APJ antibody, fusion protein of APJ antibody and Elabela, and pharmaceutical composition and application of APJ antibody and Elabela

Abstract

Provided herein are APJ antibodies and their fusion proteins with Elabela. Also provided herein are pharmaceutical compositions of an APJ antibody and an Elabela fusion protein. Further provided herein are methods of using APJ antibodies and Elabela fusion proteins for treating, preventing, or ameliorating one or more symptoms of pulmonary arterial hypertension, pulmonary hypertension, or heart failure.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of the Chinese invention patent application with application number 201810606914.0, application date June 13, 2018, and invention name “APJ antibody and its fusion protein with Elabela, as well as pharmaceutical compositions and applications thereof”. The contents of the Chinese invention patent application are incorporated herein by reference in their entirety. Technical Field

[0003] Provided herein are APJ antibodies and fusion proteins thereof with Elabela. Also provided herein are pharmaceutical compositions of APJ antibodies and Elabela fusion proteins. Further provided herein are 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. Background of the Invention

[0005] APJ contains 7 transmembrane units and 308 amino acids, and is a member of the G protein coupled receptor (GPCR) family. Apelin, a vasoactive peptide, is the earliest discovered 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 lungs, heart, and breast (Kawamata et al., 2001, Biochem. Biophys. Acta. 1538: 162-71; Medhurst et al., 2003, J. Neurochem. 84: 1162-72), and are particularly expressed in cardiovascular endothelial cells and heart tissue (Kleiz et al., 2005, Regul. Pept. 126: 233-40). Many studies have shown that the Apelin / APJ signaling system has the functions of enhancing myocardial contractility, lowering blood pressure, promoting angiogenesis, regulating the body's immune response and the release of pituitary-related hormones, and regulating insulin secretion. It is also involved in the pathophysiological processes of diabetic vascular disease, heart failure, atrial fibrillation, and ischemia-reperfusion injury.

[0006] Elabela is another endogenous ligand of APJ receptor that has attracted much attention 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 a non-coding RNA. However, studies have found that Elabela contains a conserved ORF that can encode a 54-amino acid protein, and its mature form is composed of only 32 amino acids. The Elabela / APJ signaling pathway has been shown to play a very critical role in the development of the embryonic heart and vascular system. Studies have shown that Elabela and Elabela mutants can activate the Gαi1 and β-arrestin2 signaling pathways of the APJ receptor. Furthermore, mutations at the C-terminus of the Elabela polypeptide can cause a preference for APJ receptor signaling pathway conduction (Murza et al., 2016, J. Med. Chem. 59: 2962-72), and the use of receptor signaling pathway conduction preference is an important direction for new drug development (Bologna et al., 2017, Biomol. Ther. 25: 12-25). By using isolated perfused hearts and in vivo hemodynamic and echocardiographic measurements, it was found that Elabela or Elabela mutants can reduce arterial pressure and produce a positive inotropic effect on the heart (Yang et al., 2017, Circulation 135: 1160–1173; Murza et al., 2016, J. Med. Chem. 59: 2962-72).

[0007] The fusion administration of Elabela and APJ antibodies can significantly prolong the half-life of Elabela to retain the biological activity of the Elabela molecule. At the same time, the fusion protein formed by the antibody that specifically binds Elabela and APJ has the molecular targeting provided by the antibody, which helps to improve the drugability of the Elabela fusion protein and play a role in treating one or more diseases including pulmonary arterial hypertension, pulmonary hypertension, and heart failure. Summary of the invention

[0008] Provided herein are antibodies that specifically bind to APJ.

[0009] The present invention also provides an antibody that can specifically bind to APJ, wherein the antibody 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:

[0010] a. Light chain CDR1 amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 10;

[0011] b. Light chain CDR2 amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 8;

[0012] c. Light chain CDR3 amino acid sequences: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 11;

[0013] d. Heavy chain CDR1 amino acid sequences: SEQ ID NO: 12, SEQ ID NO: 15, and SEQ ID NO: 18;

[0014] e. Heavy chain CDR2 amino acid sequences: SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21; and

[0015] f. Heavy chain CDR3 amino acid sequences: SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO: 22.

[0016] The present invention provides an Elabela fusion protein, which comprises an antibody that can specifically bind 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 to the carboxyl terminus of an APJ antibody light chain or heavy chain through a peptide linker sequence (Linker), wherein each Elabela fragment is independently a forward Elabela fragment or a mutant thereof; or the fusion protein connects the carboxyl terminus of an Elabela fragment to the amino terminus of an APJ antibody light chain or heavy chain through a peptide linker sequence (Linker), wherein each Elabela fragment is independently a reverse Elabela fragment or a mutant thereof.

[0017] The present invention provides an Elabela fusion protein, which comprises an antibody that can specifically bind to APJ, two Elabela fragments and two peptide linker sequences (Linker); the fusion protein connects the amino terminus of an Elabela fragment to the carboxyl terminus of an APJ antibody light chain through a peptide linker sequence (Linker): N'-R-Linker-Elabela-C'; or connects the amino terminus of an Elabela fragment to the carboxyl terminus of an APJ antibody heavy chain through a peptide linker sequence (Linker): N'-R-Linker-Elabela-C'; wherein: 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 chain or heavy chain of the APJ antibody, and Linker represents the peptide linker sequence.

[0018] Provided herein is a polynucleotide encoding an Elabela fusion protein described herein.

[0019] Provided herein is a vector comprising a polynucleotide encoding an Elabela fusion protein described herein.

[0020] Provided herein is a host cell comprising a vector described herein.

[0021] Provided herein is a pharmaceutical composition comprising an APJ antibody or an Elabela fusion protein described herein, and a pharmaceutically acceptable carrier.

[0022] Provided herein is a use of an APJ antibody or an Elabela fusion protein described herein in the preparation of a medicament for treating, preventing or ameliorating pulmonary hypertension and pulmonary hypertension-related disorders.

[0023] Provided herein is a use of an APJ antibody or an Elabela fusion protein described herein in the preparation of a medicament for treating, preventing or ameliorating pulmonary hypertension and pulmonary hypertension-related disorders.

[0024] Provided herein is a use of an APJ antibody or an Elabela fusion protein described herein in the preparation of a medicament for treating, preventing or ameliorating heart failure and heart failure-related disorders.

[0025] Provided herein is a use of an APJ antibody or an Elabela fusion protein described herein in the preparation of a medicament for simultaneously treating, preventing or ameliorating two or more diseases of pulmonary arterial hypertension, pulmonary hypertension or heart failure.

[0026] Provided herein are methods 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.

[0027] Provided herein are methods of 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.

[0028] Provided herein are methods 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figures 1A to 1D : Shows the results of flow cytometry (FACS) detection of the specific binding of recombinantly expressed hAPJ antibodies 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) to hAPJ (at an antibody concentration of 500 nM or 4 nM), wherein the gray peak is a negative control, representing the binding curve of L1H1 or L4H4 to CHO-DHFR-, and the solid line peak represents the binding curve of L1H1 ( Figure 1A , Figure 1B ) or L4H4( Figure 1C , Figure 1D ) and CHO-DHFR-hAPJ binding curve.

[0031] Figure 2 : Shows the activation curves of the reporter gene experiment detecting the fusion protein of the recombinant hAPJ antibody 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), and the fusion protein of L5H5 and Linker2-EA5 (which contains SEQ ID NO: 123 and SEQ ID NO: 94) to activate the Elabela / APJ signaling pathway, EC 50 Respectively: 3.6 and 2.6nM.

[0032] Figure 3: Shows the activation curves of the Elabela / APJ signaling pathway activated by reporter gene experiments detecting the fusion protein of the recombinant hAPJ antibody L5H5 and Linker2-EA1 (which contains SEQ ID NO: 123 and SEQ ID NO: 103), and the fusion protein of L5H5 and Linker2-EA2 (which contains SEQ ID NO: 123 and SEQ ID NO: 107). 50 Respectively: 5.0 and 2.2nM.

[0033] Figure 4 : Shows the activation curves of the reporter gene experiment detecting the fusion protein of the recombinant hAPJ antibody L5H5 and Linker2-EA3 (which contains SEQ ID NO: 123 and SEQ ID NO: 109), and the fusion protein of L5H5 and Linker2-EA4 (which contains SEQ ID NO: 123 and SEQ ID NO: 116) to activate the Elabela / APJ signaling pathway, EC 50 Respectively: 20.3 and 5.4nM. DETAILED DESCRIPTION

[0034] definition

[0035] Unless otherwise defined herein, scientific and technical terms associated with this document shall have the meanings understood by those of ordinary skill in the art. Generally, the nomenclature and techniques associated with pharmacology, biology, biochemistry, cell and tissue culture, biology, molecular biology, immunology, microbiology, genetics and protein nucleic acid chemistry and hybridization described herein are well known and frequently used in the art.

[0036] Standard single-letter and three-letter abbreviations are used herein to refer to polynucleotide and polypeptide sequences. When the polypeptide sequence is written, the first amino acid residue with an amino group (N') is at the leftmost and the last amino acid residue with a carboxyl group (C') is at the rightmost, for example, the forward Elabela fragment and its mutant sequences referred to herein: 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 NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121.The reverse polypeptide sequence refers to a sequence formed by reversing the order of appearance of the amino acids in the polypeptide sequence, for example, 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 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, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, and SEQ ID NO:155. The 5' end of the top strand of the Elabela single-stranded nucleic acid sequence and the double-stranded nucleic acid sequence is on the left and their 3' end is on the right. Specific parts of the polypeptide can be represented by amino acid residue numbers, such as amino acids 67 to 134, or by the actual residues at that position, such as Lys67 to Lys134. Specific polypeptide or polynucleotide sequences can also be described by explaining their differences from a reference sequence.

[0037] The term "subject" refers to an animal, including but not limited to primates (e.g., humans), cows, pigs, sheep, goats, horses, dogs, cats, rabbits, rats or mice. The terms "subject" and "patient" are used interchangeably, for example, to refer to a mammalian subject, such as a human subject, and in one embodiment, to a human.

[0038] The terms "treat", "treat", "treat" and "treat" include reducing or eliminating a disorder, disease or condition, or one or more symptoms associated with a disorder, disease or condition; or reducing or eliminating the lesions of a disorder, disease or condition.

[0039] The term "prevent" or "preventing" is meant to include delaying and / or ameliorating the onset of a disorder, disease or condition and / or its attendant symptoms; preventing a subject from acquiring a disorder, disease or condition; or reducing the risk of a subject acquiring a disorder, disease or condition.

[0040] The term "control" refers to preventing or slowing the progression, spread, or worsening of a condition, disorder, or disease, or one or more symptoms thereof (e.g., pain). Sometimes, an individual benefits from the beneficial effects of a preventive or therapeutic agent without resulting in a cure of the condition, disorder, or disease. In one embodiment, the term control refers to preventing or slowing the progression, spread, or worsening of pain of osteolysis.

[0041] The terms "therapeutically effective amount" and "effective amount" are meant to include an amount of a compound or combination of compounds that, when administered, is sufficient to prevent the development of or alleviate to some extent one or more symptoms of the disorder, disease or condition being treated. The term "therapeutically effective amount" or "effective amount" also refers to an amount of a compound that is sufficient to elicit the biological or medical response of a biomolecule (e.g., protein, enzyme, RNA or DNA), cell, tissue, system, animal or human being that is being sought by a researcher, veterinarian, medical doctor or clinician.

[0042] The term "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", "physiologically acceptable carrier" or "physiologically acceptable excipient" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, solvent or encapsulating material. In one embodiment, each "pharmaceutically acceptable" ingredient is compatible with the other ingredients of the pharmaceutical formulation and is suitable for contact with human and animal tissues or organs without excessive toxicity, irritation, allergic reaction, immunogenicity or other problems or complications, equivalent to a reasonable benefit / risk ratio.

[0043] The term "about" or "approximately" refers to an acceptable error determined by one skilled in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" refers to within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" refers to within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0044] The terms "peptide", "polypeptide", and "protein" all refer to molecules comprising two or more amino acids interconnected by peptide bonds. These terms encompass, for example, polypeptide analogs (e.g., muteins, variants, and fusion proteins) of natural and artificial proteins and protein sequences, as well as proteins that are post-transcriptionally or otherwise covalently or non-covalently modified. A peptide, polypeptide, or protein may be a monomer or a polymer.

[0045] The term "polypeptide fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion compared to the corresponding full-length protein. The fragment length 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, at most 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. The fragment can further include one or more additional amino acids at one or both ends, for example, amino acid sequences from different natural proteins (e.g., Fc or leucine zipper domains) or artificial amino acid sequences (e.g., artificial linker sequences).

[0046] The polypeptides herein include polypeptides modified for any reason and by any method, for example, to: (1) reduce proteolytic susceptibility, (2) reduce oxidative susceptibility, (3) alter affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or modify other physicochemical or functional properties. Analogs include muteins of the polypeptide. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) may be made in the native sequence (e.g., in the portion of the polypeptide outside the domain that forms intramolecular contacts). "Conservative amino acid substitutions" are those that do not significantly change the structural properties of the parent sequence (e.g., the replacement amino acid should not disrupt the helices present in the parent sequence or interfere with other secondary structure types that confer properties to the parent sequence or are necessary for its function).

[0047] A "variant" of a polypeptide comprises an amino acid sequence in which one or more amino acid residues are inserted, deleted and / or substituted relative to another polypeptide sequence. Variants herein include fusion proteins.

[0048] "Derivatives" of polypeptides are polypeptides that have been chemically modified, for example by conjugation to other chemical moieties such as polyethylene glycol, albumin (eg, human serum albumin), phosphorylation, and glycosylation.

[0049] Unless otherwise indicated, the term "antibody" includes antibodies 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 described below.

[0050] The term "antibody" is a protein comprising an antigen-binding portion and optionally a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the antibody to bind to the antigen. Examples of antibodies include complete antibodies, antibody fragments (e.g., antigen-binding portions of antibodies), antibody derivatives, and antibody analogs. The antibody may comprise, for example, an optional protein scaffold or an artificial scaffold with transplanted CDRs or CDRs derivatives. The scaffold includes, but is not limited to, an antibody-derived scaffold introduced, for example, to stabilize the three-dimensional structure of the antibody and a fully synthetic scaffold comprising, for example, a biocompatible polymer. See, for example, Korndorfer et al., 2003, Proteins 53: 121-9; Roque et al., 2004, Biotechnol. Prog. 20: 639-54. In addition, the antibody may be a peptidomimetic antibody ("PAMs") or a scaffold comprising a mimetic antibody that utilizes fibrinogen as a scaffold.

[0051] Antibodies can have the structure of, for example, natural immunoglobulins. "Immunoglobulins" are tetrameric molecules. In natural immunoglobulins, each tetramer is composed of two identical polypeptide chain pairs, 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 mainly related to antigen recognition. The carboxyl terminal portion of each chain determines the constant region mainly related to effector action. Human antibody light chains are divided into κ and λ light chains. The heavy chain is divided into μ, δ, α or ε, and determines the isotype of the antigen, for example, IgM, IgD, IgG, IgA, and IgE, respectively. In the light chain and the heavy chain, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about more than 10 amino acids. See, Fundamental Immunology Ch.7 (Paul edited, 2nd edition, Raven Press, 1989). The variable regions of each light / heavy chain pair form the antibody combining site, so that a complete immunoglobulin has two binding sites.

[0052] Natural immunoglobulin chains show the same basic structure of relatively conserved framework regions (FRs) connected by three highly variable regions, also known as complementary determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The distribution of amino acids in each domain is consistent with the definition of Kabat et al. in Sequences of Proteins of Immunological Interest, 5th edition, USDept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991.

[0053] Unless otherwise indicated, "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 intact antibodies. Antigen-binding portions include, in particular, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), fragments including complementary determining regions (CDRs), single-chain antibodies (scFv), chimeric antibodies, double-chain antibodies (diabodies), three-chain antibodies (triabodies), four-chain antibodies (tetrabodies) and polypeptides that contain at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide.

[0054] Fab fragment has V L 、V H , C L , and C H1 The F(ab')2 fragment is a bivalent fragment with two Fab fragments connected by a disulfide bond in the hinge region; the Fv fragment has a V H and V L Domain; dAb fragment has V H Domain, V L domain, or V H or V L Antigen-binding fragments of the leukemia / antigen domain (U.S. Pat. Nos. 6,846,634 and 6,696,245; U.S. Patent Application Publication Nos. 2005 / 0202512, 2004 / 0202995, 2004 / 0038291, 2004 / 0009507, and 2003 / 0039958; Ward et al., 1989, Nature 341:544-6.)

[0055] Single-chain antibody (scFv) is a fusion protein in which V L and V HThe regions are connected by a linker (e.g., a synthetic sequence of amino acid residues) to form an antibody contiguous protein, wherein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen-binding site (see, e.g., Bird et al., 1988, Science 242:423-6; and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).

[0056] Diabodies are bivalent antibodies comprising two polypeptide chains, each of which comprises a V H and V L Domain, the linker is so short that it does not allow the pairing of two domains on the same chain, thus allowing each domain 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 the double-chain antibody are identical, 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. Similarly, 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.

[0057] The complementary determining regions (CDRs) and framework regions (FRs) of a given antibody are identified herein using the method described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Edition, USDept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991. One or more CDRs can be covalently or non-covalently incorporated into a molecule to make it an antibody. An antibody can incorporate CDR(s) into a larger polypeptide chain. CDR(s) can be covalently linked to another polypeptide chain, or non-covalently incorporated into CDR(s). CDRs allow antibodies to specifically bind to a specific antigen of interest.

[0058] 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. For example, natural human immunoglobulins typically have two identical binding sites, while "bispecific" or "bifunctional" antibodies have two different binding sites.

[0059] The term "murine antibody" includes antibodies having one or more variable and constant regions derived from mouse immunoglobulin sequences.

[0060] The term "humanized antibody" refers to an antibody produced by transplanting the complementarity determining region sequence of a mouse antibody molecule into the framework of a human antibody variable region.

[0061] The term "antigen binding domain", "antigen binding region" or "antigen binding site" is the portion of an antibody that contains amino acid residues that interact with an antigen and contribute to the antibody's specificity and affinity 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.

[0062] The term "epitope" is the portion of a molecule that binds to an antibody (e.g., by an antibody). An epitope may comprise a non-contiguous portion of a molecule (e.g., in a polypeptide, non-contiguous amino acid residues in the primary sequence of a polypeptide are sufficiently close to each other in the tertiary and quaternary structures of the polypeptide to be bound by an antibody).

[0063] The "percent identity" of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (part of the GCG Wisconsin Package; version 10.3 (Accelrys, San Diego, CA)) using its default parameters.

[0064] The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably throughout the text and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs generated using nucleotide analogs (e.g., peptide nucleic acids and non-natural nucleotide analogs), and hybrids thereof. Nucleic acid molecules can be single-stranded or double-stranded. In one embodiment, the nucleic acid molecules herein comprise a continuous open reading frame encoding an antibody or fragment, derivative, mutein or variant thereof provided herein.

[0065] Two single-stranded polynucleotides are "complementary" to each other if their sequences can be arranged in antiparallel order, such that each nucleotide in one polynucleotide is opposite to the complementary nucleotide in the other polynucleotide, no gaps are introduced and there are no unpaired nucleotides at the 5' or 3' ends of each sequence. A polynucleotide is "complementary" to another polynucleotide if the two polynucleotides can hybridize to each other under moderately stringent conditions. Thus, a polynucleotide can be complementary to another polynucleotide without being its complementary sequence.

[0066] The term "vector" is a nucleic acid that can be used to introduce another nucleic acid connected to it into a cell. One type of vector is a "plasmid", which refers to a linear or circular double-stranded DNA molecule to which additional nucleic acid segments can be attached. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adenovirus-associated viruses), in which additional DNA segments can be introduced into the viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors containing bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell when introduced into the host cell and are therefore replicated together with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0067] A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression of the nucleotide sequence (e.g., the level, timing, or site of expression). A "regulatory sequence" is a nucleic acid that can affect the expression (e.g., the level, timing, or site of expression) of a nucleic acid to which it is operably linked. A regulatory gene, for example, acts directly on the regulated nucleic acid or through the action of one or more other molecules (e.g., a polynucleotide that binds to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology, Volume 185, Academic Press, San Diego, CA; and Baron et al., 1995, Nucleic Acids Res. 23:3605-6.

[0068] The term "host cell" is a cell for expressing nucleic acids, such as nucleic acids provided herein. The host cell can be a prokaryotic organism, such as Escherichia coli, or it can be a eukaryotic organism, such as a unicellular eukaryotic organism (e.g., yeast or other fungi), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma. Typically, 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 a 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 can 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 connected to the nucleic acid. It should be understood that the term host cell refers not only to a specific subject cell but also to the progeny or possible progeny of the cell. Due to, for example, mutations or environmental influences, certain modifications may occur in subsequent generations, and the progeny may in fact be different from the parent cell but still fall within the scope of the term used herein.

[0069] APJ receptor

[0070] APJ is a 7-transmembrane G protein-coupled receptor containing 377 amino acids (O'Dowd et al., 1993, Gene 136:355-60). Studies to date have found that APJ is widely distributed in the human central nervous system and various peripheral tissues such as lungs, heart, and breast (Kawamata et al., 2001, Biochem. Biophys. Acta. 1538:162-71; Medhurst et al., 2003, J. Neurochem. 84:1162-72), and is particularly expressed in cardiovascular endothelial cells and heart tissue (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 important in the regulation of insulin 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). As used herein, "human APJ" and "hAPJ" refer to human APJ and can be used interchangeably. As used herein, "mouse APJ" and "mAPJ" refer to mouse APJ and can also be used interchangeably.

[0071] In one embodiment, the antibody provided herein is an antibody that specifically binds to human APJ. In another embodiment, the fusion protein provided herein is an Elabela fusion protein that specifically binds to APJ on the cell membrane, and the fusion protein can activate the conduction of Elabela / APJ signals in these cells. In a further embodiment, 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 (e.g., monkeys and mice) and activate the signal transduction of Elabela / APJ in these species.

[0072] In one embodiment, the amino acid and polynucleotide sequences of APJ are listed below, and the sequence data are derived from the GeneBank database of the National Center for Biotechnology Information and the Uniprot database of the European Bioinformatics Institute: human (Homo sapiens) polynucleotide (SEQ ID NO: 55); accession number: X89271; human (Homo sapiens) amino acid (SEQ ID NO: 23); accession number: CAA61546; monkey (Rhesus macaque) polynucleotide (SEQ ID NO: 56); accession number: AF100206; monkey (Rhesus macaque) amino acid (SEQ ID NO: 24); accession number: AAC72404; rat (Rattus norvegicus) polynucleotide (SEQ ID NO: 57); accession number: AB033170; rat (Rattusnorvegicus) amino acid (SEQ ID NO: 25); accession number: BAA95002; mouse (Mus musculus) polynucleotide (SEQ ID NO: NO:58); Accession No.: AJ007612; and mouse (Mus musculus) amino acid (SEQ ID NO:26); Accession No.: CAB50696.

[0073] Vasoactive peptide receptor antibody (APJ antibody)

[0074] In one embodiment, provided herein is an APJ antibody. In one embodiment, provided herein is an APJ antibody. In another embodiment, provided herein is an APJ antibody fragment. In another embodiment, provided herein is an APJ antibody derivative. In another embodiment, provided herein is an APJ antibody mutein. In a further embodiment, provided herein is an APJ antibody variant.

[0075] In one embodiment, the 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:

[0076] a. Light chain CDR1 amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 10;

[0077] b. Light chain CDR2 amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 8;

[0078] c. Light chain CDR3 amino acid sequences: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 11;

[0079] d. Heavy chain CDR1 amino acid sequences: SEQ ID NO: 12, SEQ ID NO: 15, and SEQ ID NO: 18;

[0080] e. Heavy chain CDR2 amino acid sequences: SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21; and

[0081] f. Heavy chain CDR3 amino acid sequences: SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO: 22.

[0082] Table 1 lists the amino acid sequences of the light chain CDRs of the APJ antibodies provided herein, and their corresponding polynucleotide encoding sequences. Table 2 lists the amino acid sequences of the heavy chain CDRs of the APJ antibodies provided herein, and their corresponding polynucleotide encoding sequences.

[0083] Table 1: Amino acid sequences and polynucleotide encoding sequences of light chain CDRs

[0084]

[0085] Table 2: Amino acid sequences of heavy chain CDRs and their polynucleotide encoding sequences

[0086]

[0087]

[0088] In one embodiment, the antibodies provided herein comprise 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 additions, substitutions, and / or deletions. In another embodiment, the antibodies provided herein comprise 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 additions, substitutions, and 5 / or deletions.

[0089] 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 3, 2, or 1 single amino acid additions, substitutions and / or deletions.

[0090] In another embodiment, an antibody provided herein comprises a sequence that differs from one of the CDR 10 amino acid sequences listed in Tables 1 and 2 by 2 or 1 single amino acid additions, substitutions and / or deletions.

[0091] In further embodiments, the antibodies provided herein comprise a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by 1 single amino acid addition, substitution and / or deletion.

[0092] In one embodiment, the APJ antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences: 15a. Light chain CDR1 amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 10; and

[0093] b. Heavy chain CDR1 amino acid sequences: SEQ ID NO: 12, SEQ ID NO: 15, and SEQ ID NO: 18.

[0095] In another embodiment, the 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:

[0096] a. Light chain CDR2 amino acid sequence: SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 8; and

[0097] b. Heavy chain CDR2 amino acid sequences: SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, and SEQ ID NO:21.

[0098] In another embodiment, the 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:

[0099] a. Light chain CDR3 amino acid sequences: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 11; and

[0100] b. Heavy chain CDR3 amino acid sequences: SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO: 22.

[0101] In another embodiment, the 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:

[0102] a. Light chain CDR1 amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 10;

[0103] b. Heavy chain CDR1 amino acid sequences: SEQ ID NO: 12, SEQ ID NO: 15, and SEQ ID NO: 18;

[0104] c. Light chain CDR2 amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 8; and

[0105] d. Heavy chain CDR2 amino acid sequences: SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21.

[0106] In another embodiment, the 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:

[0107] a. Light chain CDR1 amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 10;

[0108] b. Heavy chain CDR1 amino acid sequences: SEQ ID NO: 12, SEQ ID NO: 15, and SEQ ID NO: 18;

[0109] c. Light chain CDR3 amino acid sequences: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 11; and

[0110] d. Heavy chain CDR3 amino acid sequences: SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO: 22.

[0111] In further embodiments, the APJ antibodies provided herein comprise one, two, three, or four amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0112] a. Light chain CDR2 amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 8;

[0113] b. Heavy chain CDR2 amino acid sequences: SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21;

[0114] c. Light chain CDR3 amino acid sequences: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 11; and

[0115] d. Heavy chain CDR3 amino acid sequences: SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO: 22.

[0116] In one embodiment, the APJ antibody provided herein comprises one, two, or three amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of 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, and SEQ ID NO: 11.

[0117] In another embodiment, the APJ antibody provided herein comprises one, two, or three amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of 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.

[0118] In one embodiment, the APJ antibody provided herein comprises a combination of light chain 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 and SEQ ID NO: 15, SEQ ID NO: 7 and SEQ ID NO: 18, and SEQ ID NO: 10 and SEQ ID NO: 18.

[0119] In another embodiment, the APJ antibody provided herein comprises a combination of light chain 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: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.

[0120] In a further embodiment, the APJ antibody provided herein comprises a combination of light chain 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: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.

[0121] In one embodiment, the APJ antibody provided herein comprises:

[0122] a. a combination of light chain and heavy chain CDR1 amino acid sequences independently selected from the following: SEQ ID NO: 1 and SEQ ID NO: 12, SEQ ID NO: 4 and SEQ ID NO: 15, SEQ ID NO: 7 and SEQ ID NO: 18, and SEQ ID NO: 10 and SEQ ID NO: 18; and

[0123] b. A combination of light chain and heavy chain CDR2 amino acid sequences 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 NO:8 and SEQ ID NO:19, and SEQ ID NO:5 and SEQ ID NO:21.

[0124] In another embodiment, the APJ antibody provided herein comprises:

[0125] a. a combination of light chain and heavy chain CDR1 amino acid sequences independently selected from the following: SEQ ID NO: 1 and SEQ ID NO: 12, SEQ ID NO: 4 and SEQ ID NO: 15, SEQ ID NO: 7 and SEQ ID NO: 18, and SEQ ID NO: 10 and SEQ ID NO: 18; and

[0126] b. A combination of light chain and heavy chain CDR3 amino acid sequences 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 NO:9 and SEQ ID NO:20, and SEQ ID NO:11 and SEQ ID NO:22.

[0127] In another embodiment, the APJ antibody provided herein comprises:

[0128] a. a combination of light chain and heavy chain CDR2 amino acid sequences 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 NO: 8 and SEQ ID NO: 19, and SEQ ID NO: 5 and SEQ ID NO: 21; and

[0129] b. A combination of light chain and heavy chain CDR3 amino acid sequences 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 NO:9 and SEQ ID NO:20, and SEQ ID NO:11 and SEQ ID NO:22.

[0130] In a further embodiment, the APJ antibody provided herein comprises:

[0131] a. a combination of light chain and heavy chain CDR1 amino acid sequences independently selected from the following: SEQ ID NO: 1 and SEQ ID NO: 12, SEQ ID NO: 4 and SEQ ID NO: 15, SEQ ID NO: 7 and SEQ ID NO: 18, and SEQ ID NO: 10 and SEQ ID NO: 18;

[0132] b. a combination of light chain 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: 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; and

[0133] c. a combination of light chain 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: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.

[0134] In one embodiment, the 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:

[0135] a. 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 the sequences thereof; and

[0136] b. 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 the sequences thereof.

[0137] In another embodiment, the polynucleotide coding sequence of the APJ antibody provided herein comprises one or two polynucleotide coding sequences, wherein each polynucleotide coding sequence is independently selected from the polynucleotide coding sequences listed below:

[0138] a. light chain variable domain polynucleotide encoding sequence: SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; and a polynucleotide encoding sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the sequences; and

[0139] b. Heavy chain variable domain polynucleotide encoding sequences: SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78; and polynucleotide encoding sequences that are at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the sequences.

[0140] In one embodiment, 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.

[0141] In another embodiment, 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.

[0142] In one embodiment, the APJ antibody provided herein comprises a combination of light chain and heavy chain variable domain amino acid sequences 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 NO:63 and SEQ ID NO:68 (L5H5).

[0143] The APJ antibodies provided herein may also be referred to by the notation "LxHy", wherein "x" corresponds to the light chain variable region sequence number; "y" corresponds to the heavy chain variable region sequence number. For example, L2H2 refers to an intact 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).

[0144] In one embodiment, the APJ antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences: a. light chain constant amino acid sequence: SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:81; and b. heavy chain constant amino acid sequence: SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84.

[0145] In another embodiment, the APJ antibody provided herein comprises a combination of light chain 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: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.

[0146] In one embodiment, the APJ antibody provided herein comprises the amino acid sequences of the light chain and heavy chain CDRs and FRs (framework) listed herein. The amino acid sequence of FRs is contained in the amino acid sequence of the light chain or heavy chain variable domain and is not separately displayed. In one embodiment, the antibody comprises a light chain CDR1 sequence listed herein. In another embodiment, the antibody comprises a light chain CDR2 sequence listed herein. In another embodiment, the antibody comprises a light chain CDR3 sequence listed herein. In another embodiment, the antibody comprises a heavy chain CDR1 sequence listed herein. In another embodiment, the antibody comprises a heavy chain CDR2 sequence listed herein. In another embodiment, the antibody comprises a heavy chain CDR3 sequence listed herein. In another embodiment, 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 light chain FR3 sequence herein. In another embodiment, the antibody comprises a light chain FR4 sequence herein. In another embodiment, the antibody comprises a heavy chain FR1 sequence described herein. In another embodiment, the antibody comprises a heavy chain FR2 sequence described herein. In another embodiment, the antibody comprises a heavy chain FR3 sequence described herein. In a further embodiment, the antibody comprises a heavy chain FR4 sequence described herein.

[0147] In one embodiment, 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 deletion. In another embodiment, 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 deletion. In a further embodiment, 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 deletion, and 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 deletion.

[0148] In one embodiment, the APJ antibody provided herein comprises a light chain variable domain amino acid sequence, which is a light chain variable domain sequence of L4 (SEQ ID NO: 62) or L5 (SEQ ID NO: 63) listed herein. In one embodiment, the light chain variable domain amino acid sequence of the APJ antibody differs from the light chain variable domain amino acid sequence of L4 (SEQ ID NO: 62) or L5 (SEQ ID NO: 63) by 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, wherein the difference of each sequence is independently a deletion, insertion or substitution of an amino acid residue. In another embodiment, the light chain variable domain amino acid sequence of the APJ antibody 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 to the light chain variable domain amino acid sequence of L4 (SEQ ID NO: 62) or L5 (SEQ ID NO: 63). In another embodiment, the light chain variable domain polynucleotide coding sequence of the APJ antibody comprises a polynucleotide coding sequence that 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 to the polynucleotide coding sequence of L4 (SEQ ID NO: 72) or L5 (SEQ ID NO: 73). In another embodiment, the light chain variable domain polynucleotide coding sequence of the APJ antibody comprises a polynucleotide sequence that hybridizes under moderate conditions to the complement of the polynucleotide coding sequence of the light chain variable domain of L4 (SEQ ID NO: 72) or L5 (SEQ ID NO: 73). In a further embodiment, the light chain variable domain polynucleotide coding sequence of the APJ antibody comprises a polynucleotide sequence that hybridizes under stringent conditions to the complement of the polynucleotide coding sequence of the light chain variable domain of L4 (SEQ ID NO: 72) or L5 (SEQ ID NO: 73).

[0149] In one embodiment, the APJ antibody provided herein comprises a heavy chain variable domain amino acid sequence, which is a H4 (SEQ ID NO: 67) or H5 (SEQ ID NO: 68) heavy chain variable domain sequence listed herein. In another embodiment, the heavy chain variable domain amino acid sequence of the APJ antibody differs from the heavy chain variable domain sequence of H4 (SEQ ID NO: 67) or H5 (SEQ ID NO: 68) by 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, wherein the difference in each sequence is independently a deletion, insertion or substitution of an amino acid residue. In another embodiment, the heavy chain variable domain amino acid sequence of the APJ antibody 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 to the heavy chain variable domain sequence of H4 (SEQ ID NO: 67) or H5 (SEQ ID NO: 68). In another embodiment, the APJ antibody heavy chain variable domain 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 to the heavy chain variable domain polynucleotide coding sequence of H4 (SEQ ID NO: 77) or H5 (SEQ ID NO: 78). In another embodiment, the APJ antibody heavy chain variable domain polynucleotide coding sequence comprises a polynucleotide sequence that hybridizes under moderate stringency conditions to the complement of the polynucleotide coding sequence of the heavy chain variable domain of H4 (SEQ ID NO: 77) or H5 (SEQ ID NO: 78). In one embodiment, the APJ antibody heavy chain variable domain polynucleotide coding sequence comprises a polynucleotide sequence that hybridizes under stringent conditions to the complement of the polynucleotide coding sequence of the heavy chain variable domain of H4 (SEQ ID NO: 77) or H5 (SEQ ID NO: 78).

[0150] In one embodiment, the antibody provided herein is an antibody comprising a combination of 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 and 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 a desired phenotype (e.g., IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE, or IgD) of one of them, or a Fab or F(ab')2 fragment thereof.

[0151] In one embodiment, 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-switched antibody thereof (e.g., IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE, and IgD), or a Fab or F(ab')2 fragment thereof.

[0152] 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 κ or λ type light chain constant region, such as a mouse κ or λ type light chain constant region. The heavy chain constant region may be, for example, an α, δ, ε, γ, or μ type heavy chain constant region, such as a mouse α, δ, ε, γ, or μ type heavy chain constant region. In one embodiment, the light chain or heavy chain constant region is a fragment, derivative, variant, or mutant protein of a natural constant region.

[0153] In one embodiment, the antibody provided herein further comprises a human constant light chain κ or a fragment thereof. The amino acid sequence of the light chain constant region is as follows:

[0154] Human constant light chain kappa domain amino acid sequence: SEQ ID NO: 80; and

[0155] Human constant light chain lambda domain amino acid sequence: SEQ ID NO:81.

[0156] In another embodiment, the antibody provided herein further comprises a human heavy chain constant domain or a fragment thereof. The amino acid sequence of the heavy chain constant region is as follows:

[0157] Human heavy chain constant region amino acid sequence (hIgG2): SEQ ID NO: 83; and

[0158] Human heavy chain constant region amino acid sequence (hIgG4): SEQ ID NO:84.

[0159] In one embodiment, the APJ antibody provided herein is selected from a murine antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single-chain antibody, a double-chain antibody, a three-chain antibody, a four-chain antibody, a Fab fragment, a F(ab')x fragment, a domain antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0160] In one embodiment, the APJ antibodies provided herein are APJ monoclonal antibodies.

[0161] In another embodiment, 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.

[0162] In one embodiment, the APJ antibody provided herein is a murine APJ antibody. In another embodiment, the APJ antibody provided herein is a humanized APJ antibody.

[0163] In one embodiment, the APJ antibodies provided herein have a Kd value of about 1 nM to about 200 nM or about 1 nM to about 100 nM.

[0164] Antibodies and Antibody Fragments

[0165] In one embodiment, the antibodies provided herein are complete antibodies (including polyclonal, monoclonal, chimeric, humanized, or human antibodies with full-length heavy and / or light chains). In another embodiment, 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, maximum antibodies (maxibodies), micro antibodies (minibodies), intrabodies (intrabodies), two-chain antibodies, three-chain antibodies, four-chain antibodies, v-NAR, or bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23: 1126-36). In another embodiment, the antibodies provided herein also include antibodies including antibody polypeptides disclosed in U.S. Patent No. 6703199, including fibronectin polypeptide monoclonal antibodies. In a further embodiment, the antibodies provided herein include single-chain polypeptides disclosed in U.S. Patent Publication 2005 / 0238646.

[0166] In one embodiment, the variable region of the monoclonal antibody gene is amplified using nucleotide primers for expression in the hybridoma. These primers can be synthesized by one of ordinary skill in the art or purchased from commercial sources. Mouse and human variable region primers include V Ha 、V Hb 、V Hc 、V Hd , C H1 、V L , and C LPrimers for the heavy chain and light chain variable regions can be purchased from commercial sources. These primers can be used to amplify the heavy chain or light chain variable region, which can then be inserted into a vector such as IMMUNOZAP. TM H or IMMUNOZAP TM L (Stratagene). These vectors are then introduced into E. coli, yeast, or mammalian based expression systems. These methods can be used to produce large quantities of V H and V L Domain fusion single-chain protein (see Bird et al., 1988, Science 242:423-6).

[0167] It will be appreciated by those skilled in the art that some proteins, such as antibodies, may undergo a variety of 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. Such modifications include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation. The carboxyl-terminal basic residues (e.g., lysine or arginine) of antibodies may be lost due to frequent modification by carboxypeptidases (see, Harris, 1995, Journal of Chromatography 705: 129-34).

[0168] Mouse monoclonal antibodies can be produced using a common hybridoma cell method. The monoclonal antibody can be separated and purified by a variety of established techniques. Such separation techniques include affinity chromatography, size exclusion chromatography, and ion exchange chromatography using protein A-agarose (see, e.g., Coligan pp. 2.7.1-2.7.12 and pp. 2.9.1-2.9.3; Baines et al., "Purification of Immunoglobulin G (IgG), " Methods in Molecular Biology, Vol. 10, pp. 79-104 (The Humana Press, Inc., 1992)). The monoclonal antibody can be purified by affinity chromatography using a suitable ligand screened based on the specific properties of the antibody (e.g., heavy chain or light chain isotype, binding specificity, etc.). Examples of suitable ligands for affinity chromatography include protein A, protein G, anti-constant region (light chain or heavy chain) antibodies, anti-idiotypic antibodies, and TGF-binding proteins or fragments or variants thereof.

[0169] The complementary determining regions (CDRs) in the center of the antibody binding site can be used to perform affinity maturation on the molecule to obtain antibodies with increased affinity, such as antibodies with increased affinity for c-erbB-2 (Schier et al., 1996, J. Mol. Biol. 263:551-67). Therefore, this type of technology can be used to prepare antibodies to human APJ.

[0170] For example, antibodies against human APJ can be used in in vitro or in vivo assays to detect the presence or absence of human APJ.

[0171] Antibodies can also be prepared by any conventional technique. For example, they can be purified from cells that naturally express these antibodies (e.g., they can be purified from hybridomas that produce the antibodies) or produced in a recombinant expression system using any technique known in the art. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al., ed., Plenum Press (1980); and Antibodies: A Laboratory Manual, Harlow and Land, ed., Cold Spring Harbor Laboratory Press (1988). This is discussed in the nucleic acid section below.

[0172] Antibodies can be prepared and screened for desired properties by any known technique. Some techniques involve isolating a nucleic acid encoding a polypeptide chain (or portion thereof) of an antibody of interest (e.g., an anti-APJ antibody) and manipulating the nucleic acid by recombinant DNA techniques. The nucleic acid can be fused to another related nucleic acid or modified (e.g., by mutagenesis or other conventional techniques) to add, delete or replace one or more amino acid residues.

[0173] When it is necessary to improve the affinity of an antibody according to the present invention comprising one or more of the above CDRs, it can be achieved by a variety of affinity maturation schemes including maintenance of CDRs (Yang et al., 1995, J. Mol. Biol. 254:392-403), chain replacement (Marks et al., 1992, Bio / Technology 10:779-83), use of mutant strains of E. coli (Low et al., 1996, J. Mol. Biol. 250:350-68) DNA shuffling (Patten et al., 1997, Curr. Opin. Biotechnol. 8:724-33), phage display (Thompson et al., 1996, J. Mol. Biol. 256:7-88) and other PCR techniques (Crameri et al., 1998, Nature 391:288-91). All of these affinity maturation methods are discussed in Vaughan et al., 1998, Nature Biotechnology 16:535-39.

[0174] In one embodiment, the antibodies provided herein are anti-APJ fragments. The fragments may consist entirely of antibody-derived sequences or may contain additional sequences. Examples of antigen-binding fragments include Fab, F(ab')2, single-chain antibodies, double-chain antibodies, three-chain antibodies, four-chain antibodies, and domain antibodies, other examples of which are provided in Lunde et al., 2002, Biochem. Soc. Trans. 30:500-06.

[0175] A single-chain antibody can be formed by connecting the heavy chain and light chain variable domains (Fv region) via an amino acid bridge (short peptide linker), thereby obtaining a single polypeptide chain. L and V H ) DNAs. The resulting polypeptides can fold back on themselves to form antigen-binding monomers, or they can form multimers (e.g., 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. 2001, Biomol. Eng. 18:95-108). By combining different V domains containing polypeptides L and V H , can form multimeric scFvs with different phenotypes (Kriangkum et al., 2001, Biomol. Eng. 18: 31-40). Technologies developed for producing single-chain antibodies include those described in U.S. Pat. No. 4,946,778; Bird, 1988, Science 242: 423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-83; Ward et al., 1989, Nature 334: 544-6; de Graaf et al., 2002, Methods Mol Biol. 178: 379-87. Single-chain antibodies derived from antibodies provided herein include, but are not limited to, scFvs comprising the variable domain combination L1H1, all of which are encompassed herein.

[0176] Antigen-binding fragments derived from antibodies can also be obtained by proteolysis of the antibody, for example, by digestion of intact antibodies with pepsin or papain according to conventional methods. For example, an antibody fragment can be produced by cleaving an antibody with pepsin to provide an SS fragment called F(ab')2. This fragment can be further cleaved using a sulfhydryl reducing agent to produce a 3.5S Fab' monovalent fragment. Optionally, the cleavage reaction can be performed using a sulfhydryl protecting group to obtain cleavage of the disulfide bonds; alternatively, enzymatic cleavage using papain can be used to directly produce two monovalent Fab fragments and an Fc fragment. These methods are described in, for example, Goldenberg, U.S. Pat. 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 (Coligan et al., ed., John Wiley & Sons, 2003), pp. 2.8, 1-2.8.10 and pp. 2.10A.1-2.10A.5. Other methods of cleaving antibodies, such as preparing heavy chains to form monovalent heavy and light chain fragments (Fd), further cleaving fragments or using other enzymes, chemicals or genetic techniques, as long as the fragments bind to an antigen that can be recognized by the intact antibody.

[0177] Another form of antibody fragment is a peptide comprising one or more antibody complementary determining regions (CDRs). CDRs can be obtained by constructing a polypeptide encoding the relevant CDR. For example, such polypeptides can be prepared by using the polymerase chain reaction to synthesize the variable region using the mRNA of antibody-producing cells 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., ed., 166 pages (Cambridge University Press, 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," Monoclonal Antibodies: Principles and Applications, Birch et al., ed., 137 pages (Wiley-Liss, Inc., 1995). The antibody fragment may further comprise at least one variable domain of an antibody described herein. Thus, for example, the V region domain may be a monomer and is a V H or V L domain, which may be at least equal to 1x10 -7 M or higher affinity binds independently to APJ.

[0178] The variable region domain may be any natural variable domain or a genetically engineered form thereof. The genetically engineered form refers to a variable region domain produced using recombinant DNA engineering techniques. The genetically engineered form includes, for example, one produced from a specific antibody variable region by inserting, deleting or altering the amino acid sequence of the 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 a variable region domain from another antibody and are assembled by genetic engineering.

[0179] The variable region domain may be covalently linked to at least one other antibody domain or fragment thereof at the C-terminal amino acid. Thus, for example, V present in the variable region domain H Immunoglobulin C H1 Similarly, V L The domain can be combined with C KIn this manner, for example, the antibody may be a Fab fragment, wherein the antigen binding domains comprise their C-termini and C- H1 and C K Domain covalently linked joint V H and V L Other amino acids can be used to extend the C H1 domains, for example to provide a hinge region or a portion of a hinge domain such as in a Fab' fragment or to provide other domains, such as an antibody C H2 and C H3 Structural domain.

[0180] Antibody derivatives and variants

[0181] For example, the nucleotide sequences encoding the amino acid sequences L1 and H1 can be altered by random mutagenesis or by site-directed mutagenesis (e.g., oligonucleotide-induced site-directed mutagenesis) to produce an altered polynucleotide comprising one or more specific nucleotide substitutions, deletions, or insertions compared to the non-mutated polynucleotide. Examples of techniques for producing such changes are described in Walder et al., 1986, Gene 42:133; Bauer et al., 1985, Gene 37:73; Craik, 1985, BioTechniques 3:12-9; Smith et al., 1981, Genetic Engineering: Principles and Methods, Plenum Press; and U.S. Pat. Nos. 4,518,584 and 4,737,462. These and other methods can be used to produce derivatives of anti-APJ antibodies that have desired properties, such as increased affinity, avidity, or specificity for APJ, increased in vivo or in vitro activity or stability, or reduced in vivo side effects compared to the underivatized antibody.

[0182] Other anti-APJ antibody derivatives in the art include covalent or aggregated conjugates of anti-APJ antibodies or fragments thereof with other proteins or polypeptides, for example, by expressing a recombinant fusion protein comprising a heterologous polypeptide fused to the N-terminus or C-terminus of an anti-APJ antibody polypeptide. For example, 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. The antibody comprising the fusion protein may comprise a peptide (e.g., polyhistidine) added to assist in the purification or identification of the antibody. The antibody may also be linked to a FLAG peptide, as described in Hopp et al., 1988, Bio / Technology 6:1204 and U.S. Pat. No. 5,011,912. The FLAG peptide has high antigenicity and provides an epitope that is reversibly bound by a specific monoclonal antibody (mAb), allowing rapid detection and convenient purification of expressed recombinant proteins. Reagents for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma-Aldrich, St. Louis, MO). In another embodiment, oligomers comprising one or more antibodies can be used as APJ antagonists or higher oligomers can be used. The oligomers can be in the form of covalently or non-covalently linked dimers, trimers, or higher oligomers. Oligomers comprising 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.

[0183] One embodiment is directed to oligomers comprising multiple antibodies, which are linked by covalent or non-covalent interactions between peptide moieties fused to the antibodies. Such peptides may be peptide spacers or peptides having properties that promote oligomerization. Leucine zippers and certain polypeptides derived from antibodies are peptides that promote antibody oligomerization, as described in detail below.

[0184] In a specific embodiment, the oligomer comprises two to four antibodies. The antibodies of the oligomer may be in any form, such as any form described above, such as a variant or fragment. Preferably, the oligomer comprises an antibody having APJ binding activity.

[0185] In one embodiment, oligomers are prepared using polypeptides derived from immunoglobulins. Preparations comprising heterologous polypeptides fused to different parts of antibody-derived polypeptides (including Fc domains) have 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 in Immunology, Suppl. 4, pp. 10.19.1-10.19.11. One embodiment herein is directed to a dimer comprising two fusion proteins produced by fusing an Elabela fragment of an anti-APJ antibody or a mutant binding fragment thereof to the Fc region of an antibody. The dimer can be prepared by, for example, inserting a gene encoding the fusion protein into an appropriate expression vector, expressing the fusion gene in a host cell transformed with the recombinant expression vector and allowing the expressed fusion protein to assemble like an antibody molecule, wherein the interchain disulfide bonds between the Fc parts form a dimer.

[0186] As used herein, the term "Fc polypeptide" includes polypeptides derived from the natural and mutant protein forms of the Fc region of an antibody. It also includes truncated forms of such polypeptides that include a hinge region that promotes dimerization. Fusion proteins containing the Fc portion (and oligomers formed therefrom) provide the advantage of convenient purification by affinity chromatography on a protein A or protein G column.

[0187] One suitable Fc polypeptide in PCT application WO 93 / 10151 (incorporated herein by reference) is a single chain polypeptide extending 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 mutant protein described in U.S. Pat. No. 5,457,035 and Baum et al., 1994, EMBO J. 13: 3992-4001. The amino acid sequence of the mutant protein is identical to the amino acid sequence of the native Fc sequence shown in WO 93 / 10151, except that amino acid 19 is changed from leucine to alanine, amino acid 20 is changed from leucine to glutamine, and amino acid 22 is changed from glycine to alanine. The mutant protein exhibits reduced affinity for Fc receptors. In other embodiments, the heavy chain and / or light chain of the anti-APJ antibody can be substituted with the variable portion of the heavy chain and / or light chain of the antibody.

[0188] Alternatively, the oligomer is a fusion protein comprising multiple antibodies, with or without spacer peptides. Suitable spacer peptides are described in US Pat. Nos. 4,751,180 and 4,935,233.

[0189] Another method for preparing oligomeric antibodies involves the use of leucine zippers. Leucine zipper domains are peptides that promote the oligomerization of proteins in which they are present. Leucine zippers were initially found to be present in several DNA binding proteins (Landschulz et al., 1988, Science 240: 1759), and have since been found to be present in a variety of different proteins. Among the known leucine zippers are natural peptides or derivatives thereof that can be dimerized or trimerized. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94 / 10308, and leucine zippers derived from lung surfactant protein D (SPD) are described in Hoppe et al., 1994, FEBS Letters 344: 191, incorporated herein by reference. The use of modified leucine zippers that allow stable trimerization of heterologous proteins fused thereto is described in Fanslow et al., 1994, Semin. Immunol. 6: 267-78. In one method, a recombinant fusion protein comprising an anti-APJ antibody fragment or derivative fused to a leucine zipper peptide is expressed in a suitable host cell, and soluble oligomeric anti-APJ antibody fragments or derivatives thereof are collected from the culture supernatant.

[0190] In another embodiment, the antibody derivative may comprise at least one of the CDRs disclosed herein. For example, one or more CDRs may be integrated into known antibody framework regions (IgG1, IgG2, etc.) or combined with an appropriate carrier to enhance its half-life. Suitable carriers include, but are not limited to, Fc, albumin, transferrin and the like. These and other suitable carriers are known in the art. The CDR-binding peptide may be a monomer, dimer, tetramer or other form. In one embodiment, one or more water-soluble polymers are bound at one or more specific sites of the binding agent, for example at the amino terminus. In one example, the antibody derivative comprises one or more water-soluble polymer attachments including, but not limited to, polyethylene glycol, polyoxyethylene glycol or polypropylene glycol. See, e.g., U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337. In some embodiments, the derivatives include one or more monomethoxy polyethylene glycols, dextran, cellulose or other carbohydrate-based polymers, poly(N-vinyl pyrrolidone), polyethylene glycol, polyoxyethylene polyols (e.g., glycerol), and polyvinyl alcohol, as well as mixtures of such polymers. In some embodiments, one or more water-soluble polymers are randomly bound to one or more side chains. In some embodiments, PEG can enhance the therapeutic effect of binding agents such as antibodies. Some such methods are described, e.g., in U.S. Pat. No. 6,133,426, which is incorporated herein by reference for any purpose.

[0191] It should be understood that the antibodies provided herein may have at least one amino acid replacement, as long as the antibody retains binding specificity. Therefore, the modification of antibody structure is included in the scope of this article. These may include amino acid replacements that do not destroy the APJ binding ability of the antibody, which may be conservative or non-conservative. Conservative amino acid replacements may include non-natural amino acid residues, which are usually integrated through chemical peptide synthesis rather than synthesized by biological systems. These include peptidomimetics and other reverse or inverted forms of amino acid parts. Conservative amino acid replacements may also involve replacing natural amino acid residues with non-natural residues so that the polarity or charge of the amino acid residues at the site has little or no effect. Non-conservative replacements may involve a member of a class of amino acids or amino acid analogs exchanged with a member of another class of amino acids with different physical properties (e.g., volume, polarity, hydrophobicity, charge).

[0192] Moreover, those skilled in the art can generate variants to be tested that include amino acid substitutions on each desired amino acid residue. Such variants can be screened using activity assays known to those skilled in the art. Such variants can be used to collect information about appropriate variants. For example, if a certain amino acid residue is found to cause activity destruction, undesirable reduction or inappropriate activity, variants with such changes can be avoided. In other words, based on the information collected from these routine tests, those skilled in the art can easily determine that further replacements (alone or in combination with other mutations) should be avoided.

[0193] The skilled person can use known techniques to determine the appropriate variants of the polypeptides as listed herein. In some embodiments, those skilled in the art can identify appropriate regions of the molecule that will not destroy the activity after the change by targeting regions that are not important for activity. In some embodiments, residues or molecular parts that are conserved in similar polypeptides can be identified. In some embodiments, even conservative replacements can be made for regions that are important for biological activity or structure without destroying biological activity or having adverse effects on the polypeptide structure. In addition, those skilled in the art can examine residues in similar polypeptides that are important for activity or structure in structure. In view of this comparison, the importance of amino acid residues in proteins corresponding to amino acid residues that are important for activity or structure in similar proteins can be predicted. Those skilled in the art can select chemically similar amino acid replacements for these predicted important amino acid residues.

[0194] Those skilled in the art can also analyze the three-dimensional structure and amino acid sequence related to the structure of similar polypeptides. In view of this type of information, those skilled in the art can predict the amino acid residue alignment of the antibody in terms of the three-dimensional structure. In some embodiments, those skilled in the art may choose not to significantly change the amino acid residues predicted to be on the surface of the protein because such residues may be involved in important interactions with other molecules. Many scientific publications are devoted to the prediction of secondary structure. See, Moult, 1996, Curr. Op. Biotech. 7: 422-7; Chou et al., 1974, Biochemistry 13: 222-45; Chou et al., 1974, Biochemistry 113: 211-22; Chou et al., 1978, Adv. Enzymol. Relat. Areas Mol. Biol. 47: 45-148; Chou et al., 1979, Ann. Rev. Biochem. 47: 251-76 and Chou et al., Biophys. J. 26: 367-84. In addition, computer programs are currently available to assist in predicting secondary structure. For example, two polypeptides or proteins with a sequence identity greater than 30% or a similarity greater than 40% generally have similar higher-order structures. The recent growth of the protein structure database (PDB) has enhanced the predictability of secondary structure, including the potential number of folds in a polypeptide or protein structure. See, Holm et al., 1999, Nucl. Acid. Res. 27: 244-7. It has been shown (Brenner et al., 1997, Curr. Op. Struct. Biol. 7: 369-76) that there are a limited number of folds in a given polypeptide or protein and once a critical number of structures are determined, structural predictions will become significantly more accurate.

[0195] Other methods for predicting secondary structure include "threading" (Jones, 1997, Curr. Opin. Struct. Biol. 7:377-87; Sippl et al., 1996, Structure 4:15-9), "profile analysis" (Bowie et al., 1991, Science 253:164-70; Gribskov et al., 1990, Meth. Enzym. 183:146-59; Gribskov et al., 1987, Proc. Nat. Acad. Sci. USA 84:4355-8), and "evolutionary linkage" (see Holm, supra (1999), and Brenner, supra (1997)). In some embodiments, antibody variants include glycosylation variants in which the number and / or type of glycosylation sites are altered 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 compared to the native protein. Alternatively, the replacement of the sequence can remove existing N-linked sugar chains. The rearrangement of N-linked sugar chains is also provided, wherein one or more N-linked sugar chain sites (usually those occurring naturally) are removed and one or more new N-linked sites are created. Other preferred antibody variants include cysteine ​​variants, wherein one or more cysteine ​​residues are deleted or replaced by another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine ​​variants can be used when the antibody must be folded into a biologically active conformation (e.g., after separating soluble inclusion bodies). Cysteine ​​variants generally have fewer cysteine ​​residues than native proteins, and generally have an even number of cysteines to minimize the interactions caused by unpaired cysteines.

[0196] One skilled in the art can determine the desired amino acid substitutions (conservative or non-conservative) when such substitutions are desired. In some embodiments, amino acid substitutions can be used to identify important residues for human APJ antibodies or to increase or decrease the affinity of the human APJ antibodies described herein.

[0197] According to some embodiments, preferred amino acid substitutions are those that: (1) reduce proteolytic susceptibility, (2) reduce oxidative susceptibility, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity and / or (5) confer or modify other physicochemical or functional properties on such polypeptides. According to some embodiments, single or multiple amino acid substitutions (in some embodiments, conservative amino acid substitutions) may be made in the naturally occurring sequence (in some embodiments, in the portion of the polypeptide outside the domain that forms intermolecular contacts). In some embodiments, conservative amino acid substitutions generally do not substantially alter the structural properties of the parent sequence (e.g., the replacement amino acid should not disrupt helices present in the parent sequence or interfere with other types of secondary structures that characterize the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles, Creighton, ed., WH Freeman and Company (1984); Introduction to Protein Structure, Branden and Tooze, eds., Garland Publishing (1991); and Thornton et al., 1991, Nature 354:105, which are incorporated herein by reference.

[0198] In some embodiments, the antibodies provided herein may be chemically bonded to polymers, lipids, or other moieties.

[0199] The antigen binding agent may comprise at least one CDRs described herein, which is incorporated into a biocompatible framework structure. In one example, the biocompatible framework structure comprises a polypeptide or portion thereof sufficient to form a conformationally stable structural support or framework or scaffold, which can display one or more amino acid sequences (e.g., CDRs, variable regions, etc.) that can bind to an antigen in a confined surface area. Such structures may be naturally occurring polypeptides or polypeptide "folds" (structural motifs), or may have one or more modifications relative to natural polypeptides or folds, such as amino acid additions, deletions, or substitutions. These scaffolds may be derived from polypeptides of any species (or more than one species), for example, humans, other mammals, other vertebrates, invertebrates, bacteria, or viruses.

[0200] Biosoluble backbone structures are usually based on protein scaffolds or backbones rather than immunoglobulin domains. For example, fibronectin, ankyrin, lipocalin, neosuppressor protein, cytochrome b, CP1 zinc finger protein, PST1, coiled coil, LACI-D1, Z domain and amylase inhibitory peptide domain (see, e.g., Nygren and Uhlen, 1997, Current Opinion in Structural Biology 7: 463-9) can be used.

[0201] In addition, those skilled in the art will recognize that suitable binding agents include portions of these antibodies, such as one or more heavy chain CDR1, CDR2, CDR3, light chain CDR1, CDR2 and CDR3, as specifically disclosed herein. At least one heavy chain CDR1, CDR2, CDR3, CDR1, CDR2 and CDR3 region has at least one amino acid substitution, as long as the antibody retains the binding specificity of the non-substituted CDR. The non-CDR portion of the antibody may be a non-protein molecule, wherein the binding agent cross-blocks the binding of the antibody disclosed herein to human APJ. The non-CDR portion of the antibody may be a non-protein molecule, wherein the antibody shows a similar binding type to the human Elabela peptide shown by antibody L4H4 in a competitive binding assay. The non-CDR portion of the antibody may be composed of amino acids, wherein the antibody is a recombinant binding protein or a synthetic peptide, and the recombinant binding protein cross-blocks the binding of the antibody disclosed herein to human APJ. The non-CDR portion of the antibody may consist of amino acids, wherein the antibody is a recombinant antibody and the recombinant antibody exhibits a similar binding pattern to human APJ peptide as exhibited by at least antibody L4H4 in a competition binding assay.

[0202] Fusion protein of APJ antibody and Elabela (Elabela fusion protein)

[0203] In one embodiment, provided herein is an Elabela fusion protein comprising an APJ antibody provided herein and an Elabela fragment.

[0204] In another embodiment, provided herein is an Elabela fusion protein, which comprises an APJ antibody provided herein, and one, two, three, four, five, six, seven, or eight Elabela fragments and a peptide linker; the fusion protein connects the amino terminus of an Elabela fragment to the carboxyl terminus of an APJ antibody light chain 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 terminus of an Elabela fragment to the amino terminus of an APJ antibody light chain or heavy chain through a peptide linker sequence, wherein each Elabela fragment is independently a reverse Elabela fragment or a mutant thereof.

[0205] In another embodiment, the present invention provides an Elabela fusion protein, which comprises an APJ antibody provided herein, and one, two, three, or four Elabela fragments and a peptide linker; the fusion protein connects the amino terminus of an Elabela fragment to the carboxyl terminus of an APJ antibody light chain or heavy chain through a peptide linker sequence (Linker), wherein each Elabela fragment is independently a forward Elabela fragment or a mutant thereof.

[0206] In another embodiment, the present invention provides an Elabela fusion protein, which comprises an APJ antibody provided herein, and one, two, three, or four Elabela fragments and a peptide linker; the fusion protein connects the carboxyl terminus of an Elabela fragment to the amino terminus of an APJ antibody light chain or heavy chain through a peptide linker sequence, wherein each Elabela fragment is independently an inverted Elabela fragment or a mutant thereof.

[0207] In another embodiment, the present invention provides an Elabela fusion protein, which comprises an APJ antibody provided herein, and two Elabela fragments and a peptide linker; the fusion protein connects the amino terminus of an Elabela fragment to the carboxyl terminus of an APJ antibody light chain or heavy chain through a peptide linker sequence (Linker), wherein each Elabela fragment is independently a forward Elabela fragment or a mutant thereof.

[0208] In another embodiment, the present invention provides an Elabela fusion protein, which comprises an APJ antibody provided herein, and two Elabela fragments and a peptide linker; the fusion protein connects the carboxyl terminus of an Elabela fragment to the amino terminus of an APJ antibody light chain or heavy chain through a peptide linker sequence (Linker), wherein each Elabela fragment is independently an inverted Elabela fragment or a mutant thereof.

[0209] In another embodiment, the present invention provides an Elabela fusion protein, which comprises an APJ antibody and two Elabela fragments and a peptide linker provided herein; the fusion protein connects the amino terminus of an Elabela fragment to the carboxyl terminus of an APJ antibody light chain through a peptide linker sequence (Linker): N'-R-Linker-Elabela-C'; or connects the amino terminus of an Elabela fragment to the carboxyl terminus of an APJ antibody heavy chain through a peptide linker sequence (Linker): N'-R-Linker-Elabela-C'; wherein: 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 the amino acid sequence of the light chain or heavy chain of an APJ antibody, and Linker represents a peptide linker sequence.

[0210] In another embodiment, the present invention provides an Elabela fusion protein, which comprises an APJ antibody and two Elabela fragments and a peptide linker provided herein; the fusion protein connects the carboxyl terminus of an Elabela fragment to the amino terminus of an APJ antibody light chain through a peptide linker sequence (Linker): N'-Elabela-Linker-RC'; or connects the carboxyl terminus of an Elabela fragment to the amino terminus of an APJ antibody heavy chain: N'-Elabela-Linker-R-C'; wherein: 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, R is the amino acid sequence of the light chain or heavy chain of an APJ antibody, and Linker represents a peptide linker sequence.

[0211] In another embodiment, the present invention provides an Elabela fusion protein, which comprises an APJ antibody and two Elabela fragments and a peptide linker provided herein; the fusion protein connects the amino terminus of an Elabela fragment to the carboxyl terminus of an APJ antibody light chain through a peptide linker sequence (Linker): N'-R-Linker-Elabela-C'; wherein: 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 the amino acid sequence of the light chain of an APJ antibody, and Linker represents a peptide linker sequence.

[0212] In a further embodiment, the present invention provides an Elabela fusion protein, which comprises an APJ antibody and two Elabela fragments and a peptide linker provided herein; the fusion protein connects the amino terminus of an Elabela fragment to the carboxyl terminus of an APJ antibody heavy chain through a peptide linker sequence (Linker): N'-R-Linker-Elabela-C'; wherein: 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 the amino acid sequence of the heavy chain of an APJ antibody, and Linker represents a peptide linker sequence.

[0213] In one embodiment, in the Elabela fusion protein provided herein, the forward Elabela fragment or its mutant is each independently selected from the amino acid sequence of one of the following: 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 NO:118, SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121.

[0214] In one embodiment, in the Elabela fusion protein provided herein, the inverted Elabela fragment or its mutant is each independently selected from the amino acid sequence of one of the following: 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, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160 NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, and SEQ ID NO:155.

[0215] In one embodiment, in the Elabela fusion protein provided herein, the sequences of the peptide linkers each independently comprise 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.

[0216] In another embodiment, in the Elabela fusion protein provided herein, the sequence of the peptide linker independently comprises 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.

[0217] In another embodiment, in the Elabela fusion protein provided herein, the 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.

[0218] Nucleic Acids

[0219] In one aspect, isolated nucleic acid molecules are provided herein. The nucleic acid molecules include, for example, polynucleotides encoding all or part of an Elabela fusion protein, such as one or both chains of an Elabela fusion protein herein, or fragments, derivatives, mutants or variants thereof; polynucleotides sufficient for use as hybridization probes; PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying polynucleotides encoding polypeptides; antisense nucleic acids for inhibiting expression of polynucleotides and their complementary sequences. The nucleic acids can be of any length. For example, they 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 in length, and / or include one or more additional sequences, such as regulatory sequences, and / or be part of a larger nucleic acid, such as a vector. The nucleic acid may be single-stranded or double-stranded and comprise RNA and / or DNA nucleotides as well as artificial variants thereof (eg, peptide nucleic acids).

[0220] Nucleic acids encoding antibody polypeptides (e.g., heavy or light chains, variable domains only or full length) can be isolated from B cells of mice immunized with APJ antigens. Nucleic acids encoding antibodies or Elabela fusion proteins can be isolated by conventional methods such as polymerase chain reaction (PCR).

[0221] The nucleic acid sequences encoding the heavy and light chain variable regions are shown above. A skilled artisan will appreciate that due to the degeneracy of the genetic code, each polypeptide sequence disclosed herein can 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.

[0222] Further provided herein are nucleic acids that hybridize to other nucleic acids (e.g., nucleic acids comprising the nucleotide sequence of any Elabela fusion protein) under specific hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. See, e.g., Current Protocols in Molecular Biology, John Wiley & Son (1989), 6.3.1-6.3.6. As defined herein, for example, moderately stringent conditions use a pre-wash solution comprising 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer of about 50% formamide, 6x SSC, and a hybridization temperature of 55°C (or other similar hybridization solutions, e.g., containing 50% formamide, hybridizing at 42°C), and an elution condition of 60°C using 0.5xSSC, 0.1% SDS. Stringent hybridization conditions hybridize in 6x SSC at 45°C, followed by one or more washes in 0.1xSSC, 0.2% SDS at 68°C. In addition, those skilled in the art can manipulate hybridization and / or washing conditions to increase or decrease hybridization stringency so that nucleic acids containing nucleotide sequences that are at least 65, 70, 75, 80, 85, 90, 95, 98 or 99% homologous to each other can still hybridize to each other. Basic parameters affecting the selection of hybridization conditions and guidance for designing appropriate conditions are listed in, for example, Sambrook, Fritsch and Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Chapters 9 and 11; Current Protocols in Molecular Biology, 1995, Ausubel et al., ed., John Wiley & Sons, Inc., Sections 2.10 and 6.3-6.4) and can be easily determined by a person with ordinary skills in the art based on, for example, the length and / or base composition of DNA. Changes can be introduced into nucleic acids by mutation, thereby resulting in changes in the amino acid sequence of the polypeptide (e.g., antigen binding protein) encoded therein. Mutations can be introduced using any technique known in the art. In one embodiment, one or more specific amino acid residues are changed using, for example, a site-directed mutagenesis scheme. In another embodiment, one or more randomly selected residues are altered using, for example, a random mutagenesis protocol. Regardless of how they are generated, the mutant polypeptides can be expressed and screened for desired properties.

[0223] Mutations can be introduced into nucleic acids without significantly changing the biological activity of the polypeptides encoded thereby. For example, nucleotide substitutions can be made that result in amino acid substitutions at non-essential amino acid residues. In one embodiment, the nucleotide sequences provided herein for the Elabela fusion protein, or fragments, variants, or derivatives thereof, are mutated such that one or more deletions or substitutions of amino acid residues encoding the Elabela fusion protein described herein are formed into two or more residues that differ in sequence. In another embodiment, mutagenesis inserts an amino acid near one or more amino acid residues of the Elabela fusion protein described herein into two or more residues that differ in sequence. Alternatively, one or more mutations can be introduced into nucleic acids to selectively alter the biological activity of the polypeptides encoded thereby (e.g., binding to APJ). For example, the mutations can quantitatively or qualitatively alter the biological activity. Examples of quantitative changes include increasing, decreasing, or eliminating the activity. Examples of qualitative changes include altering the binding specificity of the Elabela fusion protein.

[0224] In another aspect, nucleic acid molecules suitable for use as primers or hybridization probes for detecting nucleic acid sequences herein are provided herein. The nucleic acid molecules herein may 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 encodes an active portion of a polypeptide herein (e.g., an APJ binding portion).

[0225] Probes based on the nucleic acid sequences herein can be used to detect the nucleic acid or similar nucleic acids, such as transcripts encoding the polypeptides herein. The probes can include labeling groups, such as radioisotopes, fluorescent compounds, enzymes, or enzyme cofactors. Such probes can be used to identify cells expressing the polypeptides.

[0226] In another aspect, provided herein are vectors comprising a nucleic acid encoding a polypeptide herein or a portion thereof. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors.

[0227] The recombinant expression vectors herein may contain the nucleic acid herein in a form suitable for expression of the nucleic acid in a host cell. The recombinant expression vectors include one or more regulatory sequences, selected based on the host cell for expression, which are operably linked to the pre-expressed nucleic acid sequence. Regulatory sequences include those that direct constitutive expression of the nucleotide sequence in a variety of host cells (e.g., SV40 early gene enhancer, Rous sarcoma virus promoter, and cytomegalovirus promoter), those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences, see Voss et al., 1986, Trends Biochem. Sci. 11:287, Maniatis et al., 1987, Science 236:1237, the entire contents of which are incorporated herein by reference), and those that direct inducible expression of the nucleotide sequence in response to a specific treatment or condition (e.g., metallothionein promoters in mammalian cells and tetracycline response (tet-sesponsive) promoters and / or streptomycin response promoters in both prokaryotic and eukaryotic systems (supra)). Those skilled in the art will appreciate that the design of the expression vector depends on factors such as the choice of host cells for transformation, the desired protein expression level, etc. The expression vector herein can be introduced into a host cell to thereby produce a protein or peptide encoded by a nucleic acid described herein, including a fusion protein or peptide.

[0228] On the other hand, the present invention provides a host cell that can be introduced into the expression vector herein. The host cell can be any prokaryotic or eukaryotic cell. Prokaryotic host cells include gram-negative or gram-positive organisms, such as Escherichia coli or bacillus. More advanced 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 medium (see Rasmussen et al., 1998, Cytotechnology 28: 31) or CHO strain DXB-11, which lacks DHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77: 4216-20). Other 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 U.S. Pat. No. 6,210,924), HeLa cells, BHK (ATCC CRL-10) cell line, CV1 / EBNA cell line derived from African green monkey kidney cell line CV1 (ATCC CCL-70) (see McMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cells such as 293, 293EBNA or MSR 293, human epithelial A431 cells, human C010205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from primary tissue in vitro culture, primary transplants, HL-60, U937, HaK or Jurkat cells. Appropriate cloning and expression vectors for bacterial, fungal, yeast and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, 1985).

[0229] The vector DNA can be introduced into prokaryotic or eukaryotic cells by traditional transformation or transfection techniques. For stable mammalian transfection, depending on the expression vector and transfection technique used, it is known that only a small portion of cells can incorporate exogenous DNA into their genome. In order to identify and screen these integrants, genes encoding screening markers (e.g., antibiotic resistance) are usually introduced into host cells together with the gene of interest. Preferred screening markers include those that can confer resistance to drugs (e.g., G418, hygromycin, and methotrexate). In other methods, stable transfected cells containing nucleic acids introduced can be identified by drug screening (e.g., cells that have integrated the screening gene can survive, while other cells die).

[0230] The transformed cells can be cultured under conditions that increase 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 contemplated for use herein include substantially homologous recombinant mammalian Elabela fusion protein polypeptides that are substantially free of contaminating endogenous materials.

[0231] Activity of APJ Antibodies

[0232] The activity of the APJ antibody refers to the antibody provided herein having specific binding activity with APJ. In one embodiment, provided herein is a murine antibody or a humanized antibody that can specifically bind to human APJ.

[0233] In another embodiment, the K of an antibody provided herein when binding to human APJ is d In another embodiment, the K of an antibody provided herein when binding 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 100 nM. d In another embodiment, the K of the antibodies provided herein when binding to human APJ is about 1 nM to about 200 nM. d In another embodiment, the K of the antibodies provided herein when binding to human APJ is about 1 nM to about 100 nM. d 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.

[0234] In one embodiment, the reference antibody comprises a combination of the light chain variable domain amino acid sequence of SEQ ID NO:62 and the heavy chain variable domain amino acid sequence of SEQ ID NO:67.

[0235] In this context, the term "substantially similar" means that the K d Comparable or approximately the K of the reference antibody d In one embodiment, the reference antibody comprises, for example, an antibody having a light chain of SEQ ID NO: 62 and a heavy chain of SEQ ID NO: 67.

[0236] Biological activity of APJ antibody-Elabela fusion protein

[0237] The biological activity of the APJ antibody and Elabela fusion protein (Elabela fusion protein) includes two aspects: the biological activity of Elabela and the activity of the APJ antibody. The activity of the APJ antibody is as described above. "The biological activity of Elabela" refers to the Elabela fusion protein binding to and activating the Elabela receptor (e.g., APJ) in vivo and causing a cell stress response. The aforementioned cell stress response includes, but is not limited to, enhancing myocardial contractility, vasodilation, lowering blood pressure, diuresis (reducing the release of antidiuretic hormone), regulating the body's immune response and the release of pituitary-related hormones. Combining the biological activities of Elabela and APJ antibodies, the Elabela fusion protein described herein can be used to treat a variety of diseases and conditions associated with Elabela and APJ. The fusion protein exerts its biological effects by acting on Elabela and / or APJ, so the Elabela fusion protein described herein can be used to treat subjects with diseases and conditions that respond favorably to "increasing Elabela stimulation." These subjects are referred to as "subjects in need of Elabela stimulation treatment." These subjects include those with acute heart failure, chronic heart failure, pulmonary hypertension and pulmonary arterial hypertension, as well as those with diabetic vascular disease, heart failure, atrial fibrillation, and ischemia-reperfusion injury.

[0238] In one embodiment, the biological activity of the Elabela fusion protein is detected using a reporter gene assay to quantify the function of the Elabela fusion protein in activating APJ in vitro.

[0239] In one embodiment, the antibodies provided herein and the Elabela fusion protein are activated in ECs of the Elabela / APJ signaling pathway. 50From about 0.1 nM to about 100 nM, from about 0.5 nM to about 20 nM, from about 1 nM to about 10 nM, or from about 1 nM to about 5 nM.

[0240] Pharmaceutical composition

[0241] In one embodiment, provided herein is a pharmaceutical composition comprising a combination of the Elabela fusion protein provided herein and one or more pharmaceutically acceptable carriers.

[0242] In one embodiment, the pharmaceutical composition comprises the Elabela fusion protein provided herein and one or more substances selected from the following: a buffer having a pH suitable for the Elabela fusion protein, an antioxidant (e.g., ascorbic acid), a low molecular weight polypeptide (e.g., a polypeptide containing less than 10 amino acids), a protein, an amino acid, a sugar example (e.g., dextrin), a complex (e.g., EDTA), glutathione, a stabilizer, and an excipient. In one embodiment, the pharmaceutical composition provided herein may also comprise a preservative. In one embodiment, the pharmaceutical composition provided herein may be formulated as a lyophilized powder using an appropriate excipient solution as a diluent. Further examples of components that can be used for pharmaceutical formulations are found in Remington's Pharmaceutical Sciences, 16th edition (1980) and 20th edition (2000), Mack Publishing Company.

[0243] Treatment

[0244] In one embodiment, provided herein is a method for treating, preventing, or ameliorating pulmonary hypertension, comprising administering to a subject a therapeutically effective amount of an Elabela fusion protein provided herein or a pharmaceutical composition thereof.

[0245] In another embodiment, provided herein is a method for treating, preventing, or ameliorating pulmonary hypertension, comprising administering to a subject a therapeutically effective amount of an Elabela fusion protein provided herein or a pharmaceutical composition thereof.

[0246] In another embodiment, provided herein is a method for treating, preventing, or ameliorating heart failure, comprising administering to a subject a therapeutically effective amount of an Elabela fusion protein provided herein or a pharmaceutical composition thereof.

[0247] In another embodiment, provided herein is a method for treating, preventing, or ameliorating two or more of pulmonary hypertension, pulmonary hypertension, or heart failure, comprising administering to a subject a therapeutically effective amount of the Elabela fusion protein provided herein or a pharmaceutical composition thereof.

[0248] In any of the uses provided herein, the pharmaceutical composition is for intravenous or subcutaneous injection.

[0249] The Elabela fusion protein pharmaceutical composition may be administered by any suitable technique including, but not limited to, parenteral, topical or inhalation administration. If injected, the pharmaceutical composition may be administered, for example, by intraarticular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes, by rapid injection or continuous infusion. For example, local administration at the site of disease or injury, such as transdermal administration and implant sustained release administration, may be considered. Inhalation administration includes, for example, nasal or oral inhalation, the use of a spray, inhalation of the Elabela fusion protein in the form of an aerosol, and the like. Other options include oral formulations including tablets, syrups or lozenges.

[0250] The dosage and frequency of administration may vary depending on factors such as 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 condition of the patient. Appropriate dosages may be determined by methods well known in the art, for example, in clinical trials including dose escalation studies.

[0251] The Elabela fusion proteins provided herein can be administered once or multiple times, for example, at regular intervals over a period of time. In specific embodiments, the Elabela fusion protein is administered once at least once a month or longer, for example, one, two or three months or even indefinitely. For the treatment of chronic symptoms, long-term treatment is generally most effective. However, for the treatment of acute symptoms, short-term administration, for example, from one to six weeks, is sufficient. Generally, the human Elabela fusion protein is administered until the patient shows a medically relevant improvement in the selected sign or indicator above the baseline level.

[0252] An example of a treatment regimen provided herein includes subcutaneous injection of Elabela fusion protein once a week or longer at an appropriate dose to treat symptoms caused by acute heart failure, chronic heart failure, pulmonary hypertension or pulmonary hypertension, etc. Elabela fusion protein can be administered weekly or monthly until the desired result is achieved, such as the patient's symptoms subside. Treatment can be re-administered as needed, or, alternatively, a maintenance dose can be administered.

[0253] The patient's blood BNP or Pro-BNP concentration and body weight can be monitored before, during and / or after treatment with the Elabela fusion protein to detect any changes in pressure. For certain conditions, changes in BNP or Pro-BNP may vary with factors such as the course of the disease. The concentration of BNP or Pro-BNP can be determined using known techniques.

[0254] Specific embodiments of the methods and compositions herein involve the use of, for example, 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 agonists. In further embodiments, the Elabela fusion protein is administered alone or in combination with other agents used to treat the symptoms that cause the patient pain. Examples of such agents include proteins as well as non-protein drugs. When multiple drugs are administered in combination, their dosages should be adjusted accordingly as is well known in the art. "Combined administration" combination therapy is not limited to simultaneous administration, but also includes treatment regimens in which the antigen and protein are administered at least once during a course of treatment involving administration of at least one other therapeutic agent to the patient.

[0255] In another aspect, the present invention provides a method for preparing a medicament for treating heart failure, pulmonary hypertension and related diseases, which comprises a mixture of the Elabela fusion protein provided herein and a pharmaceutically acceptable excipient for treating the related diseases of the above diseases. The medicament preparation method is as described above.

[0256] Further provided herein are compositions, kits, and methods related to Elabela fusion proteins that can specifically bind to human APJ. Also provided are nucleic acid molecules and derivatives and fragments thereof, which contain polynucleotides encoding all or part of a polypeptide that binds to APJ, such as nucleic acids encoding all or part of an Elabela fusion protein or an Elabela fusion protein derivative. Further provided herein are vectors and plasmids containing such nucleic acids, and cells and cell lines containing such nucleic acids and / or vectors and plasmids. The methods provided include, for example, methods for preparing, identifying, or isolating Elabela fusion proteins that bind to human APJ, methods for determining whether the Elabela fusion protein binds to APJ, and methods for administering Elabela fusion proteins that bind to APJ to animal models.

[0257] The present invention also includes the following embodiments:

[0258] Embodiment 1. An antibody that can specifically bind to human APJ, the antibody comprising 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:

[0259] a. Light chain CDR1 amino acid sequences: SEQ ID NO: 10, SEQ ID NO: 7, SEQ ID NO: 4, and SEQ ID NO: 1;

[0260] b. Light chain CDR2 amino acid sequences: SEQ ID NO: 8, SEQ ID NO: 5, and SEQ ID NO: 2;

[0261] c. Light chain CDR3 amino acid sequences: SEQ ID NO: 11, SEQ ID NO: 9, SEQ ID NO: 6, and SEQ ID NO: 3;

[0262] d. Heavy chain CDR1 amino acid sequences: SEQ ID NO: 18, SEQ ID NO: 15, and SEQ ID NO: 12;

[0263] e. Heavy chain CDR2 amino acid sequence: SEQ ID NO:21, SEQ ID NO:19, SEQ ID NO:16, and SEQ ID NO:13; and

[0264] f. Heavy chain CDR3 amino acid sequences: SEQ ID NO:22, SEQ ID NO:20, SEQ ID NO:17, and SEQ ID NO:14.

[0265] Embodiment 2. The antibody according to embodiment 1, wherein the antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0266] a. Light chain CDR1 amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 10; and

[0267] b. Heavy chain CDR1 amino acid sequences: SEQ ID NO: 12, SEQ ID NO: 15, and SEQ ID NO: 18.

[0268] Embodiment 3. The antibody according to embodiment 1 or 2, wherein the antibody comprises or further comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0269] a. Light chain CDR2 amino acid sequence: SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 8; and

[0270] b. Heavy chain CDR2 amino acid sequences: SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, and SEQ ID NO:21.

[0271] Embodiment 4. The antibody according to any one of embodiments 1 to 3, wherein the antibody comprises or further comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0272] a. Light chain CDR3 amino acid sequences: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 11; and

[0273] b. Heavy chain CDR3 amino acid sequences: SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO: 22.

[0274] Embodiment 5. An antibody according to any one of embodiments 1 to 4, wherein the antibody comprises or further comprises one or two 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 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, and SEQ ID NO: 11.

[0275] Embodiment 6. An antibody according to any one of embodiments 1 to 5, wherein the antibody comprises or further comprises one or two 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.

[0276] Embodiment 7. An antibody according to any one of embodiments 1 to 6, wherein the antibody comprises or further comprises a combination of light chain and heavy chain CDR1 amino acid sequences independently selected from the following: SEQ ID NO: 1 and SEQ ID NO: 12, SEQ ID NO: 4 and SEQ ID NO: 15, SEQ ID NO: 7 and SEQ ID NO: 18, and SEQ ID NO: 10 and SEQ ID NO: 18.

[0277] Embodiment 8. An antibody according to any one of embodiments 1 to 7, wherein the antibody comprises or further comprises a combination of light chain and heavy chain CDR2 amino acid sequences 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 NO:8 and SEQ ID NO:19, and SEQ ID NO:5 and SEQ ID NO:21.

[0278] Embodiment 9. An antibody according to any one of embodiments 1 to 8, wherein the antibody comprises or further comprises a combination of light chain and heavy chain CDR3 amino acid sequences 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 NO:9 and SEQ ID NO:20, and SEQ ID NO:11 and SEQ ID NO:22.

[0279] Embodiment 10. The antibody according to any one of embodiments 1 to 9, wherein the antibody comprises a combination of amino acid sequences of one of the following:

[0280] a. A combination of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14;

[0281] b. a combination of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17;

[0282] c. a combination of SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 22; or

[0283] d. A combination of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0284] Embodiment 11. The antibody according to any one of embodiments 1 to 10, wherein the antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0285] a. light chain variable domain amino acid sequence: SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63; and an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the sequences thereof; and

[0286] b. Heavy chain variable domain amino acid sequences: SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68; and amino acid sequences that are at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the sequences.

[0287] Embodiment 12. The antibody according to any one of embodiments 1 to 11, wherein the polynucleotide coding sequence of the antibody comprises one or two polynucleotide coding sequences, wherein each polynucleotide coding sequence is independently selected from the polynucleotide sequences listed below:

[0288] a. light chain variable domain polynucleotide encoding sequence: SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; and a polynucleotide encoding sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the sequences; and

[0289] b. Heavy chain variable domain polynucleotide encoding sequences: SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78; and polynucleotide encoding sequences that are at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the sequences.

[0290] Embodiment 13. The antibody according to any one of embodiments 1 to 12, wherein the antibody comprises or further 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.

[0291] Embodiment 14. The antibody according to any one of embodiments 1 to 13, wherein the antibody comprises or further 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.

[0292] Embodiment 15. An antibody according to any one of embodiments 1 to 14, wherein the antibody comprises a combination of light chain and heavy chain variable domain amino acid sequences independently selected from the following: 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.

[0293] Embodiment 16. The antibody of any one of embodiments 1 to 15, wherein the antibody further comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0294] a. Light chain constant amino acid sequence: SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:81; and

[0295] b. Heavy chain constant amino acid sequences: SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84.

[0296] Embodiment 17. The antibody according to any one of embodiments 1 to 16, characterized in that: the antibody is a murine antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single-chain antibody, a double-chain antibody, a three-chain antibody, a four-chain antibody, a Fab fragment, a F(ab')x fragment, a domain antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgGl antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0297] Embodiment 18. The antibody of any one of Embodiments 1 to 17, wherein the antibody is a murine APJ antibody or a humanized APJ antibody.

[0298] Embodiment 19. The antibody of any one of Embodiments 1 to 18, wherein the antibody is an APJ monoclonal antibody.

[0299] Embodiment 20. The antibody according to any one of embodiments 1 to 19, wherein the K of the antibody is d Values ​​are from about 1 nM to about 200 nM or from about 1 nM to about 100 nM.

[0300] Embodiment 21. An Elabela fusion protein, characterized in that the fusion protein comprises an APJ antibody and an Elabela fragment according to any one of Embodiments 1 to 20.

[0301] Embodiment 22. An Elabela fusion protein, characterized in that: the fusion protein comprises an APJ antibody according to any one of Embodiments 1 to 20, and one, two, three, four, five, six, seven, or eight Elabela fragments and a peptide linker; the fusion protein connects the amino terminus of an Elabela fragment to the carboxyl terminus of an APJ antibody light chain 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 terminus of an Elabela fragment to the amino terminus of an APJ antibody light chain or heavy chain through a peptide linker sequence, wherein each Elabela fragment is independently a reverse Elabela fragment or a mutant thereof.

[0302] Embodiment 23. A fusion protein according to Embodiment 22, wherein the fusion protein comprises an APJ antibody, and one, two, three, or four Elabela fragments and a peptide linker; the fusion protein connects the amino terminus of an Elabela fragment to the carboxyl terminus of an APJ antibody light chain or heavy chain via a peptide linker sequence, wherein each Elabela fragment is independently a forward Elabela fragment or a mutant thereof.

[0303] Embodiment 24. The fusion protein of Embodiment 22, wherein the fusion protein comprises an APJ antibody, and one, two, three, or four Elabela fragments and a peptide linker; the fusion protein connects the carboxyl terminus of an Elabela fragment to the amino terminus of an APJ antibody light chain or heavy chain via a peptide linker sequence, wherein each Elabela fragment is independently an inverted Elabela fragment or a mutant thereof.

[0304] Embodiment 25. The fusion protein described in Embodiment 22, wherein the fusion protein comprises an APJ antibody, and two Elabela fragments and a peptide linker; the fusion protein connects the amino terminus of an Elabela fragment to the carboxyl terminus of an APJ antibody light chain or heavy chain through a peptide linker sequence (Linker), wherein each Elabela fragment is independently a forward Elabela fragment or a mutant thereof.

[0305] Embodiment 26. The fusion protein described in Embodiment 22, wherein the fusion protein comprises an APJ antibody, and two Elabela fragments and a peptide linker; the fusion protein connects the carboxyl terminus of an Elabela fragment to the amino terminus of an APJ antibody light chain or heavy chain through a peptide linker sequence (Linker), wherein each Elabela fragment is independently a reverse Elabela fragment or a mutant thereof.

[0306] Embodiment 27. A fusion protein according to any one of embodiments 22 to 26, wherein the APJ antibody, the Elabela fragment, and the peptide linker sequence are fused to form the fusion protein in one of the following ways:

[0307] Connecting the amino terminus of an Elabela fragment and the carboxyl terminus of an APJ antibody light chain via a peptide linker sequence: N'-R-Linker-Elabela-C'; and

[0308] The amino terminus of an Elabela fragment and the carboxyl terminus of an APJ antibody heavy chain were connected via a peptide linker sequence: N′-R-Linker-Elabela-C′;

[0309] Wherein: N' represents the amino terminus of the polypeptide chain, C' represents the carboxyl terminus of the polypeptide chain, Elabela represents a forward Elabela fragment or a mutant thereof, R is the amino acid sequence of the light chain or heavy chain of an APJ antibody described in any one of Embodiments 1 to 20, and Linker represents a peptide linker.

[0310] Embodiment 28. The Elabela fusion protein according to any one of claims 22 to 27, wherein the sequence of the peptide linker independently comprises one of the following amino acid sequences: SEQ ID NO: 122, SEQ ID NO: 123, and SEQ ID NO: 124.

[0311] Embodiment 29. The Elabela fusion protein according to any one of claims 22 to 28, wherein the peptide linker comprises SEQ ID NO: 123.

[0312] Embodiment 30. The Elabela fusion protein of any one of claims 21 to 29, wherein the Elabela fragment comprises an amino acid sequence independently selected from the group consisting of 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, NO:118, SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121.

[0313] Embodiment 31. The Elabela fusion protein of claim 28, wherein the Elabela fragment comprises an amino acid sequence independently selected from one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:103, SEQ ID NO:107, SEQ ID NO:109, and SEQ ID NO:116.

[0314] Embodiment 32. The Elabela fusion protein of any one of claims 21 to 29, wherein the Elabela fragment comprises an amino acid sequence independently selected from the group consisting of 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, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160 NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, and SEQ ID NO:155.

[0315] Embodiment 33. The Elabela fusion protein of any one of claims 21 to 31, wherein the fusion protein comprises: (i) a combination of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; (ii) an amino acid sequence independently selected from one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:103, SEQ ID NO:107, SEQ ID NO:109, and SEQ ID NO:116; and (iii) SEQ ID NO:123.

[0316] Embodiment 34. A polynucleotide encoding an Elabela fusion protein according to any one of embodiments 21 to 33.

[0317] Embodiment 35. A vector comprising a polynucleotide as described in embodiment 34.

[0318] Embodiment 36. A host cell comprising a vector as described in embodiment 35.

[0319] Embodiment 37. A pharmaceutical composition comprising the antibody of any one of Embodiments 1 to 20 and a pharmaceutically acceptable carrier.

[0320] Embodiment 38. A pharmaceutical composition comprising an Elabela fusion protein according to any one of Embodiments 21 to 33 and a pharmaceutically acceptable carrier.

[0321] Embodiment 39. Use of an Elabela fusion protein according to any one of Embodiments 21 to 33 in the preparation of a medicament for preventing or treating pulmonary arterial hypertension.

[0322] Embodiment 40. Use of an Elabela fusion protein according to any one of Embodiments 21 to 33 in the preparation of a medicament for preventing or treating pulmonary hypertension.

[0323] Embodiment 41. Use of an Elabela fusion protein according to any one of Embodiments 21 to 33 in the preparation of a medicament for preventing or treating heart failure.

[0324] Embodiment 42. Use of an Elabela fusion protein according to any one of Embodiments 21 to 33 in the preparation of a medicament for preventing or treating two or more of pulmonary arterial hypertension, pulmonary hypertension or heart failure.

[0325] Embodiment 43. The use according to any one of embodiments 39 to 42, wherein the medicament is for intravenous or subcutaneous injection.

[0326] Example

[0327] The technical solution of this article is further illustrated below through specific examples.

[0328] Herein, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following examples, unless otherwise specified, are all conventional methods in the art.

[0329] 1. Preparation of Antigens for Immunization

[0330] CHO-DHFR- cells were inoculated into 6-well plates. After 24 hours (hr) of culture, pTM15 plasmid cloned with hAPJ gene (nucleotide sequence see SEQ ID NO:55 and amino acid sequence see SEQ ID NO:23) was transfected into cells in 6-well plates. Transfection was performed according to the transfection conditions of Lipofectamine 2000 recommended by Invitrogen. After 48 hr, the culture medium was replaced with complete medium containing 300 μg / mL hygromycin, and the medium was changed every 3 days (d). After about two weeks of culture, stable growing clones appeared. Digestion and dispersion of cell colonies, cell passage, and continued cell culture until the passaged cells grew to 100% healing degree. The constructed stable cell lines were respectively subjected to FACS detection using antibodies to the V5 tag (Life Technologies), and the cell population after pressure was identified based on the FACS detection results. A large amount of hAPJ was expressed on the membrane of the screened CHO-DHFR-hAPJ cells. Finally, after subcloning and further identification, three APJ cell lines were selected as high-expressing stable cell lines. These cell lines that highly express hAPJ can be used as immunogens for preparing antibodies (see Example 2).

[0331] In addition, the fusion protein of hAPJ extracellular region and hIgG Fc can also be used as an immunogen for preparing antibodies, and the preparation method is as follows: subclone the fusion protein sequence gene of hAPJ extracellular region, hIgG2 Fc and peptide linker into pTM5 plasmid. Suspend HEK293 cells for large-scale transient expression to obtain cell supernatant, and then purify by affinity chromatography to obtain APJ extracellular region fusion protein.

[0332] 2. Preparation of APJ Antibody

[0333] The immunogen and aluminum hydroxide adjuvant were mixed and injected subcutaneously into BALB / c mice (6-8 weeks old), and the mice were boosted once a week. After a total of 6 immunizations, blood was collected by tail cutting. Serum was separated by centrifugation and serum titer was detected by FACS. When the antibody titer reached a suitable level, the mice were killed by cervical dislocation and spleen cells were obtained under sterile 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 culture until resuspended, centrifuged again-resuspended, and counted. Spleen cells and SP2 / 0 cells were mixed to ensure that the number of SP2 / 0 and spleen cells was close. After mixing, "wash-centrifuge" was performed 3 times. Gently disperse the cell pellet after the last centrifugation, add pre-warmed PEG-1500 dropwise, and after blowing up and down, slowly add 30mL of pre-warmed serum-free culture medium to terminate the fusion effect of PEG. After centrifugation again, the cell pellet was dispersed, and the fusion culture medium was added. The spleen cells and feeder layer cells were plated in a 96-well plate, and 100μL of culture medium was added to each well. The fused hybridoma cells and feeder layer cells were cultured in 96-well plates and screened with HAT (hypoxanthine, methotrexate and thymidine) to remove non-fused cells. After 10 days, the supernatant of the hybridoma cells in the culture plate was collected for ELISA detection.

[0334] 3. ELISA screening of APJ antibodies

[0335] CHO-DHFR-hAPJ cells overexpressing hAPJ and CHO-DHFR- cells not expressing hAPJ were inoculated into 96-well plates. When the cells grew to 90% confluence, the cell culture supernatant was removed, washed twice with PBS, fixed with 100% methanol at 4°C, and then 100 μL of prepared H2O2-PBS was added, treated at room temperature for 20 minutes, and washed twice with PBS. After blocking with BSA (dissolved in PBS), the hybridoma cell supernatant was added and incubated at 4°C for 90 minutes. After multiple washings, 100 μL of diluted goat anti-mouse Fc-HRP secondary antibody was added to each well and incubated at 37°C for 30 minutes. After washing 5 times, 100 μL of TMB color substrate was added to each well, reacted at 37°C for 15 minutes, 50 μL of 2M H2SO4 was added to stop the color development, and the OD 450 value was read. After blocking with 1% casein (dissolved in PBS), the hybridoma cell supernatant was added and incubated at 4°C for 90 minutes. The subsequent steps are the same as the above-mentioned ELISA method to screen anti-hAPJ monoclonal antibodies. The positive control is the serum of immunized mice; the negative control is the supernatant of the cell culture medium. After preliminary ELISA testing, several positive hybridoma cell lines that secrete anti-hAPJ antibodies were screened. These hybridoma lines that secrete anti-hAPJ antibodies were selected and cloned to obtain cell lines that can stably secrete anti-hAPJ antibodies. Finally, the ascites antibodies prepared by the positive hybridoma cells were selected, and the affinity of the hAPJ antibodies contained therein was verified and ranked using the flow cytometry experimental method (reference Example 9).

[0336] 4. Cloning and subcloning of APJ antibody gene

[0337] The hybridoma cells secreting antibodies were collected, and the mRNA of the hybridoma cells was extracted according to the operating procedures of the QIAGEN mRNA extraction kit. The extracted mRNA was then reverse transcribed into cDNA. The reverse transcription primers were specific primers for the constant regions of the mouse light and heavy chains. The heavy chain reverse transcription primer was (5'-TTTGGRGGGAAGATGAAGAC-3'), and the light chain reverse transcription primers were (5'-TTAACACTCTCCCCTGTTGAA-3') and (5'-TTAACACTCATTCCTGTTGAA-3'). The reaction conditions for RT-PCR were: 25℃5min; 50℃60min; 70℃15min. The reverse transcribed cDNA was diluted to 500 μL with 0.1 mM TE, added to an ultrafiltration centrifuge tube (Amicon Ultra-0.5), and centrifuged at 2000 g for 10 min; the filtrate was discarded, and 500 μL of 0.1 mM TE was added, and the mixture was centrifuged at 2000 g for 10 min; the filtrate was discarded, and the preparation tube was inverted into a new centrifuge tube, and the mixture was centrifuged at 2000 g for 10 min to obtain the purified cDNA; 10 μL of the purified cDNA was taken as a template, and 4 μL of 5x tailing buffer (Promega), 4 μL of dATP (1 mM) and 10 U of terminal transferase (Promega) were added, mixed, incubated at 37° C. for 5 min, and then incubated at 65° C. for 5 min; the light and heavy chain variable region genes of the antibody were then amplified by PCR using the cDNA with the Poly A tail as a template. The upstream primers were all OligodT, the heavy chain downstream primers were (5'-TGGACAGGGATCCAGAGTTCC-3') and (5'-TGGACAGGGCTCCATAGTTCC-3'), and the light chain downstream primer was (5'-ACTCGTCCTTGGTCAACGTG-3'). PCR reaction conditions: 95℃5min; 95℃30s, 56℃30s, 72℃1min40cycles; 72℃7min; PCR products were connected to PMD 18-T vector (Takara Bio) and sequenced. PCR primers were designed based on the DNA sequence of the sequenced antibody, so that the complete light chain, heavy chain signal peptide and variable domain, and mouse IgG1 constant region were connected to the expression vector pTM5.

[0338] 5. Humanization, optimization and subcloning of APJ antibodies

[0339] First, according to the mouse antibody light and heavy chain variable region sequences obtained by screening, the NCBI database is used to search for human antibody germline gene sequences (Ig Germline GeneSequence) that are homologous to the mouse antibody variable region sequences obtained by screening, and the human gene sequence with the highest homology except the CDR sequence is used as a template sequence for CDR grafting to obtain a humanized antibody variable region sequence. The genes of the light and heavy chains of the humanized antibody are synthesized, and the complete recombinant humanized antibody sequence is obtained after splicing with the human IgG2 or IgG4 constant region sequence. The recombinant antibody is expressed according to Example 8, and the affinity of the recombinant antibody for APJ is verified according to the FACS technology in step 10, and the antibody with the best affinity performance is selected. Finally, the variable region sequence is further modified by site-directed mutagenesis to further improve its affinity for APJ.

[0340] 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 connected to the expression vector pTM5 that had been loaded with the heavy chain constant region; similarly, the complete light chain variable region sequence was connected to the expression vector pTM5 that had been loaded with the light chain constant region.

[0341] 6. Construction of fusion protein of humanized APJ antibody and Elabela

[0342] The optimized humanized antibody was fused with the Elabela fragment sequence at the C-terminus of the heavy chain to form the Elabela fusion protein. The two sequences were connected by a peptide linker sequence (Linker) as a bridge. The humanized APJ antibody heavy chain nucleic acid sequence was connected to the "Linker-Elabela fragment" part by overlapping PCR, and Nhe1 and Not1 restriction sites were added to both ends of the primers to connect the complete fusion protein sequence to the expression vector pTM5. After the Elabela fusion protein was connected to the expression vector, sequencing analysis was performed to confirm the correctness of the construction.

[0343] 7. Transient Expression of APJ Antibody and Elabela Fusion Protein

[0344] Inoculate 5×10 5 / mL suspended HEK293 or CHO expression cell line into a spinner flask, and after 24 hours of rotation culture at 37°C and 5% CO2, the density reached 1×10 6 / mL was used for transfection. Polyethylenimine (PEI) was used as a transfection medium during the transfection process and mixed with DNA. The mixture was added to the cell culture after 15 minutes of static incubation. After the cells were treated with the PEI and DNA mixture, they were continued to be rotated and cultured at 37°C and 5% CO2 for 24 hours. Tryptone was added to the cell culture medium as an additive required for expression. Finally, after the expression was completed (more than 96 hours), the cell supernatant was collected for purification and separation of antibodies or fusion proteins.

[0345] 8. Purification and Isolation of APJ Antibody and Elabela Fusion Protein

[0346] The cell supernatant collected in Example 8 was centrifuged at high speed (8000rpm) to remove cells and cell debris, and then filtered with a 0.22um filter membrane for clarification. The clarified supernatant was used for purification. The purification process was completed by a chromatograph. The supernatant first flowed through a protein A / G affinity chromatography column. The antibody or fusion protein contained in the supernatant was retained in the column after binding to the ligand of the protein A / G affinity chromatography column during this period. The chromatography column was then irrigated with an elution buffer with a low pH value (less than or equal to 3.0) to dissociate the antibody or fusion protein bound to the chromatography column. The collected antibody eluate was rapidly neutralized with 1M Tris-HCl. The resulting antibody or fusion protein eluate was replaced with PBS or other buffer systems after dialysis.

[0347] 9. Verification of the Binding Activity of Functional APJ Antibodies by Flow Cytometry

[0348] Digest with PBS containing EDTA and collect 10 5 CHO-DHFR-hAPJ cells were added to 1.5mL EP tubes respectively, and the supernatant was discarded after centrifugation. The negative control samples were resuspended with flow cytometry buffer (PBS, 2% FBS). 200μL of hAPJ antibody of a specific concentration was added to each tube of the positive treatment group cells and incubated at room temperature; after incubation, centrifuged at 1500rpm, the supernatant was discarded, the cell pellet was washed once with flow cytometry buffer, centrifuged again, and the cells were resuspended; 1:50 diluted FITC-labeled goat anti-mouse fluorescent secondary antibody was added to the cell resuspension, 200μL / well, and incubated at room temperature in the dark for 30min; centrifuged, discarded the supernatant, washed once with flow cytometry buffer, centrifuged, and finally resuspended the cell pellet with flow cytometry buffer for detection. Figures 1A to 1D In the experimental results shown, the gray peak on the left is the negative control of 500nM mouse ascites antibody L1H1 and blank cell CHO-DHFR-binding, and the solid line peaks are 500nM ( Figure 1A ) and 4 nM ( Figure 1BThe binding curve of mouse ascites antibody L1H1 and CHO-DHFR-hAPJ in the right side is significantly shifted to the right relative to the gray peak negative control, proving the specific binding of L1H1 and hAPJ. The gray peak on the right is the negative control of 500nM mouse ascites antibody L4H4 and blank cell CHO-DHFR-binding, and the solid peaks are 500nM ( Figure 1C ) and 4 nM ( Figure 1D ) showed a significant rightward shift in the binding curve of the mouse ascites antibody L4H4 and CHO-DHFR-hAPJ relative to the gray peak negative control, which also demonstrated the specific binding of L4H4 and hAPJ.

[0349] 10. Reporter gene assay to detect the biological activity of Elabela and its Elabela fusion protein in stimulating the Elabela / APJ signaling pathway in vitro

[0350] CHO-DHFR- cells co-expressing hAPJ-CRE-Luciferase were inoculated into 96-well cell culture plates at 35,000 cells per well and cultured overnight at 37°C. The next day, the culture supernatant was removed, the cell surface was washed twice with serum-free medium, and the residual liquid was aspirated. 50 μL of 0.6 μM forskolin was pre-added to the 96-well plate, and then 50 μL of Elabela fusion protein or Elabela-11 peptide diluted in serum-free medium was added and incubated at 37°C for 6 hours. After the stimulation, 100 μL of Promega's Bright Glo chemiluminescent substrate was added, and finally the cell lysate was transferred to a white 96-well plate, and the relative fluorescence intensity was read on the SpectraMax L microplate reader from Molecular Devices. The data were fitted with 5 parameters using Prism5.0 to calculate the EC 50 L1H1 and L4H4 antibodies neither block nor activate APJ receptors. Figure 2 The activation curves of the Elabela / APJ signaling pathway activated by the reporter gene assay of the recombinantly expressed L5H5 and Linker2-Elabela-11 fusion protein and the L5H5 and Linker2-EA5 fusion protein. 50 Respectively: 3.61 and 2.55nM. Figure 3 The activation curves of the Elabela / APJ signaling pathway activated by the reporter gene assay of the recombinantly expressed L5H5 and Linker2-EA1 fusion protein and the L55H5 and Linker2-EA2 fusion protein. 50 Respectively: 4.95 and 2.16nM. Figure 4The activation curves of the Elabela / APJ signaling pathway activated by the reporter gene assay of the recombinantly expressed L5H5 and Linker2-EA3 fusion proteins and the L5H5 and Linker2-EA4 fusion proteins. 50 They are: 20.27 and 5.43nM respectively.

[0351] The above examples are provided to fully disclose and illustrate to those of ordinary skill in the art how to make and use the claimed embodiments, and are not meant to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art are within the scope of the claims herein. All publications, patents, and patent applications cited in this specification are incorporated herein by reference, just as if each publication, patent, or patent application was specifically and individually incorporated herein by reference.

Claims

1. An antibody that can specifically bind to human APJ, the antibody comprising 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: a. Light chain CDR1 amino acid sequences: SEQ ID NO: 10, SEQ ID NO: 7, SEQ ID NO: 4, and SEQ ID NO: 1; b. Light chain CDR2 amino acid sequences: SEQ ID NO: 8, SEQ ID NO: 5, and SEQ ID NO: 2; c. Light chain CDR3 amino acid sequences: SEQ ID NO: 11, SEQ ID NO: 9, SEQ ID NO: 6, and SEQ ID NO: 3; d. Heavy chain CDR1 amino acid sequences: SEQ ID NO: 18, SEQ ID NO: 15, and SEQ ID NO: 12; e. Heavy chain CDR2 amino acid sequences: SEQ ID NO:21, SEQ ID NO:19, SEQ ID NO:16, and SEQ ID NO:13; and f. Heavy chain CDR3 amino acid sequences: SEQ ID NO:22, SEQ ID NO:20, SEQ ID NO:17, and SEQ ID NO:

14.

2. The antibody according to claim 1, wherein the antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below: a. Light chain CDR1 amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 10; and b. Heavy chain CDR1 amino acid sequences: SEQ ID NO: 12, SEQ ID NO: 15, and SEQ ID NO:

18.

3. The antibody according to claim 1 or 2, wherein the antibody comprises or further comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below: a. Light chain CDR2 amino acid sequence: SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 8; and b. Heavy chain CDR2 amino acid sequences: SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO:

21.

4. The antibody according to any one of claims 1 to 3, wherein the antibody comprises or further comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below: a. Light chain CDR3 amino acid sequences: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 11; and b. Heavy chain CDR3 amino acid sequences: SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO:

22.

5. The antibody according to any one of claims 1 to 4, wherein the antibody comprises or further comprises one or two 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 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, and SEQ ID NO:

11.

6. The antibody of any one of claims 1 to 5, wherein the antibody comprises or further comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of 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.

7. The antibody according to any one of claims 1 to 6, wherein the antibody comprises or further comprises a combination of light chain and heavy chain CDR1 amino acid sequences independently selected from the following: SEQ ID NO: 1 and SEQ ID NO: 12, SEQ ID NO: 4 and SEQ ID NO: 15, SEQ ID NO: 7 and SEQ ID NO: 18, and SEQ ID NO: 10 and SEQ ID NO:

18.

8. The antibody according to any one of claims 1 to 7, wherein the antibody comprises or further comprises a combination of light chain and heavy chain CDR2 amino acid sequences 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 NO:8 and SEQ ID NO:19, and SEQ ID NO:5 and SEQ ID NO:

21.

9. The antibody according to any one of claims 1 to 8, wherein the antibody comprises or further comprises a combination of light chain and heavy chain CDR3 amino acid sequences 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 NO: 9 and SEQ ID NO: 20, and SEQ ID NO: 11 and SEQ ID NO:

22.

10. The antibody according to any one of claims 1 to 9, wherein the antibody comprises a combination of one of the following amino acid sequences: a. a combination of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14; b. a combination of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; c. a combination of SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 22; or d. A combination of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

Images