RELAXIN-2 FUSION PROTEIN ANALOGS AND METHODS OF USING SAME
The present disclosure provides relaxin-2 fusion protein analogs with enhanced in vivo half-life and methods for making the same. Also disclosed herein are methods of treating relaxin-2-associated disorders or diseases using the relaxin-2 fusion protein analogs described herein.
1 - 65 . (canceled)
66 . A method of producing a fusion protein, comprising culturing a host cell comprising a polynucleotide comprising a nucleotide sequence encoding a fusion protein, under conditions such that the fusion protein is produced, wherein the fusion protein comprises, from N-terminus to C-terminus:
a first peptide;
a linker peptide; and
a second peptide,
wherein:
(a) the first peptide comprises an amino acid sequence that differs at 0, 1, 2, 3, 4, or 5 amino acids when compared to the amino acid sequence of SEQ ID NO: 7 and the second peptide comprises an amino acid sequence that differs at 0, 1, 2, 3, 4, or 5 amino acids when compared to the amino acid sequence of SEQ ID NO: 9; or
the first peptide comprises an amino acid sequence that differs at 0, 1, 2, 3, 4, or 5 amino acids when compared to the amino acid sequence of SEQ ID NO: 9 and the second peptide comprises an amino acid sequence that differs at 0, 1, 2, 3, 4, or 5 amino acids when compared to the amino acid sequence of SEQ ID NO: 7; and
(b) the linker peptide comprises an amino acid sequence with 12-15 amino acids, comprising 2-5 acidic amino acids and 10-13 non-acidic amino acids.
67 . The method of claim 66 , wherein the linker peptide comprises an amino acid sequence selected from the group consisting of:
R 1 R 1 R 1 R 2 R 1 R 1 R 1 R 2 R 1 R 1 R 1 R 2 R 1 ;
R 1 R 1 R 1 R 2 R 1 R 1 R 1 R 2 R 1 R 1 R 1 R 2 R 1 R 1 R 1 ;
R 1 R 1 R 2 R 1 R 1 R 1 R 2 R 2 R 1 R 1 R 1 R 2 R 1 R 1 ;
R 1 R 1 R 1 R 2 R 2 R 1 R 1 R 1 R 2 R 2 R 1 R 1 R 1 ; and
R 1 R 1 R 2 R 1 R 2 R 1 R 1 R 2 R 1 R 2 R 1 R 1 R 1 ,
wherein R 1 is a non-acidic amino acid and R 2 is an acidic amino acid.
68 . The method of claim 66 , wherein the linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-23.
69 . The method of claim 66 , wherein each of the acidic amino acids in the linker peptide is aspartate or glutamate, each of the acidic amino acids in the linker peptide is aspartate, or each of the acidic amino acids in the linker peptide is glutamate; and/or the non-acidic amino acid(s) are glycine, proline, or serine.
70 . The method of claim 66 , wherein the first peptide comprises:
the amino acid sequence of DSWX 3 EEVIKLCGRELVRAQIAICGX 4 ST (SEQ ID NO: 3), wherein X 3 is methionine, lysine, or glutamine, and wherein X 4 is methionine or lysine;
the amino acid sequence of X 5 QX 6 YSALANKCCHVGCTKRSLAX 7 FC (SEQ ID NO: 4), wherein X 5 is arginine or absent, X 6 is leucine or aspartate, and wherein X 7 is arginine, glutamine, or glutamate;
an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-7; or
an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-13.
71 . The method of claim 66 , wherein the second peptide comprises:
the amino acid sequence of DSWX 3 EEVIKLCGRELVRAQIAICGX 4 ST (SEQ ID NO: 3), wherein X 3 is methionine, lysine, or glutamine, and wherein X 4 is methionine or lysine;
the amino acid sequence of X 5 QX 6 YSALANKCCHVGCTKRSLAX 7 FC (SEQ ID NO: 4), wherein X 5 is arginine or absent, X 6 is leucine or aspartate, and wherein X 7 is arginine, glutamine, or glutamate;
an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-7; or
an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-13.
72 . The method of claim 66 , wherein:
the first peptide comprises the amino acid sequence of SEQ ID NO: 5 and the second peptide comprises the amino acid sequence of SEQ ID NO: 8;
the first peptide comprises the amino acid sequence of SEQ ID NO: 5 and the second peptide comprises the amino acid sequence of SEQ ID NO: 9;
the first peptide comprises the amino acid sequence of SEQ ID NO: 5 and the second peptide comprises the amino acid sequence of SEQ ID NO: 10;
the first peptide comprises the amino acid sequence of SEQ ID NO: 5 and the second peptide comprises the amino acid sequence of SEQ ID NO: 11;
the first peptide comprises the amino acid sequence of SEQ ID NO: 5 and the second peptide comprises the amino acid sequence of SEQ ID NO: 12;
the first peptide comprises the amino acid sequence of SEQ ID NO: 5 and the second peptide comprises the amino acid sequence of SEQ ID NO: 13;
the first peptide comprises the amino acid sequence of SEQ ID NO: 6 and the second peptide comprises the amino acid sequence of SEQ ID NO: 8;
the first peptide comprises the amino acid sequence of SEQ ID NO: 6 and the second peptide comprises the amino acid sequence of SEQ ID NO: 9;
the first peptide comprises the amino acid sequence of SEQ ID NO: 6 and the second peptide comprises the amino acid sequence of SEQ ID NO: 10;
the first peptide comprises the amino acid sequence of SEQ ID NO: 6 and the second peptide comprises the amino acid sequence of SEQ ID NO: 11;
the first peptide comprises the amino acid sequence of SEQ ID NO: 6 and the second peptide comprises the amino acid sequence of SEQ ID NO: 12;
the first peptide comprises the amino acid sequence of SEQ ID NO: 6 and the second peptide comprises the amino acid sequence of SEQ ID NO: 13;
the first peptide comprises the amino acid sequence of SEQ ID NO: 7 and the second peptide comprises the amino acid sequence of SEQ ID NO: 8;
the first peptide comprises the amino acid sequence of SEQ ID NO: 7 and the second peptide comprises the amino acid sequence of SEQ ID NO: 9;
the first peptide comprises the amino acid sequence of SEQ ID NO: 7 and the second peptide comprises the amino acid sequence of SEQ ID NO: 10;
the first peptide comprises the amino acid sequence of SEQ ID NO: 7 and the second peptide comprises the amino acid sequence of SEQ ID NO: 11;
the first peptide comprises the amino acid sequence of SEQ ID NO: 7 and the second peptide comprises the amino acid sequence of SEQ ID NO: 12;
the first peptide comprises the amino acid sequence of SEQ ID NO: 7 and the second peptide comprises the amino acid sequence of SEQ ID NO: 13;
the second peptide comprises the amino acid sequence of SEQ ID NO: 5 and the first peptide comprises the amino acid sequence of SEQ ID NO: 8;
the second peptide comprises the amino acid sequence of SEQ ID NO: 5 and the first peptide comprises the amino acid sequence of SEQ ID NO: 9;
the second peptide comprises the amino acid sequence of SEQ ID NO: 5 and the first peptide comprises the amino acid sequence of SEQ ID NO: 10;
the second peptide comprises the amino acid sequence of SEQ ID NO: 5 and the first peptide comprises the amino acid sequence of SEQ ID NO: 11;
the second peptide comprises the amino acid sequence of SEQ ID NO: 5 and the first peptide comprises the amino acid sequence of SEQ ID NO: 12;
the second peptide comprises the amino acid sequence of SEQ ID NO: 5 and the first peptide comprises the amino acid sequence of SEQ ID NO: 13;
the second peptide comprises the amino acid sequence of SEQ ID NO: 6 and the first peptide comprises the amino acid sequence of SEQ ID NO: 8;
the second peptide comprises the amino acid sequence of SEQ ID NO: 6 and the first peptide comprises the amino acid sequence of SEQ ID NO: 9;
the second peptide comprises the amino acid sequence of SEQ ID NO: 6 and the first peptide comprises the amino acid sequence of SEQ ID NO: 10;
the second peptide comprises the amino acid sequence of SEQ ID NO: 6 and the first peptide comprises the amino acid sequence of SEQ ID NO: 11;
the second peptide comprises the amino acid sequence of SEQ ID NO: 6 and the first peptide comprises the amino acid sequence of SEQ ID NO: 12;
the second peptide comprises the amino acid sequence of SEQ ID NO: 6 and the first peptide comprises the amino acid sequence of SEQ ID NO: 13;
the second peptide comprises the amino acid sequence of SEQ ID NO: 7 and the first peptide comprises the amino acid sequence of SEQ ID NO: 8;
the second peptide comprises the amino acid sequence of SEQ ID NO: 7 and the first peptide comprises the amino acid sequence of SEQ ID NO: 9;
the second peptide comprises the amino acid sequence of SEQ ID NO: 7 and the first peptide comprises the amino acid sequence of SEQ ID NO: 10;
the second peptide comprises the amino acid sequence of SEQ ID NO: 7 and the first peptide comprises the amino acid sequence of SEQ ID NO: 11;
the second peptide comprises the amino acid sequence of SEQ ID NO: 7 and the first peptide comprises the amino acid sequence of SEQ ID NO: 12; or
the second peptide comprises the amino acid sequence of SEQ ID NO: 7 and the first peptide comprises the amino acid sequence of SEQ ID NO: 13.
73 . The method of claim 66 , wherein the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-48.
74 . The method of claim 66 , wherein the fusion protein further comprises an IgG Fc.
75 . The method of claim 74 , wherein:
the IgG Fc comprises the amino acid alanine at EU positions 234 and 235;
the IgG Fc comprises the amino acid alanine at EU position 329;
the IgG Fc comprises the amino acid alanine at EU positions 234, 235, and 329;
the IgG Fc comprises the amino acids alanine, alanine, alanine, leucine, and serine at EU positions 234, 235, 329, 428, and 434, respectively;
the IgG Fc comprises the amino acids lysine, phenylalanine, and tyrosine at EU positions 433, 434, and 436, respectively;
the IgG Fc comprises the amino acids tyrosine, threonine, and glutamate at EU positions 252, 254, and 256, respectively; and/or
the IgG Fc comprises the amino acids leucine and serine at EU positions 428 and 434, respectively.
76 . The method of claim 74 , wherein the IgG Fc comprises an amino acid sequence at least 85% identical to the amino acid sequence of a human IgG1 Fc.
77 . The method of claim 74 , wherein the IgG Fc comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-52 and 201-203.
78 . The method of claim 74 , wherein the IgG Fc is linked to the N-terminus of the first peptide or to the C-terminus of the second peptide.
79 . The method of claim 66 , wherein the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-118, 204-211, and 214-221.
80 . The method of claim 66 , wherein the first and second peptides do not comprise the amino acid sequence of a peptide selected from the group consisting of SEQ ID NOs:187-190.
81 . The method of claim 66 , wherein the fusion protein has a pI from 6.0 to 8.2.
82 . The method of claim 66 , wherein the polynucleotide is an RNA molecule or a DNA molecule.
83 . The method of claim 66 , wherein the polynucleotide is comprised within an expression vector, optionally wherein the expression vector is a plasmid or a viral vector.
84 . The method of claim 66 , wherein the host cell is prokaryotic cell, optionally wherein the prokaryotic cell is an E. coli cell or a Bacillus cell.
85 . The method of claim 66 , wherein the host cell is a eukaryotic cell, optionally wherein the eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell, and a mammalian cell, optionally wherein the mammalian cell is selected from the group consisting of a CHO cell, a HeLa cell, and a 293 cell.