Mutant gnps and uses thereof

Abstract

This invention discloses a mutant G-type natriuretic peptide (GNP) and its applications. The mutant GNP contains an amino acid substitution at position 14 of the mature GNP peptide or its truncated form as shown in SEQ ID NO:1, wherein the amino acid substitution is P14R. This invention also discloses immunoconjugates, nucleic acids, recombinant vectors, host cells, pharmaceutical compositions, and uses containing this mutant GNP. Compared to the mature GNP peptide, this mutant GNP exhibits enhanced natriuretic peptide receptor A activity and / or natriuretic peptide receptor B activity, and better vasodilatory induction.

Description

[0001] This application claims priority to Chinese patent application 2024119963300, filed on 2024 / 12 / 31. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedical technology, and in particular to a mutant GNP and its applications. Background Technology

[0003] The natriuretic peptide system (NPs) is a class of polypeptides crucial for controlling cardiovascular, endocrine, renal, and cardiovascular homeostasis. Among them, atrial natriuretic peptide (ANP) was the first natriuretic peptide with natriuretic and vasodilatory effects isolated and identified from atrial extracts. Subsequently, numerous other natriuretic peptides have been discovered, including brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), initially isolated from porcine brain extracts; snake natriuretic peptide (DNP) isolated from the venom glands of the East African green mamba (Dendoaspis angusticeps); and taipan natriuretic peptides (TNPs) isolated from the venom of the taipan.

[0004] The invention patent application with application number CN201310127277.6 discloses a natural polypeptide isolated from the venom gland tissue of the East African green mamba snake, named G-type natriuretic peptide (GNP), which can be used to treat acute heart failure, acute decompensated heart failure, patients after coronary intervention for acute myocardial infarction, chronic heart failure, and elderly patients with acute anterior wall myocardial infarction complicated with systolic heart failure.

[0005] As a ligand, GNP can bind to the extracellular ligand-binding domains of its corresponding receptors, natriuretic peptide receptor A (NPR-A) and natriuretic peptide receptor B (NPR-B), activating the intracellular guanylate cyclase domain of the receptors. This catalyzes the cyclization of intracellular guanosine triphosphate (GTP) to form cyclic guanosine monophosphate (cGMP), regulating numerous downstream physiological effects such as vasodilation, blood pressure reduction, and sodium excretion and diuresis. GNP has been reported to stimulate cGMP secretion in cellular experiments, activating both NPR-A and NPR-B. Specifically, in PC12 cells (predominantly NPR-A), GNP's stimulatory capacity is moderate, lower than ANP, BNP, and DNP, but higher than CNP. In RASMC cells (predominantly NPR-B), GNP's stimulatory capacity is moderate, lower than CNP, but higher than ANP, BNP, and DNP.

[0006] Therefore, in order to improve the utilization efficiency of GNPs, it is necessary to develop mutant GNPs with enhanced NPR-A and / or NPR-B activities, providing potential new drugs for the clinical application of natriuretic peptides to treat diseases. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a mutant GNP (G-type natriuretic peptide) and its applications. This mutant GNP exhibits enhanced natriuretic peptide receptor A activity and / or natriuretic peptide receptor B activity compared to wild-type GNP.

[0008] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0009] The present invention provides a mutant GNP, wherein the mutant GNP contains an amino acid substitution at the P14 position of the mature GNP peptide or a truncated form thereof as shown in SEQ ID NO:1, and the amino acid substitution at the P14 position enhances the natriuretic peptide receptor A activity and / or natriuretic peptide receptor B activity of the mutant GNP compared to the mature GNP peptide; wherein the amino acid site is relative to the amino acid sequence of the mature GNP peptide.

[0010] In some embodiments, the amino acid at position P14 is replaced with P14R.

[0011] In some embodiments, the mutant GNP further includes an amino acid substitution at the H18 position of the mature GNP peptide as shown in SEQ ID NO:1.

[0012] In some embodiments, the amino acid at the H18 position is substituted with H18S, H18V, or H18T.

[0013] In some embodiments, the truncated form of the mature GNP peptide comprises at least 25 consecutive amino acid residues from position 1 to position 38 of the mature GNP peptide.

[0014] A truncated sequence is a sequence shorter than a reference sequence. A truncated sequence can be created by shortening one or more amino acid residues at the N-terminus and / or C-terminus of the reference sequence. C-terminal or N-terminal truncation allows the mutant GNP to maintain essentially the same or similar biological activity as the parent.

[0015] In some embodiments, the truncated form of the mature GNP peptide has 3-13 amino acid residues missing from its C-terminus or N-terminus, relative to the mature GNP peptide.

[0016] In some embodiments, the truncated form of the mature GNP peptide has 6, 9, 11, or 13 amino acid residues missing at the C-terminus, or 3 amino acid residues missing at the N-terminus, relative to the mature GNP peptide.

[0017] In some embodiments, the mutant GNP has an amino acid sequence as shown in any one of SEQ ID NO: 2-5 and 6-10.

[0018] In another aspect, the present invention provides an immunoconjugate comprising the aforementioned mutant GNP and a coupling domain, wherein the coupling domain comprises at least one of the following: a half-life extension portion, a drug portion, a detectable portion, and an antigen-binding portion.

[0019] In some embodiments, the coupling domain is coupled to the mutant GNP via covalent bonds, non-covalent bonds, or other stable binding mechanisms.

[0020] In some embodiments, the coupling domain includes at least one of the following: Fc fragment, serum albumin, transferrin, chorionic gonadotropin β subunit carboxyl-terminal peptide (CTP), VHH, PEG polymer, fatty acid, and glycosylation modification.

[0021] The Fc fragment can be derived from human immunoglobulins, such as the native Fc sequences selected from IgG1, IgG2, IgG3, and IgG4. The Fc fragment can also contain variant Fc sequences, which are modified by mutations in the native Fc sequences derived from human immunoglobulins. Mutations in the native Fc sequences endow the fusion protein with at least the following characteristics: i) providing increased FcRn affinity compared to native IgG; ii) reducing or eliminating Fc-mediated effector function, thereby reducing the likelihood of inducing anti-drug antibody formation in the organism after fusion protein administration; iii) improving stability and / or reducing aggregation; and / or, iv) prolonging the drug's half-life.

[0022] The Fc fragment may be an Fc sequence containing human IgG1, and the human IgG1 Fc sequence contains at least one of the following amino acid mutation combinations:

[0023] i) LALA: The leucine Leu at position 234 is mutated to alanine Ala (L234A), and the leucine Leu at position 235 is mutated to alanine Ala (L235A);

[0024] ii) LALA-PG: Leucine at position 234 is mutated to alanine (L234A), Leucine at position 235 is mutated to alanine (L235A), and proline at position 329 is mutated to glycine (P329G).

[0025] iii) AAA: Leucine at position 234 is mutated to alanine (L234A), leucine at position 235 is mutated to alanine (L235A), and glycine at position 237 is mutated to alanine (G237A).

[0026] iv) L235E: The leucine residue at position 235 (Leu) is mutated to glutamate (Glu) (L235E);

[0027] v) YTE: Methionine at position 252 is mutated to tyrosine (Tyr) (M252Y), serine at position 254 is mutated to threonine (Thr) (S254T), and threonine at position 256 is mutated to glutamate (Glu) (T256E).

[0028] ⅵ) YTE-KF: Methionine at position 252 is mutated to tyrosine (Tyr) (M252Y), serine at position 254 is mutated to threonine (Thr) (S254T), threonine at position 256 is mutated to glutamic acid (Glu) (T256E), histidine at position 434 is mutated to lysine (Lys) (H433K), and asparagine at position 434 is mutated to phenylalanine (Phe) (N434F);

[0029] ⅶ)LS: Methionine at position 428 is mutated to leucine (Leu) (M428L), and asparagine at position 434 is mutated to serine (Ser) (N434S);

[0030] ⅷ) ΔK: Lysine deletion at position 447 (K447del);

[0031] ⅸ) CS: Cysteine ​​at position 220 is mutated to serine Ser (C220S);

[0032] The amino acid residues are numbered according to the EU index, such as in Kabat.

[0033] In some implementations, the sequence information of the Fc fragment of human IgG1 is as follows:

[0034] Fc segment name amino acid residue differences in the Fc sequence relative to wild-type human IgG1 IgG1_Fc-CS-AAA-LS C220S / L234A / L235A / G237A / M428L / N434S IgG1_Fc-CS-AAA-YTE C220S / L234A / L235A / G237A / M252Y / S254T / T256E IgG1_Fc-CS-AAA-LS-ΔK C220S / L234A / L235A / G237A / M428L / N434S / K447del IgG1_Fc-CS-AAA-YTE-ΔK C220S / L234A / L235A / G237A / M252Y / S254T / T256E / K447del

[0035] The " / " indicates that the differences in amino acid residues before and after the symbol coexist.

[0036] In some implementations, the Fc sequence of wild-type human IgG1 is as follows:

[0037] PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:11).

[0038] The Fc fragment may be an Fc sequence containing human IgG4, and the human IgG4 Fc sequence contains at least one of the following amino acid mutation combinations:

[0039] i) Pro: The serine Ser at position 228 is mutated to proline Pro (S228P);

[0040] ii) FALA: The phenylalanine Phe at position 234 is mutated to alanine Ala (F234A), and the leucine Leu at position 235 is mutated to alanine Ala (L235A);

[0041] iii) FALA-PG: Phenylalanine at position 234 is mutated to alanine Ala (F234A), leucine at position 235 is mutated to alanine Ala (L235A), and proline at position 329 is mutated to glycine Gly (P329G).

[0042] ⅳ)AAA: Leucine at position 234 is mutated to alanine (L234A), leucine at position 235 is mutated to alanine (L235A), and glycine at position 237 is mutated to alanine (G237A).

[0043] v) L235E: The leucine residue at position 235 (Leu) is mutated to glutamate (Glu) (L235E);

[0044] ⅵ) YTE: Methionine at position 252 is mutated to tyrosine (Tyr) (M252Y), serine at position 254 is mutated to threonine (Thr) (S254T), and threonine at position 256 is mutated to glutamate (Glu) (T256E).

[0045] ⅶ) YTE-KF: Methionine at position 252 is mutated to tyrosine (Tyr) (M252Y), serine at position 254 is mutated to threonine (Thr) (S254T), threonine at position 256 is mutated to glutamic acid (Glu) (T256E), histidine at position 434 is mutated to lysine (Lys) (H433K), and asparagine at position 434 is mutated to phenylalanine (Phe) (N434F).

[0046] ⅷ)LS: Methionine at position 428 is mutated to leucine (Leu) (M428L), and asparagine at position 434 is mutated to serine (Ser) (N434S);

[0047] ⅸ) ΔK: Lysine deletion at position 447 (K447del);

[0048] The amino acid residues are numbered according to the EU index, such as in Kabat.

[0049] In some implementations, the sequence information of the Fc fragment of human IgG4 is as follows:

[0050]

[0051]

[0052] The " / " indicates that the differences in amino acid residues before and after the symbol coexist.

[0053] In some implementations, the Fc sequence of wild-type human IgG4 is as follows:

[0054] AESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:12).

[0055] In some embodiments, the Fc fragment, the serum albumin, the transferrin, the β subunit C-terminal peptide of human chorionic gonadotropin, or the VHH is linked to the mutant GNP via a peptide bond or linker.

[0056] In some embodiments, the linker comprises an amino acid sequence as shown in SEQ ID NO:13.

[0057] In another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the aforementioned mutant GNP.

[0058] In another aspect, the present invention provides a recombinant vector comprising the aforementioned nucleic acid molecules.

[0059] In another aspect, the present invention provides a host cell comprising the aforementioned nucleic acid molecule or recombinant vector, or expressing the aforementioned mutant GNP.

[0060] In another aspect, the present invention provides a method for preparing mutant GNPs, the method comprising culturing the aforementioned host cells and obtaining the mutant GNPs from the culture.

[0061] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned mutant GNP or immunoconjugate, and a pharmaceutically acceptable carrier.

[0062] In another aspect, the present invention provides the use of the aforementioned mutant GNP, immunoconjugate, or pharmaceutical composition in the preparation of NPRA and / or NPRB agonist drugs.

[0063] In another aspect, the present invention provides the use of the aforementioned mutant GNP, immune conjugate, or pharmaceutical composition in the preparation of a medicament for treating or preventing a disease, wherein the disease is a cardiovascular disease, a lung disease, a bone disease, or an autoimmune disease.

[0064] In some embodiments, the disease is selected from: myocardial hypertrophy, acute heart failure (decompensated heart failure, acute pulmonary edema, cardiogenic shock, isolated right ventricular failure, ACS-related heart failure), chronic heart failure (heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction), pulmonary hypertension, asthma, bronchitis, achondroplasia, growth disorder, multiple sclerosis, or myasthenia gravis.

[0065] Similar to NPs from classic mammals, the NP family derived from snake venom possesses a relatively conserved 17-residue ring structure, along with variable N-terminal and C-terminal extensions (Sridharan S, Kini RM, Richards AM. Venom natriuretic peptides guide the design of heart failure therapeutics. Pharmacol Res. 2020 May; 155:104687. doi:10.1016 / j.phrs.2020.104687). However, the activation abilities of different snake venom NPs for natriuretic peptide receptors still vary considerably. In our research on GNPs derived from snake venom, we attempted to modify certain conserved amino acids in the ring structure, discovering that specific modifications enhanced the activity of NPR-A and NPR-B to varying degrees. Attached Figure Description

[0066] This specification will be further illustrated by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein:

[0067] Figure 1 The activity assays of mature GNP and GrNP peptides in the aortic ring of New Zealand rabbits are shown; where "*" indicates p < 0.05 and N = 3. Detailed Implementation

[0068] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words “a,” “an,” “an,” and / or “the” are not specifically singular and may also include plural forms.

[0069] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words “comprising” or “including” will be understood to imply the inclusion of the stated elements, but not to exclude any other elements.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0071] In this article, natriuretic peptide receptors A, NPR-A, NPRA, NPR-1, and NPR1 can be used interchangeably. Natriuretic peptide receptors B, NPR-B, NPRB, NPR-2, and NPR2 can be used interchangeably. Receptor activity and receptor activation capacity can be used interchangeably. Mature GNP peptide and GNP can be used interchangeably.

[0072] NPR-A activity refers to the ability to bind to NPR-A and trigger NPR-A-mediated intracellular signal transduction.

[0073] NPR-B activity refers to the ability to bind to NPR-B and trigger NPR-B-mediated intracellular signal transduction.

[0074] Natriuretic peptide receptor activity can be measured in vivo and / or in vitro. It can be determined by measuring the binding of a compound (such as mutant GNPs and their derivatives) to the natriuretic peptide receptor, and / or by measuring downstream events following the binding of the compound (such as mutant GNPs and their derivatives) to the natriuretic peptide receptor. In some embodiments, natriuretic peptide receptor activity can be determined by measuring the amount and / or presence of downstream signals generated by the activation of the natriuretic peptide receptor. For example, natriuretic peptide receptor activity can be determined by measuring the amount of cGMP generated by the activation of the natriuretic peptide receptor. Methods for detecting cGMP generation are known in the art, such as cGMP ELISA assays. In other embodiments, natriuretic peptide receptor activity can be determined by measuring downstream effects generated by the activation of the natriuretic peptide receptor. For example, in animal models of disease, natriuretic peptide receptor activity can be determined by measuring improvements in cardiac remodeling, including increased ejection fraction, decreased resistance artery blood pressure, etc.

[0075] The terms “mutant,” “variant,” and “mutant” are used interchangeably in this article. The term “mutant” refers to a change in the amino acid sequence of a peptide or protein, specifically the presence of one or more amino acids. The terms “mutant GNP” and “GNP mutant” are used interchangeably in this article.

[0076] In this article, the terms "bioactivity" and "functional activity" are used interchangeably. The term "bioactivity" refers to the ability of a natural natriuretic peptide to perform its biological function in vivo or in vitro.

[0077] Mutant GNPs may also include substitutions, insertions, or deletions of up to 6, 5, 4, 3, 2, or 1 amino acid at positions 1-6, 10-12, 16-17, and 24-38 of the mature GNP peptide sequence shown in SEQ ID NO:1. Preferably, mutant GNPs include substitutions, insertions, or deletions of up to 2 amino acids at the aforementioned sites of the mature GNP peptide sequence shown in SEQ ID NO:1. In this document, "amino acid" and "amino acid residue" generally have the same meaning and can be used interchangeably.

[0078] The term "amino acid deletion" refers to the removal of one or more amino acids from the amino acid sequence of a peptide or protein, at a position other than the amino terminus and carboxyl terminus.

[0079] The term "amino acid insertion" refers to the addition of one or more amino acids to the amino acid sequence of a peptide or protein. The amino acid can be added at any position, including the amino terminus and carboxyl terminus of the peptide or protein. The added amino acid can be a native amino acid (e.g., an α-amino acid, an L-amino acid, or a D-amino acid) or a non-native amino acid.

[0080] The term "amino acid substitution" refers to the replacement of one or more amino acids at specific positions in the amino acid sequence of a peptide or protein with one or more other amino acids. The substitution sites can include the amino terminus, carboxyl terminus, and any position within the sequence of the peptide or protein; the substituted amino acids can be native (e.g., α-amino acids, L-amino acids, or D-amino acids) or non-native amino acids. The total number of amino acids (residues) at the substituted site typically remains unchanged.

[0081] The substitution of amino acids is preferably conservative substitution. Conservative substitution of amino acids includes substitution in which an amino acid residue is replaced by another amino acid residue having a similar side chain, such as substitution of physically or functionally similar residues (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residue is preferably replaced by another amino acid residue from the same side chain family.

[0082] Mutant GNPs can be linked into rings via disulfide bonds formed between two cysteine ​​residues. For example, the mutant GNP shown in SEQ ID NO:2 forms the desired ring structure via a disulfide bond between its cysteine ​​residues at positions 7 and 23.

[0083] The mutant GNP of this invention, compared to the mature GNP peptide, exhibits enhanced ability to induce cGMP generation via the NPR-A / cGMP pathway and / or via the NPR-B / cGMP pathway. Furthermore, the enhanced cGMP activation capacity of the mutant GNP improves one or more of the cellular function regulation functions involved in cGMP-dependent downstream signaling pathways.

[0084] The mutant GNP of this invention, compared to mature GNP peptides, can achieve the same or similar disease therapeutic effects with lower dosages and achieve greater economic benefits with equivalent production volume. Furthermore, reducing the dosage requirements for treatment may reduce or avoid side effects (e.g., additional side effects caused by the ADA effect (Anti-Drug Antibody Effect)).

[0085] According to another aspect of this specification, a nucleic acid molecule is also provided. This nucleic acid molecule comprises a nucleotide sequence capable of encoding any of the aforementioned mutant GNPs. The nucleotide sequence of this nucleic acid molecule may be codon-optimized, and this codon optimization can improve the expression level of the target polypeptide or protein in host cells.

[0086] According to another aspect of this specification, a recombinant vector is also provided. This recombinant vector comprises any of the aforementioned nucleic acid molecules. It will be understood that the recombinant vector of this specification may also include functional elements for expressing or suitable for expressing polypeptides or proteins, such as regulatory sequences, promoters, and enhancers. Exemplarily, the recombinant vector further includes a regulatory sequence operatively linked to the aforementioned nucleic acid molecule, and regulating the expression of the product encoded by the nucleic acid molecule in a host cell or cell-free expression system.

[0087] Recombinant vectors can be constructed using any suitable vector to introduce target peptides or proteins into receptors. Suitable vectors for constructing recombinant vectors include viral vectors and non-viral vectors. Exemplary examples include adeno-associated virus (AAV) vectors, lentiviral vectors, retroviral vectors, adenovirus vectors, herpesvirus vectors, herpes simplex virus vectors, cytomegalovirus vectors, vaccinia virus vectors, modified vaccinia virus Ankara strain (MVA) vectors, baculovirus vectors, vesicular stomatitis virus vectors, human papillomavirus vectors, fowlpox virus vectors, Sindbis virus vectors, Venezuelan equine encephalitis virus (VEE) vectors, measles virus vectors, influenza virus vectors, hepatitis B virus vectors, integration-deficient lentivirus (IDLV) vectors, bacteriophages, plasmids, granules, episomes, and artificial chromosomes. In some embodiments, the vector used to construct the recombinant vector is a plasmid (e.g., pET28a(+)).

[0088] According to another aspect of this specification, a host cell is also provided. This host cell comprises the nucleic acid molecules or recombinant vectors of any of the foregoing embodiments.

[0089] The term "host cell" refers to a cell that can introduce foreign genes and maintain their stability.

[0090] In some embodiments, the host cell may be a eukaryotic cell or a prokaryotic cell. Exemplarily, the host cell may be selected from mammalian cells, plant cells, insect cells, yeast cells, and bacteria. In some embodiments, the host cell is a mammalian cell. Suitable mammalian cells as host cells include Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK293) cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS) derived from CV-1 cells transformed with SV40, human hepatocellular carcinoma cells, NK cells, T cells, macrophages, and myeloma cells. Suitable prokaryotic cells as host cells include, but are not limited to, *Escherichia coli*, *Bacillus*, and *Salmonella*. In some embodiments, the host cell is a CHO cell or *Escherichia coli* (e.g., BL21(DE3)).

[0091] According to another aspect of this specification, an immunoconjugate is provided. The immunoconjugate comprises any of the aforementioned mutant GNPs and a coupling domain, the coupling domain comprising at least one of the following: a half-life extension portion, a pharmaceutical portion, a detectable portion, and an antigen-binding portion.

[0092] The coupling domain can be attached to any position of the mutant GNP by covalent or non-covalent coupling, such as to the main chain group, side chain group, N-terminus or C-terminus of the mutant GNP.

[0093] The half-life extension portion can prolong the blood half-life of the immunoconjugate in vivo. In some embodiments, the half-life extension portion is selected from at least one of the following: Fc fragment, serum albumin, transferrin, human chorionic gonadotropin β-subunit carboxyl-terminal peptide (CTP), VHH, PEG polymer, albumin-binding ligand, fatty acid, and glycosylation modification.

[0094] In some embodiments, the mutant GNP and the extended half-life portion (e.g., at least one of the Fc fragment, serum albumin, transferrin, CTP, and VHH) form a fusion protein, which may optionally be further coupled with other coupling domains. Mature GNP peptides from the East African green mamba have in vivo half-lives in the minute range, limiting their administration methods, for example, suitability for intravenous infusion under medical supervision. Compared to mature GNP peptides, the fusion protein provided by this invention allows for safer and more convenient administration, such as extending dosing intervals and reducing dosage. The fusion protein can be administered to subjects via intravenous injection, intramuscular injection, or subcutaneous injection. In a preferred embodiment, the fusion protein is administered to subjects via subcutaneous injection. Subcutaneous injection provides subjects with the possibility of administering the fusion protein outside of a medical monitoring environment, such as self-administration outside of a hospital.

[0095] The extended half-life portion can be an Fc fragment, particularly a human Fc fragment. Depending on the immunoglobulin subtype, the human Fc fragment can include human IgG1-Fc, human IgG2-Fc, human IgG3-Fc, and human IgG4-Fc fragments, or variants thereof.

[0096] Optionally, the affinity for FcRn can be enhanced and / or antibody-dependent cytotoxicity (ADCC) reduced by mutating the Fc fragment. The extended half-life portion can be a variant of the human IgG1-Fc fragment.

[0097] The mutant GNP and the extended half-life portion can be linked by peptide bonds. Alternatively, they can be linked by linkers. The linking order between the mutant GNP and the extended half-life portion is arbitrary. For example, the C-terminus of the extended half-life portion can be directly or indirectly linked to the N-terminus of the mutant GNP, or vice versa.

[0098] The pharmaceutical part can be a small molecule compound, peptide, or protein, or a combination thereof, that has a therapeutic effect on a disease. For example, the pharmaceutical part could be a small molecule compound for treating cardiovascular disease, which can be linked to a mutant GNP via a cleavable linker.

[0099] The detectable components include at least one of biotin, streptavidin, enzymes or their catalytically active fragments, radionuclides, nanoparticles, paramagnetic metal ions, fluorescent molecules, phosphorescent molecules, chemiluminescent molecules, nucleic acid probes, and contrast agents.

[0100] The antigen-binding portion can be an antibody or an antibody fragment. The term "antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen bound by the complete antibody. Exemplary antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, bisomatic antibodies, linear antibodies, single-chain antibody molecules (e.g., scFv), single-domain antibodies, aptamers, affinities, antibody heavy chain variable region fragments (VH), and antibody light chain variable region fragments (VL).

[0101] According to another aspect of this specification, a pharmaceutical composition is also provided. This pharmaceutical composition comprises a mutant GNP or immunoconjugate of any of the foregoing embodiments, and a pharmaceutically acceptable carrier.

[0102] In some embodiments, suitable carriers include, but are not limited to, diluents, fillers, excipients, binders, wetting agents, disintegrants, effervescent agents, surfactants, absorption enhancers, protease inhibitors, lubricants, implanting agents, bioadhesives, and carriers (such as sustained-release microspheres, microspheres, liposomes, microemulsions, hydrogels, nanoparticles, lipid nanoparticles (LNPs), viruses, virus-like particles (VLPs), cell-derived vesicles (CDVs), etc.).

[0103] In some embodiments, the pharmaceutical composition may further include other active ingredients that have therapeutic effects on the disease.

[0104] According to another aspect of this specification, the use of any of the foregoing mutant GNPs, immunoconjugates, or pharmaceutical compositions in the preparation of medicaments for the treatment or prevention of diseases, namely cardiovascular diseases, lung diseases, bone diseases, or autoimmune diseases, is also provided.

[0105] The drug is suitable for treating or alleviating one or more of the following conditions: acute heart failure, chronic heart failure, myocardial hypertrophy, myocardial ischemia, myocardial infarction, myocardial fibrosis, stroke, transient ischemic attack, inflammatory cardiovascular disease, coronary and peripheral artery spasm, edema due to heart failure, peripheral circulatory disturbances, reperfusion injury, arterial and venous thrombosis, heart failure, restenosis after thrombolytic therapy, arterial and pulmonary hypertension, refractory and intractable hypertension, coronary artery disease, bronchiolitis obliterans syndrome (BOS), graft-versus-host disease, stable and unstable angina, peripheral and cardiovascular disorders, and heart disease. Arrhythmia, atrial fibrillation, atrial flutter, ventricular fibrillation, ventricular flutter, atrial and ventricular premature contractions, AV junction premature contractions, premature ventricular contractions (PVC), sick sinus syndrome, syncope, atrioventricular nodal reentrant tachycardia, Wolff-Parkinson-White syndrome (WPW), acute coronary syndrome (ACS), autoimmune heart disease, shock, asthma, bronchitis, achondroplasia, chondrodysplasia, cartilage degeneration, skeletal dysplasia, osteoarthritis, osteogenesis imperfecta, osteomyelitis, osteonecrosis, osteoporosis, rickets, vasculitis, excessive angiogenesis, systemic sclerosis, multiple sclerosis, and myasthenia gravis.

[0106] Preferably, the drug is suitable for treating or alleviating one or more of the following diseases: myocardial hypertrophy, acute heart failure, chronic heart failure (heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, and heart failure with intermediate ejection fraction), pulmonary hypertension, asthma, bronchitis, achondroplasia, growth disorder, multiple sclerosis, or myasthenia gravis.

[0107] In some implementations, in cells expressing hNPR-A, the mature GNP peptide induces EC production of cGMP. 50 EC produced by cGMP induced by mutant GNP 50 The ratio is greater than or equal to 10.

[0108] EC 50 The half-maximal effect concentration (CMP) is the concentration sufficient to cause a half-maximal (50%) change in a measurable parameter. 50 The concentration at which the activity of a compound (such as mutant GNPs and their derivatives) is observed to produce 50% of its maximum effect can be used as a measure of its activity. The natriuretic peptide receptor activity of the mutant GNPs and their derivatives of this invention can be determined based on measured EC50. 50 To determine. EC 50 The lower the value, the better the activity.

[0109] In some implementations, in cells expressing hNPR-B, the mature GNP peptide induces EC production of cGMP. 50EC produced by cGMP induced by mutant GNP 50 The ratio is greater than or equal to 1.5.

[0110] In some embodiments, the mutant GNP is linked into a ring via a disulfide bond formed between two cysteine ​​residues. As an example, the mutant GNP shown in SEQ ID NO:2 forms a disulfide bond between cysteine ​​residues at position 7 and position 23, resulting in a ring structure.

[0111] Compared to mature GNP peptides, the mutant GNPs provided by this invention exhibit enhanced cGMP activation capacity, specifically an enhanced ability to induce cGMP generation via the NPR-A / cGMP pathway and / or the NPR-B / cGMP pathway. This enhanced cGMP activation capacity of the mutant GNPs improves one or more of the cellular function regulation processes involved in cGMP-dependent downstream signaling pathways. Furthermore, the mutant GNPs can achieve the same or similar disease therapeutic effects as mature GNP peptides at lower dosages. In addition, reducing the required dosage for treatment may reduce or avoid side effects (e.g., additional side effects caused by ADA effects).

[0112] The following embodiments are more specific descriptions of embodiments related to the above embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent companies. It should be understood that the following embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0113] Example 1: Preparation of GNP mutants

[0114] To enhance the NPR-A and / or NPR-B activities of GNP, mutations were performed at the P14 and H18 positions of the mature GNP peptide to obtain GNP mutants. In addition, to study the effect of N-terminal and / or C-terminal truncation on GNP mutants, truncated GNP mutants were also designed in this embodiment (see Table 1, where the single underlined part indicates the mutation site).

[0115] Table 1. Mature GNP peptides and mutants

[0116] name amino acid sequence SEQ ID NO GrNP <![CDATA[KSTPDGCFGHKLD R IGSHSGLGCPGAGPHPKPTPGAGR]]> 2 GrsNP <![CDATA[KSTPDGCFGHKLD R IGS S SGLGCPGAGPHPKPTPGAGR]]> 3 GrvNP <![CDATA[KSTPDGCFGHKLD R IGS V SGLGCPGAGPHPKPTPGAGR]]> 4 GrtNP <![CDATA[KSTPDGCFGHKLD R IGS T SGLGCPGAGPHPKPTPGAGR]]> 5 <![CDATA[GrNP C-6 ]]> <![CDATA[KSTPDGCFGHKLD R IGSHSGLGCPGAGPHPKP]]> 6 <![CDATA[GrNP C-9 ]]> <![CDATA[KSTPDGCFGHKLD R IGSHSGLGCPGAGPH]]> 7 <![CDATA[GrNP C-11 ]]> <![CDATA[KSTPDGCFGHKLD R IGSHSGLGCPGAG]]> 8 <![CDATA[GrNP C-13 ]]> <![CDATA[KSTPDGCFGHKLD R IGSHSGLGCPG]]> 9 <![CDATA[GrNP N-3 ]]> <![CDATA[PDGCFGHKLD R IGSHSGLGCPGAGPHPKPTPGAGR]]> 10 GNP mature peptides KSTPDGCFGHKLDPIGSHSGLGCPGAGPHPKPTPGAGR 1

[0117] 1. Recombinant gene sequence design and synthesis

[0118] Recombinant proteins were designed by fusing the tag protein Trx (sulfur redox protein) with the target peptides shown in Table 1. These recombinant proteins were linked by a linker peptide containing a thrombin restriction site. Based on the amino acid sequence of the recombinant proteins and the codon bias of the host cell (E. coli), recombinant gene sequences were designed (see Table 2). Nanjing Genscript Biotech Co., Ltd. was commissioned to synthesize the recombinant gene sequences, which were then inserted into cloning vectors. Specifically, an NcoI restriction endonuclease site and a start codon were added to the 5' end of the coding sequence of the recombinant proteins. A stop codon and a HindIII restriction endonuclease site were added to the 3' end of the coding sequence of the recombinant proteins.

[0119] Table 2. Detailed information on recombinant gene sequences

[0120]

[0121]

[0122]

[0123] Note: The underlined part indicates the enzyme cleavage site, and the italic part indicates the start codon or stop codon.

[0124] 2. Construction of recombinant expression vectors

[0125] After amplifying the target fragment on the cloning vector in step 1, the fragment was digested with NcoI and HindIII restriction endonucleases and cloned into the expression vector pET28a(+) to obtain the recombinant expression vector.

[0126] 3. Recombinant protein expression

[0127] The recombinant expression vectors were transformed into BL21(DE3) expression strains, and after recovery, they were plated on Kana antibiotic plates and incubated overnight at 37°C. Five single clones from each strain were then subjected to colony PCR. The primer sequences for colony PCR are shown in Table 3, and the reaction system and conditions are shown in Tables 4 and 5, respectively. Positive clones were induced to express expression with IPTG (1 mM), and the expression was detected by SDS-PAGE (80V, 30 min; then 120V, 45 min).

[0128] Table 3. Primer Sequences

[0129] name nucleotide sequence SEQ ID NO T7 TAATACGACTCACTATAGGG 24 T7T CCGCTGAGCAATAACTAGC 25

[0130] Table 4. Colony PCR reaction system

[0131] reagents Dosage (μL) Final concentration template / / T7 primer 0.2 0.2μM T7T primers 0.2 0.2μM Taq enzyme (containing buffer) 5 5U dNTPs 0.8 0.2mM <![CDATA[ddH2O]]> Add to 10 /

[0132] Note: The template is a single-clonal colony and the dosage is not calculated.

[0133] Table 5. Colony PCR reaction conditions

[0134]

[0135] Positive clones were cultured overnight, and the bacterial culture was collected and sent to a sequencing company for sequencing. Sequencing primers are shown in Table 3. The expression strains were collected by centrifugation (8000 rpm, 10 min), and the recombinant protein expressed by fusion was isolated by affinity chromatography. The tag protein Trx on the recombinant protein was removed by thrombin digestion. Finally, the target peptides were isolated and purified, including GNP mutants GrNP, GrsNP, GrvNP, GrrtNP, and GrNP. C-6 GrNP C-9 GrNP C-11 GrNP C-13 GrNP N-3 , and GNP mature peptide as a control.

[0136] Example 2: Activity assay of GNP mutant in cells

[0137] The biological activity of GNP mutants was determined using cell lines stably transfected with the natriuretic peptide receptor (including the stable cell line HEK293-hNPRA overexpressing hNPR-A and the stable cell line HEK293-hNPRB overexpressing hNPR-B. Methods for constructing stable transfected natriuretic peptide receptor cell lines are known in the art; see, for example, the invention patent application CN201110004553.0). In stable transfected natriuretic peptide receptor cell lines, the binding of the active substance to the natriuretic peptide receptor causes an increase in cGMP levels; the biological activity of the active substance can be determined by detecting changes in cGMP using a competitive ELISA method.

[0138] The specific experimental procedure is as follows:

[0139] 1. Determination of NPR-A activity

[0140] 1.1 Cell sample preparation

[0141] HEK293-hNPRA cells were routinely cultured in complete medium (DMEM + 10% FBS) (using 500 μg / mL G418 (Merck, catalog number A1720) during routine culture; no antibiotics were added during viability testing). Cells were digested and counted using trypsin (Cytiva, catalog number SH30042.01). The cell density was adjusted to 0.9 × 10⁻⁶ cells / mL. 6 Cells / mL. The cell solution was seeded at a rate of 100 μL / well in 96-well plates and cultured at 37°C for 19 hours in 5% CO2.

[0142] IBMX stock solution (Sigma, catalog number I7018) was added to the complete culture medium to a final concentration of 0.1 mM, which served as the sample diluent. The GNP mutant sample and the control sample GNP mature peptide were diluted with the sample diluent to appropriate starting concentrations, followed by 5-fold serial dilutions, for a total of 12 dilutions.

[0143] Discard the culture medium in the 96-well plate, and add each gradient sample to the corresponding well, in duplicate. Incubate at 37°C with 5% CO2 for 1.5 hours, and collect the cell culture supernatant.

[0144] 1.2 ELISA detection

[0145] Solution preparation: Washing buffer (PBST), 1×PBS + 0.05% Tween-20; Dilution buffer, PBST + 0.5% BSA.

[0146] Coating: Dilute the Anti-cGMP antibody (Invitrogen, catalog number MA5-4455) 1:20000 with Dilution Buffer and set aside. Wash the Protein G coated plate (ThermoFisher, catalog number 15133) 4 times with PBST at 200 μL / well. Add 100 μL of the diluted Anti-cGMP antibody to the Protein G coated plate, mix well, seal the plate with a sealing membrane, and incubate at room temperature for 1 h.

[0147] Sample addition: Dilute HRP-cGMP (Genscript, catalog number M01058) 1:20000 using Dilution Buffer; store protected from light for later use. Wash the protein G-coated plate 4 times with PBST at 250 μL / well. Add 50 μL / well each of the cell culture supernatant from step 1.1 and the diluted HRP-cGMP to the protein G-coated plate, and incubate at room temperature with shaking for 2 hours.

[0148] Color development: Wash the protein G-coated plate four times with PBST at 250 μL / well. Add 100 μL TMB to each well and incubate at room temperature in the dark for 20 min. Stop the color development by adding 50 μL stop solution to each well.

[0149] Detection: Detection was performed using an ELISA reader: λ = 450 nm. A four-parameter equation was fitted to obtain the EC50 of the sample. 50 value.

[0150] 2. Determination of NPR-B activity

[0151] The method for determining NPR-B activity is the same as the method for determining NPR-A activity in step 1 of this embodiment, except that HEK293-hNPRB cells are used for cell loading treatment.

[0152] 3. Results

[0153] The biological activity results of GNP mutants (GrNP, GrsNP, GrvNP, and GrtNP) and mature GNP peptides as controls for NPR-A and NPR-B are shown in Table 6.

[0154] Table 6. Results of NPR-A and NPR-B activity assays

[0155]

[0156] Note: NA indicates no activity detected. Due to the low activation capacity of natriuretic peptide receptors, EC could not be fitted within the concentration range used in the experiment. 50 .

[0157] Based on the above test results, it can be seen that, compared with the mature GNP peptide used as a control, the GNP mutant reflects NPR-A activity and / or EC, which reflects NPR-B activity. 50 A decrease in the value corresponds to a significant increase / enhancement in receptor activity. Specifically, GNP mutants, which incorporate amino acid substitutions of P14R in mature GNP peptides (GrNP), and further include amino acid mutations at other positions or truncated mutant GNPs, can still maintain their enhanced natriuretic peptide receptor activity relative to mature GNP peptides.

[0158] It is known that the GNP mutant of the present invention exhibits enhanced cGMP activation capacity, which can increase the level of cGMP production induced in cells through the NPR-A / cGMP pathway and / or through the NPR-B / cGMP pathway. Compared with mature GNP peptides, the enhanced cGMP activation capacity can reduce the dosage requirement of GNP mutants, improve economic efficiency under the same yield conditions, and may reduce or avoid some side effects (e.g., additional side effects caused by ADA effects).

[0159] Example 3: Activity assay of GNP mutant in rabbit aortic ring

[0160] In the isolated rabbit aortic rings, the contraction of the aortic rings was first stimulated with phenylephrine (PE), and then the tension changes of the rabbit aortic rings after the action of the active substance were detected to determine the biological activity of the active substance. The specific experimental steps and results are as follows:

[0161] Male New Zealand rabbits (weighing 2-2.5 kg) were anesthetized by intravenous injection of 5.0 mL of a compound anesthetic (containing 10 mg / mL of acetaminophen and 2 mg / mL of styrosine) via the marginal ear vein, followed by exsanguination and euthanasia. The thoracic aorta was removed and placed in Tyrode's solution (8.0 g sodium chloride, 0.2 g potassium chloride, 0.2 g calcium chloride, 0.05 g sodium dihydrogen phosphate, 0.1 g magnesium sulfate heptahydrate, 1.0 g sodium bicarbonate, and 1.0 g glucose dissolved in distilled water to make a 1000 mL solution). Fat and connective tissue were removed, and the aortic rings were cut into rings approximately 3-4 mm in length. The aortic rings were vertically fixed in an 8 mL bath containing Tyrode's solution at 37°C with continuous oxygenation. Changes in isometric force were recorded using an RM6240XC multichannel physiological signal acquisition and processing system.

[0162] After equilibration for 1 hour, the thallium solution was changed every 15 minutes. A preload of 1 g was applied to the blood vessels, and after another 1 hour of equilibration, the aortic ring was stimulated with phenylephrine (1 μM) for 20 minutes to examine tissue viability. Next, to verify the responsiveness of the blood vessels pre-constricted by phenylephrine (1 μM) to acetylcholine (ACh, 1 μM), the endothelial integrity of the aortic ring tissue was determined by stimulating the aortic ring with acetylcholine (ACh, 1 μM) for 10 minutes. After elution and equilibration for 15 minutes, vasoconstriction was induced with phenylephrine (1 μM) for 20 minutes until the vasoconstriction reached a plateau. Then, the blood vessels were stimulated with a concentration gradient (0.001 μM, 0.005 μM, 0.01 μM, 0.05 μM) of mature GNP peptides or GrNP for 25 minutes to observe the degree of vasodilation.

[0163] See results Figure 1 It can be seen that both mature GNP peptides and GrNP can induce dose-dependent vasodilation of the aortic rings that have PE contraction, and GrNP induces vasodilation to a greater extent.

[0164] Example 4: Preparation of GNP mutant-Fc fusion protein

[0165] To prolong the half-life of the GNP mutant in vivo, the Fc domain IgG1_Fc-CS-AAA-LS was fused to its C-terminus via a linker (GGGGS)n (n is 1-5) (SEQ ID NO:13) to obtain the GNP mutant-Fc fusion protein, making the drug regimen safer and more convenient. The preparation method of the GNP mutant-Fc fusion protein is as follows:

[0166] 1. Synthesis of the target gene for recombinant protein

[0167] Based on the amino acid sequence of the recombinant protein shown in Table 7 and the codon bias of the host cell, recombinant gene sequences were designed (see Table 8). These sequences were synthesized by Nanjing Genscript Biotech Co., Ltd., and then inserted into cloning vectors. Specifically, an XbaI restriction endonuclease site, a start codon (methionine), a Kozak sequence, and a signal peptide sequence were sequentially added to the 5' end of the coding sequence of the recombinant protein. The signal peptide is a sequence located at the N-terminus of secretory proteins, typically 15-30 amino acids in length, responsible for guiding the transport of nascent proteins to the endoplasmic reticulum for continued synthesis. This sequence is ultimately cleaved by a signal peptidase. A stop codon and an AgeI restriction endonuclease site were sequentially added to the 3' end of the coding sequence of the recombinant protein.

[0168] Table 7. GNP mutant-Fc fusion protein

[0169]

[0170]

[0171] Table 8. Nucleotide sequence of GNP mutant-Fc fusion protein

[0172]

[0173] Note: Bold text indicates the recombinant protein coding sequence, single underline indicates the restriction enzyme site, single wavy line indicates the Kozak sequence, dashed line indicates the signal peptide sequence, italic text indicates the start codon or stop codon, and lowercase text indicates the protective base.

[0174] 2. Construction of recombinant expression vectors

[0175] After amplifying the target fragment on the above cloning vector, it was digested with XbaI and AgeI restriction endonucleases and cloned into the expression vector pcDNA3.4 to obtain the recombinant expression vector.

[0176] 3. Cell line selection and recombinant protein expression

[0177] The recombinant expression vector obtained above was transfected into CHO cells. Stable, high-expression CHO cell lines were obtained by screening with selection markers and amplification-selective markers. After the obtained stable, high-expression CHO cell lines were fully adapted for serum-free suspension culture, they were transferred to a larger bioreactor. Scale-up culture was performed using standard culture media (CHO MaxX serum-free basal medium (McBang, MB1113.102) and fed media (MaxFA and MaxFB)) at 37±0.5℃, dissolved oxygen 30-50%, and pH 7.0±0.1. When the cell density reached 15×10⁶ cells / year... 6When the cell density is reduced to 32°C, the culture medium is continued for 14 days to obtain the cell culture solution.

[0178] 4. Recombinant protein purification

[0179] Based on the characteristic that recombinant proteins contain Fc fragments, affinity chromatography was performed on the obtained cell culture medium using a packing material equipped with ligands that can bind to the Fc fragment. After clarifying the cell culture medium, it was loaded onto a column and washed first with 20 mM sodium phosphate, 150 mM sodium chloride, pH 7.2, followed by elution with 20 mM sodium phosphate (pH 7.2) containing 1 M NaCl to further remove adsorbed protein impurities. The bound recombinant protein was then eluted with 20 mM sodium citrate (pH 3.5). The collected recombinant protein was adjusted to pH 7.2 ± 0.2 using 1 mol / L Tris buffer. The purified recombinant protein achieved a purity of over 95%.

[0180] It can be seen that the present invention prepares a high-purity GNP mutant-Fc fusion protein.

[0181] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mutant GNP, characterized in that, The mutant GNP contains an amino acid substitution at position P14 of the mature GNP peptide or its truncated form as shown in SEQ ID NO:1, and the amino acid substitution at position P14 enhances the natriuretic peptide receptor A activity and / or natriuretic peptide receptor B activity of the mutant GNP compared to the mature GNP peptide.

2. The mutant GNP as described in claim 1, characterized in that, The amino acid at position P14 is replaced with P14R.

3. The mutant GNP as described in claim 1 or 2, characterized in that, The mutant GNP also includes an amino acid substitution at the H18 position of the mature GNP peptide as shown in SEQ ID NO:1; preferably, the amino acid substitution at the H18 position is H18S, H18V or H18T.

4. The mutant GNP as described in claim 1 or 2, characterized in that, The truncated form of the GNP mature peptide includes at least 25 consecutive amino acid residues from positions 1 to 38 of the GNP mature peptide; preferably, the truncated form of the GNP mature peptide has 3-13 amino acid residues missing from its C-terminus or N-terminus relative to the GNP mature peptide; more preferably, the truncated form of the GNP mature peptide has 6, 9, 11 or 13 amino acid residues missing from its C-terminus, or 3 amino acid residues missing from its N-terminus relative to the GNP mature peptide.

5. The mutant GNP as described in claim 1, characterized in that, The mutant GNP has an amino acid sequence as shown in any one of SEQ ID NO:2-5 and 6-10.

6. An immunoconjugate, characterized in that, The immunoconjugate comprises a mutant GNP as described in any one of claims 1 to 5 and a conjugated domain, wherein the conjugated domain comprises at least one of the following: a half-life extension portion, a drug portion, a detectable portion, and an antigen-binding portion; Preferably, the coupling domain is coupled to the mutant GNP via covalent bonds, non-covalent bonds, or other stable binding mechanisms.

7. The immunoconjugate as described in claim 6, characterized in that, The coupling domain includes at least one of the following: Fc fragment, serum albumin, transferrin, CTP, VHH, PEG polymer, fatty acid, and glycosylation modification.

8. The immunoconjugate as described in claim 7, characterized in that, The amino acid sequence of the Fc fragment as shown in SEQ ID NO:11, or compared to the amino acid sequence shown in SEQ ID NO:11, includes any of the following combinations of amino acid residue differences: (1)C220S / L234A / L235A / G237A / M428L / N434S; (2)C220S / L234A / L235A / G237A / M252Y / S254T / T256E; (3)C220S / L234A / L235A / G237A / M428L / N434S / K447del; and, (4)C220S / L234A / L235A / G237A / M252Y / S254T / T256E / K447del; or, The amino acid sequence of the Fc fragment as shown in SEQ ID NO:12, or compared to the amino acid sequence shown in SEQ ID NO:12, includes any or a combination of the following amino acid residue differences: (1) S228P; (2)S228P / F234A / L235A; (3)S228P / F234A / L235A / M428L / N434S; (4)S228P / F234A / L235A / M252Y / S254T / T256E; (5)S228P / F234A / L235A / M428L / N434S / K447del; and, (6)S228P / F234A / L235A / M252Y / S254T / T256E / K447del; The amino acid residues are numbered according to the EU index in Kabat, and " / " indicates that the differences between the preceding and following amino acid residues exist simultaneously.

9. The immunoconjugate as described in claim 7 or 8, characterized in that, The Fc fragment, the serum albumin, the transferrin, the CTP, or the VHH are linked to the mutant GNP via peptide bonds or linkers; Preferably, the linker comprises an amino acid sequence as shown in SEQ ID NO:

13.

10. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleotide sequence encoding a mutant GNP as described in any one of claims 1 to 5.

11. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule as described in claim 10.

12. A host cell, characterized in that, The host cell contains the nucleic acid molecule as described in claim 10 or the recombinant vector as described in claim 11, or expresses the mutant GNP as described in any one of claims 1 to 5.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a mutant GNP as described in any one of claims 1 to 5 or an immunoconjugate as described in any one of claims 6 to 9, and a pharmaceutically acceptable carrier.

14. Use of the mutant GNP as described in any one of claims 1 to 5, or the immunoconjugate as described in any one of claims 6 to 9, or the pharmaceutical composition as described in claim 13, in the preparation of NPRA and / or NPRB agonist drugs.

15. The use of the mutant GNP of any one of claims 1 to 5, or the immunoconjugate of any one of claims 6 to 9, or the pharmaceutical composition of claim 13 in the preparation of a medicament for treating or preventing a disease, wherein the disease is a cardiovascular disease, a lung disease, a bone disease, or an autoimmune disease; Preferably, the disease is selected from: myocardial hypertrophy, acute heart failure (decompensated heart failure, acute pulmonary edema, cardiogenic shock, isolated right ventricular failure, ACS-related heart failure), chronic heart failure (heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, heart failure with intermediate ejection fraction), pulmonary hypertension, asthma, bronchitis, achondroplasia, growth disorder, multiple sclerosis, or myasthenia gravis.

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