Neuregulin-1 / Anti-her3 antibody fusion protein for use in the treatment of heart failure

Abstract

Provided herein are recombinant fusion proteins comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb), and methods of using same to treat cardiovascular disease.

Description

NEUREGULIN-1 / ANTI-HER3 ANTIBODY FUSION PROTEIN FOR USE IN THE TREATMENT OF HEART FAILURECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to, and benefit of, U.S. Provisional Application No 63 / 650,131, filed on May 21, 2024, the content of which is incorporated by reference in its entirety herein.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy, created on May 7, 2025, is named SBTI-006_001WO_SeqList_ST26.xml, and is 23,515 bytes in size.BACKGROUND

[0003] Heart failure (HF) is a chronic, progressive condition and a major global health concern, affecting 60 million people worldwide (Savarese G. et al., Card Fail Rev. 2017; 3:7- 11; Benjamin E.J. et al., Circulation. 2018; 137:e67-e492). Prevalence in the United States alone is approximately 6.5 million patients with an annual incidence of approximately> 650,000 cases, which increases with age (Yancy C.W. et al., Circulation. 2017; 136:el37- el 61). Furthermore, prevalence in the US is predicted to increase 35% by 2060. HF is a growing health and economic burden for the US, predominantly because of the aging population (Heidenreich P.A. et al., Circulation. 2022; 145:e895-el032; Virani S.S. et al., Circulation. 2021; 143:e254-e743). There thus exists a need for additional therapies to treat heart failure.SUMMARY

[0004] The disclosure provides methods of treating a subject with a cardiovascular disease, comprising administering to the subject a therapeutically effective dose of a recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb), whereby blood serum concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP) is increased at least 2days after administration of the recombinant fusion protein when compared to a baseline blood serum concentration of NT-proBNP in the subject prior to administration of the recombinant fusion protein.

[0005] In some embodiments of the methods of the disclosure, the concentration of NT- proBNP is increased about 24 hours after administration of the recombinant fusion protein. In some embodiments of the methods of the disclosure, the concentration of NT-proBNP is increased at least 2, 7, or 15 days after administration of the recombinant fusion protein. In some embodiments, about 30 days after administration of the recombinant fusion protein, a blood serum concentration of NT-proBNP is similar or substantially similar to the blood serum concentration of NT-proBNP in the subject prior to administration of the recombinant fusion protein. In some embodiments, the concentration of NT-proBNP is increased by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900% or at least 1,000%. In some embodiments, (a) the concentration of NT-proBNP is increased by at least 30%, at least 50%, at least 150%, at least 300%, at least 500%, at least 600%, at least 800%, or at least 1400% about 2 days after administration of the recombinant fusion protein; (b) the concentration of NT-proBNP is increased by at least 30%, at least 50%, at least 100%, m at least 200%, at least 500%, at least 1000%, or at least 1500% about 7 days after administration of the recombinant fusion protein; or (c) the concentration of the NT-proBNP is increased by at least 30%, at least 50%, at least 70%, at least 100%, or at least 200% about 15 days after administration of the recombinant fusion protein.

[0006] In some embodiments of the methods of the disclosure, the cardiovascular disease comprises heart failure with reduced ejection fraction (HFrEF) or heart failure with preserved ejection fraction (HFpEF).

[0007] In some embodiments of the methods of the disclosure, the therapeutically effective dose comprises between 0.03 mg / kg and 0.27 mg / kg. In some embodiments, the therapeutically effective dose comprises 0.045 mg / kg or 0.09 mg / kg of the recombinant fusion protein.

[0008] In some embodiments of the methods of the disclosure, administration of a second or further therapeutically effective dose of the recombinant fusion protein results in a blood serum concentration of NT-proBNP that is increased about 24 hours after administration of the second or further therapeutically effective dose of the recombinant fusion protein. In some embodiments, a relative increase in a blood serum concentration of NT-proBNP after administration of a second or further therapeutically effective dose is similar, or substantiallysimilar, to a relative increase in a blood serum concentration of NT -proBNP after administration of the first therapeutically effective dose. In some embodiments, (a) the relative increase in blood serum concentration of NT -proBNP after the first therapeutically effective dose is relative to a baseline concentration of NT-proBNP in the subject prior to administration of the first therapeutically effective dose, and / or (b) the relative increase in blood serum concentration of NT-proBNP after the second or further therapeutically effective dose is relative to a baseline concentration of NT-proBNP in the subject prior to administration of the second or further therapeutically effective dose.

[0009] In some embodiments, the baseline concentration of NT-proBNP in the subject prior to administration of the second or further therapeutically effective dose is determined at least 14 days after administration of any previous therapeutically effective dose of the recombinant fusion protein to the subject. In some embodiments, the baseline concentration of NT- proBNP in the subject prior to administration of the first therapeutically effective dose and / or the baseline concentration of NT-proBNP in the subject prior to administration of the second or further therapeutically effective dose is determined within 24 hours prior to administration of the first therapeutically effective dose and / or second or further therapeutically effective dose.

[0010] In some embodiments, the relative increase in blood serum concentration of NT- proBNP after administration of the first dose and second or further therapeutically effective dose is at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900% or at least about 1,000%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and second or further therapeutically effective dose is between about 50% and about 1000%, between about 100% and about 800%, between about 100% and about 500%, between about 50% and about 300%, between about 100% and about 300%, between about 100% and about 200%, between about 200% and about 800%, between about 300% and about 800%, or between about 500% and about 900%

[0011] The disclosure provides methods of treating a subject with a cardiovascular disease, comprising administering to the subject a therapeutically effective dose of a recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb), whereby administration of therecombinant fusion protein results in an area under the time-concentration curve (in hours*ng / mL) from 0 to infinity (AUCo-inf) of between about 1,500 and about 200,000.

[0012] In some embodiments of the methods of the disclosure, AUCo-inf is determined by plotting serum concentration of the recombinant fusion protein in the subject after administration over time. In some embodiments of the methods of the disclosure, administration of the recombinant fusion protein results in an AUCo-inf of between about 2,000 and about 180,000. In some embodiments, administration of the recombinant fusion protein results in an AUCo-inf of between about 8,000 and about 160,000.

[0013] In some embodiments of the methods of the disclosure, administration of the recombinant fusion protein results in a Cmax (in ng / mL) of between about 250 and about 6,500. In some embodiments, administration of the recombinant fusion protein results in a Cmax (in ng / mL) of between about 450 and about 2,500.

[0014] In some embodiments of the methods of the disclosure, administration of the recombinant fusion protein results in a serum concentration of between about 200 and about 2,000 pg / L of the recombinant fusion protein approximately 12 hours after administration.

[0015] In some embodiments of the methods of the disclosure, the recombinant fusion protein has a half-life (ti / 2) of between about 5 and about 25 hours. In some embodiments, the recombinant fusion protein has a half-life (ti / 2) of between about 8 and about 19 hours.

[0016] In some embodiments of the methods of the disclosure, the cardiovascular disease comprises heart failure with reduced ejection fraction (HFrEF) or preserved ejection fraction (HFpEF).

[0017] In some embodiments of the methods of the disclosure, the therapeutically effective dose comprises between 0.03 mg / kg and 0.27 mg / kg. In some embodiments, the therapeutically effective dose comprises 0.045 mg / kg or 0.09 mg / kg of the recombinant fusion protein.

[0018] The disclosure provides methods of treating a subject with heart failure with reduced ejection fraction (HFrEF), comprising administering to the subject between 0.03 mg / kg and 0.27 mg / kg of a recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb).

[0019] In some embodiments of the methods of the disclosure, the subject has New York Heart Association (NYHA) Class II or Class III heart failure. In some embodiments, the subject has a left ventricular ejection fraction (LVEF) of less than or equal to 40%. In some embodiments, the subject has an LVEF of greater than or equal to 30%, and less than or equal to 40%, and a level of N-terminal pro-brain natriuretic peptide (NT -proBNP) level that isgreater than or equal to 300 pg / mL. In some embodiments, the subject has a left ventricular ejection fraction (LVEF) of greater than 40% and less than or equal to 65%. In some embodiments, the subject has an NT -proBNP level that is greater than or equal to 600 pg / mL prior to administration of the recombinant fusion protein.

[0020] In some embodiments of the methods of the disclosure, the subject has a history of atrial fibrillation and / or atrial flutter.

[0021] In some embodiments of the methods of the disclosure, the subject does not have hemodynamically significant and / or severe valvular disease other than mitral regurgitation or aortic stenosis.

[0022] In some embodiments of the methods of the disclosure, the subject has a hemoglobin level of greater than or equal to 9.0 g / dL prior to administration of the recombinant fusion protein.

[0023] In some embodiments of the methods of the disclosure, the subject is administered 0.045 mg / kg of the recombinant fusion protein. In some embodiments, the subject is administered 0.09 mg / kg of the recombinant fusion protein.

[0024] In some embodiments of the methods of the disclosure, the subject is administered the recombinant fusion protein once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 4 months, once every 5 months, once every 6 months, once every 8 months, once every 10 months or once every year. In some embodiments, the subject is administered the recombinant fusion protein once every 4 weeks. In some embodiments, the subject is administered the recombinant fusion protein once every 12 weeks. In some embodiments, the subject is administered the recombinant fusion protein once every 6 months. In some embodiments, the subject is administered the recombinant fusion protein once a year.

[0025] In some embodiments of the methods of the disclosure, the methods comprise a dosing holiday and / or a maintenance dose. In some embodiments, the subject administered the recombinant fusion protein every 4 weeks for an initial period, followed by a maintenance dose administered less frequently than every 4 weeks. In some embodiments, the maintenance dose comprises administering less of the recombinant fusion protein than an initial dose. In some embodiments, the maintenance dose is administered every 8 to 16 weeks. In some embodiments, the maintenance dose is administered every 8, 10, 12 or 14 weeks. In some embodiments, the maintenance dose is administered every 3 months, every 4 months, every 5 months, every 6 months, every 8 months, every 10 months or every year. Insome embodiments, the maintenance dose is administered every 2-6 months. In some embodiments, the maintenance dose is administered every 3-8 months. In some embodiments, the maintenance dose is administered every year.

[0026] In some embodiments of the methods of the disclosure, the methods comprise a planned recovery period between the initial period and the maintenance dose. In some embodiments, the planned recovery period is between about 10-18 weeks. In some embodiments, the planned recovery period is between about 3-6 months. In some embodiments, the planned recovery period is between about 2-8 months. In some embodiments, the planned recovery period is between about 6 months to one year.

[0027] In some embodiments of the methods of the disclosure, the subject is administered the recombinant fusion protein for at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, or at least 36 months.

[0028] In some embodiments of the methods of the disclosure, the administration comprises intravenous infusion. In some embodiments, the infusion occurs over a period of between 60 to 75 minutes.

[0029] In some embodiments of the methods of the disclosure, the NRG-1 active fragment comprises the ERBB3 / 4 binding domain. In some embodiments, the NRG-1 active fragment binds to and induces signaling through ErbB4 (HER4). In some embodiments, the mAb inhibits NRG-1 signaling through ErbB3 (HER3). In some embodiments, the NRG-1 fragment is fused via its N-terminal amino acid to the C-terminus of the antibody heavy chain using a linker. In some embodiments, the linker comprises at least one copy of a Gly-Gly- Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser linker set forth in SEQ ID NO: 5. In some embodiments, the C-terminus of the antibody heavy chain comprises the Fc domain of the antibody. In some embodiments, the NRG-1 fragment comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the mAb comprises a heavy chain amino acid sequence of SEQ ID NO: 2. In some embodiments, the mAb comprises a substitution mutation in at least one of amino acids 234, 239 and 434 numbered relative to SEQ ID NO: 2. In some embodiments, the at least one substitution mutation comprises an L234F mutation, a S239A mutation, a N434A mutation, or a combination thereof. In some embodiments, the mAb comprises a light chain amino acid sequence of SEQ ID NO: 3. In some embodiments, the recombinant fusion protein comprises the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 14. In some embodiments, the recombinant fusion protein promotes HER2 / 4 signaling over HER2 / 3 signaling relative to the signal induction potential of recombinant NRG-1.

[0030] In some embodiments of the methods of the disclosure, administration of the recombinant fusion protein results in an improvement in one or more parameters selected from the group consisting of LVEF, left ventricular end-diastolic volume index (LVEDVi), left ventricular end-systolic volume index (LVESVi), stroke volume index cardiac output (SVi CO), cardiac index (CI), left ventricular mass index (LVMi), diastolic function (E / e’), right ventricular systolic pressure (RVSP), left ventricular assist (LAV), left atrial ejection fraction (LAEF), and valvular function relative to a baseline measurement from before administration of the recombinant fusion protein.

[0031] In some embodiments of the methods of the disclosure, administration of the recombinant fusion protein leads to a reduced concentration of NT -proBNP at least 180 days after administration compared to a baseline concentration of NT -proBNP. In some embodiments, administration of the recombinant fusion protein reduces risk of death from cardiovascular failure.

[0032] The disclosure provides a method of treating cardiovascular disease in a subject, the method comprising administering a therapeutically effective dose of a recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb) to the subject, wherein the subject is administered the recombinant fusion protein every 4 weeks for an initial period, followed by a planned recovery period and then a maintenance dose administered less frequently than every 4 weeks.

[0033] In some embodiments of the methods of the disclosure, the maintenance dose is administered every 8 to 16 weeks, optionally wherein the maintenance dose is administered every 8, 10, 12 or 14 weeks. In some embodiments, the planned recovery period is between about 10-18 weeks. In some embodiments, the therapeutically effective dose comprises between 0.03 mg / kg and 0.27 mg / kg. In some embodiments, the therapeutically effective dose comprises 0.045 mg / kg or 0.09 mg / kg of the recombinant fusion protein.

[0034] The disclosure provides a recombinant fusion protein for use in the treatment of a cardiovascular disease in a subject, the recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb), wherein the treatment comprises administering to the subject a therapeutically effective dose of a recombinant fusion protein, and whereby blood serum concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP) is increased at least 2, 7, or 15 days after administration of the recombinant fusion protein when compared to ablood serum concentration of NT-proBNP in the subject prior to administration of the recombinant fusion protein.

[0035] The disclosure provides a recombinant fusion protein for use in the treatment of a cardiovascular disease in a subject, the recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb), wherein the treatment comprises administering to the subject a therapeutically effective dose of a recombinant fusion protein, and whereby blood serum concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP) is increased about 24 hours after administration of the recombinant fusion protein when compared to a blood serum concentration of NT-proBNP in the subject prior to administration of the recombinant fusion protein.

[0036] The disclosure provides a recombinant fusion protein for use in the treatment of cardiovascular disease in a subject, the recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb), wherein the treatment comprises administering a therapeutically effective dose of the recombinant fusion protein to the subject, and whereby administration of the recombinant fusion protein results in an area under the time-concentration curve (in hours*ng / mL) from 0 to infinity (AUCo-inf) of between 1,500 and 200,000.

[0037] The disclosure provides a recombinant fusion protein for use in the treatment of heart failure with reduced ejection fraction (HFrEF) in a subject, the use comprising administering to the subject between 0.03 mg / kg and 0.27 mg / kg of the recombinant fusion protein, wherein the recombinant fusion protein comprises an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb).

[0038] The disclosure provides a recombinant fusion protein for use in the treatment of a cardiovascular disease in a subject, the recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb), wherein the treatment comprises administering to the subject a therapeutically effective dose of a recombinant fusion protein, and wherein the subject is administered the recombinant fusion protein every 4 weeks for an initial period, followed by a planned recovery period and then a maintenance dose administered less frequently than every 4 weeks.

[0039] The disclosure provides a recombinant fusion protein for use in the manufacture of a medicament for the treatment of a cardiovascular disease in a subject, the recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecificErbB3 (HER3) monoclonal antibody (mAb), wherein the treatment comprises administering to the subject a therapeutically effective dose of a recombinant fusion protein, and whereby blood serum concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP) is increased at least 2, 7, or 15 days administration of the recombinant fusion protein when compared to a blood serum concentration of NT-proBNP in the subject prior to administration of the recombinant fusion protein.

[0040] The disclosure provides a recombinant fusion protein for use in the manufacture of a medicament for the treatment of a cardiovascular disease in a subject, the recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb), wherein the treatment comprises administering to the subject a therapeutically effective dose of a recombinant fusion protein, and whereby blood serum concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP) is increased about 24 hours after administration of the recombinant fusion protein when compared to a blood serum concentration of NT-proBNP in the subject prior to administration of the recombinant fusion protein.

[0041] The disclosure provides a recombinant fusion protein for use in the manufacture of a medicament for the treatment of cardiovascular disease in a subject, the recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb), wherein the treatment comprises administering a therapeutically effective dose of the recombinant fusion protein to the subject, and whereby administration of the recombinant fusion protein results in an area under the timeconcentration curve (in hours*ng / mL) from 0 to infinity (AUCo-inf) of between 1,500 and 200,000.

[0042] The disclosure provides a recombinant fusion protein for use in the manufacture of a medicament for the treatment of heart failure with reduced ejection fraction (HFrEF) in a subject, the use comprising administering to the subject between 0.03 mg / kg and 0.27 mg / kg of the recombinant fusion protein, wherein the recombinant fusion protein comprises an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb).

[0043] The disclosure provides a recombinant fusion protein for use in the manufacture of a medicament for the treatment of a cardiovascular disease in a subject, the recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb), wherein the treatment comprises administering to the subject a therapeutically effective dose of a recombinant fusion protein, and whereinthe subject is administered the recombinant fusion protein every 4 weeks for an initial period, followed by a planned recovery period and then a maintenance dose administered less frequently than every 4 weeks.BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1A shows a schematic of the Phase 2 trial described in Example 1. Approx.: approximately; q4w: every 4 weeks.

[0045] FIG. IB shows a schematic of an exemplary alternative Phase 2 trial, as described in Example 1. In this trial design, subjects are administered JK07 at 4 doses of 0.045 or 0.09 mg / kg at 4 week intervals, followed by 2 maintenance doses at .045 mg. / kg approximately 12 weeks apart, the first of which is administered approximately 14 weeks after the last of the initial 4 doses at 0.045 or 0.09 mg / kg.

[0046] FIG. 2 is a table showing a Schedule of Activities for the Phase 2 clinical trial described in Example 1. 2D-TTE: 2-dimensional transthoracic echocardiography; 6MWT : 6- minute walk test; ACM: all-cause mortality; AE: adverse event; AESI: adverse event of special interest; BP: blood pressure; C: clinical visit; CMR: cardiac magnetic resonance imaging; ECG: electrocardiogram; EOI: end of infusion; EOS: end of study; ET: early termination; HF: heart failure; hs-cTnT: high-sensitivity cardiac troponin T; IP: investigational product; IRT: Interactive Response Technology; IV: intravenous; KCCQ-12: Kansas City Cardiomyopathy Questionnaire 12; LVEF: left ventricular ejection fraction; NT -proBNP: N-terminal pro-brain natriuretic peptide; PK: pharmacokinetics; TSS: total symptom score; V: virtual visit; WoCBP: women of childbearing potential.

[0047] FIG. 3 is a diagram of an exemplary neuregulin 1 (NRG-1) erb-b2 receptor tyrosine kinase 3 (ErbB3, also called HER3) antibody fusion protein of the disclosure.

[0048] FIG. 4 is a diagram showing selective agonism of erb-b2 receptor tyrosine kinase 4 (ErbB4, also called HER4) via the NRG-1 HER3 antibody fusion proteins of the disclosure.

[0049] FIG. 5 A is a table showing descriptive statistics for serum NRG-1 HER3 antibody fusion protein (JK07) concentrations (in pg / L) by scheduled time for a human cohort administered 0.03 mg / kg JK07. For FIGS. 5A-5C abbreviations are BLQ: below the lower limit of quantitation, CV: coefficient of variance, Geom.: geometric, n: number of subjects with non-missing values, SD: standard deviation, SOI: start of infusion, EOI: end of the infusion, NA: not applicable, ND: not determined, %CV: 100 x (SD / Mean), where SD and Mean are the standard deviation and arithmetic mean of untransformed data, respectively,Geom. %CV: 100 x Sqrt(eSD**2 -1), where SD is the standard deviation of natural log- transformed data, concentration that are below the limit of quantification is treated as zero, Program: t-pc.sas. (a) indicates that at 24 hours (day 2) and 48 hours (day 3), samples were collected no more than 10 minutes following completion of ECG assessments.

[0050] FIG. 5B is a table showing descriptive statistics for serum JK07 concentrations (in pg / L) by scheduled time for a human cohort administered 0.09 mg / kg JK07.

[0051] FIG. 5C is a table showing descriptive statistics for serum JK07 concentrations (in pg / L) by scheduled time for a human cohort administered 0.27 mg / kg JK07.

[0052] FIG. 6 is table showing descriptive statistics for serum JK07 pharmacokinetic parameters for cohorts administered 0.03 mg / kg, 0.09 mg / kg or 0.27 mg / kg JK07. Abbreviations are CV: coefficient of variance, GeoMean: geometric mean, n: number of subjects with non-missing values, SD: standard deviation, %CV: 100 x (SD / Mean), where SD and Mean are the standard deviation and arithmetic mean of untransformed data, respectively, Geom.%CV: 100 x Sqrt(eSD**2 -1), where SD is the standard deviation of natural log-transformed data, Program: t-pc.sas.

[0053] FIG. 7 is a table showing descriptive statistics for dose normalized exposure parameters for cohorts administered 0.03 mg / kg, 0.09 mg / kg or 0.27 mg / kg JK07. Abbreviations are %CV: 100 x (SD / Mean), where SD and Mean are the standard deviation and arithmetic mean of untransformed data, respectively, Geom.%CV = 100 x Sqrt(eSD**2 - 1), where SD is the standard deviation of natural log-transformed data, (a) indicates that area under the serum concentration-time curve from time zero to the time of the last quantifiable concentration (AUC(O-last)) will be presented if AUC(O-inf) is not calculable in most subjects, Program: t-pkc.sas.

[0054] FIG. 8 is a table showing a summary of dose proportionality assessment for JK07 systemic exposure. 1: estimated slope, CI: confidence interval of the slope, the critical interval is the pre-specified acceptable 90% CI of the slope, (a) area under the serum concentration-time curve from time zero to the time of the last quantifiable concentration (AUC(O-last)) will be evaluated if AUC(O-inf) is not calculable in most subjects, (b) based on a mixed-effects model using the log-transformed Cmax or AUC as response, log-transformed dose as a fixed effect, and subject as random effect. Program: t-sdpk.sas.

[0055] FIG. 9A is a pair of plots showing JK07 serum concentration in pg / L (y-axis) as a function of time (hours, x-axis) in individuals administered 0.03 mg / kg JK07. The left plot shows linear scale, while the right plot shows the same data using a semi -logarithmic scale. 101-002, 103-003, 103-001 and 104-001 refer to individual subjects.

[0056] FIG. 9B is a pair of plots showing JK07 serum concentration in pg / L (y-axis) as a function of time (hours, x-axis) in individuals administered 0.09 mg / kg JK07. The left plot shows linear scale, while the right plot shows the same data using a semi -logarithmic scale. 103-006, 103-008, 104-002 and 107-001 refer to individual subjects.

[0057] FIG. 9C is a pair of plots showing JK07 serum concentration in pg / L (y-axis) as a function of time (hours, x-axis) in individuals administered 0.27 mg / kg JK07. The left plot shows linear scale, while the right plot shows the same data using a semi -logarithmic scale. 101-004, 103-010 and 107-006 refer to individual subjects.

[0058] FIG. 10 is a pair of plots showing mean JK07 serum concentration in pg / L (y-axis) as a function of time (hours, x-axis) in cohorts administered 0.03 mg / kg, 0.09 mg / kg or 0.27 mg / kg JK07. The left plot shows linear scale, while the right plot shows the same data using a semi -logarithmic scale.

[0059] FIG. 11 is a pair of plots that show individual and geometric mean Cmax (y-axis) versus dose (x axis) in the left plot, and individual and geometric mean Cmax / Dose (y-axis) versus dose (x-axis) in the right plot.

[0060] FIG. 12 is a pair of plots that show individual and geometric mean AUCo-inf (y-axis) versus dose (x axis) in the left plot, and individual and geometric mean AUCo-inf / Dose (y- axis) versus dose (x-axis) in the right plot.

[0061] FIG. 13 is a pair of plots showing dose proportionality for JK07 over the administered dose range (0.03 - 0.27 mg / kg).

[0062] FIG. 14A is a table showing baseline levels of N-Terminal ProB-type Natriuretic Peptide (pmol / L) prior to administration of JK07 or placebo. Abbreviations for FIGS 14A- 141 are N: number of subjects in respective treatment in biomarkers evaluation, n: number of subjects with non-missing value at the specific visit, SD: standard deviation. The baseline value is defined as the last value observed prior to first administration of study drug, the change from baseline is defined as the post-baseline value minus the baseline value. Change from baseline compared with placebo (%) refers to the mean differences of the change from baseline between active treatment and placebo. Program: t-pd-bio.sas.

[0063] FIG. 14B is a table showing levels of N-Terminal ProB-type Natriuretic Peptide (pmol / L) 2 days after administration of JK07 or placebo.

[0064] FIG. 14C is a table showing levels of N-Terminal ProB-type Natriuretic Peptide (pmol / L) 7 days after administration of JK07 or placebo.

[0065] FIG. 14D is a table showing levels of N-Terminal ProB-type Natriuretic Peptide (pmol / L) 15 days after administration of JK07 or placebo.

[0066] FIG. 14E is a table showing levels of N-Terminal ProB-type Natriuretic Peptide (pmol / L) 30 days after administration of JK07 or placebo.

[0067] FIG. 14F is a table showing levels of N-Terminal ProB-type Natriuretic Peptide (pmol / L) 60 days after administration of JK07 or placebo.

[0068] FIG. 14G is a table showing levels of N-Terminal ProB-type Natriuretic Peptide (pmol / L) 90 days after administration of JK07 or placebo.

[0069] FIG. 14H is a table showing levels of N-Terminal ProB-type Natriuretic Peptide (pmol / L) 135 days after administration of JK07 or placebo.

[0070] FIG. 141 is a table showing levels of N-Terminal ProB-type Natriuretic Peptide (pmol / L) 180 days after administration of JK07 or placebo.

[0071] FIG. 14J is a plot summarizing changes in N-Terminal Pro-B-type Natriuretic Peptide (pmol / L) between 0 and 30 days after administration of JK07 or placebo. Cohort 1 : 0.03 mg / kg; cohort 2: 0.09 mg / kg; cohort 3: 0.27 mg / kg.

[0072] FIG. 14K is a series of plots showing percent increase or decrease in ProB-type Natriuretic Peptide relative to baseline between 0 and 180 days after administration of placebo or JK07, in placebo (upper left), cohort 1 administered 0.03 mg / kg JK07 (upper right), cohort 2 administered 0.09 mg / kg JK07 (lower left), and cohort 3 administered 0.27 mg / kg JK07 (lower right).

[0073] FIG. 15A is a table showing baseline left ventricular ejection fraction as measured by 2D-TTE, prior to administration of JK07 or placebo. Abbreviations for FIGS. 15A-15J are N: number of subjects in respective treatment in 2D-TTE evaluation, n: number of subjects with non-missing value at the specific visit, SD: standard deviation. The baseline value is defined as the last value observed prior to first administration of study drug. The change from baseline is defined as the post-baseline value minus the baseline value. Change from Baseline Compared with Placebo (%) refers to the mean differences of the change from baseline between active treatment and placebo. Program: t-pd-tte.sas.

[0074] FIG. 15B is a table showing baseline left ventricular ejection fraction as measured by 2D-TTE, 6 hours after administration of JK07 or placebo.

[0075] FIG. 15C is a table showing baseline left ventricular ejection fraction as measured by 2D-TTE, 30 hours after administration of JK07 or placebo.

[0076] FIG. 15D is a table showing baseline left ventricular ejection fraction as measured by 2D-TTE, 7 days after administration of JK07 or placebo.

[0077] FIG. 15E is a table showing baseline left ventricular ejection fraction as measured by 2D-TTE, 15 days after administration of JK07 or placebo.

[0078] FIG. 15F is a table showing baseline left ventricular ejection fraction as measured by 2D-TTE, 30 days after administration of JK07 or placebo.

[0079] FIG. 15G is a table showing baseline left ventricular ejection fraction as measured by 2D-TTE, 60 days after administration of JK07 or placebo.

[0080] FIG. 15H is a table showing baseline left ventricular ejection fraction as measured by 2D-TTE, 90 days after administration of JK07 or placebo.

[0081] FIG. 151 is a table showing baseline left ventricular ejection fraction as measured by 2D-TTE, 135 days after administration of JK07 or placebo.

[0082] FIG. 15J is a table showing baseline left ventricular ejection fraction as measured by 2D-TTE, 180 days after administration of JK07 or placebo.

[0083] FIG. 16 is a table showing Cohort 1 (NYHA Class II-III with LVEF < 40%) objectives and endpoints. X2: elimination rate constant; 2D-TTE: 2-dimensional transthoracic echocardiography; 6MWT: 6-minute walk test; AUC(o-inf): area under the concentration-time curve from time 0 to infinity; AUC(o-iast): area under the concentration-time curve from time 0 to last quantifiable concentration; CI: cardiac index; CL: systemic clearance; CmaX: maximum concentration; CO: cardiac output; CV: cardiovascular; E / e’: parameter to measure early diastolic peak velocity and early diastolic mitral annular velocity; EDV: end diastolic volume; ESV: end systolic volume; HF: heart failure; hs-cTnT: high-sensitivity cardiac troponin T; IV: intravenous; KCCQ-12: Kansas City Cardiomyopathy Questionnaire 12; LV: left ventricular; LVEDVi: left ventricular end-diastolic volume index; LVEF: left ventricular ejection fraction; LVESVi: left ventricular end-systolic volume index; LVMi: left ventricular mass index; MACCE: major adverse cardiac and cerebrovascular event; NT-proBNP: N- terminal pro-brain natriuretic peptide; RVSP: right ventricular systolic pressure; SV: stroke volume; sVI CO: stroke volume index cardiac output; ti / 2: half-life; TEAE: treatment- emergent adverse event; tmaX: time to Cmax; TSS: total symptom score; Vz: volume of distribution.

[0084] FIG. 17 is a table showing Cohort 2 (NYHA Class II-III with LVEF > 40% and < 65%) Objectives and Endpoints. Abbreviations are the same as FIG. 16.

[0085] FIG. 18 is a table summarizing statistical toxicokinetic parameters from Sprague Dawley rats administered JK07 in a two-week toxicity study.

[0086] FIG. 19 is a table comparing exposure between male and female Sprague Dawley rats administered JK07 in a two-week toxicity study.

[0087] FIG. 20 is a table showing the relationship of exposure ratio on dose levels after repeated intravenous administration in Sprague Dawley rats administered JK07 in a two- week toxicity study.

[0088] FIG. 21 is a table showing linear regression of exposure on dose levels using a power model.

[0089] FIG. 22 is a table showing the drug exposure comparison of the dosing on Days 11 and 1 in Sprague Dawley rats administered JK07 in a two-week toxicity study.

[0090] FIG. 23 is a pair of plots showing concentration-time curves in Sprague Dawley rat serum after repeated intravenous administration with JK07 on days 1 and 11, in a two-week toxicity study.

[0091] FIG. 24 is a plot showing concentration-time curves in Sprague Dawley rat serum after 4 repeated intravenous administrations (days 1, 4, 8 and 11) with JK07 in a two-week toxicity study.

[0092] FIG. 25 is a table showing anti-drug antibody results for Sprague Dawley rats administered JK07 in a two-week toxicity study.

[0093] FIG. 26 is a table summarizing the design of a 13-week toxicity study in Sprague Dawley rats.

[0094] FIG. 27 is a table showing toxicokinetic parameters of JK07 in male and female serum following IV bolus injection of JK07, in a 13-week toxicity study in Sprague Dawley rats.

[0095] FIGS. 28A-28E are tables summarizing serum concentrations of JK07 in male and female serum following IV bolus injection of JK07, in a 13-week toxicity study in Sprague Dawley rats.

[0096] FIGS. 29A-29D are a series of plots showing toxicokinetic parameters versus dose response curves of JK07 in rat serum following IV bolus injection of JK07. Data is from males and females combined, the key at right indicates day for all panels.

[0097] FIGS. 30A-30D are a series of plots showing dose normalized toxicokinetic parameters versus dose response curves of JK07 in rat serum following IV bolus injection of JK07. Data is from males and females combined, the key at right indicates day for all panels.

[0098] FIGS. 31A-31D are a series of plots showing toxicokinetic parameters versus dose response curves of JK07 in rat serum following IV bolus injection of JK07. Data from males and females is presented separately, the key at right indicates day and sex for all panels.

[0099] FIGS. 32A-32D series of plots showing dose normalized toxicokinetic parameters versus dose response curves of JK07 in rat serum following IV bolus injection of JK07. Datafrom males and females is presented separately, the key at right indicates day and sex for all panels.

[0100] FIG. 33 is a table summarizing study groups in a four-week toxicology in Cynomolgus monkeys.

[0101] FIGS. 34A-34B are plots showing mean concentration of JK07 in serum of male monkeys following IV infusion administration at 3, 10 or 30 mg / kg, on day 1 (FIG. 35A) or day 22 (FIG. 35B).

[0102] FIGS. 35A-35B are plots showing mean concentration of JK07 in serum of female monkeys following IV infusion administration at 3, 10 or 30 mg / kg, on day 1 (FIG. 36A) or day 22 (FIG. 36B).

[0103] FIGS. 36A-36B are plots showing Area Under the Serum Concentration-Time Curves (AUC[0-t]) for JK07 following IV infusion administration of JK07 at a 3, 10, or 30 mg / kg / dose administered to male and female monkeys.

[0104] FIGS. 37A-37B are plots showing dose-normalized area under the serum concentration-time Curves (AUC[0-t]) for JK07 following IV infusion administration of JK07 at 3, 10, or 30 mg / kg / dose administered to male and female monkeys.

[0105] FIG. 38 is a table summarizing study groups in a thirteen-week toxicology in Cynomolgus monkeys.

[0106] FIG. 39 is a table that summarizes (Mean± SD) toxicokinetic parameters of JK07 in male and female monkey serum following IV Infusion of JK07.

[0107] FIGS. 40A-40B are plots showing toxicokinetic parameters versus dose curves of JK07 in monkey serum following IV administration of JK07 (males and females combined).

[0108] FIGS. 41A-41B are plots showing dose normalized toxicokinetic parameters versus dose curves of JK07 in monkey serum following IV administration of JK07 (males and females combined).

[0109] FIGS. 42A-42C show mean (+ / -SD) concentration versus time curves for JK07 in monkey serum following IV administration of JK07 (males and females combined) for the indicated days. Serum concentration is indicated in ng / mL. Key at right applies to all panels.

[0110] FIGS. 43A-43C show mean (+ / -SD) concentration versus time curves for JK07 in male and female monkey serum following IV administration of JK07 for the indicated days. Serum concentration is indicated in ng / mL. Key at lower right applies to all panels.[OHl] FIG. 44 is a plot showing that repeat dosing with JK07 has a cumulative effect in Rhesus monkeys.

[0112] FIG. 45 is a table summarizing microscopic findings at day 29 from a 4-week study in Cynomolgus monkeys with weekly administration of JK07.

[0113] FIG. 46 is a table summarizing microscopic findings at day 57 from a 4-week study in Cynomolgus monkeys with weekly administration of JK07 followed by a 4-week recovery period.

[0114] FIG. 47 is a table summarizing microscopic findings after a 13 week dosing period from a study in Cynomolgus monkeys with biweekly administration of JK07.

[0115] FIG. 48 is a table summarizing microscopic findings after a 13 week recovery period, following a 13 week dosing period, from a study in Cynomolgus monkeys with biweekly administration of JK07.DETAILED DESCRIPTIONIntroduction

[0116] Neuregulin-1 (NRG-1) has an indispensable role in cardiac development and homeostasis, as supported by in vivo and clinical evidence. For example, deletion of any of the NRG-1, erythroblastic oncogene B (ErbB) 4, or ErbB2 genes in mice results in profound defects in development of the heart, and death during embryogenesis (Santoro F. and Sahara M., Ann. Transl. Med. 2015; 3:249; Gassmann M. et al., Nature 1995; 378:390-394; Lee K.F. et al., Nature 1995; 378:394-398; Meyer D. and Birchmeier C., Nature 1995; 378:386-390). Along with its receptor, ErbB4 (but not the receptor ErbB3), NRG-1 is also expressed in adult heart tissues, where the NRG-l / ErbB2-ErBB4 signaling pathway is active (Sundaresan S. et al., Endocrinology 1998; 139:4756-4764; Zhao Y.Y. et al., J Biol Chem. 1998; 273: 10261-10269; Rohrbach S. et al., Circulation. 1999; 100:407-12). In humans, levels of NRG-1 decrease in the heart after development, but it continues to have a critical role in the maintenance of adult heart structural and functional integrity through cardiomyocyte differentiation, promotion of cardiac function, and protection of cardiomyocytes from apoptosis (Odiete O. et al., Circulation Res. 2012; 111 : 1376-1385). NRG-1 also plays a prominent role in the pathogenesis of heart failure (HF) and plays a role in cardiac regeneration. NRG-l / ErbB4 signaling is activated in the early stages of HF, together with a prominent upregulation of NRG-1 expression in the left ventricular (LV) chamber (De Keulenaer G.W. et al., Circ Heart Fail. 2019; 12:e006288). However, in the later stages of pump failure, both NRG-1 expression and NRG-l / ErbB4 signaling are inhibited (Id.).

[0117] Initial clinical evidence for the potential role of NRG-l / ErbB4 axis-mediated cardiovascular (CV) function improvements comes from observations of adverse cardiaceffects in clinical trials of trastuzumab for human epidermal growth factor receptor 2 (HER2) positive breast cancer. In clinical trials of trastuzumab, the rate of adverse cardiac effects, including significant increase in the risk of both left ventricular ejection fraction (LVEF) decline and symptomatic HF, ranged from 8% to 30% (Onitilo A. A. Et al., Ther Adv Drug Saf. 2014; 5: 154-166). Without wishing to be bound by theory, it is thought that trastuzumab therapy-induced HF may be due to the inhibition of ErbB2ZErbB4 signaling in cardiac muscle. The inducement of HF following exposure to anti-ErbB2 provides strong clinical evidence for the role of ErbB4 activation in cardio-protection, and supports the development of ErbB4-agonist therapeutic products for the treatment of HF.

[0118] Several clinical studies of recombinant NRG- 1 (rNRG-l)-based therapeutic products in HF patients have shown positive cardiac effects. A recombinant peptide encompassing the epidermal growth factor (EGF)-like domain of NRG- 1 (Neucardin, Zensun, Shanghai, China) was studied in humans with stable chronic HF, where it improved hemodynamics acutely (Jabbour A. et al., Eur J Heart Fail. 2011; 13:83-92) and when given by prolonged infusion over many days, yielded sustained increases in LV systolic function (Gao R. et al., J Am Coll Cardiol. 2010; 55: 1907-1914). A larger, full-length rNRG-1 known as cimaglermin was also examined as a possible treatment for HF. A single intravenous (IV) dose of cimaglermin in patients with systolic LV dysfunction demonstrated a sustained increase in LVEF over 90 days at the higher doses (Lenihan D.J. et al., JACC Basic Transl Sci. 2016; 1 :576-586). However, in the case of both Neucardin and cimaglermin, the co-stimulation of the ErbB3 pathways have limited their clinical utility. Specifically, both Neucardin and cimaglermin are associated with significant gastrointestinal (GI) side effects. Concerns about possible oncogenic effects of ErbB3 stimulation have also limited NRG- 1 -related therapeutic product development. Cimaglermin also exhibited hepatotoxicity, including a case of Hy’s law, in a first-in-human (FIH) dose-escalation study in HF patients with reduced ejection fraction. Hepatotoxicity of cimaglermin was also observed in nonclinical studies and was anticipated at higher doses in clinical studies (Id.).

[0119] The ability of the fusion proteins described herein to selectively agonize ErbB4 via fusion of rNRG-1 with an ErbB3 antibody, allows for harnessing the therapeutic effects of ErbB4 activation while mitigating the potential toxicities associated with ErbB3 activation.

[0120] HF is a chronic, progressive condition and a major global health concern, affecting 60 million people worldwide. HF may present with reduced, mildly reduced or preserved left ventricular ejection fraction (LVEF). Ejection fraction (EF) is thus a continuous variable, as opposed to a dichotomous variable. Despite phenotype variance in cardiac morphology, HFsigns and symptoms present similarly across the ranges of LVEF seen in clinical practice. Treatment of patients with HF is targeted towards a reduction in mortality and HF hospitalizations, symptom relief, and adequate management of comorbidities such as hypertension and AF.

[0121] HF with reduced ejection fraction (HFrEF; LVEF < 40%) represents half of all HF cases, and is associated with considerable morbidity and mortality (Kittleson M.M. et al., J Am Coll Cardiol. 2023; 81 : 1835-1878). The latest 2022 American Heart Association / American College of Cardiology / Heart Failure Society of America (AHA / ACC / HFSA) Class I recommendations for initial treatments to address congestion in patients with chronic HFrEF include beta-blockers, renin-angiotensin system inhibitors (angiotensin-converting enzyme [ACE] inhibitor, angiotensin II receptor blocker [ARB], or angiotensin receptor neprilysin [ARN] inhibitor), mineralocorticoid receptor antagonists (MRAs), sodium-glucose cotransporter-2 (SGLT2) inhibitors, and diuretics as needed. According to the patient’s clinical symptoms and status, additional Class I recommendations include hydralazine / isosorbide dinitrate as pharmacological therapy in African-Americans, and implantable cardioverter-defibrillator and cardiac resynchronization therapy (CRT) with defibrillation as device therapy (Heidenreich P.A. et al., Circulation. 2022;l 45:e895-el032). Other HF therapies approved to reduce morbidity and / or mortality in patients with HFrEF include digoxin, ivabradine, and vericiguat.

[0122] Recent interventional clinical trials have grouped those with mildly reduced LVEF (between 40% and 49%) and those with preserved LVEF (at or above 50%) together. For those with LVEF > 40%, evidence-based therapeutic options are fewer and according to the 2022 AHA / ACC / HFSA Class I recommendations for initial treatments whose LVEF > 40% include blood pressure (BP) control via ACE inhibitors / ARBs or ARN inhibitors and diuretics as needed, followed by SGLT2 inhibitors and then MRAs. However, for patients with atrial fibrillation (AF) or atrial flutter and presence of HF with preserved EF (HFpEF), adequate implementation of rate or rhythm control strategies along with guideline-based anti coagulation is a Class I recommendation (January C.T. et al., Circulation. 2019;140:el25-el51).

[0123] Despite the increasing uptake of such guideline-directed therapies, the rate of CV death and HF hospitalization remains unacceptably high in this patient population. The median survival for patients with HF is similar across all ranges of LVEF but declines with advancing age. Even among patients aged 65 to 69 years, median survival is < 4.0 years. Based on the National Vital Statistics Report for the general US population, individuals aged65 to 69 years have an expected median survival of 18.7 years. The 5-year composite of mortality and HF readmission in HF is approximately 96% (Shah A. et al., P&T: A Peer- reviewed Journal for Formulary Management. 2017; 42:464-472.). The clinical course for HF patients is variable, but acute episodes of clinical decompensation requiring hospitalization and / or IV diuretic use are common, and decompensation is associated with a poor long-term prognosis (Buddeke J. et al., BMC Public Health. 2020; 20:36).

[0124] Epidemiological studies demonstrate a strong link between atrial fibrillation (AF) and heart failure with preserved ejection fraction (HFpEF). AF is one of the primary precedents and predictors of the development of HFpEF. Conversely, most patients with HFpEF are destined to develop new onset AF, if the arrhythmia has not previously been documented (Zakeri R. & Cowie M.R., Heart. 2018; 104:377-384.). The evolution and progression of AF and HFpEF can be so strikingly in tandem that the determination of which disorder came first may be a matter of diagnostic diligence, rather than a reflection of a causal sequence from one disorder to the other (Packer M. Circ Arrhythm Electrophysiol. 2019; 12:e007222).

[0125] HF management is complex, with a need to balance medication management, psychosocial needs, and physical activity. Unfortunately, most HF patients are not prescribed optimal goal-directed- medical therapy and a minority remain on optimized therapy for more than a year. Underdosing / slow up-titration, low target dose achievement, and early discontinuation of ACE inhibitors / ARB s / ARN inhibitors, beta-blockers, and MRAs are common during treatment. This is commonly caused by tolerability, daily pill burden, and hemodynamic changes associated with these medications (Savarese G. et al., JACC Heart Fail. 2023; 11 : 1-14.). The Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist Trial (TOPCAT) indicated that approximately 38% of HF patients were on 5 to 9 prescribed medications, 36% were on 10 to 14 medications, and 19% were on > 15 prescribed medications (Wu C.-K. et al., Eur J Heart Fail. 2020;22:445-454.). Even though patients on more medications had reduced all-cause mortality, there was a statistically significant increase in HF hospitalizations (hazard ratios of 2.12 and 3 respectively) as well as all-cause hospitalizations (hazard ratios of 1.51, 1.81, and 2.29, respectively).

[0126] Health-related quality of life (HRQoL) of patients with HF continues to be poor and has been associated with a worse prognosis. The correlation between HRQoL and overall mortality has been well established in numerous trials in several types of patients and from different geographic regions. HRQoL was a strong predictor of mortality, even after adjusting for factors related to physical symptoms and socioeconomic parameters known to affect HF prognosis. Thus, an agent that could subjectively and objectively improve HRQoL would bevery well received from a patient’s perspective. Moreover, there is robust evidence to suggest that enhancing HRQoL also reduces mortality (Johansson I. et al., Circulation. 2021; 143:2129-2142.).

[0127] Thus, a high unmet clinical need remains for a therapeutic agent that does not add to the pill burden, has fewer drug-drug interaction liabilities, result in little or no BP reduction, and is able to improve the HRQoL of patients with HF.

[0128] Peptide rNRG-1 has a number of disadvantages, including co-stimulation of the ErbB3 pathway as described above, and a short half-life in vivo, which may limit its utility as a therapeutic. In a Phase 2 study using healthy human volunteers, rhNRG-1 was rapidly cleared from the human body, and was undetectable in serum within 45 minutes after ceasing intravenous administration (Jabbour A. et al., Eur J Heart Fail. 2011; 13:83-92). Similarly, the half-life of full-length rNRG-1 (cimaglermin / GGF2) half-life was found to be 1.3 and 1.7 hours in rats (Parry et al., European J. Pharmacol. (2017) 796: 76-89). There thus exists a need for therapeutic compositions incorporating NRG-1 that can extend the half-life of the NRG-1 peptide in vivo.

[0129] The recombinant fusion proteins disclosed herein, in which NRG-1 is fused to an ErbB3 monoclonal antibody, have been unexpectedly found to have a half-life that ranges from about 8 to 19 hours when administered to a human subject (see FIG. 6). In addition, the recombinant fusion proteins are able to induce long-lasting changes in biomarker expression indicative of NRG-1 signal induction, such as changes in pro-brain natriuretic peptide (NT- proBNP) serum levels that can persist up to 30 days after administration (FIGS. 14A-14K), and can result in improvement clinical parameters such as LVEF that can last up to 180 days after administration (FIGS. 15A-15J). In addition, the sustained pharmacologic activity of the recombinant fusion proteins was found to result in potentially adverse effects, such as increased nerve cellularity, that were independent of the maximum concentration of the recombinant fusion protein in blood serum (see FIGS. 45-48), and which could be ameliorated by a recovery period after administration of the recombinant fusion protein. These advantages of the recombinant fusion proteins of the disclosure can lead to lower doses, and / or less frequent administration of the recombinant fusion proteins, without reducing efficacy. These lower doses and / or less frequent administration of the recombinant fusion proteins disclosed herein can increase safety, and reduce side effects and toxicity. In addition, the recombinant fusion proteins of the disclosure can be administered using dosing regimens that incorporate loading and maintenance doses, as well as planned recovery periods, without loss of efficacy as a therapeutic for treating cardiac diseases.Definitions

[0130] Unless defined otherwise, 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.

[0131] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also 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.

[0132] “Neuregulin or neuregulin analogs” are molecules that can activate ErbB2ZErbB4 or ErbB2 / ErbB3 heterodimer protein tyrosine kinases, such as all neuregulin isoforms, neuregulin EGF domain alone, neuregulin mutants, and any kind of neuregulin-like gene products that also activate the above receptors. The preferred “neuregulin” used in this invention is a polypeptide fragment of human neuregulin 1 P2 isoform containing the EGF- like domain and the receptor binding domain. In some embodiments, the neuregulin fragment is an active fragment. Neuregulin-1 (NRG-1) and isoforms thereof are also known in the art as neuregulin 1 (NRG1), glial growth factor (GGF), Heregulin (HGL), HRG, new differentiation factor (NDF), ARIA, GGF2, HRG1, HRGA, SMDF, MST131, MSTP131 and NRG1 intronic transcript 2 (NRG1-IT2).

[0133] The terms “ErbB3”, “ErbB3 (HER3)”, “HER3” refer to the same protein (or the same gene when in reference thereto) and are used interchangeably herein. In some embodiments, the recombinant fusion comprises a monoclonal antibody portion that is 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, pl80-ErbB3, p45-sErbB3 and p85-sErbB3.

[0134] In one embodiment, the terms “ErbB4”, “ErbB4 (HER4)”, “HER4” refer to the same protein (or the same gene when in reference thereto) and are used interchangeably herein. ErbB4 (erb-b2 receptor tyrosine kinase 4) is also known in the art as ALS19 and pl80erbB4.

[0135] In one embodiment, the terms “ErbB2”, “ErbB2 (HER2)”, “HER2” refer to the same protein (or the same gene when in reference thereto) 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.

[0136] The term “active,” as used herein, refers to a fragment having a biological activity or biological function. In some embodiments, the activity is equal to or approximates the activity of the wild-type protein.

[0137] The term “subject” as used herein includes, but is not limited to, a mammal, including, e.g., a human, non-human primate (e.g., monkey), mouse, pig, cow, goat, rabbit, rat, guinea pig, hamster, horse, monkey, sheep, or other non-human mammal, a non-mammal, including, e.g., a non-mammalian vertebrate, such as a bird (e.g., a chicken or duck) or a fish; and a non-mammalian invertebrate. In some embodiments, the methods and compositions of the invention are used to treat (both prophylactically and / or therapeutically) non-human animals. In some embodiments, the methods and compositions of the invention are used to treat (both prophylactically and / or therapeutically) humans. The term “subject” can also refer to patients, i.e. individuals awaiting or receiving medical care.

[0138] The term “pharmaceutical composition” herein means a composition suitable for pharmaceutical use in a subject, including an animal or human. A pharmaceutical composition generally comprises an effective amount of an active agent (e.g., the recombinant fusion proteins of the invention) and a pharmaceutically acceptable carrier, diluent or excipient (e.g., a buffer, adjuvant, or the like).

[0139] The term “effective amount” means a dosage or amount sufficient to produce a desired result. The desired result may comprise an objective or subjective improvement in the recipient of the dosage or amount (e.g., long-term survival, decrease in number and / or size of tumors, effective prevention of a disease state, etc.).

[0140] A “prophylactic treatment” is a treatment administered to a subject who does not display signs or symptoms of a disease, pathology, or medical disorder, or displays only early signs or symptoms of a disease, pathology, or disorder, such that treatment is administered for the purpose of diminishing, preventing, or decreasing the risk of developing the disease, pathology, or medical disorder. A prophylactic treatment functions as a preventative treatment against a disease or disorder. A “prophylactic activity” is an activity of an agent, such as the recombinant fusion proteins disclosed herein, that, when administered to a subject who does not display signs or symptoms of a pathology, disease or disorder (or who displays only early signs or symptoms of a pathology, disease, or disorder) diminishes, prevents, or decreases the risk of the subject developing the pathology, disease, or disorder. A “prophylactically useful” agent or compound (e.g., a recombinant fusion protein of the invention) refers to an agent that is useful in diminishing, preventing, treating, or decreasing development of a pathology, disease or disorder.

[0141] A “therapeutic treatment” is a treatment administered to a subject who displays symptoms or signs of pathology, disease, or disorder, in which treatment is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms ofpathology, disease, or disorder. A “therapeutic activity” is an activity of an agent, such a recombinant fusion protein of the invention, or a composition thereof, that eliminates or diminishes signs or symptoms of a pathology, disease or disorder, when administered to a subject suffering from such signs or symptoms. A “therapeutically useful” agent (e.g., a recombinant fusion protein of the invention) indicates that an agent is useful in diminishing, treating, or eliminating such signs or symptoms of the pathology, disease or disorder.

[0142] The term “treating cancer” as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing, either partially or completely, the growth of tumors, tumor metastases, or other cancer-causing or neoplastic cells in a subject. The term “treatment” as used herein, unless otherwise indicated, refers to the act of treating.

[0143] The term “treating cardiovascular disease” as used herein, unless otherwise indicated, means preventing, inhibiting, suppressing, delaying, reversing, or alleviating, either partially or completely, the onset of a cardiovascular disease or condition in a subject, or the progression of a pre-existing cardiovascular disease or condition, or a symptom thereof, in a subject. Non-limiting examples of cardiovascular diseases that can be treated by the methods of the disclosure include chronic heart failure / congestive heart failure (CHF), acute heart failure / myocardial infarction (MI), left ventricular systolic dysfunction, reperfusion injury associated with MI, chemotherapy -induced cardiotoxicity (adult or pediatric), radiation- induced cardiotoxicity, adjunct to surgical intervention in pediatric congenital heart disease, atrial fibrosis, heart failure with preserved ejection fraction (HFpEF), and heart failure with reduced ejection fraction (HFrEF). Non-limiting examples of symptoms of cardiovascular disease include shortness of breath, cough, rapid weight gain, swelling in legs, ankles and abdomen, dizziness, fatigue, weakness, dizziness, chest pain, fainting (syncope), tachycardia, bradycardia, arrhythmia such as atrial fibrillation, and atrial flutter. Methods of determining the progression of cardiovascular disease and the effectiveness of treatment will be readily apparent to one of ordinary skill in the art. For example, the progression of various cardiovascular diseases can be determined by ejection fraction, electrocardiogram (ECG), Holter monitoring, echocardiogram, stress test, cardiac catheterization, cardiac computerized tomography (CT) scan, cardiac magnetic resonance imaging (MRI), and two-dimensional transthoracic echocardiography (2D-TTE).

[0144] By the term “heart failure” is meant an abnormality of cardiac function where the heart does not pump blood at the rate needed for the requirements of metabolizing tissues. Heart failure includes a wide range of disease states such as congestive heart failure,myocardial infarction, tachyarrhythmia, familial hypertrophic cardiomyopathy, ischaemic heart disease, idiopathic dilated cardiomyopathy, and myocarditis. The heart failure can be caused by any number of factors, including ischaemic, congenital, rheumatic, or idiopathic forms. Chronic cardiac hypertrophy is a significantly diseased state which is a precursor to congestive heart failure and cardiac arrest.

[0145] Heart failure can be classified according to the New York Heart Association (NYHA) Classification system, a classification system that is known in the art. The NYHA Classification divides heart failure into four classes or stages. Subjects with Class I heart failure exhibit no symptoms and no limitation in ordinary physical activity such as shortness of breath when walking or climbing stairs. Subjects with class II heart failure exhibit mild symptoms (mild shortness of breath and / or angina) and slight limitation during ordinary activity. Class III heart failure is characterized by a marked limitation in activity due to symptoms, even during less-than-ordinary activity, e.g. walking short distances (20 — 100 m). subjects are comfortable only at rest. Class IV heart failure is characterized by severe limitations. Subjects experiences symptoms even while at rest, and are mostly bed bound.

[0146] As used herein, “fibrosis” refers the immoderate formation and deposition of extracellular matrix (ECM) components, for example collagen. ECM normally surrounds parenchymal cells, and supports their migration, differentiation, proliferation and normal function. Fibrotic ECM compromises tissue homeostasis, and can lead to organ dysfunction due to loss of architectural integrity and aberrant remodeling. Fibrosis is characterized by the proliferation of fibroblasts, which can differentiate into myofibroblasts which secrete ECM proteins. “Cardiac fibrosis” refers to fibrosis of the heart, generally, and includes atrial fibrosis, as well as fibrosis affecting other regions of the heart, such as, but not limited to, the ventricles, myocardium, pericardium, endocardium and valves.

[0147] In the heart, replacement or reparative fibrosis occurs after cardiac injury, and is associated with cardiomyocyte death and the replacement of necrotic myocardial areas with fibrotic scar tissue. In reactive fibrosis, there is increased deposition of collagen and other ECM proteins in the interstitial space that surrounds cardiac cells and vessels, causing this space to expand, without replacement of injured or dead cardiomyocytes.

[0148] As used herein, “atrial fibrosis” refers to fibrosis of the atrium. Atrial fibrosis is strongly associated with atrial fibrillation (AF), one of the most common arrhythmias in humans. Without wishing to be bound by theory, it is thought that atrial fibrosis causes abnormal electrical conduction through the atrium, leading to atrial fibrillation. Fibrosis can be detected by any suitable means known in the art, including, but not limited to DE-MRimaging (MRI), circulating biomarkers (e.g., Galactin-3, MMP-3, MMP-9, high-sensitivity cardiac troponin T, Osteopontin, suppression of tumorigenicity 2, connective tissue growth factor (CTGF), resistin (RETN), Periostin, and midregional pro-atrial natriuretic peptide, as well as microRNAs such as miRNA-15, miR-21, miR-29c, miR-328, miR-30a, miR-214, miR-503 and miR-133a) and electroanatomic voltage mapping.

[0149] As used herein, “arrhythmia” refers to an irregular heartbeat, and includes both tachycardia (abnormally fast heartbeats, including atrial flutter) and bradycardia (abnormally slow heartbeats). Atrial fibrillation (AF) is an irregular and often very rapid heart rhythm that can lead to blood clots in the heart. In AF, the normal beating of the atrium is irregular, impeding blood flow from the atria to the ventricles. AF may be acute, or chronic. AF can be assessed in terms of the duration of AF episodes, and the number of episodes that occur in a given unit of time (e.g., AF / episodes per day, week or month). Paroxysmal AF begins suddenly and ends spontaneously within 7 days. In contrast, persistent AF occurs for longer than 7 days and ends spontaneously or with treatment. Long-standing persistent AF refers to uninterrupted AF for more than a year. Permanent AF refers to AF that persists despite treatment to restore normal sinus rhythm. Symptoms of AF include irregular heartbeat, heart palpitations, lightheadedness, extreme fatigue, shortness of breath and chest pain.

[0150] The terms “identical” or “percent identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence. To determine the percent identity, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions / total # of positions (e.g., overlapping positions)* 100). In some embodiments, the two sequences are the same length.

[0151] The term “substantially identical,” in the context of two nucleic acids or polypeptides, refers to 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 least98% identity, or at least 99% identity (e.g., as determined using one of the methods set forth infra).

[0152] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. 7 / 5490:5873-5877. 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 homologous to a nucleic acid encoding a protein of interest. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein of interest. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSLBlast can be used to perform an iterated search which detects distant relationships between molecules (id.). When utilizing BLAST, Gapped BLAST, and PSLBLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CAB IOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as 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. If ktup=2, similar regions in the two sequences being compared are found by looking at pairs of aligned residues; if ktup=l, single aligned amino acids are examined. Ktup can be set to 2 or 1 for protein sequences, or from 1 to 6 for DNA sequences. The default if ktup is not specified is 2 for proteins and 6 for DNA. Alternatively, protein sequence alignment may be carried out using the CLUSTAL W algorithm, as described by Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0153] As used herein, the term “binds,” “specifically binds to,” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and anantibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that specifically binds to a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and / or with greater duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, for example, by a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM.

[0154] In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species. In another embodiment, specific binding can include, but does not require exclusive binding.

[0155] As used in this specification, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references 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 of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0156] The term “polypeptide” refers to a polymer of amino acids and its equivalent and does not refer to a specific length of a product; thus, “peptides” and “proteins” are included within the definition of a polypeptide. Also included within the definition of polypeptides are “antibodies” as defined herein. A “polypeptide region” refers to a segment of a polypeptide, which segment may contain, for example, one or more domains or motifs (e.g., a polypeptide region of an antibody can contain, 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 or at least 50 contiguous amino acids of the polypeptide.

[0157] A “fusion protein,” “fusion polypeptide,” “recombinant fusion protein,” or “recombinant polypeptide” refers to a hybrid polypeptide which comprises polypeptide portions from at least two different polypeptides. A “fusion protein” as defined herein, is a fusion of a first amino acid sequence (protein) comprising, for example an NRG-fragment, joined via a linker to the C-terminus of a second amino acid sequence comprising a heavy chain of an antibody that binds specifically to ERBB3 (HER3).

[0158] Unless otherwise indicated by context, 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 is modified by covalent attachment of a second molecule such as, e.g., by attachment of a heterologous polypeptide, or by glycosylation, acetylation, phosphorylation, and the like. Further included within the definition of “derivative” are, for example, polypeptides containing one or more analogs of an amino acid (e.g., unnatural amino acids and the like), polypeptides with unsubstituted linkages, as well as other modifications known in the art, both naturally and non-naturally occurring.

[0159] An “isolated” polypeptide is one which has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. An isolated polypeptide includes an isolated antibody, or a fragment or derivative thereof.

[0160] The term “about” as used herein 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%.

[0161] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0162] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, z.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, z.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements 5 other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0163] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarilyincluding at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0164] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0165] All publications mentioned herein are incorporated herein by reference for the purpose of disclosing and describing material for which the reference was cited in connection with. The contents of WO 2019200033 and WO 2023 / 178086, are incorporated by reference in their entireties herein.NRG-1 HER3 Antibody Fusion Proteins

[0166] The disclosure provides recombinant fusion proteins comprising an NRG-1 fragment fused to a HER3 antibody, for use in treating adults with heart failure (HF). In some embodiments, the NRG-1 fragment comprises an active fragment of NRG-1. In some embodiments, the HER3 antibody is monospecific for HER3. In some embodiments, the HF comprises New York Heart Association (NYHA) Class II-IV HF, and the fusion proteins described herein reduce the risk of HF hospitalization and improve functional capacity and symptoms.

[0167] In some embodiments, the NRG-1 HER3 antibody fusion proteins described herein comprise 2 domains: (1) an active polypeptide fragment of the human growth factor NRG-1, and (2) a fully human monoclonal antibody that antagonizes human ErbB3. The NRG-1 fragment comprises a sequence corresponding to the epidermal growth factor (EGF)-likedomain of NRG- 1, and is fused to the C-terminus of the a-ErbB3 heavy chain via a linker in a homodimeric configuration (FIG. 3). In some embodiments, the NRG-1 HER3 antibody fusion proteins described herein comprise 2 of a peptide comprising an EGF-like domain of NRG-1 fused to the C-terminus of the HER2 antibody heavy chain, and 2 of a peptide comprising the HER3 antibody light chain. NRG-1 is a member of the EGF family known to be the ligand for ErbB3 and ErbB4 (Parodi E.M. et al., Cardiovascular Research. 2014; 102: 194-204). Binding of NRG-1 to either ErbB3 or ErbB4 enables heterodimerization of the bound receptor with ErbB2, and subsequent tyrosine protein kinase activation which transduces intracellular signaling cascades. The NRG-1 HER3 antibody fusion proteins of the disclosure are designed to selectively agonize ErbB4 and maximize NRG-1 therapeutic benefit across a variety of CV indications, including HF (FIG. 4).Antibody

[0168] The disclosure provides recombinant fusion proteins comprising an NRG-1 active fragment fused to a HER3 antibody, for use in the methods described herein.

[0169] As used herein, an “antibody” refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin (e.g., antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

[0170] Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab')2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab')2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab')2dimer into an Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, New York (1999), for a more detailed description of other antibody fragments). Whilevarious antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments, etc. may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the 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 a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide. Antibodies include single domain antibodies, which comprise an antibody fragment consisting of a single monomeric variable antibody domain that is able to bind selectively to an antigen domain. Exemplary single domain antibodies include VHH fragments, which were originally isolated from camelids.

[0171] The antibody domain of the recombinant fusion proteins described herein optionally comprises all or part of an immunoglobin molecule and optionally contains all or part of an immunoglobin variable region (z.e., the area of specificity for the disease related antigen) and optionally comprises region(s) encoded by a V gene, and / or a D gene and / or a J gene.

[0172] The antibodies used herein optionally comprise F(ab)2, F(ab')2, Fab, Fab', scFv, single domain antibodies, etc. depending upon the specific requirements of the embodiment. Some embodiments utilize fusion proteins comprising IgG domains. However, other embodiments comprise alternate immunoglobins such as IgM, IgA, IgD, and IgE. Furthermore, all possible isotypes of the various immunoglobins are also encompassed within the current embodiments. Thus, IgGl, IgG2, IgG3, etc. are all possible molecules in the antibody domains of the antibody-immunostimulant fusion proteins used in the invention. In addition to choice in selection of the type of immunoglobin and isotype, different embodiments of the invention comprise various hinge regions (or functional equivalents thereof). Such hinge regions provide flexibility between the different domains of the antibody-immunostimulant fusion proteins. See, e.g., Penichet, et al. 2001 “Antibodycytokine fusion proteins for the therapy of cancer” J Immunol Methods 248:91-101.

[0173] In some embodiments, the mAb comprised by the recombinant fusion proteins of the disclosure is monospecific for ErbB3 (HER3). Human HER3 (also called ErbB-3, ERBB3, c- erbB-3, c-erbB3, erb-b2 receptor tyrosine kinase 3 and receptor tyrosine-protein kinase erbB- 3) encodes a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases which also includes HER1 (also known as EGFR), HER2, and HER4 (Kraus, M.H. et al, PNAS 86 (1989) 9193-9197; Plowman, G.D. et al, PNAS 87 (1990) 4905-4909; Kraus, M.H. et al, PNAS 90 (1993) 2900-2904). Like the prototypical epidermal growthfactor 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. This membrane-bound HER3 protein has a Heregulin (HRG) binding domain within the extracellular domain but not an active kinase domain. It therefore can bind this ligand but not convey the signal into the cell through protein phosphorylation. However, it does form heterodimers with other HER family members which do have kinase activity.Heterodimerization leads to the activation of the receptor-mediated signaling pathway and transphosphorylation of its intracellular domain. Dimer formation between HER family members expands the signaling potential of HER3 and is a means not only for signal diversification but also signal amplification. For example the HER2 / HER3 heterodimer induces one of the most important mitogenic signals via the PI3K and AKT pathway among HER family members (Sliwkowski M.X., et al, J. Biol. Chem. 269 (1994) 14661-14665; Alimandi M, et al, Oncogene. 10 (1995) 1813- 1821; Hellyer, N.J., J. Biol. Chem. 276 (2001) 42153-4261; Singer, E., J. Biol.

[0174] In some embodiments, the human ERBB3 protein comprises the following amino acid sequence provided in GenBank AAH02706.1 and set forth in SEQ ID NO: 1 : MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYER CEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVY DGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWRDIVR DRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPN QCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTFQLEPNPHTKY QYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKAF (SEQ ID NO: 1). It is to be understood that the ERBB3 (HER3) sequence targeted by the antibody of the present methods and compositions may be an isomer, homolog, or variant of SEQ ID NO: 1.

[0175] In some embodiments, the mAb of the recombinant fusion protein provided herein is an anti-Her3 mAb that inhibits NRG-1 signaling through ErbB3 (HER3).

[0176] In particular embodiments, the mAb comprised by the recombinant fusion proteins of the disclosure comprises an anti-HER3 mAb. Such anti-HER3 antibodies, and their sequences, are known in the art and may include, but are not limited to the following: patritumab, seribantumab (fully human mAb), LJM716, KTN3379, AV-203, REGN1400, GSK2849330, or MM-141. Such antibodies may also be selected from any of the following forms, including, chimeric, bi-specific, non-human, fully human, or humanized form, so longas they bind to and inhibit signaling from human ERBB3 (HER3). In some embodiments, the anti-HER.3 antibody is of human origin.

[0177] The antibody according to the disclosure is preferably a human antibody, humanized antibody, chimeric antibody, or further genetically engineered antibody as long as the characteristic properties according to the invention are retained. “Antibody fragments” comprising a portion of a full-length antibody, preferably the variable domain thereof, or at least the antigen binding site thereof are also envisaged as within the scope of the disclosure. Examples of antibody fragments include diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are, e.g., described in Huston, J.S., Methods in Enzymol. 203 (1991) 46-88. In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain binding to the respective antigen being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the properties of an antibody according to the invention. The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of a single amino acid composition.

[0178] In some embodiments, a chimeric antibody may be used in the compositions and methods provided herein. The term “chimeric antibody” refers to a monoclonal antibody comprising a variable region, z.e., binding region, from mouse and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a mouse variable region and a human constant region are especially preferred. Such rat / human chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding rat immunoglobulin variable regions and DNA segments encoding human immunoglobulin constant regions. Other forms of “chimeric antibodies” encompassed by the present invention are those in which the class or subclass has been modified or changed from that of the original antibody. Such “chimeric” antibodies are also referred to as “class-switched antibodies.” Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. See, e.g., Morrison, S.L., et al, Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244.

[0179] In some embodiments, a humanized antibody may be used in the compositions and methods provided herein. The term “humanized antibody” or “humanized version of an antibody” refers to antibodies in which the framework or “complementarity determining regions” (CDR) have been modified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In other embodiments, the CDRs of the VH and VL are grafted into the framework region of human antibody to prepare the “humanized antibody.” See e.g. Riechmann, L., et al, Nature 332 (1988) 323-327; and Neuberger, M.S., 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 the sequences of naturally occurring human antibodies. Human heavy and light chain variable framework regions are listed e.g. in Lefranc, M.-P., Current Protocols in Immunology (2000) - Appendix IP A. IP.1-A. IP.37 and are accessible via IMGT, the international ImMunoGeneTics information system® (imgt.cines.fr) or via vbase.mrc-cpe.cam.ac.uk. Optionally the framework region can be modified by further mutations. Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies. The term “humanized antibody” as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to complement component Iq (Clq) binding and / or Fc Receptor (FcR) binding, e.g. by “class switching” i.e. change or mutation of Fc parts (e.g. from IgGl to IgG4 and / or IgGl / IgG4 mutation). The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M.A., and van de Winkel, J.G., 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 or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant 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, M.D., et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, H.R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., 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, A.L., p. 77 (1985); and Boerner, P., et al, J. Immunol. 147 (1991) 86-95). As already mentioned for humanized antibodies according to the invention the term “human antibody” as used herein also comprises such antibodieswhich are modified in the constant region to generate the properties according to the invention.

[0180] In particular embodiments, the mAh comprised by the recombinant fusion proteins provided herein comprises at least one mutation in the Fc domain or region.

[0181] The term “recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell, for example a NSO or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. 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 sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.

[0182] The terms "which binds to human HER3", "which specifically binds to human HER3", or "anti-HER3 antibody" are interchangeable and refer, in some embodiments, to an antibody which specifically binds to the human HER3 antigen with a KD-value of about 4.81x'10mol / L or lower at 25°C. The binding affinity is determined with a standard binding assay at 25°C, such as surface plasmon resonance technique (BIAcore®, GE-Healthcare Uppsala, Sweden). Thus an "antibody which binds to human HER3" as used herein refers to an antibody or portion thereof specifically which binds to the human HER3 antigen with a binding affinity within a range of KD 1.0 x 10'8mol / L — 1.0 x 10'13mol / L) at 25°C, and preferably with a KD-value of 4.8 lx'10mol / L or lower at 25°C.

[0183] In some embodiments, an anti-HER3 antibody comprised by the recombinant fusion protein disclosed herein comprises a variable region heavy (VH) chain and a variable region light (VL) chain. In some embodiments, the antibody comprises the VH and VL sequences in 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 the proliferation of tumor cells.

[0184] In some embodiments, the anti-HER3 mAb provided herein comprises a VH amino acid sequence set forth in SEQ ID NO: 2:Heavy Chain:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGS TNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPEFLGGPAVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPGK (SEQ ID NO: 2). In some embodiments the anti-HER3 mAb comprises a VH amino acid sequence of SEQ ID NO: 2, or a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments the anti-HER3 mAb comprises a VH amino acid sequence of SEQ ID NO: 2, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions or substitutions relative thereto.

[0185] In some embodiments, the anti-HER3 mAb provided herein comprises a VL amino acid sequence of SEQ ID NO: 3:Light Chain:DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3). In some embodiments the anti-HER3 mAb comprises a VL amino acid sequence of SEQ ID NO: 3, or a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the anti-HER3 mAb comprises a VL amino acid sequence of SEQ ID NO: 3, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions or substitutions relative thereto.

[0186] In some embodiments, the anti-HER3 antibody of the present invention comprises at least one mutation in the Fc region. In another embodiment, the mature anti-HER3 antibody (z.e.- lacking a signal peptide) of the present invention comprises at least one mutation in amino acids 234, 239, 434, numbered relative to SEQ ID NO: 2, or a combination thereof, where in other embodiments, the amino acid mutations comprise at least one of the following substitution mutations: L234F, S239A, N434A or a combination thereof. In some embodiments, mutations to amino acids 234 and / or 239 knock down effector functions of theanti-HER3 antibody. In some embodiments, a mutation to amino acid 434 extends the halflife of the antibody in a subject.

[0187] In some embodiments, the 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 for one or more Fc Receptors. The FcRs can be FcyRI, FcyRIIa, FcyRIIb, FcyRIIIa (158F), FcyRIIIa (158V) and Clq. In some embodiments, the reduced affinity comprises an increase in dissociation constant of about 1 order of magnitude or greater. In some embodiments, introducing one or more Fc mutations increases the KD of the anti-HER3 antibody of fusion protein comprising same for FcyRI from 2.81xl0'9M to 1.03X10'8M. In some embodiments, introducing one or more Fc mutations increases the KD of the anti-HER3 antibody of fusion protein comprising same for FcyRIIa from 3.95xl0'7M to 1.35X10'6M. In some embodiments, introducing one or more Fc mutations increases the KD of the anti-HER3 antibody of fusion protein comprising same for FcyRIIb from 1.03xl0'7M to 1.52X10'6M. In some embodiments, introducing one or more Fc mutations increases the KD of the anti-HER3 antibody of fusion protein comprising same for FcyRIIIa (158F) from 6.37xl0'8M to 1.18X10'7M. In some embodiments, introducing one or more Fc mutations increases the KD of the anti-HER3 antibody of fusion protein comprising same for FcyRIIIa (158V) from 3.41 xlO’8M to 9.10xl0’8M.

[0188] In some embodiments, the anti-HER3 antibody or recombinant fusion protein comprising same binds to FcyRI with an equilibrium dissociation constant (KD) higher than or equal to 1.03xl0'8M. In some embodiments, the anti-HER3 antibody or recombinant fusion protein comprising same comprises one or more Fc mutations and binds to FcyRIIa with a KD higher than or equal to 1.35xl0'6M. In some embodiments, the anti-HER3 antibody or recombinant fusion protein comprising same comprises one or more Fc mutations and binds to FcyRIIb with a KD higher than or equal to 1.5 xlO'6M. In some embodiments, the anti-HER3 antibody or recombinant fusion protein comprising same comprises one or more Fc mutations and binds to FcyRIIIa (158F) with a KD higher than or equal to 1.18xl0'7M. In some embodiments, the anti-HER3 antibody or recombinant fusion protein comprising same comprises one or more Fc mutations and binds to FcyRIIIa (158V) with a KD higher than or equal to 9.10x1 O'8M.

[0189] The term “antibody effector function(s)” as used herein refers to a function contributed by an Fc region(s) of an Ig. Such function can be affected by, for example, binding of an Fc effector region (s) to an Fc receptor on an immune cell with phagocytic orlytic activity or by binding of an Fc effector region(s) to components of the complement system.

[0190] In some embodiments, the anti-HER3 antibody does not induce antibody-dependent cellular cytotoxicity (ADCC). The term "antibody-dependent cellular cytotoxicity (ADCC)" refers to lysis of human target cells by an antibody according to the invention in the presence of effector cells.

[0191] In some embodiments, the antibody according to the invention is glycosylated. The glycosylation can be N-glycosylation, O-glycosylation or a combination thereof.

[0192] The antibodies and the recombinant fusion proteins comprising same of the disclosure may be produced via recombinant means. Such methods are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody polypeptide and usually purification to a pharmaceutically acceptable purity. For protein expression, nucleic acids encoding light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells, such as CHO cells, NSO cells, SP2 / 0 cells, HEK293 cells, COS cells, yeast, or E. coli cells, and the antibody is recovered from the cells (from the supernatant or after cells lysis). Recombinant production of antibodies and fusion proteins comprising antibodies is well-known in the state of the art and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al, Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., Mol. Biotechnol. 16 (2000) 151-161; Werner, R.G., Drug Res. 48 (1998) 870-880. The recombinant fusion protein comprising an antibody may be present in whole cells, in a cell lysate, or in a partially purified, or substantially pure form. Purification is performed in order to eliminate other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including, column chromatography and others well known in the art (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 by, e.g., Barnes, L.M., et al, Cytotechnology 32 (2000) 109-123; Barnes, L.M., et al, Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g., Durocher, Y., et al, Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described by 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 (HEK 293) is described by Schlaeger, E.-J. and Christensen, K., in Cytotechnology 30 (1999) 71-83, and by Schlaeger, E.-J., in J. Immunol.Methods 194 (1996) 191-199. Monoclonal antibodies, and recombinant fusion proteins comprising monoclonal antibodies, are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures. The hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA may be inserted into expression vectors, which are then transfected into host cells, such as HEK 293 cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of recombinant monoclonal antibodies in the host cells.

[0193] The heavy and light chain variable domains can be combined with sequences of promoter, translation initiation, constant region, 3' untranslated region, polyadenylation, and / or transcription termination to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells which are then fused to form a single host cell expressing both chains.Neuregulin

[0194] The disclosure provides recombinant fusion proteins comprising a fragment of an NRG-1 protein. NRG-1 proteins can bind to the ErbB4 receptor on the surface of myocardial cells, continuously activate the MARKZERK, PI3K / AKT, and JAK / STAT signal pathways in the cell, and change the structure of the myocardial cells, thereby improving the function of myocardial cells.

[0195] As used herein, “neuregulin” or “NRG” refers to proteins or peptides that can bind and activate ErbB3, ErbB4 or heterodimers or homodimers thereof, including neuregulin isoforms, neuregulin EGF-like domain, polypeptides comprising neuregulin EGF-like domain, neuregulin mutants or derivatives, and any kind of neuregulin-like gene products that can activate the above receptors Neuregulin also includes NRG-1, NRG-2, NRG-3 and NRG- 4 proteins, peptides, fragments and compounds that have the functions of neuregulin. In preferred embodiments, neuregulin is a protein or peptide that can bind to and activate ErbB2ZErbB4 or ErbB2ZErbB3 heterodimers, for example, but not for the purpose of restriction, peptides of the present invention includes a fragment of the NRG-1P2 isoform, i.e., the 177-237 amino acid fragment, which contains the EGF-like domain having the following amino acid sequence:SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ (SEQ ID NO: 4). The person of ordinary skill in the art will appreciate that sequences similar to SEQ ID NO: 4, e.g. sequences with 1, 2, 3, 4, or 5 substitutions, deletions or insertions relative thereto, that retain the ability of SEQ ID NO: 4 to bind to and induce signaling through HER4, are also within the scope of the instant disclosure. The NRG proteins of the present disclosure can activate the receptors above and regulate their biological functions, for example, stimulate the synthesis of acetylcholine receptors in skeletal muscle cells, promote the differentiation and survival of cardiomyocytes and DNA synthesis. It is well known to those of skill in this art that a mutation of a single amino acid in a non-critical region generally would 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 Benjamin / Cummings Pub. co., p. 224). The NRG proteins of the disclosure can be isolated from natural sources, may be modified through recombination technology, artificial synthesis or other means.

[0196] 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 and including heterodimers with ErbB2, and structurally similar to the EGF receptor binding region as described in WO 00 / 64400, Holmes et al., Science, 256: 1205-1210 (1992); U.S. Pat. Nos. 5,530,109 and 5,716,930; Hijazi et al., Int. J. Oncol., 13: 1061-1067 (1998); Chang et al., Nature, 387:509-512 (1997); Carraway et al., Nature, 387:512-516 (1997); Higashiyama et al., J. Biochem., 122:675-680 (1997); and WO 97 / 09425, the contents of which are all incorporated herein by reference. In some embodiments, EGF-like domain binds to and activates ErbB2ZErbB4 or ErbB2ZErbB3 heterodimers. In some embodiments, EGF-like domain comprises the amino acid sequence of the receptor binding domain of NRG-1. In some embodiments, EGF-like domain refers to amino acid residues 177-226, 177-237, or 177-240 of NRG-1. In some embodiments, EGF- like domain comprises the amino acid sequence of the receptor binding domain of neuregulin-2 (NRG-2, also known in the art as DONI, HRG2 and NTAK). In some embodiments, an EGF-like domain of NRG-2 comprises a sequence of HARKCNETAKSYCVNGGVCYYIEGINQLSCKCPNGFFGQRCL (SEQ ID NO: 15), or a sequencing having 1, 2, 3, 4, or 5 substitutions, deletions or insertions relative thereto. In some embodiments, 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 some embodiments, the EGF-like domain of NRG-3 comprises a sequence of HFKPCRDKDLAYCLNDGECFVIETLTGSHKHCRCKEGYQGVRCD (SEQ ID NO: 16),or a sequencing having 1, 2, 3, 4, or 5 substitutions, deletions or insertions relative thereto. In some embodiments, 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, an EGF-like domain of NRG-4 comprises a sequence of HEEPCGPSHKSFCLNGGLCYVIPTIPSPFCRCVENYTGARCE (SEQ ID NO: 17), or a sequencing having 1, 2, 3, 4, or 5 substitutions, deletions or insertions relative thereto. In certain embodiments, EGF-like domain comprises the amino acid sequence of AEKEKTFCVNGECFMVKDLSNP (SEQ ID NO: 18), or a sequencing having 1, 2, 3, 4, or 5 substitutions, deletions or insertions relative thereto, as described in U.S. Pat. No. 5,834,229.

[0197] In some embodiments, the NRG-1 protein provided in the recombinant fusion protein disclosed herein is the NRG-1 B2a isoform.

[0198] In some embodiments, the active NRG-1 fragment comprises the ERBB3 / 4 binding domain. In other related embodiments, the NRG-1 binds to and induces signaling through ErbB4 (HER4). In some embodiments, the mAb inhibits NRG-1 signaling through ErbB3 (HER3). In some embodiments, the active protein fragment of NRG-1 comprises the active domain of NRG- 1.

[0199] In some embodiments, the recombinant fusion protein promotes HER2 / 4 signaling over HER2 / 3 signaling relative to the signal induction potential of recombinant NRG-1.

[0200] In some embodiments, the NRG-1 fragment comprises SEQ ID NO: 4, or a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity thereto, which is capable of binding to and inducing signaling through ErbB4. In some embodiments, the NRG-1 fragment comprises SEQ ID NO: 4, or a sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or insertions relative thereto.Linkers

[0201] The disclosure provides recombinant fusion proteins in which the NRG-1 fragment is fused to the anti-HER3 antibody heavy chain using a linker. In some embodiments, the N- terminus of the NRG-1 fragment is fused to the C-terminus of the anti-HER3 antibody heavy chain. In some embodiments, for example those embodiments in which the HER3 antibody is a full-length antibody, NRG-1 fragments are fused to the C-termini of both antibody heavy chains using linkers.

[0202] In some embodiments, NRG-1 is attached to the linker via the first (1st) amino acid on the N-terminus of NRG-1, which in one embodiment is a Serine (S or Ser) amino acid. In some embodiments, the N-terminus of NRG-1 is attached to the C-terminus of the antibodyheavy chain via a linker. In some embodiments, the linker comprises a GGGGSGGGGS (G4S) linker (SEQ ID NO: 5) and NRG-1 comprises SEQ ID NO: 4, or a sequencing having 1, 2, 3, 4 or 5 substitutions, insertions or deletions relative thereto. In some embodiments, one or more copies of the linker may be used. In other embodiments, 2, 3, 4, or 5 copies of the G4S linker or any other linker known in the art as being suitable for the composition disclosed herein may be used herein.

[0203] 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 (for example, 2 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) connecting two compounds, such as two polypeptides. The linker may be comprised of a single linking molecule or may comprise a linking molecule and at least one spacer molecule, intended to separate the linking molecule and a compound by a specific distance.Fusion Protein

[0204] The disclosure provides recombinant fusion proteins comprising an NRG-1 fragment fused to an anti-HER3 monoclonal antibody. Optionally, the NRG-1 fragment is fused to the antibody using a linker, as described supra.

[0205] In one embodiment, the heavy chain of the anti-HER3 antibody comprised by the recombinant fusion protein provided herein comprises the following amino acid sequence: MEFGLSWVFLVAIIKGVQCQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWI RQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYC ARDKWTWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKGGGGSGGGGSSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLC KCPNEFTGDRCQNYVMASFYKAEELYQ (SEQ ID NO: 9). In some embodiments, the heavy chain of the anti-HER3 antibody comprises SEQ ID NO: 9, or a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the heavy chain of the anti-HER3 antibody comprises SEQ ID NO: 9, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions or substitutions relative thereto.

[0206] In some embodiments, the heavy chain of the anti-HER3 antibody comprised by the recombinant fusion protein provided herein comprises the following amino acid sequence: MEFGLSWVFLVAIIKGVQCQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWI RQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYC ARDKWTWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKSCDKTHTCPPCPAPEFLGGPAVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAH YTQKSLSLSPGKGGGGSGGGGSSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLC KCPNEFTGDRCQNYVMASFYKAEELYQ (SEQ ID NO: 10). In some embodiments, the heavy chain of the anti-HER3 antibody comprises SEQ ID NO: 10, or a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the heavy chain of the anti-HER3 antibody comprises SEQ ID NO: 10, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions or substitutions relative thereto.

[0207] In some embodiments, the anti-HER3 mAb heavy chain sequence comprises a signal peptide sequence. In some embodiments, the signal peptide sequence comprises the amino acid sequence of MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 11). In some embodiments, the signal peptide comprises SEQ ID NO: 11, or a sequence having 1, 2 or 3 amino acid insertions, deletions or substitutions relative thereto.

[0208] In some embodiments, the light chain of the anti-HER3 antibody comprised by the recombinant fusion protein comprises the following amino acid sequence: MVLQTQVFISLLLWISGAYGDIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNY L AW YQQNPGQPPKLLIYWASTRESGVPDRF SGSGSGTDFTLTIS SLQAED VAVYYCQ QYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC (SEQ ID NO: 12). In some embodiments, the light chain of the anti-HER3 antibody comprises SEQ ID NO: 12, or a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the heavy chain of the anti-HER3 antibody comprises SEQ ID NO: 10, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions or substitutions relative thereto.

[0209] In some embodiments, the anti-HER3 mAb light chain sequence comprises a signal peptide sequence. In some embodiments, the signal peptide sequence comprises the amino acid sequence of MVLQTQVFISLLLWISGAYG (SEQ ID NO: 13). In some embodiments, the signal peptide comprises SEQ ID NO: 13, or a sequence having 1, 2 or 3 amino acid insertions, deletions or substitutions relative thereto.

[0210] In some embodiments, a mature polypeptide such as an antibody heavy chain or light chain amino acid sequence disclosed herein lacks a signal peptide.

[0211] In some embodiments, the recombinant fusion protein comprises the following amino acid sequences: Heavy chain QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGS TNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPEFLGGPAVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPGKGGG'GSG'G' GGSSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVM ASFYKAEELYQ (SEQ ID NO: 14, wherein bold italics indicate the linker, and bold indicates the NRG-1 fragment); and Light chain DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3). In some embodiments, the recombinant fusion protein comprises SEQ ID NO: 14, or a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto; and SEQ ID NO: 3, or a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the recombinant fusion protein comprises SEQ ID NO: 14, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions or deletions relative thereto; and SEQ ID NO: 3, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions or deletions relative thereto.

[0212] In some embodiments, each of the heavy chain sequence and light chain sequence in the mature recombinant fusion protein lack a signal peptide amino acid sequence.

[0213] In some embodiments, the heavy chain of the anti-HER3 antibody provided herein is fused via the C-terminus linker sequence to the NRG-lB2a isoform provided herein. In some embodiments, the C-terminus of the antibody heavy chain comprises the Fc domain of the antibody.

[0214] In some embodiments, the anti-HER.3 antibody and the NRG-1 fragment described herein are recombinantly or chemically fused / operably linked via a linker to form a fusion protein.

[0215] In some embodiments, the fusion protein is recombinantly encoded and produced. In some embodiments, the recombinant fusion protein is encoded by a nucleic acid sequence encoding the antibody of the invention that is operably linked via a nucleic acid sequence encoding a linker, to a nucleic acid sequence encoding an NRG-lB2a isoform of the invention.

[0216] In some embodiments, the recombinant fusion protein amino acid sequence is homologous to SEQ ID NO: 14 and SEQ ID NO: 3. The term “homology” may refer to identity to recombinant fusion protein amino acid or nucleic acid sequence (e.g. to any of SEQ ID NO: 1-18) of greater than 70%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 72%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 75%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 78%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 80%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 82%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 83%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 85%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 87%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 88%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 90%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 92%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 93%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 95%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 96%. In another embodiment, “homology” refers toidentity to any of SEQ ID NO: 1-18 of greater than 97%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 98%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of greater than 99%. In another embodiment, “homology” refers to identity to any of SEQ ID NO: 1-18 of 100%.Nucleic Acids

[0217] The disclosure provides nucleic acids (also referred to as polynucleotides) encoding the recombinant fusion proteins described herein.

[0218] Nucleic acids encoding the recombinant fusion proteins of the disclosure may be optionally obtained or created by any method known in the art (including purchase from commercial sources). For example, nucleic acid sequences encoding the appropriate antibody framework are optionally cloned and ligated into appropriate vectors (e.g., expression vectors for, e.g., prokaryotic or eukaryotic organisms). Additionally, nucleic acid sequences encoding the NRG-1 fragment are optionally cloned into the same vector in the appropriate orientation and location so that expression from the vector produces an antibody -NRG-1 fusion protein. Some optional embodiments also require post-expression modification, e.g., assembly of antibody subunits, etc. The techniques and art for the above (and similar) manipulations are well known to those skilled in the art. Pertinent instructions are found in, e.g., Sambrook et al., Molecular Cloning — A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented through 1999). In alternate embodiments, the antibody domain and NRG-1 fragment are assembled post-expression through, e.g., chemical means. In one embodiment, the present invention provides a composition, e.g. a pharmaceutical composition comprising the recombinant fusion protein of the present invention.

[0219] In some embodiments, polynucleotides are prepared using PCR techniques using procedures and methods known to one skilled in the art. In some embodiments, the procedure involves the ligation of two different DNA sequences (See, for example, “Current Protocols in Molecular Biology”, eds. Ausubel et al., John Wiley & Sons, 1992).

[0220] 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 isoperably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the nucleic acid sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, 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, the “operably linked” also refers to the functional pairing of distinct amino acid sequences, peptides or proteins, as in the pairing of the antibody and NRG-1 fragment described herein via a linker sequence also described herein.

[0221] In some embodiments, the anti-HER.3 mAb heavy chain comprised by the recombinant fusion protein provided herein is encoded by SEQ ID NO: 6:ATGGAGTTTGGGCTGAGCTGGGTTTTCCTTGTTGCTATAATAAAAGGTGTCCAGTGTCAGGT GCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAAGCCAAGCGAGACCCTGTCTCTGACATGCG CCGTGTACGGAGGATCCTTCAGCGGATACTATTGGTCTTGGATCAGGCAGCCACCTGGCAAG GGACTGGAGTGGATCGGCGAGATCAACCACTCTGGCTCCACCAACTACAATCCCTCTCTGAA GTCCCGGGTGACCATCTCCGTGGAGACAAGCAAGAATCAGTTTTCCCTGAAGCTGTCCAGCG TGACCGCCGCTGACACAGCCGTGTACTATTGCGCTAGGGACAAGTGGACCTGGTATTTCGAT CTGTGGGGAAGGGGCACCCTGGTGACAGTGTCTTCCGCCTCTACAAAGGGCCCCTCCGTGTT TCCTCTGGCTCCAAGCTCTAAGAGCACCTCTGGAGGAACAGCCGCTCTGGGATGTCTGGTGA AGGATTACTTCCCTGAGCCAGTGACCGTGAGCTGGAACTCTGGCGCCCTGACCTCCGGAGTG CATACATTTCCCGCTGTGCTGCAGTCCAGCGGCCTGTATAGCCTGTCTTCCGTGGTGACCGT GCCTAGCTCTTCCCTGGGCACCCAGACATACATCTGCAACGTGAATCACAAGCCCTCCAATA CAAAGGTGGACAAGAGAGTGGAGCCTAAGAGCTGTGATAAGACCCATACATGCCCACCATGT CCAGCTCCTGAGCTGCTGGGAGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCAAAGGACAC CCTGATGATCTCTCGCACCCCTGAGGTGACATGCGTGGTGGTGGACGTGTCCCACGAGGATC CAGAGGTGAAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCTAAGACCAAGCCT AGGGAGGAGCAGTACAACAGCACCTATCGGGTGGTGTCTGTGCTGACAGTGCTGCACCAGGA CTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAATAAGGCCCTGCCAGCTCCCATCG AGAAGACCATCTCTAAGGCCAAGGGCCAGCCCAGAGAGCCTCAGGTGTATACACTGCCCCCT AGCCGCGAGGAGATGACCAAGAACCAGGTGTCTCTGACATGTCTGGTGAAGGGCTTCTACCC ATCTGACATCGCTGTGGAGTGGGAGTCCAATGGCCAGCCCGAGAACAATTATAAGACCACAC CACCCGTGCTGGACTCCGATGGCAGCTTCTTTCTGTACTCCAAGCTGACCGTGGATAAGAGC AGGTGGCAGCAGGGCAACGTGTTTTCCTGCAGCGTGATGCACGAGGCCCTGCACAATCATTA TACACAGAAATCTCTGTCCCTGAGCCCAGGCAAGGGAGGAGGAGGAAGCGGAGGAGGAGGCA GCTCTCATCTGGTGAAGTGTGCTGAGAAGGAGAAGACCTTCTGCGTGAACGGCGGCGAGTGT TTTATGGTGAAGGACCTGTCTAATCCATCCAGATACCTGTGCAAGTGTCCCAACGAGTTCAC AGGCGATCGCTGCCAGAATTACGTGATGGCCTCTTTTTATAAGGCTGAGGAGCTGTACCAGT AA (SEQ ID NO: 6).

[0222] In one embodiment, the sequence set forth in SEQ ID NO: 6 comprises no Fc mutations. In one embodiment, SEQ ID NO: 6 is also referred to as “NPCF”.

[0223] 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:ATGGAGTTTGGGCTGAGCTGGGTTTTCCTTGTTGCTATAATAAAAGGTGTCCAGTGTCAGGT GCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAAGCCAAGCGAGACCCTGTCTCTGACATGCG CCGTGTACGGAGGATCCTTCAGCGGATACTATTGGTCTTGGATCAGGCAGCCACCTGGCAAG GGACTGGAGTGGATCGGCGAGATCAACCACTCTGGCTCCACCAACTACAATCCCTCTCTGAA GTCCCGGGTGACCATCTCCGTGGAGACAAGCAAGAATCAGTTTTCCCTGAAGCTGTCCAGCG TGACCGCCGCTGACACAGCCGTGTACTATTGCGCTAGGGACAAGTGGACCTGGTATTTCGAT CTGTGGGGAAGGGGCACCCTGGTGACAGTGTCTTCCGCCTCTACAAAGGGCCCCTCCGTGTT TCCTCTGGCTCCAAGCTCTAAGAGCACCTCTGGAGGAACAGCCGCTCTGGGATGTCTGGTGA AGGATTACTTCCCTGAGCCAGTGACCGTGAGCTGGAACTCTGGCGCCCTGACCTCTGGAGTG CATACATTTCCCGCTGTGCTGCAGTCCAGCGGCCTGTATAGCCTGTCTTCCGTGGTGACCGT GCCTAGCTCTTCCCTGGGCACCCAGACATACATCTGCAACGTGAATCACAAGCCCTCCAATA CAAAGGTGGACAAGAGAGTGGAGCCTAAGAGCTGTGATAAGACCCATACATGCCCACCATGT CCAGCTCCTGAGTTCCTGGGAGGACCTGCCGTGTTCCTGTTTCCTCCAAAGCCAAAGGACAC CCTGATGATCTCTCGCACCCCTGAGGTGACATGCGTGGTGGTGGACGTGTCCCACGAGGATC CAGAGGTGAAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCTAAGACCAAGCCT AGGGAGGAGCAGTACAACAGCACCTATCGGGTGGTGTCTGTGCTGACAGTGCTGCACCAGGA CTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAATAAGGCCCTGCCAGCTCCCATCG AGAAGACCATCTCTAAGGCCAAGGGCCAGCCCAGAGAGCCTCAGGTGTATACACTGCCCCCT AGCCGCGAGGAGATGACCAAGAACCAGGTGTCTCTGACCTGTCTGGTGAAGGGCTTCTACCC ATCTGACATCGCTGTGGAGTGGGAGTCCAATGGCCAGCCCGAGAACAATTATAAGACCACAC CACCCGTGCTGGACTCCGATGGCAGCTTCTTTCTGTACTCCAAGCTGACCGTGGATAAGAGC AGGTGGCAGCAGGGCAACGTGTTTTCCTGCAGCGTGATGCACGAGGCCCTGCACGCTCATTA TACACAGAAATCTCTGTCCCTGAGCCCAGGCAAGGGAGGAGGAGGAAGCGGAGGAGGAGGCA GCTCTCATCTGGTGAAGTGTGCTGAGAAGGAGAAGACCTTCTGCGTGAACGGCGGCGAGTGT TTTATGGTGAAGGACCTGTCTAATCCATCCAGATACCTGTGCAAGTGTCCCAACGAGTTCAC AGGCGATCGCTGCCAGAATTACGTGATGGCCTCTTTTTATAAGGCTGAGGAGCTGTACCAGT AA (SEQ ID NO: 7).

[0224] In one embodiment, SEQ ID NO: 7 is also referred to as “NPCFA”. In one embodiment, SEQ ID NO: 7 comprises one or more mutations that encode for one or more mutations in the constant (Fc) region of the anti-HER3 mAb provided herein. In one embodiment, the mature anti-HER3 antibody of the present invention comprises at least one mutation in amino acids 234, 239, 434, or a combination thereof, numbered relative to SEQ ID NO: 2. In another embodiment, the amino acid mutations comprise at least one of the following substitution mutations: L234F, S239A, N434A or a combination thereof.

[0225] 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):ATGGTGTTGCAGACCCAGGTCTTCATTTCTCTGTTGCTCTGGATCTCTGGTGCCTACGGGGACATCGAGATGACCCAGTCTCCAGATTCCCTGGCCGTGAGCCTGGGAGAGAGGGCTACAATCAACTGCCGGTCCAGCCAGTCTGTGCTGTACTCTTCCAGCAACAGGAATTACCTGGCCTGGTATCAGCAGAATCCCGGCCAGCCCCCTAAGCTGCTGATCTAT TGGGCTAGCACCAGAGAGTCTGGAGTGCCTGACCGCTTCTCTGGATCCGGAAGCGGCACAGACTTCACCCTGACA ATCTCTTCCCTGCAGGCCGAGGACGTGGCCGTGTACTATTGCCAGCAGTATTACTCTACCCCTAGGACATTCGGC CAGGGCACCAAGGTGGAGATCAAGCGGACAGTGGCCGCTCCATCCGTGTTCATCTTTCCACCCTCCGACGAGCAG CTGAAGTCCGGAACCGCTAGCGTGGTGTGCCTGCTGAACAACTTCTACCCAAGAGAGGCCAAGGTGCAGTGGAAG GTGGATAACGCTCTGCAGAGCGGCAATTCTCAGGAGTCCGTGACCGAGCAGGACAGCAAGGATTCTACATATTCC CTGAGCTCTACCCTGACACTGTCCAAGGCCGATTACGAGAAGCACAAGGTGTATGCTTGCGAGGTGACCCATCAG GGCCTGTCCAGCCCCGTGACAAAGAGCTTCAACCGCGGCGAGTGTTAA (SEQ ID NO: 8).

[0226] In one embodiment, SEQ ID NO: 8 is also referred to as “PAL”.

[0227] In one embodiment, polynucleotides of the present invention are inserted into expression vectors (z.e., a nucleic acid construct) to enable expression of the recombinant polypeptide. In one embodiment, the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector of the present invention includes a shuttle vector which renders this vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, cloning vectors comprise transcription and translation initiation sequences (e.g., promoters, enhancer) and transcription and translation terminators (e.g., polyadenylation signals).

[0228] In some embodiments, a variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptides of the present invention. In some embodiments, these include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence.

[0229] In some embodiments, non-bacterial expression systems are used (e.g., mammalian expression systems such as CHO cells) to express the polypeptide of the present invention. In one embodiment, the expression vector used to express polynucleotides of the present invention in mammalian cells is pCI-DHFR vector comprising a CMV promoter and a neomycin resistance gene.

[0230] In some embodiments, in bacterial systems of the present invention, a number of expression vectors can be advantageously selected depending upon the use intended for the polypeptide expressed. In one embodiment, large quantities of polypeptide are desired. In one embodiment, vectors that direct the expression of high levels of the protein product, possibly as a fusion with a hydrophobic signal sequence, which directs the expressed product into theperiplasm of the bacteria or the culture medium where the protein product is readily purified are desired. In one embodiment, certain fusion protein engineered with a specific cleavage site to aid in recovery of the polypeptide. In one embodiment, vectors adaptable to such manipulation include, but are not limited to, the pET series of E. coli expression vectors (Studier et al., Methods in Enzymol. 185:60-89 (1990)).

[0231] In one embodiment, yeast expression systems are used. In one embodiment, a number of vectors containing constitutive or inducible promoters can be used in yeast as disclosed in U.S. Pat. No. 5,932,447. In another embodiment, vectors which promote integration of foreign DNA sequences into the yeast chromosome are used.

[0232] In one embodiment, the expression vectors of the present invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.

[0233] In some embodiments, the expression vectors of the present invention include elements that increase the expression of the recombinant fusion proteins of the invention. Such features include, but are not limited to, choice of promoter and polyadenylation. In some embodiments, the polyadenylation sequence is a bovine growth hormone (BGH) polyadenylation sequence. In some embodiments, the promoter comprises a constitutively active promoter. In some embodiments, the promoter comprises a cytomegalovirus promoter (pCMV).

[0234] 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, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK- RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.

[0235] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Barr virus include pHEBO, and p205. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murinemammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0236] In some embodiments, recombinant viral vectors are useful for in vivo expression of the polypeptides of the present invention since they offer advantages such as lateral infection and targeting specificity. In one embodiment, lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

[0237] In one embodiment, various methods can be used to introduce the expression vector encoding the recombinant fusion protein of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0238] In some embodiments, introduction of nucleic acid by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.

[0239] In one embodiment, it will be appreciated that the polypeptides of the present invention can also be expressed from a nucleic acid construct administered to the individual employing any suitable mode of administration, described hereinabove (z.e., in-vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle / method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (z.e., ex-vivo gene therapy).

[0240] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), theexpression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.Methods of Making NRG-1 HER3 Antibody Fusion Proteins

[0241] The disclosure provides methods of making the NRG-1 HER3 antibody fusion proteins described herein. Exemplary methods include transforming host cells with one or more vectors encoding the heavy and light chains of the recombinant fusion protein, as described supra, culturing the host cell under conditions sufficient to produce the recombinant fusion protein, and purifying the recombinant fusion protein.

[0242] Suitable host cells will be known to persons of ordinary skill in the art, and include, without limitation, a variety of prokaryotic or eukaryotic cells. Suitable prokaryotic cells include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence. Suitable eukaryotic cells include yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence and non-bacterial expression systems are used (e.g., mammalian expression systems such as CHO cells).

[0243] In some embodiments, in bacterial systems of the present invention, a number of expression vectors can be advantageously selected depending upon the use intended for the polypeptide expressed. In one embodiment, large quantities of polypeptide are desired. In one embodiment, vectors that direct the expression of high levels of the protein product, possibly as a fusion with a hydrophobic signal sequence, which directs the expressed product into the periplasm of the bacteria or the culture medium where the protein product is readily purified are desired. In one embodiment, certain fusion protein engineered with a specific cleavage site to aid in recovery of the polypeptide. In one embodiment, vectors adaptable to such manipulation include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol. 185:60-89 (1990)].

[0244] In one embodiment, yeast expression systems are used. In one embodiment, a number of vectors containing constitutive or inducible promoters can be used in yeast as disclosed in U.S. Pat. No. 5,932,447. In another embodiment, vectors which promote integration of foreign DNA sequences into the yeast chromosome are used.

[0245] In one embodiment, mammalian expression systems are used. Suitable mammalian cells will be known to persons of ordinary skill in the art, and include, inter alia, Chinese hamster ovary (CHO) cells and human embryonic kidney 293 (HEK293) cells.

[0246] In some embodiments, transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant fusion protein or polypeptide. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. In one embodiment, an effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptide of the present invention. In some embodiments, a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. In some embodiments, cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. In some embodiments, culturing is carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. In some embodiments, culturing conditions are within the expertise of one of ordinary skill in the art.

[0247] In some embodiments, depending on the vector and host system used for production, resultant polypeptides of the present invention either remain within the recombinant cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or retained on the outer surface of a cell or viral membrane.

[0248] In one embodiment, following a predetermined time in culture, recovery of the recombinant polypeptide is effected.

[0249] In one embodiment, the phrase “recovering the recombinant polypeptide” used herein refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification.

[0250] In one embodiment, polypeptides of the present invention are purified using a variety of standard protein purification techniques, such as, 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.

[0251] In one embodiment, to facilitate recovery, the expressed coding sequence can be engineered to encode the polypeptide of the present invention and fused cleavable moiety. In one embodiment, a fusion protein can be designed so that the polypeptide can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the cleavable moiety. In one embodiment, a cleavage site is engineered between the polypeptide and the cleavable moiety and the polypeptide can be released from the chromatographiccolumn by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265: 15854-15859 (1990)].

[0252] In one embodiment, the polypeptide of the present invention is retrieved in “substantially pure” form.

[0253] In one embodiment, the phrase “substantially pure” refers to a purity that allows for the effective use of the protein in the applications described herein.

[0254] In one embodiment, the polypeptide of the present invention can also 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 system are commercially available.

[0255] In some embodiments, the recombinant polypeptides are synthesized and purified; their therapeutic efficacy can be assayed in vivo or in vitro.Pharmaceutical Compositions

[0256] Provided herein are pharmaceutical compositions comprising the recombinant fusion proteins disclosed herein, formulated together with a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition comprises a recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a ErbB3 (HER3) antibody (mAb), and a pharmaceutically acceptable carrier or diluent. In some embodiments, the recombinant fusion protein comprises the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 14.

[0257] As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).

[0258] Pharmaceutical compositions must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier preferably is an isotonic buffered saline solution. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.

[0259] A pharmaceutical composition comprising the recombinant fusion protein of the present disclosure can be formulated for parenteral administration, or for administration by avariety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. To administer a pharmaceutical composition of the disclosure by certain routes of administration, it may be necessary to co-administer the recombinant fusion protein with a material to prevent its inactivation. For example, the recombinant fusion protein may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.

[0260] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.

[0261] In typical embodiments, preparations for administration to subjects 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 oils), organic esters (e.g., ethyl oleate) and other solvents known to those of skill in the art. Physiologically acceptable carriers (or excipients) are optionally used in certain embodiments of the invention. Examples of such include, e.g., saline, PBS, Ringer's solution, lactated Ringer's solution, etc. Additionally, preservatives and additives are optionally added to the compositions to help ensure stability and sterility. For example, antibiotics and other bacteriocides, antioxidants, chelating agents, and the like are all optionally present in various embodiments of the compositions herein.

[0262] In some embodiments, the pharmaceutical composition comprising the recombinant fusion protein comprises histidine (e.g., histidine-HCl). In some embodiments, the pharmaceutical composition comprises between about 5 mM and 50 mM histidine, between about 5 mM and 30 mM histidine, between about 10 mM and 30 mM histidine, or between about 15 and 25 mM histidine. In some embodiments, the pharmaceutical composition comprises about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM or about 50 mM histidine. In some embodiments, the pharmaceutical composition comprises about 25 mM histidine. In someembodiments, the pharmaceutical composition comprises about 20 mM histidine. In some embodiments, the pharmaceutical composition comprises about 30 mM histidine.

[0263] In some embodiments, the pharmaceutical composition comprising the recombinant fusion protein comprises sucrose. In some embodiment, the pharmaceutical composition comprises between about 50 mM and 500 mM, between about 100 mM and 400 mM, between about 200 mM and 300 mM or between about 200 and 250 mM sucrose. In some embodiments, the pharmaceutical composition comprises about 150 mM, about 175 mM, about 190 mM, about 195 mM, about 200 mM, about 205 mM, about 210 mM, about 215 mM, about 220 mM, about 250 mM, about 275 mM, about 300 mM or about 325 mM sucrose.

[0264] In some embodiments, the pharmaceutical composition comprising the recombinant fusion protein comprises an emulsifier such as a polysorbate, for example polysorbate 20, polysorbate 60 and / or polysorbate 80. In some embodiments, the pharmaceutical composition comprises between about 0.01% weight / volume (w / v) and 0.2% w / v, between about 0.02% w / v and 0.15% w / v, between about 0.03% w / v and 0.1% w / v, between about 0.04% w / v and 0.08% w / v, or between about 0.03% w / v and 0.06% w / v polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.01% w / v, about 0.02% w / v, about 0.03% w / v, about 0.04% w / v, about 0.05% w / v, about 0.06% w / v, about 0.07% w / v, about 0.08% w / v, about 0.09% w / v or about 0.1% w / v polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.02% w / v polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.03% w / v polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.04% w / v polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.05% w / v polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.06% w / v polysorbate 80.

[0265] In some embodiments, the pharmaceutical composition comprising the recombinant fusion protein comprises a sterile aqueous solution comprising between about 10 mM and 30 mM histidine, between about 200 mM and 250 mM sucrose, and between about 0.02% w / v and 0.06% w / v polysorbate 80. In some embodiments, the pharmaceutical composition comprising the recombinant fusion protein comprises a sterile aqueous solution comprising 25 mM histidine, 205 mM sucrose, and 0.04% w / v polysorbate 80.

[0266] In some embodiments, the pharmaceutical composition comprising the recombinant fusion protein does not comprise a preservative.

[0267] In some embodiments, the pharmaceutical composition comprises between about 0.1 mg / mL to 50 mg / mL, between about 1 mg / mL to 50 mg / mL, between about 10 mg / mL to 50 mg / mL, between about 5 mg / mL to 40 mg / mL, between about 10 mg / mL to 30 mg / mL, or between about 10 mg / ml to 20 mg / mL of the recombinant fusion protein. In some embodiments, the pharmaceutical composition comprises about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL or about 50 mg / mL of the recombinant fusion protein. In some embodiments, the pharmaceutical composition comprises about 10 mg / mL of the recombinant fusion protein. In some embodiments, the pharmaceutical composition comprises about 20 mg / mL of the recombinant fusion protein. In some embodiments, the pharmaceutical composition comprises about 30 mg / mL of the recombinant fusion protein. In some embodiments, the pharmaceutical composition comprises about 40 mg / mL of the recombinant fusion protein.

[0268] The pharmaceutical composition comprising the recombinant fusion protein can be disposed in any suitable container known in the art, including vials, syringes and the like, and in any suitable volume. In an exemplary embodiment, about 1.0 to about 3.0 mL (e.g., about 1.5 mL, about 1.8 mL, about 2.0 mL, about 2.2 mL, about 2.4 mL, about 2.8 mL, or about 3.0 mL) is disposed in a 5 mL glass vial.

[0269] Regardless of the route of administration selected, the recombinant fusion proteins of the present disclosure, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0270] Actual dosage levels and concentrations of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts.Therapeutic Methods

[0271] The disclosure provides methods of treating a cardiovascular disease or condition in a subject in need thereof, the method comprising administering a therapeutically effective amount of the recombinant fusion protein of the disclosure or a pharmaceutical composition comprising the same.

[0272] The disclosure provides methods of preventing, inhibiting, suppressing or delaying the onset of a cardiovascular disease or condition in a subject, the method comprising administering an effective amount of the recombinant fusion protein of the disclosure or the pharmaceutical composition described herein.

[0273] The disclosure provides methods of treating a subject with heart failure, the method comprising administering a therapeutically effective amount of the recombinant fusion protein or the pharmaceutical composition comprising the recombinant fusion protein disclosed herein. In some embodiments, the heart failure comprises heart failure with reduced ejection fraction (HFrEF).

[0274] The disclosure provides methods of treating a subject with heart failure with reduced ejection fraction (HFrEF), comprising administering to the subject between 0.03 mg / kg and 0.27 mg / kg of a recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb). In some embodiments, the subject has a left ventricular ejection fraction (LVEF) of less than or equal to 40%. In some embodiments, the subject has a left ventricular ejection fraction (LVEF) of greater than 40% and less than or equal to 65%. In some embodiments, the subject has left ventricular ejection fraction of greater than 40% and atrial fibrillation.

[0275] In some embodiments, the subject has NYHA Class II, Class III or Class IV heart failure. In some embodiments, the subject has NYHA Class II or Class III heart failure.

[0276] In some embodiments, the subject has a left ventricular ejection fraction (LVEF) of less than or equal to 30%. In some embodiments, the subject has a left ventricular ejection fraction (LVEF) of less than or equal to 40%. In some embodiments, the subject has a LVEF of less than or equal to 50%. In some embodiments, the subject has a LVEF of less than or equal to 60%. In some embodiments, the subject has a LVEF of less than or equal to 65%. In some embodiments, the subject has an LVEF of greater than or equal to 30%, but less than or equal to 70%. In some embodiments, the subject has an LVEF of greater than or equal to 40%, but less than or equal to 65%. In some embodiments, the subject has an LVEF of greater than or equal to 40%, but less than or equal to 60%. In some embodiments, the subject has an LVEF of greater than or equal to 30%, but less than or equal to 40%. In someembodiments, the subject has an LVEF of greater than or equal to 30%, but less than or equal to 50%. In some embodiments, the subject has an LVEF of greater than or equal to 30%, but less than or equal to 65%.

[0277] “Ejection fraction,” “EV,” “left ventricular ejection fraction” or “LVEF,” typically expressed as a percentage, refers to measure how much blood the left ventricle pumps out with each contraction. LVEF is the fraction of chamber volume ejected in systole (stroke volume) in relation to the volume of blood in the ventricle at the end of diastole (end-diastolic volume). For example, an ejection fraction of 50% means that 50% of the total amount of blood in the left ventricle is pushed out with each heartbeat. LVEF can be determined by any suitable methods known in the art, including echocardiography (including two-dimensional transthoracic echocardiography, or 2D-TTE), radionuclide ventriculogram, cardiac computed tomography (CT), and cardiac magnetic resonance imaging (CMR).

[0278] In some embodiments, the methods disclosed herein comprise measuring a level of N-terminal pro-brain natriuretic peptide (NT-proBNP) prior to administration of the recombinant fusion protein of the disclosure. The NT-proBNP prohormone, and its cleavage product brain natrieuretic peptide 32 (BNP) are used for screening and diagnosis of acute heart failure, such as congestive heart failure. Plasma concentrations of NT-proBNP and BNP may also be increased in subjects with asymptomatic or symptomatic left ventricular dysfunction, and are associated with coronary artery disease and myocardial ischemia. In general, higher levels of NT-proBNP or BNP are correlated with heart failure, although a healthy level of NT-proBNP or BNP varies with the age and sex of the subject. Thus, for men, a level of NT-proBNP of 93 pg / mL or less at 19-44 years of age, a level of 138 pg / mL or less at 45-54 years of age, a level of 177 pg / mL or less at 55-64 years of age, a level of 229 pg / mL or less at 65-74 years of age, or a level of 852 or less at greater than 75 years of age is indicative of heart health. For women, a level of NT-proBNP of 178 pg / mL or less at 19-44 years of age, a level of 192 pg / mL or less at 45-54 years of age, a level of 226 pg / mL or less at 55-64 years of age, a level of 353 pg / mL or less at 65-74 years of age, or a level of 624 or less at greater than 75 years of age is indicative of heart health. Levels of NT-proBNP can be determined using any suitable assays known in the art, including, but not limited to enzyme- linked immunosorbent assay (ELISA) and Sandwich ELISA.

[0279] In some embodiments, the subject has a level of N-terminal pro-brain natriuretic peptide (NT-proBNP) level that is greater than or equal to 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, or 700 pg / mL. In some embodiments, the subject has a level of NT-proBNP that is greater than or equal to 300 pg / mL. In some embodiments, the subject hasa level of NT-proBNP that is greater than or equal to 600 pg / mL. In specific embodiments, the subject has an LVEF of greater than or equal to 30% and less than or equal to 40%, and a level of NT-proBNP that is greater than or equal to 300 pg / mL. In specific embodiments, the subject has a left ventricular ejection fraction (LVEF) of greater than 40% and less than or equal to 65% and an NT-proBNP level that is greater than or equal to 600 pg / mL. In specific embodiments, the subject has a left ventricular ejection fraction (LVEF) of greater than 40% and less than or equal to 65%, an NT-proBNP level that is greater than or equal to 600 pg / mL and a history of fibrillation and / or atrial flutter.

[0280] In some embodiments, the methods disclosed herein comprise measuring a level of hemoglobin prior to administration of the recombinant fusion protein of the disclosure. In some embodiments, the subject has a hemoglobin (Hb) level of greater than or equal to 11 g / dL, 10 g / dL, 9 g / dL, or 8 g / dL. In some embodiments, the subject has a Hb level of greater than or equal to 9 g / dL. Hemoglobin levels can be measured using any suitable methods known in the art, including spectrophotometric methods.

[0281] In some embodiments, the subject does not have hemodynamically significant and / or severe valvular disease. In some embodiments, the subject does not have hemodynamically significant and / or severe valvular disease other than mitral and / or tricuspid regurgitation. Valvular disease refers to any disease where any valve of the heart is damaged or diseased. Exemplary valvular diseases include mitral regurgitation, and aortic stenosis. Valvular diseases can be characterized by any suitable methods known in the art, including 2D-TTE as described herein.

[0282] In some embodiments, the cardiovascular disease or condition comprises atrial fibrillation. In some embodiments, the cardiovascular disease or condition comprises cardiac fibrosis. In some embodiments, the cardiovascular disease or condition comprises atrial fibrillation and cardiac fibrosis. Atrial fibrillation or flutter can be measured by any suitable methods known in the art, including by electrocardiogram, Holter, implantable device, or telemetry based event monitoring.

[0283] In some embodiments, the cardiovascular disease comprises a chronic heart failure / congestive heart failure (CHF), acute heart failure / myocardial infarction (MI), left ventricular systolic dysfunction, reperfusion injury associated with MI, chemotherapy- induced cardiotoxicity (adult or pediatric), radiation-induced cardiotoxicity, adjunct to surgical intervention in pediatric congenital heart disease. In some embodiments, the cardiovascular disease comprises heart failure with reduced ejection fraction (HFrEF), asdescribed above. In some embodiments, the cardiovascular disease comprises heart failure with preserved ejection fraction (HFpEF).

[0284] In some embodiments, the cardiovascular disease is the result of chemotherapy- induced cardiotoxicity. In some embodiments, the chemotherapy-induced cardiotoxicity results from a subject receiving anthracy clines, alkylating agents, antimicrotubule agents, and / or antimetabolite agents used as chemotherapy.

[0285] In some embodiments, the cardiovascular condition is cardiotoxicity as a result of a subject receiving a cancer therapy. In some embodiments, the cancer therapy is a HER-2 targeted therapy. In other embodiments, the HER-2 targeted therapy comprises use of trastuzumab, ado-trastuzumab, emtansine, lapatinib, neratinib, and pertuzumab, any anti- HER2 antibody, any anti-HER2 agent or a combination thereof.

[0286] In some embodiments, the subject has heart failure that is not due to hypertrophic cardiomyopathy, restrictive and / or infiltrative cardiomyopathy, arrhythmogenic right ventricular dysplasia, Fabry disease, or Noonan syndrome with LV hypertrophy or a positive serum immunofixation result. In some embodiments, the subject has not been diagnosed with stress-induced (Takotsubo) cardiomyopathy, myocarditis, or peripartum cardiomyopathy. In some embodiments, the subject has not been diagnosed with chemotherapy- or radiation- induced cardiomyopathy. In some embodiments, the subject has not been diagnosed with stroke or transient ischemic attack (TIA) within 8-16 weeks prior to administering the recombinant fusion protein. In some embodiments, the subject has not had a history of syncope within the last 12 weeks prior to administering the recombinant fusion protein. In some embodiments, the subject has not had sustained ventricular tachycardia without an implantable cardioverter-defibrillator prior to administering the recombinant fusion protein. In some embodiments, the subject does not have moderate or severe aortic and / or mitral valve stenosis. In some embodiments, the subject has not had unstable angina, acute coronary syndrome (e.g., myocardial infarction, troponin-positive with symptoms of angina or unstable angina) within 6-16 weeks prior to administering the recombinant fusion protein. In some embodiments, the subject has not had unstable ST-elevation myocardial infarction within 10- 18 weeks prior to administering the recombinant fusion protein. In some embodiments, the subject does not have any narrow complex tachycardia (atrial fibrillation or atrial flutter) with a resting ventricular rate > 110 beats per minute prior to administration of the recombinant fusion protein. In some embodiments, for example those embodiments where the subject has a history of atrial fibrillation (AF) or atrial flutter (e.g., a CHA2DS2-VASc score of > 2 inmen or > 3 in women), the subject is taking anti coagulation medications (e.g., non-vitamin K oral anticoagulants or warfarin).

[0287] In some embodiments, the disclosure relates to a method of inducing remodeling of muscle cell sarcomeric and cytoskeleton structures, or cell-cell adhesions, the method comprising treating the cells of the subject with the recombinant fusion protein disclosed herein.

[0288] In some embodiments, the therapeutic method is directed to treating heart failure resulting from disassociation of cardiac muscle cell-cell adhesion and / or the disarray of sarcomeric structures in the subject.

[0289] “ Treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) heart hypertrophy. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in which the disorder is to be prevented. The heart hypertrophy may be from any cause which is responsive to retinoic acid, including congenital, viral, idiopathic, cardiotrophic, or myotrophic causes, or as a result of ischaemia or ischaemic insults such as myocardial infarction. Typically, the treatment is performed to stop or slow the progression of hypertrophy, especially after heart damage, such as from ischaemia, has occurred. Preferably, for treatment of myocardial infarctions, the recombinant fusion protein provided herein is given immediately after the myocardial infarction, to prevent or lessen hypertrophy.

[0290] In some embodiments, treating a subject with the recombinant fusion proteins described herein improves one or more parameters associated with cardiac efficacy, quality of life, and / or biomarkers associated cardiac function relative to a baseline measurement from before administration of the recombinant fusion protein.

[0291] Parameters associated with cardiac efficacy include, but are not limited to, LVEF, left ventricular end-diastolic volume index (LVEDVi), left ventricular end-systolic volume index (LVESVi), stroke volume index cardiac output (sVi CO), cardiac index (CI), left ventricular mass index (LVMi), E / e’ (a parameter to measure early diastolic peak velocity and early diastolic mitral annular velocity), right ventricular systolic pressure (RVSP), left ventricular assist (LAV), left atrial ejection fraction (LAEF), and valvular function. Such indicia can be measured using any suitable methods known in the art, including 2D-TTE and cardiac magnetic resonance imaging (CMR). In some embodiments, administration of the recombinant fusion protein improves the parameter relative to a baseline measurement from before administration of the recombinant fusion protein.

[0292] In some embodiments, the parameter comprises LVEF. In some embodiments, administration of the recombinant fusion protein increases LVEF by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 8%, by at least 10%, by at least 12%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35% or by at least 40% when compared to a baseline measurement from before administration of the recombinant fusion protein. In some embodiments, administration of the recombinant fusion protein increases LVEF by between about 2% to about 30%, by between about 2% to about 20%, by between about 2% to 15%, by between about 2% to about 12%, by between about 2% to about 8%, by between about 5% to about 30%, by between about 10% to about 30%, or by between about 10% to about 20% when compared to a baseline measurement from before administration of the recombinant fusion protein.

[0293] In some embodiments, improvement of LVEF in the subject is present at least about 7 days, at least about 15 days, at least about 30 days, at least about 60 days, at least about 90 days, at least about 120 days, at least about 135 days or at least about 180 days after administration of the recombinant fusion protein.

[0294] Indicia of quality of life include, but are not limited to, the 6 minute walk test (6MWT) and Kansas City Cardiomyopathy Questionnaire 12 (KCCQ-12). The 6MWT is a test to measure the distance a participant can walk for a total of 6 minutes, and is described in Example 1. The KCCQ-12 is a 12-item self-administered questionnaire developed to qualitatively and quantitatively measure the patient’s perception of their health status, which includes HF symptoms, impact on physical and social function, and how their HF impacts their quality of life within a 2-week recall period, and is described in Example 1.

[0295] Biomarkers associated cardiac function include, but are not limited to, brain natriuretic peptide, N-Terminal ProB-type Natriuretic Peptide, high-sensitivity C-reactive protein (hs-CRP), creatine phosphokinase (CPK), CPK -muscle / brain (CPK-MB), lactate dehydrogenase (LDH), blood glucose, cardiac troponin I (cTnl), and high-sensitivity cardiac troponin T (hs-cTnT). Levels of such biomarkers can be measured using any suitable methods known in the art, including ELISA and spectrographic methods.

[0296] In some embodiments, treating a subject with the recombinant fusion protein described herein can result in decreasing a sign or a symptom of atrial fibrillation or fibrosis. For example, treating a subject with the recombinant fusion protein can reduce the duration and / or frequency of atrial fibrillation, or reduce a sign or a symptom of atrial fibrillation, such as reducing irregular heartbeat, heart palpitations, lightheadedness, extreme fatigue, shortness of breath, chest pain or a combination thereof. In some embodiments, treating a subject withthe recombinant fusion protein reduces collagen content or deposition in cardiac tissue, for example in atrial tissue.

[0297] In some embodiments, treating a subject with the recombinant fusion protein promotes cardiomyocyte proliferation, differentiation, and survival. In other embodiments, treating a subject with the recombinant fusion protein promotes proliferation, differentiation and survival of cardiac tissue. In some embodiments, the recombinant fusion protein promotes cardiomyocyte proliferation, differentiation, and survival without promoting cancer and / or tumor growth. In some embodiments, the recombinant fusion protein promotes proliferation, differentiation and survival of cardiac tissue without promoting cancer or tumor growth.

[0298] In some embodiments, treating a subject with the recombinant fusion proteins provided herein can result in increase in average survival time of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a recombinant fusion protein of the disclosure. Preferably, after treatment with the strategies, treatment modalities, methods, combinations, and compositions provided herein, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90, 120, or 365 days; more preferably, by more than 365 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with the pharmaceutical composition disclosed herein.

[0299] In some embodiments, treating a subject with the recombinant fusion proteins provided herein can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving carrier alone. Treating cardiac disease can result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. Preferably, after treatment with the strategies, treatment modalities, methods, combinations, and compositions provided herein, the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%. A decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means. A decrease in the mortality rate of a population may be measured, for example, by calculating for a population theaverage number of disease-related deaths per unit time following initiation of treatment with an active compound. A decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with the pharmaceutical composition disclosed herein.

[0300] In some embodiments, administration of the recombinant fusion protein reduces risk of death from cardiovascular failure.Administration and Dosing

[0301] The recombinant fusion proteins and pharmaceutical compositions comprising same of the present disclosure can be parenterally administered to a subject in need thereof, or can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results.

[0302] In some embodiments, a pharmaceutical composition comprising the recombinant fusion protein is administered parenterally.

[0303] In some embodiments, a pharmaceutical composition comprising the recombinant fusion protein is administered via intravenous infusion. In some embodiments, the infusion takes place over about 45 to about 90 minutes. In some embodiments, the infusion takes place over a period of between 60 to 75 minutes. In some embodiments, the infusion takes place over about 60 minutes. In some embodiments, the intravenous administration comprises use of a syringe pump or an infusion pump.

[0304] In some embodiments, the pharmaceutical compositions are administered by intravenous, intra-arterial, subcutaneous or intramuscular injection of a liquid preparation. In some embodiments, the pharmaceutical compositions are administered intravenously via infusion, and are thus formulated in a form suitable for intravenous administration. In other embodiments, the pharmaceutical compositions are administered intra-arterially, and are thus formulated in a form suitable for intra-arterial administration.

[0305] In some embodiments, compositions comprising the recombinant fusion protein for use in the methods disclosed herein comprise solutions or emulsions, which in some embodiments are aqueous solutions or emulsions comprising a safe and effective amount of the recombinant fusion proteins disclosed herein and optionally, other compounds, intended for intravenous administration.

[0306] Regardless of the route of administration selected, the compositions comprising the recombinant fusion proteins of the disclosure can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0307] The recombinant fusion proteins, or pharmaceutical compositions comprising same, are optionally administered to subjects in need of treatment (either therapeutically or prophylactically) in any appropriate sterile pharmaceutical carrier. Such pharmaceutical carrier acts to maintain the solubility and action of the recombinant fusion protein. In some embodiments, it may be desired to administer additional components in conjunction with the recombinant fusion protein. For example, in some treatment regimes, chemotherapeutic agents, antibiotics, additional formulations comprising the recombinant fusion protein of the disclosure and one or more standard of care agents, etc. are all optionally included.

[0308] In some embodiments, the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject once every 2 to 12 weeks, once every 2 to 10 weeks, once every 2 to 8 weeks, once every 2 to 6 weeks, once every 3 to 12 weeks, once every 3 to 10 weeks, once every 3 to 8 weeks, once every 3 to 6 weeks, once every 3 to 5 weeks, once every 4 to 12 weeks, once every 4 to 10 weeks, once every 4 to 8 weeks, once every 4 to 6 weeks, once every 5 to 10 weeks, once every 6 to 12 weeks, once every 2 to 6 months, once every 3 to 6 months, once every 3 to 9 months, once every 4 to 12 months, once every 6 to 18 months or once every 6 to 12 months. In some embodiments, the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject once every 2 to 8 weeks. In some embodiments, the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject once every 3 to 6 weeks.

[0309] In some embodiments, the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject once every week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every 6 weeks, once every seven weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 4 months, once every 5 months, once every 6 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months, once every year, once every 14 months, once every 16 months or once every 18 months. In some embodiments, the subject is administered the recombinant fusion protein once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, or once every 9 weeks. In some embodiments, the recombinant fusion protein or pharmaceuticalcomposition comprising same is administered to a subject once every three weeks. In some embodiments, the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject once every four weeks. In some embodiments, the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject once every five weeks. In some embodiments, the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject once every six weeks. In some embodiments, the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject once every eight weeks. In some embodiments, the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject once every nine weeks. In some embodiments, the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject once every ten weeks. In some embodiments, the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject once every eleven weeks. In some embodiments, the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject once every twelve weeks. In some embodiments, the subject is administered the recombinant fusion protein once every 6 months. In some embodiments, the subject is administered the recombinant fusion protein once a year. In some embodiments, the subject is administered the recombinant fusion protein once every 18 months.

[0310] In some embodiments, a dose of the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject at least once every 2 weeks. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject at least once every 3 weeks. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject at least once every 4 weeks. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject at least once every 5 weeks. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject at least once every 6 weeks. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject at least once every 8 weeks. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject at least once every 10 weeks. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical compositioncomprising same is administered to a subject at least once every 12 weeks. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising the same is administered to a subject two or more times a year. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising the same is administered to a subject two or more times every two years. In another embodiment, a dose of the recombinant fusion protein or pharmaceutical composition comprising the same is administered to a subject two or more times every two or more years.

[0311] In some embodiments, for example those embodiments wherein the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject once 3 to 6 weeks, an individual dose may be delayed. For example, upon an adverse event in a subject, an individual dose may be delayed by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days. In some embodiments, the recombinant fusion protein or pharmaceutical composition comprising same is administered to a subject once 4 weeks, and an individual dose can be delayed up to 7 days if an adverse even occurs in the subject.

[0312] In some embodiments, administration of the recombinant fusion protein or pharmaceutical composition comprising same comprises a dosing “holiday” For example, if the subject’s condition deteriorates, or the subject experiences an adverse event, administration of the recombination fusion protein or pharmaceutical composition comprising same may be temporarily suspended, or the total amount of recombinant fusion protein administered at each dose and / or the frequency of dosage reduced until the subject improves or the adverse event is resolved. Alternatively, is a subject shows significant improvement in one or more of the parameters associated with cardiac efficacy, quality of life, and / or biomarkers associated cardiac function described herein, dosing may be temporarily suspended, or the total amount of recombinant fusion protein administered at each dose and / or the frequency of dosage reduced, until a change in the one or more parameters associated with cardiac efficacy, quality of life, and / or biomarkers associated cardiac function indicates that dosing should be resumed or increased in amount or frequency. In some embodiments, the dosing holiday is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 14 weeks, about 16 weeks, about 18 weeks, about 20 weeks, about 22 weeks, about 24 weeks, or more between doses. In some embodiments, the dosing holiday is 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 10 months or 1 year between doses. In some embodiments, the dosing holiday is 8 weeks between doses. In some embodiments, the dosing holiday is 10weeks between doses. In some embodiments, the dosing holiday is 12 weeks between doses. In some embodiments, the dosing holiday is 14 weeks between doses. In some embodiments, the dosing holiday is 16 weeks between doses. In some embodiments, the dosing holiday is 18 weeks between doses.

[0313] In some embodiments, administration of the recombinant fusion protein or pharmaceutical composition comprising same comprises administering a maintenance dose. As used herein, a “maintenance dose” refers to a dose that is lower, and / or less frequent, than one or more initial doses, sometimes referred to as a loading dose or loading doses, of the recombinant fusion protein. In some embodiments, administration of the recombinant fusion protein or pharmaceutical composition comprising same comprises administering one or more maintenance doses that are lower than one or more initial doses. In some embodiments, administration of the recombinant fusion protein or pharmaceutical composition comprising same comprises administering one or more maintenance doses that are administered less frequently than one or more initial doses. In some embodiments, the loading (initial) dose and maintenance dose comprise administering the same amount of the recombinant fusion protein, but the maintenance dose is administered less frequently than the loading dose. As an example, if a subject administered initial doses comprising 0.09 mg / kg of the recombinant fusion protein, the maintenance dose comprises less than 0.09 mg / kg, e.g. 0.045 mg / kg, of the recombinant fusion protein. Alternatively, or in addition, the subject is administered a plurality (1, 2, 3, 4, 5, 6 or more) of initial doses of the recombinant fusion protein at a shorter interval (e.g., every 1, 3, 4, or 5 weeks) and a maintenance dose at a longer interval (e.g., every 6, 8, 10, 12, 14, 16 or 18 weeks). For example, if a subject is administered initial doses comprising 0.09 mg / kg of the recombinant fusion protein every 4 weeks, the maintenance dose comprises 0.09 mg / kg of the recombinant fusion protein administered less frequently than every 4 weeks (e.g., every 6, every 8, every 10, every 12 or every 14 weeks).

[0314] In some embodiments, the subject is administered a maintenance dose every 8 to 16 weeks. In some embodiments, the maintenance dose is administered every 8, 10, 12, 14, 18, 20, 22 or 24 weeks. In some embodiments, the maintenance dose is administered every 8, 10, 12 or 14 weeks. In some embodiments, the maintenance dose is administered every 8 weeks. In some embodiments, the maintenance dose is administered every 9 weeks. In some embodiments, the maintenance dose is administered every 10 weeks. In some embodiments, the maintenance dose is administered every 11 weeks. In some embodiments, the maintenance dose is administered every 12 weeks. In some embodiments, the maintenance dose is administered every 14 weeks. In some embodiments, the maintenance dose isadministered every 16 weeks. In some embodiments, the maintenance dose is administered every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every year, every 14 months, every 16 months or every 18 months. In some embodiments, the maintenance dose is administered every 2-6 months. In some embodiments, the maintenance dose is administered every 3-8 months, every 2 to 6 months, every 3 to 6 months, every 3 to 9 months, every 4 to 12 months, every 6 to 18 months or every 6 to 12 months. In some embodiments, the maintenance dose is administered every 6 months. In some embodiments, the maintenance dose is administered every year. In some embodiments, the maintenance dose is administered every 18 months.

[0315] In some embodiments, administration of the recombinant fusion protein comprises a planned recovery period. In some embodiments, administration of the recombinant fusion protein comprises administration of one or more initial (loading) doses, a planned recovery period without administration of the recombinant fusion protein, followed by administration of one or more maintenance doses that are lower and / or less frequent than the one or more initial doses. In some embodiments, the planned recovery period is about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 7 months, about 8 months, about 9 months, about 10 months or about 1 year. In some embodiments, the planned recovery period is between about 8 weeks and about 30 weeks, between about 8 weeks and about 24 weeks, between about 8 weeks and about 20 weeks, between about 10 weeks and about 24 weeks, between about 10 weeks and about 18 weeks, between about 12 weeks and about 20 weeks, between about 12 weeks and about 18 weeks, or between about 12 weeks and about 16 weeks. In some embodiments, the planned recovery period is between about 2 months and about 18 months, between about 3 months and about 12 months, between about 3 months and about 8 months, between about 4 months and about 12 months, between about 2 months and about 6 months, or between about 6 months and about 1 year. In some embodiments, the planned recovery period is about 10 weeks. In some embodiments, the planned recovery period is about 12 weeks. In some embodiments, the planned recovery period is about 14 weeks. In some embodiments, the planned recovery period is about 16 weeks. In some embodiments, the planned recovery period is about 18 weeks. In some embodiments, the planned recovery period is about 6 months. In some embodiments, the planned recovery period is about 1 year. In some embodiments, the length of the plannedrecovery period is adjusted based on one or more clinical parameters, such as levels of NT- proBNP, LVEF or other echocardiographic parameters, toxicity or adverse effects, where improvements, such as improvements in LVEF or other echocardiographic parameters, or adverse effects, lead to an increased planned recovery period, while less improvement, and lack of adverse effects, lead to a shorter planned recovery period.

[0316] In an exemplary dosing regimen, one or more initial (loading doses) are administered (e.g., between 2-10 loading doses) at an interval of 2-8 weeks (e.g., 4 weeks) apart. This is followed by a planned recovery period of between about 12 to 16 weeks. After the planned recovery period, the subject is administered one or more maintenance doses, which are less frequent than the loading doses, e.g. the maintenance doses are administered 10-14 weeks apart. In a further exemplary dosing regimen, the subject is administered 4 initial doses about 4 weeks apart, followed by a 14 week planned recovery period, and 2 or more maintenance doses that are about 12 weeks apart. The initial doses can be same as the maintenance doses (e.g., both 0.045 mg / kg or 0.09 mg / kg), or the maintenance dose(s) can be reduced compared to the initial dose(s) (e.g., the initial dose or doses are at 0.09 mg / kg, while the maintenance dose or doses are at 0.045 mg / kg).

[0317] In some embodiments, the subject is administered the recombinant fusion protein or pharmaceutical composition comprising same for at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, or at least 36 months. In some embodiments, the subject is administered the recombinant fusion protein or pharmaceutical composition comprising same for at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years or at least 10 years.

[0318] In some embodiments, the subject is administered between 0.03 milligrams / kilogram (mg / kg) and 0.27 mg / kg of the recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a ErbB3 (HER3) antibody (mAb). In some embodiments, the subject is administered between 0.03 milligrams / kilogram mg / kg and 0.27 mg / kg of the recombinant fusion protein in a single dose (i.e., in a single intravenous infusion). In some embodiments, the subject is administered 0.03 mg / kg of the recombinant fusion protein in a single dose. In some embodiments, the subject is administered 0.045 mg / kg of the recombinant fusion protein in a single dose. In some embodiments, the subject is administered 0.09 mg / kg of the recombinant fusion protein in a single dose. In some embodiments, the subject is administered 0.27 mg / kg of the recombinant fusion protein in a single dose.

[0319] In some embodiments, a dose of the recombinant fusion protein comprises from 0.03 mg / kg to 0.30 mg / kg. In some embodiments, a dose of the recombinant fusion protein comprises from 0.03 mg / kg to 0.27 mg / kg. In some embodiments, a dose of the recombinant fusion protein comprises from 0.03 mg / kg to 0.050 mg / kg. In some embodiments, a dose of the recombinant fusion protein comprises from 0.03 mg / kg to 0.09 mg / kg. In some embodiments, a dose of the recombinant fusion protein comprises from 0.045 mg / kg to 0.27 mg / kg. In some embodiments, a dose of the recombinant fusion protein comprises from 0.045 mg / kg to 0.20 mg / kg. In some embodiments, a dose of the recombinant fusion protein comprises from 0.045 mg / kg to 0.09 mg / kg. In some embodiments, a dose of the recombinant fusion protein comprises 0.03 mg / kg. In some embodiments, a dose of the recombinant fusion protein comprises 0.045 mg / kg. In some embodiments, a dose of the recombinant fusion protein comprises 0.09 mg / kg. In some embodiments, a dose of the recombinant fusion protein comprises 0.27 mg / kg. In some embodiments, the dose is formulated as an injectable solution.

[0320] In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the same is administered to a subject in a dose ranging from 0.2 mg to 2 mg. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the same is administered to a subject in a dose ranging from 2 mg to 6 mg. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the same is administered to a subject in a dose ranging from 4 mg to 10 mg. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the same is administered to a subject in a dose ranging from 5 mg and 15 mg. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the same is administered to a subject in a dose ranging from 1 mg and 30 mg. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the same is administered to a subject in a dose ranging from 5 mg and 20 mg.

[0321] In one embodiment, a single one time dose of the recombinant fusion protein or pharmaceutical composition comprising the same is administered to a subject. In another embodiment, a total of 2 doses are administered to the subject. In another embodiment, a total of 2 or more doses are administered to the subject. In another embodiment, a total of 4 or more doses are administered to the subject. In another embodiment, a total of 10 or more doses are administered to the subject. In another embodiment, a total of 20 or more doses are administered to the subject. In another embodiment, a total of 30 or more doses are administered to the subject.

[0322] In some embodiments, repeat administrations (doses) of compositions of this invention may be undertaken immediately following the first course of treatment or after an interval of days, weeks, or years to achieve the desired effect as further provided herein (e.g. to prevent or treat a cardiovascular disease or condition).

[0323] In some embodiments, the subject is administered a dose of the recombinant fusion protein that results in a serum concentration of between about 100 pg / L and about 2,400 pg / L of the recombinant fusion protein approximately 12 hours after administration. In some embodiments, administration of the recombinant fusion protein results in a serum concentration of between about 200 pg / L and about 2,000 pg / L of the recombinant fusion protein approximately 12 hours after administration. In some embodiments, administration of the recombinant fusion protein results in a serum concentration of between about 200 pg / L and about 1,500 pg / L of the recombinant fusion protein approximately 12 hours after administration. In some embodiments, administration of the recombinant fusion protein results in a serum concentration of between about 200 pg / L and about 300 pg / L approximately 12 hours after administration. In some embodiments, administration of the recombinant fusion protein results in a serum concentration of between about 1,000 pg / L and about 1,500 pg / L approximately 12 hours after administration. In some embodiments, administration of the recombinant fusion protein results in a serum concentration of between about 1,000 pg / L and about 1,800 pg / L approximately 12 hours after administration.

[0324] In some embodiments, administration of the recombinant fusion protein results in an area under the time-concentration curve (in hours*ng / mL) from 0 to infinity (AUCo-inf) of between about 1,500 and about 200,000. In some embodiments, administration of the recombinant fusion protein results in an area under the time-concentration curve from 0 to infinity (AUCo-inf) of between about 2,000 and about 180,000. In some embodiments, administration of the recombinant fusion protein results in an AUCo-inf of between about 8,000 and about 160,000. In some embodiments, administration of the recombinant fusion protein results in an AUCo-inf of between about 8,000 and about 50,000. In some embodiments, administration of the recombinant fusion protein results in an AUCo-inf of between about 2,000 and about 40,000. In some embodiments, administration of the recombinant fusion protein results in an AUCo-inf of between about 8,000 and about 40,000. In some embodiments, administration of the recombinant fusion protein results in an AUCo-inf of about 4,000, about 8,000, about 10,000, about 20,000, about 30,000, about 35,000, about 40,000, about 50,000, about 80,000, about 100,000, about 120,000 or about 150,000. AUCo-infcan be determined by plotting serum concentration of the recombinant fusion protein in the subject after administration over time.

[0325] In some embodiments, administration of the recombinant fusion protein results in a maximum serum concentration (CMax) of between about 250 ng / mL and about 6,500 ng / mL. In some embodiments, administration of the recombinant fusion protein results in a CMax of between about 300 ng / mL and about 6,000 ng / mL. In some embodiments, administration of the recombinant fusion protein results in a Cxiax of between about 400 ng / mL and about 6,000 ng / mL. In some embodiments, administration of the recombinant fusion protein results in a CMax of between about 400 ng / mL and about 5,000 ng / mL. In some embodiments, administration of the recombinant fusion protein results in a CMax of between about 400 ng / mL and about 2,000 ng / mL. In some embodiments, administration of the recombinant fusion protein results in a CMax of between about 450 ng / mL and about 2,500 ng / mL. In some embodiments, administration of the recombinant fusion protein results in a CMax of between about 200 ng / mL and about 2,000 ng / mL.

[0326] In some embodiments, administration of the recombinant fusion protein results in a serum concentration of between about 200 and about 4,000 pg / L of the recombinant fusion protein in the subject approximately 12 hours after administration. In some embodiments, administration of the recombinant fusion protein results in a serum concentration of between about 200 and about 2,000 pg / L of the recombinant fusion protein approximately 12 hours after administration. In some embodiments, administration of the recombinant fusion protein results in a serum concentration of between about 200 and about 1,500 pg / L of the recombinant fusion protein approximately 12 hours after administration. In some embodiments, administration of the recombinant fusion protein results in a serum concentration of between about 100 and about 1,500 pg / L of the recombinant fusion protein approximately 12 hours after administration. In some embodiments, administration of the recombinant fusion protein results in a serum concentration of between about 500 and about 1,500 pg / L of the recombinant fusion protein approximately 12 hours after administration. In some embodiments, administration of the recombinant fusion protein results in a serum concentration of between about 1,000 and about 1,500 pg / L of the recombinant fusion protein approximately 12 hours after administration. In some embodiments, administration of the recombinant fusion protein results in a serum concentration of between about 1,000 and about 1,800 pg / L of the recombinant fusion protein approximately 12 hours after administration.

[0327] In some embodiments, the recombinant fusion protein has a half-life of between about 5 and about 25 hours in a human subject. In some embodiments, the recombinant fusionprotein has a half-life of between about 5 and about 20 hours in a human subject. In some embodiments, the recombinant fusion protein has a half-life of between about 8 and about 19 hours in a human subject. In some embodiments, the recombinant fusion protein has a halflife of about 5 hours, about 8 hours, about 10 hours, about 11 hours, about 15 hours, about 18 hours or about 20 hours in a human subject.

[0328] In some embodiments, administration of the recombinant fusion protein to the subject increases blood serum concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP). In some embodiments, blood serum concentration of NT-proBNP is increased about 20 to about 30 hours, about 22 to about 28 hours, about 24 to about 26 hours, or any range therebetween, after administration of the recombinant fusion protein when compared to a blood serum concentration of NT-proBNP in the subject prior to administration of the recombinant fusion protein. In some embodiments, blood serum concentration of NT-proBNP is increased about 24 hours after administration of the recombinant fusion protein when compared to a blood serum concentration of NT-proBNP in the subject prior to administration of the recombinant fusion protein. In some embodiments, blood serum concentration of NT-proBNP is increased at least 2 days after administration of the recombinant fusion protein when compared to a blood serum concentration of NT-proBNP in the subject prior to administration of the recombinant fusion protein. In some embodiments, blood serum concentration of NT-proBNP is increased at least 7 days after administration of the recombinant fusion protein when compared to a blood serum concentration of NT- proBNP in the subject prior to administration of the recombinant fusion protein. In some embodiments, blood serum concentration of NT-proBNP is increased at least 10 days after administration of the recombinant fusion protein when compared to a blood serum concentration of NT-proBNP in the subject prior to administration of the recombinant fusion protein. In some embodiments, blood serum concentration of NT-proBNP is increased at least 15 days after administration of the recombinant fusion protein when compared to a blood serum concentration of NT-proBNP in the subject prior to administration of the recombinant fusion protein. In some embodiments, the concentration of NT-proBNP is increased at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after administration of the recombinant fusion protein. In some embodiments, the concentration of NT-proBNP is increased at least 2, 7, or 15 days after administration of the recombinant fusion protein.

[0329] In some embodiments, the blood serum concentration of NT-proBNP in the subject is increased by at least 30% at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least900% or at least 1,000% after administration of the recombinant fusion protein. In some embodiments, the blood serum concentration of NT -proBNP in the subject is increased by at least 30% at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900% or at least 1,000% about 20 hours to about 30 hours (e.g., about 22 hours, about 24 hours or about 26 hours) after administration of the recombinant fusion protein. In some embodiments, the blood serum concentration of NT-proBNP in the subject is increased by at least 30% at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900% or at least 1,000% about 24 hours after administration of the recombinant fusion protein.

[0330] In some embodiments, the blood serum concentration of NT-proBNP in the subject is increased by about 1.2X, by about 1.5X, by about 2X, by about 2.5X, by about 3X, by about 3.5X, by about 4X, by about 4.5X, or by about 5X after administration of the recombinant fusion protein. In some embodiments, the blood serum concentration of NT-proBNP in the subject is increased by about 1. 2X, by about 1.5X, by about 2X, by about 2.5X, by about 3X, by about 3.5X, by about 4X, by about 4.5X, or by about 5X about 20 hours to about 30 hours (e.g., about 22 hours, about 24 hours or about 26 hours) after administration of the recombinant fusion protein.

[0331] In some embodiments, the concentration of NT-proBNP is increased by at least 30% at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 1,100% or at least 1,400% at least 24 hours, at least 2 days, at least 7 days or at least 15 days after administration of the recombinant fusion protein. In some embodiments, the concentration of NT-proBNP is increased by at least 150%, at least 300%, at least 500% at least 600%, at least 800% or at least 1400%, or at least 1600% at least 24 hours after administration of the recombinant fusion protein. In some embodiments, the concentration of NT-proBNP is increased by at least 30%, at least 50%, at least 150%, at least 300%, at least 500%m at least 600%, at least 800% or at least 1400% 2 days after administration of the recombinant fusion protein. In some embodiments, the blood serum concentration of NT-proBNP is increased by at least 30%, at least 50%, at least 100%, at least 500%, at least 700% or at least 900% 5 days after administration of the recombinant fusion protein. In some embodiments, the blood serum concentration of the NT-proBNP is increased by at least 500% or at least 700% 10 days after administration of the recombinant fusion protein. In some embodiments, the bloodserum concentration of the NT -proBNP is increased by at least 100% or at least 150% 15 days after administration of the recombinant fusion protein.

[0332] In some embodiments, the blood serum concentration of NT-proBNP is increased by at least 30%, at least 50%, at least 150%, at least 300%, at least 500%, at least 600%, at least 800%, or at least 1400% 2 days after administration of the recombinant fusion protein. In some embodiments, the blood serum concentration of NT-proBNP is increased by at least 30%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, or at least 1500% 7 days after administration of the recombinant fusion protein. In some embodiments, the blood serum concentration of NT-proBNP is increased by at least 30%, at least 50%, at least 70%, at least 100%, or at least 200% 15 days after administration of the recombinant fusion protein.

[0333] In some embodiments, the blood serum concentration of NT-proBNP returns to a baseline concentration after about 20 to 60 (e.g., about 25 to about 55, about 25 to about 50, about 30 to about 50, about 25 to about 35, or any range therebetween) days after administration of the recombinant fusion protein, i.e. the blood serum concentration of NT- proBNP is similar, or substantially similar to the blood serum concentration of NT-proBNP in the subject prior to administration of the recombinant fusion protein. In some embodiments, the blood serum concentration of NT-proBNP returns to within about 2%, within about 5%, within about 7% or within about 10% of the blood serum concentration of NT-proBNP in the subject prior to administration of the recombinant fusion protein. In some embodiments, the blood serum concentration of NT-proBNP returns to a baseline concentration after about 30 days after administration of the recombinant fusion protein.

[0334] In some embodiments, the blood serum concentration of NT-proBNP is reduced long term after administration of the recombinant fusion protein, when compared to a baseline level prior to administration of the recombinant fusion protein. For example, the level of NT- proBNP in the subject 100 days, 150 days, 180 days, 200 days or more after administration of the recombinant fusion protein is less than a baseline level before administration of the recombinant fusion protein. In some embodiments, the level of NT-proBNP is at least 2%, at least 5%, at least 7%, at least 10%, at least 12%, at least 15% or at least 20% less than a baseline level of NT-proBNP.

[0335] In some embodiments, for example those embodiments in which the subject is administered multiple therapeutically effective doses of the recombinant fusion protein disclosed herein, the blood serum concentration of NT-proBNP (e.g., when measured about 20 hours to 2 days after administration of the recombinant fusion protein) shows little or littleor no tachyphylaxis. In some embodiments, the blood serum concentration of NT-proBNP shows little or no tachyphylaxis when measured about 24 hours after administration of the recombinant fusion protein.

[0336] In some embodiments, the blood serum concentration of NT-proBNP increases after administration of a 2nd, 3rd, 4th, 5th, 6thor further therapeutically effective dose of the recombinant fusion protein, as well as after administration of a 1sttherapeutically effective dose of the recombinant fusion protein. In some embodiments, the blood serum concentration of NT-proBNP increases after one or more of a 2nd, 3rd, 4th, 5th, 6thor further therapeutically effective dose by a similar, or substantially similar amount, as observed after administration of the 1sttherapeutically effective dose of the recombinant fusion protein. In some embodiments, if the blood serum concentration of NT-proBNP increases by about 1. 2X, by about 1.5X, by about 2X, by about 2.5X, by about 3X, by about 3.5X, by about 4X, by about 4.5X, or by about 5X after administration of the first therapeutically effective dose recombinant fusion protein, the blood serum concentration of NT-proBNP increases by similar, or substantially similar amount after administration of one or more of a 2nd, 3rd, 4th, 5th, 6thor further therapeutically effective dose of the recombinant fusion protein. In some embodiments, when the blood serum concentration of NT-proBNP is increased about 24 hours after administration of a 2ndor further (e.g., 2nd, 3rd, 4th, 5thor 6th) therapeutically effective dose of the recombinant fusion protein, the blood serum concentration is within about 2%, within about 5%, within about 7%, within about 10%, within about 15%, within about 20%, or within about 30% of the blood serum concentration of NT-proBNP observed about 24 hours after administration of the first therapeutically effective dose of the recombinant fusion protein. As a further non-limiting example, when measured about 24 hours after administration a first therapeutically effective dose, and after administration of a second, third, or fourth therapeutically effective dose of the recombinant fusion protein, each dose being administered 4 weeks apart, the increase in blood serum concentration of NT- proBNP is similar, or substantially similar, to the increase blood serum concentration of NT- proBNP observed about 24 hours after administration of the first dose. As a still further nonlimiting example, in a series of doses administered every 4 weeks, the NT-proBNP blood serum concentration observed about 24 hours after administration of the fourth dose is within about 2%, within about 5%, within about 7%, within about 10%, within about 15%, within about 20%, or within about 30% of the blood serum concentration of NT-proBNP observed about 24 hours after administration of the first dose.

[0337] Without wishing to be bound by theory, it is thought that overall levels of NT- proBNP in the subject will decrease with repeated administration of the recombinant fusion protein described herein, as cardiovascular health improves. Thus, comparing relative increases of NT-proBNP blood serum concentration, determined by measuring NT-proBNP blood serum concentration before and after administration of a first therapeutically effective dose of the recombinant fusion protein, and before and after administration of a second or further therapeutically effective dose of the recombinant fusion protein, is indicative of efficacy of the recombinant fusion protein.

[0338] In some embodiments, for example those embodiments comprising repeated administration of a therapeutically effective amount of the recombinant fusion protein described herein, a relative increase in a blood serum concentration of NT-proBNP after administration of a second or further therapeutically effective dose is similar, or substantially similar, to a relative increase in a blood serum concentration of NT-proBNP after administration of a first therapeutically effective dose. Methods of calculating relative increase are within skill of the ordinarily skilled artisan, and include, for example taking the difference between the increased value and a baseline value, and normalizing to the baseline value. Such relative increase can be presented as, e.g., a percentage, or a fold change relative to the baseline value.

[0339] In some embodiments, the relative increase in blood serum concentration of NT- proBNP after the first therapeutically effective dose is relative to a baseline blood serum concentration of NT-proBNP in the subject prior to administration of the first therapeutically effective dose. Baseline blood serum concentration of NT-proBNP in the subject prior to administration of the first therapeutically effective dose can be determined at any suitable time, for example weeks, days, hours or minutes prior to administration of the first dose of the recombinant fusion protein. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after the second or further therapeutically effective dose is relative to a baseline concentration blood serum of NT-proBNP in the subject prior to administration of the second or further therapeutically effective dose of the recombinant fusion protein. When a subject is administered multiple doses of the recombinant fusion protein disclosed herein, each dose can result in a transient increase in blood serum concentration of NT-proBNP. Thus, the baseline concentration of NT-proBNP prior to administration of a second or further dose of the recombinant fusion protein is determined after the subject’s NT-proBNP concentration returns to baseline after administration of the previous dose of the recombinant protein. The time needed for the NT-proBNP concentrationto return to baseline will depend on the dose administered, and determination of an appropriate timeframe is within the skill of the ordinarily skilled artisan. In some embodiments, the baseline concentration of NT-proBNP in the subject prior to administration of the second or further therapeutically effective dose is determined at least 14 days (e.g., at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more days) after administration of any previous therapeutically effective dose of the recombinant fusion protein to the subject. In some embodiments, the baseline concentration of NT-proBNP in the subject prior to administration of the second or further therapeutically effective doses is determined within 24 hours prior to administration of the second or further therapeutically effective dose (e.g., within 1-24 hours of administering the second or further therapeutically effective dose). In some embodiments, the baseline concentration of NT-proBNP in the subject prior to administration of the second or further therapeutically effective dose is determined immediately prior to (e.g., within minutes to several hours) administering the second or further therapeutically effective dose to the subject. In some embodiments, baseline NT-proBNP levels are determined approximately the same period of time before administration of the first dose, and administration of the second or further dose (e.g., both are determined within 24 hours of administration). In some embodiments, the baseline concentration of NT-proBNP in the subject prior to administration of the first therapeutically effective dose and / or the baseline concentration of NT-proBNP in the subject prior to administration of the second or further therapeutically effective dose is determined within 24 hours prior to (e.g., within minutes to several hours) administration of the first therapeutically effective dose and / or second or further therapeutically effective dose.

[0340] The skilled artisan will also appreciate that two observed relative increases need not be identical to be substantially similar. For example, the relative increase observed upon administration of the second or further therapeutically effective dose can be within about 2%, within about 5%, within about 7%, within about 10%, within about 15%, within about 20%, or within about 30% of the relative increase observed upon administration of the first therapeutically effective dose.

[0341] In some embodiments, the relative increase in blood serum concentration of NT- proBNP after administration of the first therapeutically effective dose and after administration of the second or further therapeutically effective dose is at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 600%, at least about 700%, at least about800%, at least about 900% or at least about 1,000%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is at least about 50%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is at least about 100%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is at least about 150%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is at least about 200%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is at least about 250%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is at least about 300%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is at least about 400%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is at least about 500%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is at least about 600%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is at least about 700%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is at least about 800%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is at least about 900%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is at least about 1,000%.

[0342] In some embodiments, the relative increase in blood serum concentration of NT- proBNP after administration of the first dose and after administration of the second or further dose is between about 50% and about 1,000%, between about 50% and about 500%, between about 50% and about 300%, between about 100% and about 800%, between about 100% andabout 500%, between about 100% and about 300%, between about 100% and about 200%, between about 150% and about 700%, between about 150% and about 500%, between about 150% and about 250%,, between about 200% and about 800%, between about 300% and about 800%, between about 500% and about 900%, between about 400% and about 700%, between about 300% and about 600%, or between about 400% and about 600%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is between about 50% and about 1000%, between about 100% and about 800%, between about 100% and about 500%, between about 50% and about 300%, between about 100% and about 200%, between about 100% and about 300%, between about 200% and about 800%, between about 300% and about 800%, or between about 500% and about 900%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is between about 50% and about 250%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration second or further dose is between about 70% and about 300%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is between about 100% and about 200%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is between about 100% and about 300%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is between about 100% and about 500%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is between about 100% and about 150%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is between about 200% and about 800%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is between about 400% and about 800%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is between about 500% and about 700%. In some embodiments, the relative increase in bloodserum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is between about 500% and about 1,00%. In some embodiments, the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and after administration of the second or further dose is between about 700% and about 1,000%.

[0343] Without wishing to be bound by theory, it is thought that consistent pharmacodynamic activity of markers such as NT-proBNP can result in consistent, and cumulative, efficacy with repeated administration of the recombinant fusion proteins described herein.

[0344] In some embodiments, the recombinant fusion protein or pharmaceutical composition comprising same is administered as part of a combination therapy.

[0345] As used herein, the terms “combination treatment,” “combination therapy,” and “cotherapy” are used interchangeably and generally refer to treatment modalities featuring a recombinant fusion protein or pharmaceutical composition comprising the same as provided herein and an additional therapeutic agent. Typically, combination treatment modalities are part of a specific treatment regimen intended to provide a beneficial effect from the concurrent action of the therapeutic agent combination. The beneficial effect of the combination may include, but is not limited to, pharmacokinetic or pharmacodynamic coaction resulting from the combination of therapeutic agents. Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected). In some embodiments, combination treatment comprises administration of two or more therapeutic agents in a sequential manner, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. 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 in multiple, separate dosage forms for the therapeutic agents. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. The therapeutic agents can be administered according to the same or to a different administration interval. For example, a first therapeutic agent of the combination selected may be administered by intravenous injection while the other therapeutic agents of the combinationmay be administered orally. Alternatively, for example, all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection.

[0346] In some embodiments, combination therapy also embraces the administration of the therapeutic agents as described above in further combination with other biologically active ingredients and non-drug therapies (e.g., surgery, radiation treatment or implantation of a medical device). Where the combination therapy further comprises a non-drug treatment, the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.

[0347] In some embodiments, the additional therapeutic agent is a chemotherapeutic agent (also referred to as an anti-neoplastic agent or anti -proliferative agent), e.g., an alkylating agent; an antibiotic; an anti-metabolite; a detoxifying agent; an interferon; a polyclonal or monoclonal antibody; an EGFR inhibitor; a HER2 inhibitor; a histone deacetylase inhibitor; a hormone; a mitotic inhibitor; an MTOR inhibitor; a multi-kinase inhibitor; a serine / threonine kinase inhibitor; a tyrosine kinase inhibitors; 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 a protein methyltransferase), a cytidine analogue drug or any chemotherapeutic, an immune checkpoint inhibitor, a platinum based antineoplastic agent, a CDK inhibitor, a PARP inhibitor or any anti -neoplastic or anti -proliferative agent known to those of skill in the art.

[0348] Exemplary alkylating agents suitable for use according to 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).

[0349] 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).

[0350] Exemplary anti-metabolites 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 (T arabine PFS).

[0351] Exemplary detoxifying agents include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).

[0352] Exemplary interferons include, but are not limited to, interferon alfa-2b (Intron A) or interferon alfa-2a (Roferon-A).

[0353] 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.

[0354] Exemplary EGFR inhibitors include, but are not limited to, gefitinib (Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva); panitumumab (Vectibix); PKL166; canertinib (CI-1033); matuzumab (EMD 72000) or EKB-569.

[0355] Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin); lapatinib (Tykerb) or AC-480.

[0356] Histone Deacetylase Inhibitors include, but are not limited to, vorinostat (Zolinza).

[0357] 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).

[0358] 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).

[0359] Exemplary MTOR inhibitors include, but are not limited to, everolimus (Afinitor) or temsirolimus (Torisel); rapamune, ridaforolimus; or AP23573.

[0360] Exemplary multi-kinase inhibitors include, but are not limited to, sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474; PKC-412; motesanib; or AP24534.

[0361] 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; Azdl l52; Arry- 142886 (AZD-6244); SCIO-469; GW681323; CC-401; CEP-1347 or PD 332991.

[0362] 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 (Vectibix); 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.

[0363] Exemplary VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin), sorafenib (Nexavar), sunitinib (Sutent), ranibizumab, pegaptanib, or vandetinib.

[0364] Exemplary microtubule targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristin, vinblastin, nocodazole, epothilones and navelbine.

[0365] Exemplary topoisomerase poison drugs include, but are not limited to, teniposide, etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin and idarubicin.

[0366] Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxol.

[0367] Exemplary immune checkpoint inhibitors include programmed cell death 1 (PD-1), CD274 molecule (PD-L1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4) inhibitors. Exemplary PD-1 inhibitors include Pembrolizumab, Nivolumab and Cemiplimab. Exemplary PD-L1 inhibitors include Atezolizumab, Avelumab and Durvalumab. Exemplary CLTA4 inhibitors include Ipilimumab.

[0368] Exemplary platinum based antineoplastic agents include Cisplatin and Carboplatin.

[0369] Exemplary cyclin dependent kinase (CDK) inhibitors include abemaciclib, palbociclib, and riboci clib.

[0370] Exemplary poly (ADP-ribose) polymerase (PARP) inhibitors include talazoparib, olaparib, rucaparib, niraparib and veliparib.

[0371] Exemplary general chemotherapeutic, anti -neoplastic, anti -proliferative agents include, but are not limited to, altretamine (Hexalen); isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin (Vesanoid); azacitidine (Vidaza); bortezomib (Velcade) asparaginase (Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine (Matulane); pegaspargase (Oncaspar); denileukin diftitox (Ontak); porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid); bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel); arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosine (Leucenol); (IM tegafur - 0.4 M 5-chloro-2,4-dihydroxypyrimidine - I M potassium oxonate) or lovastatin.

[0372] 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, a pharmaceutical composition provided herein may be administered in combination with radiation therapy. Radiation therapy can also be administered in combination with a pharmaceutical composition provided herein and another chemotherapeutic agent described herein as part of a multi-agent therapy. In yet another aspect, a pharmaceutical composition provided herein may be administered in combination with standard chemotherapy combinations such as, but not restricted to, CMF (cyclophosphamide, methotrexate and 5 -fluorouracil), CAF (cyclophosphamide, adriamycin and 5 -fluorouracil), AC (adriamycin and cyclophosphamide), FEC (5-fluorouracil, epirubicin, and cyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide, and paclitaxel), rituximab, Xeloda (capecitabine), Cisplatin (CDDP), Carboplatin, TS-1 (tegafur, gimestat and otastat potassium at a molar ratio of 1 :0.4: 1), Camptothecin-11 (CPT-11, Irinotecan or Camptosar™), CHOP (cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisone or prednisolone), R-CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin, oncovin, prednisone or prednisolone), or CMFP (cyclophosphamide, methotrexate, 5-fluorouracil and prednisone).

[0373] In some preferred embodiments, a pharmaceutical composition provided herein may 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. Kinase inhibitors described herein are small molecules, polynucleic acids, polypeptides, or antibodies.

[0374] Exemplary kinase inhibitors include, but are not limited to, Bevacizumab (targets VEGF), BIBW 2992 (targets EGFR and Erb2), Cetuximab / Erbitux (targets Erbl), Imatinib / Gleevec (targets Bcr-Abl), Trastuzumab (targets Erb2), Gefitinib / Iressa (targets EGFR), Ranibizumab (targets VEGF), Pegaptanib (targets VEGF), Erlotinib / Tarceva (targets Erbl), Nilotinib (targets Bcr-Abl), Lapatinib (targets Erbl and Erb2 / Her2), GW- 572016 / lapatinib ditosylate (targets HER2 / Erb2), Panitumumab / Vectibix (targets EGFR), Vandetinib (targets RET / VEGFR), E7080 (multiple targets including RET and VEGFR), Herceptin (targets HER2 / Erb2), PKI-166 (targets EGFR), Canertinib / CI-1033 (targets EGFR), Sunitinib / SU-11464 / Sutent (targets EGFR and FLT3), Matuzumab / Emd7200 (targets EGFR), EKB-569 (targets EGFR), Zd6474 (targets EGFR and VEGFR), PKC-412 (targets VEGR and FLT3), Vatalanib / Ptk787 / ZK222584 (targets VEGR), CEP-701 (targets FLT3), SU5614 (targets FLT3), MLN518 (targets FLT3), XL999 (targets FLT3), VX-322 (targets FLT3), Azd0530 (targets SRC), BMS-354825 (targets SRC), SKI-606 (targets SRC), CP-690 (targets JAK), AG-490 (targets JAK), WHI-P154 (targets JAK), WHI-P131 (targets JAK), sorafenib / Nexavar (targets RAF kinase, VEGFR- 1, VEGFR-2, VEGFR-3, PDGFR- 13, KIT, FLT-3, and RET), Dasatinib / Sprycel (BCR / ABL and Src), AC-220 (targets Flt3), AC-480 (targets all HER proteins, “panHER”), Motesanib diphosphate (targets VEGF 1-3, PDGFR, and c-kit), Denosumab (targets RANKL, inhibits SRC), AMG888 (targets HER3), and AP24534 (multiple targets including Flt3).

[0375] In some embodiments, for example those embodiments wherein the recombinant fusion protein is administered to treat atrial fibrillation, the combination therapy can include administration of a beta blocker (e.g, bisprolol or metoprolol succinate), a calcium channel blocker (e.g, diltiazem or verapamil), digoxin, an anti-arrhythmic medication (e.g, propafenone, flecainide, sotalol, dofetilide, amiodarone, and dronedarone), or a blood thinner (e.g., warfarin, apixabab, dabigatran, edoxaban, or rivaroxaban). In some embodiments, the combination therapy can include cardioversion therapy to reset the heart sinus rhythm (e.g., electrical or drug cardioversion). In some embodiments, the combination therapy can include ablation, e.g. AV node ablation or Maze procedure. Ablation uses a scalpel, or heat or cold to create small scars on the heart that block faulty electrical signals and restore a normal heart rhythm.Kits and Articles of Manufacture

[0376] The disclosure provides kits comprising the recombinant fusion proteins described herein. For example, the various constituents of the pharmaceutical compositions comprisingthe recombinant fusion proteins described herein can pre-measured and / or prepackaged and / or ready for use without additional measurement or preparation. Alternatively, the pharmaceutical compositions comprising the recombinant fusion proteins described herein may be in concentrated form, and ready for dilution into a suitable carrier for intravenous infusion.

[0377] The present invention also comprises kits for conducting / using the methods and / or the compositions described herein. In particular, these kits optionally include, e.g., appropriate recombinant fusion protein (and optionally mixtures of a number of such proteins and / or additional compounds for performing synergistic treatments, see, above). Additionally, such kits can also comprise appropriate excipients (e.g., pharmaceutically acceptable excipients) for performing therapeutic and / or prophylactic treatments of the invention. Such kits optionally contain additional components for the assembly and / or use of the compositions of the invention including, but not limited to, e.g., diluents, etc.

[0378] The compositions described herein are optionally packaged to include all (or almost all) necessary components for performing the methods of the disclosure or for using the compositions of the disclosure (optionally including, e.g., written instructions for the use of the methods / compositions of the invention). For example, the kits can optionally include such components as, e.g., buffers, reagents, serum proteins, antibodies, substrates, etc. In the case of prepackaged reagents, the kits optionally include pre-measured or pre-dosed amounts that are ready to incorporate into the methods without measurement, e.g. , pre-measured fluid aliquots, or pre-weighed or pre-measured solid reagents that can be easily reconstituted by the end-user of the kit.

[0379] Such kits also typically include appropriate instructions for performing the methods of the disclosure and / or using the compositions of the disclosure. In some embodiments, the components of the kits / packages are provided in a stabilized form, so as to prevent degradation or other loss during prolonged storage, e.g, from leakage. A number of stabilizing processes / agents are widely used for reagents, etc. that are to be stored, such as the inclusion of chemical stabilizers (z.e., enzymatic inhibitors, microbicides / bacteriostats, anticoagulants), etc. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route ofadministration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts.

[0380] The present invention further provides a kit for preventing, treating or delaying a cardiovascular disease or condition in a human, wherein the kit comprises one or more doses of pharmaceutical composition comprising a recombinant fusion protein disclosed herein used for preventing, treating or delaying a cardiovascular disease or condition, and instructions on how to use the pharmaceutical preparation or composition. Exemplary dosages include between 0.03 mg / kg and 0.30 mg / kg, between 0.03 mg / kg and 0.27 mg / kg, between 0.03 mg / kg and 0.050 mg / kg, between 0.03 mg / kg and 0.09 mg / kg, between 0.045 mg / kg and 0.27 mg / kg, between 0.045 mg / kg and 0.20 mg / kg, or between 0.045 mg / kg and 0.09 mg / kg of the recombinant fusion proteins disclosed herein. Further exemplary dosages include 0.03 mg / kg, 0.045 mg / kg, 0.09 mg / kg or 0.27 mg / kg of the recombinant fusion proteins disclosed herein. In some embodiments, the dose is formulated as an injectable solution. As a further example, the recombinant fusion protein or pharmaceutical composition comprising the same can be formulated in a dose ranging from about 0.2 mg to about 2 mg, from about 2 mg to about 6 mg, from about 4 mg to about 10 mg, from about 5 mg to about 15 mg, or from about 1 mg to about 30 mg of the recombinant fusion protein. In another embodiment, the recombinant fusion protein or pharmaceutical composition comprising the same can be formulated in a dose ranging from about 5 mg to about 20 mg of the recombinant fusion protein.

[0381] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / 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 inventive embodiments described herein. It is, therefore, to beunderstood that the foregoing embodiments are presented by way of example only and that within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present 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, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0382] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.EXAMPLESExample 1: Phase 2 Study in Adults with Chronic Heart FailureObjectives and Endpoints

[0383] A Phase 2, randomized, double-blind, placebo-controlled, multiple dose study is currently being conducted to assess the safety, tolerability, and efficacy of NRG- 1 HER3 antibody fusion protein (JK07, comprising SEQ ID NOS: 3 and 14) in participants 18 to 85 years of age with HF. A schematic of the study is shown as FIG. 1.

[0384] This study includes heart failure (HF) patients with left ventricular ejection fraction (LVEF) < 65%. In many respects, regardless of LVEF status, patients with HF have several features in common, including shared risk factors (e.g., age, obesity, type II diabetes mellitus, chronic kidney disease), shared cardiac remodeling features (e.g., LV hypertrophy, endothelial dysfunction, cardiac fibrosis), and shared symptomatic features (e.g., reduced cardiac output, reduced exercise tolerance, dyspnea). While there are many distinct pathogenic features between patients with HF across different ranges of LVEF, an agent such as JK07, which contains the known pleiotropic growth factor NRG- 1 in a fully-human, recombinant form, has the potential to broadly mediate cell signaling pathways therapeutically implicated in all strata of HF pathogenesis.

[0385] Single-dose administration of JK07 was safe and well tolerated at the 0.03 and 0.09 mg / kg dose levels (see Example 5 below). The timeline to maximal effect of JK07 varied (between days 15 and 180) for the 0.03 and 0.09 mg / kg doses, respectively. In addition, levels of NT-proBNP returned to baseline by 30 days after administration (FIGS. 14A-14K),and a cumulative benefit was observed with repeated dosing in Rhesus monkey studies (Example 6, FIG. 44). All of these results support an every four week repeat dosing interval. Findings from GLP (good laboratory practice) repeat dose toxicity studies with JK07 suggest that sustained pharmacologic activity of JK07 can result in toxicity independent of Cmax. In addition, adverse findings generally exhibit greater reversibility with longer recovery periods. As such, q4w (every four weeks) dosing is being evaluated in this study to achieve cumulative efficacy while mitigating potential total exposure driven toxicity.

[0386] This study is designed to examine the safety and tolerability of 0.045 mg / kg and 0.09 mg / kg JK07 dosed q4w for 4 total doses in participants with New York Heart Association (NYHA) Class II-III heart failure. The study design is based on nonclinical and clinical data on the characteristics of JK07 as a potential therapeutic agent in heart failure.

[0387] There are 2 cohorts in this study:Cohort 1 : HF participants with left ventricular ejection fraction (LVEF) < 40%; n = 204 (“n” refers to the number of participants); andCohort 2: HF participants with LVEF > 40% and < 65%; n= 78.

[0388] Participants in Cohorts 1 and 2 are randomized to the following low dose treatment arms:Cohort 1 (LVEF < 40%) :- Treatment Arm 1 : Four, once every 4 weeks (q4w) doses of low dose JK07 (0.045 mg / kg); n = 68- Treatment Arm 2: Four, q4w doses of low dose placebo; n = 34Cohort 2 (LVEF > 40% and < 65%) :- Treatment Arm 1 : Four, q4w doses of low dose JK07 (0.045 mg / kg); n = 26- Treatment Arm 2: Four, q4w doses of low dose placebo; n = 13

[0389] The primary objectives and endpoints of Cohorts 1 and 2 are presented in Table 1.1 and Table 1.2, respectively. Full objectives and endpoints for the two cohorts are presented as FIGS. 16 and 17.Table 1.1 Cohort 1 (New York Heart Association (NYHA) Class II-III with LVEF < 40%) Primary Objectives and EndpointsTable 1.2 Cohort 2 (NYHA Class II-III with LVEF > 40% and < 65%) Primary Objective and Endpoint

[0390] A descriptive (no formal statistical testing) interim analysis is conducted after 50% of participants have been enrolled at the low dose level, and is described below. Randomization to high dose (Treatment Arm 3) or matching high dose placebo (Treatment Arm 4) in both Cohort 1 (LVEF < 40%) and Cohort 2 (LVEF > 40% and < 65%) will open following review of interim analysis data.Participants in Cohorts 1 and 2 may be randomized to the high dose treatment arms:Cohort 1 (LVEF < 40%) :- Treatment Arm 3: Four, q4w doses of high dose JK07 (0.09 mg / kg); n = 68- Treatment Arm 4: Four, q4w doses of high dose placebo; n = 34Cohort 2 (LVEF > 40% and < 65%) :- Treatment Arm 3: Four, q4w doses of high dose JK07 (0.09 mg / kg); n = 26- Treatment Arm 4: Four, q4w doses of high dose placebo; n = 13

[0391] All participants are receiving a total of 4 doses of their assigned dose q4w (Weeks 1, 5, 9, and 13). All participants are followed for a total of 52 weeks, with the primary and exploratory efficacy endpoints of the study being evaluated at Weeks 26 and 52, respectively. Participants undergo protocol-specified safety assessments (e.g., safety laboratory assessments, physical examinations) and efficacy assessments (e.g., blood sampling forpharmacodynamic [PD] biomarkers, and 2-dimensional transthoracic echocardiography [2D- TTE]).

[0392] The dosing day for the participant may be delayed by up to 7 days from the time of the original planned dosing day (including within the protocol-defined dosing day visit window) if an adverse event occurs. In instances where dosing cannot safely resume within 7 days from the original planned dosing day, the dose will be skipped and dosing should resume at the next protocol-defined dosing day, if applicable. In the event of any delayed or skipped dosing, all other planned dosing should remain on the original protocol-defined schedule.Participants

[0365] Approximately 204 participants in Cohort 1 and approximately 78 participants in Cohort 2 will be enrolled (to be enrolled means participants who have provided informed consent and received at least one dose of JK07 or placebo).

[0366] Key inclusion and exclusion criteria are listed below.Key Inclusion Criteria• Participants, 18 to 85 years of age, inclusive, with heart failure.• Participants with NYHA Class II-III for at least 8 weeks prior to screening and:Cohort 1 (LVEF < 40%): A screening LVEF < 40% in the absence of hemodynamically significant and / or severe valvular disease (other than mitral and / or tricuspid regurgitation) on 2D-TTE, with at least one other documented LVEF measurement < 40% within the last 52 weeks prior to screening determined by echocardiography, radionuclide ventriculogram, cardiac computed tomography (CT), or cardiac magnetic resonance imaging (CMR).■ In participants with a screening LVEF of > 30% and < 40%, elevated level of N-terminal pro-brain natriuretic peptide (NT-proBNP > 300 pg / mL).■ Participants who have undergone coronary revascularization (percutaneous coronary intervention or coronary artery bypass grafting) or implantation of a cardiac resynchronization therapy (CRT) device must have a measurement of LVEF < 40% at least 12 weeks after the intervention to be eligible for screening. orCohort 2 (LVEF > 40% and < 65%) : A screening LVEF between 40% and 65% in the absence of hemodynamically significant and / or severe valvular disease on 2D-TTE,with at least one other documented LVEF measurement between 40% and 65% within 52 weeks prior to screening as confirmed by echocardiography, radionuclide ventriculogram, cardiac CT, or CMR, and■ Elevated level of NT-proBNP (> 600 pg / mL).■ Documented evidence of paroxysmal (on > 2 occasions > 1 week apart), persistent or permanent atrial fibrillation / flutter by electrocardiogram, Holter, implantable device, or telemetry -based event monitoring of at least 2 hours duration at one of these timepoints within the last 5 years.• Stable HF is defined as no hospitalizations for cardiac-related issues within 4 weeks prior to the initial screening visit or between screening and randomization, other than for routine percutaneous procedures such as device battery / generator changes or new pacemaker lead insertion.• Participants should be established on background optimal medical therapy for HF and be treated according to locally recognized guidelines with both drugs and devices, as appropriate.- Guideline-recommended HF medications should be used at recommended doses unless contraindicated or not tolerated.- HF medications should be at stable doses for at least 8 weeks prior to screening, other than diuretics, which should be stable for at least 2 weeks prior to screening.• Screening hemoglobin > 9.0 g / dL.

[0367] In addition, sexually active male participants whose partner(s) are of childbearing potential must agree to use a medically accepted method of effective contraceptive from the start of the study until 90 days after the last dose of study drug. Women of childbearing potential (WoCBP; i.e., those who are not chemically or surgically sterilized or who are not postmenopausal) must present with a negative serum pregnancy test, must not be breastfeeding; and if sexually active with a biologically male partner(s), must be willing and able to adhere to the contraceptive methods from the start of the study until 90 days after the last dose of study drug. Subjects must be capable of providing signed informed consent, and be willing and able to comply with the requirements of the protocol. For participants with a history of cancer, for participants with in situ or resected cancers (other than adequately treated cutaneous basal or squamous cell carcinoma with no evidence of recurrence), theparticipant must have no cancer within the last 5 years. For participants with metastatic cancer, the participant must have no cancer within the last 10 years. Participants with diagnosed pre-malignancies are eligible (e.g., colonic polyps, skin lesions).Key Exclusion Criteria• Uncontrolled hypertension (defined as systolic blood pressure > 180 mmHg or in the opinion of the Investigator) or during the screening visit.• Sustained systolic blood pressure (BP) < 90 mmHg and / or diastolic BP < 50 mmHg (confirmed by a duplicate seated reading) on at least 3 consecutive readings (office visits).• Participants with poor echo windows that preclude accurate repeat assessments of LVEF.• HF due to hypertrophic cardiomyopathy, restrictive and / or infiltrative cardiomyopathy, arrhythmogenic right ventricular dysplasia, Fabry disease, or Noonan syndrome with LV hypertrophy or a positive serum immunofixation result.• Diagnosis of stress-induced (Takotsubo) cardiomyopathy, myocarditis, or peripartum cardiomyopathy.• Diagnosis of chemotherapy- or radiation-induced cardiomyopathy.• Diagnosed with stroke or transient ischemic attack (TIA) within 12 weeks of screening.• History of syncope within the last 12 weeks prior to screening or sustained ventricular tachycardia without an implantable cardioverter-defibrillator.• Moderate or severe aortic and / or mitral valve stenosis.• Medically documented unstable angina, acute coronary syndrome (e.g., myocardial infarction, troponin-positive with symptoms of angina or unstable angina) within the last 8 weeks prior to start of screening.• Medically documented ST-elevation myocardial infarction within 12 weeks of screening.• Any narrow complex tachycardia (AF or atrial flutter) with a resting ventricular rate > 110 beats per minute at screening.• For participants with a history of atrial fibrillation (AF) or atrial flutter, and a CHA2DS2- VASc score of > 2 in men or > 3 in women or per local guidelines, anti coagulation via non-vitamin K oral anticoagulants or warfarin is required.- Percutaneous occlusion of the left atrial appendage alone is not adequate.• AF ablation within the last 12 weeks or planned during the study duration.• Symptomatic bradycardia or second (Mobitz Type II)- or third-degree heart block without a pacemaker.• Cardiac surgery, coronary artery revascularization or indication for coronary artery revascularization, percutaneous coronary intervention, valve repair / replacement or valvuloplasty within 12 weeks prior to screening.• Implantation of a cardiac resynchronization therapy (CRT) device within 12 weeks prior to screening, or intent to implant a CRT device during the course of the study.• Previous cardiac transplantation, or any use of mechanical circulatory support or similar device, or implantation expected after randomization.• Receiving mechanical hemodynamic support (e.g., intra-aortic balloon pump counter pulsation) or invasive mechanical ventilation within the last 8 weeks prior to screening.• Receiving IV inotropes (e.g., dobutamine, milrinone, levosimendan) or IV vasopressors (e.g., epinephrine, norepinephrine, dopamine, or vasopressin) within the last 8 weeks prior to screening.• Receiving IV vasodilators within the last 4 weeks prior to screening.• Receiving noninvasive mechanical ventilation (e.g., bilevel positive airway pressure or continuous positive airway pressure within the last 4 weeks prior to screening (the use of noninvasive ventilation for sleep disordered breathing is permitted).

[0368] In addition, participants must not be participating in any other study and have received any other investigational drug, biologic, or device within 30 days prior to screening or 5-half-lives, whichever is longer, or any other investigational implanted device within 30 days prior to screening, or are taking part in a nonmedication study which would interfere with study compliance or outcome assessments. The following subjects are also excluded: subjects with any past participation in a study that has investigated the NRG-1 pathway (e.g., Neucardin, cimaglermin, JK07), pregnant or breastfeeding women, participants with orthopedic impairment that would prohibit credible or adequate assessment of 6-minute walk test (6MWT), who have severe chronic obstructive pulmonary disease, or other severe pulmonary disease, requiring home oxygen, chronic nebulizer therapy, chronic oral steroid therapy or hospitalized for pulmonary decompensation within 12 months of informed consent, who have had any major surgical procedure that would compromise participant safety and / or study integrity, within 4 weeks prior to screening or any planned major surgical procedure during the study period, who have had clinically significant renal dysfunction as measured by the estimated glomerular filtration (eGFR) rate of < 30 mL / min / 1.73 m2(Cockcroft-Gault equation for estimation of creatinine clearance; Cockcroft D.W. et al., Nephron. 1976; 16:31-41) at screening, or a clinically significant change in renal functionbetween screening and baseline, who have received noninvasive mechanical ventilation (e.g., bilevel positive airway pressure or continuous positive airway pressure) within the last 8 weeks prior to screening (the use of noninvasive ventilation for sleep disordered breathing is permitted), or who have clinically significant liver dysfunction at screening as measured by alanine aminotransferase (ALT) > 3.0 x upper limit of normal (ULN), aspartate aminotransferase (AST) > 3.0 * ULN, or total bilirubin > 2 * ULN, other than diagnosed Gilbert’s syndrome, at time of screening. Subjects who actively abuse alcohol or illicit drugs, or who have done so within 6 months prior to randomization (excluding medical or recreational use of marijuana or cannabidiol -based products) are also excluded. Subjects with other medical or psychiatric conditions that would preclude obtaining voluntary consent / assent or would confound the secondary objectives of study may also be excluded.

[0369] A schedule of activities is presented as FIG. 2. In the schedule of activities (a) indicates that on all dosing days, the sequence of assessments prior to and including dosing is recommended to be carried out as follows: (1) physical examination, (2) concomitant medications, (3) urine pregnancy (women of childbearing potential (WoCBP) only), (4) eligibility re-confirmation (Day 1 only), (5) 2D-TTE (this can also be performed up to 1 day prior to dosing), (6) Kansas City Cardiomyopathy Questionnaire 12 (KCCQ-12), (7) vital signs (including BP orthostasis), (8) blood sampling for all required predose laboratory assessments (IV placement in cases where IV is being used to facilitate blood draws), (9) 6- minute walk test (6MWT), (10) predose Electrocardiogram (ECG acquisition, (11) predose pharmacokinetic(s) (PK) sample, and (12) study treatment administration (via IV infusion for approximately 60 minutes, per Pharmacy Manual). Randomization (b) can occur up to 3 days prior to the scheduled Day 1 but only after the participant’s eligibility has been confirmed, (c) indicates that when vital signs and dosing are on the same day, vital signs will be conducted predose, and 30 minutes, 1 hour, and 2 hours post-EOI (end of infusion). A ± 15 minute window is allowed for all vital sign timepoints. On dosing days, assessment of orthostasis must be predose and at 30 minutes and 2 hours post-EOI. Assessment of orthostasis should also be conducted on Days 2 and 86. On non-dosing days, vital signs can be completed at any time during the visit. For the 12-lead ECG (d), single ECG needs to be obtained at all ECG timepoints. For Days 1 and 85, ECGs will be obtained pre-dose, at EOI, 30 minutes post EOI, and once between 2 - 4 hours post EOI, all within 10 minutes of the pharmacokinetic (PK) samples being collected at the same timepoints. PK collection windows on Days 1 and 85 apply to ECG collections on Days 1 and 85. On days 2 and 86, ECGs will be collected once between 18 - 30 hours after EOI. On Days 29 and 57, ECGs will be collected predose, atEOI, and once between 1 - 4 hours after EOI. PK collection windows on Days 29 and 57 apply to ECG collections on Days 29 and 57. Full physical examination (e) will be at screening, Day 180, and Day 360. All other visits will include a brief physical examination. Blood samples (f) will be collected for future biomarker and / or genetic analysis, described below. The VI blood samples for future biomarker and / or genetic analysis will be collected predose. A single 2mL sample will be collected at all timepoints indicated in the Schedule of Activities (FIG. 2) with the exception of VI predose (three, 2 mL samples to be collected) and V12 and EOSZET (Two, 2mL samples to be collected). Blood sampling for future biomarker and / or genetic analysis applies to participants in the United States only. When 2D- TTE (g) and dosing are on the same day, 2D-TTE can be completed up to 1 day before the dosing day. When the 6 minute walk test (6MWT) (h) and dosing are on the same day, the 6MWT should be completed after the KCCQ-12 TSS and within 3 hours prior to dosing. Continuous cardiac rhythm monitoring (i) will be conducted in Cohort 2 (LVEF > 40% and < 65%) participants only. Continuous cardiac rhythm monitoring will be conducted for 14 consecutive days during screening, for 14 continuous days between Day 166 (± 7 days) and Day 180 (± 3 days), and for 14 continuous days between Day 324 (± 3 days) and Day 360 (± 3 days). CMR (j) is optional for a subset of participants in Cohort 2 (LVEF > 40% and < 65%) who elect to participate. The baseline CMR should occur during the screening period as a separate visit only after the participant’s eligibility has been confirmed. Immunogenicity samples (k) will be collected pre-dose on days 1, 29, 57, 85. Immunogenicity samples are to be collected in the same timeframe as PK sample on Day 2 (between 18 - 36 hours from EOI). Immunogenicity samples can be collected at any time during the visit on Day 150. For PK sampling (1), sampling is conducted on dosing Days 1 and 85. PK sampling will be conducted predose and EOI (± 5 minute window), 30 minutes (± 5 minute window), 1 hour (± 10 minute window), and once between 2 - 4 hours from the EOI. On Days 2 and 86, PK samples are collected once between 18 - 30 hours from the EOI. On dosing Days 29 and 57, PK collections should occur at EOI (± 5 minute window), and once between 1 - 4. NT- proBNP (m) will be collected before the 6MWT on days when NT -proBNP collections and 6MWT occur on the same day. On days 2 (V2) and 86 (V9), NT-proBNP will be collected between 24 - 36 hours post-infusion. Laboratory assessments (n) will include serum chemistry, liver, kidney, lipid, hematologic, coagulation blood sampling, and urinalysis, and are described below, (o) On all dosing days, participants may be kept overnight at clinical trial sites with appropriate facilities. For all participants who are hospitalized overnight, additional PK samples will be collected at 4 and 12 hours post EOI (± 45 minute window).Also, blood sampling for NT -proBNP and high-sensitivity cardiac troponin T (hs-cTnT) will be collected between 24 - 36 hours post-infusion at all dosing visits where overnight hospitalization has occurred. A single collection of vital signs will also be captured just before blood sampling for NT -proBNP and hs-cTnT in the 24-36 hours post-infusion for participants who have been kept overnight in the hospital.Study Treatments

[0370] The study treatments administered in this study are summarized in Table 1.3.Treatment and placebo infusions are identical in physical appearance. All relevant individuals will be blinded to treatment assignment, with the exception of the pharmacy study staff, in order to maintain the double-blinded conduct of the study.Table 1.3 Study Treatment Descriptions

[0371] The infusion is managed through a syringe pump or infusion pump. A needle or catheter between 16G and 21G is required by this protocol (choice as per site procedures). The total infusion time, inclusive of saline flush, should be 60 minutes and should not exceed 75 minutes. Saline flush should be completed within 1-2 minutes following completion of the infusion. The date / time of start and time of stop of the infusion, infusion rate, volume infused, any interruption, and stop / restart are documented. In the event of a presumed acute infusion reaction, institutional standard of care treatment should be undertaken. If the infusion must be stopped due to an AE, the infusion may not be restarted.

[0372] Participants are randomized to study treatment via randomization. Separate randomization schedules are used within each cohort.

[0373] During infusion, should an infusion-related reaction occur, the infusion may be paused and resumed. At any time during the dosing period, if an AE occurs that requires an adjustment to the dosing schedule, the dosing day for the participant may be delayed by up to 7 days from the time of the original planned dosing day (including within the protocol- defined dosing day visit window). In instances where dosing cannot safely resume within 7 days from the original planned dosing day, the dose will be skipped and dosing should resume at the next protocol-defined dosing day, if applicable. In the event of any delayed or skipped dosing, all other planned dosing should remain on the original protocol-defined schedule.Efficacy AssessmentsTwo-Dimensional Transthoracic Echocardiography (2D-TTE)

[0374] Changes in 2D-TTE performance indices, including but not limited to LVEF, left ventricular end-diastolic volume index (LVEDVi), left ventricular end-systolic volume index (LVESVi), stroke volume index cardiac output (SVi CO), cardiac index (CI), left ventricular mass index (LVMi), E / e’ (a parameter to measure early diastolic peak velocity and early diastolic mitral annular velocity), right ventricular systolic pressure (RVSP), left ventricular assist (LAV), left atrial ejection fraction (LAEF), and valvular function, are determined. Potential effects of JK07 are evaluated as change from baseline and are compared with changes from baseline in participants receiving placebo. 2D-TTE will be performed locallyfollowing American Society of Echocardiography guidelines at the time points described in the Schedule of Activities (FIG. 2).Cardiac Magnetic Resonance Imaging (CMR Sub-study)

[0375] Up to 12 participants in Cohort 2 (LVEF > 40% and < 65%) may be enrolled in a CMR sub-study. Participants in the sub-study will undergo CMR at the time points specified in the Schedule of Activities (FIG. 2). Convention, at-rest imaging will be assessed using steady-state free precession cine images acquired in multiple short-axis (every 1 cm throughout the entire LV) and 2 to 3 long-axis planes. To characterize cardiac tissue, gadolinium (0.1 to 0.15 mmol / kg) will be administered intravenously, and contrast-enhanced T1 -weighted images will be acquired after 10 minutes with a segmented inversion-recovery technique in the identical planes (Backhaus S.J. et al., Circulation. 2021; 143: 1484-1498). Function and Quality of Life 6-Minute Walk Test (6MWT)

[0376] The 6MWT is a test to measure the distance a participant can walk for a total of 6 minutes. The participant is instructed to walk as far as possible in 6 minutes on a hard, flat surface. If needed, the participant may rest and pace themselves during this time as they move back and forth along a marked path. The test will be administered according to the American Thoracic Society Guidelines (American Thoracic Society. Am J Respir Crit Care Med. 2002; 166: 111-117) at the time points specified in the Schedule of Activities (FIG. 2). In brief, the 6MWT should be performed indoors, along a long, flat, straight, enclosed corridor with a hard surface with little foot traffic. Any prescribed inhaled bronchodilator medication should be taken within one hour of testing or when the patient arrives for testing. The patient should rest for at least 10 minutes before beginning a 6MWT, and for at least one hour before beginning a 6MWT if any exercise / cardiac rehabilitation has occurred on the same day. Blood pressure (BP) and heart rate (HR) are recorded before the test, and the patient is asked to assess their shortness of breath and fatigue using the Borg scale before and after the test. The patient walks as far as possible for 6 minutes, with standardized verbal encouragement, and distance and post-walk Borg dyspnea and fatigue levels are recorded. Kansas City Cardiomyopathy Questionnaire 12 (KCCQ-12)

[0377] The KCCQ-12 is a 12-item self-administered questionnaire developed to qualitatively and quantitatively measure the patient’s perception of their health status, which includes HF symptoms, impact on physical and social function, and how their HF impacts their quality of life within a 2-week recall period (Dai S. et al., Cardiol Res Pract. 2016; 2016:4571201; Green C.P. et al., J Am Coll Cardiol. 2000; 35: 1245-1255; Spertus J. A. et al., J Am CollCardiol. 2020; 76:2379-2390.). The KCCQ-12 will be administered as shown in the Schedule of Activities (FIG. 2).Safety AssessmentsPhysical Examinations

[0378] Complete physical examinations include, at a minimum, assessments of the CV, respiratory, gastrointestinal, and neurological systems, and will be performed at the time points specified in the Schedule of Activities (FIG. 2). Brief physical examinations include assessments of the skin, lungs, CV system, and abdomen (liver and spleen), and are performed at the time points specified in the Schedule of Activities.Medical History

[0379] A complete evaluation of past medical history is performed at screening and includes details of participants with a stable NYHA Class II or III HF diagnosis at least 8 weeks prior to screening and at the time of enrolment.Vital Signs

[0380] Temperature, respiration rate, heart rate (HR), and BP measurements are assessed. BP and HR measurements will be assessed in resting position, with a completely automated device. Manual techniques are used only if an automated device is not available or when an automated device cannot provide a reliable measurement. BP should be measured using the arm not used for study treatment administration. BP and HR measurements should be preceded by at least 5 minutes of resting position for the participant in a quiet setting without distractions.Assessment of Orthostatic Hypotension

[0381] Assessment of potential orthostatic hypotension occurs (per the Schedule of Activities) following the measurement of BP and HR in the resting position for 5 minutes. After resting BP and HR are taken, the participant is asked to stand for 3 minutes (with or without support), and the BP and HR measurements taken upon standing again at the end of the 1 and 3 minute periods. Orthostatic hypotension is defined as an increase in HR by >30 bpm, and / or decrease in systolic BP of at least 20 mmHg and / or diastolic BP of at least 10 mmHg with or without clinical symptoms. Any observation of orthostatic hypotension, with or without accompanying clinical symptoms, must be documented as an AE in the EDC system.Clinical Laboratory Tests

[0382] Any clinically relevant changes occurring during the study are recorded. Clinically significant abnormal laboratory findings are those that are not related to HF or any other pre-existing condition at the time of enrollment, unless judged to be more severe than expected for the participant’s condition. All laboratory tests with values considered clinically significantly abnormal, relative to baseline values, within 28 days after the dose of study treatment should be repeated until the values return to normal or baseline or are no longer considered clinically significant.Natriuretic Peptide Assessment

[0383] Blood samples for NT-proBNP levels are collected at the time points described in the Schedule of Activities. All analysis is conducted from baseline through end of study by a central laboratory.Adverse Events

[0384] All adverse events and severe adverse events are recorded through end of study.

[0385] Events that meet the AE definition include the following: any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) or other safety assessments (e.g., ECG, radiological scans, vital signs measurements), including those that worsen from baseline, considered clinically significant using medical and scientific judgment (i.e., not related to progression of underlying disease, or more severe than expected for the participant’s condition); exacerbation of a chronic or intermittent pre-existing condition including either an increase in frequency and / or intensity of the condition; new condition detected or diagnosed after study intervention administration even though it may have been present before the start of the study; signs, symptoms, or the clinical sequelae of a suspected drug-drug interaction; and signs, symptoms, or the clinical sequelae of a suspected overdose of either study intervention or a concomitant medication. Overdose per se will are reported as an AE / SAE unless it is an intentional overdose taken with possible suicidal / self-harming intent.

[0386] The following do not meet the definition of an adverse event: any abnormal laboratory findings or other abnormal safety assessments that are associated with the underlying disease, unless judged to be more severe than expected for the participant’s condition; the disease / disorder being studied or expected progression, signs, or symptoms of the disease / disorder being studied, unless more severe than expected for the participant’s condition; medical or surgical procedures (e.g., endoscopy, appendectomy): the condition that leads to the procedure is the AE; situations in which an untoward medical occurrence did not occur (social and / or convenience admission to a hospital); anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present or detected at the start of the study that do not worsen.

[0387] A severe adverse event (SAE) is defined as any untoward medical occurrence that, at any dose, meets one or more of the following criteria: the event results in death, is life threatening (the participant is at risk of death at the time of the event), requires inpatient hospitalization or prolongation of existing hospitalization, results in persistent or significant disability / incapacity (substantial disruption of a person’s ability to conduct normal life functions), is a congenital anomaly or birth defect, or events that may not be immediately life-threatening or result in death or hospitalization but may jeopardize the participant or may require medical or surgical intervention to prevent one of the other outcomes listed above.

[0388] Adverse events are assessed for the relationship between the study intervention and each occurrence of each AE / SAE using clinical judgement to determine the relationship.

[0389] Details of all pregnancies and reports of breastfeeding in female participants and female partners of male participants are collected until the end of study. While pregnancy itself is not considered to be an AE or SAE, any pregnancy complication or elective termination of a pregnancy for medical reasons will be reported as an AE or SAE. Any female participant who becomes pregnant while participating in the study will be withdrawn from the study.

[0390] Adverse events of special interest (AESI) include elevations from baseline in hs-cTnT that are >30% (change from baseline) and > 2 / ULN (male ULN = 16 ng / L; female ULN = 10 ng / L) will be recorded as AESI. Aggregate hs-cTnT AESIs will be reviewed throughout the duration of the study.Pharmacodynamics

[0391] Exploratory biomarkers and changes in 2D-TTE performance indices are evaluated in this study, and are described in further detail below.Pharmacokine tics

[0392] JK07 is evaluated in serum samples collected from all participants according to the Schedule of Activities (FIG. 2) so that the PK profile of multiple doses of JK07 is characterized.Immunogenicity

[0393] Antibodies to JK07 are evaluated in serum samples collected from all participants according to the Schedule of Activities. Serum samples are screened for antibodies binding to JK07. Samples which show a positive response in the screening assay are evaluated using a confirmatory anti-drug antibody (ADA) assay, and the confirmed positive samples are reported and their potential for neutralizing activity will be explored.Immunogenicity samples may be stored for a maximum of 10 years (or according to local regulations) following end of study to enable further analysis of immune responses to JK07. Genetic and Biomarker Analysis

[0394] Samples for future analysis are collected and banked for participants in regions where local laws and regulations allow. Blood sampling for exploratory genetic and biomarker analysis at a future timepoint are collected per the Schedule of Activities or on any leftover samples. Samples will be stored for up to 10 years following the end of study and analyzed or discarded within that time period.Statistical HypothesesCohort 1 (LVEF < 40%)Week 26 Primary Analysis

[0395] An informal interim analysis was conducted after about 50% of participants in the low dose sub-cohort are enrolled. The goal of the interim analysis is to assess safety. No formal hypothesis testing for efficacy or futility was conducted. Therefore, no alpha adjustment is necessary for the final analysis. A full analysis will be conducted when all participants have completed the Week 26 visit.

[0396] Two independent hypotheses will be tested and therefore no adjustment for multiple comparisons is necessary.1. Hoi: change from baseline to Week 26 in LVEF for 0.045 mg / kg (low) dose = change from baseline to Week 26 in LVEF for placebo versus Hlathat change from baseline to Week 26 in LVEF for 0.045 mg / kg dose change from baseline to Week 26 in LVEF for placebo.2. H02: change from baseline to Week 26 in LVEF for 0.09 mg / kg (high) dose = change from baseline to week 26 in LVEF for placebo versus H2athat change from baseline to Week 26 in LVEF for 0.09 mg / kg dose change from baseline to Week 26 in LVEF for placebo.

[0397] The week 26 analysis will be conducted using mixed model repeated measures (MMRM) adjusted for covariates. Missing data will be handled via multiple imputation methods.Week 52 Exploratory Analysis

[0398] A final analysis will be conducted once all participants complete the final end of study (Week 52) visit. Participants in the 0.09 mg / kg arm will be compared to their respectiveplacebo arm, participants in 0.045 mg / kg arm will be compared to their respective placebo arm. The hypothesis tested at the Week 52 full analysis for the low dose sub-cohort will be: HBO: change from baseline to Week 52 in LVEF for 0.045 mg / kg dose = change from baseline to 52 in LVEF for placebo versus HBathat change from baseline to 52 in LVEF for 0.045 mg / kg dose change from baseline to 52 in LVEF for respective placebo.The hypothesis tested at the Week 52 analysis for the high dose sub-cohort will be:HBO: change from baseline to Week 52 in LVEF for 0.09 mg / kg dose = change from baseline to 52 in LVEF for placebo versus HBathat change from baseline to 52 in LVEF for 0.09 mg / kg dose change from baseline to 52 in LVEF for respective placebo.

[0399] The Week 52 analysis will be conducted using MMRM adjusted for covariates. Missing data will be handled via multiple imputation methods.Cohort 2 (LVEF > 40% and < 65%)

[0400] Due to a small sample size all analyses will be descriptive.Multiplicity Adjustment

[0401] Multiplicity adjustment is not necessary. Low and high dose sub-cohorts in Cohort 1 are analyzed independently. Cohort 2 analyses are descriptive.Analysis Sets

[0402] The analysis sets are defined in 1.4 below.Table 1.4 Analysis setsPD = pharmacodynamics

[0403] Data from participants who withdraw from the study, including AEs and any followup, will be included in the analyses of all outcomes.Primary Endpoint Analyses

[0404] Analyses of efficacy endpoints will be conducted at Week 26 (primary) and Week 52, both individually for each cohort and as an aggregate analysis, resulting in 6 total statistical analyses, as outlined below:1. Cohort 1 (LVEF < 40%) Week 26 primary analysis for low dose (0.045mg / kg) versus its respective placebo and high dose (0.09 mg / kg) versus its respective placebo.2. Cohort 2 (LVEF > 40% and < 65%) Week 26 primary analysis.3. Cohorts 1 and 2 Week 26 aggregate exploratory analysis independently for the low and high doses.4. Cohort 1 (LVEF < 40%) Week 52 exploratory analysis.5. Cohort 2 (LVEF > 40% and < 65%) Week 52 exploratory analysis.6. Cohorts 1 and 2 Week 52 aggregate exploratory analysis.Safety Analyses

[0405] All safety analyses are performed on the Safety Evaluation Population. AEs will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) (version detailed in the SAP). The number of AEs and incidence rates are tabulated by preferred term and system organ class. The AEs will be coded by maximum severity, relationship to study treatment, and as AESI / SAEs as appropriate.

[0406] All laboratory test results, vital signs measurements, weight, and body mass index are summarized for each treatment group using descriptive statistics at each visit for raw numbers and change from baseline. The incidence of treatment-emergent abnormal laboratory results and vital signs are also summarized using descriptive statistics. All data will be provided in data listings sorted by treatment group, participant number, and visit. Interim Analyses

[0407] A descriptive (no formal statistical testing) Interim Analysis of available data took place during the Treatment Period of the study. After enrollment 50% of participants at the low dose (Treatment Arms 1 and 2) in Cohort 1 (LVEF < 40%), an interim analysis of all available data was conducted.Sample Size Determination Cohort 1 (LVEF <40%)

[0408] Sample size calculation: Based on the results of a Phase lb Study (see Example 5) in HFrEF patients, the absolute difference between 0.09 mg / kg dose and placebo for the change in LVEF from baseline to Day 60 was 9.5%. Assuming a conservative estimate (standard deviation of 10%), the effect size was high at 0.95. In the previous clinical work with NRG-1 in HFrEF (Lenihan D.J. et al., JACC Basic Transl Sci. 2016; 1 :576-586), the absolute difference in LVEF between placebo and high dose was 6.6% with a standard deviation of approximately 10%, yielding a moderate effect size of 0.66. Thus, a conservative effect size of 0.6 was used to determine the sample size in the Phase lb study. An independent testing strategy is planned for the low and high dose sub-cohorts for the primary analysis of the data, so no adjustment for multiple comparisons is necessary. Using a 2-sided t-test for independent samples and significance level alpha of 0.1, 58 participants in the low-dose and high-dose arms and 29 participants in their respective placebo arms (Cohort 1 [LVEF < 40%]) results in 83.45% power assuming independent testing in each sub-cohort. Thus, accounting for a 15% dropout rate, approximately 204 participants in total will be enrolled in the study.

[0409] The primary safety endpoint is not statistically powered, but the sample size of the study is sufficient to allow for meaningful descriptive statistics to be used to evaluate these endpoints.Cohort 2 (LVEF >40% and <65%)

[0410] No formal sample size calculation has been conducted for Cohort 2 (LVEF > 40% and < 65%) as the primary objective of the study in this cohort is safety and tolerability.

[0411] Clinical laboratory tests that will be performed during the study are summarized in Table 1.5 below. All blood collections will be conducted as outlined in the Schedule of Activities (FIG. 2).Table 1.5 Protocol-required laboratory assessmentsaAll samples for clinical chemistry are to be drawn fasting after 8 hours.ALP: alkaline phosphatase; ALT: alanine aminotransferase; aPTT: activated partial thromboplastin time; AST : aspartate aminotransferase; P-hCG: beta human chorionic gonadotrophin; BUN: blood urea nitrogen; GGT: gamma glutamyl transferase; Hb: hemoglobin;; HCT: hematocrit;; HDL: high-density lipoprotein;; hs-CRP: high sensitivity C- reactive protein; INR: international normalized ratio; LDL: low-density lipoprotein; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; MCV: mean corpuscular volume; PCR: polymerase chain reaction; PT: prothrombin time; RBC: red blood cell; SGOT: serum glutamic-oxaloacetic transferase; SGPT: serum glutamic-pyruvic transaminase; T3: free triiodothyronine; T4: free thyroxine; TSH: thyroid stimulating hormone; WBC: white blood cell.

[0412] An alternative dosing schedule to the one described above is presented in FIG. IB. In this alternative dosing schedule, subjects in 3 cohorts are administered 0.045 mg / kg, 0.09mg / kg JK07 or placebo, as shown in FIG. IB. Subjects are administered 4 initial doses 4 weeks apart (loading doses), as in the first version of the trial plan shown in FIG. 1 A. After a planned interval of approximately 14 weeks, the initial 4 doses are followed by two maintenance doses of 0.045 mg / kg JK07 or placebo that are approximately 12 weeks apart. Of note, the planned approximately 14 week interval between administration of the loading and maintenance doses can be adjusted based on clinical parameters such as NT-proBNP levels, LVEF or toxicity. Improvement in clinical parameters such as LVEF, and / or adverse effects, such as toxicity, would support an increase in the planned time period before the first maintenance dose, e.g., to greater than 14 weeks, such as 15 to 24 weeks. Conversely, less improvement in clinical parameters and / or no toxicity support a shorter planned time period before the administration of the first maintenance dose, (such as 4-13 weeks). As a still further alternative, the 4 initial doses are followed by two maintenance doses at 0.09 mg / kg, that are approximately 12 weeks apart, and approximately 14 weeks after the initial 4 doses.Example 2: Rhesus Macaque Model of Spontaneous Chronic Heart Failure

[0413] The NRG-1 HER3 antibody fusion protein (JK07) was investigated in a rhesus macaque model of spontaneous chronic HF.

[0414] A total of 26 animals with HFrEF (heart failure with reduced ejection fraction) and 26 diagnosed with HFpEF (heart failure with preserved ejection fraction) were evaluated in these studies, which included placebo and Entresto® (sacubitril / valsartan) as control cohorts. JK07 demonstrated clear efficacy over placebo following repeat dose administration in all JK07 fusion protein cohorts, superior efficacy compared with Entresto in HFpEF cohorts, and comparable efficacy compared with Entresto in HFrEF cohorts.

[0415] Specifically, in one study, in the HFrEF group, at 7 weeks post-treatment (6 weeks after the last dosing of JK07) LVEF remained significantly increased over baseline in 2 of 5 animals.

[0416] In the HFpEF group, the grade of diastolic dysfunction was improved by at least 1 grade in 3 of 5 animals receiving JK07, according to the clinical criteria used for the diagnosis of diastolic dysfunction.

[0417] In a second study, JK07-treated animals had statistically significant increases in LVEF compared to their corresponding baseline values (3 of 5 animals at 4 weeks posttreatment; 4 of 5 animals at 9 weeks) accompanied by significant improvement in cardiac remodeling. All 5 JK07-treated animals showed improvement at Week 9 relative to Week 4. At 13 weeks post-treatment (4 weeks after the dosing period), 3 of 5 monkeys treated withJK07 exhibited a sustained significant improvement in LVEF relative to their baseline values. JK07 significantly improved the diastolic function in HFpEF monkeys at 4, 9, and 13 weeks post-treatment, as reflected by an improvement of at least 1 level of diastolic function in 1 of 5 animals at 4 weeks post-treatment, 2 of 5 animals at 9 weeks post-treatment, and 3 of 5 animals at 13 weeks post-treatment with JK07. This study demonstrated that a series of 9 weekly doses administered over 9 weeks, at dose levels of 0.1 to 0.3 mg / kg, yields comparable efficacy to a series of 2 doses per week of 1.0 mg / kg administered over 2 weeks (as in the first study).Example 3: Single and Multiple Dose Pharmacokinetic (PK) Studies in Sprague-Dawley Rats and Cynomolgus Macaques

[0418] Single and multiple dose pharmacokinetic (PK) studies with JK07 were conducted in Sprague-Dawley rats and cynomolgus macaques. A greater than dose-proportional increase in exposure was observed in both rats and monkeys after repeated administration. However, repeated doses of JK07 did not result in accumulation in either rats or monkeys following administration of their respective treatment regimen. Of note, in both the 4- and 13-week Good Laboratory Practice (GLP) toxicology studies in nonhuman primates (NHPs), anti- JK07 anti drug antibodies (AD As) were abundantly present, with 100% of animals having ADA positivity by the end of the 13-week study. These AD As impacted overall exposure levels in all animals, noticeable from Day 29 and onward in the 13-week study.Example 4: Toxicity Studies

[0419] Toxicity of JK07 was evaluated in 6 studies: a non-GLP single-dose maximum tolerated dose (MTD) study in cynomolgus monkeys, a 2-week GLP repeat-dose study in Sprague-Dawley rats with a 2-week recovery period, a 4-week GLP repeat-dose study in cynomolgus monkeys with a 4-week recovery period, a 13-week GLP repeat-dose study in Sprague-Dawley rats with a 12-week recovery period, a 13-week repeat-dose study in cynomolgus monkeys with a 13-week recovery period, and a GLP tissue cross-reactivity study in healthy human tissues.2-week rat study (twice weekly repeat dose)

[0420] In the two-week rat toxicity study, Sprague Dawley rats were divided into 4 groups of 5 male and 5 female animals each: vehicle control (0 mg / kg), low dose group (1 mg / kg at 0.2 mg / mL), middle dose group (3 mg / kg at...

Claims

CLAIMSWhat is claimed is:

1. A recombinant fusion protein for use in the treatment of a cardiovascular disease in a subject, the recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb), wherein the treatment comprises administering to the subject a therapeutically effective dose of a recombinant fusion protein, and whereby blood serum concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP) is increased about 24 hours after administration of the recombinant fusion protein when compared to a baseline blood serum concentration of NT- proBNP in the subject prior to administration of the recombinant fusion protein.

2. The recombinant fusion protein for use according to claim 1, wherein about 30 days after administration of the recombinant fusion protein, a blood serum concentration of NT- proBNP is similar or substantially similar to the blood serum concentration of NT- proBNP in the subject prior to administration of the recombinant fusion protein.

3. The recombinant fusion protein for use according to claims 1 or 2, wherein the concentration of NT-proBNP is increased by at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900% or at least 1,000%.

4. The recombinant fusion protein for use according to claim 1 or 2, wherein: a. the concentration of NT-proBNP is increased by at least 30%, at least 50%, at least 150%, at least 300%, at least 500%, at least 600%, at least 800%, or at least 1400% 2 days after administration of the recombinant fusion protein; b. the concentration of NT-proBNP is increased by at least 30%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, or at least 1500% 7 days after administration of the recombinant fusion protein; or c. the concentration of the NT-proBNP is increased by at least 30%, at least 50%, at least 70%, at least 100%, or at least 200% 15 days after administration of the recombinant fusion protein.

5. The recombinant fusion protein for use according to of any one of claims 1-4, wherein the cardiovascular disease comprises heart failure with reduced ejection fraction (HFrEF) or heart failure with preserved ejection fraction (HFpEF).

6. The recombinant fusion protein for use according to any one of claims 1-5, wherein the therapeutically effective dose comprises between 0.03 mg / kg and 0.27 mg / kg of the recombinant fusion protein, optionally wherein the therapeutically effective dose comprises 0.045 mg / kg or 0.09 mg / kg of the recombinant fusion protein.

7. The recombinant fusion protein for use according to any one of claims 1-6, wherein administration of a second or further therapeutically effective dose of the recombinant fusion protein results in a blood serum concentration of NT-proBNP that is increased about 24 hours after administration of the second or further therapeutically effective dose of the recombinant fusion protein.

8. The recombinant fusion protein for use according to any one of claims 1-7, wherein a relative increase in a blood serum concentration of NT-proBNP after administration of a second or further therapeutically effective dose of the recombinant fusion protein is similar, or substantially similar, to a relative increase in a blood serum concentration of NT-proBNP after administration of a first therapeutically effective dose.

9. The recombinant fusion protein for use according to claim 8, wherein: a. the relative increase in blood serum concentration of NT-proBNP after the first therapeutically effective dose is relative to a baseline concentration of NT- proBNP in the subject prior to administration of the first therapeutically effective dose, and / or b. the relative increase in blood serum concentration of NT-proBNP after the second or further therapeutically effective dose is relative to a baseline concentration of NT-proBNP in the subject prior to administration of the second or further therapeutically effective dose.

10. The recombinant fusion protein for use according to claim 9, wherein the baseline concentration of NT-proBNP in the subject prior to administration of the second orfurther therapeutically effective dose is determined at least 14 days after administration of any previous therapeutically effective dose of the recombinant fusion protein to the subject.

11. The recombinant fusion protein for use according to claim 9 or 10, wherein the baseline concentration of NT-proBNP in the subject prior to administration of the first therapeutically effective dose and / or the baseline concentration of NT-proBNP in the subject prior to administration of the second or further therapeutically effective dose is determined within 24 hours prior to administration of the first therapeutically effective dose and / or second or further therapeutically effective dose.

12. The recombinant fusion protein according to any one of claims 8-11, wherein the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and second or further therapeutically effective dose is at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900% or at least about 1,000%.

13. The recombinant fusion protein according to any one of claims 8-11, wherein the relative increase in blood serum concentration of NT-proBNP after administration of the first dose and second or further therapeutically effective dose is between about 50% and about 1000%, between about 100% and about 800%, between about 100% and about 500%, between about 50% and about 300%, between about 100% and about 300%, between about 100% and about 200%, between about 200% and about 800%, between about 300% and about 800%, or between about 500% and about 900%.

14. A recombinant fusion protein for use in the treatment of cardiovascular disease in a subject, the recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb), wherein the treatment comprises administering a therapeutically effective dose of the recombinant fusion protein to the subject, andwhereby administration of the recombinant fusion protein results in an area under the time-concentration curve (in hours*ng / mL) from 0 to infinity (AUCo-inf) of between about 1,500 and about 200,000.

15. The recombinant fusion protein according to claim 14, wherein AUCo-inf is determined by plotting serum concentration of the recombinant fusion protein in the subject after administration over time.

16. The recombinant fusion protein according to claim 14 or 15, wherein administration of the recombinant fusion protein results in an AUCo-inf of between about 2,000 and about 180,000.

17. The recombinant fusion protein according to claim 14 or 15, wherein administration of the recombinant fusion protein results in an AUCo-inf of between about 8,000 and about 160,000.

18. The recombinant fusion protein according to any one of claims 14-17, wherein administration of the recombinant fusion protein results in a Cmax (in ng / mL) of between about 250 and about 6,500.

19. The recombinant fusion protein according to any one of claims 14-17, wherein administration of the recombinant fusion protein results in a Cmax (in ng / mL) of between about 450 and about 2,500.

20. The recombinant fusion protein according to any one of claims 14-19, wherein administration of the recombinant fusion protein results in a serum concentration of between about 200 and about 2,000 pg / L of the recombinant fusion protein in the subject approximately 12 hours after administration.

21. The recombinant fusion protein according to any one of claims 14-20, wherein the recombinant fusion protein has a half-life (ti / 2) of between about 5 and about 25 hours.

22. The recombinant fusion protein according to any one of claims 14-20, wherein the recombinant fusion protein has a half-life (ti / 2) of between about 8 and about 19 hours.

23. The recombinant fusion protein according to any one of claims 14-22, wherein the cardiovascular disease comprises heart failure with reduced ejection fraction (HFrEF) or heart failure with preserved ejection fraction (HFpEF).

24. The recombinant fusion protein according to any one of claims 14-23, wherein the therapeutically effective dose comprises between 0.03 mg / kg and 0.27 mg / kg, optionally wherein the therapeutically effective dose comprises 0.045 mg / kg or 0.09 mg / kg of the recombinant fusion protein.

25. A recombinant fusion protein for use in the treatment of heart failure with reduced ejection fraction (HFrEF) in a subject, the use comprising administering to the subject between 0.03 mg / kg and 0.27 mg / kg of the recombinant fusion protein, wherein the recombinant fusion protein comprises an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb).

26. The recombinant fusion protein according to claim 25, wherein the subject has New York Heart Association (NYHA) Class II or Class III heart failure.

27. The recombinant fusion protein according to claim 25 or 26, wherein the subject has a left ventricular ejection fraction (LVEF) of less than or equal to 40%.

28. The recombinant fusion protein according to claim 27, wherein the subject has an LVEF of greater than or equal to 30% and less than or equal to 40%, and a level of N-terminal pro-brain natriuretic peptide (NT -proBNP) level that is greater than or equal to 300 pg / mL.

29. The recombinant fusion protein according to claim 25 or 26, wherein the subject has a left ventricular ejection fraction (LVEF) of greater than 40% and less than or equal to 65%.

30. The recombinant fusion protein according to claim 29, wherein the subject has anNT -proBNP level that is greater than or equal to 600 pg / mL prior to administration of the recombinant fusion protein.

31. The recombinant fusion protein according to claim 25 or 26, wherein the subject has a history of atrial fibrillation and / or atrial flutter.

32. The recombinant fusion protein according to any one of claims 29-31, wherein the subject does not have hemodynamically significant and / or severe valvular disease other than mitral regurgitation or aortic stenosis.

33. The recombinant fusion protein according to any one of claims 25-32, wherein the subject has a hemoglobin level of greater than or equal to 9.0 g / dL prior to administration of the recombinant fusion protein.

34. The recombinant fusion protein according to any one of claims 25-33, wherein the subject is administered 0.045 mg / kg of the recombinant fusion protein.

35. The recombinant fusion protein according to any one of claims 25-34, wherein the subject is administered 0.09 mg / kg of the recombinant fusion protein.

36. The recombinant fusion protein according to any one of claims 1-35, wherein the subject is administered the recombinant fusion protein about once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, every 10 weeks, every 11 weeks or every 12 weeks.

37. The recombinant fusion protein according to any one of claims 1-35, wherein the subject is administered the recombinant fusion protein once every 4 weeks.

38. The recombinant fusion protein according to any one of claims 1-37, comprising a dosing holiday and / or a maintenance dose.

39. The recombinant fusion protein according to 38, wherein the subject administered the recombinant fusion protein every 4 weeks for an initial period, followed by a maintenance dose administered less frequently than every 4 weeks, optionally wherein the maintenance dose comprises administering less of the recombinant fusion protein than an initial dose.

40. The recombinant fusion protein according to claim 39, wherein the maintenance dose is administered every 8 to 16 weeks, optionally wherein the maintenance dose is administered every 8, 10, 12 or 14 weeks.

41. The recombinant fusion protein according to claim 39 or 40, comprising a planned recovery period between the initial period and the maintenance dose.

42. The recombinant fusion protein according to claim 41, wherein the planned recovery period is between about 10-18 weeks.

43. The recombinant fusion protein according to any one of claims 1-42, wherein the subject is administered the recombinant fusion protein for at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, or at least 36 months.

44. The recombinant fusion protein according to any one of claims 1-43, wherein the administration comprises intravenous infusion, optionally wherein the infusion occurs over a period of between 60 to 75 minutes.

45. The recombinant fusion protein according to any one of claims 1-44, wherein the NRG-1 active fragment comprises the ERBB3 / 4 binding domain.

46. The recombinant fusion protein according to any one of claims 1-45, wherein the NRG-1 active fragment binds to and induces signaling through ErbB4 (HER4).

47. The recombinant fusion protein according to any one of claims 1-46, wherein the mAb inhibits NRG-1 signaling through ErbB3 (HER3).

48. The recombinant fusion protein according to any one of claims 1-47, wherein the NRG-1 fragment is fused via its N-terminal amino acid to the C-terminus of the antibody heavy chain using a linker.

49. The recombinant fusion protein according to claim 48, wherein the linker comprises at least one copy of a Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser linker set forth in SEQ ID NO: 5.

50. The recombinant fusion protein according to any one of claims 1-49, wherein the C- terminus of the antibody heavy chain comprises the Fc domain of the antibody.

51. The recombinant fusion protein according to any one of claims 1-50, wherein the NRG-1 fragment comprises the amino acid sequence of SEQ ID NO: 4.

52. The recombinant fusion protein according to any one of claims 1-51, wherein the mAb comprises a heavy chain amino acid sequence of SEQ ID NO: 2.

53. The recombinant fusion protein according to any one of claims 1-51, wherein the mAb comprises a substitution mutation in at least one of amino acids 234, 239 and 434 numbered relative to SEQ ID NO: 2.

54. The recombinant fusion protein according to claim 53, wherein the at least one substitution mutation comprises an L234F mutation, a S239A mutation, a N434A mutation, or a combination thereof.

55. The recombinant fusion protein according to any one of claims 1-54, wherein the mAb comprises a light chain amino acid sequence of SEQ ID NO: 3.

56. The recombinant fusion protein according to any one of claims 1-55, wherein the recombinant fusion protein comprises the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 14.

57. The recombinant fusion protein according to any one of claims 1-56, wherein the recombinant fusion protein promotes HER2 / 4 signaling over HER2 / 3 signaling relative to the signal induction potential of recombinant NRG-1.

58. The recombinant fusion protein according to one of claims 1-57, wherein administration of the recombinant fusion protein results in an improvement in one or more parameters selected from the group consisting of LVEF, left ventricular end-diastolic volume index (LVEDVi), left ventricular end-systolic volume index (LVESVi), stroke volume index cardiac output (SVi CO), cardiac index (CI), left ventricular mass index (LVMi), diastolic function (E / e’), right ventricular systolic pressure (RVSP), left ventricular assist (LAV), left atrial ejection fraction (LAEF), and valvular function relative to a baseline measurement from before administration of the recombinant fusion protein.

59. The recombinant fusion protein according to any one of claims 1-58, wherein administration of the recombinant fusion protein leads to a reduced blood serum concentration of NT-proBNP at least 180 days after administration compared to a baseline blood serum concentration of NT-proBNP.

60. The recombinant fusion protein according to any one of claims 1-59, wherein administration of the recombinant fusion protein reduces risk of death from cardiovascular failure.

61. A recombinant fusion protein for use in the treatment of a cardiovascular disease in a subject, the recombinant fusion protein comprising an active fragment of neuregulin-1 (NRG-1) fused to a monospecific ErbB3 (HER3) monoclonal antibody (mAb), wherein the treatment comprises administering to the subject a therapeutically effective dose of a recombinant fusion protein, and wherein the subject is administered an initial dose the recombinant fusion protein every 4 weeks for an initial period, followed by a planned recovery period and then a maintenance dose administered less frequently than every 4 weeks.

62. The recombinant fusion protein according to claim 61, wherein the maintenance dose is administered every 8 to 16 weeks, optionally wherein the maintenance dose is administered every 8, 10, 12 or 14 weeks.

63. The recombinant fusion protein according to claim 61 or 62, wherein the planned recovery period is between about 10-18 weeks.

4. The recombinant fusion protein of any one of claims 61-63, wherein the therapeutically effective dose comprises between 0.03 mg / kg and 0.27 mg / kg, optionally wherein the therapeutically effective dose comprises 0.045 mg / kg or 0.09 mg / kg of the recombinant fusion protein.

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