Chimeric natriuretic peptide or variant thereof and use thereof

Abstract

Provided is a chimeric natriuretic peptide or a variant thereof. The chimeric natriuretic peptide comprises or consists of an N-terminal extension fragment, a cyclic fragment and a C-terminal extension fragment from the N-terminus to the C-terminus. The N-terminal extension fragment is truncated from an N-terminal linear region of DNP or an N-terminal linear region of ANP; the cyclic fragment is formed by truncating an N-terminal linear region and a C-terminal linear region of BNP while retaining a cyclic region thereof; and the C-terminal extension fragment is truncated from a C-terminal linear region of DNP. The chimeric natriuretic peptide has enhanced cGMP activation ability. Further provided are a fusion protein, nucleic acid molecule, recombinant vector, host cell, immunoconjugate and pharmaceutical composition of the chimeric natriuretic peptide or the variant thereof, and the related use thereof.

Description

A chimeric natriuretic peptide or a variant thereof and its applications

[0001] This application claims priority to the following two Chinese patent applications, the entire contents of which are incorporated herein by reference:

[0002] On October 16, 2024, a Chinese patent application was filed with the Chinese Patent Office with application number 202411511635.8 and invention title "A chimeric natriuretic peptide or a variant thereof and its application".

[0003] On December 26, 2024, a Chinese patent application was filed with the Chinese Patent Office with application number 202411941687.9 and invention title "A chimeric natriuretic peptide or a variant thereof and its application". Technical Field

[0004] This specification relates to the field of recombinant peptide technology, and in particular to a chimeric natriuretic peptide or a variant thereof and its applications. Background Technology

[0005] The natriuretic peptide system (NPs) is a class of polypeptides crucial for controlling cardiovascular, endocrine, renal, and cardiovascular homeostasis. Members of the natriuretic peptide family include atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), B-type natriuretic peptide (CNP), dendroaspis natriuretic peptide (DNP), and urodilatin (URO). The natriuretic peptide family shares a similar structure, including a 17-amino acid ring structure formed by disulfide bonds between two cysteine ​​residues. This ring structure is the receptor binding site.

[0006] Natriuretic peptide receptors discovered in mammals include NPR-A, NPR-B, and NPR-C. Among them, NPR-A and NPR-B belong to guanylate cyclase (GC) receptors and are therefore also known as GCA and GCB. Both NPR-A and NPR-B contain an extracellular ligand-binding domain, a transmembrane domain, an intracellular protein kinase homology domain, and an intracellular GC domain. After the extracellular ligand-binding domain of NPR-A and NPR-B binds to the natriuretic peptide, the conformation of NPR-A and NPR-B changes, the GC is activated, leading to the conversion of guanosine triphosphate (CTP) to cyclic guanosine monophosphate (cGMP). cGMP acts as a second messenger to trigger cellular physiological effects and mediate biological activity (Levin ER, Gardner DG, Samson WK. Natriuretic peptides. New England Journal of Medicine, 1998, 339(5):321-328). NPR-A and NPR-B exhibit ligand selectivity for natriuretic peptides. ANP, BNP, DNP, and URO primarily bind to NPR-A, while CNP primarily binds to NPR-B.

[0007] Previous studies have revealed the cGMP activation levels of the natriuretic peptide family. For example, Kim SM et al. evaluated the bioactivation of GC receptors by various natriuretic peptides in rabbit glomeruli. The results showed that ANP, BNP, CNP, DNP and URO could increase cGMP production through particulate GC in the glomerular membrane. Among them, ANP, BNP, DNP and URO caused similar increases in cGMP production, while CNP caused a lower increase in cGMP production. The order of cGMP production efficacy was: ANP≥URO≥DNP≥BNP>>CNP (Kim SM, Kim YA, Kim SY, et al. Presence of dendroaspis natriuretic peptide and its binding to NPR-A receptor in rabbit kidney. Regulatory peptides, 2011, 167(1):42-49).

[0008] Currently, recombinant human BNP has been successfully promoted and applied in clinical practice in China. BNP mainly regulates the function of various organs (such as the heart, brain, kidneys, and liver) through signal transduction of the NPR-A / cGMP pathway and participates in disease treatment.

[0009] To provide more drug options, achieve better disease treatment outcomes, and / or reduce the difficulty of developing peptide or protein modification products, there is a need to develop peptide and / or protein drugs with enhanced cGMP activation capabilities based on existing research. Summary of the Invention

[0010] This specification provides one or more embodiments of a chimeric natriuretic peptide or a variant thereof. The chimeric natriuretic peptide comprises, or is composed of, the following fragments from its N-terminus to its C-terminus: i) an N-terminal extension fragment truncated from the N-terminal linear region of a DNP as shown in SEQ ID NO:1 or the N-terminal linear region of an ANP as shown in SEQ ID NO:16; ii) a cyclic fragment formed by truncating the N-terminal and C-terminal linear regions of a BNP as shown in SEQ ID NO:2 while retaining the cyclic region; and iii) a C-terminal extension fragment truncated from the C-terminal linear region of the DNP.

[0011] In some embodiments, the cyclic fragment has an amino acid sequence as shown in SEQ ID NO:3, and the cyclic fragment is linked into a ring by a disulfide bond formed between two cysteine ​​residues.

[0012] In some embodiments, the N-terminal extension has an amino acid sequence as shown in any of the following:

[0013] (1) The amino acid sequence as shown in SEQ ID NO:4;

[0014] (2) An amino acid sequence obtained by truncating one amino acid from the N-terminus of the amino acid sequence shown in SEQ ID NO:4;

[0015] (3) The amino acid sequence as shown in SEQ ID NO:17;

[0016] (4) The amino acid sequence obtained by truncating one amino acid at the N-terminus of the amino acid sequence shown in SEQ ID NO:17.

[0017] In some embodiments, the C-terminal extension fragment has an amino acid sequence as shown in SEQ ID NO:5; or has an amino acid sequence obtained by truncating one or more amino acids at the C-terminus of the sequence shown in SEQ ID NO:5.

[0018] The C-terminal extension fragment can be taken from the C-terminal straight chain region of the DNP, or from the C-terminal straight chain region of the ANP or the C-terminal straight chain region of the BNP.

[0019] In some embodiments, the C-terminal extension fragment has an amino acid sequence as shown in SEQ ID NO:25 or SEQ ID NO:26, or has an amino acid sequence obtained by truncating one or more amino acids at the C-terminus of any of the above amino acid sequences.

[0020] In some embodiments, the number of C-terminal extension segments may be 2, 3, 4, 5, 6, 7, 8, or 9.

[0021] In some embodiments, the chimeric natriuretic peptide has an amino acid sequence as shown in SEQ ID NO:6.

[0022] In some embodiments, the chimeric natriuretic peptide variant comprises at least one of the following amino acid alterations in the sequence shown in SEQ ID NO:6: substitution, deletion, and / or insertion of up to 6 amino acid residues in the sequence shown in SEQ ID NO:6; truncation of up to 3 amino acid residues at the N-terminus of the sequence shown in SEQ ID NO:6; and truncation of up to 12 amino acid residues at the C-terminus of the sequence shown in SEQ ID NO:6.

[0023] In some embodiments, the chimeric natriuretic peptide has an amino acid sequence as shown in SEQ ID NO:18; variants of the chimeric natriuretic peptide include at least one of the following amino acid alterations in the sequence shown in SEQ ID NO:18: substitution, deletion, and / or insertion of up to 6 amino acid residues in the sequence shown in SEQ ID NO:18; truncation of up to 3 amino acid residues at the N-terminus of the sequence shown in SEQ ID NO:18; and truncation of up to 12 amino acid residues at the C-terminus of the sequence shown in SEQ ID NO:18.

[0024] In some embodiments, the chimeric natriuretic peptide has the amino acid sequence shown in SEQ ID NO:27; variants of the chimeric natriuretic peptide include at least one of the following amino acid alterations in the sequence shown in SEQ ID NO:27: substitution, deletion, and / or insertion of up to 6 amino acid residues in the sequence shown in SEQ ID NO:27; truncation of up to 3 amino acid residues at the N-terminus of the sequence shown in SEQ ID NO:27; and truncation of up to 12 amino acid residues at the C-terminus of the sequence shown in SEQ ID NO:27.

[0025] In some embodiments, the chimeric natriuretic peptide has an amino acid sequence as shown in SEQ ID NO:28; variants of the chimeric natriuretic peptide include at least one of the following amino acid alterations in the sequence shown in SEQ ID NO:28: substitution, deletion, and / or insertion of up to 6 amino acid residues in the sequence shown in SEQ ID NO:28; truncation of up to 3 amino acid residues at the N-terminus of the sequence shown in SEQ ID NO:28; and truncation of up to 12 amino acid residues at the C-terminus of the sequence shown in SEQ ID NO:28.

[0026] In some embodiments, the N-terminus of the chimeric natriuretic peptide is truncated by up to three amino acids, that is, the number of amino acid residues truncated at the N-terminus is 1 to 3. In some specific embodiments, the number of amino acid residues truncated at the N-terminus is 1.

[0027] In some embodiments, the chimeric natriuretic peptide has a C-terminus truncated by up to 9 amino acid residues, i.e., the number of amino acid residues truncated at the C-terminus is 1 to 9.

[0028] This specification also provides a fusion protein through one or more embodiments. The fusion protein includes a first domain and a second domain. The first domain is a chimeric natriuretic peptide or a variant of a chimeric natriuretic peptide from any of the above embodiments. The second domain (also known as a heterologous domain) prolongs the half-life of the fusion protein in vivo.

[0029] In some embodiments, the second domain includes at least one of the following: Fc fragment, serum albumin, transferrin, human chorionic gonadotropin β subunit carboxyl-terminal peptide (CTP), and VHH.

[0030] In some embodiments, the second domain is an Fc fragment or a variant thereof, wherein the Fc fragment is selected from the Fc fragments of human IgG1, human IgG2, human IgG3 or human IgG4.

[0031] In some embodiments, the Fc fragment comprises the Fc sequence of human IgG1; the variant of the Fc sequence of human IgG1 contains at least one of the following combinations of mutations i) to x):

[0032] i) L234A and L235A;

[0033] ii) L234A, L235A, and P329G;

[0034] iii), L234A, L235A, and G237A;

[0035] ⅳ)L235E;

[0036] v) M252Y, S254T and T256E;

[0037] ⅵ) M252Y, S254T, T256E, H433K and N434F;

[0038] (vii) M428 and LN434S;

[0039] ⅷ) K447 is missing;

[0040] ⅸ)C220S;

[0041] x) E356D and M358L.

[0042] In some embodiments, the Fc fragment is the Fc sequence of human IgG1, and its variants have an amino acid sequence as shown in SEQ ID NO:8.

[0043] In some embodiments, the Fc fragment comprises the Fc sequence of human IgG4; the variant of the Fc sequence of human IgG4 contains at least one of the following combinations of mutations i) to ⅸ):

[0044] i)S228P;

[0045] ii) F234A and L235A;

[0046] iii) F234A, L235A, and P329G;

[0047] iv) L234A, L235A, and G237A;

[0048] (v)L235E;

[0049] ⅵ)M252Y, S254T and T256E;

[0050] (vii) M252Y, S254T, T256E, H433K and N434F;

[0051] ⅷ) M428L and N434S;

[0052] ⅸ) K447 is missing.

[0053] In some embodiments, the Fc fragment is the Fc sequence of human IgG4. A variant of the Fc fragment is abbreviated as hIgG4_Fc-Pro-FALA-LS-ΔK, and its amino acid sequence is shown in SEQ ID NO: 37.

[0054] In some embodiments, the fusion protein has an amino acid sequence as shown in SEQ ID NO:10 or SEQ ID NO:19, denoted as DBDNP-Linker-Fc and ABDNP-Linker-Fc, respectively.

[0055] One or more embodiments of this specification also provide a nucleic acid molecule. The nucleic acid molecule comprises a nucleotide sequence encoding a chimeric natriuretic peptide, a variant of the chimeric natriuretic peptide, or a fusion protein of any of the above embodiments.

[0056] In some embodiments, the nucleic acid molecule is a nucleic acid molecule encoding the fusion protein (DBDNP-Linker-Fc or ABDNP-Linker-Fc). In some specific embodiments, the nucleic acid molecule encoding DBDNP-Linker-Fc has a nucleotide sequence as shown in SEQ ID NO:12. In some specific embodiments, the nucleic acid molecule encoding ABDNP-Linker-Fc has a nucleotide sequence as shown in SEQ ID NO:12.

[0057] This specification also provides a recombinant vector in one or more embodiments. The recombinant vector comprises the nucleic acid molecules of any of the above embodiments.

[0058] This specification also provides a host cell in one or more embodiments. The host cell comprises the nucleic acid molecule or recombinant vector of any of the above embodiments.

[0059] One or more embodiments of this specification also provide an immunoconjugate. The immunoconjugate comprises: a) a chimeric natriuretic peptide, a variant of the chimeric natriuretic peptide, or a fusion protein of any of the above embodiments; and, b) other compounds conjugated to a).

[0060] This specification also provides a pharmaceutical composition through one or more embodiments. The active ingredient of the pharmaceutical composition includes the chimeric natriuretic peptide, a variant of the chimeric natriuretic peptide, a fusion protein, a recombinant vector, or an immunoconjugate of any of the above embodiments.

[0061] In some embodiments, the active ingredient may also include other therapeutic agents.

[0062] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0063] This specification provides one or more embodiments that also provide the use of the chimeric natriuretic peptide, variants of the chimeric natriuretic peptide, fusion protein, nucleic acid molecule, recombinant vector, host cell, immune conjugate, or pharmaceutical composition of any of the above embodiments. The stated use is for the preparation of a medicament for the prevention, treatment, or relief of cardiovascular, renal, hepatic, or bone-related diseases or conditions.

[0064] In some embodiments, the disease or condition is at least one of the following: hypertrophic cardiomyopathy, heart failure, pulmonary hypertension, hypertension, renal failure, nephrotic syndrome, cirrhosis, achondroplasia, and skeletal dysplasia. Attached Figure Description

[0065] 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:

[0066] Figure 1 illustrates the GCA biological activity of DBDNP as detected by ELISA in some embodiments of this specification;

[0067] Figure 2 illustrates some embodiments of the DBD described in this specification. -1 NP was used to detect the biological activity of GCA by ELISA.

[0068] Figure 3 illustrates some embodiments of the DBD described in this specification. -6 NP was used to detect the biological activity of GCA by ELISA.

[0069] Figure 4 illustrates some embodiments of the DBD described in this specification. -11 NP was used to detect the biological activity of GCA by ELISA.

[0070] Figure 5 illustrates some embodiments of the DBD described in this specification. -12 NP was used to detect the biological activity of GCA by ELISA.

[0071] Figure 6 illustrates the GCA biological activity of ABDNP as detected by ELISA in some embodiments of this specification;

[0072] Figure 7 illustrates the GCA biological activity of BNP detected by ELISA in some embodiments of this specification;

[0073] Figure 8 illustrates the GCA biological activity of DBDNP-Linker-Fc as detected by ELISA in some embodiments of this specification;

[0074] Figure 9 illustrates the GCA biological activity of ABDNP-Linker-Fc as detected by ELISA in some embodiments of this specification;

[0075] Figure 10 illustrates the GCA biological activity of BNP-Linker-Fc as detected by ELISA in some embodiments of this specification;

[0076] Figure 11 shows the dose-response relationship of ABDNP, DBDNP and BNP inducing vasodilation in rabbit aortic rings with PE contraction. The three consecutive bars at each concentration, from left to right, represent the results of BNP, ABDNP and DBDNP, respectively.

[0077] Figure 12 shows the resistance of ABDNP, DBDNP, BNP and DNP to NEP enzyme;

[0078] Figure 13 shows the effect of ABDNP on cell area in the ISO-induced H9c2 cardiomyocyte hypertrophy model, where ISO represents the model group, NSB represents the blank control group, and * indicates a significant difference from the positive control group (P<0.01).

[0079] Figure 14 shows the effect of DBDNP on cell area in the ISO-induced H9c2 cardiomyocyte hypertrophy model, where ISO represents the model group, NSB represents the blank control group, and * indicates a significant difference from the positive control group (P<0.01).

[0080] Figure 15 shows the change of plasma ΔcGMP over time in rabbits that were intravenously administered ABDNP, DBDNP, and BNP. Detailed Implementation

[0081] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0082] As shown in this application and claims, unless the context clearly indicates an exception, words such as “comprising” or “including” will be understood to imply the inclusion of the stated elements, but not to exclude any other elements.

[0083] 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.

[0084] According to one aspect of this specification, a chimeric natriuretic peptide is provided. The chimeric natriuretic peptide comprises, or is composed of, the following fragments from its N-terminus (amino terminus) to its C-terminus (carboxyl terminus): i) an N-terminal extension fragment truncated from the N-terminal linear region of a DNP or from the N-terminal linear region of an ANP; ii) a cyclic fragment formed by truncating the N-terminal and C-terminal linear regions of a BNP while retaining the cyclic region; and iii) a C-terminal extension fragment truncated from the C-terminal linear region of a DNP.

[0085] In this document, the terms "bioactivity" and "functional activity" are used interchangeably. "Bioactivity" refers to the ability of a natural natriuretic peptide to perform its biological function in vivo or in vitro. In some cases, the bioactivity of the peptides, proteins, or variants thereof described in this specification enables them to bind to natriuretic peptide receptors and activate / induce downstream cGMP production.

[0086] The circular fragments can have different species origins. In some embodiments, the circular fragments are derived from human BNP. In other embodiments, the circular fragments can be derived from porcine BNP, canine BNP, rat BNP, mouse BNP, or chimpanzee BNP.

[0087] The number of amino acid residues truncated in the N-terminal and / or C-terminal linear regions of the BNP by the cyclic fragment can affect the functional activity of the chimeric natriuretic peptide. In some embodiments, the cyclic fragment truncates at least 5, 6, 7, 8, or 9 amino acid residues in the N-terminal linear region of the BNP. In some embodiments, the cyclic fragment truncates at least 3, 4, 5, or 6 amino acid residues in the C-terminal linear region of the BNP. Exemplarily, the cyclic fragment has the amino acid sequence shown at positions 6 to 26, 7 to 27, 8 to 28, 9 to 29, 10 to 30, 10 to 29, 10 to 28, 10 to 27, or 10 to 26 of the BNP sequence in SEQ ID NO:2 (SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH). In some preferred embodiments, the cyclic fragment has the amino acid sequence shown in SEQ ID NO:3 (CFGRKMDRISSSSGLGC).

[0088] In some embodiments, the cyclic fragments are linked together by disulfide bonds formed between two cysteine ​​residues. For example, the desired cyclic structure can be formed by introducing a disulfide bond between the cysteine ​​residues at positions 1 and 17 of the cyclic fragment shown in SEQ ID NO:3.

[0089] It is understood that, in other embodiments, any other peptide cyclization method known in the art can be used to form the desired cyclic structure in the chimeric natriuretic peptide or its variants. For example, lactonelation, thiolactoneation, or amidation can be performed between reactive groups at the desired two amino acid sites to form the desired cyclic structure.

[0090] In a preferred embodiment, the sequence of the N-terminal extension fragment is one of the following:

[0091] SLRRSS (SEQ ID NO:17), or a sequence formed by truncating one amino acid from the N-terminus of this sequence;

[0092] EVKYDP (SEQ ID NO:4), or a sequence formed by truncating one amino acid from the N-terminus of this sequence.

[0093] Extracting N-terminal extensions of different positions and / or lengths from the N-terminal linear region of DNP can affect the functional activity of chimeric natriuretic peptides. In some embodiments, the N-terminal extension consists of at least 3, 4, 5, or 6 consecutive amino acid residues from the C-terminus of the N-terminal linear region of DNP. Exemplarily, the N-terminal extension has the amino acid sequence shown at positions 4 to 6, 3 to 6, 2 to 6, or 1 to 6 of the DNP sequence shown in SEQ ID NO:1 (EVKYDPCFGHKIDRINHVSNLGCPSLRDPRPNAPSTSA). In some embodiments, the N-terminal extension consists of at least 3, 4, 5, or 6 consecutive amino acid residues from the N-terminus of the N-terminal linear region of DNP. Exemplarily, the N-terminal extension has the amino acid sequence shown at positions 1 to 3, 1 to 4, 1 to 5, or 1 to 6 of the DNP sequence shown in SEQ ID NO:1. In some preferred embodiments, the N-terminal extension fragment has the amino acid sequence shown in SEQ ID NO:4 (EVKYDP).

[0094] Extracting N-terminal extensions of different positions and / or lengths from the N-terminal linear region of an ANP can affect the functional activity of the chimeric natriuretic peptide. In some embodiments, the N-terminal extension consists of at least 3, 4, 5, or 6 consecutive amino acid residues from the C-terminus of the N-terminal linear region of the ANP. Exemplarily, the N-terminal extension has the amino acid sequence shown at positions 4 to 6, 3 to 6, 2 to 6, or 1 to 6 of the DNP sequence shown in SEQ ID NO:16 (SLRRSSCFGGRMDRIGAQSGLGCNSFRY). In some embodiments, the N-terminal extension consists of at least 3, 4, 5, or 6 consecutive amino acid residues from the N-terminus of the N-terminal linear region of the ANP. Exemplarily, the N-terminal extension has the amino acid sequence shown at positions 1 to 3, 1 to 4, 1 to 5, or 1 to 6 of the ANP sequence shown in SEQ ID NO:16. In some preferred embodiments, the N-terminal extension fragment has the amino acid sequence shown in SEQ ID NO:17 (SLRRSS).

[0095] In a preferred embodiment, the sequence of the circular fragment is as shown in SEQ ID NO:3.

[0096] In a preferred embodiment, the sequence of the C-terminal extension fragment is one of the following groups:

[0097] NSFRY (SEQ ID NO:25) is extracted from the C-terminal linear region of ANP;

[0098] KVLRRH (SEQ ID NO:26) is extracted from the C-terminal linear region of BNP;

[0099] PSLRDPRPNAPSTSA (SEQ ID NO:5) is a sequence derived from, or formed by shortening the C-terminus of, this sequence by 1 to 9 amino acids.

[0100] The above is the preferred embodiment. The inventors believe that shortening the N-terminus of all these chimeric natriuretic peptides by 1, 2 or 3 amino acids generally results in higher GCA activity, which is still higher than that of the control peptide BNP.

[0101] Extracting C-terminal extensions of different positions and / or lengths from the C-terminal linear region of DNP can affect the functional activity of the chimeric natriuretic peptide. In some embodiments, the C-terminal extension consists of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acid residues from the C-terminal linear region of DNP, starting from the N-terminus. Exemplarily, the C-terminal extension has the amino acid sequence shown at positions 24-28, 24-29, 24-30, 24-31, 24-32, 24-33, 24-34, 24-35, 24-36, 24-37, or 24-38 of the DNP sequence shown in SEQ ID NO:1. In some embodiments, the C-terminal extension fragment consists of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acid residues starting from the C-terminus of the C-terminal linear region of the DNP. Exemplarily, the C-terminal extension fragment has amino acid sequences at positions 34-38, 33-38, 32-38, 31-38, 30-38, 29-38, 28-38, 27-38, 26-38, 25-38, or 24-38 as shown in SEQ ID NO:1. In some preferred embodiments, the C-terminal extension fragment has an amino acid sequence as shown in SEQ ID NO:5 (PSLRDPRPNAPSTSA).

[0102] In a more preferred embodiment, the sequence of the chimeric natriuretic peptide is one of the following:

[0103] ABDNP:SLRRSSCFGRKMDRISSSSGLGCPSLRDPRPNAPSTSA (SEQ ID NO: 18), or a sequence formed by truncating one amino acid at the N-terminus and / or truncating one to nine amino acids at the C-terminus (ABDNP truncated peptide);

[0104] DBANP: EVKYDPCFGRKMDRISSSSGLGCNSFRY (SEQ ID NO: 27), or a sequence formed by truncating one amino acid from the N-terminus of this sequence (DBANP truncated peptide);

[0105] DBBNP: EVKYDPCFGRKMDRISSSSGLGCKVLRRH (SEQ ID NO: 28), or a sequence formed by shortening the N-terminus of this sequence by one amino acid (DBBNP truncated peptide);

[0106] DBDNP: EVKYDPCFGRKMDRISSSSGLGCPSLRDPRPNAPSTSA (DBDNP, SEQ ID NO: 6), or a sequence formed by truncating one amino acid at the N-terminus and / or truncating one to nine amino acids at the C-terminus of this sequence (DBDNP truncated peptide).

[0107] In some embodiments, the chimeric natriuretic peptide is composed of partial fragments from BNP and DNP. In some preferred embodiments, the chimeric natriuretic peptide has an amino acid sequence as shown in SEQ ID NO:6.

[0108] In some embodiments, the chimeric natriuretic peptide is composed of partial fragments from ANP, BNP, and DNP. In some preferred embodiments, the chimeric natriuretic peptide has an amino acid sequence as shown in SEQ ID NO:18.

[0109] Experimental results showed that the cGMP activation capacity of truncated peptides formed by shortening the N-terminus of the above-mentioned chimeric natriuretic peptides by one amino acid was basically the same as that of the full-length peptides. The inventors speculate that truncated peptides formed by shortening the N-terminus by 1 to 3 amino acids should all have acceptable cGMP activation capacity. For chimeric peptides with relatively long C-terminal extensions (ABDNP and DBDNP), the cGMP activation capacity of truncated peptides formed by shortening the C-terminus by 11 amino acids was still superior to the control peptide BNP; the preferred number of amino acids shortened at the C-terminus was 1 to 9.

[0110] In some embodiments, the chimeric natriuretic peptide further includes fragments from other natriuretic peptides, such as CNP and VNP. Exemplarily, the chimeric natriuretic peptide includes at least one fragment of the N-terminal linear region of CNP and the C-terminal linear region of CNP, which may be independently attached to the N-terminus of the N-terminal extension fragment or the C-terminus of the C-terminal extension fragment of the chimeric natriuretic peptide.

[0111] According to another aspect of this specification, a variant of the chimeric natriuretic peptide is also provided. The variant of the chimeric natriuretic peptide includes one or more changes to the amino acid sequence of the chimeric natriuretic peptide, each of these changes being independently selected from amino acid substitutions, deletions, insertions, and truncations.

[0112] The term "variant" refers to a change in the amino acid sequence of a peptide or protein, involving one or more amino acids. Compared to a reference peptide or protein, a variant of the peptide or protein essentially retains the biological activity of the original peptide or protein.

[0113] The term "amino acid substitution" refers to the replacement of one or more amino acids in the amino acid sequence of a peptide or protein with other amino acids. The substituted amino acids can be natural amino acids (e.g., α-amino acids, L-amino acids, or D-amino acids) or non-natural amino acids.

[0114] 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.

[0115] 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.

[0116] The term "amino acid truncation" refers to the removal of one or more amino acids from the N-terminus and / or C-terminus of the amino acid sequence of a peptide or protein.

[0117] In some embodiments, variants of chimeric natriuretic peptides include conserved changes in amino acids. These conserved changes do not affect or alter the fundamental functional activity of the peptide or protein (e.g., the chimeric natriuretic peptide).

[0118] In some embodiments, 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.

[0119] In some embodiments, the deletion of amino acids is preferably performed in regions that do not affect the biological activity of the peptide or protein (e.g., chimeric natriuretic peptides).

[0120] In some embodiments, the insertion of amino acids is preferably performed in a region that does not affect the biological activity of the peptide or protein (e.g., a chimeric natriuretic peptide).

[0121] In some embodiments, the position and length of the truncated amino acid preferably do not affect the bioactivity of the peptide or protein (e.g., chimeric natriuretic peptide).

[0122] In some embodiments, variants of chimeric natriuretic peptides may also include non-conservative changes to amino acids. Non-conservative changes can affect or alter the fundamental functional activity of a peptide or protein (e.g., a chimeric natriuretic peptide). For example, non-conservative changes may include substituting corresponding amino acid residues with physically or functionally dissimilar amino acid residues.

[0123] In some embodiments, the chimeric natriuretic peptide variants are those containing at least one of the following amino acid alterations in the sequence shown in SEQ ID NO:6: i) substitution, deletion, and / or insertion of up to 6, 5, 4, 3, 2, or 1 amino acid residues in the sequence shown in SEQ ID NO:6, that is, the sequence contains a mutation of up to 6, 5, 4, 3, 2, or 1 consecutive or discontinuous amino acid residues, the mutation at each site being independently selected from substitution, deletion, and insertion; ii) truncation of up to 3, 2, or 1 amino acid residues at the N-terminus of the sequence shown in SEQ ID NO:6; and iii) truncation of up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the C-terminus of the sequence shown in SEQ ID NO:6.

[0124] In some embodiments, the chimeric natriuretic peptide variant is a truncated peptide that is truncated by 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues at the C-terminus of the sequence shown in SEQ ID NO:6.

[0125] In some embodiments, the chimeric natriuretic peptide variants are variations in the sequence of the truncated peptide described above that include up to 6, 5, 4, 3, 2, or 1 amino acid changes, each independently selected from the substitution, deletion, and insertion of amino acid residues.

[0126] In some embodiments, the chimeric natriuretic peptide variants are those containing at least one of the following amino acid alterations in the sequence shown in SEQ ID NO:18: i) substitution, deletion, and / or insertion of up to 6, 5, 4, 3, 2, or 1 amino acid residues in the sequence shown in SEQ ID NO:18, that is, the sequence contains a mutation of up to 6, 5, 4, 3, 2, or 1 consecutive or discontinuous amino acid residues, the mutation at each site being independently selected from substitution, deletion, and insertion; ii) truncation of up to 3, 2, or 1 amino acid residues at the N-terminus of the sequence shown in SEQ ID NO:18; and iii) truncation of up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the C-terminus of the sequence shown in SEQ ID NO:18.

[0127] In some embodiments, the chimeric natriuretic peptide variant is a truncated peptide that is truncated by 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues at the C-terminus of the sequence shown in SEQ ID NO:18.

[0128] In some embodiments, the chimeric natriuretic peptide variants are variations in the sequence of the truncated peptide described above that include up to 6, 5, 4, 3, 2, or 1 amino acid changes, each independently selected from the substitution, deletion, and insertion of amino acid residues.

[0129] The chimeric natriuretic peptides and their variants described in this specification have enhanced cGMP activation capacity compared to reference peptides (e.g., BNP), particularly their ability to induce cGMP production via the NPR-A / cGMP pathway. Based on this, the chimeric natriuretic peptides described in this specification can improve one or more downstream physiological functions induced by cGMP activation. The chimeric natriuretic peptides and their variants described in this specification can show improvement in at least one of the following indicators: increased plasma cGMP levels, increased urinary cGMP excretion, increased urine flow, increased urinary sodium excretion, increased renal blood flow, decreased renal perfusion pressure, decreased renal vascular resistance, increased glomerular filtration rate (GFR), decreased proximal sodium reabsorption fraction, decreased distal sodium reabsorption fraction, increased urinary potassium excretion, decreased mean arterial pressure, decreased pulmonary capillary wedge pressure (PCWP), decreased intracardiac pressure, reduced left ventricular hypertrophy, decreased right atrial pressure, decreased ventricular fibrosis, increased ejection fraction, decreased pulmonary artery pressure, decreased systemic vascular resistance, decreased pulmonary vascular resistance, increased hematocrit, decreased plasma cardiac fibroblast proliferation, decreased plasma renin activity, decreased plasma angiotensin II levels, decreased plasma aldosterone levels, and decreased plasma renin activity (PRA).

[0130] The chimeric natriuretic peptides and their variants described in this specification have lower dosage requirements to achieve the desired therapeutic effect compared to reference peptides (such as BNP), while potentially reducing or avoiding some side effects (such as additional side effects caused by the ADA effect).

[0131] Modification of peptides and proteins typically leads to varying degrees of decrease in their activity. The chimeric natriuretic peptides and their variants described in this specification present a relatively lower level of difficulty in developing peptide-modified products compared to reference peptides (e.g., BNP).

[0132] Furthermore, the chimeric natriuretic peptides and their variants described in this specification contain fragments derived from DNP, including N-terminal and C-terminal extensions, which enhance their resistance to neutral endopeptidase degradation. Compared to reference peptides (e.g., BNP), the chimeric natriuretic peptides and their variants described in this specification exhibit enhanced stability and prolonged plasma half-life.

[0133] According to another aspect of this specification, a fusion protein is also provided. The fusion protein includes a first domain and a second domain. The first domain is the aforementioned chimeric natriuretic peptide or a variant thereof; the second domain (also known as a heterologous domain) prolongs the half-life of the fusion protein in vivo.

[0134] Non-limiting examples of the second domain that prolongs the half-life of a fusion protein in vivo include, but are not limited to, immunoglobulin Fc fragments, serum albumin (such as human serum albumin), transferrin, carboxyl-terminal peptide of the β subunit of human chorionic gonadotropin (CTP), VHH, polyglutamic acid (Glu) or polyaspartic acid (Asp) sequences and PAS-modified sequences (conformally disordered polypeptide sequences composed of proline (Pro), alanine (Ala) and serine (Ser)).

[0135] In some embodiments, the second domain includes at least one of the following: Fc fragment, serum albumin, transferrin, CTP, and VHH.

[0136] In some embodiments, the second domain is an Fc fragment, particularly a human Fc fragment. Depending on the immunoglobulin subtype, the human Fc fragment may include human IgG1-Fc, human IgG2-Fc, human IgG3-Fc, and human IgG4-Fc fragments. In some embodiments, the Fc fragment is a human IgG1-Fc fragment, preferably having the amino acid sequence shown in SEQ ID NO:7 (PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK).

[0137] Optionally, mutations in the Fc fragment can enhance affinity for FcRn and / or reduce antibody-dependent cytotoxicity (ADCC). Mutations in the native Fc sequence 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; and / or, iii) improving stability and / or reducing aggregation. In some embodiments, the Fc fragment is a variant of the human IgG1-Fc fragment (SEQ ID NO:7), preferably having the amino acid sequence shown in SEQ ID NO:8 (PKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK).

[0138] In some embodiments, the Fc fragment may comprise the Fc sequence of human IgG1. The variant contains at least one of the following amino acid mutation combinations based on the Fc sequence of human IgG1:

[0139] 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);

[0140] 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).

[0141] 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).

[0142] iv) L235E: The leucine residue at position 235 (Leu) is mutated to glutamate (Glu) (L235E);

[0143] 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).

[0144] 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);

[0145] ⅶ)LS: Methionine at position 428 is mutated to leucine (Leu) (M428L), and asparagine at position 434 is mutated to serine (Ser) (N434S);

[0146] ⅷ) ΔK: Lysine deletion at position 447 (K447del);

[0147] ⅸ) CS: Cysteine ​​at position 220 is mutated to serine Ser (C220S);

[0148] x) DL: Glutamic acid at position 356 is mutated to aspartic acid (E356D), and methionine at position 358 is mutated to leucine (M358L).

[0149] The amino acid residues are numbered according to the EU index, such as in Kabat.

[0150] In some embodiments, the Fc fragment has an amino acid sequence as shown in SEQ ID NO:8.

[0151] In some embodiments, the Fc fragment may comprise the Fc sequence of human IgG4, and the human IgG4 Fc sequence contains at least one of the following amino acid mutation combinations:

[0152] i) Pro: The serine Ser at position 228 is mutated to proline Pro (S228P);

[0153] 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);

[0154] 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).

[0155] 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).

[0156] v) L235E: The leucine residue at position 235 (Leu) is mutated to glutamate (Glu) (L235E);

[0157] 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).

[0158] 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).

[0159] ⅷ)LS: Methionine at position 428 is mutated to leucine (Leu) (M428L), and asparagine at position 434 is mutated to serine (Ser) (N434S);

[0160] ⅸ) ΔK: Lysine deletion at position 447 (K447del).

[0161] In the above mutation combinations, the amino acid residues are numbered according to the EU index, such as in Kabat.

[0162] In some embodiments, the amino acid sequence of the Fc fragment variant hIgG4_Fc-Pro-FALA-LS-ΔK is as follows:

[0163] As an explanation of the experimental part, this invention experimentally verified the feasibility of fusing chimeric natriuretic peptides with a variant of the human IgG1-Fc fragment (IgG1_Fc-CS-AAA-DL-LS, SEQ ID NO:8). The recombinant Fc fusion protein exhibited good cGMP activation ability and possessed the potential to be a candidate drug. The inventors believe that the fusion of chimeric natriuretic peptides with other heterologous peptides / proteins should also have similar potential.

[0164] The domains that make up the fusion protein can be linked in various ways. In some embodiments, the domains of the fusion protein can be linked by peptide bonds. In some embodiments, the domains of the fusion protein can be linked by linkers. In some embodiments, the linker is 1-5 repeating GGGGS sequences (SEQ ID NO:9).

[0165] In this specification, fusion proteins are typically constructed by linking the C-terminus and N-terminus of the domains that make up the fusion protein. Alternatively, fusion proteins can be constructed by linking the C-terminus and C-terminus or the N-terminus and N-terminus of the domains that make up the fusion protein.

[0166] The connection order of the domains that make up the fusion protein can be arbitrary. In some embodiments, the C-terminus of the second domain of the fusion protein is directly or indirectly connected to the N-terminus of the first domain, or the N-terminus of the second domain of the fusion protein is directly or indirectly connected to the C-terminus of the first domain.

[0167] In some embodiments, the N-terminus of the second domain of the fusion protein is directly or indirectly connected to the C-terminus of the first domain.

[0168] In some preferred embodiments, the fusion protein (DBDNP-Linker-Fc) comprises the amino acid sequence shown in SEQ ID NO:10 (EVKYDPCFGRKMDRISSSSGLGCPSLRDPRPNAPSTSAGG GGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK). In some even preferred embodiments, the fusion protein has the amino acid sequence shown in SEQ ID NO:10.

[0169] In some preferred embodiments, the fusion protein (ABDNP-Linker-Fc) comprises the amino acid sequence shown in SEQ ID NO:19 (SLRRSSCFGRKMDRISSSSGLGCPSLRDPRPNAPSTSAGG GGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK). In some even preferred embodiments, the fusion protein has the amino acid sequence shown in SEQ ID NO:19.

[0170] The fusion protein described in this specification, compared to reference peptides (such as chimeric natriuretic peptides and their variants), has a prolonged plasma half-life while maintaining effective biological activity. Compared to reference proteins (such as BNP-Fc fusion proteins), the fusion protein described in this specification has enhanced cGMP activation capacity, particularly the ability to induce cGMP generation via the NPR-A / cGMP pathway.

[0171] 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 the aforementioned chimeric natriuretic peptide, a variant of the chimeric natriuretic peptide, or a fusion protein.

[0172] In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized, which can improve the expression level of the target peptide or protein in the host cell.

[0173] According to another aspect of this specification, a recombinant vector is also provided. This recombinant vector comprises the aforementioned nucleic acid molecules.

[0174] It is understood that the recombinant vectors of this specification may also include functional elements for expressing or suitable for expressing peptides or proteins, such as regulatory sequences, promoters, and enhancers. For example, the recombinant vector may also include 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.

[0175] 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. Exemplarily, recombinant vectors can be selected from adeno-associated virus (AAV) vectors, lentiviral vectors, retroviral vectors, adenovirus vectors, herpesvirus vectors, herpes simplex virus vectors, cytomegalovirus vectors, vaccinia virus vectors, MVA vectors, baculovirus vectors, vesicular stomatitis virus vectors, human papillomavirus vectors, fowlpox virus vectors, Sindbis virus vectors, 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 recombinant vector is AAV.

[0176] According to another aspect of this specification, a host cell is also provided. This host cell contains the aforementioned nucleic acid molecules or recombinant vector.

[0177] The term "host cell" refers to a cell that can introduce foreign genes and maintain their stability.

[0178] 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) cells, 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.

[0179] According to another aspect of this specification, an immunoconjugate is provided. The immunoconjugate comprises: a) the aforementioned chimeric natriuretic peptide, a variant of the chimeric natriuretic peptide, or a fusion protein; and b) other compounds conjugated to a).

[0180] In some embodiments, other compounds may be small molecule compounds, peptides, proteins, antibodies, nucleic acids, or lipids, or any combination thereof. These compounds may each independently have therapeutic, diagnostic, and / or tracing effects.

[0181] Other compounds can be attached to any position of part a) by covalent or non-covalent coupling, such as to the main chain group, side chain group, N-terminus, or C-terminus of a chimeric natriuretic peptide, a variant of a chimeric natriuretic peptide, or a fusion protein.

[0182] According to another aspect of this specification, a pharmaceutical composition is also provided. The active ingredient of the pharmaceutical composition includes the above-described chimeric natriuretic peptide, a variant of the chimeric natriuretic peptide, a fusion protein, a recombinant vector, or an immunoconjugate.

[0183] In some embodiments, the pharmaceutical composition optionally comprises at least one pharmaceutically acceptable carrier. Suitable carriers include, but are not limited to, diluents, fillers, excipients, binders, wetting agents, disintegrants, effervescent agents, surfactants, absorption enhancers, protease inhibitors, lubricants, implants, 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.).

[0184] In some embodiments, the active ingredient of the pharmaceutical composition may further include other therapeutic agents. Exemplary other therapeutic agents may be selected from at least one of the following: therapeutic agents for hypertrophic cardiomyopathy, therapeutic agents for heart failure, therapeutic agents for pulmonary hypertension, therapeutic agents for hypertension, therapeutic agents for renal failure, therapeutic agents for nephrotic syndrome, therapeutic agents for cirrhosis, therapeutic agents for achondroplasia, therapeutic agents for skeletal dysplasia, and therapeutic agents for inflammation, etc.

[0185] The pharmaceutical composition can be administered via various routes. In some embodiments, the pharmaceutical composition is administered via injection, oral administration, inhalation, or transdermal administration. The pharmaceutical composition can be prepared into dosage forms suitable for different routes of administration by methods known in the art, such as tablets, pills, capsules, solutions, emulsions, gels, and powders.

[0186] According to another aspect of this specification, a method for preventing, treating, or alleviating a disease or symptom is also provided. This method comprises administering to an individual an effective amount of the aforementioned chimeric natriuretic peptide, or a variant or fusion protein of the chimeric natriuretic peptide, or a nucleic acid molecule, or a recombinant vector, or a host cell, or an immune conjugate, or a pharmaceutical composition (hereinafter referred to as the active substance of this specification).

[0187] The term "effective amount" refers to the amount by which an individual exhibits a preventive, therapeutic, or alleviating effect on a disease or symptom when the active substance is applied. The term "individual" refers to the object receiving treatment with the active substance. In some embodiments, the individual is a vertebrate, such as a fish, amphibian, reptile, bird, or mammal. In some embodiments, the individual is a mammal, such as a primate, rodent, carnivore, or lagomorph. In some embodiments, the individual is a human.

[0188] In some embodiments, the diseases or conditions that the active substances of this specification can prevent, treat, or alleviate are cardiovascular-related diseases or conditions. Cardiovascular-related diseases or conditions suitable for prevention, treatment, or alleviation with the active substances of this specification include heart failure (acute or chronic), 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, microalbuminuria, heart failure, endothelial dysfunction, restenosis after thrombolytic therapy, arterial and pulmonary hypertension, refractory and intractable hypertension, coronary artery disease, Bronchiolitis obliterans syndrome (BOS), tumor-related and oncological diseases, graft-versus-host disease, sickle cell disease, stable and unstable angina, peripheral and cardiovascular disorders, arrhythmias, atrial fibrillation, atrial flutter, ventricular fibrillation, ventricular flutter, atrial and ventricular premature contractions, AV junction premature contractions, premature ventricular contractions (PVCs), sick sinus syndrome, syncope, atrioventricular nodal reentrant tachycardia, Wolff-Parkinson-White syndrome (WPW), acute coronary syndrome (ACS), autoimmune heart disease, shock, and aneurysms. In some embodiments, the active substance of this specification can prevent, treat, or alleviate one or more of the following cardiovascular-related diseases or conditions: myocardial hypertrophy, heart failure, pulmonary hypertension, and hypertension.

[0189] In some embodiments, the active substances of this specification can prevent, treat, or alleviate kidney-related diseases or conditions. Kidney-related diseases or conditions suitable for prevention, treatment, or alleviation with the active substances of this specification include renal insufficiency, renal failure, kidney injury (e.g., caused by hydral insufficiency, myocardial hypertrophy, or myocardial ischemia), cardiorenal syndrome, diabetic nephropathy, non-diabetic nephropathy, glomerulonephritis, acute glomerulonephritis, glomerulosclerosis, tubulointerstitial disease, pyelonephritis, nephritis, renal cysts, and nephrosclerosis. In some embodiments, the active substances of this specification can prevent, treat, or alleviate kidney-related diseases or conditions such as renal failure and / or nephrotic syndrome.

[0190] In some embodiments, the active substances of this specification can prevent, treat, or alleviate liver-related diseases or conditions. Liver-related diseases or conditions suitable for prevention, treatment, or alleviation with the active substances of this specification include hepatitis, cirrhosis, and liver fibrosis. In some embodiments, the active substances of this specification can prevent, treat, or alleviate cirrhosis.

[0191] In some embodiments, the diseases or conditions that the active substances of this specification can prevent, treat, or alleviate are skeletal-related diseases or conditions. Suitable skeletal-related diseases or conditions for prevention, treatment, or alleviation using the active substances of this specification include achondroplasia, chondrodysplasia, cartilage degeneration, skeletal dysplasia, osteoarthritis, osteogenesis imperfecta, osteomyelitis, osteonecrosis, osteoporosis, and rickets. In some embodiments, the skeletal-related diseases or conditions that the active substances of this specification can prevent, treat, or alleviate are achondroplasia and skeletal dysplasia.

[0192] According to another aspect of this specification, the use of the above-described chimeric natriuretic peptide or its variants or fusion proteins or nucleic acid molecules or recombinant vectors or host cells or immune conjugates or pharmaceutical compositions in the preparation of medicaments for the prevention, treatment or relief of cardiovascular, renal, hepatic or bone-related diseases or conditions is also provided.

[0193] In some embodiments, the disease or condition for which the drug is suitable for prevention, treatment or relief is at least one of myocardial hypertrophy, heart failure, pulmonary hypertension, hypertension, renal failure, nephrotic syndrome, cirrhosis, achondroplasia and skeletal dysplasia.

[0194] The polypeptide sequence of this invention is as follows:

[0195] Table 1

[0196] Note: The amino acid residues shown underlined form disulfide bonds.

[0197] 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.

[0198] Example 1: Peptide Design and Synthesis

[0199] In this embodiment, a series of peptides containing BNP ring structures and other natriuretic peptide fragments were designed using a chimeric approach, and their chemical synthesis was entrusted to Nanjing Genscript Biotech Co., Ltd. HPLC analysis showed that the purity of the synthesized peptides was higher than 95%. The amino acid sequences of some of the synthesized peptides are shown in Table 2.

[0200] Table 2. Peptide sequence information

[0201] Note: Underlined cysteine ​​residues form intrapeptide bridges.

[0202] In addition, control peptides BNP and DNP were chemically synthesized as references:

[0203] BNP:SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH;

[0204] DNP: EVKYDPCFGHKIDRINHVSNLGCPSLRDPRPNAPSTSA.

[0205] Example 2: Activity of the peptide in GCA stable cell lines

[0206] In the constructed stable transfected cell line of human natriuretic peptide receptor, the active substance binds to the human natriuretic peptide receptor, causing an increase in cGMP levels. The in vitro activity of the active substance can be determined by detecting changes in cGMP using a competitive ELISA method.

[0207] Cell sample processing

[0208] HEK293-hGCA 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 100 μL / well in 96-well plates and cultured at 37°C for 19 hours in 5% CO2.

[0209] 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 peptide samples described in Table 2 of Example 1 were diluted with the sample diluent to a suitable initial concentration, followed by 5-fold serial dilutions, for a total of 12 serial dilutions.

[0210] 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.

[0211] Competitive ELISA detection

[0212] Solution preparation: Washing buffer (PBST), 1×PBS + 0.05% Tween-20; Dilution buffer, PBST + 0.5% BSA.

[0213] Coating: Dilute the Anti-cGMP antibody (Invitrogen, catalog number MA5-4455) 1:20000 using 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 with a sealing membrane, and incubate at room temperature for 1 h.

[0214] 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.

[0215] 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.

[0216] Detection: Detection was performed using an ELISA reader: λ = 450 nm. A four-parameter equation was fitted to obtain the EC50 value of the sample.

[0217] Table 3. Activity measured in GCA stable cell lines

[0218] The GCA activity assay results of the candidate peptides are shown in Table 3. The experimental results indicate that, compared with the reference BNP, the EC50 values ​​of candidate peptides ABDNP, DBANP, DBBNP, and DBDNP were decreased, and their GCA activity was significantly increased. These candidate peptides exhibit enhanced cGMP activation capacity, particularly increasing the level of cGMP induced in cells via the GCA / cGMP pathway. It can be inferred that enhanced cGMP activation capacity may reduce the dosage requirements of the candidate peptides and potentially reduce or avoid some side effects (such as additional side effects caused by the ADA effect). Furthermore, enhanced cGMP activation capacity may reduce the difficulty of developing related modified products of the candidate peptides.

[0219] Example 3: Effect of different truncated forms of peptides on their bioactivity

[0220] To investigate the effects of different truncated forms of the candidate peptides (ABDNP, DBDNP) in Example 2 on their biological activity, truncated peptides as shown in Table 4 were designed, chemically synthesized by Nanjing Genscript Biotech Co., Ltd., and their activity in GCA stable cell lines was measured.

[0221] Table 4 Sequence information of truncated peptides

[0222] Note: Underlined cysteine ​​residues form intrapeptide bridges.

[0223] The GCA activity assay results for the candidate peptides and their truncated peptides are shown in Table 5. The results indicate that the GCA activity of candidate peptides ABDNP and DBDNP decreased significantly when the C-terminus was truncated by 12 amino acid residues. When the C-terminus was truncated by 1 to 9 amino acid residues and / or the N-terminus was truncated by 1 amino acid residue, the GCA activity of the truncated peptides remained essentially at the same level as that of the full-length peptides.

[0224] Table 5. Activity measured in GCA stable cell lines

[0225] Example 4: Binding affinity of the peptide to the natriuretic peptide receptor GCA

[0226] When the active substance binds to and dissociates from the natriuretic peptide receptor immobilized on the surface of the BLI biosensor, it causes a change in the thickness of the optical layer at the sensor tip, resulting in a change in the interference spectrum. This embodiment determines the binding affinity between the active substance and the natriuretic peptide receptor by measuring the change in the interference spectrum.

[0227] BLI affinity determination was performed using the Octet R8 system (Sartorius) according to the manufacturer's instructions. Biotin-labeled human GCA (AcroBiosysterms, NP-H82E9) was immobilized onto the SA sensor and reacted thoroughly with serially diluted samples. The immobilized conjugate was then dissociated in PBST buffer, and the dissociation constant (KD) was calculated using Octet analysis software. A smaller KD value indicates a stronger binding affinity between the two substances.

[0228] Table 6. GCA binding affinity determined by BLI method

[0229] The binding affinity of the candidate peptides, determined by the BLI method, is shown in Table 6. The experimental results indicate that the candidate peptides ABDNP and DBDNP in this embodiment have higher binding affinity to GCA than BNP, which was used as a reference.

[0230] Example 5: Activity of the polypeptide in rabbit aortic rings

[0231] In the isolated rabbit aortic ring, the rabbit aortic ring was first stimulated to contract with adrenaline, and then the tension change of the rabbit aortic ring after the action of the active substance was detected to determine the biological activity of the active substance.

[0232] 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.

[0233] After equilibration, the aortic rings were stimulated with phenylephrine (PE, 1 μM) to examine tissue viability. Next, the endothelial integrity of the vascular ring tissue was determined by verifying the responsiveness of PE (1 μM) pre-constricted vessels to acetylcholine (ACh, 1 μM). After elution and equilibration for a period, vasoconstriction was induced with PE (1 μM), and the degree of vasodilation stimulated by the sample was then assessed.

[0234] The experimental results are shown in Figure 11. The candidate peptides ABDNP and DBDNP induced dose-dependent vasodilation of the rabbit thoracic aortic rings that had previously contracted. At low doses, the candidate peptides induced greater vasodilation than BNP.

[0235] Example 6: Resistance of the peptide to neutral endopeptidase NEP

[0236] Neutral endopeptidase (NEP) is involved in the clearance of natriuretic peptides in vivo. In this embodiment, the stability of the active substance in vivo is assessed by detecting the resistance of the active substance to NEP.

[0237] The enzyme digestion buffer was prepared using 100 mM Tris-HCl + 150 mM NaCl, and the pH was adjusted to 7.3. The sample was diluted with water to 1 mg / ml. A 100 μL reaction system was prepared using the enzyme digestion buffer, the diluted sample, and NEP (SinoBiological, catalog number 10805-HNCH), containing 50 μg of peptide sample and 100 ng of NEP. The reaction system was digested at 37 °C. Samples were taken before the reaction and at 10 min, 30 min, 60 min, 120 min, and 180 min after the reaction. The reaction was terminated by heating at 100 °C for 2 min after sampling, and the sample degradation rate was then detected by HPLC.

[0238] The therapeutic application of natriuretic peptides is limited by their in vivo stability. Natriuretic peptides are rapidly cleared in vivo, primarily via NEP and the natriuretic peptide clearance receptor NPRC. Previous studies have reported that DNP exhibits higher resistance to NEP compared to ANP, BNP, and CNP. In this embodiment, DNP and BNP were used as control peptides. Experimental results showed that the candidate peptides ABDNP and DBDNP demonstrated superior NEP resistance compared to DNP and BNP, exhibiting a significant advantage in stability (see Figure 12).

[0239] Example 7: The effect of peptides on inhibiting cardiomyocyte hypertrophy

[0240] In the assay for inhibiting cardiomyocyte hypertrophy, cells were first stimulated with an active substance, and then cardiomyocyte hypertrophy was induced with ISO. The effect of the active substance on inhibiting cardiomyocyte hypertrophy was assessed by measuring cell area.

[0241] Reagent preparation

[0242] Complete culture medium: DMEM + 10% FBS + antibiotics;

[0243] Starvation medium: DMEM + 0.5% FBS + antibiotics;

[0244] Diluent: DMEM + 0.5% FBS.

[0245] Experimental methods

[0246] On day 1, H9C2 cells were digested and resuspended to 2500 cells / ml. The cell suspension was added at 1 ml / well to 12-well cell culture plates and incubated at 37°C with 5% CO2 saturated humidity for 24 h. On day 2, the culture medium in the 12-well plates was replaced with starvation medium and cultured for another 24 h. On day 3, the samples were diluted to 1000 nM, 500 nM, and 100 nM using diluent. The 12-well plates were removed, the starvation medium was discarded, and the cells were randomly assigned to a test group, a blank control group (NSB), and a model group (ISO). In the test group, 1 ml of the diluted sample was added to each well and incubated at 37°C with 5% CO2 saturated humidity for 30 min. ISO was then added to the test group at a final concentration of 20 μM to induce cell hypertrophy. The 12-well plates were then incubated at 37°C with 5% CO2 saturated humidity for approximately 48 h. The blank control group received diluent treatment without ISO-induced cell hypertrophy, while the model group received only ISO-induced cell hypertrophy without any drug administration.

[0247] Remove the 12-well plate, discard the solution, wash the plate twice with PBST, fix the cells with 4% paraformaldehyde for 15 min, wash the plate twice with PBST, permeate with 0.2% Triton X-100 for 5 min, wash again, block with 1% BSA solution for 1 h, wash the plate, add phalloidin to a final concentration of 100 nM for 30 min in the dark, wash the plate, stain with 0.1 μg / ml DAPI for 5 min in the dark, wash the plate, take pictures with an inverted fluorescence microscope and synthesize the images, calculate the cell area using Image Pro Plus 6.0 software, and plot and analyze the data.

[0248] As shown in Figures 13 and 14, the cross-sectional area of ​​cardiomyocytes in the test group was significantly smaller than that in the positive control group, indicating that the candidate peptides ABDNP and DBDNP alleviated or inhibited ISO-induced hypertrophy of mouse cardiomyocytes.

[0249] Example 8: Pharmacokinetics of the peptide in rabbits

[0250] The persistence of the test substance in rabbits was evaluated by measuring changes in cGMP concentration in rabbit plasma through a single intravenous injection.

[0251] New Zealand rabbits were randomly divided into three treatment groups (n=3), and ABDNP, DBDNP, and BNP were administered via marginal ear vein injection at a dose of 30 μg / kg, respectively. Blood samples were collected from the marginal ear vein before administration and at 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours after administration. The whole blood was mixed with the anticoagulant EDTA, and the plasma was collected after centrifugation at 12000×g for 3 minutes over 30 minutes.

[0252] The concentration of cGMP in rabbit plasma was quantitatively detected using LC-MS / MS. The change in cGMP concentration compared to baseline is shown in Figure 15. At the same administered dose, intravenous injection of ABDNP, DBDNP, and the control peptide BNP increased the level of the pharmacodynamic marker cGMP up to 1 hour post-administration. The increase in cGMP induced by ABDNP and DBDNP at 1 hour post-administration was more than three times that of the control peptide BNP. Notably, DBDNP maintained a ΔcGMP level greater than 10 ng / ml even 2 hours post-administration.

[0253] Example 9: Design, Synthesis, and In Vitro Activity Assay of Recombinant Fc Fusion Protein

[0254] 1. Synthesis of the target gene for recombinant protein

[0255] To prepare the recombinant protein of this embodiment (see Table 7), a recombinant gene sequence (see Table 8) was designed based on the amino acid sequence of the recombinant protein and the codon preference of the host cell. This sequence was synthesized by Nanjing Genscript Biotech Co., Ltd., and then inserted into the cloning vector. Specifically, an XbaI restriction endonuclease cleavage 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 the secretory protein, typically 15-30 amino acids in length, responsible for guiding the transport of the nascent protein to the endoplasmic reticulum for further synthesis. This sequence is ultimately cleaved by a signal peptidase. A stop codon and an AgeI restriction endonuclease cleavage site were sequentially added to the 3' end of the coding sequence of the recombinant protein.

[0256] Table 7 Recombinant protein sequence information

[0257] Note: The bold and underlined text is for chimeric natriuretic peptides, the italic text is for Linker, and the regular text is for Fc segments.

[0258] Table 8. Detailed information on recombinant gene sequences.

[0259] 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.

[0260] 2. Construction of recombinant expression vectors

[0261] Using the above cloning vector as a template (dissolved in ddH2O to 100 ng / μL), the target fragment was amplified by PCR. The amplification primers, reaction system, and reaction conditions for the PCR reaction are shown in Tables 4, 5, and 6, respectively.

[0262] Table 9 Amplification Primers

[0263] Table 10 PCR Reaction System

[0264] Table 11 PCR reaction conditions

[0265] PCR products were recovered by 1% agarose gel electrophoresis (100V, 40 min), and the electrophoresis results showed that the product size was consistent with the theoretical size. The recovered PCR products and the expression vector pcDNA3.4 were digested with XbaI and AgeI restriction endonucleases at 37℃ for 15 min. The digestion reaction system is shown in Table 7. After digestion, ligation was performed using T4 ligase at 25℃ for 60 min. The ligation reaction system is shown in Table 8. The recombinant expression vector was obtained after ligation.

[0266] Table 12 Enzyme digestion reaction system

[0267] Note: X represents the concentration of PCR product or pcDNA3.4.

[0268] Table 13 Connection Reaction Conditions

[0269] Note: The concentrations of the PCR product and the enzyme digestion product of pcDNA3.4 are X1 and X2, respectively, based on the concentrations measured after enzyme digestion. Therefore, (Y*X1):(Z*X2)=3:2 is more appropriate.

[0270] 3. Recombinant protein expression and purification

[0271] 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... 6 When the cell density is reduced to 32°C, the culture medium is continued for 14 days to obtain the cell culture solution.

[0272] 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 then adjusted to pH 7.2 ± 0.2 using 1 mol / L Tris buffer. The purified recombinant protein achieved a purity of over 95%.

[0273] 4. Activity of recombinant protein in GCA stable cell lines

[0274] The results of GCA activity assays for the recombinant proteins are shown in the table below. The experimental results indicate that, compared to the reference BNP-Linker-Fc, the EC50 values ​​of DBDNP-Linker-Fc and ABDNP-Linker-Fc were significantly reduced, their GCA activity was significantly increased, and they exhibited enhanced cGMP activation capabilities.

[0275] DBDNP-Linker-Fc and ABDNP-Linker-Fc can prolong plasma half-life while effectively maintaining biological activity, especially maintaining a high cGMP activation capacity.

[0276] Table 14. Activity measured in GCA stable cell lines

[0277] Those skilled in the art should understand that the above embodiments are merely illustrative of the present invention and do not constitute a limitation thereof. Any modifications, equivalent substitutions, and variations made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0278] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0279] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0280] For each patent, patent application, patent application publication, and other material, such as articles, books, specifications, publications, and documents, referenced in this specification, the entire contents of which are incorporated herein by reference. This excludes historical application documents that are inconsistent with or conflict with the content of this specification, as well as documents that limit the broadest scope of the claims in this specification (currently or subsequently appended to this specification). It should be noted that in the event of any inconsistency or conflict between the descriptions, definitions, and / or terminology used in the supplementary materials to this specification and the content of this specification, the descriptions, definitions, and / or terminology used in this specification shall prevail.

[0281] 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; and these 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 chimeric natriuretic peptide or variant thereof, characterized in that, The chimeric natriuretic peptide comprises, or consists of, fragments from the following group from the N-terminus to the C-terminus. composition: i) The N-terminal extension fragment is extracted from the N-terminal straight-chain region of snake natriuretic peptide (DNP) as shown in SEQ ID NO:1 or the N-terminal straight-chain region of atrial natriuretic peptide (ANP) as shown in SEQ ID NO:

16. ii) Circular fragments, formed by truncating the N-terminal and C-terminal linear regions of the brain natriuretic peptide (BNP) sequence as shown in SEQ ID NO:2 while retaining the circular region; and, iii) C-terminal extension fragment, which is extracted from the C-terminal straight chain region of the DNP.

2. The chimeric natriuretic peptide or variant thereof of claim 1, wherein, The cyclic fragment has an amino acid sequence as shown in SEQ ID NO:3, and the cyclic fragment is linked into a ring by a disulfide bond formed between two cysteine ​​residues.

3. The chimeric natriuretic peptide or variant thereof of claim 1, wherein, The N-terminal extension fragment has an amino acid sequence as shown in any of the following: (1) The amino acid sequence as shown in SEQ ID NO:4; (2) An amino acid sequence obtained by truncating one amino acid from the N-terminus of the amino acid sequence shown in SEQ ID NO:4; (3) The amino acid sequence as shown in SEQ ID NO:17; (4) The amino acid sequence obtained by truncating one amino acid at the N-terminus of the amino acid sequence shown in SEQ ID NO:

17.

4. The chimeric natriuretic peptide or variant thereof of claim 1, wherein The C-terminal extension fragment has an amino acid sequence as shown in SEQ ID NO:5, or has an amino acid sequence obtained by truncating one or more amino acids at the C-terminus of any of the above amino acid sequences. The "multiple" can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

5. The chimeric natriuretic peptide or variant thereof of claim 1, wherein The chimeric natriuretic peptide has the amino acid sequence shown in SEQ ID NO:6; variants of the chimeric natriuretic peptide contain at least one of the following amino acid changes in the sequence shown in SEQ ID NO:6: The sequence shown in SEQ ID NO:6 may be substituted, deleted, and / or inserted with up to 6 amino acid residues; The sequence shown in SEQ ID NO:6 is truncated at the N-terminus by up to 3 amino acid residues; and, The sequence shown in SEQ ID NO:6 is truncated by up to 12 amino acid residues at its C-terminus.

6. The chimeric sodium channel peptide or variant thereof of claim 1 of claim 5, wherein, The variants of the chimeric natriuretic peptide have an amino acid sequence as described in any one of SEQ ID NO:21-24 or SEQ ID NO:

33.

7. The chimeric natriuretic peptide or variant thereof of claim 1, wherein The chimeric natriuretic peptide has the amino acid sequence shown in SEQ ID NO:18; variants of the chimeric natriuretic peptide contain at least one of the following amino acid changes in the sequence shown in SEQ ID NO:18: The sequence shown in SEQ ID NO:18 may be substituted, deleted, and / or inserted with up to 6 amino acid residues; The sequence shown in SEQ ID NO:18 is truncated at the N-terminus by up to 3 amino acid residues; and, The sequence shown in SEQ ID NO:18 is truncated by up to 12 amino acid residues at its C-terminus.

8. The chimeric natriuretic peptide or variant thereof according to claim 7, wherein The variants of the chimeric natriuretic peptide have an amino acid sequence as described in any one of SEQ ID NO:29-32.

9. A chimeric natriuretic peptide or variant thereof, characterized in that, The chimeric natriuretic peptide comprises, or consists of, fragments from the following group from the N-terminus to the C-terminus. composition: i) The N-terminal extension fragment is extracted from the N-terminal straight-chain region of snake natriuretic peptide (DNP) as shown in SEQ ID NO:1 or the N-terminal straight-chain region of atrial natriuretic peptide (ANP) as shown in SEQ ID NO:

16. ii) Circular fragments, formed by truncating the N-terminal and C-terminal linear regions of the brain natriuretic peptide (BNP) sequence as shown in SEQ ID NO:2 while retaining the circular region; and, iii) C-terminal extension fragment, which is taken from the C-terminal straight chain region of the ANP or the C-terminal straight chain region of the BNP.

10. The chimeric natriuretic peptide or variant thereof as set forth in claim 9, wherein, The C-terminal extension fragment has an amino acid sequence as shown in SEQ ID NO:25 or SEQ ID NO:26, or has an amino acid sequence obtained by truncating one or more amino acids at the C-terminus of any of the above amino acid sequences. The "multiple" can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

11. The chimeric natriuretic peptide or variant thereof of claim 9, wherein The chimeric natriuretic peptide has the amino acid sequence shown in SEQ ID NO:27; variants of the chimeric natriuretic peptide include at least one of the following amino acid changes in the sequence shown in SEQ ID NO:27: The sequence shown in SEQ ID NO:27 may be substituted, deleted, and / or inserted with up to 6 amino acid residues; The sequence shown in SEQ ID NO:27 is truncated at the N-terminus by up to 3 amino acid residues; and, The sequence shown in SEQ ID NO:27 is truncated by up to 12 amino acid residues at its C-terminus.

12. The chimeric natriuretic peptide or variant thereof of claim 9, wherein The chimeric natriuretic peptide has the amino acid sequence shown in SEQ ID NO:28; variants of the chimeric natriuretic peptide include at least one of the following amino acid changes in the sequence shown in SEQ ID NO:28: The sequence shown in SEQ ID NO:28 may be substituted, deleted, and / or inserted with up to 6 amino acid residues; The sequence shown in SEQ ID NO:28 is truncated at the N-terminus by up to 3 amino acid residues; and, The sequence shown in SEQ ID NO:28 is truncated by up to 12 amino acid residues at its C-terminus.

13. A fusion protein, characterized in that, The fusion protein includes: A first structural domain, wherein the first structural domain is a chimeric natriuretic peptide or a variant thereof as described in any one of claims 1 to 12; and The second domain extends the half-life of the fusion protein in vivo.

14. The fusion protein of claim 13, wherein, The second domain includes at least one of the following: Fc fragment, serum albumin, transferrin, carboxy-terminal peptide of human chorionic gonadotropin β subunit (CTP), and VHH.

15. The fusion protein of claim 14, wherein, The second domain is an Fc fragment or a variant of an Fc fragment, wherein the Fc fragment is selected from the Fc fragments of human IgG1, human IgG2, human IgG3 or human IgG4.

16. The fusion protein of claim 15, wherein, The Fc fragment comprises the Fc sequence of human IgG1; the variant of the Fc sequence of human IgG1 contains at least one of the following combinations of mutations i) to x): i) L234A and L235A; ii) L234A, L235A, and P329G; iii), L234A, L235A, and G237A; ⅳ)L235E; v) M252Y, S254T and T256E; ⅵ) M252Y, S254T, T256E, H433K and N434F; (vii) M428 and LN434S; ⅷ) K447 is missing; ⅸ)C220S; x) E356D and M358L.

17. The fusion protein of claim 15, wherein, The Fc fragment comprises the Fc sequence of human IgG4; the variant of the Fc sequence of human IgG4 contains at least one of the following combinations of mutations i) to ⅸ): i)S228P; ii) F234A and L235A; iii) F234A, L235A, and P329G; iv) L234A, L235A, and G237A; (v)L235E; ⅵ)M252Y, S254T and T256E; (vii) M252Y, S254T, T256E, H433K and N434F; ⅷ) M428L and N434S; ⅸ) K447 is missing.

18. The fusion protein of claim 15, wherein, The variant of the Fc fragment has an amino acid sequence as shown in SEQ ID NO:

8.

19. The fusion protein according to any one of claims 13 to 18, characterized in that, It has an amino acid sequence as shown in SEQ ID NO:10 or SEQ ID NO:

19.

20. A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric natriuretic peptide or a variant thereof as described in any one of claims 1 to 12, or a fusion protein as described in any one of claims 13 to 19.

21. The nucleic acid molecule of claim 20, wherein, The nucleic acid molecule encoding the fusion protein has a nucleotide sequence as shown in SEQ ID NO:12 or SEQ ID NO:

20.

22. A recombinant vector comprising the nucleic acid molecule as described in claim 20 or 21.

23. A host cell comprising a nucleic acid molecule as described in claim 20 or 21 or a recombinant vector as described in claim 22.

24. An immunoconjugate, comprising, a monoclonal antibody of claim 1, and a cytotoxic agent. The immunoconjugate comprises: a) the chimeric natriuretic peptide or a variant thereof as described in any one of claims 1 to 12, or the fusion protein as described in any one of claims 13 to 19; and, b) Other compounds coupled to a).

25. A pharmaceutical composition comprising, The active ingredient of the pharmaceutical composition includes: a chimeric natriuretic peptide or a variant thereof as described in any one of claims 1 to 12, a fusion protein as described in any one of claims 13 to 19, a recombinant vector as described in claim 22, or an immunoconjugate as described in claim 24.

26. The pharmaceutical composition of claim 25, wherein, The active ingredients also include other therapeutic agents.

27. The pharmaceutical composition of claim 25, wherein The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

28. Use of the chimeric natriuretic peptide or variant thereof as described in any one of claims 1 to 12, the fusion protein as described in any one of claims 13 to 19, the nucleic acid molecule as described in claim 20 or 21, the recombinant vector as described in claim 22, the host cell as described in claim 23, the immunoconjugate as described in claim 24, or the pharmaceutical composition as described in any one of claims 25 to 27, wherein the use is for the preparation of a medicament for the prevention, treatment, or relief of cardiovascular, renal, hepatic, or skeletal-related diseases or conditions.

29. The use of claim 28, wherein, The disease or condition is at least one of the following groups: myocardial hypertrophy, heart failure, pulmonary hypertension, hypertension, renal failure, nephrotic syndrome, cirrhosis, achondroplasia, and skeletal dysplasia.

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