Human neuregulin-1 (NRG-1) recombinant fusion protein compositions and methods of use thereof

Abstract

To provide human neuregulin-1 (NRG-1) recombinant fusion protein compositions and use methods thereof.SOLUTION: The present invention relates to a recombinant fusion protein comprising a fragment of the cardioprotective protein neuregulin-1 (NRG-1) fused to a monoclonal antibody (mAb) backbone and to a method of treating a disease or condition in a subject in need thereof comprising administering a therapeutically effective amount of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein disclosed herein.SELECTED DRAWING: Figure 2A-B

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Application No. 62 / 656,246, filed April 11, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] Incorporation by reference of sequence listing This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. Said ASCII copy was created on March 16, 2019, is named SBTI-001-001WO_SeqList.txt, and is 31,328 bytes in size. [Background technology]

[0003] Neuregulin (NRG; heregulin, HRG), also known as glial growth factor (GGF) or neu (new) differentiation factor, is a type of glycoprotein with a molecular weight of 44 KD. The NRG protein family has four members: 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, followed by intracellular signaling. In animal models, expression of NRG induces paracrine signaling to promote growth and differentiation in cardiac tissue, and ablation 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, demonstrating that ErbB2-mediated signaling in humans is essential not only for the development but also for the homeostasis of healthy cardiac tissue.

[0004] Evidence also indicates that NRG-1 signaling plays a role in the development and function of other organ systems and in the pathogenesis of human diseases, including schizophrenia and head and neck cancer. NRG-1 has many isoforms. Studies in 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 in various 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 development and / or progression, raising significant concerns for any application that requires chronic administration or is not associated with significant cardiovascular (CV) risk factors. Second, overactivation of HER3 by NRG-1 may disrupt the integrity and homeostasis of the gastrointestinal (GI) epithelium, resulting in severe GI toxicity, thereby losing the therapeutic window of NRG-1. Third, both clinically-stage active protein fragments of rhNRG-1 exhibit short half-lives, which may require burdensome dosing and administration schedules to achieve desired therapeutic levels of exposure. Thus, there is a need to provide NRG-1-based therapeutics that retain clinically meaningful therapeutic potential in various cardiovascular indications, but with lower risk of tumor formation or promotion of cancer progression, better GI tolerability, and more favorable pharmacokinetic (PK) profiles.

[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 in a manner that reduces the tumorigenic risk and GI toxicity of rhNRG-1, while the antibody backbone format provides the molecular half-life of a typical monoclonal antibody, allowing the product to be more conveniently dosed and administered. Summary of the Invention

[0007] In one embodiment, the invention relates to a recombinant fusion protein comprising a fragment of the cardioprotective protein Neuregulin-1 (NRG-1) fused to a related monoclonal antibody (mAb) scaffold. In a related embodiment, the NRG-1 fragment is fused to the C-terminus of the heavy chain of the antibody via a linker. In another related embodiment, NRG-1 is attached to the linker via the first amino acid on the N-terminus of NRG-1, which in one embodiment is a serine (S or Ser) amino acid. In a related embodiment, the fragment is an active fragment comprising the active domain of NRG-1. In another related embodiment, the mAb is monospecific for ErbB3 (HER3). In another related embodiment, the NRG-1 is the NRG-1 β2a isoform.

[0008] In another aspect, the present invention relates to a pharmaceutical composition comprising a recombinant fusion protein comprising a fragment of the cardioprotective protein Neuregulin-1 (NRG-1) fused to an anti-HER3 monoclonal antibody scaffold, and a pharma- ceutical acceptable carrier, diluent, or excipient.

[0009] In another aspect, the present invention relates to a method of treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein disclosed herein.

[0010] In another aspect, the present invention relates to a method for preventing, inhibiting, suppressing, or delaying the onset of a cardiovascular disease or condition in a subject, comprising administering an effective amount of a recombinant fusion protein disclosed herein.

[0011] In another aspect, the present invention relates to a method of treating a CNS-related disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant fusion protein.

[0012] In another aspect, the present invention relates to a method for preventing, inhibiting, suppressing, or delaying the onset of a CNS-related disease or condition in a subject, comprising administering an effective amount of a recombinant fusion protein.

[0013] In another related embodiment, NRG-1 binds to and induces signaling through ErbB4 (HER4). In another related embodiment, the mAb inhibits NRG-1 signaling through ErbB3 (HER3).

[0014] In another aspect, the present invention relates to a kit comprising an effective amount of a recombinant fusion protein of the present invention or a pharmaceutical composition comprising a recombinant fusion protein of the present invention.

[0015] Other features and advantages of the present invention will become apparent from the following detailed description and drawings. However, it should be understood that the detailed description and specific examples are given by way of illustration only, while illustrating embodiments of the present invention, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the detailed description.

[0016] Various objects and advantages of the present invention as well as a more complete understanding will become apparent and will be more readily appreciated by reference to the following detailed description and appended claims, taken in conjunction with the accompanying drawings, as described below. [Brief description of the drawings]

[0017] [Figure 1] 1 shows the construction of expression plasmids for expressing the recombinant fusion proteins disclosed herein.

[0018] [Figure 2A-B] Figures 2A-D show schematic structures of recombinant fusion proteins disclosed herein. Figure 2A shows a molecular schematic of the anti-HER3 mAb / NRG-1 fusion protein of the present disclosure. Figure 2B shows representative data generated by SDS-PAGE analysis. [Figure 2C-D] Figures 2A-D show schematic structures of recombinant fusion proteins disclosed herein. Figure 2C shows Western blot results detected with a primary antibody specific for a 61 amino acid active fragment of NRG-1 ("NRG-1" (R&D Systems, Minneapolis, Minn.)) that contains the HER3 / 4 binding domain. Figure 2D shows Western blot results detected with a primary antibody specific for IgG.

[0019] [Diagram 3] Figure 3 shows the binding analysis, which shows that the recombinant fusion protein disclosed herein can bind to HER3 protein (curve 1, step 2) and also bind to anti-NRG-1 antibody (curve 1, step 3). It is noted that Fc mutations were 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 reduce undesired cytotoxicity against normal tissues expressing the HER3 receptor.

[0020] [Figure 4A-B]Figures 4A-D show representative graphs showing the mean relative growth rate ± SEM (n=3) in different cancer cell lines treated with anti-HER3 mAb / NRG-1 fusion protein or control. Figure 4A shows the mean relative growth rate in NCI-N87 gastric cancer cell line. Figure 4B shows the mean relative growth rate in MCF-7 breast cancer cell line. [Figure 4C-D] Figures 4A-D show representative graphs showing the mean relative growth rate ± SEM (n=3) in different cancer cell lines treated with anti-HER3 mAb / NRG-1 fusion protein or control. Figure 4C shows the mean relative growth rate in RT-112 bladder cancer cell line. Figure 4D shows the mean relative growth rate in T47D breast cancer cell line. Compared to the control NRG-1 peptide and GP120 mAb / NRG-1 fusion protein, the recombinant fusion proteins provided herein show significantly lower activity in promoting cancer cell proliferation.

[0021] [Diagram 5] Figures 5A-B show that the recombinant fusion proteins provided herein fully retain the ability to induce the PI3K / AKT signaling pathway in cardiomyocytes despite reduced potential for cancer cell growth, demonstrating comparable activity to recombinant NRG-1 and GP120 mAb / NRG-1 fusion proteins. Figure 5A is a plot showing the relative ratio of phospho-AKT (pAKT):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.

[0022] [Figure 6A-B]Figures 6A-C 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 enzyme donor PK and target protein 2 fused to enzyme acceptor EA. A ligand is induced to interact with one target protein by binding to the other target protein, forcing the complementation of the two enzyme fragments, resulting in an enzymatic reaction that releases a chemiluminescent signal, which is detected as relative fluorescent units or RFU. FIG. 6B is a plot showing that the recombinant fusion proteins provided herein can induce HER2 / HER4 dimerization with comparable potency as NRG-1. [Figure 6C] Figures 6A-C 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 induce HER2 / HER3 dimerization with significantly lower potency than NRG-1. These findings further confirm that the recombinant fusion proteins provided herein retain the full HER2 / 4 signaling capacity of NRG-1 and significantly reduce HER2 / 3 signaling.

[0023] [Figure 7]Figure 7 shows the binding affinity of the anti-HER3 mAb / NRG-1 fusion protein of the present invention to the HER3 antigen in different species including human, monkey, rat and mouse. The equilibrium dissociation rates (KD) quantified by BIAcore analysis are 3.13 x 10-10 (human), 3.97 x 10-10 (monkey), 2.68 x 10-9 (rat) and 2.77 x 10-9 (mouse), respectively. These data indicate that the recombinant fusion protein of the present invention has similar binding affinity to human and monkey HER3, while its affinity to rodent (rat and mouse) HER3 is approximately one order of magnitude lower.

[0024] [Figure 8] FIG. 8 is a plot showing the effect of recombinant fusion proteins on ejection fraction (EF) in a rat model of myocardial infarction induced by coronary artery ligation.

[0025] [Figure 9] Figure 9A-F is a series of six images showing the histopathological changes of the myocardium in a systolic heart failure rat model induced by coronary artery ligation. Heart tissue adjacent to the surgical site was collected and fixed in 4% formaldehyde, then paraffin sections were prepared and stained with H&E. Figure 9A shows the heart tissue from a sham-operated control rat. Figure 9B shows the heart tissue from a systolic heart failure model rat treated with vehicle control. Figure 9C shows the heart tissue from a systolic heart failure model rat treated with GP120 mAb / NRG-1 (10 mg / kg). Figure 9D shows the heart tissue from a systolic heart failure model rat treated with anti-HER3 mAb / NRG-1 (1 mg / kg). Figure 9E shows the heart tissue from a systolic heart failure model rat treated with anti-HER3 mAb / NRG-1 (3 mg / kg). FIG. 9F shows cardiac tissue from a rat model of systolic heart failure treated with anti-HER3 mAb / NRG-1 (10 mg / kg).

[0026] [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.

[0027] [Figure 11] FIG. 11 is a graph showing the weight change of tumor-bearing mice treated with recombinant fusion proteins of the present disclosure and controls.

[0028] [Figure 12] FIG. 12 is a graph showing the pharmacokinetic profile of the recombinant fusion protein in cynomolgus monkeys (Macaca fascicularis). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention utilizes recombinant fusion proteins comprising fusions of monoclonal antibodies fused to active fragments of neuregulin-1 protein isoforms across a variety of cardiovascular and central nervous system (CNS) indications. definition

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0031] For purposes of interpreting the present invention, the following definitions shall apply, and wherever appropriate, terms used in the singular shall include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0032] A "neuregulin or neuregulin analog" is a molecule capable of activating ErbB2 / ErbB4 or ErbB2 / ErbB3 heterodimeric protein tyrosine kinases, including all neuregulin isoforms, the neuregulin EGF domain alone, neuregulin variants, and any type of neuregulin-like gene product that activates the above receptors. A preferred "neuregulin" for use in the present invention is a polypeptide fragment of the human neuregulin-1 β2 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, neu (new) differentiation factor (NDF), ARIA, GGF2, HRG1, HRGA, SMDF, MST131, MSTP131, and NRG1 intronic transcript 2 (NRG1-IT2).

[0033] 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 protein 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.

[0034] 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.

[0035] 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, MLN 19, and HER-2 / neu.

[0036] As used herein, the term "active" refers to a fragment that has a biological activity or function. In some embodiments, the activity is equal to or approximates the activity of the wild-type protein.

[0037] As used herein, the term "subject" includes, but is not limited to, mammals (including, for example, 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 (including, for example, non-mammalian vertebrates such as birds (e.g., chickens or ducks) or fish, and non-mammalian invertebrates). In some embodiments, the methods and compositions of the invention are used in the treatment (both prophylactic and / or therapeutic) of non-human animals. The term "subject" also refers to a patient, i.e., an individual awaiting or receiving medical care.

[0038] The term "pharmaceutical composition" as used herein means 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, etc.).

[0039] The term "effective amount" refers to a dose or amount sufficient to effect a desired result. The desired result can include an objective or subjective improvement in the recipient of the dose or amount (e.g., prolonged survival, reduction in the number and / or size of tumors, effective prevention of a disease condition, etc.).

[0040] "Prophylactic treatment" refers to treatment administered to a subject who does not exhibit signs or symptoms of a disease, condition, or medical disorder, or who only exhibits early signs or symptoms of a disease, condition, or medical disorder, with the intent of attenuating, preventing, or reducing the risk of developing the disease, condition, or medical disorder. Prophylactic treatment acts as a preventative treatment for a disease or disorder. "Prophylactic activity" refers to the activity of an agent (e.g., a recombinant fusion protein of the invention or a composition thereof) that, when administered to a subject who does not exhibit signs or symptoms of a condition, disease, or disorder (or who only exhibits early signs or symptoms of a condition, disease, or disorder), attenuates, prevents, or reduces the risk of the subject developing the condition, 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 attenuating, preventing, treating, or reducing the development of a condition, disease, or disorder.

[0041] A "therapeutic treatment" is a treatment administered to a subject exhibiting a symptom or sign of a condition, disease, or disorder, where the treatment is administered to the subject for the purpose of reducing or eliminating such sign or symptom of the condition, disease, or disorder. A "therapeutic activity" is the activity of an agent (e.g., a recombinant fusion protein of the invention or a composition thereof) that, when administered to a subject suffering from a sign or symptom of a condition, disease, or disorder, eliminates or reduces such sign or symptom. A "therapeutically useful" agent or compound (e.g., a recombinant fusion protein of the invention) indicates that the agent or compound is useful for reducing, treating, or eliminating such sign or symptom of a condition, disease, or disorder.

[0042] As used herein, the term "treating cancer" means, unless otherwise indicated, partially or completely ameliorating, reducing, inhibiting the progression of, or preventing tumor growth, tumor metastasis, or other cancer-causing or neoplastic cells in a subject. As used herein, the term "treatment" means, unless otherwise indicated, the act of treating.

[0043] As used herein, the term "treating cardiovascular disease" means, unless otherwise indicated, to partially or completely prevent, inhibit, suppress, delay, reverse, or reduce the onset of a cardiovascular disease or condition in a subject, or the progression of an existing cardiovascular disease or condition or a symptom thereof in a subject. 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, and adjuncts to surgical intervention in pediatric congenital heart disease. Non-limiting examples of symptoms of cardiovascular disease include shortness of breath, cough, rapid weight gain, swelling of the legs, ankles, and abdomen, dizziness, fatigue, weakness, chest pain, syncope (fainting), tachycardia, and bradycardia. Methods for determining the progression of cardiovascular disease and the effectiveness of treatment will be readily apparent to those skilled in the art. For example, the progression of various cardiovascular diseases can be determined by ejection fraction, electrocardiogram (ECG), Holter monitor, echocardiogram, stress test, cardiac catheterization, cardiac computed tomography (CT) scan, and cardiac magnetic resonance imaging (MRI).

[0044] As used herein, the term "treating a central nervous system (CNS)-related disease" refers to a method of partially or completely preventing, inhibiting, suppressing, delaying, reversing, or reducing the onset of a CNS-related disease or condition in a subject, unless otherwise indicated. The term "treating a CNS-related disease" also refers to reversing, slowing, or otherwise reducing 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 with 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. Treating a CNS-related disease can improve or prevent symptoms such as tremors, bradykinesia, muscle rigidity, loss of balance, impaired posture, speech changes, loss of motor control, impaired swallowing, muscle spasms, seizures, memory loss, and confusion.

[0045] In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "percent identity" 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 purposes (e.g., gaps can be introduced into a first amino acid or nucleic acid sequence to obtain optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at 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 at that position are identical. 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.

[0046] The term "substantially identical" in the context of two nucleic acids or polypeptides means two or more sequences or subsequences that have 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 (e.g., as quantified using one of the methods described below).

[0047] As used herein, the terms "bind to", "specifically bind to", or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target with greater affinity, avidity, more readily, and / or with longer duration than to other targets. In one embodiment, the extent to which an antibody binds 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, the dissociation constant (Kd) of an antibody that specifically binds to a target is <1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM.

[0048] In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species, hi another embodiment, specific binding can include, but is not required to include, exclusive binding.

[0049] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "neuregulin" or "a neuregulin peptide" includes mixtures of such neuregulins, neuregulin isoforms, and / or neuregulin-like polypeptides. Reference to "the formulation" or "the method" includes one or more formulations, methods, and / or steps described herein and / or that will be apparent to those skilled in the art upon reading this disclosure.

[0050] The term "polypeptide" refers to a polymer of amino acids and equivalents thereof, and does not refer to a specific length of the product. Thus, "peptides" and "proteins" are included within the definition of a polypeptide. Also included within the definition of a polypeptide are "antibodies," as defined herein. A "polypeptide region" refers to a segment of a polypeptide, which segment includes, for example, one or more domains or motifs (e.g., a polypeptide region of an antibody can 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 of the polypeptide, or at least 50 contiguous amino acids of the polypeptide.

[0051] Unless the context indicates otherwise, a "derivative" is a polypeptide or fragment thereof having one or more non-conservative or conservative amino acid substitutions relative to a second polypeptide (also referred to as a "variant"), or a polypeptide or fragment thereof that has been modified by the covalent attachment of a second molecule, e.g., by the attachment of a heterologous polypeptide, or by glycosylation, acetylation, phosphorylation, etc. Also included within the definition of "derivative" are, for example, polypeptides that contain one or more analogs of an amino acid (e.g., unnatural amino acids, etc.), polypeptides with unsubstituted linkages, as well as other modifications, both natural and unnatural, known in the art.

[0052] An "isolated" polypeptide is one that has been identified, separated, and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that may interfere with diagnostic or therapeutic uses for the polypeptide, and such components may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. Isolated polypeptides include isolated antibodies, or fragments or derivatives thereof.

[0053] As used herein, the term "about" means in quantitative terms, 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%.

[0054] 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. In carrying out or testing the present invention, any method and material similar or equivalent to the method and material described herein can be used, but the preferred method and material are described herein. All publications mentioned in this specification are incorporated herein by reference for the purpose of disclosing and describing the material to which the reference is related.

[0055] Recombinant fusion proteins-antibodies The present invention utilizes recombinant fusion proteins comprising fusions of monoclonal antibodies fused to fragments of Neuregulin-1 protein isoforms for use across a variety of cardiovascular and neurological indications. In an exemplary embodiment, the antibody is specific for ERBB3 (HER3).

[0056] As used herein, "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 a myriad of 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 comprises 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 primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to such light and heavy chains respectively.

[0057] 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 under the disulfide bond in the hinge region to generate a dimer of Fab, F(ab')2, which is itself a light chain linked to VH-CH1 by a disulfide bond. F(ab')2 can be reduced under mild conditions to cleave the disulfide bond in the hinge region, 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)). While various antibody fragments have been defined in terms of digestion of intact antibodies, one skilled in the art will appreciate that such Fab' fragments and the like can be synthesized de novo, either chemically or by utilizing recombinant DNA methodology. Thus, as used herein, the term antibody also includes antibody fragments produced by modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Antibodies include single chain antibodies, including single chain Fv (sFv or scFv), antibodies in which one variable heavy chain and one variable light chain are linked together (directly or via a peptide linker) to form a contiguous 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, which were originally isolated from camelids.

[0058] 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 with specificity for a disease-associated antigen), optionally comprising regions encoded by V genes and / or D genes and / or J genes.

[0059] 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 utilize fusion proteins that include an IgG domain. However, other embodiments include alternative immunoglobulins such as IgM, IgA, IgD, and IgE. Furthermore, all possible isotypes of the various immunoglobulins are encompassed within the current embodiments. Thus, IgG1, IgG2, IgG3, etc. are all possible molecules in the antibody domain of the antibody-immunostimulant fusion protein used in the present invention. Various embodiments of the present invention include various hinge regions (or functional equivalents thereof) in addition to the choice of immunoglobulin type and isotype. Such hinge regions provide flexibility between the various 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.

[0060] In some embodiments, the mAb comprised in the recombinant fusion protein of the invention is monospecific for ErbB3 (HER3).

[0061] Human HER3 (ErbB-3, ERBB3, c-erbB-3, c-erbB3, receptor tyrosine 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, GS et al, PNAS 87 (1990) 4905-4909; Kraus, MH et al, PNAS 90 (1993) 2900-2904). Like the prototypic epidermal growth factor receptor, the transmembrane receptor HER3 consists 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 its extracellular domain but not in its active kinase domain. Therefore, it can bind to this ligand but cannot transduce signals intracellularly via protein phosphorylation. However, it forms heterodimers with other HER family members that have kinase activity. Heterodimerization activates receptor-mediated signaling pathways and transphosphorylates its intracellular domain. Dimerization between HER family members expands the signaling potential of HER3, providing a means for signal diversification as well as signal amplification. For example, HER2 / HER3 heterodimers induce one of the most important mitogenic signals among HER family members via the PI3K and AKT pathways. (Sliwkowski MX, et al.

[0062] In one embodiment, the human ERBB3 protein comprises the amino acid sequence shown in GenBank AAH02706.1 and set forth in SEQ ID NO:1.

[0063] MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKAF (SEQ ID NO: 1). It is understood that the ERBB3 (HER3) sequence targeted by the antibodies of methods and compositions of the invention may be an isomer, homolog, or variant of SEQ ID NO: 1.

[0064] 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).

[0065] In certain embodiments, the mAb contained in the recombinant fusion protein of the present invention comprises an anti-HER3 mAb. Such anti-HER3 antibodies can include, but are not limited to: patritumab, serivantumab (fully human mAb), LJM716, KTN3379, AV-203, REGN1400, GSK2849330, or MM-141. Such antibodies can also be selected from any form, including chimeric, bispecific, non-human, fully human, or humanized, so long as they bind to human ERBB3 (HER3) and inhibit signaling from it. In some embodiments, the anti-HER3 antibody is of human origin.

[0066] In some embodiments, the term "antibody" encompasses various forms of antibody structures, including, but not limited to, whole antibodies and antibody fragments. The antibodies according to the present invention are preferably human, humanized, chimeric, or further engineered antibodies, so long as the unique properties are retained. An "antibody fragment" comprises a portion of a full-length antibody, preferably comprising the variable domains 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 may comprise any of the VFv, ... H The properties of the domain, i.e. V L A property that can be aggregated with a domain, or V H V that binds to the respective antigen and can assemble with the domain L It comprises a single polypeptide chain having the properties of a domain to provide a functional antigen-binding site and to provide the properties of an antibody according to the invention. As used herein, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to a preparation of antibody molecules of a single amino acid composition.

[0067] 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 contains 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, usually prepared by recombinant DNA techniques. Chimeric antibodies that contain 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 contain a DNA segment encoding a rat immunoglobulin variable region and a DNA segment encoding a human immunoglobulin constant region. Other forms of "chimeric antibodies" encompassed by the present invention are those that have been modified or changed from the class or subclass of the original antibody. Such "chimeric" antibodies are also referred to as "class-switched antibodies." Methods for producing chimeric antibodies involve 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.

[0068] 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 with a different specificity compared to that of the parent immunoglobulin. In other embodiments, the CDRs of the 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 derived from the same or different human antibody sequences. The human antibody sequences can be sequences of natural human antibodies. Human heavy and light chain variable framework regions are listed, for example, in Lefranc, M.-P., Current Protocols in Immunology (2000)-Appendix IP A.1P.1-A.1P.37 and can be accessed via IMGT (international ImMunoGeneTics information system®) (http: / / imgt.cines.fr) or http: / / vbase.mrc-cpe.cam.ac.uk. Optionally, the framework regions may be modified by further mutations. Particularly preferred CDRs correspond to the CDRs that correspond to the sequences recognizing the antigens indicated above for the chimeric antibodies. As used herein, the term "humanized antibody" also includes antibodies whose constant region has been modified to provide the properties according to the invention, in particular those related to complement component 1q (Clq) binding and / or Fc receptor (FcR) binding, for example by "class switching", i.e. alteration or mutation of the Fc part (e.g. IgG1 to IgG4 and / or IgG1 / IgG4 mutation). As used herein, the term "human antibody" 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 (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies, or selected human antibodies, in the absence of endogenous immunoglobulin production. Transfer of human germline immunoglobulin gene arrays into such germline mice will result in the production of human antibodies upon antigen challenge (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. are also available for the preparation of 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 with respect to the humanized antibodies according to the invention, the term "human antibody", as used herein, also includes antibodies in which the constant region has been modified to confer the properties according to the invention.

[0069] 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.

[0070] As used herein, the term "human antibody" is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, e.g., antibodies isolated from host cells (e.g., NS0 or CHO cells) or from transgenic animals (e.g., mice) with human immunoglobulin genes or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived 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.

[0071] In some embodiments, the terms "binds to human HER3," "specifically binds to human HER3," or "anti-HER3 antibody" are used interchangeably and have a HER3 antibody binding capacity of about 4.81x at 25° C. -10 "Antibody that specifically binds to human HER3 antigen" refers to an antibody that specifically binds to human HER3 antigen with a KD value of 1.0×10 mol / L or less. Binding affinity is determined by standard binding assays at 25° C., such as surface plasmon resonance techniques (BIAcore® (GE-Healthcare Uppsala, Sweden)). Thus, as used herein, "antibody that binds to human HER3" refers to an antibody that specifically binds to human HER3 antigen with a KD value of 1.0×10 mol / L or less at 25° C. -8 mol / L~1.0×10 -13 4.81× at 25° C. with a binding affinity in the range of mol / L -10 It means an antibody or a portion thereof that binds to human HER3 antigen with a KD value of 0.1 mol / L or less.

[0072] In another aspect, the anti-HER3 antibody contained in the recombinant fusion protein disclosed herein comprises a variable heavy (VH) chain and a variable light (VL) chain. 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.

[0073] In one embodiment, the anti-HER3 mAb provided herein comprises the VH amino acid sequence set forth in SEQ ID NO:2.

[0074] 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)

[0075] In one embodiment, the anti-HER3 mAb provided herein comprises the VL amino acid sequence of SEQ ID NO:3.

[0076] Light chain: DIEMTQSPDS LAVSLGERAT INCRSSQSVL YSSSNRNNYLA WYQQNPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQQYYST PRTFGQGTKV EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 3)

[0077] In one embodiment, the anti-HER3 antibody of the invention comprises at least one mutation in the Fc region. In another embodiment, the mature anti-HER3 antibody of the invention (i.e., lacking a signal peptide) comprises at least one mutation at amino acid 234, 239, 434, or a combination thereof, and in other embodiments, 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.

[0078] In some embodiments, one or more mutations in the Fc region reduce effector function. In some embodiments, the reduced effector function comprises a reduced affinity of the anti-HER3 antibody to one or more Fc receptors. The FcRs can be FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa(158F), FcγRIIIa(158V), and C1q. In some embodiments, the reduced affinity comprises an increase in the dissociation constant of about one order of magnitude or more. In some embodiments, the introduction of one or more Fc mutations reduces the KD of the fusion protein comprising the anti-HER3 antibody to FcγRI of the anti-HER antibody by 2.81×10 -9 M to 1.03 x 10 -8In some embodiments, the introduction of one or more Fc mutations increases the KD of the anti-HER antibody for FcγRIIa of the fusion protein comprising an anti-HER3 antibody to 3.95×10 -7 M to 1.35 x 10 -6 In some embodiments, the introduction of one or more Fc mutations increases the KD of the fusion protein comprising an anti-HER3 antibody to FcγRIIb of the anti-HER antibody to 1.03×10 -7 M to 1.52 x 10 -6 By introducing one or more Fc mutations, the KD of the fusion protein containing the anti-HER3 antibody for the anti-HER antibody FcγRIIIa (158F) increases 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 fusion protein comprising an anti-HER3 antibody to FcγRIIIa(158V) of the anti-HER antibody to 3.41×10 -8 M to 9.10 x 10 -8 Increase to M.

[0079] In some embodiments, the anti-HER3 antibody or a recombinant fusion protein comprising the same is administered in 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 FcγRI with an equilibrium dissociation constant (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.

[0080] As used herein, the term "antibody effector function" refers to a function provided by the Fc region of an Ig. Such a function may be affected, for example, by binding of the Fc effector region to Fc receptors on immune cells that have phagocytic or lytic activity, or by binding of the Fc effector region to components of the complement system.

[0081] In one embodiment, the anti-HER3 antibody does not induce antibody-dependent cellular cytotoxicity (ADCC). The term "antibody-dependent cellular cytotoxicity (ADCC)" means that the antibody according to the invention in the presence of effector cells lyses human target cells.

[0082] In one embodiment of the invention, the antibody according to the invention is glycosylated. In some embodiments, the glycosylation is N-glycosylation. In other embodiments, the glycosylation is O-glycosylation.

[0083] In the context of the recombinant fusion proteins provided herein and according to the invention, the antibodies comprised in the recombinant fusion proteins can be produced via recombinant means. Such methods are well known in the art and include protein expression in prokaryotic and eukaryotic cells followed by isolation of the antibody polypeptides and usually purification 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, for example, CHO, NS0, SP2 / 0, HEK293, COS, yeast, or E. coli cells, and the antibodies are recovered from such cells (from the supernatant or after cell lysis). Recombinant production of antibodies is well known in the art and is 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 performed by standard techniques, including column chromatography and other techniques well known in the art, to remove other cellular components or other contaminants (e.g., 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 NSO 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. (Cytotechnology 30 (1999) 71-83) and Schlaeger, E.-J. (J. Immunol. Methods 194 (1996) 191-199). The monoclonal antibodies are suitably separated from the culture medium by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced using conventional procedures. Hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA can be inserted into an expression vector and then transfected into host cells, such as HEK293 cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to synthesize recombinant monoclonal antibodies in the host cells.

[0084] 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 host cells, which are then fused to form a single host cell expressing both chains.

[0085] It will be appreciated that the antibodies are 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 being sought by a researcher, veterinarian, physician, or other clinician.

[0086] Recombinant fusion protein - neuregulin In one embodiment, the recombinant fusion protein provided herein comprises a fragment of NRG-1 protein. NRG protein can bind to ErbB4 receptor on the surface of cardiomyocytes, persistently activate the PI3K / AKT signal pathway in cells, and modify the structure of cardiomyocytes, thereby improving the function of cardiomyocytes.

[0087] As used herein, "neuregulin" or "NRG" refers to a protein or peptide capable of binding to ErbB3, ErbB4, or heterodimers or homodimers thereof, including neuregulin isoforms, neuregulin EGF-like domains, polypeptides containing the neuregulin EGF-like domain, neuregulin variants or derivatives, and any type of neuregulin-like gene product capable of activating the above receptors. Neuregulin also includes NRG-1, NRG-2, NRG-3, and NRG-4 proteins, peptides, fragments, and compounds having neuregulin function. In a preferred embodiment, neuregulin is a protein or peptide capable of binding to and activating ErbB2 / ErbB4 or ErbB2 / ErbB3 heterodimers, for example, and not by way of limitation, the peptide of the present invention comprises a fragment of the NRG-1β2 isoform, i.e., a 177-237 amino acid fragment containing an EGF-like domain having the following amino acid 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, and promoting cardiomyocyte differentiation, survival, and DNA synthesis. It is well known to those skilled in the art that mutations of single amino acids in non-critical regions generally do not alter the biological activity of the resulting protein or polypeptide (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin / Cummings Pub.co., p. 224). The NRG proteins of the present invention can be isolated from natural sources or modified via recombinant technology, artificial synthesis, or other means.

[0088] 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 region as described in WO 00 / 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., Nature, 387:512-516 (1997); al., J. Biochem., 122:675-680 (1997); and WO 97 / 09425, the entire contents of which are 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. Pat. No. 5,834,229.

[0089] In one embodiment, the NRG-1 protein provided in a recombinant fusion protein disclosed herein is the NRG-1 β2a isoform.

[0090] In some embodiments, the active NRG-1 fragment comprises an ERBB3 / 4 binding domain. In another related embodiment, NRG-1 binds to and induces signaling through ErbB4 (HER4). In other embodiments, the mAb inhibits NRG-1 signaling through ErbB3 (HER3). In some embodiments, the active protein fragment of NRG-1 comprises an activity domain of NRG-1.

[0091] Recombinant fusion proteins - compositions In one embodiment, in the recombinant fusion proteins disclosed herein, NRG-1 is fused to the C-terminus of the heavy chain of an anti-HER3 antibody using a linker. In another related aspect, NRG-1 is attached to the linker via the first amino acid on 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, e.g., an expression vector for prokaryotic or eukaryotic organisms. In addition, a nucleic acid sequence encoding an NRG-1 β2a isoform molecule is optionally cloned into the same vector in the appropriate orientation and position such that expression from the vector produces an antibody-NRG-1 β2a isoform fusion protein. Some optional embodiments require post-expression modifications, e.g., assembly of antibody subunits. Techniques and techniques for the above (and similar) manipulations are well known to those of skill in the art. Relevant teachings can be found, 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, FMAusubel et al., eds., Current Protocols (a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.) (supplemented to 1999). In some alternative embodiments, the antibody domain and the NRG-1 β2a isoform are assembled after expression, for example, via chemical means. In one embodiment, the invention provides a composition (e.g., a pharmaceutical composition) comprising a recombinant fusion protein of the invention.

[0092] 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 growth of cancer and / or tumors. In another embodiment, the recombinant fusion protein promotes the proliferation, differentiation, and survival of cardiac tissue without promoting the growth of cancer or tumors.

[0093] 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), cholangiocarcinoma, extrahepatic bile duct cancer, intrahepatic cholangiocarcinoma, bladder cancer, urinary 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 system nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasms, mycosis fungoides, Sezary 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 (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, ovarian germ cell tumors, gestational trophoblastic tumor glioma, head and neck cancer, Hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular cancer, eyeball cancer, islet cell tumor (endocrine pancreas), Kaposi's sarcoma, kidney cancer, renal cancer, kidney cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid 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 malignant tumor, mesothelioma, metastatic squamous cell neck cancer, oral cancer, tongue cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disease, chronic myeloid leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorder, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oralpulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing's sarcoma family of tumors, Kaposi's sarcoma, soft tissue sarcoma, epithelioid sarcoma, synovial ... melanoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, pharyngeal cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter and other urinary tract, gestational trophoblastic neoplasm, urethral cancer, endometrial cancer, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, or Wilms' tumor.

[0094] In another embodiment, the recombinant fusion protein promotes central nervous system (CNS) cell proliferation, differentiation, and survival. In another embodiment, the recombinant fusion protein promotes central nervous system (CNS) cell proliferation, differentiation, and survival without promoting cancer / or tumor growth. In another embodiment, the recombinant fusion protein has a reduced ability to induce antibody-dependent cellular cytotoxicity (ADCC).

[0095] In some embodiments, the recombinant fusion protein promotes HER2 / 4 signaling over HER2 / 3 signaling relative to the signaling induction capacity of recombinant NRG-1.

[0096] 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 the heavy chain of the antibody via a GGGGSGGGGS (G4S) linker (SEQ ID NO: 5) to the NRG-1 β2a isoform of SEQ ID NO: 4. In some embodiments, one or more copies of the linker can be used. In other embodiments, two, three, four, or five copies of the G4S linker can be used herein, or any other linker known in the art as suitable for the compositions disclosed herein.

[0097] The term "linker" is art-recognized and refers to a molecule (including but not limited to unmodified or modified nucleic acids or amino acids) or group of molecules (e.g., two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more) that connects two compounds (e.g., two polypeptides). A linker may consist of a single binding molecule or may include a binding molecule and at least one spacer molecule intended to separate the binding molecule and the compounds by a particular distance.

[0098] 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 coding 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, although enhancers are optionally contiguous. Linking 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 in the pairing of an antibody described herein with an NRG-1 fragment via a linker sequence, also described herein.

[0099] In another embodiment, the anti-HER3 mAb heavy chain comprised in a recombinant fusion protein provided herein is encoded by SEQ ID NO:6.

[0100] In one 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.

[0101] 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). (SEQ ID NO:8) In one embodiment, SEQ ID NO:8 is also referred to as "PAL."

[0102] 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:

[0103] (SEQ ID NO:9)

[0104] 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: (SEQ ID NO:10)

[0105] 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 of MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 11).

[0106] In one embodiment, the anti-HER3 antibody light chain comprised in the recombinant fusion protein comprises the following amino acid sequence:

[0107] MVLQTQVFISLLLWISGAYGDIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12)

[0108] 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 amino acid sequence of an antibody heavy or light chain disclosed herein, lacks a signal peptide.

[0109] In one embodiment, the recombinant fusion protein comprises the following amino acid sequence:

[0110] Heavy chain [ka] (SEQ ID NO: 14; in the sequence, bold italics indicate linkers, bold indicates NRG-1 fragments) Light chain DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3)

[0111] In one embodiment, each of the heavy and light chain sequences in the mature recombinant fusion protein lacks a signal peptide amino acid sequence.

[0112] In certain embodiments of the invention, the heavy chain of an anti-HER3 antibody provided herein is fused to an NRG-1 β2a isoform provided herein via a C-terminal linker sequence. In another embodiment, the C-terminus of the heavy chain of the antibody comprises the Fc domain of the antibody.

[0113] In some embodiments, a pharmaceutical composition is provided that comprises a recombinant fusion protein disclosed herein formulated with a pharmaceutical carrier.

[0114] In some embodiments, the anti-HER3 antibodies and NRG-1 fragments described herein are recombinantly or chemically fused / operably linked via a linker to form a fusion protein. By "fusion protein", "fusion polypeptide", "recombinant fusion protein" or "recombinant polypeptide" is meant 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-1 β2a 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).

[0115] In one embodiment, the fusion protein is recombinantly encoded and produced, hi 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-1 β2a isoform of the invention via a nucleic acid sequence encoding a linker.

[0116] 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 mean greater than 70% identity to the sequence of the recombinant fusion protein (e.g., to any of SEQ ID NOs: 1-18). In another embodiment, "homology" means greater than 72% identity to any of SEQ ID NOs: 1-18. In another embodiment, "homology" means greater than 75% identity to any of SEQ ID NOs: 1-18. In another embodiment, "homology" means greater than 78% identity to any of SEQ ID NOs: 1-18. In another embodiment, "homology" means greater than 80% identity to any of SEQ ID NOs: 1-18. In another embodiment, "homology" means greater than 82% identity to any of SEQ ID NOs: 1-18. In another embodiment, "homology" means greater than 83% identity to any of SEQ ID NOs: 1-18. In another embodiment, "homology" means greater than 85% identity to any of SEQ ID NOs: 1-18. In another embodiment, "homology" means greater than 87% identity to any of SEQ ID NOs: 1 to 18. In another embodiment, "homology" means greater than 88% identity to any of SEQ ID NOs: 1 to 18. In another embodiment, "homology" means greater than 90% identity to any of SEQ ID NOs: 1 to 18. In another embodiment, "homology" means greater than 92% identity to any of SEQ ID NOs: 1 to 18. In another embodiment, "homology" means greater than 93% identity to any of SEQ ID NOs: 1 to 18. In another embodiment, "homology" means greater than 95% identity to any of SEQ ID NOs: 1 to 18. In another embodiment, "homology" means greater than 96% identity to any of SEQ ID NOs: 1 to 18. In another embodiment, "homology" means greater than 97% identity to any of SEQ ID NOs: 1 to 18. In another embodiment, "homology" means greater than 98% identity to any of SEQ ID NOs: 1 to 18.In another embodiment, "homology" means greater than 99% identity to any of SEQ ID NOs: 1 to 18. In another embodiment, "homology" means 100% identity to any of SEQ ID NOs: 1 to 18.

[0117] The determination of percent identity between two sequences can be accomplished 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 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 with the NBLAST program (score=100, wordlength=12) to obtain nucleotide sequences that are homologous to the nucleic acid encoding the target protein. BLAST protein searches can be performed with the XBLAST program (score=50, wordlength=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 used for sequence comparison 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.Further 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 in the two compared sequences are found by looking at pairs of aligned residues, and when ktup=1, single aligned amino acids are examined. ktup should be set to 2 or 1 for protein sequences and 1-6 for DNA sequences. If ktup is not specified, the default value is 2 for proteins and 6 for DNA. Alternatively, protein sequence alignments may be performed using the CLUSTAL W algorithm, which is described in Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0118] 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).

[0119] In one embodiment, the polynucleotide of the invention is inserted into an expression vector (i.e., a nucleic acid construct) to allow for expression of a recombinant polypeptide. In one embodiment, the expression vector of the invention comprises additional sequences that make the vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector of the invention comprises additional sequences that make the vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector of the 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 factors (e.g., polyadenylation signals).

[0120] In one embodiment, a variety of prokaryotic and eukaryotic cells can be used as host expression systems for expressing the polypeptides of the invention. In some embodiments, such cells 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, and yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence.

[0121] 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.

[0122] In some embodiments, the bacterial system of the present invention allows for the advantageous selection of multiple expression vectors depending on the intended use of the polypeptide to be expressed. In one embodiment, large quantities of the polypeptide are desired. In one embodiment, vectors directing high levels of protein product expression are desired, possibly as fusions with hydrophobic signal sequences directing the expression product into the periplasm of the bacteria or into the medium where the protein is easily purified. In one embodiment, certain fusion proteins are engineered with specific cleavage sites that aid in the recovery of the polypeptide. In one embodiment, vectors amenable to such engineering include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol. 185:60-89 (1990)].

[0123] In one embodiment, yeast expression system is used. In one embodiment, as disclosed in U.S. Patent No. 5,932,447, multiple 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.

[0124] In one embodiment, the expression vector of the invention can further comprise additional polynucleotide sequences, e.g., allowing translation of multiple proteins from a single mRNA, such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.

[0125] 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).

[0126] 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.

[0127] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses, such as retroviruses, are used in 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 / I, pAV009 / N, pAV009 / N, pAV009 / P ... + , pMTO10 / A +, pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of a protein 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 have effective expression in eukaryotic cells.

[0128] 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 lateral infection and targeting specificity. In one embodiment, lateral infection is inherent, for example, in the retroviral life cycle, a process in which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, this results in the rapid infection of large areas that were initially largely uninfected by the original viral particle. In one embodiment, viral vectors are produced that cannot spread laterally. In one embodiment, this property can be useful when the desired goal is to introduce a specified gene into only a localized number of target cells.

[0129] In one embodiment, various methods can be used to introduce an expression vector encoding a recombinant fusion protein of the present invention into a cell. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992); 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 US Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0130] In some embodiments, introduction of nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, as the infectivity of the virus allows for higher transfection efficiencies.

[0131] It will be appreciated that in one embodiment, the polypeptides of the invention may 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 suitable cells via an appropriate gene delivery vehicle / method (transfection, transduction, homologous recombination, etc.) and, if desired, an expression system, and the modified cells are then expanded in culture and returned to the individual (i.e., ex vivo gene therapy).

[0132] It will be understood that the expression constructs of the present invention, in addition to containing elements necessary for the transcription and translation of the inserted coding sequence (encoding a polypeptide), may also contain sequences engineered to optimize stability, production, purification, yield, or activity of the expressed polypeptide.

[0133] In some embodiments, the transformed cells are cultured under effective conditions, which allow for the expression of recombinant fusion proteins or polypeptides in large quantities. 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 invention. In some embodiments, media typically include aqueous solutions having assimilable carbon, nitrogen, and phosphate sources, appropriate salts, minerals, metals, and other nutrients (e.g., vitamins). In some embodiments, the cells of the invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri dishes. In some embodiments, the culture is performed at a temperature, pH, and oxygen content appropriate for the recombinant cells. In some embodiments, the culture conditions are within the expertise of one of ordinary skill in the art.

[0134] In some embodiments, depending on the vector and host system used for production, the resulting polypeptide of the invention remains within the recombinant cell, is secreted into the fermentation medium, is secreted into the space between two cell membranes (e.g., the periplasmic space in E. coli), or is retained on the outer surface of a cellular or viral membrane.

[0135] In one embodiment, after a period of time in culture, the recombinant polypeptide is recovered.

[0136] In one embodiment, the phrase "recovery of recombinant polypeptide" as used herein means recovering the whole fermentation medium containing the polypeptide and does not necessarily imply further steps of separation or purification.

[0137] 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 differential solubilization.

[0138] 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 can be engineered between the polypeptide and the cleavable moiety, and the polypeptide can 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)].

[0139] In one embodiment, the polypeptides of the present invention are recovered in "substantially pure" form.

[0140] In one embodiment, the term "substantially pure" refers to a purity that allows the protein to be effectively used in the applications described herein.

[0141] In one embodiment, the polypeptides of the present invention can be synthesized using in vitro expression systems. In one embodiment, in vitro synthesis methods are well known in the art and the components of the expression systems are commercially available.

[0142] In some embodiments, recombinant polypeptides can be synthesized and purified, and their therapeutic effectiveness assayed either in vivo or in vitro.

[0143] 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 solvent, dispersion medium, coating, antibacterial agent, antifungal agent, isotonic agent, absorption delaying agent, and physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[0144] Treatment method In one embodiment, the present invention provides a method of treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant fusion protein or a pharmaceutical composition comprising a recombinant fusion protein disclosed herein.

[0145] In one embodiment, the present invention provides a method of treating a cardiovascular disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein.

[0146] In one embodiment, the present invention provides a method for preventing, inhibiting, suppressing, or delaying the onset of a cardiovascular disease or condition in a subject, comprising administering an effective amount of a recombinant fusion protein or pharmaceutical composition described herein.

[0147] 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, and adjuncts to surgical intervention in pediatric congenital heart disease.

[0148] In some embodiments, chemotherapy-induced cardiotoxicity results from subjects receiving anthracyclines, alkylating agents, microtubule inhibitors, and antimetabolites used as chemotherapy.

[0149] In some embodiments, the cardiovascular condition is cardiotoxicity as a result of the subject receiving cancer therapy. In other embodiments, the cancer therapy is a HER-2 targeted therapy. In other embodiments, the HER-2 targeted therapy includes the use of trastuzumab, ado-trastuzumab, emtansine, lapatinib, neratinib, and pertuzumab, any anti-HER2 antibody, any anti-HER2 agent, or a combination thereof.

[0150] In another aspect, the present invention relates to a method of inducing remodeling of muscle cell sarcomeres and cytoskeletal structures or cell-cell adhesion, comprising treating cells with the recombinant fusion proteins disclosed herein.

[0151] In one embodiment, the therapeutic method is directed to the treatment of heart failure resulting from dissociation of cardiac muscle cell-cell adhesion and / or disruption of sarcomere structure in a mammal.

[0152] In another aspect, the present invention provides a method for preventing, treating, or delaying heart failure with preserved ejection fraction in a human comprising administering a pharmaceutical composition comprising a recombinant fusion protein disclosed herein.

[0153] As used herein, the term "ejection fraction" refers to the ejection fraction (EF), which is a measurement, typically expressed as a percentage, of the amount of blood the left ventricle pumps with each contraction. For example, an ejection fraction of 50 percent means that 50 percent of the total blood volume in the left ventricle is pumped out with each heartbeat.

[0154] The present invention is directed to methods of treating a subject having or at risk for heart disease and related conditions (eg, heart failure).

[0155] The term "heart failure" refers to an abnormality in cardiac function in which the heart does not pump blood at a rate required for tissue metabolic requirements. Heart failure includes a wide range of disease states, such as congestive heart failure, myocardial infarction, tachyarrhythmia, familial hypertrophic cardiomyopathy, ischemic heart disease, idiopathic dilated cardiomyopathy, and myocarditis. Heart failure can be caused by any factor, including ischemic, rheumatic, or idiopathic forms. Chronic cardiac hypertrophy is a significant morbid condition that is a precursor to congestive heart failure and cardiac arrest.

[0156] In one embodiment, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, where the purpose is to prevent or slow (alleviate) cardiac hypertrophy. Those in need of treatment include those who are prone to have the disorder or those in need of preventing the disorder, as well as those who already have the disorder. Cardiac hypertrophy may result from any cause that responds to retinoic acid, including congenital, viral, idiopathic, cardiotrophic, or myotrophic causes, or may result from an ischemic attack, such as ischemia or myocardial infarction. Typically, treatment is performed to stop or slow the progression of hypertrophy, especially after the occurrence of cardiac damage (e.g., ischemic damage). Preferably, in the treatment of myocardial infarction, the pharmaceutical composition provided herein is administered immediately after myocardial infarction to prevent or alleviate hypertrophy.

[0157] In some embodiments, by treating a subject with a pharmaceutical composition comprising a recombinant fusion protein provided herein, the treated subject population may have an increased mean survival time compared to a population administered a monotherapy with a drug other than the compound disclosed herein, or a pharma- ceutically acceptable salt, solvate, analog, or derivative thereof. Preferably, after treatment with the strategies, treatment modalities, methods, combinations, and compositions provided herein, the mean survival time is increased by more than 30 days, more preferably by more than 60 days, more preferably by more than 90, 120, 365 days, more preferably by more than 365 days. The increase in the mean survival time of a population can be measured by any reproducible means. The increase in the mean survival time of a population can be measured, for example, by calculating the mean survival time of a population after starting treatment with an active compound. The increase in the mean survival time of a population can also be measured, for example, by calculating the mean survival time of a population after completing a first round of treatment with a pharmaceutical composition disclosed herein.

[0158] In some embodiments, by treating a subject with a pharmaceutical composition comprising a recombinant fusion protein provided herein, the treated subject population may have a reduced mortality rate compared to a population administered with only a carrier. By treating cancer, the treated subject population may have a reduced mortality rate compared to a population administered with a non-treated population. By treating cancer, the treated subject population may have a reduced mortality rate compared to a population administered with a 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 modalities, methods, combinations, and compositions provided herein, the mortality rate is reduced by more than 2%, more preferably by more than 5%, more preferably by more than 10%, and most preferably by more than 25%. The reduction in mortality rate of the treated subject population may be measured by any reproducible means. The reduction in mortality rate of the population may be measured, for example, by calculating the average number of disease-related deaths per unit time after the population begins 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 a population after completing a first round of treatment with a pharmaceutical composition disclosed herein.

[0159] In one embodiment, the present invention provides a method of treating a central nervous system (CNS) related disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant fusion protein or pharmaceutical composition described herein.

[0160] 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, comprising administering an effective amount of a recombinant fusion protein or pharmaceutical composition described herein.

[0161] 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-Barré syndrome, stroke, traumatic brain injury, multiple sclerosis, or a combination.

[0162] Administration The composition of the present invention can be administered parenterally to a subject in need thereof, or can be administered by various methods known in the art. As one skilled in the art will understand, the route and / or mode of administration varies depending on the desired result. 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, for example, in liposomes or in 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 pharmaceutical active substances is well known in the art.

[0163] 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. Physiologically acceptable carriers (or excipients) are optionally used in certain embodiments of the invention. Examples of such include, for example, saline, PBS, Ringer's solution, lactated Ringer's solution, and the like. In addition, preservatives and additives are optionally added to help 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.

[0164] As used herein, the expressions "parenteral administration" and "administering parenterally" refer to 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.

[0165] Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable wettable 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.

[0166] The recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is optionally administered to a subject in need of treatment (therapeutic or prophylactic) in any suitable sterile pharmaceutical carrier. Such pharmaceutical carrier serves to maintain the solubility and activity of the fusion protein. In some embodiments, it may be desirable to administer additional components together with the fusion protein. For example, in some treatment regimes, chemotherapeutic agents, antibiotics, additional formulations comprising the recombinant fusion protein of the present invention, and one or more standard treatment agents, etc., are all optionally included with the composition of the present invention.

[0167] As used herein, the terms "combination treatment", "combination therapy" and "combination therapy" are used interchangeably and generally refer to a treatment modality featuring a recombinant fusion protein or a pharmaceutical composition comprising a recombinant fusion protein provided herein and an additional therapeutic agent. Typically, a combination treatment modality is part of a specific treatment regimen intended to produce a beneficial effect from the simultaneous action of a combination of therapeutic agents. The beneficial effect of the combination can include, but is not limited to, pharmacokinetic or pharmacodynamic synergy resulting from the combination of therapeutic agents. The administration of these therapeutic agents in combination is typically performed over a defined period of time (usually minutes, hours, days, or weeks depending on the combination selected). In some embodiments, combination treatment includes sequential administration of two or more therapeutic agents, with each therapeutic agent administered at a different time, and substantially simultaneous administration of these therapeutic agents, or at least two of these therapeutic agents. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single dosage form having a fixed ratio of each therapeutic agent, or by administering the therapeutic agents in multiple separate dosage forms. The sequential or substantially simultaneous administration of each therapeutic agent may be effected by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents may be administered by the same or different routes. The therapeutic agents may be administered according to the same or different administration intervals. For example, a first therapeutic agent of the combination may be administered by intravenous injection, and the other therapeutic agent of the combination may be administered orally. Alternatively, for example, all therapeutic agents may be administered orally, and all therapeutic agents may be administered by intravenous injection.

[0168] In some embodiments, the combination therapy also includes the administration of the above-mentioned therapeutic agent further combined with other biologically active ingredients and non-drug therapy (e.g., surgery or radiation therapy). When the combination therapy further includes a non-drug therapy, the non-drug therapy can be administered at any suitable time, as long as a beneficial effect is achieved from the synergistic action of the combination of the therapeutic agent and the non-drug therapy. For example, in appropriate cases, the beneficial effect is still achieved even if the non-drug therapy is temporarily removed from the administration of the therapeutic agent (possibly for days or even weeks).

[0169] In some embodiments, the additional therapeutic agent is a chemotherapeutic agent (also called antineoplastic or antiproliferative agent), such as an alkylating agent; an antibiotic: antimetabolite; an antidote: interferon; a polyclonal or monoclonal antibody; an EGFR inhibitor; an HER2 inhibitor; a histone deacetylase inhibitor; a hormone; a mitosis inhibitor; an MTOR inhibitor; a multi-targeted kinase 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 drug, a topoisomerase poison drug, 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 antineoplastic agent, a CDK inhibitor, a PARP inhibitor, or any antineoplastic or antiproliferative agent known to one of skill in the art.

[0170] Exemplary alkylating agents suitable for use in accordance with the combination treatment modalities provided 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).

[0171] 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 (Valstar).

[0172] 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).

[0173] Exemplary antidotes include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).

[0174] Exemplary interferons include, but are not limited to, interferon alpha-2b (Intron A) or interferon alpha-2a (Roferon-A).

[0175] 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.

[0176] 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.

[0177] Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin); lapatinib (Tykerb) or AC-480.

[0178] Histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza).

[0179] 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 Depot); exemestane (Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole (Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone (Provera; Depo-Provera); estramustine (Emcyt); flutamide (Eulexin); toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron); abarelix (Plenaxis); or testolactone (Teslac).

[0180] Exemplary mitotic inhibitors include, but are not limited to, paclitaxel (Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin; Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos; VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole; epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan (Camptosar); topotecan (Hycamtin); amsacrine or lamellarin D (LAM-D).

[0181] Exemplary MTOR inhibitors include, but are not limited to, everolimus (Afinitor) or temsirolimus (Torisel); rapamune, ridaforolimus; or AP23573.

[0182] Exemplary multi-targeted kinase inhibitors include, but are not limited to, sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474; PKC-412; motesanib; or AP24534.

[0183] 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 PD 332991.

[0184] Exemplary tyrosine kinase inhibitors include, but are not limited to, erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib (Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab (Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux); panitumumab (Vecti bix); everolimus (Afinitor); alemtuzumab (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.

[0185] Exemplary VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin), sorafenib (Nexavar), sunitinib (Sutent), ranibizumab, pegaptanib, or vandetinib.

[0186] Exemplary microtubule targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epothilones, and navelbine.

[0187] Exemplary topoisomerase poisons include, but are not limited to, teniposide, etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin, and idarubicin.

[0188] Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxol.

[0189] Exemplary immune checkpoint inhibitors include programmed cell death 1 (PD-1) inhibitors, CD274 molecule (PD-L1) inhibitors, 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.

[0190] Exemplary platinum-based anti-neoplastic agents include cisplatin and carboplatin.

[0191] Exemplary cyclin-dependent kinase inhibitors include abemaciclib, palbociclib, and ribociclib.

[0192] Exemplary poly(ADP-ribose) polymerase (PARP) inhibitors include talazoparib, olaparib, rucaparib, niraparib, and veliparib.

[0193] Exemplary general chemotherapeutic, antineoplastic, and antiproliferative 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 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.

[0194] In some embodiments, combination treatment modalities are provided in which the additional therapeutic agent is a cytokine (e.g., G-CSF (granulocyte colony stimulating factor)). In another aspect, the pharmaceutical compositions 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 compositions provided herein and another chemotherapeutic agent provided herein. In yet another aspect, the pharmaceutical compositions provided herein can be administered in combination with standard chemotherapy combinations, such as, 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 (CDP), cyclophosphamide ... It can be administered in combination with rituximab, TS-1 (tegafur, gimestat, and otastat potassium in a 1:0.4:1 molar ratio), 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).

[0195] In some preferred embodiments, the pharmaceutical compositions provided herein can be administered with an inhibitor of an enzyme, such as a receptor or non-receptor kinase. 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.

[0196] Exemplary kinase inhibitors include, but are not limited to, bevacizumab (targets VEGF), BIBW 2992 (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), lapatinib (targets Erb1 and Erb2 / Her2), GW-572016 / lapatinib-2016, and / or other kinase inhibitors. tosylate (targets HER2 / Erb2), panitumumab / Vectibix (targets EGFR), vandetinib (targets RET / VEGFR), E7080 (multi-targeting (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), matuzumab / Emd7200 (targets EGFR), EKB-569 (targets EGFR (targets EGFR and VEGFR), Zd6474 (targets EGFR and VEGFR), PKC-412 (targets VEGR and FLT3), vatalanib / Ptk787 / ZK222584 (targets taGR), CEP-701 (targets FLT3), SU5614 (targets FLT3), MLN518 (targets FLT3), XL999 (targets FLT3), VX-322 (targets FLT3), Azd0530 (targets SRC), BMS-354825 (targets SRC), SKI-606 (targets SRC), CP-690 (targets JAK (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), AC-480 (targets all HER proteins (pan-HER)), motesanib diphosphate (targets VEGF1-3, PDGFR,and c-kit), denosumab (targets RANKL and inhibits SRC), AMG888 (targets HER3), and AP24534 (multiple targets including Flt3).

[0197] In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the same polypeptide disclosed herein is administered to the subject once per day. In some embodiments, the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered to the subject once every two days. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered to the subject once every three days. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered to the subject once every four days. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered to the subject once every five days. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered to the subject once every six days. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered to the subject once per week. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered to the subject once every seven to fourteen days. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered to the subject once every ten to twenty days. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to a subject once every 5 to 15 days. In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to a subject once every 15 to 30 days.

[0198] In one embodiment, the dose of the recombinant fusion protein of the invention comprises 0.005-0.1 milligrams / kg in injectable solution. In another embodiment, the dose comprises 0.005-0.5 milligrams / kg of recombinant fusion protein. In another embodiment, the dose comprises 0.05-0.1 micrograms of recombinant fusion protein. In another embodiment, the dose comprises 0.005-0.1 milligrams / kg of recombinant fusion protein in injectable solution.

[0199] In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein 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 the pharmaceutical composition comprising the recombinant fusion protein 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 the pharmaceutical composition comprising the recombinant fusion protein 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 the pharmaceutical composition comprising the recombinant fusion protein 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 the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 0.2 mg to 0.6 mg.

[0200] In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 1 to 100 mcg / kg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 10 to 80 mcg / kg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 20 to 60 mcg / kg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 10 to 50 mcg / kg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 40 to 80 mcg / kg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 10 to 30 mcg / kg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 30 to 60 mcg / kg.

[0201] In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 0.1 mcg / kg to 100 mg / kg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 0.1 mcg to 50 mg / kg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 0.1 mcg to 25 mg / kg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 0.1 mcg to 10 mg / kg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 0.1 mcg to 5 mg / kg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 0.1 mcg to 1 mg / kg. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered to a subject at a dose ranging from 0.1 mcg to 0.1 mg / kg. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered to a subject at a dose ranging from 10 mg / kg to 60 mg / kg.

[0202] In another embodiment, the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to a subject at a dose of about 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.

[0203] In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 0.2 mg to 2 mg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 2 mg to 6 mg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 4 mg to 10 mg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose ranging from 5 mg to 15 mg.

[0204] In one embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein 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 the pharmaceutical composition comprising the recombinant fusion protein 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 the pharmaceutical composition comprising the recombinant fusion protein 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 the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose of about 25 μg / kg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose of about 50 μg / kg. In another embodiment, the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose of about 100 μg / kg. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at a dose in the range of about 200 μg / kg. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein 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 recombinant fusion protein 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 recombinant fusion protein 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 recombinant fusion protein is administered to the subject at a dose of about 600 μg / kg.

[0205] In one embodiment, a single dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to the subject. In another embodiment, a total of two doses are administered to the subject. In another embodiment, a total of two or more doses are administered to the subject.

[0206] In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to a subject at least once a day. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to a subject at least once every two days. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to a subject at least once on more than one day. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to a subject once a week, once every two weeks, or once every three weeks. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to a subject at least once a week. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to a subject at least once every two weeks. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to a subject at least once every three weeks. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to a subject at least once per week for three or more weeks. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to a subject two or more times per week. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to a subject two or more times per month. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to a subject two or more times per year. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to a subject two or more times per year for two or more years.

[0207] In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered at least once every 36 hours. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered at least once every 48 hours. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered at least once every 60 hours. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered at least once every 72 hours. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered at least once every 84 hours. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered at least once every 96 hours. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered at least once every 5 days. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising the recombinant fusion protein is administered at least once every 6 days. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered at least once every 7 days. In another embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein 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 recombinant fusion protein 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 recombinant fusion protein 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 recombinant fusion protein 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 recombinant fusion protein is administered at least once every 20 to 30 days.

[0208] In one embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered to the subject at least once per month. In one embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered at least once per two months. In one embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered at least once per three months. In one embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered at least once per four months. In one embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered at least once per five months. In one embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein is administered at least once per six months. In one embodiment, a dose of the recombinant fusion protein or a pharmaceutical composition comprising the recombinant fusion protein 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 recombinant fusion protein is administered four times per year. In another embodiment, the 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 two years, every three years, every four years, or at least every five years.

[0209] In one embodiment, repeated administrations (doses) of the compositions of the invention can be administered immediately after an initial course of treatment, or after intervals of days, weeks, or years, to achieve a desired effect (e.g., to prevent or treat a cardiovascular disease or condition, or a CNS-related disease or condition) as further provided herein.

[0210] In one embodiment, the pharmaceutical composition is administered by intravenous, intraarterial, subcutaneous, or intramuscular injection of a liquid preparation. In another embodiment, the liquid preparation comprises a solution, a suspension, a dispersion, an emulsion, an oil, or the like. In one embodiment, the pharmaceutical composition is administered intravenously and is therefore formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical composition is administered intraarterially and is therefore formulated in a form suitable for intraarterial administration.

[0211] In some embodiments, compositions for use in the methods disclosed herein include solutions or emulsions, which in some embodiments are aqueous solutions or emulsions containing a safe and effective amount of a compound disclosed herein and optionally other compounds, intended for intravenous or subcutaneous administration.

[0212] In some embodiments, the various constituents of the composition are pre-measured and / or pre-packaged and / or provided in a ready-to-use state without further measurement, etc. The present invention also optionally includes kits for carrying out / using the methods and / or compositions of the present invention. In particular, such kits optionally include, for example, a suitable recombinant fusion protein (and optionally a mixture of a plurality of such proteins (see above) for carrying out a synergistic treatment, and further optionally, a suitable disease-associated antigen). In addition, such kits may also include a suitable excipient (e.g., a pharma- ceutically acceptable excipient) for carrying out the therapeutic and / or prophylactic treatment of the present invention. Such kits optionally include further components for the assembly and / or use of the compositions of the present invention, including, but not limited to, a diluent, etc.

[0213] The compositions described herein are optionally packaged to include all (or nearly all) of the components necessary to practice the methods or use the compositions of the invention (optionally including, e.g., instructions for use of the methods / compositions of the invention). For example, the kits can optionally include components such as, e.g., buffers, reagents, serum proteins, antibodies, substrates, etc. In the case of prepackaged reagents, the kits optionally include premeasured or predosed amounts that can be incorporated into the method without measuring, e.g., premeasured liquid aliquots, or preweighed or measured solid reagents that can be easily reconstituted by the end user of the kit.

[0214] Such kits also typically include suitable instructions for carrying out the methods of the invention and / or using the compositions of the invention. In some embodiments, the components of the kit / package are provided in a stabilized form to prevent deterioration or other loss (e.g., due to leakage) during long-term storage. Several stabilization processes / agents, such as the inclusion of chemical stabilizers (i.e., enzyme inhibitors, microbicides / bacteriostats, anticoagulants), are commonly used for reagents to be preserved, etc. The dosage level of the actual active ingredient in the pharmaceutical composition of the invention can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response in a particular subject, composition, and route of administration without toxicity to the subject. The dosage level selected will depend on various pharmacokinetic factors, including the activity of the particular compound 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 combination with the particular compound used, the age, sex, weight, condition, the overall health and medical history of the subject being treated, and similar factors well known in the medical field.

[0215] The composition must be sterile and fluid enough to be deliverable by syringe.The carrier is preferably an isotonic buffered saline solution other than water.Proper fluidity can be maintained by, for example, using a coating such as lecithin, maintaining the required particle size when dispersed, and using surfactants.In many cases, it is preferable to include an isotonic agent in the composition, for example, sugar, polyalcohol such as mannitol, sorbitol, or sodium chloride.

[0216] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response in a particular subject, composition, and mode of administration without toxicity to the subject. The selected dosage level depends on various pharmacokinetic factors, including the activity of the particular compound of the present invention used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular compound used, the age, sex, weight, condition, overall health and medical history of the subject being treated, and similar factors well known in the medical field.

[0217] Although several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and such variations and / or modifications are deemed to be within the scope of the embodiments of the present 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(s) in which the teaching(s) of the present 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 present invention described herein. As such, it should be understood that the foregoing embodiments are presented by way of example only, and that within the scope of the appended claims and equivalents thereto, embodiments of the present invention may be practiced otherwise than as specifically described and claimed. The inventive embodiments in this disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the inventive scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0218] All definitions defined and used herein should be understood to supersede any dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0219] All references, patents, and patent applications disclosed herein are hereby incorporated by reference with respect to the subject matter for which each is cited, which may include the entire document.

[0220] As used herein, the indefinite articles "a" and "an" in the specification and claims should be understood to mean "at least one" unless clearly indicated to the contrary.

[0221] As used herein, the term "and / or" in the specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so conjoined. Other elements, other than the element expressly identified by the "and / or" clause, can optionally be present, whether related to the element expressly identified or not. Thus, as a non-limiting example, a reference to "A and / or B," when used with an open-ended term such as "comprising," can in one embodiment refer to only A (optionally including elements other than B), in another embodiment refer to only B (optionally including elements other than A), in another embodiment refer to both A and B (optionally including other elements), and so forth.

[0222] As used herein, the phrase "at least one" in the specification and claims with reference to a list of one or more elements should be understood to mean at least one element selected from any one or more in the list of elements, but not necessarily including at least one of every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that elements other than those specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present, whether or not related to the specifically identified elements. 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 mean in one embodiment at least one (optionally including more than one) A, but no B (optionally including elements other than B), in another embodiment at least one (optionally including more than one) B, but no A (optionally including elements other than A), in yet another embodiment at least one (optionally including more than one) A and at least one (optionally including more than one) B (optionally including other elements), or otherwise.

[0223] The present invention further provides a kit for preventing, treating, or delaying a cardiovascular disease or condition in a human, comprising 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 preparation or composition.

[0224] The present invention further provides a kit for preventing, treating, or delaying a CNS-related disease or condition in a human, comprising 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 preparation or pharmaceutical composition.

[0225] The present invention further provides a kit for preventing, treating, or delaying heart failure with preserved ejection fraction in a human, 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 preparation or pharmaceutical composition.

[0226] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention, but should not be construed in any way as limiting the broad scope of the invention. EXAMPLES

[0227] Example 1: Cloning and construction of expression plasmids DNA sequences encoding the heavy chain (designated NPCFA and NPCF for sequences with or without Fc mutation, respectively) and light chain (designated PAL) of the recombinant fusion protein were synthesized by GENEWIZ (Suzhou, China). The expression vector pCHOGUN was obtained under license agreement from Horizon Discovery (Cambridge, UK). The construction of expression plasmids was performed as outlined in Figure 1. Briefly, pCHOGUN vector was linearized by restriction enzyme BfuAI, and gene insert fragments such as NPCF, NPCFA, and PAL were purified after double restriction enzyme digestion with NcoI and AscI. The linearized pCHOGUN / BfuAI and purified gene insert fragments 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, and plasmids containing the light chain insert (pCHOGUN-PAL) were digested with restriction enzymes NgoMIV and PciI. After restriction enzyme digestion, fragments carrying the heavy or light chain insert were purified, ligated, and then 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.

[0228] Example 2: Antibody production, purification, and characterization Glutamine synthase-null (GS) mutant - / -HD-BIOP3, a 4-HTLV-1 (H3-dependent chromatin-binding protein) cell line, was obtained under license from Horizon Discovery (Cambridge, UK). Plasmid DNA was isolated using the commercially available Qiagen plasmid kit. Transfection of plasmid DNA into HD-BIOP3 cells was performed using the commercially available electroporation system from Lonza. Transfected cells were plated in 96-well plates and subjected to pool selection using standard procedures. Cells from the selection pool 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.

[0229] Figure 2A illustrates 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 primary antibodies specific for NRG-1 ("NRG-1" (R&D Systems, Minneapolis, Minn.)) or a 61 amino acid active fragment of IgG containing the HER3 / 4 binding domain are shown in Figures 2C and 2D, respectively.

[0230] Example 3: Molecular integrity assessed by SPR-based binding assays The molecular structure integrity of the recombinant fusion protein disclosed herein was evaluated by evaluating the 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), and then the samples (recombinant fusion protein disclosed herein, recombinant fusion protein disclosed herein without Fc mutation, anti-HER3 mAb) were injected (step 2), and anti-NRG-1 antibody (R&D Systems, Minneapolis, MN) was injected (step 3). The binding of His-HER3 on the sensor chip can be visualized by the signal increase of all six channels in step 1. Both the recombinant fusion protein disclosed herein and the recombinant fusion protein disclosed herein without Fc mutations produced significant responses by binding to HER3 in step 2 and by binding to the injected anti-NRG-1 antibody in step 3 (Ch1, 3), indicating the presence of the 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), confirming the absence of NRG-1 binding activity in the anti-HER3 mAb molecule. Buffer was injected in steps 2 and 3 as a blank control. Thus, both the HER3 binding epitope and NRG-1 are present in the recombinant fusion protein of the present invention.

[0231] The binding sensorgrams and the sequence of sample injections are shown in FIG.

[0232] Example 4: Effect on tumor cell line proliferation in vitro Tumor cells were seeded in 96-well plates at 2,500-20,000 cells per well depending on the growth kinetics of each cell line. Cells were then treated with a stepwise 1:4 dilution series of recombinant fusion proteins disclosed herein, antibodies, or control proteins for 5 days. Cell viability was assessed using the Cell Counting Kit-8 from Dojindo Molecular Technologies (Kumamoto, Japan). Data were analyzed with GraphPad Prism software. The data are presented as percent growth relative to untreated controls.

[0233] Figure 4 includes representative graphs showing the mean relative growth rate ± SEM (n=3) in different cancer cell lines: (A) NCI-N87 (gastric cancer); (B) MCF-7 (breast cancer); (C) RT-112 (bladder cancer); and (D) T47D (breast cancer). Compared to control NRG-1 and GP120 mAb / NRG-1 fusion proteins, the recombinant fusion proteins disclosed herein show significantly lower activity in promoting cancer cell proliferation.

[0234] Example 5: PI3K / AKT signaling pathway activation in human cardiomyocytes Human cardiomyocytes obtained from Cellular Dynamics (Madison, WI) were seeded on 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 use in the 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 a stepwise 1:4 dilution series of recombinant fusion proteins or control agents (NRG-1, GP120 mAb / NRG-1, anti-HER3 mAb, or GP120 mAb) for 15 minutes. At the end of the treatment, the cells were lysed and analyzed for AKT phosphorylation using Abcam's Phospho-AKT / Total AKT ELISA Kit (Cambridge, MA) according to the manufacturer's instructions. Data were analyzed with GraphPad Prism software. The data are presented as the ratio of phospho-AKT:total AKT relative to untreated controls.

[0235] 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 control drug. At the end of treatment, cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors. SDS-PAGE and Western blot were performed according to standard protocols. Total AKT and phosphor-AKT were blotted with AKT rabbit antibody and p-AKT (S473) rabbit antibody, respectively (Cell Signaling; Danvers, MA).

[0236] 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 a potency comparable to that of NRG-1.

[0237] 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 assay principle is illustrated in FIG. 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. A ligand is induced to interact with one target protein by binding to the other, forcing complementation of the two enzyme fragments, resulting in an enzymatic reaction that releases a chemiluminescent signal that is detected as relative fluorescent units or RFUs.

[0238] 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 agents were prepared in a stepwise 1:4 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).

[0239] As shown in Figures 6B and 6C, the recombinant fusion proteins disclosed herein can induce HER2 / HER4 dimerization with potency comparable to that of NRG-1, while their ability to induce HER2 / HER3 dimerization is much lower than that of NRG-1. Neither the isotype control antibody GP120 mAb nor anti-HER3 mAb, which served as a negative control in this study, induced receptor dimerization.

[0240] Although the embodiments of the present invention have been described with reference to the accompanying drawings, it should be understood that the present invention is not limited to the detailed embodiments, and various changes and modifications may be effected by those skilled in the art without departing from the scope or spirit of the present invention as defined in the appended claims.

[0241] Example 7: In vivo efficacy of recombinant fusion proteins in a rat model of systolic heart failure To evaluate the cardiac function regeneration ability of recombinant fusion proteins in disease models, a Sprague-Dawley rat model of myocardial infarction and systolic heart failure was employed. To establish the disease model, the surgical procedure involved ligating the left anterior descending artery (LAD) 3-4 mm below the left atrial appendage 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% decrease in EF was used for inclusion in the following study. Sham control animals underwent an identical surgery without LAD ligation.

[0242] 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 to administer two tail vein injections per week for a period of four weeks, or a total of eight injections, to each group. Both the sham-operated and vehicle negative control groups received saline, three groups received 1, 3, or 10 mg / kg of recombinant fusion protein, and the last group received a positive control of GP120 mAb / NRG-1 fusion protein (10 mg / kg).

[0243] Due to weight loss observed during the study, treatment was stopped prior to the full sequence of 8 injections in the recombinant fusion protein groups administered 3 mg / kg and 10 mg / kg, resulting in these groups receiving only 6 and 3 injections, respectively. All other groups received the full set of 8 injections.

[0244] 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 in the 1, 3, and 10 mg / kg groups, respectively. The GP120 mAb / 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.

[0245] After collecting ECG values ​​28 days 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. Paraffin sections of cardiac tissue with a thickness of 5 μm were stained with hematoxylin-eosin dye, and histopathological changes were observed under an optical microscope. As shown in Figure 9, in the sham-operated group, cardiomyocytes were regularly arranged, and the cytoplasm and myocardial fibers were evenly stained. No inflammatory cell infiltration was observed in the interstitial space, and no myocardial necrosis was observed. In contrast, the vehicle control group showed enlarged gaps between cardiomyocytes in the myocardial infarction border zone, nuclei were condensed and shattered, myocardial fiber arrangement lost its orderly structure, cell size was enlarged, and interstitial edema was observed. Treatment with the recombinant fusion protein partially alleviated the pathological changes in the myocardial infarction zone, including a significant reduction in necrotic cells, a reduction in the interstitial space between cardiomyocytes, and a restoration of myocardial fiber arrangement to a normal structure.

[0246] Example 8: Attenuation of tumor growth by recombinant fusion proteins in a NOD / SCID mouse subcutaneous FaDu cancer xenograft model To evaluate the potential risk of the recombinant fusion protein in promoting tumor growth, an in vivo study was performed in the FaDu cancer xenograft model. NOD / SCID mice (Beijing AK Bio-Technology Co.Ltd.) were housed in the SPF facility of CrownBio international R&D center (Beijing, China) in accordance with the institutional guidelines. All experiments were performed in accordance with the requirements of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and with the permission of the CrownBio IACUC committee.

[0247] 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 inoculated subcutaneously. 3 Once the tumor reached tumor size, the mice were randomized and divided into six test groups with eight animals per group. Test articles were administered intravenously into the tail vein twice weekly for three consecutive weeks for a total of six treatments. Tumor growth was monitored by caliper measurements. The study was terminated 21 days after treatment.

[0248] Tumor growth in response to the different treatments is summarized in Figure 10. Anti-HER3 mAb at 10 mg / 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 a statistically significant TGI at the end of the study with 19.2% at the 1 mg / kg dose (p=0.048 vs. vehicle group) and 56.2% at the 10 mg / kg dose (p<0.001 vs. vehicle group). The control molecule, GP120 mAb / NRG-1 fusion protein, showed no antitumor activity at either high or low doses. No animal deaths occurred during the study. All test agents were well tolerated by tumor-bearing mice. No significant weight loss was observed in any of the experimental groups (Figure 11). These data demonstrate that under conditions of active in vivo tumor growth, the recombinant fusion protein exhibits dose-dependent tumor growth inhibition, suggesting that the recombinant fusion protein poses a lower risk of enhancing or promoting tumor growth in vivo than the native NRG-1 protein.

[0249] Example 9: No significant gastrointestinal toxicity was observed in cynomolgus monkeys administered recombinant fusion protein It has previously been reported that in a Phase 1 clinical trial (NCT01258387) in which subjects received placebo or a single dose of 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 total 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 common treatment-emergent adverse event, occurring in 20% of study subjects (Jabbour et al. European Journal of Heart Failure (2011)13: 83-92). Finally, in a Phase 2 trial of Nucardin (ChiCTR-TRC-00000414), published results indicate that 48.4% of observed adverse events were gastrointestinal in nature, which was 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).

[0250] Two studies were conducted to evaluate the safety and tolerability of the recombinant fusion protein in cynomolgus macaques (Macaca fascicularis): a single-dose non-GLP (Good Laboratory Practice) study and a repeat-dose GLP study. Gastrointestinal toxicity was closely monitored. 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 compared to vehicle control, with one male and one female animal included in each cohort. In this single-dose study, there were no test drug-related effects on body weight or qualitative dietary assessments, and no evidence of vomiting or diarrhea throughout the 2-week post-treatment evaluation period. In a multiple-dose GLP study, the safety and tolerability of the recombinant fusion protein was evaluated after four consecutive weekly doses at dose levels of 3, 10, and 30 mg / kg compared to vehicle control, with three males and three females in each cohort during the main 28-day study period, followed by an additional 2 males and 2 females in the 30 mg / kg and vehicle control cohorts after a subsequent 28-day recovery period. No test drug-related effects on food consumption were observed in this multiple-dose study. Test drug-related emesis was observed in this multiple-dose study, but clinical findings of emesis were only associated with infusion reactions and were transient in nature, observed in only one animal (17%) in the 10 mg / kg cohort and two animals (20%) in the 30 mg / kg cohort. Diarrhea was observed only in the vehicle control cohort and in one animal (10%) and three animals (30%) in the 30 mg / kg recombinant fusion protein cohort, 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 weights were reduced by >10% from 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 with respect to food intake, vomiting, or diarrhea other than during the acute infusion reaction, 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 gastrointestinal effects of NRG-1 recombinant protein.

[0251] Blood samples (approximately 1 ml) were collected from cynomolgus monkeys at different time points after administration of a single dose of 60 mg / kg of recombinant fusion protein, and serum was extracted and stored at -80°C until testing. Recombinant fusion protein concentrations in serum samples were assayed 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 IgG Fc antibody and then detected with TMB substrate. Figure 12 shows that the pharmacokinetic profile of the recombinant fusion protein is similar to that of the IgG antibody.

[0252] Example 10: Summary of kinetic constants for Fc receptor binding Label-free SPR technique was used to measure the binding affinity between recombinant anti-HER3 mAb / NRG-1 fusion protein and Fc receptors. 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) using HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v Surfactant P20) as the running buffer. Specifically, anti-His antibodies were coupled by amine coupling in both the active and reference flow cells of a CM5 sensor chip. Purified His-tagged Fc receptors were captured on the active flow cells of each individual channel by binding to the immobilized anti-His antibodies. The capture level of each Fc receptor was maintained between 80 and 120 RU. For kinetic analysis, the 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 sequentially injected into both flow cells of each channel. Multiple analyses were completed during the same run by injecting samples simultaneously into multiple channels.

[0253] The resulting sensorgrams were fitted to a two-state binding model to extract kinetic constants using Biacore 8K evaluation software. The equilibrium dissociation rates (KD) of all analyses are summarized in Table 1 below. The kinetically derived KD values ​​for the binding of the recombinant fusion proteins 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 designated mutations in the Fc region of the recombinant fusion proteins resulted in much lower affinities. 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 very weak and was not detected in all samples.

[0254] To confirm that the recombinant fusion proteins had limited Fc effector function, they were tested for antibody-dependent cellular cytotoxicity (ADCC) using an ADCC reporter bioassay from Promega (Madison, WI). This assay used an engineered Jurkat cell line as effector cells that stably expressed the FcγRIIIa (V158) receptor and an NFAT response element driving 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).

[0255] [Table 1]

Claims

1. A recombinant fusion protein comprising a fragment of the cardioprotective protein Neuregulin-1 (NRG-1) fused to a monoclonal antibody (mAb) backbone, A recombinant fusion protein, wherein said mAb is monospecific for ErbB3 (HER3) and said NRG-1 fragment comprises an EGF-like domain that binds to and induces signaling through ErbB4 (HER4).

2. The recombinant fusion protein of claim 1, wherein the NRG-1 fragment comprises the NRG-1 β2a isoform.

3. The recombinant fusion protein of claim 1 or 2, wherein said mAb inhibits NRG-1 signaling through ErbB3 (HER3).

4. The recombinant fusion protein according to any one of claims 1 to 3, wherein the NRG-1 fragment is fused to the C-terminus of the heavy chain of the antibody via its N-terminal amino acid using a linker.

5. the linker comprises at least one copy of the Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser linker set forth in SEQ ID NO:5; or The recombinant fusion protein of claim 4 , wherein the C-terminus of the heavy chain of the antibody comprises the Fc domain of the antibody.

6. the monoclonal antibody is glycosylated; The recombinant fusion protein according to any one of claims 1 to 5, wherein the glycosylation is N-glycosylation or O-glycosylation.

7. The recombinant fusion protein of any one of claims 1 to 6, wherein the NRG-1 fragment comprises the amino acid sequence of SEQ ID NO:

4.

8. The recombinant fusion protein of any one of claims 1 to 7, wherein the mAb comprises a heavy chain and a light chain, and the heavy chain comprises the sequence of SEQ ID NO:

2.

9. 9. The recombinant fusion protein of claim 8, wherein the light chain comprises the sequence of SEQ ID NO:

3.

10. The recombinant fusion protein of any one of claims 1 to 7, wherein the mAb comprises a substitution mutation at least one amino acid at positions 234, 239, and 434 relative to SEQ ID NO:

2.

11. 11. The recombinant fusion protein of claim 10, wherein the at least one substitution mutation comprises a L234F mutation, a S239A mutation, a N434A mutation, or a combination thereof.

12. The recombinant fusion protein according to any one of claims 1 to 11, wherein the recombinant fusion protein comprises the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:

14.

13. The recombinant fusion protein of any one of claims 1 to 12, wherein the recombinant fusion protein attenuates tumor or cancer cell proliferation more than recombinant NRG-1.

14. The recombinant fusion protein of any one of claims 1 to 13, wherein the recombinant fusion protein promotes proliferation, differentiation, and survival of cardiomyocytes or cardiac tissue.

15. The recombinant fusion protein according to any one of claims 1 to 14, wherein the recombinant fusion protein has a reduced ability to induce antibody-dependent cellular cytotoxicity (ADCC).

16. 16. The recombinant fusion protein of any one of claims 1 to 15, wherein the recombinant fusion protein promotes HER2 / 4 signaling over HER2 / 3 signaling as compared to the signaling induction ability of recombinant NRG-1.

17. A recombinant nucleic acid molecule encoding the recombinant fusion protein of any one of claims 1 to 16.

18. 18. The recombinant fusion protein of claim 17, wherein the heavy chain of the mAb is encoded by SEQ ID NO:6 or SEQ ID NO:7, and the light chain of the mAb is encoded by SEQ ID NO:

8.

19. A recombinant vector comprising the nucleic acid molecule of claim 17 or 18.

20. A recombinant cell comprising the recombinant vector of claim 19.

21. A pharmaceutical composition comprising a recombinant fusion protein according to any one of claims 1 to 16.

22. 22. The pharmaceutical composition of claim 21 for treating a cardiovascular disease or condition in a subject.

23. 22. The pharmaceutical composition of claim 21 for preventing, inhibiting, suppressing, or delaying the onset of a cardiovascular disease or condition in a subject.

24. 24. The pharmaceutical composition of claim 22 or 23, wherein the pharmaceutical composition is administered such that 0.1 mcg / kg to 5 mg / kg of recombinant fusion protein is administered to the subject.

25. The pharmaceutical composition of any one of claims 22 to 24, wherein said pharmaceutical composition alleviates a sign or symptom of a cardiovascular disease or condition in said subject.

26. A pharmaceutical composition according to any one of claims 22 to 25, suitable for intravenous administration.

27. 27. The pharmaceutical composition of any one of claims 22 to 26, wherein the cardiovascular disease or condition is heart failure with preserved ejection fraction, 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, an adjunct to surgical intervention in pediatric congenital heart disease, or cardiotoxicity as a result of a subject undergoing cancer therapy.

28. 28. The pharmaceutical composition of claim 27, wherein the chemotherapy-induced cardiotoxicity results from the subject receiving an anthracycline, an alkylating agent, a microtubule inhibitor, or an antimetabolite used in chemotherapy, and the cancer therapy is a HER-2 targeted therapy.

29. 29. The pharmaceutical composition of claim 28, wherein the HER-2 targeted therapy comprises the use of trastuzumab, adotrastuzumab, emtansine, lapatinib, neratinib, and pertuzumab, any anti-HER2 antibody, any anti-HER2 agent, or a combination thereof.

30. A kit comprising an effective amount of a recombinant protein according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 21.

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