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 fusion protein, comprising a human IL-2 variant protein domain, a peptide linker segment domain of between 6 and 35 amino acids and an IgG Fc domain where each domain has an amino-terminus (N-terminus) and a carboxy terminus (C-terminus); and where 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 and the N-terminus of the IgG Fc protein moiety is fused through a peptide bond to the C-terminus of the peptide linker;
 wherein said IL-2 fusion protein has the ability to selectively activate the high affinity IL-2 receptor and thus selectively activate human regulatory T cells.   
     
     
         2 . The fusion protein of  claim 1 , wherein the IL-2 variant protein comprises human IL-2 with a substitution selected from the group consisting of: N88R, N88G, D20H, Q126L, and Q126F, relative to human IL2 protein (SEQ ID NO:1). 
     
     
         3 . The fusion protein of  claim 1 , wherein the IL-2 variant protein comprises human IL-2 with the substitution C125S. 
     
     
         4 . The fusion protein of  claim 1 , wherein both the IL-2 variant protein is N88R provided in SEQ ID NO: 1. 
     
     
         5 . The fusion protein of  claim 1 , where the linker comprises glycine and serine residues and where the linker is between 10 and 30 amino acids. 
     
     
         6 . The fusion protein of  claim 1 , wherein the IgG Fc protein contains one or more amino acid substitutions that reduce the effector functions of the Fc portion of the fusion protein. 
     
     
         7 . A fusion protein, comprising:
 a. a IL-2 variant protein having amino acid substitutions N88R and C125S relative to human IL-2 (SEQ ID NO:1);   b. a linker peptide as set forth in SEQ ID NO: 15; and,   c. a human IgG1 Fc variant protein as set forth in SEQ ID NO:2,   wherein said fusion protein has the ability to selectively activate the high affinity IL-2 receptor and thus selectively activate human regulatory T cells.   
     
     
         8 . A fusion protein, comprising
 a. a IL-2 variant protein having amino acid substitutions N88R and C125S relative to human IL-2 (SEQ ID NO:1);   b. a linker peptide as set forth in SEQ ID NO:15; and,   c. a human IgG2 Fe protein as set forth in SEQ ID NO:3.   
     
     
         9 . A pharmaceutical composition comprising the fusion protein of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         10 . A method of selectively activating human regulatory T cells, the method comprising administering a pharmaceutical composition comprising: a IL-2 variant protein having amino acid substitutions N88R and C125S relative to human IL-2 (SEQ ID NO:1), a linker peptide as set forth in SEQ ID NO: 15, and a human IgG1 Fc protein as set forth in SEQ ID NO: 2, wherein said pharmaceutical composition is administered at therapeutically effective dose until human regulatory T cell concentrations reach desired levels. 
     
     
         11 . A method of selectively activating human regulatory T cells, the method comprising administering a pharmaceutical composition comprising: a IL-2 variant protein of  claim 2 ; and a human IgG Fc protein selected from group consisting of
 a. a human IgG1 Fc protein as set forth in SEQ ID NO:2;   b. a human IgG2 Fc protein as set forth in SEQ ID NO:3; and,   c. a human IgG4 Fc protein domain as set forth in SEQ ID NO: 24   wherein said pharmaceutical composition is administered at therapeutically effective dose until human regulatory T cell concentrations reach desired levels.   
     
     
         12 . A method of measuring the numbers of Treg cells in a human blood sample by 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. 
     
     
         13 . A dimeric protein, comprising two identical chains, where each chain comprises a N-terminal human IL-2 variant protein moiety and a C-terminal IgG Fc protein moiety wherein:
 the N-terminal human IL-2 variant protein moiety   a. has a N-terminus and a C-terminus;   b. varies from the human IL-2 wildtype as in SEQ ID NO 1 by at least one substitution selected from the group consisting of: N88R, N88G, D20H, Q126L, and Q126F;   c. has at least a 97% sequence identify to Sequence ID No. 1; and,   d. has the ability activate Treg cells by binding to a IL2Rαβγ on those cells;   the N-terminal human IL-2 variant protein is joined at its C-terminal to a N-terminus of an amino acid linker of between 6 to 30 amino acid residues where said linker also has a C-terminus; and,   the C-terminus of the amino acid linker is joined to the N-terminus of IgG Fc protein moiety having 95% sequence identify to sequence ID No. 2 and comprising cysteine residues; and where the two chains are linked to each other through the cysteine residues of the IgG Fc protein moiety.   
     
     
         14 . The dimeric protein of  claim 13 , wherein the IL-2 variant protein further comprises human IL-2 with the substitution C125S. 
     
     
         15 . The protein of  claim 13  wherein the amino acid linker consists of a linker selected from the group of glycine residues, serine residues, and a mix of glycine and serine residues. 
     
     
         16 . The protein of  claim 13 , wherein the IL-2 variant protein moiety has the substitution N88R. 
     
     
         17 . The protein of  claim 13  wherein the linker comprises a mix of between 12 and 17 serine and glycine residues. 
     
     
         18 . The fusion protein of  claim 13  wherein the linker comprises a 4:1 ratio of Glycine residues to serine residues. 
     
     
         19 . A nucleic acid encoding the fusion protein of  claim 1 .