Molecules that selectively activate regulatory T cells for the treatment of autoimmune diseases
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. a human IL-2 variant protein domain comprising a C125S substitution relative to SEQ ID NO: 1 and a substitution selected from the group consisting of: N88R, N88G, D20H, Q126L, and Q126F;
b. a peptide linker domain of from 5 to 30 amino acid residues, 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; 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, wherein protein yield of the fusion protein is increased by at least two-fold relative to a fusion protein that comprises the human IL-2 variant protein domain and the IgG Fc protein domain, but does not comprise the peptide linker domain.
2. The fusion protein of claim 1 , wherein the human IL-2 variant protein domain comprises the N88R substitution.
3. The fusion protein of claim 1 , wherein the peptide linker domain consists of 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.
4. A dimeric protein comprising two identical chains, wherein each chain comprises a fusion protein comprising:
a. a human IL-2 variant protein domain comprising a C125S substitution relative to SEQ ID NO: 1 and a substitution selected from the group consisting of: N88R, N88G, D20H, Q126L, and Q126F;
b. a peptide linker domain of from 5 to 30 amino acid residues, 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; 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, wherein protein yield of the fusion protein is increased by at least two-fold relative to a fusion protein that comprises the human IL-2 variant protein domain and the IgG Fc protein domain, but does not comprise the peptide linker domain.
5. The dimeric protein of claim 4 , wherein the IgG Fc protein domain comprises cysteine residues, and the two identical chains are linked to each other through the cysteine residues of the IgG Fc protein domain.
6. The dimeric protein of claim 4 , wherein the human IL-2 variant protein domain comprises the N88R substitution.
7. The dimeric protein of claim 4 , wherein the peptide linker domain consists of 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.
8. A pharmaceutical composition comprising a fusion protein comprising:
a. a human IL-2 variant protein domain comprising a C125S substitution relative to SEQ ID NO: 1 and a substitution selected from the group consisting of: N88R, N88G, D20H, Q126L, and Q126F;
b. a peptide linker domain of from 5 to 30 amino acid residues, 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; 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, wherein protein yield of the fusion protein is increased by at least two-fold relative to a fusion protein that comprises the human IL-2 variant protein domain and the IgG Fc protein domain, but does not comprise the peptide linker domain.
9. The pharmaceutical composition of claim 8 , wherein the human IL-2 variant protein domain comprises the N88R substitution.
10. The pharmaceutical composition of claim 8 , wherein the peptide linker domain consists of 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.
11. A fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 31.
12. A dimeric protein comprising two identical chains, wherein each chain comprises the fusion protein of claim 11 .
13. A pharmaceutical composition comprising the fusion protein of claim 11 .