Patent Publication Number: US-2021162045-A1

Title: Human endothelin receptor antibody and use thereof

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. application Ser. No. 16/538,509, filed Aug. 12, 2019, which is a continuation of U.S. application Ser. No. 15/537,190, filed Jun. 16, 2017, now U.S. Pat. No. 10,376,581, which is a U.S. National Stage Application under 35 U.S.C. § 371 of International Patent Application No. PCT/CN2016/090960, filed Jul. 22, 2016, which claims the priority to Chinese Patent Application No. 201510867288.7, filed Dec. 1, 2015, the disclosure of each of which is incorporated herein by reference in its entirety. 
    
    
     REFERENCE TO A SEQUENCE LISTING 
     This application incorporates by reference in its entirety the Computer Readable Form of a Sequence Listing in ASCII text format submitted via EFS-Web. The Sequence Listing text file submitted via EFS-Web is entitled “14254-016-999_SEQLIST.txt,” was created on Nov. 13, 2020 and is 91,800 bytes in size. 
     FIELD 
     The present invention relates to an antibody, especially an antibody specifically binding to a human endothelin receptor, and uses thereof. 
     BACKGROUND 
     Endothelin (ET) is a vasoconstriction peptide hormone, important to the homeostasis and regulation of the biological functions of the cardiovascular system. ET is found not only in the endothelium but also in many other tissues and cell types (Barton et al., 2008,  Can. J. Physiol. Pharmacol.  86:485-498). ET is a 2,400 Da peptide of 21 amino acids, having 2 disulfide bonds at its N-terminus, linking the 1st and 15th cysteine residues and the 3rd and 11th cysteine residues, respectively. Its C-terminus contains hydrophobic amino acid residues. Its N-terminal structure is important for binding to its receptor, while its C-terminal structure is important as to where on the receptor to bind. ET has three isoforms: ET-1, ET-2 and ET-3. They differ by a few amino acid residues. ET-1 plays a major role in the regulation of the biological functions of the cardiovascular system. Upon stimulation, endothelial cells synthesize and release ET-1. ET-1 is mainly regulated at the transcription level. 
     Endothelin receptors (ETR) has two isoforms: ET A R and ET B R, which belong to the G protein coupled receptor (GPCR) family. Upon stimulation, ET A R activates membrane Na + /Ca 2+  exchanger (NCX) and Na + /H +  exchanger (NHE) to increase cellular Ca 2+  concentrations and to sensitize muscle fibers to Ca 2+ , resulting in the constriction of vascular smooth muscle and cardiac muscle (Neylon, 1999,  Clin. Exp. Pharmacol. Physiol.  26:149-153). Unlike ET A R, ET B R mainly relaxes the vascular smooth muscle cells and cardiac muscle cells (Nelson et al., 2003,  Nat. Rev. Cancer  3:110-113). 
     Pulmonary artery hypertension (PAH) is due to the vasoconstriction of the lung or lung related vasculature, resulting in lung artery insufficiency and a compensatory increase in the blood pressure of the heart. On the microscopic scale, there appear to be changes in the small pulmonary arteries, including intimal fibrosis, medial hypertrophy, and plexiform lesions, causing in situ thrombosis of elastic and small pulmonary arteries, and resulting in increased blood circulation resistance in the whole lung vasculature (Simonneau et al., 2004,  J. Am. Coll. Cardiol.  43:5S-12S; Barst et al., 2004,  J. Am. Coll. Cardiol.  43:40S-47S). It has been shown that an ET A R antagonist can effectively block the vasoconstrictive signaling of ET-1, ameliorate PAH symptoms, and improve exercise capability and hemodynamics in PAH patients (Serasli et al., 2010,  Recent Pat. Cardiovasc. Drug Discov.  5:184-95). 
     The present invention provides antibodies specifically binding to ET A R, which can inhibit ET-1 signaling. The antibodies can be used as a monotherapy or combination therapy to treat PAH. The antibodies can also be used in diagnostic applications. 
     SUMMARY 
     The objective of the present invention is to provide an antibody specifically binding to a human endothelin receptor, which can inhibit its biological functions. The antibody of the present invention can be used for treating PAH. 
     ET A R, a member of class A of the GPCR family, comprises seven transmembrane domains. Its extracellular domain is very small, only 1/7 of the complete protein sequence. However, GPCR antibodies focus on its extracellular domain. Because of the structural characteristics and low abundance of GPCR, it is difficult to prepare a biologically active GPCR immunogen. GPCR as a transmembrane protein is also difficult to purify. To obtain a GPCR monoclonal antibody, it cannot be accomplished by using its N-terminal peptide only, and a whole cell or cell membrane is usually used and followed by binding and functional characterization with whole cells. The present invention provides a unique immunization method with whole cells and cell membranes and a murine monoclonal antibody that can inhibit ET A R biological functions. The present invention further provides a method to humanize the murine monoclonal antibody for therapeutic applications in human. 
     The present invention provides: 
     An antibody specifically binding to a human endothelin receptor comprises an amino acid sequence selected from: 
     (a) a light chain CDR3 sequence selected from:
         light chain CDR3 sequences differing by no more than three amino acid additions, substitutions, and/or deletions in total from one of L1-L12 light chain CDR3 sequences: SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, and SEQ ID NO: 68;       

     (b) a heavy chain CDR3 sequence selected from:
         heavy chain CDR3 sequences differing by no more than four amino acid additions, substitutions, and/or deletions in total from one of H1-H12 heavy chain CDR3 sequences: SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, and SEQ ID NO: 136; and       

     (c) light chain CDR3 sequence (a) and heavy chain CDR3 sequence (b). 
     Preferably, the antibody further comprises one or more amino acid sequences selected from: 
     (a) a light chain CDR1 sequence selected from:
         light chain CDR1 sequences differing by no more than three amino acid additions, substitutions, and/or deletions from one of L1-L12 light chain CDR1 sequences: SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30;       

     (b) a light chain CDR2 sequence selected from:
         light chain CDR2 sequences differing by no more than two amino acid additions, substitutions, and/or deletions from one of L1-L12 light chain CDR2 sequences: SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48;       

     (c) a heavy chain CDR1 sequence selected from:
         heavy chain CDR1 sequences differing by no more than two amino acid additions, substitutions, and/or deletions in total from one of H1-H12 heavy chain CDR1 sequences: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and SEQ ID NO: 90; and       

     (d) a heavy chain CDR2 sequence selected from:
         heavy chain CDR2 sequences differing by no more than three amino acid additions, substitutions, and/or deletions in total from one of H1-H12 heavy chain CDR2 sequences: SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114.       

     An antibody specifically binding to a human endothelin receptor comprises an amino acid sequence selected from: 
     (a) one or more light chain variable regions selected from:
         i. light chain CDR1 sequences: SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30;   ii. light chain CDR2 sequences: SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48; and   iii. light chain CDR3 sequences: SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, and SEQ ID NO: 68;       

     (b) one or more heavy chain variable regions selected from:
         i. heavy chain CDR1 sequences: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and SEQ ID NO: 90;   ii. heavy chain CDR2 sequences: SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114; and   iii. heavy chain CDR3 sequences: SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, and SEQ ID NO: 136; and       

     (c) light chain variable region (a) and heavy chain variable region (b). 
     An antibody specifically binding to a human endothelin receptor comprises an amino acid sequence selected from: 
     (a) a light chain variable domain selected from:
         i. amino acid sequences that are at least 80% identical to any of L1-L14 light chain variable domain sequences: SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, and SEQ ID NO: 164; and   ii. amino acid sequences encoded by polynucleotide sequences that are at least 80% identical to any of the polynucleotide sequences encoding for L1-L14 light chain variable domain sequences: SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, and SEQ ID NO: 163;       

     (b) a heavy chain variable domain sequence selected from:
         i. amino acid sequences that are at least 80% identical to any of H1-H14 heavy chain variable domain sequences: SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, and SEQ ID NO: 192; and   ii. an amino acid sequences encoded by polynucleotide sequences that are at least 80% identical to any of the polynucleotide sequences encoding for H1-H14 heavy chain variable domain sequences: SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, and SEQ ID NO: 191; and       

     (c) light chain variable domain sequence (a) and heavy chain variable domain sequence (b). 
     Preferably, the antibody comprises an amino acid sequence selected from: 
     (a) L1-L14 light chain variable domain sequences: SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, and SEQ ID NO: 164; 
     (b) H1-H14 heavy chain variable domain sequences: SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, and SEQ ID NO: 192 and 
     (c) light chain variable domain sequence (a) and heavy chain variable domain sequence (b). 
     Preferably, combination (c) of light chain variable domain sequence (a) and heavy chain variable domain sequence (b) is selected from: L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, and L14H14. 
     Preferably, the antibody further comprises an amino acid sequence selected from: 
     (a) light chain constant region amino acid sequence: SEQ ID NO: 194; 
     (b) light chain constant region amino acid sequence: SEQ ID NO: 196; 
     (c) heavy chain constant region amino acid sequence: SEQ ID NO: 198; 
     (d) light chain constant region amino acid sequence: SEQ ID NO: 194 and heavy chain constant region amino acid sequence: SEQ ID NO: 198; and 
     (e) light chain constant region amino acid sequence: SEQ ID NO: 196 and heavy chain constant region amino acid sequence of SEQ ID NO: 198; 
     Preferably, the antibody is selected from murine antibodies, human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, double-chain antibodies, triple-chain antibodies, tetra-chain antibodies, Fab fragments, F(ab′)x fragments, domain antibodies, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, and IgG4 antibodies. 
     Preferably, the antibody: 
     (a) has substantially the same K d  as a reference antibody in binding to the human endothelin receptor; 
     (b) has substantially the same IC 50  as the reference antibody in inhibiting the activation of the human endothelin receptor; or 
     (c) cross-competes with the reference antibody for binding to the human endothelin receptor. 
     Preferably, the reference antibody comprises a combination of a light chain variable domain sequence: SEQ ID NO: 138 and a heavy chain variable domain sequence: SEQ ID NO: 166. 
     Preferably, the antibody is a murine antibody or humanized antibody. When binding to a human endothelin receptor, the antibody: 
     (a) has an IC 50  of 500 nM or less in reducing the signal transduction of the human endothelin receptor; 
     (b) reduces pulmonary arterial hypertension in a rat or monkey pulmonary arterial hypertension model; or 
     (c) both (a) and (b). 
     A pharmaceutical composition comprises an antibody of the present invention that can be mixed with a pharmaceutically acceptable excipient. 
     Preferably, the antibody is a murine antibody or humanized antibody. 
     An isolated nucleic acid comprises a polynucleotide sequence encoding the light chain variable domain, the heavy chain variable domain, or both the light chain variable domain and the heavy chain variable domain of an antibody of the present invention. 
     Preferably, the polynucleotide sequence encoding the light chain variable domain of the antibody is selected from: SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, and SEQ ID NO: 163; 
     the polynucleotide sequence encoding the heavy chain variable domain of the antibody is selected from: SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, and SEQ ID NO: 191. 
     A recombinant expression vector comprises a nucleic acid of the present invention. 
     A host cell comprises a recombinant expression vector of the present invention. 
     A method for producing an antibody of the present invention, comprises culturing the host cells under the conditions that promotes the production of the antibody. A method of treating PAH in a human subject in need thereof comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition of the present invention. 
     A kit for treating PAH, a disease associated with PAH, a disease associate with vascular smooth muscle, a disease associated with smooth muscle, sickle cell disease, cardiac inefficiency, heart failure, and a reproductive organ cancer, comprises the pharmaceutical composition of the present invention. 
     A pharmaceutical composition of the present invention is for use in the preparation of a medicament for treating PAH, a disease associated with PAH, a disease associate with vascular smooth muscle, a disease associated with smooth muscle, sickle cell disease, cardiac inefficiency, heart failure, and a reproductive organ cancer. 
     The present invention provides the following superior properties: 
     (a) The monoclonal antibody of the present invention has a high affinity and functional activity towards a human endothelin receptor. 
     (b) The antibody of the present invention is a new functional antibody against ET A R. A humanized antibody of the murine antibody was obtained and ready for human therapeutic application. 
     (c) The antibody of the present invention is different from the existing small molecule drugs and offers the unique advantages of biological macromolecule drugs. 
     Therefore, the monoclonal antibody of the present invention with high affinity and specificity is valuable clinically. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  shows the ELISA screening results of the supernatants of hybridomas for binding to CHO-DHFR-ET A R cells (labeled as CHO-ET A R in the figure), where anti-ET A R antibody A-1 was obtained from hybridoma clone 15F3. 
         FIG. 2  shows the specific bindings of recombinant anti-ET A R antibodies (A-1, A-2, A-7, and A-12) to a human ET A R as determined by FACS, where the blue curves are the bindings of the anti-ET A R antibodies to CHO-DHFR- and the red curves are the bindings of the anti-ET A R antibodies to CHO-DHFR-ET A R. 
         FIG. 3  shows the inhibitory effects of the supernatants of hybridomas on cellular ET A R-mediated Ca 2+  changes as determined using a calcium flux assay. 
         FIG. 4  shows the dose responses of recombinant anti-ET A R antibodies on the inhibition of human ET A R as determined using a calcium flux assay (IC 50 =37.91 nM, R 2 =0.97) (A-1); (IC 50 =87.84 nM, R 2 =0.97) (A-9). 
         FIG. 5  shows the in vivo activity of the recombinant anti-ET A R (A-1) in a hypoxia-induced PAH monkey model. A1 was found to be able to reduce the systolic arterial pressure significantly, and also to be effective throughout the 96-hr window as measured by area under the curve of systolic arterial pressure versus time. 
     
    
    
     DETAILED DESCRIPTION 
     Through the following specific embodiments and examples in combination with figures, the present invention is further elaborated. 
     In this invention, unless specified otherwise, the raw materials and equipment are all available commercially or are commonly used. The methods described below, if not specified otherwise, are all conventional methods. 
     The present invention provides an antibody, for example, an antibody specifically binding to human ET A R, including antibodies that inhibit or block the interaction between ET-1 and ET A R, and downregulate the ET-1/ET A R single pathway. In one embodiment, the antibodies provided herein include an inhibitory murine antibody that can reduce the pulmonary artery pressure in a PAH animal model. 
     The present invention further provides a pharmaceutical composition, a kit, and a method, which comprise, contain, or employ an antibody that specifically binds to the human ET A R. The present invention also provides a polynucleotide encoding the amino acid sequence of the antibody, or a fragment thereof; e.g., a polynucleotide encoding the complete amino acid sequence or a partial amino acid sequence of the antibody, a polynucleotide encoding the complete amino acid sequence or a partial amino acid sequence of a segment or a derivative of the antibody. Furthermore, the present invention provides a plasmid and a vector comprising such a polynucleotide; and a host cell line comprising such a plasmid and vector. The methods provided herein include, for example, preparation, purification, or characterization of the antibody that binds to the human ET A R, such as a method for preparing an anti-ET A R antibody, a method for determining the binding of such an antibody to ET A R, and a method for administering an antibody that binds to ET A R in an animal model. 
     Definitions 
     Polynucleotide and polypeptide sequences are described using standard one- or three-letter abbreviations. Unless otherwise specified, polypeptide sequences have their amino termini at the left and their carboxyl termini at the right, and single-stranded nucleic acid sequences and the top strands of double-stranded nucleic acid sequences have their 5′ termini at the left and their 3′ termini at the right. A particular section of a polypeptide can be designated by amino acid residue numbers such as amino acids 80 to 130, or in combination with the corresponding actual residues such as Lys80 to Lys130. A particular polypeptide or polynucleotide sequence also can be described by showing its differences from a reference sequence. Polynucleotide and polypeptide sequences of particular light and heavy chain variable domains may be expressed as L1 (“light chain variable domain 1”) and H1 (“heavy chain variable domain 1”). An antibody comprising a light chain and heavy chain can be identified by combining the names of the light chain and heavy chain variable domains. For example, “L4H7” represents an antibody comprising light chain variable domain L4 and heavy chain variable domain H7. 
     Unless otherwise defined herein, scientific and technical terms used in the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used herein in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), each of which is incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer&#39;s specifications, as commonly accomplished in the art or as described herein. The terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. 
     The following terms, unless otherwise specified, shall be understood to have the following meanings: The term “isolated molecule” (where the molecule is, for example, a polypeptide, a polynucleotide, or an antibody) is a molecule that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized, or expressed in a cellular system different from the cell from which it naturally originates, will be “isolated” from its naturally associated components. A molecule also can be rendered substantially free of naturally associated components by isolation, using purification techniques well-known in the art. For example, the purity of a polypeptide sample can be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well-known in the art. For certain purposes, higher resolution can be provided by using HPLC or other means well-known in the art for purification. 
     The terms “peptide” “polypeptide” and “protein” each refers to a molecule comprising two or more amino acid residues joined to each other by peptide bonds. These terms encompass, e.g., native and artificial proteins, protein fragments and polypeptide analogs (such as muteins, variants, and fusion proteins) of a protein sequence as well as post translationally, or otherwise covalently or non-covalently, modified proteins. A peptide, polypeptide, or protein may be monomeric or polymeric. 
     The term “polypeptide fragment” as used herein refers to a polypeptide that has an amino-terminal and/or carboxyl-terminal deletion as compared to a corresponding full-length protein. Fragments 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 in length. Fragments can also be, for example, at most 1,000, 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 in length. A fragment can further comprise, at either or both of its ends, one or more additional amino acids, for example, a sequence of amino acids from a different naturally-occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence). 
     Polypeptides of the invention include polypeptides that have been modified in any way and for any reason, for example, to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter susceptibility to form a protein complex, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties. Analogs include muteins of a polypeptide. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in the naturally occurring sequence (e.g., in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). A “conservative amino acid substitution” is one that does not substantially change the structural characteristics of the parent sequence (e.g., replacement of an amino acid should not break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterize the parent sequence or are necessary for its functionality). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., 1991,  Nature  354:105, each of which is incorporated herein by reference. 
     The present invention also provides non-peptide analogs of anti-ET A R antibodies. Non-peptide analogs are commonly used to provide drugs with properties analogous to those of the corresponding peptides. These types of non-peptide compounds are termed “peptide mimetics” or “peptidomimetics.” Fauchere, 1986,  J. Adv. Drug Res.  15:29-69; Veber and Preidinger, 1985,  Treads Neurosci.  8:392-396; and Evans et al., 1987,  J. Med. Chem.  30:1229-1239; each of which is incorporated herein by reference in its entirety. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce an equivalent therapeutic or prophylactic effect. Generally, a peptidomimetic is structurally similar to its corresponding polypeptide (i.e., a polypeptide with the desired biochemical properties or pharmacological parameters), such as a human antibody, but has one or more peptide linkages optionally replaced by a linkage selected from: —CH 2 NH—, —CH 2 S—, —CH 2 —CH 2 —, —CH═CH— (cis and trans), —COCH 2 —, —CH(OH)CH 2 —, and —CH 2 SO— by the methods well-known in the art. Substitution of one or more amino acids or all of a peptide with a D-amino acid of the same type (e.g., D-lysine replacing L-lysine) may also be used to generate more stable peptides. In addition, methods known in the art can be applied (Rizo and Gierasch, 1992,  Ann. Rev. Biochem.  61:387-418, which is incorporated herein by reference in its entirety), for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide. 
     A “variant” of a polypeptide (e.g., an antibody) comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence. Variants of the invention include fusion proteins. 
     A “derivative” of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, e.g., via conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation. Unless otherwise indicated, the term “antibody” includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below. 
     An “antibody” is a protein comprising a portion that binds to an antigen and optionally a scaffold or framework portion that allows the antibody to adopt a conformation that promotes the binding of the antibody to the antigen. Examples of antibodies include antibodies, antibody fragments (e.g., an antigen binding portion of an antibody), antibody derivatives, and antibody analogs. The antibody can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDRs derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced, for example, to stabilize the three-dimensional structure of the antibody as well as completely synthetic scaffolds comprising, for example, a biocompatible polymer. See, for example, Korndorfer et al., 2003,  Proteins: Structure, Function, and Bioinformatics,  53:121-129; Roque et al., 2004,  Biotechnol. Prog.  20:639-654. In addition, peptide antibody mimetics (“PAMs”) can be used, as well as scaffolds based on antibody mimetics utilizing fibronectin components as a scaffold. 
     An antibody can have, for example, the structure of a naturally occurring immunoglobulin. An “immunoglobulin” is a tetrameric molecule. In a naturally occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxyl terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody&#39;s isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, The heavy chain also includes a “D” region of about 10 more amino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated herein by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites. 
     Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each region is in accordance with the definitions of Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991. 
     Unless otherwise specified, an “antibody” refers to an intact immunoglobulin or to an antigen binding portion thereof that competes with the intact antibody for specific binding. Antigen binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen binding portions include, inter alia, Fab, Fab′, F(ab′) 2 , Fv, domain antibodies (dAbs), fragments including complementarity determining regions (CDRs), single-chain antibodies (scFv), chimeric antibodies, diabodies, tribodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. 
     A Fab fragment is a monovalent fragment having the V L , V H , C L  and C H1  regions; a F(ab′) 2  fragment is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment has the V H  and C H1  regions; and a dAb fragment has a V H  region, a V L  region, or an antigen-binding fragment of a V H  or V L  region (U.S. Pat. Nos. 6,846,634, 6,696,245, US App. Pub. Nos. 05/0202512, 04/0202995, 04/0038291, 04/0009507, 03/0039958, Ward et al., 1989,  Nature  341:544-546). 
     A single-chain antibody (scFv) is an antibody in which a V L  and a V H  region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain 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  242:423-26 and Huston et al., 1988,  Proc. Natl. Acad. Sci. USA  85:5879-83). Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises V H  and V L  regions joined by a linker that is too short to allow for pairing between two regions on the same chain, thus allowing each region to pair with a complementary region on another polypeptide chain (see, e.g., Holliger et al., 1993,  Proc. Natl. Acad. Sci. USA  90:6444-48, and Poljak et al., 1994,  Structure  2:1121-23). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites. Similarly, tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different. 
     Complementarity determining regions (CDRs) and frame work regions (FR) of a given antibody can be identified using the system described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. One or more CDRs can be incorporated into a molecule either covalently or noncovalently to make it an antibody. An antibody can incorporate the CDR(s) as part of a larger polypeptide chain, can covalently link the CDR(s) to another polypeptide chain, or can incorporate the CDR(s) noncovalently. The CDRs permit the antibody to specifically bind to a particular antigen of interest. 
     An antibody can have one or more binding sites. If there are more than one binding sites, the binding sites can be identical to one another or can be different. For example, a naturally occurring human immunoglobulin typically has two identical binding sites, while a “bispecific” or “bifunctional” antibody has two different binding sites. 
     The term “murine antibody” includes all antibodies that have one or more variable and constant regions derived from a murine immunoglobulin sequence. 
     The term “humanized antibody” refers to an antibody that produced by grafting the complementarity determining region sequence of a murine antibody molecule into a human antibody variable region framework. 
     “Antigen-binding domain,” “antigen-binding region,” or “antibody-binding site” is a portion of an antibody that comprises amino acid residues (or other portion) interacting with an antigen and contributing to the specificity and affinity of the antibody for the antigen. For antibodies that specifically bind to their antigen, this will include at least a portion of at least one of its CDR regions. 
     An “epitope” is the portion of a molecule that is bound by an antibody (e.g., by an antibody). An epitope can comprise non-contiguous portions of the molecule (e.g., in a polypeptide, amino acid residues that are not contiguous in the polypeptide&#39;s primary sequence but that, in the context of the polypeptide&#39;s tertiary and quaternary structure, are near enough to each other to be bound by an antibody). 
     The “percent identity” of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. 
     The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” are used interchangeably throughout and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. The nucleic acid molecule can be single-stranded or double-stranded. In one embodiment, the nucleic acid molecules of the invention comprise a contiguous open reading frame encoding an antibody of the invention, or a fragment, derivative, mutein, or variant thereof. 
     Two single-stranded polynucleotides are “the complement” of each other if their sequences can be aligned in an anti-parallel orientation such that every nucleotide in one polynucleotide is opposite its complementary nucleotide in the other polynucleotide, without the introduction of gaps and without unpaired nucleotides at the 5′ or the 3′ end of either sequences. A polynucleotide is “complementary” to another polynucleotide if the two polynucleotides can hybridize to one another under moderately stringent conditions. Thus, a polynucleotide can be complementary to another polynucleotide without being its complement. 
     A “vector” is a nucleic acid that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a “plasmid,” which refers to a linear or circular double stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), wherein additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. An “expression vector” is a type of vectors that can direct the expression of a chosen polynucleotide. 
     A nucleotide sequence is “operably linked” to a regulatory sequence if the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the nucleotide sequence. A “regulatory sequence” is a nucleic acid that affects the expression (e.g., the level, timing, or location of expression) of a nucleic acid to which it is operably linked. The regulatory sequence can, for example, exert its effects directly on the regulated nucleic acid, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and/or the 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 in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. and Baron et al., 1995,  Nucleic Acids Res.  23:3605-06. 
     A “host cell” is a cell that can be used to express a nucleic acid, e.g., a nucleic acid of the invention. A host cell can be a prokaryote, for example,  E. coli,  or it can be an eukaryote, for example, a single-celled eukaryote (e.g., a yeast or other fungus), 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 the host cell. The phrase “recombinant host cell” can be used to denote a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell also can be a cell that comprises the nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to, e.g., mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. 
     Endothelin Receptor 
     Endothelin A receptor (ET A R) belongs to family A of 7-transmembrane receptors that are coupled to one or more intracellular signaling pathways via heterotrimeric guanine nucleotide-binding proteins (G proteins) (Jelinek et al., 1993,  Science  259:1614-1616, Segre et al., 1993,  Trends Endocrinol. Metab.  4:309-314). As used herein, “endothelin receptor” and “ET A R” are used interchangeably. 
     In one embodiment, the antibody of the present invention can be selected to bind to membrane bound endothelin receptors as expressed on cells, and inhibit or block endothelin signaling through the endothelin receptors. In one embodiment, the antibody of the present invention specifically binds to the human endothelin receptor. In a further embodiment, the antibody binding to the human endothelin receptor can also bind to the endothelin receptors of other species, e.g., rat. The examples below provide one method of generating murine antibodies which bind to human membrane-bound endothelin receptors, and in a further embodiment, bind to endothelin receptors of other species. 
     The polynucleotide and polypeptide sequences for several species of the endothelin receptors are known. SEQ ID NO: 1-SEQ ID NO: 6 present sequences for human, monkey, and rat. The sequence data were obtained from the GeneBank database of the National Center for Biotechnology Information. 
     Endothelin A Receptor 
     
         
         Human ( Homo sapiens ) polynucleotides (SEQ ID NO: 1) 
         Accession No. S63938. 
         Human ( Homo sapiens ) amino acid (SEQ ID NO: 2) 
         Accession No. AAB20278. 
         Cynomolgus ( Homo sapiens ) polynucleotides (SEQ ID NO: 3) 
         Accession No. JV635771. 
         Cynomolgus ( Homo sapiens ) amino acid (SEQ ID NO: 4) 
         Accession No. AFJ71111. 
         Rat ( Rattus norvegicus ) polynucleotides (SEQ ID NO: 5) 
         Accession No. M60786. 
         Rat ( Rattus norvegicus ) amino acid (SEQ ID NO: 6) 
         Accession No. AAA41114. 
       
    
     Antibodies 
     In one aspect, the present invention provides antibodies (e.g., antibodies, antibody fragments, antibody derivatives, antibody muteins, and antibody variants) that specifically bind to a human endothelin A receptor. In one embodiment the antibody is a murine antibody or a humanized antibody. 
     Antibodies in accordance with the present invention include antibodies that specifically bind to the human endothelin receptor and inhibit endothelin signaling through the endothelin receptor. In one embodiment, the IC 50  value of the antibody is 200 nM or less. In another aspect, the antibodies specifically bind the endothelin receptor, inhibit signaling, and exhibit therapeutic biological effects, such as lowering pulmonary hypertension in animal models. In one embodiment, the antibodies are murine antibodies that specifically bind the endothelin receptor, and inhibit signaling through the endothelin receptor. In another embodiment, the antibodies are murine antibodies that specifically bind to the endothelin receptor and inhibit signaling through the endothelin receptor, and can lower pulmonary hypertension. 
     In one embodiment, the antibody comprises a sequence that differs from a CDR sequence of one of A1-A14 listed in Table 1 below by 5, 4, 3, 2, 1, or 0 single amino acid addition(s), substitution(s), and/or deletion(s). As used herein, a CDR sequence that differs by no more than, for example, four amino acid additions, substitutions and/or deletions from a CDR sequence listed in Table 1 below refers to a sequence with 4, 3, 2, 1 or 0 single amino acid addition(s), substitution(s), and/or deletion(s) as compared with the sequence in Table 1. 
     In another embodiment, the antibody comprises one or more CDR consensus sequences shown below. Provided below are consensus sequences for light chain CDR1, CDR2, CDR3 and heavy chain CDR1, CDR2, and CDR3. 
     The light chain CDRs of antibodies A1/A14 and the heavy chain CDRs of exemplary antibodies A1/A14 are shown in Table 1. A-1 to A-14 correspond to L1 to L14 as well as H1 to H14 below. Also shown are polynucleotide sequences which encode the amino acid sequences of the CDRs. 
     In another aspect, the present invention provides antibodies that comprise a light chain variable region selected from the group consisting of L1-L14 or a heavy chain variable region selected from the group consisting of H1-H14, and fragments, derivatives, muteins, and variants thereof. Such an antibody can be designated using the nomenclature “LxHy”, wherein “x” corresponds to the sequence number of the light chain variable region and “y” corresponds to the sequence number of the heavy chain variable region. For example, L2H1 refers to an antibody with a light chain variable region comprising the amino acid sequence of L2 and a heavy chain variable region comprising the amino acid sequence of H1 as shown in Table 2 below. Antibodies of the invention include, for example, antibodies having a combination of light chain and heavy chain variable regions selected from the combinations L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13 and L14H14. In one embodiment, the antibodies are murine antibodies or humanized antibodies. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Ab 
                 CDR1 
                 CDR2 
                 CDR3 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 Light Chain L1-L12 
               
            
           
           
               
               
               
               
            
               
                 A-1 
                 agggccagtcagaacattggcac 
                 tatgcttctaagtctatatct 
                 caacatagttatagctggcc 
               
               
                 Nucleic 
                 aagcatacac 
                 (SEQ ID NO: 31) 
                 gtggacg 
               
               
                 Acid 
                 (SEQ ID NO: 7) 
                   
                 (SEQ ID NO: 49) 
               
               
                   
               
               
                 Amino 
                 RASQNIGTSIH 
                 YASKSIS 
                 QHSYSWPWT 
               
               
                 Acid 
                 (SEQ ID NO: 8) 
                 (SEQ ID NO: 32) 
                 (SEQ ID NO: 50) 
               
               
                   
               
               
                 A-2 
                 cgagcaagtgaaaatatttacagtt 
                 aatgcaaaaaccttagcagaa 
                 cagcatcattatggtattccgt 
               
               
                 Nucleic 
                 atttagca 
                 (SEQ ID NO: 33) 
                 tcacg 
               
               
                 Acid 
                 (SEQ ID NO: 9) 
                   
                 (SEQ ID NO: 51) 
               
               
                   
               
               
                 Amino 
                 RASENIYSYLA 
                 NAKTLAE 
                 QHHYGIPFT 
               
               
                 Acid 
                 (SEQ ID NO: 10) 
                 (SEQ ID NO: 34) 
                 (SEQ ID NO: 52) 
               
               
                   
               
               
                 A-3 
                 cagagcctctttgatattgatggaa 
                 ctggtgtctgaattggactct 
                 tggcaaggtacacattttccg 
               
               
                 Nucleic 
                 agacatatttgaat (SEQ ID 
                 (SEQ ID NO: 35) 
                 ctcacg 
               
               
                 Acid 
                 NO: 11) 
                   
                 (SEQ ID NO: 53) 
               
               
                   
               
               
                 Amino 
                 QSLFDIDGKTYLN 
                 LVSELDS 
                 WQGTHFPLT 
               
               
                 Acid 
                 (SEQ ID NO: 12) 
                 (SEQ ID NO: 36) 
                 (SEQ ID NO: 54) 
               
               
                   
               
               
                 A-4 
                 cgggcaagtcaggacattggtgg 
                 gccacatccagcttagattct 
                 ctacaatatgctagttctccgt 
               
               
                 Nuncleic 
                 tagcttaaac 
                 (SEQ ID NO: 37) 
                 atacg 
               
               
                 Acid 
                 (SEQ ID NO: 13) 
                   
                 (SEQ ID NO: 55) 
               
               
                   
               
               
                 Amino 
                 RASQDIGGSLN 
                 ATSSLDS 
                 LQYASSPYT 
               
               
                 Acid 
                 (SEQ ID NO: 14) 
                 (SEQ ID NO: 38) 
                 (SEQ ID NO: 56) 
               
               
                   
               
               
                 A-5 
                 agggccagccagactattagcga 
                 tatgatcccaatccatctct 
                 caaagtggtaacacctttccg 
               
               
                 Nucleic 
                 cttcttacac 
                 (SEQ ID NO: 39) 
                 tggacg 
               
               
                 Acid 
                 (SEQ ID NO: 15) 
                   
                 (SEQ ID NO: 57) 
               
               
                   
               
               
                 Amino 
                 RASQTISDFLH (SEQ 
                 YASQSIS 
                 QSGNTFPWT 
               
               
                 Acid 
                 ID NO: 16) 
                 (SEQ ID NO: 40) 
                 (SEQ ID NO: 58) 
               
               
                   
               
               
                 A-6 
                 agggcaagtgaggacatacacac 
                 ggtgcagccagtttgaaaagt 
                 caacagtataggagtattcc 
               
               
                 Nucleic 
                 tcaattagcc 
                 (SEQ ID NO: 41) 
                 gtggacg 
               
               
                   
               
               
                 Acid 
                 (SEQ ID NO: 17) 
                   
                 (SEQ ID NO: 59) 
               
               
                 Amino 
                 RASEDIHTQLA (SEQ 
                 GAASLKS 
                 QQYRSIPWT 
               
               
                 Acid 
                 ID NO: 18) 
                 (SEQ ID NO: 42) 
                 (SEQ ID NO: 60) 
               
               
                   
               
               
                 A-7 
                 agatctagtcagtacattgttcatag 
                 aaagtttccaaccgattttct 
                 tttcaaggttcacattttccatt 
               
               
                 Nucleic 
                 tactggaaccacctatttagaa 
                 (SEQ ID NO: 43) 
                 cacg 
               
               
                 Acid 
                 (SEQ ID NO: 19) 
                   
                 (SEQ ID NO: 61) 
               
               
                   
               
               
                 Amino 
                 RSSQYIVHSTGTTYLE 
                 KVSNRFS 
                 FQGSHFPFT 
               
               
                 Acid 
                 (SEQ ID NO: 20) 
                 (SEQ ID NO: 44) 
                 (SEQ ID NO: 62) 
               
               
                   
               
               
                 A-8 
                 agatctagtcattaccttgttcatga 
                 aaggtttccaaccgattttct 
                 tttcaaggttcacatttcccatt 
               
               
                 Nucleic 
                 taacggaaacacctatgttgaa 
                 (SEQ ID NO: 43) 
                 cacg 
               
               
                 Acid 
                 (SEQ ID NO: 21) 
                   
                 (SEQ ID NO: 63) 
               
               
                   
               
               
                 Amino 
                 RSSHYLVHDNGNTYV 
                 KVSNRFS 
                 FQGSHFPFT 
               
               
                 Acid 
                 E 
                 (SEQ ID NO: 44) 
                 (SEQ ID NO: 62) 
               
               
                   
                 (SEQ ID NO: 22) 
                   
                   
               
               
                   
               
               
                 A-9 
                 agatctagtcagaacattgtccata 
                 aaagtttccaaccgattttct 
                 tttcaaggttcacattttccatt 
               
               
                 Nucleic 
                 gtactggaaacacctatttagaa 
                 (SEQ ID NO: 43) 
                 cacg 
               
               
                 Acid 
                 (SEQ ID NO: 23) 
                   
                 (SEQ ID NO: 61) 
               
               
                   
               
               
                 Amino 
                 RSSQNIVHSTGNTYLE 
                 KVSNRFS 
                 FQGSHFPFT 
               
               
                 Acid 
                 (SEQ ID NO: 24) 
                 (SEQ ID NO: 44) 
                 (SEQ ID NO: 62) 
               
               
                   
               
               
                 A-10 
                 agtgtcagctcaagtgtaagttaca 
                 gacacatccaaactggcttct 
                 caccagtggagtactaaccc 
               
               
                 Nucleic 
                 tacac 
                 (SEQ ID NO: 45) 
                 acccacg 
               
               
                 Acid 
                 (SEQ ID NO: 25) 
                   
                 (SEQ ID NO: 63) 
               
               
                   
               
               
                 Amino 
                 SVSSSVSYIH 
                 DTSKLAS 
                 HQWSTNPPT 
               
               
                 Acid 
                 (SEQ ID NO: 26) 
                 (SEQ ID NO: 46) 
                 (SEQ ID NO: 64) 
               
               
                   
               
               
                 A-11 
                 agtgccagctcaagtgtaagttac 
                 gacacatccaaactggcttct 
                 cagcagtggagtagtaaccc 
               
               
                 Nucleic 
                 atgtgc 
                 (SEQ ID NO: 45) 
                 acccacg 
               
               
                 Acid 
                 (SEQ ID NO: 27) 
                   
                 (SEQ ID NO: 65) 
               
               
                   
               
               
                 Amino 
                 SASSSVSYMC 
                 DTSKLAS 
                 QQWSSNPPT 
               
               
                 Acid 
                 (SEQ ID NO: 28) 
                 (SEQ ID NO: 46) 
                 (SEQ ID NO: 66) 
               
               
                   
               
               
                 A-12 
                 cagggcattaacaattat 
                 tatacatcaactttacagtca 
                 cagcagtttagtaaacttcgg 
               
               
                 Nucleic 
                 (SEQ ID NO: 29) 
                 (SEQ ID NO: 47) 
                 aca 
               
               
                 Acid 
                   
                   
                 (SEQ ID NO: 67) 
               
               
                   
               
               
                 Amino 
                 QGINNY 
                 YTSTLQS 
                 QQFSKLRT 
               
               
                 Acid 
                 (SEQ ID NO: 30) 
                 (SEQ ID NO: 48) 
                 (SEQ ID NO: 68) 
               
               
                   
               
            
           
           
               
            
               
                 Heavy Chain H1-H12 
               
            
           
           
               
               
               
               
            
               
                 A-1 
                 gggttctcactgaccacttct 
                 cacatttggtcggatggtgac 
                 atgaaggatgatagtctttactttga 
               
               
                 Nucleic 
                 ggcttgggtgttgcc 
                 acgcgctattacccagccctg 
                 caac 
               
               
                 Acid 
                 (SEQ ID NO: 69) 
                 aagaac 
                 (SEQ ID NO: 115) 
               
               
                   
                   
                 (SEQ ID NO: 91) 
                   
               
               
                   
               
               
                 Amino 
                 GFSLTTSGLGVA 
                 HIWSDGDTRYYPA 
                 MKDDSLYFDN 
               
               
                 Acid 
                 (SEQ ID NO: 70) 
                 LKN 
                 (SEQ ID NO: 116) 
               
               
                   
                   
                 (SEQ ID NO: 92) 
                   
               
               
                   
               
               
                 A-2 
                 ggctacacctttactagctac 
                 tacattaatcctgacactgatta 
                 gcaagtgctggttattatttttttgac 
               
               
                 Nucleic 
                 tggatacac 
                 tagtgagtacaat 
                 ttc 
               
               
                 Acid 
                 (SEQ ID NO: 71) 
                 (SEQ ID NO: 93) 
                 (SEQ ID NO: 117) 
               
               
                   
               
               
                 Amino 
                 GYTFTSYWIH 
                 YINPDTDYSEYN 
                 ASAGYYFFDF 
               
               
                 Acid 
                 (SEQ ID NO: 72) 
                 (SEQ ID NO: 94) 
                 (SEQ ID NO: 118 
               
               
                   
               
               
                 A-3 
                 ggcctcaacattaaagacat 
                 aggattgatcctgcgaacggt 
                 ggtaggggggcccac (SEQ 
               
               
                 Nucleic 
                 ctatattcac 
                 aagactgcatatgac 
                 ID NO: 119) 
               
               
                 Acid 
                 (SEQ ID NO: 73) 
                 (SEQ ID NO: 95) 
                   
               
               
                   
               
               
                 Amino 
                 GLNIKDIYIH 
                 RIDPANGKTAYD 
                 GRGAH 
               
               
                 Acid 
                 (SEQ ID NO: 74) 
                 (SEQ ID NO: 96) 
                 (SEQ ID NO: 120) 
               
               
                   
               
               
                 A-4 
                 ggttactcattcaccaactac 
                 atgattgatccttccgatgctg 
                 gcaagaattggcgattactataata 
               
               
                 Nucleic 
                 tggatacac 
                 aaactgggttaaat 
                 tggactac 
               
               
                 Acid 
                 (SEQ ID NO: 75) 
                 (SEQ ID NO: 97) 
                 (SEQ ID NO: 121) 
               
               
                   
               
               
                 Amino 
                 GYSFTNYWIH 
                 MIDPSDAETGLN 
                 ARIGDYYNMDY (SEQ 
               
               
                 Acid 
                 (SEQ ID NO: 76) 
                 (SEQ ID NO: 98) 
                 ID NO: 122) 
               
               
                   
               
               
                 A-5 
                 ggattcactttcagtgactat 
                 gttagtgatggtggtggttcca 
                 acaagacatgcttcctactatagct 
               
               
                 Nucleic 
                 cccatgtct 
                 cc 
                 acgaccattctatggactac 
               
               
                 Acid 
                 (SEQ ID NO: 77) 
                 (SEQ ID NO: 99) 
                 (SEQ ID NO: 123) 
               
               
                   
               
               
                 Amino 
                 GFTFSDYPMS 
                 VSDGGGST 
                 TRHASYYSYDHSMD 
               
               
                 Acid 
                 (SEQ ID NO: 78) 
                 (SEQ ID NO: 100) 
                 Y 
               
               
                   
                   
                   
                 (SEQ ID NO: 124) 
               
               
                   
               
               
                 A-6 
                 ggattcactttcagtagcttt 
                 attagtagtgctggtagtttcac 
                 gcaagacgggggtacgacgttgg 
               
               
                 Nucleic 
                 ggcatgtct 
                 c 
                 gtgctttgaccac 
               
               
                 Acid 
                 (SEQ ID NO: 79) 
                 (SEQ ID NO: 101) 
                 (SEQ ID NO: 125) 
               
               
                   
               
               
                 Amino 
                 GFTFSSFGMS 
                 ISSAGSFT 
                 ARRGYDVGCFDH 
               
               
                 Acid 
                 (SEQ ID NO: 80) 
                 (SEQ ID NO: 102) 
                 (SEQ ID NO: 126) 
               
               
                   
               
               
                 A-7 
                 ggattcactttcagtacctat 
                 accattaatactaatggtggta 
                 gcaagagactacggggctatgga 
               
               
                 Nucleic 
                 ggcatgtct 
                 ccacctattatcgagacagtgt 
                 ctac 
               
               
                 Acid 
                 (SEQ ID NO: 81) 
                 gaagggc 
                 (SEQ ID NO: 127) 
               
               
                   
                   
                 (SEQ ID NO: 103) 
                   
               
               
                   
               
               
                 Amino 
                 GFTFSTYGMS 
                 TINTNGGTTYYRDS 
                 ARDYGAMDY 
               
               
                 Acid 
                 (SEQ ID NO: 82) 
                 VKG 
                 (SEQ ID NO: 128) 
               
               
                   
                   
                 (SEQ ID NO: 104) 
                   
               
               
                   
               
               
                 A-8 
                 ggattcactttcagtacctat 
                 accataaatactaatggtggta 
                 gcaagagactacggggctatgga 
               
               
                 Nucleic 
                 ggcatgtct 
                 acacctattattcagacaatgt 
                 ctac 
               
               
                 Acid 
                 (SEQ ID NO: 81) 
                 gaagggc 
                 (SEQ ID NO: 127) 
               
               
                   
                   
                 (SEQ ID NO: 105) 
                   
               
               
                   
               
               
                 Amino 
                 GFTFSTYGMS 
                 TINTNGGNTYYSD 
                 ARDYGAMDY 
               
               
                 Acid 
                 (SEQ ID NO: 82) 
                 NVKG 
                 (SEQ ID NO: 128) 
               
               
                   
                   
                 (SEQ ID NO: 106) 
                   
               
               
                   
               
               
                 A-9 
                 ggattcactttcagtagttat 
                 accattagtactaatggtgcca 
                 gcaactgaaaagggagctatggg 
               
               
                 Nucleic 
                 ggcatgtct 
                 ccgccaattatccagacagtg 
                 ctac 
               
               
                 Acid 
                 (SEQ ID NO: 83) 
                 tgaagggc 
                 (SEQ ID NO: 129) 
               
               
                   
                   
                 (SEQ ID NO: 107) 
                   
               
               
                   
               
               
                 Amino 
                 GFTFSSYGMS 
                 TISTNGATANYPDS 
                 ATEKGAMGY 
               
               
                 Acid 
                 (SEQ ID NO: 84) 
                 VKG 
                 (SEQ ID NO: 130) 
               
               
                   
                   
                 (SEQ ID NO: 108) 
                   
               
               
                   
               
               
                 A-10 
                 gggttttcactgaccacttct 
                 cacatttggtgggatgatgata 
                 gctcgaagaactgagactatgatt 
               
               
                 Nucleic 
                 ggtatgggtgtaggc 
                 agtactataatccatccctgaa 
                 acgacagtgctatattactatgctat 
               
               
                 Acid 
                 (SEQ ID NO: 85) 
                 gagc 
                 ggactac 
               
               
                   
                   
                 (SEQ ID NO: 109) 
                 (SEQ ID NO: 131) 
               
               
                   
               
               
                 Amino 
                 GFSLTTSGMGVG 
                 HIWWDDDKYYNPS 
                 ARRTETMITTVLYYY 
               
               
                 Acid 
                 (SEQ ID NO: 86) 
                 LKS 
                 AMDY 
               
               
                   
                   
                 (SEQ ID NO: 110) 
                 (SEQ ID NO: 132) 
               
               
                   
               
               
                 A-11 
                 ggattttcactgagcacttct 
                 cacatttggtgggatgatgata 
                 gctcgaaggagggaagttaacttc 
               
               
                 Nucleic 
                 ggtttgggtgtaggc 
                 agtactataatccatcccttaa 
                 ggtattaactattactattctatgga 
               
               
                 Acid 
                 (SEQ ID NO: 87) 
                 gaga 
                 ctac 
               
               
                   
                   
                 (SEQ ID NO: 111) 
                 (SEQ ID NO: 133) 
               
               
                   
               
               
                 Amino 
                 GFSLSTSGLGVG 
                 HIWWDDDKYYNPS 
                 ARRREVNFGINYYYS 
               
               
                 Acid 
                 (SEQ ID NO: 88) 
                 LKR 
                 MDY 
               
               
                   
                   
                 (SEQ ID NO: 112) 
                 (SEQ ID NO: 134) 
               
               
                   
               
               
                 A-12 
                 ggattcaccttcagtgattatt 
                 attagaaatcgggctaatggtt 
                 gtaagagattcctatcactacgggt 
               
               
                 Nucleic 
                 ac 
                 acacaaca 
                 acttcgatgtc 
               
               
                 Acid 
                 (SEQ ID NO: 89) 
                 (SEQ ID NO: 113) 
                 (SEQ ID NO: 135) 
               
               
                   
               
               
                 Amino 
                 GFTFSDYY 
                 IRNRANGYTT 
                 VRDSYHYGYFDV 
               
               
                 Acid 
                 (SEQ ID NO: 90) 
                 (SEQ ID NO: 114) 
                 (SEQ ID NO: 136) 
               
               
                   
               
            
           
         
       
     
     Table 2 provides polynucleotide (DNA) sequences encoding the amino acid sequences of the variable light and variable heavy domains of exemplary ET A R antibodies. (SEQ ID NO: 137-192). 
     Polynucleotide and amino acid sequences of light chain variable domains:
     L1 (A-1): polynucleotide sequence SEQ ID NO: 137, amino acid sequence SEQ ID NO: 138.   L2 (A-2): polynucleotide sequence SEQ ID NO: 139, amino acid sequence SEQ ID NO: 140.   L3 (A-3): polynucleotide sequence SEQ ID NO: 141, amino acid sequence SEQ ID NO: 142.   L4 (A-4): polynucleotide sequence SEQ ID NO: 143, amino acid sequence SEQ ID NO: 144.   L5 (A-5): polynucleotide sequence SEQ ID NO: 145, amino acid sequence SEQ ID NO: 146.   L6 (A-6): polynucleotide sequence SEQ ID NO: 147, amino acid sequence SEQ ID NO: 148.   L7 (A-7): polynucleotide sequence SEQ ID NO: 149, amino acid sequence SEQ ID NO: 150.   L8 (A-8): polynucleotide sequence SEQ ID NO: 151, amino acid sequence SEQ ID NO: 152.   L9 (A-9): polynucleotide sequence SEQ ID NO: 153, amino acid sequence SEQ ID NO: 154.   L10 (A-10): polynucleotide sequence SEQ ID NO: 155, amino acid sequence SEQ ID NO: 156.   L11 (A-11): polynucleotide sequence SEQ ID NO: 157, amino acid sequence SEQ ID NO: 158.   L12 (A-12): polynucleotide sequence SEQ ID NO: 159, amino acid sequence SEQ ID NO: 160.   L13 (A-13): polynucleotide sequence SEQ ID NO: 161, amino acid sequence SEQ ID NO: 162.   L14 (A-14): polynucleotide sequence SEQ ID NO: 163, amino acid sequence SEQ ID NO: 164.   

     Polynucleotide and amino acid sequences of heavy chain variable domains:
     H1 (A-1): polynucleotide sequence SEQ ID NO: 165, amino acid sequence SEQ ID NO: 166.   H2 (A-2): polynucleotide sequence SEQ ID NO: 167, amino acid sequence SEQ ID NO: 168.   H3 (A-3): polynucleotide sequence SEQ ID NO: 169, amino acid sequence SEQ ID NO: 170.   H4 (A-4): polynucleotide sequence SEQ ID NO: 171, amino acid sequence SEQ ID NO: 172.   H5 (A-5): polynucleotide sequence SEQ ID NO: 173, amino acid sequence SEQ ID NO: 174.   H6 (A-6): polynucleotide sequence SEQ ID NO: 175, amino acid sequence SEQ ID NO: 176.   H7 (A-7): polynucleotide sequence SEQ ID NO: 177, amino acid sequence SEQ ID NO: 178.   H8 (A-8): polynucleotide sequence SEQ ID NO: 179, amino acid sequence SEQ ID NO: 180.   H9 (A-9): polynucleotide sequence SEQ ID NO: 181, amino acid sequence SEQ ID NO: 182.   H10 (A-10): polynucleotide sequence SEQ ID NO: 183, amino acid sequence SEQ ID NO: 184.   H11 (A-11): polynucleotide sequence SEQ ID NO: 185, amino acid sequence SEQ ID NO: 186.   H12 (A-12): polynucleotide sequence SEQ ID NO: 187, amino acid sequence SEQ ID NO: 188.   H13 (A-13): polynucleotide sequence SEQ ID NO: 189, amino acid sequence SEQ ID NO: 190.   H14 (A-14): polynucleotide sequence SEQ ID NO: 191, amino acid sequence SEQ ID NO: 192.   

     Particular embodiments of antibodies of the present invention comprise one or more amino acid sequences that are identical to the amino acid sequences of the CDRs and/or FRS (framework regions) illustrated above. In one embodiment, the antibody comprises a light chain CDR1 sequence illustrated above. In another embodiment, the antibody comprises a light chain CDR2 sequence illustrated above. In another embodiment, the antibody comprises a light chain CDR3 sequence illustrated above. In another embodiment, the antibody comprises a heavy chain CDR1 sequence illustrated above. In another embodiment, the antibody comprises a heavy chain CDR2 sequence illustrated above. In another embodiment, the antibody comprises a heavy chain CDR3 sequence illustrated above. In another embodiment, the antibody comprises a light chain FR1 sequence illustrated above. In another embodiment, the antibody comprises a light chain FR2 sequence illustrated above. In another embodiment, the antibody comprises a light chain FR3 sequence illustrated above. In another embodiment, the antibody comprises a light chain FR4 sequence illustrated above. In another embodiment, the antibody comprises a heavy chain FR1 sequence illustrated above. In another embodiment, the antibody comprises a heavy chain FR2 sequence illustrated above. In another embodiment, the antibody comprises a heavy chain FR3 sequence illustrated above. In another embodiment, the antibody comprises a heavy chain FR4 sequence illustrated above. 
     In another embodiment, a CDR3 sequence of the antibody differs from a CDR3 sequence of A-1 by no more than 6, 5, 4, 3, 2, 1 or 0 single amino acid addition(s), substitution(s), and/or deletion(s). In another embodiment, a light chain CDR3 sequence of the antibody differs from the light chain CDR3 sequence of A-1 described above by no more than 6, 5, 4, 3, 2, 1 or 0 single amino acid addition(s), substitution(s), and/or deletion(s) and the heavy chain CDR3 sequence of the antibody differs from a heavy chain CDR3 sequence of A-1/A-2 described above by no more than 6, 5, 4, 3, 2, 1 or 0 single amino acid addition(s), substitution(s), and/or deletion(s). In another embodiment, the antibody further comprises 1, 2, 3, 4, or 5 CDR sequences, each of which independently differs from a CDR sequence of A-1 by 6, 5, 4, 3, 2, 1, or 0 single amino acid addition(s), substitution(s), and/or deletion(s). In another embodiment, the antibody comprises the CDRs of a light chain variable domain and the CDRs of a heavy chain variable domain set forth above. In another embodiment, the antibody comprises 1, 2, 3, 4, 5, and/or 6 consensus CDR sequence(s) shown above. In one embodiment, the antibody is a murine antibody. 
     In one embodiment, the antibody (such as an antibody or antibody fragment) comprises an amino acid sequence of a light chain variable domain that differs from the sequence of light chain variable domain L 1 by 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 single amino acid deletion(s), insertion(s), or substitution(s). In another embodiment, the light-chain variable domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% , 97% or 99% identical to the sequence of light chain variable domain L1. In another embodiment, the light chain variable domain comprises an amino acid sequence that is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identical to the L1 polynucleotide sequence listed above. In another embodiment, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide encoding light chain variable domain L1. In another embodiment, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide encoding light chain variable domain L1. 
     In another embodiment, the present invention provides an antibody comprising a heavy chain variable domain that comprises an amino acid sequence that differs from the sequence of heavy chain variable domain H1 by 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 single amino acid deletion(s), insertion(s), or substitution(s). In another embodiment, the heavy chain variable domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identical to the sequence of heavy chain variable domain H1. In another embodiment, the heavy chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide encoding heavy chain variable domain H1. In another embodiment, the heavy chain variable domain comprises an amino sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide encoding heavy chain variable domain H1. 
     Antibodies (e.g., antibodies, antibody fragments, and antibody derivatives) of the invention can comprise any constant region known in the art. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a murine kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon- gamma-, or mu-type heavy chain constant regions, e.g., a murine alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. In one embodiment, the light or heavy chain constant region is a fragment, derivative, variant, or mutein of a naturally occurring constant region. 
     Subclass switching techniques are known to derive an antibody of a different subclass or isotype. Thus, IgG antibodies can be derived from an IgM antibody, for example, and vice versa. Such techniques allow the preparation of new antibodies that possess the antigen-binding properties of a given antibody (the parent antibody), but also exhibit biological properties associated with an isotype or subclass different from that of the parent antibody. Recombinant DNA techniques can be employed. Cloned DNA encoding particular antibody polypeptides can be employed, e.g., DNA encoding the constant region of an antibody of the desired isotype. See also Lanitto et al., 2002,  Methods Mol. Biol.  178:303-16. 
     In one embodiment, an antibody of the invention further comprises a constant light chain κ or λ region or a fragment thereof. Sequences of light chain constant regions and their coding polynucleotides are provided below. 
     Light Chain Constant Region: 
     
         
         polynucleotide (κ), (SEQ ID NO: 193) 
         amino acid (κ), (SEQ ID NO: 194) 
         polynucleotide (λ), (SEQ ID NO: 195) 
         amino acid (λ), (SEQ ID NO: 196) 
       
    
     In another embodiment, an antibody of the invention further comprises a heavy chain constant region or a fragment thereof as shown below. 
     Heavy Chain Constant Region: 
     
         
         Polynucleotide (IgG1), (SEQ ID NO: 197) 
         amino acid (IgG1), (SEQ ID NO: 198) 
       
    
     The antibodies of the present invention include those comprising, for example, combination L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13 or L14H14; or an isotype thereof (for example, IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE, or IgD) or a Fab or F(ab′)2 fragment thereof. 
     Antibodies and Antibody Fragments 
     In one embodiment, the antibody is an antibody. The term “antibody” refers to an intact antibody or an antigen binding fragment thereof, as described generally in the definition section. An antibody can comprise a complete antibody molecule (including polyclonal, monoclonal, chimeric, humanized, or human versions having full length heavy and/or light chains), or comprise an antigen binding fragment thereof. Antibody fragments include F(ab′)2, Fab, Fab′, Fv, Fc, and Fd fragments, and can be incorporated into single domain antibodies, single-chain antibodies, maxibodies, minibodies, intrabodies, diabodies, tribadies, tetrabodies, v-NAR and bis-scFv (see e.g., Hollinger and Hudson, 2005,  Nature Biotechnology,  23, 9, 1126-1136). Also included are antibody polypeptides such as those disclosed in U.S. Pat. No. 6703199, including fibronectin polypeptide monobodies. Other antibody polypeptides are disclosed in U.S. Patent Publication 2005/0238646, which are single-chain polypeptides. In one embodiment, the antibodies of the present invention comprise at least one CDR or consensus CDR as set forth in Table 2 above. 
     In one embodiment, the variable regions of a gene expressing a monoclonal antibody of interest are amplified using nucleotide primers in a hybridoma. These primers can be synthesized by one of ordinary skill in the art, or can be purchased from commercially available sources (see, e.g., Stratagene, La Jolla, Calif.), which sells primers for mouse and human variable regions including, among others, primers for V Ha , V Hb , V Hc , V Hd , C H1 , V L  and C L  regions. These primers can be used to amplify heavy or light chain variable regions, which can then be inserted into vectors such as ImmunoZAP™H or ImmunoZAP™L (Stratagene), respectively. These vectors can then be introduced into  E. coli,  yeast, or mammalian-based systems for expression. Large amounts of a single-chain protein containing a fusion of the V H  and V L  regions can be produced using these methods (see Bird el al., 1988,  Science  242:423-426). 
     Once antibody-producing cells of the instant invention have been obtained using any above-described immunization and other techniques, the genes of the specific antibodies can be cloned by isolating and amplifying DNA or mRNA therefrom according to standard procedures described herein. The antibodies produced therefrom can be sequenced to identify CDRs, and the coding DNA of the CDRs can be manipulated as described above to generate other antibodies of the present invention. 
     Antibodies of the present invention preferably modulate endothelin signaling in the cell-based assay described herein and/or in the in vivo assay described herein and/or cross-block the binding of one of the antibodies described herein and/or are cross-blocked from binding ET A R by one of the antibodies described herein. Accordingly, such binding agents can be identified using the assays described herein. 
     In certain embodiments, antibodies are generated by first identifying antibodies that bind to cells overexpressing ET A R and/or neutralize in the cell-based and/or in vivo assays described herein and/or cross-block the antibodies described herein and/or are cross-blocked from binding ET A R by one of the antibodies described herein. 
     It should be understood by one skilled in the art that certain proteins, such as antibodies, can undergo a variety of post-translational modifications. The types and extents of these modifications often depend on the host cell lines used to express the protein as well as the culture conditions. Such modifications can include variations in glycosylation, methionine oxidation, diketopiperizine formation, aspartate isomerization and asparagine deamidation. A frequent modification is the loss of a carboxyl-terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, R. J., 1995,  Journal of Chromatography  705:129-134). 
     An alternative method for production of a murine monoclonal antibody is to inject hybridoma cells into the peritoneal cavity of a syngeneic mouse, for example, a mouse that has been treated (e.g., pristine-primed) to promote formation of ascites fluid containing the monoclonal antibody. Monoclonal antibodies can be isolated and purified by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, e.g., Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., “Purification of Immunoglobulin G (IgG),” in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)). A monoclonal antibody can be purified by affinity chromatography using an appropriate ligand selected based on particular properties of the antibody (e.g., heavy or light chain isotype, binding specificity, etc.). Examples of suitable ligands immobilized on a solid support include Protein A, Protein G, an anti-constant region (light chain or heavy chain) antibody, an anti-idiotype antibody, and a TGF-β binding protein, or a fragment or variant thereof. 
     Molecular evolution of the complementarity determining regions (CDRs) in the center of the antibody binding site also has been used to isolate antibodies with increased affinities, for example, antibodies having increased affinities for c-erbB-2, as described by Schier et al., 1996,  J. Mol. Biol.  263:551-567 Accordingly, such techniques are useful in preparing antibodies to human endothelin A receptor. 
     Antibodies against human endothelin A receptor can be used, for example, in assays to detect the presence of the endothelin A receptor, either in vitro or in vivo. 
     Antibodies can also be prepared by any of the conventional techniques. For example, they can be purified from cells that naturally express them (e.g., an antibody can be purified from a hybridoma that produces it) or produced in recombinant expression systems using any technique known in the art. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988). This is discussed in the nucleic acid section below. 
     Antibodies can be prepared and screened for desired properties by any known techniques. Some techniques relate to the isolation of nucleic acids encoding polypeptide chains (or portions thereof) of related antibodies (e.g., anti-ET A R antibodies) and manipulation of nucleic acid. Nucleic acids can be fused with another relevant nucleic acid or modified by recombinant DNA techniques (e.g., induced mutations or other conventional techniques) to add, delete or replace one or more amino acid residues. 
     Where it is desired to improve the affinity of antibodies according to the invention containing one or more of the above-mentioned CDRs, such antibodies can be obtained by a number of affinity maturation protocols, including maintaining the CDRs (Yang et al., 1995,  J. Mol. Biol.,  254:392-403), chain shuffling (Marks et al., 1992,  Bio/Technology,  10:779-783), use of mutation strains of  E. coli.  (Low et al., 1996,  J. Mol. Biol.,  250:350-368), DNA shuffling (Patten et al., 1997,  Curr. Opin. Biotechnol.,  8:724-733), phage display (Thompson et al., 1996,  J. Mol. Biol.,  256:7-88) and additional PCR techniques (Crameri et al., 1998,  Nature,  391:288-291). These methods or affinity maturation are discussed in Vaughan et al., 1998,  Nature Biotechnology,  16:535-539). 
     Antibody Fragments 
     In another aspect, the present invention provides fragments of an anti-ET A R antibody of the invention. Such fragments can comprises entirely antibody-derived sequences or additional sequences. Examples of antigen binding fragments include Fab, F(ab′)2, single chain antibodies, diabodies, tribodies, tetrabodies, and domain antibodies. Other examples are provided in Lunde et al., 2002,  Biochem. Soc. Trans.  30:500-06. 
     Single chain antibodies can be formed by linking heavy and light chain variable domain (Fv region) fragments via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain. Such single-chain Fvs (scFvs) have been prepared by fusion DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (V L  and V H ). 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 a 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 L  and V H -comprising polypeptides, multimeric scFvs that bind to different epitopes can be formed (Kriangkum et al., 2001,  Biomol. Eng.  18:31-40). Techniques developed for the production of single chain antibodies include those described in U.S. Pat. No. 4946778; Bird, 1988,  Science  242:423; Huston et al., 1988,  Proc. Natl. Acad. Sci. USA  85:5879; Ward et al., 1989,  Nature  334:544; de Graaf et al., 2002,  Methods Mol. Biol.  178:379-87. Single chain antibodies derived from antibodies provided herein including, but not limited to, scFvs comprising the variable domain combination L1H1, are encompassed by the present invention. 
     Antibodies derived from an antibody can also be obtained, for example, by proteolytic hydrolysis of the antibody, for example, pepsin or papain digestion of a whole antibody according to conventional methods. By way of example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a SS fragment termed F(ab′)2. This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab′ monovalent fragments. Optionally, the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages. As an alternative, an enzymatic cleavage using papain produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. No. 4,331,647, Nisonoffet 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 by Andrews, S. M. and Titus, J. A. in Current Protocols in Immunology (Coligan J. E., et al., eds), John Wiley &amp; Sons, New York (2003), pages 2.8.1-2.8.10 and 2.10A.1-2.10A.5. Other methods for cleaving antibodies, such as separating heavy chains to form monovalent light-heavy chain fragments (Fd), further cleaving of fragments, or other enzymatic, chemical, or genetic techniques can also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. 
     Another form of an antibody fragment is a peptide comprising one or more complementarity determining regions (CDRs) of an antibody. CDRs can be obtained by constructing polynucleotides that encode the CDRs. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA or 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,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); and Ward el al., “Genetic Manipulation and Expression or Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995). The antibody fragment further can comprise at least one variable region domain of an antibody described herein. Thus, for example, the V region domain can be monomeric and be a V H  or V L  domain, which can bind to ET A R with an affinity of 10 −7  M or less as described below. 
     The variable region domain can be any naturally occurring variable domain or an engineered version thereof. By engineered version is meant a variable region domain that has been created using recombinant DNA engineering techniques. Such engineered versions include those created, for example, from a specific antibody variable region by insertions, deletions, or changes in or to the amino acid sequences of the specific antibody. Particular examples include engineered variable region domains containing at least one CDR and optionally one or more framework amino acids from a first antibody and the remainder of the variable region domain from a second antibody. 
     The variable region domain can be covalently attached at a C-terminal amino acid to at least one other antibody domain or a fragment thereof. Thus, for example, a V H  domain that is present in the variable region domain can be linked to an immunoglobulin C H1  domain or a fragment thereof. Similarly, a V L  domain can be linked to a C K  domain or a fragment thereof. In this way, for example, the antibody can be a Fab fragment, wherein the antigen binding domain contains associated V H  and V L  domains covalently linked at their C-termini to a C H1  and C κ  domain, respectively. The C H1  domain can be extended with further amino acids, for example to provide a hinge region or a portion of a hinge region domain as found in a Fab′ fragment, or to provide further domains, such as antibody C H2  and C H3  domains. 
     Derivatives and Variants of Antibodies 
     The nucleotide sequences of L1 and H1, encoding the corresponding amino acid sequences of A1/A2, can be altered, for example, by random mutagenesis or by site-directed mutagenesis (e.g., oligonucleotide-directed site-specific mutagenesis) to create an altered polynucleotide comprising one or more particular nucleotide substitutions, deletions, or insertions as compared to the non-mutated polynucleotide. Examples of techniques for making such alterations are described in Walder et al., 1986,  Gene  42:133; Bauer et al., 1985,  Gene  37:73; Craik, 1985,  BioTechniques,  3:12-19; 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 make, for example, derivatives of anti-endothelin A receptor antibodies that have a desired property, for example, an increase in affinity, avidity, or specificity for an endothelin receptor or in vivo or in vitro stability, or reduced in vivo side-effects as compared to the underivatized antibody. 
     Other derivatives of anti-endothelin receptor antibodies within the scope or this invention include covalent or aggregative conjugates or anti-endothelin receptor antibodies, or fragments thereof, with other proteins or polypeptides, such as by expression or recombinant fusion proteins comprising heterologous polypeptides fused to the N-terminus or C-terminus or an anti-endothelin receptor antibody polypeptide. For example, the conjugated peptide can be a heterologous signal (or leader) polypeptide, e.g., the yeast alpha-factor leader or a peptide such as an epitope tag. An antibody containing fusion proteins can comprise peptides added to facilitate purification or identification of antigen binding protein (e.g., poly-His). An antibody also can be linked to the FLAG peptide as described in Hopp et al., 1988,  Bio/Technology  6:1204, and U.S. Pat. No. 5,011,912. The FLAG peptide is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody (mAb), enabling rapid assay and facile purification of an expressed recombinant protein. Reagents useful for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, Mo.). In another embodiment, oligomers that contain one or more antibodies can be employed as endothelin receptor antagonists. Oligomers can be in the form of covalently-linked or non-covalently-linked dimers, trimers, or higher oligomers. Oligomers comprising two or more antibodies are contemplated for use, with one example being a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc. 
     One embodiment is directed to oligomers comprising multiple antibodies joined via covalent or non-covalent interactions between peptide moieties fused to the antibodies. Such peptides can be peptide linkers (spacers), or peptides that have the property of promoting oligomerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of antibodies attached thereto, as described in more detail below. 
     In particular embodiments, the oligomers comprise from two to four antibodies. The antibodies of the oligomer can be in any form, such as any of the forms described above, e.g., variants or fragments. Preferably, the oligomers comprise antibodies that have endothelin receptor binding activity. 
     In one embodiment, an oligomer is prepared using polypeptides derived from immunoglobulins. Preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al., 1991,  PNAS USA  88:10535; Byrn et al., 1990,  Nature  344:677; and Hollenbaugh et al., 1992 “Construction of Immunoglobulin Fusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11. One embodiment of the present invention is directed to a dimer comprising two fusion proteins created by fusing an endothelin receptor binding fragment of an anti-endothelin A receptor antibody to the Fc region of an antibody. The dimer can be made by, for example, inserting a gene fusion encoding the fusion protein into an appropriate expression vector, expressing the gene fusion in host cells transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble much like antibody molecules, whereupon inter-chain disulfide bonds form between the Fc moieties to yield the dimer. 
     The term “Fc polypeptide” as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization also are included. Fusion proteins comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Protein A or Protein G columns. 
     One suitable Fc polypeptide, described in PCT application WO 93/10151 (hereby incorporated 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 mutein described in U.S. Pat. No. 5,457,035 and in Baum et al., 1994,  EMBO J.  13:3992-4001. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors. In other embodiments, the variable portion of the heavy and/or light chains of an anti-endothelin receptor antibody can be substituted for the variable portion of an antibody heavy and/or light chain. 
     Alternatively, the oligomer is a fusion protein comprising multiple antibodies, with or without peptide linkers (spacer peptides). Among the suitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and 4,935,233. 
     Another method for preparing oligomeric antibodies involves use of a leucine zipper. Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., 1988,  Science  240:1759), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94/10308, and the leucine zipper derived from lung surfactant protein D (SPD) described in Hoppe et al., 1994,  FEBS Letters  344:191, hereby incorporated by reference. The use of a modified leucine zipper that allows for stable trimerization of a heterologous protein fused thereto is described in Fanslow et al., 1994,  Semin. Immunol.  6:267-78. In one method, recombinant fusion proteins comprising an anti-endothelin receptor antibody fragment or derivative fused to a leucine zipper peptide are expressed in suitable host cells, and the soluble oligomeric anti-endothelin receptor antibody fragments or derivatives that form are recovered from the culture supernatant. 
     In another embodiment, the antibody derivatives can comprise at least one of the CDRs disclosed herein. For example, one or more CDR can be incorporated into known antibody framework regions (IgG1, IgG2, etc.), or conjugated to a suitable vehicle to enhance the half-life thereof. Suitable vehicles include, but are not limited to Fc, albumin, transferrin, and the like. These and other suitable vehicles are known in the art. Such conjugated CDR peptides can be in monomeric, dimeric, tetrameric, or other form. In one embodiment, one or more water-soluble polymer is bonded at one or more specific position, for example at the amino terminus, of a binding agent. In an example, an 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 certain embodiments, a derivative comprises one or more of monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of such polymers. In certain embodiments, one or more water-soluble polymer is randomly attached to one or more side chains. In certain embodiments, PEG can act to improve the therapeutic capacity for a binding agent, such as an antibody. Certain such methods are discussed, for example, in U.S. Pat. No. 6,133,426, which is hereby incorporated by reference for any purpose. 
     It will be appreciated that an antibody of the present invention can have at least one amino acid substitution, providing that the antibody retains binding specificity. Therefore, modifications to the antibody structures are encompassed within the scope of the invention. These can include amino acid substitutions, which may be conservative or non-conservative, that do not destroy the human endothelin receptor binding capability of an antibody. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties. A conservative amino acid substitution can also involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Non-conservative substitutions can involve the exchange of a member of one class of amino acids or amino acid mimetics for a member from another class with different physical properties (e.g., size, polarity, hydrophobicity, charge). 
     Moreover, one skilled in the art may generate variants to be tested, which contain a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change may be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations. 
     A skilled artisan will be able to determine suitable variants of the polypeptide as set forth herein using well-known techniques. In certain embodiments, one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not to be important for activity. In certain embodiments, one can identify residues and portions of the molecules that are conserved among similar polypeptides. In certain embodiments, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure. Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues which are important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues. 
     One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three dimensional structure. In certain embodiments, one skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. A number of scientific publications have been devoted to the prediction of secondary structure. See Moult, 1996,  Curr. Op. Biotech.  7:422-427; Chou et al., 1974,  Biochemistry  13:222-245; Chou et al., 1974,  Biochemistry  113:211-222; Chou et al., 1978,  Adv. Enzymol. Relat. Areas Mol. Biol.  47:45-148; Chou et al., 1979,  Ann. Rev. Biochem.  47:251-276 and Chou et al.,  Biophys. J.  26:367-384. Moreover, computer programs are currently available to assist with predicting secondary structure. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural database (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within the structure of a polypeptide or protein. See Holm et al., 1999,  Nucl. Acid. Res.  27:244-247. It has been suggested (Brenner et al., 1997,  Curr. Op. Struct. Biol.  7:369-376) that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved, structural prediction will become dramatically more accurate. 
     Additional methods of predicting secondary structure include “threading” (Jones, 1997,  Curr. Opin. Struct. Biol.  7:377-87; Sippl et al., 1996,  Structure  4:15-19), “profile analysis” (Bowie et al., 1991,  Science  253:164-170; Gribskov et al., 1990,  Meth. Enzym.  183:146-159; Gribskov et al., 1987,  Proc. Nat. Acad. Sci.  84:4355-4358), and “evolutionary linkage” (see Holm, supra (1999), and Brenner, supra (1997)). In certain embodiments, variants of antibodies include glycosylation variants, wherein the number and/or type of glycosylation sites have been altered compared to the amino acid sequences of a parent polypeptide. In certain embodiments, variants comprise a greater or lesser number of N-linked glycosylation sites than the native protein. Alternatively, elimination of such a sequence by substitutions removes an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains, wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created. Additional preferred antibody variants include cysteine variants, wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the parent amino acid sequence. Cysteine variants can be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines. 
     Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. In certain embodiments, amino acid substitutions can be used to identify important residues of antibodies to human endothelin receptor, or to increase or decrease the affinity of the antibodies to human endothelin receptor described herein. 
     According to certain embodiments, preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and/or (4) confer or modify other physiochemical or functional properties on such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be made in the naturally-occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). In certain embodiments, a conservative amino acid substitution typically cannot substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (Branden and Tooze, Eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., 1991,  Nature  354:105, each of which is incorporated herein by reference. 
     In certain embodiments, antibodies of the invention can be chemically bonded with polymers, lipids, or other moieties. 
     The antigen binding agents can comprise at least one of the CDRs described herein incorporated into a biocompatible framework structure. In one example, the biocompatible framework structure comprises a polypeptide or portion thereof that is sufficient to form a conformationally stable structural support, or framework, or scaffold, which is able to present one or more sequences of amino acids that bind to an antigen (e.g., CDRs, a variable region, etc.) in a localized surface region. Such structures can be a naturally occurring polypeptide or polypeptide “fold” (a structural motif), or can have one or more modifications, such as additions, deletions or substitutions of amino acids, relative to a naturally occurring polypeptide or fold. These scaffolds can be derived from a polypeptide of any species (or of more than one species), such as a human, other mammal, other vertebrate, invertebrate, plant, bacteria or virus. 
     Typically, the biocompatible framework structures are based on protein scaffolds or skeletons other than immunoglobulin domains. For example, those based on fibronectin, ankyrin, lipocalin, neocarzinostain, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain and tendamistat domains can be used (see, e.g., Nygren and Uhlen, 1997,  Current Opinion in Structural Biology  7:463-469). 
     Additionally, one skilled in the art will recognize that suitable binding agents include portions of these antibodies, such as one or more of heavy chain CDR1, CDR2, CDR3, light chain CDR1, CDR2 and CDR3 as specifically disclosed herein. At least one of the regions of heavy chain CDR1, CDR2, CDR3, CDR1, CDR2 and CDR3 can have at least one amino acid substitution, provided that the antibody retains the binding specificity of the non-substituted CDR. The non-CDR portion of the antibody can be a non-protein molecule, wherein the binding agent cross-blocks the binding of an antibody disclosed herein to human endothelin receptor and/or inhibits the activity of endothelin-1 signaling through the receptor. The non-CDR portion of the antibody can be a non-protein molecule in which the antibody exhibits a similar binding pattern to human endothelin receptor peptides in a competition binding assay as that exhibited by at least one of antibodies A1/A2, and/or neutralizes the activity of endothelin-1. The non-CDR portion of the antibody can 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 an antibody disclosed herein to human ET A R and/or neutralizes endothelin-1 activity in vitro or in vivo. The non-CDR portion of the antibody can be composed of amino acids, wherein the antibody is a recombinant antibody, and the recombinant antibody exhibits a similar binding pattern to human ET A R peptides in a competition binding assay as exhibited by at least one of the antibodies A1/A2, and/or neutralizes endothelin-1 signaling. 
     Nucleic Acids 
     In one aspect, the present invention provides isolated nucleic acid molecules that encode the antibodies of the present invention. The nucleic acids comprise, for example, polynucleotides that encode all or part of an antibody, for example, one or both chains of an antibody of the invention, or a fragment, derivative, mutein, or variant thereof; polynucleotides sufficient for use as hybridization probes; PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide; anti-sense nucleic acids for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing. The nucleic acids can be any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleotides in length, and/or can comprise one or more additional sequences, for example, regulatory sequences, and/or be part of a larger nucleic acid, for example, a vector. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and/or DNA nucleotides, and artificial variants thereof (e.g., peptide nucleic acids). 
     Nucleic acids encoding antibody polypeptides (e.g., heavy or light chain, variable domain only, or full length) can be isolated from B-cells of mice that have been immunized with ET A R antigen. The nucleic acid can be isolated by conventional procedures such as polymerase chain reaction (PCR). 
     Nucleic acid sequences encoding the variable regions of the heavy and light chain variable regions are shown above. The skilled artisan will appreciate that, due to the degeneracy of the genetic code, each of the polypeptide sequences disclosed herein is encoded by a large number of other nucleic acid sequences. The present invention provides each degenerate nucleotide sequence encoding each antibody of the invention. 
     The invention further provides nucleic acids that hybridize to other nucleic acids (e.g., nucleic acids comprising a nucleotide sequence of any of A-1/A-2) under particular hybridization conditions. Methods for hybridizing nucleic acids are well-known in the art. See, e.g., Current Protocols in Molecular Biology, John Wiley &amp; Sons, N.Y. (1989), 6.3.1-6.3.6. As defined herein, for example, a moderately stringent hybridization condition uses a prewashing solution containing 5× sodium chloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6×SSC, and a hybridization temperature of 55° C. (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of 42° C.), and washing conditions of 60° C., in 0.5×SSC, 0.1% SDS. A stringent hybridization condition hybridizes in 6×SSC at 45° C., followed by one or more washes in 0.1×SSC, 0.2% SDS at 68° C. Furthermore, one of skill in the art can manipulate the hybridization and/or washing conditions to increase or decrease the stringency of hybridization such that nucleic acids comprising nucleotide sequences that are at least 65, 70, 75, 80, 85, 90, 95, 98 or 99% identical to each other typically remain hybridized to each other. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by, for example, Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, Ausubel et al., Eds., John Wiley &amp; Sons, Inc., sections 2.10 and 6.3-6.4), and can be readily determined by those having ordinary skill in the art based on, for example, the length and/or base composition of the DNA. Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an antibody) that it encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more particular amino acid residues are changed using, for example, a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues is changed using, for example, a random mutagenesis protocol. No matter how it is made, a mutant polypeptide can be expressed and screened for a desired property. 
     Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues. In one embodiment, nucleotide sequences provided herein for L1 to L2 and H1 to H2, or fragments, variants, or derivatives thereof, are mutated such that they encode amino acid sequences provided herein for L1 to L2 and H1 to H2, comprising one or more deletions or substitutions of amino acid residues to result in sequences bearing two or more different amino acid residues. In another embodiment, the mutagenesis inserts an amino acid adjacent to one or more amino acid residues shown herein for L1 to L2 and H1 to H2 to result in sequences with two or more different amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively change the biological activity. (e.g., binding to ET A R) of a polypeptide that it encodes. For example, the mutation can quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity. Examples of qualitative changes include changing the antigen specificity of an antibody. 
     In another aspect, the present invention provides nucleic acid molecules that are suitable for use as primers or hybridization probes for the detection of nucleic acid sequences of the invention. A nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide of the invention, for example, a fragment that can be used as a probe or primer or a fragment encoding an active portion (e.g., an ET A R binding portion) of a polypeptide of the invention. 
     Probes based on the sequence of a nucleic acid of the invention can be used to detect the nucleic acid or similar nucleic acids, for example, transcripts encoding a polypeptide of the invention. The probe can comprise a label group, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used to identify a cell that expresses the polypeptide. 
     In another aspect, the present invention provides vectors comprising a nucleic acid encoding a polypeptide of the invention or a portion thereof. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors and expression vectors, for example, recombinant expression vectors. 
     The recombinant expression vectors of the invention can comprise a nucleic acid of the invention 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 on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types 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 disclosure of each of which is incorporated by reference herein in its entirety), and those that direct inducible expression of a nucleotide sequence in response to particular treatment or condition (e.g., the metallothionin promoter in mammalian cells and the tet-responsive and/or streptomycin responsive promoter in both prokaryotic and eukaryotic systems (see Id.). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein. 
     In another aspect, the present invention provides host cells into which a recombinant expression vector of the invention has been introduced. A host cell can be any prokaryotic cell or eukaryotic cell. Prokaryotic host cells include gram negative or gram positive organisms, for example,  E. coli  or bacilli. Higher eukaryotic cells include insect cells, yeast cells, and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines which grow in serum-free media (see Rasmussen et al., 1998,  Cytotechnology  28:31) or CHO strain DXB-11, which is deficient in DHFR (see Urlaub et al., 1980,  Proc. Natl. Acad. Sci. USA  77:4216-20). Additional CHO cell lines include CHO-K1 (ATCC #CCL-61), EM9 (ATCC #CRL-1861), and W20 (ATCC #CRL-1862). Additional 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 lines, the CV1/EBNA cell line derived from the 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, 293 EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985). 
     Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells can integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods. 
     The transformed cells can be cultured under conditions that promote expression of a polypeptide, and the polypeptide recovered by conventional protein purification procedures. One such purification procedure is described in the Examples below. Polypeptides contemplated for use herein include substantially homogeneous recombinant mammalian anti-endothelin receptor antibody polypeptides substantially free of contaminating endogenous materials. 
     Activity of Antibodies 
     In one aspect, the present invention provides murine or humanized antibodies that specifically bind to a human endothelin receptor. Such antibodies include antagonizing or neutralizing antibodies capable of reducing or neutralizing endothelin-1 signaling. In one embodiment, the antibodies, such as the antibodies of the present invention have an IC 50  of 100 nM or less, in another embodiment, an IC 50  of 80 nM or less, in another embodiment, 60 nM or less. In another embodiment, the antibodies such as the murine antibodies of the present invention are capable of specifically binding to a human endothelin receptor, and have an IC 50  that is substantially similar to that of a reference antibody. In another embodiment, the antibodies have a Kb (or Kd) as measured by the assay described in the examples below (or similar assays), that is substantially similar to that of a reference antibody. As used herein, the term “substantially similar” means comparable to, or about 100%, 99%, 98%, 97%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 50% identical to the IC 50  or Kb (or Kd) value of a reference antibody. Reference antibodies include, for example, antibodies having a heavy chain and light chain combination L1H1, L2H2. In one embodiment, the reference antibodies include A-1. In another embodiment, the antibodies such as the murine antibodies or humanized antibodies of the present invention are capable of specifically binding to a human endothelin receptor, and lowering pulmonary arterial pressure in an animal model. In one embodiment, the pulmonary arterial pressure is lowered by 2% compared with untreated animals, in another embodiment, the pulmonary arterial pressure is lowered by 5% compared with untreated animals, in another embodiment, the pulmonary arterial pressure is lowered by 10% compared to untreated animal, in another embodiment, the pulmonary arterial pressure is lowered by 15%, in another embodiment, by 20%, in another embodiment, by 25% or more. The amount of reduction of pulmonary arterial pressure is controlled by dosage. A therapeutically effective dosage is the dosage required to reduce pulmonary arterial pressure into the normal range for the animal or human patient. 
     Indications 
     Pulmonary arterial hypertension (PAH) is caused by various reasons and characteristic with a group of pathological or physiological symptoms. Under a resting state, PAH patients have a mean pulmonary arterial pressure (mPAP) higher than or equal to 25 mmHg. PAH results in alteration in the blood circulation hemodynamics of the lung vasculature and eventually leads to right heart failure, even death. PAH is a fairly common disease in China and the rates of disability and fatality among patients are fairly high. It is one of the most devastating diseases with a huge negative impact both on the patient&#39;s well beings and life, and it burdens the society with towering medical care and expense. 
     The severity of PAH depends on relevant cardiac deformities. Common congenital heart diseases that can cause secondary PAH include: aortic stenosis, aortopulmonary window, atrial septal defect, complete atrioventricular septal defect, arterial coarctation, dilated cardiomyopathy, right ventricular double outlet, hypertrophic cardiomyopathy, mitral stenosis, patent ductus arteriosus, persistent arterial trunk, ventricular septal defect. Pulmonary arterial hypertension mainly involves the pulmonary artery and right heart with right ventricular hypertrophy and right atrial dilatation. It can result in pulmonary artery dilatation and peripheral pulmonary artery sparse, proliferation of pulmonary arterial endothelial cells, hypertrophy of smooth muscle cells, thickening of vascular intimal fibrosis, mesenteric hypertrophy, stenosis, occlusion, distortion and plexiform changes. Intimal fibroelastosis and in-situ thrombosis in pulmonary small veins can occur as well. 
     The present invention provides antibodies that can specifically bind to human ET A R, reduce the pulmonary arterial pressure in different animal models and significantly ameliorate their symptoms of PAH. 
     Methods of Treatment 
     In another aspect, provided herein is a method of treating a subject, comprising administering a therapeutically effective amount of an antibody of the present invention. In one embodiment, the antibody is a murine antibody or a humanized antibody. As used herein, the term “subject” refers to a mammal, including humans, and is used interchangeably with the term “patient.” The murine antibody or humanized antibody, can be used to treat, control or prevent a disorder or condition characterized by an excessive level of pulmonary hypertension in a subject. The term “treatment” encompasses alleviation or prevention of at least one symptom or other aspect of a disorder, or reduction of disease severity, and the like. An antibody of the present invention needs not to provide a complete cure, or to eradicate every symptom or manifestation of a disease, to be an effective therapeutic agent. As is recognized in the pertinent field, therapeutic agents can reduce the severity of a given disease state, but need not to abolish every manifestation of the disease to be effective. Similarly, a prophylactic agent needs not to prevent the onset of a condition completely in order to be effective. Simply reducing the impact of a disease (for example, by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood that the disease will occur or worsen in a subject, is sufficient. One embodiment of the invention is directed to a method comprising administering to a patient an antibody in an amount and for a time sufficient to induce a sustained improvement over baseline of an indicator that reflects the severity of a particular disorder. 
     As is understood in the pertinent field, a pharmaceutical composition comprising an antibody of the invention is administered to a subject in a manner appropriate to the indication and the composition. In one embodiment, a pharmaceutical composition comprises a murine antibody or a humanized antibody of the present invention. A pharmaceutical compositions can be administered by any suitable technique, including, but not limited to, parenterally, topically, or by inhalation. If injected, the pharmaceutical composition can be administered, for example, via an intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous route, by bolus injection or continuous infusion. It is considered, for example, localized administration at the disease or injury site, such as transdermal administration and sustained release of an implant. Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation of an antibody in aerosol form, and the like. Other alternatives include oral preparations, including pills, syrups, or lozenges. 
     Advantageously, the antibodies of the invention, are administered in a composition comprising one or more additional components such as a physiologically acceptable carrier, excipient or diluent. The composition additionally comprises one or more physiologically active agents as described below. In many particular embodiments, the composition comprises one, two, three, four, five, or six physiologically active agents in addition to one or more antibodies (e.g., murine antibodies or humanized antibodies) of the present invention. 
     In one embodiment, the pharmaceutical composition comprises a murine antibody or humanized antibody of the invention together with one or more substances selected from the group consisting of a buffer suitable for the antibody at a suitable pH, an antioxidant such as ascorbic acid, a low molecular weight polypeptide (such as those having fewer than 10 amino acids), a protein, an amino acid, a carbohydrate such as dextrin, a chelating agent such as EDTA, glutathione, a stabilizer, and an excipient. In accordance with appropriate industry standards, preservatives can also be added. The composition can be formulated as a lyophilizate using appropriate excipient solutions as diluents. Suitable components are nontoxic to recipients at the dosages and concentrations employed. Further examples of components that can be employed in pharmaceutical formulations are presented in Remington&#39;s Pharmaceutical Sciences, 16th Ed. (1980) and 20th Ed. (2000). Mack Publishing Company kits for use by medical practitioners are provided, including one or more antibodies of the invention and a label or other instructions for use in treating any of the conditions discussed herein. In one embodiment, the kit includes a sterile preparation of one or more human antibodies, which can be in the form of a composition as disclosed above, and can be in one or more vials. 
     Dosages and the frequency of administration can vary according to such factors as the route of administration, the particular antibodies employed, the nature and severity of the disease to be treated, whether the condition is acute or chronic, and the size and general condition of the subject. Appropriate dosages can be determined by procedures known in the pertinent art, e.g., in clinical trials that can involve dose escalation studies. 
     An antibody of the invention can be administered, for example, once or more than once, e.g., at regular intervals over a period of time. In particular embodiments, a murine antibody or humanized antibody is administered over a period of at least once a month or more, e.g., for one, two, or three months or even indefinitely. For treating chronic conditions, long-term treatment is generally most effective. However, for treating acute conditions, administration for shorter periods, e.g., from one to six weeks, can be sufficient. In general, the humanized antibody is administered until the patient manifests a medically relevant degree of improvement over baseline for the chosen indicator or indicators. 
     One example of therapeutic regimens provided herein comprise subcutaneous injection of an antibody once a week, at an appropriate dosage, to treat a condition in which pulmonary arterial pressure levels play a role. Weekly or monthly administration of antibody would be continued until a desired result is achieved, e.g., the subject&#39;s symptoms subside. Treatment can resume as needed, or, alternatively, maintenance doses can be administered. 
     A subject&#39;s levels of pulmonary arterial pressure can be monitored before, during and/or after treatment with an antibody such as a humanized antibody, to detect changes, if any, in their levels. For some disorders, the incidence of elevated pulmonary arterial pressure can vary according to such factors as the stage of the disease. Known techniques can be employed for measuring pulmonary arterial pressure levels. 
     Particular embodiments of methods and compositions of the invention involve the use of an antibody and one or more ET A R antagonists for example, two or more antibodies of the invention, or an antibody of the invention and one or more other ET A R antagonists. In further embodiments, the antibody is administered alone or in combination with other agents useful for treating the condition with which the patient is afflicted. Examples of such agents include both proteinaceous and non-proteinaceous drugs. When multiple therapeutics are co-administered, dosages can be adjusted accordingly, as is recognized in the pertinent art. “Co-administration” and combination therapy are not limited to simultaneous administration, but also include treatment regimens in which an antibody is administered at least once during a course of treatment that involves administering at least one other therapeutic agent to the patient. 
     In another aspect, the present invention provides a method of preparing medicine for the treatment of pulmonary arterial hypertension and related disorders, including a mixture of an antibody of the present invention and pharmaceutically acceptable excipients. The pharmaceutical preparation method is as described above. 
     EXAMPLES 
     1. Construction of a Stable Antigen Cell Line for Immunization 
     CHO-DHFR- cells were seeded into a 6-well plate. After 24 h culture, the cells were transfected with a pIRES plasmid (Clontech, commercial) modified to carry hET A R gene (see SEQ ID NO: 1 for the nucleotide sequence, and SEQ ID NO: 2 for the amino acid sequence). The medium was changed before transfection and the transfection was carried out by following the transfection conditions recommended by Invitrogen for Lipofectamine 2000. Forty eight hours after transfection, the medium was replaced with a complete medium containing 10 nM MTX (methotrexate). The medium was changed every 3 days for about two weeks until stable clones appeared. The dispersed cell colonies were detached from the plate and collected. After cells grew to about 50% confluence, the concentration of MTX was graduately increased for pressure selection up to 10 μM. The constructed stable cell lines were analyzed by FACS using a polyclonal antibody (Abcam) against hET A R to identify cell clones after pressure selection. A large amount of hET A R expression were detected on the selected CHO-DHFR-hET A R cell membranes. Finally through subcloning, six high-ET A R expression and stable cell lines were identified and obtained. 
     2. Preparation of Antibodies 
     An emulsion of the CHO-DHFR-hET A R whole cells and Freund&#39;s adjuvant was injected subcutaneously into BALB/c mice (6-8 weeks) at 2×10 6  cells/mouse. After 2 weeks, the mice were boosted with incomplete Freund&#39;s adjuvant emulsified immunogen and then boosted once every week. After immunization for 6 times in total, blood samples were collected from the clipped tail ends and centrifuged to collect the serum. The serum was analyzed for serum titers by FACS. After the acceptable antibody titers were achieved, the mice were sacrificed and their spleen cells were harvested under aseptic conditions. SP2/0 cells were collected at the logarithmic phase of growth with 3 min centrifugation at 2,000 rpm. The cell pellets were resuspended with serum-free culture medium, then centrifuged, resuspended for a second time and counted. Spleen cells and SP2/0 cells were mixed at ratio of SP2/0 cells:spleen cells≥1:1, followed by 3 rounds of washing-centrifugation. After the pellets from the last centrifugation were detached, 1 mL of pre-warmed PEG-1350 was added dropwise (finished in 30 s), after pipette-mixing for 1 min, 30 mL of the pre-warmed serum-free medium (Invitrogen) was added slowly to terminate the PEG fusion. After 5 min centrifugation at 1,500 rpm, the cell pellets were resuspended in the fusion culture medium. Spleen cells (20,000) and feeder layer cells (5,000) in 100 μL were plated into each well of 96-well plates. Fused hybridoma cells and feeder layer cells were co-cultured in 96-well plates with HAT (sarcine, amethopterin and thymidine) selection to get rid of the non-fused cells. After 10 days, the supernatants of the hybridoma cells in the culture plates were collected for ELISA analysis. 
     3. ELISA Screening of Whole Cells 
     CHO-DHFR-hET A R cells over-expressing hET A R and CHO-DHFR- cells not expressing hET A R were separately transferred into a 96-well plate and allowed to grow to 90% confluent. The supernatant of the culture medium was removed and attached cells were washed twice with PBS, then 100 μL, 100% methanol was added to fix the cells for 10 min at 4° C. Then 100 μL freshly made 0.6% H 2 O 2 -PBS was added, and after incubation at room temperature for 20 min, the cells were washed twice with PBS. After blocked with PBS-1% BSA solution, the hybridoma supernatant was added and incubated for 90 min at 4° C. After several washes, 100 μL of the secondary antibody GxM-HRP-Fc (Sigma-Aldrich) was added into each well and incubated at 37° C. for 0.5 h. After five washings, 100 μL of TMB chromogenic substrate was added into each well and incubated at 37° C. for 15 min, and then 2M H 2 SO 4  was added to terminate, read for OD 450  values. The positive control was the mouse serum after immunization; the negative control was the cell culture supernatant. As shown in  FIG. 1 , after initial analysis by ELISA, several hybridoma clones secreting anti-hET A R antibodies were selected, and the stable secretory cell lines against hET A R were obtained after cell cloning. Lastly, antibody supernatant secreted by hybridoma was verified by FACS analysis. 
     4. Cloning and Subcloning of Antibody Genes 
     Hybridoma cells secreting antibodies were collected. Hybridoma mRNA was extracted according to the manufacturer protocol of QIAGEN mRNA extraction kit. Then the extracted mRNA was transcribed reversely into cDNA. The reverse transcription primers were specific primers for the light and heavy chain constant regions of a mouse, with the heavy chain reverse transcription primer being (5′-TTTGGRGGGAAGATGAAGAC-3′) (SEQ ID NO: 199), the light chain reverse transcription primers being (5′-TTAACACTCTCCCCTGTTGAA-3′) (SEQ ID NO: 200) and (5′-TTAACACTCATTCCTGTTGAA-3′) (SEQ ID NO: 201). RT-PCR reaction conditions were as following: 25° C. for 5 min, 50° C. for 60 min, and 70° C. for 15 min. Reversely transcribed cDNA was diluted with 0.1 mM TE to 500 μL, added into the ultrafiltration centrifuge tube (Amicon Ultra-0.5) and centrifuged at 2,000 g for 10 min. The filtrate was removed, 500 μL of 0.1 mM TE were added and centrifuged at 2,000 g for 10 min. The filtrate was removed and the preparation tube was placed in inversion to the new centrifugal tube, and centrifuged at 2,000 g for 10 min to obtain the purified cDNA. Purified cDNA (10 μL) was taken as a template, followed by addition of 4 μL 5× tailing buffer (Promega), 4 μL dATP (1 mM) and 10 U terminal transferase (Promega), mixing uniformly, and incubation at 37° C. for 5 min and then at 65° C. for 5 min. The PolyA tail cDNA was used as a template and PCR was performed to amplify light and heavy chain variable region genes of antibodies. Upstream primers were all oligodT, with heavy chain downstream primers being (5′-TGGACAGGGATCCAGAGTTCC-3′) (SEQ ID NO: 202) and (5′-TGGACAGGGCTCCATAGTTCC-3′) (SEQ ID NO: 203), and light chain downstream primer being (5′-ACTCGTCCTTGGTCAACGTG-3′) (SEQ ID NO: 204). The PCR reaction conditions were: 95° C. for 5 min; 95° C. for 30 s, 56° C. for 30 s, 72° C. for 1 min, 40 cycles; and 72° C. for 7 min. The PCR products were connected to the PMD 18-T vector (Takara Bio) for sequencing. The sequences of the antibody clones were listed in Table 2. 
     PCR primers were designed based on the DNA sequences of the antibodies, thus the complete light chain, heavy chain signal peptides and variable domains and mouse IgG1 constant region were ligated into expression vector pTM5. 
     5. Antibody Humanization and Optimization 
     First of all, the sequences of light and heavy chain variable regions of the screened mouse antibodies were aligned with the homologous antibodies, using NCBI online antibody variable region sequence alignment tool (Ig Blast) to search the germline gene sequences of a humanized antibody (Ig Germline Gene sequence) homologous to the selected antibodies variable region sequence for humanization, and the humanized gene sequence with highest homology except CDR sequences was used as a template for CDR grafting to obtain humanized antibody variable region sequences and to synthesize humanized antibody light and heavy chain genes through a CRO. According to the sequences, PCR primers were designed and appropriate restriction enzyme sites were introduced at the 5′ ends and 3′ ends. By PCR, the humanized antibody variable regions were amplified and then combined with the human IgG2 or IgG4 constant region sequence to obtain whole recombinant humanized antibody sequences. The expression of the recombinant antibodies was achieved according to step 7, and their affinities to ET A R was analyzed by FACS as described in step 9. The best humanized antibody candidate retaining affinity to ET A R was selected from the group, and its variable region sequence was further improved by site-specific mutagenesis for improved affinity to ET A R. 
     6. Subcloning of Genes of a Humanized Anti-hET A R Antibody 
     The heavy and light chain variable region gene sequences of an optimized humanized antibody were synthesized by Genscript Biotechnology CO., LTD by introducing two restriction sites of NheI at the 5′-end and SalI at the 3′-end. The whole heavy chain variable region was ligated with a heavy chain constant region in an expression vector of pTM5. Similarly, by introducing NheI at the 5′-end and BsiwI at the 3′-end, the light chain variable region was ligated with a light chain constant region in the expression vector of pTM5. 
     7. Transient Expression of Anti-ET A R Antibodies 
     A suspension of a HEK293 or CHO expressing cell line (5×10 5 /mL) was inoculated to a shaker flask. After 24 h rotation at 37° C., the cell density reached 1×10 6 /mL and were ready for transfection. Polyethylenimine (PEI) was used as a transfection reagent with an optimal mixing ratio of 3:1 for PEI to DNA (DNA amount, 0.5 μg/L×10 6  cells; the ratio of the antibody light chain DNA and antibody heavy chain DNA, 3:2). A mixture of both was added into the cell culture after 15 min incubation. The cells after treated with the PEI/DNA mixture were rotated for more than 24 h at 37° C. and 5% CO 2 . Then 0.5% of tryptone was added into the cell culture as a supplement required by expression, and after the completion of expression (more than 96 h), the cell supernatant was collected for the antibody purification and separation. 
     8. Purification and Preparation of Antibody 
     Cells and cellular debris were removed from the culture after centrifugation (8000 rpm, 15 min), and the supernatant was filtered through 0.45 μm filter for purification. The purification process was done through chromatography. First, the supernatant was passed through a G protein coupled affinity chromatography column, and antibodies bound to the G proteins remained in the column. The antibodies were eluted from the chromatography column using an eluent with pH of 3.0 or less. The low pH eluent was neutralized immediately with 1M Tris-HCl to keep the antibodies from denaturation and loss of activity. The antibody solution was then dialyzed over 16 h into a PBS buffer. 
     9. FACS Analysis of a Functional Antibody 
     PBS containing 10 mM EDTA was used to detach and collect 10 5  CHO-DHFR-hET A R cells into a 1.5 mL EP tube. The supernatant was removed after centrifugation and the negative control sample was resuspended with a loading buffer (PBS, 2% FBS). For the positive control, 200 μL antibody supernatant was added to resuspension cells and incubation at room temperature; the cells were then centrifuged at 1500 rpm to remove the supernatant, washed with a FACS loading buffer and centrifuged again. The cells were resuspended with addition (200 μL/well) of a FITC labeled goat anti-mouse fluorescent antibody at 1:50 dilution (BD Pharmingen) and incubated at room temperature for 30 min in the dark. Supernatant was removed after centrifugation, cells were washed with FACS loading buffer, centrifuged again and resuspended with the loading buffer for analysis. As shown in  FIG. 2 , the recombinant antibody supernatant and CHO-DHFR-hET A R cells had specific binding: gray peak and dotted line peak were negative controls; the solid line peak, corresponding to the antibody supernatant, moved to the right significantly. 
     10. Calcium Influx Assay for a Functional Antibody 
     CHO-DHFR cells co-expressing hET A R-Aequorin were seeded into a 96-well cell culture plate with 25000 cells per well and cultured at 37° C. overnight. The next day the culture supernatant was removed. Coelenterazine (50 μL) (Promega) was added in the dark and incubated at 37° C. for 2 h, and then 50 μL of a hybridoma supernatant or a purified antibody were added and incubated at 37° C. for 30 min. After the incubation, 50 μL endothelin 1 was added and the changes of calcium influx within 40 s were recorded by a SpectraMax L microplate reader (Molecular Devices). As shown in  FIG. 3 , different hybridoma supernatants inhibited the calcium influx mediated through hET A R differently, and A-1 antibody significantly inhibited the calcium influx mediated through hET A R. As shown in  FIG. 4 , the recombinant anti-hET A R functional antibody significantly inhibited calcium influx mediated through hET A R, increasingly inhibitory with an increase in the antibody concentration. 
     11. Establishment of Hypoxia-Induced PAH Cynomolgus Model to Study the In Vivo Activity of an Antibody 
     The acute hypoxia-induced pulmonary arterial hypertension (PAH) model of cynomolgus was codeveloped with Crown Bioscience Inc. (Taicang), and the efficacy of A-1 antibody as a single intravenous injection was evaluated in this PAH model. All animals were fasted overnight and weighed, and then received a single intravenous injection of 10 mg/kg of A-1 antibody. Three hours later, the animals were anesthetized. The tricuspid regurgitation velocity by Doppler color echocardiography along with heart rate and oxygen saturation were monitored simultaneously. The baseline was obtained and the induction of 12% hypoxia was followed and at the same time the tricuspid regurgitation velocity was measured; Analysis was made to determine if the antibody would improve hypoxia-induced pulmonary arterial pressure under 12% hypoxia. After 48 h of administration, the tests were performed again. The animals were anesthetized, the tricuspid regurgitation velocity by Doppler color echocardiography along with heart rate and oxygen saturation were monitored simultaneously. The baseline was obtained and the induction of 12% hypoxia was followed and at the same time the tricuspid regurgitation velocity was measured. Analysis was made to determine if the antibody would still improve hypoxia-induced pulmonary arterial pressure. If the efficacy maintained after 48 h, 96 h later, hypoxia induction experiment was performed again. As shown in  FIG. 5 , the area under the curve of pulmonary artery systolic pressure versus time was calculated, and by comparing the area under the curve, it was found that A-1 maintained the efficacy of reducing pulmonary artery pressure within 96 h. 
     The examples set forth above are to give those of ordinary skill in the art with a complete disclosure and description of how to make and use the claimed embodiments, and are not intended to limit the scope of what is disclosed herein. Modifications that are obvious to persons of skill in the art are intended to be within the scope of the following claims.