WO2019238093A1 - APJ抗体及其与Elabela的融合蛋白质，以及其药物组合物和应用 - Google Patents

APJ抗体及其与Elabela的融合蛋白质，以及其药物组合物和应用 Download PDF

Info

Publication number
WO2019238093A1
WO2019238093A1 PCT/CN2019/091090 CN2019091090W WO2019238093A1 WO 2019238093 A1 WO2019238093 A1 WO 2019238093A1 CN 2019091090 W CN2019091090 W CN 2019091090W WO 2019238093 A1 WO2019238093 A1 WO 2019238093A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
antibody
elabela
acid sequence
Prior art date
Application number
PCT/CN2019/091090
Other languages
English (en)
French (fr)
Inventor
张�成
孙蕾
王荣柱
药晨江
汪笑峰
章华
景书谦
Original Assignee
鸿运华宁（杭州）生物医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 鸿运华宁（杭州）生物医药有限公司 filed Critical 鸿运华宁（杭州）生物医药有限公司
Priority to JP2020569130A priority Critical patent/JP2021526833A/ja
Priority to KR1020217000999A priority patent/KR20210019535A/ko
Priority to US17/251,663 priority patent/US20210163614A1/en
Priority to CA3103585A priority patent/CA3103585A1/en
Priority to EP19819592.7A priority patent/EP3808847A4/en
Priority to AU2019284320A priority patent/AU2019284320A1/en
Publication of WO2019238093A1 publication Critical patent/WO2019238093A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • This article provides APJ antibodies and fusion proteins with Elabela. Also provided herein are pharmaceutical compositions of APJ antibodies and Elabela fusion proteins. This article further provides methods for treating, preventing or ameliorating one or more symptoms of pulmonary hypertension, pulmonary hypertension, or heart failure using APJ antibodies and Elabela fusion proteins.
  • APJ contains 7 transmembrane units and 308 amino acids, and belongs to the family of G protein coupled receptors (GPCRs).
  • GPCRs G protein coupled receptors
  • the vasoactive peptide Apelin is the earliest endogenous ligand of the APJ receptor.
  • Apelin and APJ are widely distributed in the human central nervous system and various peripheral tissues such as the lung, heart, and breast (Kawamata et al., 2001, Biochem. Biophys. Acta. 1538: 162-71; Medhurst et al., 2003, J. Neurochem. 84: 1162-72), especially in cardiovascular endothelial cells and cardiac tissues (Kleiz et al., 2005, Regul. Pept. 126: 233-40).
  • Apelin / APJ signaling system has the effects of enhancing myocardial contractility, reducing blood pressure, promoting new blood vessel formation, regulating the body's immune response and the release of pituitary-related hormones, and regulating insulin secretion. Pathophysiology of atrial fibrillation and ischemia-reperfusion injury.
  • Elabela is another endogenous ligand that has attracted much attention in the APJ receptors in recent years (Chng et al., 2013, Dev. Cell 27: 672-680; Pauli et al., 2014, Science 343: 1248636). Elabela is encoded by three exons on human chromosome 4 and was previously considered to be non-coding RNA. However, Elabela contains a conserved ORF that encodes a protein of 54 amino acids, and its mature body consists of only 32 amino acids. . The Elabela / APJ signaling pathway has been shown to play a critical role in the development of the heart and vasculature of the embryo.
  • Elabela and Elabela mutants can activate the G ⁇ i1 and ⁇ -arrestin2 signaling pathways of APJ receptors. Further, mutations at the C-terminal end of Elabela polypeptides can cause APJ receptor signaling pathway preferences (Murza et al., 2016, J Med.Chem. 59: 2962-72), and the use of receptor signaling pathways is an important direction for new drug development (Bologna et al., 2017, Biomol. Ther. 25: 12-25).
  • Fusion administration of both Elabela and APJ antibodies can significantly extend the half-life of Elabela to retain the biological activity of the Elabela molecule.
  • the fusion protein formed by the antibody that Elabela specifically binds to APJ has the molecular targeting provided by the antibody. Helps improve the drugability of the Elabela fusion protein, and plays a role in the treatment of pulmonary hypertension, pulmonary hypertension, type 2 diabetes and its related metabolic syndrome, and one or more diseases in heart failure.
  • antibodies that specifically bind to APJ are provided herein.
  • an antibody capable of specifically binding to APJ comprising one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the following Column amino acid sequence:
  • amino acid sequence of heavy chain CDR1 SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 18;
  • Amino acid sequence of heavy chain CDR2 SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21;
  • an Elabela fusion protein comprising an antibody capable of specifically binding to APJ and one, two, three, four, five, six, seven, or eight Elabela fragment peptide linker sequences ( Linker);
  • the fusion protein connects the amino terminus of an Elabela fragment with the carboxyl terminus of an APJ antibody light or heavy chain through a peptide linker sequence, wherein each Elabela fragment is independently a forward Elabela fragment or A mutant thereof; or the fusion protein connects the carboxyl end of an Elabela fragment with the amino end of an APJ antibody light or heavy chain through a peptide linker sequence, wherein each Elabela fragment is independently a reverse Elabela Fragments or mutants thereof.
  • This article provides an Elabela fusion protein, which contains an antibody that specifically binds to APJ, two Elabela fragments, and two peptide linker sequences (Linker); the fusion protein uses a peptide linker sequence (Linker) to link an Elabela fragment
  • the amino terminus is connected to the carboxyl terminus of the light chain of an APJ antibody: N'-R-Linker-Elabela-C '; or the amino terminus of an Elabela fragment is connected to the carboxyl terminus of an APJ antibody heavy chain through a peptide linker sequence Link: N'-R-Linker-Elabela-C '; where: N' represents the amino terminus of the fusion protein polypeptide chain, C 'represents the carboxyl terminus of the fusion protein polypeptide chain, Elabela represents the forward Elabela fragment or its mutant, R Represents the amino acid sequence of the light or heavy chain of the APJ antibody, and Linker represents the peptide linker sequence.
  • polynucleotide that encodes an Elabela fusion protein described herein.
  • a vector comprising a polynucleotide encoding an Elabela fusion protein described herein.
  • a host cell comprising a vector described herein.
  • composition comprising an APJ antibody or an Elabela fusion protein as described herein, and a pharmaceutically acceptable carrier.
  • an APJ antibody or an Elabela fusion protein described herein in the manufacture of a medicament for the treatment, prevention, or improvement of pulmonary hypertension and conditions associated with pulmonary hypertension.
  • an APJ antibody or an Elabela fusion protein described herein in the manufacture of a medicament for the treatment, prevention, or improvement of pulmonary hypertension and conditions associated with pulmonary hypertension.
  • an APJ antibody or an Elabela fusion protein described herein for the manufacture of a medicament for the treatment, prevention or amelioration of heart failure and conditions related to heart failure.
  • an APJ antibody or an Elabela fusion protein described herein in the manufacture of a medicament for the treatment of type 2 diabetes and its related metabolic syndrome.
  • an APJ antibody or an Elabela fusion protein described herein for use in the simultaneous treatment, prevention or improvement of pulmonary hypertension, pulmonary hypertension, type 2 diabetes and its related metabolic syndrome, or two or more disorders of heart failure Use in medicine.
  • kits for treating, preventing or ameliorating one or more symptoms of pulmonary hypertension which comprises administering to a subject a therapeutically effective amount of an APJ antibody or an Elabela fusion protein described herein.
  • kits for treating, preventing or ameliorating one or more symptoms of pulmonary hypertension comprising administering to a subject a therapeutically effective amount of an APJ antibody or an Elabela fusion protein described herein.
  • kits for treating, preventing or ameliorating one or more symptoms of heart failure comprising administering to a subject a therapeutically effective amount of an APJ antibody or an Elabela fusion protein described herein.
  • kits for treating one or more symptoms of type 2 diabetes and its related metabolic syndrome which comprise administering to a subject a therapeutically effective amount of an APJ antibody or an Elabela fusion protein described herein
  • FIG. 1A to FIG. 1D shows flow cytometry (FACS) detection of recombinantly expressed hAPJ antibody L1H1 (which contains SEQ ID NO: 59 and SEQ ID NO: 64) and L4H4 (which contains SEQ ID NO: 62 and SEQ ID NO: 67)
  • L1H1 which contains SEQ ID NO: 59 and SEQ ID NO: 64
  • L4H4 which contains SEQ ID NO: 62 and SEQ ID NO: 67
  • Figure 2 Shows the detection of recombinantly expressed hAPJ antibodies L5H5 (SEQ ID NO: 63 and SEQ ID NO: 68) and Linker2-Elabela-11 (which contains SEQ ID NO: 123 and SEQ ID NO: 93) by reporter gene experiments.
  • the activation curves of the fusion protein, L5H5 and Linker2-EA5 (which contains SEQ ID NO: 123 and SEQ ID NO: 94) activate the Elabela / APJ signaling pathway, with EC 50 of 3.6 and 2.6 nM, respectively.
  • Figure 3 Shows reporter gene experiments to detect recombinantly expressed fusion proteins of hAPJ antibody L5H5 and Linker2-EA1 (which contains SEQ ID NO: 123 and SEQ ID NO: 103), L5H5 and Linker2-EA2 (which contains SEQ ID NO: The activation curve of the Elabela / APJ signaling pathway activated by the fusion protein of 123 and SEQ ID NO: 107), EC 50 : 5.0 and 2.2 nM, respectively.
  • Figure 4 Shows reporter gene experiments to detect recombinantly expressed hAPJ antibodies L5H5 and Linker2-EA3 (containing SEQ ID NO: 123 and SEQ ID NO: 109) fusion proteins, L5H5 and Linker2-EA4 (containing SEQ ID NO: The activation curve of the Elabela / APJ signaling pathway by the fusion protein of 123 and SEQ ID NO: 116), EC 50 : 20.3 and 5.4 nM, respectively.
  • polypeptide sequences are indicated herein using standard one-letter or three-letter abbreviations.
  • the first amino acid residue (N ') with an amino group is on the far left and the last amino acid residue (C') with a carboxyl group is on the far right, such as the forward Elabela fragment and
  • the mutant sequence SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO : 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 , SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:
  • a reverse polypeptide sequence refers to a sequence formed by rearranging the amino acid sequence of a polypeptide sequence, such as the reverse Elabela fragment sequence formed by the above-mentioned forward Elabela fragment and its mutant sequence: SEQ ID NO: 125, SEQ ID NO: 126 , SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO : 143, SEQ IDNO: 144, SEQ IDNO: 145, SEQ IDNO: 146, SEQ IDNO: 147, SEQ IDNO: 148, SEQ IDNO: 149
  • the 5 'end of the upstream strand of the Elabela single-stranded and double-stranded nucleic acid sequences is on the left and their 3' ends are on the right.
  • Particular parts of a polypeptide can be represented by amino acid residue numbers, such as amino acids 67 to 134, or by actual residues at this site, such as Lys67 to Lys134.
  • Specific polypeptide or polynucleotide sequences can also be described by explaining their differences from the reference sequence.
  • the term "individual” refers to animals including, but not limited to, primates (eg, humans), cattle, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, or mice.
  • the terms "individual” and “patient” are used interchangeably, for example, to refer to a mammalian individual, such as a human individual, and in one embodiment, a human.
  • treatment includes reducing or eliminating a disorder, disease, or condition, or one or more symptoms associated with the disorder, disease, or condition; or reducing or eliminating the focus of the disorder, disease, or condition.
  • prevention includes delaying and / or relieving the onset of a disorder, disease or disorder, and / or its accompanying symptoms; preventing an individual from acquiring the disorder, disease, or disorder; or reducing the risk of an individual acquiring the disorder, disease, or disorder.
  • control refers to preventing or slowing the progression, spread, or worsening of a disorder, disorder, or disease, or one or more symptoms thereof (eg, pain). Sometimes individuals benefit from the beneficial effects of prophylactic or therapeutic agents without causing a cure for a disorder, disorder, or disease. In one embodiment, the term control refers to preventing or slowing the progression, spread or worsening of the pain of osteolysis.
  • therapeutically effective amount and “effective amount” include an amount of a compound or combination of compounds that, when administered, is sufficient to prevent the development of one or more symptoms of a disorder, disease, or condition being treated or to a certain extent reduce it.
  • therapeutically effective amount or “effective amount” also refers to a biological molecule (e.g., protein, enzyme, RNA, or DNA), cell, tissue, system, animal, or human that is sufficient to cause a researcher, veterinarian, doctor, or clinician to seek The amount of biologically or medically responsive compounds.
  • each ingredient “pharmaceutically acceptable” means compatible with the other ingredients of the pharmaceutical formulation and suitable for contact with human or animal tissues or organs without excessive toxicity, irritation, allergies Response, immunogenicity, or other problems or complications, equivalent to a reasonable benefit / risk ratio.
  • the term “about” or “approximately” refers to an acceptable error determined by those skilled in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “about” refers to within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “about” refers to 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5% of a given value or range , 4%, 3%, 2%, 1%, 0.5% or 0.05%.
  • peptide each refer to a molecule comprising two or more amino acids linked to each other by a peptide bond.
  • proteins encompass, for example, polypeptide analogs of natural and artificial proteins and protein sequences (such as muteins, variants, and fusion proteins) as well as proteins that are co- or non-covalently modified after transcription or otherwise.
  • the peptide, polypeptide or protein may be a monomer or a multimer.
  • polypeptide fragment refers to a polypeptide having an amino-terminal and / or carboxy-terminal deletion compared to the corresponding full-length protein. Fragment lengths can be, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 80, 90, 100, 150, or 200 amino acids. The fragment length can be, for example, up to 1000, 750, 500, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11, or 10 amino acids. A fragment may further include one or more additional amino acids at one or both ends, for example, amino acid sequences (eg, Fc or leucine zipper domains) or artificial amino acid sequences (eg, artificial linker sequences) from different natural proteins.
  • amino acid sequences eg, Fc or leucine zipper domains
  • artificial amino acid sequences eg, artificial linker sequences
  • polypeptides herein include polypeptides modified for any reason and by any method, for example, to: (1) reduce the sensitivity to proteolysis, (2) reduce the sensitivity to oxidation, (3) change the affinity for forming a protein complex, ( 4) Change binding affinity, and (5) confer or modify other physicochemical or functional properties.
  • Analogs contain mutant proteins of the polypeptide.
  • single or multiple amino acid substitutions e.g., conservative amino acid substitutions
  • a "conservative amino acid substitution” is one that does not significantly alter the structural characteristics of the parent sequence (e.g., replacement of amino acids should not disrupt the helix that appears in the parent sequence or interfere with other secondary structure types that confers or functions on the parent sequence).
  • a "variant" of a polypeptide comprises an amino acid sequence that has one or more amino acid residues inserted, deleted, and / or replaced in the amino acid sequence relative to another polypeptide sequence. Variants herein include fusion proteins.
  • a “derivative" of a polypeptide is a chemically modified polypeptide, for example, by binding to other chemical moieties such as polyethylene glycol, albumin (eg, human serum albumin), phosphorylation, and glycosylation.
  • antibody includes antibodies other than those comprising two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and muteins thereof, examples of which are provided below.
  • antibody is a protein comprising a portion that binds to an antigen and optionally a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antibody to the antigen.
  • antibodies include whole antibodies, antibody fragments (such as the antigen-binding portion of an antibody), antibody derivatives, and antibody analogs.
  • the antibody may comprise, for example, a selectable protein scaffold or an artificial scaffold with grafted CDRs or CDRs derivatives.
  • the scaffold includes, but is not limited to, an antibody-derived scaffold comprising, for example, a three-dimensional structure that is introduced to stabilize the antibody, and a fully synthetic scaffold comprising, for example, a biocompatible polymer.
  • the antibody may be a mimetic peptide antibody ("PAMs") or a scaffold comprising a mimetic antibody, which uses fibronectin like a scaffold.
  • PAMs mimetic peptide antibody
  • the antibody may have a structure such as a natural immunoglobulin.
  • "Immunoglobulin” is a tetrameric molecule. In natural immunoglobulins, each tetramer is composed of two identical pairs of polypeptide chains, each pair having a "light” chain (about 25 kDa) and a "heavy” chain (about 50-70 kDa). The amino terminus of each chain includes a variable domain of about 100 to 110 amino acids, which is primarily relevant for antigen recognition. The carboxy-terminal portion of each chain defines a constant region mainly related to effector effects. Human antibody light chains are divided into kappa and lambda light chains.
  • the heavy chain is divided into ⁇ , ⁇ , ⁇ , or ⁇ , and the isotype of the antigen is determined, such as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D” region of about 10 amino acids. See, Fundamental Immunology Ch. 7 (ed. Paul, 2nd edition, Raven Press, 1989).
  • the variable regions of each light / heavy chain pair form an antibody binding site, such that a complete immunoglobulin has two binding sites.
  • Natural immunoglobulin chains show the same basic structure of relatively conserved backbone regions (FRs) connected by three highly variable regions, also known as complementarity determining regions or CDRs. From the N-terminus to the C-terminus, the light and heavy chains each contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids in each domain is in accordance with the definitions of Kabat et al. In Sequences of Proteins of Immunological Interface, 5th Edition, U.S. Dept. of Health and Human Services, PHS, NIH, NIH, Publication No. 91-3242, 1991.
  • antibody refers to an intact immunoglobulin or an antigen-binding portion thereof that can compete with an intact antibody for specific binding.
  • Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of whole antibodies.
  • Antigen-binding portions include, in particular, Fab, Fab ', F (ab') 2 , Fv, domain antibodies (dAbs), including fragments of complementarity determining regions (CDRs), single chain antibodies (scFv), chimeric antibodies, Diabodies, triabodies, tetrabodies and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer polypeptide-specific antigen binding.
  • Fab fragments are monovalent fragments with V L , V H , C L , and C H1 domains; F (ab ') 2 fragments are bivalent fragments with two Fab fragments connected by disulfide bonds in the hinge region; Fv fragment having V H and V L, domains; a dAb fragment having a V H domain, V L domain, or a V H or V L domain antigen binding fragment (U.S. Patent No. US 6,846,634 and US 6,696,245; U.S. Patent application Publication No. (US2005 / 0202512, US 2004/0202995, US 2004/0038291, US 2004/0009507, and US2003 / 0039958; Ward et al., 1989, Nature 341: 544-6.)
  • Single-chain antibody is a fusion protein in which the V L and V H region by a linker (e.g., a synthetic amino acid residue sequences) linked to form antibodies continuous protein, wherein the linker is long enough to allow the protein chain folds Back to itself and form a monovalent antigen binding site (see, eg, Bird et al., 1988, Science 242: 423-6; and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-83).
  • a linker e.g., a synthetic amino acid residue sequences
  • a double-chain antibody is a bivalent antibody comprising two polypeptide chains, where each polypeptide chain contains V H and V L domains connected by a linker that is so short that pairing of the two domains on the same chain is not allowed, Each domain is thus allowed to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-8; and Poljak et al., 1994, Structure 2: 1121-3). If the two polypeptide chains of a double-chain antibody are the same, then the double-chain antibody obtained by pairing them will have the same antigen-binding site.
  • Polypeptide chains with different sequences can be used to prepare double-chain antibodies with different antigen-binding sites.
  • three-chain antibodies and four-chain antibodies are antibodies comprising three and four polypeptide chains, respectively, and form three and four antigen-binding sites, respectively, which may be the same or different.
  • CDRs complementarity determining region of a given antibody
  • FR framework regions
  • One or more CDRs can be covalently or non-covalently incorporated into a molecule to make it an antibody.
  • Antibodies can be incorporated into CDR (s) in larger polypeptide chains.
  • the CDR (s) can be covalently linked to another native peptide chain, or non-covalently incorporated into the CDR (s).
  • CDRs allow antibodies to specifically bind to specific related antigens.
  • An antibody may have one or more binding sites. If there is more than one binding site, the binding site may be the same as or different from the other.
  • natural human immunoglobulins typically have two identical binding sites, while "bispecific” or “bifunctional” antibodies have two different binding sites.
  • murine antibody includes antibodies having one or more variable and constant regions derived from mouse immunoglobulin sequences.
  • humanized antibody is an antibody made by transplanting the complementarity determining region sequence of a mouse antibody molecule into the framework of a human antibody variable region.
  • antigen-binding domain portions of an antibody that contain amino acid residues that interact with the antigen and contribute to the specificity and affinity of the antibody for the antigen. For an antibody that specifically binds to its antigen, this will include at least a portion of at least one of its CDR domains.
  • epitope is the portion of a molecule that binds to an antibody (eg, by an antibody).
  • An epitope may comprise a discontinuous portion of a molecule (e.g., in a polypeptide, discontinuous amino acid residues in the primary sequence of the polypeptide are sufficiently close to each other in the tertiary and quaternary structure of the polypeptide to be bound by an antibody) .
  • the "percent identity" of two polynucleotides or two polypeptide sequences is determined by comparing sequence using a GAP computer program (GCG Wisconsin Package; version 10.3 (Accelrys, San Diego, CA)) using its default parameters.
  • GAP computer program GAP computer program
  • nucleic acid molecules e.g., cDNA or genomic DNA
  • RNA molecules e.g., mRNA
  • Analogs eg, peptide nucleic acids and non-natural nucleotide analogs
  • Nucleic acid molecules can be single-stranded or double-stranded.
  • a nucleic acid molecule herein comprises a continuous open reading frame encoding an antibody or fragment, derivative, mutein or variant thereof provided herein.
  • two single-stranded polynucleotides are "complementary" to each other, such that each nucleotide in one polynucleotide is complementary to the other polynucleotide In contrast, nucleotides do not introduce gaps and there are no unpaired nucleotides at the 5 'or 3' end of each sequence. If two polynucleotides can hybridize to each other under moderately stringent conditions, one polynucleotide is "complementary" to the other. Thus, one polynucleotide may be complementary to another polynucleotide, but not its complementary sequence.
  • vector is a nucleic acid that can be used to introduce another nucleic acid linked to it into a cell.
  • plasmid refers to a linear or circular double-stranded DNA molecule to which additional nucleic acid segments can be joined.
  • viral vector eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses
  • viral vectors can autonomously replicate in the host cells into which they are introduced (eg, bacterial vectors containing bacterial origins of replication and episomal mammalian vectors).
  • vectors when introduced into a host cell, integrate into the host cell's genome and therefore replicate with the host genome.
  • An "expression vector” is a type of vector that directs expression of a selected polynucleotide.
  • a nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., expression level, time, or site) of the nucleotide sequence.
  • a "regulatory sequence” is a nucleic acid that can affect the expression (e.g., expression level, time, or site) of a nucleic acid to which it is operatively linked. Regulatory genes, for example, act directly on a regulated nucleic acid or through one or more other molecules (eg, polynucleotides that bind to regulatory sequences and / or nucleic acids). Examples of regulatory sequences include promoters, enhancers and other expression control elements (eg, polyadenylation signals).
  • the term "host cell” is a cell used to express a nucleic acid, such as a nucleic acid provided herein.
  • the host cell may be a prokaryote, such as E. coli, or it may be a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., Human cells, monkey cells, hamster cells, rat cells, mouse cells or insect cells) or hybridomas.
  • a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell.
  • the phrase "recombinant host cell” can be used to describe a host cell transformed or transfected with a nucleic acid that is expected to be expressed.
  • a host cell may also be a cell that contains the nucleic acid but is not expressed at a desired level unless a regulatory sequence is introduced into the host cell so that it is operably linked to the nucleic acid.
  • the term host cell refers not only to a particular subject cell but also to the progeny or possible progeny of that cell. Due to, for example, mutations or environmental influences that may occur in subsequent generations, the progeny may in fact be different from the mother cell but still fall within the scope of terminology used herein.
  • APJ is a 7-pass transmembrane G protein-coupled receptor containing 377 amino acids (O'Dowd et al., 1993, Gene 136: 355-60). Studies so far have found that APJ is widely distributed in the human central nervous system and various peripheral tissues such as the lung, heart, and breast (Kawamata et al., 2001, Biochem. Biophys. Acta. 1538: 162-71; Medhurst et al., 2003, J. Neurochem. 84: 1162-72), especially in cardiovascular endothelial cells and cardiac tissues (Kleiz et al., 2005, Regul. Pept. 126: 233-40).
  • APJ is mainly involved in the regulation of the cardiovascular system and has also been reported to be of great significance in insulin regulation and the regulatory mechanisms of diabetes and obesity-related diseases (Boucher et al., 2005, Endocrinology 146: 1764-71; Yue et al., 2010, Am.J. Physiol. Endocrinol. Metab. 298: E59-67).
  • Human APJ and “hAPJ” as used herein refer to APJ of human origin and can be used interchangeably.
  • rat APJ and “mAPJ” refer to rat-derived APJ, and can also be used interchangeably.
  • the antibodies provided herein are antibodies that specifically bind to human APJ.
  • the fusion protein provided herein is an Elabela fusion protein that specifically binds to APJ on the cell membrane, and the fusion protein is capable of activating the conduction of Elabela / APJ signals in these cells.
  • the fusion protein provided herein is an Elabela fusion protein that binds to human APJ, and the fusion protein can bind to APJ of other species (such as monkeys and mice) and activate Elabela / APJ in these species Signalling.
  • amino acid and polynucleotide sequences of APJ are listed below.
  • sequence data is derived from the GeneBank database of the National Center for Biotechnology Information and the Uniprot database of the European Bioinformatics Institute:
  • Rat (Rattus norvegicus) polynucleotide SEQ ID NO: 57); accession number: AB033170;
  • Rat (Rattus norvegicus) amino acid SEQ ID NO: 25); accession number: BAA95002;
  • Vasoactive peptide receptor antibody APJ antibody
  • APJ antibodies In one embodiment, provided herein are APJ antibodies. In one embodiment, the APJ antibodies provided herein are whole APJ antibodies. In another embodiment, the APJ antibodies provided herein are APJ antibody fragments. In another embodiment, the APJ antibodies provided herein are APJ antibody derivatives. In another embodiment, the APJ antibody provided herein is an APJ antibody mutein. In a further embodiment, the APJ antibodies provided herein are APJ antibody variants.
  • an APJ antibody provided herein comprises one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • amino acid sequence of heavy chain CDR1 SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 18;
  • Amino acid sequence of heavy chain CDR2 SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21;
  • Table 1 lists the amino acid sequences of the light chain CDRs of the APJ antibodies provided herein, and their corresponding polynucleotide coding sequences.
  • Table 2 lists the amino acid sequences of the heavy chain CDRs of the APJ antibodies provided herein, as well as their corresponding polynucleotide coding sequences.
  • an antibody provided herein comprises a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by 5, 4, 3, 2, or 1 single amino acid addition, substitution, and / or deletion. . In another embodiment, an antibody provided herein comprises a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by 4, 3, 2, or 1 single amino acid addition, substitution, and / or deletion.
  • an antibody provided herein comprises a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by 3, 2, or 1 single amino acid addition, substitution, and / or deletion.
  • the antibodies provided herein comprise sequences that differ from one of the CDR amino acid sequences listed in Tables 1 and 2 by one or one single amino acid addition, substitution, and / or deletion.
  • the antibodies provided herein comprise sequences that differ from one of the CDR amino acid sequences listed in Tables 1 and 2 by a single amino acid addition, substitution, and / or deletion.
  • an APJ antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • an APJ antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • Amino acid sequence of heavy chain CDR2 SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21.
  • an APJ antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • Amino acid sequence of heavy chain CDR3 SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO: 22.
  • an APJ antibody provided herein comprises one, two, three, or four amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • Amino acid sequence of heavy chain CDR2 SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21.
  • an APJ antibody provided herein comprises one, two, three, or four amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • Heavy chain CDR3 amino acid sequence SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO: 22.
  • an APJ antibody provided herein comprises one, two, three, or four amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • Amino acid sequence of heavy chain CDR2 SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21;
  • Amino acid sequences of heavy chain CDR3 SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO: 22.
  • the APJ antibody provided herein comprises one, two, or three amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below: SEQ ID NO: 1, SEQ ID NO: 2.SEQ IDNO: 3, SEQ IDNO: 4, SEQ IDNO: 5, SEQ IDNO: 6, SEQ IDNO: 7, SEQ IDNO: 8, SEQ IDNO: 9, SEQ IDNO: 10, And SEQ ID NO: 11.
  • the APJ antibody provided herein comprises one, two, or three amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below: SEQ ID NO: 12, SEQ ID NO : 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , And SEQ ID NO: 22.
  • an APJ antibody provided herein comprises a combination of light and heavy chain CDR1 amino acid sequences independently selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 12, SEQ ID NO: 4 With SEQ ID NO: 15, SEQ ID NO: 7 and SEQ ID NO: 18, and SEQ ID NO: 10 and SEQ ID NO: 18.
  • the APJ antibody provided herein comprises a combination of light and heavy chain CDR2 amino acid sequences independently selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 13, SEQ ID NO: 13 5 and SEQ ID NO: 16, SEQ ID NO: 8 and SEQ ID NO: 19, and SEQ ID NO: 5 and SEQ ID NO: 21.
  • the APJ antibody provided herein comprises a combination of light and heavy chain CDR3 amino acid sequences independently selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 14, SEQ ID NO: 14 6 and SEQ ID NO: 17, SEQ ID NO: 9 and SEQ ID NO: 20, and SEQ ID NO: 11 and SEQ ID NO: 22.
  • an APJ antibody provided herein comprises:
  • a light and heavy chain CDR2 amino acid sequence combination independently selected from the following: SEQ ID NO: 2 and SEQ ID NO: 13, SEQ ID NO: 5 and SEQ ID NO: 16, SEQ ID ID NO : 8 and SEQ ID NO: 19, and SEQ ID NO: 5 and SEQ ID NO: 21.
  • an APJ antibody provided herein comprises:
  • a light and heavy chain CDR3 amino acid sequence combination independently selected from the following: SEQ ID NO: 3 and SEQ ID NO: 14, SEQ ID NO: 6 and SEQ ID NO: 17, SEQ ID ID NO : 9 and SEQ ID NO: 20, and SEQ ID NO: 11 and SEQ ID NO: 22.
  • an APJ antibody provided herein comprises:
  • a light and heavy chain CDR3 amino acid sequence combination independently selected from the following: SEQ ID NO: 3 and SEQ ID NO: 14, SEQ ID NO: 6 and SEQ ID NO: 17, SEQ ID ID NO : 9 and SEQ ID NO: 20, and SEQ ID NO: 11 and SEQ ID NO: 22.
  • the APJ antibodies provided herein comprise:
  • a light and heavy chain CDR2 amino acid sequence combination independently selected from the following: SEQ ID NO: 2 and SEQ ID NO: 13, SEQ ID NO: 5 and SEQ ID NO: 16, SEQ ID ID NO : 8 and SEQ ID NO: 19, and SEQ ID NO: 5 and SEQ ID NO: 21; and
  • a light and heavy chain CDR3 amino acid sequence combination independently selected from the following: SEQ ID NO: 3 and SEQ ID NO: 14, SEQ ID NO: 6 and SEQ ID NO: 17, SEQ ID ID NO : 9 and SEQ ID NO: 20, and SEQ ID NO: 11 and SEQ ID NO: 22.
  • an antibody described herein comprises:
  • Heavy chain CDR1 amino acid sequence SEQ ID NO: 12;
  • Heavy chain CDR2 amino acid sequence SEQ ID NO: 13;
  • Heavy chain CDR3 amino acid sequence SEQ ID NO: 14;
  • Heavy chain CDR1 amino acid sequence SEQ ID NO: 15;
  • Heavy chain CDR2 amino acid sequence SEQ ID NO: 16;
  • Heavy chain CDR3 amino acid sequence SEQ ID NO: 17;
  • Heavy chain CDR1 amino acid sequence SEQ ID NO: 18;
  • Heavy chain CDR2 amino acid sequence SEQ ID NO: 19;
  • Heavy chain CDR3 amino acid sequence SEQ ID NO: 20;
  • Heavy chain CDR1 amino acid sequence SEQ ID NO: 18;
  • Heavy chain CDR2 amino acid sequence SEQ ID NO: 21;
  • Heavy chain CDR3 amino acid sequence SEQ ID NO: 22.
  • an antibody described herein comprises:
  • Heavy chain CDR1 amino acid sequence SEQ ID NO: 18;
  • Heavy chain CDR2 amino acid sequence SEQ ID NO: 21;
  • Heavy chain CDR3 amino acid sequence SEQ ID NO: 22.
  • an APJ antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • Light chain variable domain amino acid sequence SEQ ID NO: 59 (L1), SEQ ID NO: 60 (L2), SEQ ID NO: 61 (L3), SEQ ID NO: 62 (L4), and SEQ ID NO: 63 (L5); and an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of its sequences; and
  • Heavy chain variable domain amino acid sequence SEQ ID NO: 64 (H1), SEQ ID NO: 65 (H2), SEQ ID NO: 66 (H3), SEQ ID NO: 67 (H4), and SEQ ID NO: 68 (H5); and an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of its sequences.
  • polynucleotide coding sequence of an APJ antibody comprises one or two polynucleotide coding sequences, wherein each polynucleotide coding sequence is independently selected from the following List of polynucleotide coding sequences:
  • a light chain variable domain polynucleotide coding sequence SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 73; and any of its A sequence has a polynucleotide coding sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical;
  • Heavy chain variable domain polynucleotide coding sequence SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78; A sequence has a polynucleotide coding sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical.
  • the APJ antibody provided herein comprises an amino acid sequence independently selected from the group consisting of SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63.
  • the APJ antibody provided herein comprises an amino acid sequence independently selected from the group consisting of SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, And SEQ ID NO: 68.
  • the APJ antibody provided herein comprises a light chain and heavy chain variable domain amino acid sequence independently selected from the group consisting of SEQ ID NO: 59 and SEQ ID NO: 64 (L1H1) , SEQ ID NO: 60 and SEQ ID NO: 65 (L2H2), SEQ ID NO: 61 and SEQ ID NO: 66 (L3H3), SEQ ID NO: 62 and SEQ ID NO: 67 (L4H4), and SEQ ID ID NO : 63 and SEQ ID NO: 68 (L5H5).
  • L2H2 refers to a complete antibody having a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60 (L2) and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 65 (H2).
  • an APJ antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • an APJ antibody provided herein comprises a combination of light and heavy chain constant amino acid sequences independently selected from the group consisting of SEQ ID NO: 79 and SEQ ID NO: 82, SEQ ID NO: 82 80 and SEQ ID NO: 83, SEQ ID NO: 80 and SEQ ID NO: 84, SEQ ID NO: 81 and SEQ ID NO: 83, and SEQ ID NO: 81 and SEQ ID NO: 84.
  • an APJ antibody provided herein comprises the amino acid sequences of the light and heavy chain CDRs listed herein, as well as FRs (framework).
  • the amino acid sequences of FRs are contained in the light chain or heavy chain variable domain amino acid sequences, and are not separately displayed.
  • the antibody comprises a light chain CDR1 sequence listed herein.
  • the antibody comprises a light chain CDR2 sequence listed herein.
  • the antibody comprises a light chain CDR3 sequence listed herein.
  • the antibody comprises a heavy chain CDR1 sequence listed herein.
  • the antibody comprises a heavy chain CDR2 sequence listed herein.
  • the antibody comprises a heavy chain CDR3 sequence listed herein.
  • the antibody comprises a light chain FR1 sequence herein. In another embodiment, the antibody comprises a light chain FR2 sequence herein. In another embodiment, the antibody comprises a FR3 sequence of a light chain herein. In another embodiment, the antibody comprises a light chain FR4 sequence herein. In another embodiment, the antibody comprises a heavy chain FR1 sequence herein. In another embodiment, the antibody comprises a FR2 sequence of a heavy chain herein. In another embodiment, the antibody comprises a heavy chain FR3 sequence herein. In a further embodiment, the antibody comprises a heavy chain FR4 sequence herein.
  • the light chain CDR3 sequence of the antibody differs from the light chain CDR3 amino acid sequence SEQ ID NO: 11 listed herein by no more than 6, 5, 4, 3, 2, or 1 single amino acid addition, substitution, and / Or missing.
  • the heavy chain CDR3 sequence of the antibody differs from the heavy chain CDR3 amino acid sequence SEQ ID NO: 22 listed herein by no more than 6, 5, 4, 3, 2, or 1 single amino acid addition, substitution, and / Or missing.
  • the antibody light chain CDR3 sequence differs from the light chain CDR3 amino acid sequence SEQ ID ID NO: 11 listed herein by no more than 6, 5, 4, 3, 2, or 1 single amino acid addition, substitution, and / Or deletion
  • the antibody's heavy chain CDR3 sequence differs from the heavy chain CDR3 amino acid sequence SEQ ID ID NO: 22 listed herein by no more than 6, 5, 4, 3, 2, or 1 single amino acid addition, substitution and / or deletion .
  • the APJ antibody provided herein comprises a light chain variable domain amino acid sequence, which is the L4 (SEQ ID NO: 62) or L5 (SEQ ID NO: 63) light chain variable listed herein Domain sequence.
  • the amino acid sequence of the light chain variable domain of the APJ antibody exists with the light chain variable domain amino acid sequence of L4 (SEQ ID NO: 62) or L5 (SEQ ID NO: 63) 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid difference, wherein the difference in each sequence is independently a deletion, insertion or substitution of an amino acid residue.
  • the amino acid sequence of the light chain variable domain of the APJ antibody has at least 70% of the amino acid sequence of the light chain variable domain of L4 (SEQ ID NO: 62) or L5 (SEQ ID NO: 63) , At least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% are the same.
  • the light chain variable domain polynucleotide coding sequence of the APJ antibody comprises a polynucleotide coding for L4 (SEQ ID NO: 72) or L5 (SEQ ID NO: 73).
  • the sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical.
  • the light chain variable domain polynucleotide coding sequence of the APJ antibody comprises a light chain with L4 (SEQ ID NO: 72) or L5 (SEQ ID NO: 73) under moderate conditions.
  • the light chain variable domain polynucleotide coding sequence of the APJ antibody comprises a light chain with L4 (SEQ ID NO: 72) or L5 (SEQ ID NO: 73) under stringent conditions.
  • L4 SEQ ID NO: 72
  • L5 SEQ ID NO: 73
  • the APJ antibody provided herein comprises a heavy chain variable domain amino acid sequence, which sequence is the heavy chain variable structure of H4 (SEQ ID NO: 67) or H5 (SEQ ID NO: 68) listed herein Domain sequence.
  • the amino acid sequence of the heavy chain variable domain of the APJ antibody exists with the heavy chain variable domain sequence of H4 (SEQ ID NO: 67) or H5 (SEQ ID NO: 68) 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid difference, wherein the difference in each sequence is independently a deletion, insertion or substitution of an amino acid residue.
  • the amino acid sequence of the heavy chain variable domain of the APJ antibody has at least 70% of the heavy chain variable domain sequence of H4 (SEQ ID NO: 67) or H5 (SEQ ID NO: 68), At least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% are the same.
  • the polynucleotide encoding sequence of the heavy chain variable domain of the APJ antibody is more than the heavy chain variable domain of H4 (SEQ ID NO: 77) or H5 (SEQ ID NO: 78).
  • the polynucleotide coding sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical.
  • the heavy chain variable domain polynucleotide coding sequence of the APJ antibody comprises a heavy chain with H4 (SEQ ID NO: 77) or H5 (SEQ ID NO: 78) under moderately stringent conditions.
  • the heavy chain variable domain polynucleotide coding sequence of the APJ antibody comprises a heavy chain of H4 (SEQ ID NO: 77) or H5 (SEQ ID NO: 78) under stringent conditions.
  • the antibody provided herein is one comprising L1H1 (SEQ ID NO: 59 and SEQ ID NO: 64), L2H2 (SEQ ID NO: 60 and SEQ ID NO: 65), L3H3 (SEQ ID NO: 61 An antibody in combination with SEQ ID NO: 66), L4H4 (SEQ ID NO: 62 and SEQ ID NO: 67), or L5H5 (SEQ ID NO: 63 and SEQ ID NO: 68), or one of the desired phenotypes ( For example, IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE, or IgD), or a Fab or F (ab ') 2 fragment thereof.
  • L1H1 SEQ ID NO: 59 and SEQ ID NO: 64
  • L2H2 SEQ ID NO: 60 and SEQ ID NO: 65
  • L3H3 SEQ ID NO: 61
  • the antibody provided herein is an antibody comprising a combination of L4H4 (SEQ ID NO: 62 and SEQ ID NO: 67) or L5H5 (SEQ ID NO: 63 and SEQ ID NO: 68), or a class thereof Transformed antibodies (eg, IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE, and IgD), or a Fab or F (ab ') 2 fragment thereof.
  • the antibodies provided herein may comprise any of the constant regions known in the art.
  • the light chain constant region may be, for example, a kappa or lambda type light chain constant region, such as a mouse kappa or lambda type light chain constant region.
  • the heavy chain constant region may be, for example, an ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ -type heavy chain constant region, such as a mouse ⁇ , ⁇ , ⁇ , or ⁇ -type heavy chain constant region.
  • the light or heavy chain constant region is a fragment, derivative, variant, or mutein of a natural constant region.
  • the antibodies provided herein further comprise human constant light chain kappa or a fragment thereof.
  • the amino acid sequence of the light chain constant region is as follows:
  • the antibodies provided herein further comprise a human heavy chain constant domain or a fragment thereof.
  • the amino acid sequence of the constant region of the heavy chain is as follows:
  • Human heavy chain constant region amino acid sequence (hIgG2): SEQ ID NO: 83;
  • Human heavy chain constant region amino acid sequence (hIgG4): SEQ ID NO: 84.
  • amino acid sequences of the heavy and light chains of the APJ antibody provided herein are: SEQ ID NO: 157 and SEQ ID NO: 158, respectively.
  • the APJ antibodies provided herein are selected from murine antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, double-chain antibodies , Three-chain antibodies, four-chain antibodies, Fab fragments, F (ab ') x fragments, domain antibodies, IgD antibodies, IgE antibodies, IgM antibodies, IgGl antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies.
  • the APJ antibodies provided herein are APJ monoclonal antibodies.
  • the APJ antibody provided herein is a monoclonal antibody comprising a combination of amino acid sequences selected from the group consisting of SEQ ID NO: 59 and SEQ ID NO: 64, SEQ ID NO: 60 and SEQ ID NO: 65, SEQ ID NO: 61 and SEQ ID NO: 66, SEQ ID NO: 62 and SEQ ID NO: 67, and SEQ ID NO: 63 and SEQ ID NO: 68.
  • the APJ antibody provided herein is a mouse-derived APJ antibody. In another embodiment, the APJ antibody provided herein is a humanized APJ antibody.
  • the Kd value of an APJ antibody provided herein is about 1 nM to about 200 nM or about 1 nM to about 100 nM.
  • the antibodies provided herein are whole antibodies (including polyclonal, monoclonal, chimeric, humanized, or human antibodies with full-length heavy and / or light chains).
  • the antibodies provided herein are antibody fragments, such as F (ab ') 2 , Fab, Fab', Fv, Fc, or Fd fragments, single domain antibodies, single chain antibodies, maxibodies ), Minibodies, intrabodies, two-chain antibodies, three-chain antibodies, four-chain antibodies, v-NAR, or bis-scFv (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology 23: 1126- 36).
  • the antibodies provided herein also include antibodies including antibody polypeptides as disclosed in US Patent No. 6703199, including fibronectin polypeptide monoclonal antibodies.
  • the antibodies provided herein include a single chain polypeptide as disclosed in US Patent Publication 2005/0238646.
  • variable region of a gene of a monoclonal antibody is expressed in a hybridoma using nucleotide primer amplification.
  • nucleotide primer amplification can be synthesized by one of ordinary skill in the art or purchased from commercial sources.
  • Murine and human variable region primers including V Ha , V Hb , V Hc , V Hd , C H1 , V L , and C L region primers are commercially available. These primers can be used to amplify the heavy or light chain variable regions and then insert them into vectors such as IMMUNOZAP TM H or IMMUNOZAP TM L (Stratagene), respectively. These vectors are then introduced into E. coli, yeast or mammalian-based expression systems. These methods can be used for producing large quantities comprising V H and V L domains of a single chain fusion protein (see Bird et al., 1988, Science 242: 423-6) .
  • proteins may undergo various post-transcriptional modifications.
  • the type and extent of these modifications depend on the host cell line used to express the protein and the culture conditions.
  • modifications include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation.
  • the carboxy-terminal basic residues of antibodies may be lost due to frequent modification of carboxypeptidases (see Harris, 1995, Journal of Chromatography 705: 129-34).
  • Murine monoclonal antibodies can be produced using commonly used hybridoma cell methods. This monoclonal antibody can be isolated and purified by a variety of established techniques. Such separation techniques include affinity chromatography using protein A-sepharose, molecular exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan on pages 2.7.1-2.7.12 and 2.9.1-2.9. 3 pages; Baines et al., "Purification of Immunoglobulin G (IgG),” Methods in Molecular Biology, Vol. 10, pp. 79-104 (The Humana Press, Inc., 1992)).
  • separation techniques include affinity chromatography using protein A-sepharose, molecular exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan on pages 2.7.1-2.7.12 and 2.9.1-2.9. 3 pages; Baines et al., "Purification of Immunoglobulin G (IgG),” Methods in Molecular Biology, Vol. 10,
  • the monoclonal antibody can be purified by affinity chromatography using appropriate ligands screened based on the specific properties of the antibody (e.g., heavy or light chain isotype, binding specificity, etc.).
  • suitable ligands for affinity chromatography include protein A, protein G, anti-constant region (light or heavy chain) antibodies, anti-idiotypic antibodies, and TGF-binding proteins or fragments or variants thereof.
  • Complementary determining regions (CDRs) in the center of the antibody binding site can be used to modify the affinity maturation of the molecule to obtain antibodies with increased affinity, such as antibodies against c-erbB-2 with increased affinity (Schier et al., 1996, J. Mol. Biol. 263: 551-67). Therefore, this type of technology can be used to prepare antibodies to human APJ.
  • antibodies against human APJ can be used in in vitro or in vivo assays to detect the presence of human APJ.
  • Antibodies can also be made by any conventional technique.
  • these antibodies can be purified from cells that naturally express them (e.g., they can be purified from hybridomas that produce antibodies) or produced in recombinant expression systems using any technique known in the art. See, for example, Monoclonal Antibodies, Hybridomas: New Dimensions, Biologicals, Analyses, Kennet et al., Plenum Press (1980); and Antibodies: A Laboratory Manual, Harlow and Land editor, Cold Spring Harbor Laboratory Press (1988). This is discussed in the nucleic acid section below.
  • Antibodies can be made by any known technique and screened for the desired properties. Some techniques involve isolating a nucleic acid encoding a polypeptide chain (or a portion thereof) of a related antibody (eg, an anti-APJ antibody) and manipulating the nucleic acid by recombinant DNA technology.
  • the nucleic acid can be fused or modified (eg, by mutagenesis or other conventional techniques) with another related nucleic acid to add, delete, or replace one or more amino acid residues.
  • affinity maturation protocols can be adopted including maintenance of CDRs (Yang et al., 1995, J. Mol. Biol. 254: 392-403) Strand replacement (Marks et al., 1992, Bio / Technology 10: 779-83), DNA rearrangement using mutant strains of E. coli (Low et al., 1996, J. Mol. Biol. 250: 350-68) (Patten et al., 1997 , Curr. Opin. Biotechnol. 8: 724-33), phage display (Thompson et al., 1996, J. Mol. Biol.
  • the antibodies provided herein are anti-APJ fragments.
  • the fragment may consist entirely of antibody-derived sequences or may include additional sequences.
  • antigen-binding fragments include Fab, F (ab ') 2 , single-chain antibodies, double-chain antibodies, triple-chain antibodies, quad-chain antibodies, and domain antibodies. Other examples are provided in Lunde et al., 2002, Biochem. Soc. Trans. 30: 500-06.
  • Single chain antibodies can be formed by linking the heavy and light chain variable domains (Fv regions) via an amino acid bridge (short peptide linker), thereby obtaining a single polypeptide chain. It has DNA encoding a peptide linker between DNAs by fusing two coding variable domain polypeptides (V L and V H) for preparing the single-chain Fvs (scFvs). The resulting polypeptides can fold back to form antigen-binding monomers themselves, or they can form multimers (eg, dimers, trimers, or tetramers), depending on the length of the flexible linker between the two variable domains (Kortt et al., 1997, Prot. Eng. 10: 423; Kortt et al.
  • Single chain antibodies derived from the antibodies provided herein include, but are not limited to, scFvs comprising the variable domain combination L1H1, all of which are included herein.
  • Antigen-binding fragments derived from antibodies can also be obtained by proteolysis of antibodies, such as by digesting intact antibodies with pepsin or papain according to conventional methods.
  • an antibody fragment can be produced using a pepsin-cleaving antibody to provide an SS fragment called F (ab ') 2 .
  • F (ab ') 2 This fragment can be further cleaved using a thiol reducing agent to produce a 3.5S Fab 'monovalent fragment.
  • the alternative is to use a thiol protecting group to perform the cleavage reaction to obtain the cleavage of disulfide bonds.
  • papain can also be used to directly generate two monovalent Fab fragments and one Fc fragment. These methods are described, for example, in Goldenberg, U.S.
  • Patent No. 4,331,647 Nisonoff et al., 1960, Arch. Biochem. Biophys. 89: 230; Porter, 1959, Biochem. J. 73: 119; Edelman et al., Methods in Enzymology 1: 422 (Academic Press, 1967); and Andrews and Titus, JA Current Protocols in Immunology (edition by Coligan et al., John Wiley & Sons, 2003), pages 2.8, 1-2.8.10, and pages 2.10A.1-2.10A.5.
  • cleaving antibodies such as preparing heavy chains to form monovalent heavy, light chain fragments (Fd), further cleaving fragments, or using other enzyme, chemical or genetic techniques, as long as the fragments bind to an antigen that can be recognized by the intact antibody.
  • Fd light chain fragments
  • CDRs can be obtained by constructing polypeptides encoding related CDRs.
  • Such polypeptides can be prepared, for example, by using a polymerase chain reaction to synthesize variable regions with the mRNA of an antibody-producing cell as a template, see, for example, Larrick et al., 1991, Methods: A Companion to Methods in Enzymology 2: 106; Courtenay-Luck , "Genetic Manipulation of Monoclonal Antibodies,” Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al., 166 (Cambridge University Press, 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," Monoclonal Antibodies: Principles and Applications, edited by Birch et al., p.
  • the antibody fragment may further comprise at least one variable domain of an antibody described herein.
  • the V region domain may be a monomer and is a V H or V L domain, which may independently bind to APJ with an affinity of at least equal to 1 ⁇ 10 ⁇ 7 M or higher, as described below.
  • variable region domain may be any natural variable domain or a genetically engineered form thereof.
  • Genetically engineered form refers to a variable region domain produced using recombinant DNA engineering techniques.
  • the genetically engineered form includes, for example, produced from a specific antibody variable region by insertion, deletion, or alteration of the amino acid sequence of a specific antibody.
  • Specific examples include variable region domains that contain only one CDR and optionally one or more framework amino acids from one antibody and the remainder of the variable region domain from another antibody and are genetically assembled.
  • variable region domain may be covalently linked to at least one other antibody domain or fragment thereof at the C-terminal amino acid.
  • V H region domain may be linked to an immunoglobulin C H1 domain or fragment thereof.
  • V L domain can be linked to the C K domain or a fragment thereof.
  • the antibody may be a Fab fragment wherein the antigen binding domain comprises a V H and V L, United domains thereof are connected to the C-terminus and C K C Hl domain covalently.
  • Other amino acids can be used to extend the C H1 domain, for example to provide a hinge region or as Fab 'portions of the hinge domain fragment or provide other domains, such as an antibody C H2 and C H3 domains.
  • the nucleotide sequences encoding the amino acid sequences L1 and H1 can be altered, for example, by random mutagenesis or by site-directed mutagenesis (e.g., oligonucleotide-induced site-directed mutagenesis) to produce a compared to unmutated polynucleotides.
  • site-directed mutagenesis e.g., oligonucleotide-induced site-directed mutagenesis
  • An altered polynucleotide comprising one or more specific nucleotide substitutions, deletions, or insertions.
  • anti-APJ antibody derivatives in the art herein include covalent or aggregated conjugates of anti-APJ antibodies or fragments thereof with other proteins or polypeptides, for example, by expressing a heterologous polypeptide comprising a fusion to the N- or C-terminus of the anti-APJ antibody polypeptide Recombinant fusion protein.
  • the binding peptide may be a heterologous signal (or guide) polypeptide, such as a yeast alpha factor leader peptide or a peptide such as an epitope tag.
  • Fusion protein-containing antibodies may include peptides (e.g., polyhistidine) added to aid purification or identification of the antibody.
  • Antibodies can also be linked to FLAG peptides, as described by Hopp et al., 1988, Bio / Technology 6: 1204, and U.S. Patent 5,011,912.
  • FLAG peptides are highly antigenic and provide epitopes that are reversibly bound by specific monoclonal antibodies (mAb), allowing rapid detection and convenient purification of expressed recombinant proteins.
  • mAb monoclonal antibodies
  • Commercially available Sigma-Aldrich, St. Louis, MO
  • oligomers comprising one or more antibodies are available For APJ antagonists or higher oligomers.
  • the oligomers may be in the form of covalently linked or non-covalently linked dimers, trimers or higher oligomers. Oligomers containing two or more antibodies can be used, one example of which is a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, and the like.
  • One embodiment is directed to oligomers comprising multiple antibodies, which are linked by covalent or non-covalent interactions with the peptide portion fused to the antibody.
  • Such peptides can be peptide linkers or peptides with properties that promote oligomerization.
  • Leucine zipper and certain antibody-derived polypeptides are peptides that can promote antibody oligomerization, as described in detail below.
  • the oligomer comprises two to four antibodies.
  • the oligomer antibodies can be in any form, such as any of the forms described above, such as variants or fragments.
  • the oligomer comprises an antibody having APJ-binding activity.
  • an oligomer is prepared using an immunoglobulin-derived polypeptide.
  • the preparation of heterologous polypeptides containing some fusions with different parts of antibody-derived polypeptides (including the Fc domain) has been described, for example, in Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10535; Byrn et al., 1990, Nature 344: 677; and Hollenbaugh et al., Construction of Immunoglobulin Fusion Proteins, Current Protocols Immunology, Suppl. 4, pp. 10.19.1-10.19.11.
  • dimer comprising two fusion proteins produced from a fusion of an Elabela fragment of an anti-APJ antibody or a mutant binding fragment thereof and the Fc region of an antibody.
  • Dimers can be prepared by, for example, inserting a gene fusion encoding a fusion protein into an appropriate expression vector, expressing the fusion gene in a host cell transformed with a recombinant expression vector, and allowing the expressed fusion protein to assemble like an antibody molecule, Among them, interchain disulfide bonds between Fc moieties form dimers.
  • Fc polypeptide includes polypeptides derived from natural and mutein forms of the Fc region of an antibody. Also included are truncated forms of such polypeptides that include a hinge region that promotes dimerization. Fusion proteins containing Fc portions (and oligomers formed from them) provide the advantage of easy purification by affinity chromatography on a Protein A or Protein G column.
  • Fc polypeptide in PCT application WO 93/10151 is a single-chain polypeptide that extends from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG1 antibody.
  • Another useful Fc polypeptide is the Fc mutein described in U.S. Patent 5,457,035 and Baum et al., 1994, EMBO J. 13: 3992-4001.
  • the amino acid sequence of this mutant protein is the same as that of the natural Fc sequence shown in WO 93/10151, except that amino acid 19 changes from leucine to alanine, amino acid 20 changes from leucine to glutamine, and amino acid 22 changes from Glycine becomes alanine.
  • the mutein exhibits reduced affinity for the Fc receptor.
  • the heavy and / or light chain of an anti-APJ antibody may be replaced with a variable portion of the antibody heavy and / or light chain.
  • the oligomer is a fusion protein comprising a plurality of antibodies, with or without a spacer peptide.
  • linker peptides are described in U.S. Patents 4,751,180 and 4,935,233.
  • Leucine zipper domains are peptides that promote the oligomerization of the proteins in which they exist.
  • the leucine zipper was originally found in several DNA binding proteins (Landschulz et al., 1988, Science 240: 1759), and has since been found in a variety of different proteins.
  • the known leucine zippers are dimerizable or trimerable natural peptides or derivatives thereof.
  • leucine zipper domains suitable for the production of soluble oligomeric proteins are described in PCT application WO 94/10308, and leucine zipper derived from lung surfactant protein D (SPD) are described in Hoppe et al., 1994, FEBS Letters 344: 191, incorporated herein by reference.
  • SPD lung surfactant protein D
  • modified leucine zippers that allow stable trimerization of heterologous proteins fused to them is described in Fanslow et al., 1994, Semin. Immunol. 6: 267-78.
  • a recombinant fusion protein comprising an anti-APJ antibody fragment or derivative fused to a leucine zipper peptide is expressed in an appropriate host cell, and a soluble oligomeric anti-APJ antibody fragment is collected from the culture supernatant or Its derivatives.
  • the antibody derivative may comprise at least one of the CDRs disclosed herein.
  • one or more CDRs can be integrated into known antibody backbone regions (IgG1, IgG2, etc.) or combined with a suitable vector to enhance its half-life.
  • Suitable carriers include, but are not limited to, Fc, albumin, transferrin, and the like. These and other suitable vectors are known in the art.
  • the CDR-binding peptide may be a monomer, dimer, tetramer, or other form.
  • one or more water-soluble polymers are bound at one or more specific sites of the binding agent, such as at the amino terminus.
  • the antibody derivative comprises one or more water-soluble polymer attachments including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, for example, U.S. Patent Nos. 4640835, 4496689, 4301144, 4670417, 4791192, and 4179337.
  • the derivative comprises one or more monomethoxy.
  • one or more water-soluble polymers are randomly bound to one or more side chains.
  • PEG can enhance the therapeutic effect of binding agents such as antibodies.
  • the antibodies provided herein may have at least one amino acid substitution as long as the antibody retains binding specificity. Therefore, modification of antibody structure is included in the scope of this document. These may include amino acid substitutions that do not disrupt the antibody's APJ binding ability, which may be conservative or non-conservative. Conservative amino acid substitutions may include unnatural amino acid residues, which are typically integrated by chemical peptide synthesis rather than by biological systems. These include peptidomimetic and other amino acid moieties in inverted or inverted form. Conservative amino acid substitutions may also involve replacing natural amino acid residues with non-natural residues that have little or no effect on the polarity or charge of the amino acid residues at the site. Non-conservative substitutions may involve the exchange of a member of one class of amino acids or amino acid analogs with a member of another class of amino acids having different physical properties (eg, volume, polarity, hydrophobicity, charge).
  • test variants that include amino acid substitutions at each desired amino acid residue.
  • Such variants can be screened using activity assays known to those skilled in the art.
  • This type of variant can be used to gather information about the appropriate variant. For example, if an amino acid residue is found to cause activity disruption, undesired reduction, or inappropriate activity, variants with such changes can be avoided.
  • one skilled in the art can easily determine amino acids that should be avoided further (either alone or in combination with other mutations).
  • those skilled in the art can use known techniques to determine appropriate variants of the polypeptides as listed herein.
  • those skilled in the art can identify appropriate regions of the molecule that do not disrupt activity if altered by targeting regions that are not important for activity.
  • residues or portions of molecules that are conserved among similar polypeptides can be identified.
  • even regions that are important for biological activity or structure can be conservatively replaced without disrupting the biological activity or without adversely affecting the polypeptide structure.
  • those skilled in the art can investigate structural. Functional studies to identify residues in similar polypeptides that are important for activity or structure. In light of this comparison, the importance of amino acid residues in proteins corresponding to amino acid residues important for activity or structure in similar proteins can be predicted. Those skilled in the art can select chemically similar amino acid substitutions for these predicted important amino acid residues.
  • antibody variants include glycosylation variants in which the number and / or type of glycosylation sites is changed compared to the amino acid sequence of the parent polypeptide. In some embodiments, the variant has a greater or lesser number of N-linked glycosylation sites than the native protein. Alternatively, substitutions that remove this sequence can remove existing N-linked sugar chains. Rearrangement of N-linked sugar chains is also provided, in which one or more N-linked sugar chain sites (usually those naturally occurring) are removed and one or more new N-linked sites are created. Other preferred antibody variants include cysteine variants in which one or more cysteine residues are deleted or replaced by another amino acid (eg, serine) compared to the parent amino acid sequence.
  • another amino acid eg, serine
  • Cysteine variants can be used when the antibody must be folded into a biologically active conformation (e.g., after isolating soluble inclusion bodies). Cysteine variants generally have fewer cysteine residues than natural proteins and often have an even number of cysteine to minimize interactions caused by unpaired cysteine.
  • amino acid substitutions can be used to identify important residues of a human APJ antibody or to increase or decrease the affinity of a human APJ antibody described herein.
  • the preferred amino acid substitutions are the following: (1) reduced proteolytic sensitivity, (2) reduced oxidation sensitivity, (3) altered binding affinity to form protein complexes, and (4) altered binding affinity and / or (5) Conferring or modifying other physicochemical or functional properties on such polypeptides.
  • single or multiple amino acid substitutions may be made in a naturally occurring sequence (in some embodiments, the portion of the polypeptide outside the domain that forms the intermolecular contact) .
  • conservative amino acid substitutions generally do not substantially alter the structural characteristics of the parent sequence (eg, replacement amino acids should not break the helix present in the parent sequence or interfere with other types of secondary structures that characterize the parent sequence).
  • replacement amino acids should not break the helix present in the parent sequence or interfere with other types of secondary structures that characterize the parent sequence.
  • Examples of secondary and tertiary structures of peptides recognized in the art are described in Proteins, Structures, Molecular Principles, Editors, Creighton, WH Freeman, and Company (1984); Introduction to Protein Structure, Branden and Tooze Editor, Garland, Publishing (1991); and Thornton et al., 1991, Nature 354: 105, which is incorporated herein by reference.
  • the antibodies provided herein can be chemically bonded to polymers, lipids, or other moieties.
  • Antigen-binding reagents can include at least one CDRs described herein that are incorporated into a biocompatible backbone structure.
  • the biocompatible backbone structure comprises a polypeptide or portion thereof sufficient to form a conformationally stable structural support or backbone or scaffold, which can display one or more amino acid sequences (e.g., , CDRs, variable regions, etc.).
  • Such structures may be naturally occurring polypeptides or polypeptide "folds" (structural motifs), or may have one or more modifications, such as amino acid additions, deletions or substitutions, relative to the natural polypeptide or fold.
  • These scaffolds can be derived from polypeptides of any species (or more than one species), for example, humans, other mammals, other vertebrates, invertebrates, bacteria or viruses.
  • Biosoluble backbone structures are often based on protein scaffolds or backbones rather than immunoglobulin domains.
  • fibronectin-based, ankyrin, lipocalin, neocancer, cytochrome b, CP1 zinc finger protein, PST1, coiled coil, LACI-D1, Z domain and starch can be used Enzyme aprotin domain (see, eg, Nygren and Uhlen, 1997, Current Opinion in Structural Biology 7: 463-9).
  • suitable binding agents include portions of these antibodies, such as one or more of the heavy chain CDR1, CDR2, CDR3, light chain CDR1, CDR2, and CDR3, as specifically disclosed herein. At least one of the heavy chain CDR1, CDR2, CDR3, CDR1, CDR2, and CDR3 regions has at least one amino acid substitution as long as the antibody retains the binding specificity of the non-replaced CDRs.
  • the non-CDR portion of the antibody may be a non-proteinaceous molecule, wherein the binding agent cross-blocks the binding of the antibodies disclosed herein to human APJ.
  • the non-CDR portion of the antibody may be a non-proteinaceous molecule, wherein the antibody shows a binding type to a human Elabela peptide similar to that shown by antibody L4H4 in a competitive binding assay.
  • the non-CDR portion of an antibody may consist of amino acids, where the antibody is a recombinant binding protein or a synthetic peptide, and the recombinant binding protein cross-blocks the binding of the antibodies disclosed herein to human APJ.
  • the non-CDR portion of the antibody may consist of amino acids, where the antibody is a recombinant antibody, and the recombinant antibody shows a binding type to a human APJ peptide similar to that shown by at least antibody L4H4 in a competitive binding assay.
  • APJ antibody and Elabela fusion protein (Elabela fusion protein)
  • an Elabela fusion protein comprising an APJ antibody and an Elabela fragment provided herein.
  • an Elabela fusion protein comprising an APJ antibody provided herein and one, two, three, four, five, six, seven, or eight Elabela fragments And a peptide linker (Linker); the fusion protein connects the amino terminus of an Elabela fragment with the carboxyl end of an APJ antibody light or heavy chain through a peptide linker sequence, wherein each Elabela fragment is independently a positive To the Elabela fragment or a mutant thereof; or the fusion protein connects the carboxyl end of an Elabela fragment with the amino end of an APJ antibody light or heavy chain through a peptide linker sequence, wherein each Elabela fragment is independently A reverse Elabela fragment or a mutant thereof.
  • Linker a peptide linker
  • an Elabela fusion protein comprising an APJ antibody provided herein, and one, two, three, or four Elabela fragments and a peptide linker; the fusion protein is passed through an A peptide linker sequence (Linker) connects the amino terminus of an Elabela fragment with the carboxyl terminus of the light or heavy chain of an APJ antibody, wherein each Elabela fragment is independently a forward Elabela fragment or a mutant thereof.
  • a peptide linker sequence Linker
  • an Elabela fusion protein comprising an APJ antibody provided herein, and one, two, three, or four Elabela fragments and a peptide linker; the fusion protein is passed through an A peptide linker sequence (Linker) connects the carboxy terminus of an Elabela fragment with the amino terminus of the light or heavy chain of an APJ antibody, wherein each Elabela fragment is independently a reverse Elabela fragment or a mutant thereof.
  • a peptide linker sequence Linker
  • an Elabela fusion protein comprising an APJ antibody provided herein, two Elabela fragments and a peptide linker (Linker); the fusion protein connects an Elabela through a peptide linker sequence (Linker)
  • Linker The amino terminus of the fragment is connected to the carboxyl terminus of the light or heavy chain of an APJ antibody, wherein each Elabela fragment is independently a forward Elabela fragment or a mutant thereof.
  • an Elabela fusion protein comprising an APJ antibody provided herein, two Elabela fragments and a peptide linker (Linker); the fusion protein connects an Elabela through a peptide linker sequence (Linker)
  • the carboxy terminus of the fragment is connected to the amino terminus of the light or heavy chain of an APJ antibody, wherein each Elabela fragment is independently a reverse Elabela fragment or a mutant thereof.
  • an Elabela fusion protein which comprises an APJ antibody and two Elabela fragments and a peptide linker (Linker) provided herein; the fusion protein connects an Elabela fragment through a peptide linker sequence (Linker)
  • the N-terminus is connected to the carboxyl terminus of the light chain of an APJ antibody: N'-R-Linker-Elabela-C '; or the amino terminus of an Elabela fragment and the carboxyl group of the heavy chain of an APJ antibody are linked by a peptide linker sequence End connection: N'-R-Linker-Elabela-C '; where: N' represents the amino terminus of the fusion protein polypeptide chain, C 'represents the carboxyl terminus of the fusion protein polypeptide chain, and Elabela represents a forward Elabela fragment or a mutant thereof R is the amino acid sequence of the light or heavy chain of an APJ antibody, and Linker represents a peptide linker sequence.
  • an Elabela fusion protein comprising an APJ antibody and two Elabela fragments and a peptide linker (Linker) provided herein; the fusion protein connects an Elabela fragment with a peptide linker sequence (Linker).
  • N'-Elabela-Linker-R-C ' The carboxy terminus is connected to the amino terminus of the light chain of an APJ antibody: N'-Elabela-Linker-R-C '; or the carboxy terminus of an Elabela fragment is connected to the amino terminus of an APJ antibody heavy chain: N'-Elabela-Linker -R-C '; where: N' represents the amino terminus of the fusion protein polypeptide chain, C 'represents the carboxyl terminus of the fusion protein polypeptide chain, Elabela represents a reverse Elabela fragment or a mutant thereof, and R is the light chain of an APJ antibody Or the amino acid sequence of the heavy chain, and Linker represents a peptide linker sequence.
  • an Elabela fusion protein comprising an APJ antibody and two Elabela fragments and a peptide linker (Linker) provided herein; the fusion protein connects an Elabela fragment with a peptide linker sequence (Linker).
  • N'-R-Linker-Elabela-C ' The amino terminus is connected to the carboxyl terminus of the light chain of an APJ antibody: N'-R-Linker-Elabela-C '; where: N' represents the amino terminus of the fusion protein polypeptide chain, C 'represents the carboxyl terminus of the fusion protein polypeptide chain, Elabela Represents a forward Elabela fragment or a mutant thereof, R is an amino acid sequence of the light chain of an APJ antibody, and Linker represents a peptide linker sequence.
  • an Elabela fusion protein comprising an APJ antibody and two Elabela fragments and a peptide linker (Linker) provided herein; the fusion protein uses an peptide linker sequence (Linker) to bind an Elabela fragment
  • the amino terminus is connected to the carboxyl terminus of the heavy chain of an APJ antibody: N'-R-Linker-Elabela-C '; where N' represents the amino terminus of the fusion protein polypeptide chain, and C 'represents the carboxyl terminus of the fusion protein polypeptide chain Elabela represents a forward Elabela fragment or a mutant thereof, R is the amino acid sequence of the heavy chain of an APJ antibody, and Linker represents a peptide linker sequence.
  • the forward Elabela fragment or a mutant thereof is independently selected from one of the following amino acid sequences: SEQ ID NO: 91, SEQ ID NO: 92 , SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO : 109, SEQ IDNO: 110, SEQ IDNO: 111, SEQ IDNO: 112, SEQ IDNO: 113, SEQ IDNO: 114, SEQ IDNO: 115, SEQ IDNO: 116, SEQ IDNO: 117 , SEQ ID NO: 91, SEQ ID NO: 92 ,
  • the amino acid sequence of the forward Elabela fragment or a mutant thereof is one of the following amino acid sequences SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 166, and SEQ ID NO: 167.
  • the inverted Elabela fragments or mutants thereof are each independently selected from one of the following amino acid sequences: SEQ ID NO: 125, SEQ ID NO: 126 , SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ IDNO: 144, SEQ IDNO: 145, SEQ IDNO: 146, SEQ IDNO: 147, SEQ IDNO: 148, SEQ IDNO: 149, SEQ IDNO: 150, SEQ IDNO: 151
  • the sequence of the peptide linker each independently comprises from 1 to 200 amino acid amines, from 2 to 100 amino acid amines, from 5 to 50 amino acid amines, from 6 to 25 amino acid amines, or from 10 to 20 amino acid amines.
  • sequences of the peptide linkers each independently comprise the full length, part, or repeat of one of the following amino acid sequences: SEQ ID NO: 122 , SEQ ID NO: 123, and SEQ ID NO: 124.
  • sequences of the peptide linkers are each independently selected from the following amino acid sequences: SEQ ID NO: 122, SEQ ID NO: 123, and SEQ ID NO: 124.
  • the light chain amino acid sequence of the Elabela fusion protein provided herein is: SEQ ID NO: 158; and its heavy chain amino acid sequence is one of the following sequences: SEQ ID NO: 156, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, and SEQ ID NO: 165.
  • the amino acid sequences of the light and heavy chains of the Elabela fusion protein provided herein are: SEQ ID NO: 158 and SEQ ID NO: 156, respectively.
  • amino acid sequences of the light and heavy chains of the Elabela fusion protein provided herein are: SEQ ID NO: 158 and SEQ ID NO: 159, respectively.
  • amino acid sequences of the light and heavy chains of the Elabela fusion protein provided herein are: SEQ ID NO: 158 and SEQ ID NO: 160, respectively.
  • amino acid sequences of the light and heavy chains of the Elabela fusion protein provided herein are: SEQ ID NO: 158 and SEQ ID NO: 161, respectively.
  • amino acid sequences of the light and heavy chains of the Elabela fusion protein provided herein are: SEQ ID NO: 158 and SEQ ID NO: 162, respectively.
  • amino acid sequences of the light and heavy chains of the Elabela fusion protein provided herein are: SEQ ID NO: 158 and SEQ ID NO: 163, respectively.
  • amino acid sequences of the light and heavy chains of the Elabela fusion protein provided herein are: SEQ ID NO: 158 and SEQ ID NO: 164, respectively.
  • amino acid sequences of the light and heavy chains of the Elabela fusion protein provided herein are: SEQ ID NO: 158 and SEQ ID NO: 165, respectively.
  • nucleic acid molecules contains, for example, a polynucleotide encoding all or part of an Elabela fusion protein, such as one or two strands of the Elabela fusion protein herein, or a fragment, derivative, mutein, or variant thereof; sufficient for hybridization probing Needle polynucleotides; PCR primers or sequencing primers used to identify, analyze, mutate or amplify polynucleotides encoding polypeptides; antisense nucleic acids and their complementary sequences for inhibiting polynucleotide expression .
  • the nucleic acid can be of any length.
  • nucleic acids can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides, and / or comprise one or more additional sequences, such as regulatory sequences, and / or are part of a larger nucleic acid, such as a vector.
  • the nucleic acid may be single-stranded or double-stranded and comprises RNA and / or DNA nucleotides and artificial variants thereof (eg, peptide nucleic acids).
  • Nucleic acids encoding antibody polypeptides can be isolated from B cells immunized with APJ antigen.
  • Nucleic acids of antibodies or Elabela fusion proteins can be isolated by conventional methods such as polymerase chain reaction (PCR).
  • Nucleic acid sequences encoding the heavy and light chain variable regions are shown above. The skilled artisan will appreciate that due to the degeneracy of the genetic code, each polypeptide sequence disclosed herein may be encoded by a greater number of other nucleic acid sequences. Provided herein are degenerate nucleotide sequences encoding the antibodies or Elabela fusion proteins provided herein.
  • nucleic acids that hybridize to other nucleic acids (eg, nucleic acids comprising the nucleotide sequence of any Elabela fusion protein) under specific hybridization conditions.
  • Methods of hybridizing nucleic acids are well known in the art. See, for example, Current Protocols in Molecular Biology, John Wiley & Son (1989), 6.3.1-6.3.6.
  • medium stringent conditions use a prewash solution containing 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0mM EDTA (pH8.0), about 50% formamide hybridization buffer, 6x SSC and 55 ° C hybridization temperature (or other similar hybridization solution, such as those containing 50% formamide, hybridization at 42 ° C), and the elution condition is 60 ° C, using 0.5x SSC, 0.1% SDS.
  • Strict hybridization conditions were performed in 6xSSC at 45 ° C, and then washed one or more times at 68 ° C in 0.1xSSC, 0.2% SDS.
  • those skilled in the art can manipulate hybridization and / or washing conditions to increase or decrease the stringency of hybridization such that they contain nucleosides that are at least 65, 70, 75, 80, 85, 90, 95, 98, or 99% homologous to each other Nucleic acids of the acid sequence can usually still hybridize to each other.
  • Mutations can be introduced using any technique known in the art.
  • one or more specific amino acid residues are changed using, for example, a site-directed mutagenesis scheme.
  • one or more randomly selected residues are changed using, for example, a random mutagenesis scheme. Regardless of how it is generated, mutant polypeptides can be expressed and screened for desired properties.
  • Mutations can be introduced into a nucleic acid without significantly altering the biological activity of its encoded polypeptide.
  • nucleotide substitutions can be made that cause amino acid substitutions at non-essential amino acid residues.
  • the nucleotide sequence provided herein for the Elabela fusion protein or a fragment, variant or derivative thereof is mutated such that it encodes one or more deletions or substitutions comprising amino acid residues of the Elabela fusion protein shown herein, Become two or more residues that differ in sequence.
  • mutagenesis inserts an amino acid near one or more amino acid residues of the Elabela fusion protein shown herein to two or more residues that differ in sequence.
  • one or more mutations can be introduced into a nucleic acid to selectively alter the biological activity of its encoded polypeptide (e.g., in combination with APJ).
  • the mutation can alter biological activity quantitatively or qualitatively. Examples of quantitative changes include increasing, decreasing, or eliminating the activity. Examples of qualitative changes include altering the binding specificity of an Elabela fusion protein.
  • nucleic acid molecules suitable for use as primers or hybridization probes to detect nucleic acid sequences herein can comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide herein, for example, a fragment that can be used as a probe or primer or a fragment encoding an active portion of a polypeptide herein (eg, an APJ binding portion).
  • Probes based on the nucleic acid sequences herein can be used to detect the nucleic acid or similar nucleic acids, such as a transcript encoding a polypeptide herein.
  • the probe may include a labeling group, such as a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can be used to identify cells expressing the polypeptide.
  • vectors comprising a nucleic acid encoding a polypeptide or a portion thereof.
  • vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors.
  • a recombinant expression vector herein may comprise a nucleic acid herein in a form suitable for expression of the nucleic acid in a host cell.
  • the recombinant expression vector includes one or more regulatory sequences, and is selected based on the host cell used for expression, and is operably linked to the pre-expressed nucleic acid sequence.
  • Regulatory sequences include those that direct constitutive expression of nucleotide sequences in a variety of host cells (e.g., SV40 early gene enhancer, Rolls sarcoma virus promoter, and cytomegalovirus promoter), and direct only in certain hosts Expression of nucleotide sequences in cells (for example, tissue-specific regulatory sequences, see Voss et al., 1986, Trends Biochem.
  • inducible expression that directs the nucleotide sequence in response to specific treatments or conditions (e.g., the metal thionin promoter in mammalian cells and the tet-sesponsive response in both prokaryotic and eukaryotic systems) Promoter and / or streptomycin-responsive promoter (ibid.)).
  • specific treatments or conditions e.g., the metal thionin promoter in mammalian cells and the tet-sesponsive response in both prokaryotic and eukaryotic systems
  • Promoter and / or streptomycin-responsive promoter ibid.
  • the host cell can be any prokaryotic or eukaryotic cell.
  • Prokaryotic host cells include Gram-negative or Gram-positive organisms, such as E. coli or Bacillus.
  • Higher eukaryotic cells include insect cells, yeast cells, and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines grown in serum-free media (see Rasmussen et al., 1998, Cytotechnology 28:31) or CHO Strain DXB-11, which lacks DHFR (see Urlaub et al., 1980, Proc. Natl.
  • CHO Chinese hamster ovary
  • CHO cell lines include CHO-K1 (ATCC # CCL-61), EM9 (ATCC # CRL-1861), and UV20 (ATCC # CRL-1862).
  • Other host cells include the COS-7 line of monkey kidney cells (ATCC # CRL-1651) (see Gluzman et al., 1981, Cell 23: 175), L cells, C127 cells, 3T3 cells (ATCC CCL-163), AM-1 / D cells (described in US Patent Serial No.
  • HeLa cells BHK (ATCC CRL-10) cell line derived from the African green monkey kidney cell line CV1 (see McMahan et al., 1991, EMBO J. 10: 2821), human embryonic kidney Cells such as 293, 293, EBNA or MSR 293, human epithelial A431 cells, human C010205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro cultures of primary tissues, primary transplants, HL -60, U937, HaK or Jurkat cells. Suitable cloning and expression vectors for bacterial, fungal, yeast, and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: Laboratory, Manual, Elsevier, 1985).
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. For stable mammalian transfection, only a small percentage of cells are known to incorporate foreign DNA into their genome, depending on the expression vector and transfection technology used. To identify and screen these integrants, genes encoding a selection marker (e.g., antibiotic resistance) are usually introduced into a host cell along with the gene of interest. Preferred screening markers include those that can confer resistance to drugs such as G418, hygromycin, and methotrexate. In other methods, drug-screening can be used to identify stably transfected cells containing the introduced nucleic acid (e.g., cells that integrate the screening gene can survive, while other cells die).
  • a selection marker e.g., antibiotic resistance
  • the transformed cells can be cultured under conditions that increase the expression of the polypeptide, and the polypeptide can be recovered by conventional protein purification methods.
  • One such purification method is described in the examples below.
  • the polypeptides used herein include a substantially homologous recombinant mammalian Elabela fusion protein polypeptide, which is substantially free of contaminating endogenous materials.
  • APJ antibody activity means that the antibodies provided herein have binding activity specific to APJ.
  • provided herein is a murine or humanized antibody capable of specifically binding to human APJ.
  • the K d of an antibody provided herein when bound to human APJ is about 0.01 nM to about 1000 nM, about 0.1 nM to about 500 nM, about 0.5 nM to about 200 nM, about 1 nM to about 200 nM, or about 10 nM To about 100nM.
  • K antibody when binding to human APJ d provided herein is from about 1nM to about 200nM.
  • K antibody when binding to human APJ d provided herein is from about 1nM to about 100nM.
  • the K d of an antibody provided herein when bound to human APJ is about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, About 80 nM, about 90 nM, or about 100 nM.
  • an APJ antibody described herein is an antibody having one or more of the properties listed below:
  • the antibody cross-competitively binds to a reference antibody on human APJ.
  • the reference antibody comprises a combination of the light chain variable domain amino acid sequence SEQ ID NO: 62 and the heavy chain variable domain amino acid sequence SEQ ID NO: 67.
  • the term "substantially similar” means that the K d of the reference antibody is comparable to or about 200%, about 180%, about 160%, about 150%, about 140%, about 140% of the K d value of the reference antibody, Approximately 120%, approximately 110%, approximately 100%, approximately 99%, approximately 98%, approximately 97%, approximately 95%, approximately 90%, approximately 85%, approximately 80%, approximately 75%, approximately 70%, approximately 65% %, Or about 50%.
  • the reference antibody includes, for example, an antibody having a combination of light chain SEQ ID NO: 62 and heavy chain SEQ ID NO: 67.
  • the biological activities of the APJ antibody and Elabela fusion protein include the biological activity of Elabela and the APJ antibody activity.
  • the activity of the APJ antibody is as described above.
  • "Biological activity of Elabela” refers to an Elabela fusion protein that binds in vivo and activates Elabela receptors (such as APJ) and causes a cellular stress response.
  • the aforementioned cellular stress response includes, but is not limited to, enhancing myocardial contractility, diastolic blood vessels, lowering blood pressure, diuresis (reducing antidiuretic hormone release), regulating the body's immune response, and the release of pituitary-related hormones.
  • the Elabela fusion proteins described herein can be used to treat a variety of diseases and conditions associated with Elabela and APJ.
  • the fusion protein exerts its biological effect by acting on Elabela and / or APJ, and thus the Elabela fusion protein described herein can be used to treat subjects with diseases and conditions that respond favorably to "increased Elabela stimulation". These subjects are referred to as "subjects in need of Elabela stimulation therapy.” These subjects include subjects with acute heart failure, chronic heart failure, pulmonary hypertension, and pulmonary hypertension, as well as subjects with diabetic vascular disease, cardiac insufficiency, atrial fibrillation, and ischemia-reperfusion injury.
  • the biological activity of the Elabela fusion protein is detected using a reporter gene detection method to quantify the function of the Elabela fusion protein to activate APJ in vitro.
  • the antibody fusion proteins provided herein Elabela activation Elabela / APJ signaling pathway EC 50 of from about 0.1nM to about 10OnM, about 0.5nM to about 20 nM, from about 1nM to about 10 nM, or 1nM to about Approximately 5nM.
  • a pharmaceutical composition comprising a combination of an Elabela fusion protein provided herein and one or more pharmaceutically acceptable carriers.
  • the pharmaceutical composition comprises an Elabela fusion protein provided herein and one or more substances selected from the group consisting of a buffer suitable for the Elabela fusion protein, an antioxidant (e.g., ascorbic acid), a low molecular weight polypeptide (e.g., containing Polypeptides with less than 10 amino acids), proteins, amino acids, sugars (such as dextrin), complexes (such as EDTA), glutathione, stabilizers and excipients.
  • the pharmaceutical compositions provided herein may also include a preservative.
  • the pharmaceutical compositions provided herein can be formulated as a lyophilized powder using a suitable excipient solution as a diluent.
  • suitable excipient solution as a diluent.
  • provided herein is a method of treating, preventing, or improving pulmonary hypertension comprising administering to a subject a therapeutically effective amount of an Elabela fusion protein or a pharmaceutical composition thereof provided herein.
  • provided herein is a method of treating, preventing, or ameliorating pulmonary hypertension, comprising administering to a subject a therapeutically effective amount of an Elabela fusion protein or a pharmaceutical composition thereof provided herein.
  • provided herein is a method of treating, preventing, or ameliorating heart failure, comprising administering to a subject a therapeutically effective amount of an Elabela fusion protein or a pharmaceutical composition thereof provided herein.
  • provided herein is a method of treating type 2 diabetes and its related metabolic syndrome, comprising administering to a subject a therapeutically effective amount of an Elabela fusion protein or a pharmaceutical composition thereof provided herein.
  • provided herein is a method of treating, preventing, or ameliorating pulmonary hypertension, pulmonary hypertension, type 2 diabetes and its related metabolic syndrome, or two or more conditions of heart failure, comprising administering to an individual An amount of an Elabela fusion protein or a pharmaceutical composition thereof provided herein.
  • the pharmaceutical composition is for intravenous or subcutaneous injection.
  • Elabela fusion protein pharmaceutical compositions can be administered using any suitable technique including, but not limited to, parenteral, topical, or inhalation.
  • the pharmaceutical composition can be administered by rapid injection or continuous infusion, for example, by intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes.
  • topical administration at the site of the disease or injury such as transdermal administration and continuous release implantation, can be considered.
  • Inhalation administration includes, for example, nasal or oral inhalation, use of sprays, inhalation of Elabela fusion protein in the form of an aerosol, and the like.
  • Other options include oral formulations including tablets, syrups, or lozenges.
  • the dosage and frequency of administration may vary depending on the route of administration, the specific Elabela fusion protein used, the nature and severity of the disease being treated, whether the symptoms are acute or chronic, and the size and overall symptoms of the patient. Appropriate dosages can be determined by methods well known in the art, such as including dose escalation studies in clinical trials.
  • the Elabela fusion protein provided herein can be administered, for example, one or more times at regular intervals over a period of time.
  • the Elabela fusion protein is administered once at least one month or more, for example one, two or three months or even indeterminate.
  • long-term treatment is usually the most effective.
  • short-term administration for example, from one week to six weeks is sufficient.
  • human Elabela fusion protein is administered until the patient exhibits a medically relevant improvement in selected signs or indicators above baseline levels.
  • An example of a treatment regimen provided herein includes subcutaneous injection of Elabela fusion protein at an appropriate dose once a week or longer to treat type 2 diabetes and its related metabolic syndrome, acute heart failure, chronic heart failure, pulmonary hypertension or pulmonary hypertension symptom.
  • Elabela fusion protein can be administered weekly or monthly until the desired result is achieved, such as the patient's symptoms subsided. The treatment may be renewed as needed, or alternatively, a maintenance dose may be administered.
  • the patient's blood BNP or Pro-BNP concentration, body weight can be monitored before, during, and / or after treatment with the Elabela fusion protein to detect any changes in its pressure.
  • changes in BNP or Pro-BNP can vary with factors such as disease progression.
  • the BNP or Pro-BNP concentration can be determined by known techniques.
  • an Elabela fusion protein and one or more Elabela agonists two or more Elabela fusion proteins provided herein, or an Elabela fusion protein of the invention and one or more other Elabela Agonist.
  • the Elabela fusion protein is administered alone or in combination with other agents used to treat the symptoms that are painful to the patient. Examples of these agents include protein as well as non-protein drugs. When multiple drugs are administered in combination, the dosage should be adjusted accordingly as is well known in the art.
  • “Combined administration" combination therapy is not limited to simultaneous administration, but also includes a regimen in which the antigen and protein are administered at least once during a course of treatment involving the administration of at least one other therapeutic agent to the patient.
  • a method for preparing a medicament for treating heart failure and pulmonary hypertension and related disorders comprising a mixture of an Elabela fusion protein and a pharmaceutically acceptable excipient provided herein for use in treating a related disorder of the above-mentioned diseases.
  • the pharmaceutical preparation method is as described above.
  • Elabela fusion protein-related compositions, kits, and methods that can specifically bind to human APJ.
  • Nucleic acid molecules and derivatives and fragments thereof are also provided that comprise a polynucleotide encoding all or part of a polypeptide that binds to APJ, such as a nucleic acid encoding all or part of an Elabela fusion protein or an Elabela fusion protein derivative.
  • vectors and plasmids comprising such nucleic acids and cells and cell lines comprising such nucleic acids and / or vectors and plasmids.
  • Provided methods include, for example, a method of preparing, identifying, or isolating an Elabela fusion protein bound to human APJ, a method of determining whether the Elabela fusion protein binds to APJ, and a method of administering an Elabela fusion protein bound to APJ to an animal model.
  • CHO-DHFR- cells were seeded into 6-well plates. After culture for 24 hours (hr), the pTM15 plasmid cloned with the hAPJ gene (see SEQ ID NO: 55 for the nucleotide sequence and SEQ ID NO: 23 for the amino acid sequence) was transfected into cells in a 6-well plate. Transfection was performed according to the transfection conditions of Lipofectamine 2000 recommended by Invitrogen. After 48 hours, the culture medium was changed to a complete medium containing 300 ⁇ g / mL hygromycin, and the medium was changed every 3 days (d). After about two weeks of culture, stable growing clones appeared.
  • VACS-tagged antibodies (Life Technologies) were used to perform FACS detection on the constructed stable cell lines, and the pressurized cell population was identified based on the FACS detection results. A large amount of hAPJ was expressed on the CHO-DHFR-hAPJ cell membrane after screening. Finally, after subcloning and further identification, three APJ cells were selected as high expression stable cell lines. These hAPJ-expressing cell lines can be used as an immunogen for antibody production (Reference Example 2).
  • the fusion protein of the extracellular region of hAPJ and hIgG and Fc can also be used as an immunogen to prepare antibodies.
  • the preparation method is as follows: The fusion protein sequence gene of the extracellular region of hAPJ, hIgG2Fc and peptide linker (Linker) is in the pTM5 plasmid. A large number of transient expressions were obtained by suspending HEK293 cells, cell supernatants were obtained, and the APJ extracellular domain fusion protein was purified by affinity chromatography.
  • mice The immunogen and aluminum hydroxide adjuvant were mixed, and BALB / c mice (6-8 weeks old) were injected subcutaneously, and the mice were boosted once a week. After a total of 6 immunizations, blood was collected by tail trimming. The serum was separated by centrifugation, and the serum titer was measured by FACS. When the appropriate antibody titer was reached, the mice were sacrificed by neck dissection, and spleen cells were obtained under aseptic conditions. In addition, SP2 / 0 cells in the logarithmic growth phase were collected, the cells were centrifuged, and the precipitated cells were cultured in serum-free until resuspension, centrifuged-resuspended again, and counted.
  • the fused hybridoma cells and feeder cells were cultured together in a 96-well plate and subjected to HAT (hypoxanthine, methotrexate, and thymidine) screening to remove non-fused cells. After 10 days, the supernatant of the hybridoma cells in the culture plate was collected for ELISA detection.
  • HAT hypoxanthine, methotrexate, and thymidine
  • CHO-DHFR-hAPJ cells overexpressing hAPJ and CHO-DHFR- cells not expressing hAPJ were seeded into 96-well plates, respectively. After the cells grew to 90% healing, the cell culture supernatant was removed, washed twice with PBS, and fixed by adding 100% methanol at 4 ° C, and then 100 ⁇ L of prepared H 2 O 2 -PBS was added. The cells were treated at room temperature for 20 minutes and washed twice with PBS. After blocking with BSA (dissolved in PBS), the supernatant of hybridoma cells was added and incubated at 4 ° C for 90 minutes.
  • BSA dissolved in PBS
  • the positive control was the serum of immunized mice; the negative control was the cell culture supernatant.
  • the positive control was the serum of immunized mice; the negative control was the cell culture supernatant.
  • several positive hybridoma cell lines that secreted anti-hAPJ antibodies were screened. These hybridoma strains secreting anti-hAPJ antibodies were selected and cloned to obtain cell lines capable of stably secreting anti-hAPJ antibodies.
  • the ascites antibodies prepared from the positive hybridoma cells were selected, and the affinity of the hAPJ antibodies contained in them was verified and sorted by the flow cytometry experimental method (refer to Example 9).
  • the hybridoma cells secreting the antibodies were collected, and the mRNA of the hybridoma cells was extracted according to the QIAGEN mRNA extraction kit protocol.
  • the extracted mRNA is then reverse transcribed into cDNA.
  • the reverse transcription primers are specific primers for mouse light and heavy chain constant regions.
  • the heavy chain reverse transcription primers are (5'-TTTGGRGGGAAGATGAAGAC-3 ') and the light chain reverse transcription primers. (5'-TTAACACTCTCCCCTGTTGAA-3 ') and (5'-TTAACACTCATTCCTGTTGAA-3').
  • the reaction conditions of RT-PCR were: 25 ° C for 5min; 50 ° C for 60min; 70 ° C for 15min.
  • an ultrafiltration centrifuge tube Amicon Ultra
  • the upstream primers are all OligodT
  • the heavy chain downstream primers are (5'-TGGACAGGGATCCAGAGTTCC-3 ') and (5'-TGGACAGGGCTCCATAGTTCC-3')
  • the light chain downstream primers are (5'-ACTCGTCCTTGGTCAACGTG-3 ').
  • PCR reaction conditions 95 ° C for 5min; 95 ° C for 30s, 56 ° C for 30s, 72 ° C for 1min and 40cycles; 72 ° C for 7min; PCR products were ligated to PMD 18-T vector (Takara Bio) and then sequenced.
  • PCR primers were designed based on the DNA sequences of the sequenced antibodies to link the complete light chain, heavy chain signal peptide and variable domain, and the mouse IgG1 constant region to the expression vector pTM5.
  • the human gene sequence with the highest homology is used as a template sequence for CDR grafting to obtain a humanized antibody variable region sequence.
  • Genes of light and heavy chains of humanized antibodies were synthesized and spliced with human IgG2 or IgG4 constant region sequences to obtain complete recombinant humanized antibody sequences.
  • the recombinant antibody was expressed according to Example 8, and the affinity of the recombinant antibody to APJ was verified according to the FACS technology in step 10, and the antibody with the best affinity was selected.
  • the variable region sequence was further modified to further improve its affinity for APJ.
  • the optimized humanized antibody heavy chain and light chain variable region sequences were outsourced for synthesis, and the complete heavy chain variable region sequence was further linked to the heavy chain constant region expression vector pTM5; similarly, the complete light chain The variable region sequence is linked to the expression vector pTM5 which has been incorporated into the constant region of the light chain.
  • the optimized humanized antibody was fused to the Elabela fragment sequence at the C-terminus of the heavy chain to form an Elabela fusion protein.
  • the two sequences are linked by a peptide linker sequence (Linker).
  • the humanized APJ antibody heavy chain nucleic acid sequence was linked to the "Linker-Elabela fragment” portion by overlapping PCR. Nhe1 and Not1 restriction sites were added to the two ends of the primers, thereby linking the complete fusion protein sequence to the expression vector pTM5.
  • Elabela The fusion protein was ligated to an expression vector and sequenced to confirm that it was constructed correctly.
  • the collected cell supernatant in Example 8 was centrifuged at a high speed (8000 rpm) to remove the cells and cell debris, and then filtered through a 0.22um filter to clarify.
  • the clarified supernatant was used for purification.
  • the purification process is performed by a chromatograph.
  • the supernatant was first passed through a protein A / G affinity chromatography column.
  • the antibody or fusion protein contained in the supernatant was retained in the column after binding with the ligand of the protein A / G affinity chromatography column during this period.
  • the column is then washed with a low pH (3.0 or less) elution buffer to dissociate the antibody or fusion protein bound to the column.
  • the collected antibody eluate was quickly neutralized with 1M Tris-HCl.
  • the obtained antibody or fusion protein eluate is replaced with PBS or other buffer system after dialysis.
  • the gray peaks on the left are negative controls for 500nM mouse ascites antibody L1H1 and blank cells CHO-DHFR-binding, and the solid peaks are 500nM ( Figure 1A) and 4nM ( Figure 1B)
  • the binding curve of mouse ascites antibody L1H1 and CHO-DHFR-hAPJ has a significant right shift relative to the gray peak negative control, which proves the specific binding of L1H1 and hAPJ.
  • the right gray peak is a negative control for 500nM mouse ascites antibody L4H4 and blank cells CHO-DHFR-binding.
  • the solid peaks are 500nM (Figure 1C) and 4nM (Figure 1D) mouse ascites antibodies L4H4 and CHO-
  • the binding curve of DHFR-hAPJ has a significant right shift relative to the gray peak negative control, which also proves the specific binding of L4H4 and hAPJ.
  • CHO-DHFR- cells co-expressing hAPJ-CRE-Luciferase were seeded at 35,000 cells per well to a 96-well cell culture plate and cultured overnight at 37 ° C. The next day, the supernatant of the medium was removed, the cell surface was washed twice with serum-free medium, and the residual liquid was aspirated.
  • 50 ⁇ L of forskolin with a concentration of 0.6 ⁇ M was added in advance, and 50 ⁇ L of Elabela fusion protein or Elabela-11 polypeptide was diluted with serum-free medium, and incubated at 37 ° C. for 6 hours. After the stimulation, 100 ⁇ L of Promega's Bright Glo chemiluminescent substrate was added.
  • FIG. 1 shows the activation curve of the Elabela / APJ signaling pathway activated by the reporter gene experiment to detect the recombinantly expressed fusion protein of L5H5 and Linker2-Elabela-11, and the fusion protein of L5H5 and Linker2-EA5.
  • the EC 50 is 3.61 and 2.55nM, respectively. .
  • Figure 3 shows the activation curve of activation of the Elabela / APJ signaling pathway by the recombinant protein L5H5 and Linker2-EA1 fusion protein and the fusion protein of L55H5 and Linker2-EA2 detected by reporter gene experiments, with EC 50 of 4.95 and 2.16nM, respectively.
  • Figure 4 shows the activation curve of activation of the Elabela / APJ signaling pathway by the recombinant protein L5H5 and Linker2-EA3 fusion protein and the fusion protein of L5H5 and Linker2-EA4 detected by reporter gene experiments.
  • the EC 50 are: 20.27 and 5.43 nM, respectively.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cardiology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Endocrinology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Toxicology (AREA)
  • Diabetes (AREA)
  • Hospice & Palliative Care (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Emergency Medicine (AREA)
  • Epidemiology (AREA)
  • Inorganic Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

涉及APJ抗体及其与Elabela的融合蛋白质。还涉及APJ抗体及Elabela融合蛋白质的药物组合物，以及APJ抗体及Elabela融合蛋白质用于治疗、预防或改善肺动脉高压、肺高压、或心力衰竭的一种或多种症状方法。

Description

APJ抗体及其与Elabela的融合蛋白质，以及其药物组合物和应用 技术领域
本文提供了APJ抗体及其与Elabela的融合蛋白质。本文还提供了APJ抗体及Elabela融合蛋白质的药物组合物。本文进一步提供了APJ抗体及Elabela融合蛋白质用于治疗、预防或改善肺动脉高压、肺高压、或心力衰竭的一种或多种症状方法。
发明背景
APJ含有7个跨膜单元和308个氨基酸，属于G蛋白偶联受体(G protein coupled receptors,GPCRs)的家族成员。血管活性肽Apelin是最早发现的APJ受体的内源性配体。Apelin和APJ广泛分布于人中枢神经系统以及肺、心脏、乳腺等多种外周组织(Kawamata等，2001，Biochem.Biophys.Acta.1538:162-71；Medhurst等，2003，J.Neurochem.84:1162-72)，在心血管内皮细胞和心脏组织的表达尤为突出(Kleiz等，2005，Regul.Pept.126:233-40)。多项研究表明，Apelin/APJ信号系统具有增强心肌收缩力、降低血压、促进新生血管形成、调节机体免疫反应及垂体相关激素释放、调控胰岛素分泌等作用，并参与糖尿病血管病变、心功能不全、心房颤动、缺血再灌注损伤的病理生理过程。
Elabela是近年来发现的另外一个备受关注APJ受体的内源性配体(Chng等,2013，Dev.Cell 27:672-680；Pauli等，2014，Science 343:1248636)。Elabela由人4号染色体上的3个外显子编码，之前一直被认为是非编码RNA，然而，研究发现Elabela含有一段保守的ORF可以编码54个氨基酸的蛋白，其成熟体仅由32个氨基酸构成。Elabela/APJ信号通路被证明在胚胎的心脏和脉管系统发育过程中的起着非常关键性的作用。有研究证实，Elabela和Elabela的突变体可以激活APJ受体的Gαi1和β-arrestin2信号传导通路，进一步，Elabela多肽C端的突变可以引起APJ受体信号通路传导的偏好性(Murza等，2016，J.Med.Chem.59:2962-72)，而利用受体信号通路传导的偏好性是新药研发的一个重要方向(Bologna等，2017，Biomol.Ther.25:12-25)。通过使用分离灌注心脏和体内血流动力学和超声心动图测量，发现Elabela或Elabela的突变体可以降低动脉压，并对心脏产生正性肌力作用(Yang等，2017，Circulation 135:1160–1173；Murza等，2016，J.Med.Chem.59:2962-72)。还有研究发现，糖尿病肾病病人的Elabela含量与尿微量白蛋白肌酐比值(ACR)呈负相关(Zhang等，2018，Cell Physiol.Biochem.48:1347-1354).
把Elabela和APJ抗体两者融合施用，可以显著延长Elabela的半衰期来保留Elabela分子的生物学活性，同时Elabela与APJ特异性结合的抗体形成的融合蛋白质具有由抗体提供的分子靶向性，而有助于提高Elabela融合蛋白质的成 药性，起到治疗肺动脉高压、肺高压、二型糖尿病及其相关代谢综合症，以及心力衰竭中一种或多种疾病的作用。
发明内容
本文提供了能与APJ特异性结合的抗体。
本文还提供了一个能与APJ特异性结合的抗体，其抗体包含一个、两个、三个、四个、五个、或六个氨基酸序列，其中每个氨基酸序列都独立地选自于以下所列的氨基酸序列：
a.轻链CDR1氨基酸序列：SEQ ID NO:1、SEQ ID NO:4、SEQ ID NO:7、及SEQ ID NO:10；
b.轻链CDR2氨基酸序列：SEQ ID NO:2、SEQ ID NO:5、及SEQ ID NO:8；
c.轻链CDR3氨基酸序列：SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:9、及SEQ ID NO:11；
d.重链CDR1氨基酸序列：SEQ ID NO:12、SEQ ID NO:15、及SEQ ID NO:18；
e.重链CDR2氨基酸序列：SEQ ID NO:13、SEQ ID NO:16、SEQ ID NO:19、及SEQ ID NO:21；及
f.重链CDR3氨基酸序列：SEQ ID NO:14、SEQ ID NO:17、SEQ ID NO:20、及SEQ ID NO:22。
本文提供了一个Elabela融合蛋白质，其包含一个能与APJ特异性结合的抗体、和一个，二个，三个，四个，五个，六个，七个，或八个Elabela片段肽接头序列(Linker)；该融合蛋白质通过一肽接头序列(Linker)将一Elabela片段的氨基端与一APJ抗体轻链或重链的羧基端连接，其中每个Elabela片段各自独立地为一正向Elabela片段或其突变体；或者该融合蛋白质通过一肽接头序列(Linker)将一Elabela片段的羧基端与一APJ抗体轻链或重链的氨基端连接，其中每个Elabela片段各自独立地为一反向Elabela片段或其突变体。
本文提供了一个Elabela融合蛋白质，其包含一个能与APJ特异性结合的抗体和二个Elabela片段及二个肽接头序列(Linker)；该融合蛋白质通过一个肽接头序列(Linker)将一Elabela片段的氨基端与一APJ抗体轻链的羧基端连接：N'-R-Linker-Elabela-C'；或者通过一个肽接头序列(Linker)将一Elabela片段的氨基端与一APJ抗体重链的羧基端连接：N'-R-Linker-Elabela-C'；其中:N'代表融合蛋白质多肽链的氨基端,C'代表融合蛋白质多肽链的羧基端,Elabela代表正向Elabela片段或其突变体，R代表APJ抗体的轻链或者重链的氨基酸序列,及Linker代表肽接头序列。
本文提供了一个多核苷酸，其编码本文中所述的一个Elabela融合蛋白质。
本文提供了一个载体，其包含编码本文中所述的一个Elabela融合蛋白质的多核苷酸。
本文提供了一个宿主细胞，其包含本文中所述的一个载体。
本文提供了一个药用组合物，其包含本文所述的一个APJ抗体或一个Elabela 融合蛋白质，和一个药用可接受载体。
本文提供了本文所述的一个APJ抗体或一个Elabela融合蛋白质在制备用于治疗、预防或改善肺动脉高压以及肺动脉高压相关病症的药物中的用途。
本文提供了本文所述的一个APJ抗体或一个Elabela融合蛋白质在制备用于治疗、预防或改善肺高压以及肺高压相关病症的药物中的用途。
本文提供了本文所述的一个APJ抗体或一个Elabela融合蛋白质在制备用于治疗、预防或改善心力衰竭以及心力衰竭相关病症的药物中的用途。
本文提供了本文所述的一个APJ抗体或一个Elabela融合蛋白质在制备用于治疗二型糖尿病及其相关代谢综合症的药物中的用途。
本文提供了本文所述的一个APJ抗体或一个Elabela融合蛋白质在制备用于同时治疗、预防或改善肺动脉高压、肺高压、二型糖尿病及其相关代谢综合症或者心力衰竭二种及二种以上病症的药物中的用途。
本文提供了治疗、预防或改善肺动脉高压的一种或多种症状方法，其包括给予受试者治疗有效量的本文所述的一个APJ抗体或一个Elabela融合蛋白质。
本文提供了治疗、预防或改善肺高压的一种或多种症状方法，其包括给予受试者治疗有效量的本文所述的一个APJ抗体或一个Elabela融合蛋白质。
本文提供了治疗、预防或改善心力衰竭的一种或多种症状方法，其包括给予受试者治疗有效量的本文所述的一个APJ抗体或一个Elabela融合蛋白质。
本文提供了治疗二型糖尿病及其相关代谢综合症一种或多种症状方法，其包括给予受试者治疗有效量的本文所述的一个APJ抗体或一个Elabela融合蛋白质
附图简述
[根据细则26改正11.07.2019]　
图1A至图1D：显示了流式细胞术(FACS)检测重组表达的hAPJ抗体L1H1(其包含SEQ ID NO:59与SEQ ID NO:64)和L4H4(其包含SEQ ID NO:62与SEQ ID NO:67)与hAPJ特异性结合的结果(在500nM或4nM的抗体浓度)，其中灰色峰为阴性对照，表示L1H1或L4H4与CHO-DHFR-的结合曲线，实线峰表示L1H1(图1A、图1B)或L4H4(图1C、图1D)与CHO-DHFR-hAPJ的结合曲线。
[根据细则26改正11.07.2019]　
图2：显示了报告基因实验检测重组表达的hAPJ抗体L5H5(SEQ ID NO:63与SEQ ID NO:68)与Linker2-Elabela-11(其包含SEQ ID NO:123与SEQ ID NO:93)的融合蛋白质、L5H5与Linker2-EA5(其包含SEQ ID NO:123与SEQ ID NO:94)的融合蛋白质激活Elabela/APJ信号通路的激活性曲线，EC 50分别为：3.6和2.6nM。
[根据细则26改正11.07.2019]　
图3：显示了报告基因实验检测重组表达的hAPJ抗体L5H5与Linker2-EA1(其包含SEQ ID NO:123与SEQ ID NO:103)的融合蛋白质、L5H5与Linker2-EA2(其包含SEQ ID NO:123与SEQ ID NO:107)的融合蛋白质激活Elabela/APJ信号通路的激活性曲线，EC 50分别为：5.0和2.2nM。
[根据细则26改正11.07.2019]　
图4：显示了报告基因实验检测重组表达的hAPJ抗体L5H5与Linker2-EA3(其包含SEQ ID NO:123与SEQ ID NO:109)的融合蛋白质、L5H5与Linker2-EA4 (其包含SEQ ID NO:123与SEQ ID NO:116)的融合蛋白质激活Elabela/APJ信号通路的激活性曲线，EC 50分别为：20.3和5.4nM。
具体实施方式
定义
除非本文另外定义，与本文相关的科学和技术术语应具有本领域普通技术人员所理解的含义。通常，与本文所述药物学、生物学、生物化学、细胞和组织培养学、生物学、分子生物学、免疫学、微生物学、遗传学和蛋白质核酸化学以及杂交相关的命名法和技术为本领域熟知和经常使用的。
本文使用标准的单字母或三字母缩写表明多聚核苷酸和多肽序列。多肽序列在书写时，带有氨基的首个氨基酸残基(N')在最左而带有羧基的最末氨基酸残基(C')在最右,例如本文所涉及的正向Elabela片段和其突变体序列：SEQ ID NO:91，SEQ ID NO:92，SEQ ID NO:93，SEQ ID NO:94，SEQ ID NO:95，SEQ ID NO:96，SEQ ID NO:97，SEQ ID NO:98，SEQ ID NO:99，SEQ ID NO:100，SEQ ID NO:101，SEQ ID NO:102，SEQ ID NO:103，SEQ ID NO:104，SEQ ID NO:105，SEQ ID NO:106，SEQ ID NO:107，SEQ ID NO:108，SEQ ID NO:109，SEQ ID NO:110，SEQ ID NO:111，SEQ ID NO:112，SEQ ID NO:113，SEQ ID NO:114，SEQ ID NO:115，SEQ ID NO:116，SEQ ID NO:117，SEQ ID NO:118，SEQ ID NO:119，SEQ ID NO:120，及SEQ ID NO:121。反向多肽序列指将多肽序列的氨基酸出现顺序逆向排列后形成的序列，例如上述正向Elabela片段和其突变体序列所形成的反向Elabela片段序列：SEQ ID NO:125、SEQ ID NO:126、SEQ ID NO:127、SEQ ID NO:128、SEQ ID NO:129、SEQ ID NO:130、SEQ ID NO:131、SEQ ID NO:132、SEQ ID NO:133、SEQ ID NO:134、SEQ ID NO:135、SEQ ID NO:136、SEQ ID NO:137、SEQ ID NO:138、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141、SEQ ID NO:142、SEQ ID NO:143、SEQ ID NO:144、SEQ ID NO:145、SEQ ID NO:146、SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149、SEQ ID NO:150、SEQ ID NO:151、SEQ ID NO:152、SEQ ID NO:153、SEQ ID NO:154、及SEQ ID NO:155。Elabela单链核酸序列和双链核酸序列的上游链的5’端在左而它们的3’端在右。多肽的具体部分可由氨基酸残基编号表示，例如氨基酸67至134，或由该位点的实际残基表示例如Lys67至Lys134。也可通过解释其与参比序列的差异描述具体的多肽或多聚核苷酸序列。
术语“个体”是指动物，包括但不限于灵长类动物(例如，人类)、牛、猪、绵羊、山羊、马、狗、猫、兔、大鼠或小鼠。术语“个体”和“患者”互换使用，例如，用于指哺乳动物个体，如人个体，在一个实施方案中，指人。
术语“治疗”的含义包括减轻或消除紊乱、疾病或病症，或与紊乱、疾病或病症相关的一种或多种症状；或减轻或消除紊乱、疾病或病症的病灶。
术语“预防”的含义包括延缓和/或解除紊乱、疾病或病症和/或其伴随症状的发作；阻止个体获得紊乱、疾病或病症；或减少个体获得紊乱、疾病或病症的风 险。
术语“控制”是指防止或减缓病症、紊乱或疾病或其一种或多种症状(例如，疼痛)的进展、蔓延或恶化。有时，个体得益于预防剂或治疗剂的有益效果不会导致病症、紊乱或疾病的治愈。在一个实施方案中，术语控制是指防止或减缓骨质溶解的疼痛的进展、蔓延或恶化。
术语“治疗有效量”和“有效量”的含义包括当施用时足以防止正在接受治疗的紊乱、疾病或病症的一种或多种症状发展或在一定程度上减轻的化合物或化合物的组合的量。术语“治疗有效量”或“有效量”也指足以引起研究人员、兽医、医生或临床医师所寻求的生物分子(例如，蛋白、酶、RNA或DNA)、细胞、组织、系统、动物或人的生物或医学响应的化合物量。
术语“药学上可接受的载体”、“药学上可接受的赋形剂”、“生理上可接受的载体”或“生理上可接受的赋形剂”是指药学上接受的材料、组合物或运载体，如液体或固体填料、稀释剂、溶剂或封装材料。在一个实施方案，“药学上可接受的”每种成分是指与药剂配方的其他成分兼容，并且适用于与人和动物的组织或器官接触，而没有过多的毒性、刺激性、过敏性反应、免疫原性或其他问题或并发症，相当于合理的利益/风险比。
术语“约”或“大约”是指由本领域技术人员确定的可以接受的误差，其部分取决于如何测量或确定值。在某些实施方案中，术语“约”或“大约”是指在1、2、3或4个标准偏差之内。在某些实施方案中，术语“约”或“大约”是指在给定值或范围的50％、20％、15％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％、0.5％或0.05％之内。
术语“肽”、“多肽”、和“蛋白”均指包含两个或多个通过肽健相互连接的氨基酸的分子。这些术语涵盖例如天然和人工蛋白和蛋白序列的多肽类似物(例如突变蛋白、变异体和融合蛋白)以及转录后或否则为共价或非共价修饰的蛋白。肽、多肽或蛋白可为单体或多聚体。
术语“多肽片段”指与对应的全长蛋白相比具有氨基端和/或羧基端缺失的多肽。片段长度可为例如至少5、6、7、8、9、10、11、12、13、14、15、20、50、70、80、90、100、150、或200个氨基酸。片段长度可为，例如，最多1000、750、500、250、200、175、150、125、100、90、80、70、60、50、40、30、20、15、14、13、12、11、或10个氨基酸。片段可在其一端或两端进一步包含一个或多个附加氨基酸，例如，来自不同天然蛋白质的氨基酸序列(例如，Fc或亮氨酸拉链结构域)或人工氨基酸序列(例如，人工接头序列)。
本文的多肽包括以任何原因和经任何方法修饰的多肽，例如，以：(1)降低蛋白水解敏感性，(2)降低氧化敏感性，(3)改变形成蛋白复合物的亲和性，(4)改变结合亲和性，以及(5)赋予或修饰其它物理化学或功能性质。类似物包含多肽的突变蛋白。例如，可在天然序列(例如在形成分子内接触的结构域之外的多肽部分)中进行单个或多个氨基酸替换(例如，保守氨基酸替换)。“保守氨基酸替换”为不显著改变母体序列结构特性者(例如，替换氨基酸不应破坏母体序 列中出现的螺旋或干扰其它赋予母体序列特性或对其功能是必须的二级结构类型)。
多肽的“变异体”包含相对于另一多肽序列在氨基酸序列中插入、缺失和/或替换了一个或多个氨基酸残基的氨基酸序列。本文的变异体包括融合蛋白。
多肽的“衍生物”为经化学修饰的多肽，例如通过与其它化学部分例如聚乙二醇、白蛋白(例如人血清白蛋白)结合、磷酸化和糖基化。
除非另外说明，术语“抗体”包括除包含两个全长重链和两个全长轻链的抗体，以及其衍生物，变异体、片段、和突变蛋白，其实例见下文。
术语“抗体”为包含与抗原结合部分并任选为允许抗原结合部分采取促进该抗体与该抗原结合的构象的支架或框架部分的蛋白。抗体的实例包括完整抗体、抗体片段(例如抗体的抗原结合部分)、抗体衍生物、和抗体类似物。该抗体可包含例如可选择的蛋白支架或具有移植CDRs或CDRs衍生物的人工支架。该支架包括但不限于包含被引入的例如以稳定化该抗体的三维结构的抗体衍生支架以及包含例如生物相容性多聚体的全合成支架。参见，例如，Korndorfer等,2003,Proteins 53:121-9；Roque等,2004,Biotechnol.Prog.20:639-54。此外，该抗体可以是模拟肽抗体(“PAMs”)或包含模拟抗体的支架，其如支架一样利用纤维蛋白连接素。
抗体可具有例如天然免疫球蛋白的结构。“免疫球蛋白”为四聚体分子。在天然的免疫球蛋白中，各四聚体由两个相同的多肽链对组成，各对具有一个“轻”链(约25kDa)和一个“重”链(约50-70kDa)。各链的氨基端包括约100至110氨基酸的可变结构域，主要与抗原识别相关。各链的羧基端部分确定了主要与效应器作用相关的恒定区。人的抗体轻链分为κ和λ轻链。重链分为μ、δ、α或ε，并确定了抗原的同种型，例如分别为IgM、IgD、IgG、IgA、和IgE。在轻链和重链中，可变和恒定区由约12或更多个氨基酸的“J”区连接，重链也包括约10多个氨基酸的“D”区。参见，Fundamental Immunology Ch.7(Paul编辑，第2版，Raven Press,1989)。各轻/重链对的可变区形成抗体结合位点，这样一个完整的免疫球蛋白具有两个结合位点。
天然免疫球蛋白链显示出由三个高度可变区连接的相对保守骨架区(FR)的相同基本结构，也被称作互补决定区或CDRs。从N端到C端，轻和重链均包含结构域FR1、CDR1、FR2、CDR2、FR3、CDR3、和FR4。各结构域氨基酸的分配与Kabat等在Sequences of Proteins of Immunological Interest，第5版，U.S.Dept.of Health and Human Services,PHS,NIH,NIH Publication No.91-3242,1991中的定义一致。
除非另外指明，“抗体”指完整的免疫球蛋白或其可与完整抗体竞争特异性结合的抗原结合部分。可由重组DNA技术或通过酶或化学裂解完整抗体生产抗原结合部分。抗原结合部分包括，尤其是，Fab、Fab’、F(ab’) 2、Fv、结构域抗体(dAbs)，包括互补决定区(CDRs)的片段、单链抗体(scFv)、嵌合抗体、双链抗体(diabodies)、三链抗体(triabodies)、四链抗体(tetrabodies)和至少包 含足以赋予多肽特异抗原结合的免疫球蛋白的一部分的多肽。
Fab片段为具有V L、V H、C L、和C H1结构域的单价片段；F(ab’) 2片段为具有两个在铰链区由二硫键连接的Fab片段的二价片段；Fv片段具有V H和V L结构域；dAb片段具有V H结构域、V L结构域，或V H或V L结构域的抗原结合片段(美国专利号US 6,846,634及US 6,696,245；美国专利申请公开号US2005/0202512、US 2004/0202995、US 2004/0038291、US 2004/0009507、及US2003/0039958；Ward等,1989,Nature 341:544-6.)
单链抗体(scFv)为一融合蛋白，其中的V L和V H区由接头(例如，合成的氨基酸残基序列)连接以形成连续蛋白质的抗体，其中该接头足够长以允许该蛋白链折叠回自身并形成单价抗原结合位点(参见，例如，Bird等,1988,Science 242:423-6；和Huston等,1988,Proc.Natl.Acad.Sci.U.S.A.85:5879-83)。
双链抗体为包含两个多肽链的二价抗体，其中各多肽链包含由接头连接的V H和V L结构域，该接头很短以致于不允许两个结构域在相同链上的配对，因此允许各结构域与另一多肽链上的互补结构域配对(参见，例如，Holliger等,1993,Proc.Natl.Acad.Sci.U.S.A.90:6444-8；和Poljak等,1994,Structure 2:1121-3)。如果双链抗体的两个多肽链是相同的，那么由它们配对得到的双链抗体将具有相同的抗原结合位点。具有不同序列的多肽链可用于制备具有不同抗原结合位点的双链抗体。相似地，三链抗体和四链抗体分别为包含三个和四个多肽链的抗体并分别形成三个和四个抗原结合位点，其可相同或不同。
本文使用Kabat等在Sequences of Proteins of Immunological Interest,第5版,U.S.Dept.of Health and Human Services,PHS,NIH,NIH Publication No.91-3242,1991中描述的方法鉴定给定抗体的互补决定区(CDRs)和框架区(FR)。可向分子中共价或非共价并入一个或多个CDRs使其成为抗体。抗体可以较大多肽链并入CDR(s)。可将CDR(s)共价连接至另一乡肽链，或非共价并入CDR(s)。CDRs允许抗体与具体的相关抗原特异性结合。
抗体可有一个或多个结合位点。如果多于一个结合位点，该结合位点可与另一个相同或不同。例如，天然人免疫球蛋白通常具有两个相同的结合位点，而“双特异性”或“双功能”抗体具有两个不同的结合位点。
术语“鼠源抗体”包括具有一个或多个来源于小鼠免疫球蛋白序列的可变区和恒定区的抗体。
术语“人源化抗体”是将小鼠抗体分子的互补决定区序列移植到人抗体可变区框架中而制成的抗体。
术语“抗原结合结构域”、“抗原结合区”或“抗原结合位点”为包含与抗原相互作用的氨基酸残基并有助于抗体对抗原的特异性和亲和力的抗体的部分。对与其抗原特异性结合的抗体而言，这将包括至少部分的至少一个其CDR结构域。
术语“表位”为与抗体(例如，通过抗体)结合的分子部分。表位可包含分子的非连续部分(例如，在多肽中，在多肽的一级序列中不连续的氨基酸残基在该多肽的三级和四级结构中相互足够接近以致于被一个抗体结合)。
两个多聚核苷酸或两个多肽序列的“相同百分比”由使用GAP计算机程序(GCG Wisconsin Package；version 10.3(Accelrys，San Diego，CA)的一部分)使用其默认参数比较序列测定。
术语“多聚核苷酸”、“寡聚核苷酸”和“核酸”可在全文中交替使用并包括DNA分子(例如，cDNA或基因组DNA)、RNA分子(例如mRNA)、使用核苷酸类似物(例如，肽核酸和非天然核苷酸类似物)生成的DNA或RNA类似物及其杂交体。核酸分子可为单链或双链。在一个实施方案中，本文的核酸分子包含编码本文提供抗体或其片段、衍生物、突变蛋白或变异体连续的开放阅读框。
如果它们的序列可反向平行排列则两个单链多聚核苷酸是相互“互补的”，这样一个多聚核苷酸中的各核苷酸与另一多聚核苷酸中的互补核苷酸相反，不会引入空隙并且各序列的5’或3’端没有未配对的核苷酸。如果两个多聚核苷酸可在中等严格条件下相互杂交那么一个多聚核苷酸与另一多聚核苷酸“互补”。因此，一个多聚核苷酸可与另一多聚核苷酸互补，但并不是它的互补序列。
术语“载体”为可用于将与其相连的另一核酸引入细胞的核酸。载体的一种类型为“质粒”，其指可连接附加核酸区段的线性或环状双链DNA分子。载体的另一类型为病毒载体(例如，复制缺陷逆转录病毒、腺病毒和腺病毒伴随病毒)，其中可将附加DNA区段引入病毒基因组。某些载体可在它们被引入的宿主细胞中自主复制(例如，包含细菌复制起点的细菌载体以及游离型哺乳动物载体)。其它载体(例如，非游离型哺乳动物载体)在引入宿主细胞时整合入宿主细胞的基因组中并因此与宿主基因组一起复制。“表达载体”为可引导所选多聚核苷酸表达的载体类型。
如果调控序列影响核苷酸序列的表达(例如，表达水平、时间或位点)那么核苷酸序列与调控序列“可操作地相连”。“调控序列”为可影响与其可操作相连的核酸的表达(例如，表达水平、时间或位点)的核酸。调控基因，例如，直接对受调控核酸发挥作用或通过一个或多个其它分子(例如，与调控序列和/或核酸结合的多聚核苷酸)的作用。调控序列的实例包括启动子、增强子和其它表达控制元件(例如，多腺苷酸化信号)。调控序列的进一步实例描述于例如Goeddel,1990,Gene Expression Technology:Methods in Enzymology,Volume 185,Academic Press,San Diego,CA；和Baron等,1995,Nucleic Acids Res.23:3605-6。
术语“宿主细胞”为用于表达核酸例如本文提供核酸的细胞。宿主细胞可为原核生物，例如大肠杆菌，或者其可为真核生物，例如单细胞真核生物(例如，酵母或其它真菌)、植物细胞(例如烟草或番茄植物细胞)、动物细胞(例如，人细胞、猴细胞、仓鼠细胞、大鼠细胞、小鼠细胞或昆虫细胞)或杂交瘤。通常，宿主细胞为可用多肽编码核酸转化或转染的培养细胞，其可接着在宿主细胞中表达。短语“重组宿主细胞”可用于表述用预期表达的核酸转化或转染的宿主细胞。宿主细胞也可为包含该核酸但是不以期望水平表达的细胞，除非向该宿主细胞引入了调控序列这样其与核酸可操作地相连。应理解的是术语宿主细胞不仅指具体的受试者细胞也指该细胞的子代或可能的子代。由于例如突变或环境影响后续世代会 出现某些修饰，该子代事实上可能与母体细胞不同但是仍然属于本文使用的术语范围。
APJ受体
APJ是一个含377氨基酸的7次跨膜G蛋白偶联受体(O’Dowd等，1993，Gene 136:355-60)。截止目前的研究发现，APJ广泛分布于人中枢神经系统以及肺、心脏、乳腺等多种外周组织(Kawamata等，2001，Biochem.Biophys.Acta.1538:162-71；Medhurst等，2003，J.Neurochem.84:1162-72)，在心血管内皮细胞和心脏组织的表达尤为突出(Kleiz等，2005，Regul.Pept.126:233-40)。APJ主要参与心血管系统的调节，也被报道在胰岛素调节以及糖尿病和肥胖相关疾病的调控机制中有重要意义(Boucher等，2005，Endocrinology 146:1764-71；Yue等，2010，Am.J.Physiol.Endocrinol.Metab.298:E59-67)。本文中所使用的“人APJ”和“hAPJ”均指代人源的APJ，可交替使用。本文中所使用的“鼠APJ”和“mAPJ”均指代鼠源的APJ，也可交替使用。
在一个实施方案中，本文提供的抗体是与人APJ特异性结合的抗体。在另一个实施方案中，本文提供的融合蛋白质是与细胞膜上的APJ特异性结合的Elabela融合蛋白质，并且该融合蛋白质能激活Elabela/APJ信号在这些细胞内的传导。在进一步的实施例中，本文提供的融合蛋白质是与人APJ结合的Elabela融合蛋白质，并且该融合蛋白质能与其它物种的APJ结合(例如猴子和小鼠)，并激活在这些物种中Elabela/APJ的信号传导。
在一个实施方案中，APJ的氨基酸和多聚核苷酸序列如下所列，序列数据来源于美国国立生物技术信息中心的GeneBank数据库以及欧洲生物信息研究所的Uniprot数据库：
人(Homo sapiens)多聚核苷酸(SEQ ID NO:55)；登录号：X89271；
人(Homo sapiens)氨基酸(SEQ ID NO:23)；登录号：CAA61546；
猴子(Rhesus macaque)多聚核苷酸(SEQ ID NO:56)；登录号：AF100206；
猴子(Rhesus macaque)氨基酸(SEQ ID NO:24)；登录号：AAC72404；
大鼠(Rattus norvegicus)多聚核苷酸(SEQ ID NO:57)；登录号：AB033170；
大鼠(Rattus norvegicus)氨基酸(SEQ ID NO:25)；登录号：BAA95002；
小鼠(Mus musculus)多聚核苷酸(SEQ ID NO:58)；登录号：AJ007612；及
小鼠(Mus musculus)氨基酸(SEQ ID NO:26)；登录号：CAB50696。
血管活性肽受体抗体(APJ抗体)
在一个实施方案中，本文提供的是APJ抗体。在一个实施方案中，本文提供的APJ抗体是完整APJ抗体。在另一个实施方案中，本文提供的APJ抗体是APJ抗体片段。在另一个实施方案中，本文提供的APJ抗体是APJ抗体衍生物。在另一个实施方案中，本文提供的APJ抗体是APJ抗体突变蛋白。在进一步的实施方案中，本文提供的APJ抗体是APJ抗体变异体。
在一个实施方式中，本文提供的APJ抗体包含一个、两个、三个、四个、五个、或六个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序 列：
a.轻链CDR1氨基酸序列：SEQ ID NO:1、SEQ ID NO:4、SEQ ID NO:7、及SEQ ID NO:10；
b.轻链CDR2氨基酸序列：SEQ ID NO:2、SEQ ID NO:5、及SEQ ID NO:8；
c.轻链CDR3氨基酸序列：SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:9、及SEQ ID NO:11；
d.重链CDR1氨基酸序列：SEQ ID NO:12、SEQ ID NO:15、及SEQ ID NO:18；
e.重链CDR2氨基酸序列：SEQ ID NO:13、SEQ ID NO:16、SEQ ID NO:19、及SEQ ID NO:21；及
f.重链CDR3氨基酸序列：SEQ ID NO:14、SEQ ID NO:17、SEQ ID NO:20、及SEQ ID NO:22。
表1列出本文提供的APJ抗体的轻链CDRs的氨基酸序列，以及其相应的多聚核苷酸编码序列。表2列出本文提供的APJ抗体的重链CDRs的氨基酸序列，以及其相应的多聚核苷酸编码序列。
表1：轻链CDRs的氨基酸序列及其多聚核苷酸编码序列
Figure PCTCN2019091090-appb-000001
表2：重链CDRs的氨基酸序列及其多聚核苷酸编码序列
Figure PCTCN2019091090-appb-000002
在一个实施方案中，本文提供的抗体包含与表1和表2中所列的CDR氨基酸序列中之一相差5、4、3、2、或1个单氨基酸添加、替换和/或缺失的序列。在另一个实施方案中，本文提供的抗体包含与表1和表2中所列的CDR氨基酸序列中之一相差4、3、2、或1个单氨基酸添加、替换和/或缺失的序列。
在另一个实施方案中，本文提供的抗体包含与表1和表2中所列的CDR氨基酸序列中之一相差3、2、或1个单氨基酸添加、替换和/或缺失的序列。
在另一个实施方案中，本文提供的抗体包含与表1和表2中所列的CDR氨基酸序列中之一相差2或1个单氨基酸添加、替换和/或缺失的序列。
在进一步的实施方案中，本文提供的抗体包含与表1和表2中所列的CDR氨基酸序列中之一相差1个单氨基酸添加、替换和/或缺失的序列。
在一个实施方式中，本文提供的APJ抗体包含一个或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链CDR1氨基酸序列：SEQ ID NO:1、SEQ ID NO:4、SEQ ID NO:7、及SEQ ID NO:10；及
b.重链CDR1氨基酸序列：SEQ ID NO:12、SEQ ID NO:15、及SEQ ID NO:18。
在另一个实施方式中，本文提供的APJ抗体包含一个或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链CDR2氨基酸序列：SEQ ID NO:2、SEQ ID NO:5、及SEQ ID NO:8；及
b.重链CDR2氨基酸序列：SEQ ID NO:13、SEQ ID NO:16、SEQ ID NO:19、及SEQ ID NO:21。
在另一个实施方式中，本文提供的APJ抗体包含一个或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链CDR3氨基酸序列：SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:9、及SEQ ID NO:11；及
b.重链CDR3氨基酸序列：SEQ ID NO:14、SEQ ID NO:17、SEQ ID NO:20、及SEQ ID NO:22。
在另一个实施方案中，本文提供的APJ抗体包含一个，两个，三个，或四个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
a.轻链CDR1氨基酸序列：SEQ ID NO:1、SEQ ID NO:4、SEQ ID NO:7、及SEQ ID NO:10；
b.重链CDR1氨基酸序列：SEQ ID NO:12、SEQ ID NO:15、及SEQ ID NO:18；
c.轻链CDR2氨基酸序列：SEQ ID NO:2、SEQ ID NO:5、及SEQ ID NO:8；及
d.重链CDR2氨基酸序列：SEQ ID NO:13、SEQ ID NO:16、SEQ ID NO:19、及SEQ ID NO:21。
在另一个实施方案中，本文提供的APJ抗体包含一个，两个，三个，或四个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
a.轻链CDR1氨基酸序列：SEQ ID NO:1、SEQ ID NO:4、SEQ ID NO:7、及SEQ ID NO:10；
b.重链CDR1氨基酸序列：SEQ ID NO:12、SEQ ID NO:15、及SEQ ID NO:18；
c.轻链CDR3氨基酸序列：SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:9、及SEQ ID NO:11；及
d.重链CDR3氨基酸序列：SEQ ID NO:14、SEQ ID NO:17、SEQ ID NO:20、及SEQ ID NO:22。
在进一步的实施方案中，本文提供的APJ抗体包含一个，两个，三个，或四个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
a.轻链CDR2氨基酸序列：SEQ ID NO:2、SEQ ID NO:5、及SEQ ID NO:8；
b.重链CDR2氨基酸序列：SEQ ID NO:13、SEQ ID NO:16、SEQ ID NO:19、及SEQ ID NO:21；
c.轻链CDR3氨基酸序列：SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:9、及SEQ ID NO:11；及
d.重链CDR3氨基酸序列：SEQ ID NO:14、SEQ ID NO:17、SEQ ID NO:20、 及SEQ ID NO:22。
在一个实施方式中，本文提供的APJ抗体包含一个，两个，或三个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、及SEQ ID NO:11。
在另一个实施方案中，本文提供的APJ抗体包含一个，两个，或三个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、及SEQ ID NO:22。
在一个实施方式中，本文提供的APJ抗体包含一个独立地选自于以下所列的轻链和重链CDR1氨基酸序列的组合：SEQ ID NO:1与SEQ ID NO:12、SEQ ID NO:4与SEQ ID NO:15、SEQ ID NO:7与SEQ ID NO:18、及SEQ ID NO:10与SEQ ID NO:18。
在另一个实施方式中，本文提供的APJ抗体包含一个独立地选自于以下所列的轻链和重链CDR2氨基酸序列的组合：SEQ ID NO:2与SEQ ID NO:13、SEQ ID NO:5与SEQ ID NO:16、SEQ ID NO:8与SEQ ID NO:19、及SEQ ID NO:5与SEQ ID NO:21。
在进一步的实施方式中，本文提供的APJ抗体包含一个独立地选自于以下所列的轻链和重链CDR3氨基酸序列的组合：SEQ ID NO:3与SEQ ID NO:14、SEQ ID NO:6与SEQ ID NO:17、SEQ ID NO:9与SEQ ID NO:20、及SEQ ID NO:11与SEQ ID NO:22。
在一个实施方式中，本文提供的APJ抗体包含：
a.一个独立地选自于以下所列的轻链和重链CDR1氨基酸序列的组合：SEQ ID NO:1与SEQ ID NO:12、SEQ ID NO:4与SEQ ID NO:15、SEQ ID NO:7与SEQ ID NO:18、及SEQ ID NO:10与SEQ ID NO:18；和
b.一个独立地选自于以下所列的轻链和重链CDR2氨基酸序列的组合：SEQ ID NO:2与SEQ ID NO:13、SEQ ID NO:5与SEQ ID NO:16、SEQ ID NO:8与SEQ ID NO:19、及SEQ ID NO:5与SEQ ID NO:21。
在另一个实施方式中，本文提供的APJ抗体包含：
a.一个独立地选自于以下所列的轻链和重链CDR1氨基酸序列的组合：SEQ ID NO:1与SEQ ID NO:12、SEQ ID NO:4与SEQ ID NO:15、SEQ ID NO:7与SEQ ID NO:18、及SEQ ID NO:10与SEQ ID NO:18；和
b.一个独立地选自于以下所列的轻链和重链CDR3氨基酸序列的组合：SEQ ID NO:3与SEQ ID NO:14、SEQ ID NO:6与SEQ ID NO:17、SEQ ID NO:9与SEQ ID NO:20、及SEQ ID NO:11与SEQ ID NO:22。
在另一个实施方式中，本文提供的APJ抗体包含：
a.一个独立地选自于以下所列的轻链和重链CDR2氨基酸序列的组合：SEQ ID  NO:2与SEQ ID NO:13、SEQ ID NO:5与SEQ ID NO:16、SEQ ID NO:8与SEQ ID NO:19、及SEQ ID NO:5与SEQ ID NO:21；和
b.一个独立地选自于以下所列的轻链和重链CDR3氨基酸序列的组合：SEQ ID NO:3与SEQ ID NO:14、SEQ ID NO:6与SEQ ID NO:17、SEQ ID NO:9与SEQ ID NO:20、及SEQ ID NO:11与SEQ ID NO:22。
在进一步实施方式中，本文提供的APJ抗体包含：
a.一个独立地选自于以下所列的轻链和重链CDR1氨基酸序列的组合：SEQ ID NO:1与SEQ ID NO:12、SEQ ID NO:4与SEQ ID NO:15、SEQ ID NO:7与SEQ ID NO:18、及SEQ ID NO:10与SEQ ID NO:18；
b.一个独立地选自于以下所列的轻链和重链CDR2氨基酸序列的组合：SEQ ID NO:2与SEQ ID NO:13、SEQ ID NO:5与SEQ ID NO:16、SEQ ID NO:8与SEQ ID NO:19、及SEQ ID NO:5与SEQ ID NO:21；和
c.一个独立地选自于以下所列的轻链和重链CDR3氨基酸序列的组合：SEQ ID NO:3与SEQ ID NO:14、SEQ ID NO:6与SEQ ID NO:17、SEQ ID NO:9与SEQ ID NO:20、及SEQ ID NO:11与SEQ ID NO:22。
在一个实施方式中，本文所述的抗体包含:
(a)轻链CDR1氨基酸序列：SEQ ID NO:1；
轻链CDR2氨基酸序列：SEQ ID NO:2；
轻链CDR3氨基酸序列：SEQ ID NO:3；
重链CDR1氨基酸序列：SEQ ID NO:12；
重链CDR2氨基酸序列：SEQ ID NO:13；及
重链CDR3氨基酸序列：SEQ ID NO:14；
(b)轻链CDR1氨基酸序列：SEQ ID NO:4；
轻链CDR2氨基酸序列：SEQ ID NO:5；
轻链CDR3氨基酸序列：SEQ ID NO:6；
重链CDR1氨基酸序列：SEQ ID NO:15；
重链CDR2氨基酸序列：SEQ ID NO:16；及
重链CDR3氨基酸序列：SEQ ID NO:17；
(c)轻链CDR1氨基酸序列：SEQ ID NO:7；
轻链CDR2氨基酸序列：SEQ ID NO:8；
轻链CDR3氨基酸序列：SEQ ID NO:9；
重链CDR1氨基酸序列：SEQ ID NO:18；
重链CDR2氨基酸序列：SEQ ID NO:19；及
重链CDR3氨基酸序列：SEQ ID NO:20；
(d)轻链CDR1氨基酸序列：SEQ ID NO:10；
轻链CDR2氨基酸序列：SEQ ID NO:5；
轻链CDR3氨基酸序列：SEQ ID NO:11；
重链CDR1氨基酸序列：SEQ ID NO:18；
重链CDR2氨基酸序列：SEQ ID NO:21；及
重链CDR3氨基酸序列：SEQ ID NO:22。
在另一个实施方式中，本文所述的抗体包含:
轻链CDR1氨基酸序列：SEQ ID NO:10；
轻链CDR2氨基酸序列：SEQ ID NO:5；
轻链CDR3氨基酸序列：SEQ ID NO:11；
重链CDR1氨基酸序列：SEQ ID NO:18；
重链CDR2氨基酸序列：SEQ ID NO:21；及
重链CDR3氨基酸序列：SEQ ID NO:22。
在一个实施方式中，本文提供的APJ抗体，其包含一个或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链可变结构域氨基酸序列：SEQ ID NO:59(L1)、SEQ ID NO:60(L2)、SEQ ID NO:61(L3)、SEQ ID NO:62(L4)、及SEQ ID NO:63(L5)；及与其任一序列有至少80％、至少85％、至少90％、或至少95％相同的氨基酸序列；及
b.重链可变结构域氨基酸序列：SEQ ID NO:64(H1)、SEQ ID NO:65(H2)、SEQ ID NO:66(H3)、SEQ ID NO:67(H4)、及SEQ ID NO:68(H5)；及与其任一序列有至少80％、至少85％、至少90％、或至少95％相同的氨基酸序列。
在另一个实施方式中，本文提供的APJ抗体的多聚核苷酸编码序列包含一个或两个多聚核苷酸编码序列，其中每个多聚核苷酸编码序列独立地选自于以下所列多聚核苷酸编码序列：
a.轻链可变结构域多聚核苷酸编码序列：SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、及SEQ ID NO:73；及与其任一序列有至少80％、至少85％、至少90％、或至少95％相同的多聚核苷酸编码序列；及
b.重链可变结构域多聚核苷酸编码序列：SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77、及SEQ ID NO:78；及与其任一序列有至少80％、至少85％、至少90％、或至少95％相同的多聚核苷酸编码序列。
在一个实施方式中，本文提供的APJ抗体包含一个独立地选自于以下所列的氨基酸序列：SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、及SEQ ID NO:63。
在另一个实施方式中，本文提供的APJ抗体包含一个独立地选自于以下所列的氨基酸序列：SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、及SEQ ID NO:68。
在一个实施方式中，本文提供的APJ抗体包含一个独立地选自于以下所列的轻链与重链可变结构域氨基酸序列的组合：SEQ ID NO:59与SEQ ID NO:64(L1H1)、SEQ ID NO:60与SEQ ID NO:65(L2H2)、SEQ ID NO:61与SEQ ID NO:66(L3H3)、SEQ ID NO:62与SEQ ID NO:67(L4H4)、及SEQ ID NO:63与SEQ ID NO:68(L5H5)。
本文也可用“LxHy”符号来指代所提供的APJ抗体，其中“x”对应于轻链可 变区序列代号；“y”对应于重链可变区序列代号。例如，L2H2指具有包含SEQ ID NO:60(L2)氨基酸序列的轻链可变区和包含SEQ ID NO:65(H2)氨基酸序列的重链可变区的完整抗体。
在一个实施方式中，本文提供的APJ抗体包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
a.轻链恒定氨基酸序列：SEQ ID NO:79、SEQ ID NO:80、及SEQ ID NO:81；及
b.重链恒定氨基酸序列：SEQ ID NO:82、SEQ ID NO:83、及SEQ ID NO:84。
在另一个实施方式中，本文提供的APJ抗体包含一个独立地选自于以下所列的轻链和重链恒定氨基酸序列的组合：SEQ ID NO:79和SEQ ID NO:82、SEQ ID NO:80和SEQ ID NO:83、SEQ ID NO:80和SEQ ID NO:84、SEQ ID NO:81和SEQ ID NO:83、及SEQ ID NO:81和SEQ ID NO:84。
在一个实施方案中，本文提供的APJ抗体包含本文所列轻链及重链CDRs，以及FRs(框架)的氨基酸序列。FRs的氨基酸序列包含于轻链或者重链可变结构域氨基酸序列中，未单独陈列。在一个实施方案中，该抗体包含一个本文所列的轻链CDR1序列。在另一个实施方案中，该抗体包含一个本文所列的轻链CDR2序列。在另一个实施方案中，该抗体包含一个本文所列的轻链CDR3序列。在另一个实施方案中，该抗体包含一个本文所列的重链CDR1序列。在另一个实施方案中，该抗体包含一个本文所列的重链CDR2序列。在另一个实施方案中，该抗体包含一个本文所列的重链CDR3序列。在另一个实施方案中，该抗体包含一个本文的轻链FR1序列。在另一个实施方案中，该抗体包含一个本文的轻链FR2序列。在另一个实施方案中，该抗体包含一个本文轻链的FR3序列。在另一个实施方案中，该抗体包含一个本文的轻链FR4序列。在另一个实施方案中，该抗体包含一个本文的重链FR1序列。在另一个实施方案中，该抗体包含一个本文重链的FR2序列。在另一个实施方案中，该抗体包含一个本文的重链FR3序列。在进一步的实施方案中，该抗体包含一个本文的重链FR4序列。
在一个实施方案中，该抗体的轻链CDR3序列与本文所列轻链CDR3氨基酸序列SEQ ID NO:11相差不得超过6、5、4、3、2、或1个单氨基酸添加、替换和/或缺失。在另一个实施方案中，该抗体的重链CDR3序列与本文所列重链CDR3氨基酸序列SEQ ID NO:22相差不得超过6、5、4、3、2、或1个单氨基酸添加、替换和/或缺失。在进一步的实施方案中，该抗体轻链CDR3序列与本文所列轻链CDR3氨基酸序列SEQ ID NO:11相差不得超过6、5、4、3、2、或1个单氨基酸添加、替换和/或缺失，并且该抗体的重链CDR3序列与本文所列重链CDR3氨基酸序列SEQ ID NO:22相差不得超过6、5、4、3、2、或1个单氨基酸添加、替换和/或缺失。
在一个实施方案中，本文提供的APJ抗体包含一个轻链可变结构域氨基酸序列，该序列为本文所列的L4(SEQ ID NO:62)或L5(SEQ ID NO:63)轻链可变结构域序列。在一个实施方案中，该APJ抗体的轻链可变结构域氨基酸序 列与L4(SEQ ID NO:62)或L5(SEQ ID NO:63)的轻链可变结构域氨基酸序列存在15、14、13、12、11、10、9、8、7、6、5、4、3、2、或1个氨基酸的差异，其中各序列的差异独立为一个氨基酸残基的缺失、插入或替换。在另一个实施方案中，该APJ抗体的轻链可变结构域氨基酸序列与L4(SEQ ID NO:62)或L5(SEQ ID NO:63)的轻链可变结构域氨基酸序列有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、或至少99％相同。在另一个实施方案中，该APJ抗体的轻链可变结构域多聚核苷酸编码序列包含与L4(SEQ ID NO:72)或L5(SEQ ID NO:73)的多聚核苷酸编码序列有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、或至少99％相同。在另一个实施方案中，该APJ抗体的轻链可变结构域多聚核苷酸编码序列包含在中等条件下与L4(SEQ ID NO:72)或L5(SEQ ID NO:73)的轻链可变结构域的多聚核苷酸编码序列互补序列杂交的多聚核苷酸序列。在进一步的实施方案中，该APJ抗体的轻链可变结构域多聚核苷酸编码序列包含在严格条件下与L4(SEQ ID NO:72)或L5(SEQ ID NO:73)的轻链可变结构域的多聚核苷酸编码序列互补序列杂交的多聚核苷酸序列。
在一个实施方案中，本文提供的APJ抗体包含一个重链可变结构域氨基酸序列，该序列为本文所列H4(SEQ ID NO:67)或H5(SEQ ID NO:68)重链可变结构域序列。在另一个实施方案中，该APJ抗体的重链可变结构域氨基酸序列与H4(SEQ ID NO:67)或H5(SEQ ID NO:68)的重链可变结构域序列存在15、14、13、12、11、10、9、8、7、6、5、4、3、2、或1个氨基酸差异，其中各序列的差异独立为一个氨基酸残基的缺失、插入或替换。在另一个实施方案中，该APJ抗体的重链可变结构域氨基酸序列与H4(SEQ ID NO:67)或H5(SEQ ID NO:68)的重链可变结构域序列有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、或至少99％相同。在另一个实施方案中，该APJ抗体的重链可变结构域多聚核苷酸编码序列与H4(SEQ ID NO:77)或H5(SEQ ID NO:78)的重链可变结构域多聚核苷酸编码序列有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、或至少99％相同。在另一个实施方案中，该APJ抗体的重链可变结构域多聚核苷酸编码序列包含在中等严格条件下与H4(SEQ ID NO:77)或H5(SEQ ID NO:78)的重链可变结构域的多聚核苷酸编码序列互补序列杂交的多聚核苷酸序列。在一个实施方案中，该APJ抗体的重链可变结构域多聚核苷酸编码序列包含在严格条件下与H4(SEQ ID NO:77)或H5(SEQ ID NO:78)的重链可变结构域的多聚核苷酸编码序列互补序列杂交的的多聚核苷酸序列。
在一个实施方案中，本文提供的抗体是一包含L1H1(SEQ ID NO:59和SEQ ID NO:64)、L2H2(SEQ ID NO:60和SEQ ID NO:65)、L3H3(SEQ ID NO:61和SEQ ID NO:66)、L4H4(SEQ ID NO:62和SEQ ID NO:67)、或L5H5(SEQ ID NO:63和SEQ ID NO:68)组合的抗体，或其一的期望表型(例如，IgA、IgG1、IgG2a、IgG2b、IgG3、IgM、IgE、或IgD)，或其一Fab或F(ab') 2片段。
在一个实施方案中，本文提供的抗体是一包含L4H4(SEQ ID NO:62和SEQ ID NO:67)或L5H5(SEQ ID NO:63和SEQ ID NO:68)组合的抗体，或其一类转换的抗体(例如，IgA、IgG1、IgG2a、IgG2b、IgG3、IgM、IgE、和IgD)，或其一Fab或F(ab') 2片段。
本文提供的抗体可包含本领域已知的恒定区中任何一个。轻链恒定区可为例如κ或λ型轻链恒定区，例如小鼠κ或λ型轻链恒定区。重链恒定区可为例如α、δ、ε、γ、或μ型重链恒定区，例如小鼠α、δ、ε、γ、或μ型重链恒定区。在一个实施方案中，该轻链或重链恒定区为天然恒定区的片段、衍生物、变异体、或突变蛋白。
在一个实施方案中，本文提供的抗体进一步包含人恒定轻链κ或其片段。轻链恒定区的氨基酸序列如下：
人恒定轻链κ结构域氨基酸序列：SEQ ID NO:80；及
人恒定轻链λ结构域氨基酸序列：SEQ ID NO:81。
在另一个实施方案中，本文提供的抗体进一步包含人重链恒定结构域或其片段。重链恒定区的氨基酸序列如下：
人重链恒定区氨基酸序列(hIgG2)：SEQ ID NO:83；及
人重链恒定区氨基酸序列(hIgG4)：SEQ ID NO:84。
在一个实施方式中，本文提供的APJ抗体的重轻链氨基酸序列分别为：SEQ ID NO:157及SEQ ID NO:158。
在一个实施方式中，本文所提供的APJ抗体选自鼠源抗体、人源化抗体、嵌合抗体、单克隆抗体、多克隆抗体、重组抗体、抗原结合抗体片段、单链抗体、双链抗体、三链抗体、四链抗体、Fab片段、F(ab’)x片段、结构域抗体、IgD抗体、IgE抗体、IgM抗体、IgGl抗体、IgG2抗体、IgG3抗体、或IgG4抗体。
在一个实施方式中，本文提供的APJ抗体为APJ单克隆抗体。
在另一个实施方式中，本文提供的APJ抗体为一单克隆抗体,该单克隆抗体包含一个选自于以下所列的氨基酸序列的组合：SEQ ID NO:59与SEQ ID NO:64、SEQ ID NO:60与SEQ ID NO:65、SEQ ID NO:61与SEQ ID NO:66、SEQ ID NO:62与SEQ ID NO:67、及SEQ ID NO:63与SEQ ID NO:68。
在一个实施方式中，本文提供的APJ抗体为自鼠源APJ抗体。在另一个实施方式中，本文提供的APJ抗体为人源化APJ抗体。
在一个实施方式中，本文提供的APJ抗体的Kd值为大约1nM至大约200nM或大约1nM至大约100nM。
抗体和抗体片段
在一个实施方案中，本文提供的抗体为完整抗体(包括具有全长重和/或轻链的多克隆、单克隆、嵌合、人源化、或人类抗体)。在另一个实施方案中，本文提供的抗体为抗体片段，列如F(ab’) 2、Fab、Fab’、Fv、Fc、或Fd片段，单结构域抗体、单链抗体、最大抗体(maxibodies)、微抗体(minibodies)、内抗体(intrabodies)、二链抗体、三链抗体、四链抗体、v-NAR、或bis-scFv(参见， 例如Hollinger and Hudson,2005,Nature Biotechnology 23:1126-36)。在另一个实施方案中，本文提供的抗体也包括抗体包括如美国专利号6703199中所公开的抗体多肽，包括纤维结合素多肽单抗体。在进一步的实施方案中，本文提供的抗体包括如美国专利出版物2005/0238646中所公开的单链多肽。
在一个实施方案中，单克隆抗体的基因的可变区是使用核苷酸引物扩增在杂交瘤中表达。这些引物可由本领域普通技术人员合成或从商业来源购买。鼠和人可变区引物包括V Ha、V Hb、V Hc、V Hd、C H1、V L、和C L区的引物可从商业来源购买。这些引物可用于扩增重链或轻链可变区，然后将其分别插入载体例如IMMUNOZAP TMH或IMMUNOZAP TML(Stratagene)中。然后将这些载体引入大肠杆菌、酵母或哺乳动物为基础的表达系统。这些方法可使用于生产大量包含V H和V L结构域融合的单链蛋白(参见Bird等,1988,Science 242:423-6)。
本领域技术人员应理解的是一些蛋白质，例如抗体，可能进行可多种转录后修饰。这些修饰的类型和程度取决于用于表达该蛋白的宿主细胞系以及培养条件。该类修饰包括糖基化作用、甲硫氨酸氧化、二酮哌嗪形成、天冬氨酸异构化和天冬酰胺脱酰胺作用的变化。抗体的羧端碱性残基(例如赖氨酸或精氨酸)因羧肽酶的频繁修饰作用而可能丢失(参见，Harris,1995,Journal of Chromatography705:129-34)。
鼠单克隆抗体可使用常用的杂交瘤细胞方法来生产。该单克隆抗体可通过多种已确立的技术分离和纯化。该类分离技术包括使用蛋白A-琼脂糖的亲和色谱法、分子排阻色谱法和离子交换色谱法(参见，例如，Coligan第2.7.1-2.7.12页和第2.9.1-2.9.3页；Baines等，“Purification of Immunoglobulin G(IgG),”Methods in Molecular Biology，第10卷，第79-104页(The Humana Press，Inc.，1992))。该单克隆抗体可使用基于抗体的特殊性质(例如，重链或轻链同种型、结合特异性等)筛选的适当配基通过亲和色谱法来纯化。亲和色谱的适当配基的实例包括蛋白A、蛋白G、抗恒定区(轻链或重链)抗体、抗独特型抗体以及TGF-结合蛋白或其片段或变异体。
可使用抗体结合位点中央的互补决定区(CDRs)对分子进行亲和力成熟化的改造，来得到亲和性增加的抗体，例如针对c-erbB-2的亲和性增加的抗体(Schier等，1996，J.Mol.Biol.263:551-67)。因此，该类技术可用于制备人APJ的抗体。
例如可在检测是否存在人APJ的体外或体内测定法中使用针对人APJ的抗体。
也可通过任何传统技术制备抗体。例如，可从天然表达这些抗体的细胞将其纯化(例如，可从生产抗体的杂交瘤将其纯化)或使用本领域任何已知的技术在重组表达系统中生产。参见，例如，Monoclonal Antibodies,Hybridomas:A New Dimension in Biological Analyses,Kennet等编辑，Plenum Press(1980)；和Antibodies:A Laboratory Manual,Harlow and Land编辑,Cold Spring Harbor Laboratory Press(1988)。这在下文的核酸部分讨论。
可通过任何已知技术制备抗体并筛选期望性质。一些技术涉及分离编码相关抗体(例如，抗APJ抗体)的多肽链(或其部分)的核酸，并通过重组DNA技术操作核酸。该核酸可与另一相关核酸融合或经修饰(例如通过诱变或其它传统技术)以添加、缺失或替换一个或多个氨基酸残基。
当需要提高根据本文包含一个或多个上述CDRs的抗体的亲和性时，可通过多种亲和成熟方案包括维持CDRs(Yang等,1995,J.Mol.Biol.254:392-403)、链替换(Marks等,1992,Bio/Technology 10:779-83)、使用大肠杆菌的突变株(Low等,1996,J.Mol.Biol.250:350-68)DNA重排(Patten等,1997,Curr.Opin.Biotechnol.8:724-33)、噬菌体展示(Thompson等,1996,J.Mol.Biol.256:7-88)以及其它PCR技术(Crameri等,1998,Nature 391:288-91)。所有这些亲和力成熟方法讨论于Vaughan等,1998,Nature Biotechnology 16:535-39中。
在一个实施方案中，本文提供的抗体为抗APJ片段。该片段可完全由抗体衍生序列组成或可包含附加序列。抗原结合片段的实例包括Fab、F(ab’) 2、单链抗体、双链抗体、三链抗体、四链抗体和结构域抗体，其它实例提供于Lunde等,2002,Biochem.Soc.Trans.30:500-06。
可经氨基酸桥(短肽接头)连接重链和轻链可变结构域(Fv区)形成单链抗体，从而得到单多肽链。已通过将编码肽接头的DNA融合在编码两个可变结构域多肽(V L和V H)的DNAs之间制备该单链Fvs(scFvs)。所得多肽可折叠回自身形成抗原结合单体，或它们可形成多聚体(例如，二聚体、三聚体或四聚体)，取决于两个可变结构域之间的柔性接头的长度(Kortt等,1997,Prot.Eng.10:423；Kortt等2001,Biomol.Eng.18:95-108)。通过组合包含多肽的不同V L和V H，可形成与不同表型结合的多体scFvs(Kriangkum等,2001,Biomol.Eng.18:31-40)。已研发的用于生产单链抗体的技术包括美国专利号4946778；Bird,1988,Science242:423；Huston等,1988,Proc.Natl.Acad.Sci.USA 85:5879-83；Ward等,1989,Nature 334:544-6；de Graaf等,2002,Methods Mol Biol.178:379-87中描述的那些。来源于本文提供的抗体的单链抗体包括但不限于包含可变结构域组合L1H1的scFvs，均涵盖于本文。
也可通过抗体的蛋白水解作用例如根据传统方法用胃蛋白酶或木瓜蛋白酶消化完整的抗体获得来源于抗体的抗原结合片段。举例而言，可用胃蛋白酶裂解抗体提供称作F(ab’) 2的SS片段生产抗体片段。可使用巯基还原剂进一步裂解这一片段产生3.5S Fab’单价片段。可选择的方案有，使用巯基保护基团进行该裂解反应得到二硫键的裂解；另外还可以使用木瓜蛋白酶的酶裂解直接产生两个单价Fab片段和一个Fc片段。这些方法描述于例如Goldenberg，美国专利号4,331,647，Nisonoff等,1960,Arch.Biochem.Biophys.89:230；Porter,1959,Biochem.J.73:119；Edelman等,Methods in Enzymology l:422(Academic Press，1967)；以及Andrews和Titus,J.A.Current Protocols in Immunology(Coligan等编辑，John Wiley&Sons,2003)，第2.8,1-2.8.10页和第2.10A.1-2.10A.5页。其它裂解抗体的方法，例如制备重链以形成单价重、轻链片段(Fd)，进一步裂解片段或也可使 用其它酶、化学或基因技术，只要片段与可被该完整抗体识别的抗原结合。
另一种形式的抗体片段为包含一个或多个抗体互补决定区(CDRs)的肽。可通过构建编码相关CDR的多肽获得CDRs。例如可通过使用聚合酶链式反应用抗体生成细胞的mRNA作为模板合成可变区制备该类多肽，参见，例如，Larrick等,1991,Methods:A Companion to Methods in Enzymology 2:106；Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,”Monoclonal Antibodies:Production,Engineering and Clinical Application,Ritter等编辑,166页(Cambridge University Press,1995)；和Ward等，“Genetic Manipulation and Expression of Antibodies,”Monoclonal Antibodies:Principles and Applications,Birch等编辑,137页(Wiley-Liss,Inc.,1995)。该抗体片段可进一步包含本文所述抗体的至少一个可变结构域。因此，例如，V区结构域可为单体并且是V H或V L结构域，其可以如下文所述的至少等于1x 10 -7M或更高的亲和力独立与APJ结合。
该可变区结构域可为任何天然可变结构域或其基因工程形式。基因工程形式指使用重组DNA工程技术生产的可变区结构域。该基因工程形式包括例如通过向特异抗体的氨基酸序列插入、缺失或改变从特异抗体可变区产生的。具体实例包括包含只含一个CDR以及任选来自一个抗体的一个或多个框架氨基酸和来自另一抗体的可变区结构域剩余部分，并由基因工程组装成的可变区结构域。
可变区结构域可与至少一个其它抗体结构域或其片段在C端氨基酸共价连接。因此，举例而言，存在于可变区结构域的V H结构域可与免疫球蛋白C H1结构域或其片段相连。相似地，V L结构域可与C K结构域或其片段相连。以这种方式，例如，该抗体可为Fab片段，其中抗原结合结构域包含它们的C端分别与C H1和C K结构域共价连接的联合V H和V L结构域。可用其它氨基酸延长C H1结构域，例如以提供铰链区或如Fab’片段中的部分铰链结构域或提供其它结构域，例如抗体C H2和C H3结构域。
抗体的衍生物和变异体
例如可通过随机诱变或通过定点诱变(例如寡聚核苷酸诱导的定点诱变)改变编码对应于氨基酸序列L1和H1的核苷酸序列以产生与未突变多聚核苷酸相比包含一个或多个具体核苷酸替换、缺失或插入的经改变的多聚核苷酸。用于产生该类改变的技术实例描述于Walder等,1986,Gene 42:133；Bauer等,1985,Gene37:73；Craik,1985,BioTechniques 3:12-9；Smith等,1981,Genetic Engineering:Principles and Methods,Plenum Press；以及美国专利号4518584和4737462。这些和其它方法可用于产生例如与未衍生化抗体相比具有期望性质例如亲和性、亲和力或对APJ的特异性增强、体内或体外活性或稳定性增强或体内副作用降低的抗APJ抗体的衍生物。
本文领域的其它抗APJ抗体衍生物包括抗APJ抗体或其片段与它蛋白或多肽的共价或聚集结合物，例如通过表达包含与抗APJ抗体多肽的N端或C端融合的异源多肽的重组融合蛋白。例如，该结合肽可为异源信号(或引导)多肽，例如酵母α因子前导肽或例如表位标签的肽。包含融合蛋白的抗体可包含被添加 以辅助抗体的纯化或鉴定的肽(例如多聚组氨酸)。抗体也可与FLAG肽连接，如Hopp等,1988,Bio/Technology 6:1204和美国专利5011912所述。FLAG肽具有高抗原性并提供被特异单克隆抗体(mAb)可逆结合的表位，允许已表达重组蛋白的快速检测和方便纯化。可商业购买(Sigma-Aldrich，St.Louis，MO)用于制备其中FLAG肽与给定多肽融合的融合蛋白的试剂，在另一个实施方案中，包含一个或多个抗体的寡聚体可用作APJ拮抗剂或用更高级的寡聚体。寡聚体可以是共价连接的或非共价连接的二聚体、三聚体或更高的寡聚体形式。可使用包含两个或更多个抗体的寡具体，其中一个实例为同型二聚体。其它寡聚体包括异二聚体、同型三聚体、异三聚体、同型四聚体、杂四聚体等。
一个实施方案是针对包含多个抗体的寡聚体，它们通过与抗体融合的肽部分之间的共价或非共价相互作用连接。该类肽可为肽接头(spacers)或具有促进寡聚化作用性质的肽。亮氨酸拉链和某些来源于抗体的多肽为可促进抗体寡聚化的肽，如下文详细描述。
在具体的实施方案中，寡聚体包含两个至四个抗体。寡聚体的抗体可为任何形式，如上文所述任何形式，例如变异体或片段。优选地，该寡聚体包含具有APJ结合活性的抗体。
在一个实施方案中，使用来源于免疫球蛋白的多肽制备寡聚体。制备包含一些与抗体衍生多肽(包括Fc结构域)的不同部位融合的异源多肽已描述于例如Ashkenazi等,1991,Proc.Natl.Acad.Sci.USA 88:10535；Byrn等,1990,Nature344:677；和Hollenbaugh等,Construction of Immunoglobulin Fusion Proteins,Current Protocols in Immunology,Suppl.4,第10.19.1-10.19.11页。本文的一个实施方案是针对包含两个由融合抗APJ抗体的Elabela片段或其突变体结合片段与抗体的Fc区产生的融合蛋白的二聚体。可通过以下方式制备二聚体：例如在适当的表达载体中插入编码融合蛋白的基因融合，在用重组表达载体转化的宿主细胞中表达该融合基因并允许已表达融合蛋白像抗体分子一样组装，其中Fc部分之间的链间二硫键形成二聚体。
如本文所使用术语“Fc多肽”包括来源于抗体Fc区的天然和突变蛋白形式的多肽。也包括包含促进二聚体化的铰链区的该类多肽的截短形式。包含Fc部分(以及由其形成的寡聚体)的融合蛋白提供了在蛋白A或蛋白G柱子上进行亲和层析法方便纯化的优势。
PCT申请W0 93/10151(以参考形式并于本文)中一种适当的Fc多肽为从N端铰链区延伸至人IgG1抗体的Fc区的天然C端的单链多肽。另一种可用的Fc多肽为美国专利5457035和Baum等,1994,EMBO J.13:3992-4001中描述的Fc突变蛋白。该突变蛋白的氨基酸序列与W0 93/10151中所示天然Fc序列的氨基酸序列相同，除了氨基酸19从亮氨酸变为丙氨酸，氨基酸20从亮氨酸变为谷氨酰胺以及氨基酸22从甘氨酸变为丙氨酸。该突变蛋白表现出对Fc受体的亲和力降低。在其它实施方案中，抗APJ抗体的重链和/或轻链可被取代为抗体重链和/或轻链的可变部分。
或者，该寡聚体为包含多个抗体的融合蛋白，包含或不包含接头肽(spacer peptides)。这些适当的接头肽描述于美国专利4751180和4935233。
制备寡聚抗体的另一种方法涉及使用亮氨酸拉链。亮氨酸拉链结构域为促进它们所存在的蛋白寡聚化作用的肽。最初发现亮氨酸拉链存在于数种DNA结合蛋白中(Landschulz等,1988,Science 240:1759)，此后发现存在于各种不同蛋白中。在已知的亮氨酸拉链中为可二聚体化或三聚体化的天然肽或其衍生物。适用于生产可溶寡聚蛋白的亮氨酸拉链结构域的实例描述于PCT申请W0 94/10308，来源于肺表面活性蛋白D(SPD)的亮氨酸拉链描述于Hoppe等,1994,FEBS Letters 344:191，以参考形式并于本文。允许与其融合的异源蛋白稳定三聚体化的经修饰的亮氨酸拉链的使用描述于Fanslow等,1994,Semin.Immunol.6:267-78。在一种方法中，在适当的宿主细胞中表达包含与亮氨酸拉链肽融合的抗APJ抗体片段或衍生物的重组融合蛋白，从培养物上清中收集可溶寡聚抗APJ抗体片段或其衍生物。
在另一个实施方案中，该抗体衍生物可包含至少本文公开的CDRs之一。举例而言，可将一个或多个CDR整合入已知的抗体骨架区(IgG1，IgG2等)或与适当的载体结合以增强其半衰期。适当载体包括但不限于Fc、白蛋白、转铁蛋及类似物质。这些和其它适当的载体为本领域已知。该结合CDR肽可为单体、二聚体、四聚体或其它形式。在一个实施方案中，一个或多个水溶性多聚体在结合剂的一个或多个特异位点结合，例如在氨基端。在一个实例中，抗体衍生物包含一个或多个水溶性多聚体附着物包括但不限于聚乙二醇、聚氧乙烯二醇或聚丙二醇。参见，例如，美国专利号4640835、4496689、4301144、4670417、4791192和4179337，在一些实施方案中，衍生物包含一个或多个一甲氧基.聚乙二醇、葡聚糖、纤维素或其它基于碳水化合物的聚合物，聚(N-乙烯基吡咯酮).聚乙二醇、聚氧乙烯多元醇(例如甘油)和聚乙烯醇，以及该类聚合物的混合物。在一些实施方案中，一个或多个水溶性聚合物与一个或多个侧链随机结合。在一些实施方案中。PEG可提高结合剂例如抗体的治疗作用。一些该类方法描述于例如美国专利号6133426，其以参考形式以任何目的并于本文。
应当理解的是本文提供的抗体可具有至少一个氨基酸替换，只要该抗体保留了结合特异性。因此，抗体结构的修饰包含于本文范畴。这些可包括不破坏抗体APJ结合能力的氨基酸替换，其可为保守或非保守的。保守氨基酸替换可包括非天然氨基酸残基，其通常经化学肽合成整合而不是生物系统合成。这些包括拟肽和其它反向或倒转形式的氨基酸部分。保守氨基酸替换也可涉及用非天然残基替换天然氨基酸残基这样对该位点氨基酸残基的极性或电荷作用很小或没有作用。非保守替换可涉及一类氨基酸或氨基酸类似物的一个成员与具有不同物理性质(例如，体积、极性、疏水性、电荷)的另一类氨基酸的成员交换。
而且，本领域技术人员可生成在各期望氨基酸残基上包含氨基酸替换的待测变异体。可使用本领域技术人员已知的活性测定法筛选该类变异体。该类变异体可用于收集关于适当变异体的信息。举例而言，如果发现某一氨基酸残基可引起 活性破坏、非期望的降低或不当的活性，可避免具有该类变化的变异体。换言之，基于从这些常规试验收集的信息，本领域技术人员可轻松确定应避免进一步替换(单独或与其它突变组合)的氨基酸。
技术人员可使用已知技术确定如本文所列的多肽的适当变异体。在一些实施方案中，本领域技术人员可通过靶向对于活性不重要的区域鉴定经改变后不会破坏活性的分子适当区域。在一些实施方案中，可鉴定在相似多肽中保守的残基或分子部分。在一些实施方案中，甚至可保守替换对于生物活性或结构重要的区域而不破坏生物活性或不会有不利作用于多肽结构。此外，本领域技术人员可考察结构.功能研究鉴定对活性或结构重要的相似多肽中的残基。鉴于这一对比，可预测对应于在相似蛋白中对活性或结构重要的氨基酸残基的蛋白质中氨基酸残基的重要性。本领域技术人员可为这些经预测重要的氨基酸残基选择化学相似氨基酸替换。
本领域技术人员也可分析与相似多肽的结构相关的三维结构和氨基酸序列。鉴于该类信息，本领域技术人员可预测就三维结构而言抗体的氨基酸残基比对。在一些实施方案中，本领域技术人员可选择不对经预测在蛋白质表面的氨基酸残基进行显著改变，因为该类残基可能参与与其它分子的重要相互作用。许多科学出版物致力于二级结构的预测。参见，Moult,1996,Curr.Op.Biotech.7:422-7；Chou等,1974,Biochemistry 13:222-45；Chou等,1974,Biochemistry 113:211-22；Chou等,1978,Adv.Enzymol.Relat.Areas Mol.Biol.47:45-148；Chou等,1979,Ann.Rev.Biochem.47:251-76和Chou等,Biophys.J.26:367-84。此外，目前可使用计算机程序辅助预测二级结构。举例而言，序列同一性大于30％或相似性大于40％的两个多肽或蛋白质通常具有相似的高级结构。近期蛋白结构数据库(PDB)的增长增强了二级结构的可预测性，包括多肽或蛋白结构中潜在的折叠数量。参见，Holm等,1999,Nucl.Acid.Res.27:244-7。已表明(Brenner等,1997,Curr.Op.Struct.Biol.7:369-76)在给定多肽或蛋白质中存在有限数量的折叠并且一旦确定了临界数量的结构，结构预测将变得显著更加精确。
预测二级结构的其它方法包括“穿接(threading)”(Jones,1997,Curr.Opin.Struct.Biol.7:377-87；Sippl等,1996,Structure 4:15-9)、“图谱分析(profile analysis)”(Bowie等,1991,Science 253:164-70；Gribskov等,1990,Meth.Enzym.183:146-59；Gribskov等,1987,Proc.Nat.Acad.Sci.USA 84:4355-8)和“进化联系(evolutionary linkage)”(参见Holm,supra(1999),and Brenner,supra(1997))。在一些实施方案中，抗体变异体包括糖基化变异体，其中与母体多肽的氨基酸序列相比改变了糖基化位点的数量和/或类型。在一些实施方案中，变异体与天然蛋白质相比具有更多或更少数量的N连接糖基化位点。或者，去除该序列的替换可移除现有的N连接糖链。也提供了N连接糖链的重排，其中去除了一个或多个N连接糖链位点(通常为天然存在的那些)并创造了一个或多个新的N连接位点。其它优选抗体变异体包括半胱氨酸变异体，与母体氨基酸序列相比其中缺失或由另一氨基酸(例如丝氨酸)替换一个或多个半胱氨酸残基。当抗体必须折叠成生物 活性构象时(例如在分离可溶包涵体之后)可用半胱氨酸变异体。半胱氨酸变异体通常比天然蛋白质具有较少的半胱氨酸残基，并通常具有偶数个半胱氨酸以最小化未配对半胱氨酸引起的相互作用。
本领域技术人员可在需要该类替换时确定期望的氨基酸替换(保守或非保守)。在一些实施方案中，氨基酸替换可用于鉴定人APJ抗体的重要残基或者增加或降低本文所述人APJ抗体的亲和力。
根据一些实施方案，优选的氨基酸替换为以下：(1)降低蛋白质水解敏感性，(2)降低氧化敏感性，(3)改变形成蛋白质复合物的结合亲和力，(4)改变结合亲和力和/或(5)赋予或修饰该类多肽上的其它物理化学或功能性质。根据一些实施方案，可在天然存在序列中(在一些实施方案中，在形成分子间接触的结构域之外的多肽部分)进行单个或多个氨基酸替换(在一些实施方案中为保守氨基酸替换)。在一些实施方案中，保守氨基酸替换通常不会本质上改变母体序列的结构特性(例如，替换氨基酸不应破解存在于母体序列中的螺旋或干扰特征化母体序列的其它类型二级结构)。本领域认可的多肽二级和三级结构的实例描述于Proteins,Structures and Molecular Principles,Creighton编辑,W.H.Freeman and Company(1984)；Introduction to Protein Structure,Branden and Tooze编辑,Garland Publishing(1991)；以及Thornton等,1991,Nature 354:105，其以参考形式并于本文。
在一些实施方案中，本文提供的抗体可与多聚体、脂类或其它部分(moieties)化学键合。
抗原结合试剂可包含至少一个本文描述的CDRs，其掺入生物相容性骨架结构中。在一个实例中，该生物相容性骨架结构包含足以形成构象稳定结构支持或骨架或支架的多肽或其部分，其可在局限的表面区域展示可与抗原结合的一个或多个氨基酸序列(例如，CDRs、可变区等)。该类结构可为天然存在多肽或多肽“折叠”(结构基序)，或相对与天然多肽或折叠可具有一个或多个修饰，例如氨基酸添加、缺失或替换。这些支架可来源于任何物种(或多于一个物种)的多肽，例如，人类、其它哺乳动物、其它脊椎动物、无脊椎动物、细菌或病毒。
生物可溶性骨架结构通常是基于蛋白质支架或骨架而不是免疫球蛋白结构域。举例而言，可使用基于纤维结合素、锚蛋白、脂质运载蛋白(lipocalin)、新抑癌蛋白、细胞色素b、CP1锌指蛋白、PST1、卷曲螺旋、LACI-D1、Z结构域和淀粉酶抑肽结构域(参见，例如，Nygren和Uhlen,1997,Current Opinion in Structural Biology 7:463-9)。
此外，本领域技术人员可认识到适当的结合剂包括这些抗体的部分，例如一个或多个重链CDR1、CDR2、CDR3，轻链CDR1、CDR2和CDR3，如本文所具体公开。至少一个重链CDR1、CDR2、CDR3、CDR1，CDR2和CDR3区具有至少一个氨基酸替换，只要该抗体保留了非替换CDR的结合特异性。该抗体的非CDR部分可为非蛋白分子，其中该结合剂交叉阻断本文公开的抗体与人APJ的结合。该抗体的非CDR部分可为非蛋白质分子，其中该抗体在竞争结合测定 法中显示出与抗体L4H4所显示相似的与人Elabela肽的结合类型。抗体的非CDR部分可由氨基酸组成，其中该抗体为重组结合蛋白或合成肽，并且该重组结合蛋白交叉阻断本文公开的抗体与人APJ的结合。抗体的非CDR部分可由氨基酸组成，其中该抗体为重组抗体，并且该重组抗体在竞争结合测定法中显示出与至少抗体L4H4所显示相似的与人APJ肽的结合类型。
APJ抗体与Elabela的融合蛋白质(Elabela融合蛋白质)
在一个实施方案中，本文提供了一个Elabela融合蛋白质，其包含本文提供的一个APJ抗体和一个Elabela片段。
在另一个实施方案中，本文提供了一个Elabela融合蛋白质，其包含本文提供的一个APJ抗体，和一个，二个，三个，四个，五个，六个，七个，或八个Elabela片段和肽接头(Linker)；该融合蛋白质通过一肽接头序列(Linker)将一Elabela片段的氨基端与一APJ抗体轻链或重链的羧基端连接，其中每个Elabela片段各自独立地为一正向Elabela片段或其突变体；或者该融合蛋白质通过一肽接头序列(Linker)将一Elabela片段的羧基端与一APJ抗体轻链或重链的氨基端连接，其中每个Elabela片段各自独立地为一反向Elabela片段或其突变体。
在另一个实施方案中，本文提供了一个Elabela融合蛋白质，其包含本文提供的一个APJ抗体，和一个，二个，三个，或四个Elabela片段和肽接头(Linker)；该融合蛋白质通过一个肽接头序列(Linker)将一Elabela片段的氨基端与一APJ抗体轻链或重链的羧基端连接，其中每个Elabela片段各自独立地为一正向Elabela片段或其突变体。
在另一个实施方案中，本文提供了一个Elabela融合蛋白质，其包含本文提供的一个APJ抗体，和一个，二个，三个，或四个Elabela片段和肽接头(Linker)；该融合蛋白质通过一个肽接头序列(Linker)将一Elabela片段的羧基端与一APJ抗体轻链或重链的氨基端连接，其中每个Elabela片段各自独立地为一反向Elabela片段或其突变体。
在另一个实施方案中，本文提供了一个Elabela融合蛋白质，其包含本文提供的一个APJ抗体，和二个Elabela片段和肽接头(Linker)；该融合蛋白质通过一个肽接头序列(Linker)将一Elabela片段的氨基端与一APJ抗体轻链或重链的羧基端连接，其中每个Elabela片段各自独立地为一正向Elabela片段或其突变体。
在另一个实施方案中，本文提供了一个Elabela融合蛋白质，其包含本文提供的一个APJ抗体，和二个Elabela片段和肽接头(Linker)；该融合蛋白质通过一个肽接头序列(Linker)将一Elabela片段的羧基端与一APJ抗体轻链或重链的氨基端连接，其中每个Elabela片段各自独立地为一反向Elabela片段或其突变体。
在另一个实施方案中，本文提供了一个Elabela融合蛋白质，其包含本文提供的一个APJ抗体和二个Elabela片段和肽接头(Linker)；该融合蛋白质通过一个肽接头序列(Linker)将一Elabela片段的氨基端与一APJ抗体轻链的羧基端连接：N'-R-Linker-Elabela-C'；或者通过一个肽接头序列(Linker)将一Elabela片段的氨基端和一APJ抗体重链的羧基端连接：N'-R-Linker-Elabela-C'；其中：N'代表 融合蛋白质多肽链的氨基端，C'代表融合蛋白质多肽链的羧基端，Elabela代表一正向Elabela片段或其突变体，R为一APJ抗体的轻链或者重链的氨基酸序列,及Linker代表一肽接头序列。
另一个实施方案中，本文提供了一个Elabela融合蛋白质，其包含本文提供的一个APJ抗体和二个Elabela片段和肽接头(Linker)；该融合蛋白质通过一个肽接头序列(Linker)将一Elabela片段的羧基端与一APJ抗体轻链的氨基端连接：N'-Elabela-Linker-R-C'；或者将一Elabela片段的羧基端和一APJ抗体重链的氨基端连接：N'-Elabela-Linker-R-C'；其中：N'代表融合蛋白质多肽链的氨基端,C'代表融合蛋白质多肽链的羧基端,Elabela代表一反向Elabela片段或其突变体，R为一APJ抗体的轻链或者重链的氨基酸序列,及Linker代表一肽接头序列。
另一个实施方案中，本文提供了一个Elabela融合蛋白质，其包含本文提供的一个APJ抗体和二个Elabela片段和肽接头(Linker)；该融合蛋白质通过一个肽接头序列(Linker)将一Elabela片段的氨基端与一APJ抗体轻链的羧基端连接：N'-R-Linker-Elabela-C'；其中：N'代表融合蛋白质多肽链的氨基端，C'代表融合蛋白质多肽链的羧基端，Elabela代表一正向Elabela片段或其突变体，R为一APJ抗体的轻链的氨基酸序列,及Linker代表一肽接头序列。
在进一步的实施方案中，本文提供了一个Elabela融合蛋白质，其包含本文提供的一个APJ抗体和二个Elabela片段和肽接头(Linker)；该融合蛋白质通过一个肽接头序列(Linker)将一Elabela片段的氨基端与一APJ抗体重链的羧基端连接：N'-R-Linker-Elabela-C'；其中：N'代表融合蛋白质多肽链的氨基端，C'代表融合蛋白质多肽链的羧基端，Elabela代表一正向Elabela片段或其突变体，R为一APJ抗体的重链的氨基酸序列,及Linker代表一肽接头序列。
在一实施方案中，在本文提供的Elabela融合蛋白质中，所述的正向Elabela片段或其突变体各自独立地选自于以下之一的氨基酸序列：SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:109、SEQ ID NO:110、SEQ ID NO:111、SEQ ID NO:112、SEQ ID NO:113、SEQ ID NO:114、SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:117、SEQ ID NO:118、SEQ ID NO:119、SEQ ID NO:120、及SEQ ID NO:121、SEQ ID NO:166、及SEQ ID NO:167。
在一实施方案中，在本文提供的Elabela融合蛋白质中，所述的正向Elabela片段或其突变体的氨基酸序列为以下之一的氨基酸序列SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:103、SEQ ID NO:107、SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:166、及SEQ ID NO:167。
在一实施方案中，在本文提供的Elabela融合蛋白质中，所述的反向Elabela片段或其突变体各自独立地选自于以下之一的氨基酸序列：SEQ ID NO:125、 SEQ ID NO:126、SEQ ID NO:127、SEQ ID NO:128、SEQ ID NO:129、SEQ ID NO:130、SEQ ID NO:131、SEQ ID NO:132、SEQ ID NO:133、SEQ ID NO:134、SEQ ID NO:135、SEQ ID NO:136、SEQ ID NO:137、SEQ ID NO:138、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141、SEQ ID NO:142、SEQ ID NO:143、SEQ ID NO:144、SEQ ID NO:145、SEQ ID NO:146、SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149、SEQ ID NO:150、SEQ ID NO:151、SEQ ID NO:152、SEQ ID NO:153、SEQ ID NO:154、及SEQ ID NO:155。
在一实施方案中，在本文提供的Elabela融合蛋白质中，所述的肽接头(Linker)的序列各自独立包含从1个至200个氨基酸胺，从2个至100个氨基酸胺，从5个至50个氨基酸胺，从6个至25个氨基酸胺，或从10个至20个氨基酸胺。
在另一个实施方案中，在本文提供的Elabela融合蛋白质中，所述的肽接头(Linker)的序列各自独立地包含以下之一的氨基酸序列的全长、部分、或者重复：SEQ ID NO:122、SEQ ID NO:123、及SEQ ID NO:124。
在另一个实施方案中，在本文提供的Elabela融合蛋白质中，所述的肽接头(Linker)的序列各自独立地选自于以下的氨基酸序列:SEQ ID NO:122、SEQ ID NO:123、及SEQ ID NO:124。
在一实施方案中，本文提供的Elabela融合蛋白质的轻链氨基酸序列为：SEQ ID NO:158；及其重链氨基酸序列为以下之一的序列：SEQ ID NO:156、SEQ ID NO:159、SEQ ID NO:160、SEQ ID NO:161、SEQ ID NO:162、SEQ ID NO:163、SEQ ID NO:164、及SEQ ID NO:165。在一实施方案中，本文提供的Elabela融合蛋白质的轻重链氨基酸序列分别为：SEQ ID NO:158及SEQ ID NO:156。在另一实施方案中，本文提供的Elabela融合蛋白质的轻重链氨基酸序列分别为：SEQ ID NO:158及SEQ ID NO:159。在另一实施方案中，本文提供的Elabela融合蛋白质的轻重链氨基酸序列分别为：SEQ ID NO:158及SEQ ID NO:160。在另一实施方案中，本文提供的Elabela融合蛋白质的轻重链氨基酸序列分别为：SEQ ID NO:158及SEQ ID NO:161。在另一实施方案中，本文提供的Elabela融合蛋白质的轻重链氨基酸序列分别为：SEQ ID NO:158及SEQ ID NO:162。在另一实施方案中，本文提供的Elabela融合蛋白质的轻重链氨基酸序列分别为：SEQ ID NO:158及SEQ ID NO:163。在另一实施方案中，本文提供的Elabela融合蛋白质的轻重链氨基酸序列分别为：SEQ ID NO:158及SEQ ID NO:164。在进一步实施的方案中，本文提供的Elabela融合蛋白质的轻重链氨基酸序列分别为：SEQ ID NO:158及SEQ ID NO:165。
核酸
一方面，本文提供分离的核酸分子。该核酸分子包含例如编码全部或部分Elabela融合蛋白质的多聚核苷酸，例如本文Elabela融合蛋白质的一条链或两条链，或其片段、衍生物、突变蛋白或变异体；足以用作杂交探针的多聚核苷酸；PCR引物或用于鉴定、分析、突变或扩增编码多肽的多聚核苷酸的测序引物；用于抑制多聚核苷酸表达的反义核酸以及其互补序列。该核酸可为任何长度。例 如它们的长度可为5、10、15、20、25、30、35、40、45、50、75、100、125、150、175、200、250、300、350、400、450、500、750、1000、1500、3000、5000或更多个核苷酸，和/或包含一个或多个附加序列，例如调控序列，和/或是较大核酸例如载体的一部分。该核酸可为单链或双链并包含RNA和/或DNA核苷酸以及其人工变异体(例如，肽核酸)。
可从经APJ抗原免疫的小鼠B细胞中分离编码抗体多肽(例如，重链或轻链、仅可变结构域或全长)的核酸。可通过常规方法例如聚合酶链式反应(PCR)分离抗体或Elabela融合蛋白质的核酸。
编码重链和轻链可变区的核酸序列如上文所示。熟练的技术人员可理解由于遗传密码的简并性，本文公开的各多肽序列可由更多数量的其他核酸序列编码。本文提供编码本文提供的抗体或Elabela融合蛋白质的各简并核苷酸序列。
本文进一步提供在具体杂交条件下与其他核酸(例如，包含任何Elabela融合蛋白质的核苷酸序列的核酸)杂交的核酸。杂交核酸的方法为本领域熟知。参见，例如，Current Protocols in Molecular Biology，John Wiley&Son(1989)，6.3.1-6.3.6。如本文定义，例如，中等严格条件使用包含5x氯化钠/柠檬酸钠(SSC)的预洗溶液、0.5％SDS、1.0mM EDTA(pH 8.0)、约50％甲酰胺的杂交缓冲液、6x SSC和55℃的杂交温度(或其他相似的杂交溶液，例如包含绚50％甲酰胺的，以42℃杂交)，并且洗脱条件为60℃，使用0.5x SSC、0.1％SDS。严格杂交条件在6x SSC中于45℃杂交，然后于68℃在0.1xSSC、0.2％SDS中洗涤一次或多次。此外，本领域技术人员可操作杂交和/或洗涤条件以增加或降低杂交严格度这样包含相互之间至少65、70、75、80、85、90、95、98或99％同源的核苷酸序列的核酸通常仍可以相互杂交。影响杂交条件选择的基本参数和设计适当条件的指导列于例如Sambrook，Fritsch和Maniatis，1989，Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory Press，第9和11章；Current Protocols in Molecular Biology，1995，Ausubel等编辑，John Wiley&Sons，Inc.，第2.10和6.3-6.4节)并可由具有本领域普通技术的人员基于例如DNA的长度和/或碱基组成轻松确定。可通过突变在核酸中引入变化，藉此导致其编码的多肽(例如，抗原结合蛋白)氨基酸序列的变化。可使用本领域已知的任何技术引入突变。在一个实施方案中，使用例如定点诱变方案改变一个或多个具体氨基酸残基。在另一个实施方案中，使用例如随机诱变方案改变一个或多个随机选择的残基。无论其如何生成，可表达突变多肽并筛选期望性质。
可将突变引入核酸而不显著改变其编码多肽的生物学活性。例如，可进行引起非必需氨基酸残基处氨基酸替换的核苷酸替换。在一个实施方案中，突变本文为Elabela融合蛋白质提供的核苷酸序列或其片段、变异体或衍生物这样其编码包含本文所示Elabela融合蛋白质的氨基酸残基的一个或多个缺失或替换，成为两个或多个序列相异的残基。在另一个实施方案中，诱变作用在本文所示Elabela融合蛋白质的一个或多个氨基酸残基附近插入一个氨基酸成为两个或多个序列相异的残基。或者，可将一个或多个突变引入核酸以选择性改变其编码多肽的生 物学活性(例如，与APJ结合)。例如，该突变可在数量上或性质上改变生物学活性。量变的实例包括增加、降低或消除该活性。质变的实例包括改变Elabela融合蛋白质的结合特异性。
在另一方面，本文提供适于用做引物或检测本文核酸序列的杂交探针的核酸分子。本文的核酸分子可仅包含编码本文全长多肽的核酸序列的一部分，例如，可用作探针或引物或编码本文多肽活性部分的片段(例如，APJ结合部分)的片段。
基于本文核酸序列的探针可用于检测该核酸或相似核酸，例如编码本文多肽的转录物。该探针可包含标记基团，例如放射性同位素、荧光化合物、酶或酶辅因子。该类探针可用于鉴定表达该多肽的细胞。
在另一方面本文提供包含编码本文多肽或其部分的核酸的载体。载体的实例包括但是不限于质粒、病毒载体、非游离基因哺乳动物载体和表达载体，例如重组表达载体。
本文的重组表达载体可包含适于该核酸在宿主细胞中表达的形式的本文核酸。该重组表达载体包括一个或多个调控序列，基于用于表达的宿主细胞进行筛选，其与该预表达的核酸序列可操作性相连。调控序列包括引导核苷酸序列在多个种类宿主细胞中组成型表达的(例如，SV40早期基因增强剂、劳斯氏肉瘤病毒启动子和细胞巨化病毒启动子)，引导仅在某些宿主细胞中核苷酸序列的表达的(例如，组织特异调控序列，参见Voss等，1986，Trends Biochem.Sci.11:287，Maniatis等，1987，Science 236:1237，其完整内容以参考形式并于本文)以及引导核苷酸序列响应具体处理或条件的诱导型表达的(例如，哺乳动物细胞中的金属硫堇启动子和原核和真核系统二者中的四环霉素反应(tet-sesponsive)启动子和/或链霉素反应启动子(同前))。本领域技术人员应理解表达载体的设计取决于例如用于转化的宿主细胞的选择、所需蛋白表达水平等因素。本文的表达载体可引入宿主细胞，藉此生产由本文所述核酸编码的蛋白或肽，包括融合蛋白或肽。
另一方面，本文提供可引入本文表达载体的宿主细胞。宿主细胞可为任何原核或真核细胞。原核宿主细胞包括革兰氏阴性或革兰氏阳性生物体，例如大肠杆菌或杆菌。更高级的真核细胞包括昆虫细胞、酵母细胞以及哺乳动物源的确立细胞系。适当哺乳动物宿主细胞系的实例包括中国仓鼠卵巢(CHO)细胞或它们的衍生物例如Veggie CHO和在无血清培养基中生长的相关细胞系(参见Rasmussen等，1998，Cytotechnology 28:31)或CHO株DXB-11，其缺失DHFR(参见Urlaub等，1980，Proc.Natl.Acad.Sci.USA 77:4216-20)。其它CHO细胞系包括CHO-K1(ATCC#CCL-61)、EM9(ATCC#CRL-1861)，和UV20(ATCC#CRL-1862)，其它宿主细胞包括猴肾细胞的COS-7系(ATCC#CRL-1651)(参见Gluzman等，1981，Cell 23:175)、L细胞、C127细胞、3T3细胞(ATCC CCL-163)，AM-1/D细胞(描述于美国专利序列号6210924)、HeLa细胞、BHK(ATCC CRL-10)细胞系、来源于非洲绿猴肾细胞系CV1的CV1/EBNA细胞系(ATCC CCL-70)(参见McMahan等，1991，EMBO J.10:2821)、人胚肾细胞例如293，293 EBNA 或MSR 293、人上皮A431细胞、人C010205细胞、其它经转化灵长动物细胞系、正常二倍体细胞、来源于初生组织体外培养物的细胞株、初移植体、HL-60、U937、HaK或Jurkat细胞。用于细菌、真菌、酵母和哺乳细胞宿主的适当克隆和表达载体描述于Pouwels等(Cloning Vectors:A Laboratory Manual,Elsevier,1985)。
可通过传统转化或转染技术将载体DNA引入原核或真核细胞中。对于稳定的哺乳动物转染而言，取决于使用的表达载体和转染技术，已知只有一小部分细胞可将外源DNA鏊合入它们的基因组中。为了鉴定和筛选这些整合子，通常将编码筛选标记(例如抗生素抗性)的基因与所关注基因一起引入宿主细胞。优选的筛选标记包括可赋予药物(如G418、潮霉素和甲氨喋呤)抗性的那些。在其它方法中可通过药物筛选鉴别包含被引入核酸的稳定转染细胞(例如，整合了筛选基因的细胞可存活，而其它细胞则死亡)。
可在提高多肽表达的条件下培养已转化细胞，可通过常规蛋白纯化方法回收多肽。一种该纯化方法描述于下文实施例。预用于本文的多肽包括基本同源的重组哺乳动物Elabela融合蛋白质多肽，其基本不含污染性内源材料。
APJ抗体的活性
APJ抗体的活性是指本文提供的抗体具有与APJ特异性的结合活性。在一个实施方案中，本文提供了能与人APJ特异性结合的鼠源抗体或人源化抗体。
在另一个实施方案中，本文提供的抗体与人APJ结合时的K d为大约0.01nM至大约1000nM、大约0.1nM至大约500nM、大约0.5nM至大约200nM、大约1nM至大约200nM、或大约10nM至大约100nM。在另一个实施方案中，本文提供的抗体与人APJ结合时的K d为大约1nM至大约200nM。在另一个实施方案中，本文提供的抗体与人APJ结合时的K d为大约1nM至大约100nM。在另一个实施方案中，本文提供的抗体与人APJ结合时的K d为大约1nM、大约2nM、大约5nM、大约10nM、大约20nM、大约30nM、大约40nM、大约50nM、大约60nM、大约70nM、大约80nM、大约90nM、或大约100nM。
在一个实施方式中，本文所述的APJ抗体是一抗体具有一个或多个以下所列的性质：
a.当与人APJ结合时，其K d与一参比抗体相同或更优；及
b.该抗体在人APJ上，与一参比抗体交叉竞争结合。
在一个实施方式中,所述参比抗体包含轻链可变结构域氨基酸序列SEQ ID NO:62和重链可变结构域氨基酸序列SEQ ID NO:67的组合。
在本文中，术语“基本相似”意为与参比抗体的K d可比或大约是参比抗体的K d值的大约200％、大约180％、大约160％、大约150％、大约140％、大约120％、大约110％、大约100％、大约99％、大约98％、大约97％、大约95％、大约90％、大约85％、大约80％、大约75％、大约70％、大约65％、或大约50％。在一个实施方案中，参比抗体包括，例如，具有轻链SEQ ID NO:62和重链SEQ ID NO:67组合的抗体。
APJ抗体与Elabela融合蛋白质的生物学活性
APJ抗体与Elabela融合蛋白质(Elabela融合蛋白质)的生物学活性包含Elabela的生物学活性和APJ抗体活性两个方面。APJ抗体的活性如前文所述。“Elabela的生物学活性”指Elabela融合蛋白质在体内结合并激活Elabela受体(例如APJ)并引起细胞应激反应。前述细胞应激反应包括但是不限于增强心肌收缩力、舒张血管、降低血压、利尿(减少抗利尿激素释放)、调节机体免疫反应和垂体相关激素释放等作用。综合了Elabela和APJ抗体的生物学活性，本文所述的Elabela融合蛋白质可以用于治疗多种与Elabela和APJ相关联的疾病和病症。该融合蛋白质通过作用于Elabela和/或APJ发挥其生物学作用，因此可以用本文所述的Elabela融合蛋白质治疗对“增加Elabela刺激”做出有利应答的疾病和病症的受试者。这些受试者称为“需要Elabela刺激治疗”的受试者。这些受试者包括急性心衰、慢性心衰、肺高压和肺动脉高压的受试者，也包括糖尿病血管病变、心功能不全、心房颤动、缺血再灌注损伤的受试者。
在一个实施方案中，Elabela融合蛋白质的生物学活性是用报告基因检测方法来检测的，量化Elabela融合蛋白质在体外激活APJ的功能。
在一个实施方案中，本文提供的抗体与Elabela融合蛋白质在激活Elabela/APJ信号通路的的EC 50为大约0.1nM至大约100nM、大约0.5nM至大约20nM、大约1nM至大约10nM、或大约1nM至大约5nM。
药物组合物
在一个实施方案中，本文提供了一药物组合物，其中包含本文提供的Elabela融合蛋白质及一种或多种可药用载体的组合。
在一个实施方案中，药物组合物包含本文提供的Elabela融合蛋白质以及一个或更多选自以下的物质：pH适合于Elabela融合蛋白质的缓冲液、抗氧化剂(例如抗坏血酸)、低分子量多肽(例如含少于10个氨基酸的多肽)、蛋白质、氨基酸、糖例(如糊精)、络合物(例如EDTA)、谷胱甘肽、稳定剂和辅料。在一个实施方案中，本文提供的药物组合物也可包含防腐剂。在一个实施方案中，本文提供的药物组合物可使用适当辅料溶液作为稀释剂将组合物配制成冻干粉末。可用于药物处方组分的进一步实例见Remington's Pharmaceutical Sciences，第16版(1980)和20版(2000)，Mack Publishing Company。
治疗方法
在一个实施方案中，本文提供了治疗、预防、或改善肺动脉高压的方法，其包括给予个体治疗有效量的本文提供的Elabela融合蛋白质或其药用组合物。
在另一个实施方案中，本文提供了治疗、预防、或改善肺高压的方法，其包括给予个体治疗有效量的本文提供的Elabela融合蛋白质或其药用组合物。
在另一个实施方案中，本文提供了治疗、预防、或改善心衰的方法，其包括给予个体治疗有效量的本文提供的Elabela融合蛋白质或其药用组合物。
在另一个实施方案中，本文提供了治疗二型糖尿病及其相关代谢综合症的方法，其包括给予个体治疗有效量的本文提供的Elabela融合蛋白质或其药用组合物。
在另一个实施方案中，本文提供了治疗、预防、或改善肺动脉高压、肺高压、二型糖尿病及其相关代谢综合症或者心衰二种及二种以上病症的方法，其包括给予个体治疗有效量的本文提供的Elabela融合蛋白质或其药用组合物。
在本文提供的任意一项用途，其所述的药用组合物是用于静脉或皮下注射。
Elabela融合蛋白质药物组合物可采用任意适当技术包括但不限于肠道外、局部或吸入给药。如果是注射，可通过例如关节内、静脉内、肌肉内、损伤区内、腹膜内或皮下途径，以快速注射或连续输注给予药用组合物。可考虑例如在疾病或损伤部位局部给药，如透皮给药和埋植剂持续释放给药。吸入给药包括例如鼻腔或口腔吸入、采用喷雾剂、以气雾剂形式吸入Elabela融合蛋白质等等。其它选择包括口腔制剂包括片剂、糖浆剂或锭剂。
给药剂量和频率可根据以下因素而改变：给药途径、所用具体Elabela融合蛋白质、所治疾病的性质和严重度、症状为急性还是慢性以及患者的体积和总体症状。可通过本领域熟知的方法确定适当剂量，例如在临床试验中包括剂量放大研究。
本文提供的Elabela融合蛋白质可在例如一段时间内按规律间隔给药一次或多次。在具体实施方案中，在至少一个月或更长时间给药一次给予Elabela融合蛋白质，例如一个、两个或三个月或者甚至不确定。对于治疗慢性症状，长期治疗通常最有效。但是，对于治疗急性症状，短期给药例如从一周至六周就已足够。通常，给予人类Elabela融合蛋白质直至患者表现出所选体征或指示剂高于基线水平的医学相关改善度为止。
本文提供的治疗方案的一个实例包括以适当剂量一周一次或者更长的皮下注射Elabela融合蛋白质治疗二型糖尿病及其相关代谢综合症、急性心衰、慢性心衰、肺高压或肺动脉高压等引起的症状。可持续每周或每月给予Elabela融合蛋白质直到达到所需结果例如病人症状消退。可按需要重新治疗，或者，可选择地，给予维持剂量。
可在使用Elabela融合蛋白质治疗之前、进行中和/或之后监测病人血液BNP或Pro-BNP的浓度、体重，以检测其压力的任何变化。对于某些病症，BNP或Pro-BNP的变化可随例如疾病进程等因素而变化。可用已知技术测定其BNP或Pro-BNP的浓度。
本文的方法和组合物的具体实施方案涉及使用例如Elabela融合蛋白质和一个或多个Elabela激动剂、两个或更多本文提供的Elabela融合蛋白质，或者本发明Elabela融合蛋白质和一个或更多其它Elabela激动剂。在进一步的实施方案中，单独或与其它用于治疗使患者痛苦的症状的药剂组合给予Elabela融合蛋白质。这些药剂的实例包括蛋白质以及非蛋白质药物。当联合给予多种药物时，如本领域所熟知其剂量应相应调整。“联合给药”组合疗法不限于同时给药，也包括在涉及给予患者至少一种其它治疗剂的疗程中至少给予一次抗原和蛋白的治疗方案。
另一方面，本文提供制备治疗心衰和肺动脉高压及相关病症药剂的方法，其包含本文提供的Elabela融合蛋白质与药学可接受辅料中的混合物，用于治疗上 述疾病的相关病症。药剂制备方法如上所述。
本文进一步提供可特异性结合至人APJ的Elabela融合蛋白质相关的组合物、试剂盒和方法。也提供了核酸分子及其衍生物和片段，其包含编码与APJ结合的多肽的全部或部分的多聚核苷酸，例如编码全部或部分Elabela融合蛋白质或Elabela融合蛋白质衍生物的核酸。本文进一步提供包含该类核酸的载体和质粒以及包含该类核酸和/或载体和质粒的细胞和细胞系。所提供方法包括，例如，制备、鉴定或分离与人APJ结合的Elabela融合蛋白质的方法，测定该Elabela融合蛋白质是否与APJ结合的方法、以及将与APJ结合的Elabela融合蛋白质给予动物模型的方法。
实施例
下面通过具体实例，对本文的技术方案作进一步的说明。
本文中，若非特指，所采用的原料和设备等均可从市场购得或是本领域常用的。下述实例中的方法，如无特别说明，均为本领域的常规方法。
1.免疫用抗原的制备
接种CHO-DHFR-细胞至6孔板中。培养24小时(hr)后，转染克隆有hAPJ基因(核苷酸序列见SEQ ID NO:55及氨基酸序列见SEQ ID NO:23)的pTM15质粒入6孔板中的细胞。转染是按照Invitrogen公司推荐Lipofectamine 2000的转染条件进行。48hr后，将培养液换为含有300μg/mL hygromycin的完全培养基，并且每隔3天(d)换液。培养两周左右，稳定生长的克隆出现。消化分散细胞集落，将细胞传代，继续培养细胞,待传代细胞长至100％愈合度。利用V5标签的抗体(Life Technologies)对构建的稳定细胞株分别进行FACS检测，根据FACS检测结果鉴定加压后的细胞群体。筛选后的CHO-DHFR-hAPJ细胞膜上有大量hAPJ表达。最后经过亚克隆和进一步鉴定后，选出3株APJ细胞为高表达稳定细胞株。这些高表达hAPJ的细胞株可被作为制备抗体的免疫原(参考实施例2)。
此外，hAPJ胞外区和hIgG Fc的融合蛋白也可以作为制备抗体的免疫原，其制备方法如下：亚克隆hAPJ胞外区,hIgG2Fc和肽接头(Linker)的融合蛋白序列基因于pTM5质粒。通过悬浮HEK293细胞进行大量瞬时表达，获得细胞上清液，然后通过亲和层析纯化得到APJ胞外区融合蛋白。
2.APJ抗体的制备
将免疫原和氢氧化铝佐剂混匀，皮下注射BALB/c小鼠(6-8周龄)，每周加强免疫小鼠一次。经过总共6次免疫后，通过剪尾的方式采血。离心分离血清，用FACS检测血清效价。达到适合抗体滴度时，断颈处死小鼠，无菌状态下获取脾脏细胞。另外收集生长状态处于对数生长期的SP2/0细胞，离心细胞，将沉淀细胞用无血清培养至重悬，再次离心-重悬，计数。混合脾脏细胞和SP2/0细胞，保证SP2/0和脾脏细胞数量接近，混合以后再“洗涤-离心”3次。轻柔的分散最后一次离心后的细胞沉淀，逐滴加入预温的PEG-1500，上下吹吸后，缓慢加入30 mL预热的无血清培养基以终止PEG的融合作用。再次离心后弹散细胞沉淀，加入融合培养基，将脾细胞和饲养层细胞铺于96孔板中,每孔加入100μL培养基。融合后的杂交瘤细胞和饲养层细胞一起在96孔板中进行培养，并进行HAT(次黄嘌呤、氨甲喋呤和胸苷)筛选，以除去非融合的细胞。10天后收取培养板中的杂交瘤细胞上清进行ELISA检测。
3.ELISA筛选APJ抗体
将过表达hAPJ的CHO-DHFR-hAPJ细胞和不表达hAPJ的CHO-DHFR-细胞分别接种至96孔板。待细胞长至90％愈合度，除去细胞培养上清，PBS洗两遍，加入100％甲醇4℃固定，然后加入100μL配制的H 2O 2-PBS，室温处理20min，PBS洗两遍。经过BSA(溶于PBS中)封闭后，加入杂交瘤细胞上清4℃孵育90分钟。多次洗涤后，每孔加入100μL稀释的羊抗鼠Fc-HRP二抗，37℃孵育30分钟。洗涤5次后,每孔加入100μL TMB显色底物，37℃反应15min，加入50μL 2M H 2SO 4终止显色，读取OD 450值。经过1％酪蛋白(溶于PBS中)封闭后，加入杂交瘤细胞上清4℃孵育90min。之后步骤同上述ELISA方法筛选抗hAPJ单克隆抗体。阳性对照为免疫小鼠的血清；阴性对照为细胞培养基上清。经过ELISA的初步检测，筛选到了数个分泌抗hAPJ抗体的阳性杂交瘤细胞株。选取这些分泌抗hAPJ抗体的杂交瘤株，进行克隆化以获得能稳定分泌抗hAPJ抗体的细胞株。最后，选取阳性杂交瘤细胞制备的腹水抗体，用流式细胞仪实验方法(参考实例9)对其包含的hAPJ抗体进行亲和力的验证和排序。
4.APJ抗体基因的克隆及亚克隆
收集分泌抗体的杂交瘤细胞，按照QIAGEN的mRNA抽提试剂盒操作规程，提取杂交瘤细胞的mRNA。然后将提取后的mRNA反转录成cDNA，逆转录引物为小鼠轻、重链恒定区的特异性引物，重链逆转录引物为(5’-TTTGGRGGGAAGATGAAGAC-3’)，轻链逆转录引物为(5’-TTAACACTCTCCCCTGTTGAA-3’)和(5’-TTAACACTCATTCCTGTTGAA-3’)。RT-PCR的反应条件为：25℃5min；50℃60min；70℃15min。将反转录的cDNA用0.1mM的TE稀释至500μL，加入到超滤离心管(Amicon Ultra-0.5)中，2000g离心10min；弃滤液，再加500μL的0.1mM的TE，2000g离心10min；弃滤液，将制备管倒置到新的离心管中，2000g离心10min，得到纯化后的cDNA；取10μL的纯化后的cDNA作为模板，加入4μL的5x tailing buffer(Promega)，4μL的dATP(1mM)和10U的末端转移酶(Promega)后混匀，37℃孵育5min后再65℃孵育5min；然后以加上PolyA尾的cDNA为模板，PCR扩增抗体的轻、重链可变区基因。上游引物均为OligodT，重链下游引物为(5’-TGGACAGGGATCCAGAGTTCC-3’)和(5’-TGGACAGGGCTCCATAGTTCC-3’)，轻链下游引物为(5’-ACTCGTCCTTGGTCAACGTG-3’)。PCR反应条件：95℃5min；95℃30s，56℃30s，72℃1min 40cycles；72℃7min；PCR产物连接到PMD 18-T载体(Takara Bio)后进行测序。基于已测序得到的抗体的DNA序列设计PCR引物， 从而将完整轻链、重链信号肽和可变域以及小鼠IgG1恒定区与表达载体pTM5相连。
5.APJ抗体的人源化和优化与亚克隆
首先，根据筛选所得的鼠源抗体轻、重链可变区序列，并使用NCBI数据库搜索与筛选所得的鼠源抗体可变区序列同源的人源抗体生殖细胞系基因序列(Ig Germline Gene Sequence)，并将除CDR序列外，同源性最高的人源基因序列做为模板序列进行CDR嫁接，得到人源化的抗体可变区序列。合成人源化抗体轻、重链的基因，与人IgG2或者IgG4恒定区序列拼接后得到完整的重组人源化抗体序列。重组抗体按照实例8进行表达，并按按照步骤10中的FACS技术验证重组抗体针对APJ的亲和力，遴选出亲和力表现最优秀的抗体。最后通过定点突变，对其可变区序列进行进一步的改造，进一步提高其对APJ的亲和力。
优化后的人源化抗体重链及轻链可变区序列外包合成，进一步将完整的重链可变区序列与已装入重链恒定区的表达载体pTM5相连；同样，将完整的轻链可变区序列与已装入轻链恒定区的表达载体pTM5相连。
6.人源化APJ抗体与Elabela的融合蛋白质的构建
优化后的人源化抗体在重链的C端与Elabela片段序列进行融合组成Elabela融合蛋白质。两者的序列由肽接头序列(Linker)做为桥梁进行连接。通过overlapping PCR将人源化APJ抗体重链核酸序列与“Linker-Elabela片段”部分连接，引物两端添加Nhe1和Not1的酶切位点，从而将完整的融合蛋白质序列与表达载体pTM5相连，Elabela融合蛋白质连接到表达载体后进行测序分析，以确认构建的正确。
7.APJ抗体和Elabela融合蛋白质的瞬时表达
接种5×10 5/mL的悬浮HEK293或者CHO表达细胞株至转瓶中，经过24小时37℃，5％CO 2旋转培养后，密度达到1×10 6/mL后被用于转染。转染过程中使用polyethylenimine(PEI)作为转染介质，将其与的DNA混合。两者混合物在静置孵育15分钟后被加入到细胞培养中。细胞在接受PEI与DNA混合物后继续37℃，5％CO2旋转培24小时后，向细胞培养液中加入胰蛋白胨作为表达需要的添加物。最后在表达完成后(96小时以上)收集细胞上清用于抗体或融合蛋白质的纯化分离。
8.APJ抗体和Elabela融合蛋白质的纯化分离
收集的实例8中的细胞上清经过高速(8000rpm)离心去除细胞以及细胞碎片后，再用0.22um滤膜过滤澄清。澄清后的上清被用于纯化。纯化过程由层析仪完成。上清首先流过蛋白A/G亲和层析柱。上清中包含的抗体或融合蛋白质在此期间与蛋白A/G亲和层析柱的配基相结合后被滞留于柱内。然后用低pH值(小于等于3.0)的洗脱缓冲液灌洗层析柱解离与层析柱结合的抗体或融合蛋白质。收集到的抗体洗脱液用1M的Tris-HCl迅速中和。得到的抗体或融合蛋白质洗脱液经过透析后置换成PBS或者其他缓冲体系。
9.流式细胞仪验证功能性APJ抗体的结合活性
用含EDTA的PBS消化、收集10 5个CHO-DHFR-hAPJ细胞，分别加入1.5mL EP管，离心后弃上清。阴性对照样本用流式上样缓冲液(PBS，2％FBS)重悬。阳性处理组细胞每管加200μL特定浓度的hAPJ抗体，室温孵育；孵育完成后1500rpm离心，弃上清，用流式上样缓冲液洗一次细胞沉淀，再离心，将细胞重悬；向细胞重悬液加入1:50稀释的FITC标记的羊抗鼠荧光二抗，200μL/孔，室温避光孵育30min；离心，弃上清，再用流式上样缓冲液洗一次，离心，最后用流式上样缓冲液将细胞沉淀重悬，上机检测。图一A至图一D显示的实验结果中，左侧灰色峰为500nM小鼠腹水抗体L1H1和空白细胞CHO-DHFR-结合的阴性对照，实线峰分别为500nM(图一A)和4nM(图一B)的小鼠腹水抗体L1H1和CHO-DHFR-hAPJ的结合曲线，相对于灰色峰阴性对照有明显右移，证明了L1H1和hAPJ的特异性结合。右侧灰色峰为500nM小鼠腹水抗体L4H4和空白细胞CHO-DHFR-结合的阴性对照，实线峰分别为500nM(图一C)和4nM(图一D)的小鼠腹水抗体L4H4和CHO-DHFR-hAPJ的结合曲线，相对于灰色峰阴性对照有明显右移，也证明了L4H4和hAPJ的特异性结合。
10.报告基因实验检测Elabela及其Elabela融合蛋白质在体外激发Elabela/APJ信号通路的生物学活性
以每孔35000个接种共表达hAPJ-CRE-Luciferase的CHO-DHFR-细胞至96孔细胞培养板，37℃培养过夜。第二天除去培养基上清，用无血清培养基清洗细胞表面两次，吸去残液。96孔板中预先加入50μL浓度为0.6μM的弗司可林，再加入50μL用无血清培养基稀释Elabela融合蛋白或Elabela-11多肽，37℃孵育6小时。刺激结束后，加入100μL Promega的Bright Glo化学发光底物，最后将细胞裂解物转移至白色96孔板，在Molecular Devices的SpectraMax L酶标仪上读取相对萤光强度。数据采用Prism5.0进行5参数拟合，计算EC 50值。L1H1和L4H4抗体不阻断、也不激活APJ受体。图二显示了报告基因实验检测重组表达的L5H5与Linker2-Elabela-11的融合蛋白质、L5H5与Linker2-EA5的融合蛋白质激活Elabela/APJ信号通路的激活性曲线，EC 50分别为：3.61和2.55nM。图三显示了报告基因实验检测重组表达的L5H5与Linker2-EA1的融合蛋白质、L55H5与Linker2-EA2的融合蛋白质激活Elabela/APJ信号通路的激活性曲线，EC 50分别为：4.95和2.16nM。图四显示了报告基因实验检测重组表达的L5H5与Linker2-EA3的融合蛋白质、L5H5与Linker2-EA4的融合蛋白质激活Elabela/APJ信号通路的激活性曲线，EC 50分别为：20.27和5.43nM。
提供以上实例用于向本领域普通技术人员充分公开和说明如何制造和使用要求保护的实施方式，而不意味着限制本文披露的范围。对本领域技术人员而言显而易见的修饰都在本文权利要求书的范围内。将本说明书中引用的所有出版物、专利和专利申请都通过参考并入本文，就如同每个出版物、专利或专利申请均被具体和单独地通过参考并入本文一样。

Claims (50)

  1. 一个能与人APJ特异性结合的抗体，所述抗体包含一，两，三，四，五，或六个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
    a.轻链CDR1氨基酸序列：SEQ ID NO:10、SEQ ID NO:7、SEQ ID NO:4、及SEQ ID NO:1；
    b.轻链CDR2氨基酸序列：SEQ ID NO:8、SEQ ID NO:5、及SEQ ID NO:2；
    c.轻链CDR3氨基酸序列：SEQ ID NO:11、SEQ ID NO:9、SEQ ID NO:6、及SEQ ID NO:3；
    d.重链CDR1氨基酸序列：SEQ ID NO:18、SEQ ID NO:15、及SEQ ID NO:12；
    e.重链CDR2氨基酸序列：SEQ ID NO:21、SEQ ID NO:19、SEQ ID NO:16、及SEQ ID NO:13；及
    f.重链CDR3氨基酸序列：SEQ ID NO:22、SEQ ID NO:20、SEQ ID NO:17、及SEQ ID NO:14。
  2. 根据权利要求1所述的抗体，其中所述抗体包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
    a.轻链CDR1氨基酸序列：SEQ ID NO:1、SEQ ID NO:4、SEQ ID NO:7、及SEQ ID NO:10；及
    b.重链CDR1氨基酸序列：SEQ ID NO:12、SEQ ID NO:15、及SEQ ID NO:18。
  3. 根据权利要求1或2所述的抗体，其中所述抗体包含或还包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
    a.轻链CDR2氨基酸序列：SEQ ID NO:2、SEQ ID NO:5、及SEQ ID NO:8；及
    b.重链CDR2氨基酸序列：SEQ ID NO:13、SEQ ID NO:16、SEQ ID NO:19、及SEQ ID NO:21。
  4. 根据权利要求1至3中任一项所述的抗体，其中所述抗体包含或还包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
    a.轻链CDR3氨基酸序列：SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:9、及SEQ ID NO:11；及
    b.重链CDR3氨基酸序列：SEQ ID NO:14、SEQ ID NO:17、SEQ ID NO:20、及SEQ ID NO:22。
  5. 根据权利要求1至4中任一项所述的抗体，其中所述抗体包含或还包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、 及SEQ ID NO:11。
  6. 根据权利要求1至5中任一项所述的抗体，其中所述抗体包含或还包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、及SEQ ID NO:22。
  7. 根据权利要求1至6中任一项所述的抗体，其中所述抗体包含或还包含一个独立地选自于以下所列的轻链和重链CDR1氨基酸序列的组合：SEQ ID NO:1与SEQ ID NO:12、SEQ ID NO:4与SEQ ID NO:15、SEQ ID NO:7与SEQ ID NO:18、及SEQ ID NO:10与SEQ ID NO:18。
  8. 根据权利要求1至7中任一项所述的抗体，其中所述抗体包含或还包含一个独立地选自于以下所列的轻链和重链CDR2氨基酸序列的组合：SEQ ID NO:2与SEQ ID NO:13、SEQ ID NO:5与SEQ ID NO:16、SEQ ID NO:8与SEQ ID NO:19、及SEQ ID NO:5与SEQ ID NO:21。
  9. 根据权利要求1至8中任一项所述的抗体，其中所述抗体包含或还包含一个独立地选自于以下所列的轻链和重链CDR3氨基酸序列的组合：SEQ ID NO:3与SEQ ID NO:14、SEQ ID NO:6与SEQ ID NO:17、SEQ ID NO:9与SEQ ID NO:20、及SEQ ID NO:11与SEQ ID NO:22。
  10. 根据权利要求1至9中任一项所述的抗体，其中所述抗体包含以下所列之一的氨基酸序列的组合：
    a.SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:12、SEQ ID NO:13、与SEQ ID NO:14的组合；
    b.SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:15、SEQ ID NO:16、与SEQ ID NO:17的组合；
    c.SEQ ID NO:5、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:18、SEQ ID NO:21、与SEQ ID NO:22的组合；或
    d.SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:18、SEQ ID NO:19、与SEQ ID NO:20的组合。
  11. 权利要求1至9中任一项所述的抗体，其中所述的抗体包含
    (a)轻链CDR1氨基酸序列：SEQ ID NO:1；
    轻链CDR2氨基酸序列：SEQ ID NO:2；
    轻链CDR3氨基酸序列：SEQ ID NO:3；
    重链CDR1氨基酸序列：SEQ ID NO:12；
    重链CDR2氨基酸序列：SEQ ID NO:13；及
    重链CDR3氨基酸序列：SEQ ID NO:14；
    (b)轻链CDR1氨基酸序列：SEQ ID NO:4；
    轻链CDR2氨基酸序列：SEQ ID NO:5；
    轻链CDR3氨基酸序列：SEQ ID NO:6；
    重链CDR1氨基酸序列：SEQ ID NO:15；
    重链CDR2氨基酸序列：SEQ ID NO:16；及
    重链CDR3氨基酸序列：SEQ ID NO:17；
    (c)轻链CDR1氨基酸序列：SEQ ID NO:7；
    轻链CDR2氨基酸序列：SEQ ID NO:8；
    轻链CDR3氨基酸序列：SEQ ID NO:9；
    重链CDR1氨基酸序列：SEQ ID NO:18；
    重链CDR2氨基酸序列：SEQ ID NO:19；及
    重链CDR3氨基酸序列：SEQ ID NO:20；
    (d)轻链CDR1氨基酸序列：SEQ ID NO:10；
    轻链CDR2氨基酸序列：SEQ ID NO:5；
    轻链CDR3氨基酸序列：SEQ ID NO:11；
    重链CDR1氨基酸序列：SEQ ID NO:18；
    重链CDR2氨基酸序列：SEQ ID NO:21；及
    重链CDR3氨基酸序列：SEQ ID NO:22。
  12. 权利要求11所述的抗体，其中所述的抗体包含
    轻链CDR1氨基酸序列：SEQ ID NO:10；
    轻链CDR2氨基酸序列：SEQ ID NO:5；
    轻链CDR3氨基酸序列：SEQ ID NO:11；
    重链CDR1氨基酸序列：SEQ ID NO:18；
    重链CDR2氨基酸序列：SEQ ID NO:21；及
    重链CDR3氨基酸序列：SEQ ID NO:22。
  13. 根据权利要求1至12中任一项所述的抗体，其中所述抗体包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
    a.轻链可变结构域氨基酸序列：SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、及SEQ ID NO:63；及与其任一序列有至少80％、至少85％、至少90％、或至少95％相同的氨基酸序列；及
    b.重链可变结构域氨基酸序列：SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、及SEQ ID NO:68；及与其任一序列有至少80％、至少85％、至少90％、或至少95％相同的氨基酸序列。
  14. 根据权利要求1至13中任一项所述的抗体，其中所述抗体的多聚核苷酸编码序列包含一或两个多聚核苷酸编码序列，其中每个多聚核苷酸编码序列独立地选自于以下所列多聚核苷酸序列：
    a.轻链可变结构域多聚核苷酸编码序列：SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、及SEQ ID NO:73；及与其任一序列有至少80％、至少85％、至少90％、或至少95％相同的多聚核苷酸编码序列；及
    b.重链可变结构域多聚核苷酸编码序列：SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77、及SEQ ID NO:78；及与其任一序列有至少 80％、至少85％、至少90％、或至少95％相同的多聚核苷酸编码序列。
  15. 根据权利要求1至14中任一项所述的抗体，其中所述抗体包含或还包含一个独立地选自于以下所列的氨基酸序列：SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、及SEQ ID NO:63。
  16. 根据权利要求1至15中任一项所述的抗体，其中所述抗体包含或还包含一个独立地选自于以下所列的氨基酸序列：SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、及SEQ ID NO:68。
  17. 根据权利要求1至16中任一项所述的抗体，其中所述抗体包含一个独立地选自于以下所列的轻链与重链可变结构域氨基酸序列的组合：SEQ ID NO:59与SEQ ID NO:64、SEQ ID NO:60与SEQ ID NO:65、SEQ ID NO:61与SEQ ID NO:66、SEQ ID NO:62与SEQ ID NO:67、及SEQ ID NO:63与SEQ ID NO:68。
  18. 根据权利要求1至17中任一项所述的抗体，其中所述抗体还包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
    a.轻链恒定氨基酸序列：SEQ ID NO:79、SEQ ID NO:80、及SEQ ID NO:81；及
    b.重链恒定氨基酸序列：SEQ ID NO:82、SEQ ID NO:83、及SEQ ID NO:84。
  19. 一个能与人APJ特异性结合的抗体，所述的抗体具有一个或多个以下所列的性质：
    a.当与人APJ结合时，其K d与一参比APJ抗体相同或更优；和
    b.该APJ抗体在人APJ上与一参比APJ抗体交叉竞争结合。
  20. 权利要求19所述的抗体，其中所述的抗体在人APJ上与所述的参比APJ抗体交叉竞争结合。
  21. 权利要求19或20所述的抗体，其中所述的参比APJ抗体是权利要求1至18中任一项所述的抗体。
  22. 权利要求21所述的抗体，其中所述的参比APJ抗体包含轻链可变结构域氨基酸序列SEQ ID NO:62和重链可变结构域氨基酸序列SEQ ID NO:67的组合或轻链可变结构域氨基酸序列SEQ ID NO:63和重链可变结构域氨基酸序列SEQ ID NO:68的组合。
  23. 根据权利要求1至22中任一项所述的抗体，其特征在于：所述抗体为鼠源抗体、人类抗体、人源化抗体、嵌合抗体、单克隆抗体、多克隆抗体、重组抗体、抗原结合抗体片段、单链抗体、双链抗体、三链抗体、四链抗体、Fab片段、F(ab')x片段、结构域抗体、IgD抗体、IgE抗体、IgM抗体、IgGl抗体、IgG2抗体、IgG3抗体、或IgG4抗体。
  24. 根据权利要求1至23中任一项所述的抗体，其中所述抗体为鼠源APJ抗体或人源化APJ抗体。
  25. 根据权利要求1至24中任一项所述的抗体，其中所述抗体为APJ单克隆抗体。
  26. 根据权利要求1至25中任一项所述的抗体，其中所述抗体的K d值为大约1nM至大约200nM或大约1nM至大约100nM。
  27. 一个Elabela融合蛋白质，其结构特征在于：所述的融合蛋白质包含权利要求 1至26中任一项所述的一个APJ抗体和一Elabela片段。
  28. 一个Elabela融合蛋白质，其结构特征在于：所述的融合蛋白质包含权利要求1至26中任一项所述的一个APJ抗体、和一个，二个，三个，四个，五个，六个，七个，或八个Elabela片段和肽接头(Linker)；该融合蛋白质通过一肽接头序列(Linker)将一Elabela片段的氨基端与一APJ抗体轻链或重链的羧基端连接，其中每个Elabela片段各自独立地为一正向Elabela片段或其突变体；或者该融合蛋白质通过一肽接头序列(Linker)将一Elabela片段的羧基端与一APJ抗体轻链或重链的氨基端连接，其中每个Elabela片段各自独立地为一反向Elabela片段或其突变体。
  29. 根据权利要求27或28所述的融合蛋白质，其所述的融合蛋白质包含一个APJ抗体，和一个，二个，三个，或四个Elabela片段和肽接头(Linker)；该融合蛋白质通过一个肽接头序列(Linker)将一Elabela片段的氨基端与一APJ抗体轻链或重链的羧基端连接，其中每个Elabela片段各自独立地为一正向Elabela片段或其突变体。
  30. 权利要求27或28所述的融合蛋白质，其所述的融合蛋白质包含一个APJ抗体，和一个，二个，三个，或四个Elabela片段和肽接头(Linker)；该融合蛋白质通过一个肽接头序列(Linker)将一Elabela片段的羧基端与一APJ抗体轻链或重链的氨基端连接，其中每个Elabela片段各自独立地为一反向Elabela片段或其突变体。
  31. 权利要求27或28所述的融合蛋白质，其所述的融合蛋白质包含一个APJ抗体，和二个Elabela片段和肽接头(Linker)；该融合蛋白质通过一个肽接头序列(Linker)将一Elabela片段的氨基端与一APJ抗体轻链或重链的羧基端连接，其中每个Elabela片段各自独立地为一正向Elabela片段或其突变体。
  32. 权利要求27或28所述的融合蛋白质，其所述的融合蛋白质包含一个APJ抗体，和二个Elabela片段和肽接头(Linker)；该融合蛋白质通过一个肽接头序列(Linker)将一Elabela片段的羧基端与一APJ抗体轻链或重链的氨基端连接，其中每个Elabela片段各自独立地为一反向Elabela片段或其突变体。
  33. 根据权利要求27至32中任一项所述的融合蛋白质，其中所述的APJ抗体、Elabela片段、和肽接头序列(Linker)通过以下所述中一方式融合形成所述的融合蛋白质：
    通过一个肽接头序列(Linker)将一Elabela片段的氨基端和一APJ抗体轻链的羧基端连接：N'-R-Linker-Elabela-C'；及
    通过一个肽接头序列(Linker)将一Elabela片段的氨基端和一APJ抗体重链的羧基端连接：N'-R-Linker-Elabela-C'；
    其中:N'代表多肽链的氨基端,C'代表多肽链的羧基端,Elabela代表一正向Elabela片段或其突变体，R为权利要求1至26中任一项所述的一APJ抗体的轻链或者重链的氨基酸序列,及Linker代表一肽接头。
  34. 根据权力要求27至33中任一项所述的Elabela融合蛋白质，所述的肽接头 (Linker)的序列各自独立地包含以下之一的氨基酸序列：SEQ ID NO:122、SEQ ID NO:123、及SEQ ID NO:124。
  35. 根据权力要求27至34中任一项所述的Elabela融合蛋白质，所述的肽接头(Linker)包含SEQ ID NO:123。
  36. 根据权力要求27至35中任一项所述的Elabela融合蛋白质，其中所述的Elabela片段包含独立地选自于以下之一的氨基酸序列：SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:109、SEQ ID NO:110、SEQ ID NO:111、SEQ ID NO:112、SEQ ID NO:113、SEQ ID NO:114、SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:117、SEQ ID NO:118、SEQ ID NO:119、SEQ ID NO:120、SEQ ID NO:121、SEQ ID NO:166、及SEQ ID NO:167。
  37. 根据权力要求36所述的Elabela融合蛋白质，其中所述的Elabela片段包含独立地选自于以下之一的氨基酸序列：SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:103、SEQ ID NO:107、SEQ ID NO:109、及SEQ ID NO:116。
  38. 根据权力要求27至37中任一项所述的Elabela融合蛋白质，其中所述的Elabela片段包含独立地选自于以下之一的氨基酸序列：SEQ ID NO:125、SEQ ID NO:126、SEQ ID NO:127、SEQ ID NO:128、SEQ ID NO:129、SEQ ID NO:130、SEQ ID NO:131、SEQ ID NO:132、SEQ ID NO:133、SEQ ID NO:134、SEQ ID NO:135、SEQ ID NO:136、SEQ ID NO:137、SEQ ID NO:138、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141、SEQ ID NO:142、SEQ ID NO:143、SEQ ID NO:144、SEQ ID NO:145、SEQ ID NO:146、SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149、SEQ ID NO:150、SEQ ID NO:151、SEQ ID NO:152、SEQ ID NO:153、SEQ ID NO:154、及SEQ ID NO:155。
  39. 根据权力要求27至38中任一项所述的Elabela融合蛋白质，其中所述融合蛋白质包含：(i)SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:18、SEQ ID NO:19、与SEQ ID NO:20的组合；(ii)独立地选自于以下之一的氨基酸序列：SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:103、SEQ ID NO:107、SEQ ID NO:109、及SEQ ID NO:116；(iii)SEQ ID NO:123。
  40. 一种多核苷酸，其编码权利要求27至39中任一项所述的一Elabela融合蛋白质。
  41. 一种载体，其包含权利要求40中所述的一多核苷酸。
  42. 一种宿主细胞，其包含权利要求41中所述的一载体。
  43. 一种药用组合物，其包含权利要求1至26中任一项所述的抗体和一药用可接受的载体。
  44. 一种药用组合物，其包含权利要求27至39中任一项所述的一Elabela融合蛋白质和一药用可接受的载体。
  45. 一种包含权利要求27至39中任一项所述的一Elabela融合蛋白质在制备用于预防或治疗肺动脉高压的药物中的用途。
  46. 一种包含权利要求27至39中任一项所述的一Elabela融合蛋白质在制备用于预防或治疗肺高压的药物中的用途。
  47. 一种包含权利要求27至39中任一项所述的一Elabela融合蛋白质在制备用于预防或治疗心力衰竭的药物中的用途。
  48. 一种包含权利要求27至39中任一项所述的一Elabela融合蛋白质在制备用于治疗二型糖尿病及其相关代谢综合症的药物中的用途。
  49. 一种包含权利要求27至39中任一项所述的一Elabela融合蛋白质在制备用于预防或治疗肺动脉高压、肺高压、二型糖尿病及其相关代谢综合症或者心力衰竭二种及二种以上病症的药物中的用途。
  50. 根据权利要求45至49中任一项所述用途，其所述的药物是用于静脉或皮下注射。
PCT/CN2019/091090 2018-06-13 2019-06-13 APJ抗体及其与Elabela的融合蛋白质，以及其药物组合物和应用 WO2019238093A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
JP2020569130A JP2021526833A (ja) 2018-06-13 2019-06-13 Ａｐｊ抗体及びｅｌａｂｅｌａとのその融合タンパク質並びにその医薬組成物及び用途
KR1020217000999A KR20210019535A (ko) 2018-06-13 2019-06-13 Apj 항체 및 이와 엘라벨라의 융합 단백질, 및 그의 약학 조성물 및 적용
US17/251,663 US20210163614A1 (en) 2018-06-13 2019-06-13 Apj antibody, fusion protein thereof with elabela, and pharmaceutical compositions and use thereof
CA3103585A CA3103585A1 (en) 2018-06-13 2019-06-13 Apj antibody and its fusion protein with elabela, and pharmaceutical composition and application thereof
EP19819592.7A EP3808847A4 (en) 2018-06-13 2019-06-13 APJ ANTIBODY, FUSION PROTEIN THEREOF WITH ELABELA, AND PHARMACEUTICAL COMPOSITIONS AND USE THEREOF
AU2019284320A AU2019284320A1 (en) 2018-06-13 2019-06-13 APJ antibody, fusion protein thereof with Elabela, and pharmaceutical compositions and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810606914.0A CN110655577A (zh) 2018-06-13 2018-06-13 APJ抗体及其与Elabela的融合蛋白质，以及其药物组合物和应用
CN201810606914.0 2018-06-13

Publications (1)

Publication Number Publication Date
WO2019238093A1 true WO2019238093A1 (zh) 2019-12-19

Family

ID=68842717

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/091090 WO2019238093A1 (zh) 2018-06-13 2019-06-13 APJ抗体及其与Elabela的融合蛋白质，以及其药物组合物和应用

Country Status (8)

Country Link
US (1) US20210163614A1 (zh)
EP (1) EP3808847A4 (zh)
JP (1) JP2021526833A (zh)
KR (1) KR20210019535A (zh)
CN (1) CN110655577A (zh)
AU (1) AU2019284320A1 (zh)
CA (1) CA3103585A1 (zh)
WO (1) WO2019238093A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112210001A (zh) * 2020-10-25 2021-01-12 兰州大学 多肽片段ela13及其用途

Citations (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4301144A (en) 1979-07-11 1981-11-17 Ajinomoto Company, Incorporated Blood substitute containing modified hemoglobin
US4331647A (en) 1980-03-03 1982-05-25 Goldenberg Milton David Tumor localization and therapy with labeled antibody fragments specific to tumor-associated markers
US4496689A (en) 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
US4518584A (en) 1983-04-15 1985-05-21 Cetus Corporation Human recombinant interleukin-2 muteins
US4640835A (en) 1981-10-30 1987-02-03 Nippon Chemiphar Company, Ltd. Plasminogen activator derivatives
US4670417A (en) 1985-06-19 1987-06-02 Ajinomoto Co., Inc. Hemoglobin combined with a poly(alkylene oxide)
US4737462A (en) 1982-10-19 1988-04-12 Cetus Corporation Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of interferon-β
US4751180A (en) 1985-03-28 1988-06-14 Chiron Corporation Expression using fused genes providing for protein product
US4791192A (en) 1986-06-26 1988-12-13 Takeda Chemical Industries, Ltd. Chemically modified protein with polyethyleneglycol
US4935233A (en) 1985-12-02 1990-06-19 G. D. Searle And Company Covalently linked polypeptide cell modulators
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5011912A (en) 1986-12-19 1991-04-30 Immunex Corporation Hybridoma and monoclonal antibody for use in an immunoaffinity purification system
WO1993010151A1 (en) 1991-11-22 1993-05-27 Immunex Corporation Receptor for oncostatin m and leukemia inhibitory factor
WO1994010308A1 (en) 1992-10-23 1994-05-11 Immunex Corporation Methods of preparing soluble, oligomeric proteins
US5457035A (en) 1993-07-23 1995-10-10 Immunex Corporation Cytokine which is a ligand for OX40
US6133426A (en) 1997-02-21 2000-10-17 Genentech, Inc. Humanized anti-IL-8 monoclonal antibodies
CN1283228A (zh) * 1997-12-24 2001-02-07 武田药品工业株式会社 多肽、其生产方法及应用
US6210924B1 (en) 1998-08-11 2001-04-03 Amgen Inc. Overexpressing cyclin D 1 in a eukaryotic cell line
US20030039958A1 (en) 1999-12-03 2003-02-27 Domantis Limited Direct screening method
US20040009507A1 (en) 2000-10-13 2004-01-15 Domantis, Ltd. Concatenated nucleic acid sequence
US6696245B2 (en) 1997-10-20 2004-02-24 Domantis Limited Methods for selecting functional polypeptides
US6703199B1 (en) 1997-06-12 2004-03-09 Research Corporation Technologies, Inc. Artificial antibody polypeptides
US20040202995A1 (en) 2003-04-09 2004-10-14 Domantis Nucleic acids, proteins, and screening methods
US20050075275A1 (en) * 2001-08-06 2005-04-07 Bayer Aktiengesellschaft Regulation of the apj receptor for use in the treatment or prophylaxis of cardiac diseases
US20050238646A1 (en) 2001-01-17 2005-10-27 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
WO2006023893A2 (en) * 2004-08-23 2006-03-02 Arizona Board Of Regents On Behalf Of The University Of Arizona Methods for modulating angiogenesis and apoptosis with apelin compositions
WO2012102363A1 (ja) * 2011-01-28 2012-08-02 国立大学法人大阪大学 薬物送達システムおよびその利用
WO2015140296A2 (en) * 2014-03-20 2015-09-24 Centre National De La Recherche Scientifique (Cnrs) Use of compounds inhibiting apelin / apj / gp130 signaling for treating cancer
WO2016061141A1 (en) * 2014-10-13 2016-04-21 University Of Maryland, Baltimore Methods for treating cardiovascular dysfunction and improving fluid homeostasis with a peptide hormone

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005106493A1 (en) * 2004-04-30 2005-11-10 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with g protein-coupled apelin receptor (apj)
WO2013012855A1 (en) * 2011-07-18 2013-01-24 Amgen Inc. Apelin antigen-binding proteins and uses thereof
JP6317670B2 (ja) * 2011-08-15 2018-04-25 ザ・ユニバーシティ・オブ・シカゴＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｃｈｉｃａｇｏ ブドウ球菌プロテインａに対する抗体に関連した組成物および方法
AU2014236451B2 (en) * 2013-03-14 2018-08-09 Regeneron Pharmaceuticals, Inc. Apelin fusion proteins and uses thereof
CN104371019B (zh) * 2013-08-13 2019-09-10 鸿运华宁（杭州）生物医药有限公司 一种能与glp-1r特异性结合的抗体及其与glp-1的融合蛋白质
CA2931299C (en) * 2013-11-20 2024-03-05 Regeneron Pharmaceuticals, Inc. Aplnr modulators and uses thereof
CN106659774A (zh) * 2014-05-16 2017-05-10 贝勒研究院 用于治疗自身免疫和炎性病症的方法和组合物
WO2016156570A1 (en) * 2015-04-02 2016-10-06 Ablynx N.V. Bispecific cxcr4-cd-4 polypeptides with potent anti-hiv activity
MX2020002042A (es) * 2017-08-24 2020-08-13 Phanes Therapeutics Inc Anticuerpos anti-apelin y sus usos.

Patent Citations (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4301144A (en) 1979-07-11 1981-11-17 Ajinomoto Company, Incorporated Blood substitute containing modified hemoglobin
US4331647A (en) 1980-03-03 1982-05-25 Goldenberg Milton David Tumor localization and therapy with labeled antibody fragments specific to tumor-associated markers
US4640835A (en) 1981-10-30 1987-02-03 Nippon Chemiphar Company, Ltd. Plasminogen activator derivatives
US4737462A (en) 1982-10-19 1988-04-12 Cetus Corporation Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of interferon-β
US4518584A (en) 1983-04-15 1985-05-21 Cetus Corporation Human recombinant interleukin-2 muteins
US4496689A (en) 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
US4751180A (en) 1985-03-28 1988-06-14 Chiron Corporation Expression using fused genes providing for protein product
US4670417A (en) 1985-06-19 1987-06-02 Ajinomoto Co., Inc. Hemoglobin combined with a poly(alkylene oxide)
US4935233A (en) 1985-12-02 1990-06-19 G. D. Searle And Company Covalently linked polypeptide cell modulators
US4791192A (en) 1986-06-26 1988-12-13 Takeda Chemical Industries, Ltd. Chemically modified protein with polyethyleneglycol
US5011912A (en) 1986-12-19 1991-04-30 Immunex Corporation Hybridoma and monoclonal antibody for use in an immunoaffinity purification system
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
WO1993010151A1 (en) 1991-11-22 1993-05-27 Immunex Corporation Receptor for oncostatin m and leukemia inhibitory factor
WO1994010308A1 (en) 1992-10-23 1994-05-11 Immunex Corporation Methods of preparing soluble, oligomeric proteins
US5457035A (en) 1993-07-23 1995-10-10 Immunex Corporation Cytokine which is a ligand for OX40
US6133426A (en) 1997-02-21 2000-10-17 Genentech, Inc. Humanized anti-IL-8 monoclonal antibodies
US6703199B1 (en) 1997-06-12 2004-03-09 Research Corporation Technologies, Inc. Artificial antibody polypeptides
US6846634B1 (en) 1997-10-20 2005-01-25 Domantis Limited Method to screen phage display libraries with different ligands
US20050202512A1 (en) 1997-10-20 2005-09-15 Domantis Limited Method to screen phage display libraries with different ligands
US6696245B2 (en) 1997-10-20 2004-02-24 Domantis Limited Methods for selecting functional polypeptides
US20040038291A2 (en) 1997-10-20 2004-02-26 Domantis Limited Method to screen phage display libraries with different ligands
CN1283228A (zh) * 1997-12-24 2001-02-07 武田药品工业株式会社 多肽、其生产方法及应用
US6210924B1 (en) 1998-08-11 2001-04-03 Amgen Inc. Overexpressing cyclin D 1 in a eukaryotic cell line
US20030039958A1 (en) 1999-12-03 2003-02-27 Domantis Limited Direct screening method
US20040009507A1 (en) 2000-10-13 2004-01-15 Domantis, Ltd. Concatenated nucleic acid sequence
US20050238646A1 (en) 2001-01-17 2005-10-27 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US20050075275A1 (en) * 2001-08-06 2005-04-07 Bayer Aktiengesellschaft Regulation of the apj receptor for use in the treatment or prophylaxis of cardiac diseases
US20040202995A1 (en) 2003-04-09 2004-10-14 Domantis Nucleic acids, proteins, and screening methods
WO2006023893A2 (en) * 2004-08-23 2006-03-02 Arizona Board Of Regents On Behalf Of The University Of Arizona Methods for modulating angiogenesis and apoptosis with apelin compositions
WO2012102363A1 (ja) * 2011-01-28 2012-08-02 国立大学法人大阪大学 薬物送達システムおよびその利用
WO2015140296A2 (en) * 2014-03-20 2015-09-24 Centre National De La Recherche Scientifique (Cnrs) Use of compounds inhibiting apelin / apj / gp130 signaling for treating cancer
WO2016061141A1 (en) * 2014-10-13 2016-04-21 University Of Maryland, Baltimore Methods for treating cardiovascular dysfunction and improving fluid homeostasis with a peptide hormone

Non-Patent Citations (78)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", 1995, JOHN WILEY & SONS, INC.
"Proteins, Structures and Molecular Principles", 1984, W. H. FREEMAN AND COMPANY
"Remington's Pharmaceutical Sciences", 1980, MACK PUBLISHING COMPANY
ANDREWS, S. M.TITUS, J. A. ET AL.: "Current Protocols in Immunology", 2003, JOHN WILEY & SONS, pages: 281 - 2810,210A1-210A5
ASHKENAZI ET AL., PNAS USA, vol. 88, 1991, pages 10535
BAINES ET AL.: "Methods in Molecular Biology", vol. 10, 1992, THE HUMANA PRESS, INC., article "Purification of Immunoglobulin G (IgG", pages: 79 - 104
BARON ET AL., NUCLEIC ACIDS RES, vol. 23, 1995, pages 3605 - 06
BAUER ET AL., GENE, vol. 37, 1985, pages 73
BAUM ET AL., EMBO J, vol. 13, 1994, pages 3992 - 4001
BOLOGNA ET AL., BIOMOL. THER., vol. 25, 2017, pages 12 - 25
BOUCHER ET AL., ENDOCRINOLOGY, vol. 146, 2005, pages 1764 - 71
BOWIE ET AL., SCIENCE, vol. 253, 1991, pages 164 - 170
BRENNER, CURR. OP. STRUCT. BIOL., vol. 7, 1997, pages 369 - 376
BYRN ET AL., NATURE, vol. 344, 1990, pages 677
CHNG ET AL., DEV. CELL, vol. 27, 2013, pages 672 - 680
CHOU ET AL., ADV. ENZYMOL. RELAT. AREAS MOL. BIOL., vol. 47, 1978, pages 45 - 148
CHOU ET AL., ANN. REV. BIOCHEM., vol. 47, 1979, pages 251 - 276
CHOU ET AL., BIOCHEMISTRY, vol. 113, 1974, pages 211 - 222
CHOU ET AL., BIOPHYS. J., vol. 26, pages 367 - 384
COURTENAY-LUCK ET AL.: "Monoclonal Antibodies: Production, Engineering and Clinical Application", 1995, CAMBRIDGE UNIVERSITY PRESS, article "Genetic Manipulation of Monoclonal Antibodies", pages: 166
CRAIK, BIOTECHNIQUES, vol. 3, 1985, pages 12 - 19
CRAMERI ET AL., NATURE, vol. 391, 1998, pages 288 - 291
DE GRAAF ET AL., METHODS MOL. BIOL., vol. 178, 2002, pages 379 - 87
EDELMAN ET AL.: "Methods in Enzymology", vol. 1, 1967, ACADEMIC PRESS, pages: 422
FANSLOW ET AL., SEMIN. IMMUNOL., vol. 6, 1994, pages 267 - 78
GLUZMAN ET AL., CELL, vol. 23, 1981, pages 175
GRIBSKOV ET AL., METH. ENZYM., vol. 183, 1990, pages 146 - 159
GRIBSKOV ET AL., PROC. NAT. ACAD. SCI., vol. 84, 1987, pages 4355 - 4358
HARRIS, R. J., JOURNAL OF CHROMATOGRAPHY, vol. 705, 1995, pages 129 - 134
HOLLENBAUGH ET AL.: "Construction of Immunoglobulin Fusion Proteins", CURRENT PROTOCOLS IN IMMUNOLOGY, 1992, pages 10191 - 101911
HOLLIGER ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 90, 1993, pages 6444 - 48
HOLLINGERHUDSON, NATURE BIOTECHNOLOGY, vol. 23, no. 9, 2005, pages 1126 - 1136
HOLM ET AL., NUCL. ACID. RES., vol. 27, 1999, pages 244 - 247
HOPP ET AL., BIO/TECHNOLOGY, vol. 6, 1988, pages 1204
HOPPE ET AL., FEBS LETTERS, vol. 344, 1994, pages 191
HUSTON ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 85, 1988, pages 5879 - 83
HUSTON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 83
JONES, CURR. OPIN. STRUCT. BIOL., vol. 7, 1997, pages 377 - 87
KAWAMATA ET AL., BIOCHEM. BIOPHYS. ACTA., vol. 1538, 2001, pages 162 - 71
KLEIZ ET AL., REGUL. PEPT., vol. 126, 2005, pages 233 - 40
KORNDORFER ET AL., PROTEINS, vol. 53, 2003, pages 121 - 129
KORTT ET AL., PROT. ENG., vol. 10, 1997, pages 423
KRIANGKUM ET AL., BIOMOL. ENG., vol. 18, 2001, pages 31 - 108
LANDSCHULZ ET AL., SCIENCE, vol. 240, 1988, pages 1759 - 426
LARRICK ET AL., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, vol. 2, 1991, pages 106 - 3242
LUNDE ET AL., BIOCHEM. SOC. TRANS., vol. 30, 2002, pages 500 - 06
MANIATIS ET AL., SCIENCE, vol. 236, 1987, pages 1237
MARKS ET AL., BIOLTECHNOLOGY, vol. 10, 1992, pages 779 - 783
MCMAHAN ET AL., EMBO J, vol. 10, 1991, pages 2821
MEDHURST ET AL., J. NEUROCHEM., vol. 84, 2003, pages 1162 - 72
MOULT, CURR. OP. BIOTECH., vol. 7, 1996, pages 422 - 427
MURZA ET AL., J MED. CHEM., vol. 59, 2016, pages 2962 - 72
MURZA ET AL., J. MED. CHEM., vol. 59, 2016, pages 2962 - 72
NISONOFFET ET AL., ARCH. BIOCHEM. BIOPHYS., vol. 89, 1960, pages 230
NYGRENUHLEN, CURRENT OPINION IN STRUCTURAL BIOLOGY, vol. 7, 1997, pages 463 - 469
O'DOWD ET AL., GENE, vol. 136, 1993, pages 355 - 60
PATTEN ET AL., CURR. OPIN. BIOTECHNOL., vol. 8, 1997, pages 724 - 733
PAULI ET AL., SCIENCE, vol. 343, 2014, pages 1248636
POLJAK ET AL., STRUCTURE, vol. 2, 1994, pages 1121 - 23
PORTER: "73", BIOCHEM. J., 1959, pages 119
POUWELS ET AL.: "Cloning Vectors: A Laboratory Manual", 1985, ELSEVIER
RASMUSSEN ET AL., CYTOTECHNOLOGY, vol. 28, 1998, pages 31
ROQUE ET AL., BIOTECHNOL. PROG., vol. 20, 2004, pages 639 - 654
SAMBROOKFRITSCHMANIATIS: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS, pages: 631 - 636
See also references of EP3808847A4
SIPPL ET AL., STRUCTURE, vol. 4, 1996, pages 15 - 19
THOMPSON ET AL., J. MOL. BIOL., vol. 256, 1996, pages 350 - 368
THORNTON ET AL., NATURE, vol. 354, 1991, pages 105
URLAUB ET AL., PROC. NATL. ACAD. SCI. USA, vol. 77, 1980, pages 4216 - 20
VAUGHAN ET AL., NATURE BIOTECHNOLOGY, vol. 16, 1998, pages 535 - 539
VOSS ET AL., TRENDS BIOCHEM. SCI., vol. 11, 1986, pages 287
WALDER ET AL., GENE, vol. 42, 1986, pages 133
WARD ET AL., NATURE, vol. 334, 1989, pages 544 - 546
WARD ET AL.: "Monoclonal Antibodies: Principles and Applications", 1995, WILEY-LISS, INC., article "Genetic Manipulation and Expression or Antibodies", pages: 137
YANG ET AL., CIRCULATION, vol. 135, 2017, pages 1160 - 1173
YANG ET AL., J. MOL. BIOL., vol. 254, 1995, pages 392 - 403
YUE ET AL., AM. J. PHYSIOL. ENDOCRINOL. METAB., vol. 298, 2010, pages E59 - 67
ZHANG ET AL., CELL PHYSIOL BIOCHEM, vol. 48, 2018, pages 1347 - 1354

Also Published As

Publication number Publication date
JP2021526833A (ja) 2021-10-11
AU2019284320A1 (en) 2021-02-04
EP3808847A1 (en) 2021-04-21
EP3808847A4 (en) 2022-08-31
CN110655577A (zh) 2020-01-07
KR20210019535A (ko) 2021-02-22
CA3103585A1 (en) 2019-12-19
US20210163614A1 (en) 2021-06-03

Similar Documents

Publication Publication Date Title
US11712470B2 (en) Human endothelin receptor antibody and use thereof
WO2021052349A1 (zh) Gipr抗体及其与glp-1的融合蛋白质，以及其药物组合物和应用
US11780916B2 (en) GIPR antibody and GLP-1 fusion protein thereof, and pharmaceutical composition and application thereof
WO2021008519A1 (zh) ETA抗体与TGF-βTrap的融合蛋白质，以及其药物组合物和应用
US11542336B2 (en) GCGR antibody and GLP-1 fusion protein thereof, pharmaceutical composition thereof and application thereof
WO2017206840A1 (zh) Etar抗体，其药物组合物及其应用
WO2019238093A1 (zh) APJ抗体及其与Elabela的融合蛋白质，以及其药物组合物和应用
WO2020248967A1 (zh) Eta抗体与bnp的融合蛋白质，以及其药物组合物和应用
WO2024051802A1 (zh) Gipr抗体及其与fgf21的融合蛋白，以及其药物组合物和应用
WO2022206857A1 (zh) 一种能与人内皮素受体特异性结合的抗体及其在糖尿病肾病和慢性肾病治疗中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19819592

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3103585

Country of ref document: CA

Ref document number: 2020569130

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 20217000999

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2019819592

Country of ref document: EP

Effective date: 20210113

ENP Entry into the national phase

Ref document number: 2019284320

Country of ref document: AU

Date of ref document: 20190613

Kind code of ref document: A