Molecules that selectively activate regulatory t cells for the treatment of autoimmune diseases

Abstract

This invention provides for a fusion protein between an IL2αβγ Selective Agonist protein (IL2 Selective Agonist) and a IgG Fc protein using a linker. The IL2 Selective Agonist moiety provides a therapeutic activity by selectively activating the IL2αβγ form of the receptor, thus selectively stimulating Tregs. The Fc moiety provides a prolonged circulating half-life compared to the circulating half-life of IL-2 or an IL2SA protein.

1 . A nucleic acid encoding a fusion protein comprising:
 a. a human IL-2 variant protein domain;   b. a peptide linker domain; and   c. an IgG Fc protein domain,   
       wherein each domain has an amino-terminus (N-terminus) and a carboxy terminus (C-terminus); and wherein the fusion protein is configured so that the C-terminus of the human IL-2 variant protein domain is fused through a peptide bond to the N-terminus of the peptide linker domain, and the N-terminus of the IgG Fc protein domain is fused through a peptide bond to the C-terminus of the peptide linker domain. 
     
     
         2 . The nucleic acid of  claim 1 , wherein the human IL-2 variant protein domain comprises human IL-2 with a substitution selected from the group consisting of: N88R, N88G, D20H, Q126L, and Q126F. 
     
     
         3 . The nucleic acid of  claim 1 , wherein the human IL-2 variant protein domain comprises the N88R substitution. 
     
     
         4 . The nucleic acid of  claim 2 , wherein the human IL-2 variant protein further comprises a substitution selected from the group consisting of T3A and C125S. 
     
     
         5 . The nucleic acid of  claim 1 , wherein the peptide linker domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. 
     
     
         6 . The nucleic acid of  claim 1 , wherein the IgG Fc protein domain comprises an IgG1 Fc protein comprising an N297A mutation relative to the amino acid sequence of SEQ ID NO: 2. 
     
     
         7 . The nucleic acid of  claim 1 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 9. 
     
     
         8 . A method of selectively activating human regulatory T cells, the method comprising administering a pharmaceutical composition comprising a fusion protein comprising:
 a. a human IL-2 variant protein domain;   b. a peptide linker domain; and   c. an IgG Fc protein domain,   
       wherein each domain has an amino-terminus (N-terminus) and a carboxy terminus (C-terminus); and wherein the fusion protein is configured so that the C-terminus of the human IL-2 variant protein domain is fused through a peptide bond to the N-terminus of the peptide linker domain, and the N-terminus of the IgG Fc protein domain is fused through a peptide bond to the C-terminus of the peptide linker domain, 
       and wherein said pharmaceutical composition is administered at a therapeutically effective dose until human regulatory T cell concentrations reach desired levels. 
     
     
         9 . The method of  claim 8 , wherein the human IL-2 variant protein domain comprises human IL-2 with a substitution selected from the group consisting of: N88R, N88G, D20H, Q126L, and Q126F. 
     
     
         10 . The method of  claim 8 , wherein the human IL-2 variant protein domain comprises the N88R substitution. 
     
     
         11 . The method of  claim 9 , wherein the human IL-2 variant protein further comprises a substitution selected from the group consisting of T3A and C125S. 
     
     
         12 . The method of  claim 8 , wherein the peptide linker domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. 
     
     
         13 . The method of  claim 8 , wherein the IgG Fc protein domain comprises an IgG1 Fc protein comprising an N297A mutation relative to the amino acid sequence of SEQ ID NO: 2. 
     
     
         14 . The method of  claim 8 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 9. 
     
     
         15 . A method of measuring the number of regulatory T cells in a human blood sample comprising contacting human blood cells with the fusion protein of  claim 1  at a concentration of between 1 nM and 0.01 nM, and then detecting cells that bind to the protein by flow cytometry. 
     
     
         16 . The method of  claim 15 , wherein the human IL-2 variant protein domain comprises human IL-2 with a substitution selected from the group consisting of: N88R, N88G, D20H, Q126L, and Q126F. 
     
     
         17 . The method of  claim 15 , wherein the human IL-2 variant protein domain comprises the N88R substitution. 
     
     
         18 . The method of  claim 16 , wherein the human IL-2 variant protein further comprises a substitution selected from the group consisting of T3A and C125S. 
     
     
         19 . The method of  claim 15 , wherein the peptide linker domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. 
     
     
         20 . The method of  claim 15 , wherein the IgG Fc protein domain comprises an IgG1 Fc protein comprising an N297A mutation relative to the amino acid sequence of SEQ ID NO: 2. 
     
     
         21 . The method of  claim 15 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 9.