Relaxin-2 fusion protein analogs and methods of use thereof

Abstract

The present disclosure provides relaxin-2 fusion protein analogs having an enhanced in vivo half-life and methods of making the same. Also disclosed herein are methods of treating relaxin-2 related conditions or diseases using the relaxin-2 fusion protein analogs described herein.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 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 entire disclosures of which are hereby incorporated herein by reference in their entirety.

[0003] References to sequence lists

[0004] This application contains a sequence list, which has been electronically submitted in XML format and is hereby 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

[0005] Relaxin-2 exhibits potent anti-fibrotic activity. In injured tissues, the activation and proliferation of fibroblasts lead to increased collagen production and interstitial fibrosis. Biomechanical overload exacerbates cardiac fibrosis and affects ventricular dysfunction, remodeling, and arrhythmias. However, due to the limited in vivo half-life of relaxin, the compound must be administered via continuous infusion over at least 48 hours. Furthermore, the synthesis of relaxin-2 is challenging. The yield of the bioactive peptide obtained through these methods is extremely low due to the low solubility of the B chain and the need for laborious and specific introduction of cysteine ​​bridges between the A and B chains.

[0006] There is a need for engineered relaxin-2 analogues that have a longer half-life and are easier to produce. Summary of the Invention

[0007] This disclosure provides engineered fusion proteins of relaxin-2 analogues with improved pharmacokinetic properties. This disclosure also provides methods for using these fusion proteins to enhance relaxin-2-related activity in subjects and for treating or preventing relaxin-2-related diseases. The structures of the fusion proteins described herein are based, at least in part, on the surprising finding that lowering the isoelectric point (pI) of relaxin-2 fusion protein analogues prolongs their circulating half-life and improves their pharmacokinetic and pharmacodynamic properties.

[0008] Therefore, in one aspect, this disclosure provides a fusion protein comprising, from the N-terminus to the 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 acids modified relative to the amino acid sequence of SEQ ID NO: 502, and the second peptide comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acids modified relative to the amino acid sequence of SEQ ID NO: 503 or 504; or the first peptide comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acids modified relative to the amino acid sequence of SEQ ID NO: 503 or 504, and the second peptide comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acids modified relative to the amino acid sequence of SEQ ID NO: 502; and optionally, (b) the fusion protein has a pI of 6.0 to 8.2.

[0009] 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 of 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 of less than 9.0. In some embodiments, the fusion protein has a pI of 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.

[0010] In some embodiments, the first peptide comprises the amino acid sequence X 11 LCGRELVRAQIAIC (SEQ ID NO:505), where X 11 The first peptide may be 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.

[0011] In some embodiments, the first peptide comprises the amino acid sequence X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO:506), where: X 12 For K, Q, D, E, L, I, or Y; X 13 Any amino acid other than M, W, or C; X 14 For K, Q, D, E, L, I, or Y; X 15 It can be Q, D, E, L, I, Y, or R; and X 16 R or Q. In some embodiments, the first peptide comprises the amino acid sequence X. 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO:506), where: X 12 For K, Q, D, E, L, I, or Y; X 13 For H, K, Q, Y, L, N, I, S, T, or F; X 14 For K, Q, D, E, L, I, or Y; X 15 It can be Q, D, E, L, I, Y, or R; and X 16 For R or Q. In some embodiments, X 13 Let Q be the number of amino acids. In some embodiments, the first peptide consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0012] In some embodiments, the second peptide comprises the amino acid sequence X 11 LCGRELVRAQIAIC (SEQ ID NO:505), where X 11 The second peptide may be 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.

[0013] In some embodiments, the second peptide comprises the amino acid sequence X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO:506), where: X 12 For K, Q, D, E, L, I, or Y; X 13 Any amino acid other than M, W, or C; X14 For K, Q, D, E, L, I, or Y; X 15 It can be Q, D, E, L, I, Y, or R; and X 16 R or Q. In some embodiments, the second peptide comprises the amino acid sequence X. 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO:506), where: X 12 For K, Q, D, E, L, I, or Y; X 13 For H, K, Q, Y, L, N, I, S, T, or F; X 14 For K, Q, D, E, L, I, or Y; X 15 It can be Q, D, E, L, I, Y, or R; and X 16 For R or Q. In some embodiments, X 13 Let Q be the number of amino acids. In some embodiments, the second peptide consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0014] In some embodiments, the adaptor peptide comprises an amino acid sequence having 12-15 amino acids. In some embodiments, the adaptor peptide comprises the amino acid sequence ASDAAGAX8AX9AGA (SEQ ID NO:17), wherein: X8 is D, E, N, or Q; and X9 is D, E, N, or Q; or the adaptor peptide comprises the amino acid sequence GGEGSGGEGX. 10 GGG (SEQ ID NO:25), where: X 10 X8 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 adaptor peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 26 and 27.

[0015] In another aspect, this disclosure provides a fusion protein comprising, from the N-terminus to the 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 acids modified relative to the amino acid sequence of SEQ ID NO:1, wherein at least one of positions 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 acids modified relative to the amino acid sequence of SEQ ID NO:8, wherein 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 acids modified relative to the amino acid sequence of SEQ ID NO:8, wherein the amino acid at position 22 of the second peptide is not R; and the second peptide comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acids modified relative to the amino acid sequence of SEQ ID NO:1, wherein at least one of positions 4 or 25 of the first peptide is not M; and optionally, (b) the fusion protein has a pI of 6.0 to 8.2.

[0016] 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 of 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 of less than 9.0. In some embodiments, the fusion protein has a pI of 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.

[0017] In some embodiments, the adaptor peptide comprises an amino acid sequence having 12-15 amino acids. In some embodiments, the adaptor peptide comprises the amino acid sequence ASDAAGAX8AX9AGA (SEQ ID NO:17), wherein: X8 is D, E, N, or Q; and X9 is D, E, N, or Q; or the adaptor peptide comprises the amino acid sequence GGEGSGGEGX. 10 GGG (SEQ ID NO:25), where: X 10 X8 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.

[0018] On the other hand, this disclosure provides a fusion protein comprising, from the N-terminus to the 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), wherein: 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), where: X 10 It can be E or S.

[0019] 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 adaptor peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 26, and 27.

[0020] In some embodiments, the first peptide comprises the amino acid sequence DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO:7), wherein: X1 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: X1 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, X1 is Y. In some embodiments, the first peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, and 6. In some embodiments, the first peptide consists of 27, 28, or 29 amino acids.

[0021] In some embodiments, the first peptide comprises the amino acid sequence QLYSALANX4CCX5VGCTX6X7SLAQFC (SEQ ID NO: 16), wherein: X4 is K, Q, D, E, L, I, or Y; X5 is any amino acid other than 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), wherein: 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 NO: 8, 9, 10, 11, 12, 13, 14, 15, and 507. In some embodiments, the first peptide consists of 24 or 25 amino acids.

[0022] In some embodiments, the second peptide comprises the amino acid sequence DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO:7), wherein: X1 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: X1 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, X1 is Y. In some embodiments, the second peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, and 6. In some embodiments, the second peptide consists of 27, 28, or 29 amino acids.

[0023] In some embodiments, the second peptide comprises the amino acid sequence QLYSALANX4CCX5VGCTX6X7SLAQFC (SEQ ID NO: 16), wherein: X4 is K, Q, D, E, L, I, or Y; X5 is any amino acid other than 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), wherein: 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 NO: 8, 9, 10, 11, 12, 13, 14, 15, and 507. In some embodiments, the second peptide consists of 24 or 25 amino acids.

[0024] In some embodiments, 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; 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; 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; 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; 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; 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; 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; 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; 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; 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. The amino acid sequence of SEQ ID NO:8; the first peptide contains the amino acid sequence of SEQ ID NO:2 and the second peptide contains the amino acid sequence of SEQ ID NO:9; the first peptide contains the amino acid sequence of SEQ ID NO:2 and the second peptide contains the amino acid sequence of SEQ ID NO:10; the first peptide contains the amino acid sequence of SEQ ID NO:2 and the second peptide contains the amino acid sequence of SEQ ID NO:11; the first peptide contains the amino acid sequence of SEQ ID NO:2 and the second peptide contains the amino acid sequence of SEQ ID NO:12; the first peptide contains the amino acid sequence of SEQ ID NO:2 and the second peptide contains the amino acid sequence of SEQ ID NO:13; the first peptide contains the amino acid sequence of SEQ ID NO:2 and the second peptide contains the amino acid sequence of SEQ ID NO:14; the first peptide contains the amino acid sequence of SEQ ID NO:2 and the second peptide contains the amino acid sequence of SEQ ID NO:15; the first peptide contains the amino acid sequence of SEQ ID NO:2 and the second peptide contains the amino acid sequence of SEQ ID NO:507; the first peptide contains the amino acid sequence of SEQ ID NO:3 and the second peptide contains the amino acid sequence of SEQ ID NO:8; the first peptide contains the amino acid sequence of SEQ ID NO:8; The amino acid sequence of NO:3 and the second peptide contains the amino acid sequence of SEQ ID NO:9;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; 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; 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; 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; 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; 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; 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; 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; 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; 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. The amino acid sequence of SEQ ID NO:10; the first peptide contains the amino acid sequence of SEQ ID NO:4 and the second peptide contains the amino acid sequence of SEQ ID NO:11; the first peptide contains the amino acid sequence of SEQ ID NO:4 and the second peptide contains the amino acid sequence of SEQ ID NO:12; the first peptide contains the amino acid sequence of SEQ ID NO:4 and the second peptide contains the amino acid sequence of SEQ ID NO:13; the first peptide contains the amino acid sequence of SEQ ID NO:4 and the second peptide contains the amino acid sequence of SEQ ID NO:14; the first peptide contains the amino acid sequence of SEQ ID NO:4 and the second peptide contains the amino acid sequence of SEQ ID NO:15; the first peptide contains the amino acid sequence of SEQ ID NO:4 and the second peptide contains the amino acid sequence of SEQ ID NO:507; the first peptide contains the amino acid sequence of SEQ ID NO:5 and the second peptide contains the amino acid sequence of SEQ ID NO:8; the first peptide contains the amino acid sequence of SEQ ID NO:5 and the second peptide contains the amino acid sequence of SEQ ID NO:9; the first peptide contains the amino acid sequence of SEQ ID NO:5 and the second peptide contains the amino acid sequence of SEQ ID NO:10; the first peptide contains the amino acid sequence of SEQ ID NO:10; the first peptide contains the amino acid sequence of SEQ ID NO:11; the first peptide contains the amino acid sequence of SEQ ID NO:12; the first peptide contains the amino acid sequence of SEQ ID NO:4 and the second peptide contains the amino acid sequence of SEQ ID NO:13; the first peptide contains the amino acid sequence of SEQ ID NO:4 and the second peptide contains the amino acid sequence of SEQ ID NO:14; the first peptide contains the amino acid sequence of SEQ ID NO:4 and the second peptide contains the amino acid sequence of SEQ ID NO:15; the first peptide contains the amino acid sequence of SEQ ID NO:4 and the second peptide contains the amino acid sequence of SEQ ID NO:15; the first peptide contains the amino acid sequence of SEQ ID NO:10; the first The amino acid sequence of NO:5 and the second peptide contains the amino acid sequence of SEQ ID NO:11;The first peptide comprises the amino acid sequence of SEQ ID NO:5 and the second peptide comprises the amino acid sequence of SEQ ID NO:12; the first peptide comprises the amino acid sequence of SEQ ID NO:5 and the second peptide comprises the amino acid sequence of SEQ ID NO:13; the first peptide comprises the amino acid sequence of SEQ ID NO:5 and the second peptide comprises the amino acid sequence of SEQ ID NO:14; 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; 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; the first peptide comprises the amino acid sequence of SEQ ID NO:6 and the second peptide comprises the amino acid sequence of SEQ ID NO:8; the first peptide comprises the amino acid sequence of SEQ ID NO:6 and the second peptide comprises the amino acid sequence of SEQ ID NO:9; the first peptide comprises the amino acid sequence of SEQ ID NO:6 and the second peptide comprises the amino acid sequence of SEQ ID NO:10; the first peptide comprises the amino acid sequence of SEQ ID NO:6 and the second peptide comprises the amino acid sequence of SEQ ID NO:11; the first peptide comprises the amino acid sequence of SEQ ID NO:6 and the second peptide comprises the amino acid sequence of SEQ ID NO:11; The amino acid sequence of SEQ ID NO:12; the first peptide contains the amino acid sequence of SEQ ID NO:6 and the second peptide contains the amino acid sequence of SEQ ID NO:13; the first peptide contains the amino acid sequence of SEQ ID NO:6 and the second peptide contains the amino acid sequence of SEQ ID NO:14; the first peptide contains the amino acid sequence of SEQ ID NO:6 and the second peptide contains the amino acid sequence of SEQ ID NO:15; or the first peptide contains the amino acid sequence of SEQ ID NO:6 and the second peptide contains the amino acid sequence of SEQ ID NO:507.

[0025] 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; 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; 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; 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; 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; 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; 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; 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 ...1; 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; 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; the first peptide comprises the amino acid sequence of SEQ ID NO:9 and the second peptide comprises the amino acid sequence of SEQ The first peptide contains the amino acid sequence of SEQ ID NO:9 and the second peptide contains the amino acid sequence of SEQ ID NO:5; the first peptide contains the amino acid sequence of SEQ ID NO:9 and the second peptide contains the amino acid sequence of SEQ ID NO:6; the first peptide contains the amino acid sequence of SEQ ID NO:10 and the second peptide contains the amino acid sequence of SEQ ID NO:1; the first peptide contains the amino acid sequence of SEQ ID NO:10 and the second peptide contains the amino acid sequence of SEQ ID NO:2; the first peptide contains the amino acid sequence of SEQ ID NO:10 and the second peptide contains the amino acid sequence of SEQ ID NO:3; the first peptide contains the amino acid sequence of SEQ ID NO:10 and the second peptide contains the amino acid sequence of SEQ ID NO:4; the first peptide contains the amino acid sequence of SEQ ID NO:10 and the second peptide contains the amino acid sequence of SEQ ID NO:5; the first peptide contains the amino acid sequence of SEQ ID NO:10 and the second peptide contains the amino acid sequence of SEQ ID NO:6; the first peptide contains the amino acid sequence of SEQ ID NO:11 and the second peptide contains the amino acid sequence of SEQ ID NO:1; the first peptide contains the amino acid sequence of SEQ ID NO:11 and the second peptide contains the amino acid sequence of SEQ ID NO:5. The amino acid sequence of NO:2; the first peptide contains the amino acid sequence of SEQ ID NO:11 and the second peptide contains 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; 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; 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; 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; 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; 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; 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; 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; 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:12. The amino acid sequence of SEQ ID NO:1; the first peptide contains the amino acid sequence of SEQ ID NO:13 and the second peptide contains the amino acid sequence of SEQ ID NO:2; the first peptide contains the amino acid sequence of SEQ ID NO:13 and the second peptide contains the amino acid sequence of SEQ ID NO:3; the first peptide contains the amino acid sequence of SEQ ID NO:13 and the second peptide contains the amino acid sequence of SEQ ID NO:4; the first peptide contains the amino acid sequence of SEQ ID NO:13 and the second peptide contains the amino acid sequence of SEQ ID NO:5; the first peptide contains the amino acid sequence of SEQ ID NO:13 and the second peptide contains the amino acid sequence of SEQ ID NO:6; the first peptide contains the amino acid sequence of SEQ ID NO:14 and the second peptide contains the amino acid sequence of SEQ ID NO:1; the first peptide contains the amino acid sequence of SEQ ID NO:14 and the second peptide contains the amino acid sequence of SEQ ID NO:2; the first peptide contains the amino acid sequence of SEQ ID NO:14 and the second peptide contains the amino acid sequence of SEQ ID NO:3; the first peptide contains the amino acid sequence of SEQ ID NO:14 and the second peptide contains the amino acid sequence of SEQ ID NO:4; the first peptide contains the amino acid sequence of SEQ ID NO:14 and the second peptide contains the amino acid sequence of SEQ ID NO:4; the first peptide contains the amino acid sequence of SEQ ID NO:13 ...2; the first peptide contains the amino acid sequence of SEQ ID NO:14 and the second peptide contains the amino acid sequence of SEQ ID NO:3; the first peptide contains the amino acid sequence of SEQ ID NO:14 and the second peptide contains the amino acid sequence of SEQ ID NO:4; the first peptide contains The amino acid sequence of NO:14 and the second peptide contains the amino acid sequence of SEQ ID NO:5;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; 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; 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; 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; 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; 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; 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; 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; 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; 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; The amino acid sequence of SEQ ID NO:3; the first peptide contains the amino acid sequence of SEQ ID NO:507 and the second peptide contains the amino acid sequence of SEQ ID NO:4; the first peptide contains the amino acid sequence of SEQ ID NO:507 and the second peptide contains the amino acid sequence of SEQ ID NO:5; or the first peptide contains the amino acid sequence of SEQ ID NO:507 and the second peptide contains the amino acid sequence of SEQ ID NO:6.

[0026] In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 28 to 75 and 508 to 515.

[0027] In some embodiments, the fusion protein further comprises 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 glutamic acid 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.

[0028] In some embodiments, 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, IgG Fc comprises the amino acid sequence of human IgG1 Fc.

[0029] In some embodiments, the IgG Fc comprises an amino acid sequence that is at least 95% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:76-83.

[0030] In some embodiments, IgG Fc is attached to the N-terminus of the first peptide. In some embodiments, IgG Fc is attached to the C-terminus of the second peptide.

[0031] In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 84 to 138 and 516 to 523. In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 139-193, 524-531 and 549.

[0032] In another aspect, this disclosure provides a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6, 8-15, 18-24, 26-75, 84-193, 507-531 and 549-558.

[0033] In another respect, this disclosure provides a polynucleotide comprising a nucleotide sequence encoding any of the fusion proteins or polypeptides described herein.

[0034] 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 NO: 194-248, 410-464, and 532-547.

[0035] In some embodiments, the polynucleotide is an RNA molecule.

[0036] In another respect, this disclosure provides an expression vector comprising any of the polynucleotides described herein.

[0037] In some embodiments, the expression vector is a plasmid. In some embodiments, the expression vector is a viral vector.

[0038] In another respect, this disclosure provides a host cell comprising any of the polynucleotides or expression vectors described herein.

[0039] 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 yeast cells, insect cells, and mammalian cells. In some embodiments, the mammalian cell is selected from the group consisting of CHO cells, HeLa cells, and 293 cells.

[0040] On the other hand, this disclosure provides a cell population comprising two or more of any of the host cells described herein.

[0041] In another aspect, this disclosure provides a method for producing any of the fusion proteins or peptides described herein, the method comprising culturing any of the host cells described herein under conditions capable of producing the fusion protein.

[0042] In another aspect, this disclosure provides a pharmaceutical composition comprising an effective amount of any of the fusion proteins, polypeptides, polynucleotides, or expression vectors described herein.

[0043] 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 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 when administered (e.g., to a human). In some embodiments, the fusion protein 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% when administered (e.g., to a human). In some embodiments, the pharmaceutical composition is administered via intravenous or subcutaneous administration.

[0044] In another respect, this disclosure provides a method for enhancing relaxin-2-related activity in primary cells, the method comprising contacting primary cells with any of the fusion proteins described herein, thereby enhancing relaxin-2-related activity in the cells.

[0045] In some embodiments, the fusion protein activates the relaxin-2 receptor (RXFP1) on the cell surface.

[0046] In some embodiments, the method increases cAMP levels in primary cells, thereby inducing vasodilation, inducing the expression of angiogenic factors, inducing the expression of MMPs, and inducing collagen degradation.

[0047] 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.

[0048] In some embodiments, primary cells are present in the subject. In some embodiments, the subject has a relaxin-2 related condition. In some embodiments, the relaxin-2 related condition is selected from the group consisting of: kidney disease, fibrotic disease, and cardiovascular disease. In some embodiments, the condition is selected from the group consisting of: pulmonary hypertension, pulmonary arterial hypertension (PAH), pulmonary hypertension due to left ventricular disease (PH-LHD), combined precapillary and postcapillary pulmonary hypertension (CpcPH), isolated postcapillary pulmonary hypertension (IpcPH), heart failure, heart failure with preserved ejection fraction (HFpEF), heart failure with moderate ejection fraction (HFmrEF), heart failure with decreased ejection fraction (HFrEF), valvular heart disease, arthropathy, frozen shoulder (also known as adhesive capsulitis), kidney disease, chronic kidney disease, and hypertensive kidney disease.

[0049] In some embodiments, the condition is combined precapillary and postcapillary pulmonary hypertension (CpcPH) and heart failure with preserved ejection fraction (HFpEF). In some embodiments, it is isolated postcapillary pulmonary hypertension (IpcPH) and heart failure with preserved ejection fraction (HFpEF). In some embodiments, the condition is combined precapillary and postcapillary pulmonary hypertension (CpcPH) and heart failure with moderate ejection fraction (HFmrEF). In some embodiments, the condition is isolated postcapillary pulmonary hypertension (IpcPH) and heart failure with moderate ejection fraction (HFmrEF).

[0050] In another aspect, this disclosure provides a method for treating relaxin-related syndrome in a subject of need, the method comprising administering to the subject an effective amount of any of the fusion proteins, polynucleotides, expression vectors, or pharmaceutical compositions described herein, thereby treating relaxin-related syndrome.

[0051] In some embodiments, relaxin-2-related conditions are selected from the group consisting of: kidney disease, fibrotic disease, and cardiovascular disease. In some embodiments, conditions are selected from the group consisting of: pulmonary hypertension, pulmonary arterial hypertension (PAH), pulmonary hypertension due to left ventricular disease (PH-LHD), combined precapillary and postcapillary pulmonary hypertension (CpcPH), isolated postcapillary pulmonary hypertension (IpcPH), heart failure, heart failure with preserved ejection fraction (HFpEF), heart failure with moderate ejection fraction (HFmrEF), heart failure with decreased ejection fraction (HFrEF), kidney disease, chronic kidney disease, and hypertensive kidney disease. In some embodiments, the method reduces the subject's arterial pressure, increases renal artery blood flow, increases diastolic cardiac filling, eliminates existing fibrosis, and / or inhibits the development of new fibrosis.

[0052] In some embodiments, the method increases renal plasma flow in a subject. In some embodiments, the increase in renal plasma flow in the subject persists for 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 at 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% after a single administration of the fusion protein.

[0053] In some embodiments, the condition is combined precapillary and postcapillary pulmonary hypertension (CpcPH) and heart failure with preserved ejection fraction (HFpEF). In some embodiments, it is isolated postcapillary pulmonary hypertension (IpcPH) and heart failure with preserved ejection fraction (HFpEF). In some embodiments, the condition is combined precapillary and postcapillary pulmonary hypertension (CpcPH) and heart failure with moderate ejection fraction (HFmrEF). In some embodiments, the condition is isolated postcapillary pulmonary hypertension (IpcPH) and heart failure with moderate ejection fraction (HFmrEF).

[0054] In some embodiments, the fusion protein is administered to the subject via intravenous administration. In some embodiments, the fusion protein is administered to the subject at a concentration of about 0.1 mg / kg to about 20 mg / kg. In some embodiments, the fusion protein is administered to the subject at a concentration of about 0.3 mg / kg. In some embodiments, the fusion protein is administered to the subject at a concentration of about 1 mg / kg. In some embodiments, the fusion protein is administered to the subject at a concentration of about 3 mg / kg. In some embodiments, the fusion protein is administered to the subject at a concentration of about 10 mg / kg.

[0055] In some embodiments, the fusion protein is administered to the subject via intravenous infusion. In some embodiments, the fusion protein is administered to the subject via intravenous infusion over a period of 30 minutes. In some embodiments, the fusion protein is administered to the subject via intravenous infusion over a period of 60 minutes. In some embodiments, the fusion protein is administered to the subject via intravenous infusion over a period of 30 to 60 minutes.

[0056] In some embodiments, the fusion protein is administered to the subject subcutaneously. In some embodiments, about 100 mg to about 1500 mg of the fusion protein is administered to the subject. In some embodiments, about 150 mg of the fusion protein is administered to the subject. In some embodiments, at least 150 mg of the fusion protein is administered to the subject. In some embodiments, about 300 mg of the fusion protein is administered to the subject. In some embodiments, about 600 mg of the fusion protein is administered to the subject.

[0057] In some embodiments, the fusion protein is administered to the subject once every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 1 month. Attached Figure Description

[0058] Figures 1A to 1C The transient expression of human (SEQ ID NO:87 and wild-type (WT) human relaxin-2 induced by SEQ ID NO:87 was described respectively. Figure 1A ), rats ( Figure 1B ) and monkey ( Figure 1C cAMP response in HEK293 cells of RXFP1.

[0059] Figures 2A to 2C To plot the changes in pharmacokinetic (PK) values ​​over time, these values ​​were obtained by measuring the concentrations of various relaxin-2 fusion protein analogs as indicated in rat serum after intravenous (IV) administration of the corresponding protein analogs at 5 mg / kg (using human Fc levels as an indicator).

[0060] Figure 3 A graph depicting the change in renal artery blood flow (RABF) over time compared to baseline in rats administered various relaxin-2 fusion protein analogs as indicated.

[0061] Figure 4A and 4B To depict the response of RABF to the dose indicated as SEQ ID NO:87 or SEQ ID NO:497 ( Figure 4A ) and serum levels of fusion proteins ( Figure 4B A chart showing how human Fc levels change over time (using human Fc levels as an indicator). Figure 4C A graph depicting how serum PK changes with increasing RABF (minus baseline).

[0062] Figure 5A and 5B A graph depicting the effect of low-dose SEQ ID NO:87 on increasing and maintaining RABF in treated rats was shown to be significantly superior to SEQ ID NO:497. Figure 5A The increase of RABF over time was shown in rats treated with SEQ ID NO:87 or SEQ ID NO:497. Figure 5B The results showed that, by area under the curve analysis, the RABF in rats treated with SEQ ID NO:87 was significantly increased compared with that in rats treated with SEQ ID NO:497.

[0063] Figure 6A and 6B A graph depicting the effect of SEQ ID NO:87 on right ventricular systolic pressure (RVSP) after three weeks of intravenous treatment with SEQ ID NO:87 at 10 mg / kg in MCT-induced rats (MCT), where B cell depletion was performed using anti-CD20 antibody (without CD20 or +CD20). Figure 6A ) or not performing B cell depletion ( Figure 6B In animals that did not undergo B-cell depletion, sildenafil was used as a positive control.

[0064] Figure 7A and 7BA graph depicting the effect of SEQ ID NO:87 on mean pulmonary artery pressure (mPAP) after three weeks of intravenous treatment with SEQ ID NO:87 at 10 mg / kg in MCT-induced rats (MCT), where B cell depletion was performed using anti-CD20 antibody (without CD20 or +CD20). Figure 7A ) or not performing B cell depletion ( Figure 7B In animals that did not undergo B-cell depletion, sildenafil was used as a positive control.

[0065] Figure 8A and 8B A graph depicting the effect of SEQ ID NO:87 on the Fulton index after three weeks of intravenous treatment with SEQ ID NO:87 at 10 mg / kg in MCT-induced rats (MCT), where B cell depletion was performed using anti-CD20 antibodies (without CD20 or +CD20). Figure 8A ) or not performing B cell depletion ( Figure 8B In animals that did not undergo B-cell depletion, sildenafil was used as a positive control.

[0066] Figure 9A and 9B A graph depicting the effect of SEQ ID NO:87 on serum NT-pro-BNP levels after three weeks of intravenous treatment with SEQ ID NO:87 at 10 mg / kg in MCT-induced rats (MCT), where B cell depletion was performed using anti-CD20 antibody (without CD20 or +CD20). Figure 9A ) or not performing B cell depletion ( Figure 9B In animals that did not undergo B-cell depletion, sildenafil was used as a positive control.

[0067] Figure 10A and 10B To characterize the effect of SEQ ID NO:87 on lung inflammation after three weeks of intravenous treatment with SEQ ID NO:87 at 10 mg / kg in MCT-induced rats (MCT). Figure 10A ) and pulmonary artery muscularization ( Figure 10B Figures showing the histopathological results of the effect of anti-CD20 antibody (+ anti-CD20) on B cell depletion. *: p<0.05; **: p<0.01; ****: p<0.0001 after nonparametric one-way ANOVA and post-hoc Dung's multiple comparison test.

[0068] Figure 11A graph depicting the effect of SEQ ID NO:87 on mortality after three weeks of intravenous treatment with 10 mg / kg of SEQ ID NO:87 in MCT-induced rats (MCT) with or without B-cell depletion using anti-CD20 antibody (without CD20 or +CD20). Sildenafil was used as a positive control in animals without B-cell depletion.

[0069] Figure 12 A graph depicting the effects of SEQ ID NO:496 and SEQ ID NO:313 on collagen deposition in the renal parenchyma in a mouse model of unilateral ureteral obstruction (UUO) according to aspects of this disclosure. Mice underwent UUO surgery and were treated with a mordant (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), respectively. Control mice that underwent sham surgery and were treated with a mordant (PBS; n=5) are also shown. After treatment, the obstructed kidney was removed and fixed for histological examination. Collagen was detected by immunolabeling. The percentage of collagen level relative to the total immunolabeled area is depicted. *: p<0.05; ****: p<0.0001.

[0070] Figure 13 A graph depicting the effect of SEQ ID NO:87 on collagen deposition in the renal cortex of a mouse model of undescended urethritis (UUO) according to an aspect of the invention is shown. Mice underwent UUO surgery and were treated with either a mordant (PBS; n=8) or 10 mg / kg SEQ ID NO:87 (n=8). Control mice that underwent sham surgery and were treated with the mordant (PBS; n=8) are also shown. After treatment, the obstructed kidney was removed and fixed for histological examination. Collagen was detected by immunolabeling. The percentage of collagen level relative to the total immunolabeled area is depicted. ****: p<0.0001; *: p=0.02.

[0071] Figure 14 A graph depicting the effect of SEQ ID NO:87 on TNFα levels in the renal cortex of a mouse UUO model according to an aspect of the invention. (See diagram for...) Figure 13 The treated mice were analyzed, and the TNFα levels in the protein lysates were quantified by electrochemiluminescence analysis. ****: p < 0.0001; ***: p < 0.001.

[0072] Figure 15A graph depicting the effect of SEQ ID NO:87 on isoproterenol-induced cardiac hypertrophy according to an aspect of the invention. Mice were treated with a catalyst (n=10), isoproterenol (n=10), or isoproterenol and SEQ ID NO:87 (n=6). After treatment, the body weight and heart rate of each mouse were measured. Heart weight normalized to body weight (HW / BW) for each group is plotted. ****: p<0.0001.

[0073] Figure 16 A graph illustrating the effect of SEQ ID NO:87 on isoproterenol-induced fibrosis according to an aspect of the invention. (See diagram for...) Figure 15 The mice were treated, and collagen content was quantified using a hydroxyproline assay. ****: p<0.0001; ***: p<0.001.

[0074] Figure 17A and 17B To characterize the pharmacokinetic (PK) profile of healthy patients receiving a single 0.3 mg / kg IV dose of SEQ ID NO:87 Figure 17A ) and PD ( Figure 17B (Charts of data) Figure 17A The changes in the concentration of SEQ ID NO:87 in patients treated with the drug over time are shown (solid line), as well as the p-p-p-value curve of SEQ ID NO:87 as predicted using a non-human primate model (dashed line). Figure 17B The changes in renal plasma flow relative to baseline on days 2, 8, and 17 were shown in healthy patients who received SEQ ID NO:87 or placebo (PBO).

[0075] Figure 18 A graph depicting the PK data of healthy patients who received a single 150 mg SC dose of SEQ ID NO:87 shows the change in the concentration of SEQ ID NO:87 over time in the treated patients. Detailed Implementation

[0076] The therapeutic potential of relaxin-2 was highlighted in the RELAX-AHF trial (see, for example, 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 prolong its in vivo half-life, and the protein must be administered via continuous IV infusion over a 48-hour period. Extended half-life versions of relaxin-2 have been produced by fusing the peptide hormone with human IgG1 Fc or albumin-bound nanobodies, but such fusion proteins have shown extremely rapid plasma clearance. This disclosure is partly based on the inventors' findings that reducing the positive charge of relaxin-2 and its heparin binding leads to a significant improvement in the pharmacokinetic and pharmacodynamic profile.

[0077] This 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 bound via a peptide linker, wherein the fusion protein has a high in vivo circulating half-life when administered to a mammal. In some embodiments, the in vivo circulating half-life of the fusion protein provided by this disclosure is greater than 2 hours. In some embodiments, the fusion protein provided by this disclosure has a low pI. In some embodiments, the pI of the fusion protein provided by this disclosure is less than 8.5. In some embodiments, the low pI of the fusion protein provided by this disclosure 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 length of the peptide linker is a total of 10 to 15 amino acids.

[0078] definition

[0079] 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 pertains. It should be understood that the foregoing general description and the following detailed description are exemplary and illustrative only, and not limiting of any claimed subject matter. In this application, the singular is used to include the plural unless otherwise specifically stated. It must be noted that, as used in the specification and appended claims, the singular forms “a” and “the” include plural indicators unless the context clearly indicates otherwise. In this application, the use of “or” means “and / or” unless otherwise stated. Furthermore, the use of the term “including” and other forms such as “include,” “includes,” and “included” is not restrictive. Section headings used herein are for organizational purposes only and should not be construed as limiting the described subject matter.

[0080] As used herein, the term "polynucleotide" refers to a polymer of DNA or RNA. Polynucleotide sequences can be single-stranded or double-stranded; contain natural, non-natural, or modified nucleotides; and contain natural, non-natural, or modified internucleotide bonds, such as aminophosphate bonds or thiophosphate bonds, rather than phosphodiesters found between nucleotides in an unmodified polynucleotide sequence. 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 methods such as cloning polynucleotide sequences from recombinant libraries or cell genomes, using conventional cloning techniques and polymerase chain reactions, and synthetic methods.

[0081] The terms “protein” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acids linked by one or more peptide bonds. As used herein, “amino acid sequence” refers to information describing the relative order and identity of the amino acid residues that make up the polypeptide.

[0082] As used herein, the term “amino acid sequence with 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.

[0083] Mathematical algorithms can be used to determine the "percentage of identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences). Specific, non-limiting examples of mathematical algorithms used to compare two sequences are those of Karlin S and Altschul SF, (1990) PNAS 87:2264-2268, modified as in Karlin S and Altschul SF, (1993) PNAS90:5873-5877, each of which is incorporated herein by reference in its entirety. Such algorithms are incorporated in the NBLAST and XBLAST procedures of Altschul SF et al., (1990) J Mol Biol 215:403, which is also incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed using the NBLAST nucleotide procedure parameter set (e.g., fraction = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search can be performed using the XBLAST procedure parameter set (e.g., fraction = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules described herein. For gap alignments to be performed for comparative purposes, gap BLAST can be used as described in Altschul SF et al., (1997) Nuc Acids Res 25:3389-3402, which is incorporated herein by reference in its entirety. Alternatively, PSIBLAST can be used to perform an iterative search to detect distance relationships between molecules. Ibid. When using the BLAST, gap BLAST, and PSIBLAST procedures, the default parameters of the corresponding procedures (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17, which is incorporated herein by reference in its entirety. This algorithm has been incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, the PAM120 weighted residue table, vacancy length penalty of 12, and vacancy penalty of 4 can be used.

[0084] Whether or not gaps are allowed, techniques similar to those described above can be used to determine the percentage of identity between two sequences. When calculating the percentage of identity, typically only exact matches are counted.

[0085] As used herein, the term "linked to" refers to a covalent or non-covalent bond between two molecules or parts. Those skilled in the art will understand that when a first molecule or part is linked to a second molecule or part, the link need not be direct, but can be achieved through the insertion of a molecule or part.

[0086] As used herein, the terms "human relaxin-2B chain," "relaxin B chain," "relaxin B," or "rel B" refer to a peptide or derivative thereof comprising the amino acid sequence shown in DSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ ID NO:249). In some embodiments, a derivative of the relaxin B chain comprises the amino acid sequence of SEQ ID NO:154 having variations of 1, 2, 3, 4, or 5 amino acids.

[0087] As used herein, the terms "human relaxin-2A chain," "relaxin A chain," "relaxin A," or "rel A" refer to a peptide or derivative thereof comprising the amino acid sequence shown in QLYSALANKCCHVGCTKRSLARFC (SEQ ID NO: 257). In some embodiments, a derivative of the relaxin A chain comprises the amino acid sequence of SEQ ID NO: 155 having variations of 1, 2, 3, 4, or 5 amino acids.

[0088] As used herein, the term "connector peptide" refers to a peptide that links the relaxin A chain and the relaxin B chain in the fusion protein described herein.

[0089] As used herein, the term "acidic amino acid" refers to an amino acid whose side chain contains a carboxylic acid. In some embodiments, the acidic amino acid is aspartic acid, glutamic acid, 2-aminoadipic acid, 2-aminobutyric acid, or 2-aminopimelic acid. In some embodiments, the acidic amino acid includes aspartic acid and glutamic acid.

[0090] As used herein, the term "non-acidic amino acid" refers to an amino acid that is not a non-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.

[0091] 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 an IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the IgG Fc is an IgG1 Fc region.

[0092] As used herein, the term “EU numbering system” refers to the EU numbering convention for the constant regions of antibodies, 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 ed., 1991, each of which is incorporated herein by reference in its entirety.

[0093] As used herein, the terms “relaxin-2 receptor,” “human relaxin-2 receptor,” “human relaxin receptor 1,” “RXFP1,” or “LGR7” refer to the natural receptor for relaxin-2 in the human body. In some embodiments, RXFP1 comprises the amino acid sequence shown in the NCBI reference sequence: 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 herein by reference in their entirety.

[0094] As used herein, the term “treat / treating / treatment” refers to the treatment or preventive measures described herein. In some embodiments, a “treatment” method involves administering a fusion protein to a subject who has or is susceptible to a disease or condition, in order to prevent, cure, delay, or relapse a disease or condition, reduce its severity, or improve one or more of its symptoms, or to extend the subject’s survival beyond what would be expected in the absence of such treatment.

[0095] As used in this article, the term "effective amount" in the context of administering a therapy to a subject refers to the amount of therapy required to achieve the desired preventive or therapeutic effect.

[0096] As used herein, the term "subject" includes any human or non-human animal. In one embodiment, the subject is a human or a non-human animal. In one embodiment, the subject is a human.

[0097] As used herein, the term "pI" refers to the isoelectric point, that is, the pH of the solution when the fusion protein has zero charge on one side. In some embodiments, pI is a calculated or theoretical pI. In some embodiments, pI is measured experimentally using instruments.

[0098] Fusion protein

[0099] This 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 circulating half-life when administered to a mammal. In some embodiments, the fusion protein comprises, from the N-terminus to the 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 the N-terminus to the 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 IgG Fc. The IgG Fc is linked to the N-terminus or C-terminus of a human relaxin B chain-linker protein-human relaxin A chain fusion protein or a human relaxin A chain-linker protein-human relaxin B chain fusion protein. In some embodiments, the fusion protein forms a homodimer via interactions between the IgG Fc moieties. In some embodiments, the IgG Fc is replaced by PEG.

[0100] Human relaxin-2B chain derivatives

[0101] This disclosure provides a human relaxin-2B chain derivative, wherein the derivative has 1, 2, 3, 4, or 5 amino acid variations when compared with 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.

[0102] In some embodiments, human relaxin-2B chain derivatives comprise or consist of the following formula: DSWX 19 EEVIKLCGRELVRAQIAICGX 20 ST(SEQ ID NO:250), where X 19 and X 20 It is absent or consists of any amino acid. In some embodiments, X 19 The amino acid is methionine (M), glutamine (Q), glutamic acid (E), asparagine (N), aspartic acid (D), serine (S), or threonine (T). In some embodiments, X 19 It is methionine (M), lysine (K), or glutamine (Q). In some embodiments, X 20It is methionine (M), lysine (K), glutamine (Q), or asparagine (N). In some embodiments, X 20 It is methionine (M) or lysine (K). In some embodiments, X 20 For lysine (K). In some embodiments, X... 19 It is methionine (M), lysine (K), or glutamine (Q), and X 20 It is either methionine (M) or lysine (K).

[0103] This disclosure provides a human relaxin-2B 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:1, wherein the amino acid at position 4 is not methionine (M), or the amino acid at position 25 is not methionine (M). In some embodiments, 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:1, wherein the amino acid at position 4 is not methionine (M), and the amino acid at position 25 is not methionine (M). In some embodiments, 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:1, wherein the amino acid at at least one of positions 4 or 25 of the first peptide is not methionine (M).

[0104] In some embodiments, the human relaxin-2B 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-2B 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-2B chain derivative comprises or consists of the following formula: DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO:7), wherein X1 is any amino acid other than 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-2B chain derivative comprises or consists of the following formula: DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO:7), wherein X1 is any amino acid other than 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).

[0105] In some embodiments, the 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, the human relaxin-2B chain derivative comprises or consists of the following formula: X 11 LCGRELVRAQIAIC (SEQ ID NO:505), where X 11The amino acids are lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y).

[0106] In some embodiments, the human relaxin-2B chain derivative used in the fusion protein described herein does not contain the amino acid sequence shown in SEQ ID NO:251-254:

[0107] DSWKEEVIKLCGRELVRAQIAICGKSTAS(SEQ ID NO:251);

[0108] DSWKEEVIKLCGRELVRAQIAICGKSTWS(SEQ ID NO:252);

[0109] DSWMEEVIKLCGRELVRAQIAICGKSTAS (SEQ ID NO:253); and

[0110] DSWMEEVIKLCGRELVRAQIAICGKSTWS (SEQ ID NO: 254).

[0111] In some embodiments, the length of the human relaxin-2B chain derivative is 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids. In some embodiments, the length of the human relaxin-2B chain derivative is 25, 26, 27, 28, or 29 amino acids. In some embodiments, the length of the human relaxin-2B chain derivative is 27 amino acids. In some embodiments, the length of the human relaxin-2B chain derivative is 15-29 amino acids. In some embodiments, the length of 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.

[0112] In some embodiments, human relaxin-2B chain derivatives comprise or consist of the amino acid sequences shown in Table 1 below.

[0113] Table 1. Sequences of human relaxin-2B chain derivatives

[0114] SEQ ID NO: amino acid sequence 1 DSWQEEVIKLCGRELVRAQIAICGKST 2 DSWQEEVIKLCGRELVRAQIAICGQST 3 DSYQEEVIKLCGRELVRAQIAICGKST 4 DSFQEEVIKLCGRELVRAQIAICGKST 5 DSLQEEVIKLCGRELVRAQIAICGKST 6 DSIQEEVIKLCGRELVRAQIAICGKST 249 DSWMEEVIKLCGRELVRAQIAICGMSTWS 255 DSWKEEVIKLCGRELVRAQIAICGKST 256 DSWMEEVIKLCGRELVRAQIAICGKST 502 KLCGRELVRAQIAIC

[0115] In some embodiments, the human relaxin-2B chain derivative comprises or is composed of SEQ ID NO:1. In some embodiments, the human relaxin-2B chain derivative comprises or is composed of SEQ ID NO:2. In some embodiments, the human relaxin-2B chain derivative comprises or is composed of SEQ ID NO:3. In some embodiments, the human relaxin-2B chain derivative comprises or is composed of SEQ ID NO:4. In some embodiments, the human relaxin-2B chain derivative comprises or is composed of SEQ ID NO:5. In some embodiments, the human relaxin-2B chain derivative comprises or is composed of SEQ ID NO:6. In some embodiments, the human relaxin-2B chain derivative comprises or is composed of SEQ ID NO:249. In some embodiments, the human relaxin-2B chain derivative comprises or is composed of SEQ ID NO:255. In some embodiments, the human relaxin-2B chain derivative comprises or is composed of SEQ ID NO:256. In some embodiments, the human relaxin-2B chain derivative comprises or is composed of SEQ ID NO:502.

[0116] In some embodiments, the human relaxin-2B 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, the human relaxin-2B 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, the human relaxin-2B 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-2B 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-2B 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-2B 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-2B 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-2B 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-2B 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-2B 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).

[0117] In some embodiments, the human relaxin-2B chain derivative further comprises two residues at the C-terminus. For example, SEQ ID NO:1-7, 250, 255, and 256 may further comprise two residues at the C-terminus, such as tryptophan (W) and serine (S) at the C-terminus of SEQ ID NO:249. In some embodiments, the human relaxin-2B chain derivative further comprises an X amino acid and serine (S) at the C-terminus, wherein the X amino acid may be any amino acid other than cysteine ​​(C). Therefore, in some embodiments, the C-terminus of the human relaxin-2B chain derivative is XS, where X is any amino acid other than cysteine ​​(C).

[0118] 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, such as the stability of the relaxin-2B chain derivative after light or heat exposure as measured by methods known in the art, for example, the SEC method to assess aggregate formation and the CE-SDS method to assess purity. In some embodiments, the amino acid corresponding to amino acid position 3 in SEQ ID NO:3 in the human relaxin-2B chain derivative is tyrosine (Y).

[0119] Human relaxin-2A chain derivatives

[0120] This disclosure provides human relaxin-2A chain derivatives, wherein the derivatives have 1, 2, 3, 4, or 5 amino acid variations when compared with 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 tyrosine. In some embodiments, the amino acid corresponding to position 23 of SEQ ID NO:257 must be phenylalanine. In some embodiments, the amino acid corresponding to position 3 of SEQ ID NO:257 must be tyrosine; and the amino acid corresponding to position 23 of SEQ ID NO:257 must be phenylalanine.

[0121] In some embodiments, human relaxin-2A chain derivatives comprise or consist of the following formula: X 21 QX 22 YSALANKCCHVGCTKRSLAX 23 FC (SEQ ID NO:258), where X 21 X22 and X 23 It is absent or consists of any amino acid. In some embodiments, X 21 It may contain arginine (R), lysine (K), glutamine (Q), asparagine (N), histidine (H), serine (S), threonine (T), proline (P), glycine (G), or be absent. In some embodiments, X 21 It may be arginine (R), glycine (G), or absent. In some embodiments, X 21 Arginine (R) or absent. In some embodiments, X 22 It is leucine (L), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), serine (S), or threonine (T). In some embodiments, X 22 It is leucine (L) or aspartic acid (D). In some embodiments, X 23 It is arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D), asparagine (N), serine (S), or threonine (T). In some embodiments, X 23 It is arginine (R), glutamine (Q), or glutamic acid (E). In some embodiments, X 21 If arginine (R) is present or absent, X 22 It is either leucine (L) or aspartic acid (D), and X 23 It can be arginine (R), glutamine (Q), or glutamic acid (E).

[0122] This 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, wherein the amino acid at position 22 of the second peptide is not arginine (R).

[0123] 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 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 other than 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).

[0124] In some embodiments, the 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, the human relaxin-2A chain derivative comprises or consists of the following formula: X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO:506), where X 12 The amino acids are lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X 13 For histidine (H), lysine (K), glutamine (Q), tyrosine (Y), leucine (L), asparagine (N), isoleucine (I), serine (S), threonine (T), or phenylalanine (F); X 14The amino acids are lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X 15 The amino acids are glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R); and X 16 It is arginine (R) or glutamine (Q). In some embodiments, the human relaxin-2A chain derivative comprises or consists of the following formula: X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO:506), where X 12 The amino acids are lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X 13 For histidine (H), lysine (K), glutamine (Q), tyrosine (Y), leucine (L), asparagine (N), isoleucine (I), serine (S), threonine (T), or phenylalanine (F); X 14 The amino acids are lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X 15 The amino acids are glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R); and X 16 The derivative is arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D), asparagine (N), serine (S), or threonine (T). In some embodiments, the human relaxin-2A chain derivative comprises or consists of the following formula: X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO:506), where X 12 The amino acids are lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X 13 X is any amino acid except methionine (M), tryptophan (W), and cysteine ​​(C); 14 The amino acids are lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X 15The amino acids are glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R); and X 16 It is arginine (R) or glutamine (Q). In some embodiments, the human relaxin-2A chain derivative comprises or consists of the following formula: X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO: 506), where X 12 The amino acids are lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X 13 X is any amino acid except methionine (M), tryptophan (W), and cysteine ​​(C); 14 The amino acids are lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); X 15 The amino acids are glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R); and X 16 The components are arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D), asparagine (N), serine (S), or threonine (T).

[0125] In some embodiments, the length of the human relaxin-2A chain derivative is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the length of the human relaxin-2A chain derivative is 22, 23, 24, 25, or 26 amino acids. In some embodiments, the length of the human relaxin-2A chain derivative is 24 amino acids. In some embodiments, the length of the human relaxin-2A chain derivative is 25 amino acids. In some embodiments, the length of the human relaxin-2A chain derivative is 16-25 amino acids. In some embodiments, the length of the human relaxin-2A chain derivative is 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0126] In some embodiments, human relaxin-2A chain derivatives comprise or consist of the amino acid sequences shown in Table 2 below.

[0127] Table 2. Sequences of human relaxin-2A chain derivatives

[0128]

[0129]

[0130] In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:8. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:9. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:10. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:11. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:12. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:13. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:14. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:15. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:257. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:259. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:260. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:261. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:503. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:504. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:507. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:550. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:551. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:552. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:553. In some embodiments, the human relaxin-2A chain derivative comprises or is composed of SEQ ID NO:554. In some embodiments, the human relaxin-2A chain derivative comprises or is composed 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.

[0131] 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-2A 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-2A 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-2A 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-2A 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-2A 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-2A 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-2A 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-2A 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-2A 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-2A 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). In some embodiments, the human relaxin-2A 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-2A 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-2A 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-2A 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-2A 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-2A 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).

[0132] 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, such as the stability of the relaxin-2A chain derivative after light or heat exposure as measured by methods known in the art, for example, the SEC method to assess aggregate formation and the CE-SDS method to assess purity. In some embodiments, the amino acid corresponding to amino acid position 12 in SEQ ID NO:12 in the human relaxin-2A chain derivative is glutamine (Q).

[0133] Connector peptide

[0134] This disclosure provides a linker peptide having at least two acidic amino acids. In some embodiments, the acidic amino acid is glutamic acid. In some embodiments, the acidic amino acid is aspartic acid. 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.

[0135] In some embodiments, the linker peptide is 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long. In some embodiments, the linker peptide is 12, 13, 14, or 15 amino acids long. 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 long 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, the non-acidic amino acids can be any standard amino acid that is not aspartic or glutamic acid. In some embodiments, the non-acidic amino acids can be any amino acid that does not have a carboxylic acid in its side chain. In some embodiments, the 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. In some embodiments, the non-acidic amino acid is glycine.

[0136] In some embodiments, the adaptor peptide comprises acidic amino acids, wherein all acidic amino acids are the same amino acid. In some embodiments, all acidic amino acids in the adaptor peptide are glutamic acid. In some embodiments, all acidic amino acids in the adaptor peptide are aspartic acid. In some embodiments, the adaptor peptide comprises amino acids as a mixture of acidic amino acids. In some embodiments, the adaptor peptide comprises both glutamic acid and aspartic acid as acidic amino acids.

[0137] In some embodiments, the adaptor peptide comprises an amino acid sequence X selected from the group consisting of the following groups. 17 X 17 X 17 X 18 X 17 X17X 17 X 18 X 17 X 17 X 17 X 18 X 17 ;

[0138] 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 ;

[0139] 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 ;

[0140] 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

[0141] X 17 X 17 X 18 X 17 X 18 X 17 X 17 X18 X 17 X 18 X 17 X 17 X 17 , where X 17 It is a non-acidic amino acid, and X 18 It is an acidic amino acid.

[0142] In some embodiments, the adaptor peptide comprises non-acidic amino acids, wherein all non-acidic amino acids are the same amino acid. In some embodiments, all non-acidic amino acids in the adaptor peptide are glycine. In some embodiments, the adaptor peptide comprises amino acids as a mixture of non-acidic amino acids. In some embodiments, the adaptor peptide comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different types of non-acidic amino acids.

[0143] In some embodiments, the adaptor 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).

[0144] In some embodiments, the adaptor peptide comprises GGEGSGGEGX 10 GGG (SEQ ID NO:25), where X 10 It is either glutamic acid (E) or serine (S).

[0145] In some embodiments, the adaptor peptide comprises or consists of the amino acid sequences shown in Table 3 below.

[0146] Table 3. Adaptor peptide sequences

[0147] SEQ ID NO: amino acid sequence 18 ASDAAGADADAGA 19 ASDAAGADANAGA 20 ASDAAGADAQAGA 21 ASDAAGAEAEAGA 22 ASDAAGANADAGA 23 ASDAAGAQADAGA 24 ASDAAGAQAQAGA 26 GGEGSGGEGEGGG 27 GGEGSGGEGSGGG 267 GGGE 268 GEGE 269 GGEG 270 GEGG 271 GGEE 272 GGGEGGGEGGGEG 273 GGGEGGGEGGGEGGG 274 GEGGGEEGGGEGG 275 GGGEEGGGEEGGG 276 GGEGEGGEGEGGS

[0148] In some embodiments, the adaptor peptide comprises 2, 3, 4, or 5 repeating sequences of SEQ ID NO:267, 268, 269, 270, or 271. For example, the 3 repeating sequences of SEQ ID NO:267 are the amino acid sequence of GGGEGGGEGGGE (SEQ ID NO:277).

[0149] In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:18. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:19. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:20. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:21. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:22. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:23. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:24. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:26. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:27. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:267. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:268. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:269. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:270. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:271. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:272. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:273. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:274. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:275. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:276. In some embodiments, the adaptor peptide comprises or is composed of SEQ ID NO:277.

[0150] The relaxin / protocol peptide combination of fusion proteins

[0151] In some embodiments, the fusion protein comprises an N-terminal peptide or a first peptide, a linker peptide, and a C-terminal peptide or a second peptide. In some embodiments, the N-terminal peptide comprises a human relaxin-2A chain or a derivative thereof (RelA), and the C-terminal peptide comprises a human relaxin-2B chain or a derivative thereof (RelB). In some embodiments, the N-terminal peptide comprises a human relaxin-2B chain or a derivative thereof, and the C-terminal peptide comprises a human relaxin-2A chain or a derivative thereof. Any embodiment of a human relaxin-2A chain or a derivative thereof, and any combination of a human relaxin-2A chain or a derivative thereof linked by any linker peptide disclosed herein, may be used to construct embodiments of the fusion protein described herein. In some embodiments, at least one of the N-terminal peptide and the C-terminal peptide is a derivative of a human relaxin-2A chain or a 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.

[0152] 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 16-25 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 16-25 amino acids.

[0153] In some embodiments, the human relaxin-2B chain derivative consists of 15-29 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 15-29 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 15-29 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 15-29 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 15-29 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 15-29 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 15-29 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 15-29 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 15-29 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 15-29 amino acids, and the human relaxin-2A chain derivative consists of 25 amino acids.

[0154] Specific embodiments of the fusion protein provided in this disclosure are shown in Table 4 below.

[0155] Table 4. Fusion Proteins

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166] Table 4 (continued)

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177] Table 4 (continued)

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188] Table 4 (continued)

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200] Table 4 (continued)

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211] Table 4 (continued)

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222] Table 4 (continued)

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233] In some embodiments, there are additional amino acids between the N-terminal peptide and the adaptor peptide. In some embodiments, there are additional amino acids between the C-terminal peptide and the adaptor peptide. In some embodiments, there are no additional amino acids between the N-terminal peptide and the adaptor peptide. In some embodiments, there are no additional amino acids between the C-terminal peptide and the adaptor peptide.

[0234] In some embodiments, the portion of the fusion protein comprising the N-terminal peptide, the adaptor peptide, and the C-terminal peptide comprises or is composed of the amino acid sequences shown in Table 5 below.

[0235] Table 5. Peptide Combinations of Fusion Proteins

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242] IgG Fc

[0243] In some embodiments, the fusion proteins provided herein further comprise IgG Fc (or Fc region). As used herein, the term "IgG Fc" or "Fc region" refers to a portion of an immunoglobulin formed by the Fc domains of its two heavy chains. The Fc region can be a wild-type Fc region (natural Fc region) or a variant Fc region. The natural Fc region is a homodimer. In some embodiments, the fusion proteins provided herein form a dimer (e.g., a homodimer) via interactions between the Fc regions. In some embodiments, two fusion proteins are linked into a dimer (e.g., a homodimer) via two hinge regions of interchain disulfide bonds between the Fc regions of each fusion protein (e.g., located at the N-terminus). In some embodiments, the Fc region contains an intrachain disulfide bond in the CH2 domain and an intrachain disulfide bond in the CH3 domain.

[0244] The Fc region of the fusion protein described herein can be derived from any natural immunoglobulin. In some embodiments, the Fc region is formed from the constant region of the IgA, IgD, IgE, or IgG heavy chain. In some embodiments, the Fc region is formed from the constant region of the IgG heavy chain. In some embodiments, the IgG heavy chain is the constant region of the IgG1, IgG2, IgG3, or IgG4 heavy chain. In some embodiments, the Fc region is formed from the constant region of the IgG1 heavy chain. In some embodiments, the constant region of the IgG1 heavy chain contains allotypes of G1m1(a), G1m2(x), G1m3(f), or G1m17(z). See, for example, 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 entirety. The IgG Fc can be linked to the N-terminus of an N-terminal peptide or the C-terminus of a C-terminal peptide. IgG Fc can be directly linked to an N-terminal or C-terminal peptide, or it can be linked to an N-terminal or C-terminal peptide via an IgG Fc adapter. In some embodiments, the IgG Fc adapter comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the IgG Fc adapter comprises or consists of 1, 2, 3, 4, or 5 amino acids. In some embodiments, the IgG Fc adapter comprises or consists of 3 or 4 amino acids. In some embodiments, the IgG Fc adapter comprises or consists of the amino acid sequence of GGS. In some embodiments, the IgG Fc adapter comprises or consists of the amino acid sequence of EGGS (SEQ ID NO: 299).

[0245] In some embodiments, 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 typically cleaved during expression and purification without known impairment of activity. In some embodiments, the C-terminal lysine (K) is replaced by a C-terminal glutamate (E). Therefore, in some embodiments, IgG Fc comprises a C-terminal glutamate (E).

[0246] In some embodiments, the IgG Fc comprises the amino acid sequence of one of SEQ ID NO:76-83, wherein GGS serves 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 NO:76-83, wherein SEQ ID NO:299 serves as an IgG Fc linker at the C-terminus of the IgG Fc.

[0247] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the antibody described herein (e.g., the CH2 domain (residues 231 to 340 of human IgG1) and / or the CH3 domain (residues 341 to 447 of human IgG1) and / or the hinge region, numbered according to the EU numbering system, to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity.

[0248] In some 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), as in, for example, 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 to, for example, promote the assembly of light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody.

[0249] In specific embodiments, one, two, or more amino acid mutations (e.g., substitution, insertion, or deletion) are introduced into the constant domain of IgG or its FcRn-binding fragment (preferably an Fc or hinge-Fc domain fragment) to alter (e.g., reduce or increase) the in vivo half-life of the antibody. See, for example, International Publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patents 5,869,046, 6,121,022, 6,277,375, and 6,165,745, all of which are incorporated herein by reference in their entirety as examples of mutations that alter (e.g., reduce or increase) the in vivo half-life of an antibody. In some embodiments, one, two, or more amino acid mutations (e.g., substitution, insertion, or deletion) are introduced into the constant domain of IgG or its FcRn-binding fragment (preferably an Fc or hinge-Fc domain fragment) to reduce the in vivo half-life of the antibody. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into the constant domain of IgG or its FcRn binding fragment (preferably an Fc or hinge-Fc domain fragment) to increase the in vivo half-life of the antibody. 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 specific embodiments, the constant region of IgG1 of the antibody described herein includes 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 glutamate (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. Compared to the wild-type version of the same antibody, this type of mutant IgG (referred to as the “YTE mutant”) has shown a fourfold increase in half-life (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24, which is incorporated herein by reference in its entirety). In some embodiments, the antibody comprises an IgG constant domain containing one, two, three, or more amino acid substitutions at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU numbering system.

[0250] In some 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 the hinge region of the antibody described herein, numbered according to the EU numbering system, to increase or decrease the antibody's affinity for an Fc receptor (e.g., an activated Fc receptor) on the surface of effector cells. Mutations in the antibody's Fc region that decrease or increase the antibody's affinity for the Fc receptor, and techniques for introducing such mutations into the Fc receptor or fragments thereof, are known to those skilled in the art. Examples of mutations in the antibody Fc receptor that can alter the antibody’s affinity 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 International Publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, all of which are incorporated herein by reference in their entirety.

[0251] In some embodiments, the antibody comprises a variant of the 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. In some embodiments, the variant heavy chain constant region is a variant human heavy chain constant region, such as the variant human IgG1, variant human IgG2, or variant human IgG4 heavy chain constant region. In some 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 some embodiments, the variable human IgG heavy chain constant region comprises a group of amino acid mutations selected from the group consisting of the following according to the EU numbering system: 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. In some embodiments, FcγRIIB is expressed on cells selected from the group consisting of macrophages, monocytes, B cells, dendritic cells, endothelial cells and activated T cells.

[0252] In a further embodiment, one, two, or more amino acid substitutions are introduced into the Fc region of the IgG constant domain to alter the effector function of the antibody. For example, one or more amino acid residues selected from the EU numbering system 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332, and 396 may be substituted with different amino acid residues, giving the antibody a modified affinity for the effector ligand while retaining the antigen-binding ability of the parent antibody. The effector ligand whose affinity is modified may be, for example, an Fc receptor or a C1 component of complement. This method is described in more 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 some embodiments, deletion or inactivation of the constant domain (through point mutation or other means) may reduce Fc receptor binding of circulating antibodies, thereby increasing tumor localization. See, for example, U.S. Patent Nos. 5,585,097 and 8,591,886, each of which is incorporated herein by reference in its entirety, for descriptions of mutations that result in the loss or inactivation of constant domains and thereby increase tumor localization. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibody described herein to remove potential glycosylation sites on the Fc region, which may reduce Fc receptor binding (see, for example, Shields RL et al., (2001) J Biol Chem 276:6591-604, which is incorporated herein by reference in its entirety). In various embodiments, one or more of the following mutations may be made in the constant region of the antibody described herein: 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, numbered according to the EU numbering system.

[0253] In some embodiments, mutations selected from the group consisting of D265A, P329A, and combinations thereof according to EU numbering system can be performed in the constant region of the antibody described herein. In some embodiments, mutations selected from the group consisting of L235A, L237A, and combinations thereof according to EU numbering system can be performed in the constant region of the antibody described herein. In some embodiments, mutations selected from the group consisting of S267E, L328F, and combinations thereof according to EU numbering system can be performed in the constant region of the antibody described herein. In some embodiments, mutations selected from the group consisting of S239D, I332E, optionally A330L, and combinations thereof according to EU numbering system can be performed in the constant region of the antibody described herein. In some embodiments, mutations selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, and combinations thereof according to EU numbering system can be performed in the constant region of the antibody described herein. In some embodiments, mutations selected from the group consisting of S267E, L328F and combinations thereof, according to the EU numbering system, can be performed in the constant region of the antibody described herein.

[0254] In specific embodiments, the antibodies described herein comprise a constant domain of IgG1 having an amino acid substitution of N297Q or N297A according to the EU numbering system. In one embodiment, the antibodies described herein comprise a constant domain of IgG1 with a mutation selected from the group consisting of D265A, P329A, and combinations thereof according to the EU numbering system. In another embodiment, the antibodies described herein comprise a constant domain of IgG1 with a mutation selected from the group consisting of L234A, L235A (LALA), and combinations thereof according to the EU numbering system. In yet another embodiment, the antibodies described herein comprise a constant domain of IgG1 with a mutation selected from the group consisting of L234F, L235F, N297A, and combinations thereof according to the EU numbering system. In some embodiments, the amino acid residues at positions L234, L235, and D265 (according to the EU numbering system) in the constant region of the antibodies described herein are not L, L, and D, respectively. This method is described in detail in International Publication No. WO 14 / 108483, which is incorporated herein by reference in its entirety. In specific embodiments, the amino acids corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively; or A, A, and A, numbered according to the EU numbering system.

[0255] In some embodiments, one or more amino acid residues 329, 331, and 322 (according to EU numbering system) in the constant region of the antibody described herein may be replaced with different amino acid residues, such that the antibody exhibits altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This method is further described in detail in U.S. Patent No. 6,194,551 (Idusogie et al.), which is incorporated herein by reference in its entirety. In some embodiments, one or more amino acid residues at positions 231 to 238 (according to EU numbering system) in the N-terminal region of the CH2 domain of the antibody described herein are altered, thereby changing the antibody's ability to fix complement. This method is further described in International Publication No. WO 94 / 29351, which is incorporated herein by reference in its entirety. In some embodiments, the Fc region of the antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cytotoxicity (ADCC) and / or increase the antibody's affinity for the Fcγ receptor by mutating one or more amino acids at the following locations (e.g., introducing amino acid substitutions): 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 2 92, 293, 294, 295, 296, 298, 301, 303, 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, numbered according to the EU numbering system. This method is further described in international publication number WO 00 / 42072, which is incorporated herein by reference in its entirety.

[0256] In some embodiments, IgG Fc is IgG1 Fc or a derivative thereof. In some embodiments, IgG Fc or IgG1 Fc contains at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of IgG1 Fc. In some embodiments, IgG Fc or IgG1 Fc contains at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that provided in Table 6 below.

[0257] Table 6. IgG Fc amino acid sequence

[0258]

[0259]

[0260] In some embodiments, any IgG Fc or its derivative may be connected to the N-terminus or C-terminus of any embodiment described in Table 4 or 5 above, with or without an IgG Fc adapter. In some embodiments, human IgG1 Fc or its derivative may be connected to the N-terminus or C-terminus of any embodiment described in Table 4 or 5 above, with or without an IgG Fc adapter. 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, the derivative of human IgG1 Fc comprises 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% of the amino acid sequence of SEQ ID NO: 76 or 80.

[0261] In some embodiments, human IgG1 Fc containing the LALA mutation or a derivative thereof may be linked to the N-terminus or C-terminus of any embodiment described in Table 4 or 5 above, with or without the IgG Fc linker. In some embodiments, the amino acid sequence of human IgG1 Fc containing the LALA mutation comprises or is composed of the amino acid sequence of SEQ ID NO: 77 or 81. In some embodiments, the human IgG1 Fc derivative containing the LALA mutation comprises at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 77 or 81.

[0262] In some embodiments, human IgG1 Fc containing the LALAPA mutation or a derivative thereof may be linked to the N-terminus or C-terminus of any embodiment described in Table 4 or 5 above, with or without the IgGFc linker. In some embodiments, the amino acid sequence of human IgG1 Fc containing the LALAPA mutation comprises or is composed of the amino acid sequence of SEQ ID NO: 78 or 82. In some embodiments, the derivative of human IgG1 Fc containing the LALAPA mutation comprises 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 at least 99% of the amino acid sequence of SEQ ID NO: 78 or 82.

[0263] In some embodiments, human IgG1 Fc containing the LALA PALS mutation or a derivative thereof may be linked to the N-terminus or C-terminus of any embodiment described in Table 4 or 5 above, with or without the IgG Fc linker. In some embodiments, the amino acid sequence of human IgG1 Fc containing the LALA PALS mutation comprises or is composed of the amino acid sequence of SEQ ID NO: 79 or 83. In some embodiments, the derivative of human IgG1 Fc containing the LALA PALS mutation comprises 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% of the amino acid sequence of SEQ ID NO: 79 or 83.

[0264] 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 the amino acid sequence shown in Table 7 below. In some embodiments, the fusion protein comprises or is composed of the amino acid sequence shown in Table 7 below.

[0265] Table 7. Amino acid sequence of fusion protein

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294] In some embodiments, the IgG Fc comprises a mouse IgG kappa signal sequence having the amino acid sequence 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 having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 548). In some embodiments, different signal sequences are used. In some embodiments, the resulting fusion protein does not have a signal sequence.

[0295] 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 the amino acid sequence shown in Table 8 below. In some embodiments, the fusion protein comprises or is composed of the amino acid sequence shown in Table 8 below.

[0296] Table 8. Amino acid sequence of fusion protein

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347] Other half-life extension

[0348] As used herein, the term "half-life extension portion" includes non-protein half-life extension portions, such as PEG or HES, and protein half-life extension portions, such as Fc domains. In some embodiments, the non-protein half-life extension portion is linked to the fusion protein described herein. In some embodiments, the non-protein half-life extension portion is linked to the fusion protein instead of IgG Fc. In some embodiments, the non-protein half-life extension portion is linked to the fusion protein in addition to being linked to IgG Fc.

[0349] Examples of suitable polymer molecules that can serve as the non-protein half-life extension include polymer molecules selected from the group consisting of: polyalkylene oxides (PAOs), including polyalkylene glycols (PAGs), such as polyethylene glycol (PEG) and polypropylene glycol (PPG); branched PEGs; hydroxyalkyl starches (HASs), such as hydroxyethyl starch (HES); polysialic acid (PSA); polyvinyl alcohol (PVA); polycarboxylates; poly(vinylpyrrolidone); polyvinyl-co-maleic anhydride; polystyrene-co-maleic anhydride; dextran, including carboxymethyl dextran; or any other biopolymer suitable for reducing immunogenicity and / or increasing the functional half-life and / or serum half-life in vivo. Another example of a polymer molecule is human albumin or other abundant plasma proteins. Generally, polyalkylene glycol-derived polymers are biocompatible, non-toxic, non-antigenic, non-immunogenic, have a variety of water-soluble properties, and are readily secreted from the body.

[0350] Compared to polysaccharides such as dextran, PEG has the advantage of having only a small number of reactive groups capable of crosslinking. In particular, monofunctional PEGs, such as methoxy polyethylene glycol (mPEG), have attracted considerable attention due to their relatively simple coupling chemistry (only one reactive group is available for conjugation with attachment groups on the peptide). Therefore, the conjugated fusion proteins described herein are more homogeneous due to the elimination of the risk of crosslinking, and the reaction of the polymer molecules with the variant peptides is more easily controlled.

[0351] To achieve covalent attachment of polymer molecules to the fusion protein described herein, the hydroxyl terminal groups of the polymer molecules must be in an activated form, i.e., possessing active functional groups (examples of which include primary amino groups, hydrazides (HZ), thiols, succinates (SUC), succinimide succinates (SS), succinimide succinamides (SSA), succinimide propionates (SPA), succinimide butyrates (SBA), succinimide carboxymethyl esters (SCM), benzotriazole carbonates (BTC), N-hydroxysuccinimide (NHS), aldehydes, nitrophenyl carbonates (NPC), and trifluoromethanesulfonates (TRES)). Suitable activated polymer molecules are available, for example, from Shearwater Polymers, Inc., Huntsville, Ala., USA, or PolyMASC Pharmaceuticals plc, UK.

[0352] Alternatively, polymer molecules may be activated using conventional methods known in the art, such as those described in WO 90 / 13540. Specific examples of activated linear or branched polymer molecules used herein are described in the Shearwater Polymers, Inc. catalogues of 1997 and 2000 (Functionalized Biocompatible Polymers, Polyethylene Glycols and Derivatives for Research and Pharmaceutical Use, 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 NOR-PEG, BTC-PEG, EPOXPEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, as well as branched PEGs such as PEG2-NHS, and those disclosed in U.S. Patent Nos. 5,932,462 and 5,643,575, both of which are incorporated herein by reference.In addition, the following publications disclose useful polymer molecules and / or PEGylation chemistry: U.S. Patent No. 5,824,778, U.S. Patent No. 5,476,653, WO 97 / 32607, EP 229,108, EP 402,378, 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, WO 92 / 16555, WO 94 / 04193, WO 94 / 14758, WO 94 / 17039, WO 94 / 18247, WO 94 / 28024, WO 95 / 00162, WO 95 / 11924, WO 95 / 13090, WO 95 / 33490, WO 96 / 00080, WO WO 97 / 18832, WO 98 / 41562, WO 98 / 48837, WO 99 / 32134, WO 99 / 32139, WO 99 / 32140, WO 96 / 40791, WO 98 / 32466, WO 95 / 06058, EP 439 508, WO 97 / 03106, WO 96 / 21469, WO 95 / 13312, EP 921 131, US Patent No. 5,736,625, WO 98 / 05363, EP 809 996, US Patent No. 5,629,384, WO 96 / 41813, WO 96 / 07670, US Patent No. 5,473,034, US Patent No. 5,516,673, EP US Patent Nos. 5,382,657, 510,356, 400,472, 183,503, and 154,316.

[0353] Particularly preferred examples of activated PEG polymers for coupling with cysteine ​​residues include the following linear PEGs: vinyl sulfone-PEG (VS-PEG), preferably vinyl sulfone-mPEG (VS-mPEG); maleimide-PEG (MAL-PEG), preferably maleimide-mPEG (MAL-mPEG); and o-pyridyl-disulfide-PEG (OPSS-PEG), preferably o-pyridyl-disulfide-mPEG (OPSS-mPEG). Typically, the size of such PEG or mPEG polymers is about 5 kDa, about 10 kDa, about 12 kDa, or about 20 kDa.

[0354] The conjugation of the fusion protein to the activated polymer molecule described herein was performed using any conventional method, for example, as described in the following references (which also describe suitable methods for activating the polymer molecule): Harris and Zalipsky (eds.), Poly(ethylene glycol) Chemistry and Biological Applications, AZC Washington; R.F. Taylor, (1991), “Protein immobilisation. Fundamental 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.

[0355] Those skilled in the art will recognize that the activation methods and / or conjugation chemistry used depend on the attachment groups of the fusion protein (examples of which are further given above) and the functional groups of the polymer (e.g., amines, hydroxyl groups, carboxyl groups, aldehydes, thiol groups, succinimides, maleimides, vinyl sulfones, or haloacetic esters). PEGylation can be directly conjugated to all available attachment groups on the fusion protein (i.e., such attachment groups exposed on the surface of the polypeptide), or directly conjugated to one or more specific attachment groups, such as the N-terminal amino group as described in U.S. Patent No. 5,985,265, or conjugated to cysteine ​​residues. Furthermore, conjugation can be achieved in a one-step or stepwise manner (e.g., as described in WO 99 / 55377).

[0356] For the PEGylation of cysteine ​​residues (see above), fusion proteins are 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). PEG conjugation to cysteine ​​residues is usually performed in a suitable buffer at pH 6–9 at a temperature ranging from 4°C to 25°C for a period of up to 16 hours.

[0357] It should be understood that PEGylation is designed to produce optimal molecules for the number of PEG molecules attached, the size and form of such molecules (e.g., whether they are linear or branched), and the attachment sites in the fusion protein. The molecular weight of the polymer used can be selected, for example, based on the desired effect.

[0358] For cases where the fusion protein is conjugated to only a single attachment group (e.g., an N-terminal amino group), it may be advantageous to use a high molecular weight polymer molecule, which may be linear or branched, and has a high molecular weight, preferably about 10-25 kDa, such as about 15-25 kDa, for example about 20 kDa.

[0359] Typically, polymer conjugation is carried out under conditions designed to allow as many available polymeric attachment groups as possible to react with the polymeric molecules. This can be achieved by applying an appropriate molar excess of the polymer relative to the peptide. Generally, the molar ratio of the activated polymer molecule to the peptide is at most about 1000⁻¹, such as at most about 200⁻¹, or at most about 100⁻¹. However, in some cases, this ratio may be slightly lower, such as at most about 50⁻¹, 10⁻¹, 5⁻¹, 2⁻¹, or 1⁻¹, to obtain optimal reaction.

[0360] Alternatively, polymer molecules can be coupled to fusion proteins via linkers. Suitable linkers are well known to those skilled in the art. Preferred examples include 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).

[0361] After conjugation, the residual activated polymer molecules are blocked by methods known in the art, such as by adding a primary amine to the reaction mixture, and the resulting deactivated polymer molecules are removed by a suitable method.

[0362] It should be understood that, depending on the specific circumstances, such as the amino acid sequence of the fusion protein, the nature of the activating PEG compound used, and the specific PEGylation conditions (including the molar ratio of PEG to peptide), different degrees of PEGylation can be obtained. Generally, the higher the ratio of PEG to fusion protein, the higher the degree of PEGylation. However, PEGylated fusion proteins produced by any given PEGylation method typically contain a random distribution of conjugate fusion proteins with slightly different degrees of PEGylation.

[0363] To improve the biological half-life of the fusion protein described in this article, chemical modifications such as PEGylation or HES-ation can be employed.

[0364] Non-protein polymers of HAS and HES, and methods for generating HAS or HES conjugates, are disclosed, for example, in WO 02 / 080979, WO 03 / 070772, WO 057092391 and WO 057092390.

[0365] Polysialylation is another technology 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 for protein and therapeutic peptide drug delivery, polysialic acid can provide a protective microenvironment upon conjugation. This increases the active lifespan of the fusion protein in circulation and prevents it from being recognized by the immune system. PSA polymers are naturally present in the human body. It is absorbed by certain bacteria that have evolved over millions of years to use it to coat their cell walls. These naturally polysialylated bacteria are then able to bypass the human body's defense system using molecular mimicry techniques. PSA (nature's ultimate stealth technology) can be easily mass-produced from such bacteria with predetermined physical properties. Bacterial PSA is completely non-immunogenic, even when conjugated to proteins, because it is chemically identical to PSA in the human body.

[0366] Bioactivity of relaxin-2 fusion protein

[0367] In some embodiments, the relaxin-2 fusion proteins described herein exhibit a higher level of biological activity compared to natural relaxin-2. In some embodiments, any relaxin-2 fusion protein described herein exhibits about 1% to about 200% of biological activity compared to natural relaxin-2. In some embodiments, the relaxin-2 fusion protein exhibits 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 biological activity compared to natural relaxin-2.

[0368] In some embodiments, any of the relaxin-2 fusion proteins described herein exhibits approximately 1% to approximately 200% of the maximum biological activity compared to native relaxin-2. In some embodiments, the maximum biological activity is the maximum response (E) of relaxin-2 or the relaxin-2 fusion protein. max In 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 maximum biological activity compared to natural relaxin-2.

[0369] In some embodiments, any of the relaxin-2 fusion proteins described herein have an enhanced potency of at least about 0.001 to at least 1,000 times compared to natural relaxin-2. In some embodiments, potency is the concentration of relaxin-2 or relaxin-2 fusion protein that elicits the half-maximal response (EC50).50 In some embodiments, the relaxin-2 fusion protein has at least about 0.001 times, about 0.01 times, about 0.1 times, about 1 times, about 10 times, about 100 times, or about 1,000 times the potency of natural relaxin-2.

[0370] The bioactivity can be any bioactivity of natural relaxin-2. For example, the bioactivity can be the ability to bind to the natural relaxin-2 receptor RXFP1. The binding of relaxin-2 to RXFP1 can be measured by any method well known in the art, such as radioligand binding. In some embodiments, the fusion protein described herein binds to RXFP1 when expressed on the cell surface.

[0371] In some embodiments, bioactivity may be the ability to activate RXFP1 on the cell surface. The activation of RXFP1 by the relaxin-2 fusion protein described herein can be determined using any method well known in the art by increasing cAMP levels, such as measuring the activity of cAMP-driven reporter genes (e.g., β-galactosidase). The activation of RXFP1 by the relaxin-2 fusion protein described herein in cells can also be determined using a biosensor (such as the GloSensor biosensor). The activation of RXFP1 by the relaxin-2 fusion protein described herein in cells can also be determined using methods well known in the art by measuring the expression of certain genes such as angiogenic factors (e.g., VEGF) or MMPs. In some embodiments, bioactivity is the physiological activity, biochemical activity, or activity of any other inducing effect of relaxin-2. Exemplary bioactivities include, but are not limited to, vasodilation, collagen degradation, angiogenesis, lowering arterial blood pressure, increasing renal artery blood flow, increasing renal plasma flow, increasing diastolic cardiac filling, eliminating existing fibrosis, and inhibiting the development of new fibrosis.

[0372] In some embodiments, the fusion proteins described herein have an improved pharmacokinetic profile. Unbound by any theory, the structure of the fusion proteins described herein is based at least in part on the surprising finding that reducing the pI of relaxin-2 fusion protein analogs prolongs 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 observed in mammals. In some embodiments, the mammals are rodents or primates. In some embodiments, the rodents are rats or mice. In some embodiments, the primates are humans or monkeys. In some embodiments, the monkeys are cynomolgus monkeys. In some embodiments, the mammal is a human.

[0373] In some embodiments, the fusion protein described herein may have a cyclic half-life of more than about 5 hours, 10 hours, 20 hours, 50 hours, 75 hours, 100 hours, 125 hours, 150 hours, or longer. In some embodiments, the fusion protein described herein may have a cyclic 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 protein described herein may have a cyclic 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 protein 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 protein 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 protein described herein, when administered to humans, may have a circulating half-life greater than about 5 hours, 10 hours, 20 hours, 50 hours, 75 hours, 100 hours, 125 hours, 150 hours, or longer. In some embodiments, the fusion protein described herein may have a cyclic 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 when administered to humans. In some embodiments, the fusion protein described herein may have a cyclic 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 when administered to humans. In some embodiments, the fusion protein described herein may have a cyclic 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 when administered to humans. In some embodiments, the fusion protein 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 when administered to humans. Values ​​and ranges between said values ​​are also intended to form part of this disclosure. In some embodiments, the fusion protein described herein has a longer circulating half-life than natural double-stranded relaxin-2.For example, the circulating half-life of natural double-stranded relaxin-2 may be less than about 5 hours. (See, for example, 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).

[0374] This increased half-life is at least partly attributed to the reduced pI of the fusion protein described herein. In some embodiments, the fusion protein has a pI of less than about 9.4. As used herein, when referring to pI, the term "about" covers a variation of ±1% of a given value or range, appropriate for implementing the methods described herein. In some embodiments, the fusion protein has a pI of less than 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 The pI is 6.1, or 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 less than 9.0. In some embodiments, the fusion protein has a pI less than about 8.2. In some embodiments, the fusion protein has a pI from about 6.0 to about 9.4. In some embodiments, the fusion protein has a molecular weight 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, or about 6.0 to about 7.6. The pI is approximately 6.0 to approximately 7.5, approximately 6.0 to approximately 7.4, approximately 6.0 to approximately 7.3, approximately 6.0 to approximately 7.2, approximately 6.0 to approximately 7.1, approximately 6.0 to approximately 7.0, approximately 6.0 to approximately 6.9, approximately 6.0 to approximately 6.8, approximately 6.0 to approximately 6.7, approximately 6.0 to approximately 6.6, approximately 6.0 to approximately 6.5, approximately 6.0 to approximately 6.4, approximately 6.0 to approximately 6.3, approximately 6.0 to approximately 6.2, or approximately 6.0 to approximately 6.1. In some embodiments, the fusion protein has a pI of approximately 6.0 to approximately 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 pIs or theoretical pIs. In some embodiments, any of the above pIs are experimentally measured pIs.

[0375] As used herein, when referring to dosage, the term “about” covers a given value or range of ±10% variation, as appropriate for implementing the methods described herein.

[0376] As used herein, "circulating half-life" refers to the time required for a drug to decrease in plasma concentration to half its steady-state concentration in the whole blood circulation of an organism. The circulating half-life of a particular drug may vary due to a variety of factors, including but not limited to the 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 by Chen above).

[0377] In some embodiments, the fusion protein described herein has high bioavailability. In some embodiments, the fusion protein has high bioavailability when administered, for example, intravenously or subcutaneously. In some embodiments, the fusion protein has high bioavailability when administered subcutaneously. In some embodiments, the fusion protein has high subcutaneous bioavailability. In some embodiments, the fusion protein has 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 of bioavailability. In some embodiments, the fusion protein has 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% bioavailability. 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 at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% when administered subcutaneously. 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% when administered subcutaneously. 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% when administered subcutaneously.In some embodiments, the fusion protein has a bioavailability of about 50% to about 60% (e.g., 50% to 60%) when administered subcutaneously.

[0378] As used herein, “bioavailability” refers to the proportion of an administered drug that reaches systemic circulation. The bioavailability of a particular drug can vary due to a variety of factors, including but not limited to dosage, formulation, route of administration, and / or characteristics of the drug. Those skilled in the art can determine the bioavailability of a drug using methods well known in the art.

[0379] In some embodiments, the fusion protein has high and / or stable serum levels when administered to the subject, for example, intravenously, subcutaneously, and / or by any method described herein. In some embodiments, the fusion protein is present in the subject's serum at levels 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 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or longer 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 2 μg / mL, at least 3 μg / mL, at least 4 μg / mL, at least 5 μg / mL, at least 6 μg / mL, at least 7 μg / mL, at least 8 μg / mL, at least 9 μg / mL, at least 10 μg / mL, at least 11 μg / mL, at least 12 μg / mL, at least 13 μg / mL, at least 14 μg / mL, or at least 0.5 μg / mL) for at least 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 3 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 longer after administration). In some embodiments, the fusion protein is present in the subject's serum at a level of at least about 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 longer after intravenous administration, 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). In some embodiments, the fusion protein is present in the subject's serum at a level of at least about 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 longer after subcutaneous administration.

[0380] Vector and host cell

[0381] This 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.

[0382] 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, augmentation plasmids, microcircles, microstrings, and oligonucleotides (e.g., mRNA, naked DNA). In some embodiments, the vector is a DNA plasmid vector.

[0383] In some embodiments, the vector is a viral vector. The viral vector may be replicative or non-replicative. The viral vector may be integrated or non-integrated. Many virus-based systems have been developed for transferring genes into mammalian cells, and those skilled in the art can select suitable viral vectors. Exemplary viral vectors include, but are not limited to, adenovirus vectors (e.g., adenovirus 5), adeno-associated virus (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), flaviviruses (e.g., Kunzin virus, West Nile virus, 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 piconemavirus vectors (e.g., Coxsackie virus).

[0384] In some embodiments, the vector or expression cassette includes one or more additional elements. These additional elements include, but are not limited to, promoters, enhancers, polyadenylated (polyA) sequences, and selection genes.

[0385] In some embodiments, the vector comprises a polynucleotide sequence encoding an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences shown in any of Tables 1-8. In some embodiments, the vector comprises a polynucleotide sequence encoding an amino acid sequence that comprises or consists of the amino acid sequences shown 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 the sequence shown in Table 9 below. In some embodiments, the vector comprises a polynucleotide sequence that comprises or is composed of the sequence shown in Table 9 below.

[0386] Table 9. Nucleotide sequences encoding fusion proteins and peptide components

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460] 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 the sequence shown in Table 10 below. In some embodiments, the vector comprises a polynucleotide sequence that comprises or is composed of the sequence shown in Table 10 below.

[0461] Table 10. Nucleotide sequences encoding fusion proteins and peptide components

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542] In some embodiments, any nucleotide sequence shown in Table 10 further includes additional nucleotide sequences at its 5' and / or 3' ends. In some embodiments, any nucleotide sequence shown in Table 10 further includes the nucleotide sequence ACGGGACCGATCCAGCCTCCGGACTCTAGAGCCACC (SEQ ID NO:494) at its 5' end, and / or any nucleotide sequence shown in Table 10 further includes the nucleotide sequence TGATAAACCGGTTAGTAATGAGTTTGATATCTCGAC (SEQ ID NO:495) at its 3' end.

[0543] A variety of host cell and expression vector systems can be used to express the fusion proteins described herein. Such expression systems represent media that can produce and subsequently purify the coding sequences of interest, but also represent cells that can express the fusion proteins described herein in situ when transformed or transfected with appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms transformed with recombinant phage DNA, plasmid DNA, or copious DNA expression vectors containing, for example, fusion protein coding sequences, such as bacteria (e.g., *Escherichia coli* and *Bacillus subtilis*); yeast transformed with recombinant yeast expression vectors containing fusion protein coding sequences (e.g., *Pichia pastoris*); insect cell systems infected with recombinant viral expression vectors (DNA baculoviruses) containing fusion protein coding sequences; plant cell systems (e.g., green algae, such as *Chlamydomonas reinhardtii*) infected with, for example, recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with, for example, recombinant plasmid expression vectors containing fusion protein coding sequences (e.g., Ti plasmid); or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH). Cells of various types (3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10) were used to express a recombinant expression construct carrying, for example, a promoter derived from a mammalian cell genome (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). In some embodiments, the cells used to express the fusion protein described herein are human cells, such as human cell lines. In some embodiments, the mammalian expression vector is pOptiVEC. TM Or pcDNA3.3. In some embodiments, bacterial cells (such as Escherichia coli) or eukaryotic cells (e.g., mammalian cells) are used for the expression of the fusion protein. For example, mammalian cells (such as CHO or HEK293 cells) bound to a vector (such as a major intermediate early gene promoter element from human cytomegalovirus) are an efficient expression system for the fusion proteins disclosed herein.

[0544] In bacterial systems, a number of expression vectors can be advantageously selected based on the intended use of the expressed fusion protein. For example, when the production of large quantities of fusion proteins is required, a vector that directs the high-level expression of an easily purified fusion protein product may be desirable. Such vectors include, but are not limited to, the *E. coli* expression vector pUR278 (Ruether U and Mueller-Hill B (1983) EMBO J 2:1791-1794), in which the fusion protein coding sequence can be individually bound to a vector containing a lacZ coding region to produce the fusion protein; the pIN vector (Inouye S and Inouye M (1985) Nuc Acids Res13:3101-3109; Van Heeke G and Schuster SM (1989) J Biol Chem 24:5503-5509); and so on, all of which are incorporated herein by reference in their entirety. For example, the pGEX vector can also be used to express exogenous peptides as fusion proteins with glutathione 5-transferase (GST). Typically, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads, followed by elution in the presence of free glutathione. The pGEX vector is designed to include thrombin or factor Xa protease cleavage sites, allowing the cloned target gene product to be released from the GST moiety.

[0545] For example, in insect systems, the alfalfa silver-striped armyworm nucleopolyhedrovirus (AcNPV) can be used as a vector for expressing exogenous genes. This virus grows in fall armyworm cells. The fusion protein coding sequence can be cloned separately into a non-essential region of the virus (e.g., the polyhedrosis protein gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrosis protein promoter).

[0546] In mammalian host cells, numerous virus-based expression systems can be utilized. When using adenovirus as the expression vector, the coding sequence of the fusion protein of interest can be conjugated to an adenoviral transcription / translation control complex, such as a late promoter and a triple leader sequence. This chimeric gene can then be inserted into the adenoviral genome via in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., E1 or E3 regions) will produce a recombinant virus that is viable and capable of expressing the fusion protein molecule in an infected host (see, e.g., Logan J and Shenk T (1984) PNAS 81(12):3655-9, which is incorporated herein by reference in its entirety). Efficient translation of the inserted fusion protein coding sequence may also require specific initiation signals. These signals include the ATG start codon and adjacent sequences. Furthermore, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire inserted fragment. These exogenous translation control signals and start codons can have a variety of sources, including both natural and synthetic. Expression efficiency can be enhanced by including appropriate transcriptional enhancer elements, transcription terminators, etc. (See, for example, Bitter G et al. (1987) Methods Enzymol. 153:516-544, which is incorporated herein by reference in its entirety.)

[0547] Additionally, host cell lines can be selected to regulate the expression of the inserted sequence or to modify and process the gene product in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for protein function. Different host cells possess characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure proper modification and processing of expressed exogenous proteins. For this purpose, eukaryotic host cells can be used, which possess cellular mechanisms for the appropriate processing of primary transcripts, glycosylation, and phosphorylation of gene products. These 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.

[0548] For long-term, high-volume production of recombinant proteins, stable expression cells can be generated. For example, cell lines that can be engineered to stably express the fusion proteins described herein.

[0549] In some embodiments, instead of using an expression vector containing a viral replication initiation site, host cells are transformed with a polynucleotide (e.g., DNA or RNA) controlled by appropriate transcriptional regulatory elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After the introduction of the polynucleotide, engineered cells can be allowed to grow in an enrichment medium for 1–2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and amplified into cell lines. This method can be advantageously used for the engineering of cell lines expressing the fusion proteins described herein or fragments thereof.

[0550] Many selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler M et al. (1977) Cell 11(1):223-32), hypoxanthine-guanine phosphoribosyltransferase (Szybalska EH and Szybalski W (1962) PNAS 48(12):2026-2034) and adenine phosphoribosyltransferase (Lowy I et al. (1980) Cell 22(3):817-23) genes in tk-, hgprt-, or aprt- cells, all of which are incorporated herein by reference in their full text. In addition, antimetabolite resistance can be used as a basis for selecting 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 and Berg P (1981) PNAS 78(4):2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY and 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 and Anderson WF (1993) Ann Rev Biochem 62:191-217; Nabel GJ and 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 in their full text.The desired recombinant clone can be selected using methods known in the field of recombinant DNA technology, and such methods are described, for example, Ausubel FM et al. (ed.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli NC et al. (ed.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbère-Garapin F et al. (1981) J Mol Biol 150:1-14, all of which are incorporated herein by reference in their full text.

[0551] Pharmaceutical Composition

[0552] This disclosure provides pharmaceutical compositions comprising the fusion proteins or component peptides described herein, or nucleic acid molecules or expression vectors encoding them. The pharmaceutical compositions described herein are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, etc. A variety of suitable formulations can be found in all formularies known to medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, and vesicle-containing lipids (cationic or anionic) (such as LlPOFECTIN). TM See also Powell et al., “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

[0553] The dosage of the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, administered to a patient may vary depending on the patient's age, body size, target disease, symptoms, route of administration, etc. Preferred dosages are typically calculated based on body weight or body surface area. The frequency and duration of treatment may be adjusted depending on the severity of the symptoms. Effective dosages and schedules for administering the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, can be determined empirically; for example, by periodically assessing and monitoring the patient's disease progression and adjusting the dosage 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).

[0554] Various delivery systems are known and can be used to administer the pharmaceutical compositions described herein, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered via any convenient route, such as by infusion or bolus, absorption through the epithelial or mucosal lining (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0555] The pharmaceutical compositions described herein can be delivered subcutaneously or intravenously using standard needles and syringes. Additionally, regarding subcutaneous delivery, pen delivery devices are readily applicable for delivering the pharmaceutical compositions disclosed herein. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. Once all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and 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. Instead, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0556] In some cases, drug compositions can be delivered in controlled-release systems. In one embodiment, a pump can be used (see Langer, ibid.; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled-release system can be placed near the target of the composition, thus requiring only a portion of the systemic dose (see, for example, Goodson, 1984, Medical Applications of Controlled Release, ibid., Vol. 2, pp. 115-138). Other controlled-release systems are discussed in the review in Langer, 1990, Science 249:1527-1533.

[0557] Injectable formulations may comprise dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, infusion, etc. These injectable formulations can be prepared by methods known to be disclosed. For example, injectable formulations can be prepared, for instance, by dissolving, suspending, or emulsifying any fusion protein described herein in a sterile aqueous or oily medium typically used for injection. As aqueous media for injection, such as physiological saline, isotonic solutions containing glucose and other adjuvants, they can be used in combination with suitable solubilizers (such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]). As oily media, such as sesame oil, soybean oil, etc., can be used in combination with solubilizers (such as benzyl benzoate, benzyl alcohol, etc.). The resulting injectable formulation is preferably filled in a suitable ampoule.

[0558] Advantageously, the above-described pharmaceutical compositions for oral or parenteral use are formulated into dosage forms in unit doses suitable for containing a specific amount of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the fusion protein contained in each dosage form in a unit dose is generally from about 5 to about 500 mg; particularly for injection dosage forms, an amount of the fusion protein contained from about 5 to about 100 mg is preferred, while for other dosage forms, an amount of the fusion protein contained from about 10 to about 250 mg is preferred.

[0559] Therapeutic uses

[0560] Monotherapy

[0561] This disclosure provides a method for enhancing relaxin-2-related activity in primary cells, the method comprising contacting primary cells with a fusion protein or component peptide described herein. In some embodiments, contacting primary cells with the fusion protein or component peptide results in enhanced relaxin-2 activity in the cells, for example, as described above. In some embodiments, contacting primary cells with the fusion protein or component peptide results in activation of the relaxin-2 receptor (RFXP1) on the cell surface. Activation of RXFP1 on the cell surface can lead to cellular responses, including but not limited to increased cAMP levels, vasodilation, expression of angiogenic factors (including VEGF), expression of matrix metalloproteinases (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 in a subject, as described below.

[0562] In some embodiments, this disclosure provides methods for activating RXFP1 on cell surfaces, methods comprising administering an effective amount of the fusion protein or component peptide described herein, or a nucleic acid molecule or expression vector encoding them, to a subject in need, thereby activating RXFP1 on the cell surface. Activation of RXFP1 on the cell surface can lead to cellular responses, including but not limited to increased cAMP levels, vasodilation, expression of angiogenic factors (including VEGF), expression of matrix metalloproteinases (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.

[0563] This disclosure also provides methods for treating various relaxin-2 related diseases. As used herein, the term "relaxin-2 related disease" refers to a disease or condition caused by or related to the production or activity of relaxin-2 protein. The term "relaxin-2 related disease" includes diseases, conditions, or symptoms that may benefit from increased relaxin-2 protein activity. As used herein, the term "relaxin-2 related condition" has the same meaning as "relaxin-2 related disease".

[0564] In some embodiments, relaxin-2 related diseases or conditions are selected from the group consisting of: kidney disease, fibrotic disease, and cardiovascular disease. In some embodiments, relaxin-2 related diseases or conditions are pulmonary hypertension.

[0565] The World Health Organization (WHO) classifies pulmonary hypertension into five categories. Group 1 is pulmonary arterial hypertension (PAH), which requires right heart catheterization (RHC) to demonstrate a mean pulmonary artery (PA) pressure (mPAP) ≥20 mmHg and pulmonary vascular resistance (PVR) ≥2 Wood units at rest. Additional criteria for Group 1 PAH include: mean pulmonary capillary wedge pressure (PCWP) ≤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 certain other conditions, including systemic diseases (e.g., sarcoidosis, chronic renal insufficiency), hematologic disorders (e.g., myeloproliferative disorders and chronic hemolytic anemia), and metabolic disorders (e.g., glycogen storage disease). Group 1 also includes PAH caused by unknown mechanisms (idiopathic PAH) and heritable genetic defects (hereditary PAH); PAH caused by drugs and toxins; PAH associated with systemic conditions such as connective tissue diseases, human immunodeficiency virus (HIV) infection, congenital heart disease, and schistosomiasis; PAH characterized by venous / capillary involvement; and persistent PH in newborns.

[0566] Group 2 is PH-LHD caused by left ventricular disease. A clinical diagnosis can be made when echocardiography shows LHD sufficient to explain PH (regardless of whether other confirmatory tests are combined). For patients undergoing RHC, mPAP ≥20 mmHg, PCWP ≥15 mmHg, and normal or decreased cardiac output are consistent with the hemodynamic diagnosis of LHD-PH. Important supporting information is echocardiographic evidence of left atrial (LA) enlargement, and left ventricular catheterization (LHC) to confirm elevated left ventricular end-diastolic pressure. Once PH-LHD is confirmed, the patient should be classified into 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). Patients with PH in Group 2 can be divided into two subgroups: patients with combined pre- and post-capillary PH (CpcPH) and patients with isolated post-capillary hypertension (IpcPH).

[0567] Group 3 is PH caused by chronic lung disease and / or hypoxemia, with PH visualized by RHC or echocardiography and evidence of moderate to severe pulmonary dysfunction and / or hypoxemia. This group is used to diagnose PH caused by CLD and / or hypoxemia. Patients can be classified into pulmonary PH caused by obstructive pulmonary disease (Group 3.1), PH caused by restrictive pulmonary disease (Group 3.2), PH caused by mixed obstructive and restrictive pulmonary disease (Group 3.3), PH with hypoventilation (Group 3.4), PH with hypoxemia but without lung disease (Group 3.5), or PH caused by developmental disorders (Group 3.6). In some cases, Group 3 PH may be caused by COPD, interstitial lung disease, or obstructive sleep apnea.

[0568] Group 4 is PH caused by pulmonary artery obstruction, and mainly includes patients with chronic thromboembolic PH (CTEPH; Group 4.1) and PH caused by PA obstruction (Group 4.2, e.g., benign or malignant tumors, arteritis without CTD, congenital PA stenosis, parasites).

[0569] Group 5 comprises PH caused by multiple factors and includes patients with PH that cannot be definitively classified into Groups 1 through 4. Group 5 PH can be further subdivided into: hematologic disorders, such as chronic hemolytic anemia (e.g., sickle cell disease, β-thalassemia, or spherocytosis) and myeloproliferative disorders; systemic or metabolic disorders, including sarcoidosis, pulmonary Langerhans cell histiocytosis X, and neurofibromatosis; metabolic disorders, including Gaucher disease and glycogen storage disease; chronic renal failure and PH associated with hemodialysis; pulmonary tumor-related thrombotic microangiopathy; and fibrotic mediastinitis.

[0570] In some embodiments, relaxin-2 related disease or condition is pulmonary hypertension, including any of the group 1, group 2, group 3, group 4 and group 5 PH as defined by the WHO.

[0571] In some embodiments, relaxin-2-related diseases or conditions are pulmonary hypertension, including but not limited to pulmonary arterial hypertension (PAH), pulmonary hypertension due to left ventricular disease (PH-LHD), combined precapillary and postcapillary pulmonary hypertension (CpcPH), and isolated postcapillary pulmonary hypertension (IpcPH). In some embodiments, relaxin-2-related diseases or conditions are pulmonary arterial hypertension (PAH). In some embodiments, relaxin-2-related diseases or conditions are pulmonary hypertension due to left ventricular disease (PH-LHD). In some embodiments, relaxin-2-related diseases or conditions are combined precapillary and postcapillary pulmonary hypertension (CpcPH). In some embodiments, relaxin-2-related diseases or conditions are isolated postcapillary pulmonary hypertension (IpcPH).

[0572] In some embodiments, relaxin-2 related disease or condition is Group 2 pulmonary hypertension. In some embodiments, relaxin-2 related disease or condition is isolated postcapillary pulmonary hypertension (IpcPH). IpcPH includes features such as: right ventricular dysfunction; left ventricular thickening and stiffening (LHD); and renal impairment. In some embodiments, relaxin-2 related disease or condition is selected from the group consisting of: right ventricular dysfunction; left ventricular thickening and stiffening (LHD); and renal impairment. In some embodiments, relaxin-2 related disease or condition is combined precapillary and postcapillary pulmonary hypertension (CpcPH). CpcPH includes features such as: pulmonary artery stenosis, thickening, stiffening, and / or fibrotic remodeling; right ventricular dysfunction; left ventricular thickening and stiffening (LHD); and renal impairment. In some embodiments, relaxin-2-related diseases or conditions are selected from the group consisting of: pulmonary artery stenosis, thickening, hardening and / or fibrotic remodeling; right ventricular dysfunction; left ventricular thickening and stiffening (LHD); and renal impairment.

[0573] In some embodiments, relaxin-2 related diseases or conditions are heart failure, including but not limited to heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF). In some embodiments, relaxin-2 related diseases or conditions are heart failure with preserved ejection fraction (HFpEF). In some embodiments, relaxin-2 related diseases or conditions are heart failure with reduced ejection fraction (HFrEF).

[0574] In some embodiments, relaxin-2-related diseases or conditions are heart diseases, including but not limited to valvular heart disease.

[0575] In some embodiments, relaxin-2 related disease or condition is Group 2 PH (CpcPH or IpcPH) and heart failure with preserved ejection fraction (HFpEF). In some embodiments, relaxin-2 related disease or condition is CpcPH with HFpEF. In some embodiments, relaxin-2 related disease or condition is IpcPH with HFpEF. In some embodiments, HFpEF is defined as the signs and symptoms of New York Heart Association (NYHA) Class II-III heart failure, with LVEF ≥50%, and at least one of the following: (i) Heart Failure Association-Pretest Assessment, Echocardiography and Natriuretic Peptide Score, Functional Testing in Unknown Conditions, and End-Cause Etiology (HFA-PEFF) score ≥5; and / or (ii) HFA-PEFF score of 2-4, and abnormal diastolic pressure testing or invasive hemodynamic measurements. In some embodiments, relaxin-2 related disease or condition is CpcPH with NYHA class II-III heart failure and LVEF ≥ 50%, and at least one of the following: (i) HFA-PEFF score ≥ 5; and / or (ii) HFA-PEFF score 2-4, and abnormal diastolic pressure test or invasive hemodynamic measurement. In some embodiments, relaxin-2 related disease or condition is IpcPH with NYHA class II-III heart failure and LVEF ≥ 50%, and at least one of the following: (i) HFA-PEFF score ≥ 5; and / or (ii) HFA-PEFF score 2-4, and abnormal diastolic pressure test or invasive hemodynamic measurement.

[0576] In some embodiments, relaxin-2 related disease or condition is Group 2 PH (CpcPH or IpcPH) and heart failure with moderate ejection fraction (HFmrEF). In some embodiments, relaxin-2 related disease or condition is CpcPH with HFmrEF. In some embodiments, relaxin-2 related disease or condition is IpcPH with HFmrEF. In some embodiments, HFmrEF is defined as New York Heart Association (NYHA) Class II-III heart failure with signs and symptoms of LVEF of 40% to 49%. In some embodiments, relaxin-2 related disease or condition is CpcPH with NYHA Class II-III heart failure and LVEF of 40% to 49%. In some embodiments, relaxin-2 related disease or condition is IpcPH with NYHA Class II-III heart failure and LVEF of 40% to 49%.

[0577] In some embodiments, relaxin-2 related disease or condition is group 2 PH (CpcPH or IpcPH) and heart failure with reduced ejection fraction (HFrEF). In some embodiments, relaxin-2 related disease or condition is CpcPH with HFrEF. In some embodiments, relaxin-2 related disease or condition is IpcPH with HFrEF.

[0578] In some embodiments, CpcPH is based on a performed right heart catheterization (RHC) showing pulmonary vascular resistance (PVR) ≥3 Wood units, mPAP >20 mm Hg, PCWP >15 mm Hg or PCWP >12 mm Hg and ≤14 mm Hg, and echocardiography showing a left atrial volume index (LAVI) ≥34 mL / m². 2 In some embodiments, IpcPH is based on a performed RHC showing PVR < 3 Wood units, mPAP > 20 mm Hg, PCWP > 15 mm Hg or PCWP > 12 mm Hg and ≤ 14 mm Hg, and echocardiography showing a left atrial volume index (LAVI) ≥ 34 mL / m³. 2 .

[0579] In some embodiments, relaxin-2-related diseases or conditions are pregnancy-related cardiovascular diseases, including but not limited to preeclampsia, postpartum hypertension, postpartum cardiomyopathy, pregnancy-related heart failure, and postpartum hypertension.

[0580] In some embodiments, the relaxin-2 related disease or condition is kidney disease. In some embodiments, the relaxin-2 related disease or condition is chronic kidney disease. In some embodiments, the relaxin-2 related disease or condition is hypertensive kidney disease.

[0581] In some embodiments, relaxin-2-related diseases or conditions are joint diseases. In some embodiments, relaxin-2-related diseases or conditions are frozen shoulder (also known as adhesive capsulitis).

[0582] Application of the composition according to the methods described herein can result in a reduction in the severity, signs, symptoms, or markers of relaxin-2-related disease or condition in patients. "Reduction" as used herein means a statistically significant decrease in the level. This reduction (an absolute reduction or a reduction in the difference between the subject's elevated level and normal levels) 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 below the detection level used in the assay.

[0583] In some embodiments, administration of the composition according to the methods described herein results in pulmonary vasodilation in a patient. In some embodiments, administration of the composition according to the methods described herein results in an anti-inflammatory effect in a patient. In some embodiments, administration of the composition according to the methods described herein results in an anti-fibrotic effect in a patient. In some embodiments, administration of the composition according to the methods described herein results in right ventricular remodeling in a patient. In some embodiments, administration of the composition according to the methods described herein results in peripheral vasodilation in a patient. In some embodiments, administration of the composition according to the methods described herein results in diastole in a patient. In some embodiments, administration of the composition according to the methods described herein results in left ventricular remodeling in a patient. In some embodiments, administration of the composition according to the methods described herein results in improved renal function in a patient.

[0584] In some embodiments, administration of the composition according to the methods described herein results in an increase in renal plasma flow. In some embodiments, administration of the composition according to the methods described herein results in an increase in renal plasma flow that persists for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 1 month after a single administration. In some embodiments, the increase in renal plasma flow in the subject is maintained at 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% after a single administration. In some embodiments, administration of the composition according to the methods described herein results in an increase in renal plasma flow that persists for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 1 month after a single administration to a human. In some embodiments, the increase in renal plasma flow in subjects at 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 1 month after a single administration to a human is maintained at 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%.

[0585] Combination therapies and formulations

[0586] This disclosure also provides compositions and therapeutic formulations comprising the fusion proteins or component peptides described herein, or nucleic acid molecules or expression vectors encoding them, in combination with one or more additional therapeutically active components (i.e., therapeutic agents), and treatment methods including administering such combinations to subjects in need.

[0587] Exemplary additional therapeutic agents include any therapeutic agents that can be used to treat any relaxin-2 related conditions described herein. Exemplary additional therapeutic agents that can be combined or administered in combination with the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, include, but are not limited to, any one or more of the following: angiotensin II receptor blockers, such as azilsartan, candesartan, eprosartan, losartan; ACE inhibitors, such as lisinopril, benazepril, captopril, enalapril, moxipril, perindopril, quinapril, quindopril; calcium channel blockers, such as amlodipine, amlodipine and benazepril, amlodipine and valsartan, sacubitril and valsartan, diltiazem, felodipine, isradipine, nicardipine, nimodipine, nisoldipine, verapamil; and diuretics, such as chlorthalidone, hydrochlorothiazide, metoprazone, indapamide, torasemide, furosemide, bumetanide, and azoxystrobin. Miloride, triamterene, spironolactone, eplerenone; aldosterone antagonists, such as spironolactone, eplerenone; digoxin, such as lanoxine; beta-blockers, such as carvedilol, metoprolol, bisoprolol; activin signaling inhibitors, such as sotacicept; sodium / glucose cotransporter 2 (SGLT2) inhibitors, such as empagliflozin, dapagliflozin, betagliflozin, canagliflozin, eleggliflozin, ilagliflozin, levogliflozin, levaggliflozin, serggliflozin, soragliflozin, togliflozin, hangaggliflozin, gaggliflozin, migliflozin, vildaggliflozin proline hydrate, enagliflozin; and glucagon-like peptide-1 (GLP-1) receptor agonists, such as exenatide, liraglutide, abiglutide, dulaglutide, liximab, smegglutide, and tesipatide.

[0588] In some embodiments, additional therapeutic agents that may be administered in combination or in combination with the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, include, but are not limited to, one or more of the following: sotascept, empagliflozin, dapagliflozin, sacubitril, valsartan, smegglutide, dulaglutide, and tesipatide.

[0589] In some embodiments, additional therapeutic agents are drugs effective in treating fibrosis, including but not limited to small molecule drugs and antibodies. Exemplary antifibrotic drugs include, but are not limited to, TGF-β inhibitors, such as small molecules (e.g., hydroxynidone, desimethasone) or antibodies (e.g., non-hematoxylin and oxaliplatin), PDGF or VEGF antagonists, such as small molecules, such as imatinib, nilotinib; or any drug that targets extracellular factors involved in the pathogenesis of fibrosis. Descriptions 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.

[0590] Other therapeutically active components may be administered before, simultaneously with, or shortly after the administration of the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them.

[0591] This disclosure provides pharmaceutical compositions wherein the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are co-formulated with one or more of other therapeutically active components as described elsewhere herein.

[0592] Application plan

[0593] In some embodiments, multiple doses of the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, may be administered to a subject over a predetermined time period. A method according to this aspect of the disclosure includes sequentially administering multiple doses of the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, to a subject. As used herein, “sequential administration” means administering each dose of the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, to a subject at different time points, such as on different days separated by predetermined intervals (e.g., hours, days, weeks, or months). The disclosure provides a method comprising: sequentially administering a single initial dose of the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, to a patient; followed by administering one or more secondary doses of the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them; and optionally, subsequently administering one or more tertiary doses of the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them.

[0594] The terms “initial dose,” “second dose,” and “third dose” refer to the temporal sequence of administration of the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them. Thus, an “initial dose” is the dose administered at the start of a treatment regimen (also known as a “baseline dose”); a “second dose” is the dose administered after the initial dose; and a “third dose” is the dose administered after the second dose. The initial, second, and third doses may all contain the same amount of the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, but may typically differ from each other in terms of administration frequency. However, in some embodiments, the amounts of the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, contained in the initial, second, and / or third doses may differ from each other during treatment (e.g., adjusted up or down as appropriate). In some embodiments, two or more doses (e.g., 2, 3, 4, or 5) are administered at the start of a treatment regimen as a “loading dose,” followed by subsequent doses (e.g., a “maintenance dose”) administered on a less frequent basis.

[0595] In one exemplary embodiment, immediately following the previous dose, 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, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2、21、21 1 / 2、22、22 1 / 2、23、23 1 / 2、24、24 1 / 2、25、25 1 / 2, 26, 26 1Each second and / or third dose is administered every 2 or more weeks. As used herein, the phrase “immediately preceding dose” refers to the dose of the fusion protein or component peptide or the nucleic acid molecule or expression vector encoding them described herein administered to the patient in a sequence of multiple administrations, prior to the next dose in the administration sequence (without inserting a dose).

[0596] In one embodiment, each second and / or third dose is administered 4 weeks after the immediately preceding dose. In another embodiment, a dose of the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered to the patient every 4 weeks (Q4W).

[0597] 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 protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them. For example, in some embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, in some embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0598] In embodiments involving multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient 1 to 2 weeks after the previous dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose may be administered to the patient 2 to 4 weeks after the previous dose. Alternatively, the frequency of administration of the second and / or third doses to the patient may vary during the treatment regimen. The physician may also adjust the administration frequency during the treatment process based on the individual patient's needs following a clinical examination.

[0599] In one embodiment, one or more of the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered to a subject in a weight-based dose. A “weight-based dose” (e.g., a dose in mg / kg) is a dose of protein or peptide that varies with the subject’s weight.

[0600] In another embodiment, one or more of the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered to the subject at a fixed dose. "Fixed dose" (e.g., a dose in mg) means that a single dose of the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is used for all subjects, regardless of any specific subject-related factors, such as weight. In one particular embodiment, the fixed dose of the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is based on a predetermined weight or age.

[0601] Generally, suitable doses of the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are in the range of about 0.001 to about 200.0 mg / kg of recipient body weight, typically in the range of about 1 to 50 mg / kg of body weight. In some embodiments, the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered at doses in the range of about 0.001 mg / kg to about 200 mg / kg. In some embodiments, the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered at doses in the range of about 0.001 mg / kg to 200 mg / kg. In some embodiments, the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered at doses in the range of about 0.01 mg / kg to about 100 mg / kg. In some embodiments, the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered at doses in the range of 0.01 mg / kg to 100 mg / kg. In some embodiments, the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered at a dose ranging from about 0.1 mg / kg to about 20 mg / kg. In some embodiments, the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered at a dose ranging from 0.1 mg / kg to 20 mg / kg. In some embodiments, the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered at a dose ranging from about 1 mg / kg to about 50 mg / kg. In some embodiments, the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered at a dose ranging from 1 mg / kg to 50 mg / kg. For example, the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, may be administered at single doses of approximately 0.1 mg / kg, approximately 0.2 mg / kg, approximately 0.3 mg / kg, approximately 0.5 mg / kg, approximately 1 mg / kg, approximately 1.5 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, approximately 15 mg / kg, approximately 20 mg / kg, approximately 25 mg / kg, approximately 30 mg / kg, approximately 40 mg / kg, or approximately 50 mg / kg. In some embodiments, the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, may be administered at single doses of 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, and 50 mg / kg. These values ​​and ranges between them are also intended to form part of this disclosure.

[0602] In some embodiments, the fusion protein or component peptide, or the nucleic acid molecule or expression vector encoding them described herein, is administered at about 0.3 mg / kg. In some embodiments, the fusion protein or component peptide, or the nucleic acid molecule or expression vector encoding them described herein, is administered at 0.3 mg / kg. In some embodiments, the fusion protein or component peptide, or the nucleic acid molecule or expression vector encoding them described herein, is administered at about 1 mg / kg. In some embodiments, the fusion protein or component peptide, or the nucleic acid molecule or expression vector encoding them described herein, is administered at 1 mg / kg. In some embodiments, the fusion protein or component peptide, or the nucleic acid molecule or expression vector encoding them described herein, is administered at about 3 mg / kg. In some embodiments, the fusion protein or component peptide, or the nucleic acid molecule or expression vector encoding them described herein, is administered at 3 mg / kg. In some embodiments, the fusion protein or component peptide, or the nucleic acid molecule or expression vector encoding them described herein, is administered at about 10 mg / kg. In some embodiments, the fusion protein or component peptide, or the nucleic acid molecule or expression vector encoding them described herein, is administered at 10 mg / kg.

[0603] In some embodiments, one or more of the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered at a fixed dose between about 10 mg and about 2500 mg. In some embodiments, one or more of the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered at a fixed dose between 10 mg and 2500 mg. In some embodiments, one or more of the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered at a fixed dose between about 100 mg and about 1500 mg. In some embodiments, one or more of the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered at a fixed dose between 100 mg and 1500 mg. In some embodiments, the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are in doses 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, about 450 mg, or about [amount missing]. Administered at fixed doses of 475 mg, approximately 500 mg, approximately 525 mg, approximately 550 mg, approximately 575 mg, approximately 600 mg, approximately 625 mg, approximately 650 mg, approximately 675 mg, approximately 700 mg, approximately 725 mg, approximately 750 mg, approximately 775 mg, approximately 800 mg, approximately 825 mg, approximately 850 mg, approximately 875 mg, approximately 900 mg, approximately 925 mg, approximately 950 mg, approximately 975 mg, approximately 1000 mg, approximately 1500 mg, approximately 2000 mg, or approximately 2500 mg. In some embodiments, the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are in doses 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, 425 mg, or 450 mg. Administer at fixed doses of g, 475mg, 500mg, 525mg, 550mg, 575mg, 600mg, 625mg, 650mg, 675mg, 700mg, 725mg, 750mg, 775mg, 800mg, 825mg, 850mg, 875mg, 900mg, 925mg, 950mg, 975mg, 1000mg, 1500mg, 2000mg, or 2500mg.The values ​​and the ranges between said values ​​are also intended to form part of this disclosure.

[0604] In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of about 150 mg. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of 150 mg. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of at least 150 mg. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of about 300 mg. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of 300 mg. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of about 600 mg. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of 600 mg.

[0605] In some embodiments, the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered intravenously. In some embodiments, the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered subcutaneously.

[0606] In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered via intravenous infusion. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at 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, 4 Administered via intravenous infusion over durations of 6, 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 minutes, 2 hours, 3 hours, 4 hours, or longer. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered via intravenous infusion over 30 minutes. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered via intravenous infusion over 60 minutes. In some embodiments, the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, are administered via intravenous infusion over 30 to 60 minutes.

[0607] In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered intravenously at a dose of about 0.3 mg / kg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered intravenously at a dose of 0.3 mg / kg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a dose of about 0.3 mg / kg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a dose of 0.3 mg / kg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a dose of about 1 mg / kg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a dose of 1 mg / kg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a dose of about 3 mg / kg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at 3 mg / kg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at about 10 mg / kg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at 10 mg / kg every 4 weeks.

[0608] In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a dose of about 150 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a dose of 150 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a dose of at least 150 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of about 150 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of 150 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of at least 150 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of about 300 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of 300 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of about 600 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of 600 mg every 4 weeks.

[0609] In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a dose of about 150 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a dose of 150 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a dose of at least 150 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a fixed dose of about 150 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a fixed dose of 150 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a fixed dose of at least 150 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a fixed dose of about 300 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a fixed dose of 300 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a fixed dose of about 600 mg every 4 weeks. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a fixed dose of 600 mg every 4 weeks.

[0610] In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered intravenously once monthly at a dose of about 0.3 mg / kg. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered intravenously once monthly at a dose of 0.3 mg / kg. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered once monthly at a dose of about 0.3 mg / kg. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered once monthly at a dose of 0.3 mg / kg. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered once monthly at a dose of about 1 mg / kg. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered once monthly at a dose of 1 mg / kg. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered once monthly at a dose of about 3 mg / kg. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at 3 mg / kg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at about 10 mg / kg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at 10 mg / kg once a month.

[0611] In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a dose of about 150 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a dose of 150 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a dose of at least 150 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of about 150 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of 150 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of at least 150 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of about 300 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of 300 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of approximately 600 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered at a fixed dose of 600 mg once a month.

[0612] In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a dose of about 150 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a dose of 150 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a dose of at least 150 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a fixed dose of about 150 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a fixed dose of 150 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a fixed dose of at least 150 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a fixed dose of about 300 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a fixed dose of 300 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a fixed dose of about 600 mg once a month. In some embodiments, the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them, is administered subcutaneously at a fixed dose of 600 mg once a month.

[0613] Reagent test kit

[0614] Any composition described herein may be included in the kit. In non-limiting examples, the kit contains one or more of the fusion proteins or component peptides described herein, or nucleic acid molecules or expression vectors encoding them.

[0615] The kit may further include reagents or instructions for use in subjects of the fusion protein or component peptide described herein, or the nucleic acid molecule or expression vector encoding them. It may also include one or more buffers.

[0616] The kit components can be packaged in an aqueous medium or in lyophilized form. The kit's container device typically includes at least one vial, test tube, flask, bottle, syringe, or other container device in which the components can be placed, and preferably suitably aliquoted. When the kit contains more than one component (labeling reagents and labels may be packaged together), the kit typically also contains a second, third, or other additional container in which the additional components can be placed individually. The kit may also include a second container device for containing sterile, pharmaceutically acceptable buffers and / or other diluents. However, vials may include various combinations of components. The kits disclosed herein typically also include a device for containing the fusion proteins or component peptides described herein, or the nucleic acid molecules or expression vectors encoding them, and any other reagent containers in a tightly sealed container for commercial sale.

[0617] When the kit components are provided as one or more liquid solutions, the liquid solutions are aqueous solutions, and sterile aqueous solutions are particularly preferred. However, the kit components may also be provided as dry powders. When reagents and / or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in another container.

[0618] Example

[0619] Examples of this disclosure are provided by way of illustration and explanation and are not intended to limit the scope of this disclosure. The results described in each example reflect the specific conditions outlined in the experiments described therein.

[0620] Example 1. Heparin chromatography of relaxin-2 fusion protein analog

[0621] Heparin chromatography is a method that can be used for early screening of candidate drugs to better understand the tendency of molecules to interact with vascular system elements after patient administration. Heparin and heparan sulfate proteoglycans are negatively charged polysaccharides present in the vascular system and tissues, where positively charged molecules can bind at physiological pH (i.e., pI > 7.4). Here, heparin chromatography is used to screen candidate drugs / variants with reduced heparin binding, which suggests good PK properties. The materials used in heparin chromatography are provided in Table 11.

[0622] Table 11. Materials

[0623] project supplier Catalog Number <![CDATA[POROS TM Heparin 2.1x30mm column]]> Thermo Fisher 4333411

[0624] method

[0625] Mobile phase A (binding): 20 mM Tris pH 7.4; Mobile phase B (elution): 20 mM Tris pH 7.4 + 1 M NaCl; Injection volume: 10 μg; Detection wavelength: 220 nm.

[0626] 1. Before analysis, equilibrate the heparin column for 10 minutes using mobile phase A at 0.5 mL / min.

[0627] 2. Dilute the sample to be analyzed to 1 mg / mL with 20 mM Tris pH 7.4 to minimize the ionic strength.

[0628] 3. Run heparin chromatography on Agilent HPLC, using the gradient shown in Table 12 below:

[0629] Table 12. HPLC gradient

[0630] Time (min) Flow rate (mL / min) ％A ％B 0 0.5 100 0 1 0.5 100 0 6 0.5 50 50 6.5 0.5 50 50 7 0.5 0 100 8 0.5 0 100 8.5 0.5 100 0 10 0.5 100 0

[0631] 4. Includes a positive control (non-heparinized, human IgG mixture) and a negative control (SE301 or AT1R).

[0632] 5. Analyze the retention time of the samples and report the relative retention time compared to the positive control (i.e., RT sample / RT positive control).

[0633] 6. Calculate the approximate NaCl concentration required for elution using the following method: [NaCl] = (RT sample - 1) * 100

[0634] The results of the calculations are shown in Table 13.

[0635] Table 13. Retention time, relative retention time and NaCl concentration of the samples

[0636] sample room temperature RRT [NaCl] Positive control 1.5 N / A 50 negative control 3.5 2.3 250 sample 2.0 1.3 100

[0637] result

[0638] Table 14 shows the results of heparin chromatography for various relaxin-2 analog fusion proteins.

[0639] Table 14. Heparin Chromatography

[0640]

[0641] *Measured using an imaging capillary isoelectric focusing experiment.

[0642] IgG was obtained from Jackson ImmunoResearch (catalog number 009-000-003). The “previous fusion protein” was a LALAIgG-RelB-adaptor-RelA fusion with a theoretical pI of 8.5, but an experimentally determined pI of 9.4. Its adaptor protein contained only one acidic amino acid. SEQ ID NO:300, 302, 303, 305, 306, and 308-311 contained adaptor proteins containing at least two acidic amino acids, as well as LALA IgG (SEQ ID NO:77 or 81). The adaptor proteins of the two final fusion proteins contained only one acidic amino acid and therefore had a higher theoretical pI. As shown in Table 14 above, a correlation was found between the lower pI and lower nonspecific binding by heparin chromatography.

[0643] Example 2. Low pI relaxin-2 fusion protein analogs, as measured by affinity-trapping self-interacting nanoparticle spectroscopy (AC-SINS), tend to exhibit reduced self-association.

[0644] Understanding the self-association tendency of molecules is crucial when evaluating the biophysical properties of development candidates. Numerous methods exist for evaluating this tendency, including concentrating molecules to high concentrations and assessing them via SEC (% monomer) or measuring turbidity changes (OD 340 nm), and calculating the second virial coefficient (B) using DLS. 22 ) or self-interaction coefficient (k d ), or use AC-SINS(Δλ) max All three methods provide useful information, but are evaluated using different amounts of material. AC-SINS has emerged as a high-throughput method for evaluating self-associations using minimal material, yet still provides locally high concentrations through affinity trapping on gold nanoparticles. In short, gold nanoparticles pre-coated with anti-human antibodies (Fc, Fab, and H+L) trap and concentrate the antibody of interest in solution when incubated with the target antibody in a diluted solution. As the immobilized molecules of interest interact, the interparticle distance between the gold nanoparticles decreases, resulting in an increase in the plasma wavelength (i.e., a redshift), which can be quantified using UV-VIS spectroscopy. The materials used for the spectroscopy are provided in Table 15.

[0645] Table 15. Materials

[0646] project supplier Catalog Number 1M sodium acetate, pH 4.3 Molecular Dimensions MD2-019-PH 1x DPBS Gibco 14190-136 Panitumumab (low-association control) MyBioSource MBS156169 Iprimumab (intermediately associated control) MyBioSource MBS156153 Mannuzub (highly associated control) MyBioSource MBS156142 20nm gold nanoparticles Ted Pella 15705 PEG-methyl ether thiol (2kDa) Sigma 729140 Goat anti-human IgG, Feγ Jackson ImmunoResearch 109-005-098 Goat nonspecific mAbs Jackson ImmunoResearch 005-000-003 Zeba desalting column 40K 5mL Thermo Fisher 87770 Zeba desalting column 40K 2mL Thermo Fisher 87768 Zeba desalting column 40K 0.5mL Thermo Fishe 87766 Costar 384-hole polystyrene board Fisher Scientific 12-565-506 96-hole polypropylene board Grenier Bio-One 652230P

[0647] method

[0648] Preparation of buffer solutionTo prepare a 20 mM sodium acetate solution at pH 4.3, 2 mL of a 1 M sodium acetate stock solution at pH 4.3 was diluted to 100 mL with MilliQ water. The pH was measured to be 4.3 ± 0.1, and the solution was aseptically filtered. The solution is stable at room temperature for one month. Add 10 mL of MilliQ water to 1 g of PEG methyl ether thiol. Briefly vortex to suspend the solid, preparing a 50 mM solution. To prepare a final 10 μM dilution, follow the dilution protocol:

[0649] a. Dilute the 50 mM stock solution to 1 mM (20 μL 50 mM stock solution + 980 μL LiMilliQ water)

[0650] b. Dilute the 1 mM step solution to 100 μM (10 μL 1 mM stock solution + 90 μL LiMilliQ water)

[0651] c. Dilute the 100 μM step solution to 10 μM (100 μL 100 μM stock solution + 900 μL LiMilliQ water)

[0652] d. The volume can be adjusted according to the number of samples to be tested.

[0653] e. The remaining 50mM stock solution should be divided into equal portions and stored at -20°C until needed.

[0654] Preparation of gold nanoparticle solutions Goat anti-human Fc IgG antibody (capture) and goat IgG antibody (non-capture) were exchanged in 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 volume ratio of capture (anti-Fc):non-capture (goat IgG) solution was prepared to obtain an 80% capture capacity coating solution for incubating gold nanoparticles (AuNP).

[0655] Prepare an AuNP coating solution at a volume ratio of 9:1. Incubate the solution overnight in the dark at room temperature. After incubation, add thiolized PEG to a final concentration of 0.1 μM from a diluted 10 μM stock solution to block blank sites on the AuNP (i.e., 5 mL AuNP solution, 50 μL 10 μM stock solution), and incubate in the dark at RT for one hour.

[0656] Preparation of AuNP solution Centrifuge 2 mL of the coated AuNP solution at 20,000 x g for 15 minutes to deposit AuNP, and carefully transfer 1800 μL of the supernatant using a 1 mL pipette. Gently resuspend the precipitated AuNP using a 200 μL pipette to obtain a stock solution of coated AuNP that is 10 times concentrated.

[0657] Prepare the target antibody solution (either method follows these steps).For each sample analyzed, 10 μL of concentrated AuNP solution was incubated with 100 μL of antibody test solution (normalized to 0.05 mg / mL) in a 96-well polypropylene plate in the dark at room temperature for 2 hours. Two blank solutions were prepared by concentrating 10 μL of 10x AuNP to 100 μL of PBS for blank assays and to determine the wavelength shift after adding the test antibody. Mannomab was included as a positive control (high association, red shift), and panitumumab was included as a negative control (no 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-clear polystyrene plate (384-well plate). Two blank solutions were transferred to accurately assess the wavelength shift, and then two aliquots of standards and samples were added for analysis. The plate was then centrifuged at 1000 x g for 1 minute to ensure uniform distribution of the solution in the wells. Absorbance data were collected from 510 nm to 570 nm in 2 nm increments to determine the wavelength shift of each sample relative to an individual AuNP.

[0658] result

[0659] The results from ASCINS are shown in Table 16 below.

[0660] Table 16. pI variants have a reduced tendency to self-associate.

[0661] sample Isoelectric point (calculated value) <![CDATA[Aλ max ]]> Previous fusion protein (control) 8.5 15.2 Previous fusion protein (LALA) 8.5 15.9 SEQ ID NO:300 8.2 7.5 SEQ ID NO:301 7.9 1.2 SEQ ID NO:302 7.9 7.0 SEQ ID NO:303 7.9 6.7 SEQ ID NO:305 7.6 1.8 SEQ ID NO:306 7.6 1.9 SEQ ID NO:308 7.2 -0.6 SEQ ID NO:309 7.2 -0.4 SEQ ID NO:310 6.8 0.4 SEQ ID NO:311 6.8 0.4 SEQ ID NO:359 8.4 10.1 SEQ ID NO:360 8.3 6.1

[0662] As shown in Table 16 above, fusion proteins with low pI also tend to exhibit lower self-aggregation.

[0663] Example 3. Relaxin-2 fusion protein analogue induces cAMP response in RXFP1 transfected cells.

[0664] This example provides data relating to the potency of the various relaxin-2 fusion protein analogs described herein. The potency of the fusion protein analogs was determined by measuring cAMP signaling to test their ability to activate RXFP1.

[0665] method

[0666] HEK293 cells were seeded into 96-well tissue culture plates and then transiently co-transfected with human RXFP1 and pGloSensor-22F plasmids. Transfected cells were stimulated by relaxin-2 or its fusion protein analogues, inducing Gs-mediated cAMP signaling. cAMP was measured using the activity of the GloSensor biosensor, a mutant luciferase fused to the cAMP-binding domain that produces light in the presence of its substrate, luciferin. Relative luminescent units (RLU) readings were used as an indicator of the cAMP response.

[0667] reagents

[0668] ■ 96-well tissue culture plate. White, transparent bottom. (Corning #3610)

[0669] ■HEK293 cells (ATCC CRL-1573)

[0670] ■ Poly-D-Lysine (Gibco A3890401)

[0671] ■DPBS (Calcium-free, Magnesium-free; Gibco 14190250)

[0672] ■DMEM (High sugar content, containing L-glutamine and sodium pyruvate; Gibco 11995065)

[0673] ■TrypLE Express(Gibco 12605010)

[0674] ■FBS (HyClone) TM (Source: Australia; Cytiva SH30084)

[0675] ■ Penicillin-Streptomycin (Gibco 15140122)

[0676] ■CO2-independent culture medium (Gibco 18045088)

[0677] ■Opti-MEM TM Low serum medium (Gibco 31985062)

[0678] ■pGloSensor TM -22F cAMP plasmid (Promega catalog number E2301)

[0679] ■D-Fluorescence Potassium Salt (GoldBio LUCK-1G)

[0680] ■FuGENE HD Transfection Reagent (Promega#E2311)

[0681] ■ Storage Container (Corning / Axygen RES-V-25-SI)

[0682] ■ Relaxin-2 (R&D Biosystems 6586-RN-025)

[0683] ■ Plasmid containing RXFP1 (pcDNA5 / FRT / TO–human RXFP1, full length)

[0684] ■ Hairy throat extract (Sigma F6886)

[0685] ■ A microplate reader capable of reading luminescent microplates (CLARIOstar Plus)

[0686] Reagent preparation

[0687] D-luciferin potassium salt D-fluorescein was reconstituted at 25 mg / mL in 10 mM HEPES (pH 7.5) (78.5 mM; MW = 318.4). The reconstituted sample was aliquoted into sterile microcentrifuge tubes of approximately 200–500 μL for single use and stored at -80°C.

[0688] Relaxin-2 peptide Relaxin-2 peptide or relaxin-2 fusion protein analogue was prepared in sterile DPBS (MW = 5,986 Da, ε = 12,865 M). -1 cm -1 Reconstituted at 0.1 mg / mL in A, and in A 280 The final concentration was determined by measurement. Aliquots were stored at -20°C.

[0689] Trichosanthes Reconstruct the sample by dissolving the trichosanthes in 100% DMSO (2.05 mg / mL, MW = 410.5) at 5 mM. Store aliquots at -20°C.

[0690] cAMP assay medium Preheat the CO2-independent medium to 37°C using a bead bath. Thaw a single aliquot of D-luciferin and add it at a final concentration of 5% (e.g., 4.75 mL cAMP assay medium + 250 μL D-luciferin stock solution; final D-luciferin concentration is 1.25 mg / mL or 3.93 mM). The medium can be used or discarded on the same day.

[0691] Cell culture and maintenance

[0692] 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 and 5% CO2 until 80%-100% confluence. Cells were typically divided at a 1:6 ratio for 3 days and maintained in sterile T-75 tissue culture flasks.

[0693] cAMP signal transduction measurement scheme

[0694] This method is adapted from Promega's GloSensor cAMP assay.

[0695] Raw data were processed on the CLARIOstar microplate reader and then exported to Excel using MARS data analysis software. These values ​​were measured in RLU or relative luminescence units.

[0696] As shown in Table 17, all low pI relaxin-2 fusion protein analogs were able to induce a cAMP response in RXFP1-transfected cells.

[0697] Table 17. cAMP response in RXFP1-transfected HEK293 cells

[0698]

[0699]

[0700] Example 4. In vitro properties of relaxin-2 fusion protein analogs

[0701] This example provides the in vitro properties of the various relaxin-2 fusion protein analogs described herein.

[0702] method

[0703] Heparin chromatography Heparin chromatography was performed to understand the interaction of relaxin-2 fusion protein analogs with angiogenesis and / or their tendency to rapidly distribute into tissues after patient administration. Analogs with weaker heparin binding were found to predict favorable pharmacokinetic properties. Briefly, prior to analysis, the heparin column was equilibrated for 10 min at 0.5 mL / min using mobile phase A (20 mM Tris, pH 7.4). Heparin chromatography was performed on an Agilent HPLC system 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), and 10 μg of each sample was analyzed.

[0704] Table 18. HPLC gradient

[0705] Time (min) Flow rate (mL / min) ％A ％B 0 0.5 100 0 6 0.5 50 50 7 0.5 0 100 8 0.5 0 100 8.5 0.5 100 0 10 0.5 100 0

[0706] This included a positive control (non-heparin-bound, pembrolizumab) and a negative control (mildly heparin-bound, adalimumab), and analyzed the retention time of the samples 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 method:

[0707] [NaCl] = (RT sample) * 100

[0708] The results of the calculation are shown in Figure 19.

[0709] Table 19. Retention time and NaCl concentration of samples

[0710] sample room temperature [NaCl] Positive control 1.5 150 negative control 3.5 350 sample 2.0 200

[0711] Hydrophobic interaction chromatography (HIC) HIC is a chromatographic method based on molecular hydrophobicity for separation. 10 μg of protein is injected into a butyl HIC column pre-equilibrated with high-ammonium sulfate buffer. The protein elutes over ten minutes in a gradient from high to low salt concentration. The hydrophobicity of the samples is compared based on retention time, where longer retention times indicate higher hydrophobicity and shorter retention times indicate lower hydrophobicity. The retention times are converted to approximate salt concentrations at elution and compared with high and low hydrophobicity standards.

[0712] Size exclusion chromatography (SEC) SEC is a liquid chromatography method used to determine the monomer and polymer levels of a given analyte in solution. SEC is used to assess the presence of fusion protein aggregates. Samples are prepared and added to a 1.7 μm particle size SEC column with an aqueous mobile phase containing 25 mM potassium phosphate and 0.5 M potassium chloride at pH 8.0. Once sample elution is validated, this method quantifies the levels of soluble aggregate species in the sample, with high resolution between peaks of monomers and high molecular weight (HMW) species. The percentage of area is determined by integrating the corresponding elution curves, thus calculating the percentage of monomers (i.e., % monomers) and other species (i.e., HMW species, low molecular weight species).

[0713] Capillary isoelectric focusing (cIEF) Molecules with differentially charged structures (i.e., relaxin-2 fusion protein analogs) were separated using imaging-based cIEF, and their isoelectric points (pI) were determined using electrophoretic mobility in a zwitterionic electrolyte solution. Molecules were loaded into a capillary and separated based on their pI by allowing them to migrate along an electric field until they reached the pH value corresponding to their pI. UV absorbance throughout the capillary was measured throughout the separation process, enabling real-time observation and final quantification.

[0714] Baculovirus Particle (BVP) ELISA The BVP ELISA was used to understand the tendency of relaxin-2 fusion protein analogs to engage with nonspecific or non-targeted interactions. BVPs are empty viral capsids, lacking a viral genome, but during production, they bud from the cell membrane, allowing them to carry cell membrane components into the cell. Therefore, BVPs possess a highly diverse cell surface with numerous components that mimic the molecules of interest (i.e., relaxin-2 fusion protein analogs) that might encounter in vivo. Briefly, BVPs were coated onto the plate by adding 25 μL of BVP solution to each well. The BVP solution was prepared by diluting BVP stock solution (Medna Scientific; catalog number E3001) to 1 x 10⁻⁶.6 BVP was prepared at 100 μL / well with 0.1 M carbonate buffer, pH 9.6. After incubation overnight at 5 °C, the BVP solution was aspirated from the wells and the wells were washed three times with PBST. The plate was blocked with 100 μL / well of 1x BSA in PBS blocking buffer (Cepham Life Sciences; catalog number 10615). The plate was incubated at 25 °C on a plate shaker for 1 hour. The blocking solution was aspirated from the wells and the wells were washed three times with PBST. The sample (i.e., relaxin-2 fusion protein analog) was prepared in duplicate to cover the dilution range from 3 μM to 0.1 nM and added to the plate. The plate was incubated at 25 °C for 1 hour, after which the wells were aspirated and washed three times with PBST. Add 25 μL / well of a 1:10,000 diluted monoclonal antibody (peroxidase AffiniPure goat anti-human IgG, Fcγ fragment specific; Jackson ImmunoResearch; catalog number 50-194-1564) and incubate the plate at 25°C for 1 hour. Afterward, aspirate the wells and wash three times with PBST. Then add 1-Step... TM Ultra TMB-ELISA substrate solution (LifeTechnologies; catalog number 34029). After approximately 2 minutes, add 25 μL of 2N HCl to quench the reaction, and analyze the plate at 450 nm using a microplate reader, performing calibration at 570 nm.

[0715] efficacy determination HEK293 cells were seeded into 96-well tissue culture plates and then transiently co-transfected with human RXFP1 and pGloSensor-22F plasmids. Transfected cells were stimulated by relaxin-2 or its fusion protein analogues, inducing Gs-mediated cAMP signaling. cAMP was measured using the activity of the GloSensor biosensor, a mutant luciferase fused to the cAMP-binding domain that produces light in the presence of its substrate, luciferin. Relative luminescent units (RLU) readings were used as an indicator of the cAMP response.

[0716] cAMP signal transduction measurement scheme This protocol is adapted from Promega's GloSensor cAMP assay. Raw data were run on a CLARIOstar microplate reader and then exported to Excel using MARS data analysis software. These values ​​are measured in RLU or relative luminescence units.

[0717] Affinity-based self-interacting nanoparticle spectroscopy (AC-SINS)AC-SINS was performed to understand the tendency of molecules (i.e., relaxin-2 fusion protein analogs) to self-associate. In short, gold nanoparticles pre-coated with anti-human antibodies (Fc, Fab, and H+L) captured and concentrated the antibody of interest in solution when incubated with the target antibody in a diluted solution. When the immobilized molecules of interest interact, the interparticle distance between the gold nanoparticles decreases, resulting in an increase in the plasma wavelength (i.e., a redshift), which can be quantified using UV-VIS spectroscopy. The materials used for the spectroscopy are provided in Table 20.

[0718] Table 20. Materials used in AC-SINS

[0719] project supplier Catalog Number 1M sodium acetate, pH 4.3 Molecular Dimensions MD2-019-PH 1x DPBS Gibco 14190-136 Panitumumab (low-association control) MyBioSource MBS156169 Iprimumab (intermediately associated control) MyBioSource MBS156153 Mannuzub (highly associated control) MyBioSource MBS156142 20nm gold nanoparticles Ted Pella 15705 PEG-methyl ether thiol (2kDa) Sigma 729140 Goat anti-human IgG, Fcγ Jackson ImmunoResearch 109-005-098 Goat nonspecific mAbs Jackson ImmunoResearch 005-000-003 Zeba desalting column 40K 5mL Thermo Fishe 87770 Zeba desalting column 40K 2mL Thermo Fisher 87768 Zeba desalting column 40K 0.5mL Thermo Fisher 87766 Costar 384-hole polystyrene board Fisher Scientific 12-565-506 96-hole polypropylene board Grenier Bio-One 652230P

[0720] Goat anti-human Fc IgG antibody (capture) and goat IgG antibody (non-capture) were buffer exchanged in 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 volume ratio of capture (anti-Fc):non-capture (goat IgG) solution was prepared to obtain an 80% capture capacity coating solution for incubating gold nanoparticles (AuNP). A 9:1 volume ratio of AuNP:coating solution was prepared. The solutions were incubated overnight in the dark at room temperature. After incubation, thiolized PEG was added to a final concentration of 0.1 μM from a diluted 10 μM stock solution to block the blank sites on the AuNP (i.e., 5 mL AuNP solution, 50 μL 10 μM stock solution), and the solution was incubated in the dark at RT for one hour.

[0721] Centrifuge 2 mL of the coated AuNP solution at 20,000 x g for 15 min to deposit AuNP, and carefully transfer 1800 μL of the supernatant using a 1 mL pipette. Gently resuspend the precipitated AuNP using a 200 μL pipette to obtain a 10-fold concentrated stock solution of coated AuNP. For each sample analyzed, incubate 5 μL of the AuNP concentrate with 45 μL of antibody test solution (normalized to 0.05 mg / mL) in a 384-well polypropylene plate in the dark at room temperature for 2 h. After 2 hours of incubation, absorbance data are collected from 450 nm to 650 nm in 1 nm increments to determine the wavelength shift of each sample relative to individual AuNP.

[0722] Nanoscale differential scanning fluorescence (NanoDSF)NanoDSF was performed using the NanoTemper Prometheus Panta to investigate the conformational stability of the relaxin-2 protein fusion analog. Conformational stability was measured by applying a thermal gradient to a solution containing the molecule of interest, measuring intrinsic fluorescence, backscattering, and using dynamic light scattering (DLS) to provide various thermal stability parameters, including the temperature at which the fusion protein begins to unfold (T0). 起始 ), the temperature at which half of the fusion protein in a given sample is expanded (T) m 1) and the temperature at which the fusion protein begins to aggregate (T) agg ).

[0723] sequence: The sequences of relaxin-2 fusion protein analogs are described in this disclosure. SEQ ID NO:496, 497, and 501 are shown below:

[0724] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGKGGSDSWKEEVIKLCGRELVRAQIAICGKSTASDAAGANANAGARQLYSALANKCCHVGCTKRSLARFC(SEQ ID NO:496);

[0725] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQLYSALANKCCHVGCTKRSLARFCGGGGSGGGGSGGGGSSWMEEVIKLCGRELVRAQIAICGMSTWS(SEQ ID NO:497); and

[0726] MPRLFFFHLLGVCLLLNQFSRAVADSWMEEVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSEMQPALPQLQQHVPVLKDSLLFEEFKKLIRNRQSEAADSSPSELKYLGLDTHSRKKRQLYSALANKCCHVGCTKRSLARFC (SEQ ID NO: 501).

[0727] result

[0728] The results are shown in Tables 21, 22 and 23 below.

[0729] Table 21. In vitro properties of relaxin-2 fusion protein analogs.

[0730]

[0731]

[0732]

[0733]

[0734]

[0735] 1 Retention time

[0736] 2 The NaCl concentration at the column elution peak.

[0737] 3Based on the power of the instantaneous hRXFP1 measurement above. ND – Undetermined.

[0738] N / A – Not applicable.

[0739] Table 22. In vitro properties of relaxin-2 fusion protein analogs.

[0740]

[0741]

[0742] 1 The effectiveness based on the above instantaneous hRXFP1 measurement.

[0743] 2 The NaCl concentration at the column elution peak.

[0744] ND – Undetermined.

[0745] Table 23. In vitro properties of relaxin-2 fusion protein analogs.

[0746]

[0747]

[0748] 1 The effectiveness based on the above instantaneous hRXFP1 measurement.

[0749] 2 The NaCl concentration at the column elution peak.

[0750] ND – Undetermined.

[0751] All samples in Tables 21, 22, and 23 were LALAPA LS IgG-RelB-linker-RelA fusions, containing LALAPA LS IgG (SEQ ID NO: 79 or 83) except for wild-type human relaxin-2 and SEQ ID NO: 497. As shown in Tables 21, 22, and 23 above, heparin chromatography revealed a correlation between lower pI and lower nonspecific binding.

[0752] A subset of relaxin-2 fusion protein analogs were subjected to confirmatory AC-SINS and BVP ELISA assays, as well as additional cAMP potency assays. The results are shown in Table 24.

[0753] Table 24. Additional potency assays of relaxin-2 fusion protein analogs.

[0754]

[0755] ND – Undetermined.

[0756] A subset of relaxin-2 fusion protein analogs (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) were subjected to various developmental evaluation tests. Under high concentration stress (target concentration of approximately 100 mg / mL), none of the samples showed UV-based protein concentration loss. Thermal stress induced an increase in turbidity / opalescence in all tested samples. Agitation stress had no effect on samples SEQ ID NO:313 and SEQ ID NO:104. Under chemical stress (target concentration of approximately 5 mg / mL), samples SEQ ID NO:496 and SEQ ID NO:90 showed concentration decreases under all tested chemical stresses, while samples SEQ ID NO:313, SEQ ID NO:87, SEQ ID NO:95, and SEQ ID NO:104 remained stable. All samples showed an oxidation-induced decrease in concentration, with SEQ ID NO:496 and SEQ ID NO:90 showing an increase in high molecular weight species as detected by size exclusion chromatography. Furthermore, based on non-reducing capillary electrophoresis (CE-SDS NR), no significant stress-induced fragmentation was observed in any of the tested samples.

[0757] The formation of stress-induced post-translational modifications (PTMs) was also tested. Stress conditions included: incubation at 40°C for 4 weeks, incubation at room temperature and high pH (Tris buffer, pH 8) for 2 weeks, incubation at room temperature and low pH (glycine buffer, pH 3) for 2 weeks, and incubation at room temperature under oxidative stress (0.02% hydrogen peroxide) for 24 hours. SEQ ID NO:313 and SEQ ID NO:87 showed no stress-induced modifications; SEQ ID NO:90 and SEQ ID NO:95 showed isomerization of aspartic acid in the linker region; SEQ ID NO:496 showed deamidation of asparagine in the linker region; and SEQ ID NO:95 and SEQ ID NO:104 showed isomerization of aspartic acid in the relaxin sequence.

[0758] During the manufacturability assessment, color changes and aggregation of various molecules were observed under certain stress conditions, which is common in oxidized proteins. The color changes were typically caused by tryptophan oxidation, which drove absorbance changes at 320 nm and 365 nm (Ambrogelly (2021) Antibodies 10(2):21). To investigate the photosensitivity of the relaxin-2 fusion protein analogs SEQ ID NO:522 and SEQ ID NO:523, a lightbox was created using a 25°C benchtop incubator with a transparent glass door. A photometer was installed inside the incubator, and a rectangular LED with an adjustable light intensity switch was placed outside the incubator, with the light intensity adjusted to 1000 lux, similar to the light intensity in a standard manufacturing suite. The samples were placed in transparent glass vials with 200 μL glass liners to maximize the illuminated surface area while maintaining a minimum sample volume requirement. The relaxin-2 fusion protein analogs SEQ ID NO:522 and SEQ ID NO:523 were incubated at 25°C and 1000 lux for 7 or 14 days. The samples were also subjected to thermal stress by incubation at 40°C for 7 or 14 days. After light or heat exposure, aggregate formation was evaluated using size exclusion high-performance liquid chromatography (SEC), and sample purity was assessed using CE-SDS. As shown in Table 25, under these conditions, both SEQ ID NO:522 and SEQ ID NO:523 demonstrated resistance to heat and light stress, as assessed by aggregate formation and purity.

[0759] Table 25. Light and thermal stress stability of relaxin-2 fusion protein analogs.

[0760]

[0761] A separate study was conducted to investigate the in vitro potency of SEQ ID NO:87 compared to wild-type (WT) human relaxin 2, using mammalian cell systems that transiently, stably, or endogenously express human RXFP1 or orthologs from cynomolgus monkeys and rats. Table 26 summarizes the cAMP responses induced by SEQ ID NO:87 and wild-type human relaxin-2 on HEK293 cells transiently expressing human, monkey, and rat RXFP1. (See Table 26 and...) Figure 1A-1C As shown, SEQ ID NO:87 is for humans ( Figure 1A ), rats ( Figure 1B ) and monkey ( Figure 1C The mean EC50s of RXFP1 were 10±4 nM, 10±9 nM and 30±20 nM, respectively.

[0762] Table 26. Summary of cAMP response in cells transiently expressing RXFP1.

[0763]

[0764] 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, SEQ ID NO:87 showed selectivity against human RXFP1 in ECMO cells. 50 The value is 40±20 nM, while the value for human RXFP2 is about 100 times lower (EC). 50 (≥2000nM), which proves that SEQ ID NO:87 is selective for RXFP1.

[0765] Table 27. Summary of cAMP response in cells that stably or endogenously express RXFP1.

[0766]

[0767] In some cases, transient or stable ectopic expression of proteins in cells can lead to target overexpression, thereby affecting the potency and efficacy of the tested product. To address this issue, the potency of SEQ ID NO:87 was tested in the human leukemia mononuclear cell line THP-1, which endogenously expresses RXFP1. As shown in Table 27, the EC50 of SEQ ID NO:87 was 10 ± 6 nM, consistent with the results of the in vitro RXFP1 potency assay described above.

[0768] Example 5. Pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of relaxin-2 fusion protein analogs

[0769] Pharmacokinetic (PK) values ​​were determined by measuring the plasma concentration of relaxin-2 fusion protein analogs over time after intravenous (IV) injection of 5 mg / kg of the corresponding protein analogs in rats. PK values ​​were determined for a subset of samples of the relaxin-2 fusion protein analogs 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). Figure 2A The rat PK parameters are shown in Table 28 below.

[0770] Table 28. Summary of PK parameters.

[0771] ND – Undetermined.

[0772] Figure 2B A separate experiment was shown, in which the method was used in conjunction with the method for targeting Figure 2AThe same method was used to determine PK values ​​for subsets of the relaxin-2 fusion protein analogs (SEQ ID NO:87, SEQ ID NO:496 and SEQ ID NO:497) described herein.

[0773] Figure 2C A separate experiment was shown, in which the method was used in conjunction with the method for targeting Figure 2A The same method was used to determine the PK values ​​of subsets of the relaxin-2 fusion protein analogs (SEQ ID NO:522 and SEQ ID NO:523) described herein.

[0774] Example 6. Hemodynamic and renal blood flow effects of relaxin-2 fusion protein analogs

[0775] The high isoelectric point (pI) of relaxin and related molecules presents significant pharmacokinetic (PK) and biophysical challenges, reflected in the rapid decline in serum concentrations of these molecules observed at the earliest time points of the PK curve. Unbound by any particular theory, this high clearance is attributed to the nonspecific binding of high-pI molecules to negatively charged heparin proteoglycans in the vascular system and tissues. These problems have been addressed through structure-guided engineering of relaxin-2 fusion protein analogs to reduce their pI, as illustrated in Examples 1–5.

[0776] To assess the effect of reducing pI changes, the PK and pharmacodynamic (PD) effects of the relaxin-2 fusion protein analog were measured in rats. One of the most easily quantifiable activities of relaxin is the production of an observable increase in renal artery blood flow (RABF) shortly after administration. This is a PD effect that has been shown to be observed in rats and human patients using serelaxin and can be modeled to establish the PK / PD relationship of the test compound.

[0777] Naïve male Sprague-Dawley rats (weight range: 0.308–0.399 kg) were anesthetized in an induction chamber using 5% isoflurane driven by 100% oxygen. Once unconscious, the animals were removed from the chamber and an endotracheal tube was inserted for mechanical positive pressure ventilation. A ventilator was connected to a vaporizer, which delivered approximately 1%–2% isoflurane driven by 100% oxygen during the experiment. The depth of anesthesia was assessed approximately every 15 minutes before surgery and during the experiment. The animals were maintained at approximately 37°C ± 0.5°C on a heating pad, and their body temperature was monitored throughout the protocol using a rectal temperature probe.

[0778] A Miller pressure catheter was inserted into the right carotid artery to measure systolic blood pressure (SAP), diastolic blood 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 incised, and a Doppler flow probe was placed around the artery. Renal artery blood flow (RABF) was continuously monitored throughout the experiment, and renal vascular resistance (RVR = MAP / RABF) was calculated.

[0779] After a equilibration period of approximately 10–15 minutes, baseline (BL) measurements were collected 15 minutes later. Individual animals were selected for the study based on their health status, body weight, hemodynamic parameters, and renal blood flow. Following BL measurement, rats received an intravenous bolus (IV) administration (dose 1) of either a mediator (10 mM histidine, 50 mM NaCl, 6.5% trehalose, deionized water, pH 6.0) or a relaxin-2 fusion protein analog (test compound). Immediately following the IV bolus dose, each group of rats received a maintenance dose of the corresponding test compound via IV infusion (dose 2) over a 180-minute dosing period.

[0780] Blood samples were collected before dose 1 (before the end of BL) and at 5 minutes, 1 hour, 2 hours, and 3 hours after the start of dose 2. Samples were obtained through a jugular vein cannula and placed in K2EDTA tubes. The tubes were stored on wet ice until centrifuged 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.

[0781] The analysis used averages obtained from 15-minute blocks and 180-minute dosing intervals during the BL period. Values ​​for each individual animal were aggregated to determine the mean for each variable in each group (if applicable). The mean percentage change of each variable relative to baseline was determined. The term “dose administration period” will describe the time interval (180 minutes) between the bolus and maintenance dose infusions and will be used in the remainder of this report.

[0782] In one experiment, relaxin-2 fusion protein analogs SEQ ID NO:313 and SEQ ID NO:87 were compared with the previously mentioned fusion protein SEQ ID NO:496 to assess their effects on rat renal blood flow. Table 29 below shows the potency of relaxin-2 fusion protein analogs in recombinant human and rat RXFP1 assays (as described in Example 4).

[0783] Table 29. Fusion protein analogs in human and rat RXFP1 potency assays

[0784]

[0785] like Figure 3As shown, administration of SEQ ID NO:313 and SEQ ID NO:87 at an intravenous bolus dose of 0.3 mg / kg and an intravenous infusion of 0.2 mg / kg / hr induced a greater increase in rat RABF than administration of the previously mentioned fusion protein SEQ ID NO:496 at an intravenous bolus dose of 0.3 mg / kg and an intravenous infusion of 0.5 mg / kg / hr. Therefore, despite the reduced in vitro potency of SEQ ID NO:313 and SEQ ID NO:87, these fusion proteins exhibited a greater increase in rat RABF.

[0786] Additional experiments were conducted to further evaluate the effects of the fusion protein analogs SEQ ID NO:87 and SEQ ID NO:497 on renal blood flow. Table 30 below shows the mean potency of the relaxin-2 fusion protein analogs in recombinant human and rat RXFP1 assays in these experiments.

[0787] Table 30. Fusion protein analogs in human and rat RXFP1 potency assays

[0788]

[0789] SEQ ID NO:87 was administered via a syringe pump through a cannulated femoral vein. The dose was administered intravenously as a bolus (1 ml / kg), followed by a continuous infusion of PBS at a rate of 0.5 mL / kh / hr to maintain circulating fluid volume.

[0790] like Figure 4B As shown, the measured serum concentration of SEQ ID NO:497 at 0.3 mg / kg (using human Fc levels as an indicator) was approximately 10-fold lower than that of SEQ ID NO:87 at 0.3 mg / kg. Given that SEQ ID NO:497 was found to be more than 30-fold more potent than SEQ ID NO:87 in in vitro assays of rat RXFP1 signaling (see Table 30), it can be expected that both molecules would produce at least a comparable increase in RABF when administered at the same dose. Conversely, the efficacy of SEQ ID NO:497 was comparable to that of SEQ ID NO:87 at a dose 10-fold lower (0.03 mg / kg). Figure 4A This means that the efficacy of SEQ ID NO:87 is more than 10 times higher than expected based on PK and in vitro efficacy data. The plasma concentration of 0.03 mg / kg SEQ ID NO:87 is almost the same as that of 0.3 mg / kg SEQ ID NO:497.

[0791] Figure 4APBS data showed that intravenous bolus injection of SEQ ID NO:87 had a slight effect on renal blood flow due to volume expansion, which returned to baseline after 90 minutes. Using the 90-minute time point to fit the dose-response of SEQ ID NO:87 from 0.03 mg / kg to 10 mg / kg, the estimated EC50 of SEQ ID NO:87 in rats was approximately 2200 ng / mL. Figure 4C ), corresponding to approximately 34 nM, consistent with the average EC50 of the SEQ ID NO:87 signal transduction shown in Table 30.

[0792] In addition, such as Figure 5A and 5B As shown, low-dose SEQ ID NO:87 increased and maintained RABF more effectively than SEQ ID NO:497. At 0.3 mg / kg, infusion of SEQ ID NO:87 increased RABF by approximately 25% from baseline and maintained this level throughout the 90-minute experimental period. Infusion of SEQ ID NO:497 at a dose of 0.3 mg / kg increased RABF by approximately 15% from baseline, and RABF levels returned to near baseline within 90 minutes after infusion. Figure 5A Quantitative analysis of the area under the curve (AUC) showed that infusion of SEQ ID NO:87 significantly increased RABF, approximately twice as much as infusion of SEQ ID NO:497. Figure 5B ).

[0793] Unbound by any theory, it is assumed that the enhancing effect of SEQ ID NO:87 is due to increased distribution of SEQ ID NO:87 to the target tissue, far exceeding the expected plasma level (due to reduced heparin binding), as well as reduced nonspecific cellular uptake and blood clearance, and that the bioavailability of SEQ ID NO:87 may be higher compared to the higher-charged SEQ ID NO:497 molecule.

[0794] Example 7. Therapeutic effect of relaxin-2 fusion protein analog in a rat model of pulmonary hypertension.

[0795] Pulmonary arterial hypertension (PAH) is characterized by progressive pulmonary vascular remodeling of the distal precapillary arteries, leading to a significant increase in right ventricular (RV) load and ultimately right heart failure and premature death. Using a rat model of limonene alkaloid (MCT) as an in vivo PAH model, rats developed repeatable pulmonary hypertension approximately 4 weeks after a single MCT administration, with a mean pulmonary pressure of approximately 40 mmHg. MCT is an 11-membered macrocyclic pyrrolidine alkaloid derived from the seeds of *Physalis alkekengi*. MCT alkaloids are activated in the liver to the reactive pyrrole metabolite dehydromonoterpenoid (MCTP), a highly cytochrome P-450 (CYP3A4)-dependent response. Following administration, MCT induces a syndrome characterized by pulmonary hypertension (PH), pulmonary mononuclear vasculitis, and right ventricular hypertrophy (due to damage to pulmonary endothelial cells). The therapeutic potential of the SEQ ID NO:87 relaxin-2 fusion protein analog in an MCT-induced PAH rat model was evaluated.

[0796] On day 1 of the study, naive young male Sprague Dawley rats (200–240 g) in groups 1–5 received MCT at a dose of 60 mg / kg (1 mL / kg subcutaneously, in 100% DMSO). Rats in groups 1 and 4 received anti-mouse CD20 antibody (20 mg / kg, ip) on days 8, 9, 10, and 17. Rats in groups 1 and 2 received SEQ ID NO:87 (10 mg / kg, ip) on days 7, 10, 14, 17, 21, and 24. Starting on day 8 of the study, rats in group 5 received sildenafil orally twice daily (positive control, 30 mg / kg, orally, BID), with the last dose administered on day 28 of the study. Rats in group 6 received DMSO as a control. Twelve rats were tested in each group from 1 to 4, and ten rats were tested in each group from 5 and 6.

[0797] Animals were observed daily at their cages for general health and appearance, mortality, and signs of pain or distress. Weight was recorded before the dose on Day 0 of the study and weekly throughout the study, with a final weight recorded on the day of the final procedure. On Day 28 of the study, animals were anesthetized with urethane (1.25 g / kg, ip). Blood samples were collected from the retroorbital plexus for pharmacokinetic (PK) and biomarker analysis (e.g., N-terminal (NT)-pro-hormone BNP (NT-proBNP)). Serum NT-proBNP was analyzed using a rat NT-proBNP assay kit – Meso Scale Discovery, MD, USA (catalog number K153JKD). Ro, from Kent Scientific Corporation, was used. Rats were ventilated using a Jr. small animal ventilator. A thoracotomy was performed, and mean right ventricular pressure and mean pulmonary artery pressure were measured via a 1.6 French solid catheter purchased from Transonic Inc. Data were recorded and analyzed using an SP200 pressure system and LabChart software (ADI instruments). Animals were then euthanized under deep anesthesia via exsanguination. At necropsy, the heart and lungs of each animal were collected and weighed. After weighing, the right ventricular and lung samples were fixed in formalin and stored at room temperature. The brain was also collected and weighed to conform to the rat's body weight. Immunohistochemical analysis was performed on the formalin-fixed lungs and ventricles.

[0798] A quantitative assay for the presence of SEQ ID NO:87 in mouse serum samples (from serum and plasma) was developed. This assay utilizes a quantitative sandwich enzyme-linked immunosorbent assay (ELISA), in which an affinity-purified polyclonal antibody specific for human Fc is coated onto a 96-well plate. Samples containing human Fc molecules are added to the wells, incubated, and washed, followed by the addition of the human Fc-specific ELISA polyclonal antibody. After incubation with the ELISA antibody, the wells are washed, and the enzyme substrate is added and allowed to develop color, followed by acid quenching. After quenching with an acidic plate, the plate is read at 450 nm and 570 nm within 30 minutes of color change. The values ​​for each animal are summed to determine the mean for each variable in each group (if applicable). Mean right ventricular pressure (mRVP) and mean pulmonary artery pressure (mPAP) are recorded, and right ventricular systolic pressure (RVSP) is analyzed.

[0799] In an MCT-induced PAH model, SEQ ID NO:87 was administered intravenously twice weekly at 10 mg / kg for 3 weeks, with therapeutic dosing starting one week after MCT injection. To mitigate the effect of reduced anti-drug antibody (ADA) production on the efficacy of the test product, studies were conducted in rats with and without B-cell depletion by injection of anti-mouse CD20 antibodies. Cardiac hemodynamics, including right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP), were measured using an open-chest approach. The Fulton index, the ratio of right ventricular weight to left ventricular and diaphragmatic weight, was measured at week 4. Blood samples were collected for serum NT pro-BNP, PK, and ADA analysis.

[0800] Compared with the juvenile group (without MCT), MCT-treated rats significantly increased RVSP ( ) over a 4-week period. Figure 6A and 6B mPAP Figure 7A and 7B ), Fulton Index ( Figure 8A and 8B ) and NT-pro-BNP ( Figure 9A and9B This treatment also reduced survival rates. As shown, rats treated with SEQ ID NO:87 exhibited RVSP ( ) in rats with B-cell depletion. Figure 6B mPAP Figure 7B ) and Fulton index value ( Figure 8B Significant improvement was observed in the survival rate of rats treated with SEQ ID NO:87. Furthermore, the survival rate was significantly improved, with the treated rats being the only group achieving a 100% survival rate.

[0801] Furthermore, histopathological analysis revealed that SEQ ID NO:87 significantly improved lung inflammation and reduced pulmonary artery muscularization (respectively). Figure 10A and 10B To assess lung inflammation, histopathological analysis was performed on tissues stained with hematoxylin and eosin. The total lung histopathological score was calculated as a total score of the analyzed parameters, including pulmonary artery hypertrophy, vasculitis / necrosis, alveolar histiocytosis, perivascular and interstitial inflammation, hemorrhage, and fibrin deposition. Compared with rats receiving the catalytic agent, rats showed significantly improved total lung histopathological scores and the degree of vasculitis / necrosis, hemorrhage, and fibrin deposition in lung tissue. To assess pulmonary artery muscularization, histopathological analysis was performed on tissues stained with anti-α-smooth muscle actin (SMA) antibody to quantify muscularization, and the internal elastic layer of arterioles was stained with Verhoeff stain. In animals exposed to MCT, thickening of the arterial walls at all levels of the pulmonary artery tree was observed, primarily due to smooth muscle hypertrophy. This was associated with an increased rate of medial hypertrophy in arteries of all five vessel sizes. Medial thickening relative to outer diameter was most pronounced in the smallest arteries and arterioles, while medial hypertrophy was less pronounced in medium and larger arteries. Rats treated with SEQ ID NO:87 had a lower rate of medial hypertrophy of small arteries, small and intermediate arteries compared to untreated animals. For small-diameter arteries, the rate in rats treated with SEQ ID NO:87 was significantly lower than in untreated animals.

[0802] Compared with the carboxyl and sildenafil treatment groups, no death was observed in B-cell depleted rats treated with SEQ ID NO:87. Figure 11 ).

[0803] Example 8. Antifibrotic effect of relaxin-2 fusion protein analog in a mouse model of renal fibrosis

[0804] 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 model of unilateral ureteral obstruction (UUO) renal fibrosis. The UUO model induces renal fibrosis, where the primary characteristic is tubular damage due to urinary obstruction. Furthermore, experimental rodent UUO is considered to mimic human chronic obstructive nephropathy in an accelerated manner. Renal fibrosis is a common pathway in most progressive kidney diseases. Since removal of the obstruction is often insufficient to reverse fibrosis, animal models may benefit from concomitant therapy. UUO is a widely used model for studying obstructive nephropathy.

[0805] Preliminary studies evaluated the effects of SEQ ID NO:496 and SEQ ID NO:313. Sixty-five male C57BL / 6 mice (aged 8–10 weeks at the start of the study) were used. On day 0, unexplained ureteral occlusion (UUO) was induced by ligation of the left renal ureter, with the contralateral kidney serving as a control. The UUO surgery was performed under deep anesthesia according to standard procedures. Briefly, after dehairing the abdomen, the kidneys and ureters were exposed through a midline abdominal incision. The left ureter was completely obstructed by two ligations. The first suture was placed 1 mm below the kidney, and the second suture was placed 1 mm below the renal pelvis, using silk or prismin sutures. The wound was sutured with 2–3 staples. The animals were returned to their cages and monitored until they began to move. Buprenorphine was administered to the animals on the day of surgery and for 72 hours post-surgery. A control group (n=5) received a sham surgery. 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 2 days before surgery (day-2) and on days 2 and 5 post-surgery. The positive control group (n=10) received enalapril, an ACE inhibitor used to treat hypertension, diabetic nephropathy, and heart failure, administered 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 PBS. The study ended on day 7 post-surgery, and fibrosis symptoms and vital parameters were assessed.

[0806] No adverse clinical symptoms were observed in any of the animals following the UUO surgery. The animals tolerated treatment with SEQ ID NO:496, SEQ ID NO:313, and enalapril well, and the recorded survival rate was 100%. On day 7 post-surgery, the left kidney was harvested, weighed, fixed, and subjected to histological examination.

[0807] Compared to the sham surgery group, all groups that underwent surgically induced UUO experienced weight loss. By day 4 post-surgery, all UUO groups began to show a weight recovery trend. Furthermore, compared to the sham surgery group, all UUO groups showed a significant increase in kidney weight, and there was no statistically significant change in kidney weight in the treatment or control UUO groups.

[0808] Histological analysis of kidney sections fixed on day 7 post-surgery showed that, compared to the sham-operated group, collagen deposition in the renal parenchyma was significantly increased in all UUO groups. Figure 12 Compared to the control group treated with catalysts, animals treated with 20 mg / kg of SEQ ID NO:496 and SEQ ID NO:313 showed a significant reduction in collagen deposition.

[0809] 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 mice treated with the mordant. Figure 13 Tissue TNFα levels were evaluated by electrochemiluminescence, and mice treated with SEQ ID NO:87 also showed a significant decrease in TNFα levels compared to mice treated with the methylphenidate. Figure 14 A reduction in IL-1β and IL-6 in fibrotic kidneys was also observed in mice treated with SEQ ID NO:87, compared to mice treated with the catalytic agent.

[0810] Example 9. Effects of relaxin-2 fusion protein analogs on isoproterenol-induced cardiac hypertrophy and fibrosis

[0811] This case describes an evaluation of the effects of the relaxin-2 fusion protein analog SEQ ID NO:87 on cardiac hypertrophy and fibrosis following an isoproterenol challenge. Isoproterenol is a drug that increases heart rate and myocardial contractility and can lead to increased cardiac hypertrophy and fibrosis.

[0812] All animals were treated and cared for in accordance with the Laboratory Animal Care and Use Guidelines (NIA Guidelines, 2011 Revised Edition), and the protocols were approved by the Institutional Animal Care and Use Committee. Male C57BL / 6J mice were obtained from mice aged 10–11 weeks, weighing between 25 and 30 grams. Animals were housed in animal enclosures using a standard 12-hour light / dark cycle. After one week of balanced feeding, mice underwent microosmotic pump implantation. Briefly, mice were anesthetized using a vaporizer with 1% to 3% isoflurane. An osmotic pump at a flow rate of 0.25 μl / h was surgically implanted subcutaneously into the subscapular region of the mouse. Each pump delivered a constant dose (0.25 μl / h) of either infusion drug (isoproterenol in PBS containing 0.002% ascorbic acid, 15 mg / kg daily) or mediator (PBS containing 0.002% ascorbic acid) for two weeks. Postoperative analgesia was achieved by administering a single dose of meloxicam SR (2.5 mg / kg) during micropump implantation and a second dose of meloxicam SR (2.5 mg / kg) one day post-operation. Mice were treated with a mediator or 10 mg / kg of SEQ ID NO:87 one day prior to micropump implantation. Mice were then administered the relaxin-2 fusion protein analog SEQ ID NO:87 every two weeks for the duration of the study (14 days in total). To reduce drug resistance, mice were intraperitoneally injected with 20 mg / kg of anti-mouse CD20 antibody one day after micropump implantation to deplete all B cells.

[0813] After 14 days of incubation, the animals were euthanized via CO2 inhalation followed by cervical dislocation. Body weight was measured. The entire heart was removed, washed in PBS, dried with paper towels, and weighed. Fresh heart tissue was immediately rapidly frozen in liquid nitrogen and stored for further collagen content analysis. The tibia was obtained through blunt limb dissection, and its length was measured using digital calipers. Heart weight (HW) was normalized to body weight (BW) and tibia length (TL). Data (HW / TL and HW / BW) were analyzed using standard software. Figure 15 As shown, compared with mice treated with the catalytic agent, isoproterenol administration resulted in a significant increase in cardiac hypertrophy, as measured by normalized heart weight (HW / BW). Co-administration of SEQ ID NO:87 with isoproterenol significantly attenuated isoproterenol-induced cardiac hypertrophy.

[0814] Collagen content in each ventricle was measured using a hydroxyproline assay kit. Frozen whole ventricle tissue was weighed, chopped, and transferred to screw-cap tubes. The tissue was hydrolyzed in 6M HCl at 100 mg / ml and incubated at 95°C for 20 hours in a calibrated oven or hot block. The hydrolyzed sample was then diluted with 4M HCl and analyzed. For 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 with shaking for 20 minutes. Then, 75 μl of assay reagent was added to each well, the plate was thoroughly mixed, and incubated at 60°C for 60 minutes in an oven or incubator. The plate was cooled to room temperature, read at 570 nm, and the hydroxyproline concentration of each sample was determined using a standard curve. Data were analyzed using standard software. Figure 16 As shown, compared to mice treated with cautery, isoproterenol administration resulted in a significant increase in fibrosis, as measured by collagen content. Co-administration of SEQ ID NO:87 with isoproterenol significantly attenuated isoproterenol-induced fibrosis.

[0815] Example 10. Evaluation of the safety, tolerability, pharmacokinetics, and pharmacodynamics of relaxin-2 fusion protein analogs.

[0816] This example describes a double-blind, randomized, placebo-controlled, single-escalation dose study designed to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity of SEQ ID NO:87 in healthy subjects.

[0817] Research Design

[0818] Approximately 48 participants were enrolled in this study. Participants were administered a single, escalating dose of SEQ ID NO:87 or a matched placebo (i.e., the same buffer without SEQ ID NO:87) via intravenous infusion (IV) or subcutaneous injection (SC) at the following doses. In each group, 6 participants received SEQ ID NO:87 and 2 participants received placebo. Infusion duration was 30 to 60 minutes. Group A received 0.3 mg / kg IV of SEQ ID NO:87 or a matched placebo. Group B received 1 mg / kg IV of SEQ ID NO:87 or a matched placebo. Group C received 300 mg SC of SEQ ID NO:87 or a matched placebo. In some cases, Group C was administered concurrently with Group B. Group D received 3 mg / kg IV of SEQ ID NO:87 or a matched placebo. Group E received 600 mg SC of SEQ ID NO:87 or a matched placebo. In some cases, Group E was administered concurrently with Group D. Group F received 10 mg / kg IV of SEQ ID NO:87 or a matched placebo.

[0819] A master randomization schedule and a password-breaking envelope were created and sent to the site's non-blind pharmacy before administration. The active and placebo products were injected via a line that was covered to conceal the active and placebo. A printed randomization schedule was generated using a permutation block fixation method. Those receiving treatment, administering treatment, and analyzing results and data were all blinded.

[0820] Main results

[0821] The primary outcome of this study was to evaluate the safety and tolerability of SEQ ID NO:87 following a single escalation dose. The primary outcome was assessed by monitoring the following: 1) the incidence of adverse events (AEs) and serious adverse events (SAEs); 2) changes in clinical laboratory safety parameters, including blood test results from hematological, serological, and coagulation studies; 3) changes in vital sign measurements; and 4) changes in electrocardiogram (ECG) results. Adverse events were assessed through clinical examination, review of patient data, and self-reporting. During the study, clinic staff collected adverse events via verbal interviews, either in person during hospital or clinic visits or by telephone during remote contact. Vital sign measurements included resting heart rate, systolic blood pressure, and diastolic blood pressure (BP) measured using standard manual or electronic clinical procedures. These procedures were performed using standard nursing practices or, in the case of ECG, according to the manufacturer's instructions. These individual data were evaluated by physicians to determine safety. Additionally, all presented data were also included in the safety determination.

[0822] Adverse events and serious adverse events were assessed at screening and on days -1, 1, 2, 3, 6, 8, 15, 29, 43, and 57 after dose administration. Laboratory safety parameters were assessed at screening and on days -1, 1, 2, 8, 15, 29, 43, and 57 after dose administration. Vital signs were assessed at screening and on days -1, 1, 2, 3, 6, 8, 15, 29, 43, and 57 after dose administration. ECG was performed at screening and on days -1, 1, and 2 after dose administration.

[0823] Secondary results

[0824] A secondary outcome of this study was the characterization of the pharmacokinetic (PK) profile of SEQ ID NO:87 following a single escalation dose in healthy participants. Evaluation criteria: PK parameter: C max T max AUC - final (AUC from 0 to the final measurable concentration), AUC - inf (AUC from 0 to infinity), t 1 / 2 CL (clearance) and Vz (terminal distribution volume). Blood samples used for PK parameter assessment were taken at: before dose, at the end of infusion, 6 and 12 hours after dose, 24 hours after dose, 48 hours after dose, 120 hours after dose, 168 hours after dose, and on days 15, 29, 43, and 57 after dose.

[0825] Another secondary outcome of this study was the evaluation of the pharmacodynamic (PD) effect of a single escalation dose of SEQ ID NO:87 in healthy participants. Changes from baseline to day 2 were assessed using the following PD parameters: renal plasma flow (RPF), measured as a function of plasma para-aminohippuric acid (PAH) over time; renal blood flow (RBF); and fractional filtration (FF), calculated as a function of glomerular filtration rate (GFR) divided by RPF. For groups IV A, B, and D, blood samples were collected on days 2,...

Claims

1. A fusion protein comprising, from N- 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: 502, 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: 503 or 504; 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: 503 or 504, 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: 502; and (b) the fusion protein has a pi of 6.0 to 8.

2.

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.

2. The fusion protein of claim 1, wherein the first peptide comprises the amino acid sequence X 11 LCGRELVRAQIAIC (SEQ ID NO: 505), wherein X 11 is K, Q, D, E, L, I, or Y.

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.

4. The fusion protein of claim 1, wherein the first peptide comprises the amino acid sequence X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO: 506), wherein: X 12 is K, Q, D, E, L, I, or Y; X 13 X is any amino acid except M, W or C; X 14 is K, Q, D, E, L, I, or Y; X 15 Q is D, E, L, I, Y or R; and X 16 R or Q.

5. The fusion protein of claim 4, wherein the first peptide comprises the amino acid sequence X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO: 506), wherein: 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 Q, D, E, L, I, Y, or R; and X 16 R or Q.

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

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.

8. The fusion protein of claim 1, wherein the second peptide comprises the amino acid sequence X 11 LCGRELVRAQIAIC (SEQ ID NO: 505), wherein X 11 is K, Q, D, E, L, I, or Y.

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.

10. The fusion protein of any one of claims 1, 8, and 9, wherein the second peptide comprises the amino acid sequence X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO: 506), wherein: 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 Q is D, E, L, I, Y or R; and X 16 R or Q.

11. The fusion protein of claim 1, wherein the second peptide comprises the amino acid sequence X 12 CCX 13 VGCTX 14 X 15 SLAX 16 FC (SEQ ID NO: 506), wherein: 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 Q is D, E, L, I, Y or R; and X 16 R or Q.

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

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

15. The fusion protein of any one of the preceding claims, wherein: the linker peptide comprises the amino acid sequence ASDAAGAX8AX9AGA (SEQ ID NO: 17), wherein: X8is D, E, N, or Q; and X9is D, E, N, or Q; or 16. The fusion protein of claim 15, wherein X8is D, E, N, or Q, and X9is D, E, or Q; or X8is D, E, or Q, and X9is D, E, N, or Q. The adaptor peptide contains the amino acid sequence GGEGSGGEGX. 10 GGG (SEQ ID NO: 25), where: X 10 E or S.

17. 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. A fusion protein comprising, from N- 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: 502, 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: 503 or 504; 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: 503 or 504, 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: 502; and (b) the fusion protein has a pi of 6.0 to 8.

2. (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 the amino acid at at least one of positions 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, wherein 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, wherein the amino acid at position 22 of the second peptide is not R; 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: 1, wherein the amino acid at at least one of 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.

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

20. The fusion protein of claim 18 or 19, wherein: the linker peptide comprises the amino acid sequence ASDAAGAX8AX9AGA (SEQ ID NO: 17), wherein: X8is D, E, N, or Q; and X9is D, E, N, or Q; or The linker peptide comprises the amino acid sequence GGEGSGGEGX 10 GGG (SEQ ID NO: 25), wherein: X 10 E or S.

21. The fusion protein of claim 20, wherein X8is D, E, N, or Q and X9is D, E, or Q; or X8is D, E, or Q and X9is D, E, N, or Q.

22. A fusion protein comprising, from N- 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), wherein: X8is D, E, N, or Q; and X9is D, E, N, or Q; or The linker peptide comprises the amino acid sequence GGEGSGGEGX 10 GGG (SEQ ID NO: 25), wherein: X 10 E or S.

23. The fusion protein of claim 22, wherein X8is D, E, N, or Q and X9is D, E, or Q; or X8is D, E, or Q and X9is 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 fusion protein of any one of claims 1 to 3 and 10 to 24, wherein the first peptide comprises the amino acid sequence DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO: 7), wherein: X1is not M, H, or C; X2is K, Q, D, E, L, I, or Y; and X3is K or Q.

26. The fusion protein of any one of claims 1-3 and 10-25, wherein the first peptide comprises the amino acid sequence DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO: 7), wherein: X1is W, Y, F, L, I, V, or A; X2is K, Q, D, E, L, I, or Y; and X3is K or Q.

27. The fusion protein of claim 25 or 26, wherein X1is Y.

28. The fusion protein of any one of claims 25-27, 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 1-3 and 10-28, wherein the first peptide consists of 27, 28, or 29 amino acids.

30. The fusion protein of any one of claims 1, 4-9, and 14-24, wherein the first peptide comprises the amino acid sequence QLYSALANX4CCX5VGCTX6X7SLAQFC (SEQ ID NO: 16), wherein: X4is K, Q, D, E, L, I, or Y; X5is any amino acid except M, W, or C; X6is K, Q, D, E, L, I, or Y; and X7is Q, D, E, L, I, Y, or R.

31. The fusion protein of any one of claims 1, 4-9, 14-24, and 30, wherein the first peptide comprises the amino acid sequence QLYSALANX4CCX5VGCTX6X7SLAQFC (SEQ ID NO: 16), wherein: X4is K, Q, D, E, L, I, or Y; X5is H, K, Q, Y, L, N, I, S, T, or F; X6is K, Q, D, E, L, I, or Y; and X7is Q, D, E, L, I, Y, or R.

32. The fusion protein of claim 30 or 31, wherein X5is Q.

33. The fusion protein of any one of claims 30-32, 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, 4-9, 14-24, and 30-33, wherein the first peptide consists of 24 or 25 amino acids.

35. The fusion protein of any one of claims 1, 4-9, and 14-24, wherein the second peptide comprises the amino acid sequence DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO: 7), wherein: X1is not M, H, or C; X2is K, Q, D, E, L, I, or Y; and X3is K or Q.

36. The fusion protein of any one of claims 1, 4-9, 14-24, and 35, wherein the second peptide comprises the amino acid sequence DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO: 7), wherein: X1is W, Y, F, L, I, V, or A; X2is K, Q, D, E, L, I, or Y; and X3is K or Q.

37. The fusion protein of claim 35 or 36, wherein X1is Y.

38. The fusion protein of any one of claims 35-37, 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. 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 fusion protein of any one of claims 1-3 and 10-24, wherein the second peptide comprises the amino acid sequence QLYSALANX4CCX5VGCTX6X7SLAQFC (SEQ ID NO: 16), wherein: X4is K, Q, D, E, L, I, or Y; X5is any amino acid except M, W, or C; X6is K, Q, D, E, L, I, or Y; and X7is Q, D, E, L, I, Y, or R.

41. The fusion protein of any one of claims 1-3, 10-24, and 40, wherein the second peptide comprises the amino acid sequence QLYSALANX4CCX5VGCTX6X7SLAQFC (SEQ ID NO: 16), wherein: X4is K, Q, D, E, L, I, or Y; X5is H, K, Q, Y, L, N, I, S, T, or F; X6is K, Q, D, E, L, I, or Y; and X7is Q, D, E, L, I, Y, or R.

42. The fusion protein of claim 40 or 41, wherein X5is Q.

43. The fusion protein of any one of claims 40-42, 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 1-3, 10-24, and 40-43, wherein the second peptide consists of 24 or 25 amino acids.

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

46. 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: 8 and the second peptide comprises the amino acid sequence of SEQ ID NO: 1 ; 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; 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; 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; 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; 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: 8 and the second peptide comprises the amino acid sequence of SEQ ID NO: 7; 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: 507; 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: 508; 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: 509; 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: 510; 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: 511; 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: 512; 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: 513; 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: 514; 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: 515; 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: 516; 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: 517; 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: 518; 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: 519; 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: 520; 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: 521; 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: 522; 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: 523; 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: 524; 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: 525; 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: 526; 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: 527; 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: 528; 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: 529; 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: 530; 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: 531; 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: 532; 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: 533; 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: 534; 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: 535; 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: 536; 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: 537; 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: 538; 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: 539; 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: 540; 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: 541; 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: 542; 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: 543; 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: 544; 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: 545; 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: 546; 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: 547; the first peptide comprises 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; 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; 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; 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 ; 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; 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; 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; 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 ; 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; 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; 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; 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; 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; 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 ; 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; 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; 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; 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 ; 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; 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; 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; 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; 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; 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 ; 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; 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; 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; 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; 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; 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 ; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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 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. The fusion protein of any one of claims 1-3, 10-29, and 40-45, wherein the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-75 and 508-515.

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

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

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

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

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

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

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

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

56. 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 human IgG1 Fc.

57. The fusion protein of claim 56, wherein the IgG Fc comprises the amino acid sequence of human IgG1 Fc.

58. The fusion protein of claim 56 or 57, wherein the IgG Fc comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 76-83.

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

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

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

64. The fusion protein of any one of claims 1-3, 10-24, and 40-45, 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 1-64 or the polypeptide of claim 65.

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

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

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

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

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

73. A host cell comprising the polynucleotide of any one of claims 66-69 or the expression vector of any one of claims 70-72.

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

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

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

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. 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. A population of cells comprising two or more host cells of any one of claims 73-78.

80. A method of producing the fusion protein of any one of claims 1-64 or the polypeptide of claim 65, the method comprising culturing the host cell of any one of claims 73-78 under conditions capable of producing the fusion protein.

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

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. The pharmaceutical composition of claim 81 or 82, wherein the fusion protein 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% when administered to a human.

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

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

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

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

88. The method of any one of claims 85 to 87, wherein the primary cell is selected from the group consisting of an endothelial cell, a vascular smooth muscle cell, other vascular cell, a cardiomyocyte, other cardiac cell, and a fibroblast.

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

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

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

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 mid-range ejection fraction (HFmrEF), heart failure with reduced ejection fraction (HFrEF), heart valve disease, arthropathy, periarthritis (also known as adhesive capsulitis), kidney disease, chronic kidney disease, and hypertensive kidney disease.

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

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

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

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

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

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

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

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 mid-range ejection fraction (HFmrEF), heart failure with reduced ejection fraction (HFrEF), a kidney disease, chronic kidney disease, and hypertensive kidney disease.

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

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

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

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

105. The method of any one of claims 97 to 104, wherein the method reduces arterial pressure, increases renal arterial blood flow, increases diastolic cardiac filling, eliminates already formed fibrosis, and / or arrests development of new fibrosis in the subject.

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

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

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

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

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

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

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

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

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

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

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

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

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

119. The method of any one of claims 97-118, wherein the fusion protein is administered to the subject once every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 1 month.

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

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

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

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

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

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

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

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

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