Aperin receptor agonists and their use

Abstract

The present invention provides an apelin receptor agonist having, among other things, an increased half-life, reduced desensitization, improved apelin receptor signaling characteristics (e.g., bias towards Gα signaling rather than β-arrestin signaling), enhanced stability, and / or enhanced binding to the apelin receptor. In one embodiment, the present invention provides an apelin receptor agonist comprising, among other things, an apelin analog, the apelin analog comprising one or more amino acid substitutions compared to SEQ ID NO: 1 (QRPRLSHKGPMPF), the one or more amino acid substitutions occurring at positions 1, 6, 8, 9, 11, 12, and / or 13.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 585,027, filed on 25 September 2023, and to U.S. Provisional Patent Application No. 63 / 495,388, filed on 11 April 2023, the entire contents of which are incorporated herein by reference.

[0002] Inclusion by referencing the sequence list This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The sequence listing file, named SVI-005WO1_SL, was created on April 10, 2024, and has a size of 76,321 kilobytes. [Background technology]

[0003] The Apelin system is an endogenous physiological regulator that has emerged as a potential therapeutic target for many diseases. This system includes two families of the Apelin receptor and its endogenous ligands: Apelin and Elavera / Toddler (ELA, also known as the early endogenous ligand for the Apelin receptor). The APLNR gene (also known as APJ) was identified and cloned in 1993. This gene was found to encode the Apelin receptor, a novel G protein-coupled receptor (GPCR) with approximately 50% homology to the type 1 angiotensin II (AT1) receptor. The Apelin receptor system counteracts the effects of angiotensin II (Ang II) agonism at the AT1 receptor in vitro and in vivo. The Apelin receptor is highly conserved across species, exhibiting approximately 90% sequence similarity between mouse, rat, and human proteins.

[0004] In humans, clinical trials of the apelin system are challenging due to the short half-life of apelin peptide (only a few minutes), which has led to reports of rapid receptor desensitization via coupling to β-arrestin. These challenges have led to efforts to produce apelin receptor agonists that could be used as pharmacological probes to explore the system's role in health and disease. [Overview of the project]

[0005] The present invention provides improved apelin receptor agonists and the use of such agonists in the effective treatment of diseases such as pulmonary hypertension (PAH), systemic sclerosis (SSc), heart failure with preserved ejection fraction (HfpEf), age-related sarcopenia, and acute kidney injury (AKI).

[0006] As described herein, the present invention is based in part on the identification of a new class of apelin or elavella analogs having increased half-life, reduced desensitization, improved apelin receptor signaling characteristics (e.g., bias towards Gα signaling rather than β-arrestin signaling), enhanced stability, and / or enhanced binding to the apelin receptor. This is important because the apelin receptor agonists of the present invention can effectively induce a response to a desired downstream apelin receptor pathway without significantly adversely affecting the internalization and down-transcriptional regulation of the apelin receptor after treatment with the apelin receptor agonist. The original apelin receptor agonists of the present invention promise more potent treatment of apelin receptor-mediated diseases and disorders, including pulmonary hypertension (PAH), systemic sclerosis (SSc), heart failure with preserved ejection fraction (HfpEf), age-related sarcopenia, and acute kidney injury (AKI).

[0007] In one embodiment, the present invention provides an apelin receptor agonist comprising, in particular, an apelin analog, the apelin analog comprising one or more amino acid substitutions compared to SEQ ID NO: 1 (QRPRLSHKGPMPF), the one or more amino acid substitutions occurring at positions 1, 6, 8, 9, 11, 12, and / or 13.

[0008] In some embodiments, the apelin receptor agonist comprises one or more amino acid substitutions selected from P12V, K8W, Q1E, M11H, M11A, F13Y and / or P12A, P12H, F13W, S6Q and / or G9N.

[0009] In one embodiment, the present invention provides an apelin receptor agonist comprising an apelin analog, the apelin analog comprising the amino sequence:X1-RPRLSH-X2-GP-X3-X4-X5 (SEQ ID NO: 63), where X1 is Q or E, X2 is K or W, X3 is M, H or A, X4 is P, V, A or H, and / or X5 is F, Y or W.

[0010] In one embodiment, the present invention provides an apelin receptor agonist comprising an apelin analog, the apelin analog comprising the amino sequence: QRPRLQHKNPMA-X1 (SEQ ID NO: 64), where X1 is F or W.

[0011] In one embodiment, the present invention provides an apelin receptor agonist comprising an apelin analog, the apelin analog comprising the amino sequence: QRPRLSHKGP-X1-X2-X3 (SEQ ID NO: 65), where X1 is M, H, or A, X2 is P, V, A, or H, and / or X3 is F, Y, or W.

[0012] In one embodiment, the present invention provides an apelin receptor agonist comprising an apelin analog, the apelin analog comprising the amino sequence: X1-RPRL-X2-H-X3-X4-P-X5-X6-X7 (SEQ ID NO: 66), where X1 is Q or E, X2 is S or Q, X3 is K or W, X4 is G or N, X5 is M, H or A, X6 is P, V, A or H, and / or X7 is F, Y or W.

[0013] In some embodiments, the apelin analog comprises the amino acid sequence of SEQ ID NO: 1 (QRPRLSHKGPMPF). In some embodiments, the apelin analog comprises the amino acid sequence of SEQ ID NO: 2 (QRPRLSHKGPMVF). In some embodiments, the apelin analog comprises the amino acid sequence of SEQ ID NO: 3 (QRPRLSHWGPMPF). In some embodiments, the apelin analog comprises the amino acid sequence of SEQ ID NO: 4 (ERPRLSHKGPMPF). In some embodiments, the apelin analog comprises the amino acid sequence of SEQ ID NO: 5 (QRPRLSHKGPHPF). In some embodiments, the apelin analog comprises the amino acid sequence of SEQ ID NO: 6 (QRPRLSHKGPAPY). In some embodiments, the apelin analog comprises the amino acid sequence of SEQ ID NO: 7 (QRPRLSHKGPMAF). In some embodiments, the apelin analog comprises the amino acid sequence of SEQ ID NO: 50 (QRPRLSHKGPMHW). In some embodiments, the apelin analog comprises the amino acid sequence of SEQ ID NO: 51 (QRPRLSHKGPAAW). In some embodiments, the apelin analog comprises the amino acid sequence of SEQ ID NO: 52 (QRPRLSHKGPAAY). In some embodiments, the apelin analog comprises the amino acid sequence of SEQ ID NO: 55 (QRPRLSHWGPMAW). In some embodiments, the apelin analog comprises the amino acid sequence of SEQ ID NO: 53 (QRPRLQHKNPMAF). In some embodiments, the apelin analog comprises the amino acid sequence of SEQ ID NO: 54 (QRPRLQHKNPMAW).

[0014] In one aspect, the present invention particularly provides an apelin receptor agonist comprising an elabela analog, wherein the elabela analog comprises one or more amino acid substitutions compared to SEQ ID NO: 8 (KLRKHNCLQRRCMPLHSRVPFP), and the one or more amino acid substitutions occur at positions 3, 7, 8, 9, 10, 12, 14, 15, 17, and / or 18.

[0015] In some embodiments, the apelin analog comprises one or more amino acid substitutions including R3N, P14I, S17Q, S17R, Q9K, R18Y, L15Y, C7S, C12Y, R10E, Q9I, L8A, and / or Q9T.

[0016] In one embodiment, the present invention provides an apelin receptor agonist comprising an elavella analog, the elavella analog comprising the amino sequence: KL-Z1-KHN-Z2-Z3-Z4-Z5-R-Z6-M-Z7-Z8-H-Z9-Z10-VPFP (SEQ ID NO: 67), where Z1 is R or N, Z2 is C or S, Z3 is A or L, Z4 is Q, I, K, or T, Z5 is R or E, Z6 is C or Y, Z7 is P or I, Z8 is L or Y, Z9 is S, Q, or R, and / or Z10 is R or Y.

[0017] In some embodiments, the erabeler analog contains the amino acid sequence of SEQ ID NO: 8 (KLRKHNCLQRRCMPLHSRVPFP). In some embodiments, the erabeler analog contains the amino acid sequence of SEQ ID NO: 9 (KLNKHNCLQRRCMPLHSRVPFP). In some embodiments, the erabeler analog contains the amino acid sequence of SEQ ID NO: 10 (KLRKHNCLQRRCMILHSRVPFP). In some embodiments, the erabeler analog contains the amino acid sequence of SEQ ID NO: 11 (KLRKHNCLQRRCMPLHQRVPFP). In some embodiments, the erabeler analog contains the amino acid sequence of SEQ ID NO: 12 (KLRKHNCLQRRCMPLHRRVPFP). In some embodiments, the erabeler analog contains the amino acid sequence of SEQ ID NO: 13 (KLRKHNCLKRRCMPLHSRVPFP). In some embodiments, the erabeler analog contains the amino acid sequence of SEQ ID NO: 30 (KLRKHNCLQRRCMPLHSYVPFP). In some embodiments, the erabeler analog contains the amino acid sequence of SEQ ID NO: 31 (KLRKHNCLQRRCMPYHSRVPFP). In some embodiments, the erabeler analog contains the amino acid sequence of SEQ ID NO: 32 (KLRKHNSLQRRYMPLHSRVPFP). In some embodiments, the erabeler analog contains the amino acid sequence of SEQ ID NO: 33 (KLRKHNCLQERCMPLHSRVPFP). In some embodiments, the erabeler analog contains the amino acid sequence of SEQ ID NO: 34 (KLRKHNCLIRRCMPLHSRVPFP). In some embodiments, the erabeler analog contains the amino acid sequence of SEQ ID NO: 35 (KLRKHNCATRRCMPLHSRVPFP).

[0018] In some embodiments, the apelin receptor agonist comprises an apelin analog or an elavella analog fused to a carrier moiety. In some embodiments, the N-terminus of the apelin analog or elavella analog is fused to the carrier moiety. In some embodiments, the carrier moiety extends the half-life of the apelin receptor agonist. In some embodiments, the carrier moiety comprises an Fc domain, human serum albumin (HSA), an anti-HSA antibody or a fragment thereof, a lipid, or PEG. In some embodiments, the Fc domain is monovalent Fc. In some embodiments, the Fc domain is bivalent Fc.

[0019] In some embodiments, the anti-HSA antibody or fragment thereof is an anti-HSA Fab, nanobody (VHH), scFv, or V-NAR. In some embodiments, the anti-HSA VHH includes ALB-23.

[0020] In one embodiment, the present invention provides, in particular, an Apelin analog or an Elavera analog, the N-terminus of which is fused to a carrier portion via a linker.

[0021] In some embodiments, the N-terminus of the Apelin analog or Elavera analog is fused to the C-terminus of the carrier moiety. In some embodiments, the carrier moiety extends the half-life of the Apelin receptor agonist. In some embodiments, the carrier moiety comprises an Fc domain, human serum albumin (HSA), an anti-HSA antibody or fragment thereof, a lipid, or PEG. In some embodiments, the Fc domain is monovalent Fc. In some embodiments, the Fc domain is bivalent Fc. In some embodiments, the Fc domain is IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the Fc domain contains one or more mutations compared to the wild-type Fc domain. In some embodiments, the Fc domain contains SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the anti-HSA antibody or fragment thereof is anti-HSA Fab, nanobody (VHH), scFv, or V-NAR. In some embodiments, anti-HSA VHH contains ALB-23. In some embodiments, the linker includes GGGGGSGGGGSGGGGS (SEQ ID NO: 16).

[0022] In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 17. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 18. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 19. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 20. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 21. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 22. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 23. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 24. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 25. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 26. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 27. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 28. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 29.In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 36. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 37. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 38. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 39. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 40. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 41. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 56. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 57. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 58. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 59. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 60. In some embodiments, the apelin receptor agonist contains an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO: 61.

[0023] In some embodiments, the apelin receptor agonist, when measured by cAMP depletion, has a G level of less than 60 nM, less than 30 nM, less than 20 nM, or less than 10 nM.αi The signal transduction assay EC50 values ​​are present. In some embodiments, the apelin receptor agonist has a beta-arrestin signal transduction assay EC50 value of less than 60 nM, less than 500 nM, less than 100 nM, less than 30 nM, or less than 10 nM. In some embodiments, the apelin receptor agonist has an ERK activation assay EC50 value of less than 20 nM, less than 10 nM, less than 5 nM, less than 2 nM, or less than 1 nM.

[0024] In one embodiment, the present invention provides a method for treating a disease, comprising administering an apelin receptor agonist of the present invention to a subject in need thereof. In some embodiments, the disease is pulmonary hypertension (PAH), systemic sclerosis (SSc), heart failure with preserved ejection fraction (HfpEf), age-related sarcopenia, or acute kidney injury (AKI).

[0025] In some embodiments, this disclosure includes nucleic acids encoding the apelin receptor agonists disclosed herein. In some embodiments, this disclosure includes methods for producing the apelin receptor agonists disclosed herein. [Brief explanation of the drawing]

[0026] [Figure 1] Figure 1 is an illustrative schematic diagram illustrating how apelin receptor activation leads to a wide range of physiological effects mediated by several signaling pathways. [Figure 2] Figure 2 is an exemplary graph showing the internalization scores of exemplary apelin analogs against EC50 values ​​for beta-arrestin signaling and cAMP signaling. [Figure 3] Figure 3 is an exemplary graph showing the internalization scores of exemplary Elavera analogs against EC50 values ​​for beta-arrestin signaling and cAMP signaling. [Modes for carrying out the invention]

[0027] definition To make this disclosure easier to understand, certain terms are first defined below. Additional definitions of the terms below and other terms are provided throughout this specification. Publications and other reference materials referenced herein to provide context for this disclosure and further details relating to its implementation are incorporated herein by reference.

[0028] Apelin receptor agonists – The term “apelin receptor agonists” refers to non-natural peptides or polypeptides that have improved agonist activity for the apelin receptor. In some embodiments, apelin receptor analogs have enhanced binding to the apelin receptor compared to wild-type apelin. In some embodiments, apelin receptor analogs have improved apelin receptor signaling compared to wild-type apelin. In some embodiments, apelin receptor analogs have an increased half-life compared to wild-type apelin. In some embodiments, apelin receptor analogs are more resistant to proteolysis compared to wild-type apelin. In some embodiments, the apelin receptor analog comprises a carrier and wild-type apelin. In some embodiments, the apelin receptor analog comprises a carrier and wild-type elavella.

[0029] The term "Fc" refers to a portion of the heavy chain constant region, including at least the CH2 and CH3 domains, that typically binds to Fc receptors, such as FcγR, i.e., FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), or FcRn, i.e., neonatal Fc receptors. The Fc variants of the present invention can be optimized for various properties. Fc variants that are manipulated or predicted to exhibit one or more optimized properties are referred to herein as "optimized Fc variants." In some embodiments, optimized Fc variants have reduced affinity or excision for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q, but retain binding to FcRn.

[0030] Increased half-life – as used herein, “increased half-life,” “increased serum half-life,” or “extended half-life” means a positive change in the circulating half-life of a modified biologically active molecule (e.g., apelin or elavella analog) compared to its unmodified form (or the naked form of the peptide). Serum half-life is measured, for example, by taking blood samples at various time points after administration of the biologically active molecule and determining the concentration of that molecule in each sample.

[0031] Pharmacopoeia-acceptable – as used herein, “pharmacopoeia-acceptable” means that its use in animals, more specifically in humans, is generally recognized.

[0032] pharmaceutically acceptable excipients – as used herein, “pharmaceutically acceptable excipient,” “pharmaceutically acceptable carrier,” or “pharmaceutically acceptable adjuvant” respectively refer to an excipient, carrier, or adjuvant to which at least one of the compounds of this disclosure is administered. “pharmaceutically acceptable vehicle” refers to a diluent, adjuvant, excipient, or carrier to which at least one of the compounds of this disclosure is administered.

[0033] Subject – As used herein, “subject” includes mammals and humans. The terms “human” and “subject” are used interchangeably herein.

[0034] Therapeutic dose – as used herein, “therapeutic dose” means an amount of a compound sufficient to have an effect on treating a disease, disorder, or symptom when administered to a subject to treat at least one of the clinical symptoms of a disease or disorder. “Therapeutic dose” may vary depending on the compound, the disease, disorder, and / or the symptoms of the disease or disorder, the severity of the disease, disorder, and / or symptoms or disorder, the age of the subject being treated, and / or the weight of the subject being treated. An appropriate amount in any given case may be readily apparent to those skilled in the art or can be determined by routine experimentation.

[0035] Treatment—As used herein, “to treat” or “to cure” any disease or disorder means stopping or improving the disease, disorder, or at least one of the clinical symptoms of the disease or disorder, reducing the risk of acquiring the disease, disorder, or at least one of the clinical symptoms of the disease or disorder, reducing the onset of the disease, disorder, or at least one of the clinical symptoms of the disease or disorder, or reducing the risk of developing the disease, disorder, or at least one of the clinical symptoms of the disease or disorder. “To treat” or “to cure” also means inhibiting the disease or disorder either or both physically (e.g., stabilization of identifiable symptoms) or physiologically (e.g., stabilization of physical parameters), or inhibiting at least one physical parameter which may not be identifiable to the subject. Furthermore, “to treat” or “to cure” means delaying the onset of the disease or disorder, or at least one of its symptoms, in a subject who may be exposed to or susceptible to the disease or disorder, even though the subject has not yet experienced or exhibited any symptoms of the disease or disorder.

[0036] Variant – As used herein, “variant,” “analog,” and “mutein” refer to a biologically active derivative of a reference molecule that retains a desired activity, such as apelin activity, for use in the treatment of cardiovascular or pulmonary diseases or disorders as described herein. Generally, the terms “variant” and “analog” refer to a compound having a natural polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conserved in nature) and / or deletions to a natural molecule, provided that the modification does not destroy the biological activity, and that it is “substantially homologous” to the reference molecule as defined below. Generally, the amino acid sequence of such an analog has a high degree of sequence homology to the reference sequence, e.g., more than 50%, generally more than 60%–70%, and more specifically more than 80%–85%, e.g., at least 90%–95% when the two sequences are aligned. Often, analogs contain the same number of amino acids but include substitutions as described herein. The term “mutein” includes, but is not limited to, compounds containing only amino molecules and / or imino molecules, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids), polypeptides having substitution bonds, and polypeptides having one or more amino acid-like molecules, including other modifications, cyclizations, branching molecules known in the Art, both naturally occurring and unnatural (e.g., synthetic). The term also includes molecules containing one or more N-substituted glycine residues ("peptoids") and other synthetic amino acids or peptides. (For example, see U.S. Patents 5,831,005, 5,877,278, and 5,977,301, Nguyen et al., Chem Biol. (2000) 7:463-473, and Simon et al., Proc. Natl. Acad. Sci. USA (1992) 89:9367-9371) Preferably, the analog or mutein has at least the same apelin biological activity as the natural molecule. Methods for producing polypeptide analogs and muteins are known in the art and are described further below. (Detailed description of the invention)

[0037] Aperin receptor pathway The apelin system is an endogenous physiological regulator that has emerged as a potential therapeutic target for many diseases. This system includes two families of apelin receptors and their endogenous ligands: apelin and elavella / toddler (ELA, also known as the early endogenous ligand of the apelin receptor).

[0038] The APLNR gene (also known as APJ) was identified and cloned in 1993. This gene was found to encode the apelin receptor, a novel G protein-coupled receptor (GPCR) with approximately 50% homology to the type 1 angiotensin II (AT1) receptor. The apelin receptor system counteracts the effects of angiotensin II (Ang II) agonism at the AT1 receptor both in vitro and in vivo. In the cardiovascular system, apelin and AT1 receptors are co-expressed. The apelin receptor is highly conserved across species, exhibiting approximately 90% sequence similarity between mouse, rat, and human proteins.

[0039] Aperine Apelin peptides are produced by cleaving the C-terminus of a 77-amino acid precursor, pre-properin. Peptide fragments of various lengths circulate in vivo, with the major isoforms being apelin-36, apelin-17, and apelin-13. The pyroglutaminated forms, apelin-13 and [Pyr1]apelin-13, are structurally more resistant to metabolism by aminopeptidases than apelin-13 and are the most abundant apelin isoforms in the cardiovascular system and human plasma.

[0040] The mechanism of apelin peptide metabolism is not yet fully defined. Experimental evidence suggests that furin (also known as PCSK3), plasma kallikrein, and neprilysine cleave apelin peptides, but only neprilysine completely inactivates these peptides. Carboxypeptidase angiotensin-converting enzyme 2 (ACE2) has been clearly demonstrated to cleave apelin isoforms both in vitro and in vivo, resulting in the removal of a common C-terminal phenylalanine. This enzyme is highly expressed on the surface of alveolar epithelial cells and intestinal epithelial cells, and at lower levels in most organs, including the kidneys and heart, as well as in arterial and venous endothelial cells.

[0041] Ela Bell ELA is a 54-amino acid peptide that is expected to be processed to form mature peptides, including ELA-32, ELA-21, and ELA-11. The discovery of ELA as a second endogenous ligand for the apelin receptor provided an explanation for the unexpected and striking discrepancy between the phenotypes of apelin receptor knockout mice that did not come into birth at the expected Mendelian ratio and had significant cardiovascular developmental defects, and those of normally developing apelin knockout mice. Although ELA is detectable in human plasma, its isoform expression in specific tissues has not yet been investigated.

[0042] Expression of the aperin system Apelin and its receptor are widely expressed in the central nervous system and peripheral organs. In rodents and humans, apelin receptor mRNA is detected in many tissues, particularly the brain, spinal cord, placenta, lungs, heart, kidneys, adipose tissue, and skeletal muscle. Apelin receptor proteins have been identified in the rat and human brains, spinal cords, hearts, kidneys, and lungs. In the human heart, apelin receptors are expressed in cardiomyocytes, endothelial cells of the endocardium and intramyocardial blood vessels, endothelial cells of ductal arteries and veins, and smooth muscle. In the kidney, apelin receptor proteins are localized in the cortex and vascular system.

[0043] Apelin receptor binding and signal transduction Endogenous apelin isoforms bind to their homologous receptors with nanomolar affinity in cell expression systems, as well as in rat and human cardiovascular tissue. However, structure-activity studies have shown that longer isoforms such as apelin-36, and especially apelin-17, [Pyr 1 This suggests that it has somewhat higher affinity than shorter isoforms such as apelin-13. [Pyr] is present in all longer apelin isoforms. 1 The N-terminal RPRL sequence of apelin-13 is required for receptor binding, along with some contribution from other residues such as Pro12. The smallest apelin fragment to maintain biological activity is 10 amino acids, although with low affinity, apelin-13 (2-11) That is the case.

[0044] The crystal structure has been reported for the apelin receptor complexed with a 17-amino acid non-endogenous apelin agonist. Significant mutations were required for the successful crystallization of the receptor. However, using this structure in combination with molecular mechanical simulations and molecular modeling, this study demonstrated a bisite peptide-ligand binding mode. The concept of a "message address" for this peptide bond has also been suggested by previous studies, including those showing that the amino acids of the N-terminal tail and the first extracellular loop are crucial for both receptor protein folding and apelin peptide binding. Another structural study used a 3D homology model to visualize the top surface of the apelin receptor binding site, [Pyr 1 The interaction between the basic residues in the N-terminal RPRL of apelin-13 and the acidic residues in extracellular loops I and II, as well as the interface between extracellular loop III and transmembrane VII, was investigated using site-directed mutagenesis.

[0045] Although the ELA peptide shows little sequence similarity to apelin, the predicted isoforms ELA-32, ELA-21, and ELA-11 bind to the apelin receptor in the human heart, with ELA-32 binding at sub-nanomolar affinity and ELA-21 binding at nanomolar affinity ([Pyr1 (compared to that of apelin-13), the shortest isoform, ELA-11, bound with a similarity one order of magnitude lower than the other peptides. Alanine scanning and mutagenesis analysis suggested that apelin and ELA peptides engaged different residues of the rat and human apelin receptors.

[0046] The apelin receptor binds to pertussis toxin-sensitive inhibitory G protein (Gα i / o ), subsequently activating the extracellular regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)-AKT (also known as protein kinase B (PKB)) signaling cascades. These cascades result in a wide range of physiological effects depending on the type of cell being activated. All putative apelin and ELA isoforms elicit canonical G i -mediated inhibition of adenylate cyclase, which results in the inhibition of cAMP. Similar to other GPCRs, the apelin receptor can engage other heterotrimeric G proteins, particularly G q , resulting in downstream stimulation of the phospholipase C (PLC) and AMP-activated protein kinase (AMPK) pathways.

[0047] After activation, GPCRs can be uncoupled from their G proteins and internalized via β-arrestin recruitment to their receptors. The degree and dynamics of apelin receptor-mediated internalization may be isoform-specific, at least in vitro, and it has been suggested that apelin-36 results in longer-lasting internalization than [Pyr1]apelin-13. For the successful translation of apelin receptor agonists into clinical practice, the discovery of so-called "biased signaling," where GPCR ligands preferentially activate a subset of the GPCR signaling repertoire or actually stimulate some signaling pathways while inhibiting others, is a crucial finding. Compared to [Pyr1]apelin-13, longer apelin ligand isoforms such as apelin-36, apelin-17, ELA-32, and ELA-21 have been shown to exhibit some bias in β-arrestin recruitment. The in vivo relationship between these observations and the physiology or pathophysiology of the apelin receptor has not yet been determined.

[0048] Ligand-independent signaling via the apelin receptor has also been reported and may be pathologically important. Phosphorylation of ERK1 and ERK2 downstream of Ang II-mediated activation of the AT1 receptor was inhibited in cells co-expressing the apelin receptor, and this inhibition was abolished by apelin in a pertussis toxin-sensitive manner. Subsequently, in a chronic pressure overload mouse model, genetic deficiency of the apelin receptor showed protection against cardiac hypertrophy and heart failure, while apelin deficiency had no effect. This discrepancy may be explained by later findings regarding ELA. However, cardiomyocytes isolated from apelin receptor knockout mice showed a reduced response to stretching, suggesting that the apelin receptor can act as a mechanistic sensor even in the absence of the ligand. This stretch-mediated response was abolished by β-arrestin knockdown and blunted by the addition of apelin. Therefore, the apelin receptor may exert a hypertrophic effect via β-arrestin signaling in the absence of a ligand, and an anti-hypertrophic effect via a β-arrestin-independent pathway when activated by apelin.

[0049] Aperin receptor agonists Apelin analogues The present invention provides, among other things, apelin analogs having improved agonist activity for the apelin receptor. In some embodiments, the apelin analog has enhanced binding to the apelin receptor compared to wild-type apelin. In some embodiments, the apelin analog has improved apelin receptor signaling compared to wild-type apelin. In some embodiments, the apelin analog has an increased half-life compared to wild-type apelin. In some embodiments, the apelin analog is more resistant to proteolysis compared to wild-type apelin.

[0050] In some embodiments, the apelin analog is derived from wild-type apelin-77 (MNLRLCVQALLLLWLSLTAVCGGSLMPLPDGNGLEDGNVRHLVQPRGSRNGPGPWQGGRRKFRRQRPRLSHKGPMPF) of SEQ ID NO: 42.

[0051] In some embodiments, the apelin analog is derived from wild-type apelin-36 (LVQPRGSRNGPGPWQGGRRKFRRQRPRLSHKGPMPF) of SEQ ID NO: 43. In some embodiments, the apelin analog is derived from wild-type apelin-17 (KFRRQRPRLSHKGPMPF) of SEQ ID NO: 44. In some embodiments, the apelin analog is derived from wild-type apelin-13 (QRPRLSHKGPMPF) of SEQ ID NO: 1. In some embodiments, the apelin analog is derived from [pyr-Glu] apelin-13 (ERPRLSHKGPMPF) of SEQ ID NO: 45.

[0052] In some embodiments, the apelin analog includes modifications compared to SEQ ID NO: 1, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45. In some embodiments, the modifications are substitutions, insertions, or deletions of one or more amino acids.

[0053] In some embodiments, the apelin analog contains one or more amino acid substitutions at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of SEQ ID NO: 1. In some embodiments, the apelin analog contains one or more amino acid substitutions at position 1, 6, 8, 9, 11, 12, or 13 of SEQ ID NO: 1. In some embodiments, the apelin analog contains an amino acid substitution at position 12 of SEQ ID NO: 1. In some embodiments, the apelin analog contains an amino acid substitution at position 8 of SEQ ID NO: 1. In some embodiments, the apelin analog contains an amino acid substitution at position 1 of SEQ ID NO: 1. In some embodiments, the apelin analog contains an amino acid substitution at position 11 of SEQ ID NO: 1. In some embodiments, the apelin analog contains an amino acid substitution at position 13 of SEQ ID NO: 1. In some embodiments, the apelin analog contains amino acid substitutions at positions 11 and 13 of SEQ ID NO: 1. In some embodiments, the apelin analog includes amino acid substitutions at positions 12 and 13 of SEQ ID NO: 1. In some embodiments, the apelin analog includes amino acid substitutions at positions 11, 12 and 13 of SEQ ID NO: 1. In some embodiments, the apelin analog includes amino acid substitutions at positions 8, 9 and 12 of SEQ ID NO: 1. In some embodiments, the apelin analog includes amino acid substitutions at positions 8, 9, 12 and 13 of SEQ ID NO: 1. In some embodiments, the apelin analog includes amino acid substitutions at positions 8, 12 and 13 of SEQ ID NO: 1.

[0054] In some embodiments, the apelin analog includes the P12V amino acid substitution compared to SEQ ID NO: 1. In some embodiments, the apelin analog includes the M11H amino acid substitution compared to SEQ ID NO: 1. In some embodiments, the apelin analog includes the K8W amino acid substitution compared to SEQ ID NO: 1. In some embodiments, the apelin analog includes the Q1E amino acid substitution compared to SEQ ID NO: 1. In some embodiments, the apelin analog includes the M11A and F13Y amino acid substitutions compared to SEQ ID NO: 1. In some embodiments, the apelin analog includes the P12A amino acid substitution compared to SEQ ID NO: 1. In some embodiments, the apelin analog includes the S6Q amino acid substitution compared to SEQ ID NO: 1. In some embodiments, the apelin analog includes the G9N amino acid substitution compared to SEQ ID NO: 1. In some embodiments, the apelin analog includes the P12H amino acid substitution compared to SEQ ID NO: 1. In some embodiments, the apelin analog includes the F13W amino acid substitution compared to SEQ ID NO: 1. In some embodiments, the apelin analog includes the P12H and F13W amino acid substitutions compared to SEQ ID NO: 1. In some embodiments, the apelin analog includes the M11A, P12A, and F13W amino acid substitutions compared to SEQ ID NO: 1. In some embodiments, the apelin analog includes the M11A, P12A, and F13Y amino acid substitutions compared to SEQ ID NO: 1. In some embodiments, the apelin analog includes the S6Q, G9N, and P12A amino acid substitutions compared to SEQ ID NO: 1. In some embodiments, the apelin analog includes the S6Q, G9N, P12A, and F13W amino acid substitutions compared to SEQ ID NO: 1. In some embodiments, the apelin analog includes the K8W, P12A, and F13W amino acid substitutions compared to SEQ ID NO: 1.

[0055] In some embodiments, the apelin analog comprises the amino acid sequence shown in Table 1. [Table 1-1] [Table 1-2]

[0056] In some embodiments, the apelin analog includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the apelin analog includes the amino acid sequence of SEQ ID NO: 2. In some embodiments, the apelin analog includes the amino acid sequence of SEQ ID NO: 3. In some embodiments, the apelin analog includes the amino acid sequence of SEQ ID NO: 4. In some embodiments, the apelin analog includes the amino acid sequence of SEQ ID NO: 5. In some embodiments, the apelin analog includes the amino acid sequence of SEQ ID NO: 6. In some embodiments, the apelin analog includes the amino acid sequence of SEQ ID NO: 7. In some embodiments, the apelin analog includes the amino acid sequence of SEQ ID NO: 50. In some embodiments, the apelin analog includes the amino acid sequence of SEQ ID NO: 51. In some embodiments, the apelin analog includes the amino acid sequence of SEQ ID NO: 52. In some embodiments, the apelin analog includes the amino acid sequence of SEQ ID NO: 53. In some embodiments, the apelin analog includes the amino acid sequence of SEQ ID NO: 54. In some embodiments, the apelin analog includes the amino acid sequence of SEQ ID NO: 55.

[0057] In some embodiments, the apelin analog differs from SEQ ID NO: 1 by four or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 1 by three or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 1 by two or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 1 by one or fewer amino acid residues.

[0058] In some embodiments, the apelin analog differs from SEQ ID NO: 2 by three or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 2 by two or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 2 by one or fewer amino acid residues.

[0059] In some embodiments, the apelin analog differs from SEQ ID NO: 3 by three or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 3 by two or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 3 by one or fewer amino acid residues.

[0060] In some embodiments, the apelin analog differs from SEQ ID NO: 4 by three or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 4 by two or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 4 by one or fewer amino acid residues.

[0061] In some embodiments, the apelin analog differs from SEQ ID NO: 5 by three or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 5 by two or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 5 by one or fewer amino acid residues.

[0062] In some embodiments, the apelin analog differs from SEQ ID NO: 6 by three or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 6 by two or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 6 by one or fewer amino acid residues.

[0063] In some embodiments, the apelin analog differs from SEQ ID NO: 7 by three or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 7 by two or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 7 by one or fewer amino acid residues.

[0064] In some embodiments, the apelin analog differs from SEQ ID NO: 50 by three or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 50 by two or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 50 by one or fewer amino acid residues.

[0065] In some embodiments, the apelin analog differs from SEQ ID NO: 51 by three or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 51 by two or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 51 by one or fewer amino acid residues.

[0066] In some embodiments, the apelin analog differs from SEQ ID NO: 52 by three or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 52 by two or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 52 by one or fewer amino acid residues.

[0067] In some embodiments, the apelin analog differs from SEQ ID NO: 53 by three or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 53 by two or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 53 by one or fewer amino acid residues.

[0068] In some embodiments, the apelin analog differs from SEQ ID NO: 54 by three or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 54 by two or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 54 by one or fewer amino acid residues.

[0069] In some embodiments, the apelin analog differs from SEQ ID NO: 55 by three or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 55 by two or fewer amino acid residues. In some embodiments, the apelin analog differs from SEQ ID NO: 55 by one or fewer amino acid residues.

[0070] In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 1. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 1. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 1. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 1. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 1. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 1. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 1. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 1.

[0071] In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 2. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 2. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 2. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 2. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 2. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 2. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 2. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 2.

[0072] In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 3. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 3. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 3. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 3. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 3. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 3. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 3. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 3.

[0073] In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 4. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 4. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 4. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 4. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 4. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 4. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 4. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 4.

[0074] In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 5. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 5. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 5. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 5. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 5. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 5. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 5. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 5.

[0075] In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 6. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 6. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 6. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 6. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 6. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 6. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 6. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 6.

[0076] In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 7. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 7. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 7. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 7. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 7. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 7. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 7. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 7.

[0077] In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 50. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 50. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 50. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 50. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 50. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 50. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 50. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 50.

[0078] In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 51. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 51. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 51. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 51. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 51. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 51. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 51. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 51.

[0079] In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 52. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 52. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 52. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 52. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 52. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 52. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 52. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 52.

[0080] In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 53. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 53. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 53. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 53. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 53. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 53. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 53. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 53.

[0081] In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 54. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 54. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 54. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 54. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 54. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 54. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 54. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 54.

[0082] In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 55. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 55. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 55. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 55. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 55. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 55. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 55. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 55.

[0083] In some embodiments, the apelin analog is an amino acid sequence Includes X1-RPRLSH-X2-GP-X3-X4-X5 (sequence number 63), In the array, X1 is either Q or E. X2 is either K or W. X3 is M, H, or A. X4 is P, V, A, or H, and / or X5 is F, Y, or W.

[0084] In some embodiments, the apelin analog is an amino acid sequence Includes QRPRLQHKNPMA-X1 (Sequence ID 64), In the array, X1 is either F or W.

[0085] In some embodiments, the apelin analog is an amino acid sequence Includes QRPRLSHKGP-X1-X2-X3 (Sequence ID 65), In the array, X1 is M, H, or A. X2 is P, V, A, or H, and / or X3 is F, Y, or W.

[0086] In some embodiments, the apelin analog is an amino acid sequence Includes X1-RPRL-X2-H-X3-X4-P-X5-X6-X7 (sequence number 66), In the array, X1 is either Q or E. X2 is either S or Q, X3 is either K or W. X4 is either G or N. X5 is M, H, or A. X6 is P, V, A, or H, and / or X7 is F, Y, or W.

[0087] Elabera analogue The present invention provides, among other things, an Elavera analog having improved agonist activity for the apelin receptor. In some embodiments, the Elavera analog has enhanced binding to the apelin receptor compared to wild-type Elavera. In some embodiments, the Elavera analog has improved apelin receptor signaling compared to wild-type Elavera. In some embodiments, the Elavera analog has an increased half-life compared to wild-type Elavera. In some embodiments, the apelin analog is more resistant to proteolysis compared to wild-type Elavera.

[0088] In some embodiments, the Erabera analog is derived from wild-type Erabera-54 (MRFQQFLFAFFIFIMSLLLISGQRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP) of SEQ ID NO: 46. In some embodiments, the Erabera analog is derived from wild-type Erabera-32 (QRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP) of SEQ ID NO: 47. In some embodiments, the Erabera analog is derived from wild-type Erabera-22 (KLRKHNCLQRRCMPLHSRVPFP) of SEQ ID NO: 8. In some embodiments, the Erabera analog is derived from wild-type Erabera-21 (LRKHNCLQRRCMPLHSRVPFP) of SEQ ID NO: 48. In some embodiments, the Erabera analog is derived from wild-type Erabera-11 (CMPLHSRVPFP) of SEQ ID NO: 49.

[0089] In some embodiments, the Elavera analog includes modifications compared to SEQ ID NO: 8, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49. In some embodiments, the modifications are substitutions, insertions, or deletions of one or more amino acids.

[0090] In some embodiments, the Elabera analog contains one or more amino acid substitutions at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 of SEQ ID NO: 8. In some embodiments, the Elabera analog contains one or more amino acid substitutions at position 3, 7, 8, 9, 10, 12, 14, 15, 17, or 18 of SEQ ID NO: 8. In some embodiments, the Elabera analog contains an amino acid substitution at position 3 of SEQ ID NO: 8. In some embodiments, the Elabera analog contains an amino acid substitution at position 7 of SEQ ID NO: 8. In some embodiments, the Elabera analog contains an amino acid substitution at position 8 of SEQ ID NO: 8. In some embodiments, the Elabera analog contains an amino acid substitution at position 9 of SEQ ID NO: 8. In some embodiments, the Elabera analog contains an amino acid substitution at position 10 of SEQ ID NO: 8. In some embodiments, the Elavera analog contains an amino acid substitution at position 12 of SEQ ID NO: 8. In some embodiments, the Elavera analog contains an amino acid substitution at position 14 of SEQ ID NO: 8. In some embodiments, the Elavera analog contains an amino acid substitution at position 15 of SEQ ID NO: 8. In some embodiments, the Elavera analog contains an amino acid substitution at position 17 of SEQ ID NO: 8. In some embodiments, the Elavera analog contains an amino acid substitution at position 18 of SEQ ID NO: 8.

[0091] In some embodiments, the Elabera analog includes the R3N amino acid substitution compared to SEQ ID NO: 8. In some embodiments, the Elabera analog includes the P14I amino acid substitution compared to SEQ ID NO: 8. In some embodiments, the Elabera analog includes the S17Q amino acid substitution compared to SEQ ID NO: 8. In some embodiments, the Elabera analog includes the S17R amino acid substitution compared to SEQ ID NO: 8. In some embodiments, the Elabera analog includes the Q9K amino acid substitution compared to SEQ ID NO: 8. In some embodiments, the Elabera analog includes the R18Y amino acid substitution compared to SEQ ID NO: 8. In some embodiments, the Elabera analog includes the L15Y amino acid substitution compared to SEQ ID NO: 8. In some embodiments, the Elabera analog includes the C7S and C12Y amino acid substitutions compared to SEQ ID NO: 8. In some embodiments, the Elabera analog includes the R10E amino acid substitution compared to SEQ ID NO: 8. In some embodiments, the Elabera analog includes the Q9I amino acid substitution compared to SEQ ID NO: 8. In some embodiments, the Elabera analog includes the L8A and Q9T amino acid substitutions compared to SEQ ID NO: 8.

[0092] In some embodiments, the apelin analog comprises the amino acid sequence shown in Table 2. [Table 2-1] [Table 2-2]

[0093] In some embodiments, the Elavera analog includes the amino acid sequence of SEQ ID NO: 8. In some embodiments, the Elavera analog includes the amino acid sequence of SEQ ID NO: 9. In some embodiments, the Elavera analog includes the amino acid sequence of SEQ ID NO: 10. In some embodiments, the Elavera analog includes the amino acid sequence of SEQ ID NO: 11. In some embodiments, the Elavera analog includes the amino acid sequence of SEQ ID NO: 12. In some embodiments, the Elavera analog includes the amino acid sequence of SEQ ID NO: 13. In some embodiments, the Elavera analog includes the amino acid sequence of SEQ ID NO: 30. In some embodiments, the Elavera analog includes the amino acid sequence of SEQ ID NO: 31. In some embodiments, the Elavera analog includes the amino acid sequence of SEQ ID NO: 32. In some embodiments, the Elavera analog includes the amino acid sequence of SEQ ID NO: 33. In some embodiments, the Elavera analog includes the amino acid sequence of SEQ ID NO: 34. In some embodiments, the Elavera analog includes the amino acid sequence of SEQ ID NO: 35.

[0094] In some embodiments, the Elavera analog differs from SEQ ID NO: 8 by 5 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 8 by 4 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 8 by 3 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 8 by 2 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 8 by 1 or fewer amino acid residues.

[0095] In some embodiments, the Elavera analog differs from SEQ ID NO: 9 by 5 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 9 by 4 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 9 by 3 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 9 by 2 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 9 by 1 or fewer amino acid residues.

[0096] In some embodiments, the Elavera analog differs from SEQ ID NO: 10 by 5 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 10 by 4 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 10 by 3 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 10 by 2 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 10 by 1 or fewer amino acid residues.

[0097] In some embodiments, the Elavera analog differs from SEQ ID NO: 11 by 5 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 11 by 4 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 11 by 3 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 11 by 2 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 11 by 1 or fewer amino acid residues.

[0098] In some embodiments, the Elavera analog differs from SEQ ID NO: 12 by 5 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 12 by 4 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 12 by 3 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 12 by 2 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 12 by 1 or fewer amino acid residues.

[0099] In some embodiments, the Elavera analog differs from SEQ ID NO: 13 by 5 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 13 by 4 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 13 by 3 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 3 by 2 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 13 by 1 or fewer amino acid residues.

[0100] In some embodiments, the Elavera analog differs from SEQ ID NO: 30 by 5 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 30 by 4 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 30 by 3 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 30 by 2 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 30 by 1 or fewer amino acid residues.

[0101] In some embodiments, the Elavera analog differs from SEQ ID NO: 31 by 5 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 31 by 4 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 31 by 3 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 31 by 2 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 31 by 1 or fewer amino acid residues.

[0102] In some embodiments, the Elavera analog differs from SEQ ID NO: 32 by 5 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 32 by 4 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 32 by 3 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 32 by 2 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 32 by 1 or fewer amino acid residues.

[0103] In some embodiments, the Elabera analog differs from SEQ ID NO: 33 by 5 or fewer amino acid residues. In some embodiments, the Elabera analog differs from SEQ ID NO: 33 by 4 or fewer amino acid residues. In some embodiments, the Elabera analog differs from SEQ ID NO: 33 by 3 or fewer amino acid residues. In some embodiments, the Elabera analog differs from SEQ ID NO: 33 by 2 or fewer amino acid residues. In some embodiments, the Elabera analog differs from SEQ ID NO: 33 by 1 or fewer amino acid residues.

[0104] In some embodiments, the Elavera analog differs from SEQ ID NO: 34 by 5 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 34 by 4 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 34 by 3 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 34 by 2 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 34 by 1 or fewer amino acid residues.

[0105] In some embodiments, the Elavera analog differs from SEQ ID NO: 35 by 5 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 35 by 4 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 35 by 3 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 35 by 2 or fewer amino acid residues. In some embodiments, the Elavera analog differs from SEQ ID NO: 35 by 1 or fewer amino acid residues.

[0106] In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 8. In some embodiments, the Elabera analog contains a sequence that is at least 92% identical to sequence number 8. In some embodiments, the Elabera analog contains a sequence that is at least 95% identical to sequence number 8. In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 8. In some embodiments, the Elabera analog contains a sequence that is at least 96% identical to sequence number 8. In some embodiments, the Elabera analog contains a sequence that is at least 97% identical to sequence number 8. In some embodiments, the Elabera analog contains a sequence that is at least 98% identical to sequence number 8. In some embodiments, the Elabera analog contains a sequence that is at least 99% identical to sequence number 8.

[0107] In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 9. In some embodiments, the Elabera analog contains a sequence that is at least 92% identical to sequence number 9. In some embodiments, the Elabera analog contains a sequence that is at least 95% identical to sequence number 9. In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 9. In some embodiments, the Elabera analog contains a sequence that is at least 96% identical to sequence number 9. In some embodiments, the Elabera analog contains a sequence that is at least 97% identical to sequence number 9. In some embodiments, the Elabera analog contains a sequence that is at least 98% identical to sequence number 9. In some embodiments, the Elabera analog contains a sequence that is at least 99% identical to sequence number 9.

[0108] In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 10. In some embodiments, the Elabera analog contains a sequence that is at least 92% identical to sequence number 10. In some embodiments, the Elabera analog contains a sequence that is at least 95% identical to sequence number 10. In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 10. In some embodiments, the Elabera analog contains a sequence that is at least 96% identical to sequence number 10. In some embodiments, the Elabera analog contains a sequence that is at least 97% identical to sequence number 10. In some embodiments, the Elabera analog contains a sequence that is at least 98% identical to sequence number 10. In some embodiments, the Elabera analog contains a sequence that is at least 99% identical to sequence number 10.

[0109] In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 11. In some embodiments, the Elabera analog contains a sequence that is at least 92% identical to sequence number 11. In some embodiments, the Elabera analog contains a sequence that is at least 95% identical to sequence number 11. In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 11. In some embodiments, the Elabera analog contains a sequence that is at least 96% identical to sequence number 11. In some embodiments, the Elabera analog contains a sequence that is at least 97% identical to sequence number 11. In some embodiments, the Elabera analog contains a sequence that is at least 98% identical to sequence number 11. In some embodiments, the Elabera analog contains a sequence that is at least 99% identical to sequence number 11.

[0110] In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 12. In some embodiments, the Elabera analog contains a sequence that is at least 92% identical to sequence number 12. In some embodiments, the Elabera analog contains a sequence that is at least 95% identical to sequence number 12. In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 12. In some embodiments, the Elabera analog contains a sequence that is at least 96% identical to sequence number 12. In some embodiments, the Elabera analog contains a sequence that is at least 97% identical to sequence number 12. In some embodiments, the Elabera analog contains a sequence that is at least 98% identical to sequence number 8. In some embodiments, the Elabera analog contains a sequence that is at least 99% identical to sequence number 12.

[0111] In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 13. In some embodiments, the Elabera analog contains a sequence that is at least 92% identical to sequence number 13. In some embodiments, the Elabera analog contains a sequence that is at least 95% identical to sequence number 13. In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 13. In some embodiments, the Elabera analog contains a sequence that is at least 96% identical to sequence number 13. In some embodiments, the Elabera analog contains a sequence that is at least 97% identical to sequence number 13. In some embodiments, the Elabera analog contains a sequence that is at least 98% identical to sequence number 13. In some embodiments, the Elabera analog contains a sequence that is at least 99% identical to sequence number 13.

[0112] In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 30. In some embodiments, the Elabera analog contains a sequence that is at least 92% identical to sequence number 30. In some embodiments, the Elabera analog contains a sequence that is at least 95% identical to sequence number 30. In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 30. In some embodiments, the Elabera analog contains a sequence that is at least 96% identical to sequence number 30. In some embodiments, the Elabera analog contains a sequence that is at least 97% identical to sequence number 30. In some embodiments, the Elabera analog contains a sequence that is at least 98% identical to sequence number 30. In some embodiments, the Elabera analog contains a sequence that is at least 99% identical to sequence number 30.

[0113] In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 31. In some embodiments, the Elabera analog contains a sequence that is at least 92% identical to sequence number 31. In some embodiments, the Elabera analog contains a sequence that is at least 95% identical to sequence number 31. In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 31. In some embodiments, the Elabera analog contains a sequence that is at least 96% identical to sequence number 31. In some embodiments, the Elabera analog contains a sequence that is at least 97% identical to sequence number 31. In some embodiments, the Elabera analog contains a sequence that is at least 98% identical to sequence number 31. In some embodiments, the Elabera analog contains a sequence that is at least 99% identical to sequence number 31.

[0114] In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 32. In some embodiments, the Elabera analog contains a sequence that is at least 92% identical to sequence number 32. In some embodiments, the Elabera analog contains a sequence that is at least 95% identical to sequence number 32. In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 32. In some embodiments, the Elabera analog contains a sequence that is at least 96% identical to sequence number 32. In some embodiments, the Elabera analog contains a sequence that is at least 97% identical to sequence number 32. In some embodiments, the Elabera analog contains a sequence that is at least 98% identical to sequence number 32. In some embodiments, the Elabera analog contains a sequence that is at least 99% identical to sequence number 32.

[0115] In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 33. In some embodiments, the Elabera analog contains a sequence that is at least 92% identical to sequence number 33. In some embodiments, the Elabera analog contains a sequence that is at least 95% identical to sequence number 33. In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 33. In some embodiments, the Elabera analog contains a sequence that is at least 96% identical to sequence number 33. In some embodiments, the Elabera analog contains a sequence that is at least 97% identical to sequence number 33. In some embodiments, the Elabera analog contains a sequence that is at least 98% identical to sequence number 33. In some embodiments, the Elabera analog contains a sequence that is at least 99% identical to sequence number 33.

[0116] In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 34. In some embodiments, the Elabera analog contains a sequence that is at least 92% identical to sequence number 34. In some embodiments, the Elabera analog contains a sequence that is at least 95% identical to sequence number 34. In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 34. In some embodiments, the Elabera analog contains a sequence that is at least 96% identical to sequence number 34. In some embodiments, the Elabera analog contains a sequence that is at least 97% identical to sequence number 34. In some embodiments, the Elabera analog contains a sequence that is at least 98% identical to sequence number 34. In some embodiments, the Elabera analog contains a sequence that is at least 99% identical to sequence number 34.

[0117] In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 35. In some embodiments, the Elabera analog contains a sequence that is at least 92% identical to sequence number 35. In some embodiments, the Elabera analog contains a sequence that is at least 95% identical to sequence number 35. In some embodiments, the Elabera analog contains a sequence that is at least 90% identical to sequence number 35. In some embodiments, the Elabera analog contains a sequence that is at least 96% identical to sequence number 35. In some embodiments, the Elabera analog contains a sequence that is at least 97% identical to sequence number 35. In some embodiments, the Elabera analog contains a sequence that is at least 98% identical to sequence number 35. In some embodiments, the Elabera analog contains a sequence that is at least 99% identical to sequence number 35.

[0118] In some embodiments, the apelin analog is an amino acid sequence Includes KL-Z1-KHN-Z2-Z3-Z4-Z5-R-Z6-M-Z7-Z8-H-Z9-Z10-VPFP (Sequence No. 67), Z1 is either R or N. Z2 is either C or S. Z3 is either A or L. Z4 is Q, I, K, or T. The Z5 is either R or E. Z6 is either C or Y. Z7 is either P or I. Z8 is either L or Y. Z9 is S, Q, or R, and / or Z10 is either R or Y.

[0119] Carrier portion The present invention provides, in particular, an apelin receptor agonist comprising an apelin analog or an elavella analog fused to a carrier portion.

[0120] Half-life extension In some embodiments, the carrier portion extends the half-life of the apelin receptor agonist. As peptides, apelin and elavella have half-lives of several minutes. The short half-life is partially mediated by proteolysis by numerous circulating proteases that recognize and cleave specific sequences. In addition to proteolysis, the peptides are also rapidly removed from the blood via the kidneys. In some embodiments, the carrier portion prevents renal clearance of the apelin receptor agonist. In some embodiments, the carrier portion protects the apelin receptor agonist from proteolysis.

[0121] FC Domain In some embodiments, the carrier portion is an Fc domain. In some embodiments, the carrier portion is an Fc domain of IgG1. In some embodiments, the carrier portion is an Fc domain of IgG2. In some embodiments, the carrier portion is an Fc domain of IgG3. In some embodiments, the carrier portion is an Fc domain of IgG4.

[0122] In some embodiments, the Fc domain has reduced or disabled effector function. In some embodiments, the Fc domain has reduced or disabled binding to all FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q, but retains its ability to bind to FcRn.

[0123] In some embodiments, the Fc domain is a modified Fc domain containing one or more mutations. The Fc variants of the present invention can be optimized for a variety of properties. Fc variants that are manipulated or predicted to exhibit one or more optimized properties are referred to herein as “optimized Fc variants.” Properties that can be optimized include, but are not limited to, enhanced or reduced affinity for FcγR. In some embodiments, the Fc variants of the present invention are optimized to reduce or excise affinity for human FcγR, including but not limited to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. These embodiments are expected to provide Fc polypeptides with enhanced therapeutic properties in humans, such as reduced effector function and reduced toxicity. In other embodiments, the Fc variants of the present invention provide enhanced affinity for one or more FcγR but reduced affinity for one or more other FcγR. For example, the Fc variant of the present invention may have enhanced binding to FcγRIIIa but reduced binding to FcγRIIb. Alternatively, the Fc variant of the present invention may have enhanced binding to FcγRIIa and FcγRI but reduced binding to FcγRIIb. In yet another embodiment, the Fc variant of the present invention may have enhanced affinity for FcγRIIb but reduced affinity for one or more activated FcγRs.

[0124] In some embodiments, the Fc variant has reduced or excised affinity for FcγRI. In some embodiments, the Fc variant has reduced or excised affinity for FcγRIIa. In some embodiments, the Fc variant has reduced or excised affinity for FcγRIIb. In some embodiments, the Fc variant has reduced or excised affinity for FcγRIIc. In some embodiments, the Fc variant has reduced or excised affinity for FcγRIIIa. In some embodiments, the Fc variant has reduced or excised affinity for FcγRIIIb. In some embodiments, the Fc variant has reduced or excised affinity for C1q. In some embodiments, the Fc variant has enhanced affinity for FcRn. In some embodiments, the Fc variant maintains affinity for FcRn. In some embodiments, the Fc variant has reduced or excised affinity for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q. In some embodiments, the Fc variant has reduced or excised affinity for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q, but retains binding to FcRn.

[0125] In some embodiments, the Fc domain is a monovalent Fc domain. In some embodiments, the Fc domain is a human IgG1 monovalent Fc domain. In some embodiments, the Fc domain is a human IgG2 monovalent Fc domain. In some embodiments, the Fc domain is a human IgG3 monovalent Fc domain. In some embodiments, the Fc domain is a human IgG4 monovalent Fc domain. In some embodiments, the Fc domain is a modified human IgG1 monovalent Fc domain. In some embodiments, the Fc domain is a modified human IgG2 monovalent Fc domain. In some embodiments, the Fc domain is a modified human IgG3 monovalent Fc domain. In some embodiments, the Fc domain is a modified human IgG4 monovalent Fc domain.

[0126] In some embodiments, the Fc domain is a bivalent Fc domain. In some embodiments, the Fc domain is a human IgG1 bivalent Fc domain. In some embodiments, the Fc domain is a human IgG2 bivalent Fc domain. In some embodiments, the Fc domain is a human IgG3 bivalent Fc domain. In some embodiments, the Fc domain is a human IgG4 bivalent Fc domain. In some embodiments, the Fc domain is a modified human IgG1 bivalent Fc domain. In some embodiments, the Fc domain is a modified human IgG2 bivalent Fc domain. In some embodiments, the Fc domain is a modified human IgG3 bivalent Fc domain. In some embodiments, the Fc domain is a modified human IgG4 bivalent Fc domain. In some embodiments, the modified human IgG4 bivalent Fc domain includes an S228P substitution.

[0127] In some embodiments, the carrier portion includes SEQ ID NO: 14. In some embodiments, the Fc domain of IgG1 includes SEQ ID NO: 14. In some embodiments, the monovalent Fc domain of human IgG1 includes SEQ ID NO: 14. DKTHTSPPSPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVNLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLNSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 14)

[0128] In some embodiments, the carrier portion contains an amino acid sequence that is at least 80% identical to SEQ ID NO: 14. In some embodiments, the carrier portion contains an amino acid sequence that is at least 85% identical to SEQ ID NO: 14. In some embodiments, the carrier portion contains an amino acid sequence that is at least 88% identical to SEQ ID NO: 14. In some embodiments, the carrier portion contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 14. In some embodiments, the carrier portion contains an amino acid sequence that is at least 92% identical to SEQ ID NO: 14. In some embodiments, the carrier portion contains an amino acid sequence that is at least 93% identical to SEQ ID NO: 14. In some embodiments, the carrier portion contains an amino acid sequence that is at least 94% identical to SEQ ID NO: 14. In some embodiments, the carrier portion contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 14. In some embodiments, the carrier portion contains an amino acid sequence that is at least 96% identical to SEQ ID NO: 14. In some embodiments, the carrier portion contains an amino acid sequence that is at least 97% identical to SEQ ID NO: 14. In some embodiments, the carrier portion contains an amino acid sequence that is at least 98% identical to SEQ ID NO: 14. In some embodiments, the carrier portion contains an amino acid sequence that is at least 99% identical to SEQ ID NO: 14.

[0129] In some embodiments, the carrier portion includes SEQ ID NO: 15. In some embodiments, the Fc domain of IgG1 includes SEQ ID NO: 15. In some embodiments, the monovalent Fc domain of human IgG1 includes SEQ ID NO: 15. MRAWIFFLLCLAGRALADKTHTSPPSPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVNLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLNSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 15)

[0130] In some embodiments, the carrier portion contains an amino acid sequence that is at least 80% identical to SEQ ID NO: 15. In some embodiments, the carrier portion contains an amino acid sequence that is at least 85% identical to SEQ ID NO: 15. In some embodiments, the carrier portion contains an amino acid sequence that is at least 88% identical to SEQ ID NO: 15. In some embodiments, the carrier portion contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 15. In some embodiments, the carrier portion contains an amino acid sequence that is at least 92% identical to SEQ ID NO: 15. In some embodiments, the carrier portion contains an amino acid sequence that is at least 93% identical to SEQ ID NO: 15. In some embodiments, the carrier portion contains an amino acid sequence that is at least 94% identical to SEQ ID NO: 15. In some embodiments, the carrier portion contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 15. In some embodiments, the carrier portion contains an amino acid sequence that is at least 96% identical to SEQ ID NO: 15. In some embodiments, the carrier portion contains an amino acid sequence that is at least 97% identical to SEQ ID NO: 15. In some embodiments, the carrier portion contains an amino acid sequence that is at least 98% identical to SEQ ID NO: 15. In some embodiments, the carrier portion contains an amino acid sequence that is at least 99% identical to SEQ ID NO: 15.

[0131] Other carrier parts In some embodiments, the carrier portion includes a serum protein-binding portion. In some embodiments, the carrier portion is human serum albumin (HSA). In some embodiments, the carrier portion includes a protein or peptide that binds to HSA. In some embodiments, the carrier portion includes an anti-HSA antibody or a fragment thereof. In some embodiments, the carrier portion includes an anti-HSA Fab. In some embodiments, the carrier portion includes an anti-HSA nanobody (VHH). In some embodiments, the carrier portion includes an anti-HSA scFv. In some embodiments, the carrier portion includes an anti-HSA V-NAR. In some embodiments, the carrier portion includes an anti-HSA VHH of ALB-23. ALB-23 is described in International Publication No. 2012175400A1, the contents of which are incorporated by reference.

[0132] In some embodiments, the carrier portion comprises one or more lipids. The lipids may be of various lengths, chemicals, linkers, and conjugates, or may comprise them, as will be apparent to those skilled in the art. In some embodiments, the carrier portion comprises saturated fatty acids. In some embodiments, the saturated fatty acids are linked to the peptide via their carboxylate groups. In some embodiments, the saturated fatty acids are C 6-18 Saturated fatty acids (e.g. C6, C 12 , C 16 , or C 18 Contains saturated fatty acids. In some embodiments, the carrier portion contains saturated fatty acid. In some embodiments, the saturated fatty acid is linked to the peptide via the carboxylate group at its free end. In some embodiments, the saturated fatty acid is C 6-18 Saturated fatty diacids (e.g., C6, C 12 , C 16 , or C 18 Contains saturated fatty acids.

[0133] In some embodiments, the carrier portion comprises one or more polyethylene glycols (PEGs). The PEGs may be of various lengths, have various molecular weights, and contain linkers, as will be apparent to those skilled in the art. In some embodiments, the PEGs are monodisperse. In some embodiments, the monodisperse PEGs have 12 to 48 ethylene glycol repeats (e.g., 12 to 24 repeats, 12 to 36 repeats, 24 to 48 repeats, 24 to 36 repeats, or 36 to 48 repeats). In some embodiments, the PEGs are polydisperse. In some embodiments, polydisperse PEG is 500-50,000 Daltons (e.g., 500-1,000 Da, 500-5,000 Da, 500-20,000 Da, 500-40,000 Da, 1,000-5,000 Da, 1,000-20,000 Da, 1,000-40,000 Da, 1,000-50,000 Da, 5,0 The molecular weights are 0.5kDa, 1kDa, 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, or 50kDa. In some embodiments, the polydisperse PEG has a molecular weight of 0.5kDa, 1kDa, 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, or 50kDa. In some embodiments, the carrier portion contains polydisperse PEG having a molecular weight of 1kDa. In some embodiments, the carrier portion includes polydisperse PEG having a molecular weight of 5 kDa. In some embodiments, the carrier portion includes polydisperse PEG having a molecular weight of 10 kDa. In some embodiments, the carrier portion includes polydisperse PEG having a molecular weight of 15 kDa. In some embodiments, the carrier portion includes polydisperse PEG having a molecular weight of 20 kDa. In some embodiments, the carrier portion includes polydisperse PEG having a molecular weight of 25 kDa. In some embodiments, the carrier portion includes polydisperse PEG having a molecular weight of 30 kDa. In some embodiments, the carrier portion includes polydisperse PEG having a molecular weight of 35 kDa.In some embodiments, the carrier portion includes polydisperse PEG having a molecular weight of 40 kDa. In some embodiments, the carrier portion includes polydisperse PEG having a molecular weight of 50 kDa.

[0134] In some embodiments, the carrier portion comprises a half-life extended polypeptide. The half-life extended polypeptide may be of various lengths, chemicals, linkers, and conjugates, or may contain them, as will be apparent to those skilled in the art. In some embodiments, the half-life extended polypeptide comprises repeating units of Pro-Ala-Ser (PAS). In some embodiments, the half-life extended polypeptide comprises 50 to 500 repeating PAS units (e.g., 50 to 100 PAS units, 50 to 200 PAS units, 100 to 200 PAS units, 100 to 500 PAS units, or 200 to 500 PAS units).

[0135] In some embodiments, the half-life extension polypeptide includes the XTEN® polypeptide. XTEN is described in U.S. Patent Application Publication No. 20150037359A1, which is incorporated by reference.

[0136] In some embodiments, the apelin analog or elavella analog is not fused to the carrier portion. In some embodiments, the apelin analog or elavella analog exists as a free peptide.

[0137] Exemplary apelin receptor agonists The present invention provides an apelin receptor agonist comprising, in particular, an apelin analog or an elavella analog fused to a carrier moiety via a linker. The present invention provides an apelin receptor agonist comprising an apelin analog or an elavella analog, wherein the N-terminus of the apelin analog or elavella analog is fused to a carrier moiety via a linker. In some embodiments, the N-terminus of the apelin analog of the elavella analog is fused to the C-terminus of the carrier moiety. In some embodiments, the N-terminus of the apelin analog of the elavella analog is fused to the C-terminus of the Fc domain. In some embodiments, the N-terminus of the apelin analog of the elavella analog is fused to the C-terminus of the monovalent Fc domain. In some embodiments, the N-terminus of the apelin analog of the elavella analog is fused to the C-terminus of the monovalent IgG1 Fc domain.

[0138] In some embodiments, the linker includes glycine and serine residues. In some embodiments, the linker consists of glycine and serine residues. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 16 (GGGGSGGGGSGGGGS).

[0139] In some embodiments, the apelin receptor agonist includes the sequence shown in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0140] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 17. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 17. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 17. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 17. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 17. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 17. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 17. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 17. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 17. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 17. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 17. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 17. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 17.

[0141] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 18. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 18. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 18. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 18. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 18. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 18. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 18. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 18. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 18. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 18. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 18. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 18. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 18.

[0142] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 19. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 19. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 19. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 19. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 19. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 19. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 19. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 19. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 19. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 19. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 19. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 19. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 19.

[0143] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 20. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 20. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 20. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 20. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 20. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 20. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 20. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 20. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 20. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 20. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 20. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 20. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 20.

[0144] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 21. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 21. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 21. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 21. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 21. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 21. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 21. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 21. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 21. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 21. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 21. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 21. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 21.

[0145] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 22. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 22. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 22. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 22. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 22. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 22. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 22. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 22. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 22. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 22. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 22. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 22. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 22.

[0146] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 23. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 23. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 23. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 23. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 23. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 23. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 23. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 23. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 23. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 23. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 23. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 23. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 23.

[0147] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 24. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 24. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 24. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 24. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 24. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 24. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 24. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 24. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 24. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 24. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 24. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 24. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 24.

[0148] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 25. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 25. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 25. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 25. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 25. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 25. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 25. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 25. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 25. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 25. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 25. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 25. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 25.

[0149] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 26. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 26. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 26. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 26. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 26. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 26. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 26. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 26. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 26. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 26. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 26. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 26. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 26.

[0150] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 27. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 27. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 27. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 27. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 27. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 27. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 27. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 27. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 27. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 27. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 27. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 27. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 27.

[0151] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 28. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 28. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 28. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 28. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 28. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 28. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 28. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 28. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 28. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 28. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 28. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 28. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 28.

[0152] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 29. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 29. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 29. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 29. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 29. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 29. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 29. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 29. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 29. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 29. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 29. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 29. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 29.

[0153] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 36. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 36. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 36. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 36. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 36. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 36. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 36. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 36. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 36. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 36. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 36. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 36. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 36.

[0154] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 37. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 37. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 37. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 37. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 37. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 37. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 37. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 37. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 37. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 37. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 37. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 37. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 37.

[0155] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 38. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 38. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 38. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 38. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 38. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 38. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 38. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 38. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 38. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 38. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 38. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 38. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 38.

[0156] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 39. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 39. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 39. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 39. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 39. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 39. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 39. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 39. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 39. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 39. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 9. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 39. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 39.

[0157] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 40. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 40. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 40. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 40. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 40. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 40. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 0. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 40. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 40. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 40. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 40. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 40. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 40.

[0158] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 41. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 41. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 41. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 41. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 41. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 41. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 41. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 41. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 41. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 41. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 41. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 41. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 41.

[0159] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 56. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 56. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 56. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 56. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 56. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 56. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 56. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 56. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 56. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 56. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 56. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 56. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 56.

[0160] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 57. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 57. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 57. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 57. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 57. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 57. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 57. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 57. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 57. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 57. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 57. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 57. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 57.

[0161] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 58. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 58. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 58. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 58. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 58. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 58. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 58. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 58. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 58. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 58. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 58. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 58. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 58.

[0162] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 59. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 59. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 59. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 59. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 59. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 59. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 59. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 59. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 59. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 59. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 59. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 59. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 59.

[0163] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 60. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 60. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 60. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 60. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 60. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 60. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 60. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 60. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 60. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 60. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 60. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 60. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 60.

[0164] In some embodiments, the apelin analog contains a sequence that is at least 80% identical to sequence number 61. In some embodiments, the apelin analog contains a sequence that is at least 82% identical to sequence number 61. In some embodiments, the apelin analog contains a sequence that is at least 84% identical to sequence number 61. In some embodiments, the apelin analog contains a sequence that is at least 86% identical to sequence number 61. In some embodiments, the apelin analog contains a sequence that is at least 88% identical to sequence number 61. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 61. In some embodiments, the apelin analog contains a sequence that is at least 92% identical to sequence number 61. In some embodiments, the apelin analog contains a sequence that is at least 95% identical to sequence number 61. In some embodiments, the apelin analog contains a sequence that is at least 90% identical to sequence number 61. In some embodiments, the apelin analog contains a sequence that is at least 96% identical to sequence number 61. In some embodiments, the apelin analog contains a sequence that is at least 97% identical to sequence number 61. In some embodiments, the apelin analog contains a sequence that is at least 98% identical to sequence number 61. In some embodiments, the apelin analog contains a sequence that is at least 99% identical to sequence number 61.

[0165] Leader Array In some embodiments, the apelin receptor agonist includes a leader sequence. In some embodiments, the apelin receptor agonist includes a leader sequence at the N-terminus. In some embodiments, the apelin receptor agonist includes a leader sequence at the N-terminus of the carrier portion. In some embodiments, the apelin receptor agonist includes a leader sequence at the N-terminus of the Fc domain. In some embodiments, the leader sequence is cleaved before secretion.

[0166] In some embodiments, the leader sequence includes an osteonectin leader sequence. In some embodiments, the leader sequence includes sequence number 62 (MRAWIFFLLCLAGRALA). In some embodiments, the leader sequence includes an amino acid sequence that is at least 80% identical to sequence number 62. In some embodiments, the leader sequence includes an amino acid sequence that is at least 85% identical to sequence number 62. In some embodiments, the leader sequence includes an amino acid sequence that is at least 90% identical to sequence number 62. In some embodiments, the leader sequence includes an amino acid sequence that is at least 92% identical to sequence number 62. In some embodiments, the leader sequence includes an amino acid sequence that is at least 95% identical to sequence number 62. In some embodiments, the leader sequence includes an amino acid sequence that is at least 98% identical to sequence number 62. In some embodiments, the leader sequence includes an amino acid sequence that is at least 99% identical to sequence number 62.

[0167] Characteristics of aperin receptor agonists This application provides an apelin receptor agonist comprising an apelin analog or an elavella analog that has unexpectedly superior properties, including, but not limited to, improved signaling characteristics, a longer half-life, and reduced desensitization compared to wild-type apelin or elavella. In some embodiments, the apelin receptor agonist of the present invention binds to the APJ with higher affinity and specificity compared to wild-type apelin agonist.

[0168] Improved signaling in the APJ pathway The apelin receptor agonist of the present invention exhibits improved signal transduction characteristics compared to wild-type or endogenous apelin or elavella.

[0169] In some embodiments, the apelin receptor agonist has a bias towards Gα signaling over β-arrestin signaling. In some embodiments, the apelin receptor agonist has a bias towards Gα signaling over β-arrestin signaling by at least about 1.5 times. In some embodiments, the apelin receptor agonist has a bias towards Gα signaling over β-arrestin signaling by at least about 2 times. In some embodiments, the apelin receptor agonist has a bias towards Gα signaling over β-arrestin signaling by at least about 2.5 times. In some embodiments, the apelin receptor agonist has a bias towards Gα signaling over β-arrestin signaling by at least about 3 times. In some embodiments, the apelin receptor agonist has a bias towards Gα signaling over β-arrestin signaling by at least about 4 times. In some embodiments, the apelin receptor agonist has a bias towards Gα signaling over β-arrestin signaling by at least about 5 times. In some embodiments, the apelin receptor agonist has a bias towards Gα signaling over β-arrestin signaling by at least about 7 times. In some embodiments, the apelin receptor agonist has at least about a 10-fold bias towards Gα signaling than towards β-arrestin signaling. In some embodiments, the apelin receptor agonist has at least about a 12-fold bias towards Gα signaling than towards β-arrestin signaling. In some embodiments, the apelin receptor agonist has at least about a 15-fold bias towards Gα signaling than towards β-arrestin signaling. In some embodiments, the apelin receptor agonist has at least about a 20-fold bias towards Gα signaling than towards β-arrestin signaling. In some embodiments, the apelin receptor agonist has at least about a 25-fold bias towards Gα signaling than towards β-arrestin signaling. In some embodiments, the apelin receptor agonist has at least about a 30-fold bias towards Gα signaling than towards β-arrestin signaling.In some embodiments, the apelin receptor agonist has a bias towards Gα signaling over β-arrestin signaling by at least about 40 times. In some embodiments, the apelin receptor agonist has a bias towards Gα signaling over β-arrestin signaling by at least about 50 times. In some embodiments, the apelin receptor agonist has a bias towards Gα signaling over β-arrestin signaling by at least about 50 times. In some embodiments, the apelin receptor agonist has a bias towards Gα signaling over β-arrestin signaling by at least about 100 times. In some embodiments, the apelin receptor agonist has a bias towards Gα signaling over β-arrestin signaling by at least about 200 times. In some embodiments, the apelin receptor agonist has a bias towards Gα signaling over β-arrestin signaling by at least about 500 times.

[0170] In some embodiments, the apelin receptor agonist was measured by cAMP depletion, with a G of less than 60 nM. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 80 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 75 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 70 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 65 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 60 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 55 nM, as measured by cAMP depletion.αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 50 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 45 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 40 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 35 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 30 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 25 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 20 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 15 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 10 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 5 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 2 nM, as measured by cAMP depletion. αi The signal transduction assay has an EC50 value. In some embodiments, the apelin receptor agonist has a G value of less than 1 nM when measured by cAMP depletion. αiIt has a signal transduction assay EC50 value.

[0171] In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 1000 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 600 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 500 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 450 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 400 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 350 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 300 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 250 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 200 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 150 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 100 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 80 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 60 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 50 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 40 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 30 nM. In some embodiments, the apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 20 nM.In some embodiments, the apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 10 nM. In some embodiments, the apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 5 nM.

[0172] In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 5 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 10 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 20 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 30 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 40 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 50 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 60 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 70 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 80 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 90 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 100 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 200 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 300 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 400 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 500 nM. In some embodiments, the Apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 600 nM. In some embodiments, the apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 700 nM.In some embodiments, the apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 800 nM. In some embodiments, the apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 900 nM. In some embodiments, the apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 1000 nM. In some embodiments, the apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 2000 nM. In some embodiments, the apelin receptor agonist has a beta-arrestin signaling assay EC50 value greater than 5000 nM.

[0173] In some embodiments, the apelin receptor agonist has an ERK activation assay EC50 value of less than 50 nM. In some embodiments, the apelin receptor agonist has an ERK activation assay EC50 value of less than 40 nM. In some embodiments, the apelin receptor agonist has an ERK activation assay EC50 value of less than 30 nM. In some embodiments, the apelin receptor agonist has an ERK activation assay EC50 value of less than 25 nM. In some embodiments, the apelin receptor agonist has an ERK activation assay EC50 value of less than 20 nM. In some embodiments, the apelin receptor agonist has an ERK activation assay EC50 value of less than 15 nM. In some embodiments, the apelin receptor agonist has an ERK activation assay EC50 value of less than 10 nM. In some embodiments, the apelin receptor agonist has an ERK activation assay EC50 value of less than 5 nM. In some embodiments, the apelin receptor agonist has an ERK activation assay EC50 value of less than 2 nM. In some embodiments, the apelin receptor agonist has an ERK activation assay EC50 value of less than 1 nM.

[0174] Half-life In some embodiments, the apelin receptor agonists of the present invention have higher efficacy and potency compared to wild-type or endogenous apelin or elavera. In some embodiments, the apelin receptor agonists of the present invention have an increased half-life compared to wild-type or endogenous apelin or elavera. In some embodiments, the apelin receptor agonists of the present invention are less sensitive to proteolytic cleavage compared to wild-type or endogenous apelin or elavera. In some embodiments, the apelin receptor agonists of the present invention are less sensitive to renal clearance compared to wild-type or endogenous apelin or elavera.

[0175] In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 30 minutes. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 1 hour. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 2 hours. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 3 hours. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 4 hours. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 5 hours. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 6 hours. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 7 hours. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 8 hours. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 9 hours. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 10 hours. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 12 hours. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 24 hours. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 2 days. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 3 days. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 4 days. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 5 days. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 6 days. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 7 days.In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 10 days. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about 15 days. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about two weeks. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about three weeks. In some embodiments, the apelin receptor agonist has a plasma or serum in vivo half-life of at least about four weeks.

[0176] In some embodiments, the apelin receptor agonist has a half-life at least twice as long as that of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has a half-life at least three times longer than that of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has a half-life at least four times longer than that of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has a half-life at least five times longer than that of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has a half-life at least six times longer than that of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has a half-life at least seven times longer than that of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has a half-life at least eight times longer than that of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has a half-life at least 9 times longer than that of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has a half-life at least 10 times longer than that of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has a half-life at least 11 times longer than that of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has a half-life at least 12 times longer than that of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has a half-life at least 15 times longer than that of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has a half-life at least 20 times longer than that of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has a half-life at least 25 times longer than that of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has a half-life at least 30 times longer than that of wild-type apelin or elavella. In some embodiments, the apelin receptor agonist has a half-life at least 40 times longer than that of wild-type apelin or elavella.In some embodiments, the apelin receptor agonist has a half-life at least 50 times longer than that of wild-type apelin or elavella. In some embodiments, the apelin receptor agonist has a half-life at least 100 times longer than that of wild-type apelin or elavella.

[0177] desensitization GPCR desensitization is a multifaceted problem that occurs over multiple stages. Generally, β-arrestin signaling is associated with desensitization, but these effects have not been extensively studied in the APJ.

[0178] GPCR desensitization can occur in both the short and long term. In the short term, receptors are internalized into endosomes, so they are removed from the cell membrane and can prevent interaction with native ligands or drugs. Internalized receptors are sometimes recycled back into the membrane and become available again, but in other situations, endosomes fuse with lysosomes and target the receptors for degradation, resulting in long-term desensitization.

[0179] In the long term, sustained signaling can lead to downregulation of the transcription of receptors and their downstream effectors. This can result in the removal of receptors from the cell membrane or the delinking of receptor activation to downstream effector function, thereby again reducing the efficacy of both native ligands and drugs, respectively.

[0180] In some embodiments, the apelin receptor agonists of the present invention exhibit reduced desensitization compared to wild-type or endogenous apelin or elavella.

[0181] In some embodiments, the apelin receptor agonist causes at least twice as much reduced desensitization as wild-type apelin or elavella. In some embodiments, the apelin receptor agonist causes at least three times as much reduced desensitization as wild-type apelin or elavella. In some embodiments, the apelin receptor agonist causes at least four times as much reduced desensitization as wild-type apelin or elavella. In some embodiments, the apelin receptor agonist causes at least five times as much reduced desensitization as wild-type apelin or elavella. In some embodiments, the apelin receptor agonist causes at least six times as much reduced desensitization as wild-type apelin or elavella. In some embodiments, the apelin receptor agonist causes at least seven times as much reduced desensitization as wild-type apelin or elavella. In some embodiments, the apelin receptor agonist causes at least eight times as much reduced desensitization as wild-type apelin or elavella. In some embodiments, the apelin receptor agonist causes desensitization that is at least 9 times less than that caused by wild-type apelin or elavella. In some embodiments, the apelin receptor agonist causes desensitization that is at least 10 times less than that caused by wild-type apelin or elavella. In some embodiments, the apelin receptor agonist causes desensitization that is at least 12 times less than that caused by wild-type apelin or elavella. In some embodiments, the apelin receptor agonist causes desensitization that is at least 15 times less than that caused by wild-type apelin or elavella. In some embodiments, the apelin receptor agonist causes desensitization that is at least 20 times less than that caused by wild-type apelin or elavella. In some embodiments, the apelin receptor agonist causes desensitization that is at least 30 times less than that caused by wild-type apelin or elavella. In some embodiments, apelin receptor agonists induce a reduced desensitization that is at least 40 times less than that caused by wild-type apelin or elavella.In some embodiments, the apelin receptor agonist causes desensitization that is at least 50 times less than that caused by wild-type apelin or elavella. In some embodiments, the apelin receptor agonist causes desensitization that is at least 60 times less than that caused by wild-type apelin or elavella. In some embodiments, the apelin receptor agonist causes desensitization that is at least 80 times less than that caused by wild-type apelin or elavella. In some embodiments, the apelin receptor agonist causes desensitization that is at least 100 times less than that caused by wild-type apelin or elavella.

[0182] internalization In some embodiments, the apelin receptor agonists of the present invention have reduced internalization compared to wild-type or endogenous apelin or elavella. In some embodiments, the apelin receptor agonists of the present invention are substantially not internalized. The reduced rate of internalization of apelin receptor agonists compared to wild-type ligands may be a desirable characteristic because it may allow for more efficient and sustained activation of the apelin receptor and its downstream effects.

[0183] In some embodiments, the apelin receptor agonist has at least twice the reduced internalization of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has at least three times the reduced internalization of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has at least four times the reduced internalization of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has at least five times the reduced internalization of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has at least six times the reduced internalization of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has at least seven times the reduced internalization of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has at least eight times the reduced internalization of wild-type apelin or elavera. In some embodiments, the apelin receptor agonist has reduced internalization by at least nine times compared to the internalization of wild-type apelin or elavella. In some embodiments, the apelin receptor agonist has reduced internalization by at least ten times compared to the internalization of wild-type apelin or elavella.

[0184] In some embodiments, the apelin receptor agonist is internalized at a rate at least twice as low as the internalization rate of wild-type apelin or elavella. In some embodiments, the apelin receptor agonist is internalized at a rate at least three times lower than the internalization rate of wild-type apelin or elavella. In some embodiments, the apelin receptor agonist is internalized at a rate at least four times lower than the internalization rate of wild-type apelin or elavella. In some embodiments, the apelin receptor agonist is internalized at a rate at least five times lower than the internalization rate of wild-type apelin or elavella. In some embodiments, the apelin receptor agonist is internalized at a rate at least six times lower than the internalization rate of wild-type apelin or elavella. In some embodiments, the apelin receptor agonist is internalized at a rate at least seven times lower than the internalization rate of wild-type apelin or elavella. In some embodiments, the apelin receptor agonist is internalized at a rate at least 8 times lower than the internalization rate of wild-type apelin or elavella. In some embodiments, the apelin receptor agonist is internalized at a rate at least 9 times lower than the internalization rate of wild-type apelin or elavella. In some embodiments, the apelin receptor agonist is internalized at a rate at least 10 times lower than the internalization rate of wild-type apelin or elavella.

[0185] In some embodiments, the apelin receptor agonist is not internalized. In some embodiments, the apelin receptor agonist is internalized only when present at a concentration greater than 1000 nM. In some embodiments, the apelin receptor agonist is internalized only when present at a concentration greater than 500 nM. In some embodiments, the apelin receptor agonist is internalized only when present at a concentration greater than 100 nM. In some embodiments, the apelin receptor agonist is internalized only when present at a concentration greater than 50 nM. In some embodiments, the apelin receptor agonist is internalized only when present at a concentration greater than 100 nM.

[0186] Treatment with aperin receptor agonists The present invention provides, in particular, a method for treating a disease by administering an apelin receptor agonist disclosed herein. In some embodiments, administration of an apelin receptor agonist according to the present invention improves, stabilizes, or reduces one or more symptoms of the disease.

[0187] In some embodiments, the disease is involved in apelin receptor signaling. In some embodiments, the disease is pulmonary hypertension (PAH). In some embodiments, the disease is systemic sclerosis (SSc). In some embodiments, the disease is heart failure with preserved ejection fraction (HfpEf). In some embodiments, the disease is age-related sarcopenia. In some embodiments, the disease is acute kidney injury (AKI). In some embodiments, the disease is cardiovascular disease. In one embodiment, the disease is cancer. In some embodiments, the disease is diabetes mellitus. In some embodiments, the disease is acute decompensated heart failure. In some embodiments, the disease is congestive heart failure. In some embodiments, the disease is myocardial infarction. In some embodiments, the disease is cardiomyopathy. In some embodiments, the disease is ischemia. In some embodiments, the disease is ischemia / reperfusion injury. In some embodiments, the disease is pulmonary hypertension. In some embodiments, the disease is obesity. In some embodiments, the disease is metastatic disease. In some embodiments, the disease is fluid homeostasis. In some embodiments, the disease is pathological angiogenesis. In some embodiments, the disease is retinopathy. In some embodiments, the disease is fibrosis. In some embodiments, the disease is HIV infection. In some embodiments, the disease is insulin resistance and type 2 diabetes. In some embodiments, the disease is other Apelin-related disease.

[0188] The apelin receptor agonist of the present invention (also referred to herein as the “active compound”) can be internalized in a pharmaceutical composition suitable for administration. Such a composition typically comprises the apelin receptor agonist and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders suitable for pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference in the art, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer’s solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and non-volatile oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in the composition is intended. Auxiliary active compounds may also be incorporated into the composition.

[0189] The pharmaceutical compositions of the present invention are formulated to suit their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents such as water for injection, physiological saline, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium sulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0190] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophore EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin to maintain the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Often, it is preferable to include isotonic agents in the composition, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, and sodium chloride. Sustained absorption of an injectable composition can be achieved by including absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0191] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into one or a combination of the previously listed components in a suitable solvent, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those previously listed. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and lyophilization, from which a powder of the active component and any additional desired components is obtained from the solution, which has been previously sterile filtered.

[0192] Oral compositions generally contain an inert diluent or food carrier. These can be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound is incorporated with an excipient and can be used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier for use as an oral rinse, where the compound in the fluid carrier is applied orally, gargled, spit out, or swallowed. Medicinally suitable binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or sterotes; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavor.

[0193] For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or from a nebulizer.

[0194] Systemic administration may also be by mucosal or transdermal means. For mucosal or transdermal administration, a suitable penetrating agent for the barrier to be penetrated is used in the formulation. Such penetrating agents are commonly known in the art and, for example, for mucosal administration, include surfactants, bile salts, and fusidic acid derivatives. Mucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, creams, gels, or other formulations commonly known in the art.

[0195] The compounds can also be prepared in the form of suppositories for rectal delivery (e.g., using conventional suppository bases such as cocoa butter and other glycerides) or retained enemas.

[0196] In one embodiment, the active compound may be prepared using a carrier that protects the compound from rapid release from the body, such as a controlled-release formulation, including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid, can be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes that target infected cells having monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0197] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions into unit dosage forms. As used herein, a unit dosage form refers to a physically distinct unit suitable as a unit dose for the subject to be treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications of the unit dosage forms of the present invention are determined and directly depend on the inherent characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the limitations inherent in the technique of formulating such active compounds for the treatment of an individual.

[0198] The pharmaceutical composition may be contained in a container, pack, or dispenser, along with instructions for administration. [Examples]

[0199] While specific compounds, compositions, and methods of this disclosure have been described in detail according to specific embodiments, the following examples serve solely to illustrate this disclosure and are not intended to limit it.

[0200] Example 1 - Effects of aperin receptor agonists on the aperin receptor signaling pathway This embodiment measures the effects of the apelin receptor agonist of the present invention on various downstream signaling pathways of the apelin receptor system.

[0201] The apelin receptor is a pertussis toxin-inhibiting G protein (Gα i / o ) binds, subsequently activating the extracellular regulatory kinase (ERK) signaling cascade (Figure 1). These cascades result in a wide range of physiological effects depending on the type of cell being activated. All putative apelin and ELA isoforms induce standard Gi-mediated inhibition of adenylyl cyclase, which leads to inhibition of cAMP. Signaling via β-arrestin results in desensitization and internalization of the apelin receptor. Internalized receptors are then targeted for degradation or recycled to the cell surface. β-arrestin-mediated signaling may also contribute to the action of apelin.

[0202] In this example, the commercially available HitHunter® cAMP assay was used, G ai The effects of apelin receptor agonists on signal transduction were determined, and cAMP depletion was measured. Next, the effects of apelin receptor agonists on β-arrestin signaling were determined using the commercially available PathHunter® β-arrestin assay. Furthermore, the effects of apelin receptor agonists on ERK signaling were determined.

[0203] cAMP signaling assay In a 384-well plate (Corning #3897), cAMP Hunter® CHO-K1 AGTRL1 Gi cells (Eurofins DiscoverX #95-0147C2) were seeded at a rate of 1 × 10⁴ cells / well in 20 μL of AssayComplete Cell Plating 2 Reagent (Eurofins DiscoverX #93-0563R2A) and incubated overnight at 37°C in 5% CO₂. The following day, the seeded cells were washed with assay buffer (1 × HBSS + 10 mM HEPES), and 20 μL / well of assay buffer and 1 part cAMP antibody from HitHunter® cAMP Assay for Small Molecules Kit (Eurofins DiscoverX #90-0075SM25) were added. The plates were treated with 5 μL / well of the compound, serially diluted in assay buffer + 15 μM forskolin (Millipore Sigma #F6886), and incubated at 37°C for 30 minutes in 5% CO2. Then, 20 μL / well of the detection solution, prepared as instructed in the HitHunter® cAMP Assay for Small Molecules Kit, was added, the plates were wrapped in foil, and incubated at room temperature for 1 hour. Next, 20 μL / well of solution A from the HitHunter® cAMP Assay for Small Molecules Kit was added, and the plates were incubated overnight in foil at room temperature. The plates were read on a standard luminescence plate reader after approximately 18 hours of incubation. In this assay, agonist activity was inhibited by the use of forskolin, and therefore, low luminescence signals were directly related to high cAMP activity. Four-parameter logistic regression was used for curve fitting to process the raw data files into a Z-shaped dose-response curve, from which values ​​such as EC50, hill slope, and Emax were quantified.

[0204] β-arrestin signaling assay In a 384-well plate (Corning #3897), PathHunter® CHO-K1 AGTRL1 β-arrestin cells (Eurofins DiscoverX #93-0250C2) were seeded at 5 × 10³ cells / well in 20 μL of AssayComplete Cell Plating 2 Reagent (Eurofins DiscoverX #93-0563R2A) and incubated overnight at 37°C with 5% CO2. The following day, the plate was treated with 5 μL / well of the compound, serially diluted in 1XPBS + 0.1% BSA buffer, and incubated at 37°C with 5% CO2 for 1.5 hours. Subsequently, 12.5 μL / well of detection solution, prepared as instructed by the PathHunter® detection kit (Eurofins DiscoverX #93-0001), was added, the plate was wrapped in foil, and incubated at room temperature. Plates were read on a standard luminescence plate reader after 1–2 hours of incubation. In this assay, low levels of β-arrestin in cells were directly associated with low luminescence signaling. Four-parameter logistic regression was used for curve fitting to process the raw data files into S-shaped dose-response curves, from which values ​​such as EC50, hill slope, and Emax were quantified.

[0205] The EC50 values ​​determined from the three assays described above are summarized in Table 4. Proteolysis during production was detected using mass spectrometry, which provides insights into the proteolytic stability of the apelin receptor agonist. [Table 4-1] [Table 4-2]

[0206] Example 2 - Measurement of desensitization by an apelin receptor agonist Sustained apelin receptor signaling can lead to down-transcriptional regulation of the receptor and its downstream effectors. This can result in the removal of the receptor from the cell membrane or the decoupling of receptor activation to downstream effector function, thereby again reducing the efficacy of both the intrinsic ligand and the drug, respectively. The present invention provides, among other things, an excellent apelin receptor agonist with reduced desensitization, which provides improved efficacy and potency.

[0207] The degree of desensitization caused by the native ligands and apelin receptor agonists of the present invention is evaluated by the following assay: 1. Measurement of the efficacy and efficacy of β-arrestin signaling. 2. Use of a microscope to track and measure the localization of apelin receptors, with the aim of quantifying the degree of receptor internalization that occurs after treatment. 3. The level of downtranscriptional regulation of the apelin receptor and selected downstream signaling molecules was measured using RT-PCR. 4. Repeated treatment and measurement of apelin receptor agonists in in vitro and in vivo assays to determine whether a similar level of response can be obtained in subsequent measurements.

[0208] Example 3 - Internalization of an apelin receptor agonist In this example, the internalization of exemplary apelin receptor agonists was evaluated. CHO-M1 cells expressing APJ-eGFP fusion protein were stained and seeded in 96-well plates. Apelin receptor agonists were serially diluted and added to the wells at concentrations ranging from 0.914 nM to 2000 nM. Cells were subsequently imaged using the Operetta CLS® high-content analysis system. APJ-eGFP signals were measured in the membrane and cytoplasmic regions, and the ratio of the membrane signal to the total signal was calculated (E ratio). Internalization scores were assigned based on the lowest concentration of the compound at which maximum internalization was observed. Scores were assigned according to Table 5, and the internalization scores are summarized in Table 6. As shown in Figures 2 and 3, the internalization scores for each apelin receptor agonist were plotted against EC50 values ​​for cAMP signaling and beta-arrestin signaling. [Table 5] [Table 6-1] [Table 6-2]

Claims

1. An apelin receptor agonist comprising an apelin analog, wherein the apelin analog comprises one or more amino acid substitutions compared to SEQ ID NO: 1 (QRPRLSHKGPMPF), An apelin receptor agonist wherein one or more of the aforementioned amino acid substitutions occur at positions 1, 6, 8, 9, 11, 12, and / or 13.

2. The apelin receptor agonist according to claim 1, wherein the one or more amino acid substitutions include P12V, K8W, Q1E, M11H, M11A, F13Y, P12A, P12H, F13W, S6Q, and / or G9N.

3. An apelin receptor agonist comprising an apelin analog, wherein the apelin analog comprises an amino sequence: Includes X1-R-P-R-L-S-H-X2-G-P-X3-X4-X5, In the array, X1 is either Q or E. X2 is either K or W. X3 is M, H, or A. X4 is P, V, A, or H, and / or X5 is an apelin receptor agonist, which is F, Y, or W.

4. An apelin receptor agonist comprising an apelin analog, wherein the apelin analog comprises an amino sequence: Includes Q-R-P-R-L-Q-H-K-N-P-M-A-X1, In the array, X1 is an apelin receptor agonist, which is either F or W.

5. An apelin receptor agonist comprising an apelin analog, wherein the apelin analog comprises an amino sequence: Includes Q-R-P-R-L-S-H-K-G-P-X1-X2-X3, In the array, X1 is M, H, or A. X2 is P, V, A, or H, and / or X3 is an apelin receptor agonist, which is F, Y, or W.

6. An apelin receptor agonist comprising an apelin analog, wherein the apelin analog comprises an amino sequence: Includes X1-R-P-R-L-X2-H-X3-X4-P-X5-X6-X7, In the array, X1 is either Q or E. X2 is either S or Q, X3 is either K or W. X4 is either G or N. X5 is M, H, or A. X6 is P, V, A, or H, and / or X7 is an apelin receptor agonist, which is F, Y, or W.

7. The apelin receptor agonist according to claim 3 or 5, wherein the apelin analog comprises the amino acid sequence of SEQ ID NO: 1 (QRPRLSHKGPMPF).

8. The apelin receptor agonist according to claim 3 or 5, wherein the apelin analog comprises the amino acid sequence of SEQ ID NO: 2 (QRPRLSHKGPMVF).

9. The apelin receptor agonist according to claim 3, wherein the apelin analog comprises the amino acid sequence of SEQ ID NO: 3 (QRPRLSHWGPMPF).

10. The apelin receptor agonist according to claim 3, wherein the apelin analog comprises the amino acid sequence of SEQ ID NO: 4 (ERPRLSHKGPMPF).

11. The apelin receptor agonist according to claim 3 or 5, wherein the apelin analog comprises the amino acid sequence of SEQ ID NO: 5 (QRPRLSHKGPHPF).

12. The apelin receptor agonist according to claim 3 or 5, wherein the apelin analog comprises the amino acid sequence of SEQ ID NO: 6 (QRPRLSHKGPAPY).

13. The apelin receptor agonist according to claim 3 or 5, wherein the apelin analog comprises the amino acid sequence of SEQ ID NO: 7 (QRPRLSHKGPMAF).

14. The apelin receptor agonist according to claim 3 or 5, wherein the apelin analog comprises the amino acid sequence of SEQ ID NO: 50 (QRPRLSHKGPMHW).

15. The apelin receptor agonist according to claim 3 or 5, wherein the apelin analog comprises the amino acid sequence of SEQ ID NO: 51 (QRPRLSHKGPAAW).

16. The apelin receptor agonist according to claim 3 or 5, wherein the apelin analog comprises the amino acid sequence of SEQ ID NO: 52 (QRPRLSHKGPAAY).

17. The apelin receptor agonist according to claim 3, wherein the apelin analog comprises the amino acid sequence of SEQ ID NO: 55 (QRPRLSHWGPMAW).

18. The apelin receptor agonist according to claim 4, wherein the apelin analog comprises the amino acid sequence of SEQ ID NO: 53 (QRPRLQHKNPMAF).

19. The apelin receptor agonist according to claim 4, wherein the apelin analog comprises the amino acid sequence of SEQ ID NO: 54 (QRPRLQHKNPMAW).

20. An apelin receptor agonist comprising an elavella analog, wherein the elavella analog comprises one or more amino acid substitutions compared to SEQ ID NO: 8 (KLRKHNCLQRRRCMPLHSRVPFP), An apelin receptor agonist wherein the one or more amino acid substitutions occur at positions 3, 7, 8, 9, 10, 12, 14, 15, 17, and / or 18.

21. The apelin receptor agonist according to claim 20, wherein the one or more amino acid substitutions include R3N, P14I, S17Q, S17R, Q9K, R18Y, L15Y, C7S, C12Y, R10E, Q9I, L8A, and / or Q9T.

22. An apelin receptor agonist comprising an elavella analog, wherein the elavella analog has an amino sequence: K-L-Z1-KH-N-Z2-Z3-Z4-Z5-R-Z6-M-Z7-Z8-H-Z9-Z10-V-PFP, In the array, Z1 is either R or N. Z2 is either C or S. Z3 is either A or L. Z4 is Q, I, K, or T. The Z5 is either R or E. Z6 is either C or Y. Z7 is either P or I. Z8 is either L or Y. Z9 is S, Q, or R, and / or Z10 is an apelin receptor agonist, either R or Y.

23. The apelin receptor agonist according to claim 22, wherein the Elavera analog comprises the amino acid sequence of SEQ ID NO: 8 (KLRKHNCLQRRRCMPLHSRVPFP).

24. The apelin receptor agonist according to claim 22, wherein the Elavera analog comprises the amino acid sequence of SEQ ID NO: 9 (KLNKHNCLQRRRCMPLHSRVPFP).

25. The apelin receptor agonist according to claim 22, wherein the Elavera analog comprises the amino acid sequence of SEQ ID NO: 10 (KLRKHNCLQRRCMILHSRVPFP).

26. The apelin receptor agonist according to claim 22, wherein the Elavera analog comprises the amino acid sequence of SEQ ID NO: 11 (KLRKHNCLQRRRCMPLHQRVPFP).

27. The apelin receptor agonist according to claim 22, wherein the Elavera analog comprises the amino acid sequence of SEQ ID NO: 12 (KLRKHNCLQRRRCMPLHRRVPFP).

28. The apelin receptor agonist according to claim 22, wherein the Elavera analog comprises the amino acid sequence of SEQ ID NO: 13 (KLRKHNCLKRRCMPLHSRVPFP).

29. The apelin receptor agonist according to claim 22, wherein the Elavera analog comprises the amino acid sequence of SEQ ID NO: 30 (KLRKHNCLQRRRCMPLHSYVPFP).

30. The apelin receptor agonist according to claim 22, wherein the Elavera analog comprises the amino acid sequence of SEQ ID NO: 31 (KLRKHNCLQRRRCMPYHSRVPFP).

31. The apelin receptor agonist according to claim 22, wherein the Elavera analog comprises the amino acid sequence of SEQ ID NO: 32 (KLRKHNSLQRRYMPLHSRVPFP).

32. The apelin receptor agonist according to claim 22, wherein the Elavera analog comprises the amino acid sequence of SEQ ID NO: 33 (KLRKHNCLQERCMPLHSRVPFP).

33. The apelin receptor agonist according to claim 22, wherein the Elavera analog comprises the amino acid sequence of SEQ ID NO: 34 (KLRKHNCLIRRRCMPLHSRVPFP).

34. The apelin receptor agonist according to claim 22, wherein the Elavera analog comprises the amino acid sequence of SEQ ID NO: 35 (KLRKHNCATRRCMPLHSRVPFP).

35. The apelin receptor agonist according to any one of claims 1 to 34, wherein the apelin analog or the elavella analog is fused to the carrier portion.

36. The apelin receptor agonist according to claim 35, wherein the N-terminus of the apelin analog or the elavella analog is fused to the carrier portion.

37. The apelin receptor agonist according to claim 35 or 36, wherein the carrier portion extends the half-life of the apelin receptor agonist.

38. The apelin receptor agonist according to any one of claims 35 to 37, wherein the carrier portion comprises an Fc domain, human serum albumin (HSA), an anti-HSA antibody or a fragment thereof, a lipid, or PEG.

39. The apelin receptor agonist according to claim 38, wherein the Fc domain is monovalent Fc.

40. The apelin receptor agonist according to claim 38, wherein the Fc domain is divalent Fc.

41. The apelin receptor agonist according to claim 38, wherein the anti-HSA antibody or fragment thereof is an anti-HSA Fab, nanobody (VHH), scFv, or V-NAR.

42. The apelin receptor agonist according to claim 41, wherein the anti-HSA VHH comprises ALB-23.

43. An apelin receptor agonist comprising an apelin analog or an elavella analog, wherein the N-terminus of the apelin analog or the elavella analog is fused to a carrier portion via a linker.

44. The apelin receptor agonist according to claim 43, wherein the N-terminus of the apelin analog or the elavella analog is fused to the C-terminus of the carrier portion.

45. The apelin receptor agonist according to claim 43 or 44, wherein the carrier portion extends the half-life of the apelin receptor agonist.

46. The apelin receptor agonist according to any one of claims 43 to 45, wherein the carrier portion comprises an Fc domain, human serum albumin (HSA), an anti-HSA antibody or a fragment thereof, a lipid, or PEG.

47. The apelin receptor agonist according to claim 46, wherein the Fc domain is monovalent Fc.

48. The apelin receptor agonist according to claim 46, wherein the Fc domain is divalent Fc.

49. The apelin receptor agonist according to any one of claims 46 to 48, wherein the Fc domain is an IgG1, IgG2, IgG3, or IgG4 isotype.

50. The apelin receptor agonist according to claim 49, wherein the Fc domain is human IgG1 monovalent Fc.

51. The apelin receptor agonist according to claim 49, wherein the Fc domain is human IgG1 bivalent Fc.

52. The apelin receptor agonist according to claim 49, wherein the Fc domain is a human IgG4 monovalent Fc containing the S228P mutation.

53. The apelin receptor agonist according to any one of claims 46 to 52, wherein the Fc domain contains one or more mutations compared to the wild-type Fc domain.

54. The apelin receptor agonist according to claim 53, wherein the Fc domain comprises SEQ ID NO: 14 or SEQ ID NO:

15.

55. The apelin receptor agonist according to claim 46, wherein the anti-HSA antibody or fragment thereof is an anti-HSA Fab, nanobody (VHH), scFv, or V-NAR.

56. The apelin receptor agonist according to claim 55, wherein the anti-HAS VHH comprises ALB-23.

57. The apelin receptor agonist according to any one of claims 43 to 56, wherein the linker comprises GGGGGSGGGGGGGGGS (Sequence ID 16).

58. An apelin receptor agonist according to any one of claims 1 to 57, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

17.

59. An apelin receptor agonist according to any one of claims 1 to 58, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

18.

60. An apelin receptor agonist according to any one of claims 1 to 59, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

19.

61. An apelin receptor agonist according to any one of claims 1 to 60, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

20.

62. An apelin receptor agonist according to any one of claims 1 to 61, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

21.

63. An apelin receptor agonist according to any one of claims 1 to 62, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

22.

64. An apelin receptor agonist according to any one of claims 1 to 63, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

23.

65. An apelin receptor agonist according to any one of claims 1 to 64, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

24.

66. An apelin receptor agonist according to any one of claims 1 to 65, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

25.

67. An apelin receptor agonist according to any one of claims 1 to 66, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

26.

68. An apelin receptor agonist according to any one of claims 1 to 67, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

27.

69. An apelin receptor agonist according to any one of claims 1 to 68, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

28.

70. An apelin receptor agonist according to any one of claims 1 to 69, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

29.

71. An apelin receptor agonist according to any one of claims 1 to 70, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

36.

72. An apelin receptor agonist according to any one of claims 1 to 71, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

37.

73. An apelin receptor agonist according to any one of claims 1 to 72, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

38.

74. An apelin receptor agonist according to any one of claims 1 to 73, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

39.

75. An apelin receptor agonist according to any one of claims 1 to 74, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

40.

76. An apelin receptor agonist according to any one of claims 1 to 75, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

41.

77. An apelin receptor agonist according to any one of claims 1 to 76, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

56.

78. An apelin receptor agonist according to any one of claims 1 to 77, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

57.

79. An apelin receptor agonist according to any one of claims 1 to 78, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

58.

80. An apelin receptor agonist according to any one of claims 1 to 79, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

59.

81. An apelin receptor agonist according to any one of claims 1 to 80, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

60.

82. An apelin receptor agonist according to any one of claims 1 to 81, comprising an amino acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98%, and 99% identical to SEQ ID NO:

61.

83. When the apelin receptor agonist is measured by cAMP depletion, the G levels are less than 60 nM, less than 30 nM, less than 20 nM, or less than 10 nM. αｉ An apelin receptor agonist according to any one of claims 1 to 82, having a signal transduction assay EC50 value.

84. The apelin receptor agonist according to any one of claims 1 to 83, wherein the apelin receptor agonist has a beta-arrestin signaling assay EC50 value of less than 1000 nM, less than 600 nM, less than 500 nM, less than 100 nM, less than 30 nM, or less than 10 nM.

85. The apelin receptor agonist according to any one of claims 1 to 84, wherein the apelin receptor agonist has an ERK activation assay EC50 value of less than 20 nM, less than 10 nM, less than 5 nM, less than 2 nM, or less than 1 nM.

86. A method for treating a disease, comprising administering an apelin receptor agonist according to any one of claims 1 to 85 to a subject in need thereof.

87. The method according to claim 86, wherein the disease is pulmonary hypertension (PAH), systemic sclerosis (SSc), heart failure with preserved ejection fraction (HfpEf), age-related sarcopenia, or acute kidney injury (AKI).

88. A nucleic acid encoding an apelin receptor agonist according to any one of claims 1 to 82.

89. A method for producing an apelin receptor agonist according to any one of claims 1 to 82.

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