Pharmaceutical composition containing Anti-NPR1 antibody and use thereof

Abstract

Provided are a pharmaceutical composition containing an anti-NPR1 antibody and the use thereof. Specifically, provided are a pharmaceutical composition containing an anti-NPR1 antibody and a buffer, and the use thereof in treating NPR1-related diseases.

Description

A pharmaceutical composition comprising an anti-NPR1 antibody and its use

[0001] This application claims priority to Chinese patent application CN202411816908.X, filed on December 11, 2024. Technical Field

[0002] This disclosure pertains to the field of pharmaceutical formulations, specifically relating to a pharmaceutical composition comprising an anti-NPR1 antibody and its use as a medicine. Background Technology

[0003] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0004] The natriuretic peptide system plays a crucial role in renal, cardiovascular, endocrine, nervous, and skeletal homeostasis. This system consists of peptide ligands and membrane receptors. The peptide ligands include ANP, BNP, and CNP, while the membrane receptors include NPR1 (NPRA), NPR2 (NPRB), and NPR3 (NPRC). NPR1 is primarily expressed in the heart, kidneys, lungs, and aorta. Through interaction with ligands ANP or BNP, it undergoes an allosteric change and activates downstream guanylate cyclase activity. This catalyzes the cyclization of intracellular guanosine triphosphate (GTP) to form cyclic guanosine monophosphate (cGMP), further activating cGMP-dependent protein kinase G (protein kinase G). This results in biological functions such as vasodilation, blood pressure reduction, sodium excretion and diuresis, and anti-myocardial hypertrophy and fibrosis. Therefore, activation of this pathway can be used to treat heart failure and hypertension.

[0005] Recombinant ANP and BNP have been approved and marketed in China, Japan, and the United States for the treatment of acute heart failure. These recombinant peptides are effective in lowering blood pressure and relieving heart failure symptoms, but they have not significantly improved readmission rates and mortality, possibly due to their short pharmacokinetic activity (PK) and inability to be used long-term. Therefore, they have not been approved for chronic heart failure.

[0006] Novartis and Regeneron have each developed NPR1 agonist antibodies. Regeneron's REGN-5381 (WO2020086406A2) is in a phase II clinical trial to validate its safety in patients with heart failure, while Novartis' XXB750 (WO2020250159A1) is in a phase II clinical trial to evaluate its efficacy in patients with refractory hypertension.

[0007] Antibody drugs are unstable due to their large molecular weight, complex structure, and susceptibility to degradation, polymerization, or unwanted chemical modifications. Therefore, research on stable formulations of antibody drugs is crucial to ensure their suitability for drug delivery and to maintain stability during storage and subsequent use, thereby maximizing their efficacy. Summary of the Invention

[0008] This disclosure provides a pharmaceutical composition containing an NPR1 antibody that is more conducive to manufacturing and administration and has stable performance.

[0009] This disclosure provides a pharmaceutical composition comprising an NPR1 antibody, which has good therapeutic activity, safety, pharmacokinetic properties and drugability (e.g., stability).

[0010] In some embodiments, this disclosure provides a pharmaceutical composition as shown in any of the following:

[0011] (1) A pharmaceutical composition comprising an anti-NPR1 antibody and a buffer solution;

[0012] (2) A pharmaceutical composition comprising an anti-NPR1 antibody and a surfactant;

[0013] (3) A pharmaceutical composition comprising an anti-NPR1 antibody and a sugar;

[0014] (4) A pharmaceutical composition comprising an anti-NPR1 antibody and an antioxidant;

[0015] (5) A pharmaceutical composition comprising an anti-NPR1 antibody, a buffer solution, a surfactant, a sugar, and an antioxidant;

[0016] (6) A pharmaceutical composition comprising an anti-NPR1 antibody, a buffer solution, a surfactant, and a sugar.

[0017] In some embodiments, the anti-NPR1 antibody is selected from Hu140H5L5-11, Hu102H4L6, REGN-5381, XX-16, and XXB-750, etc. In some embodiments, the anti-NPR1 antibody can be prepared by the methods described in examples WO2020086406A2 or WO2020250159A1. In some embodiments, the anti-NPR1 antibody disclosed herein can also be selected from all anti-NPR1 antibodies prepared in Examples 4 and 5, preferably Hu140H5L5-11.

[0018] In some embodiments, this disclosure provides a pharmaceutical composition comprising an anti-NPR1 antibody and a buffer solution, wherein:

[0019] The anti-NPR1 antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3; wherein:

[0020] a. The heavy chain variable region 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 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

[0021] b. The heavy chain variable region 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 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;

[0022] The buffer solution is histidine buffer, acetate buffer, citrate buffer, succinate buffer, or phosphate buffer.

[0023] In some embodiments, this disclosure provides a pharmaceutical composition comprising an anti-NPR1 antibody and a buffer solution, wherein:

[0024] The anti-NPR1 antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 29, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 40, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 41. The LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 42, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 33, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 34.

[0025] The buffer solution is histidine buffer, acetate buffer, citrate buffer, succinate buffer, or phosphate buffer.

[0026] In some embodiments, the pharmaceutical composition as described above, wherein the buffer is a histidine-histidine hydrochloride buffer, an acetate-sodium acetate buffer, a citrate-sodium citrate buffer, a succinate-sodium succinate buffer, or a phosphate-hydrochloride buffer.

[0027] In some embodiments, such as the pharmaceutical composition described in any of the preceding claims, the buffer solution is histidine-histidine hydrochloride buffer, acetate-sodium acetate buffer, citrate-sodium citrate buffer, or succinate-sodium succinate buffer.

[0028] In some embodiments, such as the pharmaceutical composition described in any of the preceding claims, the buffer solution is a histidine-histidine hydrochloride buffer solution.

[0029] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the buffer solution is from 1 mM to 100 mM. In some embodiments, the concentration of the buffer solution is from 20 mM to 75 mM. In some embodiments, the concentration of the buffer solution is from 30 mM to 60 mM. In some embodiments, the concentration of the buffer solution is from 35 mM to 55 mM. In some embodiments, the concentration of the buffer solution is from 36 mM to 54 mM. In some embodiments, the concentration of the buffer solution is from 40 mM to 50 mM. In some embodiments, the concentration of the buffer solution is from 40.5 mM to 49.5 mM. In some embodiments, the concentration of the buffer solution is about 45 mM. In some embodiments, the concentration of the buffer solution is about 20 mM. In some embodiments, the concentration of the buffer solution is about 75 mM. In some embodiments, the concentration of the buffer solution is about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 40.5 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 49.5 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM. In some embodiments, the concentration of the buffer solution is 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 40.5 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 49.5 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM, or any range between these values. In some embodiments, the concentration of the buffer solution is 45 mM. In some embodiments, the concentration of the buffer solution is 20 mM. In some embodiments, the concentration of the buffer solution is 75 mM.

[0030] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the histidine-histidine hydrochloride buffer is from 1 mM to 100 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 20 mM to 75 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 30 mM to 60 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 35 mM to 55 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 36 mM to 54 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 40 mM to 50 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 40.5 mM to 49.5 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 45 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 20 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 75 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 40.5 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 49.5 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 40.5 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 49.5 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM, or any range between these values. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 45 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 20 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 75 mM.

[0031] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the pH of the pharmaceutical composition is 4.5 to 8.0. In some embodiments, the pH of the pharmaceutical composition is 5.0 to 6.5. In some embodiments, the pH of the pharmaceutical composition is 5.5 to 6.5. In some embodiments, the pH of the pharmaceutical composition is 5.7 to 6.3. In some embodiments, the pH of the pharmaceutical composition is 5.8 to 6.2. In some embodiments, the pH of the pharmaceutical composition is 5.9 to 6.1. In some embodiments, the pH of the pharmaceutical composition is about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 7.0, about 7.5, or about 8.0. In some embodiments, the pH of the pharmaceutical composition is about 6.0. In some embodiments, the pH of the pharmaceutical composition is about 5.0. In some embodiments, the pH of the pharmaceutical composition is about 5.5. In some embodiments, the pH of the pharmaceutical composition is about 6.5. In some embodiments, the pH of the pharmaceutical composition is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 7.0, 7.5, or 8.0, or any range between these values. In some embodiments, the pH of the pharmaceutical composition is 6.0. In some embodiments, the pH of the pharmaceutical composition is 5.0. In some embodiments, the pH of the pharmaceutical composition is 5.5. In some embodiments, the pH of the pharmaceutical composition is 6.5. When point values ​​are mentioned in this disclosure, it should be understood that these point values ​​include a range of error. This range of error is due to factors such as laboratory environment, human operation, instrumentation, methodology, and measurement errors. For example, when measuring pH, a value of approximately 6.0 should be understood to include a range of error. As an example, when measuring formulations using an industrial pH meter, "approximately 6.0" means 6.0 ± 0.3 (i.e., pH from 5.7 to 6.3).

[0032] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments further comprises a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from polysorbates (e.g., polysorbate 80, polysorbate 20), poloxamer (e.g., poloxamer 188, i.e., PF68 or P188), Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-saccharide, succinate ... Betaine, myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocaramide-propyl-betaine, linoleamide-propyl-betaine, myristamide-propyl-betaine, palmitamide-propyl-betaine, isostearamide-propyl-betaine, myristamide-propyl-dimethylamine, palmitamide-propyl-dimethylamine, isostearamide-propyl-dimethylamine, sodium methyl cocoyl, sodium methyl oleate, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, etc. In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is polysorbate 80 (PS80), polysorbate 20 (PS20), and poloxamer 188. In some embodiments, the surfactant is polysorbate 80 (PS80) and poloxamer 188. In some embodiments, the surfactant is polysorbate 80 (PS80).

[0033] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the surfactant is from 0.01 mg / mL to 2.0 mg / mL. In some embodiments, the concentration of the surfactant is from 0.1 mg / mL to 1.0 mg / mL. In some embodiments, the concentration of the surfactant is from 0.1 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of the surfactant is from 0.2 mg / mL to 0.6 mg / mL. In some embodiments, the surfactant concentration is about 0.01 mg / mL, about 0.02 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, or about 2.0 mg / mL. In some embodiments, the concentration of the surfactant is about 0.4 mg / mL. In some embodiments, the concentration of the surfactant is about 0.1 mg / mL. In some embodiments, the concentration of the surfactant is about 1.0 mg / mL. In some embodiments, the surfactant concentration is 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the surfactant concentration is 0.4 mg / mL. In some embodiments, the surfactant concentration is 0.1 mg / mL. In some embodiments, the surfactant concentration is 1.0 mg / mL.

[0034] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of polysorbate 80 is from 0.01 mg / mL to 2.0 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.1 mg / mL to 1.0 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.1 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.2 mg / mL to 0.6 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.01 mg / mL, about 0.02 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, or about 2.0 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.4 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.1 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 1.0 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the concentration of polysorbate 80 is 0.4 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.1 mg / mL. In some embodiments, the concentration of polysorbate 80 is 1.0 mg / mL.

[0035] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of poloxamer 188 is from 0.01 mg / mL to 2.0 mg / mL. In some embodiments, the concentration of poloxamer 188 is from 0.1 mg / mL to 1.0 mg / mL. In some embodiments, the concentration of poloxamer 188 is from 0.1 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of poloxamer 188 is from 0.2 mg / mL to 0.6 mg / mL. In some embodiments, the concentration of poloxamer 188 is about 0.01 mg / mL, about 0.02 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, or about 2.0 mg / mL. In some embodiments, the concentration of poloxamer 188 is about 0.4 mg / mL. In some embodiments, the concentration of poloxamer 188 is about 0.1 mg / mL. In some embodiments, the concentration of poloxamer 188 is about 1.0 mg / mL. In some embodiments, the concentration of poloxamer 188 is 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the concentration of poloxamer 188 is 0.4 mg / mL. In some embodiments, the concentration of poloxamer 188 is 0.1 mg / mL. In some embodiments, the concentration of poloxamer 188 is 1.0 mg / mL.

[0036] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments includes a sugar. In some embodiments, the sugar is selected from conventional compositions (CH2O). nAnd its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. In some embodiments, the sugar is selected from sucrose, trehalose, glucose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabinitol, sylitol, sorbitol, mannitol, melitriose, maltotriose, stachyose, maltose, lactulose, maltitol, maltitol, lactitol, and isomaltulose, etc. In some embodiments, the sugar is sucrose, trehalose, mannitol, or sorbitol. In some embodiments, the sugar is sucrose.

[0037] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the sugar is from 10 mg / mL to 120 mg / mL. In some embodiments, the concentration of the sugar is from 30 mg / mL to 90 mg / mL. In some embodiments, the concentration of the sugar is from 45 mg / mL to 75 mg / mL. In some embodiments, the concentration of the sugar is from 48 mg / mL to 72 mg / mL. In some embodiments, the concentration of the sugar is from 50 mg / mL to 70 mg / mL. In some embodiments, the concentration of the sugar is from 54 mg / mL to 66 mg / mL. In some embodiments, the concentration of the sugar is from 55 mg / mL to 65 mg / mL. In some embodiments, the sugar concentration is about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 45 mg / mL, about 48 mg / mL, about 50 mg / mL, 54 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 66 mg / mL, about 70 mg / mL, about 72 mg / mL, about 75 mg / mL, about 80 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 110 mg / mL, or about 120 mg / mL. In some embodiments, the sugar concentration is about 60 mg / mL. In some embodiments, the sugar concentration is about 30 mg / mL. In some embodiments, the sugar concentration is about 70 mg / mL. In some embodiments, the sugar concentration is about 90 mg / mL. In some embodiments, the sugar concentration is 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 45 mg / mL, 48 mg / mL, 50 mg / mL, 54 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 66 mg / mL, 70 mg / mL, 72 mg / mL, 75 mg / mL, 80 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 110 mg / mL, or 120 mg / mL, or any range between these values. In some embodiments, the sugar concentration is 60 mg / mL. In some embodiments, the sugar concentration is 30 mg / mL. In some embodiments, the sugar concentration is 70 mg / mL. In some embodiments, the sugar concentration is 90 mg / mL.

[0038] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of sucrose is from 10 mg / mL to 120 mg / mL. In some embodiments, the concentration of sucrose is from 30 mg / mL to 90 mg / mL. In some embodiments, the concentration of sucrose is from 45 mg / mL to 75 mg / mL. In some embodiments, the concentration of sucrose is from 48 mg / mL to 72 mg / mL. In some embodiments, the concentration of sucrose is from 50 mg / mL to 70 mg / mL. In some embodiments, the concentration of sucrose is from 54 mg / mL to 66 mg / mL. In some embodiments, the concentration of sucrose is from 55 mg / mL to 65 mg / mL. In some embodiments, the concentration of sucrose is about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 45 mg / mL, about 48 mg / mL, about 50 mg / mL, 54 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 66 mg / mL, about 70 mg / mL, about 72 mg / mL, about 75 mg / mL, about 80 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 110 mg / mL, or about 120 mg / mL. In some embodiments, the concentration of sucrose is about 60 mg / mL. In some embodiments, the concentration of sucrose is about 30 mg / mL. In some embodiments, the concentration of sucrose is about 70 mg / mL. In some embodiments, the concentration of sucrose is about 90 mg / mL. In some embodiments, the concentration of sucrose is 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 45 mg / mL, 48 mg / mL, 50 mg / mL, 54 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 66 mg / mL, 70 mg / mL, 72 mg / mL, 75 mg / mL, 80 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 110 mg / mL, or 120 mg / mL, or any range between these values. In some embodiments, the concentration of sucrose is 60 mg / mL. In some embodiments, the concentration of sucrose is 30 mg / mL. In some embodiments, the concentration of sucrose is 70 mg / mL. In some embodiments, the concentration of sucrose is 90 mg / mL. In some implementations, when the sucrose concentration is 60 mg / mL, the osmotic pressure of the formulation is measured to be 308 mOsm / kg, which is close to isotonic.

[0039] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments further comprises an antioxidant. In some embodiments, the antioxidant is a metal ion chelating agent. In some embodiments, the antioxidant is selected from one or more of the group consisting of ethylenediaminetetraacetic acid (EDTA) or a salt thereof, EDTA hydrate or a salt thereof, glycine, DTPA (diethylenetriaminepentaacetic acid), methionine, histidine, tryptophan, cysteine, and ascorbic acid. In some embodiments, the antioxidant is EDTA or a salt thereof, or EDTA hydrate or a salt thereof. In some embodiments, the antioxidant is EDTA hydrate or a salt thereof.

[0040] In some embodiments, the antioxidant is a sodium salt of ethylenediaminetetraacetic acid hydrate. In some embodiments, the antioxidant is disodium edetate (EDTA-2Na).

[0041] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the antioxidant is from 0.01 mg / mL to 2.0 mg / mL. In some embodiments, the concentration of the antioxidant is from 0.01 mg / mL to 1.0 mg / mL. In some embodiments, the concentration of the antioxidant is from 0.01 mg / mL to 0.1 mg / mL. In some embodiments, the concentration of the antioxidant is from 0.01 mg / mL to 0.05 mg / mL. In some embodiments, the concentration of the antioxidant is from 0.01 mg / mL to 0.03 mg / mL. In some embodiments, the concentration of the antioxidant is about 0.01 mg / mL, about 0.02 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, or about 2.0 mg / mL. In some embodiments, the concentration of the antioxidant is about 0.02 mg / mL. In some embodiments, the concentration of the antioxidant is about 0.01 mg / mL. In some embodiments, the concentration of the antioxidant is about 1.0 mg / mL. In some embodiments, the concentration of the antioxidant is 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the concentration of the antioxidant is 0.02 mg / mL. In some embodiments, the concentration of the antioxidant is 0.01 mg / mL. In some embodiments, the concentration of the antioxidant is 1.0 mg / mL.

[0042] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of disodium edetate is from 0.01 mg / mL to 2.0 mg / mL. In some embodiments, the concentration of disodium edetate is from 0.01 mg / mL to 1.0 mg / mL. In some embodiments, the concentration of disodium edetate is from 0.01 mg / mL to 0.1 mg / mL. In some embodiments, the concentration of disodium edetate is from 0.01 mg / mL to 0.05 mg / mL. In some embodiments, the concentration of disodium edetate is from 0.01 mg / mL to 0.03 mg / mL. In some embodiments, the concentration of the disodium edetate is about 0.01 mg / mL, about 0.02 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, or about 2.0 mg / mL. In some embodiments, the concentration of the disodium edetate is about 0.02 mg / mL. In some embodiments, the concentration of the disodium edetate is about 0.01 mg / mL. In some embodiments, the concentration of disodium edetate is about 1.0 mg / mL. In some embodiments, the concentration of disodium edetate is 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the concentration of disodium edetate is 0.02 mg / mL. In some embodiments, the concentration of disodium edetate is 0.01 mg / mL. In some embodiments, the concentration of the disodium edetate is 1.0 mg / mL.

[0043] In some embodiments, the polysorbate 80 in the pharmaceutical compositions comprising disodium edetate disclosed herein exhibits a lower degradation rate than that in pharmaceutical compositions not containing disodium edetate. In some embodiments, the polysorbate 80 in the pharmaceutical compositions comprising disodium edetate disclosed herein exhibits a degradation rate at least two-thirds lower than that in pharmaceutical compositions not containing disodium edetate. In some embodiments, the polysorbate 80 in the pharmaceutical compositions comprising disodium edetate disclosed herein exhibits a degradation rate at least 50% lower (e.g., at least 51% lower) than that in pharmaceutical compositions not containing disodium edetate.

[0044] In some embodiments, the polysorbate 80 in pharmaceutical compositions not containing disodium edetate disclosed herein is degraded at least 3 times (e.g., at least 3.1 times, at least 3.2 times, at least 3.3 times) more than the polysorbate 80 in pharmaceutical compositions containing disodium edetate.

[0045] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the anti-NPR1 antibody is 1 mg / mL or higher. In some embodiments, the concentration of the anti-NPR1 antibody is 100 mg / mL or higher. In some embodiments, the concentration of the anti-NPR1 antibody is from 1 mg / mL to 260 mg / mL. In some embodiments, the concentration of the anti-NPR1 antibody is from 100 mg / mL to 260 mg / mL. In some embodiments, the concentration of the anti-NPR1 antibody is from 160 mg / mL to 240 mg / mL. In some embodiments, the concentration of the anti-NPR1 antibody is from 180 mg / mL to 220 mg / mL. In some embodiments, the concentration of the anti-NPR1 antibody is about 1 mg / mL, about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 195 mg / mL, about 200 mg / mL, about 205 mg / mL, about 210 mg / mL, about 215 mg / mL, about 220 mg / mL, about 230 mg / mL, about 240 mg / mL, about 250 mg / mL, or about 260 mg / mL. In some embodiments, the concentration of the anti-NPR1 antibody is about 200 mg / mL. In some embodiments, the concentration of the anti-NPR1 antibody is about 1 mg / mL. In some embodiments, the concentration of the anti-NPR1 antibody is about 100 mg / mL. In some embodiments, the concentration of the anti-NPR1 antibody is about 260 mg / mL. In some embodiments, the concentration of the anti-NPR1 antibody is 1 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 195 mg / mL, 200 mg / mL, 205 mg / mL, 210 mg / mL, 215 mg / mL, 220 mg / mL, 230 mg / mL, 240 mg / mL, 250 mg / mL, or 260 mg / mL, or any range between these values.In some embodiments, the concentration of the anti-NPR1 antibody is 200 mg / mL. In some embodiments, the concentration of the anti-NPR1 antibody is 1 mg / mL. In some embodiments, the concentration of the anti-NPR1 antibody is 100 mg / mL. In some embodiments, the concentration of the anti-NPR1 antibody is 260 mg / mL.

[0046] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-NPR1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO: 82 or 51, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and the light chain variable region comprises SEQ ID NO: 85 or 56, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith.

[0047] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-NPR1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO: 82, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and the light chain variable region comprises SEQ ID NO: 85, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith. In some embodiments, 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. In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 82, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 85.

[0048] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-NPR1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 89 or 87, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and the light chain comprises SEQ ID NO: 90 or 88, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith.

[0049] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-NPR1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 89, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and the light chain comprises SEQ ID NO: 90, or an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith. In some embodiments, the anti-NPR1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 89, and the light chain comprises the amino acid sequence of SEQ ID NO: 90.

[0050] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0051] (a) Anti-NPR1 antibody at concentrations ranging from 1 mg / mL to 260 mg / mL,

[0052] (b) Surfactants ranging from 0.01 mg / mL to 2.0 mg / mL,

[0053] (c) Sugars ranging from 10 mg / mL to 120 mg / mL,

[0054] (d) Antioxidants ranging from 0.01 mg / mL to 2.0 mg / mL, and

[0055] (e) A buffer solution of 1 mM to 100 mM, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0056] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0057] (a) Anti-NPR1 antibody at concentrations ranging from 1 mg / mL to 260 mg / mL,

[0058] (b) Polysorbate 80 or poloxamer 188 at concentrations ranging from 0.01 mg / mL to 1.0 mg / mL.

[0059] (c) Sucrose at concentrations of 30 mg / mL to 90 mg / mL,

[0060] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 1.0 mg / mL, and

[0061] (e) a 20 mM to 75 mM histidine buffer, acetate buffer, citrate buffer, succinate buffer, or phosphate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0062] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0063] (a) Anti-NPR1 antibody at concentrations ranging from 100 mg / mL to 260 mg / mL,

[0064] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 1.0 mg / mL,

[0065] (c) Sucrose at concentrations of 30 mg / mL to 90 mg / mL,

[0066] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 1.0 mg / mL, and

[0067] (e) a 20 mM to 75 mM histidine-histidine hydrochloride buffer, acetate-sodium acetate buffer, citrate-sodium citrate buffer, or succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0068] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0069] (a) Anti-NPR1 antibody at concentrations ranging from 100 mg / mL to 260 mg / mL,

[0070] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 1.0 mg / mL,

[0071] (c) Sucrose at concentrations of 30 mg / mL to 90 mg / mL,

[0072] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 1.0 mg / mL, and

[0073] (e) a 20 mM to 75 mM histidine-histidine hydrochloride buffer, acetate-sodium acetate buffer, citrate-sodium citrate buffer, or succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.5 to 6.5.

[0074] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0075] (a) Anti-NPR1 antibody at concentrations ranging from 160 mg / mL to 240 mg / mL,

[0076] (b) Polysorbate 80 at concentrations ranging from 0.2 mg / mL to 0.6 mg / mL,

[0077] (c) Sucrose at concentrations of 45 mg / mL to 75 mg / mL

[0078] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0079] (e) 40 mM to 50 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.7 to 6.3.

[0080] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0081] (a) Anti-NPR1 antibody at concentrations ranging from 180 mg / mL to 220 mg / mL,

[0082] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.5 mg / mL.

[0083] (c) Sucrose at concentrations of 55 mg / mL to 65 mg / mL

[0084] (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.05 mg / mL, and

[0085] (e) 40 mM to 50 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.8 to 6.2.

[0086] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0087] (a) Approximately 200 mg / mL of anti-NPR1 antibody,

[0088] (b) Approximately 0.4 mg / mL of polysorbate 80,

[0089] (c) Approximately 60 mg / mL of sucrose,

[0090] (d) Sodium edetate at approximately 0.02 mg / mL, and

[0091] (e) About 45 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 6.0.

[0092] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0093] (a) 200 mg / mL of anti-NPR1 antibody,

[0094] (b) 0.4 mg / mL of polysorbate 80,

[0095] (c) 60 mg / mL sucrose,

[0096] (d) 0.02 mg / mL of disodium edetate, and

[0097] (e) 45 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 6.0.

[0098] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0099] (a) Anti-NPR1 antibody at concentrations ranging from 1 mg / mL to 260 mg / mL,

[0100] (b) Surfactants ranging from 0.01 mg / mL to 2.0 mg / mL,

[0101] (c) Sugars ranging from 10 mg / mL to 120 mg / mL, and

[0102] (e) A buffer solution of 1 mM to 100 mM, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0103] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0104] (a) Anti-NPR1 antibody at concentrations ranging from 1 mg / mL to 260 mg / mL,

[0105] (b) Polysorbate 80 or poloxamer 188 at concentrations ranging from 0.01 mg / mL to 1.0 mg / mL.

[0106] (c) Sucrose at concentrations of 30 mg / mL to 90 mg / mL, and

[0107] (e) a 20 mM to 75 mM histidine buffer, acetate buffer, citrate buffer, succinate buffer, or phosphate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0108] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0109] (a) Anti-NPR1 antibody at concentrations ranging from 100 mg / mL to 260 mg / mL,

[0110] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 1.0 mg / mL,

[0111] (c) Sucrose at concentrations of 30 mg / mL to 90 mg / mL, and

[0112] (e) a 20 mM to 75 mM histidine-histidine hydrochloride buffer, acetate-sodium acetate buffer, citrate-sodium citrate buffer, or succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0113] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0114] (a) Anti-NPR1 antibody at concentrations ranging from 100 mg / mL to 260 mg / mL,

[0115] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 1.0 mg / mL,

[0116] (c) Sucrose at concentrations of 30 mg / mL to 90 mg / mL, and

[0117] (e) a 20 mM to 75 mM histidine-histidine hydrochloride buffer, acetate-sodium acetate buffer, citrate-sodium citrate buffer, or succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.5 to 6.5.

[0118] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0119] (a) Anti-NPR1 antibody at concentrations ranging from 160 mg / mL to 240 mg / mL,

[0120] (b) Polysorbate 80 at concentrations ranging from 0.2 mg / mL to 0.6 mg / mL,

[0121] (c) 45 mg / mL to 75 mg / mL of sucrose, and

[0122] (e) 40 mM to 50 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.7 to 6.3.

[0123] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0124] (a) Anti-NPR1 antibody at concentrations ranging from 180 mg / mL to 220 mg / mL,

[0125] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.5 mg / mL.

[0126] (c) sucrose at concentrations of 55 mg / mL to 65 mg / mL, and

[0127] (e) 40 mM to 50 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.8 to 6.2.

[0128] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0129] (a) Approximately 200 mg / mL of anti-NPR1 antibody,

[0130] (b) Approximately 0.4 mg / mL of polysorbate 80,

[0131] (c) Approximately 60 mg / mL of sucrose, and

[0132] (e) About 45 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 6.0.

[0133] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0134] (a) 200 mg / mL of anti-NPR1 antibody,

[0135] (b) 0.4 mg / mL of polysorbate 80,

[0136] (c) 60 mg / mL sucrose, and

[0137] (e) 45 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 6.0.

[0138] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments is a liquid formulation. In some embodiments, the solvent of the liquid formulation is water.

[0139] In another aspect, this disclosure also provides a method for preparing a lyophilized formulation, comprising the step of lyophilizing a pharmaceutical composition as described in any of the preceding claims.

[0140] In another aspect, this disclosure also provides a lyophilized formulation obtained by the method described above.

[0141] In another aspect, this disclosure also provides a lyophilized formulation comprising the pharmaceutical composition as described in any of the preceding claims.

[0142] In another aspect, this disclosure also provides a lyophilized formulation that, upon reconstitution, can form a pharmaceutical composition as described in any of the preceding claims.

[0143] In another aspect, this disclosure also provides a reconstituted solution, characterized in that the reconstituted solution is prepared by reconstituted a lyophilized formulation as described in any of the preceding claims.

[0144] In another aspect, this disclosure also provides a reconstituted solution, which is a reconstituted form of the lyophilized formulation as described in any of the preceding claims.

[0145] In some embodiments, the reconstituted solution as described in the preceding claim has the same components and contents as the pharmaceutical composition as described in the preceding claim.

[0146] In another aspect, this disclosure also provides an article of manufacture comprising a container containing a pharmaceutical composition as described in any of the preceding claims, or a lyophilized formulation as described in any of the preceding claims, or a reconstituted solution as described in any of the preceding claims.

[0147] In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution as described in any of the preceding embodiments is a subcutaneous injection formulation, an intravenous injection formulation, an intraperitoneal injection formulation, or an intramuscular injection formulation. In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution as described in any of the preceding embodiments is a subcutaneous injection formulation.

[0148] In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution described in any of the preceding embodiments is suitable for subcutaneous, intravenous, intraperitoneal, or intramuscular injection. In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution described in any of the preceding embodiments is suitable for subcutaneous injection.

[0149] In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution as described in any of the preceding embodiments is used to prepare a drug for subcutaneous, intravenous, intraperitoneal, or intramuscular injection. In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution as described in any of the preceding embodiments is used to prepare a drug for subcutaneous injection.

[0150] In another aspect, this disclosure also provides a method for treating or preventing a disease, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition as described in any of the preceding claims, a lyophilized formulation as described in any of the preceding claims, or a reconstituted solution as described in any of the preceding claims.

[0151] In another aspect, this disclosure also provides the use of the pharmaceutical compositions as described in any of the preceding claims, or the lyophilized formulations as described in any of the preceding claims, or the reconstituted solutions as described in any of the preceding claims, in the preparation of medicaments for the treatment or prevention of diseases.

[0152] On the other hand, this disclosure also provides pharmaceutical compositions as described in any of the preceding claims, or lyophilized formulations as described in any of the preceding claims, or reconstituted solutions as described in any of the preceding claims, for use as medicaments. In some embodiments, the medicament is used to treat or prevent disease.

[0153] In some implementations, the disease is an NPR1-related disease.

[0154] In some implementations, the disease 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 and ventricular arrhythmias, arrhythmias, atrial fibrillation (AF), cardiac fibrosis, atrial flutter, harmful vascular remodeling, plaque stabilization, myocardial infarction (MI), preeclampsia, obesity, renal failure, renal impairment, cytokine release syndrome, chronic kidney disease, macular edema, glaucoma, stroke, lung disease, inflammation, asthma, bone growth disorder, fracture, and diabetes.

[0155] In some implementations, the disease is heart failure or hypertension. Attached Figure Description

[0156] Figure 1A shows the results of the ELISA experiment in which anti-NPR1 antibody binds to hNPR1 antigen;

[0157] Figure 1B shows the results of the ELISA experiment in which anti-NPR1 antibody binds to hNPR2 antigen;

[0158] Figure 1C shows the results of the ELISA experiment in which anti-NPR1 antibody binds to hNPR3 antigen;

[0159] Figure 2 shows the experimental results of anti-NPR1 antibody stimulating hNPR1-CHOK1 cells to produce cGMP;

[0160] Figure 3 shows the results of the internalization experiment of anti-NPR1 antibody binding to NPR1;

[0161] Figure 4A shows the experimental results of stimulating cGMP production in cells before and after endocytosis treatment with Hu102H4L6 antibody;

[0162] Figure 4B shows the experimental results of stimulating cGMP production in cells before and after endocytosis treatment with Hu140H5L5-11 antibody.

[0163] Figure 5A shows the effect of anti-NPR1 antibody on systolic blood pressure in ANGII-induced hypertensive hNPR1 homozygous transgenic mice;

[0164] Figure 5B shows the effect of anti-NPR1 antibody on diastolic blood pressure in ANGII-induced hypertensive hNPR1 homozygous transgenic mice;

[0165] Figure 5C shows the effect of anti-NPR1 antibody on mean arterial pressure in ANGII-induced hypertensive hNPR1 homozygous transgenic mice;

[0166] Figure 5D shows the effect of anti-NPR1 antibody on heart rate in ANGII-induced hypertensive hNPR1 homozygous transgenic mice;

[0167] Figure 5E shows the effect of anti-NPR1 antibody on the concentration of cGMP in the plasma of ANGII-induced hypertensive hNPR1 homozygous transgenic mice.

[0168] Figure 5F shows the effect of anti-NPR1 antibody on the concentration of cGMP in the urine of ANGII-induced hypertensive hNPR1 homozygous transgenic mice.

[0169] Figure 5G shows the endpoint plasma drug concentration in ANGII-induced hypertensive hNPR1 homozygous transgenic mice after drug administration.

[0170] Figure 5H shows the effect of anti-NPR1 antibody on the mean NT-proBNP concentration in the plasma of ANGII-induced hypertensive hNPR1 homozygous transgenic mice.

[0171] Figure 5I shows the effect of anti-NPR1 antibody on sodium ion concentration in urine of ANGII-induced hypertensive hNPR1 homozygous transgenic mice. Detailed Implementation

[0172] the term

[0173] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0174] The singular forms “a,” “an,” and “the” used in this disclosure include plural references unless the context clearly indicates otherwise.

[0175] Unless the context clearly requires otherwise, the words “comprising,” “having,” “including,” etc., in the patent specification and claims should be understood as “including but not limited to,” rather than as exclusive or exhaustive.

[0176] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0177] Those skilled in the art will understand that when used as a reference range, cutoff value, or specific value, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range with a difference of up to 20% (i.e., ±20%). Since many of the values ​​used herein were determined experimentally, those skilled in the art will understand that such determinations can vary between different experiments and are generally true across experiments. Due to this inherent variability, the values ​​used herein should not be unduly restricted. Therefore, the term "about" is used to cover variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% or less from a specified value.

[0178] Although this disclosure provides content ranges or content values, those skilled in the art will understand that the content ranges or content values ​​cover the acceptable range of error for the specific values ​​measured.

[0179] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0180] 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 naturally occurring NPR1 proteins (e.g., but not limited to their precursors, mature, modified, or spliced ​​variants). The human NPR1 protein has the amino acid sequence SEQ ID NO: 4. Amino acid sequences of NPR1 molecules from non-human species (e.g., mice, rats, monkeys, rabbits, dogs, pigs, etc.) are available from public resources, e.g., cynomolgus monkey NPR1 protein NCBI number: XP-005541809.1; rat NPR1 protein, Uniprot number: P18910. Although specific database accession numbers are given herein, those skilled in the art will understand that NPR1 as used herein also encompasses corresponding sequences reported in other databases or literature.

[0181] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently 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., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0182] The term "antibody" is used in the broadest sense and encompasses 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 antigen-binding fragments (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. "Natural antibody" refers to a naturally occurring immunoglobulin molecule. For example, a natural IgG antibody is a heterotetraglycoprotein of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bound by disulfide bonds. From the N to C terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable region, followed by a heavy chain constant region. The IgG heavy chain constant region (CH) typically contains three constant domains (CH1, CH2, and CH3); similarly, from the N to C terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light domain (light chain constant region, CL).

[0183] The term "variable region" or "variable domain" refers to the domain in the antibody heavy or light chain involved in antibody-antigen binding. In this paper, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also called the N-terminus) to the carboxyl terminus (also called the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen-binding specificity.

[0184] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering rule, the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule, and the ImMunoGenTics (IMGT) numbering rule. The correspondence between various numbering systems is well known to those skilled in the art and is exemplified as shown in Table 1 below.

[0185] Table 1. Relationship between CDR numbering systems

[0186] Unless otherwise stated, the variable regions and CDR sequences in this disclosure embodiment are governed by the "Kabat" numbering rule. Although the Kabat numbering rule is used to define amino acid residues in specific implementations, corresponding technical solutions using other numbering systems are considered equivalent.

[0187] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, meaning that the antibody molecules contained in this group have the same amino acid sequence, except for the possible small number of naturally occurring mutations. In contrast, polyclonal antibody formulations typically contain multiple different antibodies with different amino acid sequences in their variable structural domains, and they generally specifically target different epitopes. "Monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0188] The antibodies disclosed herein may be derived from animals (such as antibodies from mice, birds, rabbits, camels, monkeys, etc.), chimeric antibodies, or humanized antibodies.

[0189] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remaining portion of the heavy and / or light chain is derived from another different source or species.

[0190] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the frame region portion of the constant region and the variable region).

[0191] The term "affinity" refers to the overall 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, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can typically be represented by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art.

[0192] The terms "anti-NPR1 antibody" and "NPR1-binding antibody" refer to antibodies capable of binding to NPR1 or its epitopes with sufficient affinity. In one embodiment, the binding can be measured by BIACORE surface plasmon resonance assay.

[0193] The term "antigen" refers to a molecule or molecular moiety that can be bound by a selective binder of an antigen-binding protein (such as an antibody). An antigen may have one or more epitopes that can interact with different antigen-binding proteins (such as antibodies).

[0194] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. Epitopes can be formed from consecutive amino acids (linear epitopes) or contain non-consecutive amino acids (conformal epitopes), for example, due to the folding of the antigen (i.e., tertiary folding of the antigen as a protein), which allows non-consecutive amino acids to be spatially close. The difference between conformational and linear epitopes is that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. An epitope contains 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 the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, Western blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen, and cross-blocking.

[0195] The terms "capable of specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than other antigens or epitopes. Typically, antibodies bind at an affinity of approximately 1 × 10⁻⁶.-7 An equilibrium dissociation constant (KD) of M or less binds to an antigen or epitope. In some embodiments, the KD of antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or an epitope within an antigen may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous), such as humans or monkeys, such as the cynomolgus (cyno), the chimpanzee (chimp), or the common marmoset (marmoset).

[0196] "Surfactant" refers to a surface-active agent, preferably a nonionic surfactant. Surfactants can reduce protein aggregation and / or particle formation in formulations. The amount of surfactant added is such that it reduces protein aggregation and minimizes particle formation in the formulation.

[0197] Poloxamer is an α-hydro-ω-hydroxy poly(ethylene oxide)a-poly(propylene oxide)b-poly(ethylene oxide)a block copolymer. It is formed by reacting propylene oxide and propylene glycol to form polypropylene glycol, followed by the addition of ethylene oxide to form the block copolymer. Here, 'a' represents the number of ethylene oxide units, and 'b' represents the number of propylene oxide units. Examples of poloxamer include, but are not limited to, poloxamer 188 (P188 or PF68). Specifically, in poloxamer 188, the copolymer has 75-85 ethylene oxide units (a), 25-30 propylene oxide units (b), an ethylene oxide (EO) content of 79.9%-83.7%, and an average molecular weight of 7680-9510.

[0198] "Disodium edetate" (CAS No.: 6381-92-6, the disodium edetate described in this disclosure was purchased from Merck). The company's website lists its alternative names as disodium ethylenediaminetetraacetate dihydrate, disodium EDTA, EDTA-Na2, ethylenediaminetetraacetate Na2, disodium ethylenediaminetetraacetate dihydrate, disodium ethylenediaminetetraacetate dihydrate, and calcium sodium edetate. Therefore, "disodium edetate" and "EDTA-2Na" are interchangeable in this disclosure, as they are both substances with CAS No.: 6381-92-6.

[0199] "Buffer solution" refers to a buffering agent that is resistant to pH changes through the action of its acid-base conjugate components. Examples of buffer solutions that maintain the pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate (also known as tartrate), tartrate, fumarate, glycylglycine, and other organic acid buffer solutions.

[0200] "Histidine buffer" is a buffer solution containing histidine. Examples of histidine buffer solutions include histidine-histidine acetate buffer, histidine-histidine hydrochloride buffer, histidine-histidine phosphate buffer, histidine-histidine sulfate buffer, etc., with histidine-histidine acetate buffer being preferred. Histidine-histidine acetate buffer can be prepared from histidine and acetic acid, or from histidine and histidine acetate.

[0201] "Displacement" refers to the replacement of the solvent system in which antibody proteins are dissolved. For example, a buffer system containing antibody proteins may be physically replaced by a high-salt or hypertonic solvent system containing antibody proteins, thereby ensuring the presence of antibody proteins within the stable formulation. Such physical operations include, but are not limited to, ultrafiltration, dialysis, or centrifugation.

[0202] "Pharmaceutical composition" means a mixture containing one or more antibodies described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0203] "Pharmaceutically acceptable carriers" or "pharmaceutically acceptable excipients" include any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated using well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, editor, Mack Publishing Co., Easton, PA, 1990; and R. Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0204] "Lyophilized formulation" refers to a pharmaceutical composition or formulation obtained by vacuum freeze-drying a liquid or solution preparation. Typically, freeze-drying includes pre-freezing, primary drying, and secondary drying. Pre-freezing aims to freeze the product to obtain a crystalline solid; in some embodiments, the pre-freezing temperature is set to -45°C, and the pre-freezing rate is set to 1°C / min. Primary drying, also known as main drying, is the main stage of sample freeze-drying and aims to remove ice from the product while maintaining its shape and minimizing damage. Improper selection of the temperature and vacuum level during primary drying can lead to product collapse; higher temperatures and vacuum levels increase freeze-drying efficiency but also increase the risk of product collapse. In some embodiments, the primary drying temperature can be a temperature conventional in the art, such as -30°C to 0°C. Secondary drying, also known as desorption drying, is the main step of removing bound water from the product by applying an ultimate vacuum (0.01 mbar) and increasing the temperature (20°C to 40°C). Because most biological products are temperature-sensitive, the secondary drying temperature is often chosen at the lower end of the temperature range, such as 25°C. The freeze-drying time depends on the freezer, the dosage of the freeze-dried formulation, and the container of the freeze-dried drug. Such adjustments are well known to those skilled in the art.

[0205] Unless otherwise specified, the solvent in the solution form of the pharmaceutical compositions described in this disclosure is water.

[0206] In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0207] The pharmaceutical compositions disclosed herein achieve a stable effect: the antibodies contained therein substantially retain their physical and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical compositions substantially retain their physical and chemical stability and their biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, there are various analytical techniques available for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0208] Stable formulations include those in which no significant changes are observed when stored at refrigerated temperatures (2°C–8°C) for at least 1 month, at least 3 months, at least 6 months, preferably 1 year, and even more preferably up to 2 years. Stable liquid formulations also include those that exhibit the desired characteristics after storage at temperatures including 25°C for periods of 1 month, 3 months, or 6 months. Furthermore, stable liquid formulations also include those that exhibit the desired characteristics after storage at 40°C for periods of 4 weeks, 1 month, 3 months, or 6 months. Typical examples of stability include antibody aggregation or degradation typically not exceeding about 10%, preferably not exceeding about 5%, as determined by SEC-HPLC. Visually, the formulation is a pale yellow, nearly colorless, clear liquid or a colorless, clear liquid, or clear to slightly milky white. The concentration, pH, weight, and molecular osmotic pressure of the formulation exhibit aggregation of not more than about 10%, preferably not more than about 5%.

[0209] If, after visual inspection of color and / or clarity, or by means of UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody does not show significant increase in aggregation, precipitation, and / or denaturation, then the antibody “retains its physical stability” in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

[0210] If an antibody does not exhibit significant chemical changes, then the antibody "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Degradation processes that frequently alter the chemical structure of a protein include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and CE-SDS), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and isofpartate measurement), and isomerization (evaluated by measuring isofpartate content, peptide mapping, etc.).

[0211] If the antibody's biological activity at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared, then the antibody "retains its biological activity" in the pharmaceutical formulation.

[0212] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo, such as cells. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0213] "Treatment" means administering an oral or topical therapeutic agent, such as a pharmaceutical composition comprising any of the substances disclosed herein, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases, in order to induce the regression of such symptoms or inhibit their progression to any clinically measurable degree. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments disclosed herein (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0214] "Effective amount" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. Effective amount also means an amount sufficient to allow or facilitate a diagnosis. The effective amount used on a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. Effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity. Subjects disclosed herein may be animal or human subjects.

[0215] The pharmaceutical compositions disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various dosing schedules are considered herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion. In some embodiments, the pharmaceutical compositions disclosed herein are administered by intravenous injection.

[0216] The pharmaceutical compositions disclosed herein will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to a medical practitioner. Optionally, the pharmaceutical composition may also be formulated with one or more other agents for the prevention or treatment of said condition. The effective amount of such other agents depends on the amount of antigen-binding molecules present in the pharmaceutical composition, the type of condition or treatment, and other factors. It may be used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and any route of administration determined empirically / clinically to be appropriate.

[0217] Details of one or more embodiments of this disclosure are set forth in the foregoing description. While any methods and materials similar to or the same as those described herein may be used to implement or test this disclosure, preferred methods and materials are described below. Other features, objects, and advantages of this disclosure will be apparent from the description and claims. In the description and claims, the singular form includes plural references unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have their general meaning as understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications referenced in the description are incorporated herein by reference. The following embodiments are presented to illustrate preferred embodiments of this disclosure more fully. These embodiments should not be construed in any way as limiting the scope of this disclosure, which is defined by the claims.

[0218] Example

[0219] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0220] Experimental methods not specifying specific conditions in the examples or test cases disclosed herein are generally performed under standard conditions or as recommended by the raw material or product manufacturer. Reagents not specifying their source are commercially available, standard reagents.

[0221] I. Antibody Preparation

[0222] PCT / CN2024 / 098066 (Application date: 2024-06-07; Priority patent application number: CN202310675148.4) is included herein by reference in its entirety.

[0223] Example 1: Preparation of NPR1 antigen

[0224] 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 disclosed herein were designed. Optionally, fusion tags such as hFc were added to the NPR1 protein. The sequences were cloned into the pTT5 vector (Biovector, CAT#102762), transiently expressed in CHO cells, purified, and the antigens and detection proteins disclosed herein were obtained.

[0225] The sequence of the extracellular domain of human NPR1 protein fusion protein with Human-IgG1-Fc (abbreviated as hNPR1-hFc) serves as an immunogenic antigen.

[0226] Note: The underlined part is the Human-IgG1-Fc portion, and the ununderlined part is the extracellular domain of the human NPR1 protein.

[0227] The extracellular domain of the cynomolgus monkey NPR1 protein and the sequence of the Human-IgG1-Fc fusion protein (abbreviated as cNPR1-hFc) serve as an immunogenic antigen.

[0228] Note: The underlined part is the Human-IgG1-Fc portion, and the ununderlined part is the extracellular domain of the cynomolgus monkey NPR1 protein.

[0229] The sequence of the rat (Rattus norvegicus) NPR1 protein extracellular domain fusion protein with Human-IgG1-Fc (abbreviated: rNPR1-hFc) was used as an immunogenic antigen.

[0230] Note: The underlined part is the Human-IgG1-Fc portion, and the ununderlined part is the extracellular domain of the rat NPR1 protein.

[0231] Example 2: Purification of NPR1-related recombinant protein

[0232] Purification steps of NPR1-Fc fusion protein:

[0233] Cell expression supernatant samples were centrifuged at high speed to remove impurities. The supernatant was then subjected to MabSelect Sure (GE, 17-5438-01) affinity chromatography. The MabSelect Sure column was first regenerated with 0.2M NaOH, washed with pure water, and then equilibrated with PBS. After binding the supernatant, the column was washed with PBS until A was reached. 280 The reading dropped to 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 appropriate concentration, the eluted sample was further purified using PBS-equilibrated Superdex 200 gel chromatography (GE, 28-9893-35). The receiving tubes containing the target protein were combined and concentrated to an appropriate concentration. This method was used to purify the NPR1-Fc fusion protein, and it can also be used to purify the antibody protein disclosed herein.

[0234] Example 3: Preparation of NPR1 recombinant cell line

[0235] Using UniProt or NCBI NPR1 antigen (human NPR1 protein, Uniprot No.: P16066; cynomolgus monkey NPR1 protein, NCBI No.: XP-005541809.1; rat NPR1 protein, Uniprot No.: P18910) as templates for NPR1, publicly available recombinant cell lines for immunization and detection were designed. 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 CHO-K1 cell line overexpressing full-length human NPR1, cynomolgus monkey NPR1, and rat NPR1 proteins.

[0236] Human NPR1 full-length amino acid sequence

[0237] Note:

[0238] Human NPR1 is a transmembrane protein that exists as a homodimer on the cell membrane surface.

[0239] The double-underlined portion represents the NPR1 extracellular domain (33 to 473);

[0240] The dotted-dash area represents the transmembrane domain (474 ​​to 494);

[0241] The underlined portion represents the intracellular region (Cytoplasmic domain: 495 to 1061);

[0242] The unlabeled portion is the signal peptide.

[0243] Full-length amino acid sequence of NPR1 in cynomolgus monkeys

[0244] Note:

[0245] Cyno NPR1 is a transmembrane protein that exists as a homodimer on the cell membrane surface.

[0246] The double-underlined portion represents the NPR1 extracellular domain (33 to 473);

[0247] The dotted-dash area represents the transmembrane domain (474 ​​to 494);

[0248] The underlined portion represents the intracellular region (Cytoplasmic domain: 495 to 1061);

[0249] The unlabeled portion is the signal peptide.

[0250] Full-length amino acid sequence of NPR1 in rat (Rattus norvegicus)

[0251] Note:

[0252] RatNPR1 is a transmembrane protein that exists as a homodimer on the cell membrane surface.

[0253] The double-underlined portion represents the NPR1 extracellular domain (29 to 469);

[0254] The dotted-dash area represents the transmembrane domain (470 to 490).

[0255] The underlined portion represents the intracellular region (Cytoplasmic domain: 491 to 1057);

[0256] The unlabeled portion is the signal peptide.

[0257] NIH-3T3 cells were stably transfected with the above proteins. After two weeks of pressure selection, subcloning was performed to select single clones. Recombinant cell lines expressing high levels of hNPR1-NIH-3T3, cNPR1-NIH-3T3, and rNPR1-NIH-3T3 were obtained by FACS detection and used to immunize mice.

[0258] The above-mentioned protein was used to stably transfect CHO-K1 cells. After two weeks of pressure screening, subcloning was performed to screen for monoclonal clones. High-expression hNPR1-CHO-K1, cNPR1-CHO-K1, and rNPR1-CHO-K1 recombinant cell lines were obtained by FACS detection and were used for the affinity and in vitro function detection of anti-NPR1 antibodies.

[0259] Example 4: Screening of Mouse Anti-hNPR1 Antibody

[0260] 1. Mouse Immunization:

[0261] Monoclonal antibodies against human NPR1 were produced by immunizing mice. SJL white mice, female, 6 - 8 weeks old (Shanghai SLAC Laboratory Animal Co., Ltd., Animal Production License No.: SCXK(Shanghai)2017 - 0005) were used for the experiment. Breeding environment: SPF level. After the mice were purchased, they were raised in the laboratory environment for 1 week, with a 12 / 12-hour light / dark cycle adjustment, temperature 20 - 25 °C; humidity 40 - 60%. The mice that had adapted to the environment were immunized according to Protocol 1 and Protocol 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 Corporation, Cat No. RP11927).

[0262] Immunization Protocol 1: Use Gold Adjuvant (Sigma Cat No. T2684) to pre-immunize mice, intraperitoneal (IP) injection of 0.1 mL / mouse (primary immunization). 15 minutes later, intraperitoneal (IP) injection of hNPR1 NIH-3T3, 1*10 ^7 cells / mouse. The antigens hNPR1 NIH-3T3 and cNPR1 NIH-3T3 were cross-immunized. The inoculation times were days 0, 14, and 28. Blood was taken on days 21 and 35, and ELISA and FACS methods were used to determine the antibody titers in mouse sera. After the third immunization, mice with high antibody titers and a titer trend towards a plateau in their sera were selected for spleen cell fusion. Three days before spleen cell fusion, booster immunization was performed, with an intraperitoneal (IP) injection of 50 μg / mouse of an antigen hNPR1-hFc solution prepared with normal saline. 5 μg / mouse of hANP(1 - 28aa) was added to each immunization inoculation.

[0263] Immunization Protocol 2: Use Mice were pre-immunized with Gold Adjuvant (Sigma Cat No. T2684) via intraperitoneal (IP) injection (0.1 mL / mouse, initial immunization). Fifteen minutes later, hNPR1NIH-3T3 was injected intraperitoneally (IP) at a dose of 1*10^7 cells / mouse. Cross-immunization was performed using hNPR1 NIH-3T3 and rNPR1 NIH-3T3 antigens. Vaccination was administered on days 0, 14, 28, 42, 56, 70, 84, and 98. Blood samples were collected on days 35, 63, 91, and 105, and serum antibody titers were determined using ELISA and FACS. After the eighth immunization, mice with high and plateauing serum antibody titers were selected for spleen cell fusion. Three days prior to spleen cell fusion, a booster immunization was administered via intraperitoneal (IP) injection of 50 μg / animal a mixture of antigens hNPR1-hFc, cNPR1-hFc, and rNPR1-hFc prepared with physiological saline. 5 μg / animal hANP (1-28 aa) was added to each immunization.

[0264] 2. Spleen cell fusion:

[0265] An optimized electrofusion method was used to fuse splenic lymphocytes with myeloma Sp2 / 0 cells (…). CRL-8287 TM Hybridoma cells are obtained by fusing them together.

[0266] Based on the spleen cell count, the fused hybridoma cells were divided into groups of 3 × 10⁻⁶. ^5 -4×10 ^5 Cells were resuspended at a density of 150 μL / well in IMDM medium (containing 20% ​​FBS, 1×HAT, and 1×OPI) and seeded into 96-well plates. After incubation at 37°C and 5% CO2 for 4–5 days, the supernatant was removed, and 200 μL / well of HT complete medium (containing 20% ​​FBS, 1×HT, and 1×OPI) was added. After incubation at 37°C and 5% CO2 for 2 days, ELISA was performed.

[0267] 3. Hybridoma cell screening:

[0268] Based on the hybridoma cell growth density, the hybridoma culture supernatant was analyzed using a cell-based binding assay and a cGMP production assay. Cells in positive wells were promptly amplified, cryopreserved, and subcloned one to two times until single-cell clones were obtained. Single-cell clones were cultured, RNA was extracted, and reverse transcription amplification (RT-PCR) was performed using degenerate primers for mouse-Ig to obtain the variable region sequence of the antibody. The amino acid sequences corresponding to the DNA sequences of mouse antibodies 102, 127, 128, 135, and 140 were determined as follows: NPR1-CHAb-SFM-102-1

[0269] HCVR:

[0270] LCVR:

[0271] NPR1-CHAb-SFM-127-1

[0272] HCVR:

[0273] LCVR:

[0274] NPR1-CHAb-SFM-128

[0275] HCVR:

[0276] LCVR:

[0277] NPR1-CHAb-SFM-135

[0278] HCVR:

[0279] LCVR:

[0280] NPR1-CHAb-SFM-140

[0281] HCVR:

[0282] LCVR: Note: In the above sequence, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the double underscores are the CDR sequences determined according to the Kabat numbering system.

[0283] The heavy and light chain CDR region sequences of murine antibodies 102, 127, 128, 135, and 140 are shown in Table 2 below:

[0284] Table 2. Antibody heavy chain and light chain CDR sequences

[0285] Note: The CDRs in the table are CDRs determined according to the Kabat numbering system.

[0286] By linking the carboxyl terminus of the heavy chain variable region of the previously screened murine antibodies 102, 127, 128, 135, and 140 to the amino terminus of the human heavy chain constant region as shown in SEQ ID NO: 43, and simultaneously linking the carboxyl terminus of the light chain variable region of the murine antibodies to the amino terminus of the human light chain constant region as shown in SEQ ID NO: 44, chimeric antibodies corresponding to 102, 127, 128, 135, and 140 can be obtained, denoted as CHI-102, CHI-127, CHI-128, CHI-135, and CHI-140, respectively.

[0287] Constructing the chimeric human IgG1 heavy chain constant region sequence:

[0288] Constructing a chimeric sequence of constant regions using human light chains (human kappa light chain constant regions):

[0289] Full-length heavy chain sequence of CHI-102(NPR1-CHAb-SFM-102-1):

[0290] Full-length light chain sequence of CHI-102(NPR1-CHAb-SFM-102-1):

[0291] Full-length heavy chain sequence of CHI-127(NPR1-CHAb-SFM-127-1):

[0292] Full-length light chain sequence of CHI-127(NPR1-CHAb-SFM-127-1):

[0293] The full-length heavy chain sequence of CHI-128 (NPR1-CHAb-SFM-128):

[0294] Full-length light chain sequence of CHI-128 (NPR1-CHAb-SFM-128):

[0295] The full-length heavy chain sequence of CHI-135 (NPR1-CHAb-SFM-135):

[0296] Full-length light chain sequence of CHI-135 (NPR1-CHAb-SFM-135):

[0297] The full-length heavy chain sequence of CHI-140 (NPR1-CHAb-SFM-140):

[0298] Full-length light chain sequence of CHI-140 (NPR1-CHAb-SFM-140):

[0299] 4. FACS binding assay of anti-NPR1 chimeric antibody:

[0300] In vitro binding experiments were performed to confirm the correctness of the variable region sequence of the obtained monoclonal antibody. 1×10⁻⁶ ppm was used. 6 Cells / mL expressing hNPR1 CHO-K1 or cNPR1 CHO-K1 were blocked with 1% BSA PBS buffer, and then incubated for 1 hour with different concentrations of diluted anti-NPR1 chimeric antibody samples (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 more with pH 7.4 PBS, the cells were resuspended in 150 μL pH 7.4 PBS, and the fluorescence signal values ​​were read using flow cytometry. The experimental results are shown in Table 3.

[0301] Table 3. FACS binding of anti-NPR1 chimeric antibodies

[0302] Conclusion: The chimeric antibodies disclosed herein can all bind to hNPR1 CHO-K1 and cNPR1 CHO-K1.

[0303] Example 5: Humanization of anti-NPR1 murine antibody

[0304] By comparing the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, germline genes of heavy and light chain variable regions with high homology to 102, 127, 128, 135, and 140 were selected as templates. The CDRs of the murine antibody were then transplanted into the corresponding human templates, forming variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Exemplarily, in the following specific embodiments, the CDR amino acid residues were determined and annotated using the Kabat numbering system.

[0305] Humanization of 102 mouse antibodies

[0306] For the humanized heavy chain variable regions of mouse antibody 102, FR1, FR2, and FR3 used IGHV3-21*01, and FR4 used IGHJ6*01 as templates; for the light chain variable regions, FR1, FR2, and FR3 used IGKV3-11*01 or IGKV1-39*01, and FR4 used IGKJ4*01 as templates. The CDRs of mouse antibody 102 were transplanted into their respective human templates. Optionally, some amino acids in the FR region of the humanized antibody were substituted, specifically amino acid residues at positions 1, 3, 9, 44, 49, 87, and / or 93 (determined according to Kabat numbering rules) in the FR region of the heavy chain variable region, and / or amino acid residues at positions 4, 43, 45, 46, 47, 58, and / or 71 (determined according to Kabat numbering rules) in the FR region of the light chain variable region. The mutation design of the humanized antibody 102 is shown in Table 4 below:

[0307] Table 4.102 Mutations in Humanized Antibodies Note: Graft represents the insertion of murine antibody CDR into the human FR region; the location of the reversion mutation site is determined according to the Kabat numbering rules, such as "T31S" which means that the 31st T is mutated to S according to the Kabat numbering system.

[0308] Table 5.102 CDRs of humanized antibodies

[0309] The light chain variable region / heavy chain variable region sequences of the 102 humanized antibody are as follows:

[0310] >hu102VH1

[0311] >hu102VH2

[0312] >hu102VH3

[0313] >hu102VH4

[0314] >hu102VH5

[0315] >hu102VL3

[0316] >hu102VL4

[0317] >hu102VL5

[0318] >hu102VL6

[0319] Note: The double-underlined part is the antibody CDR sequence, where the CDR is numbered according to Kabat.

[0320] Humanization of 127 mouse antibody

[0321] For the humanized heavy chain variable region of murine antibody 127, FR1, FR2, and FR3 were templated using IGHV1-46*01, and FR4 was templated using IGHJ6*01. For the light chain variable region, FR1, FR2, and FR3 were templated using IGKV4-1*01 or 1-39*01, and FR4 was templated using IGKJ2*01. The CDR of murine antibody 127 was then transplanted into its humanized template. Optionally, 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 according to Kabat numbering rules) of the FR region of the heavy chain variable region. A glutamic acid mutation was designed at position 1 to avoid pyroglutamic cyclization, and / or at positions 1, 4, 17, 43, 46, 60, 68, and / or 79 (determined according to Kabat numbering rules) of the FR region of the light chain variable region were substituted. The mutation design of the variable region of the humanized antibody 127 is shown in Table 6 below:

[0322] Table 6.127 Mutations in Humanized Antibodies Note: Grafted means that the murine antibody CDR is implanted into the human FR region; the location of the mutation site is determined according to the Kabat numbering rules, such as "V2A" means that the second V is mutated to A according to the Kabat numbering system.

[0323] Table 7.127 CDRs of humanized antibodies

[0324] The light / heavy chain variable region sequence of the 127 humanized antibody is as follows:

[0325] >hu127VH4

[0326] >hu127VH7

[0327] >hu127VH8

[0328] >hu127VL1

[0329] >hu127VL3

[0330] >hu127VL4

[0331] >hu127VL5

[0332] >hu127VL6

[0333] Note: The double-underlined part is the antibody CDR sequence, where the CDR is numbered according to Kabat.

[0334] Humanization of 128 mouse antibodies

[0335] For the humanized heavy chain variable region of murine antibody 128, FR1, FR2, and FR3 used IGHV1-46*01, and FR4 used IGHJ6*01 as templates; for the light chain variable region, FR1, FR2, and FR3 used IGKV3-20*02 or 6-21*02, and FR4 used IGKJ2*01 as templates. The CDR of murine antibody 128 was transplanted into its human templates. Optionally, 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 according to Kabat numbering rules) of the FR region of the heavy chain variable region, with a glutamic acid mutation at position 1 to avoid pyroglutamic cyclization, and / or at positions 22, 42, 43, 49, 57, and / or 71 (determined according to Kabat numbering rules) of the FR region of the light chain variable region. The mutation design of the variable region of the humanized antibody 128 is shown in Table 8 below:

[0336] Table 8.128 Mutations in Humanized Antibodies Note: Grafted indicates that the murine antibody CDR has been implanted into the human germline FR region; the location of the reversion mutation site is determined according to the Kabat numbering rules. For example, "G57R" means that the 57th G has been mutated to R according to the Kabat numbering system.

[0337] >hu128VH1

[0338] >hu128VH2

[0339] >hu128VH3

[0340] >hu128VH4

[0341] >hu128VL1

[0342] >hu128VL2

[0343] >hu128VL3

[0344] In addition, based on hu128VL1, certain amino acids (A55) in the FR1 region (S22) and LCDR2 region of the light chain were modified. Specifically, the amino acid sequence of LCDR2 was changed from the original DTYNLAS (SEQ ID NO: 33) to DTYNLGS (SEQ ID NO: 73), resulting in a new humanized antibody light chain variable region sequence, as follows:

[0345] >hu128VL1-6

[0346] Note: The double-underlined part is the antibody CDR sequence, where the CDR is numbered according to Kabat.

[0347] Humanization of 135 mouse antibody

[0348] For the humanized heavy chain variable regions of murine antibody 135, FR1, FR2, and FR3 used IGHV3-21*01, and FR4 used IGHJ6*01 as templates; for the light chain variable regions, FR1, FR2, and FR3 used IGKV1-39*01, and FR4 used IGKJ4*01 as templates. The CDRs of murine antibody 135 were transplanted into their respective human templates. Optionally, some amino acids in the FR region of the humanized antibody were substituted, specifically amino acid residues at positions 1, 3, 9, 28, 29, 44, 49, and / or 93 (determined according to Kabat numbering rules) in the FR region of the heavy chain variable region, and / or amino acid residues at positions 4, 43, 46, 47, and / or 71 (determined according to Kabat numbering rules) in the FR region of the light chain variable region. The mutation design of the humanized antibody variable region of antibody 135 is shown in Table 9 below:

[0349] Table 9.135 Mutations in Humanized Antibodies Note: Grafted indicates that the murine antibody CDR has been implanted into the human germline FR region; the location of the reversion mutation site is determined according to the Kabat numbering rules. For example, "M4L" means that the 4th M has been mutated to L according to the Kabat numbering system.

[0350] Table 10.135 CDRs of humanized antibodies

[0351] >hu135VH1

[0352] >hu135VH2

[0353] >hu135VH3

[0354] >hu135VL1

[0355] Note: The double-underlined part is the antibody CDR sequence, where the CDR is numbered according to Kabat.

[0356] Humanization of 140 mouse antibodies

[0357] The humanized heavy chain variable regions FR1, FR2, and FR3 of murine antibody 140 were templated using IGHV1-46*01, and FR4 was templated using IGHJ6*01; the light chain variable regions FR1, FR2, and FR3 were templated using IGKV3-11*01, and FR4 was templated using IGKJ2*01. The CDRs of murine antibody 140 were then transplanted into their human templates. Optionally, some amino acids in the FR region of the humanized antibody may be substituted, specifically at positions 1, 40, 43, 48, 69, 71, 73, 74, 75, 77, 78, 80, and / or 81 (as determined by Kabat numbering rules) of the FR region of the heavy chain variable region. In some designs, a glutamic acid mutation may be performed at position 1 to avoid pyroglutamic cyclization, and / or at positions 20, 60, 58, 70, 71, 77, and / or 100 (as determined by Kabat numbering rules) of the FR region of the light chain variable region may be substituted. The mutation design of the variable region of the humanized antibody 140 is shown in Table 11 below:

[0358] Table 11.140 Mutations in humanized antibodies Note: Grafted indicates that the murine antibody CDR has been implanted into the human germline FR region; the location of the reversion mutation site is determined according to the Kabat numbering rules. For example, "I58V" means that the 58th I has been mutated to V according to the Kabat numbering system.

[0359] >hu140VH3

[0360] >hu140VH4

[0361] >hu140VH5

[0362] >hu140VL3

[0363] >hu140VL5

[0364] In addition, based on hu140VL5, certain amino acids (A60) in the FR1 region (T20) and FR3 region of the light chain were modified.

[0365] >hu140VL5-11

[0366] Note: The double-underlined part is the antibody CDR sequence, where the CDR is numbered according to Kabat.

[0367] Construction and expression of anti-NPR1 humanized antibody IgG1 LALA form

[0368] Primers were designed for PCR to construct the VH / VK gene fragments of each humanized antibody. These fragments were then homologously recombinated with the expression vector pTT5 (containing a 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 can be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, IgG4, or their variants, and the light chain constant region can be selected from the light chain constant regions of human κ, λ chains, or their variants. For example, in the following embodiments, the antibody heavy chain constant region was selected from the human IgG1 heavy chain constant region as shown in SEQ ID NO: 86, and the light chain constant region was selected from the human light chain constant region as shown in SEQ ID NO: 44, with the LALA mutation introduced (indicated by double underscores).

[0369] Human IgG1 LALA heavy chain constant region sequence:

[0370] Human light chain constant region sequence:

[0371] The carboxyl terminus of the variable region of the humanized antibody heavy chain with phases 102, 127, 128, 135, and 140 constructed earlier is linked to the amino terminus of the constant region of the human heavy chain as shown in SEQ ID NO: 86 to form the full-length antibody heavy chain. The carboxyl terminus of the variable region of the humanized antibody light chain is linked to the amino terminus of the constant region of the human light chain as shown in SEQ ID NO: 44 to form the full-length antibody light chain. The humanized antibodies shown in Tables 12-16 below are obtained as follows:

[0372] Table 12.102 Humanized Antibodies

[0373] Note: In the table, “Hu102H1L3” indicates that the heavy chain variable region is hu102VH1 (SEQ ID NO: 48), the light chain variable region is hu102VL3 (SEQ ID NO: 53), and the heavy chain constant region is as shown in SEQ ID NO: 86, and the light chain constant region is as shown in SEQ ID NO: 44, and so on.

[0374] Humanized antibodies in Table 13.127

[0375] Note: In the table, “Hu127H4L1” indicates that the heavy chain variable region is hu127VH4 (SEQ ID NO: 58), the light chain variable region is hu127VL1 (SEQ ID NO: 61), and the heavy chain constant region is as shown in SEQ ID NO: 86, and the light chain constant region is as shown in SEQ ID NO: 44, and so on.

[0376] Table 14.128 Humanized Antibodies

[0377] Note: In the table, "Hu128H1L3" indicates a humanized antibody with a heavy chain variable region of hu128VH1 (SEQ ID NO: 66), a light chain variable region of hu128VL3 (SEQ ID NO: 72), a heavy chain constant region as shown in SEQ ID NO: 86, and a light chain constant region as shown in SEQ ID NO: 44. Others are indicated by analogy.

[0378] Table 15.135 Humanized Antibodies

[0379] Note: In the table, "Hu135H1L1" indicates that the heavy chain variable region is hu135VH1 (SEQ ID NO: 76), the light chain variable region is hu135VL1 (SEQ ID NO: 79), and the heavy chain constant region is as shown in SEQ ID NO: 86, and the light chain constant region is as shown in SEQ ID NO: 44, and so on.

[0380] Table 16.140 Humanized Antibodies

[0381] Note: In the table, "Hu140H5L5" indicates that the heavy chain variable region is hu140VH5 (SEQ ID NO: 82) and the light chain variable region is hu140VL5 (SEQ ID NO: 84). "Hu140H5L5-11" indicates that the heavy chain variable region is Hu140VH5 (SEQ ID NO: 82) and the light chain variable region is hu140VL5-11 (SEQ ID NO: 85). The heavy chain constant region is as shown in SEQ ID NO: 86, and the light chain constant region is as shown in SEQ ID NO: 44. Others are represented by the same principle.

[0382] An example of a humanized antibody light / heavy chain full-length sequence is shown below:

[0383] Heavy chain sequence of Hu102H2L6:

[0384] Heavy chain sequence of Hu102H3L6:

[0385] Heavy chain sequence of Hu102H4L6:

[0386] Heavy chain sequence of Hu102H5L6:

[0387] Light chain sequences of Hu102H2L6, Hu102H3L6, Hu102H4L6, and Hu102H5L6:

[0388] Heavy chain sequences of Hu127H4L1, Hu127H4L3, Hu127H4L5, and Hu127H4L6:

[0389] Heavy chain sequences of Hu127H7L1, Hu127H7L3, Hu127H7L5, and Hu127H7L6:

[0390] Heavy chain sequences of Hu127H8L1, Hu127H8L3, Hu127H8L5, and Hu127H8L6:

[0391] Light chain sequences of Hu127H4L1, Hu127H7L1, and Hu127H8L1:

[0392] Light chain sequences of Hu127H4L3, Hu127H7L3, and Hu127H8L3:

[0393] Light chain sequences of Hu127H4L5, Hu127H7L5, and Hu127H8L5:

[0394] Light chain sequences of Hu127H4L6, Hu127H7L6, and Hu127H8L6:

[0395] Heavy chain sequences of Hu128H2L1 and Hu128H2L2:

[0396] Heavy chain sequences of Hu128H3L1-6, Hu128H3L1, and Hu128H3L2:

[0397] Light chain sequences of Hu128H2L1 and Hu128H3L1:

[0398] Light chain sequences of Hu128H2L2 and Hu128H3L2:

[0399] Hu128H3L1-6 light chain sequence:

[0400] Heavy chain sequence of Hu135H1L1:

[0401] Heavy chain sequence of Hu135H2L1:

[0402] Heavy chain sequence of Hu135H3L1:

[0403] Light chain sequences of Hu135H1L1, Hu135H2L1, and Hu135H3L1:

[0404] Heavy chain sequence of Hu140H4L5:

[0405] Heavy chain sequences of Hu140H5L5 and Hu140H5L5-11:

[0406] Light chain sequences of Hu140H4L5 and Hu140H5L5:

[0407] The light chain sequence of Hu140H5L5-11:

[0408] Note: In the full-length antibody sequence above, the underlined portion is the antibody CDR sequence, and the italicized portion is the antibody constant region sequence. The CDR is numbered according to Kabat.

[0409] The light and heavy chain sequences of the positive control antibodies REGN-5381 (variable region sequence see WO2020086406A2) and XX16 V (variable region sequence see WO2020250159A1) in this disclosed embodiment are as follows:

[0410] REGN-5381 heavy chain sequence:

[0411] REGN-5381 light chain sequence:

[0412] REGN-5381-m heavy chain sequence (REGN-5381 variable region + mouse IgG2a heavy chain, IMGT accession No. V0085):

[0413] REGN-5381-m light chain sequence (REGN-5381 variable region + mouse kappa light chain, Uniprot accession No. P01837):

[0414] XX16 V heavy chain sequence:

[0415] XX16 V light chain sequence:

[0416] The negative control antibody RC25 V (variable region as described in US6114143A) disclosed in this disclosure has the following light and heavy chains:

[0417] RC25 V heavy chain:

[0418] RC25 V light chain:

[0419] II. Antibody Testing Examples

[0420] Test Example 1: ELISA assay to detect the binding of anti-NPR1 antibody to hNPR protein

[0421] The binding activity of anti-NPR1 antibody to hNPR1, hNPR2, and hNPR3 was detected by ELISA. The specific experimental method is as follows: 4 μg / mL hNPR antigen (hNPR1, SEQ ID NO: 1; hNPR2, Abcam, Cat. No. Ab201371; hNPR3, Abcam, Cat. No. Ab114355) was coated at 37°C for 3 hours, followed by blocking with PBS containing 5% skim milk powder and incubating overnight at 4°C. After washing three times with PBS at pH 7.4, 20 μg / mL anti-NPR1 antibody was added to the first well, and subsequent wells were serially diluted 5-fold, incubated at 37°C for 1.5 hours. After washing three times with PBS at pH 7.4, 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 colorimetric values ​​were read. The experimental results are shown in Figures 1A to 1C and Table 17. The experimental results show that the anti-NPR1 antibodies disclosed herein all bind to hNPR1, but do not bind to hNPR2 and hNPR3 of the same family.

[0422] Table 17. ELISA results of anti-NPR1 antibody binding to hNPR protein Note: NB indicates no binding.

[0423] Test Example 2: cGMP generation experiment stimulated by anti-NPR1 antibody

[0424] Anti-NPR1 antibody, upon binding to NPR1, stimulates downstream signaling pathways to produce cGMP. This experiment uses cGMP detection to clarify the antibody's agonistic activity. The specific experimental method is as follows: Prepare 1× reaction buffer: 5mM MgCl2, 10mM HEPES pH 7.4, 0.5mM IBMX in EBSS, and prepare different concentrations of anti-NPR1 antibody using the reaction buffer. The initial concentration in each well is 300 μg / mL, and subsequent wells are serially diluted 3-fold. Add 5 μL of the prepared antibody to each well of a 384-well plate. Simultaneously, wash hNPR1-CHOK1 cells twice with PBS (pH 7.4), resuspend them in reaction buffer to a concentration of 3E6 cells / mL, and add 5 μL of the solution to each well of a 384-well plate. After mixing, centrifuge at 300g for 1 minute and incubate at 37°C for 30 minutes. Then, follow the kit instructions (Cisbio, Cat. No. 62GM2PEG) for subsequent operations. The experimental results are shown in Tables 18-1 to 18-5 and Figure 2.

[0425] Table 18-1. Ability of anti-NPR1 antibody to produce cGMP by hNPR1-CHOK1 cells

[0426] Table 18-2. Ability of anti-NPR1 antibody to produce cGMP by hNPR1-CHOK1 cells

[0427] Table 18-3. Ability of anti-NPR1 antibody to produce cGMP by binding to hNPR1-CHOK1 cells

[0428] Table 18-4. Ability of anti-NPR1 antibody to produce cGMP by hNPR1-CHOK1 cells

[0429] Table 18-5. Ability of anti-NPR1 antibody to produce cGMP by hNPR1-CHOK1 cells

[0430] Table 18-6. Ability of anti-NPR1 antibody to produce cGMP by hNPR1-CHOK1 cells

[0431] Experimental results show that the anti-NPR1 antibodies disclosed herein can all stimulate hNPR1-CHOK1 cells to produce cGMP.

[0432] Test Example 3: Anti-NPR1 antibody binding to NPR1 endocytosis assay

[0433] Anti-NPR1 antibodies undergo endocytosis upon binding to NPR1 cells. This experiment detects the intensity of endocytosis by anti-NPR1 antibodies. The specific experimental method is as follows: Adjust the hNPR1-CHOK1 cell density to 4E5 cells / mL and add 50 μL / well to each 96-well cell plate. Prepare the antibody to be tested using CHO-K1 medium (DMEM / F12+10% FBS, Cat.No.Hyclone SH30023.01; Sangon Biotech, A500023-0100). Add 20 μg / mL of anti-NPR1 antibody to each 96-well cell plate and incubate for 10 min, 1 h, 7 h, or 24 h. At 10 min, 1 h, 7 h, or 24 h, discard the culture supernatant, add trypsin to digest the cells, transfer to a new 96-well plate, centrifuge at 300g for 5 min, and wash twice with 1% BSA / PBS. The detection antibody (REGN-5381-m) was prepared using 1% BSA / PBS, and cells were resuspended at 100 μL / well and incubated at 4°C for 1 hour. Cells were then washed twice with 1% BSA / PBS, and APC anti-mouse IgG (Biolegend Cat. No. 405308) was added, followed by incubation at 4°C for 40 minutes. Cells were washed twice with PBS and resuspended in 1% BSA / PBS. Fluorescence signal intensity was detected using flow cytometry (Thermo Fisher, Attune Nxt), analyzing data from 10,000 cells per well. The experimental results are shown in Figure 3 and Table 19.

[0434] Table 19. Binding and endocytosis assays of anti-NPR1 antibody with hNPR1-CHOK1

[0435] *Ratio: The ratio of the amount of NPR1 antigen remaining on the cell surface after endocytosis to the initial amount of NPR1 antigen.

[0436] Experimental results show that the internalization of the disclosed anti-NPR1 antibody is weaker than that of the positive control antibody, resulting in a higher amount of NPR1 antigen retained on the cell surface. Weaker internalization ensures that the anti-NPR1 antibody exhibits more durable and stable pharmacokinetics and efficacy.

[0437] Test Example 4: Experiment on the stimulation of cGMP production in cells after endocytosis treatment with anti-NPR1 antibody

[0438] After NPR1 antibody binds to NPR1, it undergoes endocytosis. Endocytosis reduces antigen expression, thus affecting the agonistic activity of anti-NPR1 agonist antibodies. This experiment detected the agonistic activity of NPR1 agonist antibodies on cells treated with NPR1 endocytosis. The specific experimental method is as follows: Adjust the hNPR1-CHOK1 cell density to 4E5 cells / mL and add 2 mL / well to each well of a 6-well cell plate. Prepare the antibody to be tested using culture medium and add 1 mL / well to each well of the 6-well cell plate (final concentration 20 μg / mL). Incubate overnight. After washing the cells twice with PBS at pH 7.4, use the method described in Test Example 1 to perform an anti-NPR1 antibody-stimulated cGMP production experiment on the cells treated with the anti-NPR1 antibody. The experimental results are shown in Figures 4A and 4B and Table 20. The data in the table represent the window value for the generation of cGMP in cells before and after endocytosis treatment as the sum of signal changes. That is, at each detection concentration, the sum of the absolute values ​​of the differences between the signal values ​​of cGMP detected in un-endocytinated cells and the signal values ​​of cGMP detected in endocytinated cells.

[0439] Table 20. Window values ​​for stimulating cGMP production in cells before and after endocytosis treatment with anti-NPR1 antibody

[0440] Experimental results show that the anti-NPR1 antibody disclosed herein exhibits less difference in cGMP production on cells after endocytosis compared to positive antibodies.

[0441] Test Example 5: In vivo efficacy experiment of anti-NPR1 antibody in hNPR1 transgenic mice with ANGII model

[0442] To evaluate the in vivo efficacy of the anti-NPR1 antibody, hNPR1 homozygous transgenic mice were used, and ANGII was used to induce progressive heart failure with hypertension. Blood pressure, plasma and urine cGMP levels, and urinary biochemical parameters were assessed to evaluate the antibody's antihypertensive efficacy.

[0443] The specific modeling and experimental methods are as follows: After pre-acclimatization with a sphygmomanometer and baseline blood pressure measurement, three days before the start of drug administration, a micro-osmotic pump (Alzet Micro-Osmotic Pump; model 1004, containing angiotensin II acetate, with an average pump rate set at 0.11 μL / hour to deliver 1.5 mg / kg / day AngII) was subcutaneously implanted into the scapular region of the mice. Blood pressure was measured for three consecutive days. Mice with poorly measurable blood pressure or large fluctuations were excluded. The mice were grouped into groups of eight mice, with mean arterial pressure (MAP) as the primary reference and body weight as the secondary reference. The drug volume was 10 mL / kg, administered subcutaneously on days 0, 4, 7, 14, and 21. Blood pressure was measured as planned, and plasma was collected on days 2, 6, 9, 11, 16, 18, 25, and 29, and cumulative urine was collected on days 2, 6, 9, 11, 16, 18, and 25 to measure the corresponding cGMP content.

[0444] The experimental results are shown in Figures 5A-5I and Table 21. The table shows the average indicators over 29 days.

[0445] Table 21. In vivo efficacy of anti-NPR1 antibody in hNPR1 transgenic mouse ANGII model Note: NT-proBNP is a biomarker for cardiac dysfunction.

[0446] Statistical analysis: The model group was used as a control, and a two-way ANOVA method was used for analysis.

[0447] *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

[0448] Experimental results showed that the disclosed anti-NPR1 antibodies all had significant effects on blood pressure control and cGMP production, and showed a non-statistically significant trend in accelerating urinary sodium excretion and reducing NT-proBNP. On the other hand, the weakly endocytic molecule Hu102H4L6, at the same dose and dosing frequency, had a significantly higher endpoint plasma concentration than the positive REGN-5381 (Figure 5G).

[0449] Test Example 6: In vivo pharmacokinetic experiment of anti-NPR1 antibody

[0450] Three hNPR1 transgenic mice per group were administered the drug intravenously at a dose of 3 MPk. Whole blood (0.15 mL) was collected at 5 min, 1 day, 2 days, 4 days, 7 days, 10 days, 14 days, and 21 days post-administration (without anticoagulant). After collection, the blood was left at room temperature for 30 minutes, centrifuged at 1000g for 15 minutes, and the supernatant (serum) was collected and placed in EP tubes for storage at -20℃. Standard curves for different samples were constructed using the Total IgG method. The concentration of anti-NPR1 antibody in serum at different time points was calculated from the OD450 value. The data were analyzed using Phoenix WinNonlin software to calculate pharmacokinetic parameters. The experimental results are shown in Table 22.

[0451] Table 22. Results of in vivo pharmacokinetic experiments of anti-NPR1 antibody Note: t1 / 2 = terminal elimination half-life; Cmax = peak concentration; AUC 0-t =Area under the curve during drug administration; AUC 0-∞ =AUC 0-t +Terminal concentration / terminal elimination rate; Vz = apparent volume of distribution; CL = antibody clearance over time; MRT 0-∞ = mean residence time.

[0452] Experimental results showed that 140H5L5-11 exhibited good pharmacokinetic performance in mice at 3 mpk.

[0453] III. Preparation Examples - Anti-NPR1 Antibody Formulation

[0454] Preparation Example - SEC Size Exclusion Chromatography:

[0455] An analytical method for separating solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0456] SEC% (SEC monomer content percentage) = A monomer / A total × 100% (A monomer is the peak area of ​​the main peak monomer in the sample, and A total is the sum of the peak areas of all peaks).

[0457] ΔSEC% = SEC% of the formulation before stability testing - SEC% of the formulation after stability testing.

[0458] Instrument used for SEC determination: Agilent HPLC 1260;

[0459] Pillars: Waters, BioResolve TM SEC mAb 2.5μm 7.8×300mm Column

[0460] NR-CE capillary gel electrophoresis:

[0461] An electrophoresis method in which a gel is transferred into a capillary as a supporting medium and then separated according to the molecular weight of the sample under a certain voltage.

[0462] NR-CE% (NR-CE main peak content percentage) = A main peak / A total × 100% (A main peak is the peak area of ​​the main peak in the sample, and A total is the sum of the areas of all peaks).

[0463] ΔNR-CE% = NR-CE% of the formulation before stability testing - NR-CE of the formulation after stability testing.

[0464] CE testing instrument: Beckman capillary electrophoresis apparatus, model PA800 plus

[0465] iCIEF Imaging Capillary Isofocusing Electrophoresis:

[0466] This technique separates proteins based on their different isoelectric points (pI). The percentage of neutral peak content in iCIEF is calculated as: (neutral peak area / total area) × 100% (total area is the sum of the areas of acidic, neutral, and basic peaks). The instrument used for iCIEF determination is manufactured by Simple Protein and model Muarice.

[0467] Osmotic pressure measurement:

[0468] The freezing point method for determining osmotic pressure is based on the principle that the freezing point depression is directly proportional to the molar concentration of the solution. It uses a highly sensitive temperature sensing element to measure the freezing point of the solution and converts the electrical charge into osmotic pressure.

[0469] Instrument used for osmotic pressure measurement: Loser, model OM815.

[0470] protein

[0471] The protein used in the following formulation examples is the NPR1 antibody as described above (preferably Hu140H5L5-11, hereinafter referred to as "protein").

[0472] The instrument used for protein concentration determination was a UV-Vis spectrophotometer, model: Nano Drop oneC, with a path length of 1 mm.

[0473] Formulation Example 1. Screening of pH and Buffer Systems

[0474] Formulations containing 200 mg / mL protein, 70 mg / mL sucrose, 0.05 mg / mL disodium edetate (Na₂EDTA·2H₂O, CAS: 6381-92-6, hereinafter referred to as "EDTA-2Na"), and 0.4 mg / mL polysorbate 80 (hereinafter referred to as "PS80") were prepared. Table 23 shows formulations with different pH values ​​and buffer systems. Forced degradation studies (40℃ for 4 weeks) were conducted on the samples. The effects of different pH values ​​and buffer systems on protein stability were investigated using SEC, NR-CE, and iCIEF as evaluation indicators.

[0475] The results are shown in Table 23. After 4 weeks of storage at 40℃, the formulations prepared with histidine-histidine hydrochloride (His-HCl), acetate-sodium acetate (AA), citrate-sodium citrate (CA), and succinate-sodium succinate (SA) all showed good stability at pH 5.0-6.5. SEC data showed that, at the same pH 5.5, compared to other buffer systems, the monomer purity of the formulation prepared in the His-HCl buffer system was superior to other buffer systems after 4 weeks at 40℃. Furthermore, combined with NR-CE and iCIEF results, the formulations prepared with His-HCl at pH 5.5-6.5 all showed good stability, with the formulation prepared with His-HCl at pH 6.0 exhibiting the best stability.

[0476] Table 23. Screening results for high-temperature degradation using pH and buffer systems Note: AA represents acetic acid-sodium acetate; CA represents citric acid-sodium citrate; SA represents succinic acid-sodium succinate; His-HCl represents histidine-histidine hydrochloride; PB represents phosphate hydrochloride; 40℃ W4: Store at 40℃ for 4 weeks, the same applies below.

[0477] Formulation Example 2. pH Range Confirmation

[0478] Formulations were prepared with 200 mg / mL protein, 60 mg / mL sucrose, 0.4 mg / mL PS80, 0.02 mg / mL EDTA-2Na, and 45 mM His-HCl at different pH values, as shown in Table 24. Forced degradation studies were conducted on the samples (40°C for 4 weeks), and the effects of different pH values ​​on protein stability were investigated using SEC, NR-CE, and iCIEF as evaluation indicators.

[0479] The results are shown in Table 24. The results of SEC, NR-CE and iCIEF showed that after 4 weeks of storage at 40℃, the differences between groups were small, and the stability of the formulations was good in the pH range of 5.5-6.5.

[0480] Table 24. pH Range Confirmation Results

[0481] Formulation Example 3. Screening of Buffer System Ion Concentration

[0482] Preparations were made with 200 mg / mL protein, 60 mg / mL sucrose, 0.4 mg / mL PS80, 0.02 mg / mL LEDTA-2Na, and His-HCl at pH 6.0 with different ion concentrations as shown in Table 25. Forced degradation studies were conducted on the samples (40°C for 4 weeks). The effects of different ion concentrations of buffer systems on protein stability were investigated using SEC, NR-CE, and iCIEF as evaluation indicators.

[0483] The results are shown in Table 25. SEC, NR-CE, and iCIEF data showed that after 4 weeks of storage at 40°C, there were little difference between the 20-75mM His-HCl formulation groups, and all showed good stability.

[0484] Table 25. Screening Results of His-HCl Buffer Ion Concentration

[0485] Formulation Example 4. Screening of Surfactant Types

[0486] A formulation was prepared containing 200 mg / mL protein, 60 mg / mL sucrose, 0.02 mg / mL EDTA-2Na, different surfactants as shown in Table 26, 45 mM His-HCl, and pH 6.0. Forced degradation studies were conducted on the samples (40°C for 4 weeks), and the effects of different surfactants on protein stability were investigated using SEC, NR-CE, and iCIEF as evaluation indicators.

[0487] The results are shown in Table 26. SEC, NR-CE, and iCIEF data showed that after 4 weeks of storage at 40°C, there were no significant differences between the formulations containing PS80 and poloxamer 188 (P188), and all showed good stability.

[0488] Table 26. Results of Surfactant Selection

[0489] Formulation Example 5. Surfactant Concentration Screening

[0490] Formulations were prepared with 200 mg / mL protein, 60 mg / mL sucrose, 0.02 mg / mL LEDTA-2Na, different surfactant concentrations as shown in Table 27, 45 mL M His-HCl, and pH 6.0. Forced degradation studies were conducted on the samples (40°C for 4 weeks), and the effects of different surfactant concentrations on protein stability were investigated using SEC, NR-CE, and iCIEF as evaluation indicators.

[0491] The results are shown in Table 27. SEC, NR-CE, and iCIEF data showed that after 4 weeks of storage at 40°C, there were no significant differences among the 0.1-1.0 mg / mL PS80 formulation groups, and all showed good stability.

[0492] Table 27. Screening results for different surfactant concentrations

[0493] Formulation Example 6. Excipient Screening

[0494] A formulation was prepared with 200 mg / mL protein, 60 mg / mL sucrose, 0.4 mg / mL PS80, and either 0.02 mg / mL EDTA-2Na or 45 mM His-HCl (as shown in Table 28), at pH 6.0. Forced degradation studies (40°C for 4 weeks) were conducted on the samples, and the effects of EDTA-2Na on the stability of the formulation were investigated using SEC, NR-CE, and iCIEF as evaluation indicators.

[0495] The results are shown in Table 28. SEC, NR-CE and iCIEF data showed that after 4 weeks of storage at 40℃, there was no significant difference between the EDTA-2Na group and the non-EDTA-2Na group, and the stability of both groups was good.

[0496] Table 28. Results of excipient screening Note: N / A indicates that EDTA-2Na is not added.

[0497] Formulation Example 7. Screening of EDTA-2Na Concentration

[0498] Formulations were prepared with 200 mg / mL protein, 60 mg / mL sucrose, 0.4 mg / mL PS80, different concentrations of EDTA-2Na (as shown in Table 29), and 45 mM His-HCl at pH 6.0. Forced degradation studies (40°C for 4 weeks) were conducted on the samples, and the effects of different EDTA-2Na concentrations on the stability of the formulations were investigated using SEC, NR-CE, and iCIEF as evaluation indicators.

[0499] The results are shown in Table 29. After being placed at 40℃ for 4 weeks, there was no significant difference in the stability of the 0.01-1.0 mg / mL EDTA-2Na formulations, and all formulations showed good stability.

[0500] 29. Results of excipient concentration screening

[0501] Formulation Example 8. Sugar Concentration Screening

[0502] Formulations were prepared with 200 mg / mL protein, 0.4 mg / mL PS80, 0.02 mg / mL LEDTA-2Na, different sucrose concentrations as shown in Table 30, 45 mM His-HCl, and pH 6.0. Forced degradation studies were conducted on the samples (40°C for 4 weeks), and the effects of different sucrose concentrations on the stability of the formulations were investigated using SEC, NR-CE, and iCIEF as evaluation indicators.

[0503] The results are shown in Table 30. SEC, NR-CE, and iCIEF data showed that after 4 weeks of storage at 40℃, there was no significant difference in the stability of formulations with 30-90 mg / mL sucrose, and all formulations exhibited good stability.

[0504] Table 30. Screening results for different sucrose concentrations

[0505] Formulation Example 9. Protein Concentration Screening

[0506] Formulations were prepared with 60 mg / mL sucrose, 0.4 mg / mL PS80, 0.02 mg / mL EDTA-2Na, different protein concentrations as shown in Table 31, 45 mM His-HCl, and pH 6.0. Forced degradation studies (40°C for 4 weeks) were conducted on the samples, and the effects of different protein concentrations on the stability of the formulations were investigated using SEC, NR-CE, and iCIEF as evaluation indicators.

[0507] The results are shown in Table 31. After 4 weeks of storage at 40℃, the differences between the SEC and NR-CE data groups were small and within acceptable ranges as the protein concentration increased, while there were no significant differences between the iCIEF data groups. Therefore, the formulation showed good stability when the protein concentration was between 1-260 mg / mL.

[0508] Table 31. Screening results at different protein concentrations

[0509] Formulation Example 10. Prescription Confirmation

[0510] A formulation was prepared containing 200 mg / mL protein, 60 mg / mL sucrose, 0.4 mg / mL PS80, 0.02 mg / mL EDTA-2Na, 45 mM His-HCl, and pH 6.0. The samples were stored at 2–8 °C for 3 months, and their long-term stability was assessed using appearance, SEC, NR-CE, and iCIEF as evaluation indicators.

[0511] The results are shown in Table 32. Appearance, SEC, NR-CE, and iCIEF data showed that the formulation had good long-term stability after being stored at 2-8℃ for 3 months.

[0512] Table 32. Long-term stability results Note: 2-8℃ M3: Store at 2-8℃ for 3 months.

[0513] Although the invention has been described in detail with the aid of accompanying drawings and examples for clarity of understanding, these descriptions and examples should not be construed as limiting the scope of this disclosure. All patent and scientific literature disclosures cited herein are clearly and fully incorporated by reference.

Claims

A pharmaceutical composition comprising an anti-NPR1 antibody and a buffer solution, wherein: The anti-NPR1 antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 29, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 40, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:

41. The LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 42, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 33, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:

34. The buffer solution is histidine buffer, acetate buffer, citrate buffer, succinate buffer, or phosphate buffer; Preferably, the buffer solution is histidine-histidine hydrochloride buffer, acetate-sodium acetate buffer, citrate-sodium citrate buffer, or succinate-sodium succinate buffer. More preferably, the buffer solution is a histidine-histidine hydrochloride buffer solution. The pharmaceutical composition according to claim 1, wherein the concentration of the buffer solution is from 1 mM to 100 mM; Preferably, the concentration of the buffer solution is from 20 mM to 75 mM; More preferably, the concentration of the buffer solution is 40 mM to 50 mM; Most preferably, the concentration of the buffer solution is about 45 mM. The pharmaceutical composition according to claim 1 or 2, wherein the pH of the pharmaceutical composition is 5.0 to 6.5; Preferably, the pH of the pharmaceutical composition is 5.5 to 6.5; More preferably, the pH of the pharmaceutical composition is 5.7 to 6.3; Most preferably, the pH of the pharmaceutical composition is about 6.

0. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition further comprises a surfactant; Preferably, the surfactant is polysorbate or poloxamer; More preferably, the surfactant is polysorbate 80 or poloxamer 188; Most preferably, the surfactant is polysorbate 80. The pharmaceutical composition according to claim 4, wherein the concentration of the surfactant is from 0.01 mg / mL to 2.0 mg / mL; Preferably, the concentration of the surfactant is from 0.1 mg / mL to 1.0 mg / mL; More preferably, the concentration of the surfactant is from 0.2 mg / mL to 0.6 mg / mL; Most preferably, the concentration of the surfactant is about 0.4 mg / mL. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition further comprises sugar; Preferably, the sugar is sucrose, trehalose, mannitol, or sorbitol; More preferably, the sugar is sucrose. The pharmaceutical composition according to claim 6, wherein the concentration of the sugar is from 10 mg / mL to 120 mg / mL; Preferably, the concentration of the sugar is from 30 mg / mL to 90 mg / mL; More preferably, the concentration of the sugar is from 45 mg / mL to 75 mg / mL; Most preferably, the concentration of the sugar is about 60 mg / mL. The pharmaceutical composition according to any one of claims 1 to 7, wherein the pharmaceutical composition further comprises an antioxidant; Preferably, the antioxidant is selected from one or more of the group consisting of ethylenediaminetetraacetic acid hydrate or its salt, ethylenediaminetetraacetic acid or its salt, glycine, DTPA, methionine, histidine, tryptophan, cysteine ​​and ascorbic acid; More preferably, the antioxidant is ethylenediaminetetraacetic acid hydrate or a salt thereof, or ethylenediaminetetraacetic acid or a salt thereof; More preferably, the antioxidant is ethylenediaminetetraacetic acid hydrate or a salt thereof; Most preferably, the antioxidant is disodium edetate. The pharmaceutical composition according to claim 8, wherein the concentration of the antioxidant is from 0.01 mg / mL to 2.0 mg / mL; Preferably, the concentration of the antioxidant is from 0.01 mg / mL to 1.0 mg / mL; More preferably, the concentration of the antioxidant is from 0.01 mg / mL to 0.1 mg / mL; Most preferably, the concentration of the antioxidant is about 0.02 mg / mL. The pharmaceutical composition according to any one of claims 1 to 9, wherein the concentration of the anti-NPR1 antibody is from 1 mg / mL to 260 mg / mL; Preferably, the concentration of the anti-NPR1 antibody is from 100 mg / mL to 260 mg / mL; More preferably, the concentration of the anti-NPR1 antibody is from 160 mg / mL to 240 mg / mL; Most preferably, the concentration of the anti-NPR1 antibody is about 200 mg / mL. The pharmaceutical composition according to any one of claims 1 to 10, wherein the anti-NPR1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 82, or an amino acid sequence having at least 80% sequence identity with it; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 85, or an amino acid sequence having at least 80% sequence identity with it; Preferably, 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; More preferably, the anti-NPR1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 89, or an amino acid sequence having at least 80% sequence identity with it; and the light chain comprises the amino acid sequence of SEQ ID NO: 90, or an amino acid sequence having at least 80% sequence identity with it. Most preferably, the anti-NPR1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 89, and the light chain comprises the amino acid sequence of SEQ ID NO:

90. The pharmaceutical composition according to claim 1 comprises the following components: (a) Anti-NPR1 antibody at concentrations ranging from 1 mg / mL to 260 mg / mL, (b) Surfactants ranging from 0.01 mg / mL to 2.0 mg / mL, (c) Sugars ranging from 10 mg / mL to 120 mg / mL, (d) Antioxidants ranging from 0.01 mg / mL to 2.0 mg / mL, and (e) a buffer solution of 1 mM to 100 mM, wherein the pH of the pharmaceutical composition is 5.0 to 6.5; or The pharmaceutical composition comprises the following components: (a) Anti-NPR1 antibody at concentrations ranging from 1 mg / mL to 260 mg / mL, (b) Surfactants ranging from 0.01 mg / mL to 2.0 mg / mL, (c) Sugars ranging from 10 mg / mL to 120 mg / mL, and (e) a buffer solution of 1 mM to 100 mM, wherein the pH of the pharmaceutical composition is 5.0 to 6.5; Preferably, the pharmaceutical composition comprises the following components: (a) Anti-NPR1 antibody at concentrations ranging from 100 mg / mL to 260 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 1.0 mg / mL, (c) Sucrose at concentrations of 30 mg / mL to 90 mg / mL, (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 1.0 mg / mL, and (e) a 20 mM to 75 mM histidine-histidine hydrochloride buffer, acetate-sodium acetate buffer, citrate-sodium citrate buffer, or succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.5, preferably 5.5 to 6.5; or The pharmaceutical composition comprises the following components: (a) Anti-NPR1 antibody at concentrations ranging from 100 mg / mL to 260 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 1.0 mg / mL, (c) Sucrose at concentrations of 30 mg / mL to 90 mg / mL, and (e) a 20 mM to 75 mM histidine-histidine hydrochloride buffer, acetate-sodium acetate buffer, citrate-sodium citrate buffer or succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.5, preferably 5.5 to 6.5; More preferably, the pharmaceutical composition comprises the following components: (a) Anti-NPR1 antibody at concentrations ranging from 160 mg / mL to 240 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.2 mg / mL to 0.6 mg / mL, (c) Sucrose at concentrations of 45 mg / mL to 75 mg / mL (d) Disodium edetate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and (e) a 40 mM to 50 mM histidine-histidine hydrochloride buffer solution, wherein the pH of the pharmaceutical composition is 5.7 to 6.3; or The pharmaceutical composition comprises the following components: (a) Anti-NPR1 antibody at concentrations ranging from 160 mg / mL to 240 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.2 mg / mL to 0.6 mg / mL, (c) 45 mg / mL to 75 mg / mL of sucrose, and (e) 40 mM to 50 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.7 to 6.3; Most preferably, the pharmaceutical composition comprises the following components: (a) Approximately 200 mg / mL of anti-NPR1 antibody, (b) Approximately 0.4 mg / mL of polysorbate 80, (c) Approximately 60 mg / mL of sucrose, (d) Sodium edetate at approximately 0.02 mg / mL, and (e) a histidine-histidine hydrochloride buffer solution of about 45 mM, wherein the pH of the pharmaceutical composition is about 6.0; or The pharmaceutical composition comprises the following components: (a) Approximately 200 mg / mL of anti-NPR1 antibody, (b) Approximately 0.4 mg / mL of polysorbate 80, (c) Approximately 60 mg / mL of sucrose, and (e) About 45 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 6.

0. The pharmaceutical composition according to any one of claims 1 to 12 is a subcutaneous injection formulation, an intravenous injection formulation, an intraperitoneal injection formulation, or an intramuscular injection formulation; preferably a subcutaneous injection formulation. A method for preparing a lyophilized formulation, comprising the step of lyophilizing the pharmaceutical composition according to any one of claims 1 to 13. A lyophilized formulation, which, upon reconstitution, can form the pharmaceutical composition according to any one of claims 1 to 13. A reconstituted solution, characterized in that The reconstituted solution is prepared by reconstituted the lyophilized formulation of claim 15. An article comprising a container containing a pharmaceutical composition as described in any one of claims 1 to 13, or a lyophilized formulation as described in claim 15, or a reconstituted solution as described in claim 16. Use of the pharmaceutical composition of any one of claims 1 to 13, or the lyophilized formulation of claim 15, or the reconstituted solution of claim 16 in the preparation of a medicament for treating or preventing a disease; Preferably, the disease is a disease related to NPR1; More preferably, the diseases 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, arrhythmia, atrial fibrillation (AF), cardiac fibrosis, atrial flutter, harmful vascular remodeling, plaque stabilization, myocardial infarction (MI), preeclampsia, obesity, renal failure, renal disorders, cytokine release syndrome, chronic kidney disease, macular edema, glaucoma, stroke, lung disease, inflammation, asthma, bone growth disorder, fracture, and diabetes. Most preferably, the disease is heart failure or hypertension. A method for treating or preventing a disease, the method comprising administering to a subject a therapeutically or preventively effective amount of the pharmaceutical composition of any one of claims 1 to 13, or the lyophilized formulation of claim 15, or the reconstituted solution of claim 16; Preferably, the disease is a disease related to NPR1; More preferably, the diseases 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, arrhythmia, atrial fibrillation (AF), cardiac fibrosis, atrial flutter, harmful vascular remodeling, plaque stabilization, myocardial infarction (MI), preeclampsia, obesity, renal failure, renal disorders, cytokine release syndrome, chronic kidney disease, macular edema, glaucoma, stroke, lung disease, inflammation, asthma, bone growth disorder, fracture, and diabetes. Most preferably, the disease is heart failure or hypertension.

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