FUSION PROTEINS OF NATURAL HUMAN PROTEIN FRAGMENTS TO CREATE ORDERLY MULTIMERIZED IMMUNOGLOBULIN Fc COMPOSITIONS

Abstract

The current invention involves a series of fully recombinant multimerized forms of immunoglobulin Fc which thereby present polyvalent immunoglobulin Fc to immune cell receptors. The fusion proteins exist as both homodimeric and highly ordered multimeric fractions, termed stradomers. In comparison to the homodimeric fraction, purified multimeric stradomers have higher affinity and avidity for Fc Rs with slower dissociation and are useful in the treatment and prevention of disease. The current invention demonstrates that directly linking IgG1 Fc regions to multimerization domains leads to enhanced multimerization and biological activity.

1 - 156 . (canceled) 
     
     
         157 . A stradomer unit comprising:
 (a) a leader sequence   (b) an IgG1 Fc domain; and   (c) a multimerization domain;   wherein the leader sequence is directly linked to the IgG1 Fc domain and wherein the IgG1 Fc domain is directly linked to the multimerization domain or wherein the leader sequence is directly linked to the multimerization domain and the multimerization domain is directly linked to the IgG1 Fc domain.   
     
     
         158 . The stradomer unit of  claim 157  wherein the IgG1 Fc domain is about 90% homologous to SEQ ID NO:2. 
     
     
         159 . The stradomer unit of  claim 157  wherein the IgG1 Fc domain comprises the CH2 and CH3 domains of IgG1. 
     
     
         160 . Stradomer unit of  claim 159  wherein the IgG1 Fc domain is at least about 90% homologous to SEQ ID NO: 19. 
     
     
         161 . The stradomer unit of  claim 157  wherein the multimerization domain is an IgG2 hinge. 
     
     
         162 . The stradomer unit of  claim 161  wherein the amino acid sequence of the multimerization domain is about 90% homologous to SEQ ID NO: 3 and is capable of multimerizing said stradomer units. 
     
     
         163 . The stradomer unit of  claim 157  wherein the multimerization domain is directly linked to the amino terminus of the IgG1 Fc domain or the carboxy terminus of the IgG1 Fc domain. 
     
     
         164 . The stradomer unit of  claim 157  wherein the stradomer unit comprises: SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 18. 
     
     
         165 . The stradomer unit of  claim 157  wherein the multimerization domain creates multimers of said stradomer units. 
     
     
         166 . The stradomer unit of  claim 165  wherein the multimers of said stradomer units are high order multimers 
     
     
         167 . A stradomer composition comprising the stradomer units of  claim 157  wherein the multimers are present at a percentage of about 45% or about 55% or about 65% or about 65% or about 70% or about 73% of the total stradomer composition. 
     
     
         168 . The stradomer unit of  claim 157  wherein the IgG1 Fc domain is capable of binding FcRn, DC-SIGN, SIGN-R1 or an FcγR. 
     
     
         169 . A cluster stradomer comprising at least two stradomer units of  claim 157 . 
     
     
         170 . A method of modulating an immune response in a subject comprising administering to a subject an effective amount of the cluster stradomer of  claim 169 . 
     
     
         171 . A method for treating an inflammatory disease in a subject comprising administering to the subject an effective amount of the cluster stradomer of  claim 169 . 
     
     
         172 . The method of  claim 171  wherein the inflammatory disease is a disease selected from, multifocal mortor neuropathy, Alzheimer's disease, sepsis, arthritis, multiple sclerosis, type I diabetes, autoimmune thyroiditis, idiopathic thrombocytopenic purpura, chronic inflammatory polyneuropathy, scleroderma, autoimmune uveitis, systemic lupus erythematosus, myasthenia gravis, and atopic dermatitis or a disease associated with the transplantation of an organ from a donor to a recipient; or
 a infectious disease a bacterial infection or a viral infection. 
 
     
     
         173 . A method for blocking nonspecific binding of antibodies in an in vitro or ex vivo assay comprising incubating target tissue or target cells with a composition comprising an effective amount of the cluster stradomer of  claim 169 . 
     
     
         174 . A method for producing a cluster stradomer comprising expressing a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 18 in a host. 
     
     
         175 . The method of claim  19  further comprising
 (a) cloning a DNA encoding the stradomer selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO: 18 into an expression vector; 
 (b) transfecting the expression vector into a bacterial host; 
 (c) isolating the plasmid DNA containing cloned DNA from the bacterial culture; 
 (d) linearizing the plasmid DNA containing cloned DNA; 
 (e) transfecting the linearized DNA into mammalian cells; 
 (f) expanding the positively transfected cells to obtain a pool of stably transfected cells; 
 (g) harvesting the stradomer protein from the media; and 
 (h) purifying the stradomer protein, wherein the stradomer protein contains no extraneous sequences.