recovered in the form of the desired polymeric protein contributes to production efficiency.
[00153] If the polymeric protein is secreted by the host cell it can conveniently be recovered by affinity chromatography using its affinity for Fe binding agents, such as Protein A or Protein G columns, suitably Protein-G HiTrap (GE Healthcare). Proteins can be eluted from such low pH columns in neutral buffer. Dialysis can be performed subsequently to change the buffer. If the host cell does not secrete the polymeric protein, it can be recovered by cell lysis, followed by affinity chromatography.
[00154] The present invention will be further illustrated in the following examples, without any limitation thereto. Example 1: Production of a recombinant Fc polymeric protein
[00155] The DNA constructs were prepared as follows. The commercially available expression vector pFUSE-hlgG1-Fc2 was obtained from Invivogen, originated through Autogen BIOCLEAR, Wiltshire, United Kingdom. The expression vector comprises a coding sequence for an IL-2 signal sequence and, downstream of said, a coding sequence for the Fc portion of human IgG1. To generate a polymeric protein, two changes in the coding sequence of the human IgG1-Fc portion were made. The 18 amino acid terminal piece from IgM was sub-cloned to the C-terminal of the Fc portion, and an additional mutation was done in the Cv3 domain to convert residues 309 and 310 (in a whole EU) to cysteine and leucine, respectively.
[00156] In order to insert the IgM capping sequence into the commercially available vector, primers have been designed which, when ringed together, form a double stranded sequence with bases that tend to encode an Nhe1 restriction site to allow subcloning of the C-terminus of Fc. In order to maintain the reading frame of the protein encoded by the plasmid, an existing termination codon is removed, and to allow convenient restriction sites, an extra base of DNA has been inserted. The IgM capping sequence is preceded by a short 5 'that codes for the four amino acid ligand Leu-Val-Leu- Gly; the binder does not affect the function of the IgM cap.
[00157] Primers 1 and 2 (SEQ ID No. 1 and 2) were hybridized to each other through a temperature gradient to form the double IgM-cap chain containing the Nhe1 insert.
[00158] The pFUSE vector was then digested with the restriction enzyme Nhel, and the insertion of the IgM cap above linked to create an intermediate plasmid. In order to allow the IgM cap to be translated after the Fc region, the stop codon present was mutated in a subsequent step, through site-directed mutagenesis, using the Quick Change II kit (Stratagene, La Jolla, CA, USA ). Primers 3 and 4 (SEQ ID No. 3 and 4) are designed to remove this termination codon and create a site for the restriction enzyme Avr11 between the Fe region and the IgM cap. This created the plasmid called pFUSE-hlgG1-Fc-TP.
[00159] In human IgM, a cysteine at position 309 is involved in the formation of a disulfide bridge between two IgM monomers within the pentamer. In order to better mimic the human IgM protein sequence, primers 5 and 6 (SEQ ID NO: 5 and 6) were designed to introduce a cysteine residue at position 309, again through site-directed mutagenesis, as before . After aligning the nucleotide sequence encoding the human IgM protein sequence with that of human IgG1-Fc, it was further decided to replace the histidine residue at neighboring position 310 with a neutral leucine residue. The final plasmid incorporating both mutations was named pFUSE-hlgG1-Fc-TP-LH309 / 310CL.
[00160] Control plasmids were also prepared that encode human IgG1 Fc monomers without the mutations and the 309/310 cap.
[00161] The nucleic acid encoding the sequence of monomeric units that come together in polymers is SEQ ID No. 7.
[00162] The coding sequence has the following regions: 1-60 coding sequence for the IL2 signal peptide 61-753 sequence coding for the Fc region of human IgG1 325-330 cis-309 and Leu-310 mutations 754-810 coding sequence for IgM cap (including ligand region and termination codon)
[00163] The amino acid sequence of the monomeric units that come together in polymers is SEQ ID No. 8.
[00164] The amino acid sequence has the following regions: 1-20 IL2 signal peptide 21-247 human IgG1 Fc region 109-1 10 cis-309 and Leu-310 mutations 248-251 four 252-269 amino acid linker IgM finish
[00165] During expression, the signal peptide is cleaved IL2 and therefore, the final protein product has 249 amino acids.
[00166] Polymeric Fc proteins and monomeric Fc control proteins were prepared as follows. CHO-K1 cells (European Collection of Cell Cultures) were transfected by electroporation with plasmids and selected positive clones. The cells were cultured in complete DMEM medium supplemented with 10% ultra-low bovine IgG FCS, 100 IU / ml penicillin, and 100 gml-1 of streptomycin (PAA) at 37 ° C / 5% C02. Stable transfectants were selected in medium containing 400 μgrml-1 of Zeocin (Invivogen). Clones that secrete Fc fusion proteins were detected by Sandwich Enzyme-linked immunosorbent (ELISA) assay, using goat anti-human IgG-Fc antibody (Sigma-Aldrich: A0170). From large-scale cultures in DMEM supplemented with ultralow IgG containing FBS (Gibco), Fc fusion proteins were purified on protein-G-Sepharose (GE Healthcare, Little Chalfont, Bucks, UK). Fractions eluted from affinity purification were pooled and separated by high performance size exclusion chromatography on a Superdex-200 10 / 300GL column using an AKTAFPLC (GE Healthcare). The eluted fractions were compared to known standards for high PM gel filtration (BioRad). Protein integrity was verified by SDS-PAGE in native 6% Tris-glycine gels against SeeBlue2 pre-stained molecular weight markers (Novex-lnvitrogen).
[00167] Polymeric proteins were expressed as complexes of approximately 312 kD and about 100 kDa, consistent with expression as hexameric and dimeric entities. The proportion was about 90% versus 10% dimeric hexameric (w / w), according to size exclusion chromatography, as illustrated in Figure 1B. Example 2: Structural characterization of a recombinant Fc polymeric protein
[00168] Hexamérica-Fc were photographed by atomic force mode microscopy (AFM) in solution. Hexamérica-Fc form a highly uniform population of well-defined structures. The AFM images obtained showed the complex to be cylindrical in structure (18 ± 2 nm (n = 51) in diameter, 5.7 ± 0.4 Nm (n = 54) in height). Representative images are shown in Figure 1A. The images obtained are also consistent with the predicted dimensions from silicon modeling analyzes, also illustrated in Figure 1A.
[00169] AFM was performed as follows, and as described in Mekhaiel et al, 2011, um. The stock solutions of hlgG1-Fc-LH309 / 310CL-TP in IxHBSS buffer were diluted to 10 Mgml-1 in 0.2xHBSS buffer and then applied directly to a freshly cleaved fragment of muscovite mica. After incubation for 20 minutes, the sample was washed extensively with buffer to remove 0.2 x HBSS molecules not adsorbed. The samples were always in solution during transport to and from the image within the AFM. Imaging was performed in beat mode with an AFM multimode Nanoscope Ilia (Veeco, Santa Barbara, CA) using silicon nitride cantilevers (NP-S, Veeco Sondas, Santa Barbara, CA) with a constant source of 0.32N / m in 0.5 x HBSS buffer. The typical sweep rate and amplitude of initial oscillation were 3 Hz and 20nm, respectively, and the applied force was minimized to ~ 0.1 nN. The piezoscanner (9μm, scanner D, Veeco) was calibrated using mica and gold decision. All images reported were reproducible with different tips and different fast-scanning directions. All lateral dimensions were determined from the full width at half height.
[00170] Molecular modeling (in silico) was performed as follows, and as described in Mekhaiel et al, 2011, um. The monomer was constructed from the crystalline structure of the hlgG1 Fc-domain, using a random chain structure for both tailpiece and Hinge regions. Each monomer was minimized in energy before being assembled in the hexameric complex. All calculations were performed using VMD / NAMD energy using the CHARMM27 force field. NAMD was developed by the Theoretical Biophysics Group at the Beckman Institute for Advanced Science and Technology at the University of Illinois at Urbana-Champaign. For the construction of the hexamer, two experimental observations were obtained: on the one hand, the stoichiometry of the complex is finite (that is, oligomerization does not proceed indefinitely), and second, the oligomerization necessary for the Cys309 mutation. The observation suggests that there is some physical reason preventing further assembly, which was reasoned that, due to the complex of being ring-shaped, similar to those observed with natural IgM complexes. This latter observation suggests that the adjacent monomers are linked through these C309 residues. Because these bonds are possible in a ring-shaped complex, these residues must all be within a common plane, as is the case with homologous cysteine residues in the IgM pentamer. With these Fc-fusions, there are two types of oligomers that can satisfy these criteria: star-shaped, where the C309 plane is the same as the Fc-plane, and, barrel-shaped, where the C309 plane is perpendicular to the Fc -flat. Both models were examined to determine what produced a lower energy structure, subject to an additional criterion. Since these Fc fusions did not evolve to interact with each other, but rather because of the mutated C309 residues, we assume that the monomers do not undergo significant structural changes to form these bonds, and thus an energy penalty for such changes is attributed. This was achieved by limiting structural atoms within a harmonic well (spring constant, 1 kcal / mol / A2) centered on the positions of the monomeric structures minimized in energy. By monitoring the potential contribution of this constraint to the minimized total energy, as each conformation-mutated monomer from the monomeric form could be assessed. Without this restriction, the star-shaped and barrel-shaped structures, assessed over a range of initial distances C309-C309, were of similar minimized energy. However, with the restriction, at all distances, it was clear that the barrel-shaped structure required significantly less than the structural changes of the star-shaped model to form the C309-C309 disulfide bridge. Thus, without the constraint, the barrel-shaped structure achieves a minimized energy similar to the star-shaped structure, but requiring less changes to the monomeric shape. We therefore favor a barrel-shaped structure for hexamer. The final model was built at a distance C309-C309 closest to the initial one, in which there was not the least degree of structural changes in the monomeric structure. These calculations were performed without any of the easels attached to other monomers. With this model minimized, the easels were connected to another, randomly chosen, monomers within the complex, and then the entire complex was solvated with Tip3 water, minimized, and finally balanced, as assessed by the square root of the mean of the deviations from the protein skeleton. Example 3: The interaction of a polymeric Fc protein with components of the immune system 1. Interactions with Fc-receptors
[00171] Hexamerica Fc linked to all tested Fc receptors, namely, human FcyRI, FcyRIIA and FcyRIIB and mouse FcyRI and FcyRIIB. Hexamerica-Fc linked with greater affinity (in the nanomolar range) for low-affinity human FcyRs (FcyRIIA R131 and FcyRIIB) than dimers or monomers that bind to the same receptors in the micromolar range. This confirms that higher order polymers did not improve receptor binding kinetics that are known to be involved in the protection of ITP. In the analysis of SPR, hexameric-Fc bound to human FcyRI with a KA (1 / M) of 1.6x10 10. Dimers bound with a KA (1M) of 6.4 x 109. The binding of monomeric hexameric Fc or receptors Fc by ELISA is shown in Figure 2.
[00172] Another receiver that can play a role in ITP is FcyRIII (Park-Min et al, 2007). Hexamerica-Fc can be expected to bind human FcyRIII because the binding site for FcyRIII in IgG overlaps with FcyRIIB and FcyRI (Sondermann et al, 2001). The lower Hinge region of IgG plays a key role in the interaction with FcyRI and FcyRI I, and it seems likely that all FcyRs share a common way of interacting with IgG (Woof & Burton, 2004). In fact, it has been possible to produce complexes between the IgG Fc model and FcyRI and FcyRI I based on the crystalline structure of IgG Fc FcyRI II and, thus, to rationalize particular binding characteristics (Sondermann et al, 2001). The interaction of FcyRIII and the Fc-hexameric complex was modeled in silico using the crystal structure of FcyRIII. The model shows that the binding site on the FcyRIII hexamer is probably exposed, as it is clearly for the other FcyRs. Therefore, predict that FcyRIII would bind to hexamerica-Fc.
[00173] The binding of Fc proteins to Fc receptors was determined by enzyme linked immunosorbent assays (ELISA) as described below and in Mekhaiel et al, 1201. The microtiter wells (Nunc) were coated with titrated amounts of the Fc proteins ( 50-0.3 nM) in PBS or pH 9 carbonate buffer and incubated overnight at 4 ° C before blocking with 4% skim milk (Acumedia) for 1 h at room temperature (RT). The wells were washed four times with PBS / 0.005% Tween 20 (PBS / T) at pH 7.4, before the addition of GST or HIS-fused hFcyRI, hFcyRIIA, hFcyRIIB, mFcyRI or mFcyRIIB diluted in 4% skimmed milk PBS / 0.005% Tween 20 (PBS / T) at pH 7.4 and added to the wells. After incubation for 2 h at RT and washing as above, a polyclonal anti-GST antibody conjugated to goat HRP (1: 8000; GE Health Care) and incubated for 1 h at RT. The wells were washed as above, 100 μl of the TMB substrate (Calbiochem) was added to each well and incubated for 45 minutes before 100 μl of 0.25 M HCl was added. Absorbance was measured at 450 nm using a Sunrise TECAN spectrophotometer (TECAN, Maennedorf, Switzerland).
[00174] Binding to human FcyRI was also analyzed by surface plasma resonance analysis, exactly as described below in relation to FcRn. 2. Interactions with glycan receptors that are known to play a role in controlling autoimmunity
[00175] Human DC-SIGN and its rat homologue (SINAL-R1) have been shown to be involved in the control of ITP by binding the terminal sialic acid found in the glycane bound in N297 of IgG Fc (Anthony et al, 2011; Samuelsson et al, 2001). The sialylation of the Fc-hexameric protein was demonstrated as follows. Reduced proteins were run on 4-12% Bis-Tris gels and transferred to PVDF membranes and blocked with 1% Roche blocking reagent as previously described (Samuelsson et al, 2001). The membranes were then incubated with a dilution of biotinylated lectin bark from Sambucus nigra (Vector Laboratories) and developed with 1/200 streptavidin-HRP (Serotec). In silico modeling, it predicts that these terminal sialic acids found in hexameric Fc proteins would be available to bind to DC-SIGN or R1-SIGN (Figure 1). 3. Interactions with recently described receptors that may play a role in the control of autoimmune disease
[00176] Human B cell inhibitory receptors are important in the control of autoimmune disease (Pritchard & Smith, 2003). Two inhibitory receptors for IgG have been described on the surface of human B cells: FcyRllb (Daeron, et al, 1995) and more recently FcRL5 (Wilson et al, 2012). Although it is possible that FcRL5 is redundant with FcyRllb in human B cells, the potential for recruitment of simultaneous SHIP-1 by FcyRllb and SHP-1 for FcRL5 provides a substantial barrier to recurrent B cell activation. Interestingly, FcRL5 in innate B cells is also seen in MHC poxvirus class l-immunoevasins as, suggesting an important role in the regulation for FcRL5 immunity (Campbell et al, 2010). FcRL5 only binds IgG and not complexed monomeric IgG and therefore IVIG would be less likely that this receptor will bind with high avidity, although the polymeric fraction of IVIG can bind with high avidity. Taken together, FcyRllb FcRL5 and can limit B cell activation against chronic pathogens or auto-reactive antigen, and approaches that have the potential to target both receptors may prove beneficial in therapies aimed at controlling the activation of B cells.
[00177] Therefore, the hexameric-IgG1-Fc binding to human CD 19+ B cells was investigated (Figure 3). As a first step to assess the interaction of hexameric IgG1-Fc with the immune cells involved in autoimmune control, the ability of hexameric IgG1-Fc to bind human B cells by FACS analysis was investigated. We show that hexameric IgG1-Fc bound to CD19 + B cells purified from the peripheral circulation of healthy human volunteers. Flow cytometry was performed as described in Mekhaiel et al, 20 1-A. Human leukocytes were purified from whole blood by centrifugation on Polymorphoprep gradients, according to the manufacturer's instructions. 1 x 105 μl of cells were incubated with 200 FACS buffer (phosphate buffered saline, 0.2% bovine serum albumin, 5% goat serum) containing 50 [ig of hexa-Fc or only buffer for 1 h at room temperature. The cells were washed twice with FACS buffer and incubated for 1 h at 4 ° C with a 1/500 dilution of goat F (ab ') 2 anti-hlgG-Fc-phycoerythrin (PE) and goat isothiocyanate anti-hCD19 -fluorescein (FITC) - Abs conjugate (Southern Biotechnology) in 200 μl of FACS buffer. After washing with SCAF buffer, cells were analyzed on a FACScan (BD Biosciences). Data acquisition was performed using CELLQuest software (BD Biosciences) and analysis performed with FlowJo version 9.1. CD19 + B lymphocytes were gated taking into account their forward and lateral dispersion profiles, and binding of the Fc protein was detected with the secondary PE-labeled antibody. No binding of the secondary PE-labeled antibody was detected in the absence of the Fc protein. That Fc-hexamer was FcRL5 binding on the surface of human B cells was demonstrated by prior incubation of the cells with specific FcRL5 blocking monoclonal antibody 509F6, which ablated binding of the hexameric Fc protein. 4. Connection to neonatal FcRn
[00178] The ability of hexamerica-Fc to interact with the neonatal FcRn, responsible for maintaining the long half-life of Abs in the circulation (Mekhaiel et al, 2011a), was investigated. Although FcRn does not play a dominant role in ITP, it may play a role in controlling hexameric-mediated Fc from more chronic autoimmune disease, in which a long half-life for therapy is important. Although hexameric-Fc was unable to bind human FcRn which binds with nM affinity to rat FcRn (0.1-10 nm at pH 6), in line with the previous observation (Andersen et al, 2010). This suggests that minor modifications to the existing construct, for example, reversion of Leu310 to His310 may re-establish binding to human FcRn.
[00179] The binding of Fc proteins to FcRn was determined by surface plasma resonance analysis and, as described below and in Mekhaiel et al, 2011a. Plasmon Resonance Surface Analysis (SPR) were performed using a Biacore 3000 instrument (GE Healthcare). The flow cells of CM5 sensor chips were coupled with recombinant forms of soluble human or rat FcRn (hFcRn or mFcRn; ~ 2200-3000 RU) using amine coupling chemistry, as described in the protocol provided by the manufacturer. Coupling was performed by injecting 2 g of the protein in 10 mM sodium acetate pH 5.0 (GE Healthcare). Phosphate buffer (67 mM phosphate buffer, 0.15 M NaCl, 0.005% Tween 20) at pH 6.0, or HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA , 0.005% surfactant P20) was used as running buffer and dilution buffer. 100 nM of each of the Fc fusion proteins were injected over the immobilized receptor at a pH of 6.0 or pH 7.4, with a flow rate of 20 μl / min at 25 ° C. Surface regeneration was performed using injections of HBS-EP buffer at pH 7.4 (GE Healthcare). In all experiments, the data was adjusted to zero and the reference cell subtracted. The data were evaluated using the BIAevaluation 4.1 software (GE Healthcare). 5. Link to complement
[00180] Most of the Fc fusions studied to date do not bind C1q and are unable to activate the ensuing cascade complement (Czajkowsky et al, 2012). This inability of Fc-fusions to activate complement is probably due to the absence of residues in Fab fusion that contribute to the interactions of intact IgG with C1q (Gaboriaud et al, 2003). Because hexamerica-Fc lacks the Fab residues it is unlikely to be able to bind to C1q with the same affinity as with native antibodies. However, we did investigate the ability of hexameric -Fc to bind C1q and activate the classical pathway (Mekhaiel et al, 2011a). Hexamerica-Fc bound to C1q poorly compared to dimers, which in turn bound less than intact monomeric IgG, a gradation reflected in a proportional decrease in the detection of the C5-9 terminal complex. There was also a complete lack of an Arthus reaction, a type III hypersensitivity reaction initiated by the formation of immunocomplexes at the inoculation site, even when these reagents were administered to rats i.p. or when administered S.C. together with the alum, the observations made at the time of injection. The lack of complement activation is generally considered a desirable property of injected therapies intended for the treatment of autoimmune disease. However, binding, without the subsequent activation of the classic C1q pathway, can be advantageous, taking into account the finding that DC-SIGN, C1q, and gC1qR form a trimolecular receptor complex on the surface of immature monocyte-derived dendritic cells, that may be important in the control of autoimmune disease (Hosszu et al, Blood 2012; 120: 1228-1236).
[00181] The Arthus reaction involves the formation in situ of antigen / antibody complexes after intradermal injection of an antigen (as seen in passive immunity). If the animal / patient was previously sensitized (has circulating antibodies), an Arthus reaction occurs. Typical of most type III hypersensitivity mechanisms, Arthus manifests as local vasculitis, due to the deposition of IgG-based immune complexes in dermal blood vessels. Complement activation results mainly in the cleavage of soluble C3a and C5a anaphylotoxins, which boost the recruitment of PMN, as well as the degranulation of local mast cells (which requires the binding of the immune complex to FcyRIII), resulting in an inflammatory response. In addition, aggregation of processes related to immune complexes ensures on the walls of tissue vessels a local fibrinoid necrosis with overlapping thrombosis - aggravating ischemia. The end result is a localized area of redness, readily visible to the eye at the injection site where the duration typically lasts a day or two. Example 4: Hexamérica-Fc can restore platelet counts in a clinically relevant mouse model of ITP.
[00182] In a platelet depletion model used for ITP we can show that as little as 0.035 g / kg of hexameric Fc had a significant positive effect on platelet recovery compared to a similar dose of 0.035 g / kg of IVIG.
[00183] Balb / c mice were injected i.p. with Hexa-Fc at 0.035 g / kg, IVIG at 0.035 g / kg or 2 g / kg (Gammagard), or PBS in a final volume of 200 μl. One hour later ITP was induced in all mice by IP injection of 12.5 g specific IgG1-K for the β 3 chain / CD61 integrin mouse, clone 2C9.G2 (BD Pharmingen; Product #: 550541) in one volume 200μI of sterile PBS. Platelets were counted at 12h, 24h, 36h and 48h time points after treatment by flow cytometry using an anti-CD61 antibody conjugated to APC, n = 4 mice per group of 2 independent experiments. mean ± SD of the group were analyzed by repeated ANOVA using Dunnett's multiple comparison test in relation to the control group. Control vs hexamer: q = 3.13, * p <0.05; control vs IVIG: q = 8.66, *** P <0.0001. Data are averages of% of platelets ± SD. The mean% platelet counts in each group before exhaustion: hexamer group = 9.6% ± 1.2, high dose group IVIG = 9.4% ± 1.2, low dose group IVIG = 9.4% ± 1.2, the PBS group = 9.3% ± 1.2. Example 5: Treatment of patients with ITP
[00184] The treatment protocols for PTI with the disclosed hexameric Fc protein would be used in a tracking way to standard guidelines for ITP HIVIG therapy, as the Executive Committee of the American Society of Hematology practical guidance for the diagnosis and treatment of primary immunological thrombocytopenic purpura . See George, JN, et al. Idiopathic thrombocytopenic purpura: a practical guideline developed by explicit methods for the American Society of Hematology. Blood. July 1996. 1; 88 (1): 3-40. Alternatively, protocols for ITP may include an initial administration phase with dosages of about 0.1 to about 0.001 times the above treatment protocol dosages. The initial low dose phase is designed to minimize any short-term pro-inflammatory effects of Fc protein administration, while still sufficient to induce a long-lasting anti-inflammatory effect, which is subsequently reinforced and maintained by the second dosage. phase pattern described above. The reason for this alternative approach is that some polymeric proteins can have both a short-term inflammatory effect, as well as an anti-inflammatory effect through long-term decrease in FcyRlla expression. A low starting dose (or low starting doses) can be used to stimulate the long-term anti-inflammatory effect, while minimizing the short-term inflammatory effect.
[00185] The Fc protein would be administered intravenously and the effective dose of Fc protein is generally about 1% to about 20% of the effective dose of IVIG. The effective dose of ITP HIVIG is generally in the range of about 100 mg / kg to about 2 grams / kg administered every 10-21 days.
[00186] The intravenous formulation of Fc protein will be substantially the same as the FDA approved for HIVIG formulations, but may exclude the stabilizers present in some HIVIG formulations. Example 6: In silico models of Fc proteins
[00187] The ability of Fc proteins to modulate autoimmune or inflammatory diseases will depend on their ability to interact with the appropriate components of the immune system, while no unwanted reactions occur. This in turn will depend on the structure of the Fc protein. In the case of oligomers, the tertiary and quaternary structure will have an important impact on the availability of protein surfaces and exposed portions for binding to components of the immune system. The uniform and defined tertiary and quaternary structure can reduce the risk of undesirable reactions that could otherwise be caused by minority species in complex mixtures. The uniform and defined structure also allows easy control of the quality of medicines.
[00188] The invention has shown that hexamérica-Fc described here has a uniform and regular cylindrical shape, (Example 2), binds to several desirable receptors and components of the immune system (Example 3) and does not activate the complement (Example 3 ). These results are consistent with the in silico model of hexamerica-Fc described in Example 1.
[00189] US 2010/0239633 (University of Maryland, Baltimore; Strome) discloses IVIG replacement compounds that comprise multiples attached to the Fc moieties. Linear or pentameric arrangements are foreseen. Pentamerization is anticipated by the addition of the Cμ4 domain of IgM and the J chain (as represented in Figs 10A-D and on page 18 preferred embodiments of US 2010/0239633). In native IgM, the Cμ4 domain of the heavy chain constant region binds to the J chain to effect pentamerization. Extensive in silico modeling / molecular dynamics simulations have been carried out to determine whether the structures proposed in US 2010/0239633 are viable.
[00190] In the structures shown in Fig 10A-D of US 2010/0239633, a Cμ4 domain is linked through its N terminal to the C terminal of a domain with Cy3 (i.e., the CH3 domain of an IgG molecule), and the Cy3 domain is linked to a Cy2 domain. As domains of the monomers they are described as dimerization together to form monomeric arms of the higher order structure. However, this presents a problem for conformational structures.
[00191] In native immunoglobulins, the domains Cμ3, Cμ4, CY2 and Cy3 form homodimers that are in the form of 'v', or inverted 'v', that is, TV. A monomer forms the so-called 'V part of' V and a monomer as' part of 'the T V; or a monomer constitutes the' / 'part of TV and a monomer as forming' part V of TV. Dimmers Cy3 or Cμ4 are 'v' shape, while Cμ3 and Cy2 are TV-shaped. In a native immunoglobulin, V and TV form alternative dimers. For example, the N-terminals of the Cy3 v-shaped domains are positioned to be contiguous with the C-terminals of Cy2 in the form of TV. Likewise, the N terminals of the 0μ4 'v-shaped domains are positioned to be contiguous with the C terminals of the Cμ3 TV-shaped domains.
[00192] In the monomers represented in US2010 / 0239633, the "V-shaped Cy3 domain is directly linked to another" 0μ4 V-shaped domain. This is a problem since the C3-terminal residues of Cy3 are close to each other while the N-terminal residues of the CP4 protein are distant from each other. It is likely that a linker would be needed to join the domains together. The most likely result of this structural limitation is that, for the lengths of binders that would be most likely to be employed, the 0μ4 domains would have to be rotated by 90 degrees with respect to the OY2-OY3 domains. The Oμ4 domain would be under rotational stress. The intra-monomer disulfide bonds within the Cy3s and the intra-monomer disulfide bonds within the Cp4s are in competition with each other. One or both will be weakened. Therefore, the protein may be unstable or it may not fold properly.
[00193] To mimic the normal orientation of the OY2-OY3 domains with respect to the Ci4 domain as found in 0Y2-OY3 — Oμ4, IgM domains would require ligands in excess of 20 amino acids. The introduction of linkers (unspecified sequences) poses significant problems for these structures, not least of which is the increased likelihood of unwanted immunogenicity. There would also be a negative impact on manufacturing capacity and an increased likelihood of proteolytic cleavage. This geometric limitation of the OY3-Oμ4 bond has significant consequences for the oligomer that makes it fundamentally different from the hexametric-Fc protein.
[00194] The connection sequences between the Fcy and Wμ domains are critical. However, although the term "binder" connotes flexibility, there would be limitations to this flexibility in the present case. With a single point connection, such as between a Fab and an Fc region (at the joint), there really cannot be a great deal of rotation flexibility. The difference in the embodiments as represented in US 2010/0239633 is that there are two connection sites, one between each OY3-Oμ4 pair. Thus rotations of one Fc in relation to the other Fc results in a joint twist of two connecting elements, which is energetically unfavorable, increasing the risk of bending errors or instability.
[00195] It is unlikely that the structures described in US 2010/0239633 would form well-defined oligomers (as in IgM), with their connections only by the Cμ4 domain. The C-terminal tails of 18 amino acids found in all polymeric antibodies, being unstructured, cannot be responsible for the regular pentameric and / or hexameric structure - which must come from the presence of an Ig-domain (s) structured within the Fc . With polymerization occurring across structured Ig regions, stoichiometry is defined by the size and shape of the structured regions. Interactions between random unstructured regions must necessarily lead to a wide range of polymeric sizes. Thus, to determine whether the (few) inter-monomer contacts in the Cμ4 domains observed in IgM would be sufficient to maintain pentameric stoichiometry, we performed extensive molecular dynamics simulations of only these structured regions (ie, lack of ends). During the simulations, the structured regions of Cμ4 quickly lose their pentameric disposition, and begin (apparently) clump together (not specifically). The regular pentameric / hexameric structure is therefore very unlikely. This is to be contrasted with native IgM, in which both the Cμ4 and Cμ3 domains contribute to the regular pentameric / hexameric structure. Although US 2010/0239633 suggests the use of J chain in order to facilitate polymerization, which would not be expected to be successful. The J chain can stabilize immunoglobulin structures, but it is not possible to convert a monomer or oligomer from a higher order dimer. For example, the J-chain can facilitate dimerization of IgA, but it is not sufficient to form higher-order oligomers as in IgM, because IgA Ig domains are not suitable for the highest order of polymerization.
[00196] The most likely structure that could be formed from polymerization can occur through random tails is a 3D star-shaped structure, in which the tails are located inside the central crown and the Fc regions of the monomers to project radially to outside, in 3D. With polymerization taking place through unstructured peptides, there can be no well-defined stoichiometry and everything that can fit in three dimensions will occur. There is a possibility of dimers probably up to 20-mers depending on how many connecting tails can fit within the central sphere of interacting tails.
[00197] It is also important to note that, experimentally, it was found that the addition of an 18 amino acid cap to human IgG1 Fc is insufficient to induce multimerization (Mekhaiel, 2011a). We have also shown that with constant domain changes, the presence of the Cμ4 domain is unable to induce the formation of pentamer and / or hexamer (Ghumra et al, 2009). Thus, we consider the possibility that structures in US2010 / 0239633 may actually undergo polymerization, either by Cμ4 domains or by tails, as unlikely.
[00198] Even if the predictions resulting from the models are wrong and the Cμ4 domains of the monomers are able to interact to form IgM-like structures, the resulting structures would be very different for hexamerica-Fc. Assuming that Cμ4 interacts as they do in IgM (which is assumed by US 2010/0239633), the need for 0μ4 domains to be rotated 90 degrees with respect to OY2-OY3 domains would make OY2- OY3 to (largely) 'face' each other in any oligomer that was able to form from the monomers. That is, the OY2-OY3 side would have to face directly from the opposite side of OY2-OY3 on the neighboring arm. The overall structure would look like a closed umbrella. In contrast, in hexameric-Fc protein, these sides form the walls of the 'drum' and therefore do not face each other, but face outward. The receptors that can easily bind to these sides in the hexameric-Fc protein cannot therefore bind to the US2010 / 0239633 oligomers. The sugary regions in the OY2 OY3-FC unit are critical for binding in both Fc and sugary- receivers. In hexamérica-Fc, the sugary regions stay out, so that the receptors that bind to the sugary regions can bind. In contrast, in the US2010 / 0239633 oligomers, the receptors for these glycans would have to stay between the monomers, and, while not impossible, this would nevertheless have an impact on the overall binding forces between the oligomer and critical receptors. While a single monomer of the US 2010/0239633 oligomer can be targeted so that a single pair of Cv2-Cy3 domains is accessible to receptors, it is not clear whether more than one other monomer in the oligomer can bind to a different receptor of the same surface, due to geometric considerations described above and the physical size of the monomers. Thus, it is not clear whether the US2010 / 0239633 oligomer can bind to more than one / two receivers simultaneously. Similar issues could arise from the use of alternative polymerization of constant domains, for example, IgA. Thus, despite the presence of several monomer units in the structures designed in US 2010/0239633, binding to critical Fc receptors and other components of the immune system may be no more avid than what is observed with monomeric or dimeric immunoglobulins, if such structures can. form at all.
[00199] This building block strategy seriously limits the possibilities for interaction of the structures described in US 2010/0239633. More problematic is that, even though these molecules can be shown to multimerize, the presence of the Cμ4 domain will have a serious impact on biodistribution and on possible in vivo interactions from these chimeric molecules that are expected to be significantly different from those observed with hexamérica-Fc, as described in this patent application. These are summarized in detail in Table 1. Table 1: Differences between hexamerica-Fc and predicted structures US2010 / 0239633





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权利要求:
Claims (6)
[0001]
1. Use of an effective amount of a polymeric protein comprising six units of polypeptide monomer characterized by the fact that it is for the manufacture of a medicament for the treatment of a mammalian subject for an autoimmune or inflammatory disease; wherein each polypeptide monomer unit consists of an Fc receptor binding portion consisting of two heavy chain constant region immunoglobulins G; wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue at position 309 according to the EU numbering system which is linked via a disulfide bond to a cysteine residue of an immunoglobulin G of constant chain region weighed from an adjacent polypeptide monomer unit; and in which an end-end region is fused to each of the two regions contained in the immunoglobulin G heavy chain, where the end-end region is the μ-cap and facilitates the assembly of the monomer units within a polymer; where the monomer unit comprises a human IgG1-Fc region consisting of residues 21-247 of SEQ ID NO: 8 (except position 110 which has a His and not a Leu), and the μ-tail consists of residues 252-269 of SEQ ID NO: 8.
[0002]
2. Use according to claim 1, characterized by the fact that the end-region is an IgM end-region.
[0003]
3. Use according to claim 1, characterized by the fact that each of the immunoglobulin G heavy chain constant regions comprises an amino acid sequence comprising a cysteine residue at position 309 according to the EU numbering system, and preferably also a leucine residue at position 310, such as residues 21-247 of SEQ ID NO: 8.
[0004]
4. Use according to claim 1, characterized by the fact that the autoimmune or inflammatory disease is treatable with intravenous immunoglobulin (IVIG).
[0005]
5. Use according to claim 1, characterized by the fact that the autoimmune or inflammatory disease is an autoimmune cytopenia, idiopathic thrombocytopenic purpura, rheumatoid arthritis, systemic lupus erythematosus, asthma, Kawasaki disease, Guillain-Barre syndrome, syndrome Stevens-Johnson syndrome, Crohn's colitis, diabetes, chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, autoimmune disease Factor VIII, dermatomyositis, vasculitis and uveitis or Alzheimer's disease.
[0006]
Use according to claim 1, characterized in that each unit of polypeptide monomer consists of an Fc receptor binding portion consisting of two immunoglobulin G heavy chain constant regions; and, optionally, a polypeptide linker that links the two constant regions of the immunoglobulin G heavy chain as a single Fc chain.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2019-08-13| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-10-08| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-03-31| B25A| Requested transfer of rights approved|Owner name: CSL BEHRING LENGNAU AG. (CH) |

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2020-05-19| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-08-25| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-10-20| B09W| Decision of grant: rectification|Free format text: RETIFICA-SE O DEFERIMENTO NOTIFICADO NA RPI 2590 DE 25/08/2020. |

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2021-01-12| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 17/10/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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PCT/GB2012/052561|WO2014060712A1|2012-10-17|2012-10-17|Immunomodulatory proteins|

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