Anti-NPR1 antibodies and their pharmaceutical use

Abstract

The present invention provides an anti-NPR1 antibody, a pharmaceutical composition containing the antibody, and an immune complex. Further, it provides an isolated nucleic acid encoding the NPR1 antibody, a host cell containing the isolated nucleic acid, a method for detecting an NPR1 peptide or fragment thereof in a sample using the NPR1 antibody, and a method for treating, preventing, or improving NPR1-related diseases or conditions, including hypertension, using the NPR1 antibody, the pharmaceutical composition, and the immune complex.

Description

[Technical Field]

[0001] This application claims priority to Chinese patent application CN202310675148.4, filed on 8 June 2023.

[0002] This disclosure belongs to the field of biotechnology, and more specifically, this disclosure relates to anti-NPR1 antibodies and their pharmaceutically acceptable use. [Background technology]

[0003] The descriptions provided are not necessarily based on prior art and only provide background information relevant to this disclosure.

[0004] The natriuretic peptide system plays a crucial role in the homeostasis of the kidneys, cardiovascular system, endocrine system, nervous system, and skeletal system. This system consists of polypeptide ligands and membrane receptors. Polypeptide ligands include ANP, BNP, and CNP, while membrane receptors include NPR1 (NPRA), NPR2 (NPRB), and NPR3 (NPRC). Of these, NPR1 is primarily expressed in the heart, kidneys, lungs, and aorta. By interacting with ligands ANP or BNP, it generates allosterics and activates downstream guanylate cyclase activity, catalyzing intracellular guanosine triphosphate (GTP) cyclization to form cyclic guanosine monophosphate (cGMP). This further activates cGMP-dependent protein kinase (protein kinase G), achieving biological functions such as vasodilation, blood pressure reduction, sodium excretion / diuresis, anti-myocardial hypertrophy, and anti-fibrotic effects. Therefore, activation of this pathway can be utilized in the treatment of heart failure and hypertension. [Overview of the project] [Problems that the invention aims to solve]

[0005] Conventional recombinant ANP and BNP are already approved for the treatment of acute heart failure and are marketed in China, Japan, and the United States. While these types of recombinant polypeptides have good effects in lowering blood pressure and alleviating heart failure symptoms, they do not significantly improve readmission rates and mortality rates, which may be related to their relatively short pharmacokinetic (PK) and inability to administer them long-term. Therefore, they are not approved for the indication of chronic heart failure.

[0006] Novartis and Regeneron have each developed NPR1 agonist antibodies. Regeneron's REGN-5381 (WO2020086406A2) is currently undergoing Phase 2 safety validation clinical trials in patients with heart failure, while Novartis's XXB750 (WO2020250159A1) is undergoing Phase 2 efficacy clinical trials in patients with refractory hypertension. [Means for solving the problem]

[0007] This disclosure relates to an anti-NPR1 antibody comprising a heavy chain variable region including HCDR1, HCDR2, and HCDR3, and a light chain variable region including LCDR1, LCDR2, and LCDR3, a. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NOs. 82, 80, 81, or 15, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NOs. 85, 83, 84, or 16, or b. The heavy chain variable region HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in any one of the sequences of SEQ ID NOs. 51, 48, 49, 50, 52, or 7, and the light chain variable region LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NOs. 56, 53, 54, 55, or 8, or c. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NOs. 68, 66, 67, 69, or 11, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NOs. 74, 70, 71, 72, or 12, or d. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NOs. 78, 76, 77, or 13, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NOs. 79 or 14, or e. The heavy chain variable region HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NOs. 58, 59, 60, or 9, and the light chain variable region LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NOs. 61, 62, 63, 64, 65, or 10. We provide an anti-NPR1 antibody.

[0008] In some embodiments, the above anti-NPR1 antibody, among which, a. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 82, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 85, or b. The heavy chain variable region HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in any one of the sequences of SEQ ID NO: 51, 48, or 52, and the light chain variable region LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 56, or c. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 68, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 74 or 70, or d. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 78 or 76, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 79, or e. The heavy chain variable region HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 58 or 9, and the light chain variable region LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 61 or 10, Anti-NPR1 antibody.

[0009] In some embodiments, the above anti-NPR1 antibody, among which, a. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 82, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 85, or b. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 51, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 56, or c. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 68, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 74, or d. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 78, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 79, or e. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 58, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 61. Anti-NPR1 antibody.

[0010] In some embodiments, the above anti-NPR1 antibody, among which, a. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 82, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 85, or b. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 51, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 56, or c. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 68, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 74, or d. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 78, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 79. Anti-NPR1 antibody.

[0011] In some embodiments, the above anti-NPR1 antibody, among which, a. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 15, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 16, or b. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 7, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 8, or c. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 11, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 12, or d. The HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 13, and the LCDR1, LCDR2, and LCDR3 of the light-chain variable region each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 14, or e. The HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 9, and the LCDR1, LCDR2, and LCDR3 of the light-chain variable region each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 10, An anti-NPR1 antibody.

[0012] In some embodiments, the anti-NPR1 antibody according to any one of the above, wherein the HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region and the LCDR1, LCDR2, and LCDR3 of the light-chain variable region are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region and the LCDR1, LCDR2, and LCDR3 of the light-chain variable region are defined according to the Kabat numbering rule. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region and the LCDR1, LCDR2, and LCDR3 of the light-chain variable region are defined according to the IMGT numbering rule. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region and the LCDR1, LCDR2, and LCDR3 of the light-chain variable region are defined according to the Chothia numbering rule. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region and the LCDR1, LCDR2, and LCDR3 of the light-chain variable region are defined according to the AbM numbering rule. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region and the LCDR1, LCDR2, and LCDR3 of the light-chain variable region are defined according to the Contact numbering rule.

[0013] In some embodiments, it is an anti-NPR1 antibody according to any one of the above, among which, a. the heavy chain variable region has HCDR1 containing the amino acid sequence of SEQ ID NO: 29, HCDR2 containing the amino acid sequence of SEQ ID NO: 40, and HCDR3 containing the amino acid sequence of SEQ ID NO: 41, and the light chain variable region has LCDR1 containing the amino acid sequence of SEQ ID NO: 42, LCDR2 containing the amino acid sequence of SEQ ID NO: 33, and LCDR3 containing the amino acid sequence of SEQ ID NO: 34, or b. the heavy chain variable region has HCDR1 containing the amino acid sequence of SEQ ID NO: 45, 17 or 35, HCDR2 containing the amino acid sequence of SEQ ID NO: 46 or 18, and HCDR3 containing the amino acid sequence of SEQ ID NO: 19, and the light chain variable region has LCDR1 containing the amino acid sequence of SEQ ID NO: 20, LCDR2 containing the amino acid sequence of SEQ ID NO: 21, and LCDR3 containing the amino acid sequence of SEQ ID NO: 22, or c. the heavy chain variable region has HCDR1 containing the amino acid sequence of SEQ ID NO: 29, HCDR2 containing the amino acid sequence of SEQ ID NO: 30, and HCDR3 containing the amino acid sequence of SEQ ID NO: 31, and the light chain variable region has LCDR1 containing the amino acid sequence of SEQ ID NO: 32, LCDR2 containing the amino acid sequence of SEQ ID NO: 73 or 33, and LCDR3 containing the amino acid sequence of SEQ ID NO: 34, or d. the heavy chain variable region has HCDR1 containing the amino acid sequence of SEQ ID NO: 35, HCDR2 containing the amino acid sequence of SEQ ID NO: 75 or 36, and HCDR3 containing the amino acid sequence of SEQ ID NO: 37, and the light chain variable region has LCDR1 containing the amino acid sequence of SEQ ID NO: 38, LCDR2 containing the amino acid sequence of SEQ ID NO: 21, and LCDR3 containing the amino acid sequence of SEQ ID NO: 39, or e. the heavy chain variable region has HCDR1 containing the amino acid sequence of SEQ ID NO: 23, HCDR2 containing the amino acid sequence of SEQ ID NO: 57 or 24, and HCDR3 containing the amino acid sequence of SEQ ID NO: 25, and the light chain variable region has LCDR1 containing the amino acid sequence of SEQ ID NO: 26, LCDR2 containing the amino acid sequence of SEQ ID NO: 27, and LCDR3 containing the amino acid sequence of SEQ ID NO: 28, Anti-NPR1 antibody.

[0014] In some embodiments, an anti-NPR1 antibody as described in any one of the above, a. The heavy chain variable region may be such that HCDR1 contains the amino acid sequence of SEQ ID NO: 29, HCDR2 contains the amino acid sequence of SEQ ID NO: 40, and HCDR3 contains the amino acid sequence of SEQ ID NO: 41, and the light chain variable region may be such that LCDR1 contains the amino acid sequence of SEQ ID NO: 42, LCDR2 contains the amino acid sequence of SEQ ID NO: 33, and LCDR3 contains the amino acid sequence of SEQ ID NO: 34, or b. The heavy chain variable region may be such that HCDR1 contains the amino acid sequence of SEQ ID NO: 45, HCDR2 contains the amino acid sequence of SEQ ID NO: 46, and HCDR3 contains the amino acid sequence of SEQ ID NO: 19, and the light chain variable region may be such that LCDR1 contains the amino acid sequence of SEQ ID NO: 20, LCDR2 contains the amino acid sequence of SEQ ID NO: 21, and LCDR3 contains the amino acid sequence of SEQ ID NO: 22, or The heavy chain variable region may be such that HCDR1 contains the amino acid sequence of SEQ ID NO: 17 or 35, HCDR2 contains the amino acid sequence of SEQ ID NO: 18, and HCDR3 contains the amino acid sequence of SEQ ID NO: 19, and the light chain variable region may be such that LCDR1 contains the amino acid sequence of SEQ ID NO: 20, LCDR2 contains the amino acid sequence of SEQ ID NO: 21, and LCDR3 contains the amino acid sequence of SEQ ID NO: 22, or c. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 29, HCDR2 contains the amino acid sequence of SEQ ID NO: 30, and HCDR3 contains the amino acid sequence of SEQ ID NO: 31, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 32, LCDR2 contains the amino acid sequence of SEQ ID NO: 73 or 33, and LCDR3 contains the amino acid sequence of SEQ ID NO: 34, or d. The heavy chain variable region may be such that HCDR1 contains the amino acid sequence of SEQ ID NO: 35, HCDR2 contains the amino acid sequence of SEQ ID NO: 75 or 36, and HCDR3 contains the amino acid sequence of SEQ ID NO: 37, and the light chain variable region may be such that LCDR1 contains the amino acid sequence of SEQ ID NO: 38, LCDR2 contains the amino acid sequence of SEQ ID NO: 21, and LCDR3 contains the amino acid sequence of SEQ ID NO: 39, or e. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 23, HCDR2 contains the amino acid sequence of SEQ ID NO: 57 or 24, and HCDR3 contains the amino acid sequence of SEQ ID NO: 25, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 26, LCDR2 contains the amino acid sequence of SEQ ID NO: 27, and LCDR3 contains the amino acid sequence of SEQ ID NO: 28. Anti-NPR1 antibody.

[0015] In some embodiments, an anti-NPR1 antibody as described in any one of the above, a. The heavy chain variable region may be such that HCDR1 contains the amino acid sequence of SEQ ID NO: 29, HCDR2 contains the amino acid sequence of SEQ ID NO: 40, and HCDR3 contains the amino acid sequence of SEQ ID NO: 41, and the light chain variable region may be such that LCDR1 contains the amino acid sequence of SEQ ID NO: 42, LCDR2 contains the amino acid sequence of SEQ ID NO: 33, and LCDR3 contains the amino acid sequence of SEQ ID NO: 34, or b. The heavy chain variable region may be such that HCDR1 contains the amino acid sequence of SEQ ID NO: 45, HCDR2 contains the amino acid sequence of SEQ ID NO: 46, and HCDR3 contains the amino acid sequence of SEQ ID NO: 19, and the light chain variable region may be such that LCDR1 contains the amino acid sequence of SEQ ID NO: 20, LCDR2 contains the amino acid sequence of SEQ ID NO: 21, and LCDR3 contains the amino acid sequence of SEQ ID NO: 22, or c. The heavy chain variable region may be such that HCDR1 contains the amino acid sequence of SEQ ID NO: 29, HCDR2 contains the amino acid sequence of SEQ ID NO: 30, and HCDR3 contains the amino acid sequence of SEQ ID NO: 31, and the light chain variable region may be such that LCDR1 contains the amino acid sequence of SEQ ID NO: 32, LCDR2 contains the amino acid sequence of SEQ ID NO: 73, and LCDR3 contains the amino acid sequence of SEQ ID NO: 34, or d. The heavy chain variable region may be such that HCDR1 contains the amino acid sequence of SEQ ID NO: 35, HCDR2 contains the amino acid sequence of SEQ ID NO: 75, and HCDR3 contains the amino acid sequence of SEQ ID NO: 37, and the light chain variable region may be such that LCDR1 contains the amino acid sequence of SEQ ID NO: 38, LCDR2 contains the amino acid sequence of SEQ ID NO: 21, and LCDR3 contains the amino acid sequence of SEQ ID NO: 39, or e. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 23, HCDR2 contains the amino acid sequence of SEQ ID NO: 57, and HCDR3 contains the amino acid sequence of SEQ ID NO: 25, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 26, LCDR2 contains the amino acid sequence of SEQ ID NO: 27, and LCDR3 contains the amino acid sequence of SEQ ID NO: 28. Anti-NPR1 antibody.

[0016] In some embodiments, an anti-NPR1 antibody as described in any one of the above, a. The heavy chain variable region may be such that HCDR1 contains the amino acid sequence of SEQ ID NO: 29, HCDR2 contains the amino acid sequence of SEQ ID NO: 40, and HCDR3 contains the amino acid sequence of SEQ ID NO: 41, and the light chain variable region may be such that LCDR1 contains the amino acid sequence of SEQ ID NO: 42, LCDR2 contains the amino acid sequence of SEQ ID NO: 33, and LCDR3 contains the amino acid sequence of SEQ ID NO: 34, or b. The heavy chain variable region may be such that HCDR1 contains the amino acid sequence of SEQ ID NO: 17, HCDR2 contains the amino acid sequence of SEQ ID NO: 18, and HCDR3 contains the amino acid sequence of SEQ ID NO: 19, and the light chain variable region may be such that LCDR1 contains the amino acid sequence of SEQ ID NO: 20, LCDR2 contains the amino acid sequence of SEQ ID NO: 21, and LCDR3 contains the amino acid sequence of SEQ ID NO: 22, or c. The heavy chain variable region may be such that HCDR1 contains the amino acid sequence of SEQ ID NO: 29, HCDR2 contains the amino acid sequence of SEQ ID NO: 30, and HCDR3 contains the amino acid sequence of SEQ ID NO: 31, and the light chain variable region may be such that LCDR1 contains the amino acid sequence of SEQ ID NO: 32, LCDR2 contains the amino acid sequence of SEQ ID NO: 33, and LCDR3 contains the amino acid sequence of SEQ ID NO: 34, or d. The heavy chain variable region may be such that HCDR1 contains the amino acid sequence of SEQ ID NO: 35, HCDR2 contains the amino acid sequence of SEQ ID NO: 36, and HCDR3 contains the amino acid sequence of SEQ ID NO: 37, and the light chain variable region may be such that LCDR1 contains the amino acid sequence of SEQ ID NO: 38, LCDR2 contains the amino acid sequence of SEQ ID NO: 21, and LCDR3 contains the amino acid sequence of SEQ ID NO: 39, or e. The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 23, HCDR2 contains the amino acid sequence of SEQ ID NO: 24, and HCDR3 contains the amino acid sequence of SEQ ID NO: 25, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 26, LCDR2 contains the amino acid sequence of SEQ ID NO: 27, and LCDR3 contains the amino acid sequence of SEQ ID NO: 28. Anti-NPR1 antibody.

[0017] In some embodiments, the anti-NPR1 antibody described in any one of the above, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to Kabat numbering rules.

[0018] In some embodiments, an anti-NPR1 antibody as described in any one of the above, wherein the anti-NPR1 antibody is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0019] In some embodiments, an anti-NPR1 antibody as described in any one of the above, comprising a framework region (FR) of a human antibody.

[0020] In some embodiments, an anti-NPR1 antibody as described in any one of the above, wherein the heavy chain variable region comprises FR1, FR2, FR3 derived from IGHV1-46*01 and FR4 derived from IGHJ6*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1E, 40R, 43R, 48I, 69L, 71V, 73K, 74P, 75S, 77P, 78A, 80I, and 81Q, and / or the light chain variable region comprises FR1, FR2, FR3 derived from IGKV3-11*01 and FR4 derived from IGKJ2*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 20Q, 58V, 60D, 70S, 71Y, 77R, and 100A. In some embodiments, the anti-NPR1 antibody is one in which the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 29, HCDR2 containing the amino acid sequence of SEQ ID NO: 40, and HCDR3 containing the amino acid sequence of SEQ ID NO: 41, and the FR of the heavy chain variable region is either unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 1E, 40R, 43R, 48I, 69L, 71V, 73K, 74P, 75S, 77P, 78A, 80I, and 81Q, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 42, LCDR2 containing the amino acid sequence of SEQ ID NO: 33, and LCDR3 containing the amino acid sequence of SEQ ID NO: 34, and the FR of the light chain variable region is either unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 20Q, 58V, 60D, 70S, 71Y, 77R, and 100A. In some embodiments, the variable region and CDR are defined according to Kabat numbering rules.

[0021] In some embodiments, an anti-NPR1 antibody as described in any one of the above, wherein the heavy chain variable region comprises FR1, FR2, FR3 derived from IGHV3-21*01 and FR4 derived from IGHJ6*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1D, 3K, 9E, 44R, 49A, 87S, and 93T, and / or the light chain variable region comprises FR1, FR2, FR3 derived from IGKV3-11*01 or IGKV1-39*01 and FR4 derived from IGKJ4*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 4L, 43S, 45K, 46R, 47W, 58V, and 71Y. In some embodiments, the anti-NPR1 antibody is one in which the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 45, 17, or 35, HCDR2 containing the amino acid sequence of SEQ ID NO: 46, and HCDR3 containing the amino acid sequence of SEQ ID NO: 19, and the FR of the heavy chain variable region is either unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 1D, 3K, 9E, 44R, 49A, 87S, and 93T, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 20, LCDR2 containing the amino acid sequence of SEQ ID NO: 21, and LCDR3 containing the amino acid sequence of SEQ ID NO: 22, and the FR of the light chain variable region is either unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 4L, 43S, 45K, 46R, 47W, 58V, and 71Y.In some embodiments, the anti-NPR1 antibody is one in which the heavy chain variable region is such that HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, 17, or 35, HCDR2 comprises the amino acid sequence of SEQ ID NO: 18, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the FR of the heavy chain variable region is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1D, 3K, 9E, 44R, 49A, 87S, and 93T, and the light chain variable region is such that LCDR1 comprises the amino acid sequence of SEQ ID NO: 20, LCDR2 comprises the amino acid sequence of SEQ ID NO: 21, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 22, and the FR of the light chain variable region is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 4L, 43S, 45K, 46R, 47W, 58V, and 71Y. In some embodiments, the variable region and CDR are defined according to Kabat numbering rules.

[0022] In some embodiments, an anti-NPR1 antibody as described in any one of the above, wherein the heavy chain variable region comprises FR1, FR2, FR3 derived from IGHV1-46*01 and FR4 derived from IGHJ6*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1E, 40R, 43R, 69L, 71V, 73K, 74P, 75S, 77P, and 78A, and / or the light chain variable region comprises FR1, FR2, FR3 derived from IGKV3-20*02 or 6-21*02 and FR4 derived from IGKJ2*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 22K, 42S, 43S, 49Y, 57R, and 71Y. In some embodiments, the anti-NPR1 antibody is one in which the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 29, HCDR2 containing the amino acid sequence of SEQ ID NO: 30, and HCDR3 containing the amino acid sequence of SEQ ID NO: 31, and the FR of the heavy chain variable region is either unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 1E, 40R, 43R, 69L, 71V, 73K, 74P, 75S, 77P, and 78A, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 32, LCDR2 containing the amino acid sequence of SEQ ID NO: 73 or 33, and LCDR3 containing the amino acid sequence of SEQ ID NO: 34, and the FR of the light chain variable region is either unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 22K, 42S, 43S, 49Y, 57R, and 71Y. In some embodiments, the variable region and CDR are defined according to Kabat numbering rules.

[0023] In some embodiments, an anti-NPR1 antibody as described in any one of the above, wherein the heavy chain variable region comprises FR1, FR2, FR3 derived from IGHV3-21*01 and FR4 derived from IGHJ6*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1D, 3K, 9E, 28S, 29I, 44R, 49A, and 93T, and / or the light chain variable region comprises FR1, FR2, FR3 derived from IGKV1-39*01 and FR4 derived from IGKJ4*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 4L, 43S, 46R, 47W, and 71Y. In some embodiments, the anti-NPR1 antibody is one in which the heavy chain variable region comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 35, HCDR2 comprising the amino acid sequence of SEQ ID NO: 75 or 36, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 37, and the FR of the heavy chain variable region is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1D, 3K, 9E, 28S, 29I, 44R, 49A, and 93T, and the light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 38, LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 39, and the FR of the light chain variable region is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 4L, 43S, 46R, 47W, and 71Y. In some embodiments, the variable regions and CDRs are defined according to Kabat numbering rules.

[0024] In some embodiments, an anti-NPR1 antibody as described in any one of the above, wherein the heavy chain variable region comprises FR1, FR2, FR3 derived from IGHV1-46*01 and FR4 derived from IGHJ6*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1E, 2A, 3Y, 28S, 43K, 69L, 71V, and 73K, and / or the light chain variable region comprises FR1, FR2, FR3 derived from IGKV4-1*01 or 1-39*01 and FR4 derived from IGKJ2*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1N, 4L, 17Q, 43P, 46V, 60A, 68R, and 79E. In some embodiments, the anti-NPR1 antibody is one in which the heavy chain variable region comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 23, HCDR2 comprising the amino acid sequence of SEQ ID NO: 57 or 24, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 25, and the FR of the heavy chain variable region is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1E, 2A, 3Y, 28S, 43K, 69L, 71V, and 73K, and the light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 26, LCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 28, and the FR of the light chain variable region is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1N, 4L, 17Q, 43P, 46V, 60A, 68R, and 79E. In some embodiments, the variable region and CDR are defined according to Kabat numbering rules.

[0025] In some embodiments, an anti-NPR1 antibody as described in any one of the above, a. The heavy chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 82, 80, or 81, and the light chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 85, 83, or 84, or b. The heavy chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NOs. 51, 48, 49, 50, or 52, and the light chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NOs. 56, 53, 54, or 55, or at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, or c. The heavy chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NOs. 68, 66, 67, or 69, and the light chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NOs. 74, 70, 71, or 72, or at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, or d. The heavy chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 78, 76, or 77, and the light chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 79, or e. The heavy chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NOs. 58, 59, or 60, and the light chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NOs. 61, 62, 63, 64, or 65, or at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, or f. The heavy chain variable region contains an amino acid sequence having at least 80% (for example, at least 81%, 83%, 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 15, and the light chain variable region contains an amino acid sequence having at least 80% (for example, at least 81%, 83%, 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 16, or g. The heavy chain variable region contains an amino acid sequence having at least 80% (for example, at least 81%, 83%, 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 7, and the light chain variable region contains an amino acid sequence having at least 80% (for example, at least 81%, 83%, 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 8, or h. The heavy chain variable region contains an amino acid sequence having sequence identity with SEQ ID NO: 11 or at least 80% (for example, at least 81%, 83%, 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) thereto, and the light chain variable region contains an amino acid sequence having sequence identity with SEQ ID NO: 12 or at least 80% (for example, at least 81%, 83%, 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) thereto, or i. The heavy chain variable region contains an amino acid sequence having at least 80% (for example, at least 81%, 83%, 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 13, and the light chain variable region contains an amino acid sequence having at least 80% (for example, at least 81%, 83%, 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 14, or j. An anti-NPR1 antibody wherein the heavy chain variable region contains an amino acid sequence having at least 80% (e.g., at least 81%, 83%, 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 9, and the light chain variable region contains an amino acid sequence having at least 80% (e.g., at least 81%, 83%, 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 10.

[0026] In some embodiments, an anti-NPR1 antibody as described in any one of the above, a. The heavy chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 82, 80, or 81, and the light chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 85, 83, or 84, or b. The heavy chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NOs. 51, 48, 49, 50, or 52, and the light chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NOs. 56, 53, 54, or 55, or at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, or c. The heavy chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NOs. 68, 66, 67, or 69, and the light chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NOs. 74, 70, 71, or 72, or at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, or d. The heavy chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 78, 76, or 77, and the light chain variable region includes an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 79, or e. An anti-NPR1 antibody wherein the heavy chain variable region contains an amino acid sequence having at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NOs. 58, 59, or 60, and the light chain variable region contains an amino acid sequence having at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NOs.

[0027] In some embodiments, an anti-NPR1 antibody as described in any one of the above, a. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 82, 80, or 81, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 85, 83, or 84, or b. The heavy chain variable region includes the amino acid sequence of SEQ ID NOs. 51, 48, 49, 50, or 52, and the light chain variable region includes the amino acid sequence of SEQ ID NOs. 56, 53, 54, or 55, or c. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 68, 66, 67, or 69, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 74, 70, 71, or 72, or d. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 78, 76, or 77, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 79, or e. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 58, 59, or 60, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 61, 62, 63, 64, or 65, or f. The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 15, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 16, or g. The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 7, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 8, or h. The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 11, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 12, or i. The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 13, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 14, or j. An anti-NPR1 antibody wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 9, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 10.

[0028] In some embodiments, an anti-NPR1 antibody as described in any one of the above, a. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 82, 80, or 81, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 85, 83, or 84, or b. The heavy chain variable region includes the amino acid sequence of SEQ ID NOs. 51, 48, 49, 50, or 52, and the light chain variable region includes the amino acid sequence of SEQ ID NOs. 56, 53, 54, or 55, or c. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 68, 66, 67, or 69, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 74, 70, 71, or 72, or d. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 78, 76, or 77, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 79, or e. An anti-NPR1 antibody wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 58, 59, or 60, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 61, 62, 63, 64, or 65.

[0029] In some embodiments, an anti-NPR1 antibody as described in any one of the above, a. The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 82, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 85, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 81 or 82, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 84, or b. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 51, 49, 50, or 52, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 56, or c. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 68, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 74, 70, or 71, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 67, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 70 or 71, or d. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 78, 76, or 77, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 79, or e. The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 58, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 61, 62, 64, or 65, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 59, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 61, 62, 64, or 65, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 60, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 61, 62, 64, or 65, or f. The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 15, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 16, or g. The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 7, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 8, or h. The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 11, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 12, or i. The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 13, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 14, or j. An anti-NPR1 antibody wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 9, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 10.

[0030] In some specific embodiments, an anti-NPR1 antibody according to any one of the above, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 82, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 85.

[0031] In some specific embodiments, an anti-NPR1 antibody as described in any one of the above, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 56.

[0032] In some specific embodiments, an anti-NPR1 antibody as described in any one of the above, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 68, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 74.

[0033] In some specific embodiments, an anti-NPR1 antibody as described in any one of the above, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 79.

[0034] In some specific embodiments, an anti-NPR1 antibody as described in any one of the above, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16.

[0035] In some specific embodiments, an anti-NPR1 antibody as described in any one of the above, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8.

[0036] In some specific embodiments, an anti-NPR1 antibody as described in any one of the above, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12.

[0037] In some specific embodiments, an anti-NPR1 antibody as described in any one of the above, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 13, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14.

[0038] In some specific embodiments, an anti-NPR1 antibody as described in any one of the above, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 10.

[0039] In some embodiments, an anti-NPR1 antibody as described in any one of the above, wherein the anti-NPR1 antibody is an antibody fragment, and in some embodiments, the antibody fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, or dAb.

[0040] In some embodiments, an anti-NPR1 antibody according to any one of the above, comprising a heavy chain constant region and a light chain constant region, wherein in some embodiments, the heavy chain constant region is the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, in some embodiments, the light chain constant region is the human κ or λ light chain constant region, and in some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 86 or 43, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 44.

[0041] In some embodiments, the anti-NPR1 antibody described in any one of the above, wherein the anti-NPR1 antibody comprises a heavy chain and a light chain, of which, a. The heavy chain contains an amino acid sequence having sequence identity with at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of SEQ ID NO: 89 or 127, and the light chain contains an amino acid sequence having sequence identity with at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of SEQ ID NO: 90 or 128, or The heavy chain contains an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 110, and the light chain contains an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 111, or b. The heavy chain contains an amino acid sequence having at least 85% (for example, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 87, 112, 113, or 114, and the light chain contains an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 88, or The heavy chain contains an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 102, and the light chain contains an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 103, or c. The heavy chain contains an amino acid sequence having at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 99 or 122, and the light chain contains an amino acid sequence having at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 100, 123, or 124, or The heavy chain contains an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 106, and the light chain contains an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 107, or d. The heavy chain contains an amino acid sequence having at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 101, 125, or 126, and the light chain contains an amino acid sequence having at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 47, or The heavy chain contains an amino acid sequence having sequence identity with SEQ ID NO: 108 or at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) thereto, and the light chain contains an amino acid sequence having sequence identity with SEQ ID NO: 109 or at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) thereto, or e. The heavy chain contains an amino acid sequence having at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 115, 116, or 117, and the light chain contains an amino acid sequence having at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 118, 119, 120, or 121, or An anti-NPR1 antibody wherein the heavy chain contains an amino acid sequence having at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 104, and the light chain contains an amino acid sequence having at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 105.

[0042] In some embodiments, the anti-NPR1 antibody described in any one of the above, wherein the anti-NPR1 antibody comprises a heavy chain and a light chain, of which, a. The heavy chain contains the amino acid sequence of SEQ ID NO: 89, and the light chain contains the amino acid sequence of SEQ ID NO: 90 or 128, or The heavy chain contains the amino acid sequence of SEQ ID NO: 127, and the light chain contains the amino acid sequence of SEQ ID NO: 128, or The heavy chain contains the amino acid sequence of SEQ ID NO: 110, and the light chain contains the amino acid sequence of SEQ ID NO: 111, or b. The heavy chain contains the amino acid sequence of SEQ ID NO: 87, 112, 113, or 114, and the light chain contains the amino acid sequence of SEQ ID NO: 88, or The heavy chain contains the amino acid sequence of SEQ ID NO: 102, and the light chain contains the amino acid sequence of SEQ ID NO: 103, or c. The heavy chain contains the amino acid sequence of SEQ ID NO: 99, and the light chain contains the amino acid sequence of SEQ ID NO: 100, 123, or 124, or The heavy chain contains the amino acid sequence of SEQ ID NO: 122, and the light chain contains the amino acid sequence of SEQ ID NO: 123 or 124, or The heavy chain contains the amino acid sequence of SEQ ID NO: 106, and the light chain contains the amino acid sequence of SEQ ID NO: 107, or d. The heavy chain contains the amino acid sequence of SEQ ID NO: 101, 125, or 126, and the light chain contains the amino acid sequence of SEQ ID NO: 47, or The above heavy chain contains the amino acid sequence of SEQ ID NO: 108, and the above light chain contains the amino acid sequence of SEQ ID NO: 109, or e. The heavy chain contains the amino acid sequence of SEQ ID NO: 115, and the light chain contains the amino acid sequence of SEQ ID NO: 118, 119, 120, or 121, or The heavy chain contains the amino acid sequence of SEQ ID NO: 116, and the light chain contains the amino acid sequence of SEQ ID NO: 118, 119, 120, or 121, or The heavy chain contains the amino acid sequence of SEQ ID NO: 117, and the light chain contains the amino acid sequence of SEQ ID NO: 118, 119, 120, or 121, or An anti-NPR1 antibody wherein the heavy chain contains the amino acid sequence of SEQ ID NO: 104, and the light chain contains the amino acid sequence of SEQ ID NO: 105.

[0043] In some embodiments, the anti-NPR1 antibody described in any one of the above, wherein the anti-NPR1 antibody comprises a heavy chain and a light chain, of which, a. The heavy chain contains an amino acid sequence having sequence identity with SEQ ID NO: 89 or at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) thereto, and the light chain contains an amino acid sequence having sequence identity with SEQ ID NO: 90 or at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) thereto, or b. The heavy chain contains an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 87 (for example, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), and the light chain contains an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 88 (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), or c. The heavy chain contains an amino acid sequence having sequence identity with SEQ ID NO: 99 or at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) thereto, and the light chain contains an amino acid sequence having sequence identity with SEQ ID NO: 100 or at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) thereto, or d. An anti-NPR1 antibody wherein the heavy chain comprises an amino acid sequence having at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 101, and the light chain comprises an amino acid sequence having at least 85% (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 47.

[0044] In some embodiments, the anti-NPR1 antibody described in any one of the above, wherein the anti-NPR1 antibody comprises a heavy chain and a light chain, of which, a. The heavy chain contains the amino acid sequence of SEQ ID NO: 89, and the light chain contains the amino acid sequence of SEQ ID NO: 90, or b. The heavy chain contains the amino acid sequence of SEQ ID NO: 87, and the light chain contains the amino acid sequence of SEQ ID NO: 88, or c. The heavy chain contains the amino acid sequence of SEQ ID NO: 99, and the light chain contains the amino acid sequence of SEQ ID NO: 100, or d. An anti-NPR1 antibody wherein the heavy chain contains the amino acid sequence of SEQ ID NO: 101, and the light chain contains the amino acid sequence of SEQ ID NO: 47.

[0045] In some specific embodiments, an anti-NPR1 antibody according to any one of the above, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 89 and a light chain containing the amino acid sequence of SEQ ID NO: 90.

[0046] In some specific embodiments, an anti-NPR1 antibody according to any one of the above, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 87 and a light chain containing the amino acid sequence of SEQ ID NO: 88.

[0047] In some specific embodiments, an anti-NPR1 antibody according to any one of the above, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 89 and the amino acid sequence of the light chain is shown in SEQ ID NO: 90.

[0048] In some specific embodiments, an anti-NPR1 antibody according to any one of the above, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 87 and the amino acid sequence of the light chain is shown in SEQ ID NO: 88.

[0049] In some specific embodiments, an anti-NPR1 antibody according to any one of the above, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 99 and the amino acid sequence of the light chain is shown in SEQ ID NO: 100.

[0050] In some specific embodiments, an anti-NPR1 antibody according to any one of the above, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 101 and the amino acid sequence of the light chain is shown in SEQ ID NO: 47.

[0051] In some embodiments, the disclosure further provides isolated anti-NPR1 antibodies that compete with the anti-NPR1 antibodies described above for binding to human NPR1 or its epitope.

[0052] In some embodiments, the anti-NPR1 antibodies provided herein bind specifically to hNPR1 without binding to other members of the NPR family, hNPR2 and / or hNPR3.

[0053] In some embodiments, the isolated anti-NPR1 antibody of this disclosure has an EC of less than 30 nM (e.g., less than 27 nM, less than 10 nM, less than 9 nM, less than 7 nM, less than 5 nM, less than 4 nM, less than 2 nM, less than 1.5 nM, less than 0.1 nM, less than 0.05 nM) 50 The value binds to human NPR1, and the above EC 50 The value is measured by ELISA.

[0054] In some embodiments, the isolated anti-NPR1 antibody of this disclosure can bind to both hNPR1 CHO-K1 and cNPR1 CHO-K1, with or without 10 nM ANP.

[0055] In some embodiments, the isolated anti-NPR1 antibodies of this disclosure can all stimulate hNPR1-CHOK1 cells to produce cGMP.

[0056] In some embodiments, the isolated anti-NPR1 antibody of this disclosure exhibits less differential cGMP production in endocytotic cells compared to the positive antibody.

[0057] In another embodiment, the present disclosure provides a pharmaceutical composition comprising an anti-NPR1 antibody as described above and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition further comprises at least one second therapeutic agent.

[0058] In some embodiments, the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, or intramuscularly.

[0059] In another embodiment, the present disclosure further provides an immune complex comprising an anti-NPR1 antibody and an effector molecule as described above, wherein the effector molecule is coupled to the anti-NPR1 antibody, and preferably the effector molecule is selected from antitumor agents, immunomodulators, bioreaction modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

[0060] In another embodiment, the present disclosure provides isolated nucleic acids encoding the anti-NPR1 antibody described in any one of the above.

[0061] In another embodiment, the present disclosure provides a vector comprising an isolated nucleic acid as described in any one of the above.

[0062] In another embodiment, the present disclosure provides a host cell comprising an isolated nucleic acid as described in any one of the above.

[0063] In another embodiment, the present disclosure provides a method for detecting an NPR1 peptide or fragment thereof in a sample, comprising contacting the sample with an anti-NPR1 antibody described in any one of the above, and detecting the presence of a complex between the anti-NPR1 antibody and the NPR1 peptide or fragment thereof, wherein the detection of the complex indicates the presence of the NPR1 peptide or fragment thereof.

[0064] In another embodiment, the Disclosure provides a method for treating, preventing or improving a disease or condition related to NPR1, comprising administering to a subject an anti-NPR1 antibody, a pharmaceutical composition, or an immune complex as described in any one of the above.

[0065] In another aspect, the Disclosure provides a use in the preparation of a pharmacopoeia for treating, preventing or improving a disease or condition associated with NPR1, comprising administering to a subject a therapeutically effective amount or a preventively effective amount of any one of the anti-NPR1 antibodies, or the pharmaceutical composition, or the immune complex, described above.

[0066] In another embodiment, the disclosure provides an anti-NPR1 antibody, a pharmaceutical composition, or an immune complex as described above, to be used as a drug. In some embodiments, the drug is used to treat, prevent or improve a disease or condition associated with NPR1.

[0067] In some embodiments, the disease or condition described in any one of the above is selected from heart failure, hypertension, peripheral vascular disease, coronary artery disease (CAD), ischemic heart disease (IHD), mitral stenosis and regurgitation, angina pectoris, hypertrophic cardiomyopathy (HCM), diabetic cardiomyopathy, supraventricular arrhythmias and ventricular arrhythmias, arrhythmias, atrial fibrillation (AF), newly occurring atrial fibrillation, recurrent atrial fibrillation, cardiac fibrosis, atrial flutter, harmful vascular remodeling, plaque stabilization, myocardial infarction (MI), pre-eclampsia, obesity, renal failure, renal dysfunction, cytokine release syndrome, chronic kidney disease, macular edema, glaucoma, stroke, lung disease, pulmonary fibrosis, inflammation, asthma, bone growth disorders, fractures, diabetes, and cancer.

[0068] In some embodiments, the heart failure described above is selected from heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), heart failure with mildly reduced ejection fraction (HFmrEF), heart failure after acute myocardial infarction, or acute compensated heart failure, and in some embodiments, the heart failure described above is heart failure with preserved ejection fraction (HFpEF).

[0069] In some embodiments, the hypertension described in any one of the above is refractory hypertension. [Brief explanation of the drawing]

[0070] [Figure 1A] The results of ELISA experiments on the binding of anti-NPR1 antibodies to the hNPR1 antigen are shown. [Figure 1B] The results of an ELISA experiment on the binding of anti-NPR1 antibodies to the hNPR2 antigen are shown. [Figure 1C] The results of ELISA experiments on the binding of anti-NPR1 antibodies to the hNPR3 antigen are shown. [Figure 2] This shows experimental results demonstrating that an anti-NPR1 antibody stimulates hNPR1-CHOK1 cells to produce cGMP. [Figure 3] The results of endocytosis experiments on the binding of anti-NPR1 antibodies to NPR1 are shown. [Figure 4A]This paper shows experimental results demonstrating the stimulation of cGMP production in cells before and after endocytosis treatment with the Hu102H4L6 antibody. [Figure 4B] This paper shows experimental results demonstrating the stimulation of cGMP production in cells before and after endocytosis treatment with the Hu140H5L5-11 antibody. [Figure 5A] This study demonstrates the effect of anti-NPR1 antibodies on systolic blood pressure in ANGII-induced hypertensive hNPR1 homozygous transgenic mice. [Figure 5B] This study demonstrates the effect of anti-NPR1 antibodies on diastolic blood pressure in ANGII-induced hypertensive hNPR1 homozygous transgenic mice. [Figure 5C] This study demonstrates the effect of anti-NPR1 antibodies on mean arterial pressure in ANGII-induced hypertensive hNPR1 homozygous transgenic mice. [Figure 5D] This study demonstrates the effect of anti-NPR1 antibodies on heart rate in ANGII-induced hypertensive hNPR1 homozygous transgenic mice. [Figure 5E] This study demonstrates the effect of anti-NPR1 antibodies on plasma cGMP concentrations in ANGII-induced hypertensive hNPR1 homozygous transgenic mice. [Figure 5F] This study demonstrates the effect of anti-NPR1 antibodies on cGMP concentration in the urine of ANGII-induced hypertensive hNPR1 homozygous transgenic mice. [Figure 5G] This shows the endpoint plasma blood concentrations in ANGII-induced hypertensive hNPR1 homozygous transgenic mice after administration. [Figure 5H] This shows the effect of mean NT-proBNP concentration in plasma of ANGII-induced hypertensive hNPR1 homozygous transgenic mice induced by anti-NPR1 antibodies. [Figure 5I] This study demonstrates the effect of anti-NPR1 antibodies on sodium ion concentration in the urine of ANGII-induced hypertensive hNPR1 homozygous transgenic mice. [Modes for carrying out the invention]

[0071] term To make this disclosure more easily understood, some technical and scientific terms are defined below. Unless otherwise specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0072] The singular forms “one,” “one type,” and “above / above” used in the specification and claims refer to multiple subjects unless otherwise specified in the context.

[0073] Unless otherwise specified in the context, words such as "contain," "possess," and "include" in patent specifications and claims should be understood to mean "include, but not limited to," rather than being exclusive or exhaustive.

[0074] The term "cytokine" is a general term for proteins released from one cell population that act on other cells as intercellular mediators. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include mRNA-2, IFNγ, TNFα, CCL-2, and IL-6.

[0075] The term "and / or" refers to the fact that it includes two meanings: "and" and "or." For example, the phrase "A, B and / or C" attempts to cover A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0076] The three-letter and one-letter amino acid codes used in this disclosure are as described in J.biol.chem, 243, p3558 (1968).

[0077] The term "NPR1" refers to natriuretic peptide receptor 1, also known as atrial natriuretic peptide receptor type A (ANP-A, ANPR-A, or NPR-A) and guanylate cyclase A (GC-A). The term "NPR1" refers to the naturally occurring NPR1 protein (e.g., in its precursor, mature, modified, or splice variant forms, but not limited to these). The human NPR1 protein contains the amino acid sequence of SEQ ID NO: 4. Amino acid sequences of NPR1 molecules from non-human species (e.g., mouse, rat, monkey, rabbit, dog, pig, etc.) can be obtained from common resources, e.g., cynomolgus monkey NPR1 protein, NCBI number XP-005541809.1, rat NPR1 protein, Uniprot number P18910. While specific database registration numbers are given herein, those skilled in the art will understand that the NPR1 referred to herein also covers corresponding sequences reported in other databases or literature.

[0078] The term "amino acid" refers to naturally occurring amino acids, synthesized amino acids, and amino acid analogs and amino acid mimes that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., hydrogen, carboxyl group, amino group, and α-carbon bonded to an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide skeletons, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimes are chemical compounds that have a different structure from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0079] The term "amino acid mutation" includes amino acid substitutions (also called amino acid replacements), deletions, insertions, and modifications. The final construct can be realized by any combination of substitutions, deletions, insertions, and modifications, provided that the final construct possesses desired properties, such as reduced binding to the Fc receptor. Deletions and insertions of amino acid sequences include deletions and insertions at the amino and / or carboxyl terminals of a polypeptide chain. Specific amino acid mutations may also be amino acid substitutions. In some embodiments, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties. Amino acid substitutions include substitutions with amino acids not found in nature or derivatives of 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated by genetic or chemical methods known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods for modifying amino acid side chain groups other than genetic engineering, such as chemical modifications, are also expected to be available. In this specification, the same amino acid mutation can be represented by various names. In this specification, an amino acid residue at a specific site can be indicated using the position + amino acid residue method. For example, 102S indicates that the amino acid residue at site 102 is S. C102S indicates that the amino acid residue at site 102 has mutated from the original C to S. When a characteristic site residue is defined in a claim using the "position + amino acid residue method," the original residue at that site does not limit the protected area.

[0080] The term "antibody" is used in its broadest sense to refer to any antibody exhibiting the desired antigen-binding activity, and covers a wide range of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding moieties).

[0081] The term "natural antibody" refers to naturally occurring immunoglobulin molecules. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has one variable region (VH, also called the variable heavy chain domain or heavy chain variable region), followed by the heavy chain constant region. The natural IgG heavy chain constant region generally contains three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has one variable region (VL, also called the variable light chain domain or light chain variable domain), followed by one constant light chain domain (light chain constant region, CL).

[0082] The terms “full-length antibody,” “complete antibody,” and “whole antibody” may be used interchangeably herein and refer to antibodies having a structure substantially similar to that of a natural antibody, or having a heavy chain in the Fc region as limited herein. The light chain of a natural complete antibody comprises a variable region VL and a constant region CL, with VL being at the amino terminus of the light chain, and the constant region comprising a κ chain and a λ chain; the heavy chain comprises a variable region VH and a constant region (CH1, CH2, and CH3), with VH being at the amino terminus of the heavy chain, and the constant region being at the carboxy terminus, of which CH3 is closest to the carboxy terminus of the polypeptide; and the heavy chain may belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0083] The term "variable region" or "variable domain" of an antibody refers to a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to an antigen. In this specification, the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody each contain four conservative framework regions (FRs) and three complementarity-determining regions (CDRs), respectively. Of these, the term "complementarity-determining region" or "CDR" refers to the region in the variable domain that primarily promotes binding to the antigen, and "framework" or "FR" refers to the variable domain residues excluding the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3, and the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus (also called the N terminus) to the carboxyl terminus (also called the C terminus).

[0084] The amino acid sequence boundaries of CDRs can be determined by various known methods, such as the "Kabat" numbering system (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering system, the "ABM" numbering system, the "contact" numbering system (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering system (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003), Front Immunol. 2018 Oct 16, 9:2278), and the correspondences between various numbering systems are well known to those skilled in the art, and are exemplified as shown in Table 1 below.

[0085] [Table 1]

[0086] Unless otherwise specified, the variable regions and CDRs in the embodiments of this disclosure are all subject to the "Kabat" numbering rules. In specific embodiments, the Kabat numbering rules are employed to limit amino acid residues, but alternative numbering systems are considered equivalent.

[0087] The term "antibody fragment" refers to a molecule distinct from the complete antibody, containing the portion of the complete antibody that binds to the antigen it binds to. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-domain antibodies, single-chain Fab (scFab), bivalent antibodies, linear antibodies, single-chain antibodies (e.g., scFv), and multispecific antibodies consisting of antibody fragments.

[0088] The terms “Fc region” or “fragment crystallizable region” are used to define the C-terminal region of an antibody heavy chain and include native and modified Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined to extend from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Suitable Fc regions used in the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region may be altered, for example, by deletion of the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or by deletion of the C-terminal glycine and lysine of the Fc region (residues 446 and 447 according to the EU numbering system). Unless otherwise specified, the numbering rules for Fc regions are the EU numbering system, also known as the EU index.

[0089] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain is derived from a specific source or species, while the rest of the heavy chain and / or light chain is derived from other different sources or species.

[0090] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while simultaneously exhibiting relatively low immunogenicity in humans. This may be achieved, for example, by retaining the non-human CDR region and substituting the rest of the antibody with its human counterpart (i.e., the framework region portion of the constant and variable regions).

[0091] The terms “human antibody,” “whole human antibody,” and “fully human antibody” may be used interchangeably and refer to antibodies whose variable and constant regions are human sequences. These terms cover antibodies derived from human genes but with altered sequences, such as those with potentially reduced immunogenicity, increased affinity, or removal of cysteine ​​or glycosylation sites that may cause undesirable folding. These terms also cover antibodies produced by recombination in non-human cells (which may confer glycosylation not characteristic of human cells). Furthermore, these terms cover antibodies produced in transgenic mice containing some or all human immunoglobulin heavy and light chain loci. The meaning of “human antibody” explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0092] The term "affinity" refers to the total strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity, reflecting the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X to its ligand Y can usually be expressed by its dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0093] As used herein, the terms "kassoc" or "ka" refer to the association rate of a particular antibody-antigen interaction, and the terms "kdis" or "kd" refer to the dissociation rate of a particular antibody-antigen interaction. The term "KD" refers to the dissociation constant, obtained from the ratio of kd to ka (i.e., kd / ka) and expressed in molar concentration (M). The KD value of an antibody can be measured by methods known in the art. For example, by systematically measuring surface plasmon resonance (e.g., Biacore) using a biosensor system, or by measuring affinity in solution by solution equilibrium titration (SET).

[0094] The term "surface plasmon resonance" refers to an optical phenomenon that analyzes real-time interactions by detecting changes in protein concentration in a biosensor matrix, for example, using the BIAcore™ system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

[0095] The term "effector function" can refer to biological activity that is altered by the antibody isotype, as well as by the antibody's Fc region (either the natural sequence Fc region or a mutated amino acid sequence Fc region). Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0096] The term "monoclonal antibody" essentially refers to a homogeneous population of antibodies, meaning that the amino acid sequences of the antibody molecules in that population are identical, with the exception of a few possible native variations. In contrast, polyclonal antibody preparations generally contain multiple different antibodies, each with different amino acid sequences that are typically specific to different epitopes in their variable domains. "Monoclonal" describes an antibody characteristic of being obtained from a homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0097] The term "antigen" refers to a molecule or molecular portion that can be bound by, for example, an antigen-binding protein (e.g., an antibody). An antigen may have one or more epitopes that can interact with different antigen-binding proteins (e.g., antibodies).

[0098] The term "epitope" refers to an area or region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. Epitopes are formed by a continuous sequence of amino acids (linear epitopes) or may include discontinuous amino acids that are spatially close together, for example, due to antigen folding (i.e., tertiary folding of the antigen due to the properties of the protein) (contaxial epitopes). Contaxial epitopes and linear epitopes differ in that binding of the antibody to the conformational epitope is lost in the presence of a denaturing solvent. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed by methods commonly used in this field, including, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, antigen chemical modification (see Prot.Sci.9(2000)487-496), and cross-inhibition.

[0099] The terms "specifically bindable," "specifically bind," or "bind" mean that an antibody can bind to a particular antigen or its epitope with higher affinity than other antigens or epitopes. Generally, antibodies have an affinity of approximately 1 × 10⁻⁶. -7 M or less (for example, approximately 1 × 10) -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 The antibody binds to the antigen or an epitope within the antigen with an equilibrium dissociation constant (KD) of M or less. In some embodiments, the KD of the antibody that binds to the antigen is 10% or less (e.g., 1%) of the KD of the antibody that binds to a nonspecific antigen (e.g., BSA, casein). The KD can be measured by known methods, for example, by BIACORE® surface plasmon resonance assay. However, antibodies that specifically bind to an antigen or an epitope within an antigen may cross-react with other relevant antigens, for example, with the corresponding antigens from other species (homozygous) (e.g., humans, or monkeys such as cynomolgus monkeys (Macaca fascicularis) (cynomolgus, cyno), chimpanzees (Pan troglodytes) (chimpanzee, chimp)) or marmosets (Callithrix jacchus) (commonmarmoset, marmoset).

[0100] The term "does not bind" means that the antibody cannot bind to a certain antigen or its epitope in the specific binding manner described above. For example, an antibody can bind to approximately 1 × 10⁻⁶ antigens. -6 It binds to the antigen or its epitope with an equilibrium dissociation constant (KD) of M or greater.

[0101] The terms "anti-NPR1 antibody" and "antibody that binds to NPR1" refer to antibodies that can bind to NPR1 or its epitope with sufficient affinity.

[0102] The terms “activating antibody” or “agonist antibody” (or “antibody that increases or enhances NPR1 activity” or “antibody that stabilizes the activated conformation”) are intended to describe an antibody that, when bound to NPR1, activates at least one biological activity of NPR1. For example, the antibodies of this disclosure can reduce mean arterial pressure when administered to a target requiring it.

[0103] The terms "antibody-dependent cell cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to a mechanism that induces cell death, which depends on the interaction between antibody-coated target cells and effector cells with lytic activity (e.g., natural killer cells (NK), monocytes, macrophages, and neutrophils) mediated by Fcγ receptors (FcγR) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. The ADCC activity of the antibodies provided herein can be evaluated in vitro using antigen-expressing cells as target cells and NK cells as effector cells. Cell lysis is detected by markers released from the lysed cells (e.g., radioactive substrates, fluorescent dyes, or innate intracellular proteins).

[0104] The term "antibody-dependent cell phagocytosis (ADCP)" refers to a mechanism by which antibody-coated target cells are removed through the internalization of phagocytic cells (e.g., macrophages or dendritic cells).

[0105] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death, in which the Fc effector domain of a target-binding antibody binds to and activates complement component C1q, which in turn activates the complement cascade, leading to the death of target cells. Complement activation can also cause the deposition of complement components on the surface of target cells, and these complement components promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0106] The term "ANGII" refers to angiotensin II, a polypeptide substance produced by the hydrolysis of angiotensin I under the action of angiotensin-converting enzyme. Angiotensin receptors are present in vascular smooth muscle, adrenal cortical zona glomerulosa cells and parts of the brain, as well as in the cells of the heart and kidneys. Angiotensin II binds to angiotensin receptors and produces corresponding physiological effects, including (1) constriction of arterioles and veins throughout the body, increasing blood pressure and venous return; (2) increased release of neurotransmitters from sympathetic vasoconstrictor fibers; (3) activation of the sympathetic vasoconstrictor center; (4) stimulation of the adrenal gland to synthesize and release aldosterone; and (5) inducing or enhancing thirst to induce drinking behavior. In some embodiments, ANGII modeling is used to induce increased blood pressure in mice. In some embodiments, ANGII modeling is used to induce the development of progressive heart failure in mice.

[0107] The term “nucleic acid” may be used interchangeably with the term “polynucleotide” herein and means deoxyribonucleotides or ribonucleotides and polymers thereof, exhibiting single-stranded or double-stranded forms. The above terms cover nucleic acids containing known nucleotide analogs or modified skeletal residues or linkers, which are synthetic, naturally occurring, or not naturally occurring, and which have similar binding properties to a reference nucleic acid and are metabolized in a similar manner to a reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). “Isolated” nucleic acid means a nucleic acid molecule isolated from components of its natural environment. Isolated nucleic acids include nucleic acid molecules found in the following cells, which generally contain such nucleic acid molecules, but which are extrachromosomal or located at chromosomal locations different from their natural chromosomal locations. Isolated nucleic acids encoding polypeptides or fusion proteins refer to one or more nucleic acid molecules encoding polypeptides or fusion proteins, and include one or more nucleic acid molecules in a single vector or separate vectors, and one or more nucleic acid molecules present at one or more locations in a host cell. Unless otherwise specified, a particular nucleic acid sequence implicitly covers its conservedly modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequences explicitly stated. Specifically, as detailed below, degenerate codon substitutions can be obtained by producing sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue.

[0108] The terms “polypeptide” and “protein” may be used interchangeably herein and refer to polymers of amino acid residues. These terms apply to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, and also apply to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence implicitly covers its conservedly modified variants.

[0109] The term "sequence identity" refers to the degree (percentage) to which the amino acids / nucleic acids of two sequences are homologous at equivalent positions, and when optimal alignment is performed for two sequences, gaps are introduced as necessary to obtain the maximum percentage of sequence identity, and any conservative substitutions are not considered part of sequence identity. To measure the percentage of sequence identity, alignment can be performed using known techniques in this art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters applied to the measurement and alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0110] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide to which it is ligated. One type of vector is a "plasmid," which refers to a circular double-stranded DNA ring that can be ligated to an attached DNA segment. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), in which another DNA segment can be ligated to a viral genome. Some vectors can self-replicate in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can replicate with the host genome after being introduced into the host cell by being aligned with the host cell's genome. The term "expression vector" or "expression construct" refers to a vector containing nucleic acid sequences that can transform a host cell and guide and / or control the expression of one or more heterogeneous coding regions that are manipulably ligated to it (together with the host cell). An expression construct may include, but is not limited to, sequences that affect or control transcription, translation, and the RNA splice of coding regions that are manipulably ligated to introns, if present.

[0111] The terms “host cell,” “host cell line,” and “host cell culture” may be used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” and without regard to passage number, include primary transformed cells and their derived offspring. Offspring may contain mutations, but may not be exactly the same as the parent cells in nucleic acid contents. In this specification, this includes offspring of mutants having the same function or biological activity as those screened or selected in the initial transformed cells. Host cells include prokaryotic and eukaryotic host cells, of which eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include, but are not limited to, human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells, including Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, such as Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, and Pichia stiptis. Pichia methanolica, Pichia genus, Saccharomyces cerevisiae, Saccharomyces genus, Hansenula polymorpha, Kluyveromyces genus, Kluyveromyces lactis, Candida albicans, Aspergillus genus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium genus This includes Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.

[0112] "Optional" or "optionally" means that the event or situation described thereafter may occur, but does not necessarily have to occur, and the description includes both cases in which the event or situation occurs and cases in which it does not.

[0113] The term "pharmaceutical composition" refers to a mixture comprising one or more anti-NPR1 antibodies described herein and other chemical components, the other components being, for example, physiologically / pharmaceutically acceptable carriers and excipients.

[0114] The term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation that, unlike the active ingredient, is non-toxic to the target substance. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0115] The terms “subject” or “individual” include humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals), such as non-human primates, sheep, dogs, cattle, chickens, amphibians and reptiles. Unless otherwise noted, the above terms “patient” or “subject” may be used interchangeably herein. In some embodiments, the individual or subject is human.

[0116] "Administer" or "give" means contact between an exogenous drug, therapeutic agent, diagnostic agent or composition and an animal, human, subject, cell, tissue, organ or biofluid when applied to an animal, human, subject, cell, tissue, organ or biofluid.

[0117] The term "sample" means a fluid, cell, or tissue-like sample isolated from a subject, and fluids, cells, or tissue present within the body of the subject. Exemplary samples are biological fluids, such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excrement, sputum, mucosal secretions from secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids from the abdominal cavity and other body cavities, fluids collected from bronchial lavage fluid, synovial fluid, liquid solutions that have come into contact with the subject or biological source, such as cell and organ culture media (including cell or organ-conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration tissue, surgically excised tissue, organ cultures, or cell cultures.

[0118] "Treatment" and "processing" (and their grammatical variations) refer to clinical interventions aimed at altering the natural processes of the individual being treated, and which may be carried out for preventive purposes or in the course of a clinicopathological process. The desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, reduction / decreasation of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or mitigation of the disease state, and regression or improvement of the prognosis. In some embodiments, the use of the antibodies of this disclosure delays the formation of disease or delays the progression of disease.

[0119] The terms "recurrence," "relapse," and "relapsed" refer to the reappearance of cancer or disease after it has been clinically determined to have disappeared. A diagnosis of distant metastasis or local recurrence of cancer can be considered a recurrence.

[0120] The terms "refractory" or "resistant" refer to cancer or disease that does not respond to treatment.

[0121] An "effective dose" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or their potential causes, prevent the appearance of symptoms and / or their potential causes, and / or improve or improve damage caused by or associated with the disease state (e.g., lung disease). In some examples, the effective dose is a therapeutic effective dose or a prophylactic effective dose. A "therapeutic effective dose" is an amount sufficient to treat a disease state or symptoms, in particular a state or symptoms associated with the disease state, or to prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptoms otherwise associated with the disease. A "prophylactic effective dose" is an amount, when administered to a subject, that provides a predetermined prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. Complete therapeutic or prophylactic effects do not necessarily occur with a single dose, but may occur after a series of doses have been administered. Therefore, a therapeutic or prophylactic effective dose can be administered in one or multiple doses. The "therapeutic dose" and "preventive dose" can vary depending on several factors, such as the individual's disease state, age, sex, and weight, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, the patient's improved health status.

[0122] The anti-NPR1 antibody disclosed herein This disclosure provides an anti-NPR1 antibody or its antigen-binding fragment having many advantageous properties, such as good therapeutic activity, safety, pharmacokinetic properties, and drug discovery potential (e.g., solubility, viscosity, purity, and stability).

[0123] Exemplary anti-NPR1 antibody Examples of the present disclosure include antibody series 102, 127, 128, 135, and 140. Hereinafter, the antibodies of the present disclosure will be described using antibodies 140 and 102 as examples.

[0124] Exemplary, the anti-NPR1 antibody of this disclosure comprises a heavy chain variable region and a light chain variable region, of which, a. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 82, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 85, or b. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in any one of sequence numbers 51, 48, or 52, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in sequence number 56.

[0125] Exemplary, the anti-NPR1 antibody of this disclosure comprises a heavy chain variable region and a light chain variable region, of which, a. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 82, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 85, or b. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 51, and the light chain variable regions LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 56.

[0126] Exemplary, the anti-NPR1 antibody of this disclosure comprises a heavy chain variable region and a light chain variable region, of which, a. The heavy chain variable region may be such that HCDR1 contains the amino acid sequence of SEQ ID NO: 29, HCDR2 contains the amino acid sequence of SEQ ID NO: 40, and HCDR3 contains the amino acid sequence of SEQ ID NO: 41, and the light chain variable region may be such that LCDR1 contains the amino acid sequence of SEQ ID NO: 42, LCDR2 contains the amino acid sequence of SEQ ID NO: 33, and LCDR3 contains the amino acid sequence of SEQ ID NO: 34, or b. The heavy chain variable region includes, in HCDR1, the amino acid sequence of SEQ ID NO: 45, 17, or 35; in HCDR2, the amino acid sequence of SEQ ID NO: 46 or 18; and in HCDR3, the amino acid sequence of SEQ ID NO: 19; and the light chain variable region includes, in LCDR1, the amino acid sequence of SEQ ID NO: 20; in LCDR2, the amino acid sequence of SEQ ID NO: 21; and in LCDR3, the amino acid sequence of SEQ ID NO: 22.

[0127] Exemplary, the anti-NPR1 antibody of this disclosure comprises a heavy chain variable region and a light chain variable region, of which, The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 29, HCDR2 contains the amino acid sequence of SEQ ID NO: 40, and HCDR3 contains the amino acid sequence of SEQ ID NO: 41, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 42, LCDR2 contains the amino acid sequence of SEQ ID NO: 33, and LCDR3 contains the amino acid sequence of SEQ ID NO: 34.

[0128] Exemplary, the anti-NPR1 antibody of this disclosure comprises a heavy chain variable region and a light chain variable region, of which, The above heavy chain variable region is represented by the amino acid sequence of HCDR1 shown in SEQ ID NO: 29, the amino acid sequence of HCDR2 shown in SEQ ID NO: 40, and the amino acid sequence of HCDR3 shown in SEQ ID NO: 41, and the above light chain variable region is represented by the amino acid sequence of LCDR1 shown in SEQ ID NO: 42, the amino acid sequence of LCDR2 shown in SEQ ID NO: 33, and the amino acid sequence of LCDR3 shown in SEQ ID NO: 34.

[0129] Exemplary, the anti-NPR1 antibody of this disclosure comprises a heavy chain variable region and a light chain variable region, of which, The heavy chain variable region is such that HCDR1 contains the amino acid sequence of SEQ ID NO: 45, HCDR2 contains the amino acid sequence of SEQ ID NO: 46, and HCDR3 contains the amino acid sequence of SEQ ID NO: 19, and the light chain variable region is such that LCDR1 contains the amino acid sequence of SEQ ID NO: 20, LCDR2 contains the amino acid sequence of SEQ ID NO: 21, and LCDR3 contains the amino acid sequence of SEQ ID NO: 22.

[0130] Exemplary, the anti-NPR1 antibody of this disclosure comprises a heavy chain variable region and a light chain variable region, of which, The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 19, respectively, for the heavy chain variable region, while the amino acid sequences of LCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively.

[0131] Exemplary examples include the anti-NPR1 antibody of this disclosure, which is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0132] Exemplary example is the anti-NPR1 antibody of this disclosure, which comprises a framework region (FR) of a human antibody.

[0133] Exemplary, an anti-NPR1 antibody of the present disclosure, wherein the heavy chain variable region comprises FR1, FR2, FR3 derived from IGHV1-46*01 and FR4 derived from IGHJ6*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1E, 40R, 43R, 48I, 69L, 71V, 73K, 74P, 75S, 77P, 78A, 80I, and 81Q, and / or the light chain variable region comprises FR1, FR2, FR3 derived from IGKV3-11*01 and FR4 derived from IGKJ2*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 20Q, 58V, 60D, 70S, 71Y, 77R, and 100A. In some embodiments, the anti-NPR1 antibody is one in which the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 29, HCDR2 containing the amino acid sequence of SEQ ID NO: 40, and HCDR3 containing the amino acid sequence of SEQ ID NO: 41, and the FR of the heavy chain variable region is either unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 1E, 40R, 43R, 48I, 69L, 71V, 73K, 74P, 75S, 77P, 78A, 80I, and 81Q, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 42, LCDR2 containing the amino acid sequence of SEQ ID NO: 33, and LCDR3 containing the amino acid sequence of SEQ ID NO: 34, and the FR of the light chain variable region is either unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 20Q, 58V, 60D, 70S, 71Y, 77R, and 100A. In some embodiments, the variable region and CDR are defined according to Kabat numbering rules.

[0134] Exemplary, an anti-NPR1 antibody of the present disclosure, wherein the heavy chain variable region comprises FR1, FR2, FR3 derived from IGHV3-21*01 and FR4 derived from IGHJ6*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1D, 3K, 9E, 44R, 49A, 87S, and 93T, and / or the light chain variable region comprises FR1, FR2, FR3 derived from IGKV3-11*01 or IGKV1-39*01 and FR4 derived from IGKJ4*01, and is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 4L, 43S, 45K, 46R, 47W, 58V, and 71Y. In some embodiments, the anti-NPR1 antibody is such that the heavy chain variable region is such that HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, HCDR2 comprises the amino acid sequence of SEQ ID NO: 46, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, and the FR of the heavy chain variable region is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 1D, 3K, 9E, 44R, 49A, 87S, and 93T, and the light chain variable region is such that LCDR1 comprises the amino acid sequence of SEQ ID NO: 20, LCDR2 comprises the amino acid sequence of SEQ ID NO: 21, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 22, and the FR of the light chain variable region is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of 4L, 43S, 45K, 46R, 47W, 58V, and 71Y. In some embodiments, the variable region and CDR are defined according to Kabat numbering rules.

[0135] For example, the anti-NPR1 antibodies of this disclosure, among them, a. The heavy chain variable region contains an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 82, and the light chain variable region contains an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 85, or b. An anti-NPR1 antibody wherein the heavy chain variable region contains an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 51 (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), and the light chain variable region contains an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 56 (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0136] For example, the anti-NPR1 antibodies of this disclosure, among them, a. The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 82, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 85, or b. An anti-NPR1 antibody wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 51, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 56.

[0137] For example, the anti-NPR1 antibodies of this disclosure, among them, a. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 82, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 85, or b. An anti-NPR1 antibody in which the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 51, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 56.

[0138] Exemplary examples include an anti-NPR1 antibody of the present disclosure, wherein the anti-NPR1 antibody is an antibody fragment, and in some embodiments, the antibody fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, or dAb.

[0139] Exemplary, an anti-NPR1 antibody of the present disclosure comprising a heavy chain constant region and a light chain constant region, wherein in some embodiments, the heavy chain constant region is the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, wherein in some embodiments, the light chain constant region is the human κ or λ light chain constant region, wherein in some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 86 or 43, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 44.

[0140] Exemplary examples include the anti-NPR1 antibody of the present disclosure, wherein the anti-NPR1 antibody comprises a heavy chain and a light chain, of which, a. The heavy chain contains an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 89, and the light chain contains an amino acid sequence having at least 85% (for example, at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 90, or b. An anti-NPR1 antibody wherein the heavy chain contains an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 87 (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), and the light chain contains an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 88 (e.g., at least 85%, 87%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0141] In some embodiments, the anti-NPR1 antibody described in any one of the above, wherein the anti-NPR1 antibody comprises a heavy chain and a light chain, of which, a. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 89, and the amino acid sequence of the light chain is shown in SEQ ID NO: 90, or b. An anti-NPR1 antibody in which the amino acid sequence of the heavy chain is shown in SEQ ID NO: 87, and the amino acid sequence of the light chain is shown in SEQ ID NO: 88.

[0142] Exemplarily, the present disclosure further provides an isolated anti-NPR1 antibody that competes with the anti-NPR1 antibody described in any one of the above and binds to human NPR1 or its epitope.

[0143] Exemplarily, the NPR1 antibody provided by the present disclosure specifically binds to hNPR1 or its epitope without binding to hNPR2 and / or hNPR3, which are other members of the NPR family.

[0144] Exemplarily, the isolated anti-NPR1 antibody of the present disclosure binds to human NPR1 or its epitope with an EC50 value of less than 30 nM (e.g., less than 27 nM, less than 10 nM, less than 9 nM, less than 7 nM, less than 5 nM, less than 4 nM, less than 2 nM, less than 1.5 nM, less than 0.1 nM, less than 0.05 nM), and the EC50 value is measured by ELISA.

[0145] Exemplarily, the isolated anti-NPR1 antibody of the present disclosure can bind to hNPR1 CHO-K1 and cNPR1 CHO-K1 in both the presence and absence of 10 nM ANP.

[0146] Exemplarily, the isolated anti-NPR1 antibody of the present disclosure can stimulate cells expressing NPR1 (such as hNPR1-CHOK1 cells) to produce cGMP.

[0147] In some embodiments, the isolated anti-NPR1 antibody of the present disclosure has less differential cGMP production in cells where endocytosis has occurred compared to positive antibodies.

[0148] Exemplarily, the present disclosure relates to NPR1 + / +The efficacy of different doses of anti-NPR1 antibodies can be evaluated by subcutaneously disseminating a hypertensive-associated AngII-induced heart failure model using humanized mice. In some embodiments, the isolated anti-NPR1 antibodies of this disclosure are expected to improve the EDV (end-diastolic volume) and ESV (end-systolic volume) parameters of modeling mice. In some embodiments, the isolated anti-NPR1 antibodies of this disclosure are expected to reduce left ventricular weight. In some embodiments, the isolated anti-NPR1 antibodies of this disclosure are expected to significantly reduce the heart index (heart weight / mouse body weight). In some embodiments, the isolated anti-NPR1 antibodies of this disclosure are expected to delay cardiomyopathy and improve cardiac function in modeling mice.

[0149] Exemplary, the anti-NPR1 antibodies of this disclosure exhibit superior pharmacokinetic expression (e.g., longer half-life, higher bioavailability, better absorption, and lower clearance) compared to positive antibodies (particularly XX16V).

[0150] Antibody structure In some embodiments, the antibodies provided herein are full-length antibodies.

[0151] In some embodiments, the antibodies provided herein are antibody fragments.

[0152] In some embodiments, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab')2 fragment, particularly a Fab fragment. "Fab" is a monovalent fragment consisting of VL, VH, CL, and CH1 domains. "Fab fragment" may be produced by lysing the antibody with papain. "Fab'" contains VL, CL, VH, and CH1, and further includes a region between the CH1 and CH2 domains, so that an interchain disulfide bond is formed between the two heavy chains of two Fab' fragments, enabling the formation of an F(ab')2 molecule. "Fab'-SH" is a Fab' fragment in which a cysteine ​​residue in the constant region contains a free mercapto group. "F(ab')2" is a bivalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region.

[0153] In some other embodiments, the antibody fragment is a biantibody, triploat, or quadruploat. A biantibody is an antibody fragment containing two antigen-binding sites, the fragment containing linked VH and VL on the same polypeptide chain (VH-VL). By using a linker that is too short, pairing of two domains on the same chain becomes impossible, forcing those domains to pair with complementary domains on another chain, thereby producing two antigen-binding sites, the two antigens may be the same or different.

[0154] In some other embodiments, the antibody fragment is a single-stranded Fab fragment. The "single-stranded Fab fragment" or "scFab" is a polypeptide comprising VH, CH1, VL, CL and a linker, wherein the antibody domain and the linker have one of the following sequences in the direction from the N-terminus to the C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In some embodiments, the linker is a polypeptide containing at least 30 amino acids. In some other embodiments, the linker is a polypeptide containing 32 to 50 amino acids. The single-stranded Fab fragment is stabilized by a native disulfide bond between CL and CH1. Furthermore, by inserting cysteine ​​residues (for example, at position 44 in the heavy chain variable region and position 100 in the light chain variable region, numbered by Kabat), interchain disulfide bonds are produced, and these single-chain Fab molecules can be further stabilized.

[0155] In some other embodiments, the antibody fragment is an Fv fragment consisting of the VH and VL domains of a single arm of the antibody.

[0156] In some other embodiments, the antibody fragment is a single-chain variable fragment (scFv). “scFv” is a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain and heavy chain variable regions are sequentially linked by a short flexible peptide linker, and the scFv is expressible as a single-chain polypeptide, and the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, in this specification, scFv may comprise the VL and VH variable regions in any order; for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may comprise a VL-linker-VH or a VH-linker-VL.

[0157] In several other embodiments, the antibody fragment is a dsFv, which is obtained by linking polypeptides in which one amino acid residue in each VH and VL is substituted with a cysteine ​​residue via disulfide bonds between the cysteine ​​residues. The amino acid residue to be substituted with a cysteine ​​residue can be selected based on the prediction of the three-dimensional structure of the antibody by a known method (Protein Engineering. 7:697 (1994)).

[0158] In some other embodiments, the antibody fragment is a single-domain antibody (dAb). A single-domain antibody is an antibody fragment that includes all or part of the heavy chain variable domain of an antibody, or all or part of the light chain variable domain.

[0159] In some embodiments, the antibodies provided herein are chimeric antibodies. In some embodiments, the chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In some embodiments, the chimeric antibody is a "class-switched" antibody in which the class or subclass has already been changed from the class or subclass of the parent antibody.

[0160] In some embodiments, the antibody is a humanized antibody. Generally, humanization of a non-human antibody reduces its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable regions in which the CDR or a portion thereof is derived from a non-human antibody, and the FR or a portion thereof is derived from a human antibody. Optionally, the humanized antibody may further contain a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody may be substituted with corresponding residues from a non-human antibody (e.g., an antibody providing the CDR sequence).

[0161] Humanized antibodies and methods for their production are outlined in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further discussed in Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), US Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409, Kashmiri et al. This is described in publications such as al., Methods36:25-34 (2005) (regarding grafting of specific determination regions (SDRs)), Padlan, Mol.Immunol.28:489-498 (1991) (regarding "resurfacing"), Dall'Acqua et al., Methods36:43-60 (2005) (regarding "FR shuffling"), and Osbourn et al., Methods36:61-68 (2005) and Klimka et al., Br.J.Cancer83:252-260 (2000) (regarding the "induction selection" method for FR shuffling).

[0162] Human framework regions usable for humanization include framework regions selected by the "best-fit" method (see, e.g., Sims et al., J.Immunol. 151:2296 (1993)), framework regions derived from consensus sequences of human antibodies of specific subclasses of light chain variable regions or heavy chain variable regions (see, e.g., Carter et al., Proc.Natl.Acad.Sci.USA, 89:4285 (1992) and Presta et al., J.Immunol., 151:2623 (1993)), framework regions of human maturation (somatic mutation) or human germline framework regions (see, e.g., Almagro and Fransson, Front.Biosci. 13:1619-1633 (2008)), and framework regions obtained by screening the FR library (e.g., Baca et al. This includes, but is not limited to, al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0163] Mutants of anti-NPR1 antibodies In one embodiment, amino acid sequence variants of anti-NPR1 antibodies provided herein are covered. For example, these can be expected to improve the antibody's binding affinity and / or other biological properties. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, the deletion, and / or insertion, and / or substitution of residues in the amino acid sequence of the anti-NPR1 antibody. The final construct can be obtained by any combination of deletions, insertions, and substitutions, provided that the final construct has desired properties, such as antigen-binding properties.

[0164] Substitution, insertion, and deletion of mutants In one embodiment, antigen variants are provided that include one or more amino acid substitutions. The sites of interest for substitutional mutagenesis include CDR and FR. Conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions." More substantial changes are provided in Table 2 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into antibodies of interest, and the products can be screened for desired activities such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0165] [Table 2]

[0166] Amino acids can be grouped as follows based on their general side-chain characteristics: (1) Hydrophobic substances: norleucine, Met, Ala, Val, Leu, Ile (2) Neutral, hydrophilic substances: Cys, Ser, Thr, Asn, Gln, (3) Acidic substances: Asp, Glu, (4) Basic substances: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0167] Non-conservative substitution requires replacing a member in one of these classes with a member in another class.

[0168] A single substitution variant involves the substitution of one or more CDR residues in a parent antibody (e.g., a humanized or human antibody). Generally, a variant selected for further study will have a change (e.g., improvement) in certain biological properties (e.g., improved affinity, reduced immunogenicity) compared to the parent antibody, and / or will essentially retain certain biological properties of the parent antibody. One exemplary substitution variant is an affinity-matured antibody, which can be readily produced by affinity-maturation techniques based on phage display (e.g., techniques as described herein). In short, one or more CDR residues are mutated, the mutant antibody is displayed on a phage, and its specific biological activity (e.g., binding affinity) is screened. For example, a change (e.g., substitution) can be made to the CDR to improve antibody affinity. Such a change can be made to CDR "hotspots," i.e., residues encoded by codons that are frequently mutated during somatic cell maturation, and / or residues that come into contact with the antigen, and the resulting mutant VH or VL can be tested for binding affinity. In several embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of several methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-specific mutagenesis). A secondary library is then created. Furthermore, the library is screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves a CDR orientation method that randomizes several CDR residues (e.g., 4-6 residues at a time). For example, CDR residues involved in antigen binding can be specifically identified by alanine scanning mutagenesis or modeling. HCDR3 and LCDR3 are particularly often targeted.

[0169] In some embodiments, substitutions, insertions, or deletions may be made in one or more CDRs, provided that such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, a conservative change (e.g., a conservative substitution, as provided herein) can be made to a CDR that does not substantially reduce its binding affinity. Such changes may be, for example, outside the antigen-contact residue in the CDR. In some embodiments of the variant VH and VL sequences provided above, each CDR is either unchanged or contains one, two, or three or fewer amino acid substitutions.

[0170] A method that can be used to identify residues or regions in an antibody that may be target sites for mutagenesis is called "alanine scanning mutagenesis." In this method, one residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., Ala or polyalanine) to determine whether the interaction between the antibody and the antigen has been affected. Further substitutions can be introduced at amino acid positions that are functionally sensitive to the initial substitution. Contact points between the antibody and the antigen can also be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues may be targeted or removed as substitution candidates. Mutants may be screened to determine whether they contain desired properties.

[0171] Amino acid insertions include amino-terminal and / or carboxyl-terminal fusions of polypeptides, ranging in length from one residue to 100 or more residues, and intra-sequence insertions of one or more amino acid residues. An example of terminal insertion is an antibody containing an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N or C terminus of an antibody with a polypeptide that extends the serum half-life of an enzyme or antibody.

[0172] Recombination Anti-NPR1 antibodies can be produced by recombinant methods. For these methods, we provide one or more isolated nucleic acids encoding anti-NPR1 antibodies.

[0173] In some embodiments, the Disclosure provides isolated nucleic acids encoding the anti-NPR1 antibody described above. Each of these nucleic acids can independently encode any one of the polypeptide chains described above. In another embodiment, the Disclosure provides one or more vectors (e.g., expression vectors) containing such nucleic acids. In another embodiment, the Disclosure provides host cells containing such nucleic acids. In some embodiments, a method is provided for preparing a polypeptide or fusion protein, comprising culturing a host cell containing the nucleic acid encoding the polypeptide or fusion protein under conditions suitable for expression, as provided above, and optionally recovering the anti-NPR1 antibody from the host cell (or host cell medium).

[0174] To recombinantly produce anti-NPR1 antibodies, the nucleic acids encoding the protein are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using common procedures, or produced by recombinant methods, or obtained by chemical synthesis.

[0175] Suitable host cells for cloning or expressing vectors encoding anti-NPR1 antibodies include prokaryotic or eukaryotic cells as described herein. For example, they can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression, they can be isolated from bacterial cell paste in the soluble fraction and further purified.

[0176] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable clones or expression hosts for vectors encoding fusion proteins, and include fungal and yeast strains. Suitable host cells for the expression of fusion proteins may be derived from multicellular organisms (invertebrates and vertebrates), and examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have already been identified and may be used in combination with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells, or plant cell cultures may be used as hosts, such as US5959177, US6040498, US6420548, US7125978 and US6417429, or vertebrate cells, such as mammalian cell lines suitable for growth in suspension, may be used as hosts. Other suitable mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7), human fetal kidney cell line (293 or 293T cells), baby hamster kidney cell line (BHK), mouse Sertoli cell line (TM4 cell line), monkey kidney cell line (CV1), African green monkey kidney cell line (VERO-76), human cervical cancer cell line (HELA), canine kidney cell line (MDCK), buffalo rat hepatocyte cell line (BRL3A), human lung cell line (W138), human hepatocyte cell line (Hep G2), mouse mammary tumor cell line (MMT 060562), TRI cell line, MRC 5 cell line, and FS4 cell line. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cell line, including DHFR-CHO cell line, and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For an overview of several mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (eds.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0177] measurement The anti-NPR1 antibodies provided herein can be identified, screened, or characterized by their physical / chemical features and / or biological activity using several known assay methods in the art. In one embodiment, the activity of the anti-NPR1 antibodies of this disclosure is tested by known methods such as ELISA and Western blotting.

[0178] Treatment method and route of administration Any anti-NPR1 antibody provided in this disclosure may be used in therapeutic methods. In another embodiment, this disclosure provides the use of anti-NPR1 antibodies in the manufacture or preparation of pharmaceuticals. In some embodiments, the disease is a disease or condition related to NPR1. In some embodiments, the disease or condition is heart failure, hypertension, peripheral vascular disease, coronary artery disease (CAD), ischemic heart disease (IHD), mitral stenosis and regurgitation, angina pectoris, hypertrophic cardiomyopathy (HCM), diabetic cardiomyopathy, supraventricular and ventricular arrhythmias, arrhythmia, atrial fibrillation (AF), new onset of atrial fibrillation, recurrent atrial fibrillation, cardiac fibrosis, atrial flutter, harmful vascular remodeling, plaque stabilization, myocardial infarction (MI), pre-eclampsia, obesity, renal failure, renal dysfunction, cytokine release syndrome, chronic kidney disease, macular edema, glaucoma, stroke, lung disease, lung cancer The conditions are selected from vascular disease, inflammation, asthma, bone growth disorders, fractures, diabetes, and cancer, and in some embodiments, the heart failure described above is selected from heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), heart failure with mildly reduced ejection fraction (HFmrEF), heart failure after acute myocardial infarction, or acute compensated heart failure, and in some embodiments, the hypertension described above is refractory hypertension, and in some embodiments, the heart failure described above is heart failure with preserved ejection fraction (HFpEF).

[0179] In a further embodiment, a pharmaceutical composition comprising the anti-NPR1 antibody is provided, for example, for use in any of the above-described pharmaceutical uses or therapeutic methods. In some embodiments, the pharmaceutical composition comprises any anti-NPR1 antibody or its antigen-binding fragment provided herein and a pharmaceutically acceptable carrier. In some other embodiments, the pharmaceutical composition further comprises at least one other therapeutic agent.

[0180] The anti-NPR1 antibody of this disclosure can be used alone or in combination with other reagents for therapeutic purposes. For example, the antibody of this disclosure may be administered together with at least one other therapeutic agent.

[0181] The anti-NPR1 antibody (and any other therapeutic agent) of this disclosure may be administered by any suitable means, including parenteral administration, intrapulmonary administration and intranasal administration, and, if local treatment is required, into the lesion. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be carried out by any suitable route, for example, by injection such as intravenous or subcutaneous injection, which is determined in part by whether the administration is short-term or long-term. Multiple dosing schedules are considered herein, including, but not limited to, single doses or multiple doses at multiple time points, bolus doses, and pulse infusions.

[0182] The anti-NPR1 antibodies described herein are prepared, administered, and given in accordance with good medical practice. Possible factors in this context include the specific medical condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the medical condition, the site of reagent delivery, the method of administration, the administration schedule, and other factors known to healthcare professionals. The anti-NPR1 antibodies may be prepared with or without one or more reagents currently used for the prevention or treatment of the above medical conditions. The effective amount of such other reagents is determined by the amount present in the pharmaceutical composition, the type of medical condition or treatment, and other factors. They are generally used in the same doses and routes of administration as described herein, or in approximately 1% to 99% of the doses described herein, or in different doses and via any appropriate route determined empirically / clinically.

[0183] For the prevention or treatment of a disease, the appropriate dose of the anti-NPR1 antibody of this disclosure (used alone or in combination with one or more other therapeutic agents) is determined by the type of disease to be treated, the type of therapeutic molecule, the severity and course of the disease, whether the administration is for prevention or treatment, past treatments, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is administered to the patient as appropriate, either as a single dose or in a series of treatments.

[0184] product In another aspect of this disclosure, a product (e.g., a kit) is provided which comprises materials that can be used for the treatment, prevention and / or diagnosis of the above-mentioned medical condition. The product comprises a container and a label or package insert on or in combination with the container. Suitable containers include, for example, bottles, vials, syringes, and IV solution bags. The container may be formed from a variety of materials, such as glass or plastic. The container may contain a composition that effectively treats, prevents and / or diagnoses a disease, either alone or in combination with another composition, and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a plug through which a subcutaneous needle can be inserted). At least one active reagent in the composition is the anti-NPR1 antibody of this disclosure. The label or package insert indicates that the composition is used to treat a selected medical condition. The product may also comprise (a) a first container containing a composition comprising the anti-NPR1 antibody of this disclosure, and (b) a second container containing a composition comprising other therapeutic agents. The product in the embodiments of the present disclosure may further include a document indicating that the composition may be used to treat a particular medical condition. Optionally, or further, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer. From a business and user perspective, it may further include other necessary materials, including other buffers, diluents, filters, needles and syringes.

[0185] The present disclosure is further described below in accordance with examples and test examples, but these examples and test examples are not intended to limit the scope of the present disclosure. In the examples or test examples of the present disclosure, experimental methods for which specific conditions are not specified are generally performed under normal conditions, for example, according to Cold Spring Harbor's Antibody Technology Experiment Manual, Molecular Cloning Manual, or according to conditions recommended by the raw material or product manufacturer, and reagent materials for which specific sources are not specified were obtained by purchasing from the market.

[0186] Examples Example 1: Preparation of NPR1 antigen Using UniProt or NCBI NPR1 antigens (human NPR1 protein, Uniprot number: P16066; cynomolgus monkey NPR1 protein, NCBI number: XP-005541809.1; rat NPR1 protein, Uniprot number: P18910) as templates for NPR1, the amino acid sequences of the antigens and detection proteins used in this disclosure were designed, and fusion tags such as hFc were selectively added to the NPR1 protein. These were cloned into a pTT5 vector (Biovector, CAT#102762), expressed by transient transfection in CHO cells, purified, and the antigens and detection proteins of this disclosure were obtained.

[0187] The extracellular domain of the human NPR1 protein and the Human-IgG1-Fc fusion protein (abbreviated as hNPR1-hFc) sequence were used as the immunoantigen. [ka] Note: The underlined portion is the Human-IgG1-Fc portion, and the ununderlined portion is the extracellular domain of the human NPR1 protein.

[0188] The extracellular domain of the cynomolgus monkey NPR1 protein and the Human-IgG1-Fc fusion protein (abbreviated as cNPR1-hFc) sequence were used as the immunoantigen. [ka] Note: The underlined portion is the Human-IgG1-Fc portion, and the ununderlined portion is the extracellular domain of the cynomolgus monkey NPR1 protein.

[0189] The extracellular domain of the rat (Rattus norvegicus) NPR1 protein and the Human-IgG1-Fc fusion protein (abbreviated as rNPR1-hFc) sequence were used as the immunoantigen. [ka] Note: The underlined portion is the Human-IgG1-Fc portion, and the ununderlined portion is the rat NPR1 protein extracellular domain.

[0190] Example 2: Purification of recombinant protein related to NPR1 Purification steps for NPR1-Fc fusion protein: The cell expression supernatant sample was rapidly centrifuged to remove impurities, and affinity chromatography was performed on the supernatant using MabSelect Sure (GE, 17-5438-01). The MabSelect Sure chromatography column was first regenerated with 0.2 M NaOH, washed with pure water, and then the column was equilibrated with PBS. After the supernatant was attached, A 280 The sample was washed with PBS until the reading was reduced to the baseline. The target protein was eluted with 0.1 M acetate buffer at pH 3.5 and neutralized with 1 M, pH 8.0 Tris-HCl. After appropriately concentrating the eluted sample, it was further purified using a PBS-equipped gel chromatograph Superdex200 (GE, 28-9893-35), and the receiving tube containing the target protein was collected and concentrated to an appropriate concentration. This method was used for the purification of NPR1-Fc fusion protein, but this method may also be used for the purification of antibody proteins in this disclosure.

[0191] Example 3: Preparation of NPR1 recombinant cell line Using UniProt or NCBI NPR1 antigens (human NPR1 protein, Uniprot number: P16066; cynomolgus monkey NPR1 protein, NCBI number: XP-005541809.1; rat NPR1 protein, Uniprot number: P18910) as templates for NPR1, recombinant cell lines for immunization and detection used in the disclosure were designed. Among these, the recombinant cell line for immunization was NIH-3T3 cells overexpressing full-length human NPR1, cynomolgus monkey NPR1, and rat NPR1 proteins, and the recombinant cell line for detection was a CHO-K1 cell line overexpressing full-length human NPR1, cynomolgus monkey NPR1, and rat NPR1 proteins.

[0192] 1. Human NPR1 full-length amino acid sequence [ka] Note: Human NPR1 is a transmembrane protein that exists on the surface of the cell membrane in the form of homodimers. The double-underlined portion is the extracellular domain of NPR1 (33-473). The dotted line represents the transmembrane domain (474-494), The single underlined portion is the intracellular region (Cytoplasmic domain: 495-1061), The unlabeled portion is a signal peptide.

[0193] 2. Full-length amino acid sequence of NPR1 in cynomolgus macaque [ka] Note: Cyno NPR1 is a transmembrane protein that exists on the surface of the cell membrane in the form of homodimers. The double-underlined portion is the extracellular domain of NPR1 (33-473). The dotted line represents the transmembrane domain (474-494), The single underlined portion is the intracellular region (Cytoplasmic domain: 495-1061), The unlabeled portion is a signal peptide.

[0194] 3. Full-length amino acid sequence of rat (Rattus norvegicus) NPR1 [ka] Note: RatNPR1 is a transmembrane protein that exists on the surface of the cell membrane in the form of homodimers. The double-underlined portion is the extracellular domain of NPR1 (29-469). The dotted line represents the transmembrane domain (470-490). The single underlined portion is the intracellular region (Cytoplasmic domain: 491-1057), The unlabeled portion is a signal peptide.

[0195] NIH-3T3 cells were stably transformed with the above protein, and after two weeks of pressurized screening, subcloning was performed to screen monoclonal cells. FACS detection then yielded highly expressive hNPR1-NIH-3T3, cNPR1-NIH-3T3, and rNPR1-NIH-3T3 recombinant cell lines for mouse immunization.

[0196] CHO-K1 cells were stably transformed with the above protein, and after two weeks of pressurized screening, subcloning was performed to screen monoclonal cells. FACS detection was then used to obtain highly expressive hNPR1-CHO-K1, cNPR1-CHO-K1, and rNPR1-CHO-K1 recombinant cell lines for detecting the affinity and in vitro function of anti-NPR1 antibodies.

[0197] Example 4: Screening of mouse anti-hNPR1 antibodies 1. Mouse immunization: Anti-human NPR1 monoclonal antibody was produced by immunized mice. In the experiment, female SJL white mice aged 6 - 8 weeks (Shanghai SLAC Laboratory Animal Co., Ltd., Animal Production License Number: SCXK(Shanghai)2017 - 0005) were used. Breeding environment: SPF grade. After purchasing the mice, they were adjusted in a 12 / 12 hour light / dark cycle and bred in a laboratory environment with a temperature of 20°C - 25°C and a humidity of 40% - 60% for one week. The mice adapted to the environment were immunized according to Plan 1 and Plan 2. The immunization antigens were hNPR1 NIH - 3T3, cNPR1 NIH - 3T3, rNPR1 NIH - 3T3, hNPR1 - hFc, cNPR1 - hFc, rNPR1 - hFc, and hANP(1 - 28aa, GenScript Biotech Co., Ltd., Cat No.RP11927).

[0198] Immunization Plan 1: Mice were pre - immunized with TiterMax® Gold Adjuvant (Sigma Cat No.T2684) by intraperitoneal (IP) injection at 0.1 mL / mouse (primary immunization). 15 minutes later, hNPR1 NIH - 3T3 at 1×10 ^7 cells / mouse was injected IP. The antigens hNPR1 NIH - 3T3 and cNPR1 NIH - 3T3 cross - immunized. The inoculation times were on days 0, 14, and 28. Blood was collected on days 21 and 35, and the antibody titers in mouse sera were determined by ELISA and FACS methods. After the third immunization, mice with high and stable antibody titers in serum were selected for splenocyte fusion. Three days before splenocyte fusion, additional immunization was performed by injecting an antigen hNPR1 - hFc solution prepared with physiological saline at 50 μg / mouse IP. hANP(1 - 28aa) was added at 5 μg / mouse for each immunization inoculation.

[0199] Immunization Plan 2: Mice were pre-immunized with TiterMax® Gold Adjuvant (Sigma Cat No. T2684) and injected intraperitoneally (IP) at a dose of 0.1 mL / mice (primary immunization). 15 minutes later, hNPR1 NIH-3T3 was injected intraperitoneally (IP) at a dose of 1 × 10^7 cells / mice. The antigens hNPR1 NIH-3T3 and rNPR1 NIH-3T3 were cross-immunized. Inoculation times were days 0, 14, 28, 42, 56, 70, 84, and 98. Blood was collected on days 35, 63, 91, and 105, and antibody titers in mouse serum were determined by ELISA and FACS. After the 8th immunization, mice with high and stable antibody titers in their serum were selected, and splenocyte fusion was performed. Three days prior to splenocyte fusion, a booster immunization was performed by intraperitoneal (IP) injection of a mixed solution of the antigens hNPR1-hFc, cNPR1-hFc, and rNPR1-hFc, prepared with physiological saline, at a dose of 50 μg / animal. 5 μg / animal of hANP(1-28aa) was added with each subsequent immunization.

[0200] 2. Splenocyte fusion: Using an optimized electrofusion method, splenic lymphocytes were fused with myeloma cells (Sp2 / 0 cells, ATCC® CRL-8287®) to obtain hybridoma cells.

[0201] Splenocyte counting results showed that fused hybridoma cells numbered 3 × 10⁶. ^5 ~4×10 ^5 The cells were resuspended in complete medium (IMDM medium containing 20% ​​FBS, 1×HAT, and 1×OPI) at a density of cells / mL and inoculated into 96-well plates at 150 μL / well. After incubation at 37°C and 5% CO2 for 4-5 days, the supernatant was removed, and 200 μL / well of HT complete medium (IMDM medium containing 20% ​​FBS, 1×HT, and 1×OPI) was added. After incubation at 37°C and 5% CO2 for 2 days, ELISA detection was performed.

[0202] 3. Screening of hybridoma cells: Based on the growth density of hybridoma cells, the hybridoma culture supernatant was detected in cell-based binding assays and cGMP production experiments. The cells in the positive wells were amplified in a timely manner and cryopreserved as a seed until monoclonal clones were obtained, and subcloned one to two times. The monoclonal clones were expanded in culture, RNA was extracted, and reverse transcription amplification (RT-PCR) was performed using degenerate primers for mouse-Ig, resulting in the acquisition of the variable region sequences of the antibodies. When the amino acid sequences corresponding to the DNA sequences of mouse antibodies 102, 127, 128, 135, and 140 were measured, the results were as follows.

[0203] NPR1-CHAb-SFM-102-1 HCVR:

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chem.

Chem.

[0204] The CDR region sequences of the heavy and light chains of mouse antibodies 102, 127, 128, 135, and 140 are as shown in Table 3 below.

[0205]

Table 3

[0206] By linking the carboxy terminus of the heavy chain variable region of mouse antibodies 102, 127, 128, 135, and 140 screened above to the amino terminus of the human heavy chain constant region shown by SEQ ID NO: 43, and at the same time linking the carboxy terminus of the light chain variable region of the mouse antibody to the amino terminus of the human light chain constant region shown by SEQ ID NO: 44, the corresponding chimeric antibodies of 102, 127, 128, 135, and 140 can be obtained, which are designated as CHI-102, CHI-127, CHI-128, CHI-135, and CHI-140, respectively.

[0207] Construction of the human IgG1 heavy chain constant region sequence for chimeras:

Chem.

Chem.

[0208] 4. FACS binding experiment of anti-NPR1 chimeric antibody: In vitro binding experiments were performed to confirm the accuracy of the sequence of the variable region of the obtained monoclonal antibody. 1 × 10 6 Cells expressing hNPR1 CHO-K1 or cNPR1 CHO-K1 at a cell / mL concentration were isolated, blocked with 1% BSA PBS buffer, and then incubated for 1 hour with anti-NPR1 chimeric antibody samples diluted to different concentrations (pre-mixed with 10 nM ANP for in vitro binding experiments in the presence of ANP). After washing twice with pH 7.4 PBS, APC anti-human IgG (Biolegend Cat. No. 409306) was added and incubated for 45 minutes. After washing twice again with pH 7.4 PBS, the cells were resuspended in 150 μL of pH 7.4 PBS, and the fluorescence signal value was read using a flow cytometer. The experimental results are shown in Table 4.

[0209] [Table 4]

[0210] Example 5: Humanization of anti-NPR1 mouse antibody By aligning the IMGT human antibody heavy and light chain variable region germline gene database with MOE software, heavy and light chain variable region germline genes with high homology to 102, 127, 128, 135, and 140 were selected as templates. The CDRs of mouse antibodies were then transplanted into the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Exemplarily, in the following specific examples, the amino acid residues of the CDRs are determined and annotated using the Kabat numbering system.

[0211] Humanization of 102 mouse antibodies For the humanized heavy chain variable regions of mouse antibody 102, FR1, FR2, and FR3 used IGHV3-21*01 as a template, and FR4 used IGHJ6*01 as a template. For the light chain variable regions, FR1, FR2, and FR3 used IGKV3-11*01 or IGKV1-39*01 as a template, and FR4 used IGKJ4*01 as a template. The CDR of mouse antibody 102 was then transplanted into these human templates. Optionally, some amino acids in the FR regions of the humanized antibodies were substituted, specifically the amino acid residues at positions 1, 3, 9, 44, 49, 87, and / or 93 (determined by Kabat numbering rules) in the FR region of the heavy chain variable region, and / or the amino acid residues at positions 4, 43, 45, 46, 47, 58, and / or 71 (determined by Kabat numbering rules) in the FR region of the light chain variable region. The mutation design for the humanized antibody 102 is shown in Table 5 below.

[0212] [Table 5]

[0213] [Table 6]

[0214] The light chain variable region / heavy chain variable region sequences of the 102 humanized antibody are as follows: >hu102VH1 [ka] >hu102VH2 [ka] >hu102VH3 [ka] >hu102VH4 [ka] >hu102VH5 [ka] >hu102VL3 [ka] >hu102VL4 [ka] >hu102VL5 [ka] >hu102VL6 [ka] Note: The double-underlined portion is the antibody CDR sequence, and the CDRs are numbered by Kabat.

[0215] Humanization of 127 mouse antibodies For the humanized heavy chain variable regions of mouse antibody 127, FR1, FR2, and FR3 used IGHV1-46*01 as a template, and FR4 used IGHJ6*01 as a template. For the light chain variable regions, FR1, FR2, and FR3 used IGKV4-1*01 or 1-39*01 as a template, and FR4 used IGKJ2*01 as a template. The CDR of mouse antibody 127 was then transplanted into each of these human templates. For optional purposes, some amino acids in the FR region of the humanized antibody were substituted, specifically at positions 1, 2, 3, 28, 43, 69, 71 and / or 73 (determined by Kabat numbering rules) in the FR region of the heavy chain variable region. Some substitutions involved designing a glutamate mutation at position 1 to avoid glutamate cyclization, and / or substitutions at positions 1, 4, 17, 43, 46, 60, 68 and / or 79 (determined by Kabat numbering rules) in the FR region of the light chain variable region. The mutation designs for the humanized antibody variable region of antibody 127 are shown in Table 7 below.

[0216] [Table 7]

[0217] [Table 8]

[0218] The sequence of the light / heavy chain variable region of the 127 humanized antibody is as follows: >hu127VH4 [ka] >hu127VH7 [ka] >hu127VH8 [ka] >hu127VL1 [ka] >hu127VL3 [ka] >hu127VL4 [ka] >hu127VL5 [ka] >hu127VL6 [ka] Note: The double-underlined portion is the antibody CDR sequence, and the CDRs are numbered by Kabat.

[0219] Humanization of 128 mouse antibodies For the humanized heavy chain variable regions of mouse antibody 128, FR1, FR2, and FR3 used IGHV1-46*01 as a template, and FR4 used IGHJ6*01 as a template. For the light chain variable regions, FR1, FR2, and FR3 used IGKV3-20*02 or 6-21*02 as a template, and FR4 used IGKJ2*01 as a template. The CDR of mouse antibody 128 was then transplanted into each of these human templates. For optional modifications, some amino acids in the FR region of the humanized antibody were substituted, specifically at positions 1, 40, 43, 69, 71, 73, 74, 75, 77 and / or 78 (determined by Kabat numbering rules) in the heavy chain variable region FR region; a glutamate mutation at position 1 was performed to avoid glutamate cyclization; and / or at positions 22, 42, 43, 49, 57 and / or 71 (determined by Kabat numbering rules) in the light chain variable region FR region. The mutation designs for the humanized antibody variable region of antibody 128 are shown in Table 9 below.

[0220] [Table 9]

[0221] >hu128VH1 [ka] >hu128VH2 [ka] >hu128VH3 [ka] >hu128VH4 [ka] >hu128VL1 [ka] >hu128VL2 [ka] >hu128VL3 [ka]

[0222] Furthermore, based on hu128VL1, individual amino acids (A55) in the light chain FR1 region (S22) and LCDR2 region were modified, and the amino acid sequence of LCDR2 was changed from the original DTYNLAS (SEQ ID NO: 33) to DTYNLGS (SEQ ID NO: 73), thereby obtaining a new humanized antibody light chain variable region sequence, the specific sequence of which is as follows. >hu128VL1~6 [ka] Note: The double-underlined portion is the antibody CDR sequence, and the CDRs are numbered by Kabat.

[0223] Humanization of 135 mouse antibodies For the humanized heavy chain variable regions of mouse antibody 135, FR1, FR2, and FR3 used IGHV3-21*01 as a template, and FR4 used IGHJ6*01 as a template. For the light chain variable regions, FR1, FR2, and FR3 used IGKV1-39*01 as a template, and FR4 used IGKJ4*01 as a template. The CDR of mouse antibody 135 was transplanted into these human templates. Optionally, some amino acids in the FR region of the humanized antibody were substituted, specifically the amino acid residues at positions 1, 3, 9, 28, 29, 44, 49, and / or 93 (determined by Kabat numbering rules) in the FR region of the heavy chain variable region, and / or the amino acid residues at positions 4, 43, 46, 47, and / or 71 (determined by Kabat numbering rules) in the FR region of the light chain variable region. The mutation designs for the humanized antibody variable regions of antibody 135 are shown in Table 10 below.

[0224] [Table 10]

[0225] [Table 11]

[0226] >hu135VH1 [ka] >hu135VH2 [ka] >hu135VH3 [ka] >hu135VL1 [ka] Note: The double-underlined portion is the antibody CDR sequence, and the CDRs are numbered by Kabat.

[0227] Humanization of 140 mouse antibodies For the humanized heavy chain variable regions of mouse antibody 140, FR1, FR2, and FR3 used IGHV1-46*01 as a template, and FR4 used IGHJ6*01 as a template. For the light chain variable regions, FR1, FR2, and FR3 used IGKV3-11*01 as a template, and FR4 used IGKJ2*01 as a template. The CDR of mouse antibody 140 was then transplanted into each of these human templates. For optional purposes, some amino acids in the FR region of the humanized antibody were substituted, specifically at positions 1, 40, 43, 48, 69, 71, 73, 74, 75, 77, 78, 80 and / or 81 (determined by Kabat numbering rules) in the FR region of the heavy chain variable region. Some substitutions involved designing glutamate mutations at position 1 to avoid glutamate cyclization, and / or substitutions at positions 20, 60, 58, 70, 71, 77 and / or 100 (determined by Kabat numbering rules) in the FR region of the light chain variable region. The mutation designs for the humanized antibody variable region of antibody 140 are shown in Table 12 below.

[0228] [Table 12]

[0229] >hu140VH3 [ka] >hu140VH4 [ka] >hu140VH5 [ka] >hu140VL3 [ka] >hu140VL5 [ka]

[0230] Furthermore, based on hu140VL5, the individual amino acids (A60) in the light chain FR1 region (T20) and FR3 region were modified. >hu140VL5~11 [ka] Note: The double-underlined portion is the antibody CDR sequence, and the CDRs are numbered by Kabat.

[0231] Construction and expression of the LALA morphology of the anti-NPR1 humanized antibody IgG1. Primer PCR was designed to assemble each humanized antibody VH / VK gene fragment, and homologous recombination was performed with the expression vector pTT5 (containing the signal peptide and constant region gene (CH1-FC / CL) fragment, constructed in the laboratory) to construct the full-length antibody expression vector VH-CH1-FC-pTT5 / VK-CL-pTT5. The heavy chain constant region of the antibody may be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, IgG4 or their variants, and the light chain constant region may be selected from the light chain constant regions of human κ, λ chains or their variants. Exemplarily, in the following example, the antibody heavy chain constant region was selected from the human IgG1 heavy chain constant region shown in SEQ ID NO: 86, the light chain constant region was selected from the human light chain constant region shown in SEQ ID NO: 44, and an LALA mutation (indicated by double underline) was introduced.

[0232] Sequence of the constant region of the LALA heavy chain of human IgG1: [ka] Human light chain constant region sequence: [ka]

[0233] By ligating the carboxyl terminus of the variable region of the humanized antibody heavy chains 102, 127, 128, 135, and 140 constructed above to the amino terminus of the constant region of the human heavy chain shown in SEQ ID NO: 86 to form the full-length antibody heavy chain, and by ligating the carboxyl terminus of the variable region of the humanized antibody light chain to the amino terminus of the constant region of the human light chain shown in SEQ ID NO: 44 to form the full-length antibody light chain, humanized antibodies as shown in Tables 13 to 17 below can be obtained.

[0234] [Table 13]

[0235] [Table 14]

[0236] [Table 15]

[0237] [Table 16]

[0238] [Table 17]

[0239] The full-length light / heavy chain sequences of an example humanized antibody are as follows:

[0240] Heavy chain sequence of Hu102H2L6: [ka] Heavy chain sequence of Hu102H3L6: [ka] Heavy chain sequence of Hu102H4L6: [ka] Heavy chain sequence of Hu102H5L6: [ka] Light chain sequences of Hu102H2L6, Hu102H3L6, Hu102H4L6, and Hu102H5L6: [ka] Heavy chain sequences of Hu127H4L1, Hu127H4L3, Hu127H4L5, and Hu127H4L6: [ka] Heavy chain sequences of Hu127H7L1, Hu127H7L3, Hu127H7L5, and Hu127H7L6: [ka] Heavy chain sequences of Hu127H8L1, Hu127H8L3, Hu127H8L5, and Hu127H8L6: [ka] Light chain sequences of Hu127H4L1, Hu127H7L1, and Hu127H8L1: [ka] Light chain sequences of Hu127H4L3, Hu127H7L3, and Hu127H8L3: [ka] Light chain sequences of Hu127H4L5, Hu127H7L5, and Hu127H8L5: [ka] Light chain sequences of Hu127H4L6, Hu127H7L6, and Hu127H8L6: [ka] Heavy chain sequences of Hu128H2L1 and Hu128H2L2: [ka] Heavy chain sequences of Hu128H3L1-6, Hu128H3L1, and Hu128H3L2: [ka] Light chain sequences of Hu128H2L1 and Hu128H3L1: [ka] Light chain sequences of Hu128H2L2 and Hu128H3L2: [ka] Hu128H3L1~6 light chain sequence: [ka] Heavy chain sequence of Hu135H1L1: [ka] Heavy chain sequence of Hu135H2L1: [ka] Heavy chain sequence of Hu135H3L1: [ka] Light chain sequences of Hu135H1L1, Hu135H2L1, and Hu135H3L1: [ka] Heavy chain sequence of Hu140H4L5: [ka] Heavy chain sequences of Hu140H5L5 and Hu140H5L5-11: [ka] Light chain sequences of Hu140H4L5 and Hu140H5L5: [ka] Light chain sequence of Hu140H5L5-11: [ka] Note: In the full-length antibody sequence shown above, the single underlined portion is the antibody CDR sequence, and the italicized portion is the antibody constant region sequence. Of these, the CDR sequences are numbered by Kabat.

[0241] The light and heavy chain sequences of the positive control antibodies REGN-5381 (see WO2020086406A2 for the variable region sequence) and XX16V (see WO2020250159A1 for the variable region sequence) in the examples of this disclosure are as follows.

[0242] REGN-5381 heavy chain sequence: [ka] REGN-5381 light chain sequence: [ka] REGN-5381-m heavy chain sequence (REGN-5381 variable region + mouse IgG2a heavy chain, IMGT accession No. V0085): [ka] REGN-5381-m light chain sequence (REGN-5381 variable region + mouse kappa light chain, Uniprot accession No. P01837): [ka] XX16V heavy chain arrangement: [ka] XX16V light chain arrangement: [ka]

[0243] The light and heavy chains of the negative control antibody RC25V disclosed herein (see RC25 in US6114143A for the variable region) are as follows:

[0244] RC25V heavy chain: [ka] RC25V Light Chain: [ka] .

[0245] Test example Test Example 1: Detection of anti-NPR1 antibody binding to hNPR protein by ELISA experiment The binding activity of anti-NPR1 antibodies to hNPR1, hNPR2, and hNPR3 was detected by ELISA. The specific experimental procedure is as follows: Each well was coated with 4 μg / mL of hNPR1 antigen (hNPR1, SEQ ID NO. 1, hNPR2, Abcam, Cat. No. Ab201371, hNPR3, Abcam, Cat. No. Ab114355) at 37°C for 3 hours, then blocked with PBS containing 5% skim milk powder and incubated overnight at 4°C. After washing three times with pH 7.4 PBS, 20 μg / mL of anti-NPR1 antibody was added to the first well, and the subsequent wells were diluted with a 5-fold concentration gradient and incubated at 37°C for 1.5 hours. After washing three times with pH 7.4 PBS, anti-human IgG HRP (Jackson, Cat. No. 109-035-003) was added and incubated for 45 minutes. After washing six times with PBS at pH 7.4, the samples were color-developed and the values ​​were read. The experimental results are shown in Figures 1A to 1C and Table 18. The experimental results show that all of the anti-NPR1 antibodies in this disclosure bind to hNPR1, but not to hNPR2 or hNPR3, which belong to the same family.

[0246] [Table 18]

[0247] Experiment Example 2: cGMP production experiment induced by stimulation with anti-NPR1 antibody The anti-NPR1 antibody binds to NPR1 and stimulates the downstream signaling pathway to produce cGMP. This experiment revealed the antibody's agonist activity by detecting cGMP. The specific experimental procedure is as follows: A 1× reaction buffer was prepared using 5 mM MgCl2, 10 mM HEPES at pH 7.4, and 0.5 mM IBMX in EBSS. Anti-NPR1 antibodies of different concentrations were prepared in the reaction buffer. The concentration in the first well was 300 μg / mL, and subsequent wells were diluted with a 3-fold concentration gradient. The prepared antibodies were added to a 384-well plate at 5 μL / well. Simultaneously, hNPR1-CHOK1 cells were washed twice with PBS at pH 7.4, resuspended in the reaction buffer at 3E6 cells / mL, and added to the 384-well plate at 5 μL / well. After mixing was complete, the mixture was centrifuged at 300 g for 1 minute and incubated in a 37°C incubator for 30 minutes. Next, the subsequent procedures were carried out according to the instructions for the reagent kit (Cisbio, Cat. No. 62GM2PEG). The experimental results are shown in Tables 19-1 to 19-5 and Figure 2.

[0248] [Table 19]

[0249] [Table 20]

[0250] [Table 21]

[0251] [Table 22]

[0252] [Table 23]

[0253] [Table 24]

[0254] The experimental results demonstrate that all of the anti-NPR1 antibodies disclosed herein can stimulate hNPR1-CHOK1 cells to produce cGMP.

[0255] Test Example 3: Endocytosis experiment of binding of anti-NPR1 antibody to NPR1 Endocytosis occurs after the anti-NPR1 antibody binds to NPR1, and this experiment detected the strength of endocytosis caused by the anti-NPR1 antibody. The specific experimental procedure is as follows: The density of hNPR1-CHOK1 cells was adjusted to 4E5 cells / mL and added to a 96-well cell plate at 50 μL / well. Antibodies awaiting detection were prepared using CHO-K1 medium (DMEM / F12 + 10% FBS, Cat. No. Hyclone SH30023.01, Bio-Biology, A500023-0100), and 20 μg / mL of anti-NPR1 antibody was added to the 96-well cell plate and incubated in an incubator for 10 minutes, 1 hour, 7 hours, or 24 hours. At 10 minutes, 1 hour, 7 hours, or 24 hours, the culture supernatant was discarded, the cells were digested with trypsin, transferred to a new 96-well plate, centrifuged at 300 g for 5 minutes, and washed twice with 1% BSA / PBS. Detection antibody (REGN-5381-m) was prepared using 1% BSA / PBS, cells were resuspended in 100 μL / well, and incubated at 4°C for 1 hour. Then, cells were washed twice with 1% BSA / PBS, APC anti-mouse IgG (Biolegend Cat. No. 405308) was added, and incubated at 4°C for 40 minutes. Cells were washed twice with PBS, resuspended with 1% BSA / PBS, and fluorescence signal intensity was detected using a flow cytometer (Thermo fisher, Attune Nxt), with 10,000 cell data from each well analyzed. The experimental results are shown in Figure 3 and Table 20.

[0256] [Table 25] The experimental results indicate that the endocytosis of the anti-NPR1 antibody of this disclosure is weaker than that of the positive control antibody, resulting in a higher amount of NPR1 antigen retained on the cell surface. Weaker endocytosis can ensure that the anti-NPR1 antibody has more sustained and stable pharmacokinetics and efficacy.

[0257] Experiment Example 4: cGMP production experiment in cells stimulated by endocytosis with anti-NPR1 antibody After binding to NPR1, endocytosis occurs, reducing antigen expression and further affecting the agonist activity of the anti-NPR1 agonist antibody. This experiment detected the agonist activity of the NPR1 agonist antibody in cells treated with NPR1 endocytosis. The specific experimental procedure is as follows: The hNPR1-CHOK1 cell density was adjusted to 4E5 cells / mL and added to a 6-well cell plate at 2 mL / well. Antibodies awaiting detection were prepared using culture medium and added to a 6-well cell plate at 1 mL / well (final concentration 20 μg / mL). Incubation was performed overnight in an incubator. After washing the cells twice with PBS at pH 7.4, cGMP production experiments induced by anti-NPR1 antibody stimulation were performed using cells treated with endocytosis of the anti-NPR1 antibody according to the method of Test Example 1. The experimental results are shown in Figures 4A and 4B and Table 21. The data in the table characterizes the window value of cGMP production induced by stimulation in cells before and after endocytosis treatment by summing the signal changes, i.e., it is the sum of the absolute differences between the signal value detected by cGMP in untreated cells and the signal value detected by cGMP in treated cells at each detection concentration.

[0258] [Table 26] The experimental results show that the anti-NPR1 antibody of this disclosure exhibits less difference in cGMP production in cells after endocytosis compared to the positive antibody.

[0259] Test Example 5: In vivo efficacy experiment of anti-NPR1 antibody in an ANGII model using hNPR1 transgenic mice To evaluate the in vivo efficacy of anti-NPR1 antibodies, we used hNPR1 homozygous transgenic mice and modeled them using ANGII to induce progressive heart failure associated with hypertension. Parameters such as blood pressure, plasma and urine cGMP, and urine biochemical indicators were evaluated to assess the antihypertensive effect of the antibodies.

[0260] The specific modeling and experimental methods are as follows: Mice were pre-adapted using a blood pressure monitor warming tube, and their basal blood pressure was detected. Three days before the start of administration, a micro-osmotic pump (Alzet Micro-Osmotic Pump, model 1004, containing angiotensin II acetate, with an average pump rate set to 0.11 μL / hour and delivering AngII at 1.5 mg / kg / day) was subcutaneously implanted in the scapular region of the mice. Blood pressure was measured continuously for three days. Mice with difficult-to-measure or relatively large blood pressure fluctuations were excluded. Mice were then divided into groups, first by mean arterial pressure MAP, and then by body weight, with eight mice per group. The administration volume was 10 mL / kg, and the drug was administered by subcutaneous injection on days 0, 4, 7, 14, and 21, respectively. Blood pressure was measured according to the plan, and plasma was collected on days 2, 6, 9, 11, 16, 18, 25, and 29, as well as cumulative urine on days 2, 6, 9, 11, 16, 18, and 25. The corresponding cGMP content was then measured.

[0261] The experimental results are shown in Figures 5A to 5I and Table 22, where the average indicators for 29 days are statistically analyzed.

[0262] [Table 27] The experimental results showed that all of the anti-NPR1 antibodies disclosed herein had significant blood pressure control and cGMP production effects, as well as non-statistically insignificant trends in accelerating urinary sodium excretion and reducing NT-proBNP. On the other hand, the weak endocytosis molecule Hu102H4L6 had significantly higher endpoint blood concentrations than positive REGN-5381 at comparable doses and administration frequencies (Figure 5G).

[0263] Study Example 6: In vivo pharmacokinetic study of anti-NPR1 antibody Three hNPR1 transgenic mice per group were administered intravenously at a dose of 3 mpk. 0.15 mL of whole blood was collected at 5 min, 1 day, 2 days, 4 days, 7 days, 10 days, 14 days, and 21 days after administration. No anticoagulant was added. The blood was left at room temperature for 30 minutes after collection, then centrifuged at 1000 g for 15 minutes. The supernatant (serum) was collected, placed in an EP tube, and stored at -20°C. Standard curves for different samples were created using the Total IgG method. The serum concentrations of anti-NPR1 antibody at different time points were converted from OD450 values. The obtained data were analyzed using Phoenix WinNonlin software to calculate pharmacokinetic parameters. The experimental results are shown in Table 23.

[0264] [Table 28] The experimental results indicate that 140H5L5-11 exhibits relatively good in vivo pharmacokinetic expression in mice at 3 mpk. The above invention has been described in detail with drawings and examples so as to be clearly understood, but the description and examples should not be construed as limiting the scope of this disclosure. All disclosures of patents and scientific literature cited herein are incorporated in full and clearly by reference.

Claims

1. An anti-NPR1 antibody comprising a heavy chain variable region containing HCDR1, HCDR2, and HCDR3, and a light chain variable region containing LCDR1, LCDR2, and LCDR3, a. The heavy chain variable region contains the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NOs: 82, 80, 81, or 15, respectively, and the light chain variable region contains the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NOs: 85, 83, 84, or 16, respectively, or b. The heavy chain variable region contains the amino acid sequences of HCDR1, HCDR2, and HCDR3 in any one of the sequences of SEQ ID NOs: 51, 48, 49, 50, 52, or 7, and the light chain variable region contains the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NOs: 56, 53, 54, 55, or 8, or c. The heavy chain variable region contains the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NOs: 68, 66, 67, 69, or 11, respectively, and the light chain variable region contains the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NOs: 74, 70, 71, 72, or 12, respectively, or d. The heavy chain variable region contains the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NOs: 78, 76, 77, or 13, respectively, and the light chain variable region contains the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NOs: 79 or 14, respectively, or e. The heavy chain variable region HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NOs: 58, 59, 60, or 9, and the light chain variable region LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of LCDR1, LCDR2, and HCDR3 in SEQ ID NOs: 61, 62, 63, 64, 65, or 10, Preferably, a. The heavy chain variable region contains the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 82, and the light chain variable region contains the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 85, or b. The heavy chain variable region contains the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 51, and the light chain variable region contains the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 56, or c. The heavy chain variable region contains the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 68, and the light chain variable region contains the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 74, or d. The heavy chain variable region contains the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 78, and the light chain variable region contains the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 79, or e. The heavy chain variable region contains the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 58, and the light chain variable region contains the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 61, respectively. Anti-NPR1 antibody.

2. a. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 29, HCDR2 containing the amino acid sequence of SEQ ID NO: 40, and HCDR3 containing the amino acid sequence of SEQ ID NO: 41, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 42, LCDR2 containing the amino acid sequence of SEQ ID NO: 33, and LCDR3 containing the amino acid sequence of SEQ ID NO: 34, or b. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 45, 17, or 35, HCDR2 containing the amino acid sequence of SEQ ID NO: 46 or 18, and HCDR3 containing the amino acid sequence of SEQ ID NO: 19, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 20, LCDR2 containing the amino acid sequence of SEQ ID NO: 21, and LCDR3 containing the amino acid sequence of SEQ ID NO: 22, or c. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 29, HCDR2 containing the amino acid sequence of SEQ ID NO: 30, and HCDR3 containing the amino acid sequence of SEQ ID NO: 31, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 32, LCDR2 containing the amino acid sequence of SEQ ID NO: 73 or 33, and LCDR3 containing the amino acid sequence of SEQ ID NO: 34, or d. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 35, HCDR2 containing the amino acid sequence of SEQ ID NO: 75 or 36, and HCDR3 containing the amino acid sequence of SEQ ID NO: 37, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 38, LCDR2 containing the amino acid sequence of SEQ ID NO: 21, and LCDR3 containing the amino acid sequence of SEQ ID NO: 39, or e. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 23, HCDR2 containing the amino acid sequence of SEQ ID NO: 57 or 24, and HCDR3 containing the amino acid sequence of SEQ ID NO: 25, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 26, LCDR2 containing the amino acid sequence of SEQ ID NO: 27, and LCDR3 containing the amino acid sequence of SEQ ID NO:

28. Preferably, a. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 29, HCDR2 containing the amino acid sequence of SEQ ID NO: 40, and HCDR3 containing the amino acid sequence of SEQ ID NO: 41, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 42, LCDR2 containing the amino acid sequence of SEQ ID NO: 33, and LCDR3 containing the amino acid sequence of SEQ ID NO: 34, or b. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 45, HCDR2 containing the amino acid sequence of SEQ ID NO: 46, and HCDR3 containing the amino acid sequence of SEQ ID NO: 19, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 20, LCDR2 containing the amino acid sequence of SEQ ID NO: 21, and LCDR3 containing the amino acid sequence of SEQ ID NO: 22, or c. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 29, HCDR2 containing the amino acid sequence of SEQ ID NO: 30, and HCDR3 containing the amino acid sequence of SEQ ID NO: 31, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 32, LCDR2 containing the amino acid sequence of SEQ ID NO: 73, and LCDR3 containing the amino acid sequence of SEQ ID NO: 34, or d. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 35, HCDR2 containing the amino acid sequence of SEQ ID NO: 75, and HCDR3 containing the amino acid sequence of SEQ ID NO: 37, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 38, LCDR2 containing the amino acid sequence of SEQ ID NO: 21, and LCDR3 containing the amino acid sequence of SEQ ID NO: 39, or e. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 23, HCDR2 containing the amino acid sequence of SEQ ID NO: 57, and HCDR3 containing the amino acid sequence of SEQ ID NO: 25, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 26, LCDR2 containing the amino acid sequence of SEQ ID NO: 27, and LCDR3 containing the amino acid sequence of SEQ ID NO:

28. more, a. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 29, HCDR2 containing the amino acid sequence of SEQ ID NO: 40, and HCDR3 containing the amino acid sequence of SEQ ID NO: 41, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 42, LCDR2 containing the amino acid sequence of SEQ ID NO: 33, and LCDR3 containing the amino acid sequence of SEQ ID NO: 34, or b. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 45, HCDR2 containing the amino acid sequence of SEQ ID NO: 46, and HCDR3 containing the amino acid sequence of SEQ ID NO: 19, and the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 20, LCDR2 containing the amino acid sequence of SEQ ID NO: 21, and LCDR3 containing the amino acid sequence of SEQ ID NO:

22. The anti-NPR1 antibody according to claim 1.

3. The antibody is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody, preferably a humanized antibody. The anti-NPR1 antibody according to claim 1 or 2.

4. a. The heavy chain variable region includes an amino acid sequence having at least 85% sequence identity to sequence number 82, 80, or 81, and the light chain variable region includes an amino acid sequence having at least 85% sequence identity to sequence number 85, 83, or 84, or b. The heavy chain variable region includes an amino acid sequence having at least 85% sequence identity to sequence number 51, 48, 49, 50, or 52, and the light chain variable region includes an amino acid sequence having at least 85% sequence identity to sequence number 56, 53, 54, or 55, or c. The heavy chain variable region includes an amino acid sequence having at least 85% sequence identity to sequence number 68, 66, 67, or 69, and the light chain variable region includes an amino acid sequence having at least 85% sequence identity to sequence number 74, 70, 71, or 72, or d. The heavy chain variable region includes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 78, 76, or 77, and the light chain variable region includes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 79, or e. The heavy chain variable region includes an amino acid sequence having at least 85% sequence identity with SEQ ID NOs. 58, 59, or 60, and the light chain variable region includes an amino acid sequence having at least 85% sequence identity with SEQ ID NOs. 61, 62, 63, 64, or 65, or f. The heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 15, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 16, or g. The heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 7, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8, or h. The heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 11, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 12, or i. The heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 13, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 14, or j. The heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 9, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 10, Preferably, a. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 82, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 85, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 81 or 82, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 84, or b. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 51, 49, 50, or 52, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 56, or c. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 68, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 74, 70, or 71, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 67, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 70 or 71, or d. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 78, 76, or 77, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 79, or e. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 58, and the light chain variable region includes the amino acid sequence of SEQ ID NOs: 61, 62, 64, or 65, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 59, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 61, 62, 64, or 65, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 60, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 61, 62, 64, or 65, or f. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 15, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 16, or g. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 7, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 8, or h. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 11, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 12, or i. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 13, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 14, or j. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 9, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

10. more, a. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 82, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 85, or b. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 51, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 56, or c. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 68, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 74, or d. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 78, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

79. Most preferably, a. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 82, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 85, or b. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 51, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

56. The anti-NPR1 antibody according to any one of claims 1 to 3.

5. The anti-NPR1 antibody is an antibody fragment, preferably of which the antibody fragment is Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, or dAb. The anti-NPR1 antibody according to any one of claims 1 to 4.

6. The anti-NPR1 antibody comprises a heavy chain constant region and a light chain constant region. Preferably, the heavy chain constant region includes the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or their variants, and the light chain constant region is the human κ or λ light chain constant region. More preferably, the heavy chain constant region includes the amino acid sequence of SEQ ID NO: 86 or 43, and the light chain constant region includes the amino acid sequence of SEQ ID NO:

44. The anti-NPR1 antibody according to any one of claims 1 to 4.

7. The anti-NPR1 antibody comprises a heavy chain and a light chain, of which, a. The heavy chain contains an amino acid sequence of SEQ ID NO: 89 or 127, or having at least 85% sequence identity thereto, and the light chain contains an amino acid sequence of SEQ ID NO: 90 or 128, or having at least 85% sequence identity thereto, or The heavy chain contains an amino acid sequence of SEQ ID NO: 110, or one having at least 85% sequence identity thereto, and the light chain contains an amino acid sequence of SEQ ID NO: 111, or one having at least 85% sequence identity thereto, or b. The heavy chain contains an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 87, 112, 113, or 114, and the light chain contains an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 88, or The heavy chain contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 102, and the light chain contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 103, or c. The heavy chain contains an amino acid sequence of sequence number 99 or 122, or having at least 85% sequence identity thereto, and the light chain contains an amino acid sequence of sequence number 100, 123 or 124, or having at least 85% sequence identity thereto, or The heavy chain contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 106, and the light chain contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 107, or d. The heavy chain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 101, 125, or 126, and the light chain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 47, The heavy chain contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 108, and the light chain contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 109, or e. The heavy chain contains an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 115, 116, or 117, and the light chain contains an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 118, 119, 120, or 121, or The heavy chain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 104, and the light chain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 105, Preferably, a. The heavy chain contains the amino acid sequence of SEQ ID NO: 89, and the light chain contains the amino acid sequence of SEQ ID NO: 90, or b. The heavy chain contains the amino acid sequence of SEQ ID NO: 87, and the light chain contains the amino acid sequence of SEQ ID NO: 88, or c. The heavy chain contains the amino acid sequence of SEQ ID NO: 99, and the light chain contains the amino acid sequence of SEQ ID NO: 100, or d. The heavy chain contains the amino acid sequence of SEQ ID NO: 101, and the light chain contains the amino acid sequence of SEQ ID NO:

47. more, a. The heavy chain contains the amino acid sequence of SEQ ID NO: 89, and the light chain contains the amino acid sequence of SEQ ID NO: 90, or b. The heavy chain contains the amino acid sequence of SEQ ID NO: 87, and the light chain contains the amino acid sequence of SEQ ID NO:

88. The anti-NPR1 antibody according to claim 6.

8. A pharmaceutical composition comprising an anti-NPR1 antibody according to any one of claims 1 to 7, one or more pharmaceutically acceptable carriers, diluents, or excipients, and preferably further comprising at least one second therapeutic agent.

9. An immune complex comprising an anti-NPR1 antibody and an effector molecule according to any one of claims 1 to 7, The effector molecule is coupled to the anti-NPR1 antibody, and preferably, the effector molecule is selected from antitumor agents, immunomodulators, bioreaction modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof, forming an immune complex.

10. An isolated nucleic acid encoding the anti-NPR1 antibody according to any one of claims 1 to 7.

11. A host cell comprising the isolated nucleic acid described in claim 10.

12. A method for detecting NPR1 peptide or its fragments in a sample, A method comprising contacting the sample with an anti-NPR1 antibody according to any one of claims 1 to 7, and detecting the presence of a complex between the anti-NPR1 antibody and the NPR1 peptide or a fragment thereof, wherein the detection of the complex indicates the presence of the NPR1 peptide or a fragment thereof.

13. A method for treating, preventing, or improving diseases or conditions related to NPR1, A method comprising administering to a subject an effective therapeutic or preventive amount of an anti-NPR1 antibody according to any one of claims 1 to 7, or a pharmaceutical composition according to claim 8, or an immune complex according to claim 9.

14. The diseases or conditions associated with NPR1 are selected from heart failure, hypertension, peripheral vascular disease, coronary artery disease (CAD), ischemic heart disease (IHD), mitral stenosis and regurgitation, angina pectoris, hypertrophic cardiomyopathy (HCM), diabetic cardiomyopathy, supraventricular and ventricular arrhythmias, arrhythmias, atrial fibrillation (AF), cardiac fibrosis, atrial flutter, harmful vascular remodeling, plaque stabilization, myocardial infarction (MI), pre-eclampsia, obesity, renal failure, renal dysfunction, cytokine release syndrome, chronic kidney disease, macular edema, glaucoma, stroke, lung disease, inflammation, asthma, bone growth disorders, fractures, and diabetes. The method according to claim 13.

15. The aforementioned heart failure is selected from heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), heart failure with mildly reduced ejection fraction (HFmrEF), heart failure after acute myocardial infarction, or acute compensated heart failure, and among these, the aforementioned hypertension is refractory hypertension. The method according to claim 14.

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