Chimeric immunoglobulin fusion proteins
Disclosed are fusion proteins comprising a biologically active molecule and an immunoglobulin (Ig) Fc domain which is linked to the biologically active molecule. The Fc domain is a hybrid human Fc domain of (i) IgG1, IgG2 or IgG4 or (ii) IgG4 and IgD. The hybrid Fc is useful as a carrier of biologically active molecules.
1. A polypeptide represented by the following formula:
N′—(Z1) p —Y—Z2—Z3—Z4—C′
wherein:
N′ is the N-terminus and C′ is the C-terminus of the polypeptide,
Y is an amino acid sequence consisting of 5 or more consecutive amino acid residues from e C-terminus at positions 99 to 162 of SEQ ID NO: 14;
Z2 is an amino acid sequence consisting of four or more consecutive amino residues from the N-terminus at positions 163 to 199 of SEQ ID NO: 14;
Z3 is an amino acid sequence consisting of seventy , one or more consecutive amino acid residues from the C-terminus at positions 115 to 220 of SEQ ID NO: 13;
Z4 is an amino acid sequence consisting of eighty or more consecutive amino acid residues from the N-terminus at positions 221 to 327 of SEQ ID NO: 13; and
Z1 is an amino acid sequence consisting of five or more consecutive amino acid residues from the C-terminus at positions 90 to 98 of SEQ ID NO: 11 or five or more consecutive amino acid residues from the C-terminus at positions 90 to 98 of SEQ ID NO: 14 and p is an integer of 0 or 1,
wherein the total number of the amino acid residues for Z2 and Z3 is between 80 and 140, both inclusive; and
wherein the total number of the amino acid residues for the polypeptide is between 154 and 288, both inclusive.
2. The polypeptide of claim 1 , wherein Y is an amino acid sequence consisting of the amino acid residues at positions 158 to 162 of SEQ ID NO: 14, amino acid residues at positions 153 to 162 of SEQ ID NO: 14, amino acid residues at positions 143 to 162 of SEQ ID NO: 14, amino acid residues at positions 133 to 162 of SEQ ID NO: 14, or amino acid residues at positions 99 to 162 of SEQ ID NO: 14.
3. The polypeptide of claim 1 , wherein Z2 is an amino acid sequence consisting of 6 to 30 consecutive amino acid residues from the N-terminus at positions 163 to 199 of SEQ ID NO: 14.
4. The polypeptide of claim 3 , wherein Z2 is an amino acid sequence consisting of the amino acid residues at positions 163 to 170 of SEQ ID NO: 14.
5. The polypeptide of any one of claims 2 , 3 , or 4 , wherein:
(a) Z3 is an amino acid sequence consisting of the amino acid residues at positions 121 to 220 of SEQ ID NO: 13; and/or
(b) Z4 is an amino acid sequence consisting of the amino acid residues at positions 221 to 327 of SEQ ID NO: 13.
6. The polypeptide of claim 1 , wherein p is 0.
7. A chimeric polypeptide the polypeptide of any one of claim 1 , 2 , 3 , 4 , 5 , or 6 and a biologically active molecule, wherein the biologically active molecule is fused at the N-terminus or the C-terminus of the polypeptide, and wherein said biologically active molecule fused to the polypeptide shows an increased circulating half-life compared to the circulating half-life of said biologically active molecule without being fused to the polypeptide and possesses a biological activity.
8. The chimeric polypeptide of claim 7 , wherein the biologically active molecule is a hormone, cytokine, growth factor, co-stimulatory molecule, hormone receptor, cytokine receptor, growth factor receptor, or short peptide.
9. The chimeric polypeptide of claim 8 , wherein the biologically active molecule is EPO, CSF, insulin, human growth hormone, GLP-1, p40, TNF receptor, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, bone morphogenetic protein-1, interferon-alpha, IL-10, FSH, Factor VII, CTLA4, IFN-beta, PD-1, PD-L1, PD-L2, TIM-3, IL-10R, CXCL-11, TGF-beta receptor, TGF-beta, IL- 17R, BTC, OPG or RANK, or a variant or fragment thereof that has a biological activity.
10. The chimeric polypeptide of claim 9 , wherein said biologically active molecule is a variant of p40, wherein the variation consists of Asn303Gln substitution.
11. The chimeric polypeptide of claim 7 , wherein:
(a) said biologically active molecule is a secreted protein, a mature form of a secreted protein, or a Fab region of an antibody; and/or
(b) the polypeptide and the biologically active molecule are coupled to each other via a linker, wherein the linker is an albumin linker or a synthetic linker.
12. The chimeric polypeptide of claim 11 , wherein:
(a) said albumin linker comprises amino acid sequence 321 to 323, 318 to 325, 316 to 328, 313 to 330, 311 to 333, or 306 to 338 of SEQ ID NO: 25; or
(b) said synthetic linker is a peptide of 10 to 20 amino acid residues, wherein the peptide being composed of Gly and Ser residues.
13. A nucleic acid molecule encoding a polypeptide of claim 1 or 7 .
14. An expression vector the nucleic acid molecule according to claim 13 .
15. A method of producing the polypeptide according to claim 1 or 7 , comprising (i) introducing a nucleic acid molecule encoding a polypeptide of claim 1 or 8 into a mammalian host cell, (ii) growing the cell in a medium under conditions where the polypeptide can be expressed; and (iii) harvesting the expressed polypeptide from the cell or the medium.
16. A method for increasing a circulating half-life of a biologically active molecule in a subject, administering the biologically active molecule that is fused to the polypeptide of claim 1 at the N-terminus or the C-terminus of the polypeptide, to the subject, wherein the biological active molecule possesses the biological activity.
17. The method of claim 16 , wherein the biologically active molecule is a hormone, cytokine, growth factor, co-stimulatory molecule, hormone receptor, cytokine receptor, growth factor receptor, or short peptide.
18. The method of claim 17 , wherein the biologically active molecule is EPO, CSF, insulin, human growth hormone, GLP-1, p40, TNF receptor, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, bone morphogenetic protein- 1, interferon- alpha, IL-10, FSH, Factor VII, CTLA4, IFN-beta, PD-1, PD-L1, PD-L2, TIM-3, IL-10R, CXCL-11, TGF-beta receptor, TGF-beta, IL- 17R, BTC, OPG or RANK, or a variant or fragment thereof that has a biological activity.
19. The method of claim 18 , wherein said biologically active molecule is a variant of p40, wherein the variation consists of Asn303Gln substitution.
20. The method of claim 16 , wherein:
(a) said biologically active molecule is a secreted protein, a mature form of a secreted protein, or a Fab region of an antibody; and/or
(b) the polypeptide and the biologically active molecule are coupled to each other via a linker selected from the group consisting of an albumin linker and a synthetic linker.