Relaxin-2 fusion protein analogs and methods of using same

Abstract

The present disclosure provides relaxin-2 fusion protein analogs with enhanced in vivo half-lives and methods for making same. Also disclosed herein are methods of treating relaxin-2-associated disorders or diseases using the relaxin-2 fusion protein analogs described herein.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application Nos. 63 / 503,101, filed May 18, 2023, 63 / 585,849, filed September 27, 2023, 63 / 586,868, filed September 29, 2023, 63 / 611,732, filed December 18, 2023, and 63 / 617,398, filed January 3, 2024, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety (the XML copy, created on May 16, 2024, is named "209634_seqlist.xml" and is 727,648 bytes in size). [Background technology]

[0003] Relaxin-2 exhibits potent antifibrotic activity. In injured tissues, fibroblast activation and proliferation lead to increased collagen production and interstitial fibrosis. Cardiac fibrosis is increased by biomechanical overload, affecting ventricular dysfunction, remodeling, and arrhythmogenesis. However, due to the limited in vivo half-life of relaxin, the compound must be administered as a continuous infusion for at least 48 hours. Furthermore, the synthesis of relaxin-2 is difficult. Due to the low solubility of the B chain and the requirement for the tedious and specific introduction of a cysteine ​​bridge between the A and B chains, the yield of active peptide obtained by these methods is extremely low. Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need for engineered relaxin-2 analogs that have longer half-lives and are easier to manufacture. [Means for solving the problem]

[0005] The present disclosure provides fusion proteins that are engineered relaxin-2 analogs with improved pharmacokinetic properties. The disclosure also provides methods of using these fusion proteins to enhance relaxin-2-associated activity and treat or prevent relaxin-2-associated diseases in a subject. The fusion protein structures described herein are based, at least in part, on the surprising discovery that reducing the isoelectric point (pI) of a relaxin-2 fusion protein analog increases its circulating half-life and improves its pharmacokinetic and pharmacodynamic properties.

[0006] Accordingly, in one aspect, the present disclosure provides a fusion protein comprising, 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 having zero, one, two, three, four, or five amino acid modifications relative to the amino acid sequence of SEQ ID NO: 502, and the second peptide comprises an amino acid sequence having zero, one, two, three, four, or five amino acid modifications relative to the amino acid sequence of SEQ ID NO: 503 or 504, or wherein the first peptide comprises an amino acid sequence having zero, one, two, three, four, or five amino acid modifications relative to the amino acid sequence of SEQ ID NO: 503 or 504 and the second peptide comprises an amino acid sequence having zero, one, two, three, four, or five amino acid modifications relative to the amino acid sequence of SEQ ID NO: 502; and (b) the fusion protein has a pI of 6.0 to 8.2.

[0007] In some embodiments, the fusion protein has a pI of about 6.0 to about 9.4. In some embodiments, the fusion protein has a pI of about 6.0 to about 8.2. In some embodiments, the fusion protein has a pI that is less than about 9.0, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, or 6.1. In some embodiments, the fusion protein has a pI that is less than 9.0. In some embodiments, the fusion protein has a pI that is less than about 8.2. In some embodiments, the fusion protein has a pI of about 6.8. In some embodiments, the fusion protein has a pI of about 7.0. In some embodiments, the fusion protein has a pI of about 7.1. In some embodiments, the fusion protein has a pI of about 7.4. In some embodiments, the fusion protein has a pI of about 7.5. In some embodiments, the fusion protein has a pI of about 7.9. In some embodiments, the fusion protein has a pI of about 8.0. In some embodiments, the fusion protein has a pI of about 8.4. In some embodiments, the fusion protein has a pI of about 8.5. In some embodiments, the fusion protein has a pI of about 8.8. In some embodiments, the fusion protein has a pI of about 8.9.

[0008] In some embodiments, the first peptide has the amino acid sequence X 11 LCGRELVRAQIAIC (SEQ ID NO: 505), 11 is K, Q, D, E, L, I, or Y. In some embodiments, the first peptide consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids.

[0009] In some embodiments, the first peptide has the amino acid sequence X 12 CCX 13 VGCTX 14X 15 SLAX 16 FC (SEQ ID NO: 506), 12 is K, Q, D, E, L, I, or Y, and X 13 is any amino acid except M, W, or C, and X 14 is K, Q, D, E, L, I, or Y, and X 15 is Q, D, E, L, I, Y, or R, and X 16 is R or Q. In some embodiments, the first peptide has the amino acid sequence X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO: 506), 12 is K, Q, D, E, L, I, or Y, and X 13 is H, K, Q, Y, L, N, I, S, T, or F, and X 14 is K, Q, D, E, L, I, or Y, and X 15 is Q, D, E, L, I, Y, or R, and X 16 is R or Q. In some embodiments, X13 is Q. In some embodiments, the first peptide consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0010] In some embodiments, the second peptide has the amino acid sequence X 11 LCGRELVRAQIAIC (SEQ ID NO: 505), 11 is K, Q, D, E, L, I, or Y. In some embodiments, the second peptide consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids.

[0011] In some embodiments, the second peptide has the amino acid sequence X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO: 506), 12is K, Q, D, E, L, I, or Y, and X 13 is any amino acid except M, W, or C, and X 14 is K, Q, D, E, L, I, or Y, and X 15 is Q, D, E, L, I, Y, or R, and X 16 is R or Q. In some embodiments, the second peptide has the amino acid sequence X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO: 506), 12 is K, Q, D, E, L, I, or Y, and X 13 is H, K, Q, Y, L, N, I, S, T, or F, and X 14 is K, Q, D, E, L, I, or Y, and X 15 is Q, D, E, L, I, Y, or R, and X 16 is R or Q. In some embodiments, X 13 is Q. In some embodiments, the second peptide consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0012] In some embodiments, the linker peptide comprises an amino acid sequence having 12 to 15 amino acids. In some embodiments, the linker peptide comprises the amino acid sequence ASDAAGAX8AX9AGA (SEQ ID NO: 17), where X8 is D, E, N, or Q and X9 is D, E, N, or Q, or the linker peptide comprises the amino acid sequence GGEGSGGEGX 10 GGG (SEQ ID NO: 25), 10 is E or S. In some embodiments, X8 is D, E, N, or Q and X9 is D, E, or Q, or X8 is D, E, or Q and X9 is D, E, N, or Q. In some embodiments, the linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 26, and 27.

[0013] In another aspect, the disclosure provides a fusion protein comprising, 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 having 0, 1, 2, 3, 4, or 5 amino acid modifications relative to the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid at position 4 or 25 of the first peptide is not M; and the second peptide comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications relative to the amino acid sequence of SEQ ID NO: 8; the amino acid at position 22 of the first peptide is not R, or the first peptide comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications relative to the amino acid sequence of SEQ ID NO: 8, the amino acid at position 22 of the second peptide is not R, the second peptide comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications relative to the amino acid sequence of SEQ ID NO: 1, and at least one amino acid at positions 4 or 25 of the first peptide is not M; and (b) the fusion protein has a pI of 6.0 to 8.2.

[0014] In some embodiments, the fusion protein has a pI of about 6.0 to about 9.4. In some embodiments, the fusion protein has a pI of about 6.0 to about 8.2. In some embodiments, the fusion protein has a pI that is less than about 9.0, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, or 6.1. In some embodiments, the fusion protein has a pI that is less than 9.0. In some embodiments, the fusion protein has a pI that is less than about 8.2. In some embodiments, the fusion protein has a pI of about 6.8. In some embodiments, the fusion protein has a pI of about 7.0. In some embodiments, the fusion protein has a pI of about 7.1. In some embodiments, the fusion protein has a pI of about 7.4. In some embodiments, the fusion protein has a pI of about 7.5. In some embodiments, the fusion protein has a pI of about 7.9. In some embodiments, the fusion protein has a pI of about 8.0. In some embodiments, the fusion protein has a pI of about 8.4. In some embodiments, the fusion protein has a pI of about 8.5. In some embodiments, the fusion protein has a pI of about 8.8. In some embodiments, the fusion protein has a pI of about 8.9.

[0015] In some embodiments, the linker peptide comprises an amino acid sequence having 12 to 15 amino acids. In some embodiments, the linker peptide comprises the amino acid sequence ASDAAGAX8AX9AGA (SEQ ID NO: 17), where X8 is D, E, N, or Q and X9 is D, E, N, or Q, or the linker peptide comprises the amino acid sequence GGEGSGGEGX 10 GGG (SEQ ID NO: 25), 10is E or S. In some embodiments, X8 is D, E, N, or Q and X9 is D, E, or Q, or X8 is D, E, or Q and X9 is D, E, N, or Q.

[0016] In another aspect, the disclosure provides a fusion protein comprising, from N-terminus to C-terminus, a first peptide, a linker peptide, and a second peptide, wherein the linker peptide comprises the amino acid sequence ASDAAGAX8AX9AGA (SEQ ID NO: 17), X8 is D, E, N, or Q, and X9 is D, E, N, or Q; or the linker peptide comprises the amino acid sequence GGEGSGGEGX10GGG (SEQ ID NO: 25), and X 10 is E or S.

[0017] In some embodiments, X8 is D, E, N, or Q and X9 is D, E, or Q, or X8 is D, E, or Q and X9 is D, E, N, or Q. In some embodiments, the linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 26, and 27.

[0018] In some embodiments, the first peptide comprises the amino acid sequence DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO: 7), wherein Xi is not M, H, or C, X2 is K, Q, D, E, L, I, or Y, and X3 is K or Q. In some embodiments, the first peptide comprises the amino acid sequence DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO: 7), wherein Xi is W, Y, F, L, I, V, or A, X2 is K, Q, D, E, L, I, or Y, and X3 is K or Q. In some embodiments, Xi is Y. In some embodiments, the first peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, and 6. In some embodiments, the first peptide consists of 27, 28, or 29 amino acids.

[0019] In some embodiments, the first peptide comprises the amino acid sequence QLYSALANX4CCX5VGCTX6X7SLAQFC (SEQ ID NO: 16), where X4 is K, Q, D, E, L, I, or Y, X5 is any amino acid except M, W, or C, X6 is K, Q, D, E, L, I, or Y, and X7 is Q, D, E, L, I, Y, or R. In some embodiments, the first peptide comprises the amino acid sequence QLYSALANX4CCX5VGCTX6X7SLAQFC (SEQ ID NO: 16), where X4 is K, Q, D, E, L, I, or Y, X5 is H, K, Q, Y, L, N, I, S, T, or F, X6 is K, Q, D, E, L, I, or Y, and X7 is Q, D, E, L, I, Y, or R. In some embodiments, X5 is Q. In some embodiments, the first peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, and 507. In some embodiments, the first peptide consists of 24 or 25 amino acids.

[0020] In some embodiments, the second peptide comprises the amino acid sequence DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO: 7), wherein Xi is not M, H, or C, X2 is K, Q, D, E, L, I, or Y, and X3 is K or Q. In some embodiments, the second peptide comprises the amino acid sequence DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO: 7), wherein Xi is W, Y, F, L, I, V, or A, X2 is K, Q, D, E, L, I, or Y, and X3 is K or Q. In some embodiments, Xi is Y. In some embodiments, the second peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, and 6. In some embodiments, the second peptide consists of 27, 28, or 29 amino acids.

[0021] In some embodiments, the second peptide comprises the amino acid sequence QLYSALANX4CCX5VGCTX6X7SLAQFC (SEQ ID NO: 16), where X4 is K, Q, D, E, L, I, or Y, X5 is any amino acid except M, W, or C, X6 is K, Q, D, E, L, I, or Y, and X7 is Q, D, E, L, I, Y, or R. In some embodiments, the second peptide comprises the amino acid sequence QLYSALANX4CCX5VGCTX6X7SLAQFC (SEQ ID NO: 16), where X4 is K, Q, D, E, L, I, or Y, X5 is H, K, Q, Y, L, N, I, S, T, or F, X6 is K, Q, D, E, L, I, or Y, and X7 is Q, D, E, L, I, Y, or R. In some embodiments, X5 is Q. In some embodiments, the second peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, and 507. In some embodiments, the second peptide consists of 24 or 25 amino acids.

[0022] In some embodiments, a first peptide comprises the amino acid sequence of SEQ ID NO:1 and a second peptide comprises the amino acid sequence of SEQ ID NO:8, or a first peptide comprises the amino acid sequence of SEQ ID NO:1 and a second peptide comprises the amino acid sequence of SEQ ID NO:9, or a first peptide comprises the amino acid sequence of SEQ ID NO:1 and a second peptide comprises the amino acid sequence of SEQ ID NO:10, or a first peptide comprises the amino acid sequence of SEQ ID NO:1 and a second peptide comprises the amino acid sequence of SEQ ID NO:11, or a first peptide comprises the amino acid sequence of SEQ ID NO:1 and a second peptide or the first peptide comprises the amino acid sequence of SEQ ID NO:12; or the first peptide comprises the amino acid sequence of SEQ ID NO:1 and the second peptide comprises the amino acid sequence of SEQ ID NO:13; or the first peptide comprises the amino acid sequence of SEQ ID NO:1 and the second peptide comprises the amino acid sequence of SEQ ID NO:14; or the first peptide comprises the amino acid sequence of SEQ ID NO:1 and the second peptide comprises the amino acid sequence of SEQ ID NO:15; or the first peptide comprises the amino acid sequence of SEQ ID NO:1 and the second peptide comprises the amino acid sequence of SEQ ID NO:507; or the first peptide comprises the amino acid sequence of SEQ ID NO:2 or the first peptide comprises the amino acid sequence of SEQ ID NO:2 and the second peptide comprises the amino acid sequence of SEQ ID NO:9; or the first peptide comprises the amino acid sequence of SEQ ID NO:2 and the second peptide comprises the amino acid sequence of SEQ ID NO:10; or the first peptide comprises the amino acid sequence of SEQ ID NO:2 and the second peptide comprises the amino acid sequence of SEQ ID NO:11; or the first peptide comprises the amino acid sequence of SEQ ID NO:2 and the second peptide comprises the amino acid sequence of SEQ ID NO:12; or the first peptide comprises the amino acid sequence of SEQ ID NO: or the first peptide comprises the amino acid sequence of SEQ ID NO: 2 and the second peptide comprises the amino acid sequence of SEQ ID NO: 13; or the first peptide comprises the amino acid sequence of SEQ ID NO: 2 and the second peptide comprises the amino acid sequence of SEQ ID NO: 14; or the first peptide comprises the amino acid sequence of SEQ ID NO: 2 and the second peptide comprises the amino acid sequence of SEQ ID NO: 15; or the first peptide comprises the amino acid sequence of SEQ ID NO: 2 and the second peptide comprises the amino acid sequence of SEQ ID NO: 507; or the first peptide comprises the amino acid sequence of SEQ ID NO: 3 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:3 and the second peptide comprises the amino acid sequence of SEQ ID NO:9; or the first peptide comprises the amino acid sequence of SEQ ID NO:3 and the second peptide comprises the amino acid sequence of SEQ ID NO:10; or the first peptide comprises the amino acid sequence of SEQ ID NO:3 and the second peptide comprises the amino acid sequence of SEQ ID NO:11; or the first peptide comprises the amino acid sequence of SEQ ID NO:3 and the second peptide comprises the amino acid sequence of SEQ ID NO:12; or the first peptide comprises the amino acid sequence of SEQ ID NO:3 and the second peptide comprises the amino acid sequence of SEQ ID NO:13. or a first peptide comprises the amino acid sequence of SEQ ID NO:3 and a second peptide comprises the amino acid sequence of SEQ ID NO:14; or a first peptide comprises the amino acid sequence of SEQ ID NO:3 and a second peptide comprises the amino acid sequence of SEQ ID NO:15; or a first peptide comprises the amino acid sequence of SEQ ID NO:3 and a second peptide comprises the amino acid sequence of SEQ ID NO:507; or a first peptide comprises the amino acid sequence of SEQ ID NO:4 and a second peptide comprises the amino acid sequence of SEQ ID NO:8; or a first peptide comprises the amino acid sequence of SEQ ID NO:4 and a second peptide comprises the amino acid sequence of SEQ ID NO: or the first peptide comprises the amino acid sequence of SEQ ID NO: 9; or the first peptide comprises the amino acid sequence of SEQ ID NO: 4 and the second peptide comprises the amino acid sequence of SEQ ID NO: 10; or the first peptide comprises the amino acid sequence of SEQ ID NO: 4 and the second peptide comprises the amino acid sequence of SEQ ID NO: 11; or the first peptide comprises the amino acid sequence of SEQ ID NO: 4 and the second peptide comprises the amino acid sequence of SEQ ID NO: 12; or the first peptide comprises the amino acid sequence of SEQ ID NO: 4 and the second peptide comprises the amino acid sequence of SEQ ID NO: 13; or the first peptide comprises the amino acid sequence of SEQ ID NO: 4 or the first peptide comprises the amino acid sequence of SEQ ID NO: 4 and the second peptide comprises the amino acid sequence of SEQ ID NO: 15; or the first peptide comprises the amino acid sequence of SEQ ID NO: 4 and the second peptide comprises the amino acid sequence of SEQ ID NO: 507; or 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; or 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; or the first peptidea first peptide comprising the amino acid sequence of SEQ ID NO:5 and a second peptide comprising the amino acid sequence of SEQ ID NO:10; a first peptide comprising the amino acid sequence of SEQ ID NO:5 and a second peptide comprising the amino acid sequence of SEQ ID NO:11; a first peptide comprising the amino acid sequence of SEQ ID NO:5 and a second peptide comprising the amino acid sequence of SEQ ID NO:12; a first peptide comprising the amino acid sequence of SEQ ID NO:5 and a second peptide comprising the amino acid sequence of SEQ ID NO:13; a first peptide comprising the amino acid sequence of SEQ ID NO:5 and a second peptide comprising the amino acid sequence of SEQ ID NO:14; a first peptide comprising the amino acid sequence of SEQ ID NO:5 and a second peptide comprising the amino acid sequence of SEQ ID NO:15; a first peptide comprising the amino acid sequence of SEQ ID NO:5 and a second peptide comprising the amino acid sequence of SEQ ID NO:507; a first peptide comprising the amino acid sequence of SEQ ID NO:6 and a second peptide comprising the amino acid sequence of SEQ ID NO:8; a first peptide comprising the amino acid sequence of SEQ ID NO:5 and a second peptide comprising the amino acid sequence of SEQ ID NO:5 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; or 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; or 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; or 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; or 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; or 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:14; or 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:15; or 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:507.

[0023] In some embodiments, the first peptide comprises the amino acid sequence of SEQ ID NO:8 and the second peptide comprises the amino acid sequence of SEQ ID NO:1; or the first peptide comprises the amino acid sequence of SEQ ID NO:8 and the second peptide comprises the amino acid sequence of SEQ ID NO:2; or the first peptide comprises the amino acid sequence of SEQ ID NO:8 and the second peptide comprises the amino acid sequence of SEQ ID NO:3; or the first peptide comprises the amino acid sequence of SEQ ID NO:8 and the second peptide comprises the amino acid sequence of SEQ ID NO:4; or the first peptide comprises the amino acid sequence of SEQ ID NO:8 and the second peptide , comprising the amino acid sequence of SEQ ID NO:5, or a first peptide comprising the amino acid sequence of SEQ ID NO:8 and a second peptide comprising the amino acid sequence of SEQ ID NO:6, or a first peptide comprising the amino acid sequence of SEQ ID NO:9 and a second peptide comprising the amino acid sequence of SEQ ID NO:1, or a first peptide comprising the amino acid sequence of SEQ ID NO:9 and a second peptide comprising the amino acid sequence of SEQ ID NO:2, or a first peptide comprising the amino acid sequence of SEQ ID NO:9 and a second peptide comprising the amino acid sequence of SEQ ID NO:3, or a first peptide comprising the amino acid sequence of SEQ ID NO:9 and a second peptide comprising the amino acid sequence of SEQ ID NO: or the first peptide comprises the amino acid sequence of SEQ ID NO: 4; or the first peptide comprises the amino acid sequence of SEQ ID NO: 9 and the second peptide comprises the amino acid sequence of SEQ ID NO: 5; or the first peptide comprises the amino acid sequence of SEQ ID NO: 9 and the second peptide comprises the amino acid sequence of SEQ ID NO: 6; or the first peptide comprises the amino acid sequence of SEQ ID NO: 10 and the second peptide comprises the amino acid sequence of SEQ ID NO: 1; or the first peptide comprises the amino acid sequence of SEQ ID NO: 10 and the second peptide comprises the amino acid sequence of SEQ ID NO: 2; or the first peptide comprises the amino acid sequence of SEQ ID NO: 10. or the first peptide comprises the amino acid sequence of SEQ ID NO: 10 and the second peptide comprises the amino acid sequence of SEQ ID NO: 4; or the first peptide comprises the amino acid sequence of SEQ ID NO: 10 and the second peptide comprises the amino acid sequence of SEQ ID NO: 5; or the first peptide comprises the amino acid sequence of SEQ ID NO: 10 and the second peptide comprises the amino acid sequence of SEQ ID NO: 6; or the first peptide comprises the amino acid sequence of SEQ ID NO: 11 and the second peptide comprises the amino acid sequence of SEQ ID NO: 1; or the first peptideThe first peptide comprises the amino acid sequence of SEQ ID NO:11 and the second peptide comprises the amino acid sequence of SEQ ID NO:2; the first peptide comprises the amino acid sequence of SEQ ID NO:11 and the second peptide comprises the amino acid sequence of SEQ ID NO:3; the first peptide comprises the amino acid sequence of SEQ ID NO:11 and the second peptide comprises the amino acid sequence of SEQ ID NO:4; the first peptide comprises the amino acid sequence of SEQ ID NO:11 and the second peptide comprises the amino acid sequence of SEQ ID NO:5; or the first peptide comprises the amino acid sequence of SEQ ID NO:11 and the second peptide comprises the amino acid sequence of SEQ ID NO:6. or a first peptide comprises the amino acid sequence of SEQ ID NO:12 and a second peptide comprises the amino acid sequence of SEQ ID NO:1; or a first peptide comprises the amino acid sequence of SEQ ID NO:12 and a second peptide comprises the amino acid sequence of SEQ ID NO:2; or a first peptide comprises the amino acid sequence of SEQ ID NO:12 and a second peptide comprises the amino acid sequence of SEQ ID NO:3; or a first peptide comprises the amino acid sequence of SEQ ID NO:12 and a second peptide comprises the amino acid sequence of SEQ ID NO:4; or a first peptide comprises the amino acid sequence of SEQ ID NO:12 and a second peptide The first peptide comprises the amino acid sequence of SEQ ID NO:5, or the first peptide comprises the amino acid sequence of SEQ ID NO:12 and the second peptide comprises the amino acid sequence of SEQ ID NO:6, or the first peptide comprises the amino acid sequence of SEQ ID NO:13 and the second peptide comprises the amino acid sequence of SEQ ID NO:1, or the first peptide comprises the amino acid sequence of SEQ ID NO:13 and the second peptide comprises the amino acid sequence of SEQ ID NO:2, or the first peptide comprises the amino acid sequence of SEQ ID NO:13 and the second peptide comprises the amino acid sequence of SEQ ID NO:3, or the first peptide comprises the amino acid sequence of SEQ ID NO:13. and the second peptide comprises the amino acid sequence of SEQ ID NO:4; or the first peptide comprises the amino acid sequence of SEQ ID NO:13 and the second peptide comprises the amino acid sequence of SEQ ID NO:5; or the first peptide comprises the amino acid sequence of SEQ ID NO:13 and the second peptide comprises the amino acid sequence of SEQ ID NO:6; or the first peptide comprises the amino acid sequence of SEQ ID NO:14 and the second peptide comprises the amino acid sequence of SEQ ID NO:1; or the first peptide comprises the amino acid sequence of SEQ ID NO:14 and the second peptide comprises the amino acid sequence of SEQ ID NO:2; or the first peptideor a first peptide comprises the amino acid sequence of SEQ ID NO: 14 and a second peptide comprises the amino acid sequence of SEQ ID NO: 3; or a first peptide comprises the amino acid sequence of SEQ ID NO: 14 and a second peptide comprises the amino acid sequence of SEQ ID NO: 4; or a first peptide comprises the amino acid sequence of SEQ ID NO: 14 and a second peptide comprises the amino acid sequence of SEQ ID NO: 5; or a first peptide comprises the amino acid sequence of SEQ ID NO: 14 and a second peptide comprises the amino acid sequence of SEQ ID NO: 6; or a first peptide comprises the amino acid sequence of SEQ ID NO: 15 and a second peptide comprises the amino acid sequence of SEQ ID NO: 1; or a first peptide comprises the amino acid sequence of SEQ ID NO: 15 and a second peptide comprises the amino acid sequence of SEQ ID NO: 2; or a first peptide comprises the amino acid sequence of SEQ ID NO: 15 and a second peptide comprises the amino acid sequence of SEQ ID NO: 3; or a first peptide comprises the amino acid sequence of SEQ ID NO: 15 and a second peptide comprises the amino acid sequence of SEQ ID NO: 4; or a first peptide , wherein the first peptide comprises the amino acid sequence of SEQ ID NO: 15 and the second peptide comprises the amino acid sequence of SEQ ID NO: 5, or wherein the first peptide comprises the amino acid sequence of SEQ ID NO: 15 and the second peptide comprises the amino acid sequence of SEQ ID NO: 6, or wherein the first peptide comprises the amino acid sequence of SEQ ID NO: 507 and the second peptide comprises the amino acid sequence of SEQ ID NO: 1, or wherein the first peptide comprises the amino acid sequence of SEQ ID NO: 507 and the second peptide comprises the amino acid sequence of SEQ ID NO: 2, or wherein the first peptide comprises the amino acid sequence of SEQ ID NO: 3, or wherein the first peptide comprises the amino acid sequence of SEQ ID NO: 507 and the second peptide comprises the amino acid sequence of SEQ ID NO: 4, or 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: 5, or wherein the first peptide comprises the amino acid sequence of SEQ ID NO: 507 and the second peptide comprises the amino acid sequence of SEQ ID NO: 6.

[0024] In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-75 and 508-515.

[0025] In some embodiments, the fusion protein further comprises an IgG Fc. In some embodiments, the IgG Fc comprises the amino acid alanine at each of EU positions 234 and 235. In some embodiments, the IgG Fc comprises the amino acid alanine at EU position 329. In some embodiments, the IgG Fc comprises the amino acid alanine at each of EU positions 234, 235, and 329. In some embodiments, the IgG Fc comprises the amino acids alanine, alanine, alanine, leucine, and serine at EU positions 234, 235, 329, 428, and 434, respectively. In some embodiments, the IgG Fc comprises the amino acids lysine, phenylalanine, and tyrosine at EU positions 433, 434, and 436, respectively. In some embodiments, the IgG Fc comprises the amino acids tyrosine, threonine, and glutamate at EU positions 252, 254, and 256, respectively. In some embodiments, the IgG Fc comprises the amino acids leucine and serine at EU positions 428 and 434, respectively.

[0026] In some embodiments, the IgG Fc comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of human IgG1 Fc. In some embodiments, the IgG Fc comprises the amino acid sequence of human IgG1 Fc.

[0027] In some embodiments, 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: 76 to 83. In some embodiments, the IgG Fc comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 76 to 83.

[0028] In some embodiments, the IgG Fc is linked to the N-terminus of the first peptide. In some embodiments, the IgG Fc is linked to the C-terminus of the second peptide.

[0029] In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 84-138 and 516-523. In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 139-193, 524-531, and 549.

[0030] In another aspect, the present disclosure provides a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 8-15, 18-24, 26-75, 84-193, 507-531, and 549-558.

[0031] In another aspect, the disclosure provides a polynucleotide comprising a nucleotide sequence encoding any one of the fusion proteins described herein or any one of the polypeptides described herein.

[0032] In some embodiments, the polynucleotide is a DNA molecule. In some embodiments, the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 194-248, 410-464, and 532-547.

[0033] In some embodiments, the polynucleotide is an RNA molecule.

[0034] In another aspect, the disclosure provides an expression vector comprising any one of the polynucleotides described herein.

[0035] In some embodiments, the expression vector is a plasmid. In some embodiments, the expression vector is a viral vector.

[0036] In another aspect, the disclosure provides a host cell comprising any one of the polynucleotides described herein or any one of the expression vectors described herein.

[0037] In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is an E. coli cell or a Bacillus cell. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell, and a mammalian cell. In some embodiments, the mammalian cell is selected from the group consisting of a CHO cell, a HeLa cell, and a 293 cell.

[0038] In another aspect, the disclosure provides a population of cells comprising two or more of any of the host cells described herein.

[0039] In another aspect, the disclosure provides a method of producing any one of the fusion proteins described herein or any one of the polypeptides described herein, comprising culturing any one of the host cells described herein under conditions such that the fusion protein is produced.

[0040] In another aspect, the present disclosure provides a pharmaceutical composition comprising an effective amount of any one of the fusion proteins described herein, any one of the polypeptides described herein, any one of the polynucleotides described herein, or any one of the expression vectors described herein.

[0041] In some embodiments, the fusion protein has a circulating half-life of at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, or at least 23 days. In some embodiments, the fusion protein, when administered (e.g., to a human), has a circulating half-life of at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, or at least 23 days. In some embodiments, the fusion protein, when administered (e.g., to a human), has a bioavailability of at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70%. In some embodiments, administration of the pharmaceutical composition is via intravenous or subcutaneous administration.

[0042] In another aspect, the present disclosure provides a method of enhancing relaxin-2-related activity in a primary cell, the method comprising contacting the primary cell with any one of the fusion proteins described herein, thereby enhancing relaxin-2-related activity in the cell.

[0043] In some embodiments, the fusion protein activates the relaxin-2 receptor (RXFP1) on the cell surface.

[0044] In some embodiments, the methods increase cAMP levels, induce vasodilation, induce expression of angiogenic factors, induce expression of MMPs, and induce collagen degradation in primary cells.

[0045] In some embodiments, the primary cells are selected from the group consisting of endothelial cells, vascular smooth muscle cells, other vascular cells, cardiomyocytes, other cardiac cells, and fibroblasts.

[0046] In some embodiments, the primary cells are located within a subject. In some embodiments, the subject has a relaxin-2-related disorder. In some embodiments, the relaxin-2-related disorder is selected from the group consisting of renal disease, fibrotic disease, and cardiovascular disease. In some embodiments, the disorder is selected from the group consisting of pulmonary hypertension, pulmonary arterial hypertension (PAH), pulmonary hypertension due to left heart disease (PH-LHD), combined pre- and post-capillary pulmonary hypertension (CpcPH), isolated post-capillary pulmonary hypertension (IpcPH), heart failure, heart failure with preserved ejection fraction (HFpEF), heart failure with intermediate ejection fraction (HFmrEF), heart failure with reduced ejection fraction (HFrEF), valvular heart disease, joint disease, shoulder periarthritis (also known as adhesive capsulitis), renal disease, chronic kidney disease, and hypertensive kidney disease.

[0047] In some embodiments, the disorder is combined pre- and post-capillary pulmonary hypertension (CpcPH) with heart failure with preserved ejection fraction (HFpEF). In some embodiments, the disorder is isolated post-capillary pulmonary hypertension (IpcPH) with heart failure with preserved ejection fraction (HFpEF). In some embodiments, the disorder is combined pre- and post-capillary pulmonary hypertension (CpcPH) with heart failure with intermediate ejection fraction (HFmrEF). In some embodiments, the disorder is isolated post-capillary pulmonary hypertension (IpcPH) with heart failure with intermediate ejection fraction (HFmrEF).

[0048] In another aspect, the disclosure provides a method of treating a relaxin-related disorder in a subject in need thereof, comprising administering to the subject an effective amount of any one of the fusion proteins described herein, any one of the polynucleotides described herein, any one of the expression vectors described herein, or any one of the pharmaceutical compositions described herein, thereby treating the relaxin-related disorder.

[0049] In some embodiments, the relaxin-2-related disorder is selected from the group consisting of renal disease, fibrotic disease, and cardiovascular disease. In some embodiments, the disorder is selected from the group consisting of pulmonary hypertension, pulmonary arterial hypertension (PAH), pulmonary hypertension due to left heart disease (PH-LHD), combined pre- and post-capillary pulmonary hypertension (CpcPH), isolated post-capillary pulmonary hypertension (IpcPH), heart failure, heart failure with preserved ejection fraction (HFpEF), heart failure with moderate ejection fraction (HFmrEF), heart failure with reduced ejection fraction (HFrEF), renal disease, chronic kidney disease, and hypertensive kidney disease. In some embodiments, the method reduces arterial pressure, increases renal artery blood flow, increases cardiac diastolic filling, resolves established fibrosis, and / or inhibits the development of new fibrosis in a subject.

[0050] In some embodiments, the method increases renal plasma flow in a subject. In some embodiments, the increase in renal plasma flow in the subject is sustained 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 1 month after a single administration of the fusion protein. In some embodiments, the increase in renal plasma flow in the subject is maintained by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 1 month after a single administration of the fusion protein.

[0051] In some embodiments, the disorder is combined pre- and post-capillary pulmonary hypertension (CpcPH) with heart failure with preserved ejection fraction (HFpEF). In some embodiments, the disorder is isolated post-capillary pulmonary hypertension (IpcPH) with heart failure with preserved ejection fraction (HFpEF). In some embodiments, the disorder is combined pre- and post-capillary pulmonary hypertension (CpcPH) with heart failure with intermediate ejection fraction (HFmrEF). In some embodiments, the disorder is isolated post-capillary pulmonary hypertension (IpcPH) with heart failure with intermediate ejection fraction (HFmrEF).

[0052] In some embodiments, the subject is administered the fusion protein by intravenous administration. In some embodiments, the subject is administered about 0.1 mg / kg to about 20 mg / kg of the fusion protein. In some embodiments, the subject is administered about 0.3 mg / kg of the fusion protein. In some embodiments, the subject is administered about 1 mg / kg of the fusion protein. In some embodiments, the subject is administered about 3 mg / kg of the fusion protein. In some embodiments, the subject is administered about 10 mg / kg of the fusion protein.

[0053] In some embodiments, the subject is administered the fusion protein by intravenous infusion. In some embodiments, the subject is administered the fusion protein by intravenous infusion over 30 minutes. In some embodiments, the subject is administered the fusion protein by intravenous infusion over 60 minutes. In some embodiments, the subject is administered the fusion protein by intravenous infusion over 30-60 minutes.

[0054] In some embodiments, the subject is administered the fusion protein by subcutaneous administration. In some embodiments, the subject is administered about 100 mg to about 1500 mg of the fusion protein. In some embodiments, the subject is administered about 150 mg of the fusion protein. In some embodiments, the subject is administered at least 150 mg of the fusion protein. In some embodiments, the subject is administered about 300 mg of the fusion protein. In some embodiments, the subject is administered about 600 mg of the fusion protein.

[0055] In some embodiments, the subject is administered the fusion protein once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once a month. [Brief explanation of the drawings]

[0056] [Figure 1A]Graph showing cAMP responses induced by SEQ ID NO: 87 and wild-type (WT) human relaxin-2 in HEK293 cells transiently expressing human RXFP1. [Figure 1B] Graph showing cAMP responses induced by SEQ ID NO: 87 and wild-type (WT) human relaxin-2 in HEK293 cells transiently expressing rat RXFP1. [Figure 1C] Graph showing cAMP responses induced by SEQ ID NO: 87 and wild-type (WT) human relaxin-2 in HEK293 cells transiently expressing monkey RXFP1. [Figure 2A] A graph showing pharmacokinetic (PK) values ​​obtained by measuring the concentrations of various relaxin-2 fusion protein analogs in rat serum over time (using human Fc levels as a proxy) following intravenous (IV) injection of 5 mg / kg of each protein analog. [Figure 2B] A graph showing pharmacokinetic (PK) values ​​obtained by measuring the concentrations of various relaxin-2 fusion protein analogs in rat serum over time (using human Fc levels as a proxy) following intravenous (IV) injection of 5 mg / kg of each protein analog. [Figure 2C] A graph showing pharmacokinetic (PK) values ​​obtained by measuring the concentrations of various relaxin-2 fusion protein analogs in rat serum over time (using human Fc levels as a proxy) following intravenous (IV) injection of 5 mg / kg of each protein analog. [Figure 3] Graph showing the change in renal artery blood flow (RABF) over time compared to baseline in rats administered various relaxin-2 fusion protein analogs, as indicated. [Figure 4A] Graph showing change in RABF over time in response to dose of SEQ ID NO: 87 or SEQ ID NO: 497, as indicated. [Figure 4B]Graph showing change in serum levels of fusion protein (using human Fc levels as a proxy) over time in response to doses of SEQ ID NO:87 or SEQ ID NO:497, as indicated. [Figure 4C] 1 is a graph showing serum PK as a function of increase in RABF (baseline subtracted). [Figure 5A] Graph showing that low doses of SEQ ID NO: 87 increase and maintain RABF in treated rats significantly more than SEQ ID NO: 497. Graph showing the increase in RABF over time in rats treated with SEQ ID NO: 87 or SEQ ID NO: 497. [Figure 5B] Graph showing that low doses of SEQ ID NO: 87 increase and maintain RABF in treated rats significantly more than SEQ ID NO: 497. Shows that rats treated with SEQ ID NO: 87 show a significant increase in RABF by area under the curve analysis compared to SEQ ID NO: 497. [Figure 6A] Graph showing the effect of SEQ ID NO: 87 on right ventricular systolic pressure (RVSP) after 3 weeks of intravenous treatment at 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCT) with B cell depletion using anti-CD20 antibody (without or +CD20). Sildenafil was used as a positive control in non-B cell depleted animals. [Figure 6B] Graph showing the effect of SEQ ID NO: 87 on right ventricular systolic pressure (RVSP) after 3 weeks of intravenous treatment at 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCT) without B cell depletion using anti-CD20 antibody (without or +CD20). Sildenafil was used as a positive control in non-B cell depleted animals. [Figure 7A] Graph showing the effect of SEQ ID NO: 87 on mean pulmonary artery pressure (mPAP) after 3 weeks of intravenous treatment at 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCT) with B cell depletion using anti-CD20 antibody (without or +CD20). Sildenafil was used as a positive control in non-B cell depleted animals. [Figure 7B]Graph showing the effect of SEQ ID NO: 87 on mean pulmonary artery pressure (mPAP) after 3 weeks of intravenous treatment at 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCT) without B cell depletion using anti-CD20 antibody (without or +CD20). Sildenafil was used as a positive control in non-B cell depleted animals. [Figure 8A] Graph showing the effect of SEQ ID NO: 87 on the Fulton index after 3 weeks of intravenous treatment at 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCT) with B cell depletion using anti-CD20 antibody (without or +CD20). Sildenafil was used as a positive control in non-B cell depleted animals. [Figure 8B] Graph showing the effect of SEQ ID NO: 87 on the Fulton index after 3 weeks of intravenous treatment at 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCT) without B cell depletion using anti-CD20 antibody (without or +CD20). Sildenafil was used as a positive control in non-B cell depleted animals. [Figure 9A] Graph showing the effect of SEQ ID NO: 87 on serum NT-pro-BNP levels after 3 weeks of intravenous treatment at 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCT) with B cell depletion using anti-CD20 antibody (without or +CD20). Sildenafil was used as a positive control in non-B cell depleted animals. [Figure 9B] Graph showing the effect of SEQ ID NO: 87 on serum NT-pro-BNP levels after 3 weeks of intravenous treatment at 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCT) without B cell depletion using anti-CD20 antibody (without or +CD20). Sildenafil was used as a positive control in non-B cell depleted animals. [Figure 10A]Graph showing the results of histopathological analysis of the effect of SEQ ID NO: 87 on pulmonary inflammation after 3 weeks of intravenous treatment with 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats with B cell depletion (MCT) using anti-CD20 antibody (+CD20). *: p<0.05, **: p<0.01, ****p<0.0001 using non-parametric one-way ANOVA with post-hoc Dunn's multiple comparison test. [Figure 10B] Graph showing the results of histopathological analysis of the effect of SEQ ID NO: 87 on pulmonary artery muscularization after 3 weeks of intravenous treatment with 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCT) with B cell depletion using anti-CD20 antibody (+CD20). *: p<0.05, **: p<0.01, ****p<0.0001 using non-parametric one-way ANOVA with post-hoc Dunn's multiple comparison test. [Figure 11] Graph showing the effect of SEQ ID NO: 87 on mortality after 3 weeks of intravenous treatment at 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCT) with or without B cell depletion using anti-CD20 antibody (without or +CD20). Sildenafil was used as a positive control in non-B cell depleted animals. [Figure 12] Graph showing the effect of SEQ ID NO: 496 and SEQ ID NO: 313 on collagen deposition in the renal parenchyma in a mouse unilateral ureteral obstruction (UUO) model, according to an embodiment of the present disclosure. Mice underwent UUO surgery and were treated with vehicle (PBS, n=10), 20 mg / kg SEQ ID NO: 496 (n=10), 10 mg / kg SEQ ID NO: 313 (n=10), or 20 mg / kg SEQ ID NO: 313 (n=10). Also shown are control mice that underwent sham surgery and were treated with vehicle (PBS, n=5). After treatment, the obstructed kidneys were harvested and fixed for histology. Collagen was detected via immunolabeling. Shown is quantification of collagen levels as a percentage of total immunolabeled area. *: p<0.05, ****: p<0.0001. [Figure 13]Graph showing the effect of SEQ ID NO: 87 on collagen deposition in the kidney cortex in a mouse UUO model, according to an embodiment of the present disclosure. Mice underwent UUO surgery and were treated with vehicle (PBS, n=8) or 10 mg / kg of SEQ ID NO: 87 (n=8). Also shown are control mice that underwent sham surgery and were treated with vehicle (PBS, n=8). After treatment, the obstructed kidneys were harvested and fixed for histology. Collagen was detected via immunolabeling. Shown is quantification of collagen levels as a percentage of total immunolabeled area. ****: p<0.0001, *: p=0.02. [Figure 14] Graph showing the effect of SEQ ID NO: 87 on TNFα levels in the kidney cortex in a mouse UUO model, according to an embodiment of the present disclosure. Mice were treated as described in FIG. 13, and TNFα levels in protein lysates were quantified via electrochemiluminescence assay. ****: p<0.0001, ***: p<0.001. [Figure 15] Graph showing the effect of SEQ ID NO: 87 on isoproterenol-induced cardiac hypertrophy according to an embodiment of the present disclosure. Mice were treated with vehicle (n=10), isoproterenol (n=10), or isoproterenol and SEQ ID NO: 87 (n=6). After treatment, body weight and heart rate were measured for each mouse. Heart weight normalized by body weight (HW / BW) for each group is shown. ****: p<0.0001. [Figure 16] Graph showing the effect of SEQ ID NO: 87 on isoproterenol-induced fibrosis, according to an embodiment of the present disclosure. Mice were treated as described in Figure 15, and collagen content was quantified using a hydroxyproline assay. ****: p<0.0001, ***: p<0.001. [Figure 17A] 1 shows PK data for a healthy human patient administered a single 0.3 mg / kg IV dose of SEQ ID NO: 87. Shown are the concentrations of SEQ ID NO: 87 over time in the dosed patient (solid line) and the predicted PK profile of SEQ ID NO: 87 using non-human primate modeling (dashed line). [Figure 17B]Figure 1 shows PD data for healthy human patients administered a single 0.3 mg / kg IV dose of SEQ ID NO: 87. Figure 2 shows the change in renal plasma flow relative to baseline on days 2, 8, and 17 in healthy patients dosed with SEQ ID NO: 87 or placebo (PBO). [Figure 18] Graph showing PK data for healthy human patients administered a single 150 mg SC dose of SEQ ID NO:87, showing the concentration of SEQ ID NO:87 over time in dosed patients. DETAILED DESCRIPTION OF THE INVENTION

[0057] The therapeutic potential of relaxin-2 was highlighted in the RELAX-AHF trial (see, e.g., Teerlink et al., (2013) Lancet 381(9860):29-39). However, the therapeutic protein used, human relaxin-2 (serelaxin), was not modified in any way to extend its half-life in vivo, and the protein had to be administered by continuous IV infusion over 48 hours. Half-life-extended forms of relaxin-2 have been generated via fusion of the peptide hormone to human IgG1 Fc or albumin-binding nanobodies, but such fusion proteins have shown very rapid clearance from plasma. The present disclosure is based, in part, on the discovery by the inventors that reducing the positive charge and heparin binding of relaxin-2 results in significantly improved pharmacokinetic and pharmacodynamic profiles.

[0058] The present disclosure provides a fusion protein comprising a human relaxin-2B chain, or a derivative thereof, and a human relaxin-2A chain, or a derivative thereof, linked by a peptide linker, wherein the fusion protein has a high in vivo circulatory half-life when administered to a mammal. In some embodiments, the in vivo circulatory half-life of the fusion protein provided herein exceeds 2 hours. In some embodiments, the fusion protein provided herein has a low pI. In some embodiments, the pI of the fusion protein provided herein is less than 8.5. In some embodiments, the low pI of the fusion protein provided herein is caused by acidic amino acid residues present in the peptide linker. In some embodiments, the peptide linker of the fusion protein comprises two or more acidic amino acids. In some embodiments, the peptide linker is 10 to 15 amino acids in total length.

[0059] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory and are not intended to limit any claimed subject matter. In this application, the use of the singular includes the plural unless expressly stated otherwise. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless expressly stated otherwise. Furthermore, the use of the term "including," as well as other forms of "include," "includes," and "included," are not limiting. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0060] The term "polynucleotide," as used herein, refers to a polymer of DNA or RNA. Polynucleotide sequences may be single- or double-stranded and may contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, instead of the phosphodiester linkages found between nucleotides in unmodified polynucleotide sequences. Polynucleotide sequences include, but are not limited to, all polynucleotide sequences obtained by any means available in the art, including, but not limited to, recombinant means, such as cloning polynucleotide sequences from recombinant libraries or cellular genomes, using conventional cloning techniques and the polymerase chain reaction, and synthetic means.

[0061] The terms "amino acid sequence" and "polypeptide" are used interchangeably herein and refer to a polymer of amino acids joined by one or more peptide bonds. As used herein, "amino acid sequence" refers to information describing the relative order and identities of the amino acid residues that make up a polypeptide.

[0062] As used herein, with respect to an amino acid sequence, the term "0, 1, 2, 3, 4, or 5 amino acid modifications" refers to an amino acid sequence that contains up to 5 amino acid substitutions, alterations, inversions, additions, or deletions compared to a reference amino acid sequence.

[0063] The determination of "percent identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin S & Altschul SF, (1990) PNAS 87:2264-2268, modified as in Karlin S & Altschul SF, (1993) PNAS 90:5873-5877, each of which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215:403, which is incorporated herein by reference in its entirety. To obtain nucleotide sequences homologous to the nucleic acid molecules described herein, BLAST nucleotide searches can be performed using the NBLAST nucleotide program parameters set, for example, to score=100 and word length=12. To obtain amino acid sequences homologous to the protein molecules described herein, BLAST protein searches can be performed with the XBLAST program parameters set, for example, to score 50 and word length = 3. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nucleic Acids Res 25:3389-3402, the entire contents of which are incorporated herein by reference. Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules. When utilizing Id.BLAST, Gapped BLAST, and PSI BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov).Another specific, non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0064] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0065] As used herein, the term "linked to" refers to a covalent or non-covalent bond between two molecules or moieties. Those skilled in the art will understand that when a first molecule or moiety is linked to a second molecule or moiety, the linkage need not be direct, but instead may be through an intervening molecule or moiety.

[0066] As used herein, the terms "human relaxin-2 B chain" or "relaxin B chain" or "relaxin B" or "relB" refer to a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 249, or a derivative thereof. In some embodiments, a derivative of the relaxin B chain comprises the amino acid sequence of SEQ ID NO: 154 with 1, 2, 3, 4, or 5 amino acid changes.

[0067] As used herein, the terms "human relaxin-2A chain" or "relaxin A chain" or "relaxin A" or "relA" refer to a peptide comprising or consisting of the amino acid sequence set forth in QLYSALANKCCHVGCTKRSLARFC (SEQ ID NO: 257), or a derivative thereof. In some embodiments, a derivative of the relaxin A chain comprises the amino acid sequence of SEQ ID NO: 155 with 1, 2, 3, 4, or 5 amino acid changes.

[0068] As used herein, the term "linker peptide" refers to a peptide that connects the relaxin A chain and the relaxin B chain in the fusion proteins described herein.

[0069] As used herein, the term "acidic amino acid" refers to an amino acid having a carboxylic acid in its side chain. In some embodiments, the acidic amino acid is aspartate, glutamate, 2-aminoadipic acid, 2-aminobutyric acid, or 2-aminopimelic acid. In some embodiments, the acidic amino acid includes aspartate and glutamate.

[0070] As used herein, the term "non-acidic amino acid" refers to an amino acid that is not an acidic amino acid. In some embodiments, non-acidic amino acids include glycine, proline, and serine. In some embodiments, non-specific amino acids also include arginine, histidine, lysine, threonine, asparagine, glutamine, cysteine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0071] As used herein, the term "IgG Fc" refers to the crystallizable (Fc) region of an immunoglobulin G (IgG) fragment. In some embodiments, the IgG Fc is a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the IgG Fc is an IgG1 Fc region.

[0072] As used herein, the term "EU numbering system" refers to the EU numbering rules for antibody constant regions as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

[0073] As used herein, the terms "relaxin-2 receptor," "human relaxin-2 receptor," "human relaxin receptor 1," "RXFP1," or "LGR7" refer to the native receptor for relaxin-2 in humans. In some embodiments, RXFP1 comprises the amino acid sequence set forth in the NCBI reference sequences: NP_067647.2, NP_001240656.1, NP_001240657.1, NP_001240658.1, NP_001240659.1, NP_001240661.1, NP_001240662.1, or NP_001350705.1, which are incorporated by reference in their entireties.

[0074] As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or prophylactic measures as described herein. In some embodiments, the method of "treatment" employs administering a fusion protein to a subject having or previously diagnosed with a disease or disorder to prevent, cure, delay, reduce the severity of, or ameliorate a disease or disorder, or one or more symptoms of a recurrent disease or disorder, or to extend the subject's survival beyond that expected in the absence of such treatment.

[0075] As used herein, in the context of administering therapy, the term "effective amount" refers to the amount of therapy that achieves the desired prophylactic or therapeutic effect.

[0076] As used herein, the term "subject" includes any human or non-human animal. In one embodiment, the subject is a human or non-human mammal. In one embodiment, the subject is a human.

[0077] As used herein, the term "pI" refers to the isoelectric point, i.e., the pH of a solution at which the next charge on the fusion protein is zero. In some embodiments, the pI is a calculated pI or a theoretical pI. In some embodiments, the pI is experimentally measured by instrumentation.

[0078] fusion proteins The present disclosure provides a fusion protein comprising a human relaxin-2B chain, or a derivative thereof, and a human relaxin-2A chain, or a derivative thereof, linked by a peptide linker, wherein the fusion protein has a long in vivo circulatory half-life when administered to a mammal. In some embodiments, the fusion protein comprises, from N- to C-terminus, a human relaxin-2B chain, or a derivative thereof, a peptide linker, and a human relaxin-2A chain, or a derivative thereof. In some embodiments, the fusion protein comprises, from N- to C-terminus, a human relaxin-2A chain, or a derivative thereof, a peptide linker, and a human relaxin-2B chain, or a derivative thereof. In some embodiments, the fusion protein further comprises an IgG Fc. The IgG Fc is linked to the N- or C-terminus of the human relaxin B chain linker protein-human relaxin A chain fusion protein or the human relaxin A chain linker protein-human relaxin B chain fusion protein. In some embodiments, the fusion protein forms a homodimer through interactions between the IgG Fc portions. In some embodiments, the IgG Fc is replaced with PEG.

[0079] Human relaxin-2B chain derivative The present disclosure provides human relaxin-2 B chain derivatives, wherein the derivatives have 1, 2, 3, 4, or 5 amino acid changes compared to the amino acid sequence of SEQ ID NO: 249. In some embodiments, the amino acid corresponding to position 13 of SEQ ID NO: 249 must be arginine. In some embodiments, the amino acid corresponding to position 17 of SEQ ID NO: 249 must be arginine. In some embodiments, the amino acid corresponding to position 20 of SEQ ID NO: 249 must be isoleucine. In some embodiments, the amino acid corresponding to position 13 of SEQ ID NO: 249 must be arginine, the amino acid corresponding to position 17 of SEQ ID NO: 249 must be arginine, and the amino acid corresponding to position 20 of SEQ ID NO: 249 must be isoleucine.

[0080] In some embodiments, the human relaxin-2B chain derivative has the following formula: DSWX 19 EEVIKLCGRELVRAQIAICGX 20 ST (SEQ ID NO: 250), 19 and X 20 is absent or any amino acid. In some embodiments, X 19 is methionine (M), glutamine (Q), glutamic acid (E), asparagine (N), aspartic acid (D), serine (S), or threonine (T). 19 is methionine (M), lysine (K), or glutamine (Q). 20 is methionine (M), lysine (K), glutamine (Q), or asparagine (N). 20 is methionine (M) or lysine (K). In some embodiments, X 20 is lysine (K). In some embodiments, X 19 is methionine (M), lysine (K), or glutamine (Q), and X 20 is methionine (M) or lysine (K).

[0081] The present disclosure provides human relaxin-2 B chain derivatives, wherein the derivatives comprise an amino acid sequence having zero, one, two, three, four, or five amino acid modifications relative to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at position 4 is not a methionine (M) or wherein the amino acid at position 25 is not a methionine (M). In some embodiments, the derivatives comprise an amino acid sequence having zero, one, two, three, four, or five amino acid modifications relative to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at position 4 is not a methionine (M) and wherein the amino acid at position 25 is not a methionine (M). In some embodiments, the derivatives comprise an amino acid sequence having zero, one, two, three, four, or five amino acid modifications relative to the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid at positions 4 or 25 of the first peptide is not a methionine (M).

[0082] In some embodiments, the human relaxin-2 B chain derivative comprises or consists of the following formula: DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO: 7), wherein X1 is tryptophan (W), tyrosine (Y), phenylalanine (F), leucine (L), isoleucine (I), valine (V), or alanine (A), X2 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y), and X3 is lysine (K) or glutamine (Q). In some embodiments, the human relaxin-2 B chain derivative comprises or consists of the following formula: DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO: 7), wherein X1 is tryptophan (W), tyrosine (Y), phenylalanine (F), leucine (L), isoleucine (I), valine (V), or alanine (A); X2 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and X3 is methionine (M), lysine (K), glutamine (Q), or asparagine (N). In some embodiments, the human relaxin-2 B chain derivative comprises or consists of the following formula: DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO: 7), wherein X1 is any amino acid except methionine (M), histidine (H), and cysteine ​​(C), X2 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y), and X3 is lysine (K) or glutamine (Q). In some embodiments, the human relaxin-2 B chain derivative comprises or consists of the following formula: DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO: 7), wherein X1 is any amino acid except methionine (M), histidine (H), and cysteine ​​(C), X2 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y), and X3 is methionine (M), lysine (K), glutamine (Q), or asparagine (N).

[0083] In some embodiments, a human relaxin-2B chain derivative comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications relative to the amino acid sequence of SEQ ID NO: 502. In some embodiments, a human relaxin-2B chain derivative has the following formula: X 11 LCGRELVRAQIAIC (SEQ ID NO: 505), 11 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y).

[0084] In some embodiments, the human relaxin-2 B chain derivative used in the fusion proteins described herein does not comprise the amino acid sequence of SEQ ID NOs: 251-254 set forth below: DSWKEEVIKLCGRELVRAQIAICGKSTAS (SEQ ID NO: 251), DSWKEEVIKLCGRELVRAQIAICGKSTWS (SEQ ID NO: 252), DSWMEEVIKLCGRELVRAQIAICGKSTAS (SEQ ID NO: 253), and DSWMEEVIKLCGRELVRAQIAICGKSTWS (SEQ ID NO: 254).

[0085] In some embodiments, the human relaxin-2B chain derivative is 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids in length. In some embodiments, the human relaxin-2B chain derivative is 25, 26, 27, 28, or 29 amino acids in length. In some embodiments, the human relaxin-2B chain derivative is 27 amino acids in length. In some embodiments, the human relaxin-2B chain derivative is 15-29 amino acids in length. In some embodiments, the human relaxin-2B chain derivative is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids in length.

[0086] In some embodiments, the human relaxin-2 B chain derivative comprises or consists of the amino acid sequence shown in Table 1 below. [Table 1]

[0087] In some embodiments, the human relaxin-2B chain derivative comprises or consists of SEQ ID NO: 1. In some embodiments, the human relaxin-2B chain derivative comprises or consists of SEQ ID NO: 2. In some embodiments, the human relaxin-2B chain derivative comprises or consists of SEQ ID NO: 3. In some embodiments, the human relaxin-2B chain derivative comprises or consists of SEQ ID NO: 4. In some embodiments, the human relaxin-2B chain derivative comprises or consists of SEQ ID NO: 5. In some embodiments, the human relaxin-2B chain derivative comprises or consists of SEQ ID NO: 6. In some embodiments, the human relaxin-2B chain derivative comprises or consists of SEQ ID NO: 249. In some embodiments, the human relaxin-2B chain derivative comprises or consists of SEQ ID NO: 255. In some embodiments, the human relaxin-2B chain derivative comprises or consists of SEQ ID NO: 256. In some embodiments, the human relaxin-2B chain derivative comprises or consists of SEQ ID NO: 502.

[0088] In some embodiments, a human relaxin-2 B chain derivative comprises or consists of SEQ ID NO: 1, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y). In some embodiments, a human relaxin-2 B chain derivative comprises or consists of SEQ ID NO: 2, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y). In some embodiments, a human relaxin-2 B chain derivative comprises or consists of SEQ ID NO: 3, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y). In some embodiments, the human relaxin-2 B chain derivative comprises or consists of SEQ ID NO:4, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y). In some embodiments, the human relaxin-2 B chain derivative comprises or consists of SEQ ID NO:5, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y). In some embodiments, the human relaxin-2 B chain derivative comprises or consists of SEQ ID NO:6, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y). In some embodiments, the human relaxin-2 B chain derivative comprises or consists of SEQ ID NO: 249, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y). In some embodiments, the human relaxin-2 B chain derivative comprises or consists of SEQ ID NO: 255, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y).In some embodiments, the human relaxin-2 B chain derivative comprises or consists of SEQ ID NO: 256, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y). In some embodiments, the human relaxin-2 B chain derivative comprises or consists of SEQ ID NO: 502, wherein the amino acid at position 1 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y).

[0089] In some embodiments, human relaxin-2B chain derivatives further comprise two residues at the C-terminus. For example, SEQ ID NOs: 1-7, 250, 255, and 256 may further comprise two residues at the C-terminus, e.g., tryptophan (W) and serine (S) at the C-terminus of SEQ ID NO: 249. In some embodiments, human relaxin-2B chain derivatives further comprise an X amino acid and serine (S) at the C-terminus, where X amino acid can be any amino acid except cysteine ​​(C). Thus, in some embodiments, the C-terminus of human relaxin-2B chain derivatives is XS, where X is any amino acid except cysteine ​​(C).

[0090] In some embodiments, the human relaxin-2B chain derivative further comprises one or more substitutions that improve the stability of the relaxin-2B chain derivative, e.g., the stability of the relaxin-2B chain derivative after light or heat exposure, as measured by methods known in the art, e.g., SEC to assess aggregate formation and CE-SDS to assess purity. In some embodiments, the amino acid in the human relaxin-2B chain derivative corresponding to amino acid position 3 of SEQ ID NO:3 is tyrosine (Y).

[0091] Human relaxin-2A chain derivative The present disclosure provides human relaxin-2A chain derivatives, which have 1, 2, 3, 4, or 5 amino acid changes when compared to the amino acid sequence of SEQ ID NO: 257. In some embodiments, the amino acid corresponding to position 3 of SEQ ID NO: 257 must be a tyrosine. In some embodiments, the amino acid corresponding to position 23 of SEQ ID NO: 257 must be a phenylalanine. In some embodiments, the amino acid corresponding to position 3 of SEQ ID NO: 257 must be a tyrosine and the amino acid corresponding to position 23 of SEQ ID NO: 257 must be a phenylalanine.

[0092] In some embodiments, the human relaxin-2A chain derivative has the following formula: X 21 QX 22 YSALANKCCHVGCTKRSLAX 23 FC (SEQ ID NO: 258), 21 , X 22 , and X 23 is absent or any amino acid. In some embodiments, X 21 is arginine (R), lysine (K), glutamine (Q), asparagine (N), histidine (H), serine (S), threonine (T), proline (P), glycine (G), or absent. 21 is arginine (R), glycine (G), or absent. 21 is arginine (R) or absent. In some embodiments, X 22 is leucine (L), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), serine (S), or threonine (T). 22 is leucine (L) or aspartic acid (D). 23 is arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D), asparagine (N), serine (S), or threonine (T). 23is arginine (R), glutamine (Q), or glutamic acid (E). 21 is arginine (R) or absent, and X 22 is leucine (L) or aspartic acid (D), and X 23 is arginine (R), glutamine (Q), or glutamic acid (E).

[0093] The present disclosure provides a human relaxin-2A chain derivative, wherein the derivative comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications relative to the amino acid sequence of SEQ ID NO: 8, and wherein the amino acid at position 22 of the second peptide is not arginine (R).

[0094] In some embodiments, the human relaxin-2 A chain derivative comprises or consists of the following formula: QLYSALANX4CCX5VGCTX6X7SLAQFC (SEQ ID NO: 16), wherein X4 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and X5 is histidine (H), lysine (K), glutamine (Q), tyrosine (Y), leucine (L). , asparagine (N), isoleucine (I), serine (S), threonine (T), or phenylalanine (F); X6 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and X7 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2A chain derivative comprises or consists of the following formula: QLYSALANX4CCX5VGCTX6X7SLAQFC (SEQ ID NO: 16), wherein X4 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X5 is any amino acid except methionine (M), tryptophan (W), and cysteine ​​(C); X6 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and X7 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R).

[0095] In some embodiments, a human relaxin-2A chain derivative comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications relative to the amino acid sequence of SEQ ID NO: 503 or 504. In some embodiments, a human relaxin-2A chain derivative has the following formula: X 12 CCX 13 VGCTX 14 X 15 SLAX 16FC (SEQ ID NO: 506), 12 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X is 13 is histidine (H), lysine (K), glutamine (Q), tyrosine (Y), leucine (L), asparagine (N), isoleucine (I), serine (S), threonine (T), or phenylalanine (F); X 14 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X is 15 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R); X 16 is arginine (R), or glutamine (Q). In some embodiments, the human relaxin-2 A chain derivative has the following formula: X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO: 506), 12 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X is 13 is histidine (H), lysine (K), glutamine (Q), tyrosine (Y), leucine (L), asparagine (N), isoleucine (I), serine (S), threonine (T), or phenylalanine (F); X 14 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X is 15 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R); X 16is arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D), asparagine (N), serine (S), or threonine (T). In some embodiments, the human relaxin-2 A chain derivative has the following formula: X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO: 506), 12 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X is 13 is any amino acid except methionine (M), tryptophan (W), and cysteine ​​(C), and X 14 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X is 15 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R); X 16 is arginine (R) or glutamine (Q). In some embodiments, the human relaxin-2 A chain derivative has the following formula: X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO: 506), 12 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X is 13 is any amino acid except methionine (M), tryptophan (W), and cysteine ​​(C), and X 14 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X is 15is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R); X 16 is arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D), asparagine (N), serine (S), or threonine (T).

[0096] In some embodiments, the human relaxin-2A chain derivative is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. In some embodiments, the human relaxin-2A chain derivative is 22, 23, 24, 25, or 26 amino acids in length. In some embodiments, the human relaxin-2A chain derivative is 24 amino acids in length. In some embodiments, the human relaxin-2A chain derivative is 25 amino acids in length. In some embodiments, the human relaxin-2A chain derivative is 16-25 amino acids in length. In some embodiments, the human relaxin-2A chain derivative is 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0097] In some embodiments, the human relaxin-2A chain derivative comprises or consists of the amino acid sequence shown in Table 2 below. [Table 2]

[0098] In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 8. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 9. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 10. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 11. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 12. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 13. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 14. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 15. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 257. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 259. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 260. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 261. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 503. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 504. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 507. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 550. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 551. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 552. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 553. In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO:554.In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 555. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 556. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 557. In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 558.

[0099] In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 8, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 17 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 18 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 9, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 17 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 18 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 10, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 17 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 18 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R).In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 11, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 17 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 18 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 12, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 17 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 18 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 13, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 17 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 18 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R).In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 14, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 17 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 18 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2A chain derivative comprises or consists of SEQ ID NO: 15, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 17 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 18 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO: 257, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 17 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 18 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R).In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO: 259, wherein the amino acid at position 10 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 18 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 19 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO: 260, wherein the amino acid at position 10 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 18 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 19 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO: 261, wherein the amino acid at position 10 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 18 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 19 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R).In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO: 503, wherein the amino acid at position 1 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 10 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO: 504, wherein the amino acid at position 1 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 10 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO: 507, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 17 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 18 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R).In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO: 550, wherein the amino acid at position 9 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 17 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 18 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO: 550, wherein the amino acid at position 10 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 18 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 19 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO: 551, wherein the amino acid at position 10 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y), the amino acid at position 18 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y), and the amino acid at position 19 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). The laxin-2 A chain derivative comprises or consists of SEQ ID NO: 553, wherein the amino acid at position 10 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 18 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 19 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO: 554, wherein the amino acid at position 10 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 18 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 19 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO: 555, wherein the amino acid at position 10 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 18 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 19 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R).In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO: 556, wherein the amino acid at position 10 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 18 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 19 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO: 557, wherein the amino acid at position 10 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 18 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 19 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R). In some embodiments, the human relaxin-2 A chain derivative comprises or consists of SEQ ID NO: 558, wherein the amino acid at position 10 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 18 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 19 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R).

[0100] In some embodiments, the human relaxin-2A chain derivative further comprises one or more substitutions that improve the stability of the relaxin-2A chain derivative, e.g., the stability of the relaxin-2A chain derivative after light or heat exposure, as measured by methods known in the art, e.g., SEC to assess aggregate formation and CE-SDS to assess purity. In some embodiments, the amino acid in the human relaxin-2A chain derivative corresponding to amino acid position 12 of SEQ ID NO: 12 is glutamine (Q).

[0101] Linker peptide The present disclosure provides linker peptides, wherein the peptide has at least two acidic amino acids. In some embodiments, the acidic amino acid is glutamate. In some embodiments, the acidic amino acid is aspartate. In some embodiments, the acidic amino acid is a non-standard amino acid. In some embodiments, the acidic amino acid is 2-aminoadipic acid, 2-aminobutyric acid, or 2-aminopimelic acid. In some embodiments, the linker peptide has 2, 3, 4, 5, 6, 7, 8, 9, or 10 acidic amino acids.

[0102] In some embodiments, the linker peptide is 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In some embodiments, the linker peptide is 12, 13, 14, or 15 amino acids in length. In some embodiments, the linker peptide has 2, 3, 4, or 5 acidic amino acids. In some embodiments, the linker peptide is 12, 13, 14, or 15 amino acids in length and has 2, 3, 4, or 5 acidic amino acids. In some embodiments, the remaining amino acids are non-acidic amino acids. In some embodiments, a non-acidic amino acid can be any standard amino acid that is not aspartate or glutamate. In some embodiments, a non-acidic amino acid can be any amino acid that does not have a carboxylic acid in its side chain. In some embodiments, a non-acidic amino acid is glycine, proline, serine, arginine, histidine, lysine, threonine, asparagine, glutamine, cysteine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In some embodiments, the non-acidic amino acid is glycine, proline, or cysteine, hi some embodiments, the non-acidic amino acid is glycine.

[0103] In some embodiments, the linker peptide comprises acidic amino acids, and all of the acidic amino acids are the same amino acid. In some embodiments, the acidic amino acids in the linker peptide are both / all glutamate. In some embodiments, the acidic amino acids in the linker peptide are both / all aspartate. In some embodiments, the linker peptide comprises amino acids that are a mixture of acidic amino acids. In some embodiments, the linker peptide comprises both glutamate and aspartate as acidic amino acids.

[0104] In some embodiments, the linker peptide is X 17 X 17 X 17 X 18 X 17 X 17 X 17 X18 X 17 X 17 X 17 X 18 X 17 , X 17 X 17 X 17 X 18 X 17 X 17 X 17 X 18 X 17 X 17 X 17 X 18 X 17 X 17 X 17 , X 17 X 17 X 18 X 17 X 17 X 17 X 18 X 18 X 17 X 17 X 17 X 18 X 17 X 17 , X 17 X 17 X 17 X 18 X 18 X 17 X 17 X 17 X 18 X 18 X 17 X 17 X 17 , and X 17 X 17 X 18 X 17 X 18 X 17 X 17 X 18 X 17 X 18 X 17 X 17 X 17 and comprising an amino acid sequence selected from the group consisting of: X 17 is a non-acidic amino acid, and X 18 is an acidic amino acid.

[0105] In some embodiments, the linker peptide comprises non-acidic amino acids, and all of the non-acidic amino acids are the same amino acid. In some embodiments, all of the non-acidic amino acids in the linker peptide are glycine. In some embodiments, the linker peptide comprises amino acids that are a mixture of non-acidic amino acids. In some embodiments, the linker peptide comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different types of non-acidic amino acids.

[0106] In some embodiments, the linker peptide comprises the amino acid sequence ASDAAGAX8AX9AGA (SEQ ID NO: 17), wherein X8 is aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q), and X9 is aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q). In some embodiments, X8 is aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q), and X9 is aspartic acid (D), glutamic acid (E), or glutamine (Q). In some embodiments, X8 is aspartic acid (D), glutamic acid (E), or glutamine (Q), and X9 is aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q).

[0107] In some embodiments, the linker peptide is GGEGSGGEGX 10 GGG (SEQ ID NO: 25), 10 is glutamic acid (E) or serine (S).

[0108] In some embodiments, the linker peptide comprises or consists of an amino acid sequence shown in Table 3 below. [Table 3]

[0109] In some embodiments, the linker peptide comprises 2, 3, 4, or 5 repeats of SEQ ID NO: 267, 268, 269, 270, or 271. For example, 3 repeats of SEQ ID NO: 267 are the amino acid sequence GGGEGGGEGGGE (SEQ ID NO: 277).

[0110] In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 18. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 19. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 20. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 21. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 22. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 23. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 24. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 26. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 27. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 267. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 268. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 269. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 270. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 271. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 272. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 273. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 274. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 275. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 276. In some embodiments, the linker peptide comprises or consists of SEQ ID NO: 277.

[0111] Relaxin / linker peptide combinations for fusion proteins In some embodiments, the fusion protein comprises an N-terminal or first peptide, a linker peptide, and a C-terminal or second peptide. In some embodiments, the N-terminal peptide comprises the human relaxin-2A chain or a derivative thereof (RelA), and the C-terminal peptide comprises the human relaxin-2B chain or a derivative thereof (RelB). In some embodiments, the N-terminal peptide comprises the human relaxin-2B chain or a derivative thereof, and the C-terminal peptide comprises the human relaxin-2A chain or a derivative thereof. Any combination of any of the embodiments of the human relaxin-2A chain or a derivative thereof and the human relaxin-2A chain or a derivative thereof linked by any of the linker peptides disclosed herein can be used to construct the fusion protein embodiments described herein. In some embodiments, at least one of the N-terminal peptide and the C-terminal peptide is a derivative of the human relaxin-2A chain or the human relaxin-2B chain. In some embodiments, the N-terminal peptide comprises a human relaxin-2A chain derivative, and the C-terminal peptide comprises a human relaxin-2B chain derivative. In some embodiments, the N-terminal peptide comprises a human relaxin-2B chain derivative and the C-terminal peptide comprises a human relaxin-2A chain derivative.

[0112] In some embodiments, the human relaxin-2B chain derivative consists of 15 amino acids, and the human relaxin-2A chain derivative consists of 16-25 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 16 amino acids, and the human relaxin-2A chain derivative consists of 16-25 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 17 amino acids, and the human relaxin-2A chain derivative consists of 16-25 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 18 amino acids, and the human relaxin-2A chain derivative consists of 16-25 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 19 amino acids, and the human relaxin-2A chain derivative consists of 16-25 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 20 amino acids, and the human relaxin-2A chain derivative consists of 16-25 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 21 amino acids, and the human relaxin-2A chain derivative consists of 16-25 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 22 amino acids, and the human relaxin-2A chain derivative consists of 16-25 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 23 amino acids, and the human relaxin-2A chain derivative consists of 16-25 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 24 amino acids, and the human relaxin-2A chain derivative consists of 16-25 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 25 amino acids, and the human relaxin-2A chain derivative consists of 16-25 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 26 amino acids, and the human relaxin-2A chain derivative consists of 16-25 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 27 amino acids, and the human relaxin-2A chain derivative consists of 16-25 amino acids.In some embodiments, the human relaxin-2B chain derivative consists of 28 amino acids and the human relaxin-2A chain derivative consists of 16 to 25 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 29 amino acids and the human relaxin-2A chain derivative consists of 16 to 25 amino acids.

[0113] In some embodiments, the human relaxin-2B chain derivative consists of 15 to 29 amino acids, and the human relaxin-2A chain derivative consists of 16 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 15 to 29 amino acids, and the human relaxin-2A chain derivative consists of 17 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 15 to 29 amino acids, and the human relaxin-2A chain derivative consists of 18 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 15 to 29 amino acids, and the human relaxin-2A chain derivative consists of 19 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 15 to 29 amino acids, and the human relaxin-2A chain derivative consists of 20 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 15 to 29 amino acids, and the human relaxin-2A chain derivative consists of 21 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 15 to 29 amino acids, and the human relaxin-2A chain derivative consists of 22 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 15 to 29 amino acids, and the human relaxin-2A chain derivative consists of 23 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 15 to 29 amino acids, and the human relaxin-2A chain derivative consists of 24 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 15 to 29 amino acids, and the human relaxin-2A chain derivative consists of 25 amino acids.

[0114] Specific embodiments of the fusion proteins provided in this disclosure are shown in Table 4 below. Table 4 TIFF2026501550000005.tif254170TIFF2026501550000006.tif250170TIFF2026501550000007.tif249170TIFF2026501550000008.tif251170TIFF2026501550000009.tif191170TIFF2026501550000010.tif254170TIFF2026501550000011.tif251170TIFF2026501550000012.tif250170TIFF2026501550000013.tif250170TIFF2026501550000014.tif250170TIFF2026501550000015.tif186170TIFF2026501550000016.tif254170TIFF2026501550000017.tif251170TIFF2026501550000018.tif250170TIFF2026501550000019.tif251170TIFF2026501550000020.tif250170TIFF2026501550000021.tif186170TIFF2026501550000022.tif254170TIFF2026501550000023.tif251170TIFF2026501550000024.tif250170TIFF2026501550000025.tif250170TIFF2026501550000026.tif250170TIFF2026501550000027.tif187170TIFF2026501550000028.tif253170TIFF2026501550000029.tif250170TIFF2026501550000030.tif250170TIFF2026501550000031.tif250170TIFF2026501550000032.tif251170TIFF2026501550000033.tif187170TIFF2026501550000034.tif254170TIFF2026501550000035.tif249170TIFF2026501550000036.tif249170TIFF2026501550000037.tif250170TIFF2026501550000038.tif249170TIFF2026501550000039.tif186170TIFF2026501550000040.tif254170TIFF2026501550000041.tif24 9170TIFF2026501550000042.tif250170TIFF2026501550000043.tif250170TIFF2026501550000044.tif250170TIFF2026501550000045.tif187170.

[0115] In some embodiments, there are additional amino acids between the N-terminal peptide and the linker peptide. In some embodiments, there are additional amino acids between the C-terminal peptide and the linker peptide. In some embodiments, there are no additional amino acids between the N-terminal peptide and the linker peptide. In some embodiments, there are no additional amino acids between the C-terminal peptide and the linker peptide.

[0116] In some embodiments, the portion of the fusion protein comprising the N-terminal peptide, the linker peptide, and the C-terminal peptide comprises or consists of the amino acid sequence shown in Table 5 below. [Table 5] TIFF2026501550000047.tif254170TIFF2026501550000048.tif254170TIFF2026501550000049.tif101170

[0117] IgG Fc In some embodiments, the fusion proteins provided herein further comprise an IgG Fc (or Fc region). As used herein, the term "IgG Fc" or "Fc region" refers to the portion of an immunoglobulin formed by the Fc domains of the two heavy chains of the immunoglobulin. The Fc region can be a wild-type Fc region (native Fc region) or a variant Fc region. Native Fc regions are homodimers. In some embodiments, the fusion proteins provided herein form dimers (e.g., homodimers) through interactions between the Fc regions. In some embodiments, the two fusion proteins are linked into a dimer (e.g., homodimer) through two hinge region interchain disulfide bonds between the Fc regions of each fusion protein (e.g., at the N-terminus). In some embodiments, the Fc region comprises one intrachain disulfide bond in the CH2 domain and one intrachain disulfide bond in the CH3 domain.

[0118] The Fc region of the fusion proteins provided herein can be derived from any native immunoglobulin. In some embodiments, the Fc region is formed from an IgA, IgD, IgE, or IgG heavy chain constant region. In some embodiments, the Fc region is formed from an IgG heavy chain constant region. In some embodiments, the IgG heavy chain is an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the Fc region is formed from an IgG1 heavy chain constant region. In some embodiments, the IgG1 heavy chain constant region comprises the G1m1(a), G1m2(x), G1m3(f), or G1m17(z) allotype. See, e.g., Jefferis and Lefranc (2009) mAbs 1(4):332-338 and de Taeye et al. (2020) Front Immunol. 11:740, which are incorporated herein by reference in their entireties. The IgG Fc can be linked to the N-terminus of the N-terminal peptide or the C-terminus of the C-terminal peptide. The IgG Fc can be linked directly to the N-terminal peptide or the C-terminal peptide, or they can be linked to the N-terminal peptide or the C-terminal peptide via an IgG Fc linker. In some embodiments, the IgG Fc linker comprises or consists of a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the IgG Fc linker comprises or consists of 1, 2, 3, 4, or 5 amino acids. In some embodiments, the IgG Fc linker comprises or consists of 3 or 4 amino acids. In some embodiments, the IgG Fc linker comprises or consists of the amino acid sequence of GGS. In some embodiments, the IgG Fc linker comprises or consists of the amino acid sequence of EGGS (SEQ ID NO: 299).

[0119] In some embodiments, the IgG Fc comprises a C-terminal lysine (K). It is known in the art that the C-terminal lysine (K) in many monoclonal antibodies is flexible and is often cleaved during expression and purification without any known impairment to activity. In some embodiments, the C-terminal lysine (K) is replaced with a C-terminal glutamic acid (E). Thus, in some embodiments, the IgG Fc comprises a C-terminal glutamic acid (E).

[0120] In some embodiments, the IgG Fc comprises the amino acid sequence of one of SEQ ID NOs: 76-83 together with GGS as an IgG Fc linker at the C-terminus of the IgG Fc. In some embodiments, the IgG Fc comprises the amino acid sequence of one of SEQ ID NOs: 76-83 together with SEQ ID NO: 299 as an IgG Fc linker at the C-terminus of the IgG Fc.

[0121] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced in the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1)) and / or hinge region of an antibody described herein, numbered according to the EU numbering system, to alter one or more functional properties of the antibody, e.g., serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0122] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), e.g., as described in U.S. Patent No. 5,677,425, which is incorporated herein by reference in its entirety. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, for example, to facilitate assembly of the light and heavy chains or to alter (e.g., increase or decrease) the stability of the antibody.

[0123] In a specific embodiment, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into an IgG constant domain, or an FcRn-binding fragment thereof (preferably, an Fc or hinge-Fc domain fragment) to alter (e.g., decrease or increase) the half-life of the antibody in vivo. For examples of mutations that alter (e.g., decrease or increase) the half-life of an antibody in vivo, see, e.g., WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, all of which are incorporated by reference in their entirety. In certain embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into an IgG constant domain, or an FcRn-binding fragment thereof (preferably, an Fc or hinge-Fc domain fragment), to decrease the half-life of the antibody in vivo. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into an IgG constant domain, or an FcRn-binding fragment thereof (preferably, an Fc or hinge-Fc domain fragment), to increase the half-life of the antibody in vivo. In specific embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1), numbered according to the EU numbering system. In a specific embodiment, the IgG1 constant region of the antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU numbering system. See U.S. Patent No. 7,658,921, which is incorporated herein by reference in its entirety.Mutant IgGs of this type, termed "YTE variants," have been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24, which is incorporated herein by reference in its entirety). In certain embodiments, the antibody comprises an IgG constant domain comprising one, two, three, or more amino acid substitutions at amino acid residues 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU numbering system.

[0124] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1)) and / or hinge region of an antibody described herein, numbered according to the EU numbering system, to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activating Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that decrease or increase the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or a fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptors of antibodies that can be made to alter the affinity of the antibody for the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, all of which are incorporated herein by reference in their entireties.

[0125] In certain embodiments, the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to FcγRIIB with a higher affinity than the wild-type heavy chain constant region binds to FcγRIIB. In certain embodiments, the variant heavy chain constant region is a variant human heavy chain constant region, e.g., a variant human IgG1, variant human IgG2, or variant human IgG4 heavy chain constant region. In certain embodiments, the variant human IgG heavy chain constant region comprises one or more of the following amino acid mutations according to the EU numbering system: G236D, P238D, S239D, S267E, L328F, and L328E. In certain embodiments, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of S267E and L328F, P238D and L328E, P238D, and one or more substitutions selected from the group consisting of E233D, G237D, H268D, P271G, and A330R, P238D, E233D, G237D, H268D, P271G, and A330R, G236D and S267E, S239D and S267E, V262E, S267E, and L328F, and V264E, S267E, and L328F, numbered according to the EU numbering system. In certain embodiments, FcγRIIB is expressed on cells selected from the group consisting of macrophages, monocytes, B cells, dendritic cells, endothelial cells, and activated T cells.

[0126] In further embodiments, one, two, or more amino acid substitutions are introduced into the IgG constant domain Fc region to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332, and 396, numbered according to the EU numbering system, can be replaced with a different amino acid residue such that the antibody has altered affinity for an effector ligand, while retaining the antigen-binding ability of the parent antibody. The affinity-altered effector ligand can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, each of which is incorporated herein by reference in its entirety. In certain embodiments, deletion or inactivation of the constant region domain (by point mutation or other means) may reduce Fc receptor binding of circulating antibodies, thereby increasing tumor localization. See, e.g., U.S. Patent Nos. 5,585,097 and 8,591,886, each of which is incorporated by reference in its entirety, for a description of mutations that delete or inactivate constant domains, thereby increasing tumor localization. In certain embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region, which may reduce Fc receptor binding (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604, which is incorporated by reference in its entirety).In various embodiments, one or more of the following mutations in the constant region of the antibodies described herein, numbered according to the EU numbering system, can be made: N297A substitution, N297Q substitution, L234A substitution, L234F substitution, L235A substitution, L235F substitution, L235V substitution, L237A substitution, S239D substitution, E233P substitution, L234V substitution, L235A substitution, C236 deletion, P238A substitution, S239D substitution, F243L substitution, D265A substitution, S267E substitution, L328F substitution, R292P substitution, Y300L substitution, A327Q substitution, P329A substitution (PA), A332L substitution, I332E substitution, or P396L substitution.

[0127] In certain embodiments, a mutation selected from the group consisting of D265A, P329A, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein. In certain embodiments, a mutation selected from the group consisting of L235A, L237A, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein. In certain embodiments, a mutation selected from the group consisting of S267E, L328F, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein. In certain embodiments, a mutation selected from the group consisting of S239D, I332E, optionally A330L, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein. In certain embodiments, a mutation selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein. In certain embodiments, a mutation selected from the group consisting of S267E, L328F, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein.

[0128] In specific embodiments, the antibodies described herein comprise an IgG1 constant domain with an N297Q or N297A amino acid substitution, numbered according to the EU numbering system. In one embodiment, the antibodies described herein comprise an IgG1 constant domain with a mutation selected from the group consisting of D265A, P329A, and combinations thereof, numbered according to the EU numbering system. In another embodiment, the antibodies described herein comprise an IgG1 constant domain with a mutation selected from the group consisting of L234A, L235A (LALA), and combinations thereof, numbered according to the EU numbering system. In another embodiment, the antibodies described herein comprise an IgG1 constant domain with a mutation selected from the group consisting of L234F, L235F, N297A, and combinations thereof, numbered according to the EU numbering system. In certain embodiments, the amino acid residues in the constant region of the antibodies described herein at positions corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain, numbered according to the EU numbering system, are not L, L, and D, respectively. This approach is described in detail in WO 14 / 108483, which is incorporated herein by reference in its entirety. In certain embodiments, the amino acids corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, or A, A, and A, respectively, numbered according to the EU numbering system.

[0129] In certain embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 in the constant region of an antibody described herein, numbered according to the EU numbering system, can be replaced with a different amino acid residue such that the antibody has altered C1q binding and / or reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 (Idusogie et al.), which is incorporated herein by reference in its entirety. In certain embodiments, one or more amino acid residues within amino acid positions 231-238 in the N-terminal region of the CH2 domain of an antibody described herein, numbered according to the EU numbering system, are altered to thereby alter the antibody's ability to fix complement. This approach is further described in WO 94 / 29351, which is incorporated herein by reference in its entirety. In certain embodiments, the Fc region of an antibody described herein may comprise one or more of the following amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 352, 354, 355, 356, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 372, 376, 37 , 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439, which are numbered according to the EU numbering system. This approach is further described in WO 00 / 42072, which is incorporated by reference in its entirety.

[0130] In some embodiments, the IgG Fc is an IgG1 Fc, or a derivative thereof. In some embodiments, the IgG Fc or IgG1 Fc comprises an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, or 99% identical to the amino acid sequence of an IgG1 Fc. In some embodiments, the IgG Fc or IgG1 Fc comprises an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequence provided in Table 6 below. [Table 6] TIFF2026501550000051.tif88170

[0131] In some embodiments, any IgG Fc, or derivative thereof, with or without an IgG Fc linker, may be linked to the N- or C-terminus of any of the embodiments set forth in Tables 4 or 5 above. In some embodiments, human IgG1 Fc, or derivative thereof, with or without an IgG Fc linker, may be linked to the N- or C-terminus of any of the embodiments set forth in Tables 4 or 5 above. In some embodiments, the amino acid sequence of human IgG1 Fc comprises or consists of the amino acid sequence of SEQ ID NO: 76 or 80. In some embodiments, derivatives where the human IgG1 Fc comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 76 or 80.

[0132] In some embodiments, the human IgG1 Fc comprising the LALA mutation, or a derivative thereof, may be linked to the N- or C-terminus of any of the embodiments described above in Tables 4 or 5, with or without an IgG Fc linker. In some embodiments, the amino acid sequence of the human IgG1 Fc comprising the LALA mutation comprises or consists of the amino acid sequence of SEQ ID NO: 77 or 81. In some embodiments, the derivative of the human IgG1 Fc comprising the LALA mutation comprises an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 77 or 81.

[0133] In some embodiments, the human IgG1 Fc comprising the LALA PA mutation, or a derivative thereof, may be linked to the N-terminus or C-terminus of any of the embodiments described above in Tables 4 or 5, with or without an IgG Fc linker. In some embodiments, the amino acid sequence of the human IgG1 Fc comprising the LALA PA mutation comprises or consists of the amino acid sequence of SEQ ID NO: 78 or 82. In some embodiments, a derivative is where the human IgG1 Fc comprising the LALA PA mutation comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 78 or 82.

[0134] In some embodiments, the human IgG1 Fc comprising the LALA PA LS mutation, or a derivative thereof, may be linked to the N-terminus or C-terminus of any of the embodiments described above in Tables 4 or 5, with or without an IgG Fc linker. In some embodiments, the amino acid sequence of the human IgG1 Fc comprising the LALA PA LS mutation comprises or consists of the amino acid sequence of SEQ ID NO: 79 or 83. In some embodiments, a derivative is where the human IgG1 Fc comprising the LALA PA LS mutation comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 79 or 83.

[0135] In some embodiments, the fusion protein comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical to an amino acid sequence set forth in Table 7. In some embodiments, the fusion protein comprises or consists of an amino acid sequence set forth in Table 7. [Table 7] TIFF2026501550000053.tif223170TIFF2026501550000054.tif222149TIFF20265015500000 55.tif221149TIFF2026501550000056.tif223148TIFF2026501550000057.tif223150TIFF202 6501550000058.tif223148TIFF2026501550000059.tif223147TIFF2026501550000060.tif22 2149TIFF2026501550000061.tif225170TIFF2026501550000062.tif224170TIFF20265015500 00063.tif223170TIFF2026501550000064.tif225170TIFF2026501550000065.tif223170TIF F2026501550000066.tif222170TIFF2026501550000067.tif225170TIFF2026501550000068.t if225170TIFF2026501550000069.tif222170TIFF2026501550000070.tif223170TIFF2026501 550000071.tif224170TIFF2026501550000072.tif222148TIFF2026501550000073.tif100152

[0136] In some embodiments, the IgG Fc comprises a mouse IgG kappa signal sequence comprising the amino acid sequence of METDTLLLWVLLLWVPGSTG (SEQ ID NO: 329). In some embodiments, the IgG Fc comprises a mouse IgG heavy chain signal sequence. In some embodiments, the IgG Fc comprises a signal sequence comprising the amino acid sequence of MGWSCIILFLVATATGVHS (SEQ ID NO: 548). In some embodiments, a different signal sequence is used. In some embodiments, no signal sequence is present on the fusion protein produced.

[0137] In some embodiments, the fusion protein comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical to an amino acid sequence set forth in Table 8. In some embodiments, the fusion protein comprises or consists of an amino acid sequence set forth in Table 8. [Table 8] TIFF2026501550000075.tif222149TIFF2026501550000076.tif222148TIFF20265015500 00077.tif222148TIFF2026501550000078.tif221148TIFF2026501550000079.tif222148 TIFF2026501550000080.tif221147TIFF2026501550000081.tif222148TIFF20265015500 00082.tif222148TIFF2026501550000083.tif222149TIFF2026501550000084.tif221149 TIFF2026501550000085.tif223150TIFF2026501550000086.tif223149TIFF20265015500 00087.tif223149TIFF2026501550000088.tif222147TIFF2026501550000089.tif222149 TIFF2026501550000090.tif221148TIFF2026501550000091.tif221147TIFF20265015500 00092.tif221147TIFF2026501550000093.tif222148TIFF2026501550000094.tif181149

[0138] Other half-life extending moieties As used herein, the term "half-life extending moiety" includes non-proteinaceous half-life extending moieties, such as PEG or HES, and proteinaceous half-life extending moieties, such as an Fc domain. In some embodiments, a non-proteinaceous half-life extending moiety is linked to a fusion protein described herein. In some embodiments, a non-proteinaceous half-life extending moiety is linked to a fusion protein in place of the IgG Fc. In some embodiments, a non-proteinaceous half-life extending moiety is linked to a fusion protein in addition to the IgG Fc.

[0139] Examples of suitable polymer molecules that function as non-proteinaceous half-life extending moieties include polymer molecules selected from the group consisting of polyalkylene glycols (PAGs), such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEGs, hydroxyalkyl starches (HASs), e.g., hydroxyethyl starch (HES), polysialic acid (PSA), polyvinyl alcohol (PVA), polycarboxylates, poly(vinylpyrrolidone), polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, dextrans, including carboxymethyl-dextran, or any other biopolymer suitable for reducing immunogenicity and / or increasing functional in vivo and / or serum half-life. Another example of a polymer molecule is human albumin or another abundant plasma protein. In general, polyalkylene glycol-derived polymers are biocompatible, non-toxic, non-antigenic, non-immunogenic, have various water-soluble properties, and are readily excreted from the body.

[0140] PEG has the advantage of having only a few crosslinkable reactive groups compared to polysaccharides such as dextran. In particular, monofunctional PEG, such as methoxypolyethylene glycol (mPEG), is of interest because its coupling chemistry is relatively simple (only one reactive group is available for conjugation with the linking group on the polypeptide). Therefore, as the risk of crosslinking is eliminated, the resulting conjugated fusion proteins described herein are more homogeneous, and the reaction between the polymer molecule and the variant polypeptide is easier to control.

[0141] To effect covalent attachment of a polymer molecule(s) to the fusion proteins described herein, the hydroxyl end groups of the polymer molecules must be provided in activated form, i.e., with a reactive functional group (examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl propionate (SPA), succinimidyl butyrate (SBA), succinimidyl carboxymethylate (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS), aldehyde, nitrophenyl carbonate (NPC), and tresylate (TRES)). Suitable activated polymer molecules are commercially available, for example, from Shearwater Polymers, Inc., Huntsville, Ala., USA, or PolyMASC Pharmaceuticals plc, UK.

[0142] Alternatively, the polymer molecules can be activated by conventional methods known in the art, such as those disclosed in WO 90 / 13540. Specific examples of activated linear or branched polymer molecules for use herein are described in the Shearwater Polymers, Inc. 1997 and 2000 Catalogs (Functionalized Biocompatible Polymers for Research and Pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference). Specific examples of activated PEG polymers include the following linear PEGs: NHS-PEG (e.g., SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM-PEG), and branched PEGs such as NOR-PEG, BTC-PEG, EPOXPEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and PEG2-NHS, as well as those disclosed in U.S. Pat. Nos. 5,932,462 and 5,643,575, both of which are incorporated herein by reference. Additionally, the following publications disclose useful polymer molecules and / or PEGylation chemistries:U.S. Patent No. 5,824,778, U.S. Patent No. 5,476,653, WO97 / 32607, EP229,108, EP402,378, U.S. Patent No. 4,902,502, U.S. Patent No. 5,281,698, U.S. Patent No. 5,122,614, U.S. Patent No. 5,219,564, WO92 / 16555, WO9 4 / 04193, WO94 / 14758, WO94 / 17039, WO94 / 18247, WO94 / 28024, WO95 / 00162, WO95 / 119 24, WO95 / 13090, WO95 / 33490, WO96 / 00080, WO97 / 18832, WO98 / 41562, WO98 / 48837, WO 99 / 32134, WO99 / 32139, WO99 / 32140, WO96 / 40791, WO98 / 32466, WO95 / 06058, EP439508, WO97 / 03106, WO96 / 21469, WO95 / 13312, EP921131, U.S. Patent No. 5,736,625, WO98 / 05363, EP 809996, U.S. Pat. No. 5,629,384, WO 96 / 41813, WO 96 / 07670, U.S. Pat. No. 5,473,034, U.S. Pat. No. 5,516,673, EP 605963, U.S. Pat. No. 5,382,657, EP 510356, EP 400472, EP 183503, and EP 154316.

[0143] Specific examples of activated PEG polymers that are particularly preferred for coupling to cysteine ​​residues include the following linear PEGs: vinylsulfone-PEG (VS-PEG), preferably vinylsulfone-mPEG (VS-mPEG), maleimide-PEG (MAL-PEG), preferably maleimide-mPEG (MAL-mPEG), and orthopyridyl-disulfide-PEG (OPSS-PEG), preferably orthopyridyl-disulfide-mPEG (OPSS-mPEG). Typically, such PEG or mPEG polymers have a size of about 5 kDa, about 10 kDa, about 12 kDa, or about 20 kDa.

[0144] Conjugation of the fusion proteins and activated polymer molecules described herein can be carried out using any conventional method, for example, as described in the following references, which also describe suitable methods for activating polymer molecules: Harris and Zalipsky, eds., Poly(ethylene glycol) Chemistry and Biological Applications, AZC Washington; RF Taylor, (1991), "Protein immobilization: Fundamentals and applications," Marcel Dekker, NY; S.S. Wong, (1992), "Chemistry of Protein Conjugation and Crosslinking," CRC Press, Boca Raton; G.T. Hermanson et al., (1993), "Immobilized Affinity Ligand Techniques," Academic Press, NY

[0145] Those skilled in the art will recognize that the activation method and / or conjugation chemistry used will depend on the attachment group(s) on the fusion protein (examples of which are further provided above), as well as the functional groups on the polymer (e.g., amine, hydroxyl, carboxyl, aldehyde, sulfhydryl, succinimidyl, maleimide, vinylsulfone, or haloacetate). PEGylation can be directed to conjugation to all available attachment groups on the fusion protein (i.e., those attachment groups exposed on the surface of the polypeptide), or can be directed to one or more specific attachment groups, such as the N-terminal amino group as described in U.S. Pat. No. 5,985,265, or cysteine ​​residues. Furthermore, conjugation can be achieved in one step or in a stepwise manner (e.g., as described in WO 99 / 55377).

[0146] For PEGylation to cysteine ​​residues (see above), the fusion protein is typically treated with a reducing agent, such as dithiothreitol (DDT), prior to PEGylation. The reducing agent is then removed by any conventional method, such as desalting. Conjugation of PEG to cysteine ​​residues is typically carried out in a suitable buffer solution, pH 6-9, at temperatures ranging from 4°C to 25°C for up to 16 hours.

[0147] It will be understood that PEGylation will be designed to produce an optimal molecule with respect to the number of PEG molecules attached, the size and form of such molecules (e.g., whether they are linear or branched), and the attachment site(s) in the fusion protein. The molecular weight of the polymer used can be selected based, for example, on the desired effect to be achieved.

[0148] In connection with conjugation to only a single binding group on the fusion protein (e.g., the N-terminal amino group), it may be advantageous for the polymer molecule, which may be linear or branched, to have a high molecular weight, preferably about 10-25 kDa, for example about 15-25 kDa, for example about 20 kDa.

[0149] Typically, polymer conjugation is carried out under conditions designed to allow many of the available polymer binding groups to react with the polymer molecule. This is achieved by a suitable molar excess of polymer relative to the polypeptide. Typically, the molar ratio of activated polymer molecules to polypeptide is up to about 1000-1, for example, up to about 200-1, or up to about 100-1. However, in some cases, the ratio may be somewhat lower, such as up to about 50-1, 10-1, 5-1, 2-1, or 1-1, to achieve optimal reaction.

[0150] It is also contemplated that the polymer molecule may be coupled to the fusion protein via a linker. Suitable linkers are well known to those skilled in the art. A preferred example is cyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; U.S. Patent No. 4,179,337; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375-378).

[0151] Following conjugation, any remaining activated polymer molecules are blocked according to methods known in the art, for example, by adding a primary amine to the reaction mixture, and the resulting unactivated polymer molecules are removed by an appropriate method.

[0152] It will be understood that depending on the circumstances, e.g., the particular PEGylation conditions, including the amino acid sequence of the fusion protein, the nature of the activated PEG compound used, and the molar ratio of PEG to polypeptide, various degrees of PEGylation can be obtained, with higher degrees of PEGylation generally being obtained with higher ratios of PEG to fusion protein. However, the PEGylated fusion proteins resulting from any given PEGylation process will usually contain a stochastic distribution of conjugated fusion proteins having slightly different degrees of PEGylation.

[0153] To improve the biological half-life of the fusion proteins described herein, chemical modifications such as PEGylation or HESylation can be applied.

[0154] HAS and HES non-proteinaceous polymers and methods for producing HAS or HES conjugates are disclosed, for example, in WO02 / 080979, WO03 / 070772, WO057092391 and WO057092390.

[0155] Polysialylation is another technique that uses the natural polymer polysialic acid (PSA) to extend the half-life and improve the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (a sugar). When used in protein and therapeutic peptide drug delivery, polysialic acid provides a protective microenvironment upon conjugation. This increases the active lifespan of the fusion protein in the circulation and prevents it from being recognized by the immune system. PSA polymers are naturally found in the human body. They were introduced by certain bacteria that evolved over millions of years to coat their walls. These naturally polysialylated bacteria were then able to compromise the body's defense system through molecular mimicry. PSA, the ultimate in natural stealth technology, can be easily produced from such bacteria in large quantities with defined physical properties. Bacterial PSA, even when conjugated to proteins, is completely non-immunogenic because it is chemically identical to human PSA.

[0156] Biological activity of relaxin-2 fusion proteins In some embodiments, the relaxin-2 fusion proteins described herein have an increased level of biological activity compared to native relaxin-2. In some embodiments, any of the relaxin-2 fusion proteins described herein have about 1% to about 200% of the biological activity compared to native relaxin-2. In some embodiments, the relaxin-2 fusion proteins have at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, or about 200% of the biological activity compared to native relaxin-2.

[0157] In some embodiments, any of the relaxin-2 fusion proteins described herein has about 1% to about 200% of the maximal biological activity compared to native relaxin-2. In some embodiments, the maximal biological activity is measured by the maximal response (E) of relaxin-2 or relaxin-2 fusion protein. maxIn some embodiments, the relaxin-2 fusion protein has at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, or about 200% of the maximal biological activity compared to native relaxin-2.

[0158] In some embodiments, any of the relaxin-2 fusion proteins described herein has an enhanced potency of at least about 0.001-fold to at least about 1,000-fold compared to native relaxin-2. In some embodiments, potency is measured as the half-maximal response (EC 50 In some embodiments, the relaxin-2 fusion protein is at least about 0.001-fold, about 0.01-fold, about 0.1-fold, about 1-fold, about 10-fold, about 100-fold, or about 1,000-fold more potent than native relaxin-2.

[0159] The biological activity can be any biological activity of natural relaxin-2. For example, the biological activity can be the ability to bind to the receptor of natural relaxin-2, RXFP1. The binding of relaxin-2 to RXFP1 can be measured by any known method in the art, such as radioligand binding. In some embodiments, the fusion protein described herein binds to RXFP1 when RXFP1 is expressed on the cell surface.

[0160] In some embodiments, the biological activity can be the ability to activate RXFP1 on the cell surface. Activation of RXFP1 by a relaxin-2 fusion protein described herein can be determined by an increase in cAMP using any method known in the art, such as measuring the activity of a cAMP-driven reporter gene, e.g., β-galactosidase. Activation of RXFP1 by a relaxin-2 fusion protein described herein in a cell can also be determined by using a biosensor, such as a GloSensor biosensor. Activation of RXFP1 by a relaxin-2 fusion protein described herein in a cell can also be determined by measuring the expression of a specific gene, such as the expression of an angiogenic factor, e.g., VEGF, or MMP, using methods known in the art. In some embodiments, the biological activity is a physiological, biochemical, or any other effect-inducing activity of relaxin-2. Exemplary biological activities include, but are not limited to, vasodilation, collagen degradation, angiogenesis, lowering arterial blood pressure, increasing renal artery blood flow, increasing renal plasma flow, increasing cardiac filling during diastole, resolving established fibrosis, and inhibiting the development of new fibrosis.

[0161] In some embodiments, the fusion proteins described herein have improved pharmacokinetic profiles. Without wishing to be bound by any theory, the structures of the fusion proteins described herein are based, at least in part, on the surprising discovery that lowering the pI of relaxin-2 fusion protein analogs increases their circulating half-life. In some embodiments, the fusion proteins described herein have high bioavailability. In some embodiments, the fusion proteins described herein have high and / or stable serum levels. In some embodiments, the circulating half-life, bioavailability, high serum levels, and / or stable serum levels are in a mammal. In some embodiments, the mammal is a rodent or a primate. In some embodiments, the rodent is a rat or a mouse. In some embodiments, the primate is a human or a monkey. In some embodiments, the monkey is a cynomolgus monkey. In some embodiments, the mammal is a human.

[0162] In some embodiments, the fusion proteins described herein may have a circulating half-life of about 5 hours, 10 hours, 20 hours, 50 hours, 75 hours, 100 hours, 125 hours, 150 hours, or more. In some embodiments, the fusion proteins described herein may have a circulating half-life of 5-10 hours, 10-20 hours, 20-50 hours, 50-75 hours, 75-100 hours, 100-125 hours, or 125-150 hours. In some embodiments, the fusion proteins described herein may have a circulating half-life of about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, or about 23 days. In some embodiments, the fusion proteins described herein may have a circulating half-life of 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, or 23 days. In some embodiments, the fusion proteins described herein may have a circulating half-life of at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, or at least 23 days. In some embodiments, the fusion proteins described herein may have a circulating half-life of about 5 hours, 10 hours, 20 hours, 50 hours, 75 hours, 100 hours, 125 hours, 150 hours, or more when administered to a human. In some embodiments, the fusion proteins described herein, when administered to a human, may have a circulating half-life of 5 to 10 hours, 10 to 20 hours, 20 to 50 hours, 50 to 75 hours, 75 to 100 hours, 100 to 125 hours, or 125 to 150 hours. In some embodiments, the fusion proteins described herein, when administered to a human, may have a circulating half-life of about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, or about 23 days.In some embodiments, the fusion proteins described herein, when administered to a human, may have a circulating half-life of 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, or 23 days. In some embodiments, the fusion proteins described herein, when administered to a human, may have a circulating half-life of at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, or at least 23 days. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the present disclosure. In some embodiments, the fusion proteins described herein have a longer circulating half-life than native two-chain relaxin-2. For example, the circulating half-life of native two-chain relaxin-2 may be less than about 5 hours. (See, e.g., Chen et al., The Pharmacokinetics of Recombinant Human Relaxin in Non-Pregnant Women after Intravenous, Intravaginal, and Intracervical Administration, Pharm. Res. 10:834038 (1993), incorporated herein by reference.)

[0163] This increased half-life may be due, at least in part, to the reduced pI of the fusion proteins described herein. In some embodiments, the fusion proteins have a pI that is less than about 9.4. As used herein, the term "about" when referring to a pI encompasses a ±1% variation of a given value or range, as appropriate for practicing the methods disclosed herein. In some embodiments, the fusion proteins have a pI of 9.0, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, or 6.1. In some embodiments, the fusion protein has a pI that is less than about 9.0, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, or 6.1. In some embodiments, the fusion protein has a pI that is less than 9.0. In some embodiments, the fusion protein has a pI that is less than about 8.2. In some embodiments, the fusion protein has a pI of about 6.0 to about 9.4. In some embodiments, the fusion protein has a nucleotide sequence of about 6.5 to about 8.5, about 6.6 to about 8.4, about 6.7 to about 8.3, about 6.8 to about 8.2, about 6.8 to about 8.1, about 6.8 to about 8.0, about 6.8 to about 7.9, about 6.0 to about 8.2, about 6.0 to about 8.1, about 6.0 to about 8.0, about 6.0 to about 7.9, about 6.0 to about 7.8, about 6.0 to about 7.7, about 6.0 to about 7.6 , about 6.0 to about 7.5, about 6.0 to about 7.4, about 6.0 to about 7.3, about 6.0 to about 7.2, about 6.0 to about 7.1, about 6.0 to about 7.0, about 6.0 to about 6.9, about 6.0 to about 6.8, about 6.0 to about 6.7, about 6.0 to about 6.6, about 6.0 to about 6.5, about 6.0 to about 6.4, about 6.0 to about 6.3, about 6.0 to about 6.2, or about 6.0 to about 6.1. In some embodiments, the fusion protein has a pI of about 6.0 to about 8.2.In some embodiments, the fusion protein has a pI of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, or 8.2, or about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, or 8.2, or between any two such values. In some embodiments, the fusion protein has a pI of about 6.8. In some embodiments, the fusion protein has a pI of about 7.0. In some embodiments, the fusion protein has a pI of about 7.1. In some embodiments, the fusion protein has a pI of about 7.4. In some embodiments, the fusion protein has a pI of about 7.5. In some embodiments, the fusion protein has a pI of about 7.9. In some embodiments, the fusion protein has a pI of about 8.0. In some embodiments, the fusion protein has a pI of about 8.4. In some embodiments, the fusion protein has a pI of about 8.5. In some embodiments, the fusion protein has a pI of about 8.8. In some embodiments, the fusion protein has a pI of about 8.9. In some embodiments, any of the above pIs are calculated or theoretical pIs. In some embodiments, any of the above pIs are experimentally determined pIs.

[0164] As used herein, the term "about" when referring to a dosage encompasses a ±10% variation of a given value or range, as appropriate for practicing the methods disclosed herein.

[0165] As used herein, "circulating half-life" refers to the time it takes for the plasma concentration of a drug to decrease by half from its steady state when circulating in the whole blood of an organism. The circulating half-life of a particular drug can vary depending on numerous factors, including, but not limited to, the drug's dosage, formulation, and / or route of administration. Those skilled in the art can determine the circulating half-life of a drug using methods well known in the art, such as the method described in Chen, supra.

[0166] In some embodiments, the fusion proteins described herein have high bioavailability. In some embodiments, the fusion proteins have high bioavailability when administered, for example, intravenously or subcutaneously. In some embodiments, the fusion proteins have high bioavailability when administered subcutaneously. In some embodiments, the fusion proteins have high subcutaneous bioavailability. In some embodiments, the fusion proteins have a bioavailability of at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. In some embodiments, the fusion protein has a bioavailability of about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100%. In some embodiments, the fusion protein has a bioavailability of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%. In some embodiments, the fusion protein has a bioavailability of about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 60%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 80%, or about 70% to about 80%. In some embodiments, the fusion protein has a bioavailability of about 50% to about 60% (e.g., 50% to 60%). In some embodiments, the fusion protein has a bioavailability of at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more when administered subcutaneously.In some embodiments, the fusion protein has a bioavailability of about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% when administered subcutaneously. In some embodiments, the fusion protein has a bioavailability of about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% when administered subcutaneously. In some embodiments, the fusion protein, when administered subcutaneously, has a bioavailability of about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 60%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 80%, or about 70% to about 80%. In some embodiments, the fusion protein, when administered subcutaneously, has a bioavailability of about 50% to about 60% (e.g., 50% to 60%).

[0167] As used herein, "bioavailability" refers to the proportion of an administered drug that reaches the systemic circulation. The bioavailability of a particular drug can vary depending on numerous factors, including, but not limited to, the drug's dosage, formulation, route of administration, and / or properties. Those skilled in the art can determine the bioavailability of a drug using methods well known in the art.

[0168] In some embodiments, the fusion protein has high and / or stable serum levels when administered to a subject, e.g., intravenously, subcutaneously, and / or according to any of the methods described herein. In some embodiments, the fusion protein is present in the subject's serum at a level of at least about 0.5 μg / mL, at least about 1 μg / mL, at least about 2 μg / mL, at least about 3 μg / mL, at least about 4 μg / mL, at least about 5 μg / mL, at least about 6 μg / mL, at least about 7 μg / mL, at least about 8 μg / mL, or at least about 9 μg / mL for 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after administration. In some embodiments, the fusion protein is present in the subject's serum at a level of at least about 0.5 μg / mL (e.g., at least about 1 μg / mL, at least about 2 μg / mL, at least about 3 μg / mL, at least about 4 μg / mL, at least about 5 μg / mL, at least about 6 μg / mL, at least about 7 μg / mL, at least about 8 μg / mL, or at least about 9 μg / mL) for at least 0.5 days, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or more after administration. In some embodiments, the fusion protein is present in the subject's serum at a level of at least about 0.5 μg / mL (e.g., at least about 1 μg / mL, at least about 2 μg / mL, at least about 3 μg / mL, at least about 4 μg / mL, at least about 5 μg / mL, at least about 6 μg / mL, at least about 7 μg / mL, at least about 8 μg / mL, or at least about 9 μg / mL) for at least 0.5 days, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or more following intravenous administration.In some embodiments, the fusion protein is present in the subject's serum at a level of at least about 0.5 μg / mL (e.g., at least about 1 μg / mL, at least about 2 μg / mL, at least about 3 μg / mL, at least about 4 μg / mL, at least about 5 μg / mL, at least about 6 μg / mL, at least about 7 μg / mL, at least about 8 μg / mL, or at least about 9 μg / mL) for at least 0.5 days, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or more following subcutaneous administration.

[0169] Vectors and host cells The present disclosure also provides nucleic acid molecules encoding any of the fusion proteins or peptides described herein. In some embodiments, the nucleic acid molecules described herein are DNA molecules. In some embodiments, the nucleic acid molecules described herein are RNA molecules.

[0170] The nucleic acid molecules described herein can be transcribed from a promoter in an expression vector. In some embodiments, the vector is a non-viral vector. Exemplary non-viral vectors include, but are not limited to, plasmid DNA, transposons, episomal plasmids, minicircles, ministrings, and oligonucleotides (e.g., mRNA, naked DNA). In some embodiments, the vector is a DNA plasmid vector.

[0171] In some embodiments, the vector is a viral vector. Viral vectors can be replication-competent or replication-incompetent. Viral vectors can be integrating or non-integrating. Several viral-based systems have been developed for gene transfer into mammalian cells, and a suitable viral vector can be selected by one of skill in the art. Exemplary viral vectors include, but are not limited to, adenoviral vectors (e.g., adenovirus 5), adeno-associated viral (AAV) vectors (e.g., AAV2, 3, 5, 6, 8, 9), retroviral vectors (MMSV, MSCV), lentiviral vectors (e.g., HIV-1, HIV-2), gamma retroviral vectors, herpesvirus vectors (e.g., HSV1, HSV2), alphavirus vectors (e.g., SFV, SIN, VEE, M1), flavivirus (e.g., Kunjin, West Nile, Dengue virus), rhabdovirus vectors (e.g., rabies virus, VSV), measles virus vectors (e.g., MV-Edm), Newcastle disease virus vectors, poxvirus vectors (e.g., VV), measles virus, and picornavirus vectors (e.g., coxsackievirus).

[0172] In some embodiments, a vector or expression cassette comprises one or more additional elements, including, but not limited to, a promoter, an enhancer, a polyadenylation (polyA) sequence, and a selection gene.

[0173] In some embodiments, the vector comprises a polynucleotide sequence that encodes an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical to an amino acid sequence listed in any of Tables 1-8. In some embodiments, the vector comprises a polynucleotide sequence that encodes an amino acid sequence that comprises or consists of an amino acid sequence listed in any of Tables 1-8. In some embodiments, the vector comprises a polynucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical to a sequence listed below in Table 9. In some embodiments, the vector comprises a polynucleotide sequence that comprises or consists of a sequence listed below in Table 9. [Table 9] TIFF2026501550000096.tif241148TIFF2026501550000097.tif241146TIFF2026501550000098.tif242147TIFF2026501550000099.tif243145TIFF2026501550000100.tif242147TIFF2026501550000101.tif242148TIFF2026501550000102.tif244149TIFF2026501550000103.tif242146TIFF2026501550000104.tif243148TIFF2026501550000105.tif242147TIFF2026501550000106.tif242147TIFF2026501550000107.tif243147TIFF2026501550000108.tif242146TIFF2026501550000109.tif243146TIFF2026501550000110.tif242146TIFF2026501550000111.tif243146TIFF2026501550000112.tif241146TIFF2026501550000113.tif243148TIFF2026501550000114.tif242146TIFF2026501550000115.tif243146TIFF2026501550000116.tif243147TIFF2026501550000117.tif242147TIFF2026501550000118.tif243146TIFF2026501550000119.tif243146TIFF2026501550000120.tif242148TIFF2026501550000121.tif242146TIFF2026501550000122.tif242147TIFF2026501550000123.tif244147TIFF2026501550000124.tif242146TIFF2026501550000125.tif242146TIFF2026501550000126.tif242146TIFF2026501550000127.tif243146TIFF2026501550000128.tif242146TIFF2026501550000129.tif241146TIFF2026501550000130.tif242147TIFF2026501550000131.tif243146T IFF2026501550000132.tif242146TIFF2026501550000133.tif242144TIFF2026501550000134.tif242147TIFF2026501 550000135.tif242146TIFF2026501550000136.tif242146TIFF2026501550000137.tif248147TIFF2026501550000138. tif243147TIFF2026501550000139.tif242147TIFF2026501550000140.tif243146TIFF2026501550000141.tif129146.

[0174] In some embodiments, the vector comprises a polynucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical to a sequence listed below in Table 10. In some embodiments, the vector comprises a polynucleotide sequence that comprises or consists of a sequence listed below in Table 10. [Table 10] TIFF2026501550000143.tif253145TIFF2026501550000144.tif252145TIFF2026501550000145.tif252146TIFF2026501550000146.tif253146TIFF2026501550000147.tif252145TIFF2026501550000148.tif253146TIFF2026501550000149.tif252145TIFF2026501550000150.tif252145TIFF2026501550000151.tif252145TIFF2026501550000152.tif252146TIFF2026501550000153.tif252146TIFF2026501550000154.tif252146TIFF2026501550000155.tif252145TIFF2026501550000156.tif252145TIFF2026501550000157.tif253145TIFF2026501550000158.tif252146TIFF2026501550000159.tif253147TIFF2026501550000160.tif252145TIFF2026501550000161.tif251145TIFF2026501550000162.tif252146TIFF2026501550000163.tif252146TIFF2026501550000164.tif253144TIFF2026501550000165.tif252146TIFF2026501550000166.tif253146TIFF2026501550000167.tif252145TIFF2026501550000168.tif253146TIFF2026501550000169.tif252145TIFF2026501550000170.tif252146TIFF2026501550000171.tif252146TIFF2026501550000172.tif252146TIFF2026501550000173.tif252146TIFF2026501550000174.tif252146TIFF2026501550000175.tif252145TIFF2026501550000176.tif252146TIFF2026501550000177.tif252147TIFF2026501550000178.tif252146T IFF2026501550000179.tif252146TIFF2026501550000180.tif252145TIFF2026501550000181.tif252145TIFF2026501 550000182.tif252147TIFF2026501550000183.tif251145TIFF2026501550000184.tif247145TIFF2026501550000185. tif252145TIFF2026501550000186.tif252145TIFF2026501550000187.tif253146TIFF2026501550000188.tif134147.

[0175] In some embodiments, any of the nucleotide sequences set forth in Table 10 further comprise additional nucleotide sequences at the 5' and / or 3' end. In some embodiments, any of the nucleotide sequences set forth in Table 10 further comprise the nucleotide sequence ACGGGACCGATCCAGCCTCCGGACTCTAGAGCCACC (SEQ ID NO:494) at the 5' end, and / or any of the nucleotide sequences set forth in Table 10 further comprise the nucleotide sequence TGATAAACCGGTTAGTAATGAGTTTGATATCTCGAC (SEQ ID NO:495) at the 3' end.

[0176] A variety of host cell and expression vector systems can be utilized to express the fusion proteins described herein. Such expression systems represent vehicles in which a coding sequence of interest may be produced and subsequently purified, and also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of expressing the fusion proteins described herein in situ. These include, for example, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the protein coding sequence; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the protein coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the protein coding sequence; and plant cell systems (e.g., Chlamydomonas spp.) infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) containing the protein coding sequence or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the protein coding sequence. reinhardtii); or mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells) harboring a recombinant expression construct containing, for example, a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter; a vaccinia virus 7.5K promoter). In certain embodiments, the cells for expressing the fusion proteins described herein are human cells, e.g., human cell lines. In certain embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3.In certain embodiments, bacterial cells, such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells) are used for expression of fusion proteins. For example, mammalian cells such as CHO or HEK293 cells, in combination with vectors such as the major immediate early gene promoter element from human cytomegalovirus, are effective expression systems for the fusion proteins disclosed herein.

[0177] In bacterial systems, several expression vectors can be advantageously selected depending on the intended use of the expressed fusion protein. For example, if a large amount of fusion protein is to be produced, a vector that directs the expression of a high-level fusion protein product that is easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2:1791-1794), in which the fusion protein coding sequence can be individually ligated in frame with the lac Z coding region to produce the fusion protein, and the pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13:3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24:5503-5509), all of which are incorporated herein by reference in their entirety. Also, for example, pGEX vectors can be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads, followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0178] In an insect system, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The fusion protein coding sequence can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0179] Several viral-based expression systems can be used in mammalian host cells. When adenovirus is used as an expression vector, the fusion protein coding sequence of interest can be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) results in a recombinant virus that is viable and capable of expressing the fusion protein molecule in infected hosts (see, e.g., Logan J & Shenk T (1984) PNAS 81(12):3655-9, incorporated herein by reference in its entirety). Specific initiation signals may also be required for efficient translation of the inserted fusion protein coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol. 153:516-544, which is incorporated herein by reference in its entirety).

[0180] In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. An appropriate cell line or host system may be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells.

[0181] For long-term, high-yield production of recombinant proteins, stable expression cells can be generated, for example, cell lines that stably express the fusion proteins described herein can be engineered.

[0182] In certain embodiments, rather than using expression vectors containing viral origins of replication, host cells may be transformed with a polynucleotide (e.g., DNA or RNA) controlled by appropriate transcriptional regulatory elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the polynucleotide, engineered cells may be grown in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker on the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to form foci that can then be cloned and expanded into cell lines. This method may be advantageously used to engineer cell lines that express the fusion proteins or fragments thereof described herein.

[0183] Several selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler M et al. (1977) Cell 11(1):223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12):2026-2034), and adenine phosphoribosyltransferase (Lowy I et al. (1980) Cell 22(3):817-23), in tk-, hgprt-, or aprt- cells, respectively, all of which are incorporated herein by reference in their entirety. Antimetabolite resistance can also be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al. (1980) PNAS 77(6):3567-70; O'Hare K et al. (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4):2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3:87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32:573-596; Mulligan RC (1993) Science 260:926-932; Morgan RA & Anderson WF (1993) Ann Rev Biochem 62:191-217, Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5):211-5), and hygro, which confers resistance to hygromycin (Santerre RF et al. (1984) Gene 30(1-3):147-56), all of which are incorporated herein by reference.Methods commonly known in the field of recombinant DNA technology can be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel FM et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993), Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990), and Chapters 12 and 13, Dracopoli NC et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994), Colbere-Garapin F et al. (1981) J Mol Biol 150:1-14, all of which are incorporated herein by reference in their entirety.

[0184] Pharmaceutical Composition The present disclosure provides pharmaceutical compositions comprising the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them. The pharmaceutical compositions described herein are formulated with suitable carriers, excipients, and other agents that provide improved mobility, delivery, tolerability, etc. Many suitable formulations can be found in formularies known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., 11POFECTIN™, LifeTechnologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0185] The dose of the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein administered to a patient may vary depending on the patient's age and size, the target disease, condition, route of administration, etc. Preferred doses are typically calculated according to body weight or body surface area. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective dosages and schedules for administering the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein, can be determined empirically, e.g., by monitoring the patient's progress through periodic evaluations and adjusting the dose accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0186] Various delivery systems, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis, are known and can be used to administer the pharmaceutical compositions disclosed herein (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0187] Any pharmaceutical composition described herein can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device facilitates application when delivering the pharmaceutical compositions disclosed herein. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and easily replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0188] In certain circumstances, pharmaceutical compositions can be delivered in a sustained-release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a sustained-release system can be placed near the target of the composition, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other sustained-release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0189] Injectable preparations may include dosage forms for intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip infusion, etc. These injectable preparations may be prepared by publicly known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying any of the fusion proteins described herein in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injections include, for example, saline, isotonic solutions containing glucose, and other adjuvants, which may be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)). Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection solution prepared in this manner is preferably filled into an appropriate ampule.

[0190] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in a suitable unit dosage form to accommodate the dose of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the fusion protein contained is generally about 5 to about 500 mg per dosage form in a unit dosage. In particular, in the form of injection, the amount of the fusion protein contained is about 5 to about 100 mg, and for other dosage forms, it is preferably about 10 to about 250 mg.

[0191] therapeutic use Monotherapy The present disclosure provides methods for enhancing relaxin-2-related activity in primary cells, comprising contacting the primary cells with a fusion protein or component peptide described herein. In some embodiments, contacting the primary cells with the fusion protein or component peptide results in enhanced relaxin-2 activity in the cells, e.g., as described above. In some embodiments, contacting the primary cells with the fusion protein or component peptide results in activation of the relaxin-2 receptor (RFXFP1) on the cell surface. Activation of RFXFP1 on the cell surface can result in cellular responses including, but not limited to, elevated cAMP levels, vasodilation, expression of angiogenic factors including VEGF, expression of MMPs, and collagen degradation. In some embodiments, the cells are selected from the group consisting of endothelial cells, vascular smooth muscle cells, other vascular cells, cardiomyocytes, other cardiac cells, and fibroblasts. In some embodiments, the primary cells are present in a subject, as described below.

[0192] In certain embodiments, the present disclosure provides methods for activating RXFP1 on the cell surface, comprising administering to a subject in need thereof an effective amount of a fusion protein or component peptide, or nucleic acid molecule, or expression vector encoding the same. Activation of RXFP1 on the cell surface can result in cellular responses, including, but not limited to, elevated cAMP levels, vasodilation, expression of angiogenic factors including VEGF, expression of MMPs, and collagen degradation. In some embodiments, the cells are selected from the group consisting of endothelial cells, vascular smooth muscle cells, other vascular cells, cardiomyocytes, other cardiac cells, and fibroblasts.

[0193] The present disclosure also provides methods for treating various relaxin-2-related disorders. As used herein, the term "relaxin-2-related disorder" is a disease or disorder caused by or associated with relaxin-2 protein production or relaxin-2 protein activity. The term "relaxin-2-related disorder" includes diseases, disorders, or conditions that would benefit from increased relaxin-2 protein activity. As used herein, the term "relaxin-2-related disorder" has the same meaning as "relaxin-2-related disorder."

[0194] In certain embodiments, the relaxin-2 associated disease or disorder is selected from the group consisting of a renal disease, a fibrotic disease, and a cardiovascular disease, hi certain embodiments, the relaxin-2 associated disease or disorder is pulmonary hypertension.

[0195] There are five groups of pulmonary hypertension defined by the World Health Organization (WHO). Group 1 is pulmonary arterial hypertension (PAH), the diagnosis of which requires right heart catheterization (RHC) to demonstrate a resting mean pulmonary artery (PA) pressure (mPAP) of ≥ 20 mmHg and a pulmonary vascular resistance (PVR) of ≥ 2 Wood units. Additional criteria for meeting Group 1 PAH include a mean pulmonary capillary wedge pressure (PCWP) of ≤ 15 mmHg, mild or absent chronic lung disease (CLD) and other causes of hypoxemia, absence of venous thromboembolic disease and PA obstruction, and absence of a wide range of specific disorders, including systemic disorders (e.g., sarcoidosis, chronic renal failure), blood disorders (e.g., myeloproliferative disorders and chronic hemolytic anemia), and metabolic disorders (e.g., glycogen storage diseases). Group 1 also includes PAH due to unknown mechanisms (idiopathic PAH) and inherited genetic defects (hereditary PAH); PAH resulting from drugs and toxins; PAH associated with systemic disorders such as connective tissue disease, human immunodeficiency virus (HIV) infection, congenital heart disease, and blood somnosis; PAH with clear features of venous / capillary involvement; and persistent PH in newborns.

[0196] Group 2, PH due to left heart disease (PH-LHD), may be diagnosed clinically if there is sufficient LHD to explain the PH on echocardiography (with or without other confirmatory tests). For patients in whom RHC is performed, mPAP ≥ 20 mmHg, PCWP ≥ 15 mmHg, and normal or reduced cardiac output are consistent with the hemodynamic diagnosis of LHD-PH. Important supporting information is the presence of left atrial (LA) enlargement on echocardiography and left heart catheterization (LHC) to confirm elevated left ventricular end-diastolic pressure. Once PH-LHD is confirmed, patients should be assigned to one of the following categories: PH-LHD due to heart failure with preserved or reduced ejection fraction (group 2.1), heart failure with reduced ejection fraction (group 2.2), valvular heart disease, or congenital or acquired conditions leading to postcapillary PH (group 2.3, e.g., restrictive cardiomyopathy, constrictive pericarditis, LA myxoma, congenital or acquired inflow / outflow tract obstruction, and congenital cardiomyopathy). There are two subgroups of group 2 PH; patients can be differentiated into those with combined pre- and postcapillary PH (CpcPH) and those with isolated postcapillary hypertension (IpcPH).

[0197] Group 3 is PH due to chronic lung disease and / or hypoxemia, which is a diagnosis of PH due to chronic lung disease and / or hypoxemia, made by demonstration of PH on RHC or echocardiography and evidence of moderate to severe pulmonary dysfunction and / or hypoxemia. Patients are assigned to PH due to obstructive pulmonary disease (Group 3.1), restrictive pulmonary disease (Group 3.2), mixed obstructive and restrictive pulmonary disease (Group 3.3), PH with hypoxia (Group 3.4), hypoxia without pulmonary disease (Group 3.5), or PH due to developmental disorders (Group 3.6). In some cases, PH in Group 3 may be due to COPD, interstitial lung disease, or obstructive sleep apnea.

[0198] Group 4 is PH due to pulmonary artery obstruction and includes mostly patients with chronic thromboembolic PH (CTEPH, group 4.1), as well as PH due to PA obstruction (group 4.2, e.g., benign or malignant tumors, arteritis in the absence of CTD, congenital PA stenosis, parasites).

[0199] Group 5 is PH due to multifactorial mechanisms and includes patients with PH that does not clearly fit into Groups 1 to 4. Group 5 PH can be further classified as having blood disorders such as chronic hemolytic anemia (e.g., sickle cell disease, beta-thalassemia, or spherocytosis) and myeloproliferative disorders; systemic or metabolic disorders including sarcoidosis, pulmonary Langerhans histiocytosis X, and neurofibromatosis; metabolic disorders including Gaucher disease and glycogen storage disorders; hemodialysis-associated chronic renal failure and PH; pulmonary tumor thrombotic microangiopathy; and fibrosing mediastinitis.

[0200] In certain embodiments, the relaxin-2 related disease or disorder is pulmonary hypertension, which includes any of WHO defined Group 1, Group 2, Group 3, Group 4, and Group 5 PH.

[0201] In certain embodiments, the relaxin-2 related disease or disorder is pulmonary hypertension, including but not limited to pulmonary hypertension (pulmonary arterial hypertension (PAH), pulmonary hypertension due to left heart disease (PH-LHD), combined pre- and post-capillary pulmonary hypertension (CpcPH), and isolated post-capillary pulmonary hypertension (IpcPH). In certain embodiments, the relaxin-2 related disease or disorder is pulmonary arterial hypertension (PAH). In certain embodiments, the relaxin-2 related disease or disorder is pulmonary hypertension due to left heart disease (PH-LHD). In certain embodiments, the relaxin-2 related disease or disorder is combined pre- and post-capillary pulmonary hypertension (CpcPH). In certain embodiments, the relaxin-2 related disease or disorder is isolated post-capillary pulmonary hypertension (IpcPH).

[0202] In certain embodiments, the relaxin-2 related disease or disorder is pulmonary hypertension of Group 2. In certain embodiments, the relaxin-2 related disease or disorder is isolated post-capillary pulmonary hypertension (IpcPH). IpcPH includes features such as right ventricular dysfunction, left ventricular thickening and stiffening (LHD), and renal dysfunction. In certain embodiments, the relaxin-2 related disease or disorder is selected from the group consisting of right ventricular dysfunction, left ventricular thickening and stiffening (LHD), and renal dysfunction. In certain embodiments, the relaxin-2 related disease or disorder is combined pre- and post-capillary pulmonary hypertension (CpcPH). CpcPH includes features such as pulmonary artery narrowing, thickening, stiffening, and / or fibrotic remodeling, right ventricular dysfunction, left ventricular thickening and stiffening (LHD), and renal dysfunction. In certain embodiments, the relaxin-2 related disease or disorder is selected from the group consisting of pulmonary artery narrowing, thickening, stiffening, and / or fibrotic remodeling, right ventricular dysfunction, left ventricular thickening and stiffening (LHD), and renal dysfunction.

[0203] In certain embodiments, the relaxin-2 related disease or disorder is heart failure, including but not limited to heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF). In certain embodiments, the relaxin-2 related disease or disorder is heart failure with preserved ejection fraction (HFpEF). In certain embodiments, the relaxin-2 related disease or disorder is heart failure with reduced ejection fraction (HFrEF).

[0204] In certain embodiments, the relaxin-2 related disease or disorder is a cardiac disease, including but not limited to, valvular heart disease.

[0205] In certain embodiments, the relaxin-2-related disease or disorder is Group 2 PH (CpcPH or IpcPH) with heart failure with preserved ejection fraction (HFpEF). In certain embodiments, the relaxin-2-related disease or disorder is CpcPH with HFpEF. In certain embodiments, the relaxin-2-related disease or disorder is IpcPH with HFpEF. In certain embodiments, HFpEF is defined as signs and symptoms of New York Heart Association (NYHA) Class II-III heart failure and an LVEF of ≥ 50%, and at least one of: (i) a Heart Failure Association-Pretest Assessment, Echocardiography, and Natriuretic Peptide Score, Functional Test in Case of Uncertainty, End-of-Life Etiology (HFA-PEFF) score of ≥ 5; and / or (ii) an HFA-PEFF score of 2-4 and an abnormal diastolic stress test or invasive hemodynamic measurement. In certain embodiments, the relaxin-2 associated disease or disorder is CpcPH with NYHA class II-III heart failure and an LVEF > 50%, and at least one of (i) an HFA-PEFF score > 5, and / or (ii) an HFA-PEFF score of 2-4 and an abnormal diastolic stress test or invasive hemodynamic measurement. In certain embodiments, the relaxin-2 associated disease or disorder is IpcPH with NYHA class II-III heart failure and an LVEF > 50%, and at least one of (i) an HFA-PEFF score > 5, and / or (ii) an HFA-PEFF score of 2-4 and an abnormal diastolic stress test or invasive hemodynamic measurement.

[0206] In certain embodiments, the relaxin-2-related disease or disorder is group 2 PH (CpcPH or IpcPH) with heart failure with intermediate ejection fraction (HFmrEF). In certain embodiments, the relaxin-2-related disease or disorder is CpcPH with HFmrEF. In certain embodiments, the relaxin-2-related disease or disorder is IpcPH with HFmrEF. In certain embodiments, HFmrEF is defined as signs and symptoms of New York Heart Association (NYHA) class II-III heart failure and an LVEF of 40%-49%. In certain embodiments, the relaxin-2-related disease or disorder is CpcPH with NYHA class II-III heart failure and an LVEF of 40%-49%. In certain embodiments, the relaxin-2-related disease or disorder is IpcPH with NYHA class II-III heart failure and an LVEF of 40%-49%.

[0207] In certain embodiments, the relaxin-2 related disease or disorder is Group 2 PH (CpcPH or IpcPH) with heart failure with reduced ejection fraction (HFrEF). In certain embodiments, the relaxin-2 related disease or disorder is CpcPH with HFrEF. In certain embodiments, the relaxin-2 related disease or disorder is IpcPH with HFrEF.

[0208] In certain embodiments, CpcPH is defined as a pulmonary vascular resistance (PVR) of ≥ 3 Wood units, a mPAP of > 20 mmHg, a PCWP > 15 mmHg, or 34 mL / m 2 Based on a performed right heart catheterization (RHC) showing PCWP > 12 mmHg and ≤ 14 mmHg with echocardiographic evidence of a left atrial volume index (LAVI) of ≥ 12 mmHg. In certain embodiments, IpcPH is defined as PVR < 3 Wood units, mPAP > 20 mmHg, PCWP > 15 mmHg, or 34 mL / m 2 Based on performed RHC showing PCWP >12mmHg and ≤14mmHg with evidence of left atrial volume index (LAVI) on echocardiography.

[0209] In certain embodiments, the relaxin-2 related disease or disorder is a pregnancy-related cardiovascular disease, including but not limited to, pre-eclampsia, postpartum hypertension, postpartum cardiomyopathy, pregnancy-induced heart failure, and maternal hypertension complicating the postpartum period.

[0210] In certain embodiments, the relaxin-2 related disease or disorder is a kidney disease. In certain embodiments, the relaxin-2 related disease or disorder is a chronic kidney disease. In certain embodiments, the relaxin-2 related disease or disorder is a hypertensive kidney disease.

[0211] In certain embodiments, the relaxin-2 related disease or disorder is a joint disease, hi certain embodiments, the relaxin-2 related disease or disorder is periarthritis of the shoulder (also known as adhesive capsulitis).

[0212] Administration of a composition according to the methods described herein may result in a reduction in the severity, signs, symptoms, or markers of a relaxin-2-related disease or disorder in a patient having the disease or disorder. "Reduction" in this context means a statistically significant decrease in such level. The decrease (absolute decrease or reduction in the difference between elevated and normal levels in a subject) can be, for example, at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or less than the detection level of the assay used.

[0213] In certain embodiments, administering a composition according to the methods described herein results in pulmonary vasodilation in a patient. In certain embodiments, administering a composition according to the methods described herein results in an anti-inflammatory effect in a patient. In certain embodiments, administering a composition according to the methods described herein results in an anti-fibrotic effect in a patient. In certain embodiments, administering a composition according to the methods described herein results in right ventricular remodeling in a patient. In certain embodiments, administering a composition according to the methods described herein results in peripheral vasodilation in a patient. In certain embodiments, administering a composition according to the methods described herein results in cardiac relaxation in a patient. In certain embodiments, administering a composition according to the methods described herein results in left ventricular remodeling in a patient. In certain embodiments, administering a composition according to the methods described herein results in improved renal function in a patient.

[0214] In certain embodiments, administration of a composition according to the methods described herein results in an increase in renal plasma flow. In certain embodiments, administration of a composition according to the methods described herein results in an increase in renal plasma flow that is sustained 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 1 month after a single administration. In certain embodiments, the increase in renal plasma flow in a subject is maintained by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 1 month after a single administration. In certain embodiments, administration of a composition according to the methods described herein results in an increase in renal plasma flow that is sustained 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 1 month after a single administration to a human. In certain embodiments, the increase in renal plasma flow in a subject is maintained by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% at 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 1 month after a single administration to a human.

[0215] Combination Therapies and Formulations The present disclosure also provides compositions and therapeutic formulations comprising the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein in combination with one or more additional therapeutically active ingredients (e.g., therapeutic agents), as well as methods of treatment comprising administering such combinations to a subject in need thereof.

[0216] Exemplary additional therapeutic agents include any therapeutic agent that can be used to treat any of the relaxin-2-associated disorders described herein. Exemplary additional therapeutic agents that can be combined or administered in combination with the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein include, but are not limited to, angiotensin II receptor blockers, such as azilsartan, candesartan, eprosartan, and losartan; ACE inhibitors, such as lisinopril, benazepril, captopril, enalapril, moexipril, and perindopril; Quinapril, Trandolapril, Calcium channel blockers such as Amlodipine, Amlodipine and Benazepril, Amlodipine and Valsartan, Sacubitril and Valsartan, Diltiazem, Felodipine, Isradipine, Nicardipine, Nimodipine, Nisoldipine, Verapamil, Diuretics such as Chlorthalidone, Hydrochlorothiazide, Metolazone, Indapamide, Torsemide, Furosemide, Bumetanide, Amiloride, Triamterene, Spironolac aldosterone antagonists, e.g., spironolactone, eplerenone, digoxin, e.g., lanoxin, beta-blockers, e.g., carvedilol, metoprolol, bisoprolol, activin signaling inhibitors, e.g., sotatercept, sodium / glucose cotransporter 2 (SGLT2) inhibitors, e.g., empagliflozin, dapagliflozin, bexagliflozin, canagliflozin, ertugliflozin, ipragliflozin velagliflozin, luseogliflozin, remogliflozin etabonate sergliflozin etabonate, sotagliflozin, tofogliflozin, henagliflozin, janagliflozin, mizagliflozin, velagliflozin proline hydrate, enavogliflozin, and glucagon-like peptide-1 (GLP-1) receptor agonists such as exenatide, liraglutide, albiglutide, dulaglutide, lixisenatide, semaglutide, and tirzepatide.

[0217] In some embodiments, additional therapeutic agents that may be combined or administered in combination with the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein include, but are not limited to, any one or more of sotatercept, empagliflozin, dapagliflozin, sacubitril, valsartan, semaglutide, dulaglutide, and tirzepatide.

[0218] In some embodiments, the additional therapeutic agent is a drug effective in treating fibrosis, including, but not limited to, small molecule drugs and antibodies. Exemplary anti-fibrotic drugs include, but are not limited to, small molecules such as TGF-β inhibitors, e.g., hydronidone, distiertide, or antibodies such as fresolimumab, PDGF or VEGF antagonists, e.g., imatinib, nilotinib, or any drug that targets extracellular factors involved in the pathogenesis of fibrosis. A description of exemplary drugs for fibrosis can be found, for example, in Li et al., "Drugs and Targets in Fibrosis, Frontiers in Pharm." 8:Article 855 (2007), which is incorporated herein by reference.

[0219] The additional therapeutically active ingredient(s) may be administered immediately before, simultaneously with, or immediately after administration of the fusion proteins or component peptides described herein, or nucleic acid molecules, or expression vectors encoding them.

[0220] The present disclosure provides pharmaceutical compositions in which the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are co-formulated with one or more additional therapeutically active ingredient(s) described elsewhere herein.

[0221] Dosing regimen In some embodiments, multiple doses of the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, can be administered to a subject over a predetermined time course. A method according to this aspect of the disclosure includes sequentially administering multiple doses of the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them. As used herein, "sequentially administering" means that each dose of the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, is administered to a subject at different time points, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The disclosure provides methods that include sequentially administering to a patient a single primary dose of the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, followed by one or more secondary doses of the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, optionally followed by one or more tertiary doses of the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them.

[0222] The terms "primary dose," "secondary dose," and "tertiary dose" refer to the time sequence for administration of the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein. Thus, a "primary dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the primary dose, and a "tertiary dose" is a dose administered after the secondary dose. The primary, secondary, and tertiary doses may all contain the same amount of the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein, but generally may differ from one another with respect to frequency of administration. However, in certain embodiments, the amounts of the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, contained in the primary, secondary, and / or tertiary doses may differ from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., two, three, four, or five) doses are administered at the beginning of the treatment regimen as a "loading dose," with subsequent doses administered on a less frequent basis (e.g., "maintenance doses").

[0223] In an exemplary embodiment, each secondary and / or tertiary dose is administered within 1 to 26 (e.g., 1, 1 1 / 2, 2, 2 1 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7, 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2) of the immediately preceding dose. 1 / 2, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks later. The phrase "immediately preceding dose," as used herein, refers to a dose of a fusion protein or component peptide, or nucleic acid molecule, or expression vector encoding thereof, as described herein, in a series of multiple doses, that is administered to a patient prior to administration of the immediately following dose series with no intervening doses.

[0224] In one embodiment, each secondary and / or tertiary dose is administered four weeks after the immediately preceding dose. In another embodiment, doses of the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered to a patient once every four weeks (Q4W).

[0225] Methods according to this aspect of the disclosure may include administering to a patient any number of secondary and / or tertiary doses of the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein. For example, in certain embodiments, only a single secondary dose is administered to a patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) secondary doses are administered to a patient. Similarly, in certain embodiments, only a single tertiary dose is administered to a patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) tertiary doses are administered to a patient.

[0226] In embodiments involving multiple secondary doses, each secondary dose may be administered with the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered with the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The administration frequency may also be adjusted by the physician during the course of treatment depending on the needs of the individual patient after clinical testing.

[0227] In one embodiment, one or more of the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered to a subject as a weight-based dose. A "weight-based dose" (e.g., a dose in mg / kg) is a dose of a protein or peptide that varies depending on the weight of the subject.

[0228] In another embodiment, one or more of the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered to a subject as a fixed dose. A "fixed dose" (e.g., a dose in mg) means that one dose of the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein is used for all subjects, regardless of any particular subject-related factors, such as body weight. In one particular embodiment, the fixed dose of the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein, is based on a predetermined weight or age.

[0229] Generally, suitable doses of the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, can be in the range of about 0.001 to about 200.0 milligrams per kilogram of recipient body weight, generally in the range of about 1 to 50 mg per kilogram of recipient body weight. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered in the range of about 0.001 mg / kg to about 200 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered in the range of 0.001 mg / kg to 200 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered in the range of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered in the range of 0.01 mg / kg to 100 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered in the range of about 0.1 mg / kg to about 20 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered in the range of 0.1 mg / kg to 20 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered in the range of about 1 mg / kg to about 50 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered in the range of 1 mg / kg to 50 mg / kg.For example, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, can be administered at about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg per single dose. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, may be administered at 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg per single dose. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of this disclosure.

[0230] In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered at about 0.3 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered at 0.3 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered at about 1 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered at 1 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered at about 3 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered at 3 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered at about 10 mg / kg. In certain embodiments, the fusion proteins or component peptides described herein, or nucleic acid molecules, or expression vectors encoding them, are administered at 10 mg / kg.

[0231] In some embodiments, one or more of the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered as a fixed dose of about 10 mg to about 2500 mg. In some embodiments, one or more of the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered as a fixed dose of 10 mg to 2500 mg. In some embodiments, one or more of the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered as a fixed dose of about 100 mg to about 1500 mg. In some embodiments, one or more of the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered as a fixed dose of 100 mg to 1500 mg. In some embodiments, the fusion proteins or component peptides described herein, or nucleic acid molecules, or expression vectors encoding them, are administered in an amount of about 10 mg, about 15 mg, about 20 mg, 25 mg, about 30 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, g, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1500 mg, about 2000 mg, or about 2500 mg.In some embodiments, the fusion proteins or component peptides described herein, or nucleic acid molecules, or expression vectors encoding them, are administered in amounts of 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 4 and administered as a fixed dose of 25 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, 1000 mg, 1500 mg, 2000 mg, or 2500 mg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of this disclosure.

[0232] In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of about 150 mg. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of 150 mg. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of at least 150 mg. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of about 300 mg. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of 300 mg. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of about 600 mg. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of 600 mg.

[0233] In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered by intravenous administration. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered by subcutaneous administration.

[0234] In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered by intravenous infusion. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered by intravenous infusion for 1 minute, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered by intravenous infusion over a period of 30 minutes. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered by intravenous infusion over a period of 60 minutes. In some embodiments, the fusion proteins or component peptides described herein, or nucleic acid molecules, or expression vectors encoding them, are administered by intravenous infusion over a period of 30 to 60 minutes.

[0235] In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered intravenously at a dose of about 0.3 mg / kg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered intravenously at a dose of 0.3 mg / kg once every four weeks. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered intravenously at a dose of 0.3 mg / kg once every four weeks. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered at 0.3 mg / kg once every four weeks. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered at about 1 mg / kg once every four weeks. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered at 1 mg / kg once every four weeks. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered at about 3 mg / kg once every four weeks. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered at 3 mg / kg once every four weeks. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered at about 10 mg / kg once every four weeks. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered at 10 mg / kg once every four weeks.

[0236] In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered at a dose of about 150 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered at a dose of 150 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered at a dose of at least 150 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of about 150 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of at least 150 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of at least 150 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of about 300 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of 300 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of about 600 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of 600 mg once every four weeks.

[0237] In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered subcutaneously at a dose of about 150 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered subcutaneously at a dose of 150 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered subcutaneously at a dose of at least 150 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered subcutaneously as a flat subcutaneous dose of about 150 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered subcutaneously as a flat subcutaneous dose of 150 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered as a subcutaneous flat dose of at least 150 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered as a subcutaneous flat dose of about 300 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered as a subcutaneous flat dose of 300 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered as a subcutaneous flat dose of about 600 mg once every four weeks. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered as a subcutaneous flat dose of 600 mg once every four weeks.

[0238] In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered intravenously at a dose of about 0.3 mg / kg once per month. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered intravenously at a dose of 0.3 mg / kg once per month. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered intravenously at a dose of about 0.3 mg / kg once per month. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered once per month at 0.3 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered once per month at about 1 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered once per month at 1 mg / kg. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered at about 3 mg / kg once a month. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered at 3 mg / kg once a month. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered at about 10 mg / kg once a month. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered at 10 mg / kg once a month.

[0239] In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered once a month in a dose of about 150 mg. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered once a month in a dose of 150 mg. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered once a month in a dose of at least 150 mg. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered once a month as a fixed dose of about 150 mg. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered once a month as a fixed dose of 150 mg. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered once a month as a fixed dose of at least 150 mg. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of about 300 mg once per month. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of 300 mg once per month. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of about 600 mg once per month. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered as a fixed dose of 600 mg once per month.

[0240] In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered subcutaneously at a dose of about 150 mg once per month. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered subcutaneously at a dose of 150 mg once per month. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered subcutaneously at a dose of at least 150 mg once per month. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered subcutaneously as a flat subcutaneous dose of about 150 mg once per month. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, are administered subcutaneously as a flat subcutaneous dose of 150 mg once per month. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered as a subcutaneous fixed dose of at least 150 mg once per month. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered as a subcutaneous fixed dose of about 300 mg once per month. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered as a subcutaneous fixed dose of 300 mg once per month. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered as a subcutaneous fixed dose of about 600 mg once per month. In some embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, described herein are administered as a subcutaneous fixed dose of 600 mg once per month.

[0241] kit Any of the compositions described herein can be included in a kit. In a non-limiting example, the kit includes one or more of the fusion proteins or component peptides described herein, or nucleic acid molecules, or expression vectors encoding them.

[0242] The kit may further include reagents or instructions for using the fusion proteins or component peptides, or nucleic acid molecules described herein, or expression vectors encoding them, in a subject. It may also include one or more buffers.

[0243] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit (such as when the labeling reagent and label are packaged together), the kit will also generally include a second, third, or other additional container into which the additional component may be placed separately. The kit may also include a second container means for containing a sterile, pharmaceutically acceptable buffer and / or other diluent. However, various combinations of components may also be included in the vial. The kits of the present disclosure will also typically include a means for containing the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them, as described herein, and any other reagent containers in close confinement for commercial sale.

[0244] When the components of the kit are provided in one or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. However, the components of the kit may also be provided as dry powder(s). When the reagents and / or components are provided as dry powders, the powders can be reconstituted by adding a suitable solvent. It is contemplated that the solvent may also be provided in a separate container means. [Example]

[0245] The examples of the present disclosure are provided by way of illustration and description and are not intended to limit the scope of the disclosure. The results described in each example reflect the specific conditions outlined in the experiments described herein.

[0246] Example 1. Heparin chromatography for relaxin-2 fusion protein analogs Heparin chromatography is a method that can be used in early candidate screening to better understand the tendency of molecules to interact with elements of the vasculature when administered to patients. Heparin and heparin sulfate proteoglycans are negatively charged polysaccharides present in the vasculature and tissues, to which positively charged molecules can bind at physiological pH (i.e., pI > 7.4). Here, heparin chromatography was used to screen candidates / variants with reduced heparin binding, which predicts favorable PK properties. The materials used for heparin chromatography are provided in Table 11. [Table 11]

[0247] method Mobile phase A (binding): 20 mM Tris pH 7.4, Mobile phase B (elution): 20 mM Tris pH 7.4 + 1 M NaCl, injection: 10 μg, detection: 220 nm. 1. Prior to analysis, the heparin column was equilibrated with mobile phase A at 0.5 mL / min for 10 min. 2. Samples for analysis were diluted to 1 mg / mL in 20 mM Tris pH 7.4 to minimize ionic strength. 3. The heparin chromatography method was performed on an Agilent HPLC using the gradient shown in Table 12 below. [Table 12] 4. A positive control (no heparin binding, human IgG pool) and a negative control (SE301 or AT1R) were included. 5. Samples were analyzed for retention time and reported relative retention time compared to the positive control (i.e., RT sample / RT positive control). 6. The approximate concentration of NaCl required for elution was calculated using the following calculation:

number

[0248] The results of the calculations are shown in Table 13. [Table 13]

[0249] result Table 14 shows the heparin chromatography results for various relaxin-2 analog fusion proteins. [Table 14]

[0250] The IgG was from Jackson ImmunoResearch (catalog number 009-000-003). The "previous fusion protein" is a LALA IgG-RelB-linker-RelA fusion with a theoretical pI of 8.5 but an experimentally determined pI of 9.4. The linker protein contains only one acidic amino acid. SEQ ID NOS: 300, 302, 303, 305, 306, and 308-311 have linker proteins containing at least two acidic amino acids, as well as LALA IgG (SEQ ID NOS: 77 and 81). The last two fusion proteins have linker proteins containing only one acidic amino acid and have higher theoretical pIs. As shown in Table 14 above, there is a correlation between a lower pI found through heparin chromatography and lower nonspecific binding.

[0251] Example 2. Low pI Relaxin-2 Fusion Protein Analogs Prone to Reduced Self-Association as Measured by Affinity Capture Self-Interacting Nanoparticle Spectroscopy (AC-SINS) When assessing the biophysical properties of development candidates, it is important to understand the tendency of a molecule to self-associate. To assess the tendency of a molecule to self-associate, concentrate the molecule to a high concentration and assess it by SEC (% monomer) or measure the change in turbidity (OD 340 nm) and the second virial coefficient (B) using DLS. 22 ) or the self-interaction coefficient (k d ) or AC-SINS(Δλ max There are many methods for using ion capture (AC-SINS). All three methods provide useful information but use different amounts of material to perform the assessment. AC-SINS has emerged as a high-throughput method for assessing self-association using minimal materials, yet still provides high localized concentrations by using affinity capture on gold nanoparticles. Briefly, gold nanoparticles are pre-coated with anti-human antibodies (Fc, Fab, and H+L), which, when incubated with the target antibody in dilute solution, capture the antibody of interest and concentrate it in solution. When the immobilized molecules of interest interact, the interparticle distance between the gold nanoparticles decreases, resulting in an increased plasmon wavelength (i.e., red-shift) that can be quantified using UV-VIS spectroscopy. The materials used for spectroscopy are provided in Table 15. [Table 15]

[0252] method Preparation of buffer solution: To prepare 20 mM sodium acetate pH 4.3, 2 mL of 1 M sodium acetate pH 4.3 stock was diluted to 100 mL with MilliQ water. The pH was measured at 4.3 ± 0.1, and the solution was sterile filtered. The solution remained stable at room temperature for one month. To 1 g of PEG methyl ether thiol, 10 mL of MilliQ water was added. This was vortexed briefly to suspend the solid, creating a 50 mM solution. To prepare a 10 μM solution for the final dilution, the following dilution scheme was followed: a. Dilute the 50 mM stock to 1 mM (20 μL of 50 mM stock + 980 μL of MilliQ water) b. Dilute the 1 mM step to 100 μM (10 μL of 1 mM stock + 90 μL of MilliQ water) c. Dilute the 100 μM step to 10 μM (100 μL of 100 μM stock + 900 μL of MilliQ water) d. Volumes can be scaled depending on the number of samples to be assayed e. The remaining 50 mM stock should be aliquoted and kept at -20°C until needed.

[0253] Preparation of gold nanoparticle solution: Goat anti-human Fc IgG antibody (capture) and goat IgG antibody (non-capture) were buffer-exchanged into 20 mM sodium acetate, pH 4.3. After buffer exchange, the concentrations of both antibodies were normalized to 0.4 mg / mL. A 4:1 volumetric mixture of capture (anti-Fc):non-capture (goat IgG) solutions was prepared for an 80% capture capacity coating solution used to incubate gold nanoparticles (AuNPs).

[0254] A 9:1 volume ratio of AuNP to coating solution was prepared. The solution was incubated overnight at room temperature in the dark. After incubation, thiolated PEG was added to a final concentration of 0.1 μM from a diluted 10 μM stock to block vacant sites on the AuNP (i.e., 5 mL of AuNP solution was added to 50 μL of the 10 μM stock) and incubated for 1 hour at room temperature in the dark.

[0255] Preparation of AuNP solution: 2 mL of coated AuNP solution was centrifuged at 20,000 × g for 15 min to precipitate the AuNPs, and 1800 μL of the supernatant was carefully removed using a 1 mL pipette. The pelleted AuNPs were gently resuspended using a 200 μL pipette to produce a 10x concentrated stock of coated AuNPs.

[0256] Preparation of target antibody solutions (follow this procedure for both methods): For each sample analyzed, 10 μL of AuNP concentrate was incubated with 100 μL of antibody test solution (normalized to 0.05 mg / mL) in a 96-well polypropylene plate for 2 hours in the dark at room temperature. For the purpose of blanking the assay and determining the wavelength shift upon addition of the test antibody, two blank solutions were prepared: 10 μL of 10× AuNP concentrate in 100 μL of PBS. Ganitumab was included as a positive control (high association, red shift), and panitumumab was included as a negative control (non-association, no UV shift). Each sample was prepared in duplicate for analysis. After 2 hours of incubation, 100 μL of the resulting solution was transferred to a UV-transparent polystyrene plate (384-well format). The two blank solutions were transferred to properly assess the wavelength shift, and then duplicate standards and samples were added for analysis. The plate was then centrifuged at 1000 × g for 1 min to level the solution in the wells. Absorbance data were collected from 510 nm to 570 nm in 2 nm steps, and the wavelength shift was determined for each sample relative to AuNPs alone.

[0257] result The results from ASCINS are shown in Table 16 below. [Table 16]

[0258] As shown in Table 16 above, fusion proteins with low pI also have a reduced tendency to exhibit self-aggregation.

[0259] Example 3. Relaxin-2 fusion protein analogs induce cAMP responses in RXFP1 transfected cells This example provides data on the efficacy of various relaxin-2 fusion protein analogs described herein. The efficacy of the fusion protein analogs was assayed by testing their ability to activate RXFP1 by measuring cAMP signaling.

[0260] method HEK293 cells were seeded in 96-well tissue culture plates and subsequently transiently co-transfected with human RXFP1 and pGloSensor-22F plasmids. Transfected cells were stimulated with relaxin-2 or its fusion protein analog to induce Gs-mediated cAMP signaling. cAMP was assayed using the activity of the GloSensor biosensor, a mutant luciferase fused to a cAMP-binding domain, leading to the production of light in the presence of its substrate, luciferin. This relative light unit (RLU) readout was used as a proxy for the cAMP response. reagent 96-well tissue culture-treated plate, white with clear bottom. (Corning #3610) ·HEK293 cells (ATCC CRL-1573) Poly-D-lysine (Gibco A3890401) DPBS (calcium-free, magnesium-free, Gibco 14190250) DMEM (high glucose with L-glutamine and sodium pyruvate, Gibco 11995065) ·TrypLE Express(Gibco 12605010) FBS (HyClone™, Australian source, Cytiva SH30084) Penicillin-streptomycin (Gibco 15140122) ·CO2 independent medium (Gibco 18045088) Opti-MEM™ I Reduced Serum Medium (Gibco 31985062) pGloSensor™-22F cAMP Plasmid (Promega Catalog No. E2301) D-Luciferin, potassium salt (GoldBio LUCK-1G) FuGENE HD Transfection Reagent (Promega #E2311) Reservoir (Corning / Axygen RES-V-25-SI) Relaxin-2 (R&D Biosystems 6586-RN-025) RXFP1-containing plasmid (pcDNA5 / FRT / TO-human RXFP1, full-length) Forskolin (Sigma F6886) Plate reader capable of reading luminescence (CLARIOstar Plus)

[0261] Preparation of reagents D-Luciferin, potassium salt: D-luciferin was reconstituted in 10 mM HEPES, pH 7.5 at 25 mg / mL (78.5 mM; MW = 318.4), aliquoted into disposable aliquots of approximately 200–500 µL in sterile microfuge tubes, and stored at -80 °C.

[0262] Relaxin-2 peptide: Sterile DPBS (MW = 5,986 Da, ε = 12,865 M -1 cm -1 ) and reconstitute the relaxin-2 peptide or relaxin-2 fusion protein analog at 0.1 mg / mL in A 280 The final concentration was determined by measuring the aliquots at -20°C.

[0263] Forskolin: Forskolin was reconstituted in 100% DMSO at 5 mM (2.05 mg / mL, MW=410.5). Aliquots were stored at -20°C.

[0264] cAMP Assay Medium: CO2-independent medium was pre-warmed to 37°C using a bead bath. A single aliquot of D-luciferin was thawed and added to a final concentration of 5% (e.g., 4.75 mL of cAMP Assay Medium + 250 μL of D-luciferin stock, yielding a final D-luciferin concentration of 1.25 mg / mL or 3.93 mM). This was used the same day or discarded.

[0265] Cell culture and maintenance HEK293 cells (ATCC CRL-1573) were cultured in DMEM + 10% FBS, 1% (1X or 10 U / mL) Pen-Strep in a humidified CO2 incubator at 37°C with 5% CO2 to 80-100% confluency. Cells were typically split 1:6 for 3 days and maintained in sterile T-75 tissue culture flasks.

[0266] cAMP Signaling Assay Protocol This protocol was adapted from the GloSensor cAMP assay by Promega.

[0267] Raw data was exported to Excel using MARS data analysis software, which was opened after a run on the CLARIOstar plate reader. These values ​​are measured in RLU, or relative luminescence units.

[0268] As shown in Table 17, all of the low pI relaxin-2 fusion protein analogs were able to induce a cAMP response in RXFP1 transfected cells. [Table 17]

[0269] Example 4. In vitro characterization of relaxin-2 fusion protein analogs This example provides in vitro characterization of various relaxin-2 fusion protein analogs described herein.

[0270] method Heparin Chromatography: Heparin chromatography was performed to understand the propensity of relaxin-2 fusion protein analogs to interact with elements of the vasculature and / or rapidly distribute to tissues when administered to patients. Analogs found to bind weakly to heparin may predict favorable pharmacokinetic properties. Briefly, prior to analysis, the heparin column was equilibrated at 0.5 mL / min for 10 minutes using mobile phase A (20 mM Tris pH 7.4). 10 μg per sample was run using the heparin chromatography method on an Agilent HPLC with detection at 280 nm using the gradient shown in Table 18 below (mobile phase B: 20 ​​mM Tris pH 7.4, 1 M NaCl). [Table 18]

[0271] A positive control (no heparin binding, pembrolizumab) and a negative control (mild heparin binding, adalimumab) were included and samples were analyzed for retention time and relative retention time compared to the positive control (i.e., RT sample / RT positive control). The approximate concentration of NaCl required for elution was calculated using the following calculation:

number

[0272] Hydrophobic Interaction Chromatography (HIC): HIC is a chromatographic method that separates molecules based on their hydrophobicity. 10 μg of protein was injected onto a butyl HIC column pre-equilibrated with a high ammonium sulfate buffer. Protein was eluted with a gradient from high to low salt over 10 minutes. Samples were compared for hydrophobicity based on retention time, with higher retention times indicating higher hydrophobicity and lower retention times indicating lower hydrophobicity. Retention times were converted to approximate salt concentrations at elution and compared against high and low hydrophobicity standards.

[0273] Size Exclusion Chromatography (SEC): SEC is a liquid chromatography method used to determine the levels of monomeric and multimeric species in a solution of a given analyte. SEC was used to assess the presence of fusion protein aggregates. Samples were prepared and loaded onto a 1.7 μm particle SEC column with an aqueous mobile phase composed of 25 mM potassium phosphate and 0.5 M potassium chloride (pH 8.0). Once the sample eluted, the method was able to quantify the level of soluble aggregate species in the sample with high resolution between the monomer peak and high molecular weight (HMW) species. The percentage of monomer (i.e., % monomer) and other species (e.g., HMW species, low molecular weight species) were calculated by integrating the corresponding elution curves to determine area percent.

[0274] Capillary isoelectric focusing (cIEF): Imaged cIEF was used to separate differentially charged molecules (i.e., relaxin-2 fusion protein analogs) using electrophoretic mobility in ampholyte solutions to determine their isoelectric points (pI). Molecules were loaded into a capillary and separated based on their pI by allowing them to migrate along an electric field until they reached a pH corresponding to their pI. UV absorption across the capillary was measured throughout the separation, allowing for real-time observation and eventual quantification.

[0275] Baculovirus particle (BVP) ELISA: BVP ELISA was used to understand the propensity of relaxin-2 fusion protein analogs for nonspecific or nontarget interactions. BVPs are empty viral capsids that lack the viral genome, but during production, they can bud from the cell membrane, taking with them membrane components. Therefore, BVPs have a highly diverse cell surface with many moieties that mimic those that the molecule of interest (i.e., the relaxin-2 fusion protein analog) may encounter in vivo. Briefly, BVP was coated onto plates by adding 25 μL of BVP solution to each well. BVP solution was made by diluting BVP stock (Medna Scientific; catalog number E3001) to 1 × 10 PFU / mL with 0.1 M carbonate buffer, pH 9.6. After overnight incubation at 5°C, the BVP solution was blotted from the wells, and the wells were washed three times with PBST. Plates were blocked with 100 μL / well of 1× BSA in PBS blocking buffer (Cepham Life Sciences; catalog number 10615). Plates were incubated at 25°C on a plate shaker for 1 hour. The blocking solution was blotted from the wells, and the wells were washed three times with PBST. Samples (i.e., relaxin-2 fusion protein analogs) were prepared in duplicate to cover a dilution range of 3 μM to 0.1 nM and added to the plate. Plates were incubated at 25°C for 1 hour, after which the wells were blotted and washed three times with PBST. 25 μL / well of a 1:10,000 diluted detection monoclonal antibody (Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ fragment specific; Jackson ImmunoResearch; catalog number 50-194-1564) was added, and the plate was incubated for 1 hour at 25°C, after which the wells were blotted and washed three times with PBST. 1-Step™ Ultra TMB-ELISA Substrate Solution (Life Technologies, catalog number 34029) was then added.After approximately 2 minutes, the reaction was quenched by adding 25 μL of 2N HCl and the plate was analyzed using a plate reader at 450 nm with correction at 570 nm.

[0276] Potency Assay: HEK293 cells were seeded in 96-well tissue culture plates and subsequently transiently co-transfected with human RXFP1 and pGloSensor-22F plasmids. Transfected cells were stimulated with relaxin-2 or its fusion protein analog to induce Gs-mediated cAMP signaling. cAMP was assayed using the activity of the GloSensor biosensor, a mutant luciferase fused to a cAMP-binding domain, leading to the production of light in the presence of its substrate, luciferin. This relative light unit (RLU) readout was used as a proxy for the cAMP response.

[0277] cAMP Signaling Assay Protocol: This protocol is adapted from the GloSensor cAMP assay by Promega. Raw data was exported to Excel using MARS data analysis software, which was opened after running on a CLARIOstar plate reader. These values ​​are measured in RLU, or relative luminescence units.

[0278] Affinity Capture Self-Interaction Nanoparticle Spectroscopy (AC-SINS): AC-SINS was performed to understand the tendency of molecules (i.e., relaxin-2 fusion protein analogs) to self-associate. Briefly, gold nanoparticles were pre-coated with anti-human antibodies (Fc, Fab, and H+L), which, when incubated with target antibodies in dilute solution, capture the antibodies of interest and concentrate them in solution. When the immobilized molecules of interest interact, the interparticle distance between the gold nanoparticles decreases, resulting in an increased plasmon wavelength (i.e., a red shift) that can be quantified using UV-VIS spectroscopy. The materials used for spectroscopy are provided in Table 20. [Table 20]

[0279] Goat anti-human Fc IgG antibody (capture) and goat IgG antibody (non-capture) were buffer-exchanged into 20 mM sodium acetate, pH 4.3. After buffer exchange, the concentrations of both antibodies were normalized to 0.4 mg / mL. A 4:1 volumetric mixture of capture (anti-Fc):non-capture (goat IgG) solutions was prepared for an 80% capture capacity coating solution used to incubate gold nanoparticles (AuNPs). A 9:1 volumetric ratio of AuNPs:coating solution was created. The solution was incubated overnight at room temperature in the dark. After incubation, thiolated PEG was added from a diluted 10 μM stock to a final concentration of 0.1 μM to block vacant sites on the AuNPs (i.e., 5 mL of AuNP solution was added to 50 μL of the 10 μM stock) and incubated in the dark for 1 hour at room temperature.

[0280] Two milliliters of the coated AuNP solution was centrifuged at 20,000 x g for 15 minutes to precipitate the AuNPs, and 1,800 µL of the supernatant was carefully removed using a 1 mL pipette. The pelleted AuNPs were gently resuspended using a 200 µL pipette to generate a 10x concentrated stock of coated AuNPs. For each sample analyzed, 5 µL of the AuNP concentrate was incubated with 45 µL of antibody test solution (normalized to 0.05 mg / mL) in a 384-well polypropylene plate for 2 hours at room temperature in the dark. After the 2-hour incubation, absorbance data were collected from 450 nm to 650 nm in 1 nm steps to determine the wavelength shift for each sample compared to AuNPs alone.

[0281] Nanoscale Differential Scanning Fluorescence (NanoDSF): NanoDSF was performed using a NanoTemper Prometheus Panta to investigate the conformational stability of relaxin-2 protein fusion analogs. A heat lamp was applied to a solution containing the molecule of interest, and intrinsic fluorescence, backscattering, and dynamic light scattering (DLS) were used to determine the temperature at which the fusion protein begins to unfold (T onset ), the temperature at which half of the fusion protein in a given sample is unfolded (Tm 1), and the temperature at which fusion protein aggregation begins (T agg The structural stability was measured by providing various thermal stability parameters including

[0282] Sequences: The sequences of relaxin-2 fusion protein analogs are set forth throughout this disclosure. SEQ ID NOs: 496, 497, and 501 are shown below: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGKGGSDSWKEEVIKLCGRELVRAQIAICGKSTASDAAGANANAGARQLYSALANKCCHVGCTKRSLARFC (SEQ ID NO: 496), DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQLYSALANKCCHVGCTKRSLARFCGGGGSGGGGSGGGGSSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ ID NO: 497), and MPRLFFFHLLGVCLLLNQFSRAVADSWMEEVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSEMQPALPQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSSPSELKYLGLDTHSRKKRQLYSALANKCCHVGCTKRSLARFC (SEQ ID NO: 501).

[0283] result The results are shown in Tables 21, 22, and 23 below. [Table 21] TIFF2026501550000202.tif193145 [Table 22] [Table 23]

[0284] All samples in Tables 21, 22, and 23 are LALA PA LS IgG-RelB-linker-RelA fusions containing wild-type human relaxin-2 and LALA PA LS IgG (SEQ ID NO: 79 or 83), except for SEQ ID NO: 497. As shown above in Tables 21, 22, and 23, there is a correlation between the lower pI and lower nonspecific binding found via heparin chromatography.

[0285] Confirmatory AC-SINS and BVP ELISA assays, and additional cAMP potency assays, were performed on a subset of relaxin-2 fusion protein analogs and the results are shown in Table 24. [Table 24]

[0286] A subset of relaxin-2 fusion protein analog samples (SEQ ID NO:496, SEQ ID NO:313, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:95, and SEQ ID NO:104) was tested under various development feasibility assessments. Under high concentration stress (target concentration of approximately 100 mg / mL), none of the samples showed a loss of UV-based protein concentration. Heat stress induced an increase in turbidity / opalescence for all samples tested. Agitation stress had no effect on SEQ ID NO:313 and SEQ ID NO:104 samples. Under chemical stress (target concentration of approximately 5 mg / mL), SEQ ID NO:496 and SEQ ID NO:90 samples showed a decrease in concentration for all chemical stresses tested, while SEQ ID NO:313, SEQ ID NO:87, SEQ ID NO:95, and SEQ ID NO:104 samples appeared stable. All samples showed an oxidation-induced decrease in concentration, with the oxidized SEQ ID NO:496 and SEQ ID NO:90 samples showing an increase in high molecular weight species detected by size exclusion chromatography. Additionally, based on the non-reduced capillary electrophoresis samples (CE-SDS NR), no significant fragmentation was observed in response to any stress in any of the samples tested.

[0287] The formation of stress-induced post-translational modifications (PTMs) was also examined. Stresses included 4 weeks of incubation at 40°C, 2 weeks of incubation at room temperature and high pH (Tris buffer pH 8), 2 weeks of incubation at room temperature and low pH (glycine buffer pH 3), and 24 hours of incubation under room temperature oxidative stress (0.02% hydrogen peroxide). SEQ ID NOs: 313 and 87 did not show any stress-induced modifications, SEQ ID NOs: 90 and 95 showed aspartate isomerization in the linker region, SEQ ID NO: 496 showed asparagine deamidation in the linker region, and SEQ ID NOs: 95 and 104 showed aspartate isomerization in the relaxin sequence.

[0288] During manufacturability evaluation, color changes and aggregation with multiple molecules were observed under certain stress conditions, which are common in oxidized proteins. The color change typically occurs due to tryptophan oxidation, which causes changes in absorbance at 320 nm and 365 nm (Ambrogelly (2021) Antibodies 10(2):21). To investigate the light sensitivity of the relaxin-2 fusion protein analogs SEQ ID NO:522 and SEQ ID NO:523, a light box was created using a benchtop 25°C incubator with a clear glass door. A photometer was attached inside the incubator, and a rectangular LED with an adjustable light intensity switch was placed outside the incubator. The light intensity was adjusted to 1000 lux, similar to the light intensity in a standard manufacturing room. Samples were placed in clear glass vials with 200 μL glass inserts to allow for maximum surface area for light exposure while maintaining minimal sample volume requirements. Relaxin-2 fusion protein analogs SEQ ID NO:522 and SEQ ID NO:523 were incubated at 25°C, 1000 lux for 7 or 14 days. Samples were subjected to heat stress via incubation at 40°C for 7 or 14 days. After exposure to light or heat, samples were evaluated using size-exclusion high performance liquid chromatography (SEC) to assess aggregate formation and CE-SDS to assess purity. As shown in Table 25, under these conditions, both SEQ ID NO:522 and SEQ ID NO:523 are resistant to heat and light stress, as assessed by aggregate formation and purity. [Table 25]

[0289] Another study was conducted to examine the in vitro potency of SEQ ID NO: 87 compared to wild-type (WT) human relaxin-2 using mammalian cell lines either transiently, stably, or endogenously expressing human RXFP1 or orthologues from cynomolgus monkey and rat. Table 26 shows a summary of the cAMP responses induced by SEQ ID NO: 87 and wild-type human relaxin-2 in HEK293 cells transiently expressing human, monkey, and rat RXFP1. As shown in Table 26 and Figures 1A-1C, the average EC50 of SEQ ID NO: 87 for human (Figure 1A), rat (Figure 1B), and monkey (Figure 1C) RXFP1 was 10±4 nM, 10±9 nM, and 30±20 nM, respectively. [Table 26]

[0290] To test the selectivity of SEQ ID NO: 87, CHO-K1 cells stably expressing human RXFP1 or human RXFP2 were used. As shown in Table 27, the EC of SEQ ID NO: 87 for human RXFP1 50 The EC value was 40±20 nM, approximately 100-fold less potent for human RXFP2 (EC 50 is ≧2000 nM), indicating that SEQ ID NO: 87 is selective for RXFP1. [Table 27]

[0291] In some cases, transient or stable ectopic expression of protein in cells may result in overexpression of target, which may affect the efficacy and effectiveness of test samples.To address this, the efficacy of SEQ ID NO: 87 was tested in the human leukemia monocytic cell line THP-1, which endogenously expresses RXFP1.As shown in Table 27, the EC50 of SEQ ID NO: 87 was found to be 10±6nM, which is consistent with that found in the above-mentioned in vitro RXFP1 efficacy assay.

[0292] Example 5. Pharmacokinetic (PK) and Pharmacodynamic (PD) Properties of Relaxin-2 Fusion Protein Analogs Pharmacokinetic (PK) values ​​were determined by measuring the concentration of relaxin-2 fusion protein analogs in rat plasma over time after intravenous (IV) injection of 5 mg / kg of each protein analog. PK values ​​were determined for a subset of relaxin-2 fusion protein analog samples described herein (SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO:367, SEQ ID NO:313, SEQ ID NO:87, SEQ ID NO:95, SEQ ID NO:90, and SEQ ID NO:104) ( FIG. 2A ). PK parameters in rats are shown in Table 28 below. [Table 28]

[0293] Figure 2B shows another experiment with PK values ​​determined using the same method as described in Figure 2A for a subset of relaxin-2 fusion protein analog samples described herein (SEQ ID NO: 87, SEQ ID NO: 496, and SEQ ID NO: 497).

[0294] Figure 2C shows another experiment with PK values ​​determined using the same method as described in Figure 2A for a subset of relaxin-2 fusion protein analog samples described herein (SEQ ID NO: 522 and SEQ ID NO: 523).

[0295] Example 6. Hemodynamic and renal blood flow effects of relaxin-2 fusion protein analogs The high isoelectric point (pI) of relaxin and related molecules presents significant pharmacokinetic (PK) and biophysical challenges, reflected in the rapid decline observed in serum concentrations of these molecules at the earliest time points in the PK curve. Without being bound by any theory, this high clearance phenomenon has been attributed to nonspecific binding of high pI molecules to negatively charged heparin proteoglycans in the vasculature and tissues. These issues were resolved through structure-guided engineering of relaxin-2 fusion protein analogs to reduce their pI, as shown in Examples 1-5.

[0296] To assess the impact of the change to a lower pI, the PK and pharmacodynamic (PD) effects of relaxin-2 fusion protein analogs were measured in rats. One of the most easily quantifiable activities of relaxin is that it produces an observable increase in renal artery blood flow (RABF) immediately after administration. This is a PD effect that has been shown to be observable in both rats and human patients administered serelaxin, and can be modeled to establish the PK / PD relationship of test compounds.

[0297] Naive male Sprague-Dawley rats (weight range: 0.308-0.399 kg) were anesthetized via 5% isoflurane driven by 100% oxygen in an induction chamber. Once unconscious, the animals were removed from the chamber and an endotracheal tube was inserted for mechanical positive pressure ventilation. The ventilator was connected to a vaporizer delivering approximately 1-2% isoflurane driven by 100% oxygen for the duration of the experiment. Depth of anesthesia was assessed preoperatively and approximately every 15 minutes during the experimental procedure. Animals were maintained at approximately 37 ± 0.5 °C on a heating pad, and body temperature was monitored throughout the protocol using a rectal temperature probe.

[0298] A Millar pressure catheter was placed in the right carotid artery to measure systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and heart rate (HR). Mean arterial pressure (MAP) was calculated. A small incision was made along the linea alba to access the abdominal cavity. The left renal artery was dissected, and a Doppler flow probe was placed around the artery. Renal artery blood flow (RABF) was continuously monitored throughout the experimental period, and renal vascular resistance (RVR = MAP / RABF) was calculated.

[0299] After an approximately 10-15 minute equilibration period, baseline (BL) measurements were collected for 15 minutes. Individual animals were deemed acceptable for use in the study based on health status, body weight, hemodynamic parameters, and renal blood flow. After BL measurements, rats received a bolus intravenous (IV) administration (Dose 1) of either vehicle (10 mM histidine, 50 mM NaCl, 6.5% trehalose in deionized water, pH 6.0) or a relaxin-2 fusion protein analog (test compound). Immediately following the bolus IV dose, rats in each group received a maintenance dose via IV infusion (Dose 2) of the respective test compound over a 180-minute dosing period.

[0300] Blood samples were collected prior to Dose 1 (before the end of BL) and 5 minutes, 1 hour, 2 hours, and 3 hours after the start of Dose 2. Samples were obtained from the jugular vein cannula into K2EDTA tubes. The tubes were stored on wet ice until centrifugation in a refrigerated centrifuge. The resulting plasma was frozen on dry ice and stored at -80°C. At the end of the study, rats were euthanized by exsanguination.

[0301] Mean values ​​obtained from the 15-minute block during BL and the 180-minute dose period were used for analysis. Values ​​from each individual animal were pooled to determine the mean for each variable for each group (where applicable). The mean percent change from baseline values ​​was determined for each variable. The term "dosing period" refers to the period during the bolus and maintenance dose infusions (180 minutes) and is used throughout the remainder of this report.

[0302] In one experiment, the effects of relaxin-2 fusion protein analogs SEQ ID NO: 313 and SEQ ID NO: 87 on renal blood flow in rats were compared to the previous fusion protein SEQ ID NO: 496. Table 29 below shows the efficacy of relaxin-2 fusion protein analogs in recombinant human and rat RXFP1 assays (as described in Example 4). [Table 29]

[0303] As shown in Figure 3, administration of SEQ ID NO: 313 and SEQ ID NO: 87 at a 0.3 mg / kg bolus intravenous dose and 0.2 mg / kg / hour intravenous infusion caused a greater increase in rat RABF than administration of the previous fusion protein SEQ ID NO: 496 at a 0.3 mg / kg bolus intravenous dose and 0.5 mg / kg / hour intravenous infusion. Thus, despite the reduced in vitro potency observed for SEQ ID NO: 313 and SEQ ID NO: 87, these fusion proteins exhibit a greater increase in rat RABF.

[0304] Additional experiments were performed to further evaluate the effect of fusion protein analogs SEQ ID NO: 87 and SEQ ID NO: 497 on renal blood flow. In these experiments, the mean potencies of relaxin-2 fusion protein analogs in the recombinant human and rat RXFP1 assays are shown in Table 30 below. [Table 30]

[0305] SEQ ID NO: 87 was infused via a cannulated femoral vein using a syringe pump. The dose was infused intravenously (1 ml / kg) as a bolus, followed by a continuous infusion of PBS at a rate of 0.5 mL / kh / h to maintain circulating fluid volume.

[0306] As shown in Figure 4B, the measured serum concentration (using human Fc levels as a proxy) for 0.3 mg / kg SEQ ID NO:497 was approximately 10-fold lower than that of 0.3 mg / kg SEQ ID NO:87. Given the fact that the in vitro potency of SEQ ID NO:497 in the rat RXFP1 signaling assay was found to be more than 30-fold higher than that of SEQ ID NO:87 (see Table 30), it is expected that the two molecules would produce at least comparable increases in RABF when administered at the same dose. Instead, the efficacy of SEQ ID NO:497 was equivalent to a 10-fold lower (0.03 mg / kg) dose of SEQ ID NO:87 (Figure 4A), meaning that the efficacy of SEQ ID NO:87 was more than 10-fold higher than expected based on the PK and in vitro efficacy data. The plasma concentration of SEQ ID NO:87 at 0.03 mg / kg was nearly identical to that of SEQ ID NO:497 at 0.3 mg / kg.

[0307] The PBS data in Figure 4A show that there was a small effect on renal blood flow from the volume expansion from the intravenous bolus that returned to baseline by 90 minutes. Using the 90 minute time point to fit a dose response of 0.03 mg / kg to 10 mg / kg of SEQ ID NO: 87, the EC50 of SEQ ID NO: 87 in vivo in rats was estimated to be approximately 2200 ng / mL (Figure 4C), corresponding to approximately 34 nM, consistent with the average EC50 of signaling for SEQ ID NO: 87 shown in Table 30.

[0308] Furthermore, as shown in Figures 5A and 5B, low doses of SEQ ID NO: 87 increased and maintained RABF more than SEQ ID NO: 497. Infusion of SEQ ID NO: 87 at 0.3 mg / kg resulted in an approximately 25% increase in RABF over baseline, which was maintained over the 90-minute experimental time course. Infusion of SEQ ID NO: 497 at 0.3 mg / kg resulted in an approximately 15% increase in RABF over baseline, with RABF levels declining toward baseline within 90 minutes after infusion (Figure 5A). Quantitation of the area under the curve (AUC) indicated that infusion of SEQ ID NO: 87 significantly increased RABF by approximately two-fold more than infusion of SEQ ID NO: 497 (Figure 5B).

[0309] Without being bound by any theory, it is hypothesized that the enhanced effect of SEQ ID NO:87 is due to reduced heparin binding, as well as increased distribution of SEQ ID NO:87 to target tissues far beyond that expected from plasma levels, due to reduced non-specific cellular uptake, blood clearance, and potentially higher bioavailability of SEQ ID NO:87 compared to the more highly charged molecule SEQ ID NO:497.

[0310] Example 7. Therapeutic effects of relaxin-2 fusion protein analogs in a rat pulmonary arterial hypertension model Pulmonary arterial hypertension (PAH) is characterized by progressive pulmonary vascular remodeling of peripheral precapillary arteries, causing a significant increase in right ventricular (RV) load, ultimately leading to right heart failure and premature death. Using the monocrotaline (MCT) rat model as an in vivo PAH model, rats reproducibly develop pulmonary hypertension with a mean pulmonary pressure of approximately 40 mmHg approximately 4 weeks after a single MCT administration. MCT is an 11-membered macrocyclic pyrrolizidine alkaloid derived from the seeds of the Crotalaria spectabilis plant. MCT alkaloids are activated in the liver to the reactive pyrrole metabolite dehydromonocrotaline (MCTP), and this reaction is highly dependent on cytochrome P-450 (CYP3A4). Upon administration, MCT induces a syndrome characterized by pulmonary hypertension (PH), pulmonary mononuclear vasculitis, and right ventricular hypertrophy via damage to pulmonary endothelial cells, among other symptoms. The therapeutic potential of the relaxin-2 fusion protein analog of SEQ ID NO: 87 was evaluated in a rat model of MCT-induced PAH.

[0311] On study day 1, naive young (200-240 g) male Sprague Dawley rats in groups 1-5 received a 60 mg / kg dose of MCT (1 mL / kg sc in 100% DMSO). Rats in groups 1 and 4 received anti-mouse CD20 antibody (20 mg / kg, i.p.) on days 8, 9, 10, and 17. Rats in groups 1 and 2 received SEQ ID NO: 87 (10 mg / kg, i.v.) on days 7, 10, 14, 17, 21, and 24. Starting on study day 8, rats in group 5 were orally dosed twice daily with sildenafil (positive control, 30 mg / kg, po, BID), with the final dose administered on study day 28. Rats in group 6 were administered DMSO as a control. Twelve rats were tested in each of groups 1-4, and 10 rats were tested in each of groups 5 and 6.

[0312] Cageside observations were performed daily for general health and appearance, mortality, and signs of pain or distress. Body weights were recorded pre-dose on study day 0, weekly throughout the study, and on the day of terminal treatment. On study day 28, animals were anesthetized with urethane (1.25 g / kg, i.p.). Blood samples were collected from the retroorbital plexus for PK and biomarker analysis (e.g., N-terminal (NT) prohormone BNP (NT-proBNP)). Serum NT-proBNP was analyzed using the Rat NT-proBNP Assay Kit - Meso Scale Discovery, MD, USA (catalog number K153JKD). Rats were mechanically ventilated using a RoVent® Jr. Small Animal Ventilator from Kent Scientific Corporation. Mean right ventricular pressure and mean pulmonary artery pressure were measured via a 1.6 French solid-state catheter purchased from Transonic Inc. using an open-chest approach. Data were recorded and analyzed using an SP200 pressure system and LabChart software (ADI Instruments). The animals were then humanely euthanized by exsanguination under deep anesthesia. At necropsy, the heart and lungs were collected and weighed from each animal. After weighing, both the right ventricle and lung samples were fixed in formalin and then stored at room temperature. The brain was also collected, weighed, and normalized to the rat's body weight. Immunohistochemical analysis was performed on the formalin-fixed lungs and ventricles.

[0313] An assay was developed to quantify the presence of SEQ ID NO:87 in mouse serum samples, both from serum and plasma. The assay utilizes a quantitative sandwich enzyme immunoassay using an affinity-purified polyclonal antibody specific for human Fc coated onto a 96-well plate. Samples containing molecules with human Fc were added to the wells, incubated, and washed, followed by the addition of an enzyme-linked polyclonal antibody specific for human Fc. After incubation with the enzyme-linked antibody, the wells were washed, enzyme substrate was added, color was developed, and the plate was quenched with acid. After quenching with the acid plate, the plate was read at 450 and 570 nm within 30 minutes of the color change. Values ​​from each animal were pooled to determine the mean of each variable for each group (if applicable). Mean right ventricular pressure (mRVP) and mean pulmonary artery pressure (mPAP) were recorded, and right ventricular systolic pressure (RVSP) was analyzed.

[0314] SEQ ID NO:87 was administered intravenously at 10 mg / kg twice weekly for 3 weeks in the MCT-induced PAH model, with treatment starting 1 week after MCT injection. To mitigate the effectiveness of reducing anti-drug antibody (ADA) production to the test substance, this study was conducted with and without B cell depletion by injecting rats with anti-mouse CD20 antibody. Cardiac hemodynamics, including right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP), were measured using an open-chest approach. The Fulton index, which is the ratio of right ventricular mass to left ventricular and septal mass, was measured at week 4. Blood samples were collected for serum NT pro-BNP, PK, and ADA analysis.

[0315] MCT treatment of rats significantly increased RVSP (FIGS. 6A and 6B), mPAP (FIGS. 7A and 7B), Fulton index values ​​(FIGS. 8A and 8B), and NT-pro-BNP (FIGS. 9A and 9B) while decreasing survival compared to the naive group (no MCT) over a 4-week period. As shown, rats treated with SEQ ID NO: 87 showed significant improvements in RVSP (FIG. 6B), mPAP (FIG. 7B), and Fulton index values ​​(FIG. 8B) in B cell-depleted rats. In addition, there was a clear survival benefit after treatment with SEQ ID NO: 87, with treated rats being the only group to show 100% survival.

[0316] Furthermore, histopathological analysis revealed that SEQ ID NO:87 significantly improved lung inflammation and reduced pulmonary arterial muscularization (Figures 10A and 10B, respectively). To assess lung inflammation, histopathological analysis was performed on tissues stained with hematoxylin and eosin. The lung histopathology total score was calculated as the sum of the analyzed parameters, including pulmonary arterial hypertrophy, vasculitis / necrosis, alveolar histiocytosis, perivascular and interstitial inflammation, hemorrhage, and fibrin deposition. Compared with rats receiving vehicle control, significant improvements were observed in the overall lung histopathology total score and the degree of vasculitis / necrosis, hemorrhage, and fibrin deposition in the lung tissue of rats. To assess pulmonary arterial muscularization, histopathological analysis was performed using tissue stained with anti-alpha smooth muscle actin (SMA) antibody to quantify muscularization and Verhoeff staining to stain the internal elastic lamina of arterioles. Thickening of the arterial wall, primarily due to smooth muscle hypertrophy, was observed at all levels of the pulmonary arterial tree in animals exposed to MCT. This was associated with an increase in the ratio of arterial medial hypertrophy across the five vessel sizes quantified. Medial thickening relative to outer diameter was most pronounced in the smallest caliber arteries and arterioles, with less dramatic medial hypertrophy seen in medium and larger caliber arteries. Lungs from rats treated with SEQ ID NO: 87 had lower arteriolar, small arteriole, and medium arterial medial hypertrophy ratios than untreated animals. For small caliber arteries, rats treated with SEQ ID NO: 87 had significantly lower ratios than untreated animals.

[0317] No mortality was observed in B cell depleted rats treated with SEQ ID NO: 87 compared to vehicle and sildenafil treated groups (Figure 11).

[0318] Example 8. Antifibrotic effects of relaxin-2 fusion protein analogs in a mouse model of renal fibrosis This example describes the evaluation of the antifibrotic effects of relaxin-2 fusion protein analogs SEQ ID NO:496, SEQ ID NO:313, and SEQ ID NO:87 in a mouse unilateral ureteral obstruction (UUO) model of renal fibrosis. The UUO model induces renal fibrosis, and the primary characteristic of UUO is tubular injury due to obstruction of urine flow. Furthermore, experimental UUO in rodents is thought to mimic human chronic obstructive nephropathy in an accelerated manner. Renal fibrosis is a common pathway for most forms of progressive renal disease. Because removal of obstruction is generally not sufficient to reverse fibrosis, model animals may benefit from concomitant therapy. UUO is a widely used model for studying obstructive nephropathy.

[0319] The first study evaluated the effects of SEQ ID NO:496 and SEQ ID NO:313. Sixty-five male C57BL / 6 mice (8-10 weeks old at the start) were used in the study. UUO was induced on day 0 by ligation of the ureter of the left kidney, while the contralateral kidney served as a control. UUO surgery was performed according to standard procedures under deep anesthesia. Briefly, after removing hair from the abdomen, the kidney and ureter were exposed through a midline abdominal incision. The left ureter was completely occluded with two ligatures. The first suture was placed 1 mm below the kidney, and the other suture was placed 1 mm below the renal pelvis using silk or prolene sutures. The wound was closed with two to three staples. The animals were returned to their cages and monitored until they began to move. The animals were given the analgesic buprenorphine on the day of surgery and for 72 hours after surgery. A control group (n=5) underwent sham surgery. Groups of UUO mice were treated with 10 mg / kg SEQ ID NO: 496 (n=10), 20 mg / kg SEQ ID NO: 496 (n=10), 10 mg / kg SEQ ID NO: 313 (n=10), or 20 mg / kg SEQ ID NO: 313 (n=10). Treatment groups received intravenous injections two days before surgery (day -2) and on postoperative days 2 and 5. The positive control group (n=10) received enalapril, an ACE inhibitor used to treat hypertension, diabetic kidney disease, and heart failure, provided via drinking water at 200 mg / L starting on day -2 and continuing until the end of the study. The negative control group (n=10) was treated with vehicle (PBS). The study was terminated on postoperative day 7, and fibrosis symptoms and survival parameters were assessed.

[0320] None of the animals after UUO surgery developed any adverse clinical symptoms. The animals tolerated SEQ ID NO: 496, SEQ ID NO: 313, and enalapril treatment well, with a 100% survival rate. On the 7th day after surgery, the left kidney was harvested, weighed, and fixed for histological examination.

[0321] All groups with surgically induced UUO showed a decrease in body weight relative to the sham-operated group. On postoperative day 4, body weight began to show a trend toward recovery for all UUO groups. Furthermore, all UUO groups showed a significant increase in kidney weight relative to the sham-operated group, and there was no statistically significant change in kidney weight across treatment or control UUO groups.

[0322] Histological analysis of kidney sections fixed 7 days after surgery showed a significant increase in collagen deposition in the kidney parenchyma in all UUO groups relative to the sham-operated group ( FIG. 12 ). Animals treated with 20 mg / kg of SEQ ID NO: 496 and SEQ ID NO: 313 showed significantly reduced collagen deposition relative to the vehicle-treated control group.

[0323] The anti-fibrotic effect of SEQ ID NO: 87 was observed following a similar procedure. Mice treated with SEQ ID NO: 87 showed a significant reduction in collagen deposition after UUO induction compared to vehicle-treated mice (FIG. 13). Tissue TNFα levels were assessed via electrochemiluminescence, and mice treated with SEQ ID NO: 87 also showed a significant reduction in TNFα levels compared to vehicle-treated mice (FIG. 14). Reductions in IL-1β and IL-6 in fibrotic kidneys were also observed in mice treated with SEQ ID NO: 87 compared to vehicle-treated mice.

[0324] Example 9. Effects of relaxin-2 fusion protein analogs on isoproterenol-induced cardiac hypertrophy and fibrosis This example describes the evaluation of the effect of relaxin-2 fusion protein analog SEQ ID NO: 87 on cardiac hypertrophy and fibrosis following isoproterenol challenge. Isoproterenol is a drug that increases heart rate and myocardial contractility, potentially leading to increased cardiac hypertrophy and fibrosis.

[0325] All animals were treated and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, revised 2011), and the protocol was approved by the Institutional Animal Care and Use Committee. Male C57BL / 6J mice, 10–11 weeks old and weighing between 25 and 30 g, were obtained. Animals were housed in an animal facility under a conventional 12-h light / dark cycle. After 1 week of acclimation, microosmotic minipump implantation was performed on the mice. Briefly, mice were anesthetized using 1%–3% isoflurane administered by inhalation through a vaporizer. Osmotic minipumps with a flow rate of 0.25 μl / h were surgically implanted subcutaneously in the subscapular space of the mice. Each pump delivered a fixed dose (0.25 μl / hour) of infusion drug (isoproterenol in PBS containing 0.002% ascorbic acid at 15 mg / kg per day) or vehicle (PBS containing 0.002% ascorbic acid) for two weeks. Postoperative analgesia was achieved with a single dose of meloxicam SR (2.5 mg / kg) during minipump implantation and another single dose of meloxicam SR (2.5 mg / kg) one day after surgery. Mice were treated with either vehicle or 10 mg / kg of SEQ ID NO: 87 one day before minipump implantation. Mice were then dosed with relaxin-2 fusion protein analog SEQ ID NO: 87 every other week for the duration of the study (14 days total). To reduce anti-drug activity, mice were intraperitoneally injected with 20 mg / kg of anti-mouse CD20 to deplete all B cells one day after minipump implantation.

[0326] After 14 days of incubation, animals were euthanized via CO2 inhalation followed by cervical dislocation. Body weight was measured. The whole heart was removed, washed in PBS, dried on a paper towel, and weighed. Fresh cardiac tissue was immediately frozen in liquid nitrogen and stored for further collagen content analysis. Tibiae were obtained by blunt limb dissection, and tibia length was measured using a digital caliper. Heart weight (HW) was normalized by body weight (BW) and tibia length (TL). Data (HW / TL and HW / BW) were analyzed using standard software. As shown in Figure 15, isoproterenol administration caused a significant increase in cardiac hypertrophy, as measured via normalized heart weight (HW / BW), relative to vehicle-treated mice. Coadministration of SEQ ID NO: 87 with isoproterenol significantly attenuated isoproterenol-induced cardiac hypertrophy.

[0327] The collagen content of each ventricle was measured using a hydroxyproline assay kit. Instant-frozen whole ventricular tissue was weighed, minced, and transferred to a screw-cap tube. The tissue was hydrolyzed at 100 mg / ml in 6 M HCl and incubated at 95°C for 20 hours in a calibrated oven or thermoblock. The hydrolyzed samples were then diluted with 4 M HCl prior to assay analysis. To perform the assay, 35 μl of hydrolysate or hydroxyproline standard was mixed with 75 μl of assay buffer in each well of a 96-well plate. The plate was incubated at room temperature for 20 minutes with shaking. After adding 75 μl of detection reagent to each well, the plate was mixed well and incubated at 60°C for 60 minutes in an oven or incubator. The plate was cooled to room temperature and read at 570 nm, and the hydroxyproline concentration of each sample was determined using a standard curve. Data were analyzed using standard software. As shown in Figure 16, isoproterenol administration caused a significant increase in fibrosis, as measured by collagen content, relative to vehicle-treated mice. Co-administration of SEQ ID NO: 87 with isoproterenol significantly attenuated isoproterenol-induced fibrosis.

[0328] Example 10. Evaluating the safety, tolerability, pharmacokinetics, and pharmacodynamics of relaxin-2 fusion protein analogs. This example describes a double-blind, randomized, placebo-controlled, single ascending dose study conducted to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity of SEQ ID NO: 87 in healthy subjects.

[0329] research design Approximately 48 participants were enrolled in this study. Single ascending doses of SEQ ID NO: 87 or a matching placebo (i.e., the same buffer without the addition of SEQ ID NO: 87) were administered to study participants via intravenous infusion (IV) or subcutaneous (SC) injection at the doses listed below. In each cohort, six participants received SEQ ID NO: 87 and two participants received a placebo. The infusion time was 30-60 minutes. Cohort A received 0.3 mg / kg IV SEQ ID NO: 87 or a matching placebo. Cohort B received 1 mg / kg IV SEQ ID NO: 87 or a matching placebo. Cohort C received 300 mg SC SEQ ID NO: 87 or a matching placebo. In some cases, Cohort C was dosed simultaneously with Cohort B. Cohort D received 3 mg / kg IV SEQ ID NO: 87 or a matching placebo. Cohort E received 600 mg SC SEQ ID NO: 87 or a matching placebo. In some cases, Cohort E is dosed simultaneously with Cohort D. Cohort F receives 10 mg / kg IV SEQ ID NO: 87 or matching placebo.

[0330] A master randomization schedule and code-break envelopes were created and delivered to the site's unblinded pharmacy prior to administration. Active and placebo products are infused through covered lines to conceal the active from the placebo. A printed randomization schedule is generated using an exchange lock securement method. Those receiving the treatment(s), those administering the treatment(s), and those analyzing the results and data are blinded.

[0331] Primary outcome The primary outcome of this study is to evaluate the safety and tolerability of SEQ ID NO: 87 after a single ascending dose. The primary outcome will be assessed by monitoring: 1) the incidence of adverse events (AEs) and serious adverse events (SAEs); 2) changes in clinical laboratory safety parameters, including blood test results for hematology, serum chemistry, and coagulation studies; 3) changes in vital sign measurements; and 4) changes in electrocardiogram (ECG) findings. Adverse events will be assessed through clinical examination, patient data review, and self-report. Adverse events will be collected via oral interviews by clinic staff during the study period, either in person during hospitalization or clinic visits, or via telephone during remote contact. Vital sign measurements will include resting heart rate, systolic, and diastolic blood pressure (BP) using standard manual or electronic clinical procedures. These procedures will be completed using standard nursing practice or, in the case of ECGs, using instructions provided by the manufacturer. These individual data will be utilized to determine safety through physician evaluation. Additionally, the totality of the presented data will also be considered in determining safety.

[0332] Adverse events and serious adverse events will be assessed at screening on days -1, 1, 2, 3, 6, 8, 15, 29, 43, and 57 after dosing. Laboratory safety parameters will be assessed at screening on days -1, 1, 2, 8, 15, 29, 43, and 57 after dosing. Vital signs will be assessed at screening on days -1, 1, 2, 3, 6, 8, 15, 29, 43, and 57 after dosing. ECGs will be performed at screening on days -1, 1, and 2 after dosing.

[0333] Secondary outcomes A secondary outcome of this study is to characterize the pharmacokinetic (PK) profile of SEQ ID NO: 87 in healthy participants after single ascending doses of SEQ ID NO: 87. The following standard PK parameters will be evaluated: max , T max , AUC-last (AUC from 0 to the last measurable concentration), AUC-inf (AUC from 0 to infinity), t1 / 2 , CL (clearance), and Vz (terminal phase volume of distribution). Blood samples for PK parameter evaluation will be taken pre-dose, at the end of the infusion, 6 and 12 hours after dosing on day 1, 24 hours after dosing on day 2, 48 hours after dosing on day 3, 120 hours after dosing on day 6, and 168 hours after dosing on days 8, 15, 29, 43, and 57.

[0334] Another secondary outcome of this study is to evaluate the pharmacodynamic (PD) effects of single ascending doses of SEQ ID NO: 87 in healthy participants. Changes from baseline to day 2 will be assessed in the following PD parameters: renal blood flow (RPF), measured by the change in plasma para-aminohippuric acid (PAH) over time; renal blood flow (RBF); and glomerular filtration rate (GFR), calculated by dividing RPF by filtration fraction (FF). Blood samples for RPF / GFR assessment will be collected on days 2, 8, and 15 for IV cohorts A, B, and D, and on days 15 and 29 for IV cohort F. Blood samples for RPF / GFR assessment will be collected on days 2, 15, and 29 for SC cohorts C and E.

[0335] Eligibility Participants met the following criteria to enroll in this study: · Men between the ages of 18 and 55 or women who are not of childbearing age. · Be in good health based on medical history, physical examination, vital signs, electrocardiogram, and routine laboratory tests. · Have a body mass index (BMI) of 18-32 kg per square meter at the time of screening. Understand the study procedures and agree to participate in the study by granting written informed consent. · Be at least 18 years old and under 55 years old.

[0336] Participants were excluded from the study if they met any of the following criteria: · Mentally or legally incompetent, or having significant emotional problems at the time of the study, or having a history of significant psychiatric illness at the investigator's discretion. Any clinically significant physical examination abnormality observed during the screening visit or, in the opinion of the investigator, is unsuitable for participation in the study. Have a clinically significant abnormal complete blood count, clinical chemistry, or urinalysis at screening or day -1 (in asymptomatic participants, abnormal test results including creatine phosphokinase within 3 times the upper limit of normal with a suspected cause due to strenuous physical activity may be repeated once during the screening period). -Having been hospitalized for any reason within 30 days of the screening visit. Have a history of any clinically significant renal, neurological, gastrointestinal, hepatic, or respiratory disease (note that subjects with completely resolved childhood asthma without recurrence in adulthood may be enrolled). -Have a history of anaphylaxis or other significant allergies in the opinion of the investigator. ·Have a history of clinically significant cardiovascular disease, including arrhythmias, conduction abnormalities, or clinically significant vital signs. Previously received relaxin or relaxin fusion protein. -Receipt of any clinical trial evaluating another investigational drug (including biologics) or therapy (including specific immunotherapy) within 90 days prior to the screening visit, or within 5 half-lives of the investigational drug (whichever is longer), or within 4 weeks in the case of another investigational drug.

[0337] Partial Results Preliminary PK and PD results were obtained from healthy human patients who received a single 0.3 mg / kg IV dose of SEQ ID NO:87 (Figures 17A and 17B, respectively). As shown in Figure 17A, SEQ ID NO:87 demonstrated a favorable PK profile in four individual healthy patients after IV administration and a terminal half-life of 17 days, more than double the predicted terminal half-life of 6 days obtained from simulations in a non-human primate model (dashed line). The PD effects of SEQ ID NO:87 in healthy human patients were investigated by assessing the change from baseline in renal plasma flow (RPF) on days 2, 8, and 15 (Figure 17B). Briefly, patients received a bolus dose of para-aminohippuric acid (PAH), and plasma PAH was measured on days 2, 8, and 15. The effective RPF at each time point was calculated using the following formula:

number

[0338] SEQ ID NO:87 also demonstrated a favorable PK profile in healthy human patients administered a single 150 mg SC dose, with a terminal half-life yet to be determined (Figure 18). Based on the data, SC bioavailability was in the range of 50-60%.

[0339] Preliminary data from healthy patients administered SEQ ID NO:87 IV and SC support a Q4W dosing schedule. PK data showed low inter-subject variability in serum concentrations (≦20%), with no evidence of immune-mediated drug clearance. SEQ ID NO:87 was found to be safe and well-tolerated, with minimal adverse events and no drug-related SAEs. Expected on-target effects were observed (e.g., mild orthostatic tachycardia without BP effects in some subjects), and no infusion or injection site reactions were reported.

[0340] Example 11. Single-dose, open-label study to evaluate the safety, tolerability, and hemodynamic effects of a relaxin-2 fusion protein analog. This predictive example describes a single-dose, open-label study to evaluate the safety, tolerability, and hemodynamics of SEQ I...

Claims

1. From the N-terminus to the C-terminus, a first peptide; a linker peptide; a second peptide, (a) the first peptide comprises an amino acid sequence that has zero, one, two, three, four, or five amino acid modifications relative to the amino acid sequence of SEQ ID NO:502, and the second peptide comprises an amino acid sequence that has zero, one, two, three, four, or five amino acid modifications relative to the amino acid sequence of SEQ ID NO:503 or 504; or the first peptide comprises an amino acid sequence having zero, one, two, three, four, or five amino acid modifications relative to the amino acid sequence of SEQ ID NO:503 or 504, and the second peptide comprises an amino acid sequence having zero, one, two, three, four, or five amino acid modifications relative to the amino acid sequence of SEQ ID NO:502; (b) a fusion protein, wherein the fusion protein has a pI of 6.0 to 8.

2.

2. The first peptide has the amino acid sequence X １１ LCGRELVRAQIAIC (SEQ ID NO: 505), １１ is K, Q, D, E, L, I, or Y.

3. 3. The fusion protein of claim 1 or 2, wherein the first peptide consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids.

4. The first peptide has the amino acid sequence X １２ CCX １３ VGCTX １４ X １５ SLAX １６ FC (SEQ ID NO: 506), X １２ is K, Q, D, E, L, I, or Y; X １３ is any amino acid except M, W, or C, X １４ is K, Q, D, E, L, I, or Y; X １５ is Q, D, E, L, I, Y, or R; X １６ is R or Q.

5. The first peptide has the amino acid sequence X １２ CCX １３ VGCTX １４ X １５ SLAX １６ FC (SEQ ID NO: 506), X １２ is K, Q, D, E, L, I, or Y; X １３ is H, K, Q, Y, L, N, I, S, T, or F; X １４ is K, Q, D, E, L, I, or Y; X １５ is Q, D, E, L, I, Y, or R; X １６ is R or Q.

6. X １３ The fusion protein of claim 4 or 5, wherein is Q.

7. The fusion protein of any one of claims 4 to 6, wherein the first peptide consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

8. The second peptide has the amino acid sequence X １１ LCGRELVRAQIAIC (SEQ ID NO: 505), １１ is K, Q, D, E, L, I, or Y.

9. 9. The fusion protein of claim 8, wherein the second peptide consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids.

10. The second peptide has the amino acid sequence X １２ CCX １３ VGCTX １４ X １５ SLAX １６ FC (SEQ ID NO: 506), X １２ is K, Q, D, E, L, I, or Y; X １３ is any amino acid except M, W, or C, X １４ is K, Q, D, E, L, I, or Y; X １５ is Q, D, E, L, I, Y, or R; X １６ is R or Q.

11. The second peptide has the amino acid sequence X １２ CCX １３ VGCTX １４ X １５ SLAX １６ FC (SEQ ID NO: 506), X １２ is K, Q, D, E, L, I, or Y; X １３ is H, K, Q, Y, L, N, I, S, T, or F; X １４ is K, Q, D, E, L, I, or Y; X １５ is Q, D, E, L, I, Y, or R; X １６ is R or Q.

12. X １３ The fusion protein of claim 10 or 11, wherein is Q.

13. The fusion protein of any one of claims 10 to 12, wherein the second peptide consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

14. The fusion protein according to any one of the preceding claims, wherein the linker peptide comprises an amino acid sequence having 12 to 15 amino acids.

15. The linker peptide has the amino acid sequence ASDAAGAX ８ AX ９ AGA (SEQ ID NO: 17), X ８ is D, E, N, or Q; X ９ is D, E, N, or Q; or The linker peptide has the amino acid sequence GGEGSGGEGX １０ GGG (SEQ ID NO: 25), X １０ A fusion protein according to any one of the preceding claims, wherein is E or S.

16. X ８ is D, E, N, or Q, and X ９ is D, E, or Q, or X ８ is D, E, or Q, and X ９ is D, E, N, or Q.

17. 10. The fusion protein of any one of the preceding claims, wherein the linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 26, and 27.

18. From the N-terminus to the C-terminus, a first peptide; a linker peptide; a second peptide, (a) the first peptide comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications relative to the amino acid sequence of SEQ ID NO: 1, and at least one amino acid at position 4 or 25 of the first peptide is not M; the second peptide comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications relative to the amino acid sequence of SEQ ID NO: 8, and the amino acid at position 22 of the second peptide is not R; or the first peptide comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications relative to the amino acid sequence of SEQ ID NO: 8, and the amino acid at position 22 of the second peptide is not R; the second peptide comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications relative to the amino acid sequence of SEQ ID NO: 1, and at least one amino acid at position 4 or 25 of the first peptide is not M; (b) a fusion protein, wherein the fusion protein has a pI of 6.0 to 8.

2.

19. The fusion protein of claim 18, wherein the linker peptide comprises an amino acid sequence having 12 to 15 amino acids.

20. The linker peptide has the amino acid sequence ASDAAGAX ８ AX ９ AGA (SEQ ID NO: 17), X ８ is D, E, N, or Q; X ９ is D, E, N, or Q; or The linker peptide has the amino acid sequence GGEGSGGEGX １０ GGG (SEQ ID NO: 25), X １０ is E or S.

21. X ８ is D, E, N, or Q, and X ９ is D, E, or Q, or X ８ is D, E, or Q, and X ９ is D, E, N, or Q.

22. From the N-terminus to the C-terminus, a first peptide; a linker peptide; a second peptide, The linker peptide has the amino acid sequence ASDAAGAX ８ AX ９ AGA (SEQ ID NO: 17), X ８ is D, E, N, or Q; X ９ is D, E, N, or Q; or The linker peptide has the amino acid sequence GGEGSGGEGX １０ GGG (SEQ ID NO: 25), X １０ is E or S.

23. X ８ is D, E, N, or Q, and X ９ is D, E, or Q, or X ８ is D, E, or Q, and X ９ is D, E, N, or Q.

24. The fusion protein of any one of claims 18 to 23, wherein the linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 26, and 27.

25. the first peptide having the amino acid sequence DSX １ QEEVIX ２ LCGRELVRAQIAICGX ３ ST (SEQ ID NO: 7), X １ but not M, H, or C, X ２ is K, Q, D, E, L, I, or Y; X ３ The fusion protein according to any one of claims 1 to 3 and 10 to 24, wherein is K or Q.

26. The first peptide has the amino acid sequence DSX １ QEEVIX ２ LCGRELVRAQIAICGX ３ ST (SEQ ID NO: 7), X １ is W, Y, F, L, I, V, or A; X ２ is K, Q, D, E, L, I, or Y; X ３ The fusion protein according to any one of claims 1 to 3 and 10 to 25, wherein is K or Q.

27. X １ is Y.

28. The fusion protein of any one of claims 25 to 27, wherein the first peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

29. The fusion protein of any one of claims 1 to 3 and 10 to 28, wherein the first peptide consists of 27, 28, or 29 amino acids.

30. the first peptide having the amino acid sequence QLYSALANX ４ CCX ５ VGCTX ６ X ７ SLAQFC (SEQ ID NO: 16), X ４ is K, Q, D, E, L, I, or Y; X ５ is any amino acid except M, W, or C, X ６ is K, Q, D, E, L, I, or Y; X ７ is Q, D, E, L, I, Y, or R.

31. the first peptide having the amino acid sequence QLYSALANX ４ CCX ５ VGCTX ６ X ７ SLAQFC (SEQ ID NO: 16), X ４ is K, Q, D, E, L, I, or Y; X ５ is H, K, Q, Y, L, N, I, S, T, or F; X ６ is K, Q, D, E, L, I, or Y; X ７ is Q, D, E, L, I, Y, or R.

32. X ５ is Q.

33. 33. The fusion protein of any one of claims 30 to 32, wherein the first peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, and 507.

34. The fusion protein of any one of claims 1, 4 to 9, 14 to 24, and 30 to 33, wherein the first peptide consists of 24 or 25 amino acids.

35. the second peptide having the amino acid sequence DSX １ QEEVIX ２ LCGRELVRAQIAICGX ３ ST (SEQ ID NO: 7), X １ but not M, H, or C, X ２ is K, Q, D, E, L, I, or Y; X ３ The fusion protein of any one of claims 1, 4 to 9, and 14 to 24, wherein is K or Q.

36. The second peptide has the amino acid sequence DSX １ QEEVIX ２ LCGRELVRAQIAICGX ３ ST (SEQ ID NO: 7), X １ is W, Y, F, L, I, V, or A; X ２ is K, Q, D, E, L, I, or Y; X ３ is K or Q.

37. X １ is Y.

38. The fusion protein of any one of claims 35 to 37, wherein the second peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

39. 39. The fusion protein of any one of claims 1, 4-9, 14-24, and 35-38, wherein the second peptide consists of 27, 28, or 29 amino acids.

40. the second peptide having the amino acid sequence QLYSALANX ４ CCX ５ VGCTX ６ X ７ SLAQFC (SEQ ID NO: 16), X ４ is K, Q, D, E, L, I, or Y; X ５ is any amino acid except M, W, or C, X ６ is K, Q, D, E, L, I, or Y; X ７ is Q, D, E, L, I, Y, or R.

41. the second peptide having the amino acid sequence QLYSALANX ４ CCX ５ VGCTX ６ X ７ SLAQFC (SEQ ID NO: 16), X ４ is K, Q, D, E, L, I, or Y; X ５ is H, K, Q, Y, L, N, I, S, T, or F; X ６ is K, Q, D, E, L, I, or Y; X ７ is Q, D, E, L, I, Y, or R.

42. X ５ is Q.

43. 43. The fusion protein of any one of claims 40 to 42, wherein the second peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, and 507.

44. The fusion protein of any one of claims 1 to 3, 10 to 24, and 40 to 43, wherein the second peptide consists of 24 or 25 amino acids.

45. the first peptide comprises the amino acid sequence of SEQ ID NO: 1 and the second peptide comprises the amino acid sequence of SEQ ID NO: 8; or the first peptide comprises the amino acid sequence of SEQ ID NO: 1 and the second peptide comprises the amino acid sequence of SEQ ID NO: 9; or the first peptide comprises the amino acid sequence of SEQ ID NO: 1 and the second peptide comprises the amino acid sequence of SEQ ID NO: 10; or the first peptide comprises the amino acid sequence of SEQ ID NO: 1 and the second peptide comprises the amino acid sequence of SEQ ID NO: 11; or the first peptide comprises the amino acid sequence of SEQ ID NO: 1 and the second peptide comprises the amino acid sequence of SEQ ID NO: 12; or the first peptide comprises the amino acid sequence of SEQ ID NO: 1 and the second peptide comprises the amino acid sequence of SEQ ID NO: 13; or the first peptide comprises the amino acid sequence of SEQ ID NO: 1 and the second peptide comprises the amino acid sequence of SEQ ID NO: 14; or the first peptide comprises the amino acid sequence of SEQ ID NO: 1 and the second peptide comprises the amino acid sequence of SEQ ID NO: 15; or the first peptide comprises the amino acid sequence of SEQ ID NO: 1 and the second peptide comprises the amino acid sequence of SEQ ID NO: 507; or the first peptide comprises the amino acid sequence of SEQ ID NO:2 and the second peptide comprises the amino acid sequence of SEQ ID NO:8; or the first peptide comprises the amino acid sequence of SEQ ID NO:2 and the second peptide comprises the amino acid sequence of SEQ ID NO:9; or the first peptide comprises the amino acid sequence of SEQ ID NO:2 and the second peptide comprises the amino acid sequence of SEQ ID NO:10; or the first peptide comprises the amino acid sequence of SEQ ID NO:2 and the second peptide comprises the amino acid sequence of SEQ ID NO:11; or the first peptide comprises the amino acid sequence of SEQ ID NO:2 and the second peptide comprises the amino acid sequence of SEQ ID NO:12; or the first peptide comprises the amino acid sequence of SEQ ID NO:2 and the second peptide comprises the amino acid sequence of SEQ ID NO:13; or the first peptide comprises the amino acid sequence of SEQ ID NO:2 and the second peptide comprises the amino acid sequence of SEQ ID NO:14; or the first peptide comprises the amino acid sequence of SEQ ID NO:2 and the second peptide comprises the amino acid sequence of SEQ ID NO:15; or the first peptide comprises the amino acid sequence of SEQ ID NO:2 and the second peptide comprises the amino acid sequence of SEQ ID NO:507; or the first peptide comprises the amino acid sequence of SEQ ID NO:3 and the second peptide comprises the amino acid sequence of SEQ ID NO:8; or the first peptide comprises the amino acid sequence of SEQ ID NO:3 and the second peptide comprises the amino acid sequence of SEQ ID NO:9; or the first peptide comprises the amino acid sequence of SEQ ID NO:3 and the second peptide comprises the amino acid sequence of SEQ ID NO:10; or the first peptide comprises the amino acid sequence of SEQ ID NO:3 and the second peptide comprises the amino acid sequence of SEQ ID NO:11; or the first peptide comprises the amino acid sequence of SEQ ID NO:3 and the second peptide comprises the amino acid sequence of SEQ ID NO:12; or the first peptide comprises the amino acid sequence of SEQ ID NO:3 and the second peptide comprises the amino acid sequence of SEQ ID NO:13; or the first peptide comprises the amino acid sequence of SEQ ID NO:3 and the second peptide comprises the amino acid sequence of SEQ ID NO:14; or the first peptide comprises the amino acid sequence of SEQ ID NO:3 and the second peptide comprises the amino acid sequence of SEQ ID NO:15; or the first peptide comprises the amino acid sequence of SEQ ID NO:3 and the second peptide comprises the amino acid sequence of SEQ ID NO:507; or the first peptide comprises the amino acid sequence of SEQ ID NO:4 and the second peptide comprises the amino acid sequence of SEQ ID NO:8; or the first peptide comprises the amino acid sequence of SEQ ID NO:4 and the second peptide comprises the amino acid sequence of SEQ ID NO:9; or the first peptide comprises the amino acid sequence of SEQ ID NO:4 and the second peptide comprises the amino acid sequence of SEQ ID NO:10; or the first peptide comprises the amino acid sequence of SEQ ID NO:4 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:4 and the second peptide comprises the amino acid sequence of SEQ ID NO:12; or the first peptide comprises the amino acid sequence of SEQ ID NO:4 and the second peptide comprises the amino acid sequence of SEQ ID NO:13; or the first peptide comprises the amino acid sequence of SEQ ID NO:4 and the second peptide comprises the amino acid sequence of SEQ ID NO:14; or the first peptide comprises the amino acid sequence of SEQ ID NO:4 and the second peptide comprises the amino acid sequence of SEQ ID NO:15; or the first peptide comprises the amino acid sequence of SEQ ID NO:4 and the second peptide comprises the amino acid sequence of SEQ ID NO:507; or 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; or 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; or 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; or 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; or 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; or 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; or 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:14; or 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:15; or 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:507; or 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; or 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; or 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; or 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; or 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; or 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; or 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:14; or 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:15; or 25. The fusion protein of any one of claims 1 to 3 and 10 to 24, wherein 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:

507.

46. the first peptide comprises the amino acid sequence of SEQ ID NO:8 and the second peptide comprises the amino acid sequence of SEQ ID NO:1; or the first peptide comprises the amino acid sequence of SEQ ID NO:8 and the second peptide comprises the amino acid sequence of SEQ ID NO:2; or the first peptide comprises the amino acid sequence of SEQ ID NO:8 and the second peptide comprises the amino acid sequence of SEQ ID NO:3; or the first peptide comprises the amino acid sequence of SEQ ID NO:8 and the second peptide comprises the amino acid sequence of SEQ ID NO:4; or the first peptide comprises the amino acid sequence of SEQ ID NO:8 and the second peptide comprises the amino acid sequence of SEQ ID NO:5; or the first peptide comprises the amino acid sequence of SEQ ID NO:8 and the second peptide comprises the amino acid sequence of SEQ ID NO:6; the first peptide comprises the amino acid sequence of SEQ ID NO:9 and the second peptide comprises the amino acid sequence of SEQ ID NO:1; or the first peptide comprises the amino acid sequence of SEQ ID NO:9 and the second peptide comprises the amino acid sequence of SEQ ID NO:2; or the first peptide comprises the amino acid sequence of SEQ ID NO:9 and the second peptide comprises the amino acid sequence of SEQ ID NO:3; the first peptide comprises the amino acid sequence of SEQ ID NO:9 and the second peptide comprises the amino acid sequence of SEQ ID NO:4; or the first peptide comprises the amino acid sequence of SEQ ID NO:9 and the second peptide comprises the amino acid sequence of SEQ ID NO:5; or the first peptide comprises the amino acid sequence of SEQ ID NO:9 and the second peptide comprises the amino acid sequence of SEQ ID NO:6; the first peptide comprises the amino acid sequence of SEQ ID NO: 10 and the second peptide comprises the amino acid sequence of SEQ ID NO: 1; or the first peptide comprises the amino acid sequence of SEQ ID NO: 10 and the second peptide comprises the amino acid sequence of SEQ ID NO: 2; or the first peptide comprises the amino acid sequence of SEQ ID NO: 10 and the second peptide comprises the amino acid sequence of SEQ ID NO: 3; or the first peptide comprises the amino acid sequence of SEQ ID NO: 10 and the second peptide comprises the amino acid sequence of SEQ ID NO: 4; or the first peptide comprises the amino acid sequence of SEQ ID NO: 10 and the second peptide comprises the amino acid sequence of SEQ ID NO: 5; or the first peptide comprises the amino acid sequence of SEQ ID NO: 10 and the second peptide comprises the amino acid sequence of SEQ ID NO: 6; or the first peptide comprises the amino acid sequence of SEQ ID NO: 11 and the second peptide comprises the amino acid sequence of SEQ ID NO: 1; or the first peptide comprises the amino acid sequence of SEQ ID NO: 11 and the second peptide comprises the amino acid sequence of SEQ ID NO: 2; or the first peptide comprises the amino acid sequence of SEQ ID NO: 11 and the second peptide comprises the amino acid sequence of SEQ ID NO: 3; or the first peptide comprises the amino acid sequence of SEQ ID NO: 11 and the second peptide comprises the amino acid sequence of SEQ ID NO: 4; or the first peptide comprises the amino acid sequence of SEQ ID NO: 11 and the second peptide comprises the amino acid sequence of SEQ ID NO: 5; or the first peptide comprises the amino acid sequence of SEQ ID NO: 11 and the second peptide comprises the amino acid sequence of SEQ ID NO: 6; or the first peptide comprises the amino acid sequence of SEQ ID NO: 12 and the second peptide comprises the amino acid sequence of SEQ ID NO: 1; or the first peptide comprises the amino acid sequence of SEQ ID NO: 12 and the second peptide comprises the amino acid sequence of SEQ ID NO: 2; or the first peptide comprises the amino acid sequence of SEQ ID NO: 12 and the second peptide comprises the amino acid sequence of SEQ ID NO: 3; or the first peptide comprises the amino acid sequence of SEQ ID NO: 12 and the second peptide comprises the amino acid sequence of SEQ ID NO: 4; or the first peptide comprises the amino acid sequence of SEQ ID NO: 12 and the second peptide comprises the amino acid sequence of SEQ ID NO: 5; or the first peptide comprises the amino acid sequence of SEQ ID NO: 12 and the second peptide comprises the amino acid sequence of SEQ ID NO: 6; or the first peptide comprises the amino acid sequence of SEQ ID NO: 13 and the second peptide comprises the amino acid sequence of SEQ ID NO: 1; or the first peptide comprises the amino acid sequence of SEQ ID NO: 13 and the second peptide comprises the amino acid sequence of SEQ ID NO: 2; or the first peptide comprises the amino acid sequence of SEQ ID NO: 13 and the second peptide comprises the amino acid sequence of SEQ ID NO: 3; or the first peptide comprises the amino acid sequence of SEQ ID NO: 13 and the second peptide comprises the amino acid sequence of SEQ ID NO: 4; or the first peptide comprises the amino acid sequence of SEQ ID NO: 13 and the second peptide comprises the amino acid sequence of SEQ ID NO: 5; or the first peptide comprises the amino acid sequence of SEQ ID NO: 13 and the second peptide comprises the amino acid sequence of SEQ ID NO: 6; or the first peptide comprises the amino acid sequence of SEQ ID NO: 14 and the second peptide comprises the amino acid sequence of SEQ ID NO: 1; or the first peptide comprises the amino acid sequence of SEQ ID NO: 14 and the second peptide comprises the amino acid sequence of SEQ ID NO: 2; or the first peptide comprises the amino acid sequence of SEQ ID NO: 14 and the second peptide comprises the amino acid sequence of SEQ ID NO: 3; or the first peptide comprises the amino acid sequence of SEQ ID NO: 14 and the second peptide comprises the amino acid sequence of SEQ ID NO: 4; or the first peptide comprises the amino acid sequence of SEQ ID NO: 14 and the second peptide comprises the amino acid sequence of SEQ ID NO: 5; or the first peptide comprises the amino acid sequence of SEQ ID NO: 14 and the second peptide comprises the amino acid sequence of SEQ ID NO: 6; or the first peptide comprises the amino acid sequence of SEQ ID NO: 15 and the second peptide comprises the amino acid sequence of SEQ ID NO: 1; or the first peptide comprises the amino acid sequence of SEQ ID NO: 15 and the second peptide comprises the amino acid sequence of SEQ ID NO: 2; or the first peptide comprises the amino acid sequence of SEQ ID NO: 15 and the second peptide comprises the amino acid sequence of SEQ ID NO: 3; or the first peptide comprises the amino acid sequence of SEQ ID NO: 15 and the second peptide comprises the amino acid sequence of SEQ ID NO: 4; or the first peptide comprises the amino acid sequence of SEQ ID NO: 15 and the second peptide comprises the amino acid sequence of SEQ ID NO: 5; or the first peptide comprises the amino acid sequence of SEQ ID NO: 15 and the second peptide comprises the amino acid sequence of SEQ ID NO: 6; or the first peptide comprises the amino acid sequence of SEQ ID NO:507 and the second peptide comprises the amino acid sequence of SEQ ID NO:1; or the first peptide comprises the amino acid sequence of SEQ ID NO:507 and the second peptide comprises the amino acid sequence of SEQ ID NO:2; or the first peptide comprises the amino acid sequence of SEQ ID NO:507 and the second peptide comprises the amino acid sequence of SEQ ID NO:3; or the first peptide comprises the amino acid sequence of SEQ ID NO:507 and the second peptide comprises the amino acid sequence of SEQ ID NO:4; or the first peptide comprises the amino acid sequence of SEQ ID NO:507 and the second peptide comprises the amino acid sequence of SEQ ID NO:5; or 25. The fusion protein of any one of claims 1, 4-9, and 14-24, wherein the first peptide comprises the amino acid sequence of SEQ ID NO:507 and the second peptide comprises the amino acid sequence of SEQ ID NO:

6.

47. 46. ​​The fusion protein of any one of claims 1 to 3, 10 to 29, and 40 to 45, wherein the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 28 to 75 and 508 to 515.

48. 48. The fusion protein of any one of claims 1 to 47, further comprising an IgG Fc.

49. 49. The fusion protein of claim 48, wherein the IgG Fc comprises the amino acid alanine at each of EU positions 234 and 235.

50. 50. The fusion protein of claim 48 or 49, wherein the IgG Fc comprises the amino acid alanine at EU position 329.

51. 51. The fusion protein of any one of claims 48 to 50, wherein the IgG Fc comprises the amino acids alanine at EU positions 234, 235, and 329.

52. 52. The fusion protein of any one of claims 48 to 51, wherein the IgG Fc comprises the amino acids alanine, alanine, alanine, leucine, and serine at EU positions 234, 235, 329, 428, and 434, respectively.

53. 52. The fusion protein of any one of claims 48 to 51, wherein the IgG Fc comprises the amino acids lysine, phenylalanine, and tyrosine at EU positions 433, 434, and 436, respectively.

54. 54. The fusion protein of any one of claims 48 to 53, wherein the IgG Fc comprises the amino acids tyrosine, threonine, and glutamate at EU positions 252, 254, and 256, respectively.

55. 55. The fusion protein of any one of claims 48 to 54, wherein the IgG Fc comprises the amino acids leucine and serine at EU positions 428 and 434, respectively.

56. 49. The fusion protein of claim 48, wherein the IgG Fc comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of a human IgGl Fc.

57. 57. The fusion protein of claim 56, wherein the IgG Fc comprises the amino acid sequence of a human IgGl Fc.

58. 58. The fusion protein of claim 56 or 57, 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: 76-83.

59. 58. The fusion protein of claim 56 or 57, wherein the IgG Fc comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 76-83.

60. 60. The fusion protein of any one of claims 48-59, wherein the IgG Fc is optionally linked to the N-terminus of the first peptide via an IgG Fc linker.

61. 60. The fusion protein of any one of claims 48-59, wherein the IgG Fc is optionally linked to the C-terminus of the second peptide via an IgG Fc linker.

62. 62. The fusion protein of claim 60 or 61, wherein the IgG Fc linker comprises or consists of the amino acid sequence GGS or EGGS (SEQ ID NO: 299).

63. 46. ​​The fusion protein of any one of claims 1 to 3, 10 to 24, and 40 to 45, wherein the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 84 to 138 and 516 to 523.

64. 46. ​​The fusion protein of any one of claims 1 to 3, 10 to 24, and 40 to 45, wherein the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 139-193, 524-531, and 549.

65. A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 8-15, 18-24, 26-75, 84-193, 507-531, and 549-558.

66. A polynucleotide comprising a nucleotide sequence encoding the fusion protein of any one of claims 1 to 64 or the polypeptide of claim 65.

67. 67. The polynucleotide of claim 66, wherein the polynucleotide is a DNA molecule.

68. 68. The polynucleotide of claim 67, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 194-248, 410-464, and 532-547.

69. 67. The polynucleotide of claim 66, wherein the polynucleotide is an RNA molecule.

70. An expression vector comprising the polynucleotide of any one of claims 66 to 69.

71. 71. The expression vector of claim 70, wherein the expression vector is a plasmid.

72. 71. The expression vector of claim 70, wherein the expression vector is a viral vector.

73. A host cell comprising a polynucleotide according to any one of claims 66 to 69 or an expression vector according to any one of claims 70 to 72.

74. 74. The host cell of claim 73, wherein the host cell is a prokaryotic cell.

75. 75. The host cell of claim 74, wherein the prokaryotic cell is an E. coli cell or a Bacillus cell.

76. 74. The host cell of claim 73, wherein the host cell is a eukaryotic cell.

77. 77. The host cell of claim 76, wherein the eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell, and a mammalian cell.

78. 78. The host cell of claim 77, wherein the mammalian cell is selected from the group consisting of a CHO cell, a HeLa cell, and a 293 cell.

79. 79. A population of cells comprising two or more of the host cells of any one of claims 73 to 78.

80. 79. A method of producing a fusion protein according to any one of claims 1 to 64, or a polypeptide according to claim 65, comprising culturing a host cell according to any one of claims 73 to 78 under conditions such that the fusion protein is produced.

81. 72. A pharmaceutical composition comprising an effective amount of a fusion protein according to any one of claims 1 to 64, a polypeptide according to claim 65, a polynucleotide according to any one of claims 66 to 69, or an expression vector according to any one of claims 70 to 72.

82. 82. The pharmaceutical composition of claim 81, wherein the fusion protein has a circulating half-life of at least 14 days when administered to a human.

83. 83. The pharmaceutical composition of claim 81 or 82, wherein the fusion protein has a bioavailability when administered to a human of at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70%.

84. 84. The pharmaceutical composition of claim 82 or 83, wherein the administration is via intravenous or subcutaneous administration.

85. 65. A method for enhancing relaxin-2 associated activity in a primary cell, comprising contacting the primary cell with a fusion protein of any one of claims 1 to 64, thereby enhancing relaxin-2 associated activity in the cell.

86. 86. The method of claim 85, wherein the fusion protein activates the relaxin-2 receptor (RXFP1) on the cell surface.

87. 87. The method of claim 85 or 86, wherein the method increases cAMP levels, induces vasodilation, induces expression of angiogenic factors, induces expression of MMPs, and / or induces collagen degradation in the primary cells.

88. 88. The method of any one of claims 85 to 87, wherein the primary cells are selected from the group consisting of endothelial cells, vascular smooth muscle cells, other vascular cells, cardiomyocytes, other cardiac cells, and fibroblasts.

89. 89. The method of any one of claims 85 to 88, wherein the primary cells are in a subject.

90. 90. The method of claim 89, wherein the subject has a relaxin-2 associated disorder.

91. 91. The method of claim 90, wherein the relaxin-2 associated disorder is selected from the group consisting of a renal disease, a fibrotic disease, and a cardiovascular disease.

92. 92. The method of claim 90 or 91, wherein the disorder is selected from the group consisting of pulmonary hypertension, pulmonary arterial hypertension (PAH), pulmonary hypertension due to left heart disease (PH-LHD), combined pre- and post-capillary pulmonary hypertension (CpcPH), isolated post-capillary pulmonary hypertension (IpcPH), heart failure, heart failure with preserved ejection fraction (HFpEF), heart failure with intermediate ejection fraction (HFmrEF), heart failure with reduced ejection fraction (HFrEF), valvular heart disease, joint disease, shoulder periarthritis (also known as adhesive capsulitis), kidney disease, chronic kidney disease, and hypertensive kidney disease.

93. 92. The method of claim 90 or 91, wherein the disorder is combined pre- and post-capillary pulmonary hypertension (CpcPH) with heart failure with preserved ejection fraction (HFpEF).

94. 92. The method of claim 90 or 91, wherein the disorder is isolated post-capillary pulmonary hypertension (IpcPH) with heart failure with preserved ejection fraction (HFpEF).

95. 92. The method of claim 90 or 91, wherein the disorder is combined pre- and post-capillary pulmonary hypertension (CpcPH) with heart failure with moderate ejection fraction (HFmrEF).

96. 92. The method of claim 90 or 91, wherein the disorder is isolated post-capillary pulmonary hypertension (IpcPH) with heart failure with intermediate ejection fraction (HFmrEF).

97. 82. A method of treating a relaxin-related disorder in a subject in need thereof, comprising administering to the subject an effective amount of a fusion protein of any one of claims 1 to 64, a polynucleotide of any one of claims 66 to 69, an expression vector of any one of claims 70 to 72, or a pharmaceutical composition of any one of claims 81 to 84, thereby treating the relaxin-related disorder.

98. 98. The method of claim 97, wherein the relaxin-related disorder is a relaxin-2-related disorder.

99. 99. The method of claim 98, wherein the relaxin-2 associated disorder is selected from the group consisting of a renal disease, a fibrotic disease, and a cardiovascular disease.

100. 100. The method of claim 98 or 99, wherein the disorder is selected from the group consisting of pulmonary hypertension, pulmonary arterial hypertension (PAH), pulmonary hypertension due to left heart disease (PH-LHD), combined pre- and post-capillary pulmonary hypertension (CpcPH), isolated post-capillary pulmonary hypertension (IpcPH), heart failure, heart failure with preserved ejection fraction (HFpEF), heart failure with intermediate ejection fraction (HFmrEF), heart failure with reduced ejection fraction (HFrEF), kidney disease, chronic kidney disease, and hypertensive kidney disease.

101. 100. The method of claim 98 or 99, wherein the disorder is combined pre- and post-capillary pulmonary hypertension (CpcPH) with heart failure with preserved ejection fraction (HFpEF).

102. 100. The method of claim 98 or 99, wherein the disorder is isolated post-capillary pulmonary hypertension (IpcPH) with heart failure with preserved ejection fraction (HFpEF).

103. 100. The method of claim 98 or 99, wherein the disorder is combined pre- and post-capillary pulmonary hypertension (CpcPH) with heart failure with moderate ejection fraction (HFmrEF).

104. 100. The method of claim 98 or 99, wherein the disorder is isolated post-capillary pulmonary hypertension (IpcPH) with heart failure with intermediate ejection fraction (HFmrEF).

105. 105. The method of any one of claims 97 to 104, wherein the method reduces arterial pressure, increases renal artery blood flow, increases cardiac filling during diastole, resolves established fibrosis, and / or inhibits the development of new fibrosis in the subject.

106. 105. The method of any one of claims 97 to 104, wherein the method increases renal plasma flow in the subject.

107. 107. The method of claim 106, wherein the increase in renal plasma flow in the subject is sustained 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 1 month after a single administration of the fusion protein.

108. 108. The method of any one of claims 97 to 107, wherein the subject is administered the fusion protein by intravenous administration.

109. 109. The method of claim 108, wherein the subject is administered about 0.1 mg / kg to about 20 mg / kg of the fusion protein.

110. 110. The method of claim 108 or 109, wherein the subject is administered about 0.3 mg / kg of the fusion protein.

111. 110. The method of claim 108 or 109, wherein the subject is administered about 1 mg / kg of the fusion protein.

112. 110. The method of claim 108 or 109, wherein the subject is administered about 3 mg / kg of the fusion protein.

113. 110. The method of claim 108 or 109, wherein the subject is administered about 10 mg / kg of the fusion protein.

114. 108. The method of any one of claims 97 to 107, wherein the subject is administered the fusion protein by subcutaneous administration.

115. 115. The method of claim 114, wherein the subject is administered about 100 mg to about 1500 mg of the fusion protein.

116. 116. The method of claim 114 or 115, wherein the subject is administered about 150 mg of the fusion protein.

117. 116. The method of claim 114 or 115, wherein the subject is administered about 300 mg of the fusion protein.

118. 116. The method of claim 114 or 115, wherein the subject is administered about 600 mg of the fusion protein.

119. 119. The method of any one of claims 97-118, wherein the subject is administered the fusion protein once per week, once per two weeks, once per three weeks, once per four weeks, once per five weeks, or once per month.

120. 120. The method of any one of claims 97-119, wherein the fusion protein is present in the serum of the subject at a level of at least about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, or about 5 μg / mL one day after administration.

121. 121. The method of any one of claims 97-120, wherein the fusion protein is present in the serum of the subject at a level of at least about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, or about 5 μg / mL two days after administration.

122. 122. The method of any one of claims 97-121, wherein the fusion protein is present in the serum of the subject at a level of at least about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, or about 5 μg / mL three days after administration.

123. 123. The method of any one of claims 97-122, wherein the fusion protein is present in the serum of the subject at a level of at least about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, or about 5 μg / mL 4 days after administration.

124. 124. The method of any one of claims 97-123, wherein the fusion protein is present in the serum of the subject at a level of at least about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, or about 5 μg / mL 5 days after administration.

125. 125. The method of any one of claims 97-124, wherein the fusion protein is present in the serum of the subject at a level of at least about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, or about 5 μg / mL 6 days after administration.

126. 126. The method of any one of claims 97-125, wherein the fusion protein is present in the serum of the subject at a level of at least about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, or about 5 μg / mL 7 days after administration.

127. 127. The method of any one of claims 97-126, wherein the fusion protein is present in the serum of the subject at a level of at least about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, or about 5 μg / mL 14 days after administration.

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