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    • 专利摘要:
    fusion proteins of natural human protein fragments to create orderly multimerized immunoglobulin fc compositions. The present invention involves a series of fully recombinant multimerized forms of immunoglobulin fc which thus present polyvalent immunoglobulin fc to immune cell receptors. fusion proteins exist as both homodimeric and highly ordered multimeric fractions, called stradomers. compared to the homodimeric fraction, purified multimeric stradomers have higher affinity and avidity for slower dissociating fc rs and are useful in disease treatment and prevention. The present invention demonstrates that directly linking the fc regions of igg1 to the multimerization domains leads to enhanced multimerization and biological activity.
    公开号:BR112013002074B1
    申请号:R112013002074-1
    申请日:2011-07-28
    公开日:2021-09-14
    发明作者:David S. Block；Henrik Olsen
    申请人:Gliknik Inc；
    IPC主号:
    专利说明:

    CROSS REFERENCE TO RELATED ORDERS:
    This application claims priority from Provisional Application US 61/368,465, filed July 28, 2011, the contents of which are incorporated herein by reference in their entirety.
    Description of the Electronically Submitted Text File
    The contents of the attached electronically-submitted text file are incorporated by reference in their entirety: A copy in computer-readable format of the Sequence Listing (filename: GLIK_006_01US_SeqList_ST25.txt, recorded data: July 26, 2011, file size 61 kilobytes ). Field of Invention
    This invention relates generally to the fields of immunology, autoimmunity, inflammation, and tumor immunology. More specifically, the present invention relates to biologically active biomimetic molecules that comprise naturally linked immunoglobulin Fc domains, compositions that comprise such biomimetic molecules, and methods for making and using such biomimetic molecules.
    The invention also relates to the treatment and prophylaxis of pathological conditions mediated by lymphocytes, NK cells, monocyte-derived cells and immune cells that interact with monocyte-derived cells, and more particularly to the use of functional stabilized portions of IgG Fc fragments linked for such treatment and prophylaxis.
    Immunoglobulin products from human plasma have been used since the early 1950s for the treatment of immunodeficiency disorders and more recently and more commonly for autoimmune and inflammatory diseases.
    Initially, immunoglobulin products were administered by intramuscular injection. More recently, intravenous immunoglobulin (IVIG) has been used and was initially shown to be effective in the treatment of idiopathic thrombocytopenic purpura (ITP) autoimmune disease (Imbach P, Barandun S, d'Apuzzo V, et al: High-dose intravenous gammaglobulin for idiopathic thrombocytopenic purpura in childhood Lancet 1981 Jun 6; 1(8232): 1228-31). Human IVIG (herein referred to as "hIVIG") is a formulation of purified, sterile immunoglobulin G (IgG) products made from pooled human plasma that typically contains more than 95% unmodified IgG, with only small and variable amounts of immunoglobulin A (IgA) or immunoglobulin M (IgM) (see, for example, Rutter A, Luger TA: High-dose intravenous immunoglobulins: an approach to treat severe immune-mediated and autoimmune diseases of the skin. J Am Acad Dermatol 2001 Jun;44(6):1010-24). Today, the most common single clinical use of hIVIG is in the treatment of Chronic Inflammatory Demyelinating Polyneuropathy.
    Although hIVIG has been an effective clinical treatment, there are several drawbacks to treatments with hIVIG, including the possibility of inadequate sterility, the presence of impurities or infectious agents, including viruses and prions, the lack of availability of this aggregated human blood product, batch variation a batch, high cost, high protein load that affects kidney function, and long administration time, usually for many hours and sometimes two consecutive days a month. In particular, hIVIG preparations can vary greatly in their immunoglobulin A (IgA) content, which may be a concern because IgA can cause allergic and anaphylactic reactions in IgA-deficient receptors. In view of the negative aspects of hIVIG, there is a need for improved means of treating autoimmune and inflammatory diseases and, in particular, a need for an abundant source of recombinantly produced product with at least equivalent efficacy, greater potency. , shorter administration time and greater purity.
    Furthermore, several pathological conditions of a wide variety of types are mediated by cells derived from monocytes, lymphocytes and NK cells. A new therapeutic and/or prophylactic agent for use in many, if not all, of these conditions would meet an important unmet clinical need and be commercially valuable.
    Many of the immunoregulatory properties of hIVIG reside in the Fc domain of IgG molecules. For example, in murine models of ITP, both unmodified hIVIG and the Fc fragment individually demonstrate therapeutic efficacy in restoring platelet counts, whereas isolated hIVIG Fab fragments are not therapeutic (Samuelsson, A., Towers, TL & Ravetch, JV Anti-inflammatory Activity of HIV Mediated Through the Inhibitory Fc Receptor. Science 291, 484-486 (2001)). In addition, Fc, but not Fab fragments of hIVIG, is also therapeutically effective in the treatment of idiopathic thrombocytopenic purpura in childhood and adults (Follea, G. et al. Intravenous plasmin-treated gamma globulin therapy in idiopathic thrombocytopenic purpura. Nouv Rev. Fr Hematol 27, 5-10 (1985); Solal-Celigny, P., Bernard, J., Herrera, A. & Biovin, P. Treatment of adult autoimmune thrombocytopenic purpura with high-dose intravenous plasmin-cleaved gammaglobulins. Haematol 31, 39-44 (1983); Debre, M. & Bonnet, M.-C. Infusion of Fc gamma fragments for treatment of children with acute immune thrombocytopenic purpura. Lancet 342, 945-49 (1993); Burdach, SE , Evers, K. & Geurson, R. Treatment of acute idiopathic thrombocytopenic purpura of childhood with intravenous immunoglobulin G: Comparative efficacy of 7S and 5S preparations. J Pediatr 109, 770-775 (1986)).
    In addition to the family of classical Fc gamma receptors, which can be distinguished into activating and inhibitory members, the neonatal Fc receptor (FcRn), which belongs to the major histocompatibility class I (MHC-I) and SignR1/DC family of molecules - SIGN, which belong to the C-type lectin family, can bind to the Fc fragment of IgG (Nimmerjahn and Ravetch, Antibody-mediated modulation of immune responses, Immunological Reviews, 236: 265-275 (2010). , there are Fc gamma receptor-like receptors to which immunoglobulin Fc molecules bind and exert the physiological effect (Davis RS. “Fc Receptor-like molecules” Annu. Rev. Immunol. 2007. 25:52560). hIVIG is initially mediated through the Fc gamma receptor (FCYR) and relies on the parallel communication of Dendritic Cell (DC) macrophages for its long-term tolerogenic effects. FcYRIIIa plays an indispensable role in the initiator phase and FcYRIIb is required for the effect phase tor in ITP murine models (Samuelsson, A., Towers, T.L. & Ravetch, J.V. Anti-inflammatory Activity of hIVIG Mediated Through the Inhibitory Fc Receptor. Science 291, 484-486 (2001); Siragam, V. et al. Intravenous immunoglobulin ameliorates ITP via activating FCY receptors on dendritic cells. Nat Med 12, 688 (2006)). Likewise, studies in humans demonstrate that anti-FcY receptor antibodies are effective in the treatment of refractory ITP (Clarkson, S. et al. Treatment of refractory immune thrombocytopenic purpura with an anti-Fc gamma-receptor antibody. N Engl J Med 314, 1236-1239 (1986)). Importantly, long-term tolerogenic effects are mediated by cell-cell interactions, as adoptive transfer of DCs treated with hIVIG is effective in treating murine models of ITP (Siragam, V. et al. Intravenous immunoglobulin ameliorates ITP via activating FCY receptors on dendritic cells. Nat Med 12, 688 (2006)).
    The immunomodulatory effects of hIVIG require aggregation of FCYR. FcR aggregation is mediated by IgG "dimers" present in hIVIG (5-15% of total hIVIG) (Bleeker, WK et al. activating factor acetylhydrolase. Blood 95, 1856-1861 (2000)). For example, the known clinical immunocirculatory hypotensive effects of IVIG correlates with the presence of "dimers" in IVIG (Kroez M et. al. Hypotension with Intravenous Immunoglobulin Therapy: importance of pH and dimer formation. Biologicals 31 (2003) 277-286 .). For example, in a murine model of ITP, treatment with hIVIG with a high platelet count content enhanced with "dimers" (dimers of whole homodimeric immunoglobulin molecules) while the "monomers" of hIVIG (homodimeric immunoglobulin molecules) were not effective ( Teeling, JL et al. Therapeutic efficacy of intravenous immunoglobulin preparations depends on the immunoglobulin G dimers: studies in experimental immune thrombocytopenia. Blood 98, 1095-1099 (2001)). Furthermore, despite the fact that ion exchange resin and polyethylene glycol fractionation are routinely used in the manufacture of hIVIG to remove IgG aggregates, the clinical efficacy of hIVIG correlates with the presence of aggregates in patient serum (Augener, W., Friedman, B. & Brittinger, G. Are aggregates of IgG the effective part of high-dose immunoglobulin therapy in adult idiopathic thrombocytopenic purpura (ITP) Blut 50, 249-252 (1985)). Importantly, the percentage of dimers also correlates with vasoactive side effects, which are treatable with acetylhydrolase (Bleeker, WK et al. Blood 95, 1856-1861 (2000)). SUMMARY OF THE INVENTION
    There is a need for an alternative to IVIG that solves the problems of protein loading, inconvenient patient dosing, infectious risk, IgA anaphylaxis, and limited availability while maintaining and enhancing the effectiveness of the IVIG aggregate fraction. The present invention relates to biomimetic biologically active fusion protein molecules comprising human immunoglobulin Fc and a single naturally occurring multimerization domain, compositions comprising the same, and methods of using the same. These biomimetics have wide application in the treatment of immunological and inflammatory disorders including, but not limited to, autoimmune diseases as well as hIVIG once these biomimetics have been modeled. In addition, some of these biomimetics also have utility as laboratory reagents, for example, for use in immunological assays to test immune cell function, in disease diagnosis, and in blocking non-specific Fc binding in antibody-based immunoassays. Furthermore, the biomimetics and compositions of the present invention have the advantage of overcoming the aforementioned limitations of hIVIG, as well as the multimerization limitations of precursor biomimetic stradomers.
    WO 2008/151088 discloses the use of linked immunoglobulin Fc domains to create orderly multimerized immunoglobulin Fc biomimetics of hIVIG (biologically active ordered multimers known as stradomers) for the treatment of pathological conditions, including autoimmune diseases and other inflammatory conditions. See WO 2008/151088, incorporated by reference in its entirety. The disclosed molecules were designed to contain sequences foreign to native immunoglobulin Fc, including the restriction sites and affinity tags on the immunoglobulin Fc monomer. These foreign sequences accounted in total for a small fraction of the total amino acid composition of stradomer (approximately 16 total amino acids) and were placed between separable domains and with distinct functions, i.e., FcR binding function and multimerization function. In general, it is common practice to include short binding sequences between domains with independent structures and/or functions in order to avoid any steric restrictions or to diminish the independent functions of the flanking domains. However, as disclosed herein, removal of these short segments can provide a dramatic enhancement of multimer formation, receptor binding, and/or biological activity in general.
    In one embodiment, the present invention relates to a stradomer unit comprising a leader sequence, an IgG1 Fc domain and a multimerization domain. In another embodiment of the present invention, the leader sequence is directly linked to the Fc domain of IgG1 and the Fc domain of IgG1 is directly linked to the multimerization domain.
    In another embodiment, the present invention relates to a stradomer unit wherein the IgG1 Fc domain amino acid sequence is at least 80% homologous to SEQ ID NO: 2. In another embodiment the Fc domain amino acid sequence of IgG1 is at least 90% homology to SEQ ID NO: 2. In yet another embodiment, the amino acid sequence of the Fc domain of IgG1 is at least 95% homology to SEQ ID NO: 2. In yet another embodiment , the amino acid sequence of the Fc domain of IgG1 is at least 99% homologous to SEQ ID
    NO: 2. In another embodiment the IgG1 Fc domain amino acid sequence of SEQ ID NO: 2 is directly linked to a leader sequence and/or a multimerization domain. In some embodiments, the stradomer unit of the present invention binds to FcYRIIIa or DC-SIGN/SIGN-R1 when multimerized.
    In another embodiment, the present invention relates to a stradomer unit wherein the amino acid sequence of the multimerization domain is at least 80% homologous to SEQ ID NO: 3. In another embodiment, the multimerization domain is at least 90% homology to SEQ ID NO: 3. In yet a further embodiment, it is a stradomer unit of the present invention wherein the amino acid sequence of the multimerization domain is at least 95% homology to SEQ ID NO: 3 In yet another embodiment, it is a stradomer unit of the present invention wherein the amino acid sequence of the multimerization domain is at least 99% homologous to SEQ ID NO: 3. In another embodiment, the multimerization domain is capable of multimerize the stradomer units. In another embodiment, the multimerization domain is directly linked to the carboxy terminus of the IgG1 Fc domain. In some embodiments, the multimerization domain is directly linked to the amino terminus of the Fc domain of IgG1.
    In another embodiment, the present invention relates to a stradomer unit wherein the amino acid sequence of the multimerization domain is at least 80% homologous to SEQ ID NO:5. In another embodiment, the multimerization domain is at least 90 % Homology to SEQ ID NO:5 In yet another embodiment, is the stradomer unit of the present invention wherein the amino acid sequence of the multimerization domain is at least 95% homology to SEQ ID NO:5. a further embodiment is the stradomer unit of the present invention wherein the amino acid sequence of the multimerization domain is at least 99% homologous to SEQ ID NO: 5. In another embodiment, the multimerization domain is capable of multimerizing the stradomer units. In another embodiment, the multimerization domain is directly linked to the carboxy terminus of the IgG1 Fc domain. In some embodiments, the multimerization domain is directly linked to the amino terminus of the Fc domain of IgG1.
    In another embodiment, the present invention relates to a stradomer unit wherein the amino acid sequence of the multimerization domain is at least 80% homologous to SEQ ID NO: 26. In a further embodiment, the multimerization domain is at least 80% homologous to SEQ ID NO: 26. minus 90% homology to SEQ ID NO: 26. In yet another further embodiment, is the stradomer unit of the present invention wherein the amino acid sequence of the multimerization domain is at least 95% homology to SEQ ID NO: 26. In yet another embodiment, the stradomer unit is the present invention wherein the amino acid sequence of the multimerization domain is at least 99% homologous to SEQ ID NO: 26. In another embodiment, the multimerization domain is able to multimerize the stradomer units. In another embodiment, the multimerization domain is directly linked to the carboxy terminus of the IgG1 Fc domain. In some embodiments, the multimerization domain is directly linked to the amino terminus of the Fc domain of IgG1.
    In one embodiment, the present invention relates to a stradomer unit comprising a leader sequence directly linked to an IgG1 Fc domain, which is directly linked to an IgG2 hinge multimerization domain, wherein the IgG2 hinge creates multimers of stradomer units. In one embodiment, the amino acid sequence of the stradomer unit is at least 80% homology to SEQ ID NO: 4 In another embodiment, the amino acid sequence of the stradomer unit is at least 90% homology to SEQ ID NO. : 4. In yet another embodiment, the stradomer unit amino acid sequence is at least 95% homologous to SEQ ID NO: 4. In yet another embodiment, the stradomer unit amino acid sequence is at least 99% homology to SEQ ID NO: 4. In another embodiment, the leader sequence (SEQ ID NO: 1) is cleaved from the mature protein.
    In one embodiment, the present invention relates to a stradomer unit comprising a leader sequence directly linked to an IgG1 Fc domain, which is directly linked to an isoleucine zipper multimerization domain, wherein the isoleucine zipper creates multimers of the stradomer units. In one embodiment, the amino acid sequence of the stradomer unit is at least 80% homology to SEQ ID NO: 10 In another embodiment, the amino acid sequence of the stradomer unit is at least 90% homology to SEQ ID NO. : 10. In yet another embodiment, the stradomer unit amino acid sequence is at least 95% homologous to SEQ ID NO: 10. In yet another embodiment, the stradomer unit amino acid sequence is at least 99% homology to SEQ ID NO: 10. In another embodiment, the leader sequence (SEQ ID NO: 1) is cleaved from the mature protein.
    In one embodiment, the present invention relates to a stradomer unit comprising a leader sequence directly linked to an IgG1 Fc domain that is directly linked to a multimerization domain, wherein the GPP multimerization domain creates multimers of the GPP units. stradomer. In one embodiment, the amino acid sequence of the stradomer unit is at least 80% homology to SEQ ID NO: 27. In another embodiment, the amino acid sequence of the stradomer unit is at least 90% homology to SEQ ID NO. :27. In yet another embodiment, the stradomer unit amino acid sequence is at least 95% homologous to SEQ ID NO: 27. In yet another embodiment, the stradomer unit amino acid sequence is at least 99% homology to SEQ ID NO: 27. In another embodiment, the leader sequence (SEQ ID NO: 1) is cleaved from the mature protein.
    In one embodiment, the present invention relates to a stradomer unit comprising a leader sequence directly linked to an IgG2 hinge multimerization domain which is, in turn, directly linked to an IgG1 Fc domain, wherein the hinge of IgG2 creates multimers of the stradomer units. In one embodiment, the amino acid sequence of the stradomer unit is at least 80% homology to SEQ ID NO: 8. In another embodiment, the amino acid sequence of the stradomer unit is at least 90% homology to SEQ ID NO: 8. In yet another embodiment, the stradomer unit amino acid sequence is at least 95% homologous to SEQ ID NO: 8. In yet another embodiment, the stradomer unit amino acid sequence is at least 99% of homology to SEQ ID NO: 8. In another embodiment, the leader sequence (SEQ ID NO: 1) is cleaved from the mature protein.
    In one embodiment, the present invention relates to a stradomer unit comprising a leader sequence directly linked to an isoleucine zipper multimerization domain which is, in turn, directly linked to an IgG1 Fc domain, wherein the zipper of isoleucine creates multimers of the stradomer units. In another embodiment, the amino acid sequence of the stradomer unit is at least 80% homology to SEQ ID NO: 9 In another embodiment, the amino acid sequence of the stradomer unit is at least 90% homology to SEQ ID NO. : 9. In yet another embodiment, the stradomer unit amino acid sequence is at least 95% homologous to SEQ ID NO: 9. In yet another embodiment, the stradomer unit amino acid sequence is at least 99% homology to SEQ ID NO: 9. In another embodiment, the leader sequence (SEQ ID NO: 1) is cleaved from the mature protein.
    In one embodiment, the present invention relates to a stradomer unit comprising a leader sequence directly linked to a GPP multimerization domain which is, in turn, directly linked to an IgG1 Fc domain, wherein the GPP multimerization domain creates multimers from the stradomer units. In one embodiment, the stradomer unit amino acid sequence is at least 80% homology to SEQ ID NO: 28. In another embodiment, the stradomer unit amino acid sequence is at least 90% homology to SEQ ID NO: 28. In yet another embodiment, the stradomer unit amino acid sequence is at least 95% homologous to SEQ ID NO: 28. In yet another embodiment, the stradomer unit amino acid sequence is at least 99% of homology to SEQ ID NO: 28. In another embodiment, the leader sequence (SEQ ID NO: 1) is cleaved from the mature protein.
    In another embodiment, the present invention relates to a stradomer unit comprising a leader sequence directly linked to an IgG1 Fc domain which, in turn, is directly linked to a multimerization domain, wherein the multimerization domain creates multimers of the stradomer units. In another embodiment, the multimerization domain creates higher order multimers from the stradomer units. In yet another embodiment, at least about 35% of the resulting stradomer composition contains multimers of the stradomer units. In yet another embodiment, at least about 55% of the resulting stradomer composition contains multimers of the stradomer units. In yet another embodiment, at least about 65% of the resulting stradomer composition contains multimers of the stradomer units. In yet another embodiment, at least about 70% of the resulting stradomer composition contains multimers of the stradomer units. In yet another embodiment, at least about 75% of the resulting stradomer composition contains multimers of the stradomer units.
    In a further embodiment, the present invention relates to a stradomer unit comprising a leader sequence, an IgG1 Fc domain with one or more mutations, and a multimerization domain. In another embodiment of the present invention, the leader sequence is directly linked to the Fc domain of IgG1 with one or more mutations and the Fc domain of IgG1 with one or more mutations is directly linked to the multimerization domain.
    In one embodiment, the present invention relates to a stradomer unit wherein the amino acid sequence of the stradomer unit comprises SEQ ID NO: 20. In a further embodiment, the invention relates to a cluster stradomer comprising at least two stradomer units comprising SEQ ID NO: 20. In another embodiment, the leader sequence (SEQ ID NO: 1) is cleaved from the mature protein.
    In one embodiment, the present invention relates to a stradomer unit wherein the amino acid sequence of the stradomer unit comprises SEQ ID NO: 24. In a further embodiment, the invention relates to a cluster stradomer comprising at least two stradomer units comprising SEQ ID NO: 24. In another embodiment, the leader sequence (SEQ ID NO: 1) is cleaved from the mature protein.
    In one embodiment, the present invention relates to a stradomer unit wherein the amino acid sequence of the stradomer unit comprises SEQ ID NO: 21. In a further embodiment, the invention relates to a cluster stradomer comprising at least two stradomer units comprising SEQ ID NO:21. In another embodiment, the leader sequence (SEQ ID NO:1) is cleaved from the mature protein.
    In one embodiment, the present invention relates to a stradomer unit comprising a leader sequence directly linked to a multimerization domain, wherein the multimerization domain is directly linked to an IgG1 Fc domain that lacks the hinge domain of IgG1, where the multimerization domain creates multimers of the stradomer units. In another embodiment, the present invention relates to a stradomer unit comprising a leader sequence directly linked to a multimerization domain, wherein the multimerization domain is directly linked to a portion of an IgG1 Fc that is capable of binding to an FcR or in which the multimerization domain directly linked to a portion of an IgG1 Fc together are capable of binding to an FcR.
    In one embodiment, the present invention relates to a stradomer unit comprising a leader sequence directly linked to a multimerization domain which, in turn, is directly linked to an IgG1 Fc domain consisting of IgG1 hinge, CH2 domains and CH3, where the multimerization domain creates multimers of the stradomer units.
    In one embodiment, the present invention relates to a stradomer unit comprising a leader sequence directly linked to a multimerization domain which, in turn, is directly linked to an IgG1 Fc domain consisting of IgG1 CH2 and CH3 domains. , in which the multimerization domain creates multimers of the stradomer units.
    In one embodiment, the present invention relates to a stradomer unit comprising a leader sequence directly linked to an Fc domain comprising an IgG2 hinge, which is directly linked to a CH2 domain of IgG1 and CH3 of IgG1 wherein the IgG2 hinge creates multimers from stradomer units. In one embodiment, the stradomer unit amino acid sequence is at least 80% homology to SEQ ID NO: 18. In another embodiment, the stradomer unit amino acid sequence is at least 90% homology to SEQ ID NO: 18. In yet another embodiment, the stradomer unit amino acid sequence is at least 95% homologous to SEQ ID NO: 18. In yet another embodiment, the stradomer unit amino acid sequence is at least 99% of homology to SEQ ID NO: 18. In another embodiment, the leader sequence (SEQ ID NO: 1) is cleaved from the mature protein.
    In one embodiment, the present invention relates to a stradomer unit that comprises an IgG1 Fc domain directly linked to a multimerization domain. In one embodiment, the Fc domain of IgG1 is at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% homology to SEQ ID NO:2. In another embodiment, the Fc domain of IgG1 is at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% homology to SEQ ID NO:19. In another embodiment, the Fc domain of IgG1 contains one or more mutations. In one embodiment, the multimerization domain is at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% homology to SEQ ID NO: 3. In another embodiment, the multimerization domain is at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% homology to SEQ ID NO: 5. In yet another embodiment , the multimerization domain is at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% homology to SEQ ID NO: 26. In one embodiment, the The multimerization domain is directly linked to the N-terminus of the Fc domain of IgG1.
    In another embodiment, the multimerization domain is directly linked to the C-terminus of the Fc domain of IgG1. In one embodiment, the present invention relates to a cluster stradomer comprising at least two stradomer monomers each, comprising a leader sequence directly linked to an IgG1 Fc domain which, in turn, is directly linked to a multimerization domain. In an additional modality, the clustered stradomer binds to FcYRIIIa. In an additional modality, the clustered stradomer binds to FcYRIIb.
    In another embodiment, the present invention relates to a method of modulating the immune response to a subject, comprising administering to the subject an effective amount of a cluster stradomer comprising at least two stradomer monomers each, comprising a directly related leader sequence with an IgG1 Fc domain which, in turn, is directly linked to a multimerization domain. In an additional modality, the clustered stradomer binds to FcYRIIIa. In an additional modality, the clustered stradomer binds to FcYRIIb. In another embodiment, modulating the immune response comprises the induction of CD86 on immature dendritic cells.
    In an additional modality, modulation of the immune response is associated with selective apoptosis of certain memory B cells with a decrease in antibody production. In another embodiment, the modulation is associated with selective apoptosis of activated memory B cells with a decrease in antibody production. In a further embodiment, the immune modulation can be the modulation of NK cells leading to an increase in antibody-dependent cellular cytotoxicity. In yet another modality, modulation of the immune response can result in an increase in cell populations that express CD8beta and CD11c. In yet another embodiment, immune modulation can lead to a decrease in pro-inflammatory cytokines or cytokines that are commonly elevated in autoimmune diseases, such as IL-6 and IL-8. In another embodiment, immune modulation can lead to NKT cell activation and TGF-beta secretion and cleavage.
    In a further embodiment, the present invention relates to a method of treating an inflammatory or autoimmune disease in a subject in need thereof, comprising administering an effective amount of a stradomer comprising at least two stradomer units each, comprising a leader sequence directly linked to an IgG1 Fc domain which, in turn, is directly linked to a multimerization domain or, alternatively, which comprises a leader sequence directly linked to a multimerization domain which, in turn, is directly linked to an Fc domain of an IgG1. In another embodiment, the inflammatory disease is an autoimmune disease. In yet another modality, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, type I diabetes mellitus, autoimmune thyroiditis, idiopathic thrombocytopenia purpura, autoimmune anemia, chronic inflammatory demyelinating polyneuropathy, scleroderma, systemic lupus erythematosus, psoriasis , inflammatory bowel disease, autoimmune uveitis, ANCA positive vasculitis, celiac disease, pemphigus, dermatopolymyositis, Goodpasture's disease, myasthenia gravis, Grave's disease, Kawasaki's disease, sickle cell crises, and atopic dermatitis. In yet another modality, autoimmune disease is associated with the transplantation of an organ from a donor to a recipient. In yet another embodiment, autoimmune disease is a disease that is not classically characterized as an autoimmune disease, but in which the cells of the immune system play an important role, such as Alzheimer's disease, Parkinson's disease, Huntingdon's disease, osteopenia , and osteoporosis.
    In an additional modality, the stradomer is administered intravenously, subcutaneously, orally, nasally, intraperitoneally, sublingually, buccally, transdermally, subcutaneously or subdermally implanted, or intramuscularly. In one modality, the cluster stradomer is administered intravenously. Because of the enhanced effectiveness of the stradomers of the present invention, in some embodiments, the stradomers can be administered at a lower dose intravenously compared to the predecessor and/or IVIG molecules. In one embodiment, the cluster stradomer is administered intravenously at a dose of about 0.01 mg/kg to about 1000 mg/kg IV. In a further embodiment, the cluster stradomer is administered at about 0.1 mg/kg to about 100 mg/kg IV. In yet a further modality, the cluster stradomer is administered at about 0.5 mg/kg to about 50 mg/kg IV. In yet another embodiment, the cluster stradomer is administered at about 1 mg/kg to about 25 mg/kg IV. In yet another embodiment, the cluster stradomer is administered at about 5 mg/kg to about 15 mg/kg IV. In one embodiment, the cluster stradomer is administered subcutaneously. Because of the enhanced effectiveness of the stradomers of the present invention, in some embodiments, the stradomers can be administered at a lower dose subcutaneously compared to the predecessor and/or IVIG molecules. In one embodiment, the cluster stradomer is administered subcutaneously at a dose of about 0.01 mg/kg to about 1000 mg/kg SQ. In a further embodiment, the cluster stradomer is administered at about 0.2 mg/kg to about 150 mg/kg SQ. In yet a further modality, the cluster stradomer is administered at about 0.5 mg/kg to about 80 mg/kg SQ. In yet another additional embodiment, agglomerated stradomer is administered at about 2 mg/kg to about 50 mg/kg SQ. In yet another additional embodiment, agglomerated stradomer is administered at about 5 mg/kg to about 30 mg/kg SQ.
    In one embodiment, the cluster stradomer is administered covalently fixed to an implantable device. In one modality, the clustered stradomer is attached to a suture. In another modality, the clustered stradomer is fixed to a graft or stent. In another modality, the cluster stradomer is attached to a heart valve, an orthopedic joint replacement, or implanted electronic conductor. In another modality, the agglomerated stradomer is fixed and embedded within an implantable matrix. In a preferred embodiment, the agglomerated stradomer is fixed and embedded within an implantable hydrogel. In one embodiment, the hydrogel is comprised of dextran, polyvinyl alcohol, sodium polyacrylate, or acrylate polymers. In an additional modality, the cluster stradomer is administered fixed in a hydrogel with pore sizes large enough to allow entry of immune cells to interact with the fixed cluster stradomer and then return to the circulation. In another embodiment, the pore size of the hydrogel is 5 to 50 microns. In a preferred embodiment, the pore size of the hydrogel is 25 to 30 microns.
    In another embodiment, the stradomer is administered before, during or after administration of one or more additional pharmaceutical and/or therapeutic agents. In another embodiment, the additional pharmaceutically active agent comprises a steroid, an anti-autoimmune biological drug, such as a monoclonal antibody, a fusion protein, or an anti-cytokine; a non-biological anti-autoimmune drug, an immunosuppressant, an antibiotic; and antiviral agent, a cytokine, or an agent otherwise capable of acting as an immune modulator. In yet an additional modality, the steroid is prednisone, cortisone, prednisolone, dexamethasone, momethesone, testosterone, estrogen, oxandrolone, fluticasone, budesonide, beclamethasone, albuterol, or levalbuterol. In yet another embodiment, the monoclonal antibody is infliximab, adalimumab, rituximab, tocilizumab, golimumab, ofatumumab, LY2127399, belimumab, veltuzumab, or certolizumab. In yet another embodiment, the fusion protein is etanercept or abatacept. In yet another embodiment, the anticytokine biological product is anakinra. In yet another embodiment, the non-biological antirheumatic drug is cyclophosphamide, methotrexate, azathioprine, hydroxychloroquine, leflunomide, minocycline, organic gold compounds, fostamatinib, tofacitinib, etoricoxib, or sulfasalazine. In yet another modality, the immunosuppressant is cyclosporin A, tacrolimus, sirolimus, mycophenolate mofetil, everolimus, OKT3, antithymocyte globulin, basiliximab, daclizumumab, or alemtuzumab. In yet another embodiment, the stradomer is administered before, during or after administration of a chemotherapeutic agent. In yet another embodiment, the stradomer and the additional therapeutic agent exhibit therapeutic synergy when administered together. In one embodiment, the stradomer is administered prior to administration of the additional therapeutic agent. In another embodiment, the stradomer is administered at the same time as administering the additional therapeutic agent. In yet another embodiment, the stradomer is administered after administration with the additional therapeutic agent.
    In yet another embodiment, the invention relates to a method of treating an infectious disease in a subject in need thereof, comprising administering an effective amount of an agglomerated stradomer comprising at least two stradomer units each, comprising a sequence leader directly linked to an IgG1 Fc domain which, in turn, is directly linked to a multimerization domain or, alternatively, which comprises a leader sequence directly linked to a multimerization domain which, in turn, is directly linked to a Fc domain of an IgG1. In yet another embodiment, the infectious disease is a bacterial infection. In yet another modality, the infectious disease is a viral infection. In another embodiment, the infectious disease is bacterial or viral sepsis. In an additional modality, the cluster stradomer is administered intravenously, subcutaneously, orally, intraperitoneally, sublingually, buccally, transdermally, subcutaneously or by subdermal implant, or intramuscularly. In one modality, the cluster stradomer is administered intravenously. In one modality, the stradomeraglomerate is administered intravenously. In one embodiment, the stradomeraglomerate is administered at a dose of from about 0.01 mg/kg to about 1000 mg/kg. In a further embodiment, agglomerated stradomer is administered at about 0.1 mg/kg to about 100 mg/kg. In yet a further modality, stradomer agglomerate is administered at about 0.5 mg/kg to about 50 mg/kg. In yet another embodiment, the cluster stradomer is administered at about 1 mg/kg to about 25 mg/kg. In yet another embodiment, the cluster stradomer is administered at about 5 mg/kg to about 15 mg/kg.
    In another embodiment, the cluster stradomer is administered to treat humans, non-human primates (eg, monkeys, baboons, and chimpanzees), mice, rats, cattle, horses, cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, gerbils, guinea pigs, hamsters, bats, birds (eg chickens, turkeys and ducks), fish and reptiles with species-specific or chimeric stradomer molecules. In yet another modality, the human is an adult or a child. In yet another modality, cluster stradomer is administered to prevent autoimmune disease. In an additional modality, pelleted stradomer is administered to prevent vaccine-associated autoimmune conditions in companion and farm animals.
    In yet another embodiment, the present invention relates to a method of blocking non-specific binding of antibodies in an in vitro or ex vivo assay which comprises incubating target cells or target tissues with a composition comprising an effective amount of a stradomer cluster comprising at least two stradomer units each, comprising a leader sequence directly linked to an IgG1 Fc domain which, in turn, is directly linked to a multimerization domain or, alternatively, which comprises a leader sequence directly linked to a domain of multimerization which, in turn, is directly linked to one of an IgG1 Fc domain. In one embodiment, the antibodies are monoclonal antibodies. In another embodiment, the antibodies are polyclonal antibodies. In one embodiment, the in vitro or ex vivo assay is an immunohistochemical, flow cytometry, Western blot, or immunofluorescence assay. In a further embodiment, the stradomer cluster is species specific for the target cell species or tissue.
    In yet another embodiment, the present invention relates to a method of reducing the endotoxin levels of a composition with an effective amount of a cluster stradomer comprising at least two stradomer units each comprising a leader sequence directly linked to an Fc domain of IgG1 which, in turn, is directly linked to a multimerization domain or, alternatively, which comprises a leader sequence directly linked to a multimerization domain which, in turn, is directly linked to an Fc domain of an IgG1. In another embodiment, the cluster stradomer complexes with the endotoxin in the composition. In yet another embodiment, the method includes removing the stradomer-complexed endotoxin from the composition. In one embodiment, the stradomer-complexed endotoxin is removed by filtration from the composition. In one embodiment, the composition is a pharmaceutical composition.
    In one embodiment, the present invention relates to a method of producing a cluster stradomer which comprises expressing a stradomer unit comprising a leader sequence directly linked to an Fc domain of IgG1 which, in turn, is directly linked to a multimerization domain or a leader sequence directly linked to a multimerization domain that is directly linked to an IgG1 Fc domain, wherein the multimerization domain creates stradomer unit multimers from a transfected cell which comprises the cloning of the stradomer sequence DNA encoding the stradomers of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 18 into an expression vector, transfection of the expression vector into a host bacterial, isolating plasmid DNA containing DNA encoding the stradomer of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 18 from the bacterial culture, linearization of plasmid DNA contains nding DNA encoding the stradomer of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 18 transfection of linearized DNA into mammalian cells, expansion of the cells positively transfected to obtain an aggregate of stably sectioned cells, collecting the stradomer protein from the medium, and purifying the stradomer protein, in which the stradomer protein does not contain foreign sequences. In one embodiment, the expression vector contains a selectable marker. In a further embodiment, the selectable marker is an antibiotic resistance gene. In yet another embodiment, the antibiotic resistance gene is a neomycin resistance gene. In one embodiment, the mammalian cells are CHO cells, HEK293 cells, PER.C6 cells, CAP cells, or other commercially relevant mammalian cells used for protein production. In one embodiment, the stradomer protein is purified by affinity chromatography. In another embodiment, the protein is further purified by gel filtration. In another embodiment, the protein is further purified by ion exchange chromatography. In another embodiment the protein is further purified by hydrophobic interaction chromatography. BRIEF DESCRIPTION OF THE FIGURES
    Figure 1 is a schematic of the preferred stradomer of the present inventions. Figure 1a is a schematic of the stradomer G045c; Figure 1b is a schematic of the stradomer G045old; Figure 1c is a schematic of stradomer G046; Figure 1d is a schematic view of the stradomer G019; Figure 1e is a schematic of the stradomer G028; Figure 1f is a schematic of the G051 stradomer; Figure 1g is a schematic of the G089 stradomer, and Figure 1h is a schematic of the G096 stradomer.
    Figure 2 A) is a protein gel showing the differences in multimerization ability between the first generation stradomer compounds and the direct naturally linked stradomer compounds of the present invention. B) is a comparison of multimer/monomer formation between the first generation stradomer compounds and the direct naturally linked stradomer compounds of the present invention.
    Figure 3 shows the greater effect of the direct naturally linked stradomers (M045c) of the present invention compared to the stradomer compound previously containing the foreign sequences (M045old) on the severity of collagen-induced arthritis.
    Figure 4 shows the synergistic effect that the direct naturally linked stradomer compounds of the present invention have when administered with prednisolone in collagen-induced arthritis.
    Figure 5 shows the enhanced binding affinity of compounds M046, M045, M019 and M028 of the present invention for FcYRIIIa, FcYRIIb and SIGN-R1 compared to IgG2a controls.
    Figure 6 shows the enhanced binding affinity of compounds of the present invention for (a) FcYRIIb and FcYRIIIa and (b) SIGN-R1. M045 F1 is the highest molecular weight multimer fraction of the stradomer while M045 F2 is the lowest molecular weight multimer fraction of the stradomer. M045 F3 is the homodimer fraction of the stradomer.
    Figure 7 shows the effect of direct naturally bound M045c stradomer on the severity of idiopathic thrombocytopenic purpura (ITP).
    Figure 8 shows that stradomers more effectively block the binding of anti-FcYR antibodies to FcYRs relative to IgG1 Fc control.
    Figure 9 shows the effect of naturally linked M045c, M019, M028, M046 and M051 stradomers on the severity of a mouse model of collagen-induced arthritis.
    Figure 10 shows the increased binding of stradomer G075 to FcYRIIIa and decreased binding of stradomer G075 to FcYRJIa and FcYRIIb. B shows the increased binding affinity of stradomer G076 for FcYRIIa and FcYRIIb and decreased binding of stradomer G076 for FcYRIIIa.
    Figure 11 shows the effect of M051 compared to IVIg and albumin controls on (A) weight loss associated with experimental autoimmune neuritis in rats (EAN); (B) clinical score in EAN rats; (C) on hip amplitude in EAN rats; (D) ankle amplitude in EAN rats, and (E) motor nerve conduction velocity in EAN rats.
    Figure 12 shows the effect of M045c compared to IVIg and albumin controls on (A) weight loss associated with experimental autoimmune neuritis in rats (EAN); (B) clinical score in EAN rats; (C) hip amplitude in EAN rats, (D) ankle amplitude in EAN rats; and (E) motor nerve conduction velocity in EAN rats.
    Figure 13 shows the greater effect of naturally bound stradomers M075 (A), M096 (B) and M098 (C), but not M076 (A) of the present invention compared to vehicle control and the positive control M045c on the severity of collagen-induced arthritis. DETAILED DESCRIPTION OF THE INVENTION
    The rational molecular model approach for the hIVIG replacement compounds described in this document includes the creation of recombinant and/or biochemical products of immunologically active biomimetic(s) that are surprisingly more effective at multimerization and, consequently, binding to receptors Fc range than the molecules described above designed to achieve this goal. Replacement compounds have utility in treating, for example, autoimmune diseases, inflammatory diseases, cancer and sepsis. Due to the superior binding affinity for Fc gamma receptors relative to native immunoglobulin, the compositions are also useful as Fc blocking reagents in antibody-based immunoassays and in removing endotoxins from pharmaceutical and laboratory composition. Each modality is described in detail below, along with certain exemplary modality.
    As used in this document, the use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "a", but is also consistent with the meaning of "one or more", "at least one" and "one or more than one".
    As used herein, the terms "biomimetic", "biomimetic molecule", "biomimetic compound", and related terms refer to a human-made compound that mimics the function of another compound, such as aggregated hIVIG, an antibody monoclonal or the Fc fragment of an antibody. "Biologically active" biomimetics are compounds that possess biological activities that are the same or similar to their naturally occurring counterparts. By "naturally occurring" we mean a molecule or portion thereof that is normally found in an organism. By “naturally occurring” is also meant substantially naturally occurring. "Immunologically active" biomimetics are or are biomimetics that exhibit the same or similar immunological activity as naturally occurring immunologically active molecules such as antibodies, cytokines, interleukins, and other immunological molecules known in the art. In preferred embodiments, the biomimetics of the present invention are stradomers as defined herein.
    By "directly linked" we mean two sequences connected to each other without intervening or extraneous sequences, for example restriction enzyme recognition sites or cloning fragments. One of ordinary skill in the art will understand that "directly linked" encompasses the addition or removal of amino acids so long as the ability to multimerize is not substantially affected.
    By "homologs" we mean the identity over the entire sequence of a particular nucleic acid or amino acid sequence. For example, by "80% homologous" we mean that a given sequence shares about 80% identity with the claimed sequence and may include insertions, deletions, substitutions, and structure changes. One of ordinary skill in the art will understand that sequence alignments can be made to account for insertions and deletions to determine identity over the entire length of a sequence.
    The immunologically active biomimetics of the present invention are designed to possess one or more immunomodulating activities of the Fc domain of IgG and have at least (i) a first Fc domain capable of binding FcRn, DC-SIGN, and SIGN- R1 and/or an FCYR including FCYRI, FCYRII, FCYRIII and FCYRIV, and (ii) a second Fc domain capable of binding to FcRn, DC-SIGN, SIGN-R1 and/or an FCYR including FcyRI, FCYRII, FCYRIII and FCYRIV. Specifically, the immunologically active compounds of the present invention are multimers of homodimers. Each homodimer having the ability to bind FcRn, DC-SIGN, SIGN-R1 and/or and FCyR. Thus, when multimerized, immunologically active biomimetics contain at least two homodimers, each having the ability to bind FcRn, DC-SIGN, SIGN-R1 and/or and FCyR.
    The following paragraphs define the building blocks of the biomimetics of the present invention, both structurally and functionally, and then define the biomimetics themselves. However, it is primarily useful to note that, as indicated above, each of the biomimetics of the present invention has at least two Fc domains. At a minimum, an Fc domain is a dimeric polypeptide (or a dimeric region of a larger polypeptide) that comprises two peptide chains or arms (monomers) that associate to form a functional Fc receptor binding site. Therefore, the functional form of the individual fragments and domains discussed here generally exist in a dimeric (or multimeric) form. The monomers of the individual fragments and domains discussed here are the single chains or arms that must associate with a second chain or arm in order to form a functional dimeric structure. Fragment Fc
    The "Fc fragment" is a term of the art that is used to describe the region of the protein or the folded structure of the protein that is commonly found at the carboxy terminus of immunoglobulins. The Fc fragment can be isolated from the Fab fragment of a monoclonal antibody through the use of enzymatic digestion, eg papain digestion, which is an incomplete and imperfect process (see Mihaesco C and Seligmann M. Papain Digestion Fragments Of Human IgM Globulins, Journal of Experimental Medicine, Vol 127, 431-453 (1968)). Together with the Fab fragment (which contains the antigen-binding domain), the Fc fragment constitutes the holo-antibody, meaning here the complete antibody. The Fc fragment consists of the carboxy terminal portions of antibody heavy chains. Each of the chains in an Fc fragment is between about 220-265 amino acids long and the chains are often linked through a disulfide bond. The Fc fragment often contains one or more independent structural folds or functional subdomains. In particular, the Fc fragment encompasses an Fc domain, defined herein as the minimal structure that binds to an Fcy receptor. An isolated Fc fragment is composed of two Fc fragment monomers (eg, two carboxy terminal portions of antibody heavy chains, further defined herein) that are dimerized. When two Fc fragment monomers associate, the resulting Fc fragment has FcY receptor binding activity. Partial Fc Fragment
    A "partial Fc fragment" is a domain that comprises less than the entire Fc fragment of an antibody, but which retains sufficient structure to have the same activity as the Fc fragment, including FCY receptor binding activity. A partial Fc fragment may, therefore, lack part or all of a hinge region, part or all of a CH2 domain, part or all of a CH3 domain, and/or part or all of a CH4 domain, depending on the antibody isotype from which the partial Fc domain is derived. An example of a partial Fc fragment includes a molecule comprising the upper, core, and lower hinge regions plus the CH2 domain of IgG3 (Tan, LK, Shopes, RJ, Oi, VT and Morrison, SL, Influence of the hinge region on complement activation, CIq binding, and segmental flexibility in chimeric human immunoglobulins, Proc Natl Acad Sci USA 1990 January; 87(1): 162-166). Thus, in this example, the partial Fc fragment lacks the CH3 domain present in the IgG3 Fc fragment. Another example of a partial Fc fragment includes a molecule comprising the CH2 and CH3 domains of IgG1. In this example, the partial Fc fragment lacks the hinge domain present in IgG1. Partial Fc fragments are composed of two partial Fc fragment monomers. As further defined herein, when two such partial Fc fragment monomers associate, the resulting partial Fc fragment has FCY receptor binding activity. domain Fc
    As used herein, the "Fc domain" describes the minimal region (in the context of a larger polypeptide) or a folded structure of the smaller protein (in the context of an isolated protein) that can bind to, or be bound by, a Fc receptor (FcR). In both the Fc fragment and the partial Fc fragment, the Fc domain is the minimal binding region that allows binding of the molecule to an Fc receptor. While an Fc domain can be limited to a discrete polypeptide that is bound by an Fc receptor, it will also be clear that an Fc domain can be a part or all of an Fc fragment, as well as part or all of a partial Fc fragment. . When the term "Fc domains" is used in the present invention, it will be recognized by one of skill in the art to mean more than one Fc domain. An Fc domain is composed of two Fc domain monomers. As further defined herein, when two such Fc domain monomers associate, a resulting Fc domain has Fc receptor binding activity. Thus, an Fc domain is a dimeric structure that can bind an Fc receptor. Partial FC domain
    As used in this document, "partial Fc domain" describes a portion of an Fc domain. The individual partial Fc domains include the heavy chain constant region domains (for example, CH1, CH2, CH3 and CH4 domains) and the hinge regions of the different classes and subclasses of immunoglobulins. Thus, the human partial Fc domains of the present invention include the CH1 domains of IgG1, IgG2, IgG3, IgG4, IgM, Igal, IgA2, IgD and IgE, the CH2 domains of IgG1, IgG2, IgG3, IgG4, IgM, Igal, IgA2 , IgD and IgE, the CH3 domains of IgG1, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD and IgE, the CH4 domains of IgM and IgE, and the hinge regions of IgG1, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD and IgE. The corresponding partial Fc domains in other species will depend on the immunoglobulins present in the species and their nomenclature. Preferably, the partial Fc domains of the present invention include CH1, CH2 and IgG1 hinge domains and IgG2 hinge domains. The partial Fc domain of the present invention may further comprise a combination of more than one of these domains and hinges. However, the individual partial Fc domains of the present invention and combinations of memos do not have the ability to bind FCYR. Therefore, partial Fc domains and combinations thereof comprise less than one Fc domain. Partial Fc domains can be linked together to form a peptide that has FCY receptor binding activity, thereby forming an Fc domain. In the present invention, partial Fc domains are used with Fc domains as building blocks to create the biomimetics of the present invention as defined herein. Each partial Fc domain is composed of two partial Fc domain monomers. When two of these partial Fc domain monomers associate, a partial Fc domain is formed.
    As indicated above, each of the Fc fragments, partial Fc fragments, Fc domains and partial Fc domains are proteins or dimeric domains. Thus, each of these molecules is made up of two monomers that associate to form the protein or dimeric domain. Although the characteristics and activity of homodimeric forms have been discussed above, monomeric peptides are discussed below. Fc Fragment Monomer
    As used herein, an "Fc fragment monomer" is a single chain protein which, when associated with another Fc fragment monomer, comprises an Fc fragment. The Fc fragment monomer is thus the carboxy terminal portion of one of the antibody heavy chains that make up the Fc fragment of a holoantibody (for example, the contiguous portion of the heavy chain that includes the hinge region, the CH2 domain and the CH3 domain of IgG). In one embodiment, the Fc fragment monomer comprises at least one chain of a hinge region (a hinge monomer), one chain of a CH2 domain (a CH2 domain monomer), and one chain of a CH3 domain (a CH3 domain monomer, contiguously linked to form a peptide. In another embodiment, the Fc fragment monomer comprises at least one chain of a hinge region, one chain of a CH2 domain, one chain of a CH3 domain, and one chain of a CH4 domain (a CH4 domain monomer) contiguously linked to form a peptide. In one embodiment, the CH2, CH3 and hinge domains are of different isotypes. In a particular embodiment, the Fc fragment monomer contains an IgG2 hinge domain and IgG1 CH2 and CH3 domains. Fc Domain Monomer
    As used herein, "Fc domain monomer" describes single-chain protein which, when associated with another Fc domain monomer, comprises an Fc domain, which can bind to an FCY receptor. The association of two Fc domain monomers creates an Fc domain. An individual Fc domain monomer, comprising only one side of an Fc domain, cannot bind to an FcY receptor. The Fc domain monomers of the present invention do not contain foreign sequences as do the previously described Fc domain monomers (see, for example, WO 2008/151088, figures 17 and 18). Instead, the Fc domain monomers of the present invention (SEQ ID NO: 2) are linked directly to the leader sequence (SEQ ID NO: 1) from one terminus (e.g., the N terminus of the Fc monomer) and to the domain of multimerization (SEQ ID NO: 3) at the other terminus (e.g., the C terminus of the Fc monomer). The resulting stradomer monomer of SEQ ID NO: 4 is herein called G045c. In another embodiment, the Fc domain monomer comprises an IgG2 hinge domain and the IgG1 CH2 and CH3 domains are linked directly to the leader sequence (SEQ ID NO: 1) at the N-terminus.
    SEQ ID NO: 18 is referred to herein as G051. Alternatively, the multimerization domain (SEQ ID NO: 3) can be placed at the amino terminus of the Fc domain monomer (SEQ ID NO: 2). The resulting stradomer monomer of SEQ ID NO: 8 is referred to herein as G019. Alternatively, the multimerization domain may comprise SEQ ID NO: 5, rather than SEQ ID NO: 3 and may be placed at the carboxy terminus of the Fc domain monomer, resulting in SEQ ID NO: 10, or may be placed at the amino terminus of the Fc domain monomer, which results in SEQ ID NO: 9. These stradomers are referred to herein as G046 and G028, respectively. Partial Fc Domain Monomer
    As used herein, "partial Fc domain monomer" describes the single chain protein which, when associated with another partial Fc domain monomer, comprises a partial Fc domain. The association of two partial Fc domain monomers creates a partial Fc domain. Stradomers
    In particular embodiments, biomimetics of the present invention include stradomers. Stradomers are biomimetic compounds capable of binding to two or more Fc receptors, preferably two or more FCY receptors, and more preferably, demonstrating significantly improved binding relative to an Fc domain and more preferably, demonstrating slow dissociation characteristic of avidity. In a preferred embodiment, the stradomers of the present invention are used to bind FcRn, DC-SIGN, SIGN-R1 and/or FCY receptors on effector cells, such as NK cells and immature dendritic cells and other cells derived from monocytes. In one embodiment, Fcy receptors are low-affinity Fcy receptors. A stradomer can have four different physical conformations: series, cluster, nucleus, or Fc fragment. The stradomers of the present invention are preferably agglomerated stradomers. As will be evident, the Fc fragments, partial Fc fragments, Fc domains and partial Fc domains discussed above are used in the construction of various conformations of stradomers. Furthermore, it is the individual Fc domain monomers and the partial Fc domain monomers, also discussed above, that are first produced, and then self-associate to form the dimeric structures that are the stradomers of the present invention.
    As used herein, a "stradomer dimer" is a specific form of a stradomer, composed of only two stradomer monomers. In one embodiment, stradomer dimers are molecules formed by self-aggregation of relevant stradomer monomers. In another embodiment, the stradomer monomers in the stradomer dimers are physically linked through an inter-stradomer monomeric bond, as defined herein. A "multimeric stradomer" is comprised of three or more stradomers formed either by self-aggregation of stradomer monomers or through an inter-stradomer monomeric bond, as defined in the present invention. Stradomer monomer
    As used herein, the term "stradomer monomer" or "stradomer unit" refers to a single, contiguous peptide molecule which, when associated with at least one second stradomer monomer, forms a polypeptide comprising at least two domains Fc. While in preferred embodiments the agglomerated stradomers are composed of two associated stradomer monomers, an agglomerated stradomer may also contain three or more stradomer monomers. Stradomer monomers can be associated to form stradomers by inter-stradomer monomer bonds or can form stradomers through self-aggregation.
    A stradomer monomer can have an amino acid sequence that forms one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more Fc domains when associated with another stradomer monomer. stradomer to form a stradomer. A stradomer monomer may further have an amino acid sequence that forms one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more partial Fc domains, when associated with another stradomer monomer to form a stradomer.
    The regions of stradomer monomers that will form the Fc domains and partial Fc domains in the context of a stradomer can simply be arranged from the carboxy terminus to the amino terminus of successive regions of the stradomer monomer molecule. The arrangement of particular Fc domain monomers and partial Fc domain monomers comprising a stradomer monomer is not critical. However, the arrangement must allow the formation of two functional Fc domains through the association of two stradomer monomers. In a preferred embodiment, the stradomer of the present invention contains a direct link between the N-terminus of the IgG1 Fc monomer (SEQ ID NO: 2), and the C-terminus of the leader peptide (SEQ ID NO: 1) and the C-terminus of IgG1 Fc (SEQ ID NO: 2), and the N-terminus of the IgG2 hinge multimerization domain (SEQ ID NO: 3), or the isoleucine zipper (SEQ ID NO: 5) or the GPP domain (SEQ ID NO: 26) to form the stradomer G045c of SEQ ID NO: 4, the stradomer G046 of SEQ ID NO: 10, or the stradomer G089 of SEQ ID NO: 27, respectively (Table 1). In another preferred embodiment, the stradomer of the present invention contains a direct link between the C-terminus of the leader peptide (SEQ ID NO: 1) and the N-terminus of the IgG2 hinge multimerization domain (SEQ ID NO: 3), isoleucine zipper (SEQ ID NO: 5) or GPP domain (SEQ ID NO: 26) and a direct link between the C-terminus of the multimerization domain (SEQ ID NOs: 3, 5, or 26) and the N-terminus of IgG1 Fc to form the stradomer G019 of SEQ ID NO: 8, the stradomer G028 of SEQ ID NO: 9, or the stradomerG096 of SEQ ID NO: 28, respectively (Table 1), or a direct link between the leader peptide (SEQ ID NO: 1 ) and the N-terminus of the multimerization domain (SEQ ID NO: 3) and direct link between the C-terminus of the multimerization domain (SEQ ID NO: 3) and the N-terminus of the partial Fc domain of IgG1 containing the CH2 and CH3 portions of IgG1 to form the G051 stradomer of SEQ ID NO: 18 (Table 1).



    As an example of clarification, one skilled in the art will understand that the stradomer molecules of the present invention can be constructed by preparing a polynucleotide molecule encoding various combinations of Fc domain monomers and partial Fc domain monomers, each with or without mutations selected points, but with a combination that will form a minimum of two Fc domain monomers. Such a polynucleotide molecule, for example, the polynucleotides encoding the stradomers of SEQ ID NO: 4, 8, 9, 10 or 18, can be inserted into an expression vector that can be used to transform a population of bacteria or to transfect a mammalian cell population. Destradomer monomers can then be produced by culturing the transformed or transfected bacteria into mammalian cells under suitable culture conditions. For example, a clonal cell line that continues a pool of stably transfected cells can be achieved through genetecin/G418 cell selection.
    Alternatively, cells can be transiently transfected with DNA encoding the stradomer of the present invention, i.e., G045c (SEQ ID NO: 4), G019 (SEQ ID NO: 8), G028 (SEQ ID NO: 9) G046 (SEQ ID NO: 10), or G051 (SEQ ID NO: 18), under the control of the CMV promoter. The expressed stradomer monomers can then form functional stradomers upon self-aggregation of the stradomer monomers or association of stradomer monomers using inter-stradomer monomeric bonds. Expressed stradomers can then be purified from cell culture media by affinity chromatography using, for example, Protein A or Protein G columns. The present invention encompasses both stradomers formed by the association of stradomer monomers having the amino acid sequences identical, stradomer monomers having substantially similar amino acid sequences, or stradomer monomers having different sequences. In the latter embodiment, the amino acid sequence of stradomer monomers comprising a stradomer need only be of similarity such that two or more functional FCYR binding sites are formed.
    Surprisingly, the G045c (SEQ ID NO: 4) produced by the processes of the present invention, which contain the claimed leader sequence sequences (SEQ ID NO: 1) directly linked to the N-terminus of IgG1 Fc (SEQ ID NO: 2) which , in turn, is directly linked through its terminus to the IgG2 hinge multimerization domain (SEQ ID NO: 3), leads to a higher order multimer formation that was observed with predecessor molecules containing foreign sequences that were previously of functional importance, such as restriction sites on the IgG1 Fc monomer, as shown in SEQ ID NOs: 6 and 7. Indeed, about 74% of the resulting stradomer G045c preparation contains multimers whereas only about 26% of the composition contains monomers. This is in contrast to preparations which contain the molecules with the foreign sequences. These preparations contain only about 27% of multimers and 72% of the composition is present in the form of monomers. Furthermore, the higher order multimers produced by the stradomer monomers of SEQ ID NO: 4 had surprisingly superior efficacy when compared to the older foreign sequence containing the stradomers described above. Therefore, in an embodiment of the present invention, the multimerization domain creates higher order multimers from the stradomer units. In yet another embodiment, at least about 35% of the resulting stradomer composition contains multimers of the stradomer units. In yet another embodiment, at least about 55% of the resulting stradomer composition contains multimers of the stradomer units. In yet another embodiment, at least about 65% of the resulting stradomer composition contains multimers of the stradomer units. In yet another further embodiment, at least about 70% of the resulting stradomer composition contains multimers of the stradomer units. In yet another further embodiment, at least about 75% of the resulting stradomer composition contains multimers of the stradomer units.
    As indicated above, an Fc domain can be functionally defined by its ability to bind FcRn, DC-SIGN, SIGN-R1 and/or an FCY receptor. The compounds of the present invention bind to cognate receptors, including, FcRIIIa, FcRIIb and/or SIGN-R1, with much higher affinity than the IgG2a controls (Figures 5 and 6). As a result, the particular amino acid sequence of an Fc domain varies according to the partial Fc domains that comprise the Fc domain. However, in one embodiment of the present invention, the Fc domain comprises the hinge region and a CH2 domain of an immunoglobulin molecule. In another preferred embodiment, the Fc domain comprises the hinge region, a CH2 domain and a CH3 domain of an immunoglobulin molecule. In a further embodiment, the Fc domain comprises the hinge region, a CH2 domain, CH3 domain and CH4 domain of an immunoglobulin molecule. In yet another embodiment, the Fc domain comprises the hinge region, a CH2 domain and CH4 domain of an immunoglobulin molecule. In another preferred embodiment, the Fc domain comprises a CH2 domain and a CH3 domain. In a preferred embodiment, the Fc domain contains the hinge region, the CH2 and CH3 domain of IgG1 (SEQ ID NO: 2). In another preferred embodiment, the Fc domain contains the CH2 and CH3 domains of IgG1 (SEQ ID NO:19). Inter-stradomer Linking Monomer
    A separate bond found in the biomimetic compounds of the present invention is the "inter-stradomer monomeric bond", which occurs between two or more individual stradomer monomers that comprise the stradomers of the present invention. Although the domain bonds are short amino acid sequences, which serve to link Fc domain monomers and partial Fc domain monomers that comprise the individual stradomer monomers of the biomimetic compounds to each other, the inter-stradomer monomeric bonds serve to join two or plus individual stradomer monomers comprising the biomimetic compounds. The inter-stradomer monomeric bond can be any bond capable of stably associating with the individual stradomer monomers. In some embodiments, the inter-stradomer monomeric bond can be a covalent bond between the stradomer monomers. Alternatively, the inter-stradomer monomeric bond between the stradomer monomers can be direct chemical crosslinking. In preferred embodiments, stradomer monomer structures take advantage of the natural self-aggregating properties among Fc domain monomers to create self-aggregating stradomers. In some such embodiments, self-aggregation occurs without the covalent disulfide bridge bonds normally found in nature. Without being bound by theory, intact IgG1, for example, has 2 cysteine bonds in the hinge region (Dorai H. Role of interheavy and light chain disulfide bonds in the effector functions of human immunoglobulin IgG1. Molecular Immunology. 29;12 , 1992,1487-1491; Meulenbroek AJ and Zeijlemaker WP. Human IgG Subclasses: Useful diagnostic markers for immunocompetence. ISBN 90-5267-011-0) while the IgG1 hinge, IgG1 CH2 domains, and IgG1 CH3 domains of G045 do not demonstrate intermonomer bonds; in G045 all intermonomer bonds occur at the C-terminus of the IgG2 hinge domain. In other of these embodiments, disulfide bonds form between the individual stradomer monomers to form the stradomers. Disulfide bonds form between the cysteine residues of the Fc domain monomers that comprise the biomimetic molecules, using either cysteine residues that occur in the natural Fc domain monomer sequence or cysteine residues incorporated into an Fc domain monomer by site-directed mutagenesis. Such natural self-aggregating properties can also be used to form the inter-stradomer monomeric bonds between the individual stradomer monomers in stradomer multimers. In a preferred embodiment, the cysteine residues that form the inter-stradomer monomeric bond are at positions 236, 237, 240 and 243 of the IgG2 hinge domain of the mature protein of G045. In a preferred embodiment, the cysteine residues that form the inter-stradomer monomeric bond are at positions 4, 5, 8, and 11 of the IgG2 hinge domain of the mature protein of G051. Alternative modalities include inter-stradomer monomeric bonds where disulfide bonds form between the introduced cysteine residues through site-directed mutagenesis in the amino acid sequence comprising the individual stradomer monomers.
    As discussed above, in a preferred embodiment, the inter-stradomer monomeric bond that forms a stradomer is a bond that results from self-aggregation of stradomer monomers. In one embodiment, the two stradomer monomers that comprise the stradomer are identical peptides, such that the two individual stradomer monomers that make up the stradomer have identical sequences. However, one of skill in the art will understand that other embodiments include stradomers in which the stradomer monomers differ from each other in amino acid sequence.
    Two stradomer monomers can form a stradomer by, for example, aligning in parallel such that pairing occurs between identical partial Fc domain monomers in the stradomer monomers. However, the present invention also includes embodiments in which the pairing occurs between the non-identical partial Fc domain monomers, and embodiments where the pairing occurs between the identical partial Fc domain monomers in the stradomer monomers, but where the alignment of the two monomers of stradomer is displaced. Agglomerated Stradomer
    The "clustered stradomer" is a biomimetic that has a radial shape with a "head" of central fraction and two or more "legs", in which each of the legs comprises one or more Fc domains, which is capable of binding to the fur. minus one Fc gamma receptor, thus creating a biomimetic capable of binding to two or more Fc gamma receptors. Each cluster stradomer is composed of more than one dimeric protein, each called a "clustered stradomer unit". Each cluster stradomer unit is comprised of a multimerizing region and a "leg" region comprising at least one functional Fc domain. The multimerization region creates a cluster stradomer "head" once multimerized with another cluster stradomer unit. The leg region is able to bind to many FCY receptors as there are Fc domains in each leg region. Thus, a cluster stradomer is a biomimetic compound capable of binding to two or more Fcy receptors, increasing binding affinity and avidity.
    The region of multimerization can be a peptide sequence that causes the dimeric proteins to further multimerize, or, alternatively, the region of multimerization can be a glycosylation that enhances the multimerization of dimeric proteins. Examples of peptide multimerization regions include the IgG2 hinge, IgE CH2 domain, isoleucine zipper, collagen Glycine-Proline-Proline repeat ("GPP") and zinc fingers. The influence of glycosylation on peptide multimerization is well described in the art (eg, Role of Carbohydrate in Multimeric Structure of Factor VIII/V on Willebrand Factor Protein. Harvey R. Gralnick, Sybil B. Williams and Margaret E. Rick. Proceedings of the National Academy of Sciences of the United States of America, Vol. 80, No. 9, [Part 1: Biological Sciences] (May 1, 1983), pp. 2771-277 '4; Multimerization and collagen binding of vitronectin is modulated by its glycosylation. Kimie Asanuma, Fumio Arisaka and Haruko Ogawa. International Congress Series Volume 1223, December 2001, Pages 97-101).
    The multimerization region can be a peptide sequence that causes peptides to dimerize or multimerize and includes the IgG2 hinge, the IgE CH2 domain, an isoleucine zipper, GPP collagen, and a zinc finger. As is known in the art, the hinge region of human IgG2 can form covalent dimers (Yoo, EM et al. J. Immunol. 170, 3134-3138 (2003); Salfeld Nature Biotech. 25, 1369-1372 (2007)) . The formation of IgG2 dimers is potentially mediated through the IgG2 hinge structure by C-C bonds (Yoo et al., 2003), suggesting that the individual hinge structure may mediate dimer formation. The amount of IgG2 dimers found in human serum, however, is limited. It is estimated that the amount of IgG2 existing as a dimer of the homodimer is less than 10% of the total IgG2 (Yoo et al. 2003). Furthermore, there is no quantitative evidence of the IgG2 multimerization domain beyond the homodimer dimer. (Yoo et al. 2003). That is, non-native IgG2 was found to form higher order multimers in human serum. Therefore, the results presented here are particularly surprising in that IgG2 hinge-containing stradomers (i.e., G045c, G019 and G051) are present in higher order multimers and in contrast to native IgG2 in human serum, in which the IgG2 hinge interactions are variable and dynamic, G045c has been shown to form highly stable multimers as evidenced by non-reducing SDS-PAGE gels, by analytical ultracentrifugation and by 3 months of stability studies at 100% humidity at 37 °C. Furthermore, it is also surprising that the amount of multimers in the IgG2 hinge-containing stradomer preparations is significantly higher than the 10% observed for IgG2 in human serum. For example, the amount of multimers including homodimer dimers in the G045c preparations is approximately 67%.
    The amino acid sequence of the human IgG2 hinge monomer is as follows: ERKCCVECPPCP (SEQ ID NO: 3). Mutation of any one of the four cysteines in SEQ ID 3 may be strongly associated with decreased stradomer multimerization. There are two C-X-X-C portions of the IgG2 hinge monomer. Thus, the stradomer monomers of the present invention can comprise either the complete 12 amino acid sequence of the IgG2 hinge monomer, or either or both of the four core amino acids, together with the Fc domain monomers. Although the X-X bond of the core structures can be any amino acid, in a preferred embodiment the X-X sequence is V-E or P-P. One of skill in the art will understand that the IgG2 hinge monomer can be made up of any portion of the hinge sequence in addition to the four core amino acid structure, including the entire IgG2 hinge sequence and some or all of the CH2 domain monomer sequences and CH3 from IgG2. Without being bound by theory, the IgG2 hinge multimerization domain can form multimers by interacting with any portion of the stradomer monomer. That is, the IgG2 hinge of one stradomer monomer can bind to the IgG2 hinge of another stradomer monomer, thus forming a higher-order homodimer dimer, or multimers, while maintaining increased functional binding to Fc receptors relative to natural IgG1 Fc. Alternatively, the IgG2 hinge domain of one stradomer monomer can bind the IgG1 hinge of another stradomer monomer, thereby forming a dimer of the higher order homodimer, or multimers, while maintaining increased functional binding to receptors Fc to natural IgG1 Fc. It is also possible that the IgG2 hinge domain of one stradomer monomer will link to another portion of the Fc domain of IgG1, i.e. the CH2 or CH3 domain of another stradomer monomer to form the homodimer dimer, or multimers of higher order, maintaining an increased functional binding to Fc receptors over natural IgG1 Fc.
    Leucine and isoleucine zippers can also be used as the multimerization region. Leucine and isoleucine zippers (coiled spiral domains) are known to facilitate the formation of protein dimers, trimers, and tetramers (Harbury et al, Science 262:1401-1407 (1993); O'Shea et al, Science 243: 538 (1989)). Taking advantage of the natural tendency of an isoleucine zipper to form a trimer, bonded stradomers can be produced.
    Although the person skilled in the art understands that the different types of leucine and isoleucine zippers can be used, in a preferred embodiment, the isoleucine zipper from the modified GCN4 transcriptional regulator as described (Morris et al., Mol. Immunol 44:3112) - 3121 (2007), Harbury et al., Science 262:1401-1407 (1993)) is used: GGGSIKQIEDKIEEILSKIYHIENEIARIKKLIGERGHGGG (SEQ ID NO:5). This isoleucine zipper sequence is just one of several possible sequences that can be used for the multimerization of Fc domain monomers. Although the entire sequence shown in SEQ ID NO:5 can be used, the underlined portion of the sequence represents the core sequence of the isoleucine zipper which can be used in the cluster stradomers of the present invention. Thus, the stradomer monomers of the present invention can comprise either the complete amino acid sequence of the isoleucine zipper, or the 28 amino acid core, along with one or more Fc domain monomers. One of skill in the art will also understand that the isoleucine zipper can be made up of any portion of the zipper in addition to the 28 amino acid backbone of the core and thus it can comprise more than 28 amino acids but less than the entire sequence.
    GPP is a sequence of amino acids found in human collagen that causes collagen protein:protein binding. Although the person skilled in the art understands that the different types of GPP repeats can be used as a multimerization domain, in a preferred embodiment, the Glycine-Proline-Proline repeats are as described (Fan et al. FASEB Journal 3796 vol 22 2008) are used: (SEQ ID NO: 26). This glycine-proline-proline repeat sequence is just one of several possible sequences that can be used for multimerization of the Fc domain monomers. Although the entire sequence shown in SEQ ID NO: 26 can be used, the different length repeats can also possibly be used to multimerize the Fc domain monomers. Likewise, repeats containing different amino acids within the GPP repeats can also be replaced.
    It is understood that stradomers and other biomimetic molecules disclosed herein can be derived from any of a variety of species. Indeed, the Fc domains, or partial Fc domains, in any of the biomimetic molecules of the present invention may be derived from immunoglobulin from more than one species (e.g., from two, three, four, five, or most). However, they will most commonly be derived from a single species. Furthermore, it should be noted that any of the methods described here (eg treatment methods) can be applied to any species. Generally, the components of a biomimetic applied to a species of interest will all be derived from that species. However, biomimetics in which all components are of a different species, or are of more than one species (including or not the species to which the method in question is applied) can also be used.
    The CH1, CH2, CH3 and CH4 specific domains and hinge regions comprising the Fc domains and partial Fc domains of the stradomers and other biomimetics of the present invention can be independently selected, either in terms of the immunoglobulin subclass, as well as in the organism, to from which they are derived. Therefore, stradomers and other biomimetics described herein may include Fc domains and partial Fc domains that independently are derived from various types of immunoglobulins, such as IgG1, IgG2, IgG3, IgG4, IgAl, IgAl, IgD, IgE, and human IgM, IgG2a mouse, or dog IgA or IGB. Preferably, for human therapeutics, the Fc domains of the present invention are of the human IgG1 isotype. Likewise, each of the Fc domain and partial Fc domain can be derived from a number of species, preferably a mammalian species, including non-human primates (eg, monkeys, baboons, and chimpanzees), humans, murine, rat, bovine , horses, felines, canines, pigs, rabbits, goats, sheep, deer, ferrets, gerbils, guinea pigs, hamsters, bats, birds (eg chickens, turkeys and ducks), fish and reptiles to produce specific stradomer molecules species or chimerical.
    Individual Fc domains and partial Fc domains can also be humanized. One skilled in the art will understand that different Fc domains and partial Fc domains will provide different types of functionality. For example, FCYRS specifically binds to IgG immunoglobulins and does not bind well to other classes of immunoglobulins. Thus, one of those skilled in the art, intending to design a stradomer with multiple FCY receptor binding capability, would design stradomer Fc domains that at least incorporate the well-characterized IgG FCY receptor binding sequences, including those in the region IgG lower hinge and/or the CH2 and CH3 domains of IgG. One of ordinary skill in the art will also understand that various deleterious consequences can be associated with the use of specific Ig domains, such as the anaphylaxis associated with IgA infusions. The biomimetics disclosed here generally must be designed to avoid such effects, although under certain circumstances such effects may be desirable.
    The present invention also encompasses stradomers comprising the Fc domains and partial Fc domains having amino acids that differ from the naturally occurring amino acid sequences of the Fc domain or partial Fc domain. Preferred Fc domains for inclusion in the biomimetic compounds of the present invention have a specific measurable binding affinity, either an FCY holoreceptor or a portion of the soluble extracellular domain of an FCYR. The primary amino acid sequences and X-ray crystallography structures of various Fc domains and Fc domain monomers are available in the art. See, for example, Woof JM, Burton DR. Human antibody-Fc receptor interactions illuminated by crystal structures. Nat Rev Immunol. 2004 Feb;4(2):89-99. Representative Fc domains capable of Fc receptor binding include the Fc domains of human IgG1 (SEQ ID NO: 2). These native sequences were subjected to extensive structure-function analysis, including site-directed mutagenesis mapping of functional sequences. Based on these prior structure-function studies and available crystallography data, one skilled in the art can design functional Fc domain sequence variants while preserving the FcR receptor binding capacity of the Fc domain. For example, cysteine residues can be added to enhance the sulfide bond between monomers or deleted to alter the interaction between stradomer homodimers.
    Amino acid changes can be found throughout the Fc domain sequence, or be isolated to particular partial Fc domains that comprise the Fc domain. Functional variants of the Fc domain used in the stradomers and other biomimetics of the present invention will be at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of sequence identity with a native Fc domain. Similarly, functional variants of the partial Fc domains used in the stradomers and other biomimetics of the present invention will be at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with a partial native Fc domain.
    One of skill in the art will appreciate that the present invention further encompasses the use of functional variants of Fc domain monomers in the construction of the monomers, Fc fragment monomers, partial Fc fragment monomers, stradomers monomers and others of the present invention. Functional variants of the Fc domain monomers will have at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with a sequence of native Fc domain monomer.
    Likewise, the present invention also encompasses the use of functional variants of partial Fc domain monomers in the construction of Fc fragment monomers, partial Fc fragment monomers, Fc domain monomers, stradomer monomers and the other monomers of the present invention. Functional variants of the partial Fc domain monomers will have at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence of native partial Fc domain monomer.
    Amino acid changes can decrease, increase or leave the binding affinity of the stradomer for the FCY receptor unchanged. Preferably, such amino acid changes will be conservative amino acid substitutions, however, these changes include deletions, additions and other substitutions. Conservative amino acid substitutions typically include changes within the following groups: glycine and alanine; valine, isoleucine and leucine, aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; histidine, lysine and arginine; and phenylalanine and tyrosine. Furthermore, changing an amino acid can enhance the strength of multimerization, for example, by adding cysteine residues.
    The amino acid changes can be naturally occurring amino acid changes resulting in Fc domain polymorphism, or the amino acid changes can be introduced, for example, by site-directed mutagenesis. Amino acid changes can occur anywhere within the Fc domain as long as the Fc domain maintains its receptor binding function and biological activity. In a preferred embodiment, the polymorphism or mutation leads to enhanced receptor binding and/or enhanced multimerization or biological function. The polymorphism/mutation preferentially occurs at one or more of amino acid positions 233-435 according to the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Specific polymorphisms/mutations at these amino acid positions are well known in the art and can be found, for example, in Shields, et al. (2001) "High Resolution Mapping of the Binding Site on Human IgG1 for FcRI, FcYRII, FcRIII and FcRn and Design of IgG1 Variants with Improved Binding to the FCYR," J. Biol. Chem., 276(9):6591-6601, which is incorporated herein by reference in its entirety.
    In a preferred embodiment, the polymorphism/mutation contains one or more amino acid substitutions at positions 233, 234, 235, 236, 237, 238, 239, 253, 254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 280, 285, 286, 288, 290, 293, 295,296, 297, 298, 301, 303, 305, 307, 309, 311, 312, 315, 317, 322, 326, 327, 329, 330, 331,332, 333, 334, 337, 338, 339, 360, 362, 376, 378, 380, 382, 392, 414, 415, 424, 430, 433,434, 435, and/or 436 of IgG1 Fc . In a further embodiment, the polymorphism/mutation contains two or more amino acid substitutions at positions 233, 234, 235, 236, 237, 238, 239, 253, 254, 255, 256, 258, 264, 265, 267, 268, 269 , 270,272, 276, 280, 285, 286, 288, 290, 293, 295, 296, 297, 298, 301, 303, 305, 307, 309, 311,312, 315, 317, 322, 326, 327, 329, 330 , 331, 332, 333, 334, 337, 338, 339, 360, 362, 376,378, 380, 382, 392, 414, 415, 424, 430, 433, 434, 435, and/or 436 of IgG1 Fc. In a further embodiment, the polymorphism/mutation contains three or more amino acid substitutions at positions 233, 234, 235, 236, 237, 238, 239, 253, 254, 255, 256, 258, 264, 265, 267, 268, 269 , 270, 272, 276, 280, 285, 286, 288, 290, 293, 295, 296, 297, 298, 301,303, 305, 307, 309, 311, 312, 315, 317, 322, 326, 327, 329 , 330, 331, 332, 333, 334, 337,338, 339, 360, 362, 376, 378, 380, 382, 392, 414, 415, 424, 430, 433, 434, 435, and/or 436 of IgG1 Fc . In a further embodiment, the polymorphism/mutation contains more than three amino acid substitutions at positions 233, 234, 235, 236, 237, 238, 239, 253,254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 280, 285, 286, 288, 290, 293,295, 296, 297, 298, 301, 303, 305, 307, 309, 311, 312, 315, 317, 322, 326, 327, 329, 330,331, 332, 333, 334, 337, 338, 339, 360, 362, 376, 378, 380, 382, 392, 414, 415, 424, 430,433, 434, 435, and/or 436 of IgG1 Fc.
    The term "functional variant" as used herein refers to a sequence related by homology to a reference sequence, which is capable of mediating the same biological effects as the reference sequence (when a polypeptide), or which encodes a polypeptide which is capable of mediating the same biological effects as a polypeptide encoded by the reference sequence (when a polynucleotide). For example, a functional variant of any of the biomimetics described herein would have a specific homology or identity and would be capable of immunomodulating monocytes or dendritic cells (DCs). Functional sequence variants include both polynucleotides and polypeptides. Sequence identity is generally evaluated using BLAST 2.0 (Local Alignment Basic Search Tool), operating with the default parameters: Filter-On, Score Matrix-BLOSUM62, word length -3, E value - 10, Gap costs - 11.1 and Alignments - 50.
    From the above, it will be appreciated that stradomers of the present invention include stradomers having: (a) only naturally occurring Fc domains, (b) a mixture of naturally occurring Fc domains and Fc domains with changes in amino acid sequences, and ( c) only Fc domains with changes in amino acid sequences. All that is needed is that the altered stradomers containing amino acid sequences are at least 25%; 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%; 96%; 97%; 98%; 99%; 99.5%; or 100% or even more of the ability of a corresponding stradomer comprising the Fc domains with naturally occurring sequences to bind two or more FCYR receptors.
    The aforementioned FCY receptor binding sites that occur in the stradomers of the present invention can be sequence-altered through genetic engineering to predictably derive binding sites altered with binding capabilities and affinities relative to a native sequence. For example, specific residues can be altered which reduce binding of the Fc domain of biomimetic compounds to FcRIIb, increasing binding to FcRIIIa. An example of a structure-function analysis based on extensive mutagenesis for hlgG Fcy receptor binding sequences is Robert L. Shields, et al. High Resolution Mapping of the Binding Site on Human IgGl for FcRI, FcRII, FcRIII, and FcRn and Design of IgGl Variants with Improved Binding to the FcR. J. Biol. Chem., Feb 2001; 276: 6591 - 6604. Similar studies were performed on murine IgG Fc (Fc mIgG). Based on the structural and primary sequence homologies of the native IgG Fc domains for all species, one of skill in the art can translate the extensive structure-function knowledge of human IgG Fc and mouse IgG Fc to rational mutagenesis of all native Fc receptor binding site sequences in the biomimetic compounds of the present invention to design binding sites with particular Fc receptor specificities and binding affinities.
    In addition to the amino acid sequence composition of native Fc domains, the carbohydrate content of the Fc domain is known to play an important role in Fc domain structure and binding interactions with FcR. See, for example, Robert L. Shields et al. Lack of Fucose on Human IgGl N-Linked Oligosaccharide Improves Binding to Human FCYRIII and Antibody-dependent Cellular Toxicity. J. Biol. Chem, Jul 2002; 277: 26733-26740 (doi: 10.1074/jbc.M202069200); Ann Wright and Sherie L. Morrison. Effect of C2-Associated Carbohydrate Structure on Ig Effector Function: Studies with Chimeric Mouse-Human IgG1 Antibodies in Glycosylation Mutants of Chinese Hamster Ovary Cells. J. Immunol, April 1998; 160: 3393-3402. Carbohydrate content can be controlled using, for example, particular protein expression systems, including particular cell lines or in vitro enzymatic modification. Thus, the present invention includes stradomers comprising the Fc domains, with the native carbohydrate content of holo-antibody from which the domains were obtained, as well as those biomimetic compounds with an altered carbohydrate content. In another embodiment, the multimer components of the stradomer are characterized by a different glycosylation pattern compared to the homodimer component of the same stradomer. In another preferred embodiment, the stradomer is enriched with multimers that comprise a glycosylation pattern that enhances binding to the Fc receptor.
    Addition to the polypeptide chain of a partial Fc domain, a region of multimerization, or glycosylation changes can create a conformational change in the Fc domain allowing enhanced binding of the Fc domain of an FCY receptor. Thus, apparently much minor alterations to the polypeptide may also create a stradomer capable of forming enhanced binding of multiple Fc receptors or a stradomer with diminished ability to bind multiple Fc receptors. Partial Domains and Partial Fragments
    One of skill in the art will further recognize that the Fc domains and partial Fc domains used in embodiments of the present invention need not be full-length versions. That is, the present invention encompasses the use of Fc domain monomers and partial Fc domain monomers that lack the amino-terminal, carboxy-terminal or intermediate amino acids of particular Fc domain monomers and partial Fc domain monomers comprising the stradomers and other biomimetics of the invention.
    For example, the binding site in human IgG immunoglobulins for Fcy receptors has been described (e.g., Radaev, S., Sun, P., 2001. Recognition of Immunoglobulins by Fcy Receptors. Molecular Immunology 38, 1073 - 1083; Shields, RL et.al., 2001. High Resolution Mapping of the Binding Site on Human IgGl for FcRI, FcRII, FcRIII, and FcRn and Design of IgGl Variants with Improved Binding to the FCYR. J. Biol. Chem. 276 (9), 6591-6604). Based on the knowledge that one can remove amino acids from the Fc domain of these immunoglobulins and determine the effects on the binding interaction between the Fc domain and the receptor. Thus, the present invention encompasses the Fc domains of IgG having at least about 90% of the amino acids spanning from bottom hinge position 233 to 338 and CH2 as defined in Radaev, S., Sun, P., 2001.
    The IgG immunoglobulin partial Fc domains of the present invention include all or part of the hinge region, all or part of the CH2 domain, and all or part of the CH3 domain.
    IgG partial Fc domains having only a part of the hinge region, part of the CH2 domain or part of the CH3 domain are constructed from partial Fc domain monomers. Thus, the present invention includes IgG hinge region monomers derived from the N-terminus of the hinge region or the C-terminus of the hinge region. They can thus contain, for example, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36.37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, or 62 (up to 15 for IgG1, up to 12 for IgG2, up to 62 for IgG3, up to 12 for IgG4) region amino acids of hinge.
    The present invention also includes IgG CH2 domain monomers derived from the N-terminus of the CH2 domain or the C-terminus of the CH2 domain. They can thus contain, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 62, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48.63, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72.73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96.97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 (up to 110 for IgG1 and IgG3, up to 109 for IgG2 and IgG4) amino acids of the CH2 domain.
    The present invention further includes IgG CH3 domain monomers derived from the N-terminus of the CH3 domain or the C-terminus of the CH3 domain. They can thus contain, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26.98, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.99, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,100, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 101, 100, 101, 102, 103, 104, 105, 106, or 107 (up to 106 for IgG1 and IgG3, up to 107 for IgG2 and IgG4) amino acids of the CH3 domain.
    From the above, it will be appreciated that different embodiments of the present invention include stradomers containing: (a) full-length Fc domains, (b) a mixture of full-length Fc domains and partial Fc domains, and (c) partial Fc domains. In each of these embodiments, stradomers can further comprise CH1 domains. As discussed herein, in each embodiment of the stradomers of the present invention, the stradomers have the ability to bind two or more Fc receptors. Preferred Stradomer Modalities and Stradomer Monomers
    The terms "FCYR" and "FCY receptor" as used herein include all members of the Fc gamma receptor family of proteins expressed on the surface of immune cells, as described in Nimmerjahn F and Ravetch JV. Fcy receivers: old friends and new family members. Immunity 2006 Jan; 24(1): 19-28, or as it can be defined later. The term "FcR" described herein is intended to encompass all members of the Fc gamma families RI, RII, and RIII. Fcy receptors include low-affinity and high-affinity Fcy receptors including, but not limited to, human FcRI (CD64); FcRII (CD32) and its isotypes and allotypes FcRIIa LR, FcRIIa HR, FcRIIb, and FcRIIc; FcRIII (CD 16) and its FcRIIIa and FcRIIIb isotypes. One of skill in the art will recognize that the present invention, which includes compounds that bind FcR and FcR homologues, such as those described in Davis, et al. (2004) "Differential B cell expression of mouse Fc receptor homologs," Int. Immunol., 16(9):1343-1353, will be applied to future FcyRs and associated isotypes and allotypes that may not yet have been discovered.
    It has been described that hIVIG binds to and fully saturates the neonatal Fc receptor ("FcRn") and that competitive inhibition of FcRn may play an important role in the biological activity of hIVIG (eg, Mechanisms of Intravenous Immunoglobulin Action in Immune Thrombocytopenic Purpura .F. Jin, J. Balthasar. Human Immunology, 2005, Volume 66, Issue 4, Pages 403-410.). Since immunoglobulins that bind strongly to Fcy receptors also bind at least to some degree to FcRn, a skilled person will recognize that stradomers that are capable of binding to more than one Fcy receptor also bind and may completely saturate the FcRn. "Able to specifically bind an FCYRX" as used herein refers to binding an FCYR such as FCYRIII. Specific binding is generally defined as the amount of labeled ligand that is displaceable by an excess of unlabeled ligand subsequent to a binding assay. However, this does not preclude other means of assessing specific binding that are well established in the art (eg, Mendel CM, Mendel DB, 'Non-specific' binding. The problem, and a solution. Biochem J. 1985 May 15; 228(1):269-72). Specific binding can be measured in a number of ways well known in the art, such as surface plasmon resonance (SPR) technology (commercially available from BIACORE®) or biolayer interferometry (commercially available from ForteBio®) to characterize the constants. of association and dissociation of the immunologically active biomimetic (Asian K, Lakowicz JR, Geddes C. Plasmon light scattering in biology and medicine: new sensing approaches, visions and perspectives. Current Opinion in Chemical Biology 2005, 9:538-544).
    The "aggregated native IgG immune activity" refers to the properties of multimerized IgG that impact the functioning of an immune system after exposure of the immune system with the IgG aggregates. Specific properties of multimerized native IgG include altered specific binding to FcRs, cross-linking of FcRs on immune system cell surfaces, or an effector functionality of multimerized IgG such as antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis (ADCP) , or complement fixation (see, eg, Nimmerjahn F, Ravetch JV. The anti-inflammatory activity of IgG: the intravenous IgG paradox. J Exp Med. 2007; 204:11-15; Augener W, Friedman B, Brittinger G Are aggregates of IgG the effective part of high-dose immunoglobulin therapy in adult idiopathic thrombocytopenic purpura (ITP) Blut. 1985;50:249-252; Arase N, Arase H, Park SY, Ohno H, Ra C, Saito T Association with FcRgamma is essential for activation signal through NKR-P1 (CD161) in natural killer (NK) cells and NKl.1+ T cells. J Exp Med. 1997;186:1957-1963; Teeling JL, Jansen-Hendriks T , Kuijpers TW, et al. ons depends on the immunoglobulin G dimers: studies in experimental immune thrombocytopenia. Blood. 2001;98:1095-1099; Anderson CF,
    Mosser DM. Cutting edge: biasing immune responses by directing antigen to macrophage Fc gamma receptors. J Immunol. 2002; 168:3697-3701; Jefferis R, Lund J. Interaction sites on human IgG-Fc for FcR: current models. Immunology Letters. 2002;82:57; Banki Z,
    Kacani L, Mullauer B, et al. Cross-Linking of CD32 Induces Maturation of Human Monocyte - Derived Dendritic Cells Via NF-{kappa}B Signaling Pathway. J Immunol. 2003;170:3963-3970; Siragam V, Brine D, Crow AR, Song S, Freedman J, Lazarus AH. Can antibodies with specificity for soluble antigens mimic the therapeutic effects of intravenous IgG in the treatment of autoimmune disease J Clin Invest. 2005; 15:155160). These properties are generally evaluated by comparison with the homodimeric properties of IgG. "Comparable or superior to an FCY receptor crosslinking or an effector functionality of a plurality of aggregated naturally occurring IgG immunoglobulins" as used herein means that the stradomer generates an Fcy receptor crosslinking assay value of about 70% or more of the value achieved using a similar dose or concentration of hIVIG. In some embodiments, the assay value is at least within the standard error range of assay values obtained using hIVIG. In other modalities, the assay value is 110% or greater than that of hIVIG at the same dose. Assays for Fcy crosslinking are well known to those of ordinary skill in the art (see, for example, Nimmerjahn and Ravetch, (2008) "Fcy receptors as regulators of immune responses", Nature Reviews Immunology, 8:34-47).
    Although higher order multimers were found to be effective in modulating the immune response, it was surprisingly found that homodimers were also effective immune modulators. Without being bound by theory, it is believed that homodimers are capable of forming higher order multimers in vivo. It has been verified through multimerization experiments that an otherwise pure population of homodimers is capable of multimerizing in the presence of low levels of blood or fetal bovine serum. Therefore, although higher order multimers are more effective than the homodimer moiety in modulating the immune response, the homodimer moiety of the naturally linked stradomers of the present invention may also be effective immune modulators, in part, by multimerizing the homodimer in the presence low levels of blood or serum. Therefore, by "higher order multimers" we mean multimers other than homodimer, which are formed in solution prior to injection into a subject, as well as multimers beyond homodimer that are formed in vivo.
    "Immune modulating activities", "immune modulating response", "immune modulating system," and "immunomodulating" mean alterations of immune systems through changes in the activities, capacities and relative members of one or more immune cells, including maturation of a cell type within its cell type or into other cell types. For example, immune modulation of immature monocytes can lead to a larger population of more mature monocytes, dendritic cells, macrophages, or osteoclasts, which are derived from immature monocytes. As another example, immune modulation of memory B cells can lead to selective apoptosis of memory B cells with concomitant decreases in the production of particular antibodies. As another example, immune modulation of NK cells can lead to enhanced Antibody-Dependent Cellular Cytotoxicity. As another example, immune modulating activities can lead to an increase in cell populations with phenotypes that cannot otherwise be expressed at high levels, such as CD8 beta + / CD11c + cells. As another example, immune modulating activities can lead to a decrease in pro-inflammatory cytokines or cytokines that are commonly elevated in autoimmune diseases, such as IL-6 and IL-8. As another example, immune modulating activities can lead to NK cell activation with cleavage and subsequent secretion of TGF-beta. For example, immune cell receptors must be bound by immunologically active biomimetics and activate intracellular signaling to induce various changes in immune cells, referred to separately as "activating immune modulation". Blocking immune cell receptors to prevent receptor activation is also encompassed within "immune modulation" and may be separately referred to as "inhibitory immune modulation".
    Modulation of dendritic cells can promote or inhibit antigen presentation to T cells, for example, by inducing the expression of CD86 and/or CD1a on the surface of dendritic cells. CD1a is a class I MHC-related glycoprotein that is expressed on the surface of antigen-presenting cells, particularly, on dendritic cells. CD1a is involved in the presentation of lipid antigens to T cells. CD86 is also expressed on the surface of antigen presenting cells and provides costimulation of T cells. CD86 is a ligand for CD28 and CTLA-4 on the surface of T cells to send activation signals and inhibitors, respectively. Therefore, the expression level of CD86 and its cognate receptors determines whether tolerance or a specific immune response will be induced. In a preferred embodiment, the stradomers of the present invention are capable of modulating the immune response, in part, by inducing the expression of CD86 and CD1a on the surface of antigen-presenting cells, particularly, dendritic cells.
    Modulation of monocyte maturation refers to the differentiation of a monocyte into a mature DC, a macrophage, or an osteoclast. Differentiation can be modulated to accelerate the rate or direction of maturation and/or to increase the number of monocytes undergoing differentiation. Alternatively, differentiation can be reduced in terms of rate of differentiation and/or number of differentiating cells.
    The term "isolated" polypeptide or peptide as used herein refers to a polypeptide or peptide which either has no naturally occurring counterpart or which has been separated or purified from naturally accompanying components, for example , in tissues such as the pancreas, liver, spleen, ovary, testes, muscle, connective tissue, neural tissue, gastrointestinal tissue, or breast tissue or tumor tissue (e.g. breast cancer tissue), or bodily fluids, such as blood, serum, or urine. Typically, the polypeptide or peptide is considered "isolated" when it is at least 70%, by dry weight, free of proteins and other naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation of a polypeptide (or peptide) of the present invention is at least 80%, more preferably, at least 90%, and most preferably at least 99%, by dry weight, the polypeptide (peptide), respectively, of the invention. Since a polypeptide or peptide that is chemically synthesized is, by its very nature, separated from the other components that naturally accompany it, the synthetic peptide or polypeptide is "isolated".
    An isolated polypeptide (or peptide) of the present invention can be obtained, for example, by extraction from a natural source (for example, from tissues or bodily fluids), by expression of a recombinant nucleic acid encoding the polypeptide, or a peptide, or by chemical synthesis. A polypeptide or peptide that is produced in a cellular system other than the source from which it naturally originates is "isolated" because it will necessarily be free of components that naturally accompany it. In a preferred embodiment, the isolated polypeptide of the present invention contains only those sequences that correspond to the leader peptide (SEQ ID NO: 1), the IgG1 Fc monomer (SEQ ID NO: 2) or (SEQ ID NO: 19) and the IgG2 hinge multimerization domain (SEQ ID NO: 3), the isoleucine multimerization domain (SEQ ID NO: 5) or the GPP multimerization domain (SEQ ID NO: 26) and there are no additional sequences that can aid in the protein cloning or purification (ie, introduced restriction enzyme recognition sites or purification tags). The degree of isolation or purity can be measured by any suitable method, for example column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Pharmaceutical Compositions
    Administration of the stradomer compositions described herein will be via any common route, orally, parenterally, or topically. Exemplary routes include, but are not limited to, oral, nasal, buccal, rectal, ophthalmic, vaginal, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intratumoral, spinal, intrathecal, intraarticular, intraarterial, subarachnoid, sublingual, oral mucosal, lymphatic, bronchial, intrauterine, subcutaneous, intratumoral, integrated into an implantable device, such as a suture or implantable device, such as an implantable polymer, intradural, intracortical, or dermal. Such compositions would normally be administered as pharmaceutically acceptable compositions as described herein. In a preferred embodiment, the stradomer alone is administered intravenously or subcutaneously.
    The term "pharmaceutically acceptable carrier", as used herein, includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except to the extent that any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
    The stradomer compositions of the present invention may be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric , mandelic, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like.
    Sterile injectable solutions are prepared by incorporating the stradomer in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for preparing sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization techniques which produce a powder of the active ingredient plus any additional desired ingredient from a solution previously sterilized by filtration.
    Furthermore, one embodiment is a stradomer composition suitable for oral administration which is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier must be assimilable or edible, and include liquid, semi-solid, ie pastes, or solid carriers. Except to the extent that any conventional means, agent, diluent or carrier is detrimental to the recipient or to the therapeutic efficacy of a stradomer preparation contained therein, its use in the oral administration of a stradomer composition for use in the practice of the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. "Oral administration" as used herein includes oral, buccal, enteral or intragastric administration.
    In one embodiment, the stradomer composition is combined with the carrier in any convenient and practical manner, namely, by suspension, solution, emulsification, mixing, encapsulation, microencapsulation, absorption, and the like. Such processes are routine for those skilled in the art.
    In a specific embodiment, the stradomer composition in powder form is combined or mixed with a solid or semi-solid carrier. Mixing can be carried out in any convenient manner such as milling. Stabilizing agents can also be added to the mixing process in order to protect the composition from loss of therapeutic activity through, for example, denaturation in the stomach. Examples of stabilizers for use in an orally administrable composition include buffers, antagonists for the secretion of stomach acids, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose , sorbitol, mannitol, etc., proteolytic enzyme inhibitors, and the like. More preferably, for an orally administered composition, the stabilizer may also include antagonists to the secretion of stomach acids.
    In addition, the stradomer composition for oral administration which is combined with a semi-solid or solid carrier can further be formulated in soft or hard shell gelatin capsules, tablets, or pills. Most preferably, gelatin capsules, tablets, or pills are enteric coated. Enteric coatings prevent denaturation of the composition in the stomach or upper intestine where the pH is acidic. See, for example, US Patent 5,629,001. Upon reaching the small intestine, the basic pH in it dissolves the lining and allows the composition to be released to interact with intestinal cells, eg Peyer's patch M cells.
    In another embodiment, the stradomer composition in powder form is combined or blended with materials that create a nanoparticle encapsulating the immunologically active biomimetic or to which the immunologically active biomimetic is bound. Each nanoparticle will have a size of less than or equal to 100 microns. Nanoparticles may have mucoadhesive properties that allow gastrointestinal absorption of an immunologically active biomimetic that is otherwise not bioavailable orally.
    In another embodiment, a powder composition is combined with a liquid carrier, such as, for example, water or saline, with or without a stabilizing agent.
    A specific stradomer formulation that can be used is an immunologically active biomimetic protein solution in a hypotonic based phosphate buffer, which is potassium free where the buffer composition is as follows: 6 mM monobasic sodium phosphate monohydrate , 9 mM sodium phosphate dibasic heptahydrate, 50 mM sodium chloride, pH 7.0 +/- 0.1. The concentration of immunologically active biomimetic protein in a hypotonic buffer can range from 10 micrograms/ml to 100 milligrams/ml. This formulation can be administered via any route of administration, for example, but not limited to, intravenous administration.
    In addition, a stradomer composition for topical administration that is combined with a semi-solid carrier can be further formulated in a cream or ointment, gel form. A preferred carrier for forming a gel ointment is a polymer gel. Preferred polymers that are used to make a gel composition of the present invention include, but are not limited to, carbopol, carboxymethyl cellulose, and pluronic polymers. Specifically, a powdered Fc multimer composition is combined with an aqueous gel containing a polymerizing agent such as Carbopol 980 with a strength of between 0.5% and 5% by weight/volume for application to the skin or under the skin to the skin. disease treatment. "Topical administration" as used herein includes application via a dermal, epidermal, subcutaneous or mucosal surface.
    In addition, a stradomer composition can be formulated into a polymer by subcutaneous or subdermal implantation. A preferred formulation for the implantable drug-infused polymer is an agent generally considered to be safe and may include, for example, cross-linked dextran (Samantha Hart, Master of Science Thesis, “Elution of Antibiotics from a Novel Cross-Linked Dextran Gel: Quantification ” Virginial Polytechnic Institute and State University, June 8, 2009) dextran-tyramine (Jin, et al. (2010) Tissue Eng. Part A. 16(8):2429-40), dextran-polyethylene glycol (Jukes, et al. (2010) Tissue Eng. Part A., 16(2):565-73), or gluteraldehyde dextran (Brondsted, et al. (1998) J. Controlled Release, 53:7-13). One of skill in the art will know that many polymers and similar hydrogels can be formed by incorporating the fixed stradomer into the polymer or hydrogel and controlling the pore size to the desired diameter.
    After formulation, the solutions are administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective to result in an amelioration or repair of symptoms. The formulations are easily administered in a variety of dosage forms such as solutions, ingestible drug release capsules and the like. Some variation in dosage may occur depending on the condition of the subject being treated. The person responsible for administration may, in any case, determine the appropriate dose for the individual subject. In addition, for human administration, the preparations meet standards of sterility, general safety and purity as required by the FDA's Center for Biological Assessments and Research Standards.
    The route of administration will, of course, vary with the location and nature of the disease being treated, and may include, for example, intradermal, transdermal, subcutaneous, intramuscular, parenteral, nasal, intravenous, intranasal, subcutaneous, percutaneous, intratracheal administration. , intraperitoneal, perfusion, lavage, intratumoral, by direct injection, and oral administration.
    The term "parenteral administration", as used herein, includes any form of administration, in which the compound is absorbed by the subject, without involving absorption through the intestine. Examples of parenteral administrations that are used in the present invention include, but are not limited to, intramuscular, intravenous, intraperitoneal, intratumoral, intraocular, nasal, or intraarticular administration.
    Furthermore, the stradomer of the present invention may optionally be administered before, during or after another pharmaceutical agent. For example, it has surprisingly been found that the concomitant administration of the stradomer of the present invention and prednisolone achieves results synergistically superior to those observed with either individual stradomer or prednisolone composition (See Figure 3). The following are specific examples of various categories of pharmaceutical formulation. and preferred routes of administration, as indicated, for specific exemplary diseases:
    Dissolvable buccal or sublingual tablet: angina, polyarteritis nodosa.Intravenous: Idiopathic Thrombocytopenic Purpura, Inclusion Body Myositis, Paraproteinemic IgM Demyelinating Polyneuropathy, Necrotizing Fasciitis, Pemphigus, Gangrene, Dermatomyositis, Granuloma, Lymphoma, Multiorgan Anemia, Sepsis , Multiple Myeloma and Monoclonal Gammopathy of Unknown Significance, Chronic Inflammatory Demyelinating Polyradiculoneuropathy, Inflammatory myopathies, Thrombotic thrombocytopenic purpura, Myositis, Anemia, Neoplasm, Haemolytic anemia, Encephalitis, Myelitis, Myelopathy especially associated with human lymphotropic cell virus-1 , Multiple Sclerosis and Optic Neuritis, Asthma, Epidermal Necrolysis, Lambert-Eaton Myasthenic Syndrome, Myasthenia Gravis, Neuropathy, Uveitis, Guillain-Barré Syndrome, Graft Versus Host Disease, Stiff Man Syndrome, Paraneoplastic Cerebellar Degeneration with Anti- yo, encephalomyelitis paraneoplastic and sensory neuropathy with anti-Hu antibodies, Systemic Vasculitis, Systemic Lupus Erythematosus, Autoimmune Diabetic Neuropathy, Acute Idiopathic Dysautonomic Neuropathy, Vogt-Koyanagi-Harada Syndrome, Multifocal Motor Neuropathy, Lower Motor Neuron Syndrome associated with anti-/GMl Demyelination, Membranoproliferative glomerulonephritis, Cardiomyopathy, Kawasaki disease, Rheumatoid arthritis, and IM syndrome - Evan's ITP, CIDP, MS, dermatomyositis, myasthenia gravis, muscular dystrophy. The term "intravenous administration" as used herein includes all techniques for delivering a compound or composition of the present invention to the systemic circulation via an intravenous infusion or injection.
    Dermal gel, lotion, cream, or transdermal patch: vitiligo, herpes zoster, acne, chelitis. Rectal suppository, gel, or infusion: ulcerative colitis, hemorrhoidal inflammation.
    Oral as pills, troche, encapsulated, or enteric coated: Crohn's disease, celiac disease, irritable bowel syndrome, inflammatory liver disease, Barrett's esophagus.Intracortical: epilepsy, Alzheimer's disease, multiple sclerosis, Parkinson's disease, Parkinson's disease Huntingdon.
    Intra-abdominal Implant or Infusion: endometriosis. Intravaginal gel or suppository: bacterial, trichomonal, or fungal vaginitis.
    Medical devices: Coronary artery stent-coated, prosthetic joints. The stradomers described herein can be administered in dosages of about 0.01 mg per kg to about 300 mg per kg of body weight, and especially 0.01 mg per kg kg of body weight to about 1000 mg per kg of body weight, and may be administered at least once a day, weekly, biweekly or monthly. A biphasic dosing regimen which can be used wherein the first dosing stage comprises about 0.1% to about 300% of the second dosing stage. Therapeutic Applications of Stradomers
    Based on the rational model and in vitro and in vivo validations, the stradomers of the present invention will serve as important biopharmaceuticals for the treatment of autoimmune diseases and for the modulation of immune function in a wide variety of contexts, such as bioimmunotherapy for cancer and inflammatory diseases. Medical conditions suitable for treatment with the immunologically active biomimetics described in the present invention include those currently routinely treated with hIVIG or where hIVIG has been found to be clinically useful such as autoimmune cytopenias, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, myasthenia gravis , anti-Factor VIII autoimmune disease, dermatomyositis, vasculitis, and uveitis (see, FG van der Meche, PI Schmitz, N. Engl. J. Med. 326, 1123 (1992); P.
    Gajdos et al, Lancet i, 406 (1984); Y. Sultan, M.D. Kazatchkine, P. Maisonneuve, U.E. Nydegger, Lancet ii, 765 (1984); M.C. Dalakas et al., N. Engl. J. Med. 329, 1993 (1993); D.R. Jayne, M.J. Davies, C.J. Fox, C.M. Black, C.M. Lockwood, Lancet 337, 1137 (1991); P. LeHoang, N. Cassoux, F. George, N. Kullmann, M.D. Kazatchkine, Ocul. Immunol. Inflamm. 8, 49 (2000)) and cancers or inflammatory disease conditions in which a monoclonal antibody can be used or is already in clinical use. Conditions included among those that can be effectively treated by compounds that are objects of the present invention include an inflammatory disease with an imbalance in cytokine networks, an autoimmune disorder mediated by pathogenic autoantibodies or autoaggressive T cells, or an acute or chronic phase of a chronic, inflammatory, or infectious recurrent autoimmune process or disease.
    In addition, other medical conditions that have an inflammatory component will benefit from treatment with stradomers such as Amyotrophic Lateral Sclerosis, Huntington's Disease, Alzheimer's Disease, Parkinson's Disease, Myocardial Infarction, Stroke, Hepatitis B, Hepatitis C, Virus of Human Immunodeficiency Associated with Inflammation, Adrenoleukodystrophy and Epileptic Disorders, especially those believed to be associated with Post-Viral Encephalitis, including Rasmussen Syndrome, West Syndrome and Lennox-Gastaut Syndrome.
    The general approach to therapy using the isolated stradomers described herein is to administer to a subject having a disease or condition, a therapeutically effective amount of the isolated immunologically active biomimetic to effect a treatment. In some modalities, the diseases or conditions can be broadly classified as inflammatory diseases with an imbalance in the cytokine network, an autoimmune disorder mediated by pathogenic autoantibodies or autoaggressive T cells, or an acute or chronic phase of a chronic relapsing disease or process.
    The term "treating" and "treatment" as used herein refers to administering to a subject a therapeutically effective amount of a stradomer of the present invention such that the subject has an improvement in a disease or condition, or a symptom of the disease or condition. Improvement is any amelioration or remediation of the disease or condition, or symptoms of the disease or condition. Improvement is an observable or measurable improvement, or it can be an improvement in the subject's general sense of well-being. Thus, one skilled in the art understands that a treatment can improve the disease condition, but it may not be a complete cure for the disease.
    Specifically, improvements in subjects can include one or more of the following: decreased inflammation; decreased inflammatory laboratory markers such as C-reactive protein, decreased autoimmunity as evidenced by one or more of the following: improvement in autoimmune markers such as antibodies or platelet count, white blood cell count, or red cell count, decreased rash or purpura, decreased weakness, numbness, or tingling, increased glucose levels in patients with hyperglycemia, decreased joint pain, inflammation, swelling or degradation, decreased frequency of cramps and diarrhea and volume, decreased angina, decreased inflammation tissue, or a decrease in the frequency of seizures; decreased cancer tumor burden, increased time to tumor progression, decreased cancer pain, increased survival or improved quality of life, or delayed progression or improved osteoporosis.
    The term "therapeutically effective amount" as used herein refers to an amount that results in an amelioration or amelioration of damage from the symptoms of the disease or condition.
    As used herein, "prophylaxis" can mean the complete prevention of symptoms of a disease, a delay in the onset of symptoms of a disease, or a reduction in the severity of symptoms of the disease that develop later.
    The term "subject" as used herein is taken to mean any mammalian patient to which the stradomers of the present invention are administered in accordance with the methods described herein. In a specific embodiment, the methods of the present disclosure are employed to treat a human subject. The methods of the present disclosure can also be used to treat non-human primates (e.g., monkeys, baboons, and chimpanzees), mice, rats, cattle, horses, cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, gerbils, guinea pigs, hamsters, bats, birds (eg chickens, turkeys and ducks), fish and reptiles to produce species-specific or chimeric stradomer molecules.
    In particular, the stradomers of the present invention can be used to treat conditions including, but not limited to, congestive heart failure (CHF), vasculitis, rosacea, acne, eczema, myocarditis and other myocardial conditions, systemic lupus erythematosus, diabetes, spondylopathies , synovial fibroblasts, and bone marrow stroma, bone loss, Paget's disease, osteoclastoma, multiple myeloma, breast cancer; disuse osteopenia, malnutrition, periodontal disease, Gaucher disease, Langerhans cell histiocytosis, spinal cord injury, acute septic arthritis, osteomalacia, Cushing's syndrome, monoostotic fibrous dysplasia, polyostotic fibrous dysplasia, periodontal reconstruction, and bone fractures; sarcoidosis; osteolytic bone cancers, lung cancer, kidney cancer and rectal cancer; bone metastasis, treatment of bone pain and humoral malignant hypercalcemia, ankylosing spondylitis and other spondyloarthropathies; transplant rejection, viral infections, hematologic malignancies, and neoplastic-like conditions, eg, Hodgkin's lymphoma, non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocytic lymphoma / chronic lymphocytic leukemia, mycosis fungoid, mantle cell lymphoma, follicular lymphoma, lymphoma of diffuse large B cell, marginal zone lymphoma, hairy cell leukemia and mononuclear leukemia), precursor cell tumors including B cell lymphoma/acute lymphoblastic leukemia and T cell lymphoma/acute lymphoblastic leukemia, thymoma, NK cell tumors and mature T cells, including peripheral T cell leukemias, adult T cell leukemia / T cell lymphoma and large granular lymphocytic leukemia, Langerhans cell histocytosis, myeloid neoplasms such as acute myeloid leukemias including AML with maturation, AML without differentiation, acute promyelocytic leukemia, acute myelomonocytic leukemia, and acute monocytic leukemia, synd myelodysplastic romes, and chronic myeloproliferative disorders, including chronic myelogenous leukemia, central nervous system tumors, eg brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma and retinoblastoma), solid tumors (nasopharyngeal cancer, basal cell carcinoma, pancreatic cancer, bile duct cancer, Kaposi's sarcoma, testicular cancer, uterine cancer, vaginal or cervical cancer, ovarian cancer, primary liver cancer or endometrial cancer, vascular system tumors (angiosarcoma and hemangiopericytoma)) or other cancer.
    "Cancer" herein refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, leiomyosarcoma, rhabdomyosarcoma, rhabdomyosarcoma, chordate, chondrosarcoma, fibrosarcoma, chondrosarcoma , synovioma, schwannoma, meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignant tumors. More particular examples of such cancers include squamous cell cancer (eg squamous epithelial cell cancer), lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma of the lung, small cell lung cancer, peritoneum cancer, hepatocellular cancer, gastric, or stomach cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, liver cancer breast, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma , testicular cancer, esophageal cancer, biliary tract tumors, Ewing's tumor, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, carcinoma. sebaceous gland cynoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilms' tumor, testicular tumor, lung carcinoma, carcinoma of bladder, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, myelodysplastic disease, myelodysplasia disease cancers, neuroendocrine tumors, schwannoma, and other carcinomas, as well as cancer of the head and neck.
    The stradomers of the present invention can be used to treat autoimmune diseases. "Autoimmune disease" as used herein refers to a diverse group of over 80 diseases and conditions. In all these diseases and conditions, the underlying problem is that the body's immune system attacks the body itself. Autoimmune diseases affect all major systems in the body, including the connective tissue, nerves, muscles, endocrine system, skin, blood, respiratory and gastrointestinal systems. Autoimmune diseases include, for example, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, type 1 diabetes.
    The disease or condition treatable using the compositions and methods of the present invention may be a hematoimmunological process, including but not limited to idiopathic thrombocytopenic purpura, alloimmune/autoimmune thrombocytopenia, acquired immune thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, red cell aplasia associated with Parvovirus PB19, Acquired Antifactor VIII Autoimmunity, Acquired von Willebrand Disease, Multiple Myeloma and Monoclonal Gammopathy of Unknown Significance, Sepsis, Aplastic Anemia, Pure Red Cell Aplasia, Diamond-Blackfan anemia, Newborn Hemolytic Disease, Immune-mediated neutropenia, platelet transfusion refractoriness, post-transfusional purpura, neonatal, hemolytic uremic syndrome, systemic vasculitis, thrombotic thrombocytopenic purpura, or Evan's syndrome.
    The disease or condition may also be a neuroimmunological process, including, but not limited to, Guillain-Barré syndrome, Inflammatory Demyelinating Polyradiculoneuropathy, Paraproteinemic IgM Demyelinating Polyneuropathy, Lambert-Eaton myasthenic syndrome, Myasthenia gravis, Multifocal Motor Neuropathy, Syndrome of Lower Motor Neuron associated with anti-/GMl, Demyelination, Multiple Sclerosis and optic neuritis, Stiff Man syndrome, Paraneoplastic cerebellar degeneration with anti-Yo antibodies, paraneoplastic encephalomyelitis, sensory neuropathy with anti-Hu antibodies, epilepsy, Encephalitis, Myelitis Myelopathy especially associated with human T-cell lymphotropic virus-1, Autoimmune Diabetic Neuropathy, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, or Acute Idiopathic Dysautonomic Neuropathy.
    The disease or condition may also be a Rheumatic disease process, including, but not limited to, Kawasaki disease, rheumatoid arthritis, Felty's syndrome, ANCA positive vasculitis, Spontaneous Polymyositis, Dermatomyositis, Antiphospholipid syndrome, recurrent miscarriages, lupus erythematosus systemic, juvenile idiopathic arthritis, Raynaud's phenomenon, CREST syndrome, or uveitis.
    The disease or condition may also be a dermatoimmunological disease process, including, but not limited to, Toxic Epidermal Necrolysis, Gangrene, Granuloma, Bullous Autoimmune Skin Diseases including Pemphigus vulgaris, Bullous Pemphigoid, Pemphigus foliaceus, Vitiligo, Toxic Shock Syndrome Streptococcal, Scleroderma, systemic sclerosis including diffuse and limited cutaneous systemic sclerosis, or atopic dermatitis (especially steroid dependent).
    The disease or condition may also be a musculoskeletal immune disease process, including, but not limited to, Inclusion Body Myositis, Necrotizing Fasciitis, Inflammatory Myopathies, Myositis, Anti-Decorine Myopathy (BJ Antigen), Paraneoplastic Necrotic Myopathy, Myopathy X-Linked Vacuolate, Penacylamine-Induced Polymyositis, Atherosclerosis, Coronary Artery Disease, or Cardiomyopathy.
    The disease or condition may also be an immunological gastrointestinal disease process, including, but not limited to, pernicious anemia, autoimmune chronic active hepatitis, primary biliary cirrhosis, Celiac disease, dermatitis herpetiformis, cryptogenic cirrhosis, Reactive Arthritis, Crohn's Disease, Whipple's disease, ulcerative colitis, or sclerosing cholangitis.
    The disease or condition may also be Graft Versus Host Disease, Antibody-mediated Graft Rejection, Post-Bone Marrow Transplant Rejection, Post-Infectious Disease Inflammation, Lymphoma, Leukemia, Neoplasm, Asthma, Type 1 Diabetes Mellitus with Antibodies anti-beta cells, Sjogren's syndrome, Mixed Connective Tissue Disease, Addison's Disease, Vogt-Koyanagi-Harada Syndrome, Membranoproliferative Glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Hashimoto's Thyroiditis, Wegener's Granulomatosis, Micropolyarteritis, Churitis Polyarteritis nodosa or Multisystem Organ Failure.
    In another embodiment, the stradomers described herein can be used in an initiation system, in which blood is drawn from a patient and transiently contacted with the stradomer(s) for a time period of from about half an hour to about three hours before being introduced back into the patient. In this form of cell therapy, the patient's own effector cells are exposed to the stradomer which is fixed onto an ex vivo matrix in order to modulate the effector cells by exposing the effector cells to the stradomer. The blood, including the effector cells, is modulated and then infused back into the patient. Such an initiation system can have numerous clinical and therapeutic applications.
    The stradomers disclosed herein can also be readily applied to alter immune system responses in a wide variety of contexts to affect specific alterations in immune response profiles. Altering or modulating an immune response in a subject refers to increasing, decreasing or altering the ratio or components of an immune response. For example, levels of cytokine production or secretion can be increased or decreased as desired by targeting the appropriate combination of FcRs with a stradomer designed to interact with the receptors. Antibody production can also be increased or decreased; the ratio of two or more cytokines or immune cell receptors can be altered, or other types of cytokines or antibodies can be produced. The immune response may also be an effector function of an immune cell expressing an FCYR, including increased or decreased phagocytic potential of monocyte macrophage-derived cells, increased or decreased osteoclast function, increased or decreased antigen presentation by antigen-presenting cells ( e.g., DCs), NK cell function increased or decreased, B cell function increased or decreased compared to an immune response that is not modulated by an immunologically active biomimetic disclosed herein.
    In a preferred embodiment, a subject with cancer or an autoimmune or inflammatory disease has its immune response altered comprising the step of administering a therapeutically effective amount of a stradomer described herein to a subject, wherein the therapeutically effective amount of the stradomer alters the immune response in the subject. Ideally, this intervention addresses the subject's illness or condition. The altered immune response can be an increase or a decrease in the response, and can include cytokine levels, including altered levels of any of IL-6, IL-10, IL-8, IL-23, IL-7, IL-4, IL-12, IL-13, IL-17, TNF-alpha and IFN-alpha. In a preferred embodiment, IL-6 or IL-8 is reduced in response to therapy. In an especially preferred embodiment, IL-6 and IL-8 are reduced in response to therapy. The invention is not, however, limited to any particular mechanism of action of the described biomimetics. The altered immune response can be a level of altered autoantibodies in the subject. The altered immune response may be a level of altered autoaggressive T cells in the subject.
    For example, reducing the amount of TNF-alpha production in autoimmune diseases can have therapeutic effects. A practical application of this is anti-TNF-alpha antibody therapy (eg REMICADE®), which is clinically proven to treat Plaque Psoriasis, Rheumatoid Arthritis, Psoriatic Arthritis, Crohn's Disease, Ulcerative Colitis and Ankylosing Spondylitis. These autoimmune diseases have distinct etiologies, but share major immunological components of disease processes related to inflammation and immune cell activity. Stradomer designed to reduce TNF-alpha production will also be effective in these and other autoimmune diseases. The altered immune response profile can also be directly or indirectly modulated to effect a reduction in antibody production, for example, autoantibodies that target the subjects' own tissues, or altered autoaggressive T cell levels in the subject. For example, Multiple Sclerosis is an autoimmune disorder involving autoreactive T-cells that can be treated by interferon beta therapy. See, for example, Zafranskaya M, et al., Interferon-beta therapy reduces CD4+ and CD8+ T-cell reactivity in multiple sclerosis, Immunology May 2007;121(l):29-39-Epub 2006 Dec 18. A project by stradomer to reduce autoreactive T cell levels will be equally effective in multiple sclerosis and other autoimmune diseases involving autoreactive T cells.
    The stradomers described herein can be used to modulate the expression of costimulatory molecules of an immune cell, including a dendritic cell, a macrophage, an osteoclast, a monocyte, or an NK cell, or to inhibit these same immune cells' differentiation, maturation, or secretion of cytokines, including interleukin-12 (IL-12), or increase secretion of cytokines, including interleukin-10 (IL-10), or interleukin-6 (IL-6). One skilled in the art can also validate the effectiveness of an immunologically active biomimetic by exposing an immune cell to the immunologically active biomimetic and modulating the measurement of immune cell function, where the cell is a dendritic immune cell, a macrophage, an osteoclast, or a monocyte. In one embodiment, the immune cell is exposed to the immunologically active biomimetic in vitro and further comprises the step of determining an amount of a cell surface receptor or cytokine production, in which a change in the amount of cell surface receptor or cytokine production indicates a modulation of immune cell function. In another embodiment, the immune cell is exposed to the immunologically active biomimetic in vivo in an animal model for an autoimmune disease further comprising a step of evaluating a degree of improvement in the autoimmune disease. Methods Employing Fixed Fc
    In order to understand the role of Fc:Fc receptor gamma (FCYR, the Fc receptor for IgG Fc) interactions and the importance of the hIVIG function of its Fc being biologically immobilized within an immunoglobulin, we compared the effects of hIVIG with both a fixed form of a recombinant IgG1 Fc fragment (rFCF) and a soluble form of a recombinant IgG1 Fc fragment (sFC), containing the hinge-CH2-CH3 domains on monocyte function during the process of differentiation from monocytes to immature dendritic cells (iDC).
    Exposure of monocytes cultured in granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to immobilized rFCF and immobilized hIVIG, but not low dose soluble hIVIG, enhanced CD86 expression , delayed the expression of CDl Ic, and suppressed the expression of CD1a on the cells. On the other hand, these changes are probably not secondary to non-specific immobilization of rFCF proteins in plastic, such as soluble aggregated heat (sHA) hIVIG, sHA rFCF or high dose hIVIG (recognized to contain multimeric Fs), induced changes similar to observed with immobilized rFCF.
    Taken together, our data indicate that iDC exposure to hIVIG immobilized on the surface of a solid, semi-solid, or gelatinous substrate results in a unique population of DCs (CD86 high, CDia low) capable of orchestrating immune tolerance, and that immobilized molecules that include the functional portion of Fc immunoglobulin G (IgG) fragments may be useful as mimetics of hIVIG for the treatment of local and systemic inflammation, as well as a wide variety of other pathological conditions that are directly or indirectly mediated by monocyte-derived cells (MDC), such as iDC. Furthermore, to immobilize the functional Fc portion of IgG in devices, described herein as "coating devices", which are implanted inside bodies or fixed to the bodies of animals (eg, human patients) with molecules containing the portion The functional Fc fragment of IgG can decrease, if not prevent, inflammatory responses to these devices, or treat systemic diseases by affecting immune cells that then pass into the circulation after being altered by contact with the stradomer fixed coated over or inside the implanted device.
    The present invention provides a method for inhibiting the activity of a monocyte-derived cell (MDC). The method includes contacting the cell with a composition comprising a substrate with an Fc reagent bound thereto. Contact can be in vitro, in vivo or ex vivo. Alternatively, the cell could be in an animal. The animal may be one that has, or is at risk of developing a condition mediated by monocyte-derived cells (MDCMC). The MDC can be, for example, a dendritic cell, a macrophage, a monocyte, or an osteoclast. The invention also provides a method of treatment or prophylaxis. The method which comprises administering to an animal a composition which contains a substrate with an Fc reagent attached thereto, the animal being one which has or is at risk of developing an MDCMC. It is also possible that the stradomers of the present invention conduct to a fixed Fc reagent in vivo. By "in vivo fixed Fc reagent" we mean Fc fixed to the surface of cells, i.e. platelets in vivo. It has been observed that platelets expressing FCYRS are efficiently coated with the stradomers of the present invention and that these coated platelets induce tolerance in an organism until they are cleared. The stradomers of the present invention, surprisingly, were observed to efficiently bind to the percentage of platelets expressing FCYRS. Insofar as roe:anti-ova aggregates and RBC:anti-RBC aggregates complexes induce tolerance and prevent platelet destruction in the ITP, these stradomer-coated platelets can induce tolerance in an organism. This may be another mechanism by which the stradomers of the present invention exert their immune modulatory functions.
    Based on the much greater binding affinity to the stradomer Fc gamma receptors compared to the native immunoglobulin Fc contained in the stradomer, it is also possible that the stradomers of the present invention lead to a fixed immunoglobulin Fc control reagent in vivo. By "in vivo fixed immunoglobulin Fc control reagent" we mean the stradomer fixed to the surface of cells including, but not limited to, monocytes, dendritic cells, T cells, regulatory T cells, Gamma Delta T cells, and platelets , in vivo. By binding to cell surface receptors, including Fc gamma receptors, stradomers block other immunoglobulins from binding to those receptors. Monoclonal or polyclonal antibodies are used in research assays such as flow cytometry and in clinical diagnostics. An important aspect of the present invention is the prevention of non-specific binding by the Fc component of these antibodies to Fc gamma receptors and other cell surface receptors to which the immunoglobulin Fc can bind.
    As used herein, the term "monocyte-derived cell-derived-mediated condition (MDCMC)" refers to a pathological condition that is directly or indirectly, wholly or partially, due to the activity of, or factors produced by, monocyte-derived cells. Monocyte-derived cells include, but are not limited to, monocytes, macrophages, interdigitating dendritic cells (generally referred to herein as follicular dendritic-like cells and dendritic-like cells comprising "dendritic cells") (mature and immature), osteoclasts, like cells microglia, monocyte-derived insulin-producing islet-like cells, monocyte-derived immature mast cells, and monocyte-derived microparticles.
    With respect to methods that use fixed Fc, the term "Fc reagent" refers to any molecule or molecular complex, which includes one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, or more) functional portions of an immunoglobulin Ig (IgG) Fc fragment. The IgG Fc fragment consists of the C-terminal portions of the two IgG heavy chains of an IgG molecule linked together and consists of hinge regions, the CH2 domains and the CH3 domains of both heavy chains linked together. The "functional portion of the IgG Fc fragment" consists of the hinge regions, the CH2 domains, and optionally all or some (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49) of the first 50 amino acids (from the N-terminus) of the CH3 domains of both heavy chains linked in set. In humans, (a) the IgG1 hinge region contains 15 amino acids, the CH2 domain contains 110 amino acids, and the CH3 domain contains 106 amino acids, (b) the IgG2 hinge region contains 12 amino acids, the CH2 domain contains 109 acidic amino acids , and the CH3 domain contains 107 amino acids; (c) the IgG3 hinge region contains 62 amino acids, the CH2 domain contains 104 amino acids, and the CH3 domain contains 106 amino acids; and (d) the IgG4 hinge region contains 12 amino acids, the CH2 domain contains 109 amino acids, and the CH3 domain contains 107 amino acids.
    As with wild-type IgG molecules, in the above-described Fc reagents the two polypeptide chains derived from IgG heavy chains are generally, but not necessarily, identical. Thus, an Fc reagent can be, without limitation, a total IgG molecule, a total IgG molecule linked to a non-immunoglobulin derived polypeptide, an IgG Fc fragment, an IgG Fc fragment linked to a non-immunoglobulin derived polypeptide. immunoglobulin, a functional portion of an IgG Fc fragment, a functional portion of an IgG Fc fragment linked to a polypeptide or multimers derived from non-immunoglobulin (e.g., dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nomers or decamers) of any of these. Fc reagents can also be the above-described stradomers and stradobodies as long as they fall within the definition of an Fc reagent above.
    In the fixed Fc, the immunoglobulin heavy chain components of the Fc reagents may have wild-type amino acid sequences or may be wild-type amino acid sequences, but with no more than 20 (eg, no more than: 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1) amino acid substitutions. Such substitutions are preferably, but not necessarily, conservative substitutions. Conservative changes typically include changes within the following groups: glycine and alanine; valine, isoleucine and leucine, aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; histidine, lysine and arginine, and phenylalanine and tyrosine.
    An "Fc reagent" of the present invention is at least 25% (e.g. at least: 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% ; 99.5%, or 100% or even more), of the ability of the IgG molecule from which the Fc reagent IgG heavy chain components were derived (the reference IgG molecule) to bind to a Fc receiver of interest. Where an "Fc reagent" has heavy chain components derived from more than one type of IgG molecule, the reference IgG molecule is the one that most avidly binds to the relevant Fc receptor of interest.
    As used herein, "Fixed Fc" refers to an Fc reagent that is bound to a "substrate" as defined below. The terms "Fc fixed", "Fc on" and "Fc stabilized" are synonymous terms. Fixed Fc is composed of the functional portion of Fc (including, but not limited to any polypeptide that includes the functional portion of Fc) attached to a substrate. Fixed Fc includes, for example, direct binding as well as indirect binding through Fc polymers to the substrate; incorporation of complete IgG Fc in alone; incorporating only functional IgG Fc domains, or incorporating complete IgG Fc or IgG Fc functional domains, as part of a larger polypeptide, such as an antibody, stradomer, or stradobody.
    As applied to fixed Fc, the term "substrate" refers to a solid, semi-solid, or gelatinous object. The substrate can be implanted into, or attached to (or adhered to) the surface of an animal's body. Substrates can include, for example, liquid or gaseous components, but at least a portion of the substrate is solid, semi-solid or gelatinous. Thus, a substrate can be a substance that is substantially insoluble in an aqueous solvent, but soluble in a non-aqueous solvent. Such substances include lipids (eg, phospholipids), fatty acids, and other fat-soluble compounds insoluble with an aqueous solvent. From this it will be clear that substrates include liposomes. The substrate can be porous or non-porous. In certain embodiments, the substrate is inert to the surface and/or the body to which it is implanted, attached, or adhered to.
    The substrate can contain or be made from a synthetic polymer, eg, nylon, Teflon, dacron, polyvinyl chloride, PEU (poly (urethane ester)), PTFE (polytetrafluoroethylene), PMMA (methyl methacrylate), PEEK, elastomers thermoplastics, radiopaque polymers, polyethersulfone, silicones, polycarbonates, polyurethanes, polyisobutylene and its copolymers, polyesters, polyolefins, polyisobutylene, ethylene-alphaolefin copolymers, acrylic copolymers and polymers, vinyl halide copolymers and polymers, such as polyvinyl chloride, polyvinyl ethers, polyvinyl methyl ether, polyvinylidene halides, polyvinylidene fluoride, polyvinylidene chloride, polyacrylonitrile, polyvinyl ketones, polyvinyl aromatics, polystyrene, polyvinyl esters, polyvinyl acetate, copolymers , vinyl monomer and olefin copolymers, ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, resin in ABS, ethylene-vinyl acetate copolymers, polyamide, Nylon 66, polycaprolactone, alkyd resins, polyoxyethylenes, polyimides, polyethers, epoxy resins, rayon-triacetate, cellulose, cellulose acetate, cellulose butyrate, cellulose acetate butyrate , cellophane, cellulose nitrate, cellulose propionate, cellulose ethers, carboxymethyl cellulose, collagens, chitins, polylactic acid, polyglycolic acid, polylactic acid-polyethylene oxide copolymers, polysiloxanes, substituted polysiloxanes, ethylene-vinyl acetate copolymers, polyolefin elastomers and EPDM rubbers and combinations thereof.
    The substrate may further contain or be made of a metal or metal alloy, for example stainless steel, platinum, iridium, titanium, tantalum, nickel-titanium alloy and cobalt-chromium. Furthermore, the substrate can include or be an animal tissue or an animal tissue product, for example, a tissue or organ graft. Animal tissue can be, for example, bone (eg osteogenic bone) or cartilage. Furthermore, the substrate may contain a protein, for example collagen, or keratin. The substrate can also be or contain a tissue matrix, for example an acellular tissue matrix. Cellular matrices of particulates and non-particulates are described in detail, for example, in US Patents 5,336,616 and 6,933,326, the disclosures of which are incorporated herein by reference in their entirety. The substrate can also be or include an animal cell (for example tissue repair cells such as fibroblasts, mesenchymal stem cells) and can be, for example, a hair transplant plug. The substrate can contain or be a polysaccharide, for example agarose. It can also contain or be a salt, preferably a relatively insoluble salt, for example calcium sulfate. The substrate can be a gel or cream. Furthermore, it can contain silicone or silastics. The substrates can also contain a natural fiber, for example silk, cotton or wool.
    Furthermore, the substrate may be an implantable medical device. It can be, for example, a stent (for example, a vascular stent such as a coronary artery stent, an airway stent such as a tracheal or nasal stent, a gastrointestinal stent such as a pancreatic or biliary stent, or a urinary stent, such as a ureteral stent) or a surgical suture (for example, a silk braid, chrome gut, nylon, plastic, or metal suture) or a surgical clip (for example, an aneurysm clip). The substrate can be, for example, an artificial hip, an artificial hip joint, an artificial knee, an artificial knee joint, an artificial shoulder, an artificial shoulder joint, an artificial finger joint or toe joint, a bone plate, a bone nail, a non-joined bone implant, an intervertebral disc implant, bone cement, or a bone cement spacer. It may also be an arteriovenous shunt, an implantable wire, a pacemaker, an artificial heart, a cardiac assist device, a cochlear implant, an implantable defibrillator, a spinal cord stimulator, a central nervous system stimulator, or a peripheral nerve implant . Other substrates are dental prostheses or dental crowns.
    In other embodiments, the substrate may be a large vessel embolic filtering device or cage, a percutaneous device, a dermal or submucosal patch, or an implantable drug delivery device. The substrate can also be a large blood vessel graft, where the blood vessel is, for example, a carotid artery, a femoral artery, or an aorta. In addition, the substrate can be a subdermal implant, a corneal implant, an intraocular lens, or a contact lens.
    The substrate can be in the form of a sheet, a granule, a mesh, a powdered particle, a yarn, a sphere or a fiber. It can also include or be a solid, semi-solid, or gelatinous substance. The substrate can also be a cell that expresses FCYR. Preferably, the substrate is a platelet.
    Polymers useful in the present invention are preferably those that are biostable, biocompatible, particularly during insertion or implantation of the device into the body, and prevent irritation to body tissues.
    Fc reagents can be coated (i.e., fixed or stabilized) onto substrates in any of a variety of ways. For example, they can be coated directly onto the substrate surface where they remain fixed, for example by hydrophobic interactions. The following describes some other methodologies ((a) - (e)) involving the use of polymers: (a) The Fc reagent is mixed with a mixture of miscible polymer which is then placed on the surface of the implantable synthetic material in order to stabilize the Fc reagent. Monomers routinely used in the art to make polymer blends include PLMA [oly(lauryl methacrylate)]; PEG [olyethylene glycol], PEO [olyethylene oxide]; alkyl-functionalized PMMA, PEMA methacrylate polymers. PPMA, and PBMA; itaconates; fumarates and styrenics. (b) A polymeric coating layer or nano-sized film is adhered to the substrate surface and then the Fc reagent is adhered to the polymeric coating layer or nano-sized film, thereby stabilizing , reagent F.(c) A thin film of a polymer monomer that is applied to the surface of the implantable substrate and the monomer is then caused to polymerize the monomers including, for example, Methane, Tetrafluoroethylene, Methanol, Benzene, Ethylene Oxide , Tetraglyme, Acrylic Acid, Allylamine, Hydroxyethyl Methacrylate, N-vinyl-pyrrolidone, and Mercaptoethanol. The Fc reagent is then affixed to the resulting monomer.(d) The substrate is coated with a protein such as protein A or albumin that attaches to the Fc reagent, thereby stabilizing Fc to the substrate surface.(e ) The Fc reagent can be labeled with a chain of hydrophobic amino acids that bind to implantable synthetic materials and cause the stabilized Fc to orient itself uniformly.
    The methods of the present invention can be applied to any animal species and IgG molecules from which IgG-derived portions of Fc reagents can be made from any animal species. Of course, the relevant animal species are those in which IgG or IgG-like molecules occur. Generally, the species to which the methods are applied and the species from which the IgG-derived portions of the Fc reagents used in the methods are the same. However, they are not necessarily the same. Relevant animal species are preferably mammals and these include, without limitation, humans, non-human primates (for example, monkeys, baboons, and chimpanzees), horses, cattle (for example, oxen, cows, or oxen), pigs, goats, sheep, dogs, cats, rabbits, gerbils, hamsters, rats and mice. Non-mammalian species include, for example, birds (for example, chickens, turkeys, and ducks) and fish.
    The terms "treating", "treatment" and "prophylaxis" have the same meaning using fixed Fc as described above for stradomers.
    Wherever the fixed Fc are implantable devices coated with Fc reagents, they can be implanted into, attached to, or adhered to noble organs or tissue or body surfaces of the relevant subjects, using methods well known in the art. Whenever they are formulated as, for example, suspensions, powders, they can be formulated and administered as described above for stradomers.
    The fixed Fc reagents of the present invention can be used to treat or prevent conditions including, but not limited to cancer, congestive heart failure (CHF), vasculitis, rosacea, acne, eczema, myocarditis and other myocardial conditions, systemic lupus erythematosus, diabetes, spondylopathies, synovial fibroblasts, and bone marrow stroma; bone loss, Paget's disease, hypertrophic bone formation; disuse osteopenia, malnutrition, periodontal disease, Gaucher disease, Langerhans cell histiocytosis, spinal cord injury, acute septic arthritis, osteomalacia, Cushing's syndrome, monoostotic fibrous dysplasia, polyostotic fibrous dysplasia, periodontal reconstruction, and bone fractures , control of bone pain and malignant humoral hypercalcemia, ankylosing spondylitis and other spondyloarthropathies; transplant rejection, and viral infections.
    All autoimmune diseases can be in whole or in part MDCMD. The term "autoimmune disease" as used herein refers to a diverse group of over 80 chronic diseases. In all these diseases, the underlying problem is that the body's immune system attacks the body itself. Autoimmune diseases affect all major systems in the body, including the connective tissue, nerves, muscles, endocrine system, skin, blood, and the respiratory and gastrointestinal systems.
    The autoimmune disease or condition may be a hematoimmunological process, including, but not limited to, idiopathic thrombocytopenic purpura, alloimmune/autoimmune thrombocytopenia, acquired immune thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, parvovirus B19-associated red cell aplasia, antifactor autoimmunity VIII acquired, acquired von Willebrand disease, Multiple Myeloma and Monoclonal Gammopathy of unknown significance, Sepsis, Aplastic Anemia, pure red cell aplasia, Diamond-Blackfan anemia, newborn hemolytic disease, immune-mediated neutropenia, refractoriness to transfusion of platelets, post-transfusion purpura, neonatal, hemolytic uremic syndrome, systemic vasculitis, thrombotic thrombocytopenic purpura, or Evan's syndrome.
    The autoimmune disease or disease may be a neuroimmunological process, including, but not limited to, Guillain-Barré syndrome, Chronic Inflammatory Demyelinating Polyradiculoneuropathy, IgM Paraproteinemic Demyelinating Polyneuropathy, Lambert-Eaton myasthenic syndrome, Myasthenia Gravis, Multifocal Motor Neuropathy, Lower Motor Neuron syndrome associated with anti-/GMl, Demyelination, Multiple Sclerosis and optic neuritis, Stiff Man Syndrome, Paraneoplastic Cerebellar Degeneration with anti-Yo antibodies, paraneoplastic encephalomyelitis, sensory neuropathy with anti-Hu antibodies, epilepsy, Encephalitis Myelopathy especially associated with human T-cell lymphotropic virus-1, Autoimmune Diabetic Neuropathy, or Acute Idiopathic Dysautonomic Neuropathy.
    The autoimmune disease or condition may be a Rheumatic disease process, including, but not limited to, Kawasaki disease, Rheumatoid Arthritis, Felty's syndrome, ANCA positive vasculitis, Spontaneous polymyositis, Dermatomyositis, Antiphospholipid syndrome, Recurrent miscarriages, Lupus erythematosus Systemic, Juvenile Idiopathic Arthritis, Raynaud's phenomenon, CREST Syndrome, or uveitis.
    The autoimmune disease or condition may be a dermatoimmunological disease process, including, but not limited to, Toxic Epidermal Necrolysis, Gangrene, Granuloma, Autoimmune Bullous Skin Diseases including Pemphigus vulgaris, Bullous Pemphigoid, and Pemphigoid Foliaceous, Vitiligo, Toxic Shock Syndrome Streptococcal, Scleroderma, Systemic Sclerosis, including limited, diffuse cutaneous systemic sclerosis and/or Atopic Dermatitis (especially steroid dependent).
    The disease or condition may also be a musculoskeletal immune disease process, including, but not limited to, Inclusion Body Myositis, Necrotizing Fasciitis, Inflammatory Myopathies, Myositis, Anti-Decorine Myopathy (BJ Antigen), Paraneoplastic Necrotic Myopathy, Myopathy X-Linked Vacuolate, Penacylamine-Induced Polymyositis, Atherosclerosis, Coronary Artery Disease, or Cardiomyopathy.
    The autoimmune disease or condition may also be an immunological gastrointestinal disease process, including, but not limited to, pernicious anemia, autoimmune chronic active hepatitis, primary biliary cirrhosis, Celiac disease, dermatitis herpetiformis, cryptogenic cirrhosis, Reactive Arthritis, Crohn's Disease , Whipple's disease, ulcerative colitis, or sclerosing cholangitis.
    The disease or condition may also be Graft Versus Host Disease, Antibody-mediated Graft Rejection, Post Bone Marrow Transplant Rejection, Post-Infectious Disease Inflammation, Lymphoma, Leukemia, Neoplasm, Asthma, Type 1 Diabetes Mellitus with Antibodies anti-beta cells, Sjogren's syndrome, Mixed Connective Tissue Disease, Addison's Disease, Vogt-Koyanagi-Harada Syndrome, Membranoproliferative Glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Hashimoto's Thyroiditis, Wegener's Granulomatosis, Micropolyarteritis, Churitis Polyarteritis nodosa or Multisystem Organ Failure.
    "Cancer" herein refers to, or describes, the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, leiomyosarcoma, rhabdomyosarcoma, osteogenic sarcoma, angiosarcoma, chondrosarcoma, fibrosarcoma, mesothelioma, chordoma, synovioma, schwannoma, meningioma, adenocarcinoma, melanoma, leukemia and malignant or lymphoid tumors. More particular examples of such cancers include squamous cell cancer (eg, squamous epithelial cell cancer), lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung , small cell lung cancer, peritoneum cancer, hepatocellular cancer, gastric, or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, cancer of the stomach breast, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, biliary tract tumors, Ewing's tumor, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, carcinoma. sebaceous gland cynoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilms' tumor, testicular tumor, lung carcinoma, carcinoma of bladder, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, myelodysplastic disease, myelodysplasia disease tumors, neuroendocrine tumors, Schwanoma and other carcinomas, head and neck cancer, myeloid neoplasms such as acute myeloid leukemias, including matured AML, non-differentiated AML, acute promyelocytic leukemia, acute myelomonocytic leukemia, and acute monocytic leukemias, myelodysplastic syndromes , and myeloprol disorders chronic iferative, including chronic myelogenous leukemia, central nervous system tumors, eg brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma and retinoblastoma), solid tumors (nasopharyngeal cancer, basal cell carcinoma, pancreatic cancer, bile duct cancer, Kaposi's sarcoma, testicular cancer, uterine, vaginal or cervical cancers, ovarian cancer, primary liver cancer or endometrial cancer, vascular system tumors (angiosarcoma and hemagiopericytoma), hematologic neoplasms, and neoplastic-like conditions, by example, Hodgkin's lymphoma, non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocytic lymphoma/chronic lymphocytic leukemia, mycosis fungoid, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, hairy cell leukemia, and lymphoplasmacytic leukemia), lymphocyte precursor cell tumors, including lymphoma/acute lymphoblastic leukemia; B-cell and T-cell lymphoma/acute lymphoblastic leukemia, thymoma, mature NK and T-cell tumors, including peripheral T-cell leukemias, adult T-cell leukemia/T-cell lymphomas and large granular lymphocytic leukemia, bone cancers osteolytic and bone metastases.
    As used herein, a subject "at risk of developing a monocyte-derived cell-mediated disease (MDCMD)" is a subject who has a predisposition to develop MDCMD, that is, a genetic predisposition to develop MDCMD or has been exposed with the conditions that can result in
    MDCM. A subject "suspected of having an MDCMD" is one having one or more symptoms of an MDCMD. From the above, it will be clear that neither the subjects "at risk of developing an MDCMC" nor the "subjects suspected of having an MDCMD" are all individuals within a species of interest. In any of the above methods, the MDCMC can be caused by the substrate and the Fc reagent serves to prevent or ameliorate MDCMC. Immunological Testing Applications
    The immunologically active biomimetics described herein can be used to perform immunological assays to test immune cell functions to which the immunologically active biomimetics are designed to modulate.
    Signaling through the low-affinity FCY receptor pathways requires aggregation and cross-linking of the receptor on the cell surface. These aggregation and cross-linking parameters are postulated to be realized through Fab binding to an antigen-specific target with subsequent interaction between the Fc region and low-affinity FcRs on the surface of responding cells. In this context, antibodies have the potential to elicit cellular responses via two different pathways: 1. Fab interaction/blocking with/from an epitope-specific target and 2. Fc interactions with FcRs. Despite this knowledge, current controls for most therapeutic studies using monoclonal antibodies used in vivo do not adequately address the potential of Fc:Fcy receptor interactions as contributors to the observed functional effects. Multiple strategies are currently employed to eliminate Fc:FcR interactions as confounding variables. For example, some studies employ Scv (single-chain variable regions) or Fab fragments, which retain epitope specificity but lack the Fc region. These approaches are limited by the short half-life of these reagents and their limited potential to induce signaling. Other studies employ fusion proteins composed of a receptor or ligand fused to an Fc fragment. Although these types of approaches help to differentiate Fab-specific effects from those seen with ligand-receptor interactions, they do not effectively control Fc-mediated effects. Antibody-based therapeutic evaluations in animal models can also use isotype control antibodies with an irrelevant Fab binding site. The justification for this choice is based on the presumed functional similarity between antibodies of the same isotype, irrespective of their specificity or Fab binding affinity. However, this use of irrelevant isotype controls has several fundamental flaws:
    1. If the Fab fragments of these antibodies cannot bind an antigenic epitope or ligand, it is likely that the Fc fragments do not stimulate signaling through low-affinity FcR interactions, due to the absence of FCY receptor crosslinking. Therefore, the functional differences observed between the experimental and control antibodies cannot be correctly attributed to the Fab interaction with an epitope-specific target without a means to cross-link the FcR.
    2. If these isotypes are produced in cells that produce different glycoforms or different relative percentages of individual glycoforms than the parent antibody, binding to both low and high affinity FcRs will be altered, even if the Fab affinity is identical.
    Although there is no perfect control to overcome this problem, one option is to use isotype-specific stradomers produced in the same cells as the parent antibodies and administered at a dose proportional to the expression levels of the target epitope by the experimental antibody. For example, the appropriate control for an epitope-specific antibody produced in mice would be a mouse isotype-specific stradomer capable of aggregating Fcy receptor on the surface of effector cells.
    In general, an immune cell is exposed to an effective amount of an immunologically active biomimetic to modulate an immune cell activity in a known manner and this immune modulation is compared to a test molecule or compound to determine whether the test compound has activity. of similar immune modulation.
    In another embodiment, heat-aggregated stradomers, and aggregated immunoglobulins can be used as reagents for laboratory controls in various immunoassays described herein and known to those of skill in the art.
    Immunological assays may be in vitro assays or in vivo assays, and may include human or non-human immune cells using a species-matched or non-species-matched stradomer. In one embodiment, an immunological assay is performed using an effective amount of the immunologically active biomimetic to modulate an immune cell activity and compare the modulation to the modulation of an immune cell by a test compound. The stradomer can serve the function of a positive control reagent in assays that involve testing other compounds for immunological effect. The assay can compare the effect of the subject's monoclonal antibody compared to the stradomer for effector cell FCY receptor binding and functional response as measured by changes in receptor expression level, cytokine release and functions, such as using a Reaction of Mixed Lymphocytes. Thus, if a stradomer (which lacks the Fab) generates a response that is, in part, similar to the monoclonal antibody, then the effect of the monoclonal antibody is, in part, not due to the specificity of its Fab, but to the general binding and cross-linking effect of more than one Fcy receptor on the effector cell.
    If the biological activity of a species-specific and isotype-specific antibody is replicated in part or in whole by a species-specific and isotype-specific stradomer, then it is clear that the activity of the Fc - Fcy receptor accounts for the biological activity portion observed attributable to species-specific and isotype-specific stradomer. Thus, species-specific and isotype-specific stradomers are useful for evaluating potential therapeutic antibodies to determine whether, and to what degree, the observed biological activity is attributable either to the Fab portion of the test antibody, or to a non-specific effect. of the Fc portion of the binding and cross-linking molecule more than one Fc receptor.
    The stradomers of the present invention are also useful in blocking non-specific binding of Fc receptors during antibody-based immunoassays such as flow cytometry, Western blot, immunohistochemistry and immunofluorescence. Traditionally, in assays such as these, non-specific antibodies of the same species as the test antibodies are used to block non-specific binding to Fc receptors. The stradomers of the present invention provide an advantage over traditional means of Fc blocking in that each stradomer has multiple FcR binding sites, and therefore much less stradomer can be used. Furthermore, because the stradomer of the present invention lacks the Fab antigen binding portion of the antibody, no non-specific binding of dead cells, for example, will be observed as is generally the case with the corresponding IgG control species.
    The stradomers of the present invention, surprisingly, have been found to bind endotoxin with very high affinity. Standard endotoxin removal kits and columns are unable to remove significant percentages of tightly bound endotoxin from stradomers. Therefore, the claimed compositions may be useful to act as a binding aggregate for endotoxin, a useful tool for endotoxin removal in pharmaceutical and laboratory preparations. This is beneficial in that the complexes formed between endotoxin and stradomers have sufficiently high affinity to efficiently remove endotoxin from a pharmaceutical preparation or composition. In one embodiment, endotoxin-stradomer complexes are removed from the composition via filtration.
    All references cited herein are incorporated by reference in their entirety. EXAMPLES Example 1 Production and Purification of Stradomers
    HEK293F cells (Invitrogen, Carlsbad, CA) or Chinese Hamster Ovary (CHO) cells were used for stable expression of G045/M045 and G051. HEK293F or CHO cells were cultured in suspension to increase protein expression.
    The genes encoding G045c (SEQ ID NO: 4), G045old (SEQ ID NO: 7) G051 (SEQ ID NO: 18), G019 (SEQ ID NO: 8), G028 (SEQ ID NO: 9), G046 ( SEQ ID NO: 10), G075 (SEQ ID NO: 20), G076 (SEQ ID NO: 21), G096 (SEQ ID NO: 28), G098 (SEQ ID NO: 24), G089 (SEQ ID NO: 27 ), or the corresponding murine sequences from the preceding stradomers were cloned into a vector containing a neomycin resistance gene, such as pcDNA3.3 from Invitrogen (Carlsbad, CA) and under the transcriptional control of the CMV promoter to facilitate level expression high from G045 or G051. Plasmid DNA for transfection was isolated from the bacterial culture using an endotoxin-free plasmid DNA isolation kit (Nucleobond, Macherey-Nagel). Plasmid DNA encoding G045c/G045old and G051 was linearized with restriction enzyme and transfected into 293-F cells or CHO cells. After transfection, positive cells expressing G045c, G045old or G051 were selected with Geneticin/G418 to obtain an aggregate of transfected cells. To obtain a clonal cell line the pool of stably transfected cells was diluted to 1-2 cells per well in a 96-well plate from which individual cell clones of the stable cell line were obtained. Isolated cell clones were tested by ELISA for protein expression. Individual cell clones are grown and protein G045c, G045old, G051, G019, G028, G046, G075, G076, G096, G098 or G089 is harvested from the medium as secreted protein.
    For the production of G045c, G045old, G051 G019, G028, G046, G075, G076, G096, G098, or G089 protein by transient transfection, HEK293 cells or CHO cells were transfected with DNA encoding proteins G045c, G045old, G051, G019, G028, G046, G075, G076, G096, G098, or G089 under the control of a promoter of
    CMV to ensure high level expression of the protein. Transfection was performed with one of several commercially available transfection reagents. Secreted protein G045c, G045old, G051, G019, G028, G046, G075, G076, G096, G098, or G089 is harvested from the cell culture medium 4-5 days after transfection.
    Cell culture media from transiently transfected cells or stable cell lines were filtered using a 0.22 µm filter and adjusted to pH 7.2 with one volume of binding buffer (20 mM sodium phosphate, pH 7.2 + 150 mM NaCl) and purified by affinity chromatography on a HiTrap Mabselet protein A affinity column using an AKTAXpress purification system. (GE life science). After elution in 0.1 M sodium citrate pH 3.3 the protein is purified and a HiPrep 26/20 desalting column for buffer exchange.
    For further purification the G045c, G045old, G051, G019, G028, G046, G075, G076, G096, G098, or G089 protein is purified by gel filtration on a HiLoad Superdex 200 (GE Lifesciences) gel filtration column in Tris-HCL 50 mM, pH 7.5 + 150 mM NaCl followed by purification on Mono S ion exchange column (GE Lifesciences). Ion exchange purification is done in 20 mM MES buffer, pH 6 with a gradient of 0-1M NaCl. After chromatography, the protein is adjusted to PBS by dialysis. A schematic of the resulting G045c stradomer is shown in Figure 1.
    In order to test the multimerization ability of the resulting G045c protein, a 10% polyacrylamide gel was run containing equal concentrations of G045c produced by the method described above, and G045old, produced by the method previously described in WO 2008/151088 and containing the extraneous cloning sequences. Surprisingly, removal of the extraneous fragments led to a dramatic increase in multimerization in G045c compared to G045old with G045c showing a much higher concentration of higher order multimers compared to G045old. (See Figure 2A).
    The formation of G045old versus G045c multimers was analyzed using a Gel-DocIT imaging system. After scanning the density, the amount of gel image proteins in each band on the gel was assessed. Surprisingly, the formation of multimers in the G045old sample was estimated to be approximately 27.9%, whereas the removal of the extraneous fragments to generate naturally associated stradomers led to the formation of multimers in the G045c sample which was significantly higher and estimated to be about 73.8% of total proteins. (See Figure 2B).Example 2: Enhanced Efficacy of M045c Compared to M045old in a Mouse Model of Arthritis
    Evaluation of the efficacy of M045c compared to M045old in collagen-induced arthritis was performed. On day 0 and day 21 DBA1/J mice were immunized with type II bovine collagen (Chondrex, Inc., Cat. 20021) with a 4mg/ml solution emulsified with Incomplete Freund's Adjuvant (Sigma, Cat #5506). Mice were weighed weekly and scored daily for signs of arthritis. Each paw was scored and the sum of all four scores was recorded as the Arthritic Index (AI). The maximum possible AI was 16 as follows: 0 = no visible effect of arthritis, 1 = edema and/or erythema of one digit 2 = edema and/or erythema of two joints, 3 = edema and/or erythema of more than 2 joints, 4 = severe arthritis of the entire paw and digits, including limb deformity and joint ankylosis. Starting on day 22 (the day 0 treatment) 10 of the collagen-immunized mice were separated into treatment groups based on the mean AI (3.3) and ten undiseased mice were designated as the undiseased group. The Arthritis Index was measured by 14 days of treatment after which the mice were sacrificed. For the positive control group 10 mice were divided into treatment based on mean AI (3.3) and orally dosed with 10 ml/kg every day of prednisolone. For the group treated with M045c or M045old, 20 mice were divided into treatment based on the mean AI (3.3) and dosed every 4th day (day 0, day 4, day 8 and day 12) with 400 μg of M045c or M045old (17.4 mg/kg).
    M045c treated mice had statistically less severe disease compared to M045old treated mice at almost all time points tested. (See Figure 3). Therefore, removing 16 amino acids from cloning the fragment between the leader sequence and IgG1 Fc leads not only to the formation of larger multimers, but also an increased efficacy against inflammatory disease.
    The evaluation of the efficacy of M045c, M019, M028, M046 and M051 in collagen-induced arthritis was similarly performed and compared with the efficacy of prednisolone. CIA was induced in mice as described above and mice were treated starting on day 22 post CIA induction with 400 µg of M045, M019, M028, M046 or M051 intravenously twice weekly or 10 mg/kg of predisolone every day and scored for two weeks for the AI as above. The mice that received each of the tested stradomers had statistically less severe disease than the PBS-treated controls (see Figure 9).Example 3: Synergistic Effect of M045 and Prednisolone in a Mouse Model of Arthritis
    Evaluation of the efficacy of M045c combined with low dose prednisolone in a collagen-induced arthritis model was performed. Briefly, on day 0 and day 21 DBA1/J mice were immunized with type II bovine collagen (Chondrex, Inc., Cat. 20021) with a 4mg/ml solution emulsified with Incomplete Freund's Adjuvant (Sigma, Cat #5506) . Mice were weighed weekly and scored daily for signs of arthritis. Each paw was scored and the sum of all four scores was recorded as the Arthritic Index (AI). The maximum possible AI was 16 as follows: 0 = no visible effect of arthritis, 1 = single-digit edema and/or erythema, 2 = edema and/or erythema of two joints, 3 = edema and/or erythema of more than 2 joints, 4 = severe arthritis of the entire paw and digits including limb deformity and joint ankylosis. Starting on day 22 (treatment day 0) 10 of the collagen-immunized mice were separated into treatment groups based on the mean AI (3.3) and ten undiseased mice were designated as the undiseased group. Arthritic index was measured during 14 days of treatment after which the mice were sacrificed. For the positive control group ten of the mice were divided into mean AI (3.3) based on treatment and dosed orally with 10 ml/kg daily of prednisolone. For the group treated with 10 M045c, the mice were divided into treatment based on the average of AI (3.3) and dosed every 4th day (day 0, day 4, day 8 and day 12) with 400 μg of M045c (17, 4 mg/kg). For the measurement of synergy between prednisolone and M045c one group was treated with a low dose of prednisolone 2 mg/kg every day one group was treated with a low dose of M045c 200 μg/dosing dose every 4th day and one group was dosed with a low dose of 2mg/kg of prednisolone and a low dose of 200 μg/dose of M045c dosing every 4th day.
    Although high doses of prednisolone (10 mg/kg) were effective as a single agent in ameliorating collagen-induced arthritis, low doses of prednisolone (2 mg/kg) were only marginally effective. In addition, low dose M045c (200 μg/dose or 9.1 mg/kg) was also ineffective in reducing disease severity compared to untreated controls. However, surprisingly, mice treated with low-dose prednisolone combined with low-dose M045c showed a synergistic (rather than additive) reduction in disease severity when compared to the individual prednisolone and M045c treatment groups. (See Figure 4).Example 4: Binding analysis of IgG2A, M045, M046, M028, M019 and G051 to mouse receptors.
    FCYRIIIA, FCYRIIB, and SIGN-R1 receptors were immobilized to a CM4 chip using amine immobilization of 560, 500, and 1000RU, respectively, to compensate for protein size.
    M045c (SEQ ID NO: 11), M046 (SEQ ID NO: 15), M019 (SEQ ID NO: 13) and M028 (SEQ ID NO: 14) were serially diluted from 500 nM to 1.9nM in buffer of HBSS-EP running and injected at 20μl/min for 180s. Regeneration was achieved by a 10 second injection of 1M MgCl at 100ul/min, followed by a brief wash with running buffer. KDs were calculated using the T100 evaluation software.
    For mouse FCYR3A and FcYR2b, mouse IgG2a Fc homodimers bind with lower affinity and faster dissociation rate compared to each of the stradomers tested. For SIGN-R1 mice, IgG2a Fc homodimers do not bind appreciably while selective stradomers associate to varying degrees and dissociate slowly. (See Figure 5).
    Fractions of M045c were evaluated in this model. The M045F was first gel fractionated using an AktaXpress protein purification system and a GE grade HiLoad 16/60 Superdex 200 prep column. Fraction 1 (M045 F1) contains the highest molecular weight component of the M045 multimers after separating the multimers according to size. M045 F2 is the lowest molecular weight multimer component and M045 F3 is the stradomer homodimer fraction. For mouse FcYRIIIa, FcYRIIb (see Figure 6a), and SIGN-R1 (see Figure 6b) the binding affinity is higher with a slow off rate for M045 F1 compared to other fractions or with the IgG2a Fc control.
    To assess G051 binding, the bilayer interferometry assay was performed on an Octet 96 Red type instrument (ForteBio, Menlo Park CA) according to the manufacturer's instructions (ForteBio Data Acquisition Guide 6.4). For mouse protein binding analysis, the His-tag mouse FcyRII (R&D system cat. #1460-CD) and FcyRIII R&D system cat. #1960) were separately uploaded to the
    Anti-penta-His biosensors (ForteBio cat #18-5077) at 10 μg/ml in 1X Kinetic Analysis Buffer (ForteBio cat #18-5032). For human protein binding analysis, His-tag human FcYRIIb (R&D system cat. #1875-CD) and FcYRIIIa R&D system cat. #4325-Fc) were separately loaded onto penta-anti-His Biosensors (ForteBio cat #18-5077) at 10ug/ml in 1X Kinetic Analysis Buffer (ForteBio cat #18-5032). After sensor tip loading, protein association was measured by transferring the tips to preparations of either monomeric fraction (over a range of concentrations), or multimeric fractions (over a range of concentrations) in 1X kinetic analysis buffer. dissociation and was measured by transferring 1X sensor tips into kinetics buffer. The analysis has been described (6.4ForteBio Data Analysis Guide). The analysis was standardized as described above assigning a molecular weight of 50 kD for homodimers and 150 kD for all other protein preparations.
    Table 2 shows that the larger stradomer multimer fractions of M051 bind with greater affinity and avidity and slower dissociation than the smaller multimer fractions, which in turn bind with greater affinity and avidity and slower dissociation than the homodimer fraction.

    To assess the binding kinetics of G045 or G051 relative to G001 (native IgG1 Fc), the bilayer interferometry assay was performed on a 96 Octet Red type instrument as described above. Binding of G045 and G001 to human FcYRIIb, human FcYRIIIa (both F and V variants), Cynomolgus FcYRIIIa, Cynomolgus FcYRIIIb and
    Cynomolgus FcRIII was measured. G045 has a significantly higher binding affinity with dissociation and evidence of slower avidity for each of the receptors tested compared to G001. (see Tables 3 and 4).

    Likewise, the binding of G051 and G001 to human FcYRIIb and FcYRIIIa was measured. G051 has significantly higher binding affinity with dissociation and evidence of slower avidity for each of the tested receptors compared to G001. (see Table 5).
    Example 5: Naturally Bound Stradomer Compounds are Effective in
    Treatment/Prevention of ITPTo elucidate the effect of stradomers on Idiopathic Thrombocytopenic Purpura (ITP) stradomers were tested in a preventive mouse model of ITP. Low platelet counts are induced after exposure to anti-mouse integrin IIb antibody that coats integrin receptors on platelets. Briefly, 8-week-old C57BL/6 mice (Charles River) were injected into the tail vein with stradomer or control on day 1 after a blood draw and platelet count. On day 2 after blood collection and platelet counts the mice are treated with MWReg30 (BD Pharmingen cat #553847) given at a concentration of 2 µg of antibody in 200 µl of phosphate-buffered saline administered by intraperitoneal injection to induce loss of platelets. Blood collection for platelet counts and MWReg30 injections continues on days 3, 4, and 5. The IVIG positive control is measured daily on days 2 to 5. Platelet counts are taken with a Drew Scientific Hemavet 950 hemocytometer. M045c e its fractions are dosed once on day 2. Blood is collected by cutting the tail vein and mixed with citrate buffer to prevent clotting. M045c is significantly protective in this model, both in the control of ITP from drug-free treatment and in the control of IgG2a Fc and is comparable with both preventive IVIG therapy and mice that did not receive the MWReg30 insult (See Figure 7). Fractions M045c were made as described in Example 4. Fraction 1 of M045 is significantly protective in this model against ITP control of drug-free treatment whereas Fraction 3 of M045 is not. Example 6: Removal of Endotoxin Complexes with Naturally Bound Stradomer Complexes
    To remove endotoxins, an endotoxin-containing protein solution containing a protein of interest will be adjusted to pH 7.2 with 1 volume of binding buffer (20 mM sodium phosphate, pH 7.2 + 150 mM NaCl) after mixing the endotoxin-binding stradomer into the solution. To remove the endotoxin the solution containing the protein will be applied to an affinity chromatography column (HiTrap Mabselect protein A affinity column, GE Life Sciences). Stradomer-bound endotoxin will bind to the affinity column and purified endotoxin-free protein will elute in the continuous flow fractions.
    In an alternative approach to using stradomers to trap endotoxin, protein A coated magnetic beads (New England Biolabs, MA) will be mixed into the endotoxin-containing protein solution along with the stradomer-bound endotoxins and the stradomer-bound endotoxin will be removed by magnetic separation. Example 7: Use of Naturally Bound Stradomer Complexes for Fc Blocking in Immunological Assays
    Improved sensitivity and specificity of antibody-based research tools and clinical diagnostics. The screening utility and clinical diagnostic utility of antibodies are limited by non-specific binding. This can occur, for example, through the binding of the Fc portion of monoclonal or polyclonal antibodies to the high-affinity Fc gamma receptors and other Fc binding receptors on cells, including immune cells and tumors, or through the binding of antibody aggregates or antibody-coated cell aggregates to low-affinity Fc gamma receptors.
    To demonstrate the ability of stradomers to block Fc gamma receptor interactions with specific anti-Fc gamma receptor antibodies, flow cytometry assays were performed and compared increasing doses of human stradomer G045c with G001, an IgG1 Fc homodimeric monomer, in its ability to block anti-FcYR antibody binding to FCYRS known to be present in specific cells. Stradomers were found to effectively block the binding of FCYRS-binding anti-FcYR antibodies relative to IgG1 Fc control and to do so in a concentration-dependent manner (see Figure 8).
    Due to their high binding affinity for Fc gamma receptors and other receptors that bind to immunoglobulin Fc regions, stradomers are surprisingly effective in blocking the binding and interaction of even specific anti-Fc gamma receptor monoclonal antibodies. Stradomers are therefore useful as control reagents to decrease the binding of non-specific antibodies to administered antibodies, both in search tool settings and in clinical diagnostic settings. Example 8: Naturally Bound Stradomer Compounds Are Effective in the Treatment of Experimental Autoimmune Neuritis
    The evaluation of the efficacy of M045c and M051 in each case compared to that of IVIG and albumin was performed in an Experimental Autoimmune Neuritis (EAN) mouse model. The EAN murine models are widely used animal models of human acute inflammatory demyelinating polyradiculoneuropathy. Briefly, 45 Lewis rats were immunized with bovine peripheral nerve myelin and randomized into three groups. At the onset of clinical deficits, which is usually weight loss starting on day 9 or 10, 15 rats per treatment group were treated with IVIg (body weight 1 gm/1 kg), M045 (20 mg/kg) or M051 ( 17.5 mg/kg) or with albumin all administered in two consecutive IV doses over two days. All drugs were administered intravenously, by injection into the tail vein.
    EAN rats were evaluated clinically, electrophysiologically and histologically. The severity of clinical disease was assessed by daily clinical classification and weight changes. Electrophysiological studies included examination of the amplitude of compound muscle action potentials (CMAPs) and motor conduction velocity (MCV). On day 15, at peak disease, five rats from each group were sacrificed, the sciatic nerves collected and histopathological changes were analyzed. Treatment efficacy was compared between the IVIg and albumin treatment groups, and the M045c or recombinant M051 and albumin groups.
    Mice that received the tested M045c stradomer had statistically less severe disease than albumin-treated controls (see Figure 12). Albumin-treated EAN rats had higher mortality (4 out of 15) compared to rats treated with IVIg-(1 out of 15) and treated with M045c (0 out of 15). Animals that received treatment with M045c and IVIg had significantly less prominent weight loss. There was a statistically significant improvement in both motor conduction velocity (MCV) and proximal and distal PBMC amplitudes in rats treated with IVIg and M045c compared to those treated with albumin. Albumin-treated rats had more severe axonal loss and active axonal degeneration in the sciatic nerves compared to rats treated with IVIg or M045c. Thus, the M045c stradomer demonstrated mortality, weight, clinical, electrophysiological and histopathological efficacy compared to albumin, comparable to the efficacy demonstrated by clinical gold standard IVIg at approximately 2% of the IgIV dose.
    In a separate experiment, with 11 rats per test group, rats that received the tested M051 stradomer had statistically less severe disease than albumin-treated controls (see Figure 11). In this study, 7 of 11 animals died in the control group (albumin) compared to 4 in the M051 group. Rats that received either IVIg or M051 demonstrated significantly less weight loss compared to rats that received albumin control. Rats that received either IVIg or M051 demonstrated significant improvement in clinical scores compared to mice that received albumin control. There was a statistically significant improvement in both MCV and the amplitude of MCs following treatment with IVIg or M051. Thus, the M051 stradomer demonstrated significant mortality, weight, clinical and electrophysiological efficacy compared to albumin, comparable to the efficacy demonstrated by the clinical gold standard of IVIg at approximately 2% of the IVIg dose. EXAMPLE 9: Naturally Linked Stradomer Compounds Containing Fc Mutations Display Enhanced Binding to FCYRS and are Effective for Treatment in a Mouse Model of Arthritis
    Binding of stradomers containing Fc mutants, G075 (SEQ ID NO: 20) and G076 (SEQ ID NO: 21) to FcYRIIIa, FcYRIIb and FcYRIIa was performed by the Biacore binding assay as described above in Example 4. G075 showed a an increase in binding to FcYRIIIa and a decrease in binding to FcYRIIa and FcYRIIb while G076 showed a decrease in binding to FcYRIIIa and an increase in binding to FcYRIIa and FcYRIIb. (See Figures 10A and B).
    Evaluation of the efficacy of stradomers containing mutant Fc M075 (SEQ ID NO: 22), M076 (SEQ ID NO: 23) and M098 (SEQ ID NO: 25) in a mouse model of arthritis was then determined as it was done in Example 3 above. Stradomers containing mutant Fc were compared with vehicle and M045c. Both M098 and M075 were significantly more effective in inhibiting CIA progression, whereas the M076 stradomer was not. (See Figures 13A and B). Other immunoglobulin Fc mutations that are known to alter individual Fc-FcR binding or alter complement-dependent cytotoxicity are expected to be similarly complementary to stradomers comprising IgG1 Fc and displaying polyvalent IgG1 Fc to Fc receptors.
    权利要求:
    Claims (20)
    [0001]
    1. Homodimeric compound characterized by comprising two monomers, each comprising an amino acid sequence from amino acid residues 21-264 of SEQ ID NO: 4, wherein the homodimeric compound comprises an Fc domain of IgG1 directly linked to its carboxy- terminal, to a multimerization domain, wherein said multimerization domain is an IgG2 hinge domain.
    [0002]
    2. Homodimeric compound according to claim 1, characterized in that said compound is glycosylated.
    [0003]
    A homodimeric compound according to claim 1, further comprising a leader sequence comprising amino acids 1-20 of SEQ ID NO: 4 directly linked to the amino terminus of at least one of said first polypeptides.
    [0004]
    4. Higher order dimer or multimer of the homodimeric compound as defined in claim 1, characterized in that said higher order dimer or multimer is capable of binding to at least two Fc gamma receptors (FCYR).
    [0005]
    Higher order multimer according to claim 4, characterized in that said higher order multimer comprises at least three, at least four, at least five, at least six, or at least seven homodimeric compounds.
    [0006]
    Higher order dimer or multimer according to claim 4, characterized in that a first compound is linked via its multimerization domain to at least one second homodimeric compound.
    [0007]
    Higher order dimer or multimer according to claim 6, characterized in that the second homodimeric compound is linked via its multimerization domain to the first homodimeric compound.
    [0008]
    Higher order dimer or multimer according to claim 6, characterized in that all linked homodimeric compounds are linked via the multimerization domains.
    [0009]
    Higher order dimer or multimer, according to claim 6, characterized in that said first and at least second homodimeric compounds are covalently linked by means of disulfide bonds between the cysteine residues present in the respective multimerization domains.
    [0010]
    10. Composition characterized in that it comprises the higher order dimer or multimer, as defined in claim 4.
    [0011]
    Composition according to claim 10, characterized in that said higher order dimers or multimers comprise at least 45%, at least 55%, at least 65%, at least 70% or at least 73% of the total amount of compound in said composition.
    [0012]
    12. Use of the higher order dimer or multimer as defined in claim 4, characterized in that it is in the preparation of a composition for modulating an immune response in an individual.
    [0013]
    Use according to claim 12, characterized in that said modulation results in induction of CD86 expression or inhibition of CD1a expression in dendritic cells.
    [0014]
    Use of the higher order dimer or multimer as defined in claim 4 for the preparation of a composition for treating an inflammatory or autoimmune disease in a subject.
    [0015]
    Use according to claim 14, characterized in that the inflammatory or autoimmune disease can be treated with human IVIG (intravenous immunoglobulin).
    [0016]
    16. Use according to claim 14, characterized in that the inflammatory or autoimmune disease is selected from the group consisting of acquired autoimmune thrombocytopenia, acquired factor VIII autoimmunity, acquired von Willebrand disease, acute idiopathic dysautonomic neuropathy, alloimmune thrombocytopenia/ autoimmune, ANCA positive vasculitis, ankylosing spondylitis, anti-decorin myopathy (BJ antigen), aplastic anemia, asthma, atopic dermatitis, autoimmune anemia, autoimmune hemolytic anemia, autoimmune neutropenia, autoimmune thyroiditis, autoimmune uveitis, bone marrow transplant rejection, celiac disease, chronic inflammatory demyenilizing polyneuropathy (CHDD), chronic inflammatory demyenilizing polyradiculoneuropathy, chronic lymphocytic leukemia (CLL), Crohn's disease, Cushing's syndrome, dermatomyositis, dermatopolymyositis, diabetic neuropathy, DiamondBlackfan anemia, epilepsy, Evan's syndrome, syndrome Felty, Gaucher's Disease, Good's Disease pasture, Grave's disease, Guillain-Barré syndrome, hemolytic disease of the newborn, hemolytic uremic syndrome, idiopathic thrombocytopenic purpura (ITP), immune mediated neutropenia, inclusion body myositis, inflammatory bowel disease, inflammatory myopathies, idiopathic arthritis juvenile, Kawasaki disease, Lambert-Eaton myasthenic syndrome, anti-/GM1 associated lower motor neuron syndrome, monoclonal gammopathy of unknown significance, multifocal motor neuropathy (MMM), multiple sclerosis, myasthenia gravis, myelitis, myositis, necrotizing fasciitis , optic neuritis, organ transplant rejection, Paget's disease, paraneoplastic cerebellar degeneration with anti-Yo antibodies, paraneoplastic encephalomyelitis, paraneoplastic necrotic myopathy, paraproteinemic IgM demyelinating polyneuropathy, pemphigus, pencilamine-induced polymyositis, post-transfusion purpura, pure red cell aplasia, reactive arthritis, refractoriness to trans. platelet sfusion, rheumatoid arthritis, sarcoidosis, scleroderma, sclerosing cholangitis, sensory neuropathy with antiHu antibodies, sepsis, sickle cell crisis, spondyloarthropathies, spontaneous polymyositis, Stiff Man syndrome, systemic lupus erythematosus, thrombocytic purpura , type 1 diabetes mellitus, ulcerative colitis, Wegener's granulomatosis, Whipple's disease, and X-linked vacuolated myopathy.
    [0017]
    17. Use according to claim 16, characterized in that the inflammatory or autoimmune disease is selected from the group consisting of chronic inflammatory demyelinating polyneuropathy (CDID), chronic lymphocytic leukemia (CLL), diabetic neuropathy, idiopathic thrombocytopenic purpura (ITP) , Guillain-Barré syndrome, multifocal motor neuropathy (NMM), and systemic lupus erythematosus (SLE).
    [0018]
    18. A compound characterized in that it comprises a multimer of linked dimers, each dimer comprising two monomers, each comprising the amino acid sequence of amino acid residues 21-264 of SEQ ID NO: 4, each of which comprises an Fc domain monomer of IgG1 directly linked at its carboxy terminus to an IgG2 hinge domain monomer, where the binding of the dimers occurs through their respective IgG2 hinge domains.
    [0019]
    19. A multimer as defined by claim 18, wherein said multimer comprises at least two, at least three, at least four, at least five, at least six, or at least seven linked dimers.
    [0020]
    Use of the compound as defined by claim 1, characterized in that it is in the preparation of a composition for treating an inflammatory or autoimmune disease in an individual, wherein said inflammatory or autoimmune disease is selected from the group consisting of acquired autoimmune thrombocytopenia , acquired factor VIII autoimmunity, acquired von Willebrand disease, acute idiopathic dysautonomic neuropathy, alloimmune/autoimmune thrombocytopenia, ANCA positive vasculitis, ankylosing spondylitis, antidecorine myopathy (BJ antigen), aplastic anemia, asthma, atopic dermatitis, autoimmune anemia , autoimmune hemolytic anemia, autoimmune neutropenia, autoimmune thyroiditis, autoimmune uveitis, bone marrow transplant rejection, celiac disease, chronic inflammatory demyenilizing polyneuropathy (CIDP), chronic inflammatory demyenilizing polyradiculoneuropathy, chronic lymphocytic leukemia (CLL), Crohn's disease, syndrome Cushing, dermatomyositis, dermatopolymyositis, ne diabetic uropathy, Diamond-Blackfan anemia, epilepsy, Evan's syndrome, Felty's syndrome, Gaucher's disease, Goodpasture's disease, Grave's disease, Guillain-Barré syndrome, newborn hemolytic disease, hemolytic uremic syndrome, thrombocytopenic purpura idiopathic (ITP), immune mediated neutropenia, inclusion body myositis, inflammatory bowel disease, inflammatory myopathies, juvenile idiopathic arthritis, Kawasaki disease, Lambert-Eaton myasthenic syndrome, lower motor neuron syndrome associated with anti-/GM1 monoclonal gammopathy of unknown significance, multifocal motor neuropathy (MMN), multiple sclerosis, myasthenia gravis, myelitis, myositis, necrotizing fascitis, optic neuritis, organ transplant rejection, Paget's disease, paraneoplastic cerebellar degeneration with anti-Yo antibodies, paraneoplastic encephalomyelitis , paraneoplastic necrotic myopathy, paraproteinemic IgM demyelinating polyneuropathy, pemphigus, induced polymyositis a by penacylamine, post-transfusion purpura, psoriasis, pure red cell aplasia, reactive arthritis, refractoriness to platelet transfusion, rheumatoid arthritis, sarcoidosis, scleroderma, sclerosing cholangitis, sensory neuropathy with antiHu antibodies, sepsis, sickle cell crisis, spondyloarthropathy , spontaneous polymyositis, Stiff Man syndrome, systemic lupus erythematosus (SLE), systemic vasculitis, thrombotic thrombocytopenic purpura, type 1 diabetes mellitus, ulcerative colitis, Wegener's granulomatosis, Whipple's disease, and X-linked vacuolated myopathy.
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    公开号 | 公开日
    EP2598533A4|2015-08-05|
    US20220056087A1|2022-02-24|
    WO2012016073A3|2012-08-09|
    MX2013001148A|2014-02-27|
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    AU2011282579A1|2013-01-24|
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    WO2012016073A2|2012-02-02|
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    法律状态:
    2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
    2019-05-21| B07E| Notification 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 |
    2019-07-16| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|
    2020-07-28| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|
    2021-03-30| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2021-07-27| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-09-14| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 28/07/2011, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |
    优先权:
    申请号 | 申请日 | 专利标题
    US36846510P| true| 2010-07-28|2010-07-28|
    US61/368,465|2010-07-28|
    PCT/US2011/045768|WO2012016073A2|2010-07-28|2011-07-28|Fusion proteins of natural human protein fragments to create orderly multimerized immunoglobulin fc compositions|
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