Methods for Treating Fibrosis and Arrhythmia with Neuregulin-1 Fusion Proteins

Abstract

The present invention relates to a recombinant fusion protein comprising a fragment of the cardioprotective protein Neuregulin-1 (NRG-1) fused to a HER3 monoclonal antibody (mAb) backbone, and a method for treating atrial fibrillation and / or cardiac fibrosis in a subject in need of such treatment comprising administering a therapeutically effective amount of the recombinant fusion protein disclosed herein or a pharmaceutical composition comprising said recombinant fusion protein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 319,886, filed March 15, 2022, and No. 63 / 385,705, filed December 1, 2022, the contents of each of which are incorporated by reference in their entirety herein.

[0002] Incorporation by reference of sequence listing This application contains a Sequence Listing that was submitted in XML format via EFS-Web and is incorporated by reference in its entirety. The XML copy, created on Mar. 12, 2023, is named SBTI-003-001WO_SeqList_ST26.xml and is 24,646 bytes in size. [Background technology]

[0003] 2. Background of the Invention Neuregulin (NRG, heregulin, HRG), also known as glial growth factor (GGF) and novel differentiation factor (NDF), is a glycoprotein with a molecular weight of 44 KD. There are four members in the NRG protein family: NRG-1, NRG-2, NRG-3, and NRG-4. NRGs (including NRG-1) play a particularly important role in cardiac development. As a ligand for the ErbB family of tyrosine kinase receptors, NRG-1 directly binds to membrane-bound ErbB3 or ErbB4 and induces dimerization to form ErbB2 / ErbB4, ErbB2 / ErbB3, ErbB3 / ErbB3, and ErbB4 / ErbB4 complexes, which then trigger intracellular signal transduction. In animal models, expression of NRG induces paracrine signaling to promote cardiac tissue proliferation and differentiation during embryonic development, and deletion of either ErbB2, ErbB4, or NRG-1 leads to embryonic lethality. Furthermore, cancer therapies that block ErbB2 receptor signaling have been shown to have significant cardiotoxic side effects, and in humans, signaling through ErbB2 has been demonstrated to be essential for the homeostasis as well as the development of healthy cardiac tissue.

[0004] Evidence also indicates that NRG-1 signaling is involved in the development and function of other organ systems, as well as the development of human diseases, including schizophrenia and head and neck cancer. There are many isoforms of NRG-1. Studies of genetically mutant mice (gene knockout mice) have shown that isoforms with different N-terminal regions or EGF-like domains have different in vivo functions. The present invention is based on the NRG-1βa2 isoform.

[0005] Endogenous NRG-1 binds to and induces signaling through both ErbB3 (HER3) and ErbB4 (HER4). Numerous preclinical and clinical studies have demonstrated the therapeutic potential of NRG-1 across a range of cardiovascular indications, primarily through its interaction with cardiomyocyte-expressed ErbB4 (HER4). However, three major factors limit the clinical application and utility of recombinant human NRG-1 (rhNRG-1). First, NRG-1 signaling through HER3 may promote cancer initiation and progression, raising major concerns for applications requiring chronic administration and / or without significant cardiovascular (CV) risk factors. Second, overactivation of HER3 by NRG-1 may disrupt the integrity and homeostasis of the gastrointestinal (GI) epithelium, causing severe GI toxicity and thus losing the therapeutic window of NRG-1. Third, both clinical-stage active protein fragments of rhNRG-1 have been shown to have short half-lives, potentially requiring burdensome dosing and administration schedules to achieve the desired therapeutic levels of exposure. There is therefore a need to provide NRG-1-based therapeutics that have lower risk of carcinogenesis or promotion of cancer progression, better GI tolerability, and more favorable pharmacokinetic (PK) profiles, while maintaining clinically important therapeutic potential across a range of cardiovascular indications.

[0006] The present invention addresses these needs by providing a recombinant protein comprising a fusion of the rhNRG-1 active domain with a HER3-specific antagonist antibody: HER3 signaling is blocked to reduce the oncogenic risk and GI toxicity of rhNRG-1, while the antibody backbone format confers the molecular half-life of a typical monoclonal antibody, allowing for more convenient dosing and administration of the product. Summary of the Invention

[0007] The present disclosure provides methods of treating atrial fibrillation and / or cardiac fibrosis in a subject, the methods comprising administering to the subject a recombinant fusion protein comprising a fragment of Neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb). In some embodiments, the cardiac fibrosis comprises atrial fibrosis.

[0008] The present disclosure provides a method of treating atrial fibrillation in a subject, the method comprising administering to the subject a recombinant fusion protein comprising a fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb). In some embodiments, the atrial fibrillation is associated with atrial fibrosis.

[0009] The present disclosure provides a method for treating atrial fibrosis in a subject, the method comprising administering to the subject a recombinant fusion protein comprising a fragment of Neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb).

[0010] The present disclosure provides a recombinant fusion protein comprising a fragment of Neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb) for use in a method for treating atrial fibrillation and / or cardiac fibrosis. In some embodiments, the cardiac fibrosis comprises atrial fibrosis.

[0011] In some embodiments of the recombinant fusion protein for the method or use of the present disclosure, the NRG-1 fragment comprises an active domain of NRG-1. In some embodiments, the NRG-1 fragment comprises an ERBB3 / 4 binding domain. In some embodiments, the NRG-1 fragment binds to ErbB4 (HER4) and induces signaling through ErbB4. In some embodiments, the mAb inhibits NRG-1 signaling through ErbB3 (HER3). In some embodiments, the NRG-1 fragment comprises the NRG-1β2a isoform.

[0012] In some embodiments of the recombinant fusion protein for the method or use of the present disclosure, the NRG-1 fragment is fused via its N-terminal amino acid to the C-terminus of the antibody heavy chain using a linker. In some embodiments, the linker comprises at least one copy of the Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser linker shown in SEQ ID NO:5. In some embodiments, the C-terminus of the antibody heavy chain comprises the Fc domain of the antibody. In some embodiments, the monoclonal antibody is glycosylated.

[0013] In some embodiments of the recombinant fusion protein for the method or use of the disclosure, the NRG-1 fragment comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the mAb comprises the heavy chain amino acid sequence of SEQ ID NO:2. In some embodiments, the mAb comprises the light chain amino acid sequence of SEQ ID NO:3. In some embodiments, the mAb comprises a substitution mutation at at least one of amino acids 234, 239, and 434 of SEQ ID NO:2. In some embodiments, the at least one substitution mutation comprises a L234F mutation, a S239A mutation, a N434A mutation, or a combination thereof. In some embodiments, the recombinant fusion protein comprises the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:14.

[0014] In some embodiments of the recombinant fusion protein for the method or use of the present disclosure, the recombinant fusion protein promotes HER2 / 4 signaling over HER2 / 3 signaling, compared to the signal induction ability of recombinant NRG-1.In some embodiments, the recombinant fusion protein promotes the proliferation, differentiation, and survival of cardiomyocytes or cardiac tissue in a subject.In some embodiments, the recombinant fusion protein attenuates the proliferation of tumor or cancer cells, compared to recombinant NRG-1.

[0015] In some embodiments of the recombinant fusion protein for the method or use of the present disclosure, administration of the recombinant fusion protein shortens the duration of atrial fibrillation episodes or reduces the frequency of occurrence of atrial fibrillation. In some embodiments, administration of the recombinant fusion protein reduces the signs or symptoms of atrial fibrillation or atrial fibrosis. In some embodiments, the symptoms of atrial fibrillation include arrhythmia, palpitations, lightheadedness, extreme fatigue, shortness of breath, chest pain, or a combination thereof. In some embodiments, the signs of atrial fibrosis include collagen deposition, and administration of the recombinant fusion protein reduces collagen deposition in atrial tissue.

[0016] In some embodiments, NRG-1 binds to ErbB4 (HER4) and induces signaling through ErbB4. In some embodiments, the mAb inhibits NRG-1 signaling through ErbB3 (HER3).

[0017] The present disclosure provides kits comprising an effective amount of a recombinant fusion protein of the invention or a pharmaceutical composition comprising a recombinant fusion protein of the invention.

[0018] The present disclosure provides a recombinant fusion protein comprising a fragment of Neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb) for use in the manufacture of a medicament for the treatment of atrial fibrillation and / or cardiac fibrosis.

[0019] Other features and advantages of the present invention will become apparent from the following detailed description, examples, and drawings. It should be understood, however, that the detailed description and specific examples illustrating embodiments of the present invention are provided for purposes of illustration only, and that various changes and modifications will become apparent to those skilled in the art from this detailed description, within the spirit and scope of the invention. [Brief description of the drawings]

[0020] Various objects and advantages of the present invention as well as a more complete understanding thereof will become apparent and will be more readily appreciated by reference to the following detailed description and appended claims when taken in conjunction with the accompanying drawings.

[0021] [Figure 1] FIG. 1 shows the construction of an expression plasmid for expressing the recombinant fusion proteins disclosed herein.

[0022] [Figure 2AB] Figure 2A shows a molecular schematic of the anti-HER3 mAb / NRG-1 fusion protein of the present disclosure, and Figure 2B shows representative data generated by SDS-PAGE analysis.

[0023] [Figure 2C] FIG. 2C shows the results of a Western blot detected with a primary antibody specific for a 61-amino acid active fragment of NRG-1 ("NRG-1," R&D Systems, Minneapolis, MN) that contains the HER3 / 4 binding domain.

[0024] [Figure 2D] FIG. 2D shows the results of a Western blot detected with a primary antibody specific for IgG.

[0025] [Diagram 3] 3 shows a binding analysis demonstrating that the recombinant fusion protein disclosed herein binds to HER3 protein (curve 1, step 2) and can simultaneously bind to anti-NRG-1 antibody (curve 1, step 3). It is noted that Fc mutations have been introduced into the recombinant fusion protein disclosed herein to knock out the Fc effector function of the parent antibody sequence encoding the HER3 specific antibody, which may mitigate undesired cytotoxicity against normal tissues expressing the HER3 receptor.

[0026] [Figure 4AB]Figures 4A-4D show representative graphs showing the mean relative proliferation rate ± SEM (n=3) of various cancer cell lines treated with anti-HER3 mAb / NRG-1 fusion protein or control. Figure 4A shows the mean relative proliferation rate of the NCI-N87 gastric cancer cell line. Figure 4B shows the mean relative proliferation rate of the MCF-7 breast cancer cell line.

[0027] [Figure 4C-D] Figures 4A-4D show representative graphs depicting the mean relative proliferation rate ± SEM (n=3) of various cancer cell lines treated with anti-HER3mAb / NRG-1 fusion protein or control. Figure 4C shows the mean relative proliferation rate of the RT-112 bladder cancer cell line. Figure 4D shows the mean relative proliferation rate of the T47D breast cancer cell line. Compared to the control NRG-1 peptide and GP120mAb / NRG-1 fusion protein, the recombinant fusion proteins provided herein show significantly less activity in promoting cancer cell proliferation.

[0028] [Diagram 5] Figures 5A-5B illustrate that despite reduced proliferation of cancer cells, the recombinant fusion proteins provided herein fully retain the ability to induce PI3K / AKT signaling in cardiomyocytes, exhibiting comparable activity to recombinant NRG-1 and GP120mAb / NRG-1 fusion proteins. Figure 5A is a plot showing the relative ratio of phosphorylated AKT (pAKT) and total AKT (tAKT) versus antibody concentration (nM) in human cardiomyocytes treated with recombinant fusion proteins of the present disclosure and controls. Figure 5B is a Western blot analysis of AKT phosphorylation in human cardiomyocytes treated with recombinant fusion proteins of the present disclosure and controls.

[0029] [Figure 6AB]Figures 6A-6C show a direct comparison of HER2 / 4 and HER2 / 3 dimerization in the presence of recombinant fusion proteins disclosed herein and controls. Figure 6A shows the assay principle for detecting ligand-induced dimerization. The PathHunter dimerization assay, developed by Eurofins DiscoverX (Fremont, CA), is used to detect ligand-induced dimerization of the two subunits of a receptor-dimer pair. The β-gal enzyme is split into two fragments, ProLink (PK) and enzyme acceptor (EA). Cells are engineered to co-express target protein 1 fused to the enzyme donor PK and target protein 2 fused to the enzyme acceptor EA. When the ligand binds to one target protein, it is induced to interact with the other target protein, forcing the complementation of the two enzyme fragments, thus causing the enzymatic reaction to emit a chemiluminescent signal, which is detected as relative fluorescent units (RFU). FIG. 6B is a plot showing that the recombinant fusion proteins provided herein can induce HER2 / HER4 dimerization with potency comparable to that of NRG-1. [Figure 6C] Figures 6A-6C show a direct comparison of HER2 / 4 and HER2 / 3 dimerization in the presence of recombinant fusion proteins disclosed herein and a control. Figure 6C is a plot showing that the recombinant fusion proteins provided herein are significantly less able to induce HER2 / HER3 dimerization than NRG-1. These findings further support that the recombinant fusion proteins provided herein significantly reduce HER2 / 3 signaling induction while maintaining the full HER2 / 4 signaling capacity of NRG-1.

[0030] [Figure 7]Figure 7 shows the binding affinity of the anti-HER3mAb / NRG-1 fusion protein of the present invention to the HER3 antigen across various species including human, monkey, rat, and mouse. The equilibrium dissociation rates (KD) determined by BIAcore analysis are 3.13x10-10 (human), 3.97x10-10 (monkey), 2.68x10-9 (rat), and 2.77x10-9 (mouse), respectively. These data indicate that the recombinant fusion protein of the present invention has similar binding affinity to human and monkey HER3, while the affinity to rodent (rat and mouse) HER3 is approximately one order of magnitude lower.

[0031] [Figure 8] FIG. 8 is a graph showing the effect of recombinant fusion proteins on ejection fraction (EF) in a rat model of systolic heart failure induced by coronary artery ligation.

[0032] [Figure 9] Figures 9A-9F are a series of six images showing histopathological changes of myocardial structure in a rat model of systolic heart failure induced by coronary artery ligation. Cardiac tissue adjacent to the surgical site was harvested and fixed in 4% formaldehyde, then paraffin sectioned and stained with H&E. Figure 9A shows the cardiac tissue of a sham-operated control rat. Figure 9B shows the cardiac tissue of a systolic heart failure model rat treated with vehicle control. Figure 9C shows the cardiac tissue of a systolic heart failure model rat treated with GP120mAb / NRG-1 (10mg / kg). Figure 9D shows the cardiac tissue of a systolic heart failure model rat treated with anti-HER3mAb / NRG-1 (1mg / kg). Figure 9E shows the cardiac tissue of a systolic heart failure model rat treated with anti-HER3mAb / NRG-1 (3mg / kg). FIG. 9F shows cardiac tissue from a rat model of systolic heart failure treated with anti-HER3 mAb / NRG-1 (10 mg / kg).

[0033] [Figure 10] FIG. 10 is a graph showing the evaluation of in vivo antitumor activity using a subcutaneous FaDu cancer xenograft model in NOD / SCID mice.

[0034] [Figure 11] FIG. 11 is a graph showing the weight change of tumor-bearing mice treated with a recombinant fusion protein of the present disclosure and a control.

[0035] [Figure 12] FIG. 12 is a graph showing the pharmacokinetic profile of the recombinant fusion protein in cynomolgus monkeys (macaques).

[0036] [Figure 13A] FIG. 13A shows a diagram illustrating a fibrosis assay in which fibrosis was induced in vitro in rat atrial tissue samples.

[0037] [Figure 13B] Figure 13B shows a graph depicting type I collagen induction in the absence of an exemplary NRG-1 / HER3 antibody fusion protein in the assay shown in Figure 13A. The x-axis indicates the day the samples were taken. The y-axis represents type I collagen (type I collagen alpha 1 chain, or Col1a1, mRNA) mRNA expression as the fold change in Col1a1 mRNA present relative to the level of Col1a1 mRNA present on day 1 of the assay.

[0038] [Figure 13C] Figure 13C shows a graph depicting induction of collagen III in the absence of an exemplary NRG-1 / HER3 antibody fusion protein in the assay shown in Figure 13A. The x-axis shows the day the sample was taken. The y-axis represents mRNA expression of collagen III (type III collagen alpha 1 chain, or Col3a1, mRNA) as the fold change in Col3a1 mRNA present relative to the level of Col3a1 mRNA present on day 1 of the assay.

[0039] [Figure 14A]Figure 14A shows a graph depicting the effect of NRG-1 / HER3 antibody fusion protein (NRG-1 / HER3) on induction of collagen type I in the assay shown in Figure 13A. The x-axis indicates the day the samples were taken. The y-axis represents collagen type I mRNA expression as the fold change in Col1a1 mRNA present relative to the level of Col1a1 mRNA present on day 2 of the assay in the absence of NRG-1 / HER3.

[0040] [Figure 14B] Figure 14B shows a graph depicting the effect of NRG-1 / HER3 antibody fusion protein (NRG-1 / HER3) on induction of collagen type III in the assay shown in Figure 13A. The x-axis indicates the day the samples were taken. The y-axis represents collagen type III mRNA expression as the fold change in Col3a1 mRNA present relative to the level of Col3a1 mRNA present on day 2 of the assay in the absence of NRG-1 / HER3.

[0041] [Figure 15] FIG. 15 shows a diagram illustrating programmed electrical stimulation (PES) via a transjugular octapolar catheter used to measure atrial fibrillation (AF) inducibility in the in vivo experimental model shown in FIGS.

[0042] [Figure 16] FIG. 16 shows an angiotensin II (Ang-II)-induced hypertensive mouse model for measuring atrial fibrillation in vivo.

[0043] [Figure 17A] Figure 17A is a graph showing the effect of NRG-1 / HER3 antibody fusion protein on the total duration of AF in the model shown in Figure 16. The x-axis indicates treatment condition: CTRL: sham-treated control; ANGII+vehicle: vehicle only control; ANGII+NRG-1 / HER3: ANGII-induced hypertensive animals treated with NRG-1 / HER3 antibody fusion protein. The y-axis indicates time in seconds (s).

[0044] [Figure 17B] Figure 17B is a graph showing the effect of NRG-1 / HER3 antibody fusion protein on AF inducibility in the model shown in Figure 16. The x-axis shows treatment. The y-axis shows the percentage of mice with AF.

[0045] [Figure 18] FIG. 18 shows a second atrial fibrillation (AF) model in which mice were fed a high-fat diet.

[0046] [Figure 19A] Figure 19A is a graph showing the change in body weight over time for three groups of mice in the model shown in Figure 18. The x-axis represents time in weeks (W). The y-axis represents body weight. CRTL: mice not fed a high-fat diet and treated with vehicle alone control; HFD+Vehicle: mice fed a high-fat diet and treated with vehicle alone control; HFD+NRG-1 / HER3: mice fed a high-fat diet and treated with NRG-1 / HER3 antibody fusion protein.

[0047] [Figure 19B] Figure 19B is a graph showing the effect of NRG-1 / HER3 antibody fusion protein on total body weight over time in the metabolic syndrome model shown in Figure 18. The x-axis represents time (weeks (W)). The y-axis represents change in body weight.

[0048] [Figure 19C] Figure 19C is a graph showing the effect of NRG-1 / HER3 antibody fusion protein on glucose tolerance in the model shown in Figure 18. The x-axis shows time after 20% glucose infusion. The y-axis shows blood glucose concentration (mg / dL).

[0049] [Figure 20A]Figure 20A is a graph showing the effect of NRG-1 / HER3 antibody fusion protein on the total duration of atrial arrhythmias in the metabolic syndrome model shown in Figure 18. The x-axis indicates treatment condition: WT: wild type, injected with vehicle alone without high fat diet; HFD+vehicle, mice fed a high fat diet and treated with vehicle only control; HFD+NRG-1 / HER3: mice fed a high fat diet and treated with NRG-1 / HER3 antibody fusion protein. The y-axis indicates time in seconds (s).

[0050] [Figure 20B] Figure 20B is a graph showing the effect of NRG-1 / HER3 antibody fusion protein on arrhythmogenicity in the metabolic syndrome model shown in Figure 18. The x-axis shows the treatment condition: CRTL, mice not fed a high fat diet and treated with a vehicle only control; HFD+vehicle, mice fed a high fat diet and treated with a vehicle only control; HFD+NRG-1 / HER3: mice fed a high fat diet and treated with NRG-1 / HER3 antibody fusion protein. The y-axis shows the percentage of mice with induced AF.

[0051] [Figure 21] 21A-21F are a series of images showing collagen I immunohistochemical staining of cardiac tissue from a rat model of systolic heart failure (or reduced ejection fraction heart failure, HFrEF) (Example 7). The rats were sham-operated (FIG. 21A), or operated with vehicle alone (FIG. 21B), treated with GP120-NRG-1 antibody fusion protein (FIG. 21C), 1 mg / kg NRG-1 / HER3 antibody fusion protein (FIG. 21D), 3 mg / kg NRG-1 / HER3 antibody fusion protein (FIG. 21E), or 10 mg / kg NRG-1 / HER3 antibody fusion protein (FIG. 21F).

[0052] [Figure 22] FIG. 22 illustrates the experimental design for testing the effect of NRG-1 / HER3 antibody fusion protein in the Aachener minipig model of deoxycorticosterone acetate (aldosterone agonist) induced hypertension and atrial fibrosis.

[0053] [Figure 23] Figure 23 shows electrocardiogram (ECG) and formula pairs for a DOCA model minipig. The top ECG is a representative ECG where 50 Hz burst pacing induced atrial fibrillation. The bottom ECG is a representative ECG where 50 Hz burst pacing did not induce atrial fibrillation. The formula shows how the ECG data was used to calculate atrial fibrillation inducibility.

[0054] [Figure 24] FIG. 24 is a plot showing mean atrial pressure (mmHg, y-axis) in control, DOCA+vehicle (VEH), and DOCA+NRG-1 / HER3 minipigs.

[0055] [Diagram 25] 25 is a plot showing atrial fibrillation (AF) inducibility in control, DOCA+vehicle, and DOCA+NRG-1 / HER3 minipigs. AF inducibility was calculated as shown in FIG.

[0056] [Figure 26] FIG. 26 is a plot (left) and three representative images (right) showing the extent of atrial fibrosis in control, DOCA+vehicle, and DOCA+NRG-1 / HER3 minipigs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Detailed Description The present invention utilizes recombinant fusion proteins comprising fusions of monoclonal antibodies with active fragments of Neuregulin-1 protein isoforms across a range of cardiovascular and central nervous system (CNS) indications.

[0058] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0059] For purposes of interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall include the plural and vice versa. In the event that a definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0060] A "neuregulin or neuregulin analog" is a molecule capable of activating ErbB2 / ErbB4 or ErbB2 / ErbB3 heterodimeric protein tyrosine kinase, such as all neuregulin isoforms, neuregulin EGF domain only, neuregulin mutants, and any type of neuregulin-like gene product that also activates the above receptors. A preferred "neuregulin" for use in the present invention is a polypeptide fragment of the human neuregulin 1β2 isoform that contains the EGF-like domain and the receptor binding domain. In one embodiment, the neuregulin fragment is an active fragment. Neuregulin-1 (NRG-1) and its isoforms are also known in the art as neuregulin 1 (NRG1), glial growth factor (GGF), heregulin (HGL), HRG, novel differentiation factor (NDF), ARIA, GGF2, HRG1, HRGA, SMDF, MST131, MSTP131, and NRG1 intronic transcript 2 (NRG1-IT2).

[0061] The terms "ErbB3", "ErbB3 (HER3)", and "HER3" refer to the same protein (or, where referred to, the same gene) and are used interchangeably herein. In some embodiments, the recombinant fusion comprises a monoclonal antibody portion specific for ErbB3. 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.

[0062] In one embodiment, the terms "ErbB4", "ErbB4 (HER4)", and "HER4" refer to the same protein (or, where referred to, 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.

[0063] In one embodiment, the terms "ErbB2", "ErbB2 (HER2)", and "HER2" refer to the same protein (or, where referred to, 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.

[0064] As used herein, the term "active" refers to a fragment that has a biological activity or function. In some embodiments, the activity is the same as or close to the activity of the wild-type protein.

[0065] The term "subject" as used herein includes, but is not limited to, mammals, such as humans, non-human primates (e.g., monkeys), mice, pigs, cows, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, or other non-human mammals, non-mammals, such as birds (e.g., chickens or ducks) or fish, non-mammalian vertebrates, and non-mammalian invertebrates. In some embodiments, the methods and compositions of the present invention are used to treat (both prophylactically and / or therapeutically) non-human animals. The term "subject" may also refer to a patient, i.e., an individual awaiting or receiving medical care.

[0066] As used herein, the term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a subject, including an animal or a human. A pharmaceutical composition generally comprises an effective amount of an active agent (e.g., a recombinant fusion protein of the invention) and a pharma- ceutical acceptable carrier, diluent, or excipient (e.g., buffers, adjuvants, and the like).

[0067] The term "effective amount" refers to a dosage or amount sufficient to produce a desired result, which may include an objective or subjective improvement in the recipient of the dosage or amount (e.g., prolonged survival, reduction in the number and / or size of tumors, effective prevention of a disease condition, etc.).

[0068] "Prophylactic treatment" means the prevention or treatment of a disease, medical condition, or medical disorder. A prophylactic treatment is a treatment administered to a subject who exhibits only early signs or symptoms of a pathology, disease, or medical disorder, and thus the treatment is administered with the intent to alleviate, prevent, or reduce the risk of developing the disease, condition, or medical disorder. A prophylactic treatment serves as a preventative treatment against a disease or disorder. "Prophylactic activity" is the activity of an agent, such as a recombinant fusion protein of the invention or a composition thereof, which, when administered to a subject who does not exhibit signs or symptoms of a pathology, disease, or disorder (or to a subject who exhibits only early signs or symptoms of a pathology, disease, or disorder), alleviates, prevents, or reduces the risk of the subject developing the pathology, disease, or disorder. A "prophylactically useful" agent or compound (e.g., a recombinant fusion protein of the invention) refers to an agent or compound that is useful for alleviating, preventing, treating, or reducing the onset of a pathology, disease, or disorder.

[0069] A "therapeutic treatment" is a treatment administered to a subject exhibiting a symptom or sign of a pathology, disease, or disorder, where the treatment is administered to the subject with the intent of reducing or eliminating the sign or symptom of the pathology, disease, or disorder. "Therapeutic activity" is the activity of an agent, such as a recombinant fusion protein of the invention or a composition thereof, to eliminate or reduce the signs or symptoms of a pathology, disease, or disorder when administered to a subject suffering from such signs or symptoms. A "therapeutically useful" agent or compound (e.g., a recombinant fusion protein of the invention) indicates that the agent or compound is useful for alleviating, treating, or eliminating the signs or symptoms of a pathology, disease, or disorder.

[0070] As used herein, the term "treating cancer" means, unless otherwise specified, partially or completely reversing, mitigating, inhibiting the progression of, or preventing the proliferation of tumors, tumor metastases, or other cancer-causing or neoplastic cells in a subject. As used herein, the term "treatment" refers to a therapeutic action, unless otherwise specified.

[0071] The term "treatment of cardiovascular disease" as used herein means, unless otherwise specified, to partially or completely prevent, inhibit, suppress, delay, reverse, or alleviate the onset of a cardiovascular disease or condition in a subject, or the progression of an existing cardiovascular disease or condition in a subject, or symptoms thereof. Non-limiting examples of cardiovascular diseases that can 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, adjunct to surgical intervention in pediatric congenital heart disease, and atrial fibrosis. 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, chest pain, fainting (syncope), tachycardia, bradycardia, and arrhythmias such as atrial fibrillation. 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), Holter monitor, echocardiogram, stress test, cardiac catheterization, cardiac computed tomography (CT) scan, and cardiac magnetic resonance imaging (MRI).

[0072] "Fibrosis" as used herein refers to the excessive formation and deposition of extracellular matrix (ECM) components, such as collagen. The ECM normally surrounds parenchymal cells and supports their migration, differentiation, proliferation, and normal function. Fibrous ECM can impair tissue homeostasis and cause organ dysfunction due to loss of structural integrity and abnormal remodeling. Fibrosis is characterized by the proliferation of fibroblasts, which can differentiate into myofibroblasts and secrete ECM proteins. "Cardiac fibrosis" generally refers to fibrosis of the heart, including, but not limited to, atrial fibrosis, as well as fibrosis affecting other areas of the heart, such as the ventricles, myocardium, pericardium, endocardium, and valves.

[0073] In the heart, replacement or reparative fibrosis occurs after cardiac injury and is associated with the death of cardiomyocytes and the replacement of areas of necrotic myocardium with fibrous scar tissue. In reactive fibrosis, the deposition of collagen and other ECM proteins increases in the interstitial space surrounding cardiac cells and blood vessels, expanding this space without replacing damaged or dead cardiomyocytes.

[0074] As used herein, "atrial fibrosis" refers to fibrosis of the atrium. Atrial fibrosis is closely related to atrial fibrillation (AF), one of the most common arrhythmias in humans. Without being bound by theory, it is believed that atrial fibrosis causes abnormal electrical conduction through the atrium, leading to atrial fibrillation. Fibrosis can be detected by any suitable means known in the art, including, but not limited to, DE-MR imaging (MRI), circulating biomarkers (e.g., galactin-3, MMP-3, MMP-9, high-sensitivity cardiac troponin T, osteopontin, suppression of tumor suppressor 2, connective tissue growth factor (CTGF), resistin (RETN), periostin, and mid-region pro-atrial natriuretic peptide, and microRNAs, e.g., miRNA-15, miR-21, miR-29c, miR-328, miR-30a, miR-214, miR-503, and miR-133a), and electroanatomical voltage mapping.

[0075] As used herein, "arrhythmia" refers to an irregular heartbeat, including both tachycardia (abnormally fast heartbeat) and bradycardia (abnormally slow heartbeat). Atrial fibrillation (AF) is an irregular, often very fast, heart rhythm that can lead to blood clots in the heart. In AF, the normal beating of the atria becomes irregular, disrupting blood flow from the atria to the ventricles. AF can be acute or chronic. AF can be assessed by the duration of AF episodes and the number of episodes occurring in a given unit of time (e.g., AF / episode per day, week, or month). Paroxysmal AF begins suddenly and ends naturally within 7 days. In contrast, persistent AF lasts for 7 days or more and ends naturally or with treatment. Long-term persistent AF refers to AF that is uninterrupted for more than a year. Persistent AF refers to AF that persists despite treatment to restore normal sinus rhythm. Symptoms of AF include arrhythmia, palpitations, lightheadedness, extreme fatigue, shortness of breath, and chest pain.

[0076] As used herein, the term "treatment of a central nervous system (CNS)-related disease" refers to a method of partially or completely preventing, inhibiting, suppressing, delaying, reversing, or alleviating the onset of a CNS-related disease or condition in a subject, unless otherwise specified. The term "treatment of a CNS-related disease" also refers to reversing, delaying, or alleviating an existing CNS-related disease or condition, or a symptom thereof. Illustrative, but non-limiting examples of CNS-related diseases or conditions that can be treated by the methods of the present disclosure include amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Bell's palsy, epilepsy and seizures, Guillain-Barre syndrome, stroke, traumatic brain injury, multiple sclerosis, or a combination thereof. Treating a CNS-related disease can improve or prevent symptoms such as tremors, bradykinesia, muscle stiffness, loss of balance, posture disorders, speech changes, loss of motor control, paralysis, swallowing disorders, muscle spasms, seizures, memory loss, and confusion.

[0077] The term "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a certain percentage of the same nucleotides or amino acid residues when compared and aligned for maximum correspondence. To determine percent identity, the sequences are aligned for optimal comparison (e.g., gaps can be introduced into the sequence of the first amino acid or nucleic acid sequence to allow optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (e.g., overlapping positions) x 100). In some embodiments, the two sequences are the same length.

[0078] The term "substantially identical" in the context of two nucleic acids or polypeptides refers to two or more sequences or subsequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% identity, or at least 99% identity (as determined, for example, using one of the methods described below).

[0079] As used herein, the terms "bind", "specifically bind to" or "specific to" refer to a measurable and reproducible interaction, such as the binding of an antibody to a target, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, ease, and / or longer duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of <1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM.

[0080] In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins of different species, hi another embodiment, specific binding can include, but is not necessarily, exclusive binding.

[0081] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "neuregulin" or a "neuregulin peptide" includes mixtures of such neuregulins, neuregulin isoforms, and / or neuregulin-like polypeptides. Reference to a "formulation" or "method" includes one or more formulations, methods, and / or steps of the type described herein and / or that will become apparent to those of skill in the art upon reading this disclosure.

[0082] The term "polypeptide" refers to a polymer of amino acids and equivalents, and does not refer to a specific length of the material. Thus, "peptide" and "protein" are included in the definition of a polypeptide. Also included in the definition of a polypeptide is an "antibody," as defined herein. A "polypeptide region" refers to a segment of a polypeptide, which may include, for example, one or more domains or motifs (e.g., a polypeptide region of an antibody may include, for example, one or more complementarity determining regions (CDRs)). The term "fragment" refers to a portion of a polypeptide, preferably having at least 20 contiguous amino acids or at least 50 contiguous amino acids of the polypeptide.

[0083] Unless the context indicates otherwise, a "derivative" is a polypeptide or a fragment thereof that has one or more non-conservative or conservative amino acid substitutions relative to a second polypeptide (also referred to as a "variant"), or that has been modified by, for example, the attachment of a heterologous polypeptide, or the covalent attachment of a second molecule, such as by glycosylation, acetylation, phosphorylation, etc. Additionally included within the definition of "derivative" are, for example, polypeptides that contain one or more analogs of an amino acid (such as, for example, unnatural amino acids), polypeptides that have unsubstituted bonds, and other modifications known in the art, both natural and unnatural.

[0084] An "isolated" polypeptide is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. Isolated polypeptides include isolated antibodies, or fragments or derivatives thereof.

[0085] The term "about" as used herein means quantitatively plus or minus 5%, or in another embodiment plus or minus 10%, or in another embodiment plus or minus 15%, or in another embodiment plus or minus 20%.

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference and are cited for the purpose of disclosing and describing the material to which the references are cited.

[0087] Recombinant fusion proteins - antibodies The present invention employs recombinant fusion proteins comprising fusions of monoclonal antibodies with fragments of Neuregulin-1 protein isoforms across a range of cardiovascular and neurological indications. In an exemplary embodiment, the antibody is specific for ERBB3 (HER3).

[0088] As used herein, an "antibody" refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. A typical immunoglobulin (e.g., antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0089] Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies at the disulfide bonds in the hinge region to produce F(ab')2, which is a dimer of Fab, a light chain itself linked to VH-CH1 by a disulfide bond. F(ab')2 can be reduced under mild conditions to cleave the disulfide bonds in the hinge region, thus converting the F(ab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region (for a more detailed description of other antibody fragments, see Fundamental Immunology, WE Paul, ed., Raven Press, New York (1999)). Although the various antibody fragments are defined in terms of digestion of the intact antibody, one skilled in the art will appreciate that such Fab' fragments and the like can be synthesized de novo, either chemically or using recombinant DNA methodology. Thus, the term antibody as used herein also includes antibody fragments generated by modification of whole antibodies or synthesized de novo using recombinant DNA methodology. Antibodies include single chain antibodies, including single chain Fv (sFv or scFv) antibodies, in which a variable heavy chain and a variable light chain are linked (directly or via a peptide linker) to form a continuous polypeptide. Antibodies include single domain antibodies, which include antibody fragments consisting of a single monomeric variable antibody domain capable of selectively binding to an antigen domain. Exemplary single domain antibodies include VHH fragments originally isolated from camelids.

[0090] The antibody domain of the fusion protein optionally comprises all or a portion of an immunoglobulin molecule, optionally comprising all or a portion of an immunoglobulin variable region (i.e., the region specific for a disease associated antigen), optionally comprising regions encoded by V genes and / or D genes and / or J genes.

[0091] As explained above (see definitions above), antibodies as used herein optionally include F(ab)2, F(ab')2, Fab, Fab', scFv, single domain antibodies, etc., depending on the specific requirements of the embodiment. Some embodiments use fusion proteins that include an IgG domain. However, other embodiments include alternating immunoglobulins such as IgM, IgA, IgD, and IgE. Furthermore, all possible isotypes of the various immunoglobulins are also included in this embodiment. Thus, IgG1, IgG2, IgG3, etc. are all possible molecules within the antibody domain of the antibody-immunostimulant fusion protein used in the present invention. In addition to the choice of immunoglobulin type and isotype, different embodiments of the present invention include different hinge regions (or functional equivalents thereof). Such hinge regions provide flexibility between the different domains of the antibody-immunostimulant fusion protein. See, e.g., Penichet, et al. 2001 "Antibody-cytokine fusion proteins for the therapy of cancer" J Immunol Methods 248:91-101.

[0092] In some embodiments, the mAb comprised in the recombinant fusion protein of the present invention is monospecific for ErbB3 (HER3).

[0093] Human HER3 (ErbB-3, ERBB3, c-erbB-3, c-erbB3, receptor tyrosine-protein kinase erbB-3) encodes a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which also includes HER1 (also known as EGFR), HER2, and HER4 (Kraus, MH et al, PNAS 86 (1989) 9193-9197; Plowman, GD et al, PNAS 87 (1990) 4905-4909; see also Kraus, MH et al, PNAS 90 (1993) 2900-2904). Like the prototypical epidermal growth factor receptor, the transmembrane receptor HER3 is composed of an extracellular ligand-binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, an intracellular protein tyrosine kinase domain (TKD), and a C-terminal phosphorylation domain. The membrane-bound HER3 protein has a Heregulin (HRG) binding domain in the extracellular domain, but no active kinase domain. Thus, it can bind to this ligand but cannot transmit signals intracellularly via protein phosphorylation. However, it forms heterodimers with other HER family members that have kinase activity. Heterodimerization activates the receptor-mediated signaling pathway and transphosphorylates the intracellular domain. Dimerization between HER family members expands the signaling capabilities of HER3, providing a means of signal diversification as well as signal amplification. For example, the HER2 / HER3 heterodimer induces one of the most important mitogenic signals among HER family members through the PI3K and AKT pathways (Sliwkowski MX, et al, J. Biol. Chem. 269 (1994) 14661-14665; Alimandi M, et al, Oncogene. 10 (1995) 1813- 1821; Hellyer, NJ, J. Biol. Chem. 276 (2001) 42153-4261; Singer, E., J. Biol.).

[0094] In one embodiment, the human ERBB3 protein comprises the following amino acid sequence as provided in GenBank AAH02706.1 and set forth in SEQ ID NO:1:

[0095] MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKAF (SEQ ID NO: 1). It is understood that the ERBB3 (HER3) sequence targeted by the antibodies of the present methods and compositions may be an isomer, homolog, or variant of SEQ ID NO: 1.

[0096] In one embodiment, the mAb of the recombinant fusion protein provided herein is an anti-Her3 mAb that inhibits NRG-1 signaling through ErbB3 (HER3).

[0097] In certain embodiments, the mAb contained in the recombinant fusion protein of the present invention comprises an anti-HER3 mAb. Such anti-HER3 antibodies and their sequences are known in the art and include, but are not limited to, patritumab, seribantumab (fully human mAb), LJM716, KTN3379, AV-203, REGN1400, GSK2849330, or MM-141. Such antibodies can also be selected from any of the following forms, including chimeric, bispecific, non-human, fully human, or humanized forms, so long as they bind to human ERBB3 (HER3) and inhibit signaling from human ERBB3 (HER3). In some embodiments, the anti-HER3 antibody is of human origin.

[0098] In some embodiments, the term "antibody" encompasses various forms of antibody structures, including, but not limited to, complete antibodies and antibody fragments. The antibody according to the invention is preferably a human antibody, a humanized antibody, a chimeric antibody, or an antibody further engineered, so long as the characteristic properties according to the invention are retained. An "antibody fragment" comprises a portion of a full-length antibody, preferably the variable domain thereof, or at least the antigen-binding site thereof. Examples of antibody fragments include diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are described, for example, in Huston, JS, Methods in Enzymol. 203 (1991) 46-88. Additionally, antibody fragments can be any of the VFv, ... H The properties of the domain, i.e. V L The property of being able to assemble with domains, or V H The V domains bind to the respective antigens and can be assembled together into a functional antigen-binding site, thereby providing the properties of the antibodies according to the invention. L The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single amino acid composition.

[0099] In some embodiments, chimeric antibodies can be used in the compositions and methods provided herein. In one embodiment, the term "chimeric antibody" refers to a monoclonal antibody that comprises a variable region (i.e., a binding region) from a mouse and at least a portion of a constant region from a different source or species, and is usually prepared by recombinant DNA technology. Chimeric antibodies that comprise a mouse variable region and a human constant region are particularly preferred. Such rat / human chimeric antibodies are the product of expressed immunoglobulin genes that comprise a DNA segment encoding a rat immunoglobulin variable region and a DNA segment encoding a human immunoglobulin constant region. Other forms of "chimeric antibodies" included in the present invention are those in which the class or subclass has been modified or changed from that of the original antibody. Such "chimeric" antibodies are also referred to as "class-switched antibodies." Methods for producing chimeric antibodies include conventional recombinant DNA and gene transfection techniques that are now well known in the art. See, e.g., Morrison, SL, et al, Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Patent No. 5,202,238 and U.S. Patent No. 5,204,244.

[0100] In one embodiment, humanized antibodies can be used in the compositions and methods provided herein. In some embodiments, the term "humanized antibody" or "humanized version of an antibody" refers to an antibody whose framework or "complementarity determining regions" (CDRs) have been modified to include CDRs of an immunoglobulin of different specificity compared to the parent immunoglobulin. In other embodiments, the CDRs of VH and VL are grafted into the framework regions of a human antibody to prepare a "humanized antibody." See, for example, Riechmann, L., et al, Nature 332 (1988) 323-327, and Neuberger, MS, et al, Nature 314 (1985) 268-270. The heavy and light chain variable framework regions can be obtained from the same or different human antibody sequences. The human antibody sequences can be sequences of naturally occurring human antibodies. Human heavy and light chain variable framework regions are described, for example, in Lefranc, M.-P., Current Protocols in Immunology (2000) - Appendix IP A.1P.1-A.1P.37 and are accessible via IMGT, the international ImMunoGeneTics information system (http: / / imgt.cines.fr) or via http: / / vbase.mrc-cpe.cam.ac.uk. Optionally, the framework regions can be modified by further mutations. Particularly preferred CDRs correspond to those representing sequences recognizing the antigens mentioned above for chimeric antibodies. The term "humanized antibody" as used herein also includes antibodies in which the constant region has been modified to produce the properties according to the invention, for example by "class switching", i.e. alteration or mutation of the Fc part (e.g. IgG1 to IgG4 and / or IgG1 / IgG4 mutation), in particular with regard to complement component 1q (Clq) binding and / or Fc receptor (FcR) binding. The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.Human antibodies are well known in the art (van Dijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (such as mice) that can be immunized to generate a full repertoire or selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of human germ-line immunoglobulin genes into such germ-line mutant mice results in the production of human antibodies upon antigenic stimulation (see, e.g., Jakobovits, A., et al, Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al, Nature 362 (1993) 255-258+Brueggemann, MD, et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, HR, and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD, et al, J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole, A. et al. and Boerner, P. et al. can also be used to prepare human monoclonal antibodies (Cole, A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, AL, p. 77 (1985), and Boerner, P., et al, J. Immunol. 147 (1991) 86-95). As already mentioned for the humanized antibodies according to the invention, the term "human antibody" as used herein also includes antibodies whose constant regions have been modified to generate the properties according to the invention.

[0101] In one specific embodiment of the present invention, the mAb comprised in the recombinant fusion protein provided herein comprises at least one mutation in the Fc domain or region.

[0102] The term "recombinant human antibody" as used herein is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, e.g., antibodies isolated from host cells such as NS0 or CHO cells, or from animals (e.g., mice) that are transgenic for human immunoglobulin genes, or expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in rearranged form. The recombinant human antibodies of the invention have undergone somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

[0103] In some embodiments, the terms "binds to human HER3," "specifically binds to human HER3," or "anti-HER3 antibody" are used interchangeably and have a molecular weight of about 4.81x at 25° C. -10 The term "antibody that specifically binds to human HER3 antigen" refers to an antibody that specifically binds to human HER3 antigen with a KD value of 1.0x10 mol / L or less. Binding affinity is determined at 25°C by standard binding assays such as surface plasmon resonance technology (BIAcore®, GE-Healthcare Uppsala, Sweden). Thus, as used herein, an "antibody that binds to human HER3" refers to an antibody that specifically binds to human HER3 antigen with a KD value of 1.0x10 mol / L or less at 25°C. -8 mol / L~1.0x10 -13 Binding affinity in the range of mol / L, preferably 4.81x at 25°C -10 This refers to an antibody or a portion thereof that specifically binds to the human HER3 antigen with a KD value of 0.01 mol / L or less.

[0104] In another embodiment, the anti-HER3 antibody comprised in the recombinant fusion protein disclosed herein comprises a variable heavy chain (VH) and a variable light chain (VL). In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and has one or more of the following properties: inhibition of HER3 phosphorylation in tumor cells, inhibition of AKT phosphorylation in tumor cells, inhibition of signaling through ErbB3 (HER3), and inhibition of tumor cell proliferation.

[0105] In one embodiment, the anti-HER3 mAb provided herein comprises the VH amino acid sequence set forth in SEQ ID NO:2: Heavy Chain: QVQLQQWGAG LLKPSETLSL TCAVYGGSFS GYYWSWIRQP PGKGLEWIGE INHSGSTNYN PSLKSRVTIS VETSKNQFSL KLSSVTAADT AVYYCARDKW TWYFDLWGRG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APEFLGGPAV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHAHYTQKS LSLSPGK (SEQ ID NO:2).

[0106] In one embodiment, the anti-HER3 mAb provided herein comprises the VL amino acid sequence of SEQ ID NO:3: Light chain: DIEMTQSPDS LAVSLGERAT INCRSSQSVL YSSSNRNYLA WYQQNPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQQYYST PRTFGQGTKV EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC (SEQ ID NO:3).

[0107] In one embodiment, the anti-HER3 antibody of the present invention comprises at least one mutation in the Fc region. In another embodiment, the mature anti-HER3 antibody of the present invention (i.e., lacking a signal peptide) comprises at least one mutation at amino acid 234, 239, 434, or a combination thereof, wherein in another embodiment, the amino acid mutation comprises at least one of the following substitution mutations: L234F, S239A, N434A, or a combination thereof. In another embodiment, the mutation at amino acid 234 and / or 239 knocks down the effector function of the anti-HER3 antibody. In another embodiment, the mutation at amino acid 434 extends the half-life of the antibody in a subject.

[0108] In some embodiments, one or more mutations in the Fc region reduce effector function. In some embodiments, the reduced effector function comprises a reduction in affinity of the anti-HER3 antibody for one or more Fc receptors. The FcRs can be FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa(158F), FcγRIIIa(158V), and C1q. In some embodiments, the reduction in affinity comprises an increase in the dissociation constant by about one order of magnitude or more. In some embodiments, the introduction of one or more Fc mutations reduces the KD of the anti-HER3 antibody or fusion protein comprising the same for FcγRI by 2.81x10 -9 M to 1.03x10 -8In some embodiments, the introduction of one or more Fc mutations increases the KD of the anti-HER3 antibody or fusion protein comprising the same against FcγRIIa to 3.95×10 -7 M to 1.35x10 -6 In some embodiments, the introduction of one or more Fc mutations increases the KD of the anti-HER3 antibody or a fusion protein comprising the same for FcγRIIb to 1.03×10 -7 M to 1.52 x 10 -6 In some embodiments, the introduction of one or more Fc mutations increases the KD of the anti-HER3 antibody or a fusion protein comprising the same to FcγRIIIa(158F) to 6.37×10 -8 M to 1.18 x 10 -7 In some embodiments, the introduction of one or more Fc mutations increases the KD of the anti-HER3 antibody or a fusion protein comprising the same to FcγRIIIa(158V) to 3.41×10 -8 M to 9.10 x 10 -8 Increase to M.

[0109] In some embodiments, the anti-HER3 antibody or a recombinant fusion protein comprising the same binds to FcγRI at a concentration of 1.03×10 -8 In some embodiments, the anti-HER3 antibody or a recombinant fusion protein comprising the same comprises one or more Fc mutations and binds to FcγRIIa with a KD of 1.35×10 -6 In some embodiments, the anti-HER3 antibody or a recombinant fusion protein comprising the same comprises one or more Fc mutations and binds to FcγRIIa with a KD of 1.5×10 -6 In some embodiments, the anti-HER3 antibody or a recombinant fusion protein comprising the same comprises one or more Fc mutations and binds to FcγRIIb with a KD of 1.18×10 -7 In some embodiments, the anti-HER3 antibody or a recombinant fusion protein comprising the same comprises one or more Fc mutations and binds to FcγRIIIa(158F) with a KD of 9.10×10 -8It binds to FcγRIIIa (158V) with a KD of ≥ M.

[0110] As used herein, the term "antibody effector function" refers to a function contributed by the Fc region of an Ig. Such a function may be affected, for example, by binding of the Fc effector region to an Fc receptor on an immune cell that has phagocytic or lytic activity, or to a component of the complement system.

[0111] In one embodiment, the anti-HER3 antibody does not induce antibody-dependent cellular cytotoxicity (ADCC). The term "antibody-dependent cellular cytotoxicity (ADCC)" refers to the lysis of human target cells by the antibodies of the invention in the presence of effector cells.

[0112] In one embodiment of the present invention, the antibody of the present invention is glycosylated. In some embodiments, the glycosylation is N-glycosylated. In other embodiments, the glycosylation is O-glycosylated.

[0113] In the context of the recombinant fusion proteins according to the invention provided herein, the antibodies comprised in the recombinant fusion proteins may be produced by recombinant means. Such methods are widely known in the art and include protein expression in prokaryotic and eukaryotic cells, followed by isolation of the antibody polypeptides and purification, usually to a pharma- ceutically acceptable degree of purity. For protein expression, nucleic acids encoding the light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is carried out in suitable prokaryotic or eukaryotic host cells, such as CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, yeast, or E. coli cells, and the antibodies are recovered from the cells (from the supernatant or after cell lysis). Recombinant production of antibodies is well known in the art and has been described, for example, in the reviews of Makrides, SC, Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al, Protein Expr. Purif. 8 (1996) 271-282; Kaufman, RJ, Mol. Biotechnol. 16 (2000) 151-161; Werner, RG, Drug Res. 48 (1998) 870-880. The antibodies may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Purification is carried out by standard techniques, including column chromatography and other methods known in the art, to remove other cellular components or other contaminants, such as other cellular nucleic acids or proteins (see Ausubel, F., et al, ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)). Expression in NS0 cells is described, for example, in Barnes, LM, et al, Cytotechnology 32 (2000) 109-123; Barnes, LM, et al, Biotech. Bioeng. 73 (2001) 261-270.Transient expression is described, for example, in Durocher, Y., et al, Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described in Orlandi, R., et al, Proc. Natl. Acad. Sci. USA 86 (1989) 3833- 3837; Carter, P., et al, Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; Norderhaug, L., et al, J. Immunol. Methods 204 (1997) 77-87. A preferred transient expression system (HEK293) is described in Schlaeger, E.-J. and Christensen, K., in Cytotechnology 30 (1999) 71-83, and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199. The monoclonal antibodies are suitably separated from the culture medium by conventional immunoglobulin purification methods, such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced using conventional methods. Hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA is inserted into an expression vector and then transfected into host cells, such as HEK293 cells, CHO cells, or myeloma cells that do not produce immunoglobulin protein, to synthesize recombinant monoclonal antibodies in the host cells.

[0114] The heavy and light chain variable domains according to the invention are combined with promoter, translation initiation, constant region, 3' untranslated region, polyadenylation, and transcription termination sequences to form an expression vector construct. The heavy and light chain expression constructs can be combined in a single vector and co-transfected, sequentially transfected, or separately transfected into a host cell and then fused to form a single host cell expressing both chains.

[0115] It will be appreciated that the antibody is administered to a subject in a therapeutically effective amount, that being that amount of the subject compound or combination that elicits the biological or medical response in a tissue, system, animal, or human that is desired by a researcher, veterinarian, physician, or other clinician.

[0116] Recombinant fusion protein - neuregulin In one embodiment, the recombinant fusion protein provided herein comprises a fragment of NRG-1 protein, which can bind to ErbB4 receptor on the surface of cardiomyocytes, continuously activate the PI3K / AKT signal pathway in cells, and change the structure of cardiomyocytes, thereby improving the function of cardiomyocytes.

[0117] As used herein, "neuregulin" or "NRG" refers to a protein or peptide that can bind to and activate ErbB3, ErbB4, or heterodimers or homodimers thereof, including neuregulin isoforms, neuregulin EGF-like domains, polypeptides containing the neuregulin EGF-like domain, mutants or derivatives of neuregulin, and any type of neuregulin-like gene product that can activate the above receptors. Neuregulin also includes NRG-1, NRG-2, NRG-3, and NRG-4 proteins, peptides, fragments, and compounds that have neuregulin function. In a preferred embodiment, neuregulin is a protein or peptide that can bind to and activate, for example, ErbB2 / ErbB4 or ErbB2 / ErbB3 heterodimers, including, but not limited to, a fragment of the NRG-1β2 isoform, i.e., the following amino acid sequence: The NRG protein of the present invention includes a 177-237 amino acid fragment containing an EGF-like domain having the sequence SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ (SEQ ID NO: 4). The NRG protein of the present invention can activate the above receptors and regulate their biological functions, for example, stimulating the synthesis of acetylcholine receptors in skeletal muscle cells, promoting the differentiation and survival of cardiomyocytes, and DNA synthesis. It is known to those skilled in the art that mutations of single amino acids in less important regions usually do not change the biological activity of the resulting protein or polypeptide (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin / Cummings Pub.co., p. 224). The NRG protein of the present invention can be isolated from natural sources and modified by recombinant technology, artificial synthesis, or other means.

[0118] As used herein, "epidermal growth factor-like domain" or "EGF-like domain" refers to a polypeptide fragment encoded by the neuregulin gene that binds to and activates ErbB3, ErbB4, or heterodimers or homodimers thereof (including heterodimers with ErbB2) and is structurally similar to the EGF receptor binding regions described in WO00 / 64400, Holmes et al., Science, 256:1205-1210 (1992); U.S. Patent Nos. 5,530,109 and 5,716,930; Hijazi et al., Int. J. Oncol., 13:1061-1067 (1998); Chang et al., Nature, 387:509-512 (1997); Carraway et al., Nature, 387:512-516 (1997); Higashiyama et al., J. Biochem., 122:675-680 (1997); and WO97 / 09425, the contents of which are all incorporated herein by reference. In certain embodiments, the EGF-like domain binds to and activates ErbB2 / ErbB4 or ErbB2 / ErbB3 heterodimers. In certain embodiments, the EGF-like domain comprises the amino acid sequence of the receptor binding domain of NRG-1. In some embodiments, the EGF-like domain refers to amino acid residues 177-226, 177-237, or 177-240 of NRG-1. In certain embodiments, the EGF-like domain comprises the amino acid sequence of the receptor binding domain of neuregulin 2 (NRG-2, also known in the art as DON1, HRG2, and NTAK). In certain embodiments, the EGF-like domain of NRG-2 comprises the sequence HARKCNETAKSYCVNGGVCYYIEGINQLSCKCPNGFFGQRCL (SEQ ID NO: 15). In certain embodiments, the EGF-like domain comprises the amino acid sequence of the receptor binding domain of neuregulin 3 (NRG-3, also known in the art as HRG3 and pro-NRG3).In certain embodiments, the EGF-like domain of NRG-3 comprises the sequence HFKPCRDKDLAYCLNDGECFVIETLTGSHKHCRCKEGYQGVRCD (SEQ ID NO: 16). In certain embodiments, the EGF-like domain comprises the amino acid sequence of the receptor binding domain of neuregulin 4 (NRG-4, also known in the art as HER4). In certain embodiments, the EGF-like domain of NRG-4 comprises the sequence HEEPCGPSHKSFCLNGGLCYVIPTIPSPFCRCVENYTGARCE (SEQ ID NO: 17). In certain embodiments, the EGF-like domain comprises the amino acid sequence Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro (SEQ ID NO: 18), as described in U.S. Patent No. 5,834,229.

[0119] In one embodiment, the NRG-1 protein provided in the recombinant fusion protein disclosed herein is the NRG-1β2a isoform.

[0120] In some embodiments, the active NRG-1 fragment comprises an ERBB3 / 4 binding domain. In another related embodiment, NRG-1 binds to ErbB4 (HER4) and induces signal transduction through ErbB4. In other embodiments, the mAb inhibits NRG-1 signal transduction through ErbB3 (HER3). In some embodiments, the active protein fragment of NRG-1 comprises an active domain of NRG-1.

[0121] In some embodiments, the NRG-1 fragment comprises SEQ ID NO:4, or a sequence having at least 70%, at least 80%, at least 90%, or at least 95% identity thereto, and capable of binding to ErbB4 and inducing signal transduction via ErbB4.

[0122] Recombinant fusion proteins - compositions In one embodiment, in the recombinant fusion proteins disclosed herein, NRG-1 is fused to the C-terminus of the anti-HER3 antibody heavy chain using a linker. In another related embodiment, NRG-1 is attached to the linker via the first (first) amino acid at the N-terminus of NRG-1, which in one embodiment is a serine (S or Ser) amino acid. The particular recombinant fusion proteins utilized in the present invention can optionally be obtained or made by any method known in the art, including purchase from a commercial source. For example, a nucleic acid sequence encoding a suitable antibody framework is optionally cloned and ligated into a suitable vector, such as a prokaryotic or eukaryotic expression vector. In addition, a nucleic acid sequence encoding an NRG-1β2a isoform molecule is optionally cloned into the same vector in a suitable orientation and position, such that expression from the vector produces an antibody-NRG-1β2a isoform fusion protein. Some optional embodiments require post-expression modifications, such as assembly of antibody subunits. The techniques and art of the above (and similar) manipulations are known to those skilled in the art. Relevant descriptions are described, for example, in Sambrook et al., Molecular Cloning-A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989 and Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented until 1999). In some alternative embodiments, the antibody domain and the NRG-1β2a isoform are assembled after expression, for example by chemical means. In one embodiment, the invention provides a composition, for example a pharmaceutical composition, comprising a recombinant fusion protein of the invention.

[0123] In one embodiment, the recombinant fusion protein shortens the duration of atrial fibrillation episodes.In one embodiment, the recombinant fusion protein reduces the frequency of atrial fibrillation.Symptoms of atrial fibrillation include, but are not limited to, arrhythmia, palpitations, dizziness, extreme fatigue, shortness of breath, chest pain, and combinations thereof.In one embodiment, the recombinant fusion protein reduces collagen content or deposition in cardiac tissue.

[0124] In one embodiment, the recombinant fusion protein promotes the proliferation, differentiation, and survival of cardiomyocytes. In another embodiment, the recombinant fusion protein promotes the proliferation, differentiation, and survival of cardiac tissue. In one embodiment, the recombinant fusion protein promotes the proliferation, differentiation, and survival of cardiomyocytes without promoting the proliferation of cancer and / or tumors. In another embodiment, the recombinant fusion protein promotes the proliferation, differentiation, and survival of cardiac tissue without promoting the proliferation of cancer or tumors.

[0125] In one embodiment, the cancer is adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, anorectal cancer, anal canal cancer, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliary tract cancer, extrahepatic bile duct cancer, intrahepatic cholangiocarcinoma, bladder cancer, bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, carcinoid tumor, gastrointestinal, nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system Lymphoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphatic tumors, mycosis fungoides, Sediary syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, ovarian germ cell tumors, gestational trophoblastic tumor glioma, head and neck cancer, hepatocellular carcinoma (liver cancer), Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, eye cancer, islet cell tumors (pancreatic endocrine), Kaposi's sarcoma, kidney cancer (kidney cancer), renal cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Waldenstrom's macroglobulinemia, medulloblastoma, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, malignant mesothelioma, mesothelioma, metastatic squamous cell cervical cancer, oral cancer, tongue cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, chronic myeloid leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasopharyngeal carcinoma, neuroblastoma, oral cancer,), oral cavity cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian low malignant potential tumor, pancreatic cancer, pancreatic islet cell cancer, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing's sarcoma family, Kaposi's sarcoma, soft tissue carcinoma sarcoma, epithelioid sarcoma, synovial sarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, pharyngeal cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis, ureter and other urinary tracts, gestational trophoblastic neoplasm, urethral cancer, endometrial cancer, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, or Wilms' tumor.

[0126] In another embodiment, the recombinant fusion protein promotes the proliferation, differentiation, and survival of central nervous system (CNS) cells. In another embodiment, the recombinant fusion protein promotes the proliferation, differentiation, and survival of central nervous system (CNS) cells without promoting the growth of cancer and / or tumors. In another embodiment, the recombinant fusion protein has a reduced ability to induce antibody-dependent cellular cytotoxicity (ADCC).

[0127] In some embodiments, the recombinant fusion protein promotes HER2 / 4 signaling over HER2 / 3 signaling relative to the signal-inducing ability of recombinant NRG-1.

[0128] In certain embodiments of the present invention, the recombinant fusion protein comprises an anti-HER3 mAb fused or operably linked to the C-terminus of an antibody heavy chain via a GGGGSGGGGS (G4S) linker (SEQ ID NO: 5) to the NRG-1β2a isoform of SEQ ID NO: 4. 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 to be suitable for the compositions disclosed herein, may be used herein.

[0129] The term "linker" is art-recognized and refers to a molecule (including, but not limited to, unmodified or modified nucleic acids or unmodified or modified 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, e.g., two polypeptides. A linker may be composed of a single linking molecule or may be composed of a linking molecule and at least one spacer molecule, where the spacer molecule is intended to separate the linking molecule and the compounds by a specific distance.

[0130] A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a nucleic acid presequence or secretory leader is operably linked to a nucleic acid encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide. A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Or, a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the nucleic acid sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. Enhancers, however, are optionally contiguous. Linkage can be accomplished, for example, by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors, linkers, or other methods known in the art can be used. In another embodiment, "operably linked" also refers to the functional pairing of different amino acid sequences, peptides, or proteins, such as the pairing of an antibody described herein with an NRG-1 fragment via a linker sequence (also described herein).

[0131] In another embodiment, the anti-HER3 mAb heavy chain comprised in the recombinant fusion protein provided herein is encoded by SEQ ID NO:6:

[0132] In another embodiment, the recombinant fusion protein provided herein comprises a heavy chain of an anti-HER3 mAb. In another embodiment, the anti-HER3 mAb heavy chain is encoded by SEQ ID NO:7:

[0133] In one embodiment, the recombinant fusion protein provided herein comprises a light chain sequence of an anti-HER3 mAb. In another embodiment, the light chain sequence is encoded by (SEQ ID NO:8): ATGGTGTTGCAGACCCAGGTCTTCATTTCTCTGTTGCTCTGGATCTCTGGTGCCTACGGGGACATCGAGATGACCCAGTCTCCAGATTCCCTGGCCGTGAGCCTGGGAGAGGGCTACAATCAACTGCCGGTCCAGCCAGTCTGTGCTGTACTCTTCCAGCAACAGGAATTACCTGGCC TGGTATCAGCAGAATCCCGGCCAGCCCCCTAAGCTGCTGATCTATTGGGCTAGCACCAGAGAGTCTGGAGTGCCTGACCGCTTCTCTGGATCCGGAAGCGGCACAGACTTCACCCTGACAATCTCTTCCCTGCAGGCCGAGGACGTGGCCGTGTACTATTGCCAGCAGTATTACTCTACCC CTAGGACATTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGACAGTGGCCGCTCCATCCGTGTTCATCTTTCCACCCTCCGACGAGCAGCTGAAGTCCGGAACCGCTAGCGTGGTGTGCCTGCTGAACAACTTCTACCCAAGAGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCTCTGCAGAGCGGCAATTCTCAGGAGTCCGTGACCGAGCAGGACAGCAAGGATTCTACATATTCCCTGAGCTCTACCCTGACACTGTCCAAGGCCGATTACGAGAAGCACAAGGTGTATGCTTGCGAGGTGACCCATCAGGGCCTGTCCAGCCCCGTGACAAAGAGCTTCAACCGCGGCGAGTGTTAA (SEQ ID NO: 8). In one embodiment, SEQ ID NO: 8 is also referred to as "PAL."

[0134] In one embodiment, the heavy chain of the anti-HER3 antibody comprised in the recombinant fusion protein provided herein comprises the following amino acid sequence: MEFGLSWVFLVAIIKGVQCQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ (SEQ ID NO: 9).

[0135] In one embodiment, the heavy chain of the anti-HER3 antibody comprised in the recombinant fusion protein provided herein comprises the following amino acid sequence: MEFGLSWVFLVAIIKGVQCQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFLGGPAVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPGKGGGGSGGGGSSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ (SEQ ID NO: 10).

[0136] In one embodiment, the anti-HER3 mAb heavy chain sequence comprises a signal peptide sequence. In another embodiment, the anti-HER3 mAb heavy chain signal peptide sequence comprises the amino acid sequence MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 11).

[0137] In one embodiment, the light chain of the anti-HER3 antibody comprised in the recombinant fusion protein comprises the following amino acid sequence: MVLQTQVFISLLLWISGAYGDIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).

[0138] In one embodiment, the anti-HER3 mAb light chain sequence comprises a signal peptide sequence. In another embodiment, the anti-HER3 mAb light chain signal peptide sequence comprises the amino acid sequence MVLQTQVFISLLLWISGAYG (SEQ ID NO: 13). In one embodiment, the mature polypeptide, such as the antibody heavy or light chain amino acid sequence disclosed herein, lacks a signal peptide.

[0139] In one embodiment, the recombinant fusion protein comprises the following amino acid sequence: Heavy chain QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE F LGGP AVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH A HYTQKSLSLSPGKGGGGSGGGGSSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ (SEQ ID NO: 14, where bold italics indicate the linker and bold indicates the NRG-1 fragment); and Light chain DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3).

[0140] In one embodiment, each of the heavy and light chain sequences in the mature recombinant fusion protein lacks a signal peptide amino acid sequence.

[0141] In certain embodiments of the present invention, the heavy chain of the anti-HER3 antibody provided herein is fused to the NRG-1β2a isoform provided herein via a C-terminal linker sequence.In another embodiment, the C-terminus of the antibody heavy chain comprises the Fc domain of the antibody.

[0142] In some embodiments, a pharmaceutical composition is provided comprising a recombinant fusion protein disclosed herein formulated with a pharmaceutical carrier.

[0143] In some embodiments, the anti-HER3 antibody and NRG-1 fragment described herein are recombinantly or chemically fused / operably linked via a linker to form a fusion protein. "Fusion protein", "fusion polypeptide", "recombinant fusion protein", or "recombinant polypeptide" refers to a hybrid polypeptide comprising polypeptide moieties from at least two different polypeptides. As defined herein, a "fusion protein" is a fusion in which a first amino acid sequence (protein) comprising, for example, the NRG-1β2a isoform of the present invention is linked via a linker to the C-terminus of a second amino acid sequence comprising the heavy chain of an antibody that specifically binds to ERBB3 (HER3).

[0144] In one embodiment, the fusion protein is recombinantly encoded and produced. In some embodiments, the recombinant fusion protein is encoded by a nucleic acid sequence encoding an antibody of the invention operably linked to a nucleic acid sequence encoding an NRG-1β2a isoform of the invention via a nucleic acid sequence encoding a linker.

[0145] In one embodiment, the amino acid sequence of the recombinant fusion protein is homologous to SEQ ID NO: 14 fused to SEQ ID NO: 3. The term "homology" may refer to greater than 70% identity with a recombinant fusion protein sequence (e.g., any of SEQ ID NOs: 1-18). In another embodiment, "homology" refers to greater than 72% identity with any of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to greater than 75% identity with any of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to greater than 78% identity with any of SEQ ID NOs: 1-18. In another embodiment, "homology" refers to greater than 80% identity with any of SEQ ID NOs: 1-18. In another embodiment, "homology" means greater than 82% identity with any of SEQ ID NOs: 1-18. In another embodiment, "homology" means greater than 83% identity with any of SEQ ID NOs: 1-18. In another embodiment, "homology" means greater than 85% identity with any of SEQ ID NOs: 1-18. In another embodiment, "homology" means that the identity with any one of SEQ ID NOs: 1 to 18 is greater than 87%. In another embodiment, "homology" means that the identity with any one of SEQ ID NOs: 1 to 18 is greater than 88%. In another embodiment, "homology" means that the identity with any one of SEQ ID NOs: 1 to 18 is greater than 90%. In another embodiment, "homology" means that the identity with any one of SEQ ID NOs: 1 to 18 is greater than 92%. In another embodiment, "homology" means that the identity with any one of SEQ ID NOs: 1 to 18 is greater than 93%. In another embodiment, "homology" means that the identity with any one of SEQ ID NOs: 1 to 18 is greater than 95%. In another embodiment, "homology" means that the identity with any one of SEQ ID NOs: 1 to 18 is greater than 96%. In another embodiment, "homology" means that the identity with any one of SEQ ID NOs: 1 to 18 is greater than 97%. In another embodiment, "homology" means that the identity with any one of SEQ ID NOs: 1 to 18 is greater than 98%.In another embodiment, "homology" means greater than 99% identity with any of SEQ ID NOs: 1 to 18. In another embodiment, "homology" means greater than 100% identity with any of SEQ ID NOs: 1 to 18.

[0146] The determination of percent identity between two sequences can be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm that can be used to compare two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed using the NBLAST program, score=100, word length=12, to obtain nucleotide sequences that are homologous to the nucleic acid that codes for the protein of interest. BLAST protein searches can be performed with the XBLAST program (score=50, word length=3) to obtain amino acid sequences homologous to the protein of interest. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When using BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM, described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5, and FASTA, described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. When ktup=2, similar regions of the two sequences being compared are found by examining pairs of aligned residues, and when ktup=1, aligned single amino acids are examined. For protein sequences, ktup can be set to 2 or 1, and for DNA sequences it can be set to 1-6. If ktup is not specified, the default is 2 for proteins and 6 for DNA. Alternatively, protein sequence alignment can be performed using the CLUSTAL W algorithm, as described in Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0147] In some embodiments, the polynucleotides of the invention are prepared using PCR techniques using procedures and methods known to those of skill in the art, which in some embodiments involve the ligation of two different DNA sequences (see, e.g., "Current Protocols in Molecular Biology", eds. Ausubel et al., John Wiley & Sons, 1992).

[0148] In one embodiment, the polynucleotide of the present invention is inserted into an expression vector (i.e., a nucleic acid construct) to allow expression of the recombinant polypeptide. In one embodiment, the expression vector of the present invention comprises additional sequences that make the vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector of the present invention comprises additional sequences that make the vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector of the present invention comprises a shuttle vector that makes the vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, the cloning vector comprises transcription and translation initiation sequences (e.g., promoters, enhancers) and transcription and translation termination elements (e.g., polyadenylation signals).

[0149] In one embodiment, a variety of prokaryotic or eukaryotic cells can be used as host expression systems for expressing the polypeptides of the invention. In some embodiments, these include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the polypeptide coding sequence, yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence.

[0150] In some embodiments, non-bacterial expression systems (e.g., mammalian expression systems such as CHO cells) are used to express the polypeptides of the invention. In one embodiment, the expression vector used to express the polynucleotides of the invention in mammalian cells is the pCI-DHFR vector, which contains the CMV promoter and the neomycin resistance gene.

[0151] In some embodiments, in the bacterial system of the present invention, several expression vectors can be advantageously selected depending on the intended use of the expressed polypeptide. In one embodiment, large quantities of the polypeptide are desired. In one embodiment, vectors that direct high levels of protein product expression, possibly as fusions with hydrophobic signal sequences, and direct the expressed product to the bacterial periplasm or culture medium (where the protein product is easily purified) are desired. In one embodiment, specific fusion proteins with engineered specific cleavage sites to aid in the recovery of the polypeptide are desired. In one embodiment, vectors amenable to such manipulations include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol. 185:60-89 (1990)].

[0152] In one embodiment, yeast expression systems are used. In one embodiment, as disclosed in U.S. Patent No. 5,932,447, a number of vectors containing constitutive or inducible promoters can be used in yeast. In another embodiment, vectors that facilitate the integration of foreign DNA sequences into yeast chromosomes are used.

[0153] In one embodiment, the expression vector of the present invention can further comprise additional polynucleotide sequences that allow translation of multiple proteins from a single mRNA, such as, for example, an internal ribosome entry site (IRES), and sequences for genomic integration of the promoter-chimeric polypeptide.

[0154] In some embodiments, the expression vector of the invention comprises elements that increase expression of the recombinant fusion protein of the invention. Such features include, but are not limited to, promoter and polyadenylation selection. In some embodiments, the polyadenylation sequence is the bovine growth hormone (BGH) polyadenylation sequence. In some embodiments, the promoter comprises a constitutively active promoter. In some embodiments, the promoter comprises the cytomegalovirus promoter (pCMV).

[0155] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV, and pBK-CMV available from Strategene, pTRES available from Clontech, and derivatives thereof.

[0156] In some embodiments, expression vectors containing regulatory elements derived from eukaryotic viruses, such as retroviruses, are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papillomavirus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p205. Other exemplary vectors include pMSG, pAV009 / A, pAV009 / B, pAV009 / C, pAV009 / D, pAV009 / E, pAV009 / F, pAV009 / G, pAV009 / H, pAV009 / I, pAV009 / L, pAV009 / N ... + , pMTO10 / A +, pMAMneo-5, baculovirus pDSVE, and other vectors that allow expression of proteins under the direction of the SV-40 early promoter, the SV-40 late promoter, the metallothionein promoter, the mouse mammary tumor virus promoter, the Rous sarcoma virus promoter, the polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells.

[0157] In some embodiments, recombinant viral vectors are useful for in vivo expression of the polypeptides of the present invention because they provide advantages such as horizontal infection and target specificity. In one embodiment, horizontal infection is inherent in, for example, the life cycle of retroviruses, and is the process by which a single infected cell produces many progeny viral particles that bud and infect neighboring cells. In one embodiment, the result is the rapid infection of a large area, most of which was not originally infected by the original viral particle. In one embodiment, the viral vectors produced cannot spread horizontally. In one embodiment, this property can be useful when the desired objective is to introduce a specific gene only into a localized number of target cells.

[0158] In one embodiment, a variety of methods can be used to introduce into cells an expression vector encoding a recombinant fusion protein of the invention. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. Additionally, see U.S. Patent Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0159] In some embodiments, introduction of nucleic acid by viral infection offers several advantages over other methods such as lipofection or electroporation, since viral infectivity results in higher transfection efficiency.

[0160] It will be appreciated that in one embodiment, the polypeptides of the invention can also be expressed from a nucleic acid construct that is administered to an individual using any suitable mode of administration as described above (i.e., in vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into a suitable cell via a suitable gene delivery vehicle / method (transfection, transduction, homologous recombination, etc.), and optionally an expression system, and the modified cells are then grown in culture and returned to the individual (i.e., ex vivo gene therapy).

[0161] It will be understood that, in addition to containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding a polypeptide), the expression constructs of the invention can also contain sequences engineered to optimize the stability, production, purification, yield, or activity of the expressed polypeptide.

[0162] In some embodiments, the transformed cells are cultured under effective conditions that allow for the expression of large amounts of recombinant fusion proteins or polypeptides. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactors, temperature, pH, and oxygen conditions that allow for protein production. In one embodiment, effective media refers to any medium in which cells are cultured to produce the recombinant polypeptides of the present invention. In some embodiments, media typically include aqueous solutions having assimilable carbon, nitrogen, and phosphate sources, as well as appropriate salts, minerals, metals, and other nutrients (e.g., vitamins). In some embodiments, the cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter plates, and petri dishes. In some embodiments, the culture is performed at a temperature, pH, and oxygen content suitable for the recombinant cells. In some embodiments, the culture conditions are within the expertise of one of ordinary skill in the art.

[0163] In some embodiments, depending on the vector and host system used for production, the resulting polypeptides of the invention remain within the recombinant cell, are secreted into the fermentation medium, are secreted into the space between two cell membranes, such as the periplasmic space of E. coli, or are retained on the outer surface of a cell or viral membrane.

[0164] In one embodiment, after a period of time in culture, recovery of the recombinant polypeptide is performed.

[0165] In one embodiment, the phrase "recovering the recombinant polypeptide" as used herein refers to collecting the entire fermentation medium containing the polypeptide and does not necessarily imply additional separation or purification steps.

[0166] In one embodiment, the polypeptides of the invention are purified using a variety of standard protein purification techniques, including, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing, and fractional solubilization.

[0167] In one embodiment, the expressed coding sequence can be engineered to encode a polypeptide of the invention and a fused cleavable moiety to facilitate recovery. In one embodiment, the fusion protein can be designed so that the polypeptide can be easily isolated by affinity chromatography, for example by immobilization on a column specific for the cleavable moiety. In one embodiment, a cleavage site is engineered between the polypeptide and the cleavable moiety, allowing the polypeptide to be released from the chromatography column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site (see, e.g., Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)).

[0168] In one embodiment, the polypeptides of the present invention are recovered in "substantially pure" form.

[0169] In one embodiment, the phrase "substantially pure" refers to a purity that allows for the effective use of the protein in the applications described herein.

[0170] In one embodiment, the polypeptides of the present invention can also be synthesized using an in vitro expression system. In one embodiment, in vitro synthesis methods are known in the art and the components of the system are commercially available.

[0171] In some embodiments, recombinant polypeptides can be synthesized and purified, and their therapeutic effects assayed in vivo or in vitro.

[0172] In one embodiment, the pharmaceutical composition provided herein, which comprises the recombinant fusion protein of the present invention, is further formulated with a pharmaceutical carrier. As used herein, "pharmaceutical carrier" includes any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

[0173] Treatment method In one embodiment, the present invention provides a method for treating a disease or condition in a subject in need of treatment, the method comprising administering a therapeutically effective amount of a recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein disclosed herein.

[0174] In one embodiment, the present invention provides a method for treating a cardiovascular disease or condition in a subject in need of treatment, the method comprising administering a therapeutically effective amount of a recombinant fusion protein or a pharmaceutical composition comprising the same.

[0175] In one embodiment, the invention provides a method for preventing, inhibiting, suppressing, or delaying the onset of a cardiovascular disease or condition in a subject, the method comprising administering an effective amount of a recombinant fusion protein or pharmaceutical composition described herein.

[0176] In some embodiments, the cardiovascular disease or condition comprises atrial fibrillation.

[0177] In some embodiments, the cardiovascular disease or condition comprises cardiac fibrosis.

[0178] In some embodiments, the cardiovascular disease or condition comprises atrial fibrillation and cardiac fibrosis.

[0179] In some embodiments, cardiovascular disease includes 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, adjunct to surgical intervention in pediatric congenital heart disease.

[0180] In some embodiments, chemotherapy-induced cardiotoxicity occurs as a result of the subject receiving anthracyclines, alkylating agents, anti-microtubule agents, and antimetabolites used as chemotherapy.

[0181] In some embodiments, the cardiovascular condition is cardiotoxicity that occurs as a result of the subject undergoing cancer treatment.In other embodiments, the cancer treatment is HER-2 targeting therapy.In other embodiments, the HER-2 targeting therapy includes the use of trastuzumab, ado-trastuzumab, emtansine, lapatinib, neratinib, and pertuzumab, any anti-HER2 antibody, any anti-HER2 agent, or combinations thereof.

[0182] In another aspect, the invention relates to a method for inducing remodeling of sarcomeric and cytoskeletal structures or cell-cell adhesion in muscle cells, comprising treating the cells with a recombinant fusion protein disclosed herein.

[0183] In one embodiment, the therapeutic method relates to the treatment of heart failure resulting from dissociation of cardiac muscle cell-cell adhesion and / or disruption of sarcomere structure in a mammal.

[0184] In another aspect, the present invention provides a method of preventing, treating, or delaying heart failure with preserved ejection fraction in a human, the method comprising administering a pharmaceutical composition comprising a recombinant fusion protein disclosed herein.

[0185] The term "ejection fraction" as used herein refers to the ejection fraction (EF), which is a measurement, usually expressed as a percentage, of the amount of blood pumped by the left ventricle with each contraction. For example, an ejection fraction of 50 percent means that 50 percent of the total blood volume of the left ventricle is pumped with each heartbeat.

[0186] The present invention relates to the treatment of subjects suffering from or at risk of developing cardiac disease and related conditions, such as heart failure.

[0187] The term "heart failure" refers to an abnormality in cardiac function in which the heart does not pump blood at a rate required for the metabolic tissue requirements. Heart failure includes various conditions 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 variety of factors, including ischemic, congenital, rheumatic, and idiopathic. Chronic cardiac hypertrophy is a significant precursor to congestive heart failure and cardiac arrest.

[0188] In one embodiment, "treatment" refers to both therapeutic and prophylactic or preventative treatment, where the purpose is to prevent or slow down (reduce) cardiac hypertrophy. Those in need of treatment include those already affected, those susceptible to disease, or those in whom the disease is to be prevented. Cardiac hypertrophy can result from any cause that responds to retinoic acid, including congenital, viral, idiopathic, cardiac hypertrophic, or myohypertrophic causes, or as a result of an ischemic insult, such as ischemia or myocardial infarction. Treatment is usually performed to stop or slow down the progression of hypertrophy, especially after cardiac damage has occurred, such as from ischemia. Preferably, for the treatment of myocardial infarction, the pharmaceutical composition provided herein is administered immediately after myocardial infarction to prevent or reduce hypertrophy.

[0189] In some embodiments, treating a subject with atrial fibrillation or cardiac fibrosis using recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein described herein may reduce the signs or symptoms of atrial fibrillation or fibrosis.For example, treating a subject with recombinant fusion protein may reduce the duration and / or frequency of atrial fibrillation, or reduce the signs or symptoms of atrial fibrillation, such as arrhythmia, palpitations, lightheadedness, extreme fatigue, shortness of breath, chest pain, or a combination thereof.In some embodiments, treating a subject with recombinant fusion protein reduces collagen content or deposition in cardiac tissue, such as atrial tissue.

[0190] In some embodiments, treating a subject with a pharmaceutical composition comprising a recombinant fusion protein provided herein may increase the average survival time of the treated subject population compared to a population receiving monotherapy with a drug that is not a compound of the present disclosure, or a pharma- ceutically acceptable salt, solvate, analog, or derivative thereof.Preferably, after treatment with the strategies, treatment regimes, methods, combinations, and compositions provided herein, the average survival time is increased by more than 30 days, more preferably more than 60 days, more preferably more than 90 days, 120 days, or more than 365 days, more preferably more than 365 days. The increase in the average survival time of a population can be measured by any reproducible means. The increase in the average survival time of a population can be measured, for example, by calculating the average survival time of the population after the start of treatment with an active compound. The increase in the average survival time of a population can also be measured, for example, by calculating the average survival time of the population after the completion of a first round of treatment with a pharmaceutical composition disclosed herein.

[0191] In some embodiments, treating a subject with a pharmaceutical composition comprising a recombinant fusion protein provided herein may reduce the mortality rate of the treated subject population compared to a population administered with carrier alone. Treating cancer may reduce the mortality rate of the treated subject population compared to an untreated population. Treating cancer may reduce the mortality rate of the treated subject population compared to a population receiving monotherapy with a drug that is not a compound of the present disclosure, or a pharma-ceutically acceptable salt, solvate, analog, or derivative thereof. Preferably, after treatment with the strategies, treatment regimes, methods, combinations, and compositions provided herein, the mortality rate is reduced by more than 2%, more preferably more than 5%, more preferably more than 10%, and most preferably more than 25%. The reduction in mortality rate of the treated subject population can be measured by any reproducible means. The reduction in mortality rate of the population can be measured, for example, by calculating the average number of disease-related deaths per unit time for the population after the start of treatment with the active compound. The reduction in mortality in a population can also be measured, for example, by calculating the average number of disease-related deaths per unit time for the population after completion of a first round of treatment with a pharmaceutical composition disclosed herein.

[0192] In one embodiment, the present invention provides a method for treating a central nervous system (CNS)-related disease or condition in a subject in need of treatment, the method comprising administering a therapeutically effective amount of a recombinant fusion protein or pharmaceutical composition described herein.

[0193] In one embodiment, the present invention provides a method for preventing, inhibiting, suppressing, or delaying the onset of a CNS-related disease or condition in a subject, the method comprising administering an effective amount of a recombinant fusion protein or pharmaceutical composition described herein.

[0194] In some embodiments, the CNS-related disease or condition is amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Bell's palsy, epilepsy and seizures, Guillain-Barre syndrome, stroke, traumatic brain injury, multiple sclerosis, or a combination thereof.

[0195] Administration The composition of the present invention can be administered parenterally to a subject in need thereof, or by various methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. To administer the compound of the present invention by a certain route of administration, it may be necessary to coat the compound with a material to prevent its inactivation, or to co-administer the compound with the material. For example, the compound can be administered to a subject in a suitable carrier, such as liposomes or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharma-ceutically active substances is well known in the art.

[0196] In typical embodiments, preparations for administration to a subject include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Some embodiments include non-aqueous solvents, such as propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), organic esters (e.g., ethyl oleate), and other solvents known to those skilled in the art. In certain embodiments of the present invention, physiologically acceptable carriers (or excipients) are optionally used. Examples of such carriers include, for example, saline, PBS, Ringer's solution, lactated Ringer's solution, and the like. In addition, preservatives and additives are optionally added to the composition to ensure stability and sterility. For example, antibiotics and other bactericides, antioxidants, chelating agents, and the like are all optionally present in various embodiments of the compositions herein.

[0197] The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0198] Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention are formulated into pharma- ceutical acceptable dosage forms by conventional methods known to those skilled in the art.

[0199] The recombinant fusion protein or pharmaceutical composition comprising the same is optionally administered to a subject in need of treatment (therapeutic or prophylactic) in any suitable sterile pharmaceutical carrier. Such pharmaceutical carriers act to maintain the solubility and activity of the fusion protein. In some embodiments, it may be desirable to administer additional components in combination with the fusion protein. For example, in some treatment regimens, chemotherapeutic agents, antibiotics, additional formulations including the recombinant fusion protein of the present invention and one or more standard therapeutic agents, etc., are all optionally included with the composition of the present invention.

[0200] As used herein, the terms "combination treatment", "combination therapy" and "co-therapy" are used interchangeably and generally refer to a treatment regimen featuring a recombinant fusion protein provided herein or a pharmaceutical composition comprising the same and an additional therapeutic agent. A combination treatment regimen is usually part of a particular treatment regimen and is intended to provide a beneficial effect from the simultaneous action of the combination of therapeutic agents. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic synergy resulting from the combination of therapeutic agents. The combined administration of these therapeutic agents is usually performed over a defined period of time (usually minutes, hours, days, or weeks, depending on the combination selected). In some embodiments, the combination treatment includes the sequential administration of two or more therapeutic agents, where each therapeutic agent is administered at a different time and where the therapeutic agent or at least two of the therapeutic agents are administered substantially simultaneously. Substantially simultaneous administration can be achieved, for example, by administering to the subject a single dosage form having a fixed ratio of each therapeutic agent or separate dosage forms of the therapeutic agents. The sequential or substantially simultaneous administration of each therapeutic agent can be by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same route or by different routes. The therapeutic agents can be administered according to the same or different dosing intervals. For example, the first therapeutic agent of a selected combination can be administered by intravenous injection, while the other therapeutic agents of the combination are administered orally. Alternatively, for example, all therapeutic agents can be administered orally, or all therapeutic agents can be administered by intravenous injection.

[0201] In some embodiments, the combination therapy also includes administering the above-mentioned therapeutic agent in combination with other biologically active ingredients and non-drug therapy (e.g., surgery or radiation therapy).When the combination therapy further includes non-drug therapy, the non-drug therapy can be administered at any appropriate time, as long as beneficial effects are achieved from the synergistic action of the combination of the therapeutic agent and the non-drug therapy.For example, in appropriate cases, the non-drug therapy can be administered temporarily, perhaps days or weeks away from the administration of the therapeutic agent, and still achieve beneficial effects.

[0202] In some embodiments, the additional therapeutic agent is a chemotherapeutic agent (also called an anti-tumor or anti-proliferative agent), such as an alkylating agent, an antibiotic, an antimetabolite, an antidote, an interferon, a polyclonal or monoclonal antibody, an EGFR inhibitor, a HER2 inhibitor, a histone deacetylase inhibitor, a hormone, a mitosis inhibitor, an MTOR inhibitor, a multikinase inhibitor, a serine / threonine kinase inhibitor, a tyrosine kinase inhibitor, a VEGF / VEGFR inhibitor, a taxane or taxane derivative, an aromatase inhibitor, an anthracycline, a microtubule targeting agent, a topoisomerase poison, an inhibitor of a molecular target or enzyme (e.g., a kinase or protein methyltransferase), a cytidine analog drug, or any chemotherapeutic agent, an immune checkpoint inhibitor, a platinum-based anti-tumor agent, a CDK inhibitor, a PARP inhibitor, or any anti-tumor or anti-proliferative agent known to one of skill in the art.

[0203] Exemplary alkylating agents suitable for use in accordance with the combination treatment regimens described herein include, but are not limited to, cyclophosphamide (Cytoxan, Neosar), chlorambucil (Leukeran), melphalan (Alkeran), carmustine (BiCNU), busulfan (Busulfex), lomustine (CeeNU), dacarbazine (DTIC-Dome), oxaliplatin (Eloxatin), carmustine (Gliadel), ifosfamide (Ifex), mechlorethamine (Mustargen), busulfan (Myleran), carboplatin (Paraplatin), cisplatin (CDDP, Platinol), temozolomide (Temodar), thiotepa (Thioplex), bendamustine (Treanda), or streptozocin (Zanosar).

[0204] Exemplary suitable anthracyclines include, but are not limited to, doxorubicin (Adriamycin), doxorubicin liposomal (Doxil), mitoxantrone (Novantrone), bleomycin (Blenoxane), daunorubicin (Cerubidine), daunorubicin liposomal (DaunoXome), dactinomycin (Cosmegen), epirubicin (Ellence), idarubicin (Idamycin), plicamycin (Mithracin), mitomycin (Mutamycin), pentostatin (Nipent), or valrubicin (Nipent).

[0205] Exemplary antimetabolites include, but are not limited to, fluorouracil (Adrucil), capecitabine (Xeloda), hydroxyurea (Hydrea), mercaptopurine (Purinethol), pemetrexed (Alimta), fludarabine (Fludara), nelarabine (Arranon), cladribine (Cladribine Novaplus), clofarabine (Clolar), cytarabine (Cytosar-U), decitabine (Dacogen), cytarabine liposomal (DepoCyt), hydroxyurea (Droxia), pralatrexate (Folotyn), floxuridine (FUDR), gemcitabine (Gemzar), cladribine (Leustatin), fludarabine (Oforta), methotrexate (MTX, Rheumatrex), methotrexate (Trexall), thioguanine (Tabloid), TS-1, or cytarabine (Tarabine PFS).

[0206] Exemplary antidotes include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).

[0207] Exemplary interferons include, but are not limited to, interferon alpha-2b (Intron A) or interferon alpha-2a (Roferon-A).

[0208] Exemplary polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin), ofatumumab (Arzerra), bevacizumab (Avastin), rituximab (Rituxan), cetuximab (Erbitux), panitumumab (Vectibix), tositumomab / iodine-131 tositumomab (Bexxar), alemtuzumab (Campath), ibritumomab (Zevalin, In-111, Y-90 Zevalin), gemtuzumab (Mylotarg), eculizumab (Soliris), or denosumab.

[0209] Exemplary EGFR inhibitors include, but are not limited to, gefitinib (Iressa), lapatinib (Tykerb), cetuximab (Erbitux), erlotinib (Tarceva), panitumumab (Vectibix), PKI-166, canertinib (CI-1033), matuzumab (EMD 72000), or EKB-569.

[0210] Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin), lapatinib (Tykerb), or AC-480.

[0211] Histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza).

[0212] Exemplary hormones include, but are not limited to, tamoxifen (Soltamox, Nolvadex), raloxifene (Evista), megestrol (Megace), leuprolide (Lupron, Lupron Depot, Eligard, Viadur), fulvestrant (Faslodex), letrozole (Femara), triptorelin (Trelstar LA, Trelstar Examples of drugs that may be used include fluoxetine (Depot), exemestane (Aromasin), goserelin (Zoladex), bicalutamide (Casodex), anastrozole (Arimidex), fluoxymesterone (Androxy, Halotestin), medroxyprogesterone (Provera, DepoProvera), estramustine (Emcyt), flutamide (Eulexin), toremifene (Fareston), degarelix (Firmagon), nilutamide (Nilandron), abarelix (Plenaxis), or testolactone (Teslac).

[0213] Exemplary mitotic inhibitors include, but are not limited to, paclitaxel (Taxol, Onxol, Abraxane), docetaxel (Taxotere), vincristine (Oncovin, Vincasar PFS), vinblastine (Velban), etoposide (Toposarol, Etopophos, VePesid), teniposide (Vumon), ixabepilone (Ixempra), nocodazole, epothilones, vinorelbine (Navelbine), camptothecin (CPT), irinotecan (Camptosar), topotecan (Hycamtin), amsacrine, or lamellarin D (LAM-D).

[0214] Exemplary MTOR inhibitors include, but are not limited to, everolimus (Afinitor) or temsirolimus (Torisel), rapamne, ridaforolimus, or AP23573.

[0215] Exemplary multikinase inhibitors include, but are not limited to, sorafenib (Nexavar), sunitinib (Sutent), BIBW2992, E7080, Zd6474, PKC-412, motesanib, or AP24534.

[0216] Exemplary serine / threonine kinase inhibitors include, but are not limited to, ruboxistaurin, eril / fasudil hydrochloride, flavopiridol, seliciclib (CYC202, Roscovitine), SNS-032 (BMS-387032), Pkc412, bryostatin, KAI-9803, SF1126, VX-680, Azd1152, Arry-142886 (AZD-6244), SCIO-469, GW681323, CC-401, CEP-1347, or PD332991.

[0217] Exemplary tyrosine kinase inhibitors include, but are not limited to, erlotinib (Tarceva), gefitinib (Iressa), imatinib (Gleevec), sorafenib (Nexavar), sunitinib (Nexavar), trastuzumab (Herceptin), bevacizumab (Avastin), rituximab (Rituxan), lapatinib (Tykerb), cetuximab (Erbitux), panitumumab (Vectibix), everolimus (Afinitor), alemtuzumab (Alemtuzumab), and cetinib (Erbitux). (Campath), gemtuzumab (Mylotarg), temsirolimus (Torisel), pazopanib (Votrient), dasatinib (Sprycel), nilotinib (Tasigna), vatalanib (Ptk787, ZK222584), CEP-701, SU5614, MLN518, XL999, VX-322, Azd0530, BMS-354825, SKI-606, CP-690, AG-490, WHI-P154, WHI-P131, AC-220, or AMG888.

[0218] Exemplary VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin), sorafenib (Nexavar), sunitinib (Sutent), ranibizumab, pegaptanib, or vandetinib.

[0219] Exemplary microtubule targeting agents include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epothilones, and navelbine.

[0220] Exemplary topoisomerase poisons include, but are not limited to, teniposide, etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin, and idarubicin.

[0221] Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxol.

[0222] Exemplary immune checkpoint inhibitors include programmed cell death 1 (PD-1), CD274 molecule (PD-L1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitors. Exemplary PD-1 inhibitors include pembrolizumab, nivolumab, and cemiplimab. Exemplary PD-L1 inhibitors include atezolizumab, avelumab, and durvalumab. Exemplary CLTA4 inhibitors include ipilimumab.

[0223] Exemplary platinum-based antitumor agents include cisplatin and carboplatin.

[0224] Exemplary cyclin-dependent kinase (CDK) inhibitors include abemaciclib, palbociclib, and ribociclib.

[0225] Exemplary poly(ADP-ribose) polymerase (PARP) inhibitors include talazoparib, olaparib, rucaparib, niraparib, and veliparib.

[0226] Exemplary common chemotherapeutic, anti-tumor, and anti-proliferative agents include, but are not limited to, altretamine (Hexalen), isotretinoin (Accutane, Amnesteem, Claravis, Sotret), tretinoin (Vesanoid), azacytidine (Vidaza), bortezomib (Velcade), asparaginase (Elspar), levamisole (Ergamisol), mitotane (Lysodren), procarbazine (Matula), and the like. ne), pegaspargase (Oncaspar), denileukin diftitox (Ontak), porfimer (Photofrin), aldesleukin (Proleukin), lenalidomide (Revlimid), bexarotene (Targretin), thalidomide (Thalomid), temsirolimus (Torisel), arsenic trioxide (Trisenox), verteporfin (Visudyne), mimosine (Leucenol), (1M tegafur-0.4M 5-chloro-2,4-dihydroxypyrimidine-1M potassium oxonate), or lovastatin.

[0227] In some embodiments, a combination treatment regimen is provided in which the additional therapeutic agent is a cytokine, such as G-CSF (granulocyte colony stimulating factor). In another embodiment, the pharmaceutical composition provided herein can be administered in combination with radiation therapy. Radiation therapy can also be administered as part of a multi-drug therapy in combination with the pharmaceutical composition provided herein and another chemotherapeutic agent described herein. In yet another embodiment, the pharmaceutical composition provided herein can be administered in combination with any of the following chemotherapeutic agents, including, but not limited to, CMF (cyclophosphamide, methotrexate, and 5-fluorouracil), CAF (cyclophosphamide, adriamycin, and 5-fluorouracil), AC (adriamycin and cyclophosphamide), FEC (5-fluorouracil, epirubicin, and cyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide, and paclitaxel), rituximab, Xeloda (capecitabine), cisplatin (CDDP), carboplatin, TS-1 (molecule), cyclophosphamide (cyclophosphamide ... It may be administered in combination with standard chemotherapy agents such as tegafur, gimestat, and otastat potassium in a ratio of 1:0.4:1, camptothecin-11 (CPT-11, irinotecan, or Camptosar™), CHOP (cyclophosphamide, hydroxydaunorubicin, Oncovin, and prednisone or prednisolone), R-CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin, Oncovin, prednisone, or prednisolone), or CMFP (cyclophosphamide, methotrexate, 5-fluorouracil, and prednisone).

[0228] In some preferred embodiments, the pharmaceutical compositions provided herein may be administered with inhibitors of enzymes, such as receptor or non-receptor kinases. Receptor and non-receptor kinases are, for example, tyrosine kinases or serine / threonine kinases. The kinase inhibitors described herein are small molecules, polynucleic acids, polypeptides, or antibodies.

[0229] Exemplary kinase inhibitors include, but are not limited to, bevacizumab (targets VEGF), BIBW2992 (targets EGFR and Erb2), cetuximab / erbitux (targets Erb1), imatinib / Gleevec (targets Bcr-Abl), trastuzumab (targets Erb2), gefitinib / iressa (targets EGFR), ranibizumab (targets VEGF), pegaptanib (targets VEGF), erlotinib / tarceva (targets Erb1), nilotinib (targets Bcr-Abl), (targets Erb1 and Erb2 / Her2), GW-572016 / lapatinib ditosylate (targets HER2 / Erb2), panitumumab / vectibix (targets EGFR), vandetinib (targets RET / VEGFR), E7080 (multiple targets including RET and VEGFR), Herceptin (targets HER2 / Erb2), PKI-166 (targets EGFR), canertinib / CI-1033 (targets EGFR), sunitinib / SU-11464 / sutent (targets EGFR and FLT3) (targets EGFR), matuzumab / Emd7200 (targets EGFR), EKB-569 (targets EGFR), Zd6474 (targets EGFR and VEGFR), PKC-412 (targets VEGR and FLT3), vatalanib / Ptk787 / ZK222584 (targets VEGR), CEP-701 (targets FLT3), SU5614 (targets FLT3), MLN518 (targets FLT3), XL999 (targets FLT3), VX-322 (targets FLT3), Azd0530 (targets SRC (targets SRC), BMS-354825 (targets SRC), SKI-606 (targets SRC), CP-690 (targets JAK), AG-490 (targets JAK), WHI-P154 (targets JAK), WHI-P131 (targets JAK), sorafenib / Nexavar (targets RAF kinase, VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-β, KIT, FLT-3, and RET), dasatinib / Sprycel (targets BCR / ABL and Src), AC-220 (targets Flt3),These include AC-480 (targets all HER proteins, "panHER"), motesanib diphosphate (targets VEGF1-3, PDGFR, and c-kit), denosumab (targets RANKL and inhibits SRC), AMG888 (targets HER3), and AP24534 (multiple targets including Flt3).

[0230] In some embodiments, such as those in which the recombinant fusion protein is administered to treat atrial fibrillation, the combination therapy may include administration of a beta-blocker (e.g., bisprolol or metoprolol succinate), a calcium channel blocker (e.g., diltiazem or verapamil), digoxin, an antiarrhythmic drug (e.g., propafenone, flecainide, sotalol, dofetilide, amiodarone, and dronedarone), or an anticoagulant (e.g., warfarin, apixabab, dabigatran, edoxaban, or rivaroxaban). In some embodiments, the combination therapy includes a defibrillation therapy (e.g., electrical or chemical cardioversion) to reset the heart's sinus rhythm. In some embodiments, the combination therapy includes ablation (e.g., AV node ablation or Maze therapy). Ablation uses a scalpel, heat, or cold to create small scars in the heart, blocking erroneous electrical signals and restoring normal heart rhythm.

[0231] In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising the same polypeptide disclosed herein is administered to a subject once a day. In some embodiments, a recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject once every two days. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject once every three days. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject once every four days. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject once every five days. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject once every six days. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject once a week. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject once every 7 to 14 days. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject once every 10 to 20 days. In another embodiment, a recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject once every 5 to 15 days. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject once every 15 to 30 days.

[0232] In one embodiment, the dosage of the recombinant fusion protein of the present invention comprises 0.005-0.1 mg / kg in injection solution. In another embodiment, the dosage comprises 0.005-0.5 mg / kg of recombinant fusion protein. In another embodiment, the dosage comprises 0.05-0.1 micrograms of recombinant fusion protein. In another embodiment, the dosage comprises 0.005-0.1 mg / kg of recombinant fusion protein in injection solution.

[0233] In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose ranging from 0.0001 mg to 0.6 mg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose ranging from 0.001 mg to 0.005 mg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose ranging from 0.005 mg to 0.01 mg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose ranging from 0.01 mg to 0.3 mg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose ranging from 0.2 mg to 0.6 mg.

[0234] In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose ranging from 1 to 100 mcg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose ranging from 10 to 80 mcg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose ranging from 20 to 60 mcg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose ranging from 10 to 50 mcg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose ranging from 40 to 80 mcg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose ranging from 10 to 30 mcg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose ranging from 30 to 60 mcg / kg.

[0235] In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 0.1 mcg / kg to 100 mg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 0.1 mcg / kg to 50 mg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 0.1 mcg / kg to 25 mg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 0.1 mcg / kg to 10 mg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 0.1 mcg / kg to 5 mg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 0.1 mcg / kg to 1 mg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 0.1 mcg / kg to 0.1 mg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 10 mg / kg to 60 mg / kg.

[0236] In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising same is administered to a subject at a dose of about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, or about 70 mg / kg. In some embodiments, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose of about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, or 1 mg / kg.

[0237] In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 0.2 mg to 2 mg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 2 mg to 6 mg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 4 mg to 10 mg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at a dose ranging from 5 mg to 15 mg.

[0238] In one embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose ranging from 10 μg / kg to 1000 μg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose ranging from 25 μg / kg to 600 μg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose ranging from 50 μg / kg to 400 μg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose of about 25 μg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose of about 50 μg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose of about 100 μg / kg. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject at a dose of about 200 μg / kg. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the same is administered to the subject at a dose of about 300 μg / kg. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the same is administered to the subject at a dose of about 400 μg / kg. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the same is administered to the subject at a dose of about 500 μg / kg. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the same is administered to the subject at a dose of about 600 μg / kg.

[0239] In one embodiment, a single dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject. In another embodiment, a total of two doses of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject. In another embodiment, a total of two or more doses of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to the subject.

[0240] In another embodiment, the dose of the recombinant fusion protein or the pharmaceutical composition comprising the same is administered to the subject at least once a day. In another embodiment, the dose of the recombinant fusion protein or the pharmaceutical composition comprising the same is administered to the subject at least once every two days. In another embodiment, the dose of the recombinant fusion protein or the pharmaceutical composition comprising the same is administered to the subject at least once every two or more days. In another embodiment, the dose of the recombinant fusion protein or the pharmaceutical composition comprising the same is administered to the subject every week, every other week, or every three weeks. In another embodiment, the dose of the recombinant fusion protein or the pharmaceutical composition comprising the same is administered to the subject at least once a week. In another embodiment, the dose of the recombinant fusion protein or the pharmaceutical composition comprising the same is administered to the subject at least once every two weeks. In another embodiment, the dose of the recombinant fusion protein or the pharmaceutical composition comprising the same is administered to the subject at least once every three weeks. In another embodiment, the dose of the recombinant fusion protein or the pharmaceutical composition comprising the same is administered to the subject at least once every three weeks or more. In another embodiment, the dose of the recombinant fusion protein or the pharmaceutical composition comprising the same is administered to the subject two or more times a week. In another embodiment, the dose of the recombinant fusion protein or the pharmaceutical composition comprising the same is administered to the subject more than once a month. In another embodiment, the dose of the recombinant fusion protein or the pharmaceutical composition comprising the same is administered to the subject more than once a year. In another embodiment, the dose of the recombinant fusion protein or the pharmaceutical composition comprising the same is administered to the subject more than once every two years. In another embodiment, the dose of the recombinant fusion protein or the pharmaceutical composition comprising the same is administered to the subject more than once every two years or more.

[0241] In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 36 hours. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 48 hours. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 60 hours. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 72 hours. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 84 hours. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 96 hours. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 5 days. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 6 days. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 7 days. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 8 to 10 days. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 10 to 12 days. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 12 to 15 days. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 15 to 25 days. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once every 20 to 30 days.

[0242] In one embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered to a subject at least once per month. In one embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once per two months. In one embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once per three months. In one embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once per four months. In one embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once per five months. In one embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once per six months. In one embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered at least once per six to twelve months. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the same is administered quarterly. In another embodiment, a dose is administered daily, weekly, biweekly, monthly, or yearly. In another embodiment, the dose is administered once, twice, or more than once daily, weekly, monthly, or yearly. In another embodiment, the dose is administered every 2, 3, 4, or at least 5 years.

[0243] In one embodiment, repeated administrations (doses) of the compositions of the invention can be performed immediately after an initial course of treatment, or at intervals of days, weeks, or years, to achieve the desired effect (e.g., preventing or treating a cardiovascular disease or condition, or a central nervous system-related disease or condition) as further provided herein.

[0244] In one embodiment, the pharmaceutical composition is administered by intravenous, intraarterial, subcutaneous or intramuscular injection of liquid formulation.In another embodiment, the liquid formulation includes solution, suspension, dispersion, emulsion, oil, etc.In one embodiment, the pharmaceutical composition is administered intravenously, and therefore formulated in a form suitable for intravenous administration.In another embodiment, the pharmaceutical composition is administered intraarterially, and therefore formulated in a form suitable for intraarterial administration.

[0245] In some embodiments, the compositions used in the methods disclosed herein comprise solutions or emulsions, which in some embodiments are aqueous solutions or emulsions comprising a safe and effective amount of a compound disclosed herein and optionally other compounds, intended for intravenous or subcutaneous administration.

[0246] In some embodiments, the various components of the composition are pre-measured and / or pre-packaged and / or ready to use without additional measurements etc. The present invention also optionally includes kits for carrying out / using the methods and / or compositions of the present invention. In particular, these kits include, for example, suitable recombinant fusion proteins (and optionally mixtures of multiple such proteins for carrying out synergistic treatments, see above), and optionally also suitable disease-related antigen(s). Furthermore, such kits may also include suitable excipients (e.g., pharma- ceutically acceptable excipients) for carrying out the therapeutic and / or prophylactic treatments of the present invention. Such kits optionally include additional components for the assembly and / or use of the compositions of the present invention, including, but not limited to, diluents etc.

[0247] The compositions described herein are optionally packaged to include all (or nearly all) components necessary to carry out the methods or use the compositions of the invention (optionally including, e.g., written instructions for using the methods / compositions of the invention). For example, the kits optionally include components such as buffers, reagents, serum proteins, antibodies, substrates, etc. In the case of prepackaged reagents, the kits optionally include premeasured or pre-portioned amounts that can be incorporated into the method without measurement, e.g., premeasured liquid aliquots, or pre-measured or pre-measured solid reagents that can be easily reconstituted by the end user of the kit.

[0248] Such kits also typically include suitable instructions for carrying out the methods of the invention and / or for using the compositions of the invention. In some embodiments, the components of the kit / package are provided in a stabilized form so that deterioration or other losses, e.g., due to leakage, can be prevented during long-term storage. Many stabilization processes / agents, such as the addition of chemical stabilizers (i.e., enzyme inhibitors, bactericides / bacteria inhibitors, anticoagulants), are widely used for reagents to be preserved. The actual dose level of the active ingredient in the pharmaceutical composition of the invention can be modified to obtain an amount of the active ingredient effective for a particular subject, composition, and method of administration to achieve a desired therapeutic response without causing toxicity to the subject. The dose level selected will depend on various pharmacokinetic factors, including the activity of the particular composition of the invention used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in conjunction with the particular composition used, the age, sex, weight, medical condition, general health, and medical history of the subject to be treated, and similar factors well known in the medical field.

[0249] The composition must be sterile and fluid enough to be delivered by syringe.Besides water, the carrier is preferably an isotonic buffered saline solution.Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants.In many cases, it is preferable to include isotonic substances in the composition, for example, sugars, polyhydric alcohols (e.g., mannitol or sorbitol), and sodium chloride.

[0250] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied for a particular subject, composition, and method of administration to obtain an amount of the active ingredient effective to achieve a desired therapeutic response without toxicity to the subject. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition being used, the age, sex, weight, medical condition, general health, and medical history of the subject being treated, and similar factors well known in the medical arts.

[0251] While several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision numerous other means and / or configurations for performing the functions and / or results and / or obtaining one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications for which the teachings of the invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that, within the scope of the appended claims and their equivalents, the embodiments of the invention may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure relate to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the scope of the invention of this disclosure, unless such features, systems, articles, materials, kits, and / or methods are mutually inconsistent.

[0252] All definitions provided and used herein should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0253] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which may include the entire document, in some cases.

[0254] The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0255] The term "and / or" as used in the specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are sometimes conjointly present and other times disjointly present. Multiple elements listed with "and / or" should be interpreted similarly, i.e., "one or more" of the elements so conjoined. Elements other than the elements specifically identified by the term "and / or" may optionally be present, whether or not in association with the elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with an open-ended expression such as "comprises", can refer in one embodiment to only A (optionally including elements 5 other than B), in another embodiment to only B (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), etc.

[0256] In this specification and claims, the phrase "at least one" as used in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or not to the specifically identified elements, may optionally be present. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one (optionally including more than one) A in which B is absent (and optionally including elements other than B), in another embodiment to at least one (and optionally including more than one) B in which A is absent (and optionally including elements other than A), in yet another embodiment to at least one (and optionally including more than one) A and at least one (and optionally including more than one) B (and optionally including other elements), etc.

[0257] The invention further provides kits for preventing, treating, or delaying a cardiovascular disease or condition in a human, wherein the kit comprises one or more doses of a pharmaceutical composition comprising a recombinant fusion protein disclosed herein for use in preventing, treating, or delaying a cardiovascular disease or condition, and instructions on how to use the pharmaceutical formulation or composition.

[0258] The present invention further provides kits for preventing, treating, or delaying a CNS-related disease or condition in a human, wherein the kit comprises one or more doses of a pharmaceutical composition comprising a recombinant fusion protein disclosed herein for use in preventing, treating, or delaying a cardiovascular disease or condition, and instructions on how to use the pharmaceutical formulation or composition.

[0259] The present invention further provides a kit for preventing, treating, or delaying heart failure with preserved ejection fraction in a human, the kit comprising one or more doses of a pharmaceutical composition comprising a recombinant fusion protein disclosed herein for use in preventing, treating, or delaying heart failure with preserved ejection fraction, and instructions on how to use the pharmaceutical formulation or composition.

[0260] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention, but should in no way be construed as limiting the broad scope of the invention. EXAMPLES

[0261] Example 1 - Cloning and construction of expression plasmids The DNA sequences encoding the heavy chain (designated NPCFA and NPCF depending on the presence or absence of Fc mutation, respectively) and light chain (designated PAL) of the recombinant fusion proteins were synthesized by GENEWIZ (Suzhou, China). The expression vector pCHOGUN was obtained from Horizon Discovery (Cambridge, UK) under a license agreement. The construction of expression plasmids is performed as outlined in Figure 1. Briefly, the pCHOGUN vector was linearized with the restriction enzyme BfuAI, and the gene inserts, such as NPCF, NPCFA, and PAL, were purified after double restriction enzyme digestion with NcoI and AscI. The linearized pCHOGUN / BfuAI and the purified gene inserts were ligated according to standard protocols and then transformed into E. coli DH5α competent cells. DH5α cells were plated and incubated at 37°C overnight. Plasmids pCHOGUN-NPCF, pCHOGUN-NPCFA, and pCHOGUN-PAL were isolated and confirmed by restriction enzyme digestion or PCR. Plasmids containing the heavy chain insert (pCHOGUN-NPCF or pCHOGUN-NPCFA) were digested with restriction enzymes BspEI and PciI, whereas plasmids containing the light chain insert (pCHOGUN-PAL) were digested with restriction enzymes NgoMIV and PciI. After restriction enzyme digestion, fragments containing the heavy or light chain insert were purified, ligated, and transformed into DH5α cells. Plasmid constructs containing both the heavy and light chain inserts (pCHOGUN-NPCF+PAL or pCHOGUN-NPCFA+PAL) were identified and confirmed by restriction enzyme digestion and DNA sequencing.

[0262] Example 2 - Antibody production, purification, and characterization Glutamine synthetase-null (GS - / -HD-BIOP3, a human ovarian tumor suppressor (HUVEC) cell line, was obtained from Horizon Discovery (Cambridge, UK) under license agreement. Plasmid DNA was isolated using the commercially available QiagenPlasmid kit. Plasmid DNA was transfected into HD-BIOP3 cells using the commercially available electroporation system from Lonza. Transfected cells were plated in 96-well plates and pool selection was performed using standard procedures. Cells from selected pools were cultured in 125 mL shake flasks for 10-14 days and media was harvested for antibody purification. Antibody proteins were purified by protein A affinity chromatography followed by size exclusion chromatography and then analyzed using SDS-PAGE and Western blot according to standard protocols.

[0263] Figure 2A shows the schematic structure of the recombinant fusion protein disclosed herein. Figure 2B shows representative data generated by SDS-PAGE analysis. Western blot results detected with a primary antibody specific for a 61 amino acid active fragment of NRG-1 containing the HER3 / 4 binding domain ("NRG-1", R&D Systems, Minneapolis, Minn.) or a primary antibody specific for IgG are shown in Figures 2C and 2D, respectively.

[0264] Example 3 - Molecular integrity assessed by SPR-based binding assays The molecular structural integrity of the recombinant fusion protein disclosed herein is evaluated by assessing its simultaneous binding ability to HER3 protein and anti-NRG-1 antibody. His-tagged HER3 recombinant protein (Sino Biological, Beijing, China) was captured on a sensor chip immobilized with anti-His antibody (Thermo Fisher, Waltham, MA) (step 1), followed by injection of samples (containing the recombinant fusion protein disclosed herein, the recombinant fusion protein disclosed herein without Fc mutation, anti-HER3 mAb (step 2), and anti-NRG-1 antibody (R&D Systems, Minneapolis, MN) (step 3)). The attachment of His-HER3 on the sensor chip can be visualized by the increase in signal in all six channels in step 1. Both the recombinant fusion protein disclosed herein and the recombinant fusion protein disclosed herein without Fc mutations generated significant responses by binding to HER3 in step 2 and by binding to the anti-NRG-1 antibody injected in step 3 (Ch1, 3), indicating the presence of a HER3 binding epitope and NRG-1 on the recombinant fusion protein disclosed herein. In contrast, the anti-HER3 mAb only bound to His-HER3 in step 2, but not to the anti-NRG-1 antibody and buffer (step 3) (Ch4, 5), proving the absence of NRG-1 binding activity for the anti-HER3 mAb molecule. Buffer was injected as a blank control in steps 2 and 3. Thus, both the HER3 binding epitope and NRG-1 are present on the recombinant fusion protein of the present invention.

[0265] The binding sensorgram and the sample injection sequence are shown in FIG.

[0266] Example 4 - Effect on tumor cell line proliferation in vitro Tumor cells were seeded at 2,500-20,000 cells per well in 96-well plates depending on the proliferation rate of each cell line. Cells were then treated with recombinant fusion proteins disclosed herein, antibodies, or control proteins in a stepwise 1:4 serial dilution series for 5 days. Cell viability was assessed using the Cell Counting Kit-8 from Dojindo Molecular Technologies (Kumamoto, Japan) according to the manufacturer's instructions. Data were analyzed with GraphPad Prism software and are presented as percent proliferation relative to untreated controls.

[0267] 4 is a representative graph showing the mean relative proliferation rate ± SEM (n=3) of various cancer cell lines [(A) NCI-N87, gastric; (B) MCF-7, breast; (C) RT-112, bladder; and (D) T47D, breast]. Compared to control NRG-1 and GP120mAb / NRG-1 fusion proteins, the recombinant fusion proteins disclosed herein show significantly reduced activity in promoting cancer cell proliferation.

[0268] Example 5 - Activation of the PI3K / AKT signaling pathway in human cardiomyocytes Human cardiomyocytes obtained from Cellular Dynamics (Madison, WI) were seeded onto 0.1% gelatin-coated 96-well plates and allowed to recover in plating medium (Cellular Dynamics) for 4 hours. The cells were then cultured in maintenance medium (Cellular Dynamics) for 96 hours before being used for experiments. To examine the ability of the recombinant fusion proteins of the present invention to activate the HER2:HER4 signaling pathway in cardiomyocytes, the cells were first starved in serum-free medium for 4 hours and then treated with recombinant fusion proteins or control substances (NRG-1, GP120mAb / NRG-1, anti-HER3mAb, or GP120mAb) in a stepwise 1:4 serial dilution series for 15 minutes. After the end of treatment, the cells were lysed and AKT phosphorylation was analyzed using Abcam's Phospho-AKT / Total AKT ELISA kit (Cambridge, MA) according to the manufacturer's instructions. Data were analyzed with raphPad Prism software and are presented as phosphorylated AKT / total AKT ratio relative to untreated control.

[0269] For Western blot analysis, cells were seeded in 6-well plates and treated with a single concentration of 16 nM of the recombinant fusion protein of the present invention or a control substance. After the end of treatment, cells were lysed with RIPA lysis buffer containing protease and phosphatase inhibitors. SDS-PAGE and Western blot were performed according to standard protocols. Total AKT and phosphorylated AKT were blotted with AKT rabbit antibody and p-AKT (S473) rabbit antibody, respectively (Cell Signaling; Danvers, MA).

[0270] Figure 5 shows AKT phosphorylation in response to stimulation in human cardiomyocytes. The results suggest that the recombinant fusion proteins disclosed herein can activate the HER2:HER4 signaling pathway in cardiomyocytes with potency comparable to that of NRG-1.

[0271] Example 6: Induction of HER2:HER3 dimerization and HER2:HER4 dimerization The PathHunter Dimerization assay, developed by Eurofins DiscoverX (Fremont, CA), detects ligand-induced dimerization of the two subunits of a receptor dimer pair. The principle of the assay is illustrated in Figure 6A. The β-gal enzyme is split into two fragments, ProLink (PK) and enzyme acceptor (EA). Cells are engineered to co-express target protein 1 fused to the enzyme donor PK and target protein 2 fused to the enzyme acceptor EA. When a ligand binds to one target protein, it induces an interaction with the other target protein, forcing the complementation of the two enzyme fragments, resulting in an enzymatic reaction that releases a chemiluminescent signal that is detected as relative fluorescence units (RFU).

[0272] PathHunter U2OS ErbB2 / ErbB4 dimerization cell line and ErbB2 / ErbB3 dimerization cell line were obtained from Eurofins DiscoverX. Cells were seeded at 4,000 cells / well in 384-well plates and incubated overnight at 37°C / 5% CO2. Test substances were prepared in a stepwise 1:4 serial dilution series starting at 28.8 nM and then added to the cells in the 384-well plates. After 4 hours of incubation, cells were assayed for receptor dimerization according to the manufacturer's instructions. Data were analyzed with GraphPad Prism software and presented as mean RFU ± SEM (n = 3).

[0273] As shown in Figures 6B and 6C, the recombinant fusion proteins disclosed herein can induce HER2 / HER4 dimerization with potency comparable to NRG-1, while their ability to induce HER2 / HER3 dimerization is much weaker. As a negative control for this study, neither the isotype control antibody GP120mAb nor anti-HER3mAb induced receptor dimerization.

[0274] Although an embodiment of the present invention has been described with reference to the accompanying drawings, it should be understood that the invention is not limited to the precise embodiment, and various changes and modifications may be made by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

[0275] Example 7: In vivo efficacy of recombinant fusion proteins in a rat model of systolic heart failure To evaluate the ability of recombinant fusion proteins to regenerate cardiac function in disease models, a Sprague Dawley rat model of myocardial infarction and systolic heart failure was employed. To establish the disease model, the left anterior descending coronary artery (LAD), located 3-4 mm below the left atrial appendage, was surgically ligated using 6-0 silk suture. Four weeks after ligation, ejection fraction (EF) was recorded by M-mode echocardiography (ECG) Doppler ultrasound to measure cardiac function relative to pre-surgery baseline EF. A threshold of a minimum 30% reduction in EF was used for inclusion in the following study. Sham control animals underwent an identical procedure without LAD ligation.

[0276] After establishing the disease model, the animals were divided into five groups of 11 rats each, with an additional 10 sham-operated rats included in a sixth group. The study was designed so that each group received tail vein injections twice a week for four weeks, for a total of eight injections. Both the sham-operated and negative control vehicle groups received saline, three groups received 1, 3, or 10 mg / kg of recombinant fusion protein, and the last group received the positive control GP120mAb / NRG-1 fusion protein (10 mg / kg).

[0277] Due to weight loss observed during the study, treatment was discontinued in the recombinant fusion protein groups receiving 3 mg / kg and 10 mg / kg before completing the full 8 injections, and these groups received only 6 and 3 injections, respectively. All other groups received the full series of 8 injections.

[0278] Four weeks after the first treatment, EF was measured again by M-mode ECG. As shown in Figure 8, compared to baseline, the recombinant fusion protein significantly increased EF in all three treatment groups. Specifically, increases of 14.7% (P<0.001), 26.9% (P<0.001), and 36.6% (P<0.001) were observed for the 1, 3, and 10 mg / kg groups, respectively. The GP120mAb / NRG-1 positive control increased EF by 28.8% (P<0.001) at the matching time point. Saline had no effect on either the sham or vehicle control groups.

[0279] After collecting ECG values ​​on the 28th day after the procedure, the mice were euthanized, and cardiac tissue adjacent to the surgical site was collected, fixed in 4% formaldehyde, and embedded in paraffin. 5 μm-thick paraffin sections of cardiac tissue were stained with hematoxylin-eosin dye, and histopathological changes were observed under an optical microscope. As shown in Figures 9A-9F, in the sham-operated group, cardiomyocytes were arranged in an orderly manner, and the cytoplasm and myocardial fibers were uniformly stained. No inflammatory cell infiltration was observed in the interstitial space, and no myocardial necrosis was observed. In contrast, in the vehicle control group, the gaps between cardiomyocytes widened in the myocardial infarction marginal zone, the nuclei were condensed and shattered, the myocardial fiber composition lost its orderly structure, the cell size was enlarged, and interstitial edema was observed. Treatment with the recombinant fusion protein partially alleviated the pathological changes in the myocardial infarction area, including a significant reduction in necrotic cells, narrowing of the interstitial space between cardiomyocytes, and restoration of the myocardial fiber composition to the normal structure.

[0280] Example 8: Recombinant fusion protein attenuated tumor growth in a subcutaneous FaDu cancer xenograft model in NOD / SCID mice To evaluate the potential risk of the recombinant fusion protein promoting tumor growth, an in vivo study in the FaDu cancer xenograft model was performed. NOD / SCID mice (Beijing AK Bio-Technology Co. Ltd.) were housed in the SPF facility of the CrownBio international R&D center (Beijing, China) in accordance with the institutional guidelines. All experiments were performed in accordance with the requirements of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and with the permission of the CrownBio IACUC committee.

[0281] FaDu tumor cells (3 × 10 ) suspended in 0.1 ml of PBS were injected into the right flank of 7- to 10-week-old female NOD / SCID mice. 6 ) was subcutaneously inoculated. The tumor was approximately 150 mm 3 At that time, mice were randomized into six test groups of eight mice per group. Test samples were administered intravenously via tail vein injection twice a week for three consecutive weeks for a total of six treatments. Tumor growth was tracked by caliper measurements. The study was terminated 21 days after treatment.

[0282] Tumor growth for the different treatments is summarized in Figure 10. Anti-HER3mAb at 10mg / kg showed significant antitumor activity with a tumor growth inhibition (TGI) of 93.5% at the end of the study (p<0.001 vs. vehicle group). The recombinant fusion protein also showed statistically significant TGI at the end of the study: 19.2% at the 1mg / kg dose (p=0.048 vs. vehicle group) and 56.2% at the 10mg / kg dose (p<0.001 vs. vehicle group). The control molecule, GP120mAb / NRG-1 fusion protein, showed no antitumor activity at either high or low doses. No animal deaths occurred during the study. All test substances were well tolerated by the tumor-bearing mice. No significant weight loss was observed in any of the experimental groups (Figure 11). These data indicate that under conditions of active tumor growth in vivo, the recombinant fusion protein exhibits dose-dependent inhibition of tumor growth and suggests that the recombinant fusion protein poses a lower risk of enhancing or accelerating tumor growth in vivo than the native NRG-1 protein.

[0283] Example 9: No significant gastrointestinal toxicity was observed in cynomolgus monkeys administered recombinant fusion proteins It was previously reported that in a Phase 1 clinical trial (NCT01258387) in which subjects received a single dose of placebo or cimaglermin (full-length recombinant NRG-1β3), nausea and diarrhea were the second and fourth most common treatment-emergent adverse events, occurring in 40% and 27% of the aggregated high-dose cohort, respectively (Lenihan et al. J Am Coll Cardiol Basic Trans Science. 2016;1(7):576-86). Similarly, in a Phase 2 trial of a recombinant NRG-1 peptide fragment (neucardin), nausea was the most commonly observed treatment-related adverse event, occurring in 20% of subjects (Jabbour et al. European Journal of Heart Failure (2011) 13: 83-92). Finally, in the second Phase 2 trial of Nucalgin (ChiCTR-TRC-00000414), published results indicate that 48.4% of observed adverse events were gastrointestinal, the most frequently observed type of adverse event in the trial and correlated with dose level (Gao et al. J Am Coll Cardiol 2010; 55:1907-14).

[0284] Two studies were conducted to evaluate the safety and tolerability of the recombinant fusion protein in cynomolgus monkeys (Macaca fascicularis): a single-dose non-GLP study and a repeat-dose GLP study. Gastrointestinal toxicity was closely followed. In the single-dose study, the safety and tolerability of the recombinant fusion protein was evaluated at dose levels of 10, 30, and 60 mg / kg, including one male and one female animal per cohort, compared to a vehicle control group. In this single-dose study, there were no test drug-related effects on body weight or qualitative food assessments, and no vomiting or diarrhea was observed throughout the 2-week post-treatment evaluation period. A repeat-dose GLP study evaluated the safety and tolerability of the recombinant fusion protein after four consecutive weekly doses at dose levels of 3, 10, and 30 mg / kg compared to vehicle control, including three males and three females in each cohort during the main 28-day study period, with two additional males and two females evaluated in the 30 mg / kg and vehicle control cohorts after a subsequent 28-day recovery period. No test article-related effects on food intake were observed in this repeat-dose study. Test article-related emesis was observed in this repeat-dose study, however, clinical observations of emesis were related to infusion reactions only, were observed in only one animal (17%) in the 10 mg / kg cohort and two animals (20%) in the 30 mg / kg cohort, and were transient in nature. Diarrhea was observed in only one (10%) and three (30%) animals in the vehicle control and 30 mg / kg recombinant fusion protein cohorts, respectively, and was considered normal for this type of procedure and unrelated to the recombinant fusion protein. Finally, in this repeated-dose study, mean body weight was reduced by >10% compared to baseline only at the 10 mg / kg and 30 mg / kg dose levels, and only after the fourth dose in the 10 mg / kg cohort and after the third and fourth doses in the 30 mg / kg cohort. In summary, treatment with recombinant fusion protein did not result in any clinically significant findings related to food intake, vomiting, or diarrhea, except during acute infusion reactions, and gastrointestinal findings did not affect the determination of no-adverse-event levels in either study. These results indicate that the design of the recombinant fusion protein reduces the adverse effects of NRG-1 recombinant protein on the gastrointestinal tract.

[0285] After a single dose of 60 mg / kg of recombinant fusion protein, blood samples (approximately 1 ml) were collected from cynomolgus monkeys at different time points, and serum was extracted and stored at -80°C until testing. The concentration of recombinant fusion protein in serum samples was measured by capture ELISA according to standard procedures. Briefly, 96-well plates were coated with recombinant human HER3 protein (R&D System), blocked with BSA, and incubated with test samples. After multiple washes, the plates were incubated with HRP-conjugated anti-human IgGFc antibody, then detected with TMB substrate. Figure 12 shows that the pharmacokinetic profile of the recombinant fusion protein is similar to that of IgG antibody.

[0286] Example 10: Summary of rate constants for Fc receptor binding The binding affinity of recombinant anti-HER3mAb / NRG-1 fusion protein to Fc receptors was measured using label-free SPR technology. A total of six Fc receptors (each fused to a His tag), including FcγRI (Abcam), FcγRIIa, FcγRIIb, FcγRIIIa(158F), FcγRIIIa(158V), and C1q (Sino Biological), were analyzed against the recombinant fusion protein, the recombinant fusion protein without Fc mutation, and the anti-HER3 antibody, respectively. All Fc receptors and test samples were purified by affinity chromatography. All experiments were performed on a Biacore 8K system (GE Healthcare), and HBS-EP+ (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.05% v / v surfactant P20) was used as the running buffer. Specifically, anti-His antibodies were coupled to both the active and reference flow cells of a CM5 sensor chip by the amine coupling method. Purified His-tagged Fc receptors were captured on the active flow cells of each individual channel via binding to immobilized anti-His antibodies. Capture levels for each Fc receptor were maintained at 80–120 RU. For kinetic analysis, recombinant fusion proteins and all other samples were serially diluted to a total of six concentrations ranging from 0.3 nM to 30 nM, and the serial dilutions were injected sequentially into both flow cells of each channel. Multiple analyses were completed in the same experiment by injecting samples simultaneously into multiple channels.

[0287] The obtained sensorgrams were fitted to a two-state binding model by Biacore 8K evaluation software to obtain kinetic constants. The equilibrium dissociation rates (KD) of all analyses are summarized in Table 1 below. The kinetically obtained KD values ​​of the recombinant fusion proteins binding to FcγRI, FcγRIIa, and FcγRIIb were more than 10-fold higher than those of the recombinant fusion proteins and anti-HER3 antibodies without Fc mutations, indicating that the affinity was significantly reduced as a result of the specific mutations within the Fc region of the recombinant fusion proteins. For FcγRIIIa(158F) and FcγRIIIa(158V), the Fc mutations reduced the binding affinity of the recombinant fusion proteins by 2-3-fold, respectively. Binding to C1q was too weak to be detected in all samples.

[0288] To confirm that the recombinant fusion proteins had limited Fc effector function, antibody-dependent cellular cytotoxicity (ADCC) was examined using an ADCC reporter bioassay from Promega (Madison, WI). This assay used an engineered Jurkat cell line as effector cells, which stably expresses the FcγRIIIa (V158) receptor and an NFAT response element that drives the expression of firefly luciferase. Rituximab, as a positive control for the assay, showed potent ADCC activity against CD20-positive Raji cells, whereas the recombinant fusion proteins showed no detectable ADCC against HER3-positive target cells (MCF7 or BT474) (data not shown). [Table 1]

[0289] Example 11: NRG-1-HER3 recombinant fusion protein exhibits anti-fibrotic effects in atrial tissue in vitro and AF prevention effects in an in vivo mouse model Introduction. Atrial fibrillation (AF) is caused by electrical and structural remodeling of the atria involving inflammation and fibrosis. Current treatments are limited to antiarrhythmic drugs and ablation and do not target the structural problem. Recent studies have demonstrated that neuregulin 1 (NRG1), a member of the epidermal growth factor family, has anti-fibrotic and anti-inflammatory effects in the myocardium.

[0290] Objective: The effect of an exemplary NRG-1 / HER3 antibody fusion protein on atrial fibrosis and AF inducibility was examined. The NRG-1 / HER3 antibody fusion protein contains an NRG-1 active fragment and an antagonistic HER3 (ERBB3) antibody, which selectively signals through ERBB4 in preference to ERBB3.

[0291] Methods. Fibrosis was induced in rat tissue samples in vitro using an in vitro fibrosis assay as shown in FIG. 13A. Atrial samples were taken from male rats (Wistar Han, 10 weeks old) and cut into small pieces (1-2 mm 2 ) and maintained in low serum medium in the presence or absence of NRG-1 / HER3 fusion protein (5 nM concentration). After 24-72 hours, Col1a1 and Col3a1 mRNA was quantified.

[0292] AF inducibility was tested in two models. In the first AF model, male mice (C57BL / 6N, 12-15 weeks old) were treated with angiotensin II (Ang-II, 4 weeks, osmotic minipump, 3000 ng / kg / min), the experimental outline of which is shown in Figure 16. In the second AF model, shown in Figure 18, mice were fed a high-fat diet (HFD, 8 weeks, 60% Kcal fat), which induced a significant weight gain (56 ± 3% increase compared to 23 ± 4% increase on normal diet), as shown in Figure 19A. AF inducibility in both models was tested by five runs of programmed electrical stimulation (PES) using a transjugular octapolar catheter (shown in Figure 15). AF inducibility (% of mice inducible with 3 or more PES runs) and duration of PES-induced AF (AF duration) were recorded. Mice were randomly assigned to treatment with vehicle or a representative NRG-1 / HER3 fusion protein (1 mg / kg, IV, twice weekly, n=5-7 mice / group).

[0293] Results. In cultured atrial samples, Col1a1 and Col3a1 mRNA expression increased gradually by 2-3 fold over 3 days, as shown in Figures 13A and 13B, respectively. NRG-1 / HER3 fusion protein robustly attenuated this effect by 59±17% (p<0.05), as shown in Figures 14A and 14B. In Ang-II mice, both Ang-II and HFD significantly increased AF inducibility and AF duration. In Ang-II mice, NRG-1 / HER3 fusion protein attenuated AF inducibility (from 57% to 20%), as shown in Figure 17B, and shortened AF duration (from 33.3±15.1 sec to 1.5±1 sec), as shown in Figure 17A. In HFD mice, NRG-1 / HER3 fusion protein attenuated AF inducibility (from 57% to 0%) as shown in Figure 20B, and significantly shortened AF duration (from 10.9 ± 3.2 sec to 0.76 ± 0.5 sec, p < 0.05) as shown in Figure 20A.

[0294] Conclusions: These results demonstrate the antifibrotic effects of selective ERBB4 stimulation with NRG-1 / HER3 fusion protein in atrial tissue in vitro, as well as AF prevention in two unrelated mouse models.

[0295] Example 12: NRG-1 / HER3 antibody fusion protein reduces type I collagen associated with myocardial stiffness Collagen type I and type III expression was measured using the Sprague-Dawley rat model of myocardial infarction and systolic heart failure (also called heart failure with reduced ejection fraction, or HFrEF) used in Example 7. After 4 weeks of treatment, cardiac tissue samples were removed, fixed and sectioned as described in Example 7, and stained for collagen type I or collagen type III protein expression using immunohistochemistry.

[0296] Representative results for collagen type I are shown in Figures 21A-21F. As can be seen from Figures 21A-21F, NRG-1 / HER3 antibody fusion protein administered at 1 mg / kg, 3 mg / kg, and 10 mg / kg reduced collagen type I expression by 6.6%, 37.1%, and 40.5%, respectively, compared to the vehicle control. Furthermore, as shown in Table 2 below, the collagen type I / collagen type III ratio was reduced by 4.7%, 40.8%, and 36.6%, respectively, compared to the vehicle control.

[0297] [Table 2]

[0298] Example 13: NRG-1 / HER3 antibody fusion protein prevents atrial fibrosis and atrial fibrillation inducibility in the porcine DOCA model Atrial fibrosis is the cornerstone of atrial fibrillation (AF) recurrence, and a higher fibrosis burden leads to resistance to treatment and poor prognosis. The extent of fibrosis predicts prognosis and treatment refractoriness. Currently, there are no therapies that target atrial fibrosis. NRG-1 / HER3 fusion protein is a long-acting neuregulin fusion protein that has been shown to reduce ventricular fibrosis (i.e., possess antifibrotic properties) in animal models of heart failure through selective stimulation of the ErbB4 receptor. In this example, the ability of NRG-1 / HER3 fusion protein to reduce atrial fibrosis and AF inducibility was tested in a miniature pig model of deoxycorticosterone acetate (DOCA, an aldosterone agonist)-induced hypertension.

[0299] Methods: Eighteen Aachener minipigs were randomly divided into three groups: control (CTRL), DOCA+vehicle (DOCA+VEH), and DOCA+NRG-1 / HER3 fusion protein. The control group received no therapeutic intervention. A diagram of the experiment is shown in Figure 22. To induce hypertension and atrial fibrosis, pellets releasing 10 mg / kg DOCA for 60 days were implanted into minipigs in the DOCA+VEH and DOCA+NRG-1 / HER3 fusion protein groups. DOCA-implanted pigs received weekly treatments with NRG-1 / HER3 fusion protein (0.3 mg / kg, DOCA+NRG-1 / HER3 fusion protein) or its vehicle (DOCA+VEH) starting on the day of implantation (total of 9 doses). After 60 days, arterial blood pressure was measured invasively and a 10-electrode catheter was placed in the right atrium. 50 burst pacing episodes were performed to test AF inducibility, quantified as the percentage of "successful" episodes out of 50 attempts that were able to induce AF execution for 5 seconds or more after the burst. Exemplary results of AF inducibility testing are shown in Figure 23. Atrial fibrosis was quantified using ImageJ software in Masson's Trichrome staining of left atrial specimens isolated after euthanasia of the animals.

[0300] Results: Mean arterial pressure was significantly higher in the DOCA+VEH (142±10 mmHg) and DOCA+NRG-1 / HER3 fusion protein (132±15 mmHg) groups than in the CTRL group (105±8 mmHg, p<0.01), without any effect of NRG-1 / HER3 fusion protein (Figure 24).

[0301] As can be seen from Figure 25, the AF inducibility of the DOCA+VEH group was significantly higher than that of the CTRL group (66 / 250 vs. 9 / 300, p<0.001) and the DOCA+NRG-1 / HER3 fusion protein group (66 / 250 vs. 8 / 300, p<0.001). Similarly, the degree of atrial fibrosis was significantly higher in the DOCA+VEH group compared to the CTRL group (14.18±1.81 vs. 8.30±2.52, p<0.001) and the DOCA+NRG-1 / HER3 fusion protein group (14.18±1.81 vs. 10.65±1.59, p=0.0049), which can be seen from Figure 26.

[0302] Conclusion: NRG-1 / HER3 fusion protein prevents atrial fibrosis and AF inducibility in the porcine DOCA model. The effect of NRG-1 / HER3 fusion protein is independent of its effect on blood pressure but may be associated with reduced atrial fibrosis.

Claims

1. A pharmaceutical composition comprising a recombinant fusion protein containing a fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb), for use in a method of treating atrial fibrillation and / or cardiac fibrosis in a subject.

2. The pharmaceutical composition according to claim 1, wherein the cardiac fibrosis includes atrial fibrosis.

3. The pharmaceutical composition according to claim 1, wherein the NRG-1 fragment comprises the active domain of NRG-1.

4. The pharmaceutical composition according to claim 1, wherein the NRG-1 fragment comprises an ERBB3 / 4 binding domain.

5. The pharmaceutical composition according to claim 1, wherein the NRG-1 fragment binds to ErbB4 (HER4) and induces signal transduction via ErbB4 (HER4).

6. The pharmaceutical composition according to claim 1, wherein the HER3mAb inhibits NRG-1 signaling via ErbB3 (HER3).

7. The pharmaceutical composition according to claim 1, wherein the NRG-1 fragment comprises the NRG-1β2a isoform.

8. The pharmaceutical composition according to claim 1, wherein the NRG-1 fragment is fused to the C-terminus of the antibody heavy chain of HER3 mAb via its N-terminal amino acid using a linker.

9. The pharmaceutical composition according to claim 8, wherein the linker comprises at least one copy of the Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser linker described in Sequence ID No.

5.

10. The pharmaceutical composition according to claim 1, wherein the C-terminus of the antibody heavy chain of the HER3 mAb comprises the Fc domain of the antibody.

11. The recombinant fusion protein for use according to the method of claim 1, or any one of the claims, wherein the HER3 mAb is glycosylated.

12. The pharmaceutical composition according to claim 1, wherein the NRG-1 fragment comprises the amino acid sequence of SEQ ID NO:

4.

13. The pharmaceutical composition according to claim 1, wherein the HER3mAb comprises the heavy chain amino acid sequence of SEQ ID NO:

2.

14. The pharmaceutical composition according to claim 1, wherein the HER3mAb comprises the light chain amino acid sequence of SEQ ID NO:

3.

15. The pharmaceutical composition according to claim 14, wherein the HER3mAb contains a substitution mutation in at least one of the amino acids 234, 239, and 434 of SEQ ID NO:

2.

16. The pharmaceutical composition according to claim 15, wherein the at least one substitution mutation includes an L234F mutation, an S239A mutation, an N434A mutation, or a combination thereof.

17. The pharmaceutical composition according to claim 1, wherein the recombinant fusion protein comprises the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO:

14.

18. The pharmaceutical composition according to claim 1, wherein the recombinant fusion protein promotes HER2 / 4 signaling more than HER2 / 3 signaling compared to the signal-inducing ability of recombinant NRG-1.

19. The pharmaceutical composition according to claim 1, wherein the recombinant fusion protein promotes the proliferation, differentiation, and survival of cardiomyocytes or cardiac tissue in the subject.

20. The pharmaceutical composition according to claim 1, wherein the recombinant fusion protein attenuates the proliferation of tumor or cancer cells compared to recombinant NRG-1.

21. The pharmaceutical composition according to claim 1, wherein administration of the recombinant fusion protein shortens the duration of atrial fibrillation episodes or reduces the frequency of atrial fibrillation episodes.

22. The pharmaceutical composition according to claim 1, wherein administration of the recombinant fusion protein reduces the signs or symptoms of atrial fibrillation or cardiac fibrosis.

23. The pharmaceutical composition according to claim 22, wherein the symptoms of atrial fibrillation include arrhythmia, palpitations, dizziness, extreme fatigue, shortness of breath, chest pain, or a combination thereof.

24. The pharmaceutical composition according to claim 22, wherein the symptoms of cardiac fibrosis include collagen quantity or collagen deposition, and the administration of the recombinant fusion protein reduces the amount or deposition of collagen in cardiac tissue.

25. The pharmaceutical composition according to claim 1, wherein the recombinant fusion protein is administered to the subject in a dose of 0.1 mcg / kg to 5 mg / kg.

26. A kit containing a therapeutically effective amount of a pharmaceutical composition comprising a recombinant fusion protein containing a fragment of neuregulin 1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb) for use in the treatment of atrial fibrillation and / or cardiac fibrosis in a subject.

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