Engineered NRG-1 variants with improved selectivity for ErbB4 but not ErbB3

Abstract

The present invention relates to modified neuregulin-1 variants that selectively activate the ErbB4 receptor but not the ErbB3 receptor. The present invention also provides methods for using such variants to treat heart failure.

Description

[Technical field]

[0001] The present invention relates to modified neuregulin-1 variants that selectively activate the ErbB4 receptor but do not or only weakly activate the ErbB3 receptor. The present invention also provides methods for using such neuregulin variants in the treatment of heart failure.

[0002] Sequence Listing This application contains, as another part of the disclosure, a sequence listing in computer readable format (filename: A-2828-WO-PCT Seq List_ST25.txt, created on June 7, 2022, size 334 KB), which is incorporated by reference in its entirety. [Background technology]

[0003] The epidermal growth factor receptor family, which includes four members, EGFR, ErbB2, ErbB3, and ErbB4, has been shown to play important roles in multiple cellular functions, including cell growth, differentiation, and survival. They are protein tyrosine kinase receptors consisting of an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. Several receptor ligands have been identified that mediate receptor homo- or heterodimerization upon binding. Specific receptor binding results in distinct patterns of phosphorylation, complex signaling cascades, and multiple biological functions, including cell proliferation, inhibition of apoptosis, and promotion of tumor cell motility, adhesion, and invasion. Representative cells that express ErbB receptors include glial cells, glioblastoma cells, Schwann cells, hepatocytes, epithelial cells, and muscle cells. Glial cells are derived from the central nervous system and include oligodendrocytes and astrocytes. Muscle cells that express ErbB receptors include muscle cell precursors (myoblasts) as well as more specialized skeletal muscle cells, cardiac muscle cells, and smooth muscle cells.

[0004] Neuregulin, also known as heregulin, glial growth factor (GGF) and novel differentiation factor (NDF), is an important growth factor, especially for the heart and nervous system. More than 15 distinct isoforms of neuregulin-1 (NRG-1) have been identified and divided into two groups, known as alpha and beta, based on differences in the sequence of their essential EGF-like domain. Neuregulin-1 is a ligand for the ErbB3 and ErbB4 receptors. The EGF-like domain of neuregulin-1, which ranges in size from 50 to 64 amino acids, has been shown to be sufficient to bind and activate these receptors. See, for example, Jones et al., 1999, FEBS Lett. 26:447:227-231. Previous studies have shown that neuregulin-1β (NRG-1β) can directly bind to ErbB3 and ErbB4 with high affinity. The orphan receptor ErbB2 retains a preactivated conformation to promote heterodimerization with ErbB3 or ErbB4 with approximately 100-fold higher affinity than the homodimers of ErbB3 and ErbB4. The heteromeric receptors act on distinct cell types: ErbB2 / ErbB3 in the peripheral nervous system and ErbB2 / ErbB4 in the heart. Studies in neural development have shown that intact NRG-1β, ErbB2 and ErbB3 signaling systems are required for the formation of the sympathetic nervous system. ErbB2 / ErbB4 receptor activation promotes cardiomyocyte growth and survival. Targeted disruption of NRG-1β or ErbB2 or ErbB4 led to embryonic lethality due to abnormal cardiac development. Recent studies also highlight the role of NRG-1β, ErbB2 and ErbB4 in cardiovascular development and in maintaining normal cardiac function in adults.

[0005] Neuregulin-stimulated ErbB2 / ErbB4 heterodimerization is crucial for myocardial function during early cardiac development and also prevents severe dysfunction in the adult heart, so ErbB4 activation by recombinant NRG-1 is a potential therapeutic option for heart failure. Short-term administration of recombinant NRG-1β EGF domain significantly improves or prevents the deterioration of myocardial performance in three separate animal models of heart failure. More importantly, NRG-1β significantly extends the survival of heart failure animals. See, for example, De Keulenaer et al., 2019, Circulation: Heart Failure 12:e006288. These effects make NRG-1β a promising broad-spectrum therapeutic or lead compound for heart failure due to various common diseases.

[0006] There are several drug candidates based on NRG-1 undergoing clinical trials for the treatment of cardiac disease through binding to ErbB4, however, binding through ErbB3 is thought to promote the development or progression of certain cancers and may also cause gastrointestinal toxicity.

[0007] A 61-mer peptide (S177-Q237 of wild-type hNRG-1) showed potent activity against both ErbB4 and ErbB3. See Liu et al., 2006, J Amer Coll Cardiol 48:1438-1447; and U.S. Patent No. 7,226,907. See also WO 2010060265; WO 2009007332; WO 2009033373; WO 2006030241; Jay et al., 2013; Circulation 128:152-161; and Wali et al., 2014, Mol Cancer Res 12:1140-1155. U.S. Patent Nos. 7,115,554 and 7,063,961 describe heregulin β1 variants that exhibit improved affinity for both the ErbB3 and ErbB4 receptors. U.S. Patent Application Publication No. 2007 / 0213264 describes neuregulin-1β variants that exhibit enhanced or decreased binding affinity to ErbB3 and / or ErbB4.

[0008] However, there remains a need for NRG-1 variants that exhibit high selectivity for ErbB4 over ErbB3 for therapeutic use in heart failure. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 7,226,907 [Patent Document 2] International Publication No. 2010060265 Brochure [Patent Document 3] International Publication No. 2009007332 Brochure [Patent Document 4] International Publication No. 2009033373 Brochure [Patent Document 5] International Publication No. 2006030241 Brochure [Patent Document 6] U.S. Patent No. 7,115,554 [Patent Document 7] U.S. Patent No. 7,063,961 [Patent Document 8] US Patent Application Publication No. 2007 / 0213264 [Non-patent literature]

[0010] [Non-Patent Document 1] Jones et al.,1999,FEBS Lett.26:447:227-231 [Non-Patent Document 2] De Keulenaer et al.,2019,Circulation:Heart Failure 12:e006288 [Non-Patent Document 3] Liu et al.,2006,J Amer Coll Cardiol 48:1438-1447 [Non-Patent Document 4] Jay et al.,2013;Circulation 128:152-161 [Non-Patent Document 5] Wali et al.,2014,Mol Cancer Res 12:1140-1155 Summary of the Invention [Means for solving the problem]

[0011] The present disclosure provides polypeptide variants of neuregulin-1β that are selective for ErbB4 and not selective for ErbB3 when compared to the wild-type sequence. In certain embodiments, the variants have increased binding affinity for ErbB4 and decreased binding affinity for ErbB3 compared to the wild-type sequence. In certain embodiments, the variants have increased binding affinity for ErbB4 and similar binding affinity for ErbB3 compared to the wild-type sequence. In certain embodiments, the variants have similar binding affinity for ErbB4 and decreased binding affinity for ErbB3 compared to the wild-type sequence.

[0012] The present disclosure also includes NRG-1 variants that are more specific for the ErbB4 receptor compared to the ErbB3 receptor than the NRG-1 from which they are derived. In certain embodiments, the NRG-1 variants have a binding affinity for ErbB4 that is 2-fold, 3-fold, or 4-fold higher than that for ErbB3.

[0013] The present disclosure also includes NRG-1 variants that have greater selectivity for the ErbB4 receptor compared to the ErbB3 receptor, in certain embodiments, the NRG-1 variants have an ErbB4 / ErbB3 selectivity of 1000 or more or 10,000 or more.

[0014] In certain embodiments, the NRG-1 variants have agonist activity that is at least 50%, 60%, 70%, or 80% of the corresponding wild-type sequence.

[0015] In one embodiment, the disclosure provides a polypeptide variant comprising an amino acid sequence of the following formula: SHLVKCX 183 EX 185 X 186 KX 188 FCVNGGECX 197 X 198 X 199 X 200 X 201 X 202 SX204 PSRX 208 LCKCPNEFTGDRCX 222 X 223 X 224 X 225 X 226 ASX 229 (SEQ ID NO:177) (In the formula, X 183 is A or G; X 185 X 186 is KD, KE, KH, ND, NE, NH, RD, RE, RH, RQ, SD, SE, or SH; X 188 is S or T; X 197 X 198 X 199 X 200 X 201 X 202 is FMIEDS (SEQ ID NO: 178), FMIEGP (SEQ ID NO: 179), FMIEHL (SEQ ID NO: 180), FMVEDL (SEQ ID NO: 181), FMVERS (SEQ ID NO: 182), FMVKRP (SEQ ID NO: 183), FVIEDP (SEQ ID NO: 184), FVIEGS (SEQ ID NO: 185), FVVEGL (SEQ ID NO: 186), YMIEDL (SEQ ID NO: 187), YMIEGL (SEQ ID NO: 188), YMIEHP (SEQ ID NO: 189), YMVEDL (SEQ ID NO: 190), YMVEGS (SEQ ID NO: 191), YMVERP (SEQ ID NO: 192), YVIEDS (SEQ ID NO: 193), YVIEGS (SEQ ID NO: 194), YVIEHL (SEQ ID NO: 195), YVVEDL (SEQ ID NO: 196), YVVEHS (SEQ ID NO: 197) or YVVERP (SEQ ID NO: 198); X 204 is I or N, X 208 is F or Y; X 222 X 223 X 224 X 225 X 226EKDVM (SEQ ID NO: 199), QAPHI (SEQ ID NO: 200), QDDFM (SEQ ID NO: 201), QDFFL (SEQ ID NO: 202), QDFFM (SEQ ID NO: 203), QDVFL (SEQ ID NO: 204), QDVFM (SEQ ID NO: 205), QEDFM (SEQ ID NO: 206), QETQI (SEQ ID NO: 207), QKDFL (SEQ ID NO: 208), QKDFM (SEQ ID NO: 209), QKDVM (SEQ ID NO: 210), QKFFL (SEQ ID NO: 211), QKFFM (SEQ ID NO: 212), QKLDI (SEQ ID NO: 213), Q KTSL (SEQ ID NO:214), QKVFL (SEQ ID NO:215), QKVFM (SEQ ID NO:216), QKVVM (SEQ ID NO:217), QNDFL (SEQ ID NO:218), QNDFM (SEQ ID NO:219), QNVFL (SEQ ID NO:220), QNVFM (SEQ ID NO:221), QNYVM (SEQ ID NO:222), QQSFP (SEQ ID NO:223), QSALT (SEQ ID NO:224), QSSEL (SEQ ID NO:225), QSTRV (SEQ ID NO:226), QTSLL (SEQ ID NO:227) or QVTRL (SEQ ID NO:228); X 229 is absent, F, or FYKAEELYQ (SEQ ID NO: 229).

[0016] In an aspect of this embodiment, the polypeptide variant comprises the amino acid sequence of any of SEQ ID NOs: 114-176.

[0017] In one embodiment, the present disclosure provides a compound comprising the formula: X 183 is A or G; X 185 X 186 is KD, KE, KH, ND, NH, RD, RE, RH, RQ, SD, SE, or SH; X 188 is S or T; X 197 X 198 X 199 X 200 X 201 X 202is FMIEDS (SEQ ID NO:178), FMIEGP (SEQ ID NO:179), FMIEHL (SEQ ID NO:180), FMVEDL (SEQ ID NO:181), FMVERS (SEQ ID NO:182), FMVKRP (SEQ ID NO:183), FVIEDP (SEQ ID NO:184), FVIEGS (SEQ ID NO:185), YMIEDL (SEQ ID NO:187), YMIEGL (SEQ ID NO:188), YMIEHP (SEQ ID NO:189), YMVEDL (SEQ ID NO:190), YMVEGS (SEQ ID NO:191), YMVERP (SEQ ID NO:192), YVIEDS (SEQ ID NO:193), YVIEGS (SEQ ID NO:194), YVIEHL (SEQ ID NO:195), YVVEHS (SEQ ID NO:197), or YVVERP (SEQ ID NO:198); X 204 is I or N, X 208 is F or Y; X 222 X 223 X 224 X 225 X 226 EKDVM (SEQ ID NO: 199), QAPHI (SEQ ID NO: 200), QDFFL (SEQ ID NO: 202), QDFFM (SEQ ID NO: 203), QDVFL (SEQ ID NO: 204), QEDFM (SEQ ID NO: 206), QETQI (SEQ ID NO: 207), QKDFL (SEQ ID NO: 208), QKDFM (SEQ ID NO: 209), QKDVM (SEQ ID NO: 210), QKFFL (SEQ ID NO: 211), QKFFM (SEQ ID NO: 212), QKLDI (SEQ ID NO: 213), Q KTSL (SEQ ID NO:214), QKVFL (SEQ ID NO:215), QNDFL (SEQ ID NO:218), QNDFM (SEQ ID NO:219), QNVFL (SEQ ID NO:220), QNVFM (SEQ ID NO:221), QNYVM (SEQ ID NO:222), QQSFP (SEQ ID NO:223), QSALT (SEQ ID NO:224), QSSEL (SEQ ID NO:225), QSTRV (SEQ ID NO:226), QTSLL (SEQ ID NO:227) or QVTRL (SEQ ID NO:228); X 229 is F or FYKAEELYQ (SEQ ID NO: 229).

[0018] In one aspect of this embodiment, the polypeptide variant comprises the amino acid sequence of any of SEQ ID NOs: 114, 115, 117-124, 126-135, 138, 139, 144-147, 149-156, 160-168, or 171-176.

[0019] In one embodiment, the present disclosure provides a compound comprising the formula: X 183 is A or G; X 185 is KE, KH, ND, RD, RH, RQ, SD, SE, or SH; X 188 is S or T; X 197 X 198 X 199 X 200 X 201 X 202 is FMIEDS (SEQ ID NO:178), FMIEGP (SEQ ID NO:179), FMVEDL (SEQ ID NO:181), FMVKRP (SEQ ID NO:183), FVIEDP (SEQ ID NO:184), FVIEGS (SEQ ID NO:185), YMIEDL (SEQ ID NO:187), YMIEHP (SEQ ID NO:189), YMVEDL (SEQ ID NO:190), YMVEGS (SEQ ID NO:191), YMVERP (SEQ ID NO:192), YVIEDS (SEQ ID NO:193), YVIEGS (SEQ ID NO:194), YVIEHL (SEQ ID NO:195), YVVEHS (SEQ ID NO:197), or YVVERP (SEQ ID NO:198); X 204 is I or N, X 208 is F or Y; X 222 X 223 X 224 X 225 X 226is EKDVM (SEQ ID NO: 199), QAPHI (SEQ ID NO: 200), QEDFM (SEQ ID NO: 206), QETQI (SEQ ID NO: 207), QKDFL (SEQ ID NO: 208), QKDFM (SEQ ID NO: 209), QKDVM (SEQ ID NO: 210), QKFFL (SEQ ID NO: 211), QKFFM (SEQ ID NO: 212), QKLDI (SEQ ID NO: 213), QKTSL (SEQ ID NO: 214), QKVFL (SEQ ID NO: 215), QNDFL (SEQ ID NO: 218), QNDFM (SEQ ID NO: 219), QNVFL (SEQ ID NO: 220), QNVFM (SEQ ID NO: 221), QNYVM (SEQ ID NO: 222), QQSFP (SEQ ID NO: 223), QSALT (SEQ ID NO: 224), QSSEL (SEQ ID NO: 225), QSTRV (SEQ ID NO: 226), QTSLL (SEQ ID NO: 227) or QVTRL (SEQ ID NO: 228); X 229 is F or FYKAEELYQ (SEQ ID NO: 229); Polypeptide variants are provided.

[0020] In one aspect of this embodiment, the polypeptide variant comprises the amino acid sequence of any of SEQ ID NOs: 117-124, 126-135, 144-147, 149-152, 154-156, 163, 167, 168, or 171-176.

[0021] In one embodiment, the present disclosure provides: During the ceremony, X 183 is A or G; X 185 X 186 is KH, ND, RD, RH or SH; X 188 is S or T; X 197 X 198 X 199 X 200 X 201 X 202is FMIEDS (SEQ ID NO: 178), FMIEGP (SEQ ID NO: 179), FMVEDL (SEQ ID NO: 181), FVIEDP (SEQ ID NO: 184), YMIEDL (SEQ ID NO: 187), YMVEGS (SEQ ID NO: 191), YVIEGS (SEQ ID NO: 194), YVIEHL (SEQ ID NO: 195) or YVVERP (SEQ ID NO: 198); X 204 is I or N, X 208 is F or Y; X 222 X 223 X 224 X 225 X 226 is QAPHI (SEQ ID NO:200), QEDFM (SEQ ID NO:206), QETQI (SEQ ID NO:207), QKDFL (SEQ ID NO:208), QKDFM (SEQ ID NO:209), QKTSL (SEQ ID NO:214), QNDFL (SEQ ID NO:218), QNDFM (SEQ ID NO:219), QQSFP (SEQ ID NO:223), QSALT (SEQ ID NO:224), QSSEL (SEQ ID NO:225), QSTRV (SEQ ID NO:226), QTSLL (SEQ ID NO:227) or QVTRL (SEQ ID NO:228); X 229 is F or FYKAEELYQ (SEQ ID NO: 229); Polypeptide variants are provided.

[0022] In one aspect of this embodiment, the polypeptide variant comprises the amino acid sequence of any of SEQ ID NOs: 117-124, 126, 128-135, 146, 147, 150, 151, 163, 167, 168, or 171.

[0023] In another aspect of this embodiment, the polypeptide variant comprises the amino acid sequence of any of SEQ ID NOs: 117, 118, 119, 120, 122, 123, 124, 126, 128, 129, 130, 131, 133, or 135.

[0024] In any of the embodiments, the polypeptide variant further comprises a second amino acid sequence to act as a signal sequence, to extend half-life, or to facilitate purification. In certain aspects, the polypeptide variant can be fused to an amino acid sequence to extend its half-life. In one aspect, the amino acid sequence is an Fc region. In one aspect, the amino acid sequence is fused to the C-terminus of the polypeptide variant. In one aspect, the amino acid sequence is fused to the N-terminus of the polypeptide variant. In certain aspects, the amino acid sequence is fused to the polypeptide variant via a linker. In certain aspects, the polypeptide variant can be fused to a His tag to aid in purification. In one aspect, the His tag is fused to the C-terminus of the polypeptide variant. In one aspect, the His tag is fused to the N-terminus of the polypeptide variant.

[0025] In another aspect, the present polypeptide variant comprises any of the amino acid sequences of SEQ ID NOs: 1 to 24, 29 to 54, 56 to 67, 69 to 80, and 82 to 111. In another aspect, the present polypeptide variant comprises any of the amino acid sequences of SEQ ID NOs: 1 to 15, 19, 20, 22, 23, 29 to 34, 37, 38, 43 to 54, 56 to 59, 61 to 67, 69 to 80, 82 to 85, 87 to 93, and 97 to 111. In one aspect, the polypeptide variant comprises any of the amino acid sequences of SEQ ID NOs: 3, 4, 6, 9, 11 to 13, 19, 22, 31 to 34, 43 to 48, 52, 54, 56 to 59, 61 to 63, 65 to 67, 71, 72, 75 to 80, 84, 85, 88, 100, or 104 to 106. In one aspect, the polypeptide variant comprises any of the amino acid sequences of SEQ ID NOs: 3, 4, 6, 9, 11 to 13, 19, 22, 31 to 34, 43 to 48, 52, 54, 56 to 59, 61 to 63, 65 to 67, 71, 72, 75 to 80, 84, 85, 88, 100, or 104 to 106. In one aspect the polypeptide variant comprises the amino acid sequence of SEQ ID NO: 4, 11, 13, 32, 34, 45, 46, 48, 52, 72, 75, 76, 77, 78, 79 or 80. In one aspect the polypeptide variant comprises the amino acid sequence of SEQ ID NO: 56, 57, 58, 59, 61, 62, 63, 65, 66 or 67.

[0026] The present disclosure also provides a pharmaceutical composition comprising a polypeptide variant described herein and a pharma- ceutically acceptable carrier.

[0027] The present disclosure also provides a method of treating a cardiovascular disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a polypeptide variant or pharmaceutical composition. In certain aspects of this embodiment, the cardiovascular disease or condition is heart failure, myocardial infarction, dilated cardiomyopathy, myocarditis or cardiotoxicity. In certain aspects, the subject is a human.

[0028] The present disclosure also provides the above-mentioned polypeptide variants for use in the treatment of a cardiovascular disease or condition or for the preparation of a medicament for treating a cardiovascular disease or condition. In one embodiment, the present disclosure provides a neuregulin polypeptide variant for use in the treatment of a cardiovascular disease or condition. In another embodiment, the present disclosure provides the use of a neuregulin polypeptide for the preparation of a medicament for the treatment of a cardiovascular disease or condition. [Brief description of the drawings]

[0029] [Figure 1-1] Figure 1A-C shows cardiac function assessment one week after ErbB4 agonist administration. Statistical significance between groups was assessed using GraphPad one-way ANOVA. A) Terminal serum exposure. B) Cardiac function by ejection fraction (EF). C) Heart rate (HR) during echocardiography. [Figure 1-2] Figure 1A-C shows cardiac function assessment one week after ErbB4 agonist administration. Statistical significance between groups was assessed using GraphPad one-way ANOVA. A) Terminal serum exposure. B) Cardiac function by ejection fraction (EF). C) Heart rate (HR) during echocardiography. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The present invention is based in part on the discovery that neuregulin variants can be designed to be highly selective for ErbB4 while having low or no binding affinity for ErbB3. Such variants are candidates for therapeutic agents for heart-related diseases while minimizing cross-reactivity to other cell types. The present invention is in part derived from efforts to improve the efficacy of heart failure drugs. Thus, the present disclosure also relates to treating subjects with or at risk for developing heart disease and related conditions, such as heart failure.

[0031] definition The terminology used in this application is standard within the art, but definitions of certain terms are provided herein to ensure clarity and clarity in the meaning of the claims. Units, prefixes, and symbols may be expressed in SI (International System of Units) accepted form. Numerical ranges recited herein are inclusive of the numbers defining the range, and include and support each integer within the defined range. Unless otherwise indicated, the methods and techniques described herein are generally carried out according to conventional methods well known in the art, and such methods and techniques are described in various general and more specific references cited and discussed throughout this specification. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992) and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990).

[0032] As used herein, the terms "a" and "an" mean one or more, unless specifically indicated otherwise. Further, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

[0033] All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, and journal articles, are expressly incorporated herein by reference. What is described in one embodiment of the invention can be combined with other embodiments of the invention.

[0034] As used herein, the term "neuregulin-1" or "NRG-1" or "neuregulin" refers to proteins or peptides that can bind to and activate ErbB2 / ErbB4 or ErbB2 / ErbB3 heterodimer protein kinases, such as all neuregulin isoforms, the neuregulin EGF domain alone, neuregulin mutations, and any type of neuregulin-like gene product that also activates the above receptors. Neuregulin also includes NRG-1, NRG-2, NRG-3, and NRG-4. These proteins and polypeptides can activate the above ErbB receptors and modulate their biological responses, such as stimulating breast cancer cell differentiation and milk protein secretion; inducing differentiation of neural crest cells into Schwann cells; stimulating acetylcholine synthesis in skeletal muscle cells; and improving cardiomyocyte survival and DNA synthesis. Neuregulin also includes variants with conservative amino acid substitutions that do not substantially change their biological activity. Suitable conservative substitutions of amino acids are known to those of skill in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art will generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987. The Benjamin / Cummings Pub. Co. p. 224). The phrase "neuregulin protein" includes full-length neuregulin proteins as well as neuregulin peptides (e.g., truncated versions of full-length neuregulin proteins). Neuregulin nucleic acids include neuregulin nucleic acids and neuregulin oligonucleotides.

[0035] As used herein, "neuregulin variant" refers to a neuregulin having a modified sequence that alters or enhances the selectivity of the neuregulin for ErbB4 and / or decreases the selectivity of the neuregulin for ErbB3.

[0036] As used herein, the term "epidermal growth factor-like domain" or "EGF-like domain" refers to the domains described in WO 00 / 64400 and WO 97 / 09425, Holmes et al., 1992, Science, 256:1205-1210; U.S. Pat. Nos. 5,530,109 and 5,716,930; Hijazi et al., 1998, Int. J. Oncol., 13:1061-1067; Chang et al., 1997, Nature, 387:509-512; Carraway et al., 1997, Nature, 387:512-516; Higashiyama et al., 1997, J. Biol. It refers to a polypeptide motif encoded by the neuregulin gene that binds to and activates ErbB2, ErbB3, ErbB4, or a combination thereof, and possesses a structure similar to the EGF receptor binding domain, as disclosed in Chem., 122:675-680. The EGF-like domain may be derived from NRG-1, NRG-2, NRG-3, or NRF-4. The EGF-like domain may be of the C or B subtype.

[0037] As used herein, the terms "ErbB2", "ErbB2 (HER2)", and "HER2" refer to the same protein (or, when referring to a gene, the same gene) and are used interchangeably herein. ErbB2 (erb-b2 receptor tyrosine kinase 2) is also known in the art as NEU, NGL, TKR1, CD340, HER-2, MLN19, and HER-2 / neu.

[0038] As used herein, the terms "ErbB3", "ErbB3 (HER3)", and "HER3" refer to the same protein (or, when referring to a gene, the same gene) and are used interchangeably herein. ErbB3 (erb-b2 receptor tyrosine kinase 3) is also known in the art as FERLK, LCCS2, ErbB-3, c-erbB3, erbB3-S, MDA-BF-1, c-erbB-3, p180-ErbB3, p45-sErbB3, and p85-sErbB3.

[0039] As used herein, the terms "ErbB4", "ErbB4 (HER4)", and "HER4" refer to the same protein (or, when referring to a gene, the same gene) and are used interchangeably herein. ErbB4 (erb-b2 receptor tyrosine kinase 4) is also known in the art as ALS19 and p180erbB4.

[0040] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dosage or amount sufficient to produce a desired result, which may include an objective or subjective improvement in the subject of the dosage or amount (e.g., prolonged survival, improved cardiac function, effective prevention of a disease state, etc.).

[0041] As used herein, the term "ejection fraction" refers to the ejection fraction (EF), a measurement generally expressed as a percentage of how much blood the left ventricle pumps with each contraction. For example, an ejection fraction of 50 percent means that 50 percent of the total volume of blood in the left ventricle is pumped out with each heartbeat.

[0042] As used herein, the term "heart failure" refers to an abnormality in cardiac function in which cardiac output does not match the metabolic tissue requirements. Heart failure includes a wide variety of disease states, such as congestive heart failure, myocardial infarction, tachyarrhythmia, familial hypertrophic cardiomyopathy, ischemic heart disease, idiopathic dilated cardiomyopathy, and myocarditis. Heart failure can be caused by a number of factors, including ischemic, congenital, rheumatic, or idiopathic. Chronic cardiac hypertrophy is an important disease state that is a precursor to congestive heart failure and cardiac arrest.

[0043] As used herein, the terms "polypeptide" and "protein" may be used interchangeably herein to refer to a polymer of amino acid residues. These terms also apply to naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acids. These terms may also encompass amino acid polymers that have been modified, for example, by the addition of carbohydrate residues to form glycoproteins, or by phosphorylation. Polypeptides and proteins may be produced by naturally occurring non-recombinant cells, or they may be produced by genetically engineered or recombinant cells. Polypeptides and proteins may also be made by synthetic means. Polypeptides and proteins may include molecules having the amino acid sequence of a naturally occurring protein, or molecules with deletions, additions and / or substitutions of one or more amino acids of the naturally occurring sequence.

[0044] The terms "polypeptide" and "protein" encompass molecules that contain only naturally occurring amino acids as well as molecules that contain non-naturally occurring amino acids. Examples of non-naturally occurring amino acids (which may be substituted, if desired, for the naturally occurring amino acids found in any sequence disclosed herein) include 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl terminal direction, in accordance with standard usage and convention.

[0045] A non-limiting list of examples of non-naturally occurring amino acids that may be inserted into a protein or polypeptide sequence or substituted for wild-type residues in a protein or polypeptide sequence include β-amino acids, homoamino acids, cyclic amino acids and side chain derivatized amino acids. Examples include the following (in either the L- or D-form;Abbreviated as in parentheses: citrulline (Cit), homocitrulline (hCit), Nα-methylcitrulline (NMeCit), Nα-methylhomocitrulline (Nα-MeHoCit), ornithine (Orn), Nα-methylornithine (Nα-MeOrn or NMeOrn), sarcosine (Sar), homolysine (hLys or hK), homoarginine (hArg or hR), homoglutamine (hQ), Nα-methylarginine (NMeR), Nα-methylleucine (Nα-MeL or NMeL), N-methylhomolysine (NMeH oK), Nα-methylglutamine (NMeQ), norleucine (Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic), octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal), 3-(2-naphthyl)alanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline (Tic), 2-indanylglycine (IgI), para-iodophenylalanine (pI-Phe), para-aminophenylalanine (4AmP or 4-amino-Phe), 4-guaialine (4-guaialine), 4-guanidinium phosphate (4-guanidinium phosphate ... Nitrophenylalanine (Guf), glycyrrhizin (abbreviated as "K(Nε-glycyl)" or "K(glycyl)" or "K(gly)"), nitrophenylalanine (nitroPhe), aminophenylalanine (aminoPhe or amino-Phe), benzylphenylalanine (benzylPhe), gamma-carboxyglutamic acid (gamma-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine (Cpa), alpha-aminoadipic acid (Aad), Nα-methylvaline (NMeVal), N-α- Methylleucine (NMeLeu), Nα-methylnorleucine (NMeNle), cyclopentylglycine (Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), α,β-diaminopropionic acid (Dpr), α,γ-diaminobutyric acid (Dab), diaminopropionic acid (Dap), cyclohexylalanine (Cha), 4-methyl-phenylalanine (MePhe), β,β-diphenyl-alanine (BiPhA), aminobutyric acid (Abu), 4-phenyl-phenylalanine (or biphenylalanine;4Bip), α-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionic acid, piperidinic acid, aminocaproic acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine, N-ethylaspargine, hydroxylysine, allo-hydroxylysine, isodesmosine, allo-isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline, 4-hydroxyproline (Hyp), γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-amino-O-phthalic acid (4APA) and other similar amino acids and any derivatized forms of those specifically mentioned;

[0046] "Fc" region, as that term is used herein, refers to the C region of an antibody. H 2 domain and C H It is composed of two heavy chain fragments each containing 3 domains. The two heavy chain fragments are separated by two or more disulfide bonds and C H The three domains are held together by hydrophobic interactions. Proteins of interest comprised of an Fc region, including antigen binding proteins and Fc fusion proteins, constitute another embodiment of the present disclosure.

[0047] NRG-1β variants The present disclosure provides a polypeptide variant of NRG-1 that is selective for ErbB4.Neuregulin variants, as disclosed herein, have an amino acid sequence that is not found in nature, in which one or more wild-type amino acid residues in native neuregulin are replaced with different amino acid residues.At least one substitution is non-conservative to change function, for example to improve selectivity for ErbB4.

[0048] A functional human NRG-1 fragment containing the EGF-like domain, corresponding to amino acids 177-237 of human NRG-1, has the amino acid sequence SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ (sequence number 112).

[0049] In some embodiments, a neuregulin fragment containing an EGF-like domain refers to amino acid residues 177-226, 177-228, 177-229, 177-237, or 177-240 of NRG-1 (SEQ ID NO:112).

[0050] Representative neuregulin variants that include modified neuregulin sequences and one or more of linkers, Fc sequences, and His tags are shown in Table 1 below.

[0051] [Table 1]

[0052] [Table 2]

[0053] [Table 3]

[0054] [Table 4]

[0055] [Table 5]

[0056] [Table 6]

[0057] [Table 7]

[0058] [Table 8]

[0059] [Table 9]

[0060] [Table 10]

[0061] [Table 11]

[0062] [Table 12]

[0063] [Table 13]

[0064] [Table 14]

[0065] [Table 15]

[0066] [Table 16]

[0067] Human neuregulin variant sequences (without linker, Fc sequence or His tag) are shown in Table 2 below.

[0068] [Table 17]

[0069] [Table 18]

[0070] [Table 19]

[0071] In certain embodiments, the polypeptide variant comprises an amino acid sequence of the following formula: SHLVKCX 183 EX 185 X 186 KX 188 FCVNGGECX 197 X 198 X 199 X 200 X 201 X 202 SX 204 PSRX 208 LCKCPNEFTGDRCX 222 X 223 X 224 X 225 X 226 ASX 229 (SEQ ID NO:177) (In the formula, X 183 teeth, [ka] or G; X 185 X 186 ,K.D., [ka] , KH, ND, NE, NH, RD, RE, RH, RQ, SD, SE or SH; X 188 is S or [ka] and; X 197 X 198 X 199 X 200 X 201 X 202 is FMIEDS (SEQ ID NO: 178), FMIEGP (SEQ ID NO: 179), FMIEHL (SEQ ID NO: 180), FMVEDL (SEQ ID NO: 181), FMVERS (SEQ ID NO: 182), FMVKRP (SEQ ID NO: 183), FVIEDP (SEQ ID NO: 184), FVIEGS (SEQ ID NO: 185), FVVEGL (SEQ ID NO: 186), YMIEDL (SEQ ID NO: 187), YMIEGL (SEQ ID NO: 188), YMIEHP (SEQ ID NO: 189), YMVEDL (SEQ ID NO: 190), YMVEGS (SEQ ID NO: 191), YMVERP (SEQ ID NO: 192), YVIEDS (SEQ ID NO: 193), YVIEGS (SEQ ID NO: 194), YVIEHL (SEQ ID NO: 195), YVVEDL (SEQ ID NO: 196), YVVEHS (SEQ ID NO: 197) or YVVERP (SEQ ID NO: 198); X 204 I or [ka] and X 208 is F or [ka] and; X 222 X 223 X 224 X 225 X 226EKDVM (SEQ ID NO: 199), QAPHI (SEQ ID NO: 200), QDDFM (SEQ ID NO: 201), QDFFL (SEQ ID NO: 202), QDFFM (SEQ ID NO: 203), QDVFL (SEQ ID NO: 204), QDVFM (SEQ ID NO: 205), QEDFM (SEQ ID NO: 206), QETQI (SEQ ID NO: 207), QKDFL (SEQ ID NO: 208), QKDFM (SEQ ID NO: 209), QKDVM ( SEQ ID NO: 210), QKFFL (SEQ ID NO: 211), QKFFM (SEQ ID NO: 212), QKLDI (SEQ ID NO: 213), QKTSL (SEQ ID NO: 214), QKVFL (SEQ ID NO: 215), QKVFM (SEQ ID NO: 216), QKVVM (SEQ ID NO: 217), QNDFL (SEQ ID NO: 218), QNDFM (SEQ ID NO: 219), QNVFL (SEQ ID NO: 220), QNVFM (SEQ ID NO: 221), [ka] (SEQ ID NO:222), QQSFP (SEQ ID NO:223), QSALT (SEQ ID NO:224), QSSEL (SEQ ID NO:225), QSTRV (SEQ ID NO:226), QTSLL (SEQ ID NO:227) or QVTRL (SEQ ID NO:228); X 229 teeth, [ka] (Sequence number 229).

[0072] In the formulae above and below, the wild type sequence is underlined.

[0073] In certain embodiments, the polypeptide variant comprises the amino acid sequence of any of SEQ ID NOs: 114-176.

[0074] In certain embodiments, the polypeptide variant comprises the amino acid sequence of any of SEQ ID NOs: 1-24, 29-54, 56-67, 69-80, or 82-111.

[0075] In certain embodiments, the polypeptide variant comprises an amino acid sequence of the following formula: SHLVKCX183 EX 185 X 186 KX 188 FCVNGGECX 197 X 198 X 199 X 200 X 201 X 202 SX 204 PSRX 208 LCKCPNEFTGDRCX 222 X 223 X 224 X 225 X 226 ASX 229 [SEQ ID NO:177] (In the formula, X 183 teeth, [ka] or G; X 185 X 186 ,K.D., [ka] , KH, ND, NH, RD, RE, RH, RQ, SD, SE or SH; X 188 is S or [ka] and; X 197 X 198 X 199 X 200 X 201 X 202is FMIEDS (SEQ ID NO: 178), FMIEGP (SEQ ID NO: 179), FMIEHL (SEQ ID NO: 180), FMVEDL (SEQ ID NO: 181), FMVERS (SEQ ID NO: 182), FMVKRP (SEQ ID NO: 183), FVIEDP (SEQ ID NO: 184), FVIEGS (SEQ ID NO: 185), YMIEDL (SEQ ID NO: 187), YMIEGL (SEQ ID NO: 188), YMIEHP (SEQ ID NO: 189), YMVEDL (SEQ ID NO: 190), YMVEGS (SEQ ID NO: 191), YMVERP (SEQ ID NO: 192), YVIEDS (SEQ ID NO: 193), YVIEGS (SEQ ID NO: 194), YVIEHL (SEQ ID NO: 195), YVVEHS (SEQ ID NO: 197) or YVVERP (SEQ ID NO: 198); X 204 I or [ka] and X 208 F or [ka] and; X 222 X 223 X 224 X 225 X 226 EKDVM (SEQ ID NO: 199), QAPHI (SEQ ID NO: 200), QDFFL (SEQ ID NO: 202), QDFFM (SEQ ID NO: 203), QDVFL (SEQ ID NO: 204), QEDFM (SEQ ID NO: 206), QETQI (SEQ ID NO: 207), QKDFL (SEQ ID NO: 208), QKDFM (SEQ ID NO: 209), QKDVM (SEQ ID NO: 210), QKFFL (SEQ ID NO: 211), QKFFM (SEQ ID NO: 212), QKLDI (SEQ ID NO: 213), QKTSL (SEQ ID NO: 214), QKVFL (SEQ ID NO: 215), QNDFL (SEQ ID NO: 218), QNDFM (SEQ ID NO: 219), QNVFL (SEQ ID NO: 220), QNVFM (SEQ ID NO: 221), [ka] (SEQ ID NO:222), QQSFP (SEQ ID NO:223), QSALT (SEQ ID NO:224), QSSEL (SEQ ID NO:225), QSTRV (SEQ ID NO:226), QTSLL (SEQ ID NO:227) or QVTRL (SEQ ID NO:228); X 229 teeth, [ka] (SEQ ID NO:229) (It is.)

[0076] In certain embodiments, the polypeptide variant comprises the amino acid sequence of any of SEQ ID NOs: 114, 115, 117-124, 126-135, 138, 139, 144-147, 149-156, 160-168, or 171-176.

[0077] In certain embodiments, the polypeptide variant comprises any of the amino acid sequences of SEQ ID NOs: 1-15, 19, 20, 22, 23, 29-34, 37, 38, 43-54, 56-59, 61-67, 69-80, 82-85, 87-93, or 97-111.

[0078] In certain embodiments, the polypeptide variant comprises an amino acid sequence of the following formula: SHLVKCX 183 EX 185 X 186 KX 188 FCVNGGECX 197 X 198 X 199 X 200 X 201 X 202 SX 204 PSRX 208 LCKCPNEFTGDRCX 222 X 223 X 224 X 225 X 226 ASX 229 (SEQ ID NO:177) (In the formula, X 183 teeth, [ka] or G; X 185 teeth, [ka] , KH, ND, RD, RH, RQ, SD, SE or SH; X 188 is S or [ka] and; X 197 X 198 X 199 X 200 X 201 X 202 is FMIEDS (SEQ ID NO: 178), FMIEGP (SEQ ID NO: 179), FMVEDL (SEQ ID NO: 181), FMVKRP (SEQ ID NO: 183), FVIEDP (SEQ ID NO: 184), FVIEGS (SEQ ID NO: 185), YMIEDL (SEQ ID NO: 187), YMIEHP (SEQ ID NO: 189), YMVEDL (SEQ ID NO: 190), YMVEGS (SEQ ID NO: 191), YMVERP (SEQ ID NO: 192), YVIEDS (SEQ ID NO: 193), YVIEGS (SEQ ID NO: 194), YVIEHL (SEQ ID NO: 195), YVVEHS (SEQ ID NO: 197) or YVVERP (SEQ ID NO: 198); X 204 I or [ka] and X 208 is F or [ka] and; X 222 X 223 X 224 X 225 X 226EKDVM (SEQ ID NO: 199), QAPHI (SEQ ID NO: 200), QEDFM (SEQ ID NO: 206), QETQI (SEQ ID NO: 207), QKDFL (SEQ ID NO: 208), QKDFM (SEQ ID NO: 209), QKDVM (SEQ ID NO: 210), QKFFL (SEQ ID NO: 211), QKFFM (SEQ ID NO: 212), QKLDI (SEQ ID NO: 213), QKTSL (SEQ ID NO: 214), QKVFL (SEQ ID NO: 215), QNDFL (SEQ ID NO: 218), QNDFM (SEQ ID NO: 219), QNVFL (SEQ ID NO: 220), QNVFM (SEQ ID NO: 221), [ka] (SEQ ID NO:222), QQSFP (SEQ ID NO:223), QSALT (SEQ ID NO:224), QSSEL (SEQ ID NO:225), QSTRV (SEQ ID NO:226), QTSLL (SEQ ID NO:227) or QVTRL (SEQ ID NO:228); The X8 is [ka] (SEQ ID NO:229) (It is.)

[0079] In certain embodiments, the polypeptide variant comprises the amino acid sequence of any of SEQ ID NOs: 117-124, 126-135, 144-147, 149-152, 154-156, 163, 167, 168, or 171-176.

[0080] In certain embodiments, the polypeptide variant comprises any of the amino acid sequences of SEQ ID NOs: 3, 4, 6, 9, 11-13, 19, 22, 31-34, 43-48, 52, 54, 56-59, 61-63, 65-67, 71, 72, 75-80, 84, 85, 88, 100, or 104-106.

[0081] In certain embodiments, the polypeptide variant comprises an amino acid sequence of the following formula: SHLVKCX 183 EX 185 X 186 KX 188 FCVNGGECX197 X 198 X 199 X 200 X 201 X 202 SX 204 PSRX 208 LCKCPNEFTGDRCX 222 X 223 X 224 X 225 X 226 ASX 229 [SEQ ID NO:177] (In the formula, X 183 teeth, [ka] or G; X 185 X 186 is KH, ND, RD, RH or SH; X 188 is S or [ka] and; X 197 X 198 X 199 X 200 X 201 X 202 is FMIEDS (SEQ ID NO: 178), FMIEGP (SEQ ID NO: 179), FMVEDL (SEQ ID NO: 181), FVIEDP (SEQ ID NO: 184), YMIEDL (SEQ ID NO: 187), YMVEGS (SEQ ID NO: 191), YVIEGS (SEQ ID NO: 194), YVIEHL (SEQ ID NO: 195) or YVVERP (SEQ ID NO: 198); X 204 I or [ka] and; X 208 is F or [ka] and; X 222 X223 X 224 X 225 X 226 is QAPHI (SEQ ID NO:200), QEDFM (SEQ ID NO:206), QETQI (SEQ ID NO:207), QKDFL (SEQ ID NO:208), QKDFM (SEQ ID NO:209), QKTSL (SEQ ID NO:214), QNDFL (SEQ ID NO:218), QNDFM (SEQ ID NO:219), QQSFP (SEQ ID NO:223), QSALT (SEQ ID NO:224), QSSEL (SEQ ID NO:225), QSTRV (SEQ ID NO:226), QTSLL (SEQ ID NO:227) or QVTRL (SEQ ID NO:228); X 229 teeth, [ka] (SEQ ID NO:229) (It is.)

[0082] In certain embodiments, the polypeptide variant comprises the amino acid sequence of any of SEQ ID NOs: 117-124, 126, 128-135, 146, 147, 150, 151, 163, 167, 168, or 171.

[0083] In certain embodiments, the polypeptide variant comprises any of the amino acid sequences of SEQ ID NOs: 3, 4, 6, 9, 11-13, 19, 22, 31-34, 43-48, 52, 54, 56-59, 61-63, 65-67, 71, 72, 75-80, 84, 85, 88, 100, or 104-106.

[0084] In certain embodiments, the polypeptide variant has enhanced binding affinity to ErbB4 compared to the polypeptide of SEQ ID NO:112.

[0085] In certain embodiments, the polypeptide variant has a reduced binding affinity to ErbB3 compared to the polypeptide of SEQ ID NO:112.

[0086] In certain embodiments, the polypeptide variants have similar binding affinity to ErbB3 while having improved binding affinity to ErbB4 compared to the polypeptide of SEQ ID NO:112.

[0087] In certain embodiments, the polypeptide variants have reduced binding affinity to ErbB3 and improved binding affinity to ErbB4 compared to the polypeptide of SEQ ID NO:112.

[0088] In certain embodiments, the polypeptide variants have reduced binding affinity to ErbB3 compared to the polypeptide of SEQ ID NO:124, while having similar binding to ErbB4.

[0089] In certain embodiments, the polypeptide variants have a selectivity for ErbB4 / ErbB3 that is at least 500, at least 1000, at least 5000, or at least 10,000. Selectivity for ErbB4 / ErbB3 can be measured by methods known to those skilled in the art, such as the Akt assay described in Example 1. For example, selectivity can be expressed as the ratio of EC50 in Schwann cells / cardiomyocytes. This ratio provides activity in neuronal cells compared to cardiac cells.

[0090] In certain embodiments, the polypeptide variants have agonist activity that is at least 50%, 60%, 70% or 80% of the activity relative to the wild-type sequence, as may be measured by phosphorylation of Akt.

[0091] Any of the above neuregulin variants may be modified by fusion to a heterologous polypeptide to generate a "chimeric neuregulin variant" or fusion protein. Generally, the heterologous polypeptide is fused to the N- or C-terminus of the neuregulin variant to preserve the biological activity of the neuregulin variant. However, a heterologous polypeptide may also be introduced into a region of the neuregulin variant that is not important for biological activity. Generally, chimeric neuregulin variants are produced by recombinant techniques or chemical synthesis. Examples of chimeric neuregulin variants include neuregulin variants fused to a "signal sequence," a "purification handle," an immunoglobulin sequence, or any combination thereof. A linker may be used to link the neuregulin polypeptide to a heterologous polypeptide.

[0092] A "signal sequence" is an amino acid sequence that directs secretion of a polypeptide fused to the signal sequence from a cell expressing the chimeric protein. Thus, fusion of a neuregulin variant to a signal sequence facilitates recombinant production of the neuregulin variant, since the chimeric neuregulin variant is secreted into the host cell medium, from which it can be relatively easily recovered. A suitable signal sequence can be obtained from any protein that has a signal sequence, and is typically (but not always) fused to the N-terminus of the neuregulin variant. DNA encoding prokaryotic signal sequences can be obtained, for example, from lamB or ompF, MalE, PhoA, and other genes. Another suitable prokaryotic signal sequence is the E. coli heat-stable enterotoxin II (STII) signal sequence. Mammalian signal sequences are discussed below.

[0093] A "purification handle" is a polypeptide or a portion of a polypeptide sequence that binds to another polypeptide, referred to as a "binding partner." Fusion of a purification handle to a neuregulin variant confers on the variant the ability to bind to a binding partner, facilitating purification of the resulting chimeric neuregulin variant. Generally, the purification handle is selected such that the binding partner does not substantially cross-react with other components present in the mixture from which the chimeric neuregulin variant is to be purified. An exemplary "purification handle" is a His tag sequence. As used herein, the term "does not substantially cross-react" means that the affinity of the binding partner for the purification handle is something that is at least about 20-fold, usually at least about 100-fold, and more usually at least about 1000-fold, greater than any other component present in the mixture.

[0094] Chimeric neuregulin variants include neuregulin variants fused to an immunoglobulin sequence. In one embodiment, the immunoglobulin sequence is an Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that extends the in vivo serum half-life of IgG. See, e.g., U.S. Patent Application Publication No. 2006 / 0140934.

[0095] In one embodiment, the neuregulin variant is linked to the linker via the first (1st) amino acid at the N-terminus of the neuregulin variant, which in one embodiment is a serine (S or Ser) amino acid.

[0096] In certain embodiments, the Fc region has at least one mutation, preferably to reduce effector function and / or extend the half-life of the molecule. In certain aspects, the Fc region comprises at least one mutation at amino acid 234, 239, 434, or a combination thereof (numbered according to EU numbering), and in certain aspects, the amino acid mutation comprises at least one of the following substitution mutations: L234F, S239A, N434A, or a combination thereof. The mutation at amino acid 234 and / or 239 knocks down the effector function of the Fc region. The mutation at amino acid 434 extends the half-life of the fusion protein in a subject. Other mutations are known in the art, such as SEFL2.0 and SEFL2.2 mutations. See, for example, Jacobsen et al., 2017, J Biol Chem 292:1865-1875 and Yang et al., 2018, Front Immunol Vol. 8, Art. 1860.

[0097] In certain embodiments, one or more mutations in the Fc region reduce effector function. In some embodiments, reduced effector function includes reducing the affinity of the fusion protein for one or more Fc receptors. The FcRs can be FcyRI, FcyRIIa, FcyRIib, FcyRIIIa(158F), FcyRIIIa(158V), and Clq.

[0098] In one embodiment, the fusion protein comprises a neuregulin variant fused or operably linked to the C-terminus of the Fc region via a GGGGSGGGGS (G4S) linker (SEQ ID NO: 113). In some embodiments, one or more copies of the linker may be used. In other embodiments, two, three, four or five copies of the G4S linker or any other linker known in the art and / or described herein as suitable for the compositions disclosed herein may be used herein.

[0099] The term "linker" is art-recognized and refers to a molecule (including but not limited to unmodified or modified nucleotides or amino acids) or group of molecules (e.g., two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more) that links two compounds, such as two polypeptides. A linker may be composed of a single linking molecule or may include a linking molecule and at least one spacer molecule that is intended to separate the linking molecule and the compounds by a specific distance.

[0100] Preparation of Neuregulin Variants The polypeptides of the present invention can be produced by chemical synthesis or recombinant methods. Methods for chemically synthesizing polypeptides are well known in the art. Synthesizing polypeptides using recombinant methods is also well known in the art and is further described herein.

[0101] For example, methods and vectors for genetically engineering cells and / or cell lines to express polypeptides are well known to those of skill in the art; for example, various techniques are exemplified in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988 and updated quarterly); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, pp. 15-69. The polypeptides produced can be tested for their binding affinity to receptors and activation of receptors using methods known in the art.

[0102] A vector can be any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, transposon, cosmid, chromosome, virus, viral capsid, virion, naked DNA, complexed DNA, etc.) suitable for use in transferring and / or transporting information-encoding proteins to a host cell and / or a specific location and / or compartment within a host cell. Vectors can include viral and non-viral vectors, non-episomal mammalian vectors. Vectors are often referred to as expression vectors, e.g., recombinant expression vectors and cloning vectors. A vector can be introduced into a host cell to allow the vector to replicate itself, thereby amplifying copies of the polynucleotide contained therein. A cloning vector can contain sequence components that generally include, but are not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements can be selected as needed by those skilled in the art.

[0103] A vector is useful for transformation of a host cell and contains nucleic acid sequences that induce and / or regulate (in cooperation with the host cell) the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, if introns are present, RNA splicing of the coding region operably linked thereto. "Operably linked" means that the components to which the term is applied are in a relationship that allows them to perform their inherent functions. For example, a control sequence in a vector that is "operably linked" to a protein coding sequence, such as a promoter, is positioned such that normal activation of the control sequence results in transcription of the protein coding sequence and recombinant expression of the encoded protein.

[0104] Vectors can be selected to be functional in the particular host cell used (i.e., the vector is compatible with the host's cellular machinery and allows gene amplification and / or expression to occur). In some embodiments, vectors are used that use protein fragment complementation assays using protein reporters such as dihydrofolate reductase (see, e.g., U.S. Pat. No. 6,270,964). Suitable expression vectors are known in the art and are commercially available.

[0105] Typically, vectors used in host cells contain sequences for maintaining the plasmid and for cloning and expressing exogenous nucleotide sequences. Such sequences typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, transcriptional and translational control sequences, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, various pre- or pro-sequences for improving glycosylation or yield, a native or heterologous signal sequence (leader sequence or signal peptide) for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, an internal ribosome entry site (IRES) sequence, an expression enhancing sequence element (EASE), a tripartite leader (TPA) and a VA gene RNA from adenovirus type 2, a polylinker region for inserting a polynucleotide encoding a polypeptide to be expressed, and a selectable marker element. Vectors can be constructed from a starting vector, such as a commercially available vector, or additional elements can be obtained separately and ligated into the vector. The methods used to obtain each of the components are well known to those skilled in the art.

[0106] Vector components can be homologous (i.e., derived from the same species and / or strain as the host cell), heterologous (e.g., derived from a species other than the host cell species or host cell strain), hybrid (i.e., a combination of adjacent sequences from two or more sources), synthetic, or natural. The sequences of components useful in vectors can be obtained by methods well known in the art, such as those previously identified by mapping and / or restriction endonucleases. Additionally, they can be obtained by polymerase chain reaction (PCR) and / or by screening genomic libraries with appropriate probes.

[0107] A ribosome binding site is usually required for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed.

[0108] Replication origins aid in the amplification of vectors in host cells. They can be included as part of commercially available prokaryotic vectors, or can be chemically synthesized and ligated into vectors based on known sequences. Various viral origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells.

[0109] Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from viral genomes. Commonly used promoter and enhancer sequences are derived from polyoma virus, adenovirus type 2, simian virus 40 (SV40) and human cytomegalovirus (CMV). For example, the human CMV promoter / enhancer of immediate early gene 1 can be used. See, for example, Patterson et al., 1994, Applied Microbiol. Biotechnol. 40:691-98. DNA sequences derived from the SV40 viral genome, for example, the SV40 origin, early and late promoters, enhancers, splices and polyadenylation sites, can be used to provide other genetic elements for expressing structural gene sequences in mammalian host cells. The viral early and late promoters are particularly useful because both are easily obtained as fragments from the viral genome and may contain the viral origin of replication (Fiers et al., 1978, Nature 273:113; Kaufman, 1990, Meth. in Enzymol. 185:487-511). Smaller or larger SV40 fragments can also be used, provided there is included the approximately 250 bp sequence extending from the HindIII site toward the BglI site located at the site of the SV40 viral origin of replication.

[0110] A transcription termination sequence is typically located 3' to the end of a polypeptide coding region and serves to terminate transcription. Usually, a transcription termination sequence in a prokaryotic cell is a GC-rich fragment followed by a poly-T sequence. This sequence can be easily cloned from a library or even purchased commercially as part of a vector, but can also be easily synthesized using methods for nucleic acid synthesis known to those skilled in the art.

[0111] A selectable marker gene encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, tetracycline, or kanamycin, to a prokaryotic host cell; (b) complement an auxotrophic deficiency of the cell; or (c) supply vital nutrients unavailable from complex or defined media. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, the neomycin resistance gene can also be used for selection in both prokaryotic and eukaryotic host cells.

[0112] Other selectable genes can be used to amplify the gene to be expressed. Amplification is the process by which genes required for the production of a protein important for growth or cell survival are repeated in tandem in the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include the glutamine synthase (GS) / methionine sulfoximine (MSX) system, dihydrofolate reductase (DHFR) and promoter-less thymidine kinase genes. The mammalian cell transformant is placed under selection pressure in which the transformant is uniquely adapted to survive due to the selectable gene present in the vector. Selection pressure is applied by culturing the transformed cells under conditions of successively increasing concentrations of the selective drug in the medium, thereby amplifying both the selection gene and the DNA encoding the protein of interest. As a result, large amounts of the polypeptide of interest are synthesized from the amplified DNA.

[0113] In some cases where glycosylation is desired in eukaryotic host cell expression systems, various pre- or pro-sequences may be engineered to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide may be modified or a pro-sequence may be added, which may also affect glycosylation. The final protein product may have one or more additional amino acids at position -1 (relative to the first amino acid of the mature protein) that are associated with expression, but this amino acid may not be completely removed. For example, the final protein product may have one or two amino acid residues found in the peptidase cleavage site attached to the amino terminus. Alternatively, the use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide when cleaved by an enzyme at such a region within the mature polypeptide.

[0114] Expression and cloning typically contain a promoter that is recognized by the host organism and operably linked to a molecule encoding a protein of interest. A promoter is a non-transcribed sequence located upstream (i.e., 5') of the start codon of a structural gene (generally within about 100-1000 bp) and controls transcription of the structural gene. Traditionally, promoters are classified into one of two classes: inducible promoters and constitutive promoters. Inducible promoters cause increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Constitutive promoters, on the other hand, transcribe the genes to which they are operably linked uniformly, i.e., with little or no control over gene expression. A large number of promoters recognized by a variety of potential host cells are well known.

[0115] Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus type 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and the actin promoter.

[0116] Additional promoters of interest include, but are not limited to, the SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310); the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. USA 81:659-663); the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1444-1445); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); the promoter and regulatory sequences from the metallothionine gene (Prinster et al., 1982, Nature 296:39-42); and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Also of interest are the following animal transcriptional control regions that exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region, which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulin gene control region, which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122); the immunoglobulin gene control region, which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444); mouse mammary tumor virus control region active in testis, breast, lymphocytes and mast cells (Leder et al., 1986, Cell 45:485-495); albumin gene control region active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); alpha-feto-protein gene control region active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 253:53-58); alpha 1-antitrypsin gene control region active in liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); beta-globin gene control region active in bone marrow cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene control region, which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); the myosin light chain-2 gene control region, which is active in skeletal muscle (Sani, 1985, Nature 314:283-286); and the gonadotropin releasing hormone gene control region, which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[0117] Enhancer sequences may be inserted into vectors to increase transcription by higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about 10-300 bp long, that act on promoters to increase transcription. Enhancers are relatively orientation and position independent, and are found at both 5' and 3' positions relative to the transcription unit. Several enhancer sequences are known that are available from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically, however, enhancers from viruses are used. SV40 enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers, and adenovirus enhancers known in the art are exemplary enhancing elements for the activation of eukaryotic promoters. Enhancers may be located either 5' or 3' of the coding sequence in the vector, but are typically located at a site 5' from the promoter.

[0118] To promote extracellular secretion of the protein of interest, a sequence encoding an appropriate native or heterologous signal sequence (leader sequence or signal peptide) may be incorporated into the expression vector. The choice of signal peptide or leader depends on the host cell type in which the protein of interest is to be produced, and a heterologous signal sequence may replace the native signal sequence. Examples of signal peptides that function in mammalian host cells include the signal sequence for interleukin-7 described in U.S. Pat. No. 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signal peptide described in European Patent No. 0367566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in European Patent No. 0460846.

[0119] Additional regulatory sequences that have been shown to improve expression of heterologous genes from mammalian expression vectors include elements such as the expression-enhancing sequence element (EASE) derived from CHO cells (Morris et al., in Animal Cell Technology, pp. 529-534 (1997); U.S. Pat. Nos. 6,312,951 B1, 6,027,915, and 6,309,841 B1), as well as the tripartite leader (TPL) and VA gene RNA derived from adenovirus type 2 (Gingeras et al., 1982, J. Biol. Chem. 257:13475-13491). Internal ribosome entry site (IRES) sequences of viral origin allow efficient translation of dicistronic mRNAs (Oh and Sarnow, 1993, Current Opinion in Genetics and Development 3:295-300; Ramesh et al., 1996, Nucleic Acids Research 24:2697-2700).

[0120] After construction, one or more vectors can be inserted into an appropriate cell for amplification and / or polypeptide expression. Transformation of the expression vector into the selected cell can be performed by well-known methods, including gene transfer, infection, calcium phosphate co-precipitation, electroporation, nucleofection, microinjection, DEAE-dextran mediated gene transfer, cationic lipid-mediated delivery, liposome-mediated gene transfer, particle bombardment, receptor-mediated gene delivery, polylysine, histone, chitosan and peptide-mediated delivery. The method selected will depend, in part, on the type of host cell to be used. These and other suitable methods are well known to those of skill in the art and are described in manuals and other technical publications, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001).

[0121] The term "transformation" refers to a change in the genetic characteristics of a cell; a cell is transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed when it has been genetically modified from its native state by introducing new genetic material via gene transfer, transduction or other techniques. Following gene transfer or transduction, the transforming DNA may recombine with the DNA of the cell by physically integrating into the cell's chromosome, or may be maintained transiently without replication as an episomal element, or may be independently replicated as a plasmid. A cell is considered to be "stably transformed" when the transforming DNA is replicated with cell division.

[0122] The term "gene transfer" refers to the uptake of foreign or exogenous DNA by a cell. Many gene transfer techniques are known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0123] The term "transduction" refers to the process by which foreign DNA is introduced into cells via a viral vector. See Jones et al., (1998). Genetics: principles and analysis. Boston: Jones & Bartlett Publ.

[0124] A wide variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and commercial suppliers. Examples of cell lines commonly used in the industry include monkey kidney CV1 transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture (Graham et al., 1977, J. Gen Virol. 36:59); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocellular carcinoma cells (Hep G2, HB 8065); mouse mammary carcinoma (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals NY Acad. Sci. 383:44-68); MRC5 cells or FS4 cells; mammalian myeloma cells, and many other cell lines, as well as Chinese hamster ovary (CHO) cells.

[0125] Large-scale production of proteins for commercial use is typically performed in suspension culture. Thus, the mammalian host cells used to generate the recombinant mammalian cells described herein may, but need not, be adapted to growth in suspension culture. A variety of host cells adapted to growth in suspension culture are known, including mouse myeloma NS0 cells, and CHO cells derived from CHO-S, DG44 and DXB11 cell lines. Other suitable cell lines include mouse myeloma SP2 / 0 cells, baby hamster kidney BF1K-21 cells, human PER.C6® cells, human embryonic kidney F1EK-293 cells, and cell lines derived from or modified from any of the cell lines disclosed herein.

[0126] CHO cells are widely used to produce complex recombinant proteins, including CHOK1 cells (ATCC CCL61). Dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al., 1980, Proc Natl Acad Sci USA 77:4216-4220), DXB11 and DG-44 are desirable CHO host cell lines because efficient DHFR-selectable and amplifiable gene expression systems allow high levels of recombinant protein to be expressed in these cells (Kaufman RJ, 1990, Meth Enzymol 185:537-566). Also included is the glutamine synthase (GS) knockout CHOK1SV cell line, which utilizes GS-based methionine sulfoximine (MSX) selection. Other suitable CHO host cells include, but are not limited to, the following (ECACC accession numbers in parentheses): CHO (85050302), CHO (protein free) (00102307), CHO-K1 (85051005), CHO-K1 / SF (93061607), CHO / dhfr- (94060607), CHO / dhFr-AC-free (05011002), RR-CHOKI (92052129).

[0127] The production of recombinant proteins begins with establishing a mammalian cell production culture of cells expressing the protein in a culture plate, flask, tube, bioreactor, or other suitable vessel. Typically smaller production bioreactors are used, in one embodiment the bioreactor is between 500L and 2000L. In another embodiment a bioreactor between 1000L and 2000L is used. The seed cell density used to inoculate the bioreactor can have a positive effect on the level of recombinant protein produced. In one embodiment, at least 0.5x10 cells are cultured in serum-free culture medium. 6 From 3.0x10 6 The bioreactor is seeded with less than or equal to 1.0x10 viable cells / mL. In another embodiment, the seeding is less than or equal to 1.0x10 6 viable cells / mL.

[0128] The mammalian cells then undergo an exponential growth phase. The cell culture may be maintained without additional feeding until a desired cell density is achieved. In one embodiment, the cell culture is maintained for up to 3 days with or without additional feeding. In another embodiment, the culture may be seeded at a desired cell density to initiate a production phase without a short growth phase. In any of the embodiments herein, the switch from the growth phase to the production phase may also be initiated by any of the methods described above.

[0129] At the transition between the growth phase and the production phase, and during the production phase, the percent packed cell volume (%PCV) is 35% or less. The desired packed cell volume to be maintained during the production phase is 35% or less. In one embodiment, the packed cell volume is 30% or less. In another embodiment, the packed cell volume is 20% or less. In yet another embodiment, the packed cell volume is 15% or less. In a further embodiment, the packed cell volume is 10% or less.

[0130] Three methods are commonly used in commercial processes for producing recombinant proteins by mammalian cell culture: batch, fed-batch, and perfusion. Batch culture is a discontinuous method in which cells are grown in a fixed volume of culture medium for a short period of time followed by a complete harvest. Cultures grown using batch methods experience an increase in cell density until a maximum cell density is reached, after which the viable cell density decreases as medium components are consumed and levels of metabolic by-products (such as lactate and ammonia) accumulate. Harvesting is typically performed once maximum cell density is reached (e.g., 5x10 depending on the medium formulation, cell line, etc.). 6 cells / mL or more). The batch process is the simplest culture method, but viable cell density is limited by nutrient availability, and once cells reach maximum density, the culture declines and production drops. The production phase cannot be extended (typically around 3-7 days) because the culture declines rapidly due to waste accumulation and nutrient depletion.

[0131] Fed-batch culture improves on the batch process by providing a bolus or continuous medium feed to replenish consumed medium components. Fed-batch cultures receive additional nutrients throughout the run, allowing for higher cell densities (>10–30x10 depending on media formulation, cell line, etc.) when compared to batch processes. 6cells / ml) and increased production titers. Unlike batch processes, biphasic cultures can be generated and maintained by manipulating the feeding regime and media formulation to differentiate between a period of cell growth to achieve a desired cell density (growth phase) and a period of cessation or stagnation of cell growth (production phase). Thus, compared to batch cultures, fed-batch cultures may achieve higher production titers. Typically, a batch regime is used during the growth phase and a fed-batch regime is used during the production phase, but fed-batch feeding regimes can be used throughout the process. However, unlike batch processes, the volume of the bioreactor is the limiting factor, which limits the feed rate. Also, similar to batch processes, the culture is reduced by accumulation of metabolic by-products, which limits the duration of the production phase to about 10-21 days. Fed-batch cultures are discontinuous and typically harvest occurs when metabolic by-product levels or viability of the culture reach a predefined level. Compared to batch cultures without feeding, fed-batch cultures may produce larger amounts of recombinant protein. See, for example, U.S. Pat. No. 5,672,502.

[0132] Perfusion techniques offer the potential for improvement over batch and fed-batch processes by simultaneously adding fresh medium and removing spent medium. In a typical large-scale commercial cell culture system, approximately one-third to more than one-half of the reactor volume is biomass, with the average volume ranging from 60-90(+) x 10 6 We aim to achieve high cell densities of >1x10 cells / mL by perfusion culture. 8Ultimate cell densities of cells / mL have been achieved, and even higher densities are predicted. A typical perfusion culture begins by initiating a batch culture lasting 1 or 2 days, after which fresh feed medium is added continuously, stepwise, and / or intermittently to the culture, while retaining the cells and additional high molecular weight compounds, such as proteins (based on the molecular weight cut-off of the filter), throughout the growth and production phase of the culture, and simultaneously removing spent medium. Various methods, such as sedimentation, centrifugation, or filtration, can be used to remove spent medium while maintaining cell density. Perfusion flow rates of a few working volumes per day up to many multiple working volumes per day have been reported.

[0133] The advantage of the perfusion process is that the production culture can be maintained for a longer period of time than batch or fed-batch cultures. However, more nutrients are also required, and increased medium preparation, use, storage and disposal are required to support perfusion cultures for a longer period of time, especially with higher cell densities, all of which lead to higher production costs compared to batch and fed-batch processes. In addition, higher cell densities can cause problems during production such as maintaining dissolved oxygen levels and problems with increased gassing, including providing more oxygen and removing more carbon dioxide, which creates more foam and requires changes to the anti-foam regime; similarly, the effort required to remove excess cell material during harvest and downstream processes can result in product losses, which negates the advantage of increased titer due to increased cell mass.

[0134] Also provided is a large-scale cell culture method that combines fed-batch feeding during a growth phase followed by continuous perfusion during a production phase, the method being directed to a production phase in which the cell culture is maintained at no more than 35% packed cell volume.

[0135] In one embodiment, fed-batch culture with a bolus feed is used to maintain the cell culture during the growth phase. Perfusion feed may then be used during the production phase. In one embodiment, perfusion begins when the cells reach the production phase. In another embodiment, perfusion begins about day 3 to about day 9 of cell culture. In another embodiment, perfusion begins about day 5 to about day 7 of cell culture.

[0136] The use of a bolus feed during the growth phase allows the cells to transition into the production phase, resulting in less reliance on temperature change as a means to initiate and control the production phase, although a temperature change of about 36° C. to about 31° C. can occur between the growth and production phases. In one embodiment, the change is from 36° C. to 32° C.

[0137] At least 0.5x10 cells / ml in serum-free culture medium as described herein 6 From 3.0x10 6 Viable cells / mL below and above, e.g., 1.0x10 6 The bioreactor may be seeded at 10 viable cells / mL.

[0138] In perfusion culture, the cell culture receives fresh perfusion feed medium while simultaneously removing spent medium. Perfusion can be continuous, stepwise, intermittent, or any or all combinations of these. The perfusion rate can be less than to many working volumes per day. The cells are retained in the culture medium, and the removed spent medium is substantially free of cells or has significantly fewer cells than the culture. Recombinant proteins expressed by the cell culture can also be retained in the culture medium. Perfusion can be achieved by many means including centrifugation, sedimentation, or filtration, see for example Voisard et al., 2003, Biotechnology and Bioengineering 82:751-65. One example of a filtration method is alternating tangential flow filtration. Alternating tangential flow is maintained by pumping the medium through a hollow fiber filter module. See, e.g., U.S. Patent No. 6,544,424; Furey, 2002, Gen. Eng. News. 22(7):62-63.

[0139] "Perfusion flow rate" is the amount of medium passing through (added and removed from) a bioreactor, typically expressed as a fraction or multiple of a working volume in a given time. "Working volume" refers to the amount of the volume of the bioreactor used for cell culture. In one embodiment, the perfusion flow rate is no more than one working volume per day. Perfusion feed medium can be formulated to maximize perfusion nutrient concentrations and minimize perfusion rates.

[0140] The cell culture may be supplemented with a concentrated feed medium that contains components such as nutrients and amino acids that are consumed during the course of the production phase of the cell culture.

[0141] Concentrated feed media can be based on almost any cell culture medium formulation. Such concentrated feed media can contain most of the components of the cell culture medium, for example, at about 5x, 6x, 7x, 8x, 9x, 10x, 12x, 14x, 16x, 20x, 30x, 50x, 100x, 200x, 400x, 600x, 800x, or even about 1000x their normal amounts. Concentrated feed media are often used in fed-batch processes.

[0142] The methods described herein can be used to improve recombinant protein production in a doubling phase culture process. In a doubling phase process, cells are cultured in two or more different phases. For example, cells can be first cultured in one or more growth phases under environmental conditions that maximize cell growth and viability, and then shifted to a production phase under conditions that maximize protein production. In commercial processes for producing proteins by mammalian cells, there are typically multiple, e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9 or 10, growth phases that are performed in different culture vessels that precede the final production culture.

[0143] The growth and production phases may be preceded by or separated by one or more transition phases. In the doubling phase process, the method according to the invention may be used at least during the growth phase and the production phase of the final production phase of the commercial cell culture, but may also precede the growth phase. The production phase may be carried out on a large scale. Large scale processes may be carried out in volumes of at least about 100, 500, 1000, 2000, 3000, 5000, 7000, 8000, 10,000, 15,000, 20,000 liters. In one embodiment, production is carried out in 500L, 1000L and / or 2000L bioreactors.

[0144] The growth phase may occur at a higher temperature than the production phase. For example, the growth phase may occur at a first temperature of about 35°C to about 38°C, and the production phase may occur at a second temperature of about 29°C to about 37°C, optionally about 30°C to about 36°C, or about 30°C to about 34°C. In addition, chemical inducers of protein production, such as, for example, caffeine, butyrate, and hexamethylene bisacetamide (HMBA), may be added simultaneously with, before, and / or after the temperature change. If inducers are added after the temperature change, they may be added 1 hour to 5 days after the temperature change, optionally 1 to 2 days after the temperature change. The cell culture may be maintained for several days or even weeks, with the cells producing the desired protein.

[0145] Any of the analytical techniques known in the art may be used to monitor and evaluate samples from the cell culture. Various parameters may be monitored during the culture period, including recombinant protein and medium quality and characteristics. Samples may be taken and monitored intermittently at any desired frequency, including continuous monitoring, real-time or near real-time.

[0146] Typically, cell cultures preceding the final production culture (Nx to N-1) are used to generate the seed cells, N-1 culture, used to inoculate the production bioreactor. Seed cell density can have a positive effect on the level of recombinant protein produced. Production levels tend to be higher as seed density increases. Improved titers are not only associated with higher seed densities, but can also affect the metabolic and cell cycle state of the cells put into production.

[0147] Seed cells may be generated by any culture method. One such method is perfusion culture with alternating tangential flow filtration. Alternating tangential flow filtration may be used to run an N-1 bioreactor to provide a high density of cells for seeding the production bioreactor. The N-1 stage may be used to grow cells >90x10 6The N-1 bioreactor may be used to generate a bolus seed culture or may be used as a rotating seed stock culture that can be maintained to inoculate multiple production bioreactors at high seed cell densities. The duration of the product growth phase may range from 7 to 14 days and may be designed to maintain the cells in an exponential growth state prior to inoculation of the production bioreactor. To grow the cells, perfusion rates, media formulations and timing are optimized to deliver the cells to the production bioreactor under conditions most conducive to optimizing production. Seed cell densities of >15x10 6 Inoculation of the production bioreactor may be achieved by using a seed cell density of 10000 cells / mL. Higher seed cell densities at inoculation may shorten or even eliminate the time required to reach a desired production density.

[0148] The present invention also provides pharmaceutical compositions comprising a polypeptide variant of neuregulin or any of the polynucleotides encoding the polypeptide variants described herein and a pharma- ceutically acceptable excipient or carrier. As used herein, a "pharma- ceutically acceptable excipient or carrier" includes any substance that, when combined with an active ingredient, allows the ingredient to retain biological activity and is not reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers, such as phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, and various types of wetting agents. A preferred diluent for aerosol or parenteral administration is phosphate buffered saline or normal (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000). Generally, an appropriate amount of a pharma- ceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of carriers include saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and the concentration of the polypeptide and polynucleotide being administered.

[0149] How to Use Neuregulin Variants The present invention also provides methods for preventing, treating or delaying the onset of disease in an individual comprising administering to the individual a pharmaceutical composition comprising a neuregulin polypeptide variant described herein via activation of the ErbB2 / ErbB4 receptor.

[0150] Neuregulin variants according to the invention can also be used to treat muscle cells and medical conditions that affect muscle cells. In particular, such neuregulin variants can be useful for treating muscle disorders, reducing muscle cell atrophy, and improving muscle cell survival, proliferation, and / or regeneration. Examples of pathophysiological conditions of the muscular system that are suitable for treatment with neuregulin variants include skeletal muscle diseases (e.g., myopathies or dystrophies), myocardial disorders (including atrial arrhythmias, cardiomyopathies, ischemic disorders, congenital diseases, and cardiac trauma), and smooth muscle disorders (such as arteriosclerosis, vascular lesions, or congenital vascular diseases). Neuregulin variants can also be used to reduce high blood pressure and increase functional acetylcholine receptors in muscle cells (e.g., individuals with myasthenia gravis or tachycardia).

[0151] The term "treating cardiovascular disease", as used herein, unless otherwise indicated, means inhibiting, suppressing, delaying, reversing or alleviating, either partially or completely, the onset of a cardiovascular disease or condition in a subject, or the progression of a pre-existing cardiovascular disease or condition or its symptoms in a subject. Non-limiting examples of cardiovascular diseases that may be treated by the methods of the present disclosure include chronic heart failure, congestive heart failure (CHF), acute heart failure, myocardial infarction (MI), left ventricular systolic dysfunction, reperfusion injury associated with MI, chemotherapy-induced cardiotoxicity (adult or pediatric), radiation-induced cardiotoxicity, and support for surgical intervention in pediatric congenital heart disease. Non-limiting examples of symptoms of cardiovascular disease include shortness of breath, cough, rapid weight gain, swelling of the legs, ankles and abdomen, dizziness, fatigue, weakness, vertigo, chest pain, fainting (asphyxia), tachycardia and bradycardia. Methods for determining the progression of cardiovascular disease and the effectiveness of treatment will be readily apparent to those skilled in the art. For example, the progression of various cardiovascular diseases can be determined by ejection fraction / electrocardiogram (ECG), ECG / Holter monitor, stress test, cardiac catheterization, cardiac computed tomography (CT) scan and cardiac magnetic resonance imaging (MRI).

[0152] In certain embodiments, cardiovascular diseases whose onset may be prevented, treated or delayed through preferentially activating ErbB2 / ErbB4 receptors include, but are not limited to, heart failure, myocardial infarction, dilated or hypertrophic cardiomyopathy and myocarditis (e.g., viral myocarditis), and cardiotoxicity.

[0153] As used herein, an "individual" or "subject" is a mammal, more preferably a human. Mammals include, but are not limited to, farm animals (such as cows, pigs, sheep, goats, etc.), sport animals, pets (such as cats, dogs, horses, etc.), primates, mice and rats.

[0154] According to the present invention, the polypeptide variants of neuregulin-1 described herein or nucleic acids encoding the polypeptide variants, alone or in combination with other agents, carriers or excipients, can be formulated for any suitable route of administration, such as subcutaneous, intravenous, intramuscular, intradermal, oral or topical administration. The method may use formulations for injectable administration in unit dosage form, ampoules or multi-dose containers, with added preservatives. The formulations may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, sterile pyrogen-free water or other solvents, before use. Topical administration in the present invention may employ the use of foams, gels, creams, ointments, transdermal patches or pastes.

[0155] The magnitude of the therapeutic dose in treatment or prevention varies with the type and severity of the condition to be treated and the route of administration. The dose and perhaps the frequency of administration also vary according to the age, weight, condition and response of the individual patient. It should be noted that the attending physician will know how and when to discontinue, interrupt or adjust the treatment to a lower dose due to toxicity or side effects. Conversely, the physician will also know how and when to adjust the treatment to a higher level if the clinical response is not sufficient (excluding toxic side effects).

[0156] The dosage of the neuregulin variant composition to be used for treatment depends, for example, on the therapeutic objectives, the route of administration, and the condition of the patient. Thus, the clinician needs to titrate the dosage and modify the route of administration as necessary to obtain the optimal therapeutic effect. A typical daily dosage can range from about 1 μg / kg to 100 mg / kg body weight per day or more, but is generally about 10 μg / kg to 10 mg / kg per day. Generally, the clinician will start with a low dosage of the pharmaceutical neuregulin variant composition and increase the dosage until the desired therapeutic effect is achieved.

[0157] In practical use, the polypeptide variant of neuregulin, the fusion protein containing the polypeptide variant of neuregulin, or the nucleic acid encoding any of the foregoing, may be combined as an active agent, alone or in combination with other drugs, intimately mixed with pharmaceutical carriers or excipients, such as beta-cyclodextrin and 2-hydroxy-propyl-beta-cyclodextrin, according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of preparation forms suitable for administration, topical or parenteral. In preparing compositions for parenteral dosage forms, such as intravenous injection or infusion, similar pharmaceutical vehicles, such as water, glycols, oils, buffers, sugars, preservatives, liposomes, etc., known to those skilled in the art may be used. Examples of such parenteral compositions include, but are not limited to, dextrose 5% w / v, saline or other solutions. The total dose of the polypeptide variant of neuregulin-1 or the nucleic acid encoding the polypeptide variant, alone or in combination with other drugs to be administered, may be administered in a vial of intravenous infusion ranging from about 1 ml to 2000 ml. The volume of diluent liquid varies according to the total dose to be administered.

[0158] Additionally, neuregulin variants may be capable of promoting survival, proliferation and / or differentiation of cells that have the appropriate ErbB receptor. The phrase "promoting survival of a cell" refers to extending the existence of a cell, either in vitro or in vivo, as compared to the existence of a cell that has not been exposed to a neuregulin variant (an "untreated cell").

[0159] The phrase "promoting cell proliferation" refers to increasing the rate or number of mitoses, either in vitro or in vivo, as compared to untreated cells. Increased cell proliferation in cell culture can be detected by counting the number of cells before and after exposure to a neuregulin variant, or by examining the degree of confluency by microscopy. Cell proliferation is the proliferation of cells by a cell. 3 It can also be quantified by measuring 3H-thymidine incorporation.

[0160] The phrase "promoting differentiation of a cell" refers to increasing the degree of specialization of a cell. Cell specialization is characterized by the acquisition of one or more characteristics that differ from those of the original cell. Thus, the degree of specialization of a cell is generally determined by screening for changes in the phenotype of the cell (e.g., identifying changes in cell morphology).

[0161] The present invention is not limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims. EXAMPLES

[0162] Example 1: Yeast display engineering and generation of NRG variants Neuregulin variant sequences were displayed on the yeast surface through fusion to alpha agglutinin. Degenerate codons were introduced at structurally identified positions to either alternate between NRG1 and NRG4 sequences or alternately replace the full 20 amino acid randomization through the use of the NNK codon. Three visualization markers were used for the selection of binding-competent / selectable sequences. The first marker was an anti-HA antibody conjugated to Brilliant Violet 421 to measure the surface display level of neuregulin variants. The second marker was a soluble recombinant ErbB3 receptor ECD conjugated to Alexafluor 488 for negative selection. The third marker was a soluble recombinant ErbB4 receptor ECD conjugated to Alexafluor 647 for positive selection. Multiple rounds of positive selection with a constant ErbB4 concentration (2.5 nM) and negative selection against increasing ErbB3 concentrations (250, 500, 750 nM) resulted in selective neuregulin variants. From the gblock DNA, selected neuregulin variants were cloned into mammalian expression vectors using standard Golden Gate cloning methods. Final constructs incorporated various NRG domain truncation points, various linkers and different fusion domains and tags (Fc, scFc, 6xhis) as shown in Table 1. Stable expression was performed in suspension adapted CHO K1 cell lines using standard Lipofectamine methods under puromycin selection. After 7 days of production, proteins from filtered conditioned media were purified through sequential individual column methods of triple tandem LFAS system (ProA, In Line Dilution, SEC) or ProA (or NiNTA for 6xHis tagged proteins), buffer exchange, CIEX and / or HIC followed by formulation into 10 mM 9% sucrose acetate pH 5.2 buffer. Protein concentration was determined by A280.

[0163] In vitro phosphorylation of Akt in neonatal rat cardiomyocytes and rat Schwann cells by neuregulin variants The specificity of neuregulin variants for cardiomyocytes (ErbB4) compared to Schwann cells (ErbB3) was tested by measuring Akt activity generated by the formation of ErbB2 / ErbB4 and ErbB2 / ErbB3 complexes. Upon activation by neuregulin, ErbB4 and ErbB3 receptors preferentially dimerize with ErbB2 coreceptors to form ErbB2 / ErbB4 complexes in cardiomyocytes and ErbB2 / ErbB3 complexes in Schwann cells, both of which signal through the Akt pathway. Neonatal rat cardiomyocytes were isolated from hearts of 0-4 day old Sprague Dawley rat pups using the Neomyt kit according to the manufacturer's protocol and plated on Surecoat plates in NS medium containing serum (all from Cellultron Life Technologies, Baltimore, MD). Rat Schwann cells were seeded on poly-D-lysine coated plates with Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (Thermo Fisher) and forskolin (Millipore Sigma). After 24 hours of incubation in a humidified incubator at 37°C and 5% CO2, cardiomyocytes and Schwann cells were washed with serum-free medium and incubated for another 16 hours in DMEM supplemented with bovine serum albumin (Sigma). Both cell lines were then treated with a titration of neuregulin variant molecules for 15 minutes, whereupon cells were lysed in lysis buffer (Meso Scale Discovery (MSD)). For detection of Akt phosphorylation, lysates were added to Multi-Spot 96-Well 4-Spot Phospho(Serine 473) / Total Akt plates (MSD), incubated according to the manufacturer's protocol, and read on a SECTOR Imager 6000 (MSD). For both cellular assays, percent phosphorylation of Akt was calculated by multiplying the phospho-Akt signal by 2, then dividing by the total Akt signal plus the phospho-Akt signal, and multiplying by 100.The Akt phosphorylation data points were fitted using log(agonist) vs response, variable slope (4 parameter) analysis in GraphPad Prism to obtain EC. 50 Curves were generated and CM% agonism reflects the top value of the data points.

[0164] The results are shown in Table 3 below.

[0165] [Table 20]

[0166] [Table 21]

[0167] [Table 22]

[0168] [Table 23]

[0169] The neuregulin variants tested generally showed improved selectivity for Erbb4, with many having selectivities, expressed as SM / CM ratios, of over 1000 and even 10000. Many of the neuregulin variants also showed agonism for cardiomyocytes that was at least 80% compared to the wild-type sequence.

[0170] These results demonstrate that neuregulin variants can be designed to be selective for cardiac cells, suggesting that these neuregulin variants may be candidates for the treatment of cardiac disease and may exhibit fewer side effects on neural cells.

[0171] Example 2: ErbB4 selective agonist efficacy in a rat myocardial infarction (MI) model An ErbB4 selective agonist (huNRG1(S177-F229)(NRGE1C)((G4E)2)::G4::huFcSEFL2(Pb)) was tested in a rat MI model to determine whether benefit might be seen in improving cardiac function.

[0172] Surgically induced myocardial infarction (MI) Sprague Dawley (SD) rats (body weight 180-200 g) were purchased from ENVIGO (Indianapolis, Indiana, USA). Fourteen days before MI surgery, rats underwent echocardiography to assess cardiac function by ejection fraction (EF%). Noninvasive parasternal long axis B-mode cine loops were obtained using a Vevo 2100 system (VisualSonics Inc., Toronto, Canada). EF was calculated using the manufacturer's recommended method. Rats were randomized into three groups based on baseline ejection fraction numbers: 1. vehicle; 2. ErbB4 agonist 10 μg / kg; 3. ErbB4 agonist 20 μg / kg. After randomization at week 2, 16 rats were subcutaneously (SC) administered vehicle or ErbB4 agonist at 10 μg / kg or 20 μg / kg.

[0173] Since exposure to anesthesia is known to affect cardiac function, isoflurane was maintained at 2.5% during echocardiography to minimize its effect on cardiac function. Duration of isoflurane exposure per animal was <10 min during echocardiography. Animals were closely monitored throughout the study. No adverse events (death / morbidity) were observed throughout the study.

[0174] After randomization at week 2, baseline cardiac function and heart rate of all groups were similar. All enrolled rats showed heart failure status. Terminal serum exposure was significantly higher in the ErbB4-treated group compared to the vehicle group (Figure 1A). Plasma exposure level of ErbB4 was significantly higher in the 20 μg / kg group than in the 10 μg / kg group (Figure 2A). One week after subcutaneous (SC) administration, ErbB4 selective agonist improved cardiac function in the ErbB4 selective agonist-treated group compared to the vehicle group (Figure 1B). No statistically significant effect of ErbB4 selective agonist on heart rate was observed (Figure 1C), suggesting that the improvement of cardiac function mediated by ErbB4 selective agonist was not due to heart rate variability between groups. Although plasma exposure is higher at 20 μg / kg than at 10 μg / kg, both treatment groups show similar cardiac function improvements. This suggests that higher plasma ErbB4 selective agonist exposure (>1.9 ng / mL) may be required to induce significantly greater cardiac function than that observed at 10 μg / kg. No adverse events (death / morbidity) were observed following ErbB4 selective agonist treatment.

[0175] Treatment with an ErbB4 selective agonist significantly improves cardiac function in a rat myocardial infarction model. No significant effect on heart rate was observed after treatment with an ErbB4 selective agonist. ErbB4 treatment was not associated with mortality or morbidity in the myocardial infarction model.

[0176] Example 3: Fc-free ErbB4 selective agonist, effects in a rat myocardial infarction (MI) model An ErbB4 selective agonist (huNRG1(S177-F229)(NRGE1C)(E47S_D48A_F49L_M50T)6xHis:SEQ ID NO:61) was tested in a rat MI model to determine whether a benefit on cardiac function could be observed in response to treatment.

[0177] Sprague Dawley (Envigo, IN) rats (body weight 142-280 g) underwent surgery to induce MI. Seven days after MI surgery, rats underwent echocardiography to assess baseline cardiac function by ejection fraction (EF%). Noninvasive parasternal long-axis B-mode cine loops were obtained using a Vevo 3100 system (VisualSonics Inc., Toronto, Canada). EF% was calculated using the manufacturer's recommended method. Rats were randomly assigned to groups based on baseline EF%: [1. Vehicle]; [2. ErbB4 agonist 50 μg / kg]; [3. ErbB4 agonist 150 μg / kg]; [4. ErbB4 agonist 500 μg / kg]. After randomization 8 days after MI surgery, all enrolled rats presented with cardiac dysfunction, underwent surgery for jugular vein cannulation for IV dose administration, and began dosing as per treatment group label: vehicle or ErbB4 agonist 50 μg / kg / day, 150 μg / kg / day, or 500 μg / kg / day administered once daily via the cannulated jugular vein for 10 days.

[0178] Serum exposure is determined in blood samples collected at 10 minutes on day 10 post-treatment, and data from echocardiograms performed on day 11 and approximately 4 weeks after the start of dosing are analyzed to assess effects on cardiac function, EF%.

Claims

**Claim 1** A neuregulin variant comprising the amino acid sequence of the following formula: SHLVKCX １８３ EX １８５ X １８６ KX １８８ FCVNGGECX １９７ X １９８ X １９９ X ２００ X ２０１ X ２０２ SX ２０４ PSR X ２０８ LCKCPNEFTGDRCX ２２２ X ２２３ X ２２４ X ２２５ X ２２６ ASX ２２９ (SEQ ID NO: 177) (wherein, X １８３ is A or G; X １８５ X １８６ is KD, KE, KH, ND, NE, NH, RD, RE, RH, RQ, SD, SE or SH; X １８８ is S or T; X １９７ X １９８ X １９９ X ２００ X ２０１ X ２０２ is FMIEDS (SEQ ID NO: 178), FMIEGP (SEQ ID NO: 179), FMIEHL (SEQ ID NO: 180), FMVEDL (SEQ ID NO: 181), FMVERS (SEQ ID NO: 182), FMVKRP (SEQ ID NO: 183), FVIEDP (SEQ ID NO: 184), FVIEGS (SEQ ID NO: 185), FVVEGL (SEQ ID NO: 186), YMIEDL (SEQ ID NO: 187), YMIEGL (SEQ ID NO: 188), YMIEHP (SEQ ID NO: 189), YMVEDL (SEQ ID NO: 190), YMVEGS (SEQ ID NO: 191), YMVERP (SEQ ID NO: 192), YVIEDS (SEQ ID NO: 193), YVIEGS (SEQ ID NO: 194), YVIEHL (SEQ ID NO: 195), YVVEDL (SEQ ID NO: 196), YVVEHS (SEQ ID NO: 197) or YVVERP (SEQ ID NO: 198); X ２０４ is I or N, X ２０８ is F or Y; X ２２２ X ２２３ X ２２４ X ２２５ X ２２６ is EKDVM (SEQ ID NO: 199), QAPHI (SEQ ID NO: 200), QDDFM (SEQ ID NO: 201), QDFFL (SEQ ID NO: 202), QDFFM (SEQ ID NO: 203), QDVFL (SEQ ID NO: 204), QDVFM (SEQ ID NO: 205), QEDFM (SEQ ID NO: 206), QETQI (SEQ ID NO: 207), QKDFL (SEQ ID NO: 208), QKDFM (SEQ ID NO: 209), QKDVDM (SEQ ID NO: 210), QKFFL (SEQ ID NO: 211), QKFFM (SEQ ID NO: 212), QKLDI (SEQ ID NO: 213), QKTSL (SEQ ID NO: 214), QKVFL (SEQ ID NO: 215), QKVFM (SEQ ID NO: 216), QKVVM (SEQ ID NO: 217), QNDFL (SEQ ID NO: 218), QNDFM (SEQ ID NO: 219), QNVFL (SEQ ID NO: 220), QNVFM (SEQ ID NO: 221), QNYVM (SEQ ID NO: 222), QQSFP (SEQ ID NO: 223), QSALT (SEQ ID NO: 224), QSSel (SEQ ID NO: 225), QSTRV (SEQ ID NO: 226), QTSLL (SEQ ID NO: 227) or QVTRL (SEQ ID NO: 228); It should be noted that there may be a misspelling in "QSSel" in the original text. It might be "QSSEL". If this is an important correction for the accurate understanding of the content, please adjust accordingly. X ２２９ either does not exist or is F or FYKAEELYQ (SEQ ID NO: 229). **Claim 2** The neuregulin variant according to claim 1, comprising any one of the amino acid sequences of SEQ ID NOs: 114 to 176. **Claim 3** (wherein, X １８３ is A or G; X １８５ X １８６ is KD, KE, KH, ND, NH, RD, RE, RH, RQ, SD, SE or SH; X １８８ is S or T; X １９７ X １９８ X １９９ X ２００ X ２０１ X ２０２ is FMIEDS (SEQ ID NO: 178), FMIEGP (SEQ ID NO: 179), FMIEHL (SEQ ID NO: 180), FMVEDL (SEQ ID NO: 181), FMVERS (SEQ ID NO: 182), FMVKRp (SEQ ID NO: 183), FVIEDP (SEQ ID NO: 184), FVIEGS (SEQ ID NO: 185), YMIEDL (SEQ ID NO: 187), YMIEGL (SEQ ID NO: 188), YMIEHP (SEQ ID NO: 189), YMVEDL (SEQ ID NO: 190), YMVEgs (SEQ ID NO: 191), YMVERP (SEQ ID NO: 192), YVIEDS (SEQ ID NO: 193), YVIEGS (SEQ ID NO: 194), YVIEHL (SEQ ID NO: 195), YVVehs (SEQ ID NO: 197) or YVVERP (SEQ ID NO: 198); X ２０４ is I or N, and X ２０８ is F or Y; X ２２２ X ２２３ X ２２４ X ２２５ X ２２６ is EKDVM (SEQ ID NO: 199), QAPHI (SEQ ID NO: 200), QDFFL (SEQ ID NO: 202), QDFFM (SEQ ID NO: 203), QDVFL (SEQ ID NO: 204), QEDFM (SEQ ID NO: 206), QETQI (SEQ ID NO: 207), QKDFL (SEQ ID NO: 208), QKDFM (SEQ ID NO: 209), QKDVDM (SEQ ID NO: 210), QKFFL (SEQ ID NO: 211), QKFFM (SEQ ID NO: 212), QKLDI (SEQ ID NO: 213), QKTSL (SEQ ID NO: 214), QKVFL (SEQ ID NO: 215), QNDFL (SEQ ID NO: 218), QNDFM (SEQ ID NO: 219), QNVFL (SEQ ID NO: 220), QNVFM (SEQ ID NO: 221), QNYVM (SEQ ID NO: 222), QQSFP (SEQ ID NO: 223), QSALT (SEQ ID NO: 224), QSSEL (SEQ ID NO: 225), QSTRV (SEQ ID NO: 226), QTLL (SEQ ID NO: 227) or QVTRL (SEQ ID NO: 228); X ２２９ is F or FYKAEELYQ (SEQ ID NO: 229), The neuregulin variant according to claim 1. **Claim 4** The neuregulin variant according to claim 3, comprising any one of the amino acid sequences of SEQ ID NOs: 114, 115, 117 to 124, 126 to 135, 138, 139, 144 to 147, 149 to 156, 160 to 168, or 171 to 176. **Claim 5** (wherein, X １８３ is A or G; X １８５ is KE, KH, ND, RD, RH, RQ, SD, SE or SH; X １８８ is S or T; X １９７ X １９８ X １９９ X ２００ X ２０１ X ２０２ is FMIE DS (SEQ ID NO: 178), FMIE GP (SEQ ID NO: 179), FMVEDL (SEQ ID NO: 181), FMVKRP (SEQ ID NO: 183), FVIEDP (SEQ ID NO: 184), FVIE GS (SEQ ID NO: 185), YMIE DL (SEQ ID NO: 187), YMIE HP (SEQ ID NO: 189), YMVEDL (SEQ ID NO: 190), YMVE GS (SEQ ID NO: 191), YMVERP (SEQ ID NO: 192), YVIED S (SEQ ID NO: 193), YVIE GS (SEQ ID NO: 194), YVIE HL (SEQ ID NO: 195), YVVEH S (SEQ ID NO: 197) or YVVERP (SEQ ID NO: 198); X ２０４ is I or N, X ２０８ is F or Y; X ２２２ X ２２３ X ２２４ X ２２５ X ２２６ is EKDVM (SEQ ID NO: 199), QAPHI (SEQ ID NO: 200), QEDFM (SEQ ID NO: 206), QETQI (SEQ ID NO: 207), QKDFL (SEQ ID NO: 208), QKDFM (SEQ ID NO: 209), QKDVM (SEQ ID NO: 210), QKFFL (SEQ ID NO: 211), QKFFM (SEQ ID NO: 212), QKLDI (SEQ ID NO: 213), QKTSL (SEQ ID NO: 214), QKVFL (SEQ ID NO: 215), QNDFL (SEQ ID NO: 218), QNDFM (SEQ ID NO: 219), QNVFL (SEQ ID NO: 220), QNVFM (SEQ ID NO: 221), QNYVM (SEQ ID NO: 222), QQSF P (SEQ ID NO: 223), QSALT (SEQ ID NO: 224), QSS EL (SEQ ID NO: 225), QSTRV (SEQ ID NO: 226), QTSLL (SEQ ID NO: 227) or QVTRL (SEQ ID NO: 228); X ２２９ The neuregulin variant according to claim 1, wherein X is F or FYKAEELYQ (SEQ ID NO: 229). **Claim 6** The neuregulin variant according to claim 5, comprising any one of the amino acid sequences of SEQ ID NOs: 117 to 124, 126 to 135, 144 to 147, 149 to 152, 154 to 156, 163, 167, 168, or 171 to 176. **Claim 7** (wherein, X １８３ is A or G; X １８５ X １８６ is KH, ND, RD, RH or SH; X １８８ is S or T; X １９７ X １９８ X １９９ X ２００ X ２０１ X ２０２ is FMIEDS (SEQ ID NO: 178), FMIEGP (SEQ ID NO: 179), FMVEDL (SEQ ID NO: 181), FVIEDP (SEQ ID NO: 184), YMIEDL (SEQ ID NO: 187), YMVEGS (SEQ ID NO: 191), YVIEGS (SEQ ID NO: 194), YVIEHL (SEQ ID NO: 195) or YVVERP (SEQ ID NO: 198); X ２０４ is I or N, and X ２０８ is F or Y; X ２２２ X ２２３ X ２２４ X ２２５ X ２２６ is QAPHI (SEQ ID NO: 200), QEDFM (SEQ ID NO: 206), QETQI (SEQ ID NO: 207), QKDFL (SEQ ID NO: 208), QKDFM (SEQ ID NO: 209), QKTSL (SEQ ID NO: 214), QNDFL (SEQ ID NO: 218), QNDFM (SEQ ID NO: 219), QQSFP (SEQ ID NO: 223), QSALT (SEQ ID NO: 224), QSSEL (SEQ ID NO: 225), QSTRV (SEQ ID NO: 226), QTSLL (SEQ ID NO: 227) or QVTRL (SEQ ID NO: 228); X ２２９ is F or FYKAEELYQ (SEQ ID NO: 229), The neuregulin variant according to claim 1. **Claim 8** The neuregulin variant according to claim 7, comprising any one of the amino acid sequences of SEQ ID NOs: 117 to 124, 126, 128 to 135, 146, 147, 150, 151, 163, 167, 168, or 171. **Claim 9** The neuregulin variant according to claim 8, comprising any one of the amino acid sequences of SEQ ID NOs: 117, 118, 119, 120, 122, 123, 124, 126, 128, 129, 130, 131, 133, or 135. **Claim 10** The neuregulin variant according to claim 1, further comprising a second amino acid sequence comprising a signal sequence, a half-life extension portion, or a purification tag. **Claim 11** The neuregulin variant according to claim 9, wherein the second amino acid sequence comprises an Fc region or a His tag. **Claim 12** The neuregulin variant according to claim 11, comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 24, 29 to 54, 56 to 67, 69 to 80, 82 to 111. **Claim 13** The neuregulin variant according to claim 11, comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 15, 19, 20, 22, 23, 29 to 34, 37, 38, 43 to 54, 56 to 59, 61 to 67, 69 to 80, 82 to 85, 87 to 93, or 97 to 111. **Claim 14** The neuregulin variant according to claim 11, comprising an amino acid sequence of any one of SEQ ID NO: 3, 4, 6, 9, 11 to 13, 19, 22, 31 to 34, 43 to 48, 52, 54, 56 to 59, 61 to 63, 65 to 67, 71, 72, 75 to 80, 84, 85, 88, 100 or 104 to 106.

15. The neuregulin variant according to claim 11, comprising an amino acid sequence of any one of SEQ ID NO: 3, 4, 6, 9, 11 to 13, 19, 22, 31 to 34, 43 to 48, 52, 54, 56 to 59, 61 to 63, 65 to 67, 71, 72, 75 to 80, 84, 85, 88, 100 or 104 to 106.

16. The neuregulin variant according to claim 15, comprising an amino acid sequence of SEQ ID NO: 4, 11, 13, 32, 34, 45, 46, 48, 52, 72, 75, 76, 77, 78, 79 or 80.

17. The neuregulin variant according to claim 15, comprising an amino acid sequence of SEQ ID NO: 56, 57, 58, 59, 61, 62, 63, 65, 66 or 67.

18. A pharmaceutical composition comprising the neuregulin variant according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to claim 18, which is used for treating a cardiovascular disease or condition in a subject in need of treatment for a cardiovascular disease or condition.

20. The pharmaceutical composition according to claim 19, wherein the cardiovascular disease or condition is heart failure, myocardial infarction, dilated or hypertrophic cardiomyopathy, myocarditis or cardiotoxicity.

21. The pharmaceutical composition according to claim 19, wherein the subject is a human.

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