Relaxin-2 fusion protein analog and method of using the same

Abstract

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

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 503,101 filed on 18 May 2023, No. 63 / 585,849 filed on 27 September 2023, No. 63 / 586,868 filed on 29 September 2023, No. 63 / 611,732 filed on 18 December 2023, and No. 63 / 617,398 filed on 3 January 2024, the entirety of which disclosures are incorporated herein by reference.

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

[0003] Relaxin-2 exhibits strong anti-fibrotic activity. In damaged tissue, fibroblast activation and proliferation lead to increased collagen production and interstitial fibrosis. Cardiac fibrosis is exacerbated by biomechanical overload and affects ventricular dysfunction, remodeling, and arrhythmogenicity. However, due to the limited in vivo half-life of relaxin, compound administration must be carried out as a continuous infusion of at least 48 hours. Furthermore, the synthesis of relaxin-2 is difficult. Due to the low solubility of the B chain and the complex and specific requirements for introducing cysteine ​​crosslinks between the A and B chains, the yield of the active peptide obtained by these methods is extremely low. [Overview of the project] [Problems that the invention aims to solve]

[0004] There is a need for manipulated relaxin-2 analogs with longer half-lives and easier manufacturing processes. [Means for solving the problem]

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

[0006] Accordingly, in one embodiment, the present disclosure provides a fusion protein comprising a first peptide, a linker peptide, and a second peptide from the N-terminus to the C-terminus, wherein (a) the first peptide comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications 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 to the amino acid sequence of SEQ ID NO: 503 or 504, or (b) the fusion protein has a pI of 6.0 to 8.2.

[0007] In some embodiments, the fusion protein has a pI of about 6.0 to about 9.4. In some embodiments, the fusion protein has a pI of about 6.0 to about 8.2. In some embodiments, the fusion protein has a pI of 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 less than 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.

[0008] In some embodiments, the first peptide is an amino acid sequence X 11 Includes LCGRELVRAQIAIC (Sequence ID 505), X 11 is K, Q, D, E, L, I, or Y. In some embodiments, the first peptide consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids.

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

[0010] In some embodiments, the second peptide has the amino acid sequence X 11 contains LCGRELVRAQIAIC (SEQ ID NO: 505), and X 11 is K, Q, D, E, L, I, or Y. In some embodiments, the second peptide consists of 15, 16, 17, 1, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids.

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

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

[0013] In another embodiment, the Disclosure provides a fusion protein comprising a first peptide, a linker peptide, and a second peptide from the N-terminus to the C-terminus, wherein (a) the first peptide comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications to the amino acid sequence of SEQ ID NO: 1, and at least one amino acid at position 4 or 25 of the first peptide is not M, and the second peptide comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications to the amino acid sequence of SEQ ID NO: 8, and the second peptide (b) The 22nd amino acid of Tide is not R, or the first peptide contains an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications to the amino acid sequence of SEQ ID NO: 8, the 22nd amino acid of the second peptide is not R, the second peptide contains an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications to the amino acid sequence of SEQ ID NO: 1, at least one amino acid at position 4 or 25 of the first peptide is not M, and (b) the fusion protein has a pI of 6.0 to 8.2.

[0014] In some embodiments, the fusion protein has a pI of about 6.0 to about 9.4. In some embodiments, the fusion protein has a pI of about 6.0 to about 8.2. In some embodiments, the fusion protein has a pI of 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 less than 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.

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

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

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

[0018] In some embodiments, the first peptide comprises the amino acid sequence DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO: 7), where 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), where 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 NOs: 1, 2, 3, 4, 5, and 6. In some embodiments, the first peptide consists of 27, 28, or 29 amino acids.

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

[0020] In some embodiments, the second peptide comprises the amino acid sequence DSX1QEEVIX2LCGRELVRAQIAICGX3ST (SEQ ID NO: 7), where 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), where 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 NOs: 1, 2, 3, 4, 5, and 6. In some embodiments, the second peptide consists of 27, 28, or 29 amino acids.

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

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

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

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

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

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

[0027] In some embodiments, IgG Fc contains 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.

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

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

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

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

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

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

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

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

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

[0037] In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is an E. coli cell or a Bacillus cell. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is selected from the group consisting of 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.

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

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

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

[0041] In some embodiments, the fusion protein has a cyclic 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, when administered (e.g., to humans), the fusion protein has a cyclic 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, when administered (e.g., to humans), 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%. In some embodiments, the pharmaceutical composition is administered intravenously or subcutaneously.

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

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

[0044] In some embodiments, the method increases cAMP levels in primary cells, induces vasodilation, induces the expression of angiogenic factors, induces the expression of MMPs, and induces collagen degradation.

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

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

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

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

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

[0050] In some embodiments, the method increases renal plasma flow in the subject. In some embodiments, the increase in renal plasma flow in the subject persists one week, two weeks, three weeks, four weeks, five weeks, or one month after a single dose of the fusion protein. In some embodiments, the increase in renal plasma flow in the subject is maintained 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% one week, two weeks, three weeks, four weeks, five weeks, or one month after a single dose of the fusion protein.

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

[0052] In some embodiments, subjects are administered the fusion protein intravenously. In some embodiments, subjects are administered approximately 0.1 mg / kg to approximately 20 mg / kg of fusion protein. In some embodiments, subjects are administered approximately 0.3 mg / kg of fusion protein. In some embodiments, subjects are administered approximately 1 mg / kg of fusion protein. In some embodiments, subjects are administered approximately 3 mg / kg of fusion protein. In some embodiments, subjects are administered approximately 10 mg / kg of fusion protein.

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

[0054] In some embodiments, subjects are administered the fusion protein subcutaneously. In some embodiments, subjects are administered approximately 100 mg to approximately 1500 mg of the fusion protein. In some embodiments, subjects are administered approximately 150 mg of the fusion protein. In some embodiments, subjects are administered at least 150 mg of the fusion protein. In some embodiments, subjects are administered approximately 300 mg of the fusion protein. In some embodiments, subjects are administered approximately 600 mg of the fusion protein.

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

[0056] [Figure 1A]A graph showing the cAMP response induced by SEQ ID NO: 87 and wild-type (WT) human relaxin-2 in HEK293 cells transiently expressing human RXFP1. [Figure 1B] A graph showing the cAMP response induced by SEQ ID NO: 87 and wild-type (WT) human relaxin-2 in HEK293 cells transiently expressing rat RXFP1. [Figure 1C] A graph showing the cAMP response induced by SEQ ID NO: 87 and wild-type (WT) human relaxin-2 in HEK293 cells transiently expressing monkey RXFP1. [Figure 2A] Graph showing pharmacokinetic (PK) values ​​obtained by measuring the concentrations of various relaxin-2 fusion protein analogs in rat serum after intravenous (IV) injection of 5 mg / kg of each protein analog over time (using human Fc levels as a proxy). [Figure 2B] Graph showing pharmacokinetic (PK) values ​​obtained by measuring the concentrations of various relaxin-2 fusion protein analogs in rat serum after intravenous (IV) injection of 5 mg / kg of each protein analog over time (using human Fc levels as a proxy). [Figure 2C] Graph showing pharmacokinetic (PK) values ​​obtained by measuring the concentrations of various relaxin-2 fusion protein analogs in rat serum after intravenous (IV) injection of 5 mg / kg of each protein analog over time (using human Fc levels as a proxy). [Figure 3] As shown, the graph illustrates the time course of renal artery blood flow (RABF) compared to baseline in rats administered various relaxin-2 fusion protein analogs. [Figure 4A] As shown, this is a graph illustrating the change in RABF over time in response to the dose of SEQ ID NO: 87 or SEQ ID NO: 497. [Figure 4B]As shown, this is a graph illustrating the change over time in serum levels of the fusion protein (using human Fc levels as a proxy) in response to the dose of SEQ ID NO: 87 or SEQ ID NO: 497. [Figure 4C] This graph shows serum PK as a function of the increase in RABF (subtracting baseline). [Figure 5A] A graph showing that low doses of SEQ ID NO: 87 significantly increased and maintained RABF in treated rats compared to SEQ ID NO: 497. The graph shows the increase in RABF over time in rats treated with SEQ ID NO: 87 or SEQ ID NO: 497. [Figure 5B] A graph showing that low doses of SEQ ID NO: 87 significantly increased and maintained RABF in treated rats compared to SEQ ID NO: 497. The graph also shows that rats treated with SEQ ID NO: 87 showed a significant increase in RABF compared to those treated with SEQ ID NO: 497, as determined by area under the curve analysis. [Figure 6A] This graph shows the effect of SEQ ID NO: 87 on right ventricular systolic pressure (RVSP) after 3 weeks of intravenous administration of 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats with B cell depletion, using anti-CD20 antibodies (without or with CD20). Sildenafil was used as a positive control in non-B cell depleted animals. [Figure 6B] This graph shows the effect of SEQ ID NO: 87 on right ventricular systolic pressure (RVSP) after 3 weeks of intravenous administration of 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCTs) without B-cell depletion, using anti-CD20 antibodies (without or with CD20). Sildenafil was used as a positive control in non-B-cell depleted animals. [Figure 7A] This graph shows the effect of SEQ ID NO: 87 on mean pulmonary artery pressure (mPAP) after 3 weeks of intravenous administration of 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats with B cell depletion, using anti-CD20 antibodies (without CD20 or with CD20). Sildenafil was used as a positive control in non-B cell depleted animals. [Figure 7B]This graph shows the effect of SEQ ID NO: 87 on mean pulmonary artery pressure (mPAP) after 3 weeks of intravenous administration of 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCTs) without B-cell depletion, using anti-CD20 antibodies (without or with CD20). Sildenafil was used as a positive control in non-B-cell depleted animals. [Figure 8A] This graph shows the effect of SEQ ID NO: 87 on the Fulton index after 3 weeks of intravenous treatment at 10 mg / kg using anti-CD20 antibodies (without or with CD20) in MCT-induced rats with B-cell depletion. Sildenafil was used as a positive control in non-B-cell depleted animals. [Figure 8B] This graph shows the effect of SEQ ID NO: 87 on the Fulton index after 3 weeks of intravenous treatment at 10 mg / kg using anti-CD20 antibodies (without CD20 or with CD20) in MCT-induced rats (MCTs) without B-cell depletion. Sildenafil was used as a positive control in non-B-cell depleted animals. [Figure 9A] This graph shows the effect of SEQ ID NO: 87 on serum NT-pro-BNP levels after 3 weeks of intravenous treatment with 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCTs) with B-cell depletion, using anti-CD20 antibodies (without or with CD20). Sildenafil was used as a positive control in non-B-cell depleted animals. [Figure 9B] This graph shows the effect of SEQ ID NO: 87 on serum NT-pro-BNP levels after 3 weeks of intravenous treatment with 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCTs) without B-cell depletion, using anti-CD20 antibodies (without or with CD20). Sildenafil was used as a positive control in non-B-cell depleted animals. [Figure 10A]Graph showing the results of histopathological analysis of the effect of SEQ ID NO: 87 on pneumonia after 3 weeks of intravenous administration of 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCTs) with B-cell depletion using anti-CD20 antibody (+CD20). *: p<0.05, **: p<0.01, ****: p<0.0001. Nonparametric one-way ANOVA with post-hoc Dunn's multiple comparison test was used. [Figure 10B] Graph showing the results of histopathological analysis of the effect of SEQ ID NO: 87 on pulmonary artery muscle formation after 3 weeks of intravenous administration of 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCTs) with B-cell depletion using anti-CD20 antibody (+CD20). *: p<0.05, **: p<0.01, ****: p<0.0001. Nonparametric one-way ANOVA with post-hoc Dunn's multiple comparison test was used. [Figure 11] A graph showing the effect of SEQ ID NO: 87 on mortality after 3 weeks of intravenous administration of 10 mg / kg of SEQ ID NO: 87 in MCT-induced rats (MCTs) with or without B-cell depletion, using anti-CD20 antibodies (without CD20 or with CD20). Sildenafil was used as a positive control in non-B-cell depleted animals. [Figure 12] Graphs showing the effects of SEQ ID NOs: 496 and 313 on collagen deposition in the renal parenchyma of a mouse unilateral ureteral obstruction (UUO) model according to aspects of this disclosure. Mice underwent UUO surgery and were treated with vehicle (PBS, n=10), 20 mg / kg of SEQ ID NOs: 496 (n=10), 10 mg / kg of SEQ ID NOs: 313 (n=10), or 20 mg / kg of SEQ ID NOs: 313 (n=10). Control mice that underwent sham surgery and were treated with vehicle (PBS, n=5) are also shown. After treatment, the obstructed kidneys were harvested and fixed for histology. Collagen was detected via immunolabeling. Shown are quantifications of collagen levels as a percentage of the total immunolabeled area. *: p<0.05, ****: p<0.0001. [Figure 13]A graph showing the effect of Sequence ID No. 87 on collagen deposition in the renal cortex in a mouse UUO model according to aspects of this disclosure. Mice underwent UUO surgery and were treated with either vehicle (PBS, n=8) or 10 mg / kg of Sequence ID No. 87 (n=8). Control mice that underwent sham surgery and were treated with vehicle (PBS, n=8) are also shown. After treatment, the obstructed kidneys were harvested and fixed for histology. Collagen was detected via immunolabeling. Shown is the quantification of collagen levels as a percentage of the total immunolabeled area. ****: p<0.0001, *: p=0.02. [Figure 14] A graph showing the effect of Sequence ID No. 87 on TNFα levels in the renal cortex of a mouse UUO model according to an aspect of this disclosure. Mice were treated as described in Figure 13, and TNFα levels in protein lysates were quantified via electrochemiluminescence assay. ****: p<0.0001, ***: p<0.001. [Figure 15] A graph showing the effect of Sequence ID No. 87 on isoproterenol-induced cardiac hypertrophy according to an aspect of this disclosure. Mice were treated with vehicle (n=10), isoproterenol (n=10), or isoproterenol and Sequence ID No. 87 (n=6). Body weight and heart rate were measured for each mouse after treatment. Cardiac weight normalized by body weight (HW / BW) for each group is shown. ****: p<0.0001. [Figure 16] A graph showing the effect of Sequence ID No. 87 on isoproterenol-induced fibrosis according to an aspect of this disclosure. Mice were treated as described in Figure 15, and collagen content was quantified using a hydroxyproline assay. ****: p<0.0001, ***: p<0.001. [Figure 17A] This shows pharmacokinetic (PK) data for healthy human patients who received a single IV dose of 0.3 mg / kg of SEQ ID NO: 87. The time-course concentrations of SEQ ID NO: 87 in the treated patients (solid line) and the predicted PK profile of SEQ ID NO: 87 using non-human primate modeling (dashed line) are shown. [Figure 17B]This shows disease progression (PD) data from healthy human patients who received a single IV dose of 0.3 mg / kg of SEQ ID NO: 87. It also shows the change in renal plasma flow relative to baseline at days 2, 8, and 17 in healthy patients who received SEQ ID NO: 87 or placebo (PBO). [Figure 18] A graph showing the time course of the concentration of SEQ ID NO: 87 in patients who received the drug, and PK data from healthy human patients who received a single 150 mg SC dose of SEQ ID NO: 87. [Modes for carrying out the invention]

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

[0058] 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, the fusion protein having 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 in this disclosure is greater than 2 hours. In some embodiments, the fusion protein provided in this disclosure has a low pI. In some embodiments, the pI of the fusion protein provided in this disclosure is less than 8.5. In some embodiments, the low pI of the fusion protein provided in this disclosure is caused by acidic amino acid residues present in the peptide linker. In some embodiments, the peptide linker of the fusion protein contains two or more acidic amino acids. In some embodiments, the peptide linker has a total length of 10 to 15 amino acids.

[0059] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the field to which the claimed subject matter belongs. It should be understood that the above general descriptions and the following detailed descriptions are merely illustrative and descriptive and do not limit any claimed subject matter. In this application, unless otherwise specified, the use of the singular includes the plural. It should be noted that, where used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context explicitly indicates otherwise. In this application, the use of "or" means "and / or" unless otherwise specified. Furthermore, the term "including," as well as the other forms "include," "includes," and "included," are not limiting. Section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described.

[0060] As used herein, the term "polynucleotide" refers to a polymer of DNA or RNA. A polynucleotide sequence may be single-stranded or double-stranded, may contain natural, unnatural, or modified nucleotides, and may contain natural, unnatural, or modified internucleotide bonds, such as phosphoramidate bonds or phosphorothioate bonds, instead of phosphodiesters found between nucleotides in unmodified polynucleotide sequences. Examples of polynucleotide sequences include, but are not limited to, all polynucleotide sequences obtained by any means available in the art, and examples of such means include, but are not limited to, recombinant means, such as cloning of polynucleotide sequences from recombinant libraries or cell genomes, using conventional cloning techniques and polymerase chain reactions, and synthetic means.

[0061] The terms “amino acid sequence” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acids linked by one or more peptide bonds. Where used herein, “amino acid sequence” refers to information describing the relative order and identity of the amino acid residues constituting the polypeptide.

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

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

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

[0065] As used herein, the term “linked to” refers to a covalent or non-covalent bond between two molecules or parts. Those skilled in the art will understand that when a first molecule or part is linked to a second molecule or part, the linkage does not have to be direct, but may instead be via an intervening molecule or part.

[0066] As used herein, the terms “human relaxin-2B chain,” “relaxin B chain,” “relaxin B,” or “relB” refer to peptides containing or comprising the amino acid sequence shown in DSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ ID NO: 249), or derivatives thereof. In some embodiments, derivatives of the relaxin B chain contain the amino acid sequence of SEQ ID NO: 154 with 1, 2, 3, 4, or 5 amino acid changes.

[0067] As used herein, the terms “human relaxin-2A chain,” “relaxin A chain,” “relaxin A,” or “relA” refer to peptides containing or comprising the amino acid sequence shown in QLYSALANKCCHVGCTKRSLARFC (SEQ ID NO: 257), or derivatives thereof. In some embodiments, derivatives of the relaxin A chain contain the amino acid sequence of SEQ ID NO: 155 with one, two, three, four, or five amino acid changes.

[0068] As used herein, the term “linker peptide” refers to the peptide that links the relaxin A chain and the relaxin B chain in the fusion protein described herein.

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

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

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

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

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

[0074] As used herein, the terms “to treat,” “to treat,” and “treatment” refer to therapeutic or preventive measures as described herein. In some embodiments, a “treatment” method involves administering a fusion protein to a subject who has or has been previously diagnosed with a disease or disorder in order to prevent, cure, delay, reduce the severity of, or improve one or more symptoms of a disease or disorder, or a recurrent disease or disorder, or to extend the survival of the subject beyond the survival expected in the absence of such treatment.

[0075] As used herein, in the context of therapy, the term “effective dose” refers to the amount of therapy that achieves the desired preventive or therapeutic effect.

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

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

[0078] Fusion protein 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, the fusion protein having a high in vivo circulating half-life when administered to a mammal. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a human relaxin-2B chain, or a derivative thereof, a peptide linker, and a human relaxin-2A chain, or a derivative thereof. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a human relaxin-2A chain, or a derivative thereof, a peptide linker, and a human relaxin-2B chain, or a derivative thereof. In some embodiments, the fusion protein further comprises 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 homodimers via interactions between IgG Fc moieties. In some embodiments, the above-mentioned IgG Fc is replaced with PEG.

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

[0080] In some embodiments, the human relaxin-2B chain derivative is given by the following formula: DSWX 19 EEVIKLCGRELVRAQIAICGX 20 ST (Sequence ID 250) is included or consists of X 19 and X 20 is either absent or any amino acid. In some embodiments, X 19 This is methionine (M), glutamine (Q), glutamic acid (E), asparagine (N), aspartic acid (D), serine (S), or threonine (T). In some embodiments, X 19 is methionine (M), lysine (K), or glutamine (Q). In some embodiments, X 20 This is methionine (M), lysine (K), glutamine (Q), or asparagine (N). In some embodiments, X 20 is methionine (M) or lysine (K). In some embodiments, X 20 This is lysine (K). In some embodiments, X 19 is methionine (M), lysine (K), or glutamine (Q), and X 20 This is either methionine (M) or lysine (K).

[0081] This disclosure provides human relaxin-2B chain derivatives, each comprising an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications to the amino acid sequence of SEQ ID NO: 1, where the amino acid at position 4 is not methionine (M), or where 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 to the amino acid sequence of SEQ ID NO: 1, where the amino acid at position 4 is not methionine (M), and where 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 to the amino acid sequence of SEQ ID NO: 1, where at least one amino acid at position 4 or 25 of the first peptide is not methionine (M).

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

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

[0084] In some embodiments, the human relaxin-2B chain derivatives used in the fusion proteins described herein do not contain the amino acid sequences of SEQ ID NOs. 251-254 described below: DSWKEEVIKLCGRELVRAQIAICGKSTAS (Sequence ID 251), DSWKEEVIKLCGRELVRAQIAICGKSTWS (Sequence ID 252), DSWMEEVIKLCGRELVRAQIAICGKSTAS (Sequence ID 253), and DSWMEEVIKLCGRELVRAQIAICGKSTWS (Sequence ID 254).

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

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

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

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

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

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

[0091] Human relaxin-2A chain derivative This disclosure provides human relaxin-2A chain derivatives, each having 1, 2, 3, 4, or 5 amino acid changes compared to the amino acid sequence of SEQ ID NO: 257. In some embodiments, the amino acid corresponding to position 3 of SEQ ID NO: 257 must be 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.

[0092] In some embodiments, the human relaxin-2A chain derivative is given by the following formula: X 21 QX 22 YSALANKCCHVGCTKRSLAX 23 Contains or consists of FC (sequence number 258), X 21 , X 22 , and X 23 is either absent or any amino acid. In some embodiments, X 21 is either arginine (R), lysine (K), glutamine (Q), asparagine (N), histidine (H), serine (S), threonine (T), proline (P), glycine (G), or absent. In some embodiments, X 21 is either arginine (R), glycine (G), or absent. In some embodiments, X 21 is either arginine(R) or absent. In some embodiments, X 22 This is leucine (L), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), serine (S), or threonine (T). In some embodiments, X 22 is leucine (L) or aspartic acid (D). In some embodiments, X 23 This is arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D), asparagine (N), serine (S), or threonine (T). In some embodiments, X 23is arginine (R), glutamine (Q), or glutamic acid (E). In some embodiments, X 21 is either arginine(R) or absent, X 22 is leucine (L) or aspartic acid (D), and X 23 These are arginine (R), glutamine (Q), or glutamic acid (E).

[0093] This disclosure provides human relaxin-2A chain derivatives, each comprising an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid modifications 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).

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

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

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

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

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

[0099] In some embodiments, the human relaxin-2A chain derivative comprises or consists of Sequence 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 Sequence 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 Sequence 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 Sequence 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 Sequence 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 Sequence 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 Sequence 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 Sequence 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 Sequence 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 Sequence 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 Sequence 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 Sequence 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 Sequence 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 Sequence 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 Sequence 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 Sequence 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 Sequence 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, human relaxin... Laxin-2A chain derivatives include or consist of Sequence 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 Sequence 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 Sequence 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 Sequence 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 Sequence 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 Sequence ID No. 558, wherein the amino acid at position 10 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); the amino acid at position 18 is lysine (K), glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), or tyrosine (Y); and the amino acid at position 19 is glutamine (Q), aspartic acid (D), glutamic acid (E), leucine (L), isoleucine (I), tyrosine (Y), or arginine (R).

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

[0101] Linker Peptide This disclosure provides linker peptides, each peptide having at least two acidic amino acids. In some embodiments, the acidic amino acids are glutamates. In some embodiments, the acidic amino acids are aspartates. In some embodiments, the acidic amino acids are non-standard amino acids. In some embodiments, the acidic amino acids are 2-aminoadipic acid, 2-aminobutyric acid, or 2-aminopimelic acid. In some embodiments, the linker peptide has 2, 3, 4, 5, 6, 7, 8, 9, or 10 acidic amino acids.

[0102] In some embodiments, the linker peptide is 8, 9, 10, 11, 12, 13, 14, or 15 amino acids 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 an aspartate or glutamate. In some embodiments, the non-acidic amino acid 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.

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

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

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

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

[0107] In some embodiments, the linker peptide is GGEGSGGEGX 10 Includes GGG (sequence number 25), X 10 This is glutamic acid (E) or serine (S).

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

[0109] In some embodiments, the linker peptide includes two, three, four, or five repeats of SEQ ID NOs: 267, 268, 269, 270, or 271. For example, the three repeats of SEQ ID NO: 267 are the amino acid sequence GGGEGGGEGGGE (SEQ ID NO: 277).

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

[0111] Relaxin / linker peptide combinations for fusion proteins In some embodiments, the fusion protein comprises an N-terminal or first peptide, a linker peptide, and a C-terminal or second peptide. In some embodiments, the N-terminal peptide comprises 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. Embodiments of the fusion protein described herein can be constructed using any combination of any embodiment of a human relaxin-2A chain or a derivative thereof linked by any of the linker peptides disclosed 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.

[0112] In some embodiments, the human relaxin-2B chain derivative consists of 15 amino acids, and the human relaxin-2A chain derivative consists of 16 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 25 amino acids.In some embodiments, the human relaxin-2B chain derivative consists of 28 amino acids, and the human relaxin-2A chain derivative consists of 16 to 25 amino acids. In some embodiments, the human relaxin-2B chain derivative consists of 29 amino acids, and the human relaxin-2A chain derivative consists of 16 to 25 amino acids.

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

[0114] Specific embodiments of the fusion protein provided in this disclosure are shown in Table 4 below. Table 4 TIFF0007835367000005.tif254170TIFF0007835367000006.tif250170TIFF0007835367000007.tif249170TIFF0007835367000008.tif251170TIFF0007835367000009.tif191170TIFF0007835367000010.tif254170TIFF0007835367000011.tif251170TIFF0007835367000012.tif250170TIFF0007835367000013.tif250170TIFF0007835367000014.tif250170TIFF0007835367000015.tif186170TIFF0007835367000016.tif254170TIFF0007835367000017.tif251170TIFF0007835367000018.tif250170TIFF0007835367000019.tif251170TIFF0007835367000020.tif250170TIFF0007835367000021.tif186170TIFF0007835367000022.tif254170TIFF0007835367000023.tif251170TIFF0007835367000024.tif250170TIFF0007835367000025.tif250170TIFF0007835367000026.tif250170TIFF0007835367000027.tif187170TIFF0007835367000028.tif253170TIFF0007835367000029.tif250170TIFF0007835367000030.tif250170TIFF0007835367000031.tif250170TIFF0007835367000032.tif251170TIFF0007835367000033.tif187170TIFF0007835367000034.tif254170TIFF0007835367000035.tif249170TIFF0007835367000036.tif249170TIFF0007835367000037.tif250170TIFF0007835367000038.tif249170TIFF0007835367000039.tif186170TIFF0007835367000040.tif254170TIFF0007835367000041.tif24 9170TIFF0007835367000042.tif250170TIFF0007835367000043.tif250170TIFF0007835367000044.tif250170TIFF0007835367000045.tif187170.

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

[0116] In some embodiments, the portion of the fusion protein comprising the N-terminal peptide, linker peptide, and C-terminal peptide contains or consists of the amino acid sequences shown in Table 5 below. [Table 5] TIFF0007835367000047.tif254170TIFF0007835367000048.tif254170TIFF0007835367000049.tif101170

[0117] IgG Fc In some embodiments, the fusion proteins provided herein further comprise an IgG Fc (or Fc region). As used herein, the terms “IgG Fc” or “Fc region” refer to the portion of immunoglobulin formed by the Fc domains of two heavy chains of immunoglobulin. The Fc region may 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 an interaction between Fc regions). In some embodiments, two fusion proteins are linked to a dimer (e.g., a homodimer) via two hinge-domain interchain disulfide bonds between the Fc regions of each fusion protein (e.g., at the N-terminus). In some embodiments, the Fc region comprises one intrachain disulfide bond in the CH2 domain and one intrachain disulfide bond in the CH3 domain.

[0118] The Fc region of the fusion proteins provided herein may be derived from any innate immunoglobulin. In some embodiments, the Fc region is formed from an IgA, IgD, IgE, or IgG heavy chain constant region. In some embodiments, the Fc region is formed from an IgG heavy chain constant region. In some embodiments, the IgG heavy chain is an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the Fc region is formed from an IgG1 heavy chain constant region. In some embodiments, the IgG1 heavy chain constant region includes G1m1(a), G1m2(x), G1m3(f), or G1m17(z) allotypes. 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. 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 they can be linked to an N-terminal or C-terminal peptide via an IgG Fc linker. In some embodiments, the IgG Fc linker contains or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid sequences. In some embodiments, the IgG Fc linker contains or consists of 1, 2, 3, 4, or 5 amino acids. In some embodiments, the IgG Fc linker contains or consists of 3 or 4 amino acids. In some embodiments, the IgG Fc linker contains or consists of the amino acid sequence of GGS. In some embodiments, the IgG Fc linker contains or consists of the amino acid sequence of EGGS (SEQ ID NO: 299).

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

[0120] In some embodiments, IgG Fc contains one amino acid sequence from SEQ ID NOs.76-83 along with GGS as the IgG Fc linker at the C-terminus of IgG Fc. In some embodiments, IgG Fc contains one amino acid sequence from SEQ ID NOs.76-83 along with SEQ ID NOs.299 as the IgG Fc linker at the C-terminus of IgG Fc.

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

[0122] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region is modified (e.g., increased or decreased) as described 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 may be modified, for example, to facilitate the assembly of the light and heavy chains, or to modify (e.g., increase or decrease) the stability of the antibody.

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

[0124] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., CH2 domain (residues 231-340 of human IgG1) and / or CH3 domain (residues 341-447 of human IgG1)) and / or hinge region of the antibodies described herein, numbered according to the EU numbering system, to increase or decrease the affinity of the antibody to an Fc receptor (e.g., an activated Fc receptor) on the surface of effector cells. Mutations in the Fc region of an antibody that decrease or increase the affinity of the antibody to an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those skilled in the art. Examples of antibody mutations at the Fc receptor that can be performed to alter the affinity of an antibody to 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 Publication Nos. 02 / 060919, 98 / 23289, and 97 / 34631, all of which are incorporated herein by reference in their entirety.

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

[0126] In further embodiments, one, two, or more amino acid substitutions are introduced into the IgG constant domain Fc region to modify the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332, and 396, numbered according to the EU numbering system, can be replaced with different amino acid residues so that the antibody has a modified affinity for the effector ligand, while the antigen-binding ability of the parent antibody is retained. The effector ligand whose affinity is modified may be, for example, the Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patents 5,624,821 and 5,648,260, each of which is incorporated herein by reference in whole. In certain embodiments, deletion or inactivation of the constant domain (by point mutation or other means) may reduce the Fc receptor binding of the circulating antibody, thereby increasing tumor localization. For example, see U.S. Patents 5,585,097 and 8,591,886, each of which is incorporated herein by reference in whole. In certain embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibodies described herein to remove a potential glycosylation site 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 whole).In various embodiments, one or more of the following mutations in the constant region of the antibodies described herein, numbered according to the EU numbering system, can be produced as N297A substitution, N297Q substitution, L234A substitution, L234F substitution, L235A substitution, L235F substitution, L235V substitution, L237A substitution, S239D substitution, E233P substitution, L234V substitution, L235A substitution, C236 deletion, P238A substitution, S239D substitution, F243L substitution, D265A substitution, S267E substitution, L328F substitution, R292P substitution, Y300L substitution, A327Q substitution, P329A substitution (PA), A332L substitution, I332E substitution, or P396L substitution.

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

[0128] In specific embodiments, the antibody described herein comprises a constant domain of IgG1 having an N297Q or N297A amino acid substitution, numbered according to the EU numbering system. In one embodiment, the antibody described herein comprises a constant domain of IgG1 having a mutation selected from the group consisting of D265A, P329A, and combinations thereof, numbered according to the EU numbering system. In another embodiment, the antibody described herein comprises a constant domain of IgG1 having a mutation selected from the group consisting of L234A, L235A(LALA), and combinations thereof, numbered according to the EU numbering system. In yet another embodiment, the antibody described herein comprises a constant domain of IgG1 having a mutation selected from the group consisting of L234F, L235F, N297A, and combinations thereof, numbered according to the EU numbering system. In certain embodiments, the amino acid residues in the constant region of the antibody described herein at positions corresponding to L234, L235, and D265 of the human IgG1 heavy chain, numbered according to the EU numbering system, are not L, L, and D, respectively. This approach is described in detail in International Publication No. 14 / 108483, which is incorporated herein by reference in its entirety. In certain embodiments, the amino acids corresponding to positions L234, L235, and D265 in the human IgG monoheavy chain are F, E, and A, or A, A, and A, respectively, as numbered according to the EU numbering system.

[0129] In certain embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 within the constant region of the antibody described herein, numbered according to the EU numbering system, can be replaced with different amino acid residues so that the antibody has modified C1q binding and / or reduced or absent complement-dependent cytotoxicity (CDC). This approach is described in more detail in U.S. Patent No. 6,194,551 (Idusogie et al.), which is incorporated herein by reference in its entirety. In certain embodiments, one or more amino acid residues within amino acid positions 231-238 in the N-terminal region of the CH2 domain of the antibody described herein are modified to modify the antibody's complement-fixing ability, numbered according to the EU numbering system. This approach is described in International Publication No. 94 / 29351, which is incorporated herein by reference in its entirety. In certain embodiments, the Fc region of the antibody described herein is positioned at the following locations, 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303 to increase the antibody's ability to mediate antibody-dependent cytotoxicity (ADCC) and / or to increase the antibody's affinity for the Fc receptor. These are modified by mutating one or more amino acids in 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 (e.g., by introducing amino acid substitutions), and these are numbered according to the EU numbering system. This approach is further described in International Publication No. 00 / 42072, which is incorporated herein by reference in its entirety.

[0130] In some embodiments, IgG Fc is IgG1 Fc or a derivative thereof. In some embodiments, IgG Fc or IgG1 Fc contains an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, or 99% identical to the amino acid sequence of IgG1 Fc. In some embodiments, IgG Fc or IgG1 Fc contains an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequences provided in Table 6 below. [Table 6] TIFF0007835367000051.tif88170

[0131] In some embodiments, any IgG Fc or its derivative may be linked to the N-terminus or C-terminus of any of the embodiments described in Table 4 or 5 above, with or without an IgG Fc linker. In some embodiments, human IgG1 Fc or its derivative may be linked to the N-terminus or C-terminus of any of the embodiments described in Table 4 or 5 above, with or without an IgG Fc linker. In some embodiments, the amino acid sequence of human IgG1 Fc includes or consists of the amino acid sequence of SEQ ID NO: 76 or 80. In some embodiments, the derivative of human IgG1 Fc includes 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 amino acid sequence to the amino acid sequence of SEQ ID NO: 76 or 80.

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

[0133] In some embodiments, human IgG1 Fc containing the LALA PA mutation or a derivative thereof may be linked to any of the N-terminus or C-terminus of the embodiments described in Table 4 or 5 above, with or without an IgG Fc linker. In some embodiments, the amino acid sequence of human IgG1 Fc containing the LALA PA mutation contains or consists of the amino acid sequence of SEQ ID NO: 78 or 82. In some embodiments, the derivative of human IgG1 Fc containing the LALA PA mutation contains 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 amino acid sequence to the amino acid sequence of SEQ ID NO: 78 or 82.

[0134] In some embodiments, human IgG1 Fc containing the LALA PA LS mutation, or a derivative thereof, may be linked to any of the N-terminus or C-terminus of the embodiments described in Table 4 or 5 above, with or without an IgG Fc linker. In some embodiments, the amino acid sequence of human IgG1 Fc containing the LALA PA LS mutation contains or consists of the amino acid sequence of SEQ ID NO: 79 or 83. In some embodiments, the derivative of human IgG1 Fc containing the LALA PA LS mutation contains 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 amino acid sequence to the amino acid sequence of SEQ ID NO: 79 or 83.

[0135] In some embodiments, the fusion protein contains amino acid sequences that are 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 sequences shown in Table 7. In some embodiments, the fusion protein contains or consists of the amino acid sequences shown in Table 7. [Table 7] TIFF0007835367000053.tif223170TIFF0007835367000054.tif222149TIFF00078353670000 55.tif221149TIFF0007835367000056.tif223148TIFF0007835367000057.tif223150TIFF000 7835367000058.tif223148TIFF0007835367000059.tif223147TIFF0007835367000060.tif22 2149TIFF0007835367000061.tif225170TIFF0007835367000062.tif224170TIFF00078353670 00063.tif223170TIFF0007835367000064.tif225170TIFF0007835367000065.tif223170TIF F0007835367000066.tif222170TIFF0007835367000067.tif225170TIFF0007835367000068.t if225170TIFF0007835367000069.tif222170TIFF0007835367000070.tif223170TIFF0007835 367000071.tif224170TIFF0007835367000072.tif222148TIFF0007835367000073.tif100152

[0136] In some embodiments, IgG Fc includes a mouse IgG kappa signal sequence containing the amino acid sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO: 329). In some embodiments, IgG Fc includes a mouse IgG heavy chain signal sequence. In some embodiments, IgG Fc includes a signal sequence containing the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 548). In some embodiments, different signal sequences are used. In some embodiments, the signal sequence is not present on the fusion protein produced.

[0137] In some embodiments, the fusion protein contains amino acid sequences that are 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 sequences shown in Table 8. In some embodiments, the fusion protein contains or consists of the amino acid sequences shown in Table 8. [Table 8] TIFF0007835367000075.tif222149TIFF0007835367000076.tif222148TIFF00078353670 00077.tif222148TIFF0007835367000078.tif221148TIFF0007835367000079.tif222148 TIFF0007835367000080.tif221147TIFF0007835367000081.tif222148TIFF00078353670 00082.tif222148TIFF0007835367000083.tif222149TIFF0007835367000084.tif221149 TIFF0007835367000085.tif223150TIFF0007835367000086.tif223149TIFF00078353670 00087.tif223149TIFF0007835367000088.tif222147TIFF0007835367000089.tif222149 TIFF0007835367000090.tif221148TIFF0007835367000091.tif221147TIFF00078353670 00092.tif221147TIFF0007835367000093.tif222148TIFF0007835367000094.tif181149

[0138] Other half-life extensions As used herein, the term “half-life extension portion” includes non-proteinaceous half-life extension portions such as PEG or HES, and proteinaceous half-life extension portions such as Fc domains. In some embodiments, the non-proteinaceous half-life extension portion is ligated to the fusion protein described herein. In some embodiments, the non-proteinaceous half-life extension portion is ligated to the fusion protein instead of IgG Fc. In some embodiments, the non-proteinaceous half-life extension portion is ligated to the fusion protein in addition to IgG Fc.

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

[0140] PEGs have the advantage of having fewer crosslinkable reactive groups compared to polysaccharides such as dextran. Monofunctional PEGs, such as methoxypolyethylene glycol (mPEG), are particularly interesting because their coupling chemistry is relatively simple (there is only one reactive group available to conjugate with the binding group on the polypeptide). As a result, as the risk of crosslinking is eliminated, the resulting conjugated fusion proteins described herein are more homogeneous, and the reaction between the polymer molecule and the variant polypeptide is easier to control.

[0141] To provide covalent bonding of polymer molecules(s) to the fusion proteins described herein, the hydroxyl-terminated groups of the polymer molecules must be provided in an activated form, i.e., having reactive functional groups (examples include primary amino groups, hydrazides (HZ), thiols, succinates (SUC), succinimidyl succinates (SS), succinimidyl succinamide (SSA), succinimidyl propionates (SPA), succinimidyl butyrates (SBA), succinimidyl carboxymethylates (SCM), benzotriazole carbonates (BTC), N-hydroxysuccinimide (NHS), aldehydes, nitrophenyl carbonates (NPC), and torecylates (TRES)). Suitable activated polymer molecules are commercially available, for example, from Shearwater Polymers, Inc., Huntsville, Ala, USA, or PolyMASC Pharmaceuticals plc, UK.

[0142] Alternatively, polymer molecules may be activated by conventional methods known in the art, such as those disclosed in WO90 / 13540. Specific examples of activated linear or branched polymer molecules for use herein are described in Shearwater Polymers, Inc. 1997 and 2000 Catalogs (Functionalized Biocompatible Polymers for Research and Pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference). Specific examples of activated PEG polymers include the following linear PEGs:NHS-PEG (e.g., SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM-PEG), as well as branched PEGs such as NOR-PEG, BTC-PEG, EPOXPEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and PEG2-NHS, as well as those disclosed in U.S. Patents 5,932,462 and 5,643,575, both of which are incorporated herein by reference. Furthermore, the following publications disclose useful polymer molecules and / or PEGylation chemistry.U.S. Patent Nos. 5,824,778, 5,476,653, WO97 / 32607, EP229,108, EP402,378, 4,902,502, 5,281,698, 5,122,614, 5,219,564, WO92 / 16555, WO9 4 / 04193, WO94 / 14758, WO94 / 17039, WO94 / 18247, WO94 / 28024, WO95 / 00162, WO95 / 119 24, WO95 / 13090, WO95 / 33490, WO96 / 00080, WO97 / 18832, WO98 / 41562, WO98 / 48837, WO 99 / 32134, WO99 / 32139, WO99 / 32140, WO96 / 40791, WO98 / 32466, WO95 / 06058, EP439508, WO97 / 03106, WO96 / 21469, WO95 / 13312, EP921131, US Patent No. 5,736,625, WO98 / 05363, EP809996, U.S. Patent No. 5,629,384, WO96 / 41813, WO96 / 07670, U.S. Patent No. 5,473,034, U.S. Patent No. 5,516,673, EP605963, U.S. Patent No. 5,382,657, EP510356, EP400472, EP183503, and EP154316.

[0143] Specific examples of activated PEG polymers particularly preferred for coupling to 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 orthopyridyl-disulfide-PEG (OPSS-PEG), preferably orthopyridyl-disulfide-mPEG (OPSS-mPEG). Typically, such PEG or mPEG polymers have sizes of about 5 kDa, about 10 kDa, about 12 kDa, or about 20 kDa.

[0144] The conjugation of fusion proteins and activated polymer molecules described herein is carried out by any conventional method, such as those described in the following references (which also describe preferred methods for activating polymer molecules): Harris and Zalipsky, eds., Poly(ethylene glycol) Chemistry and Biological Applications, AZC Washington; RF Taylor, (1991), “Protein immobilisation. Fundamental and applications,” Marcel Dekker, NY; SSWong, (1992), “Chemistry of Protein Conjugation and Crosslinking,” CRC Press; Boca Raton, GTHermanson et al., (1993), “Immobilized Affinity Link Techniques,” Academic Press, NY.

[0145] Those skilled in the art will recognize that the activation method and / or conjugation chemistry used depends on the binding group(s) of the fusion protein (examples of which are further shown above), as well as the functional groups of the polymer (e.g., amines, hydroxyls, carboxyls, aldehydes, sulfhydryls, succinimidyls, maleimides, vinyl sulfones, or haloacetates). PEGylation may be directed to conjugation to all available binding groups on the fusion protein (i.e., any such binding group exposed on the surface of the polypeptide), or to one or more specific binding groups, e.g., the N-terminal amino group or cysteine ​​residue described in U.S. Patent No. 5,985,265. Furthermore, conjugation may be achieved in one step or stepwise manner (e.g., as described in WO99 / 55377).

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

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

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

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

[0150] It is also intended to couple polymer molecules to fusion proteins via linkers. Suitable linkers are well known to those skilled in the art. A preferred example is cyanuryl chloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581, U.S. Patent No. 4,179,337, Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375-378).

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

[0152] Depending on the circumstances, such as the amino acid sequence of the fusion protein, the properties of the activated PEG compound used, and specific PEGylation conditions including the PEG-to-polypeptide molar ratio, it will be understood that varying degrees of PEGylation can be obtained, and that higher degrees of PEGylation are generally obtained with higher PEG-to-fusion proteins. However, the PEGylated fusion proteins resulting from any given PEGylation process typically contain a stochastic distribution of conjugated fusion proteins with slightly different degrees of PEGylation.

[0153] Chemical modifications such as PEGylation or HESation can be applied to improve the biological half-life of the fusion proteins described herein.

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

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

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

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

[0158] 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 about 1,000 times compared to natural relaxin-2. In some embodiments, the potency is measured in half-maximal response (EC2). 50 This is the concentration of relaxin-2 or relaxin-2 fusion protein to induce relaxin-2. 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 time, about 10 times, about 100 times, or about 1,000 times the potency of natural relaxin-2.

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

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

[0161] In some embodiments, the fusion proteins described herein have an improved pharmacokinetic profile. While we do not wish to be bound by any theory, the structures of the fusion proteins described herein are at least in part based on the surprising discovery that reducing the pI of relaxin-2 fusion protein analogs increases their circulating half-life. In some embodiments, the fusion proteins described herein have high bioavailability. In some embodiments, the fusion proteins described herein have high and / or stable serum levels. In some embodiments, the circulating half-life, bioavailability, high serum levels, and / or stable serum levels are in mammals. In some embodiments, the mammal is a rodent or primate. In some embodiments, the rodent is a rat or mouse. In some embodiments, the primate is a human or monkey. In some embodiments, the monkey is a cynomolgus macaque. In some embodiments, the mammal is a human.

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

[0163] This increase in half-life may be at least in part due 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. Where used herein, the term “about” when referring to pI includes a variation of ±1% of a given value or range, where appropriate for carrying out the methods disclosed herein. In some embodiments, the fusion protein has pIs of 9.0, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, or 6.1 The fusion protein has a pI of less than 9.0, or 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 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 6.0 to about 9.4. In some embodiments, the fusion protein has a range of approximately 6.5–8.5, 6.6–8.4, 6.7–8.3, 6.8–8.2, 6.8–8.1, 6.8–8.0, 6.8–7.9, 6.0–8.2, 6.0–8.1, 6.0–8.0, 6.0–7.9, 6.0–7.8, 6.0–7.7, and 6.0–7.6. The pI values ​​are approximately 6.0 to 7.5, 6.0 to 7.4, 6.0 to 7.3, 6.0 to 7.2, 6.0 to 7.1, 6.0 to 7.0, 6.0 to 6.9, 6.0 to 6.8, 6.0 to 6.7, 6.0 to 6.6, 6.0 to 6.5, 6.0 to 6.4, 6.0 to 6.3, 6.0 to 6.2, or 6.0 to 6.1. In some embodiments, the fusion protein has a pI value of approximately 6.0 to 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 pI values ​​are calculated or theoretical pI values. In some embodiments, any of the above pI values ​​are experimentally measured pI values.

[0164] As used herein, the term “about” when referring to a dosage includes a variation of ±10% of a given value or range, where appropriate for carrying out the methods disclosed herein.

[0165] As used herein, “circulating half-life” refers to the time it takes for the plasma concentration of a drug to be reduced by half as it circulates in the whole blood of an organism. The circulating half-life of a particular drug can vary depending on a number of factors, including, but not limited to, the dosage, formulation, and / or route of administration of the drug. 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.

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

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

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

[0169] Vectors and host cells 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.

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

[0171] In some embodiments, the vector is a viral vector. Viral vectors may be able to replicate or not. Viral vectors may be embedded or non-embedded. Several virus-based systems have been developed for gene transfer into mammalian cells, and suitable viral vectors can be selected by those skilled in the art. Examples of 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), retrovirus vectors (MMSV, MSCV), lentivirus vectors (e.g., HIV-1, HIV-2), gamma-retrovirus vectors, herpesvirus vectors (e.g., HSV1, HSV2), alphavirus vectors (e.g., SFV, SIN, VEE, M1), flaviviruses (e.g., Kunzin, West Nile, dengue virus), rhabdovirus vectors (e.g., rabies virus, VSV), measles virus vectors (e.g., MV-Edm), Newcastle disease virus vectors, poxvirus vectors (e.g., VV), measles virus, and picornavirus vectors (e.g., coxsackievirus).

[0172] In some embodiments, the vector or expression cassette includes one or more additional elements. These additional elements include, but are not limited to, promoters, enhancers, polyadenylated (poly-A) sequences, and selection genes.

[0173] In some embodiments, the vector includes a polynucleotide sequence encoding an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical to an amino acid sequence listed in any of Tables 1 to 8. In some embodiments, the vector includes an amino acid sequence listed in any of Tables 1 to 8, or a polynucleotide sequence encoding an amino acid sequence consisting of such sequences. In some embodiments, the vector contains 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 sequences listed in Table 9 below. In some embodiments, the vector contains a polynucleotide sequence that contains or consists of the sequences listed in Table 9 below. [Table 9] TIFF0007835367000096.tif241148TIFF0007835367000097.tif241146TIFF0007835367000098.tif242147TIFF0007835367000099.tif243145TIFF0007835367000100.tif242147TIFF0007835367000101.tif242148TIFF0007835367000102.tif244149TIFF0007835367000103.tif242146TIFF0007835367000104.tif243148TIFF0007835367000105.tif242147TIFF0007835367000106.tif242147TIFF0007835367000107.tif243147TIFF0007835367000108.tif242146TIFF0007835367000109.tif243146TIFF0007835367000110.tif242146TIFF0007835367000111.tif243146TIFF0007835367000112.tif241146TIFF0007835367000113.tif243148TIFF0007835367000114.tif242146TIFF0007835367000115.tif243146TIFF0007835367000116.tif243147TIFF0007835367000117.tif242147TIFF0007835367000118.tif243146TIFF0007835367000119.tif243146TIFF0007835367000120.tif242148TIFF0007835367000121.tif242146TIFF0007835367000122.tif242147TIFF0007835367000123.tif244147TIFF0007835367000124.tif242146TIFF0007835367000125.tif242146TIFF0007835367000126.tif242146TIFF0007835367000127.tif243146TIFF0007835367000128.tif242146TIFF0007835367000129.tif241146TIFF0007835367000130.tif242147TIFF0007835367000131.tif243146T IFF0007835367000132.tif242146TIFF0007835367000133.tif242144TIFF0007835367000134.tif242147TIFF0007835 367000135.tif242146TIFF0007835367000136.tif242146TIFF0007835367000137.tif248147TIFF0007835367000138. tif243147TIFF0007835367000139.tif242147TIFF0007835367000140.tif243146TIFF0007835367000141.tif129146.

[0174] In some embodiments, the vector comprises a polynucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical to the sequences listed in Table 10 below. In some embodiments, the vector comprises a polynucleotide sequence comprising or consisting of the sequences listed in Table 10 below. [Table 10] TIFF0007835367000143.tif253145TIFF0007835367000144.tif252145TIFF0007835367000145.tif252146TIFF0007835367000146.tif253146TIFF0007835367000147.tif252145TIFF0007835367000148.tif253146TIFF0007835367000149.tif252145TIFF0007835367000150.tif252145TIFF0007835367000151.tif252145TIFF0007835367000152.tif252146TIFF0007835367000153.tif252146TIFF0007835367000154.tif252146TIFF0007835367000155.tif252145TIFF0007835367000156.tif252145TIFF0007835367000157.tif253145TIFF0007835367000158.tif252146TIFF0007835367000159.tif253147TIFF0007835367000160.tif252145TIFF0007835367000161.tif251145TIFF0007835367000162.tif252146TIFF0007835367000163.tif252146TIFF0007835367000164.tif253144TIFF0007835367000165.tif252146TIFF0007835367000166.tif253146TIFF0007835367000167.tif252145TIFF0007835367000168.tif253146TIFF0007835367000169.tif252145TIFF0007835367000170.tif252146TIFF0007835367000171.tif252146TIFF0007835367000172.tif252146TIFF0007835367000173.tif252146TIFF0007835367000174.tif252146TIFF0007835367000175.tif252145TIFF0007835367000176.tif252146TIFF0007835367000177.tif252147TIFF0007835367000178.tif252146T IFF0007835367000179.tif252146TIFF0007835367000180.tif252145TIFF0007835367000181.tif252145TIFF0007835 367000182.tif252147TIFF0007835367000183.tif251145TIFF0007835367000184.tif247145TIFF0007835367000185. tif252145TIFF0007835367000186.tif252145TIFF0007835367000187.tif253146TIFF0007835367000188.tif134147.

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

[0176] The fusion proteins described herein can be expressed using a variety of host cells and expression vector systems. Such expression systems represent vehicles from which the desired coding sequence can be produced and subsequently purified, and also represent cells that can express the fusion proteins described herein in situ when transformed or transfected with a suitable nucleotide coding sequence. These include, for example, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing protein coding sequences; for example, yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing protein coding sequences; for example, insect cell lines infected with recombinant virus expression vectors containing protein coding sequences (e.g., baculovirus); for example, plant cell lines (e.g., Chlamydomonas) infected with recombinant virus expression vectors containing protein coding sequences (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors containing protein coding sequences (e.g., Ti plasmid). This includes, but is not limited to, green algae such as reinhardtii; or mammalian cell lines possessing recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., late adenovirus promoter; vaccinia virus 7.5K promoter) (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells). In certain embodiments, the cells for expressing the fusion proteins described herein are human cells, e.g., human cell lines. In certain embodiments, the mammalian expression vector is pOptiVEC® or pcDNA3.3.In certain embodiments, bacterial cells, such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells) are used for the expression of the fusion protein. For example, mammalian cells such as CHO or HEK293 cells, in combination with vectors such as the major immediate gene promoter element from human cytomegalovirus, are an effective expression system for the fusion proteins disclosed herein.

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

[0178] In insect systems, for example, the Autographa californica nuclear polyhedron disease virus (AcNPV) can be used as a vector to express foreign genes. The virus proliferates in Spodoptera frugiperda cells. Fusion protein coding sequences can be individually cloned into non-essential regions of the virus (e.g., polyhedrin genes) and placed under the control of the AcNPV promoter (e.g., the polyhedrin promoter).

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

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

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

[0182] In certain embodiments, instead of using an expression vector containing a viral replication origin, host cells may be transformed with a polynucleotide (e.g., DNA or RNA) controlled by appropriate transcriptional regulatory elements (e.g., promoters, enhancers, sequences, transcriptional terminators, polyadenylation sites, etc.) and a selectable marker. After the introduction of the polynucleotide, the engineered cells may be grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing cells to stably incorporate the plasmid into their chromosomes, grow, and form a focus, which can then be cloned and grown in a cell line. This method can advantageously be used to engineer cell lines expressing the fusion proteins or fragments thereof described herein.

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

[0184] Pharmaceutical composition This disclosure provides pharmaceutical compositions comprising fusion proteins or component peptides, or nucleic acid molecules, or encoding expression vectors as described herein. The pharmaceutical compositions described herein are formulated with suitable carriers, excipients, and other agents that provide improved mobility, delivery, tolerability, etc. Many suitable formulations can be found in the pharmacist-known formulary: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Examples of these formulations include powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., 1POFECTIN®, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water emulsions and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al., “Compendium of excipients for parenteral formulations,” PDA (1998) J Pharm Sci Technol 52:238-311.

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

[0186] Various delivery systems are known and may be used to administer the pharmaceutical compositions disclosed herein (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of delivery include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, for example, by injection or bolus injection, or by absorption through the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa), and may be administered together with other bioactive agents. Administration may be systemic or topical.

[0187] Any pharmaceutical composition described herein can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, with respect to subcutaneous delivery, pen-type delivery devices facilitate the application of the pharmaceutical compositions disclosed herein. Such pen-type delivery devices may be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and easily replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. Disposable pen-type delivery devices do not have replaceable cartridges. Rather, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is empty, the entire device is discarded.

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

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

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

[0191] therapeutic use Monotherapy This disclosure provides a method for enhancing relaxin-2-related activity in primary cells, comprising contacting primary cells with a fusion protein or component peptide described herein. In some embodiments, contacting primary cells with a fusion protein or component peptide results in enhanced relaxin-2 activity within the cell, for example, as described above. In some embodiments, contacting primary cells with a fusion protein or component peptide results in activation of the relaxin-2 receptor (RFXP1) on the cell surface. Activation of RFXP1 on the cell surface can result in cellular responses including, but not limited to, increased cAMP levels, vasodilation, expression of angiogenic factors including VEGF, expression of MMPs, and collagen degradation. In some embodiments, the cells are selected from the group consisting of endothelial cells, vascular smooth muscle cells, other vascular cells, cardiomyocytes, other cardiac cells, and fibroblasts. In some embodiments, the primary cells are within the scope as described below.

[0192] In certain embodiments, the Disclosure provides a method for activating RXFP1 on a cell surface, comprising activating RXFP1 on a cell surface by administering an effective amount of a fusion protein or component peptide described herein, or a nucleic acid molecule or encoding expression vector, to a target requiring such activation. Activation of RXFP1 on a cell surface can result in cellular responses including, but not limited to, increased cAMP levels, vasodilation, expression of angiogenic factors including VEGF, expression of MMPs, and collagen degradation. In some embodiments, the cells are selected from the group consisting of endothelial cells, vascular smooth muscle cells, other vascular cells, cardiomyocytes, other cardiac cells, and fibroblasts.

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

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

[0195] There are five classes of pulmonary hypertension as defined by the World Health Organization (WHO). Class 1 is pulmonary artery hypertension (PAH), and its diagnosis requires right heart catheterization (RHC) to show mean pulmonary artery (PA) pressure (mPAP) ≥ 20 mmHg and pulmonary vascular resistance (PVR) ≥ 2 Wood units at rest. Additional criteria for meeting Class 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 occlusion, and absence of certain multifaceted disorders including systemic disorders (e.g., sarcoidosis, chronic renal failure), hematological disorders (e.g., myeloproliferative disorders and chronic hemolytic anemia), and metabolic disorders (e.g., glycogen storage disorders). Group 1 also includes PAH due to unknown mechanisms (idiopathic PAH) and hereditary gene deficiencies (hereditary PAH); PAH caused by drugs and toxins; PAH associated with systemic disorders such as connective tissue disease, human immunodeficiency virus (HIV) infection, congenital heart disease, and hemosomiasis; PAH with clear features of venous / capillary involvement; and persistent PH in neonates.

[0196] Group 2 is PH (PH-LHD) caused by left heart disease, which may be clinically diagnosed when echocardiography shows sufficient LHD to explain the PH (with or without other confirmatory tests). In patients undergoing RHC, mPAP ≥ 20 mmHg, PCWP ≥ 15 mmHg, and normal or reduced cardiac output are consistent with the hemodynamic diagnosis of LHD-PH. Important supporting information is the presence of left atrial (LA) enlargement on echocardiography and left ventricular end-diastolic pressure (LHC) to confirm. If PH-LHD is confirmed, the patient should be assigned to one of the following categories: PH-LHD resulting from heart failure with maintained 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 post-capillary PH (Group 2.3, e.g., restrictive cardiomyopathy, constrictive pericarditis, LA myxoma, congenital or acquired inflow / outflow tract obstruction, and congenital cardiomyopathy). There are two subgroups of PH in Group 2, and patients may be distinguished into those with concomitant post-capillary PH (CpcPH) and those with isolated post-capillary hypertension (IpcPH).

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

[0198] Group 4 consists of pulmonary hypertension (PH) caused by pulmonary artery occlusion, and includes patients with chronic thromboembolic PH (CTEPH, Group 4.1), as well as PH due to PA occlusion (Group 4.2, e.g., benign or malignant tumors, arteritis in the absence of CTD, congenital PA stenosis, parasitic infections).

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

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

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

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

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

[0204] In certain embodiments, relaxin-2-related disorders or conditions include, but are not limited to, valvular heart disease.

[0205] In certain embodiments, relaxin-2-related disorder or impairment is a group 2 PH (CpcPH or IpcPH) with heart failure with preserved ejection fraction (HFpEF). In certain embodiments, relaxin-2-related disorder or impairment is CpcPH with HFpEF. In certain embodiments, relaxin-2-related disorder or impairment is IpcPH with HFpEF. In certain embodiments, HFpEF is defined as the signs and symptoms of New York Heart Association (NYHA) class II-III heart failure, and LVEF ≥ 50%, and (i) Heart Failure Association pre-test assessment, echocardiography, and natriuretic peptide score, functional tests in case of uncertainty, end-etiological (HFA-PEFF) score ≥ 5, and / or (ii) HFA-PEFF score 2-4 and abnormal diastolic stress test or invasive hemodynamic measurement. In certain embodiments, relaxin-2-related disorder or impairment is a CpcPH having NYHA class II-III heart failure, LVEF ≥ 50%, and (i) an HFA-PEFF score ≥ 5, and / or (ii) an HFA-PEFF score of 2-4 and an abnormal diastolic stress test or invasive hemodynamic measurement. In certain embodiments, relaxin-2-related disorder or impairment is an IpcPH having NYHA class II-III heart failure, LVEF ≥ 50%, and (i) an HFA-PEFF score ≥ 5, and / or (ii) an HFA-PEFF score of 2-4 and an abnormal diastolic stress test or invasive hemodynamic measurement.

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

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

[0208] In certain embodiments, CpcPH is 34 mL / m³, pulmonary vascular resistance (PVR) ≥ 3 Wood units, mPAP > 20 mmHg, PCWP > 15 mmHg, or 34 mL / m³. 2 The above is based on right heart catheterization (RHC) performed showing PCWP > 12 mmHg and ≤ 14 mmHg, accompanied by echocardiographic evidence of left atrial volume index (LAVI). In certain embodiments, IpcPH is defined as PVR < 3 Wood units, mPAP > 20 mmHg, PCWP > 15 mmHg, or 34 mL / m². 2 Based on the above echocardiographic evidence of left atrial volume index (LAVI) and performed RHC showing PCWP > 12 mmHg and ≤ 14 mmHg.

[0209] In certain embodiments, relaxin-2-related disorders or conditions include, but are not limited to, pre-eclampsia, postpartum hypertension, postpartum cardiomyopathy, pregnancy-induced heart failure, and maternal hypertension complicated with the postpartum period.

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

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

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

[0213] In certain embodiments, administration of the composition according to the method herein results in pulmonary vasodilation in the patient. In certain embodiments, administration of the composition according to the method herein results in an anti-inflammatory effect in the patient. In certain embodiments, administration of the composition according to the method herein results in an anti-fibrotic effect in the patient. In certain embodiments, administration of the composition according to the method herein results in right ventricular remodeling in the patient. In certain embodiments, administration of the composition according to the method herein results in peripheral vasodilation in the patient. In certain embodiments, administration of the composition according to the method herein results in cardiac relaxation in the patient. In certain embodiments, administration of the composition according to the method herein results in left ventricular remodeling in the patient. In certain embodiments, administration of the composition according to the method herein results in improvement of the patient's renal function.

[0214] In certain embodiments, administration of a composition according to the method herein results in an increase in renal plasma flow. In certain embodiments, administration of a composition according to the method 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 dose. In certain embodiments, the increase in renal plasma flow in a subject is maintained 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 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 1 month after a single dose. In certain embodiments, administration of a composition according to the method 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 dose in humans. In certain embodiments, the increase in renal plasma flow in subjects is maintained 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% one, two, three, four, five, or one month after a single dose to humans.

[0215] Combination therapies and formulations This disclosure also provides compositions and therapeutic formulations comprising a fusion protein or component peptide, or nucleic acid molecule, or an expression vector encoding one or more additional therapeutic active ingredients (e.g., therapeutic agents) as described herein, and methods of treatment comprising administering such combination to a subject requiring such treatment.

[0216] Exemplary additional therapeutic agents include any therapeutic agents that may be used to treat any relaxin-2 related disorder described herein. Exemplary additional therapeutic agents that may be combined with, or administered in combination with, any fusion proteins or component peptides, nucleic acid molecules, or encoding expression vectors described herein include, but are not limited to, angiotensin II receptor blockers, e.g., azilsartan, candesartan, eprosartan, losartan; ACE inhibitors, e.g., lisinopril, benazepril, captopril, enalapril, moexipril, perindopril; Quinapril, trandolapril, calcium channel blockers, e.g., amlodipine, amlodipine and benazepril, amlodipine and valsartan, sacubitril and valsartan, diltiazem, felodipine, isradipine, nicardipine, nimodipine, nisoldipine, verapamil, diuretics, e.g., chlorthalidone, hydrochlorothiazide, metrazone, indapamide, torsemide, furosemide, bumetanide, amiloride, triamterene, spironolactone Eplerenone, aldosterone antagonists, e.g., spironolactone, eplerenone, digoxin, e.g., lanoxin, beta-blockers, e.g., carvedilol, metoprolol, bisoprolol, activin signaling inhibitors, e.g., sotatercept, sodium / glucose cotransporter 2 (SGLT2) inhibitors, e.g., empagliflozin, dapagliflozin, bexagliflozin, canagliflozin, erzgliflozin, ipragliflozin Examples include flozin, luseogliflozin, remogliflozin etabonate, cergliflozin etabonate, sotagliflozin, tofogliflozin, henagliflozin, janagliflozin, mizagliflozin, veragliflozin proline hydrate, enabogliflozin, and glucagon-like peptide-1 (GLP-1) receptor agonists, such as exenatide, liraglutide, albiglutide, dulaglutide, lixisenatide, semaglutide, and tylzepatide.

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

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

[0219] Additional therapeutic active ingredients(s) may be administered immediately before, simultaneously with, or immediately after the administration of the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein.

[0220] This disclosure provides pharmaceutical compositions in which a fusion protein or component peptide, or nucleic acid molecule, or an expression vector encoding one or more additional therapeutic active ingredients as described elsewhere in this specification are co-formulated with one or more additional therapeutic active ingredients as described elsewhere in this specification.

[0221] Administration regimen In some embodiments, multiple doses of the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein can be administered to a subject over a predetermined period of time. Methods according to this aspect of the Disclosure include administering multiple doses of the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein, to a subject in succession. As used herein, “administering in succession” means that each dose of the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered to a subject on different days separated by a predetermined interval, for example, several hours, several days, several weeks, or several months. The Disclosure provides a method comprising administering a single primary dose of the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein, followed by one or more secondary doses of the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein, to a patient in succession, and then optionally one or more tertiary doses of the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein.

[0222] The terms “primary dose,” “secondary dose,” and “tertiary dose” refer to the time series of administrations of the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein. Therefore, the “primary dose” is the dose administered at the start of the treatment regimen (also referred to as the “baseline dose”), the “secondary dose” is the dose administered after the primary dose, and the “tertiary dose” is the dose administered after the secondary dose. The primary, secondary, and tertiary doses may all contain the same amount of the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein, but may generally differ from each other in terms of administration frequency. However, in certain embodiments, the amounts of the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein in the primary, secondary, and / or tertiary doses may differ from each other throughout the course of treatment (e.g., adjusted up or down as appropriate). In certain embodiments, two or more doses (e.g., two, three, four, or five) are administered as “loading doses” at the start of the treatment regimen, with subsequent doses administered on a lower frequency basis (e.g., “maintenance doses”).

[0223] In one exemplary embodiment, each secondary and / or tertiary dose is 1 to 26 times the preceding dose (e.g., 1, 1 and 1 / 2, 2, 2 and 1 / 2, 3, 3 and 1 / 2, 4, 4 and 1 / 2, 5, 5 and 1 / 2, 6, 6 and 1 / 2, 7, 7 and 1 / 2, 8, 8 and 1 / 2, 9, 9 and 1 / 2, 10, 10 and 1 / 2, 11, 11 and 1 / 2, 12, 12 and 1 / 2, 13, 13 and 1 It is administered 2, 14, 14 and 1 / 2 weeks later, 15, 15 and 1 / 2 weeks later, 16, 16 and 1 / 2 weeks later, 17, 17 and 1 / 2 weeks later, 18, 18 and 1 / 2 weeks later, 19, 19 and 1 / 2 weeks later, 20, 20 and 1 / 2 weeks later, 21, 21 and 1 / 2 weeks later, 22, 22 and 1 / 2 weeks later, 23, 23 and 1 / 2 weeks later, 24, 24 and 1 / 2 weeks later, 25, 25 and 1 / 2 weeks later, 26, 26 and 1 / 2 weeks later, or more weeks later. The phrase "immediately preceding dose," as used herein, means the dose of the fusion protein or component peptide or nucleic acid molecule or encoding expression vector described herein in a series of multiple doses, which is administered to the patient before a series of immediate doses without an intervention dose.

[0224] In one embodiment, each secondary and / or tertiary dose is administered four weeks after the immediately preceding dose. In another embodiment, the dose of the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered to the patient once every four weeks (Q4W).

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

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

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

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

[0229] In general, the preferred dose of the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein may be in the range of about 0.001 to about 200.0 milligrams per kilogram of body weight of the recipient, generally in the range of about 1 to 50 mg per kilogram of body weight. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered in the range of about 0.001 mg / kg to about 200 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered in the range of 0.001 mg / kg to 200 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered in the range of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered in the range of 0.01 mg / kg to 100 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered in a range of approximately 0.1 mg / kg to approximately 20 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered in a range of approximately 0.1 mg / kg to approximately 20 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered in a range of approximately 1 mg / kg to approximately 50 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered in a range of 1 mg / kg to approximately 50 mg / kg.For example, the fusion proteins or component peptides, nucleic acid molecules, or expression vectors encoding them described herein may be administered in single doses of approximately 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. In certain embodiments, the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them described herein may be administered in 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. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of this disclosure.

[0230] In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at approximately 0.3 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at approximately 0.3 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at approximately 1 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at approximately 1 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at approximately 3 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at approximately 3 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at approximately 10 mg / kg. In certain embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered at a dose of 10 mg / kg.

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

[0232] In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered as a fixed dose of approximately 150 mg. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered as a fixed dose of 150 mg. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered as a fixed dose of at least 150 mg. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered as a fixed dose of approximately 300 mg. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered as a fixed dose of 300 mg. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered as a fixed dose of approximately 600 mg. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered as a fixed dose of 600 mg.

[0233] In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered intravenously. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously.

[0234] In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered by intravenous infusion. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered for 1 minute, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, administered by intravenous infusion for 90 minutes, 2 hours, 3 hours, 4 hours, or longer. In some embodiments, the fusion protein or component peptide or nucleic acid molecule or encoding expression vector described herein is administered by intravenous infusion over 30 minutes. In some embodiments, the fusion protein or component peptide or nucleic acid molecule or encoding expression vector described herein is administered by intravenous infusion over 60 minutes. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered by intravenous infusion over a period of 30 to 60 minutes.

[0235] In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered intravenously at a dose of approximately 0.3 mg / kg once every four weeks. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered intravenously at a dose of 0.3 mg / kg once every four weeks. In a particular embodiment, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at a dose of approximately 0.3 mg / kg once every four weeks. In a particular embodiment, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at a dose of 0.3 mg / kg once every four weeks. In a particular embodiment, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at a dose of approximately 1 mg / kg once every four weeks. In a particular embodiment, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at a dose of 1 mg / kg once every four weeks. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at approximately 3 mg / kg once every four weeks. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at approximately 3 mg / kg once every four weeks. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at approximately 10 mg / kg once every four weeks. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at approximately 10 mg / kg once every four weeks.

[0236] In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered at a dose of approximately 150 mg once every four weeks. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered at a dose of 150 mg once every four weeks. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered at a dose of at least 150 mg once every four weeks. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered at a fixed dose of approximately 150 mg once every four weeks. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered at a fixed dose of 150 mg once every four weeks. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered at a fixed dose of at least 150 mg once every four weeks. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at a fixed dose of approximately 300 mg once every four weeks. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at a fixed dose of 300 mg once every four weeks. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at a fixed dose of approximately 600 mg once every four weeks. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered at a fixed dose of 600 mg once every four weeks.

[0237] In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously at a dose of approximately 150 mg once every four weeks. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously at a dose of 150 mg once every four weeks. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously at a dose of at least 150 mg once every four weeks. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously at a fixed dose of approximately 150 mg once every four weeks. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously at a fixed dose of 150 mg once every four weeks. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously at a fixed dose of at least 150 mg once every four weeks. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously at a fixed dose of approximately 300 mg once every four weeks. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously at a fixed dose of 300 mg once every four weeks. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously at a fixed dose of approximately 600 mg once every four weeks. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously at a fixed dose of 600 mg once every four weeks.

[0238] In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered intravenously once a month at a dose of approximately 0.3 mg / kg. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered intravenously once a month at a dose of 0.3 mg / kg. In certain embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered once a month at a dose of approximately 0.3 mg / kg. In certain embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered once a month at a dose of 0.3 mg / kg. In certain embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered once a month at a dose of approximately 1 mg / kg. In certain embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered once a month at a dose of 1 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered once a month at approximately 3 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered once a month at 3 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered once a month at approximately 10 mg / kg. In certain embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered once a month at 10 mg / kg.

[0239] In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered once a month at a dose of approximately 150 mg. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered once a month at a dose of 150 mg. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered once a month at a dose of at least 150 mg. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered once a month at a fixed dose of approximately 150 mg. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered once a month at a fixed dose of 150 mg. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered once a month at a fixed dose of at least 150 mg. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered once a month at a fixed dose of approximately 300 mg. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered once a month at a fixed dose of 300 mg. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered once a month at a fixed dose of approximately 600 mg. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered once a month at a fixed dose of 600 mg.

[0240] In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously at a dose of approximately 150 mg once a month. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously at a dose of 150 mg once a month. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously at a dose of at least 150 mg once a month. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously at a fixed dose of approximately 150 mg once a month. In some embodiments, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein is administered subcutaneously at a fixed dose of 150 mg once a month. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered once a month as a fixed subcutaneous dose of at least 150 mg. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered once a month as a fixed subcutaneous dose of approximately 300 mg. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered once a month as a fixed subcutaneous dose of 300 mg. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered once a month as a fixed subcutaneous dose of approximately 600 mg. In some embodiments, the fusion protein or component peptide, nucleic acid molecule, or encoding expression vector described herein is administered once a month as a fixed subcutaneous dose of 600 mg.

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

[0242] The kit may further include reagents or instructions for using, in a subject, the fusion protein or component peptide, or nucleic acid molecule, or encoding expression vector described herein. It may also include one or more buffers.

[0243] The components of the kit may be packaged in either an aqueous medium or a lyophilized form. The kit's container means generally include at least one vial, test tube, flask, bottle, syringe, or other container means in which the components can be placed, preferably, preferably divided equally. If the kit contains two or more components (for example, if the labeled reagent and label can be packaged together), the kit also generally includes a second, third, or other additional container in which the additional components can be placed separately. The kit may also include a second container means for containing sterile, pharmaceutically acceptable buffers and / or other diluents. However, various combinations of components may be contained in vials. The kits of this disclosure also typically include means for containing the fusion proteins or component peptides, or nucleic acid molecules, or encoding expression vectors described herein, and any other reagent containers that are tightly sealed for commercial use.

[0244] If the components of the kit are provided in one or more liquid solutions, the liquid solutions are aqueous solutions, and sterile aqueous solutions are particularly preferred. However, the components of the kit may be provided as dry powder(s). If the reagents and / or components are provided as dry powder, the powder may be reconstituted by the addition of a suitable solvent. It is also assumed that the solvent may be provided in a separate container. [Examples]

[0245] The examples provided herein are illustrative and descriptive 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 herein.

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

[0247] method Mobile phase A (bound): 20 mM Tris pH 7.4, Mobile phase B (elution): 20 mM Tris pH 7.4 + 1 M NaCl, Injection: 10 μg, Detection: 220 nm. 1. Before analysis, the heparin column was equilibrated for 10 minutes at 0.5 mL / min using mobile phase A. 2. To minimize ionic strength, the sample for analysis was diluted to 1 mg / mL with 20 mM Tris pH 7.4. 3. Heparin chromatography was performed on Agilent HPLC using the gradients shown in Table 12 below. [Table 12] 4. The study included positive controls (unbound, human IgG pool) and negative controls (SE301 or AT1R). 5. Samples were analyzed for retention time and reported relative retention time compared to positive controls (i.e., RT sample / RT positive control). 6. The approximate concentration of NaCl required for elution was calculated using the following calculation:

number

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

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

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

[0251] Example 2. Low pI relaxin-2 fusion protein analogs tend to reduce self-assembly when measured by affinity-captured self-interacting nanoparticle spectroscopy (AC-SINS). When evaluating the biophysical properties of development candidates, it is important to understand the self-association tendency of the molecules. The self-association tendency of molecules can be evaluated by concentrating the molecules to high concentrations and assessing them by SEC (monomer %), or by measuring the change in turbidity (OD340nm), and using DLS to determine a second virial coefficient (B 22 ) or self-interaction coefficient (k d ) calculate or AC-SINS(Δλ max There are many methods for using ). All three of these methods provide useful information but use different amounts of material to perform the evaluation. AC-SINS has emerged as a high-throughput method for evaluating self-assembly using minimal material, but still gives locally high concentrations by using affinity capture on gold nanoparticles. In short, gold nanoparticles are pre-coated with anti-human antibodies (Fc, Fab, and H+L), which, when incubated with the target antibody in a diluted solution, capture the antibody of interest and concentrate it in solution. When the immobilized molecules of interest interact, the interparticle distance between the gold nanoparticles decreases, resulting in an increased plasmon wavelength (i.e., redshift) that can be quantified using UV-VIS spectroscopy. The materials used for the spectroscopy are provided in Table 15. [Table 15]

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

[0253] Preparation of gold nanoparticle solution: Goat anti-human Fc IgG antibody (capture) and goat IgG antibody (non-capture) were buffered with 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 mixture of the capture (anti-Fc):non-capture (goat IgG) solutions was prepared for use as an 80% capture capacity coating solution for incubation of gold nanoparticles (AuNP).

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

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

[0256] Preparation of target antibody solutions (all methods followed this procedure): For each sample analyzed, 10 μL of AuNP concentrate was incubated in a 96-well polypropylene plate in the dark at room temperature for 2 hours with 100 μL of antibody test solution (normalized to 0.05 mg / mL). Two blank solutions were prepared by concentrating 10 μL of 10×AuNP in 100 μL of PBS to blank the assay and determine the wavelength shift upon addition of the test antibody. Ganitumumab was included as a positive control (high association, redshift), and panitumumab as a negative control (unassociated, no UV shift). Each sample was prepared in two separate batches for analysis. After 2 hours of incubation, 100 μL of the resulting solution was transferred to a UV-transparent polystyrene plate (384-well format). The two blank solutions were transferred and the wavelength shift was appropriately evaluated, and then the duplicated standard solution and sample were added for analysis. Next, the plate was centrifuged at 1000 × g for 1 minute to level the solution in the wells. Absorbance data were collected in 2 nm steps from 510 nm to 570 nm, and the wavelength shift was determined for each sample relative to AuNP alone.

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

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

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

[0260] method HEK293 cells were seeded in 96-well tissue culture plates and subsequently subjected to transient co-transfection with human RXFP1 and pGloSensor-22F plasmids. Transfected cells were stimulated with relaxin-2 or its fusion protein analogs to induce Gs-mediated cAMP signaling. cAMP was assayed using the activity of the GloSensor biosensor, a mutant luciferase fused to its cAMP-binding domain, leading to light generation in the presence of its substrate luciferin. The readout of this relative luminescence unit (RLU) is used as a proxy for the cAMP response. reagent • 96-well tissue culture processed plate. White with a clear bottom. (Corning number 3610) ·HEK293 cells (ATCC CRL-1573) • Poly-D-lysine (Gibco A3890401) • DPBS (calcium-free, magnesium-free, Gibco 14190250) • DMEM (high glucose containing L-glutamine and sodium pyruvate, Gibco 11995065) ·TrypLE Express(Gibco 12605010) • FBS (HyClone® trademark, Australian source, Cytiva SH30084) Penicillin-streptomycin (Gibco 15140122) ·CO2 independent medium (Gibco 18045088) • Opti-MEM(trademark) I reduced serum medium (Gibco 31985062) • pGloSensor(trademark)-22F cAMP plasmid (Promega catalog number E2301) • D-luciferin, potassium salt (GoldBio LUCK-1G) • FuGENE HD Transfection Reagent (Promega code E2311) • Reservoir (Corning / Axygen RES-V-25-SI) • Relaxin-2 (R&D Biosystems 6586-RN-025) • RXFP1-containing plasmid (pcDNA5 / FRT / TO-human RXFP1, full length) • Forskolin (Sigma F6886) • A plate reader (CLARIOstar Plus) that can read light emission.

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

[0262] Relaxin-2 peptide: sterile DPBS (MW=5,986Da, ε=12,865M) -1 cm -1 ) in which relaxin-2 peptide or relaxin-2 fusion protein analog is reconstituted at 0.1 mg / mL, A 280 The final concentration was determined by measurement. Aliquots were stored at -20°C.

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

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

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

[0266] cAMP signaling assay protocol This protocol was adapted from the GloSensor cAMP assay using Promega.

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

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

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

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

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

number

[0272] Hydrophobic Interaction Chromatography (HIC): HIC is a chromatography method that separates molecules based on their hydrophobicity. 10 μg of protein was injected into a butyl HIC column pre-equilibriumized with high-concentration ammonium sulfate buffer. The protein was eluted over 10 minutes in a gradient from high to low salt concentration. Samples were compared for hydrophobicity based on retention time; high retention times indicated high hydrophobicity, and low retention times indicated low hydrophobicity. Retention times were converted to approximate salt concentrations at elution and compared to highly hydrophobic and low hydrophobic standards.

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

[0274] Capillary Isoelectric Focusing (cIEF): Using imaged cIEF, differentially charged molecules (i.e., relaxin-2 fusion protein analogs) were separated using electrophoretic mobility in an amphoteric electrolyte solution to determine their isoelectric points (pI). The molecules were loaded into capillaries and separated based on their pI by allowing them to move along an electric field until the molecules reached the pH corresponding to their pI. UV absorption across the capillaries was measured throughout the separation process to allow for real-time observation and final quantification.

[0275] Baculovirus Particle (BVP) ELISA: BVP ELISA was used to understand the tendency of relaxin-2 fusion protein analogs for nonspecific or non-target interactions. BVP is an empty viral capsid without a viral genome, but during the manufacturing process, it can be extracted from the cell membrane along with the cell membrane components by budding from the cell membrane. Therefore, BVP has a highly diverse cell surface with many parts that mimic what the target molecule (i.e., relaxin-2 fusion protein analog) might encounter in vivo. Briefly, BVP was coated onto plates by adding 25 μL of BVP solution to each well. The BVP solution was prepared by diluting BVP stock (Medna Scientific; catalog number E3001) to 1 × 10⁶ PFU / mL in 0.1 M carbonate buffer, pH 9.6. After incubation overnight at 5°C, the BVP solution was blotted from the wells, and the wells were washed three times with PBST. The plate was blocked with 100 μL / well of 1×BSA in PBS blocking buffer (Cepham Life Sciences; catalog no. 10615). The plate was incubated on a plate shaker at 25°C for 1 hour. The blocking solution was blotted from the wells, and the wells were washed three times with PBST. The sample (i.e., relaxin-2 fusion protein analog) was prepared in two strips covering a dilution range of 3 μM to 0.1 nM and added to the plate. The plate was incubated at 25°C for 1 hour, then the wells were blotted and washed three times with PBST. 25 μL / well of a 1:10,000 dilution detection monoclonal antibody (Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ fragment specific; Jackson ImmunoResearch; catalog no. 50-194-1564) was added, the plate was incubated at 25°C for 1 hour, then the wells were blotted and washed three times with PBST. 1-Step® Ultra TMB-ELISA Substrate Solution (Life Technologies, catalog no. 34029) was then added.After approximately 2 minutes, the reaction was quenched by adding 25 μL of 2N HCl, and the plate was analyzed at 450 nm with a correction at 570 nm using a plate reader.

[0276] Efficacy assay: HEK293 cells were seeded in 96-well tissue culture plates and subsequently subjected to transient co-transfection with human RXFP1 and pGloSensor-22F plasmids. Transfected cells were stimulated with relaxin-2 or its fusion protein analogs to induce Gs-mediated cAMP signaling. cAMP was assayed using the activity of the GloSensor biosensor, a mutant luciferase fused to the cAMP-binding domain, which leads to the generation of light in the presence of its substrate luciferin. The readout of this relative luminescence unit (RLU) is used as a proxy for the cAMP response.

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

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

[0279] Goat anti-human Fc IgG antibody (capture) and goat IgG antibody (non-capture) were buffered with 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 mixture of capture (anti-Fc):non-capture (goat IgG) solution was prepared for an 80% capture capacity coating solution used to incubate gold nanoparticles (AuNP). A 9:1 volume ratio AuNP:coating solution was prepared. The solutions were incubated overnight at room temperature in the dark. After incubation, thiolated PEG was added to a diluted 10 μM stock solution to a final concentration of 0.1 μM to block empty areas on the AuNP (i.e., 5 mL of AuNP solution was added to 50 μL of 10 μM stock) and incubated at room temperature in the dark for 1 hour.

[0280] 2 mL of coated AuNP solution was centrifuged at 20,000 × g for 15 minutes to precipitate the AuNP, and 1800 μL of supernatant was carefully removed using a 1 mL pipette. The pelleted AuNP was gently resuspended using a 200 μL pipette to produce a 10-fold concentrated stock of coated AuNP. For each sample analyzed, 5 μL of the AuNP concentrate was incubated in a 384-well polypropylene plate with 45 μL of antibody test solution (normalized to 0.05 mg / mL) at room temperature in the dark for 2 hours. After 2 hours of incubation, absorbance data were collected in 1 nm steps from 450 nm to 650 nm, and the wavelength shift for each sample was determined by comparing it with AuNP alone.

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

[0282] Sequences: The sequences of relaxin-2 fusion protein analogs are described throughout this disclosure. Sequence IDs 496, 497, and 501 are shown below: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGKGGSDSWKEEVIKLCGRELVRAQIAICGKSTASDAAGANANAGARQLYSALANKCCHVGCTKRSLARFC (Sequence ID 496), DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQLYSALANKCCHVGCTKRSLARFCGGGGSGGGGSGGGGSSWMEEVIKLCGRELVRAQIAICGMSTWS (Sequence ID 497), and MPRLFFFHLLGVCLLLNQFSRAVADSWMEEVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSEMQPALPQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSSPSELKYLGLDTHSRKKRQLYSALANKCCHVGCTKRSLARFC (Sequence ID 501).

[0283] result The results are shown in Tables 21, 22, and 23 below. [Table 21] TIFF0007835367000202.tif193145 [Table 22] [Table 23]

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

[0285] For confirmation, AC-SINS and BVP ELISA assays, as well as an additional cAMP efficacy assay, were performed on a subset of relaxin-2 fusion protein analogs, and the results are shown in Table 24. [Table 24]

[0286] A subset of relaxin-2 fusion protein analogs (SEQ ID NOs: 496, 313, 87, 90, 95, and 104) were tested under various feasibility assessments. Under high-concentration stress (target concentration of approximately 100 mg / mL), none of the samples showed any loss of protein concentration based on UV. Thermal stress induced an increase in turbidity / emulsification for all tested samples. Stirring stress did not affect the samples of SEQ ID NOs: 313 and 104. Under chemical stress (target concentration of approximately 5 mg / mL), the samples of SEQ ID NOs: 496 and 90 showed a decrease in concentration for all chemical stresses tested, while the samples of SEQ ID NOs: 313, 87, 95, and 104 appeared stable. All samples showed an oxidation-induced decrease in concentration, and the oxidized samples of SEQ ID NOs: 496 and 90 showed an increase in high molecular weight species detected by size exclusion chromatography. In addition, based on non-reducing capillary electrophoresis samples (CE-SDS NR), no significant fragmentation was observed in any of the tested samples under any stress.

[0287] The formation of stress-induced post-translational modifications (PTMs) was also tested. Stresses 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 with oxidative stress (0.02% hydrogen peroxide) for 24 hours. Sequence IDs 313 and 87 showed no stress-induced modifications, Sequence IDs 90 and 95 showed aspartic acid isomerization in the linker region, Sequence ID 496 showed asparagine deamidation in the linker region, and Sequence IDs 95 and 104 showed aspartic acid isomerization in the relaxin sequence.

[0288] During feasibility studies, color changes and aggregation with multiple molecules under specific stress conditions, common to oxidized proteins, were observed. The color changes were typically caused by tryptophan oxidation, which resulted in changes in absorbance at 320 nm and 365 nm (Ambrogelly (2021) Antibodies 10(2):21). To investigate the photosensitivity of relaxin-2 fusion protein analogs SEQ ID NO: 522 and SEQ ID NO: 523, a lightbox was constructed using a tabletop 25°C incubator with a clear glass door. A photometer was mounted 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 of a standard manufacturing room. Samples were placed in clear glass vials with 200 μL glass inserts to allow for maximum surface area for light exposure while maintaining minimum sample volume requirements. 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 subjected to thermal stress via incubation at 40°C for 7 or 14 days. After exposure to light or heat, the samples were evaluated using size exclusion high-performance liquid chromatography (SEC) to assess aggregate formation and CE-SDS to assess purity. As shown in Table 25, under these conditions, both SEQ ID NO: 522 and SEQ ID NO: 523 are tolerant to thermal and light stress, as assessed by aggregate formation and purity. [Table 25]

[0289] Another 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 lines expressing human RXFP1 or orthologs derived from cynomolgus monkeys and rats, either transiently, stably, or endogenously. Table 26 summarizes the cAMP response induced by SEQ ID NO: 87 and wild-type human relaxin-2 in HEK293 cells transiently expressing human, monkey, and rat RXFP1. As shown in Table 26 and Figures 1A–1C, the mean EC50s of SEQ ID NO: 87 for human (Figure 1A), rat (Figure 1B), and monkey (Figure 1C) RXFP1 were 10±4 nM, 10±9 nM, and 30±20 nM, respectively. [Table 26]

[0290] To test the selectivity of SEQ ID NO: 87, CHO-K1 cells stably expressing human RXFP1 or human RXFP2 were used. As shown in Table 27, the EC of SEQ ID NO: 87 for human RXFP1 50 It is 40±20nM, and for human RXFP2, the potency is about 100 times lower (EC 50 This indicates that sequence number 87 is selective for RXFP1 (≥2000nM). [Table 27]

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

[0292] Example 5. Pharmacokinetic (PK) and pharmacodynamic (PD) properties of relaxin-2 fusion protein analogs Pharmacokinetic (PK) values ​​were determined by measuring the concentration of relaxin-2 fusion protein analogs in rat plasma over time after intravenous (IV) injection of 5 mg / kg of each protein analog. PK values ​​were determined for a subset of the relaxin-2 fusion protein analogs described herein (SEQ ID NOs: 496, 497, 367, 313, 87, 95, 90, and 104) (Figure 2A). The rat PK parameters are shown in Table 28 below. [Table 28]

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

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

[0295] Example 6. Hemodynamic and renal blood flow effects of relaxin-2 fusion protein analogs. The high isoelectric points (pI) of relaxin and related molecules present significant pharmacokinetic (PK) and biophysical challenges, reflected in the rapid decline in serum concentrations of these molecules observed at the earliest points in the PK curve. While not bound by any theory, this high clearance phenomenon is attributed to the nonspecific binding of high-pI molecules to negatively charged heparin proteoglycans in the vascular system and tissues. These problems were addressed through structural induction engineering of relaxin-2 fusion protein analogs to reduce pI, as shown in Examples 1–5.

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

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

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

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

[0300] Blood samples were collected 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 from jugular vein cannulas into K2EDTA tubing. The tubing was stored on wet ice until centrifugation in a refrigerated centrifuge. The obtained plasma was frozen on dry ice and stored at -80°C. At the end of the study, rats were euthanized by bleeding.

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

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

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

[0304] Additional experiments were conducted to further evaluate the effects of fusion protein analogs SEQ ID NO: 87 and SEQ ID NO: 497 on renal blood flow. The mean potency of relaxin-2 fusion protein analogs in recombinant human and rat RXFP1 assays in these experiments is shown in Table 30 below. [Table 30]

[0305] Sequence ID No. 87 was administered by cannula via femoral vein using a syringe pump. The dose was infused intravenously as a bolus (1 ml / kg), followed by continuous infusion of PBS at a rate of 0.5 mL / kh / hour to maintain the circulating fluid volume.

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

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

[0308] Furthermore, as shown in Figures 5A and 5B, low doses of SEQ ID NO: 87 increased and maintained RABF more effectively than SEQ ID NO: 497. Infusion of SEQ ID NO: 87 at 0.3 mg / kg resulted in an approximately 25% increase in RABF compared to baseline, which was maintained over the 90-minute experimental period. Infusion of SEQ ID NO: 497 at 0.3 mg / kg resulted in an approximately 15% increase in RABF compared to baseline, and RABF levels decreased towards baseline within 90 minutes after infusion (Figure 5A). Quantification of the area under the curve (AUC) showed that infusion of SEQ ID NO: 87 significantly increased RABF by approximately twice as much as infusion of SEQ ID NO: 497 (Figure 5B).

[0309] While not bound by any theory, the enhanced effect of SEQ ID NO: 87 is hypothesized to be due to increased distribution of SEQ ID NO: 87 to target tissues far exceeding the expected levels from plasma levels, due to reduced heparin binding, decreased nonspecific cellular uptake, blood clearance, and the potentially higher bioavailability of SEQ ID NO: 87, compared to the more charged SEQ ID NO: 497 molecule.

[0310] Example 7. Therapeutic effect of relaxin-2 fusion protein analog in a rat pulmonary hypertension model. Pulmonary hypertension (PAH) is characterized by progressive pulmonary vascular remodeling of the peripheral precapillary arteries, leading to a significant increase in right ventricular (RV) load and ultimately to right heart failure and premature death. Using a monoclotaline (MCT) rat model as an in vivo PAH model, rats reproducibly develop pulmonary hypertension with a mean pulmonary pressure of approximately 40 mmHg about 4 weeks after a single dose of MCT. MCT is an 11-membered macrocyclic pyrrolizidine alkaloid derived from the seeds of the plant Crotalaria spectabilis. The MCT alkaloid is activated in the liver to the reactive pyrrole metabolite dehydromonoclotaline (MCTP), a reaction that is highly dependent on cytochrome P-450 (CYP3A4). Upon administration, MCT induces a syndrome characterized, among other symptoms, by pulmonary hypertension (PH), pulmonary mononuclear vasculitis, and right ventricular hypertrophy via damage to pulmonary endothelial cells. The therapeutic potential of the relaxin-2 fusion protein analog of sequence number 87 was evaluated in a rat model of MCT-induced PAH.

[0311] On day 1 of the study, naive young (200-240g) male Sprague Dawley rats in groups 1-5 received MCT at a dose of 60 mg / kg (1 mL / kg sc 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, iv) on days 7, 10, 14, 17, 21, and 24. Starting on day 8 of the study, rats in group 5 were orally administered sildenafil (positive control, 30 mg / kg, po, BID) twice daily, with the final dose administered on day 28 of the study. Rats in group 6 were administered DMSO as a control. Twelve rats were tested in each of groups 1-4, and ten rats were tested in each of groups 5 and 6.

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

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

[0314] Sequence ID No. 87 was administered 10 mg / kg IV twice weekly for 3 weeks in an MCT-induced PAH model, with therapeutic administration initiated one week after MCT infusion. To mitigate the efficacy of reducing anti-drug antibody (ADA) production to the test substance, this study was conducted with and without B-cell depletion by injecting rats with anti-mouse CD20 antibody. Cardiac hemodynamics, including right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP), were measured using a thoracotomy approach. The Fulton index, the ratio of right ventricular weight to left ventricular and septal weight, was measured at week 4. Blood samples were collected for serum NT pro-BNP, PK, and ADA analysis.

[0315] MCT treatment in rats significantly increased RVSP (Figures 6A and 6B), mPAP (Figures 7A and 7B), Fulton index (Figures 8A and 8B), and NT-pro-BNP (Figures 9A and 9B) over 4 weeks compared to the naive group (no MCT), while decreasing survival. As shown, rats treated with Sequence ID No. 87 showed significant improvements in RVSP (Figure 6B), mPAP (Figure 7B), and Fulton index (Figure 8B) in B-cell depleted rats. In addition, there was a clear survival benefit after treatment with Sequence ID No. 87, and the treated rats were the only group to show 100% survival.

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

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

[0318] Example 8. Antifibrotic effect of relaxin-2 fusion protein analog in a mouse model of renal fibrosis. This example describes the evaluation of the antifibrotic effects of relaxin-2 fusion protein analogs SEQ ID NO: 496, SEQ ID NO: 313, and SEQ ID NO: 87 in a mouse unilateral ureteral obstruction (UUO) model of renal fibrosis. The UUO model induces renal fibrosis, and the main feature of UUO is tubular injury for obstruction of urinary flow. Furthermore, experimental UUO in rodents is thought to accelerate the mimicry of human chronic obstructive nephropathy. Renal fibrosis is a common pathway for most forms of progressive kidney disease. Since removal of the obstruction is generally not sufficient to reverse fibrosis, model animals may benefit from concomitant treatments. UUO is a widely used model for studying obstructive nephropathy.

[0319] The initial study evaluated the effects of Sequence ID No. 496 and Sequence ID No. 313. Sixty-five male C57BL / 6 mice were used in the study (8–10 weeks old at baseline). UUO was induced on day 0 by ligation of the ureter of the left kidney, while the contralateral kidney served as a control. The UUO surgery was performed under deep anesthesia following a standard procedure. Briefly, after removing hair from the abdomen, the kidney and ureter were exposed through a midline abdominal incision. The left ureter was completely occluded with two ligations. The first suture was placed 1 mm below the kidney, and the other suture was placed 1 mm below the renal pelvis using silk or prolene sutures. The wound was closed with 2–3 staples. The animals were returned to cages and monitored until they began to move. The animals were given the analgesic buprenorphine on the day of surgery and for 72 hours post-surgery. The control group (n=5) received a sham surgery. UUO mice were treated with either 10 mg / kg of SEQ ID NO: 496 (n=10), 20 mg / kg of SEQ ID NO: 496 (n=10), 10 mg / kg of SEQ ID NO: 313 (n=10), or 20 mg / kg of SEQ ID NO: 313 (n=10). The treatment groups received intravenous injections two days before surgery (-2 days) and on 2 and 5 days post-surgery. The positive control group (n=10) received enalapril, an ACE inhibitor used to treat hypertension, diabetic nephropathy, and heart failure, starting on -2 days and administered via 200 mg / L drinking water, and continued until the end of the study. The negative control group (n=10) was treated with a vehicle (PBS). The study concluded on 7 days post-surgery, and fibrosis symptoms and survival parameters were evaluated.

[0320] No adverse clinical symptoms developed in any of the animals after the UUO surgery. The animals tolerated treatment with SEQ ID NO: 496, SEQ ID NO: 313, and enalapril well, and a 100% survival rate was recorded. On postoperative day 7, the left kidney was harvested, weighed, and fixed for histological examination.

[0321] All groups with surgically induced unuurated urinary ostomies (UUOs) showed weight loss compared to the sham surgery group. On postoperative day 4, weight began to show a trend toward recovery in all UUO groups. Furthermore, all UUO groups showed a significant increase in kidney weight compared to the sham surgery group, and there were no statistically significant changes in kidney weight across the treatment group and the control UUO group.

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

[0323] The antifibrotic effect of SEQ ID NO: 87 was observed using 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 vehicle (Figure 13). Tissue TNFα levels were assessed via electrochemiluminescence, and mice treated with SEQ ID NO: 87 also showed a significant reduction in TNFα levels compared to mice treated with the vehicle (Figure 14). Reductions in IL-1β and IL-6 in fibrous kidneys were observed in mice treated with SEQ ID NO: 87 compared to mice treated with the vehicle.

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

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

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

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

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

[0329] research design Approximately 48 participants were enrolled in this study. A single escalating dose of SEQ ID NO: 87 or the corresponding placebo (i.e., the same buffer without the addition of SEQ ID NO: 87) was administered to the study participants via intravenous infusion (IV) or subcutaneous (SC) injection at the doses described below. In each cohort, six participants received SEQ ID NO: 87 and two participants received placebo. Infusion times ranged from 30 to 60 minutes. Cohort A received 0.3 mg / kg of IV SEQ ID NO: 87 or the corresponding placebo. Cohort B received 1 mg / kg of IV SEQ ID NO: 87 or the corresponding placebo. Cohort C received 300 mg of SC SEQ ID NO: 87 or the corresponding placebo. In some cases, Cohort C was administered concurrently with Cohort B. Cohort D received 3 mg / kg of IV SEQ ID NO: 87 or the corresponding placebo. Cohort E received 600 mg of SC SEQ ID NO: 87 or the corresponding placebo. In some cases, cohort E is administered concurrently with cohort D. Cohort F receives 10 mg / kg of IV SEQ ID NO: 87 or the corresponding placebo.

[0330] Master randomization schedules and code-break envelopes were created and delivered to the facility's open-label pharmacy prior to administration. Active and placebo products were injected via covered lines to conceal the activity from the placebo. Printed randomization schedules were generated using an exchange-lock fixing method. Individuals receiving, administering, and analyzing outcomes and data were blinded.

[0331] Primary Outcome The primary outcome of this study is to evaluate the safety and tolerability of SEQ ID NO: 87 after a single dose escalation. The primary outcome will be assessed by monitoring: 1) the incidence of adverse events (AEs) and serious adverse events (SAEs); 2) changes in clinical laboratory safety parameters, including hematological, serological, and coagulation studies; 3) changes in vital signs measurements; and 4) changes in electrocardiogram (ECG) findings. Adverse events will be assessed by clinical laboratory testing, patient data review, and self-reporting. Adverse events will be collected through oral interviews by clinic staff during the study period, either in person during hospitalization or clinic visits, or via telephone during remote contact. Measurement of vital signs will include resting heart rate, systolic, and diastolic blood pressure (BP) using standard manual or electronic clinical procedures. These procedures will be completed using standard nursing practice, or, in the case of ECG, using instructions provided by the manufacturer. These individual data will be used to determine safety by physician assessment. Furthermore, the overall data presented will also be considered in determining safety.

[0332] Adverse and serious adverse events are screened for on days -1, 1, 2, 3, 6, 8, 15, 29, 43, and 57 after administration. Laboratory safety parameters are screened for on days -1, 1, 2, 8, 15, 29, 43, and 57 after administration. Vital signs are screened for on days -1, 1, 2, 3, 6, 8, 15, 29, 43, and 57 after administration. ECG is screened for on days -1, 1, and 2 after administration.

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

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

[0335] Eligibility Participants must meet the following criteria to be enrolled in this study: • Males aged 18-55 or females who are not capable of childbirth. Based on medical history, physical examination, vital signs, electrocardiogram, and routine clinical tests, the patient is judged to be in good health. • At the time of screening, they have a body mass index (BMI) of 18-32 kg per square meter. I understand the research procedures and agree to participate in the research by providing written informed consent. • Must be at least 18 years old and no older than 55 years old.

[0336] Participants were excluded from the study if they met any of the following criteria: • The person is mentally or legally incapacitated, has significant emotional problems at the time of the study, or has a history of a serious mental illness at the discretion of the principal investigator. • Any clinically significant physical examination abnormalities were observed during the screening visit, or the principal investigator deemed the patient unsuitable for participation in the study. • Having clinically significant abnormalities in complete blood count, clinical chemistry, or urine analysis at screening or on day 1-1 (in asymptomatic participants, abnormal test results, including creatine phosphokinase within 3 times the upper limit of normal, with a suspected cause due to strenuous physical activity, may be repeated once during the screening period). • The patient was hospitalized for any reason within 30 days of their screening visit. • Having a history of any clinically significant renal, neurological, gastrointestinal, hepatic, or respiratory disease (note that subjects with completely resolved childhood asthma without recurrence in adulthood may be enrolled). • In the opinion of the principal investigator, the patient has a history of anaphylaxis or other serious allergies. • A history of clinically significant cardiovascular disease, including arrhythmias, conduction abnormalities, or clinically significant vital signs. • The patient has previously received relaxin or relaxin fusion protein. • In any clinical trial evaluating another investigational drug (including biological agents) or therapy (including specific immunotherapy), the drug was administered within 90 days prior to the screening visit, or within 5 half-lives of the investigational drug (whichever is longer), or within 4 weeks in the case of another investigational drug.

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

number

[0338] Sequence ID 87 also demonstrated a desirable PK profile in healthy human patients administered a single 150 mg SC dose, for which the terminal half-life has not yet been determined (Figure 18). Based on the data, the bioavailability of SC ranged from 50% to 60%.

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

[0340] Example 11. A single-dose, open-label study to evaluate the safety, tolerability, and hemodynamic effects of relaxin-2 fusion protein analogs. This predictive example describes a single-dose, open-label study to evaluate the safety, tolerability, and hemodynamics of Sequence ID No. 87 in subjects with combined post-capillary and pre-capillary pulmonary hypertension (CpcPH), or isolated post-capillary pulmonary hypertension (IpcPH), and heart failure with preserved ejection fraction (HFpEF).

[0341] Background and Logical Basis Sequence ID No. 87 is a relaxin peptide Fc fusion biologic being developed for the treatment of pre-capillary and post-capillary pulmonary hypertension (CpcPH) and isolated post-capillary pulmonary hypertension (IpcPH). Relaxin is an insulin-like peptide originally identified as a pregnancy-related hormone, possessing anti-fibrotic, anti-inflammatory, and vasodilatory properties in both men and women. These effects are produced by binding to and activating its G protein-binding receptor, relaxin family peptide receptor 1 (RXFP1), which then triggers various signaling pathways, including cAMP (cyclic adenosine monophosphate), cGMP (cyclic guanosine monophosphate), and MAPK (mitogen-activated protein kinase), as well as altering the gene expression of transforming growth factor beta (TGF-β), MMP (matrix metalloproteinase), angiogenic growth factor, and endothelin-1 receptor.

[0342] Pulmonary hypertension (PH) is a condition characterized by dyspnea, decreased exercise capacity, and hypoxemia, and carries a high risk of death. It is defined as mean pulmonary artery pressure (mPAP) ≥ 20 mmHg. Because this condition occurs in many different clinical settings, the World Health Organization (WHO) classifies PH into five different groups. CpcPH is a subgroup of pulmonary hypertension (PH) due to left-sided heart disease (PH-LHD), which is WHO group 2. LHD includes: heart failure with maintained ejection fraction (EF), defined as heart failure with an ejection fraction greater than 50% (HFpEF); heart failure wi...

Claims

1. From the N-terminus to the C-terminus, A first peptide containing the amino acid sequence of SEQ ID NO: 1, A first linker peptide containing the amino acid sequence of SEQ ID NO: 21, A fusion protein comprising a second peptide containing the amino acid sequence of SEQ ID NO:

8.

2. The fusion protein according to claim 1, wherein the amino acid sequence of the first peptide is 27, 28, or 29 amino acids long.

3. The fusion protein according to claim 1, wherein the amino acid sequence of the first peptide is the amino acid sequence of SEQ ID NO:

1.

4. The fusion protein according to claim 1, wherein the amino acid sequence of the first linker peptide is 13, 14, or 15 amino acids long.

5. The fusion protein according to claim 1, wherein the amino acid sequence of the first linker peptide is the amino acid sequence of SEQ ID NO:

21.

6. The fusion protein according to claim 1, wherein the amino acid sequence of the second peptide is 24 or 25 amino acids long.

7. The fusion protein according to claim 1, wherein the amino acid sequence of the second peptide is the amino acid sequence of SEQ ID NO:

8.

8. The fusion protein according to claim 1, wherein the amino acid sequence of the second peptide is the amino acid sequence of SEQ ID NO:

260.

9. The fusion protein according to claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:

31.

10. The fusion protein according to claim 1, wherein the amino acid sequence of the fusion protein consists of the amino acid sequence of SEQ ID NO:

31.

11. The fusion protein according to claim 1, further comprising an IgG Fc polypeptide.

12. The fusion protein according to claim 11, wherein the IgG Fc polypeptide comprises the amino acid sequence of human IgG1 Fc.

13. The fusion protein according to claim 11, wherein the IgG Fc polypeptide comprises alanine at EU position 329; alanine at EU positions 234 and 235, respectively; alanine at EU positions 234, 235, and 329, respectively; leucine and serine at EU positions 428 and 434, respectively; or alanine, alanine, alanine, leucine, and serine at EU positions 234, 235, 329, 428, and 434, respectively.

14. The fusion protein according to claim 11, wherein the IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 79 or 83.

15. The fusion protein according to claim 11, wherein the IgG Fc polypeptide is linked to the N-terminus of the first peptide.

16. The fusion protein according to claim 15, wherein the IgG Fc polypeptide is linked to the N-terminus of the first peptide via a second linker peptide.

17. The fusion protein according to claim 16, wherein the second linker peptide comprises the amino acid sequence GGS or EGGS (SEQ ID NO: 299).

18. The fusion protein according to claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:

87.

19. A homodimer comprising two fusion proteins according to Claim 18.

20. The fusion protein according to claim 1, wherein the amino acid sequence of the fusion protein is the amino acid sequence of SEQ ID NO:

87.

21. A homodimer comprising two fusion proteins according to claim 20.

22. A polynucleotide comprising a nucleotide sequence encoding a fusion protein according to any one of claims 1 to 18 and 20.

23. The polynucleotide according to claim 22, wherein the polynucleotide is a DNA molecule or an RNA molecule.

24. An expression vector comprising the polynucleotide described in claim 22.

25. The expression vector according to claim 24, wherein the expression vector is a plasmid vector or a viral vector.

26. A host cell comprising the polynucleotide described in claim 22.

27. The aforementioned host cells It is either a prokaryotic cell or a eukaryotic cell; E. Prokaryotic cells selected from coli cells or Bacillus cells; A eukaryotic cell selected from the group consisting of yeast cells, insect cells, and mammalian cells; or The host cell according to claim 26, which is a mammalian cell selected from the group consisting of CHO cells, HeLa cells, and HEK293 cells.

28. A method for producing the fusion protein according to any one of claims 1 to 18 and 20, comprising culturing the host cell according to claim 26 or 27 under conditions such that the fusion protein is produced.

29. A host cell comprising the expression vector described in Claim 24.

30. The host cell, It is either a prokaryotic cell or a eukaryotic cell; E. Prokaryotic cells selected from coli cells or Bacillus cells; A eukaryotic cell selected from the group consisting of yeast cells, insect cells, and mammalian cells; or The host cell according to claim 29, which is a mammalian cell selected from the group consisting of CHO cells, HeLa cells, and HEK293 cells.

31. A method for producing a fusion protein according to any one of claims 1 to 18 and 20, comprising culturing the host cell according to claim 29 or 30 under conditions such that the fusion protein is produced.

32. A pharmaceutical composition comprising an effective amount of the fusion protein described in any one of claims 1 to 18 and 20.

33. The pharmaceutical composition according to claim 32, for use in enhancing relaxin-2 related activity in cells or activating relaxin-2 receptors (RXFP1) on cells.

34. The pharmaceutical composition according to claim 33, wherein enhancement of relaxin 2-related activity in the cells or activation of RXFP1 on the cells increases the intracellular cAMP level.

35. The pharmaceutical composition according to claim 33, wherein the cells are selected from the group consisting of endothelial cells, vascular smooth muscle cells, other vascular cells, cardiomyocytes, other cardiac cells, and fibroblasts.

36. The pharmaceutical composition according to claim 33 for use in the treatment of relaxin 2-related disorders in the subject.

37. Use of the fusion protein according to any one of claims 1 to 18 and 20 for the manufacture of a pharmaceutical product for the treatment of relaxin 2-related disorders in a subject.

38. The use according to claim 37, wherein the relaxin 2-related disorder is selected from the group consisting of renal disease, fibrotic disease, and cardiovascular disease.

39. The use according to claim 37, wherein the relaxin 2-related disorder is selected from the group consisting of pulmonary hypertension, pulmonary arterial hypertension (PAH), pulmonary hypertension due to left heart disease (PH-LHD), complicated precapillary and postcapillary pulmonary hypertension (CpcPH), isolated postcapillary pulmonary hypertension (IpcPH), heart failure, heart failure with maintained ejection fraction (HFpEF), heart failure with moderate ejection fraction (HFmrEF), heart failure with reduced ejection fraction (HFrEF), valvular heart disease, joint disease, periarthritis of the shoulder (also known as adhesive capsulitis), kidney disease, chronic kidney disease, and hypertensive kidney disease.

40. The relaxin 2-related disorder described above is Precapillary and postcapillary pulmonary hypertension (CpcPH) associated with heart failure with preserved ejection fraction (HFpEF), Isolated retrocapillary pulmonary hypertension (IpcPH) with heart failure with preserved ejection fraction (HFpEF), Precapillary and postcapillary pulmonary hypertension (CpcPH) associated with heart failure with moderate ejection fraction (HFmrEF), or The use according to claim 37, which is isolated retrocapillary pulmonary hypertension (IpcPH) accompanied by heart failure with moderate ejection fraction (HFmrEF).

Images