CN112500493A

CN112500493A - Recombinant human neuregulin derivative and application thereof

Info

Publication number: CN112500493A
Application number: CN202010955006.XA
Authority: CN
Inventors: 周明东
Original assignee: Zensun Shanghai Science and Technology Ltd
Current assignee: Zensun Shanghai Science and Technology Ltd
Priority date: 2019-09-16
Filing date: 2020-09-11
Publication date: 2021-03-16

Abstract

The invention relates to an application of a recombinant human neuregulin derivative in preparing a medicament for preventing, treating or delaying cardiovascular diseases of mammals. In particular, the present invention relates to a novel recombinant human NRG-FC protein and its use in the treatment of cardiovascular diseases, which has an extended half-life and enhanced biological activity.

Description

Recombinant human neuregulin derivative and application thereof

Technical Field

The invention relates to an application of a recombinant human neuregulin derivative in preparing a medicament for preventing, treating or delaying cardiovascular diseases of mammals. In particular, the present invention relates to a novel recombinant human NRG-Fc protein and its use in the treatment of cardiovascular diseases.

Background

Neuregulin (NRG; heregulin, HRG), also known as Glial Growth Factor (GGF), Neu Differentiation Factor (NDF), are glycoproteins with a molecular weight of about 44KD, which transmit signals between cells and are ligands of the ErbB family of tyrosine kinase receptors. The neuregulin family contains 4 members: NRG1, NRG2, NRG3, NRG4(Falls et al, Exp Cell Res.284:14-30,2003). NRG1 plays an important role in the nervous system, heart and breast, and there is also evidence that NRG1 signaling plays a role in the development and function of several other organ systems and in the pathogenesis of human diseases including schizophrenia and breast cancer. There are many isomers of NRG 1. Studies on genetically mutated mice (knockout mice) have shown that the isoforms differ in the N-terminal region or Epidermal Growth Factor (EGF) -like region, and also differ in their function in vivo. The present invention is based on neuregulin 1 beta (NRG1 beta).

Neuregulin 1 β is a transmembrane protein (Holmes et al, Science 256,1205-1210, 1992). The membrane-external portion is N-terminal, and includes immunoglobulin-like region (Ig-like domain) and EGF-like region (EGF-like domain), and the membrane-internal portion is C-terminal. Under the action of metalloprotease in extracellular matrix, the extracellular part of neuregulin can be cut off by enzyme and is in free state, thus facilitating the binding with ErbB receptor on the surface of surrounding cells and activating corresponding cell signaling.

The ErbB receptor family is also classified into four classes, ErbB1, ErbB2, ErbB3 and ErbB4, which are transmembrane proteins with molecular weights around 180-185 KD. Except ErbB2, they all contain a ligand binding region at the membrane N-terminus; except for ErbB3, they all contain protein tyrosine kinase activity at the C-terminus of the membrane. Wherein ErbB1 is the receptor of epidermal growth factor, and ErbB3 and ErbB4 are the receptors of neuregulin. Among the receptors for neuregulin, only ErbB2 and ErbB4 were expressed in higher amounts in the heart (Yarden et al, Nat Rev Mol Cell Biol,2: 127-.

When neuregulin binds to the extracellular portion of ErbB3 or ErbB4, it causes ErbB3, ErbB4 to form heterodimers with other ErbB receptors (often including ErbB2), or ErbB4 itself to form homodimers, which then leads to phosphorylation of the intracellular portion of the receptor (Yarden et al, Nat Rev Mol Cell Biol,2: 127-. The phosphorylated intramembrane fraction can further bind to various signaling proteins within the cell, thereby activating downstream ERK or AKT signaling pathways, resulting in a series of cellular responses: including stimulating or inhibiting cell proliferation, apoptosis, cell migration, cell differentiation, or cell adhesion.

Neuregulins are particularly important for cardiac development (WO0037095, CN1276381, WO03099300, WO9426298, US6444642, WO9918976, WO0064400, Zhao et al, j.biol.chem.273,10261-10269,1998). In early embryonic development, neuregulin expression is primarily localized to the endocardium and subsequently released to peripheral cardiomyocytes via the paracrine pathway and binds to the extracellular portion of the protein tyrosine kinase receptor ErbB4 on the cell membrane, which in turn forms a heterodimer with ErbB2 in ErbB 4. Formation and activation of the ErbB4/ErbB2 complex is essential for early spongiform trabeculoplasty. Deletion of any of the three protein genes neuregulin, ErbB4 and ErbB2 resulted in embryos lacking trabeculae and dying of the uterus early in development. WO0037095 shows that a certain concentration of neuregulin can continuously activate an ERK signal pathway, promote growth and differentiation of cardiomyocytes, guide reconstruction of sarcomere and cytoskeleton at adhesion of the cardiomyocytes and cells, improve the structure of the cardiomyocytes, and enhance contraction of the cardiomyocytes. WO0037095 and WO003099300 also indicate that neuregulin can be used for the detection, diagnosis and treatment of various cardiovascular diseases.

Some prior art documents relating to the present invention are listed below: WO 0037095; 2. new application of growth factor neuregulin and analogues thereof: CN 1276381; neuredulin based methods and compositions for manipulating cardiac diseases WO 03099300; zhao YY, Sawyer DR, Balia RR, Opel DJ, Han X, Marchionni MA and Kelly RA.Neureegulin proton Survival and Growth of Cardiac Myocytes.J.biol.chem.273,10261-10269 (1998); methods for manipulating muscle diseases and disorders WO 9426298; methods of creating mythogenic format or subvalval or muscle cell mitogens, differentiation or subvalucing a neurogenin, US6444642.7.therapeutic Methods comprising use of a neurogenin, WO 9918976; methods for manipulating a consistent diagnostic heart failure, WO 0064400; holmes WE, Sliwkowski MX, Akita RW, Henzel WJ, Lee J, Park JW, Yansura D, Abadi N, Raab H, Lewis GD, et al.identification of heregulin, specific activator p185erbb2.science 256,1205-1210 (1992); metals DL, Neuregis, functions, Experimental Cell Research,284,14-30(2003), 11, Yarden Y, Sliwkowski X, Untingling the ErbB signaling network Nature Reviews, Molecular Cell Biology, 2127-.

One promising new therapeutic approach involves the administration of neuregulin (hereinafter "neuregulin") to cardiovascular patients. It has been shown that the EGF-like region of NRG1, about 50 to 64 amino acids, is sufficient to bind to and activate these receptors. Previous studies have shown that neuregulin-1 beta (NRG-1 beta) binds directly to ErbB3 and ErbB4 with high affinity. The orphan receptor ErbB2 is capable of forming heterodimers with ErbB3 or ErbB4 and has higher affinity than ErbB3 or ErbB4 homodimers. Results of neurodevelopment studies suggest that the formation of the sympathetic nervous system requires the entire NRG-1 β, ErbB2, and ErbB3 signaling systems. Targeted disruption of NRG-1 β, or ErbB2 or ErbB4 results in embryonic lethality due to defects in cardiac development. Recent studies have also highlighted that NRG-1 β, ErbB2 and ErbB4 have important roles in cardiovascular development and maintenance of normal adult cardiac function. Research has shown that NRG-1 β enhances the organization of the sarcomere of adult cardiomyocytes. Short term administration of a recombinant NRG-1 β EGF-like domain significantly improved or prevented deterioration of myocardial function in three different animal models of heart failure. More importantly, NRG-1 β significantly prolongs the survival of heart failure animals. However, there is still a need to further optimize or improve neuregulin, and thus to find a more effective neuregulin protein, which can be clinically used for preventing, treating or delaying cardiovascular diseases.

Summary of The Invention

The invention relates to an application of a recombinant human neuregulin derivative in preparing a medicament for preventing, treating or delaying cardiovascular diseases of mammals. In particular, the present invention relates to a novel recombinant human NRG-Fc protein and its use in the treatment of cardiovascular diseases. In certain embodiments, the mammal is a human. In certain embodiments, the subject is a human.

In a first aspect of the invention, neuregulin fusion polypeptides are provided. In certain embodiments, the neuregulin fusion polypeptide comprises an EGF domain of a neuregulin. In certain embodiments, the neuregulin fusion polypeptide comprises the EGF domain of the NRG1 β 2 isoform. In certain embodiments, the neuregulin fusion polypeptide comprises the amino acid sequence of SEQ ID NO. 1. In certain embodiments, the neuregulin fusion polypeptide comprises the analog amino acid sequence of SEQ ID NO. 1. In certain embodiments, the neuregulin fusion polypeptide comprises an immunoglobulin Fc amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an immunoglobulin Fc analog amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an immunoglobulin IgG Fc amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an IgG Fc analog amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an IgG1 Fc amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an IgG4 Fc amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an IgG1 Fc analog amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an IgG4 Fc analog amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an IL-2 signal peptide amino acid sequence, and the IL-2 signal peptide amino acid sequence is cleaved during recombinant production of the neuregulin fusion polypeptide for extracellular secretion. In certain embodiments, the neuregulin fusion polypeptide comprises an amino acid sequence of an EGF domain of a neuregulin and an IgG Fc amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises a linker peptide amino acid sequence, and the neuregulin EGF domain portion is fused to the IgG Fc portion via a peptide linker. In certain embodiments, the neuregulin fusion polypeptide comprises the amino acid sequence of SEQ ID NO. 2. In certain embodiments, the neuregulin fusion polypeptide comprises the amino acid sequence of SEQ ID NO. 3.

Certain neuregulin fusion polypeptides contain the following amino acid sequence: ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln (SEQ ID NO:1), i.e., the human NRG-1 amino acid sequence 177-237.

Certain neuregulin fusion polypeptides contain the following amino acid sequence: ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys AspLeu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys (SEQ ID NO: 2).

Certain neuregulin fusion polypeptides contain the following amino acid sequence: ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser (SEQ ID NO:3)

The neuregulin fusion polypeptide can be prepared according to any of the relevant techniques well known in the art. Typical techniques for preparing neuregulin fusion polypeptides are provided herein. In certain embodiments, the neuregulin fusion polypeptide can be recombinant.

In another aspect, the invention provides neuregulin fusion polypeptide related nucleic acids, vectors and host cells. The nucleic acid or the complement thereof encodes neuregulin fusion polypeptide or a fragment thereof. The nucleic acid may be double-stranded or single-stranded DNA or RNA, and can be inserted into a suitable vector for propagation and expression of the neuregulin fusion polypeptide. The modified vector is transferred into a suitable host cell, such as a host cell capable of expressing the recombinant neuregulin fusion polypeptide.

In another aspect of the invention, neuregulin fusion polypeptides are provided for therapeutic and non-therapeutic use. In particular to the application of neuregulin fusion polypeptide in preventing, treating or delaying various heart diseases and disorders. Accordingly, the present invention provides pharmaceutical formulations comprising neuregulin fusion polypeptides and related methods of treatment.

Another aspect of the invention provides a method of treating heart failure in a mammal. In certain embodiments, the method comprises injecting the neuregulin fusion polypeptide into a mammal.

In another aspect of the invention, methods of inducing phosphorylation of an ErbB receptor in a cell are provided. In certain embodiments, the method comprises contacting the cell with a neuregulin fusion polypeptide.

Another aspect of the invention provides for inducing and maintaining activation of the AKT signaling pathway in cardiac cells. In certain embodiments, the method comprises contacting a cardiac cell with a neuregulin fusion polypeptide.

Another aspect of the invention provides for inducing and maintaining activation of the ERK signaling pathway in cardiac cells. In certain embodiments, the method comprises contacting a cardiac cell with a neuregulin fusion polypeptide.

Detailed Description

A. Explaining the meaning

Unless defined otherwise, 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 invention belongs. All patents, applications, published applications and other publications mentioned herein are incorporated herein by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

As used herein, the terms "a" or "an" mean "at least one" or "one or more than one," unless expressly specified otherwise.

As used herein, "EGF-like domain" or "EGF-like domain" refers to a polypeptide fragment encoded by the neuregulin gene that binds to and activates ErbB2, ErbB3, ErbB4, or a heterologous or homodimer thereof, and has structural similarity to EGF receptor binding regions described in the following references: WO 00/64400; holmes et al, Science,256:1205-1210 (1992); U.S. Pat. nos. 5,530,109 and 5,716,930; hijazi et al, int.J.Oncol.,13: 1061-; chang et al, Nature, 387: 509-; carraway et al, Nature, 387:512-516 (1997); higashiyama et al, J.biochem.,122:675-680 (1997); and WO 97/09425, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the EGF-like domain binds to and activates the ErbB2/ErbB4 or ErbB2/ErbB3 heterodimer. In certain embodiments, the EGF-like domain comprises the receptor binding domain amino acids of NRG-1. In certain embodiments, the EGF-like domain refers to amino acids 177-226, 177-237, or 177-240 of NRG-1. In certain embodiments, the EGF-like domain comprises the receptor binding domain amino acids of NRG-2. In certain embodiments, the EGF-like domain comprises the receptor binding domain amino acids of NRG-3. In certain embodiments, the EGF-like domain comprises the receptor binding domain amino acids of NRG-4.

The Fc amino acid sequence used herein can be selected from the heavy chain of human immunoglobulin IgG-1, see Ellison, J.W. et al, nucleic acids research, 10: 4071-4079(1982), or any Fc sequence well known in the art (e.g., other IgG types including, but not limited to, IgG-2, IgG-3 and IgG-4, or other immunoglobulins). It is well known that the Fc portion of an antibody is composed of monomeric polypeptide fragments that can be joined by disulfide bonds or non-covalent bonds to form dimeric or multimeric forms. The number of intramolecular disulfide bonds formed between the monomeric subunits of a native Fc molecule varies from 1 to 4, depending on the type (e.g., IgG, IgA, IgE) or subtype (e.g., IgG-1, IgG-2, IgG-3, IgA-1, IgA-2) involved in the antibody. The term "Fc" as used herein may represent monomeric, dimeric and multimeric forms of an Fc molecule. It should be noted that when the appropriate cysteine residue is present, the Fc monomer will spontaneously form dimers unless specific conditions exist that prevent disulfide bond formation and thus dimer production. Monomer chains typically dimerize by non-covalent interactions even though cysteines that may normally form disulfide bonds in Fc dimers are removed or replaced with other residues. The term "Fc" is used herein to denote any of the following forms: natural monomers, natural dimers (disulfide linkages), modified dimers (disulfide linkages and/or non-covalent linkages), and modified monomers (i.e., derivatives).

Fc analogs, including variants, analogs or derivatives, as used herein, may be constructed, for example, by making various substitutions of residues or sequences.

The Fc analogs include insertion analogs, deletion analogs, substitution analogs and other analog types.

Variant (or analog) polypeptides include insertion variations in which one or more amino acid residues are appended to the Fc amino acid sequence. The insertion site may be either terminal or both terminal of the protein, or may be an internal region of the Fc amino acid sequence. Insertional variants resulting from the addition of residues at either or both termini may include, for example, fusion proteins and proteins that include amino acid tags or labels.

In a deletion variant (or analog) of Fc, one or more amino acid residues in the Fc polypeptide are removed. Deletions may occur at one or both ends of the Fc polypeptide, and one or more residues may also be removed within the Fc amino acid sequence. Thus, deletion variants include all fragments of the Fc polypeptide sequence.

In Fc substitution variants (or analogs), one or more amino acid residues of an Fc polypeptide are removed and substituted with other residues. In one aspect, naturally occurring substitutions are conservative, but non-conservative substitutions are also encompassed by the invention.

For example, to prevent some or all of the disulfide bonds from forming in the Fc sequence, cysteine residues may be removed or substituted with other amino acids. These cysteine residues may each be removed, or one or more of these cysteine residues may be substituted with other amino acids, such as alanine or serine. As another example, the modification may also be by introducing amino acid substitutions (1) to remove the Fc receptor binding site; (2) removing complement (Clq) binding sites; and/or (3) removal of antibody-dependent cell-mediated cytotoxicity (ADCC) sites. These sites are well known in the art and any well known substitution in the Fc range can be used. For example, see molecular immunology, Vol.29, No. 5, 633-639(1992), which is an ADCC site on IgG 1.

Likewise, one or more tyrosine residues may be substituted by a phenylalanine residue. In addition, other amino acid insertion, deletion and/or substitution variants are also contemplated and are included within the scope of the present invention. Conservative amino acid substitutions are generally preferred. Alternatively, the alteration may be in the form of an amino acid, such as a peptidomimetic or a D-amino acid.

The signal peptide, also called leader peptide, is usually a polypeptide fragment with a length of 15-30 amino acids, which is present at the N-terminal of the protein molecule, and makes the protein have the secretion ability through the cell membrane, and after the protein is secreted, the signal sequence is removed.

Signal peptide sequences as used herein include secretory signal peptides commonly used in expression in mammalian cells, insect cells/baculovirus expression systems. Such as melittin, IFN, IL-2 signal peptide, and the like.

The IL-2 signal peptide used herein has the amino acid sequence: met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu Val Thr Asn Ser (SEQ ID NO:4) or an analog thereof. The secretion of the fusion polypeptide is guided by adding the signal peptide sequence, so that the secretion efficiency is improved, the downstream purification process is simplified, and the stability and the activity of the fusion polypeptide are positively influenced.

Linker peptide: linker peptides (linker peptides) are attached to the fusion protein over a sequence between the fused protein segments.

Linker peptides as used herein include two classes: 1. flexible linker, as commonly known as (GGGGS) n (n < ═ 6),2. rigid linker, as commonly known as (EAAAK) n (n < ═ 6) or (XP) n wherein X is preferably alanine, glutamic acid, lysine, and the like.

The linker peptide amino acid sequence used herein is: gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO:5) or an analog thereof.

As used herein, an "effective amount" of an active ingredient to treat a particular disease is an amount sufficient to ameliorate, or in some way reduce, the symptoms associated with the disease. This dose may cure the disease, but is typically used to ameliorate the symptoms of the disease.

As used herein, an "active ingredient" is any substance used to diagnose, cure, alleviate, treat or prevent a disease in a human or other animal, or to enhance physical or mental health.

As used herein, "amelioration" of symptoms of a particular disorder refers to permanent or temporary, sustained or transient relief of symptoms by administration of a particular active agent, which relief can be attributed to or associated with administration of the agent.

As used herein, "treating" or "treatment" refers to any means by which the symptoms of a disorder, condition or disease may be ameliorated or otherwise favorably directed. The effect may be prophylactic, such as completely or partially preventing a disease or a symptom thereof, or therapeutic, such as a partial or complete cure for a disease and/or adverse effects caused by a disease. Treatment also includes any pharmaceutical use of the compositions described herein.

As used herein, "vector (or plasmid)" refers to a discrete component used to introduce heterologous DNA into a cell for expression or replication therein. The selection and use of these vectors is well known to the skilled person. Expression vectors include vectors capable of expressing DNA linked to regulatory sequences, such as promoter regions, capable of effecting the expression of such DNA fragments. Thus, an expression vector refers to a recombinant DNA or RNA component, such as a plasmid, phage, recombinant virus, or other vector, which when introduced into an appropriate host cell results in expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include those which replicate in eukaryotic and/or prokaryotic cells, as well as those which remain episomal or those which integrate into the genome of the host cell.

As used herein, "cardiomyocyte differentiation" refers to a state characterized by a 10% or greater reduction in DNA synthesis, greater than 10% inhibition of DNA synthesis stimulated by other factors, ordered myo-sarcomere binding and cell-cell adhesion, sustained activation of MAP kinase, and p21^Cip1Enhanced expression of (a). For further discussion see WO00/37095, the contents of which are incorporated herein by reference in their entirety.

As used herein, "ejection fraction" or "EF" refers to the proportion of blood pumped from a filled left ventricle with a heartbeat. Can be defined by the following equation: (left ventricular end-diastolic volume-left ventricular end-systolic volume)/left ventricular end-diastolic volume.

As used herein, "fractional contraction" or "FS" refers to the ratio of the change in diameter of the left ventricle in the systolic state to the diastolic state. Can be defined by the following equation: (left ventricular end-diastolic inner diameter-left ventricular end-systolic inner diameter)/left ventricular end-diastolic inner diameter.

As used herein, "cardiovascular disease" is heart failure, myocardial infarction, coronary atherosclerotic heart disease, arrhythmia, myocarditis, valvular heart disease, infectious endocarditis, a cardiac disease, ischemic heart disease, congenital heart disease, etc. These diseases can lead to myocardial damage.

As used herein, "myocardial injury" refers to myocardial injury resulting from a cardiac pathological condition, which often results in a reduction in cardiac function and thus health in humans. The mechanism of myocardial injury is involved. The mechanism of myocardial injury involves multiple pathophysiological changes such as the generation of oxygen free radicals, calcium ion overload, inflammatory reaction caused by infiltration of damaged areas of neutrophils, apoptosis or necrosis of myocardial cells, tissue metabolic disorder caused by energy supply disorder, abnormal conduction of cardiac electric signals, accumulation of cholesterol, formation of atherosclerotic plaques and the like.

As used herein, "heart failure" or "heart failure" refers to cardiac dysfunction in which the heart is unable to pump blood at the rate required by metabolic tissues. Heart failure includes a variety of disease states such as congestive heart failure, myocardial infarction, tachyarrhythmia, familial myocardial hypertrophy, ischemic heart disease, congenital dilated cardiomyopathy, myocarditis, and the like. Heart failure can be caused by a variety of factors, including, but not limited to: ischemic, congenital, rheumatic, or primary form. Chronic cardiac hypertrophy is an obvious disease state that is predictive of congestive heart failure and cardiac arrest.

As used herein, "myocardial infarction" refers to the massive and persistent ischemic plaque necrosis of portions of the myocardium resulting from blockage of coronary arteries or interruption of blood flow.

As used herein, "ordered, enhanced arrangement of sarcomere or sarcomere structure" refers to a state characterized by an ordered arrangement of contractile proteins as demonstrated by immunofluorescence staining for alpha-actinin in cardiomyocytes. The alignment of alpha-actinin in cells can be identified by microscopy and its associated photographic equipment. As used herein, "disorder or irregularity of the sarcomere or sarcomere structure" is the opposite of "ordered, enhanced arrangement of sarcomere or sarcomere structures".

As used herein, "ordered or enhanced arrangement of cytoskeletal structures" refers to a state characterized by ordered arrangement of actin fibers as revealed by phalloidin (phaseolin) staining in cardiomyocytes. The alignment of actin fibers in cells can be identified by a microscope and its associated camera, as exemplified in the present picture. As used herein, "disorder or irregularity of the cytoskeletal structure" refers to the opposite of "ordered, or enhanced, arrangement of the cytoskeletal structure".

As used herein, "protein" is synonymous with "polypeptide" or "peptide" unless the context clearly dictates otherwise.

As used herein, "activity unit" or "1U" refers to the amount of standard product that will cause 50% of the maximum response. In other words, to determine the unit of activity of an active agent, EC50 must be determined. For example, if the EC50 for a product is 0.067. mu.g/ml, then the amount is 1 unit. Further, if 1. mu.g of the product is used, 14.93U (1/0.067) is used. EC50 may be determined by any method known in the art, including the methods used by the inventors in the examples below. The determination of the activity units is important for the quality control of genetically engineered products and clinically used drugs, so that different drugs and/or different batches of products can be quantified with the same standard.

In certain examples, the neuregulin units are determined by measuring neuregulin activity using kinase receptor-activated enzyme-linked immunosorbent assay (KIRA-ELISA), as described in WO03/099300 and Sadick et al, 1996, Analytical Biochemistry, 235: 207-14, the contents of which are hereby incorporated by reference in their entirety.

B. Neuregulin fusion polypeptides

The invention provides neuregulin fusion polypeptide fragments. In certain embodiments, the neuregulin fusion polypeptide comprises an EGF domain of a neuregulin. In certain embodiments, the neuregulin fusion polypeptide comprises the EGF domain of a β 2 isoform of human neuregulin. In certain embodiments, the neuregulin fusion polypeptide comprises the amino acid sequence of SEQ ID NO. 1. In certain embodiments, the neuregulin fusion polypeptide comprises the analog amino acid sequence of SEQ ID NO. 1. In certain embodiments, the neuregulin fusion polypeptide comprises an immunoglobulin Fc amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an immunoglobulin Fc analog amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an immunoglobulin IgG Fc amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an IgG Fc analog amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an IgG1 Fc amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an IgG4 Fc amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an IgG1 Fc analog amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an IgG4 Fc analog amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises an IL 2-signal peptide amino acid sequence, and the IL-2 signal peptide amino acid sequence is cleaved during recombinant production of the neuregulin fusion polypeptide for extracellular secretion. In certain embodiments, the neuregulin fusion polypeptide comprises an amino acid sequence of an EGF domain of a neuregulin and an IgG Fc amino acid sequence. In certain embodiments, the neuregulin fusion polypeptide comprises a linker peptide amino acid sequence, and the neuregulin EGF domain portion is fused to the IgG Fc portion via a peptide linker. In certain embodiments, the neuregulin fusion polypeptide comprises the amino acid sequence of SEQ ID NO. 2. In certain embodiments, the neuregulin fusion polypeptide comprises the amino acid sequence of SEQ ID NO. 3.

In a more preferred embodiment, the neuregulin fusion polypeptide comprises the amino acid sequence of the EGF domain of neuregulin, the amino acid sequence of an immunoglobulin IgG Fc or Fc analog. In a more preferred embodiment, the neuregulin fusion polypeptide comprises an IL-2 signal peptide amino acid sequence, a neuregulin EGF domain amino acid sequence, an immunoglobulin IgG Fc or Fc analog amino acid sequence, and the neuregulin EGF domain portion is fused to the IgG Fc portion via a peptide linker, and the IL-2 signal peptide amino acid sequence is cleaved during recombinant production of the neuregulin fusion polypeptide for secretion to the extracellular space. In a more preferred embodiment, the neuregulin fusion polypeptide comprises the neuregulin amino acid sequence of SEQ ID NO. 1, an IgG1 or IgG4 subtype Fc amino acid sequence, and the neuregulin moiety is fused to the IgG Fc moiety via a peptide linker. In a more preferred embodiment, the neuregulin fusion polypeptide is the amino acid sequence of SEQ ID NO. 2. In a more preferred embodiment, the neuregulin fusion polypeptide is the amino acid sequence of SEQ ID NO. 3. In certain embodiments, the present invention provides a method of treating heart failure by administering an effective amount of neuregulin fusion polypeptide.

The neuregulin fusion polypeptide may be administered in the form of a pharmaceutical formulation.

The mode of administration of neuregulin fusion polypeptides will be determined by one skilled in the art and includes, but is not limited to, oral, intravenous, intragastric, rectal, intraperitoneal or intraventricular administration.

In a preferred embodiment, the composition for administration is a pharmaceutical formulation. The pharmaceutical formulation can be a composition comprising a prophylactic or therapeutic amount of one or more prophylactic or therapeutic agents (e.g., a complex comprising neuregulin fusion polypeptide and other prophylactic or therapeutic agents), and a pharmaceutically acceptable carrier or excipient. In one embodiment and as used herein, "pharmaceutically acceptable" means that the compound is one that has been approved by the relevant national authorities or is otherwise documented as being useful in the art of pharmaceutical use in animals, particularly humans. "carrier" refers to a diluent, adjuvant (e.g., Freund's complete adjuvant and incomplete adjuvant), excipient, or other carrier that aids in the administration of the therapeutic agent. The pharmaceutical carrier can be a sterile liquid such as water and oils, including petroleum, animal, vegetable, or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The most preferred carrier for intravenous injection of pharmaceutical preparations is water. In preparing injectable formulations, saline, dextrose and glycerol liquids may be employed. Examples of suitable Pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, written by E.W. Martin.

Typical pharmaceutical formulations and dosage forms contain one or more excipients. Suitable excipients are well known to those skilled in the pharmaceutical arts and include, but are not limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, mica, sodium chloride, powdered skim milk, propylene, glycol, water, alcohol, and the like. Whether a certain excipient is suitable for incorporation into a pharmaceutical formulation or dosage form depends on many factors well known in the art, including, but not limited to, the manner in which the dosage form is administered to a patient and the particular active ingredient in the dosage form. If desired, the formulation or unitary dosage form may contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents.

Pharmaceutical formulations contain excipients well known in the art or published on, for example, the United States Pharmacopeia (USP) SP (XXI)/NF (XVI). Generally, lactose-free formulations contain an active ingredient, a binder/filler, and a pharmaceutically compatible and dose-acceptable lubricant. A typical lactose-free dosage form contains the active ingredient, microcrystalline cellulose, pregelatinized starch, and magnesium stearate.

Pharmaceutical formulations and dosage forms of the invention contain one or more compounds that reduce the rate of decomposition of the active ingredient. The compound is referred to herein as a "stabilizer" and includes, but is not limited to, antioxidants such as ascorbic acid, pH buffers or salt buffers.

The pharmaceutical formulations and single dosage forms may take the form of: solutions, suspensions, emulsions, tablets, capsules, powders, sustained release forms, and the like. Oral formulations contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. The pharmaceutical agents and dosage forms contain a prophylactic or therapeutic amount of a purified prophylactically or therapeutically effective agent which is admixed with an amount of a carrier to be shaped for better administration to a patient. The dosage form should be adapted to the mode of administration. In a preferred embodiment, the pharmaceutical formulation or single dosage form should be sterile and administered in a suitable form, preferably the subject is an animal, more preferably the subject is a mammal, and most preferably the subject is a human.

The form of the pharmaceutical preparation containing neuregulin fusion polypeptide should be adapted to the mode of administration. Modes of administration include, but are not limited to, injection (such as intravenous, intramuscular, subcutaneous or intradermal), oral, buccal (such as sublingual), inhalation, intranasal, transdermal, topical, transmucosal, intratumoral, intrasynovial and rectal administration. In a particular embodiment, the formulation may be prepared by reference to a conventional procedure, such as that used to prepare pharmaceutical formulations for intravenous or subcutaneous or intramuscular administration, oral, intranasal or topical administration to humans. In a certain embodiment, the pharmaceutical formulation is in a form consistent with conventional modes of administration by subcutaneous injection. Typically, formulations for intravenous administration are sterile isotonic solutions. If desired, the formulation can also contain a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the site of injection.

Dosage forms include, but are not limited to, the following forms: tablets, caplets, capsules such as soft elastic gelatin capsules, cachets, tablets, lozenges, dispersions, suppositories, ointments, poultices (poultices), pastes, powders, dressings, emulsions, plasters, solutions, drug patches, aerosols (e.g., nasal sprays or inhalers), colloids; liquid dosage forms are suitable for oral or mucosal administration to a patient, and include suspensions (such as aqueous or non-aqueous suspensions, oil-in-water emulsions, or water-in-oil emulsions), solutions, and all-purpose drugs; the liquid dosage form is suitable for patients taking injection; sterile solids (e.g., crystalline or amorphous) can be reconstituted into liquid dosage forms suitable for administration to patients for injection.

Depending on the application, the formulation, shape and type of formulation of neuregulin fusion polypeptides will vary. For example, a dosage form for acute treatment of a disorder may contain more neuregulin fusion polypeptide than a dosage form for chronic treatment of the same disease. Similarly, the dosage forms for different cancers have different therapeutic effects. Similarly, injectable dosage forms contain smaller amounts of the active ingredient than oral dosage forms that treat the same disease or disorder. It is well known to those skilled in the art that the above formulation and other specific dosage forms included in the present invention are different. See Remington pharmacy, 18 th edition, Mack Press, Iston, Pa.1990.

Administration of neuregulin fusion polypeptides can be carried out by any route, including but not limited to, according to the judgment of one of skill in the art: oral, intravenous, intragastric, duodenal, intraperitoneal or intraventricular injection.

C. Dosage and route of administration

The amount of neuregulin fusion polypeptide used in the present invention will vary with the nature and severity of the disease or condition, and the route of administration of the active ingredient. The frequency and dosage of administration will also vary with the particular factors of each patient, depending on the particular treatment (e.g., therapeutic or prophylactic agent), disorder, disease, or severity of discomfort, route of administration, and age, weight, response, and prior medical history of the patient. Effective doses can be extrapolated from dose-response curves obtained from in vitro or animal model test systems.

Exemplary dosages for neuregulin fusion polypeptides include the amount of milligram or microgram of neuregulin per kilogram of body weight of the subject (e.g., about 1 microgram per kilogram of body weight to about 500 milligrams per kilogram of body weight, about 100 micrograms per kilogram of body weight to about 5 milligrams per kilogram of body weight, or about 1 microgram per kilogram of body weight to about 50 micrograms per kilogram of body weight). For example, the amount of active peptide administered to a patient will typically be in the range of 0.001mg/kg to 15mg/kg per kg of body weight of the patient. Suitable amounts are also: 0.001mg/kg-15mg/kg, 0.005mg/kg-10mg/kg, 0.01mg/kg-5mg/kg, 0.001mg/kg-4mg/kg, 0.005mg/kg-3mg/kg, 0.01mg/kg-2mg/kg, 0.001mg/kg-1mg/kg, 0.005mg/kg-0.5mg/kg, 0.010mg/kg-0.2mg/kg, 0.005mg/kg-0.050 mg/kg.

Exemplary dosages for neuregulin fusion polypeptides also include how many units (U) or unit amounts of neuregulin per kilogram body weight is administered to a subject (e.g., about 1U per kilogram body weight to about 5,000U per kilogram body weight, about 10U per kilogram body weight to about 1,000U per kilogram body weight, or about 100U per kilogram body weight to about 500U per kilogram body weight). As the dose to be administered to a patient, the unit of active peptide used per kg of body weight of the patient is typically from 10U/kg to 1,000U/kg. Suitable amounts are also: 1U/kg-10,000U/kg, 1U/kg-5,000U/kg, 10U/kg-1,000U/kg, 50U/kg-2,000U/kg, 50U/kg-1,000U/kg, 50U/kg-500U/kg, 100U/kg-1,000U/kg, 100U/kg-500U/kg, 100U/kg-200U/kg.

In general, for the various diseases described herein, the recommended daily dosage range of neuregulin fusion protein polypeptides in the methods of the invention is (calculated as the neuregulin-containing content): about 0.001mg to 1000mg per day. In particular instances, the total amount administered per day may range from: 0.001mg-15mg, 0.005mg-10mg, 0.01mg-5mg, 0.001mg-4mg, 0.005mg-3mg, 0.01mg-2mg, 0.001mg-1mg, 0.005mg-0.5mg, 0.010mg-0.2 mg. When a patient is scheduled for treatment, a low dose, such as about 0.1 μ g to about 1 μ g per day, may be used initially, and if necessary increased to about 20 μ g to about 1,000 μ g per day, either in single or divided doses, depending on the patient's overall response. In some cases, it may be necessary to use dosages of the active ingredient which are outside the ranges set forth herein, as will be apparent to those of ordinary skill in the art. Furthermore, it should be noted that the clinician or treating physician should know how and when to interrupt, adjust or terminate therapy depending on the individual patient response. In some embodiments, neuregulin is used in an amount of about 1U/day to about 10,000U/day. In some embodiments, neuregulin is used in an amount of about 1U/day to about 5,000U/day. In some embodiments, neuregulin is used in an amount of about 10U/day to about 2,000U/day. In some embodiments, neuregulin is used in an amount of about 10U/day to about 1,000U/day. In some embodiments, neuregulin is used in an amount of about 100U/day to about 200U/day.

Neuregulin fusion polypeptides can also be administered via a dosage schedule or "treatment cycle". The daily dosage for the treatment cycle is detailed above. The treatment cycle may last for 2 days, 5 days, 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks.

In some particular cases, the neuregulin fusion polypeptide is administered daily during the treatment cycle. In certain embodiments, the neuregulin fusion polypeptide is administered for a duration of 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days within a treatment cycle. In some embodiments, neuregulin fusion polypeptides are administered on the first day of a treatment cycle, and neuregulin is not administered for the remaining one or more days of the treatment cycle. In certain embodiments, the neuregulin fusion polypeptide is administered daily for 3, 5,7, or 10 days during a treatment cycle, and is not administered for the remainder of the cycle. Each administration of neuregulin fusion polypeptide is separated by a time interval of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, any number of days between 2 weeks and 6 weeks during a treatment cycle. .

Description of the drawings:

FIG. 1: schematic representation of expression vectors

FIG. 2: schematic representation of NRG-IgG1/IgG4-Fc fusion protein

FIG. 3: expression SDS-PAGE/Western blot detection result in IFN/IL2-eGFP 293F

FIG. 4: expression SDS-PAGE/Western blot detection result in IL2-NRG-IgG1-Fc 293F

FIG. 5: SDS-PAGE detection result after NRG-IgG1-Fc fusion polypeptide (reduction state) purification

FIG. 6: rat heart super-junction fruit for heart failure models of groups before and after administration

Examples

Example 1 construction of vectors expressing fusion Polypeptides

The full-length DNA sequence comprising NRG of human origin (SEQ ID NO:1), Linker and Fc fragment of human immunoglobulin (IgG1 or IgG4) was subcloned into pcDNA3.1(+) vector. At the 5 'end, a sequence containing the restriction enzyme Hind III site, Kozak sequence, and melittin signal peptide was introduced, and at the 3' end, a sequence containing the EcoRI site was introduced. After correct sequencing, the plasmid was amplified and stored by transformation into e.coli with CaCl 2.

DNA sequences comprising IL-2-eGFP and IFN-eGFPSubcloning into pcDNA3.1(+) vector. At the 5 'end, a sequence containing the restriction enzyme Hind III site, Kozak sequence, and at the 3' end, a sequence containing the EcoRI site were introduced. After correct sequencing, the DNA was sequenced through CaCl₂Coli and the plasmid was amplified and stored.

The full-length DNA sequence comprising NRG of human origin (SEQ ID NO:1), Linker and Fc fragment of human immunoglobulin (IgG1 or IgG4) was subcloned into pcDNA3.1(+) vector. At the 5 'end, a sequence containing a restriction enzyme Hind III site, a Kozak sequence, and an IL-2 signal peptide or IFN signal peptide was introduced, and at the 3' end, a sequence containing an EcoRI site was introduced. After correct sequencing, the DNA was sequenced through CaCl₂Coli and the plasmid was amplified and stored.

As an example, the above full-length DNA sequence containing NRG, linker peptide and Fc fragment of human immunoglobulin (IgG1 or IgG4) has the corresponding amino acid sequences of SEQ ID NO:2 and SEQ ID NO: 3. The construction vector is shown in FIG. 1, and the NRG-IgG1/IgG4-Fc fusion protein is shown in FIG. 2.

EXAMPLE 2 expression and detection of proteins

The correct plasmid was sequenced and transiently transfected into 293F cells. Preparation before transfection: 293F cells in logarithmic growth phase, activity > 95%, inoculated into fresh and 1% cyan/streptomycin mixture added SMM 293-TII medium, adjusted to density 1.2-1.5 x 10⁶cells/ml, 24h incubation. On the day of transfection, cell viability is required>Adjusting cell density to 2.0-2.5 × 10 by 90%⁶cells/mL, volume 20 mL.

melittin-NRG-IgG 1/IgG4-Fc and IFN-eGFP/IL-2-eGFP vectors: taking a 30ug plasmid reference

(100uL, Sino Biological Co.) transfection reagent transfection method for transfection operation. The feed was fed once 24h after transfection and every 48h thereafter. 37 ℃ and 8% CO₂Performing orbital shaking culture at the rotation speed of 120rpm, and collecting samples every 24 h. And after collecting the sample, carrying out SGS-PAGE and Western blot detection on the expression of the target protein in the supernatant and the cell lysate. In the melittin-NRG-IgG 1/IgG4-Fc expression system, neither cell lysate nor supernatantThe detection of the target protein indicated that the NRG-IgG1/IgG4-Fc fusion polypeptide was not expressed in 293F cells using melittin as the signal peptide.

And IFN/IL-2 is used as a signal peptide, the eGFP fusion polypeptide obtains higher expression in 293F cells, and the eGFP extracellular secretion efficiency IL2 signal peptide is obviously superior to IFN (shown in figure 3).

IL-2-NRG-IgG1/IgG4-Fc vector transfection: plasmid DNA (30ug and 45ug gradients) was added to a final volume of 0.5ml with 150mM NaCl and mixed, and allowed to stand for 5-10 min. PEI solution with molecular weight of 40K is mixed with 150mM NaCl solution to the final volume of 0.5ml, and the mixture is stood for 5-10 min. And mixing the PEI after standing with the DNA, and incubating for 20-30min at room temperature to form a DNA-PEI complex. And dropwise adding the transfection solution into the cell culture solution, slightly shaking the culture flask while dropwise adding, and after shaking uniformly, putting the culture flask back to the shaking table for continuous culture. The feed was fed once 24h after transfection and every 48h thereafter. 37 ℃ and 8% CO₂And (5) carrying out orbital shaking culture, and sampling every 24h to detect the cell transfection efficiency. Depending on the expression characteristics of the different proteins, the samples can be collected up to 6-10 days after transfection. The supernatant was harvested by centrifugation and stored at-20 ℃.

Samples with different DNA/PEI mass ratios and different time points are respectively taken, expression conditions are detected through SDS-PAGE and Western-blot (the result is shown in figure 4), and optimal transfection and sample collection conditions are determined.

EXAMPLE 3 purification of fusion proteins

An equal volume of binding buffer (0.02M disodium phosphate, pH 7.0) was added to the collected supernatant to adjust the pH to 7.0. The sample may also be pretreated by a dialysis system or desalting column. Next, the supernatant was passed through a 1um filter head and then through a 0.45um filter membrane, and then through an affinity column. Samples at the OD280 peak were collected separately and examined by SDS-PAGE (see FIG. 5).

EXAMPLE 4 binding of receptors to neuregulin polypeptides

MCF-7 cells were collected, counted, centrifuged and resuspended in DMEM (10% serum, 9. mu.g/ml insulin) at a cell density of 5X 10⁴And/ml. The plates were plated in 96-well plates and 100. mu.l of the suspension was added to each well overnight at 37 ℃. The cells were washed three times with PBS the next day, and the cell was changed with PBSSerum DMEM was cultured for 24 hours.

With coating buffer (50mM Na)₂CO₃-NaHCO₃pH9.6) diluted ErbB2 antibody H4 to 4. mu.g/ml and added to a 96-well plate at 50. mu.l per well. The antibody was allowed to bind to the plate overnight at 4 ℃.

The culture solution of DMEM in MCF-7 cells was aspirated, and NRG-IgG1-Fc were serially diluted with DMEM and added to the wells at 100. mu.l per well. Here, NRG is a neuregulin polypeptide having the recombinant amino acid sequence of SEQ ID NO. 1 and NRG-IgG1-Fc is a neuregulin fusion protein having the recombinant amino acid sequence of SEQ ID NO. 2. The blank was DMEM only. After incubation at 37 ℃ for 20 minutes, washed once with PBS buffer, 100. mu.l/well lysis buffer (50mM Hepes, pH8.0, 150mM NaCl, 2mM sodium orthovanadate, 0.01% Thimersaxon, 1% Triton X-100 and 1 protease inhibitor cocktail/25 ml) was added, lysed at 4 ℃ for 30 minutes, and the plates were gently shaken to detach the cells from the plates and centrifuged at 15,000rpm for 15 minutes.

The antibody-coated plate was washed 5 times with a wash solution (10mM PBS, pH7.4, 0.05% Tween 20), 200. mu.l of 5% skim milk wash solution was added to each well, incubated at 37 ℃ for 2 hours, and washed 3 times with the wash solution.

The lysed cell sap was added to the corresponding coated plate in an amount of 90. mu.l per well, incubated at 37 ℃ for 1 hour, then washed 5 times with a washing solution, added with 100. mu.l of horseradish catalase (HRP) -linked phosphotyrosine antibody at an appropriate concentration, and incubated at 37 ℃ for 1 hour. The wash was washed 5 times and fresh prepared HRP substrate solution (50mM citric acid, 100mM Na) was added₂HPO₄0.2mg/ml of Tetramethylbenzidine (TMB), 0.003% H, pH5.0₂O₂) Co-incubation was performed at 37 ℃ for 10 minutes. Finally 50. mu.l of 1M H was added to each well₂SO₄HRP activity was destroyed to stop the reaction. The OD of 450nm per well was measured on a microplate reader, and EC50 was the concentration of neuregulin or fusion protein that reached half the maximum absorbance. The lower the EC50 value, the higher the affinity of the receptor for neuregulin or fusion proteins.

The EC50 values of NRG, NRG-IgG1-Fc and NRG-IgG4-Fc are shown in Table 1 and Table 2, and the EC50 values of NRG-IgG1-Fc and NRG-IgG4-Fc are slightly higher than the EC50 value of NRG.

TABLE 1 EC50 values for NRG and NRG-IgG1-Fc

Sample (I)	EC₅₀(nM)
		NRG	0.9245
NRG-IgG1-Fc	2.483

TABLE 2 EC50 values for NRG and NRG-IgG4-Fc

Sample (I)	EC₅₀(nM)
		NRG	1.651
NRG-IgG4-Fc	3.659

Example 5Elisa method to determine the half-life of intravenous or subcutaneous NRG-IgG1-Fc and NRG-IgG4-Fc fusion polypeptides in rats

Rats were injected either as 250ug/kg NRG-IgG1-Fc tail vein injection or 500ug/kg NRG-IgG1-Fc subcutaneous injection or 250ug/kg NRG-IgG4-Fc tail vein injection, and were bled from the jugular vein at different time points after administration, respectively. Standing at room temperature for at least 30min, centrifuging after blood coagulation, and separating supernatant. Rat serum containing NRG-IgG1-Fc was diluted 1:1 with dilution buffer for use.

NRG-IgG1-Fc standard samples were prepared from rat serum at concentrations ranging from 5000ng/ml, 2500ng/ml, 1000ng/ml, 200ng/ml, 40ng/ml, 8ng/ml, 1.6ng/ml, 0.32ng/ml, 0.064ng/ml and 0ng/ml, and then diluted 1:1 with dilution buffer.

Plate coating and sealing: human NRG1/HRG 1-. beta.1 EGF domain antibody was diluted with coating buffer and 50uL was added to each reaction well in a 96-well plate and coated overnight at 4 ℃. The next day the coating buffer was discarded, the plates were washed, blocking buffer was added and blocked at room temperature. After the plate is drained on absorbent paper, 50uL of the corresponding standard sample or sample to be tested is added into each reaction hole, and the plate is incubated for 2 hours at room temperature. Plates were washed, Anti-Human IgG1 Fc (HRP) antibody was added and incubated for 1 hour at room temperature. Washing the plate, adding the TMB substrate solution prepared temporarily into each reaction hole, reacting for 20 minutes at 37 ℃ in a dark place, and adding 50uL of 1M sulfuric acid to terminate the reaction. The absorbance of each reaction well at 450nM was measured and the neuregulin and protein content of the sample was calculated from the standard curve. Data were analyzed using GraphPad Prism 5.0 software.

The results are shown in tables 3, 4 and 5, respectively:

table 3: half-life results for intravenous injection of NRG-IgG1-Fc

Table 4: half-life results of NRG-IgG1-Fc subcutaneous injection

Parameter(s)	Unit of	Mean value of
			AUC_(0-t)	ng/ml*h	6330.97
T_(1/2)	h	13.45
			CLz/F	L/h/kg	0.08
C_max	ng/ml	282.33

Table 5: half-life results for intravenous injection of NRG-IgG4-Fc

Parameter(s)	Unit of	Mean value of
			AUC_(0-t)	ng/ml*h	38034.83
T_(1/2)	h	8.48
			CLz/F	L/h/kg	0.01
C_max	ng/ml	9816.47

Experimental data show that compared with the intravenous half-life of an NRG rat of 10min and the subcutaneous half-life of 1.5h, the fusion polypeptides NRG-IgG1-Fc and NRG-IgG4-Fc can remarkably prolong the in vivo intravenous and subcutaneous half-life of the NRG fragment in the rat.

EXAMPLE 6 intravenous injection of NRG-IgG1-Fc fusion polypeptide for treatment of Heart failure in rats pharmacodynamic experiment

6.1 purpose of the experiment

On a rat model with heart failure caused by left coronary artery ligation, the therapeutic effects of the four administration methods on the rat heart failure model were compared by administering recombinant human neuregulin (rhNRG) by continuous intravenous drip with NRG-IgG1-Fc fusion protein and insulin infusion pump of Meidunli, which were injected intravenously once a day.

6.2 Experimental materials

6.2.1 Experimental animals

Line 6.2.1.1, source: wistar rat, supplied by Shanghai Sphall-Bikay laboratory animals Ltd

6.2.1.2 gender, weight and quantity: male, 200-

6.2.2 reagent drugs

6.2.2.1 excipients: the Shanghai Zesheng science and technology development company, the dosage form is: freeze-dried powder, specification: 2 mgAlb/bottle,

6.2.2.2 recombinant human Neuregulin (NRG) product: the Shanghai Zesheng science and technology development company, the dosage form is: freeze-dried powder, specification: 250 μ g/arm 6.2.2.3 recombinant human neuregulin-IgG 1-Fc fusion protein: the Shanghai Zesheng science and technology development company, the dosage form is: injection solution

6.2.2.4 Isoflurane: riwode Life technologies Ltd

6.3 Experimental Equipment and Equipment

6.3.1 anesthesia machine (isoflurane evaporator): MSS INTERNATIONAL LTD

6.3.2 cardiac ultrasound detector: vivid E95, probe model: 12S-D

6.3.3 insulin Pump: meidunli, type: MMT-712EWS, MMT-722NAS/L

6.3.4Power Lab multichannel physiological recorder: edd instruments (shanghai) ltd, model: ML-845

6.4 Experimental methods

6.4.1 establishment of Heart failure model caused by coronary artery ligation in rats

Rats were anesthetized with isoflurane at 4% concentration by gas anesthesia, fixed lying on their back after anesthesia, and sterilized with 75% alcohol after chest unhairing. After the left anterior skin of the chest is incised, the chest muscles are separated bluntly, the 4 th rib and the 5 th rib are exposed, the intercostal muscles of the 4 th rib and the 5 th rib are separated bluntly by hemostatic forceps, the heart is extruded from the chest by matching with the two hands for extrusion, the heart is fully exposed, the pulmonary inflation and heartbeat conditions are observed, the left auricle and the pulmonary artery cone are fully exposed, and the anterior descending branch of the left coronary artery is ligated by 6-0 operation suture between the left auricle and the pulmonary artery cone. And then forcefully squeezing the chest to exhaust air, suturing the muscles and the skin of the chest, feeding the rat in a cage after an operation, and closely observing the condition of the rat, if the occurrence of acute arrhythmia is found, massaging the heart for 3-5 minutes in an emergency. After the operation, each rat was injected intramuscularly with 8 muf of penicillin sodium for 2 consecutive days.

6.4.2 Experimental groups and dosing

6.4.2.1 Experimental grouping

The experimental groups are shown in Table 6. The heart function of the rats is detected by using a B ultrasonic machine Vivid E95 respectively 2 weeks and 4 weeks after operation, and the rats with the EF value ranging from 28% to 45% are selected for next experiment after 4 weeks of heart ultrasonic detection.

TABLE 6 grouping of Experimental animals and administration schedule

Rats were randomly divided into 4 groups based on the heart super fruits.

The intravenous injection is divided into an excipient group, an NRG-IgG1-Fc 30 mu g/kg qid group and an NRG-IgG1-Fc 6 mu g/kg qid group, and the intravenous injection is injected every day according to the table I, the continuous administration is carried out for 10 days, the administration volume is 2 ml/kg/time, and the administration concentration is 5 mu g/ml and 15 mu g/ml respectively.

NRG tail vein group is continuously administrated for 8 hours in tail vein by using insulin pump every day for 10 days continuously, the administration volume is 5ml/kg, the NRG administration dose is 6 mug/kg, and the administration concentration is 1.2 mug/ml

In the sham operation group, the coronary artery was not ligated by threading, and no drug administration was performed.

6.4.2.2 dispensing methods

1) Excipient: adding 1ml of normal saline into each bottle of 2 mgAlb/bottle to prepare mother liquor, adding 49.76ml of normal saline into 0.24ml of mother liquor to dilute into 9.6 mu g/ml Alb solution

2) NRG-IgG1-Fc, taking NRG-IgG1-Fc mother liquor with the concentration of 0.4mg-0.8mg/ml, and diluting the NRG-IgG1-Fc mother liquor with physiological saline to a specific use concentration

3) NRG: adding 1ml of normal saline into each 250 mu g NRG bottle to prepare a mother solution, adding 49.76ml of normal saline into 0.24ml of the mother solution to dilute the mother solution into 1.2 mu g/ml NRG solution

6.4.3 Observation index

6.4.3.1 cardiac function detection

After the rats were anesthetized with 4% isoflurane by a gas anesthetic machine, the left side was fixed on an operation plate in a horizontal position. Gas anesthesia machine for fixing head of ratThe respirator of (1) was maintained at a concentration of 2% isoflurane. The breast is unhaired, sterilized by 75% alcohol, smeared with a coupling agent, and detected by a rat heart ultrasonic probe. Selecting B-mode, placing the cardiotachometer probe on left side of sternum, pointing the probe at 2-3 o' clock, cutting heart vertically to the direction of sound beam and heart long axis, adjusting the probe to two papillary muscle levels to obtain papillary muscle level left ventricle short axis section, collecting a section of left ventricle papillary muscle plane dynamic image and storing. Selecting 'M-mode', keeping the probe in the short-axis section of the left ventricle of the papillary muscle, adjusting an M-shaped sampling line to pass through the weakest point of the anterior wall pulsation, adjusting the focal length, collecting an M-shaped curve (the left ventricle cavity and the front and rear walls of the left ventricle should be clearly displayed), measuring the internal diameter (D) of the left ventricle at the end diastole and the end systole, and adopting a Teichholtz formula V which is 7/(2.4+ D) D³The left ventricular end-diastolic and end-systolic volumes, EDV, ESV, were calculated, and the Ejection Fraction (EF) value was calculated, EF ═ EDV-ESV/EDV × 100%.

6.4.3.2 cardiac hemodynamic testing

Recording hemodynamic indexes such as carotid artery pressure, indoor pressure, + dp/dt, -dp/dt and the like by using a physiological recorder. The method mainly comprises the following steps: rats are anesthetized by 20% urethane intraperitoneal injection, the injection volume is 6ml/kg, the right common carotid artery is separated, the distal end of the right common carotid artery is ligated, the proximal end of the right common carotid artery is blocked by an artery clamp, a small opening is cut between the two ends, a PE50 catheter connected with an instrument probe is inserted into the common carotid artery, the waveform displayed by a Powerlab physiological recorder is observed, the carotid artery pressure is recorded after stabilization, the catheter is further inserted into the left ventricle and is left for 10 minutes, indexes such as LVSP, LVEDP, + dp/dt and-dp/dt are recorded after stabilization, and the analysis is carried out by LabChart7 software.

6.4.4 data processing

For all experimental data

Represents, one-way analysis of variance, P, using GraphPad Prism 6 software<0.05 indicated a significant difference between groups, P<0.01 indicated a very significant difference between groups.

6.5. Results of the experiment

The heart super fruits are shown in table 7 and fig. 6, respectively. The experimental result shows that after the rat heart failure modeling is randomly grouped, the cardiac hyperdata of the groups before administration have no obvious difference. After administration, the difference was significant between the groups. After the group of 10d NRG-IgG1-Fc 30ug/kg is administrated by bolus injection once a day, the heart EF and FS values (both p is less than 0.001) of rats can be remarkably improved, and the LVESV (p is less than 0.001), LVEDV (p is less than 0.05), LVDs (p is less than 0.001) and LVDd values (p is less than 0.05) can be remarkably reduced, which indicates that the heart function of heart failure rats can be remarkably improved. The 8h x 10d NRG group administered continuously daily improved EF, LVESV, FS, LVDs values. It was shown that the equimolar amount (30ug/kg dose) of NRG-IgG1-Fc treated group had better therapeutic effect on heart failure in rats than NRG. Meanwhile, NRG-IgG1-Fc is more convenient compared with NRG administration mode.

TABLE 7 Heart ultrasound results of rats before and after treatment of each group

The scope of the invention is not limited to the description of the embodiments. It will be apparent to those skilled in the art that many modifications and variations can be made to the present invention without departing from the spirit and scope thereof. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Sequence listing

<110> Shanghai Zesheng science and technology development Ltd

<120> recombinant human neuregulin derivatives and uses thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 61

<212> PRT

<213> Homo sapiens

<400> 1

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr

35 40 45

Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln

50 55 60

<210> 2

<211> 308

<212> PRT

<213> Homo sapiens

<400> 2

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr

35 40 45

Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln Gly Gly Gly

50 55 60

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser

65 70 75 80

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

85 90 95

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

100 105 110

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

115 120 125

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

130 135 140

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

145 150 155 160

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

165 170 175

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

180 185 190

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

195 200 205

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

210 215 220

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

225 230 235 240

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

245 250 255

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

260 265 270

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

275 280 285

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

290 295 300

Ser Pro Gly Lys

305

<210> 3

<211> 307

<212> PRT

<213> Homo sapiens

<400> 3

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr

35 40 45

Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln Gly Gly Gly

50 55 60

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser Lys Tyr

65 70 75 80

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro

85 90 95

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

100 105 110

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

115 120 125

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

130 135 140

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

145 150 155 160

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

165 170 175

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

180 185 190

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

195 200 205

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

210 215 220

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

225 230 235 240

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

245 250 255

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

260 265 270

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

275 280 285

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

290 295 300

Lys Ala Ser

305

<210> 4

<211> 20

<212> PRT

<213> Homo sapiens

<400> 4

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser

20

<210> 5

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 5

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Claims

1. A neuregulin fusion polypeptide comprising an amino acid sequence of a neuregulin EGF domain, an immunoglobulin IgG Fc, or an Fc analog amino acid sequence.

2. The neuregulin fusion polypeptide of claim 1, wherein the amino acid sequence of the EGF domain of neuregulin is the amino acid sequence of SEQ ID NO. 1 or an analog thereof.

3. The neuregulin fusion polypeptide of claim 1, wherein the immunoglobulin IgG Fc is an IgG1 or IgG4 subtype Fc or analog amino acid sequence thereof.

4. The neuregulin fusion polypeptide of any of claims 1-3, further comprising a linker peptide sequence, and wherein the neuregulin EGF domain portion is linked to the IgG Fc portion via a linker peptide.

5. The neuregulin fusion polypeptide of any of claims 1-3, further comprising an IL-2 signal peptide amino acid sequence at the N-terminus, wherein the IL-2 signal peptide amino acid sequence is cleaved during recombinant production of the neuregulin fusion polypeptide for extracellular secretion.

6. The neuregulin fusion polypeptide of claim 1, comprising an amino acid sequence set forth in SEQ ID NO 2 or SEQ ID NO 3.

7. Use of neuregulin fusion polypeptide for the preparation of a medicament for preventing, treating or delaying cardiovascular disease in a mammal. Use of neuregulin fusion polypeptide for the preparation of a medicament for preventing, treating or delaying cardiovascular disease in a mammal.

8. The use of claim 7, wherein the neuregulin fusion polypeptide comprises an amino acid sequence of a neuregulin EGF domain, an immunoglobulin IgG Fc, or an Fc analog.

9. A pharmaceutical formulation comprising a neuregulin fusion polypeptide comprising an amino acid sequence of a neuregulin EGF domain, an immunoglobulin IgG Fc or Fc analog, and a pharmaceutically acceptable carrier, excipient, or diluent.

10. A method of treating a cardiovascular disease comprising administering to an individual in need thereof an effective amount of a neuregulin fusion polypeptide comprising an amino acid sequence of a neuregulin EGF domain, an immunoglobulin IgG Fc, or an Fc analog amino acid sequence.