Fusion protein of ETA antibody and TGF-β trap and pharmaceutical composition and application thereof

Abstract

To provide a fusion protein of an ETA antibody and a TGF-β Trap, and a pharmaceutical composition for preventing, improving, or treating two or more diseases of pulmonary arterial hypertension, pulmonary hypertension, pulmonary fibrosis, or cardiovascular fibrosis.SOLUTION: Provided is a fusion protein of an ETA antibody and a TGF-β Trap, wherein the fusion protein comprises an ETA antibody, one or more TGF- Traps and one or more peptide linkers (Linkers); the fusion protein connects the amino terminal of a TGF-β Trap with the carboxyl terminal of a light chain or a heavy chain of the ETA antibody via a peptide linker sequence, or the fusion protein connects the carboxyl terminal of a TGF-β Trap with the amino terminal of a light chain or a heavy chain of the ETA antibody via a peptide linker sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] A fusion protein of an ETA antibody and TGF-β Trap is provided herein. Pharmaceutical compositions of fusion proteins of ETA antibodies and TGF-β Trap are also described herein. The present invention provides a method for treating TGF-β Trap by using a fusion protein of an ETA antibody and TGF-β Trap. treating one or more symptoms of pulmonary arterial hypertension, pulmonary hypertension, or pulmonary fibrosis, Further provided herein are methods for preventing or ameliorating. [Background technology]

[0002] Pulmonary arterial hypertension (PAH) is a rare, progressive disease characterized by a marked increase in pulmonary arterial blood pressure. Pulmonary arterial hypertension has become a serious disease that threatens human health. The annual incidence of various types of pulmonary arterial hypertension worldwide ranges from approximately 2.4 to 7.6 per million people. The prevalence is approximately 15-26 per million people. Hypertension is the third most common cardiovascular disease, only after ischemic heart disease and hypertension. The causes of pulmonary arterial hypertension are not yet fully understood. Therefore, most patients already have grade III to IV cardiac function due to pulmonary arterial hypertension. Symptoms of pulmonary arterial hypertension usually include shortness of breath (especially during exercise), chest pain, and intermittent fainting. Furthermore, as this condition continues, the continued high pulmonary artery pressure causes the right ventricle to The inability to maintain a continuous supply of blood ultimately leads to right heart failure. Heart failure is the most common cause of death in patients with pulmonary arterial hypertension.

[0003] Currently, there is no cure for pulmonary arterial hypertension, and drug therapy is the only treatment that can maintain pulmonary arterial hypertension. It is the first choice of therapy. Drugs approved by the FDA for the treatment of pulmonary arterial hypertension is a vasodilator, which, depending on the mechanism, may act as a calcium channel blocker, prostacyclin Icrin receptor agonists, phosphodiesterase type 5 (PDE5) inhibitors, endothelial They can be divided into steroid receptor inhibitors and others.

[0004] Pulmonary arterial hypertension occurs when the cardiac blood supply to the lungs is compromised by vasoconstriction in or associated with the lungs. caused by a compensatory increase in the pressure of the heart's blood supply to the lungs after it becomes more inadequate The microscopic signs are thickening of the intimal membrane of pulmonary arterioles and vascular congestion caused by thrombosis. This may include narrowing, remodeling, stiffening, or localized obstruction of blood vessels to the pulmonary circulation. (Simonneau et al., 2004, J. Am. Coll. .Cardiol.43:5S-12S;Barst et al.,2004,JA m. Coll. Cardiol. 43:40S-47S).

[0005] Endothelin receptors (e.g., endothelin receptor A (ETA) or ET A R)) inhibitor effectively blocks the increase in vascular pressure caused by endothelin, preventing pulmonary arterial hypertension. It can reduce the symptoms of hypertension and improve the patient's exercise capacity and hemodynamics (Seras li et al.,2010,Recent Pat.Cardiovasc.Dru g Discov.5:184-95).

[0006] Several studies have shown that transforming growth factor β1 (TGF-β1) plays a key role in various conditions, particularly TGF-β1 has been shown to play an important role in cardiovascular disease. It is involved in processes such as migration and apoptosis, and regulates multiple signaling pathways, which Therefore, by inducing the onset and progression of pulmonary arterial hypertension, smooth muscle cell proliferation, It promotes processes such as deposition of extracellular matrix and endothelial-mesenchymal transition (EndMT) (Yan et al. al.,2016,Int.J.Cardiol.,222:368-374;Gra ham et al.,2013,Circulation,128:1354-136 4;Liu et al.,2016,Int.J.Biochem.Cell.Bio I., 77:141-154). Pirfenidone as an approved drug for the treatment of pulmonary fibrosis. inhibits TGF-β expression and reduces fibroblast proliferation, induced by TGF-β Attenuating the mRNA and protein levels of α-smooth muscle actin (α-SMA) This can be done.

[0007] TGF-β Trap is composed of the extracellular terminal of the TGF-β type 2 receptor and binds to TGF- β1 or TGF-β3, thereby contributing to the development of vascular smooth muscle in the process of pulmonary arterial hypertension. TGF-β-mediated processes such as muscle cell proliferation and differentiation, extracellular matrix deposition, and epithelial-mesenchymal transition TGF-β Trap effectively inhibits pulmonary arteriolar fibrosis and lymphocyte proliferation. Reduces modeling, thereby reducing pulmonary circulation resistance and improving right heart function. (Goumans et al., 2018, Cold Spring Harb.Perspect.Biol.,10:a022210;Yung et a l.,2016,Am.J.Respir.Crit.Care Med.,194:1 140-1151). Summary of the Invention [Problem to be solved by the invention]

[0008] A fusion protein of an ETA inhibitory antibody and TGF-β Trap is provided herein. On the other hand, the fusion protein of ETA inhibitory antibody and TGF-β Trap is It can block the ETA signaling pathway and reduce vascular resistance to pulmonary circulation; On the other hand, it inhibits the TGF-β signaling pathway and prevents pulmonary arteriolar fibrosis and remodeling. The purpose of treating pulmonary arterial hypertension and improving right ventricular function is to , achieved by a dual mechanism of action. [Means for solving the problem]

[0009] A fusion protein of an ETA antibody and TGF-β Trap is provided herein. The use of fusion proteins can be used to treat pulmonary arterial hypertension, pulmonary hypertension, pulmonary fibrosis, and Methods for treating, preventing, or ameliorating one or more symptoms of cardiovascular fibrosis and cardiovascular fibrosis are also described herein. provided in the document.

[0010] A fusion protein of ETA antibody and TGF-β Trap, A fusion protein structurally characterized by containing a TA antibody and a TGF-β Trap fragment. Proteins are provided herein.

[0011] A fusion protein of an ETA antibody and TGF-β Trap, wherein the fusion protein is ETA antibody, 1, 2, 3, 4, 5, 6, 7 or 8 TGF-β Trap and corresponding the fusion protein contains a peptide linker (linker); The amino terminus is connected to the carboxyl terminus of the light or heavy chain of the ETA antibody and a peptide linker ( The fusion protein is linked via a carboxyl group of TGF-β Trap. The amino terminus of the light or heavy chain of the ETA antibody is connected to a peptide linker (linker). Fusion proteins structurally characterized by linkage via a sequence are provided herein. will be done.

[0012] A fusion protein of an ETA antibody and TGF-β Trap, wherein the fusion protein is ETA antibody, 1, 2, 3 or 4 TGF-β Traps and the corresponding number of peptide phosphorylation the fusion protein contains a linker; the amino terminus of TGF-β Trap is linked to an E The carboxyl terminus of the light or heavy chain of the TA antibody is connected to the peptide linker (linker) sequence. The carboxyl terminus of TGF-β Trap is linked to an ET A: Linked to the amino terminus of the antibody light or heavy chain via a peptide linker (linker) sequence Fusion proteins are provided herein that have the structural characteristic of:

[0013] A fusion protein of an ETA antibody and TGF-β Trap, wherein the fusion protein is Contains ETA antibody, two TGF-β Traps, and two peptide linkers (linkers). The fusion protein is composed of the amino terminus of TGF-β Trap and the light or heavy chain of the ETA antibody. The carboxyl terminus of the chain is linked or fused via a peptide linker (linker) sequence. The protein is a mixture of the carboxyl terminus of TGF-β Trap and the light or heavy chain of the ETA antibody. The structural feature is that it is linked to the amino terminus of the Provided herein is a fusion protein comprising:

[0014] A fusion protein of an ETA antibody and TGF-β Trap, wherein the fusion protein is Contains ETA antibody, TGF-β Trap, and a peptide linker (linker); fusion protein The protein binds the amino terminus of TGF-β Trap to the carboxyl group of the light or heavy chain of the ETA antibody. The syl end is connected via a peptide linker (linker) sequence, or the fusion protein The carboxyl terminus of TGF-β Trap is connected to the amino terminus of the light chain or heavy chain of the ETA antibody. and a fusion tag structurally characterized by linking the two via a peptide linker (linker) sequence. Proteins are provided herein.

[0015] A fusion protein of ETA antibody and TGF-β Trap, comprising the ETA antibody, TGF -β Trap and peptide linker sequences are prepared in the following manner: (1) The amino terminus of TGF-β Trap is linked via a peptide linker (linker) sequence. The N'-R-linker- is linked to the carboxyl terminus of the heavy / light chain of the ETA antibody. TGF-β Trap-C'; and (2) TGF-β Trap carboxylates via a peptide linker (linker) sequence. The syl terminus is linked to the amino terminus of the light or heavy chain of the ETA antibody: N'-TGF-β Trap-Linker-R-C' structurally characterized by being fused to form a fusion protein; where N' represents the amino terminus of the polypeptide chain and C' represents the carboxyl terminus of the polypeptide chain. TGF-β Trap represents the carboxyl terminus of the TGF-β Trap fragment. wherein R is the amino acid sequence of the light chain or heavy chain of the ETA antibody, and the linker is a peptide linker. Fusion proteins representing the CAR are provided herein.

[0016] Polynucleotides encoding the ETA antibodies described herein and TGF-β Tr Fusion proteins of ap are provided herein.

[0017] Polynucleotides encoding the ETA antibodies described herein and TGF-β Tr Vectors containing fusion proteins of ap are provided herein.

[0018] Provided herein are host cells comprising the vectors described herein.

[0019] The fusion protein of the ETA antibody and TGF-β Trap described herein and the pharmaceutical and a physiologically acceptable carrier.

[0020] Treatment, prevention, or amelioration of one or more symptoms of pulmonary arterial hypertension and pulmonary arterial hypertension-related diseases and a combination of an ETA antibody and a TGF-β T cell line as described herein in the preparation of a medicament for improving Uses of rap fusion proteins are provided herein.

[0021] For treating, preventing, or ameliorating one or more symptoms of pulmonary hypertension and pulmonary hypertension-related diseases The use of an ETA antibody and TGF-β Trap as described herein in the preparation of a medicament for Uses of fusion proteins are provided herein.

[0022] For treating, preventing or ameliorating one or more symptoms of pulmonary fibrosis and pulmonary fibrosis-related diseases Fusion of the ETA antibody and TGF-β Trap described herein in the preparation of a medicament Uses of the proteins are provided herein.

[0023] Therapeutic methods for treating, preventing, or ameliorating one or more symptoms of cardiovascular fibrosis and cardiovascular fibrosis-related diseases and combinations of an ETA antibody and a TGF-β Trap as described herein in the preparation of a medicament for The use of fusion proteins of p is provided herein.

[0024] One of two or more of the following diseases: pulmonary arterial hypertension, pulmonary hypertension, pulmonary fibrosis, or cardiovascular fibrosis In the preparation of a medicament for the simultaneous treatment, prevention or amelioration of one or more symptoms, The use of the fusion protein of ETA antibody and TGF-β Trap described in It is provided at.

[0025] Treatment, prevention, or amelioration of one or more symptoms of pulmonary arterial hypertension and pulmonary arterial hypertension-related diseases The method comprises administering a therapeutically effective dose of an ETA antibody described herein and A method comprising administering to a subject a fusion protein of TGF-β Trap is described herein. It is provided at.

[0026] For treating, preventing, or ameliorating one or more symptoms of pulmonary hypertension and pulmonary hypertension-related diseases The method comprises administering a therapeutically effective dose of an ETA antibody described herein and a TGF- The method described herein includes administering to a subject a fusion protein of β Trap. Provided.

[0027] For treating, preventing or ameliorating one or more symptoms of pulmonary fibrosis and pulmonary fibrosis-related diseases The method comprises administering a therapeutically effective dose of an ETA antibody described herein and TGF-β A method is provided herein that includes administering to a subject a fusion protein of Trap. will be done.

[0028] Therapeutic methods for treating, preventing, or ameliorating one or more symptoms of cardiovascular fibrosis and cardiovascular fibrosis-related diseases The method comprises administering a therapeutically effective dose of an ETA antibody and a TG described herein. The method of administering to a subject a fusion protein of F-β Trap is described herein. It is provided as follows.

[0029] One of two or more of the following diseases: pulmonary arterial hypertension, pulmonary hypertension, pulmonary fibrosis, or cardiovascular fibrosis 2. A method for treating, preventing or ameliorating one or more symptoms of a disease, comprising administering a therapeutically effective amount of A fusion protein of an ETA antibody and TGF-β Trap described herein is administered to a subject. Provided herein are methods comprising administering [Brief explanation of the drawings]

[0030] [Figure 1] 1 shows the results of the fusion protein h15F3-(G4S)4-TGF-β Trap (comprising SEQ ID NO: 162, SEQ ID NO: 190, SEQ ID NO: 207, and SEQ ID NO: 210) of ETA antibody and TGF-β Trap inhibiting Ca2+ changes mediated by human ETA. [Figure 2] 1 shows the results of ETA antibody and TGF-β Trap fusion protein h15F3-(G4S)4-TGF-β Trap blocking TGF-β1 from activating the TGF-β1 receptor. [Figure 3] This shows that the fusion protein h15F3-(G4S)4-TGF-β Trap of ETA antibody and TGF-β Trap exerts a significantly improved effect on pulmonary fibrosis. [Figure 4] This shows that the fusion protein h15F3-(G4S)4-TGF-β Trap of ETA antibody and TGF-β Trap can significantly improve the survival rate / duration of mice with pulmonary fibrosis. DETAILED DESCRIPTION OF THE INVENTION

[0031] definition Unless otherwise defined herein, scientific and technical terms are understood by those of ordinary skill in the art. Generally, the term "pharmacology," "biology," "biochemistry," "cell and tissue culture" is used in the fields of , Biology, Molecular Biology, Immunology, Microbiology, Genetics and Protein, Nucleic Acid Chemistry and Hybridization The nomenclature and techniques associated with redox synthesis are well known in the art and are generally It is intended to be used for practical purposes.

[0032] Standard one-letter or three-letter abbreviations are used to designate polynucleotide and polypeptide sequences. As used herein, unless otherwise specified, the amino terminus of a polypeptide sequence is , the upper strand of the single-stranded and double-stranded nucleic acid sequence, with its carboxyl terminus on the left and its carboxyl terminus on the right. The 5' ends of the upstream chain are on the left and their 3' ends are on the right Specific parts of polypeptides are divided into groups by the number of amino acid residues, such as 80 to 130 amino acids. or by the actual residues at that site, such as Lys80 to Lys130. A particular polypeptide or polynucleotide sequence may be identified by its differences from a reference sequence. It can also be expressed by

[0033] The terms "peptide," "polypeptide," and "protein" refer to peptide bonds. Thus, these terms refer to molecules containing two or more amino acids linked together. , natural and artificial proteins, protein fragments and polypeptides of protein sequences Analogues (such as muteins, variants and fusion proteins) and post-translational or co-translational proteins Peptides, polypeptides, and proteins that are covalently or non-covalently modified. The bonds, or proteins, can be monomeric or polymeric.

[0034] The term "polypeptide fragment" refers to an amino acid sequence from the corresponding full-length protein. refers to a polypeptide having a terminal and / or carboxyl terminal deletion. Length must be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 The fragment may be 50, 70, 80, 90, 100, 150, or 200 amino acids. The maximum length is, for example, 1000, 750, 500, 250, 200, 175, 15 0, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, The fragment may be 13, 12, 11, or 10 amino acids. and one or more additional amino acids, e.g., amino acids from different naturally occurring proteins. amino acid sequences (e.g., Fc or leucine zipper domains) or artificial amino acid sequences (e.g., The nucleic acid sequence may further comprise an artificial binding sequence.

[0035] The polypeptides herein may be prepared for any reason and by any means, e.g., 1) Reduced susceptibility to proteolysis; (2) reduced susceptibility to oxidation; (3) modifying the affinity to form a protein complex; (4) modifying the binding affinity; (4) Polypeptides modified to impart or modulate other physicochemical or functional properties Analogs include muteins of polypeptides. For example, single or multiple A number of amino acid substitutions (e.g., conservative amino acid substitutions) can be made in the native sequence (e.g., , outside the domain of the polypeptide that forms intramolecular contacts). It does not substantially alter the structural characteristics of the parent sequence (e.g., amino acid substitutions are It should not destroy any helices present in the sequence or add properties or functions that confer properties to the parent sequence. (It should not interfere with other secondary structural types that are necessary for function.)

[0036] A "variant" of a polypeptide is one that has one or more amino acid changes in sequence compared to another polypeptide sequence. The present specification also includes amino acid sequences in which multiple amino acid residues have been inserted, deleted, and / or substituted. Variants as referred to herein include fusion proteins.

[0037] "Derivatives" of polypeptides include polyethylene glycol, albumin (human serum albumin), by conjugation to other chemical moieties, such as amino acids, phosphorylation, and glycosylation. It is a chemically modified polypeptide.

[0038] Unless otherwise specified, the term "antibody" refers to an antibody that contains two full-length heavy chains and two full-length light chains. and antibodies, and derivatives, variants, fragments, and muteins thereof, comprising Examples of which are given below.

[0039] The term "antibody" refers to an antigen-binding portion and, optionally, a molecule that promotes binding of the antibody to the antigen. The antigen-binding moiety may comprise a scaffold or framework portion that enables the antigen-binding moiety to adopt a conformation that conforms to the antigen-binding moiety. Examples of antibodies include intact antibodies, antibody fragments (antibody antigens), and antibody derivatives, and antibody analogs. For example, antibodies may be grafted Alternatively, the scaffolds may include alternative protein or artificial scaffolds containing modified CDRs or derivatives of CDRs. The scaffold may be, but is not limited to, a scaffold derived from the antibody to be introduced, for example, a tertiary scaffold of the antibody. Stabilizing the parent structure and, for example, completely synthetic scaffolds for biocompatible polymers For example, Korndorfer et al., 2003, Protein s:Structure, Function and Bioinformatics 53:121-129;Roque et al.,2004,Biotechnol. Prog. 20:639-654. In addition, fibrin ligands have been used as scaffolds. Peptide antibody mimetics ("PAMs") and scaffolds based on these antibody mimetics are used. It can be used.

[0040] An antibody can have, for example, the structure of a native immunoglobulin. In native immunoglobulins, each tetramer consists of two identical polypeptide chains. Each pair contains a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The amino terminus of each chain contains approximately 100-110 or more amino acids that are primarily involved in antigen recognition. The carboxyl terminus of each chain contains a variable domain of amino acids, primarily responsible for the activity of the effector. Human antibody light chains are classified as kappa and lambda light chains. Chains are classified as μ, δ, α, or ε heavy chains, and are responsible for IgM, IgD, IgG, IgA, and Ig Within the light and heavy chains, the variable and constant regions define the isotype of the antigen, such as E. The heavy chains are joined by a "J" region of about 10 or more amino acids, and the heavy chains are joined by a "D" region of about 10 or more amino acids. " area. Fundamental Immunology Ch.7(edit d by Paul, 2nd edition, Raven Press, 1989)( See, e.g., U.S. Pat. No. 6,229,399, the disclosure of which is incorporated herein by reference in its entirety for all purposes. The variable regions of each light / heavy chain pair are what allow an intact immunoglobulin to have two binding sites. The antibody binding site is formed so as to have the following structure:

[0041] Native immunoglobulin chains consist of three superunits, also known as complementarity-determining regions or CDRs. The same basic structure of relatively conserved framework regions (FR) bounded by variable regions From the N-terminus to the C-terminus, the light chain and the heavy chain are composed of the domains FR1, CDR1, FR2, , CDR2, FR3, CDR3, and FR4. Amino acid assignments in each domain Sequences of Proteins of I mmunological interest,5th edition,US Dep t.of Health and Human Services,PHS,NIH,N Consistent with the definition in IH Publication No. 91-3242, 1991 do.

[0042] Unless otherwise specified, an "antibody" refers to an intact immunoglobulin or a specific binding Antigen-binding refers to any antigen-binding portion thereof that can compete with an intact antibody for antigen binding. Binding moieties can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. The antigen-binding portion can be produced by, among others, Fab, Fab', F(ab')2, Fv, domain antibody (dAb), fragment containing complementarity-determining regions (CDRs), single chain Antibodies (scFv), chimeric antibodies, double-chain antibodies, triple-chain antibodies, quadruplex antibodies, and polypeptides A polypeptide containing at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide. Contains lipids.

[0043] Fab fragments are V L , V H , C L , and C H1 Monovalent fragments with domains F(ab')2 fragments are fragments of the same nucleotide sequence as the F(ab')2 fragment, which is a fragment of the same nucleotide sequence as the F(ab')2 fragment. a bivalent fragment having two Fab fragments linked by the Fd flag; Mention is V H or V L domain; the dAb fragment has a V H Domain, V L Do Main or V H or V L The antigen-binding fragment of the domain (U.S. Patent No. 6,449,299) ,846,634 and U.S. Pat. No. 6,696,245; U.S. Pat. Publication No. 2005 / 0202512, U.S. Patent Application Publication No. 2004 / 0202995 No. 2004 / 0038291, U.S. Patent Application Publication No. No. 2004 / 0009507 and U.S. Patent Application Publication No. 2003 / 0039958 No. specification; Ward et al., 1989, Nature 341:544-546 ).

[0044] Single-chain antibodies (scFv) are V L and V H The region may be a linker (e.g., synthetic amino acid residues) sequence) to form a contiguous protein, where the linker is , allowing the protein chain to fold back on itself to form a monovalent antigen-binding site (e.g., Bird et al., 1988, Science 242 :423-26; and Huston et al., 1988, Proc. Natl. A See cad.Sci.USA 85:5879-83).

[0045] A double-chain antibody is a bivalent antibody containing two polypeptide chains, each of which is and connected by a linker that is too short to allow pairing of the two domains on the same chain. V H and V L domains, each of which is a separate polypeptide chain. allowing pairing with complementary domains (e.g., Holliger et al. ,1993,Proc.Natl.Acad.Sci.USA 90:6444-48; and Poljak et al., 1994, Structure 2:1121-23 (See also ). When a double-chain antibody has two identical polypeptide chains, it is Double-chain antibodies, which result from the pairing of peptide chains, have the same antigen-binding site. and generating double-chain antibodies with different antigen-binding sites using polypeptide chains having different Similarly, triple-chain antibodies and quadruplex antibodies can be made up of three and four polypeptides, respectively. Antibodies containing chains, which may be the same or different, forming three and four antigen-binding sites, respectively Complete.

[0046] Kabat et al.Sequences of Proteins of Im munological interest,5th edition,US Dept .of Health and Human Services,PHS,NIH,NI Using the method described in H Publication No. 91-3242, 1991 to identify the complementarity determining regions (CDRs) and framework regions (FRs) of a given antibody. One or more CDRs can be incorporated into a molecule either covalently or non-covalently. This can then be made into an antibody. Antibodies can be made by incorporating larger polypeptide chains into the CDRs. The CDRs can be covalently linked to another polypeptide chain or non-covalently linked thereto. The CDRs are the components that allow an antibody to specifically bind to a particular antigen of interest. This allows you to do so.

[0047] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites The positions may be identical to or different from each other. For example, in naturally occurring human immunoglobulins typically have two identical binding sites, while "bispecific" or "bifunctional" antibodies , which has two different binding sites.

[0048] The term "murine antibody" refers to one or more possible antibodies derived from murine immunoglobulin sequences. The term "antibody" includes antibodies having both variable and constant regions.

[0049] The term "humanized antibody" refers to a method for modifying the sequences of the complementarity determining regions of a murine antibody molecule to make them compatible with a human antibody. These are antibodies created by grafting variable regions onto a framework.

[0050] The terms "antigen-binding domain," "antigen-binding region," or "antigen-binding site" refer to the binding site of an antigen. and contribute to the specificity and affinity of the antibody for the antigen (or other For antibodies that specifically bind to their antigens, this comprises at least a portion of at least one of the CDR domains.

[0051] The term "epitope" refers to the portion of a molecule that binds to an antibody (e.g., by an antibody). An epitope is a non-contiguous portion of a molecule (e.g., in a polypeptide, are not adjacent in the primary sequence of the antibody, but in the tertiary and quaternary structure of the polypeptide The amino acid residues may be sufficiently close to each other to be joined by

[0052] The "percent identity" of two polynucleotide or two polypeptide sequences is determined by the GA P computer program (GCG Wisconsin Package; versi on 10.3 (part of Accelrys, San Diego, CA) default parameter The parameter is determined using sequence comparison.

[0053] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used throughout the text. and are used interchangeably, and refer to DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), nucleotide analogs (e.g., peptide nucleic acids and non-natural nucleosides DNA or RNA analogs and their hybrids produced using The nucleic acid molecule may be single-stranded or double-stranded. The nucleic acid molecule in the present invention is an antibody or a fragment, derivative, or protease thereof provided herein. and contiguous open reading frames encoding the mutant or variant proteins. nothing.

[0054] When the sequences of two single-stranded polynucleotides can be arranged in antiparallel, Nucleotides are "complementary" to one another, and each nucleotide in one polynucleotide is opposite to the complementary nucleotide in another polynucleotide, and no gap is introduced. , so that no unpaired nucleotides are found at the 5' or 3' end of each sequence. Two polynucleotides hybridize to each other under moderately stringent conditions. A polynucleotide is "complementary" to another polynucleotide if they can be fused together. Thus, one polynucleotide may be complementary to another polynucleotide, but It is not its complementary sequence.

[0055] The term "vector" is used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid," which contains additional nucleic acid sequences. A vector is a linear or circular double-stranded DNA molecule that can be ligated to a target fragment. Another type of vector is , viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adenoviruses) virus-associated viruses), in which additional DNA segments are inserted into the viral genome Some vectors are capable of autonomous replication in the host cell into which they are introduced. (eg, bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can, upon introduction into a host cell, It integrates into the host cell genome and thereby replicates along with the host genome.

[0056] An "expression vector" is a type of vector capable of directing the expression of a selected polynucleotide. It is a pu.

[0057] Regulatory sequences affect the expression (e.g., level, time, or location of expression) of a nucleotide sequence. A nucleotide sequence is "operably linked" to a regulatory sequence if it exerts an effect on the transcription of the target gene. " refers to the expression (e.g., level, time, or location of expression) of a nucleic acid to which it is operably linked. A regulatory gene is a nucleic acid that affects, for example, the regulated nucleic acid directly or The polynucleotide is linked to one or more other molecules (e.g., regulatory sequences and / or nucleic acids). Examples of regulatory sequences include promoters, enhancers, and other expression regulators. Further examples of regulatory sequences include transcription control elements (e.g., polyadenylation signals). , Goeddel, 1990, Gene Expression Technology Technology:Methods in Enzymology,Volume 185, Academic Press, San Diego, CA; and Baron et al.,1995,Nucleic Acids Res.23:3605-0 6, etc.

[0058] The term "host cell" refers to a cell in which a nucleic acid, such as those provided herein, can be expressed. The host cell is a prokaryotic organism, such as E. coli. or it may be a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi). ), plant cells (e.g., tobacco or tomato plant cells), animal cells (e.g., human cells, cells, hamster cells, rat cells, mouse cells or insect cells) or hybridomas. Typically, the host cell is transformed with a peptide-encoding nucleic acid that can then be expressed in the host cell. A cultured cell that can be transformed or transfected. The term "recombinant host cell" refers to a , used to indicate a host cell transformed or transfected with a nucleic acid of predicted expression The host cell also contains the nucleic acid, but the control sequence is operably linked to the nucleic acid. These may be cells that do not express the nucleic acid at the desired level unless introduced into the host cell as described above. The term "host cell" includes not only the cell of a particular subject, but also the progeny or potential host cell of that cell. It should be understood that the term "progeny" also refers to the potential progeny of a particular gene, e.g., Such progeny may, in fact, differ from the parent cell due to mutations or environmental influences; and are included within the scope of this term as used herein.

[0059] Endothelin receptor Endothelin receptors (ETAs) are heterotrimeric guanine nucleotide-binding proteins 7-transmembrane complexes coupled via G proteins to one or more intracellular signaling pathways It belongs to the A subfamily of the receptor family (Jelinek et al., 1999). 93, Science 259:1614-1616, and Segre et al., 1993,Trends Endocrinol.Metab.4:309-314). As used herein, "endothelin receptor" and "ETA" or "ET A R " may be used interchangeably.

[0060] In one embodiment, the antibodies described herein target membrane-bound enzymes expressed in cells. It binds to endothelin receptors and inhibits endothelin signal transduction via endothelin receptors. In one embodiment, the antibodies described herein can be selected to inhibit or block In a further embodiment, the human endothelin receptor specifically binds to the human endothelin receptor. Antibodies that bind to serine receptors also bind to endothelin receptors in other species, e.g., rats. The following example illustrates the generation of murine antibodies that bind to human membrane-bound endothelin receptors. and in a further embodiment, the murine antibody also reacts with endothelin receptors of other species. Combine with.

[0061] The polynucleotide and polypeptide sequences of several species of endothelin receptors are known. SEQ ID NO: 1 to SEQ ID NO: 6 show the sequences of human, monkey and rat. , US National Center for Biotechnology Information GenBank database of Biotechnology Information I got it from there.

[0062] The sequence information for endothelin receptor A (ETA) is as follows: Human (Homo sapiens) polynucleotide (SEQ ID NO:1 );Accession number: S63938; Human (Homo sapiens) amino acid (SEQ ID NO: 2); No.:AAB20278; Monkey (cynomolgus monkey) polynucleotide (SEQ ID NO: 3); Accession number: JV635771; Monkey (cynomolgus monkey) amino acid (SEQ ID NO: 4); Accession number: AFJ71111; Rat (Rattus norvegicus) polynucleotide (sequence No. 5); Accession No.: M60786; Rat (Rattus norvegicus) amino acid (SEQ ID NO: 6) ;Accession number: AAA41114.

[0063] Endothelin receptor A (ETA) antibodies In one embodiment, the ETA antibodies described herein are 1, 2, 3, 4, 5 or 6 and wherein each amino acid sequence independently comprises one of the amino acids listed below. Acid sequence: a. Light chain CDR1 amino acid 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: No. 26, SEQ ID NO: 28, and SEQ ID NO: 30; b. Light chain CDR2 amino acid sequences: SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO: No. 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48 ; c. Light chain CDR3 amino acid sequence: SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, Row number 68, and sequence number 205; d. Heavy chain CDR1 amino acid sequences: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, Sequence number 88, and sequence number 90; e. Heavy chain CDR2 amino acid sequences: SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 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 f. Heavy chain CDR3 amino acid 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 is selected from.

[0064] Table 1 shows the light chain CDR amino acid sequences of the ETA antibodies described herein, as well as their The corresponding polynucleotide coding sequences are listed in Table 2. Heavy chain CDR amino acid sequences of TA antibodies and their corresponding polynucleotide coding sequences Lists the columns.

[0065] [Table 1] TIFF2025143267000002.tif221170

[0066] [Table 2] TIFF2025143267000004.tif232170

[0067] In one embodiment, the antibodies described herein have 5, 4, 3, 2, or 1 amino acid residues. The amino acid additions, substitutions, and / or deletions are identical to one of the CDR amino acid sequences listed in Tables 1 and 2. In another embodiment, the antibodies described herein comprise 4, 3, 2 or more different sequences. or one amino acid addition, substitution, and / or deletion is selected from the CDR amino acids listed in Tables 1 and 2. In another embodiment, the antibody described herein comprises a sequence that differs from one of the amino acid sequences. , 3, 2 or 1 amino acid addition, substitution, and / or deletion are listed in Tables 1 and 2. In another embodiment, the DR amino acid sequence comprises a sequence different from one of the DR amino acid sequences described herein. The antibodies to be used may have two or one amino acid addition, substitution, and / or deletion as listed in Tables 1 and 2. In another embodiment, the CDR comprises a sequence different from one of the CDR amino acid sequences described herein. The antibodies described have one amino acid addition, substitution, and / or deletion that is different from the amino acid sequences listed in Tables 1 and 2. The CDR amino acid sequence may be different from that of the CDR amino acid sequence.

[0068] In another embodiment, the ETA antibody described herein (ETA-1 antibody) is one or more comprises two amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of: Amino acid sequence: a. Light chain CDR1 amino acid 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: SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30; and b. Heavy chain CDR1 amino acid sequences: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, Column number 88 and sequence number 90 is selected from.

[0069] In one embodiment, the ETA-1 antibody further comprises one or two amino acid sequences, wherein , each amino acid sequence independently being an amino acid sequence listed below: a. Light chain CDR2 amino acid sequence: SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: No. 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48 and b. Heavy chain CDR2 amino acid sequences: SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO: 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 is selected from.

[0070] In another embodiment, the ETA-1 antibody further comprises one or two amino acid sequences, wherein each amino acid sequence is independently an amino acid sequence listed below: a. Light chain CDR3 amino acid sequences: SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, Column number 68, and sequence number 205; and b. Heavy chain CDR3 amino acid 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 is selected from.

[0071] In another embodiment, the ETA antibody described herein (ETA-2 antibody) is one or more comprises two amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of: Amino acid sequence: a. Light chain CDR2 amino acid sequence: SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: No. 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48 and b. Heavy chain CDR2 amino acid sequences: SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO: 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 is selected from.

[0072] In one embodiment, the ETA-2 antibody further comprises one or two amino acid sequences, wherein , each amino acid sequence independently being an amino acid sequence listed below: a. Light chain CDR1 amino acid 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: SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30; and b. Heavy chain CDR1 amino acid sequences: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, Column number 88 and sequence number 90 is selected from.

[0073] In another embodiment, the ETA-2 antibody further comprises one or two amino acid sequences, wherein each amino acid sequence is independently an amino acid sequence listed below: a. Light chain CDR3 amino acid sequences: SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, Column number 68, and sequence number 205; and b. Heavy chain CDR3 amino acid 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 is selected from.

[0074] In another embodiment, the ETA antibody described herein (ETA-3 antibody) is one or more comprises two amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of: Amino acid sequence: a. Light chain CDR3 amino acid sequences: SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, Column number 68, and sequence number 205; and b. Heavy chain CDR3 amino acid 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 is selected from.

[0075] In one embodiment, the ETA-3 antibody further comprises one or two amino acid sequences, wherein , each amino acid sequence independently being an amino acid sequence listed below: a. Light chain CDR1 amino acid 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: SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30; and b. Heavy chain CDR1 amino acid sequences: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, Column number 88 and sequence number 90 is selected from.

[0076] In another embodiment, the ETA-3 antibody further comprises one or two amino acid sequences, wherein each amino acid sequence is independently an amino acid sequence listed below: a. Light chain CDR2 amino acid sequence: SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: No. 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48 and b. Heavy chain CDR2 amino acid sequences: SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO: 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 is selected from.

[0077] In one embodiment, the ETA antibodies described herein are selected from the group consisting of: a. 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: No. 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 3 a light chain CDR1 amino acid sequence independently selected from: b. The list of: SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO: Independently of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48 a selected light chain CDR2 amino acid sequence; c. The list of: SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO: No. 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, and SEQ ID NO: 6 a light chain CDR3 amino acid sequence independently selected from 8; d. The list of: SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO: No. 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and a heavy chain CDR1 amino acid sequence independently selected from SEQ ID NO: 90; e. The following list: SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: No. 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: Heavy chain CDR2 independently selected from SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114 the amino acid sequence; and f. The list of: SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:12 2, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 13 2, SEQ ID NO: 134, and SEQ ID NO: 136. array Includes.

[0078] In one embodiment, the ETA antibody described herein has a sequence similar to that of the antibody of the following list: SEQ ID NO:5 0, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: Independently of SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 205 In another embodiment, the light chain CDR3 amino acid sequence comprises a selected The ETA antibodies used are listed below: 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, Heavy chain CD14 independently selected from SEQ ID NO: 132, SEQ ID NO: 134, and SEQ ID NO: 136 Contains the R3 amino acid sequence.

[0079] In another embodiment, the ETA antibodies described herein are selected from the group consisting of the antibodies of the following list: SEQ ID NO: 50 and SEQ ID NO: 116, SEQ ID NO: 50 and SEQ ID NO: 205, SEQ ID NO: 62 and SEQ ID NO: 128, SEQ ID NO: 62 and SEQ ID NO: 130, SEQ ID NO: 64 and SEQ ID NO: 132, SEQ ID NO: 66 and SEQ ID NO: 134, and SEQ ID NO: 68 and SEQ ID NO: 136 It comprises a combination of light and heavy chain CDR3 amino acid sequences.

[0080] In one embodiment, the ETA antibodies described herein are (a) Light chain CDR1 amino acid sequence: SEQ ID NO: 8; Light chain CDR2 amino acid sequence: SEQ ID NO: 32; Light chain CDR3 amino acid sequence: SEQ ID NO: 50 or SEQ ID NO: 205; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 70; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 92; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 116; (b) light chain CDR1 amino acid sequence: SEQ ID NO: 10; Light chain CDR2 amino acid sequence: SEQ ID NO: 34; Light chain CDR3 amino acid sequence: SEQ ID NO: 52; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 72; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 94; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 118; (c) light chain CDR1 amino acid sequence: SEQ ID NO: 12; Light chain CDR2 amino acid sequence: SEQ ID NO: 36; Light chain CDR3 amino acid sequence: SEQ ID NO: 54; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 74; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 96; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 120; (d) light chain CDR1 amino acid sequence: SEQ ID NO: 14; Light chain CDR2 amino acid sequence: SEQ ID NO: 38; Light chain CDR3 amino acid sequence: SEQ ID NO: 56; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 76; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 98; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 122; (e) light chain CDR1 amino acid sequence: SEQ ID NO: 16; Light chain CDR2 amino acid sequence: SEQ ID NO: 40; Light chain CDR3 amino acid sequence: SEQ ID NO: 58; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 78; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 100; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 124; (f) light chain CDR1 amino acid sequence: SEQ ID NO: 18; Light chain CDR2 amino acid sequence: SEQ ID NO: 42; Light chain CDR3 amino acid sequence: SEQ ID NO: 60; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 80; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 102; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 126; (g) light chain CDR1 amino acid sequence: SEQ ID NO: 20 or SEQ ID NO: 22; Light chain CDR2 amino acid sequence: SEQ ID NO: 44; Light chain CDR3 amino acid sequence: SEQ ID NO: 62; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 82; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 104 or 106; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 128; (h) light chain CDR1 amino acid sequence: SEQ ID NO: 24; Light chain CDR2 amino acid sequence: SEQ ID NO: 44; Light chain CDR3 amino acid sequence: SEQ ID NO: 62; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 84; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 108; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 130; (i) light chain CDR1 amino acid sequence: SEQ ID NO: 26; Light chain CDR2 amino acid sequence: SEQ ID NO: 46; Light chain CDR3 amino acid sequence: SEQ ID NO: 64; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 86; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 110; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 132; (j) light chain CDR1 amino acid sequence: SEQ ID NO: 28; Light chain CDR2 amino acid sequence: SEQ ID NO: 46; Light chain CDR3 amino acid sequence: SEQ ID NO: 66; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 88; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 112; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 134; or (k) light chain CDR1 amino acid sequence: SEQ ID NO: 30; Light chain CDR2 amino acid sequence: SEQ ID NO: 48; Light chain CDR3 amino acid sequence: SEQ ID NO: 68; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 90; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 114; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 136 Includes.

[0081] In another embodiment, the ETA antibodies described herein Light chain CDR1 amino acid sequence: SEQ ID NO: 28; Light chain CDR2 amino acid sequence: SEQ ID NO: 46; Light chain CDR3 amino acid sequence: SEQ ID NO: 66; Heavy chain CDR1 amino acid sequence: SEQ ID NO: 88; Heavy chain CDR2 amino acid sequence: SEQ ID NO: 112; and Heavy chain CDR3 amino acid sequence: SEQ ID NO: 134 Includes.

[0082] In another embodiment, the ETA antibodies described herein have one or two amino acid sequences a string, wherein each amino acid sequence is independently an amino acid sequence listed below: a. Light chain variable domain amino acid sequences: SEQ ID NO: 138 (L1), SEQ ID NO: 140 (L2) ), SEQ ID NO: 142 (L3), SEQ ID NO: 144 (L4), SEQ ID NO: 146 (L5), SEQ ID NO: No. 148 (L6), SEQ ID NO: 150 (L7), SEQ ID NO: 152 (L8), SEQ ID NO: 15 4 (L9), SEQ ID NO: 156 (L10), SEQ ID NO: 158 (L11), SEQ ID NO: 160 ( L12), SEQ ID NO: 162 (L13), and SEQ ID NO: 164 (L14), and those with less at least 80%, at least 85%, at least 90%, or at least 95% identical the amino acid sequence; and b. Heavy chain variable domain amino acid sequences: SEQ ID NO: 166 (H1), SEQ ID NO: 168 (H2 ), SEQ ID NO: 170 (H3), SEQ ID NO: 172 (H4), SEQ ID NO: 174 (H5), SEQ ID NO: No. 176 (H6), SEQ ID NO: 178 (H7), SEQ ID NO: 180 (H8), SEQ ID NO: 18 2 (H9), SEQ ID NO: 184 (H10), SEQ ID NO: 186 (H11), SEQ ID NO: 188 ( H12), SEQ ID NO: 190 (H13), and SEQ ID NO: 192 (H14), and at least 80%, at least 85%, at least 90%, or at least 95% identical Amino acid sequence is selected from.

[0083] In another embodiment, the polynucleotide coding sequence for the ETA antibody described herein is The string contains one or two polynucleotide sequences, where each polynucleotide sequence is Independently, the polynucleotide sequences listed below: a. Light chain variable domain polynucleotide coding 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, and at least 80%, at least 8 5%, at least 90%, or at least 95% identical polynucleotide sequences; and to b. Heavy chain variable domain polynucleotide coding 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 at least 80%, at least 8 Polynucleotide sequences that are 5%, at least 90%, or at least 95% identical is selected from.

[0084] In another embodiment, the ETA antibodies described herein a. The following list: SEQ ID NO:138 (L1), SEQ ID NO:140 (L2), SEQ ID NO:14 2 (L3), SEQ ID NO: 144 (L4), SEQ ID NO: 146 (L5), SEQ ID NO: 148 (L6 ), SEQ ID NO: 150 (L7), SEQ ID NO: 152 (L8), SEQ ID NO: 154 (L9), SEQ ID NO: SEQ ID NO: 156 (L10), SEQ ID NO: 158 (L11), SEQ ID NO: 160 (L12), SEQ ID NO: a light chain variable domain independently selected from SEQ ID NO: 162 (L13), and SEQ ID NO: 164 (L14); The main amino acid sequence, and at least 80%, at least 85%, at least 90%, or at least 95% identical amino acid sequences; and b. The following list: SEQ ID NO: 166 (H1), SEQ ID NO: 168 (H2), SEQ ID NO: 17 0 (H3), SEQ ID NO: 172 (H4), SEQ ID NO: 174 (H5), SEQ ID NO: 176 (H6 ), SEQ ID NO: 178 (H7), SEQ ID NO: 180 (H8), SEQ ID NO: 182 (H9), SEQ ID NO: No. 184 (H10), SEQ ID NO. 186 (H11), SEQ ID NO. 188 (H12), SEQ ID NO. SEQ ID NO: 190 (H13), and SEQ ID NO: 192 (H14). The main amino acid sequence, and at least 80%, at least 85%, at least 90% or at least 95% identical amino acid sequences Includes:

[0085] In another embodiment, the ETA antibodies described herein a. The following list: SEQ ID NO:138 (L1), SEQ ID NO:140 (L2), SEQ ID NO:14 2 (L3), SEQ ID NO: 144 (L4), SEQ ID NO: 146 (L5), SEQ ID NO: 148 (L6 ), SEQ ID NO: 150 (L7), SEQ ID NO: 152 (L8), SEQ ID NO: 154 (L9), SEQ ID NO: No. 156 (L10), SEQ ID NO: 158 (L11), SEQ ID NO: 160 (L12), SEQ ID NO: a light chain variable domain independently selected from SEQ ID NO: 162 (L13), and SEQ ID NO: 164 (L14); the main amino acid sequence; and b. The following list: SEQ ID NO: 166 (H1), SEQ ID NO: 168 (H2), SEQ ID NO: 17 0 (H3), SEQ ID NO: 172 (H4), SEQ ID NO: 174 (H5), SEQ ID NO: 176 (H6 ), SEQ ID NO: 178 (H7), SEQ ID NO: 180 (H8), SEQ ID NO: 182 (H9), SEQ ID NO: No. 184 (H10), SEQ ID NO. 186 (H11), SEQ ID NO. 188 (H12), SEQ ID NO. SEQ ID NO: 190 (H13), and SEQ ID NO: 192 (H14). Main amino acid sequence Includes:

[0086] In another embodiment, the ETA antibodies described herein are selected from the group consisting of the antibodies of the following list: SEQ ID NO: 138 and SEQ ID NO: 166 (L1H1), SEQ ID NO: 140 and SEQ ID NO: 168 (L2H2 ), SEQ ID NO: 142 and SEQ ID NO: 170 (L3H3), SEQ ID NO: 144 and SEQ ID NO: 17 2 (L4H4), SEQ ID NO: 146 and SEQ ID NO: 174 (L5H5), SEQ ID NO: 148 and SEQ ID NO: 176 (L6H6), SEQ ID NO: 150 and SEQ ID NO: 178 (L7H7), SEQ ID NO: No. 152 and SEQ ID NO: 180 (L8H8), SEQ ID NO: 154 and SEQ ID NO: 182 (L9H 9), SEQ ID NO: 156 and SEQ ID NO: 184 (L10H10), SEQ ID NO: 158 and SEQ ID NO: No. 186 (L11H11), SEQ ID NO: 160 and SEQ ID NO: 188 (L12H12), SEQ ID NO: No. 162 and SEQ ID NO: 190 (L13H13), and SEQ ID NO: 164 and SEQ ID NO: 19 2 (L14H14) In another embodiment, the ETA antibodies described herein comprise a light chain and a heavy chain. Variable domain amino acid sequence: SEQ ID NO: 162 and SEQ ID NO: 190 (L13H13) This includes.

[0087] The designation "LxHy" may also be used to refer to the ETA antibodies described herein. where "x" corresponds to the light chain variable region and "y" corresponds to the heavy chain variable region. For example, L2H1 is a light chain variable region comprising the amino acid sequence SEQ ID NO: 140 (L2) and This refers to an antibody having a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 166 (H1).

[0088] In another embodiment, the ETA antibody described herein is selected from L1-L14. a light chain variable region selected from H1 to H14, or a heavy chain variable region selected from H1 to H14, and a fragment thereof , derivatives, muteins, or variants.

[0089] In another embodiment, the ETA antibodies described herein are selected from the group consisting of the antibodies of the following list: SEQ ID NO: 138 and SEQ ID NO: 166, SEQ ID NO: 150 and SEQ ID NO: 178, SEQ ID NO: 152 and SEQ ID NO: Sequence number 180, sequence number 154 and sequence number 182, sequence number 156 and sequence number 184 , SEQ ID NO: 158 and SEQ ID NO: 186, SEQ ID NO: 160 and SEQ ID NO: 188, SEQ ID NO: 1 62 and SEQ ID NO: 190, and SEQ ID NO: 164 and SEQ ID NO: 192. It comprises a combination of light chain and heavy chain CDR3 amino acid sequences that are

[0090] In one embodiment, the ETA antibodies described herein comprise the light chain variable domain amino acid sequence The heavy chain variable domain amino acid sequence includes SEQ ID NO: 138 or SEQ ID NO: 166. In embodiments, the ETA antibodies described herein comprise the light chain variable domain amino acid sequence and the heavy chain variable domain amino acid sequence SEQ ID NO: 166. In another embodiment, the ETA antibodies described herein further comprise a constant amino acid sequence. wherein each constant amino acid sequence is independently an amino acid sequence listed below: a. a. Light chain constant amino acid sequences: SEQ ID NO: 194 and SEQ ID NO: 196; and b. Heavy chain constant amino acid sequences: SEQ ID NO: 196 and SEQ ID NO: 194 Acid sequences: selected from SEQ ID NO: 198 and SEQ ID NO: 206.

[0091] In another embodiment, the ETA antibodies described herein further comprise a constant amino acid sequence. wherein each constant amino acid sequence is independently selected from the light and heavy chain constant amino acids listed below. Amino acid sequence combination: a. Light chain constant amino acid sequence SEQ ID NO: 194 and heavy chain constant amino acid sequence SEQ ID NO: 19 Combination of 8; b. Light chain constant amino acid sequence SEQ ID NO: 194 and heavy chain constant amino acid sequence SEQ ID NO: 20 Combination of 6; c. Light chain constant amino acid sequence SEQ ID NO: 196 and heavy chain constant amino acid sequence SEQ ID NO: 19 Combination of 8; d. Light chain constant amino acid sequence SEQ ID NO: 196 and heavy chain constant amino acid sequence SEQ ID NO: 20 Combination of 6 is selected from.

[0092] In one embodiment, the antibodies described herein comprise the light and heavy chains listed herein. In one embodiment, the antibody comprises the amino acid sequences of the CDRs and FRs. In another embodiment, the antibody comprises a light chain CDR1 sequence as recited herein. In another embodiment, the antibody comprises a light chain CDR2 sequence as set forth in the specification. In another embodiment, the antibody comprises a light chain CDR3 sequence listed herein. In another embodiment, the antibody comprises a heavy chain CDR1 sequence listed herein. In another embodiment, the antibody comprises a heavy chain CDR2 sequence as listed herein. In another embodiment, the antibody comprises a light chain FR1 sequence as described herein. In another embodiment, the antibody comprises a light chain FR2 sequence herein. In another embodiment, the antibody comprises a light chain FR3 sequence herein. In another embodiment, the antibody comprises the light chain FR4 sequence described herein. In another embodiment, the antibody comprises a heavy chain FR1 sequence as described herein. In another embodiment, the antibody comprises a heavy chain FR2 sequence as described herein. In a further embodiment, the antibody comprises the heavy chain FR4 sequence described herein. nothing.

[0093] In one embodiment, the CDR3 sequence of the antibody has no more than 6, 5, 4, 3, 2 or 1 amino acid residues. The light and heavy chain CDR3 amino acid additions, substitutions and / or deletions are The antibody is different from the combination of sequences SEQ ID NO: 50 and SEQ ID NO: 116. The light chain CDR3 sequence of the present invention may have no more than 6, 5, 4, 3, 2 or 1 amino acid additions, substitutions and / or The deletion differs from the light chain CDR3 amino acid sequence SEQ ID NO: 50 listed herein. In this embodiment, the light chain CDR3 sequence of the antibody has no more than 6, 5, 4, 3, 2 or 1 amino acid residues. Amino acid additions, substitutions and / or deletions are made to the light chain CDR3 amino acid sequences listed herein. The heavy chain CDR3 sequence of the antibody differs from SEQ ID NO: 50 by not more than 6, 5, 4, 3, 2, or 1 amino acid sequence. Amino acid additions, substitutions and / or deletions may be made to the heavy chain CDR3 amino acid sequences listed herein. In another embodiment, the antibody is different from SEQ ID NO: 116 or SEQ ID NO: 118 of the present specification. Further, combinations of one, two, three, four, five or six of the light and heavy chain CDR sequences listed in the document are In another embodiment, the antibody comprises one, two, three, four, five or more of the light and heavy chain CDR sequences. Further, six combinations are included, each of which may independently have no more than 6, 5, 4, 3, 2, or 1 amino acid sequence. The amino acids are SEQ ID NO: 50 and SEQ ID NO: 60 of the light and heavy chain CDR3 amino acid sequences listed herein. In another embodiment, the antibody is different from the combination of SEQ ID NO: 116. In another embodiment, the antibody comprises a light chain variable region CDR and a heavy chain variable region CDR comprising: Sets of 1, 2, 3, 4, 5 and / or 6 of the light and heavy chain CDR sequences listed herein Includes combinations.

[0094] In one embodiment, the antibody (e.g., antibody or antibody fragment) is selected from the group consisting of those listed herein. In one embodiment, the light chain variable domain comprises an L1 light chain variable domain sequence 1 light chain variable domain sequence and , 4, 3, 2 or 1 amino acid difference, wherein each sequence The differences are independently a deletion, insertion, or substitution of an amino acid residue. The light chain variable domain is at least 70% identical to the L1 light chain variable domain sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, The amino acid sequence of the present invention is at least 97%, or at least 99% identical to the amino acid sequence of the present invention. wherein the light chain variable domain polynucleotide coding sequence is an L1 polynucleotide coding sequence. At least 70%, at least 75%, at least 80%, at least 8% 5%, at least 90%, at least 95%, at least 97%, or at least 99% In another embodiment, the light chain variable domain fragments comprise nucleotide coding sequences that are identical. The polynucleotide coding sequence is the complement of the L1 light chain variable domain polynucleotide coding sequence. A polynucleotide sequence that hybridizes under moderately stringent conditions to the sequence In another embodiment, the light chain variable domain polynucleotide coding sequence comprises an L1 light chain variable domain. The complementary sequence of the chain variable domain polynucleotide coding sequence is It includes hybridizing polynucleotide sequences.

[0095] In one embodiment, the antibody (e.g., antibody or antibody fragment) is selected from the group consisting of those listed herein. In another embodiment, the variable domain comprises an H1 heavy chain variable domain sequence 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, with heavy chain variable domain sequence amino acid sequences having 4, 3, 2, or 1 amino acid difference, where each sequence The differences are independently deletions, insertions, or substitutions of amino acid residues. The heavy chain variable domain is at least 70%, at least 7%, 5%, at least 80%, at least 85%, at least 90%, at least 95%, In another embodiment, the amino acid sequence is at least 97%, or at least 99% identical. wherein the heavy chain variable domain polynucleotide coding sequence is an H1 polynucleotide coding sequence. At least 70%, at least 75%, at least 80%, at least 85% of the sequence %, at least 90%, at least 95%, at least 97%, or at least 99% identical In another embodiment, the heavy chain variable domain polypeptide comprises a nucleotide coding sequence that is identical to the heavy chain variable domain polypeptide. The nucleotide coding sequence is the complement of the H1 heavy chain variable domain polynucleotide coding sequence. A polynucleotide sequence that hybridizes under moderately stringent conditions to the sequence In one embodiment, the heavy chain variable domain polynucleotide coding sequence is an H1 heavy chain variable domain polynucleotide. The complementary sequence of the variable domain polynucleotide coding sequence is hybridized under stringent conditions. It contains a polynucleotide sequence that replicates.

[0096] In one embodiment, the antibodies described herein are L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11 H11, L12H12, L13H13, or L14H14, or a desired phenotype thereof (e.g., For example, IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE, and Ig D), or an antibody comprising a combination of Fab or F(ab')2 fragments thereof.

[0097] In one embodiment, the antibodies described herein are L1H1 or its class-switched counterparts. Antibodies with a specific function (e.g., IgA, IgG1, IgG2a, IgG2b, IgG3, Ig IgM, IgE, and IgD), or a combination of Fab or F(ab')2 fragments thereof The present invention also includes antibodies containing the above.

[0098] The antibodies (e.g., antibodies, antibody fragments, and antibody derivatives) described herein are The light chain constant region may comprise any of the constant regions known in the art. The heavy chain constant region may be a kappa or lambda light chain constant region, e.g., a murine kappa or lambda light chain constant region. can be, for example, an α, δ, ε, γ, or μ heavy chain constant region, e.g., a murine α, δ, ε, γ, or μ heavy chain constant region. can be a μ heavy chain constant region. In one embodiment, the light or heavy chain constant region is a naturally occurring constant region. It may be a fragment, derivative, variant, or mutein of the region.

[0099] In one embodiment, the antibodies described herein comprise a light chain kappa or lambda constant domain or The light chain constant region sequence and its polynucleotide coding sequence further include a fragment of , provided as follows: 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).

[0100] In another embodiment, the antibodies described herein comprise a heavy chain constant domain or fragment thereof. The heavy chain constant region sequence and its polynucleotide coding sequence are as follows: Polynucleotide (IgG4), (SEQ ID NO: 197); Amino acid ( IgG4), (SEQ ID NO: 198).

[0101] In one embodiment, the ETA antibodies described herein are murine antibodies, humanized antibodies, Chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antigen-binding antibodies fragments, single-chain antibodies, double-chain antibodies, triabodies, tetrabodies, Fab fragments F(ab') x Fragment, domain antibody, IgD antibody, IgE antibody, IgM antibody , IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, and IgG4 antibodies. In embodiments, the ETA antibodies described herein are ETA monoclonal antibodies. In another embodiment, the ETA antibody described herein is a murine ETA antibody. The ETA antibodies described herein are humanized ETA antibodies.

[0102] In one embodiment, the ETA antibody described herein is monoclonal antibody A-1 (including SEQ ID NO: 138 and SEQ ID NO: 166), A-7 (including SEQ ID NO: 150 and SEQ ID NO: 1 78), A-8 (including SEQ ID NO: 152 and SEQ ID NO: 180), A-9 (including SEQ ID NO: 154 and SEQ ID NO: 182), A-10 (including SEQ ID NO: 156 and SEQ ID NO: 184) A-11 (including SEQ ID NO: 158 and SEQ ID NO: 186), A-12 (including SEQ ID NO: 16 0 and SEQ ID NO: 188), A-13 (including SEQ ID NO: 162 and SEQ ID NO: 190) , or A-14 (including SEQ ID NO: 164 and SEQ ID NO: 192).

[0103] Antibodies and antibody fragments In one embodiment, the antibodies described herein are intact antibodies (full-length heavy chains and and / or light chains, including polyclonal, monoclonal, chimeric, humanized or human antibodies. In another embodiment, the antibodies described herein are antibody fragments, For example, F(ab')2, Fab, Fab', Fv, Fc, or Fd fragments. , single domain antibodies, single chain antibodies, maxibodies, minibodies (minibodies), intrabodies, bispecific antibodies can be incorporated into antibodies, triabodies, tetrabodies, v-NARs and bis-scFvs (e.g., Hollinger and Hudson, 2005, Nature Bi (See Biotechnology, 23:1126-1136). In another embodiment, The antibodies described herein include fibronectin polypeptide monoclonal antibodies. antibody polypeptides such as those disclosed in (U.S. Patent No. 6,703,199) In another embodiment, the antibodies described herein are single-chain polypeptides. Other antibody polymerases disclosed in (US Patent Application Publication No. 2005 / 0238646) Also contains lipids.

[0104] In one embodiment, the genes expressing the relevant monoclonal antibodies in the hybridoma are The variable regions of the clones are amplified using nucleotide primers. These primers are: It can be synthesized by one of ordinary skill in the art or purchased from commercial sources (e.g., Strat (See also: agene, La Jolla, and California), these supplies The vendor is V Ha , V Hb , V Hc , V Hd , C H1 , V L , and C L Contains region primers We sell mouse and human variable region primers containing IM MUNOZAPTMH or ILLFLUNOZAPTML (Stratagene) It can be used to amplify heavy or light chain variable regions which can then be inserted into a vector such as These vectors can then be used in E. coli, yeast, or mammalian-based expression systems. can be introduced into the system. V H and V L A large number of fusion single-chain proteins containing these domains have been (Bird et al., 1988, Science 2 42:423-426).

[0105] The antibody-producing cells of the present application may be obtained using any of the above immunization and other techniques. After this, the genes for the specific antibodies are then cloned according to standard methods described herein. The gene can be cloned by isolating it and amplifying the DNA or mRNA. The antibodies produced therefrom can be sequenced and the CDRs identified. The DNA may be engineered as described above to produce the other antibodies described herein. obtain.

[0106] The antibodies described herein are preferably used in the cell-based assays described herein. and / or in vivo assays described herein, and / or cross-block the binding of one of the antibodies described in this application. oss-block) and / or binding to ETA by one of the antibodies described in this application. Therefore, the assays described herein can be used to detect such cross-blocking. This can be used to identify such binding agents.

[0107] In certain embodiments, the antibodies are derived from cell-based and / or in vivo antibodies described herein. In a 2D assay, the antibody binds to and / or neutralizes ETA-overexpressing cells and / or cross-blocks the antibodies described herein and / or cross-blocks one of the antibodies described herein By first identifying antibodies that cross-block ETA binding, is generated.

[0108] It is well known in the art that certain proteins, such as antibodies, can undergo a variety of post-translational modifications. The type and extent of these modifications should be understood by those skilled in the art. Such modifications depend on the host cell line and culture conditions used for the synthesis of the glycoprotein. Sylation, methionine oxidation, diketopiperizine formation These include changes in aspartate isomerization and asparagine deamidation. Frequent modifications by the action of enzymes occur at the carboxy-terminal basic residues (lysine or arginine). (Harris, 1995, Journal of Chronicles As described in matography 705:129-134).

[0109] Alternative methods for the production of mouse monoclonal antibodies include monoclonal antibody-containing The mice were treated to promote the formation of ascites (e.g., pristane primed). ane primary immunization)) allogeneic mice, e.g. The first step is to inject hybridoma cells into the peritoneal cavity. Monoclonal antibodies are produced by various established Such isolation techniques include Protein-A Sepharose affinity chromatography, size exclusion chromatography and ion exchange chromatography (e.g., Coligan et al. pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3 ; and Baines et al., “Purification of Immuno globulin G(IgG)”,Methods in Molecular Bi ology,Vol.10,pages 79-104(The Humana Pre ss, Inc. 1992). Monoclonal antibodies are antibodies that are specifically designed to possess specific properties (e.g., a suitable ligand selected based on the target molecule (e.g., heavy or light chain isotype, binding specificity, etc.) The purified product can be purified by affinity chromatography using a solid support. Examples of suitable ligands include protein A, protein G, and anti-constant regions (light or heavy chains). Antibody, anti-idiotype antibody, and TGF-β binding protein, or a fragment thereof Examples include mutants or variants.

[0110] Schier et al.,1996,J.Mol.Biol.263:551-5 67, the complementarity determining region (CDR) molecules in the center of the antibody binding site. Evolution can lead to antibodies with increased affinity, e.g., increased affinity for c-erbB-2. Such techniques have therefore been used to isolate antibodies with human It is useful for preparing antibodies against the human endothelin receptor. The antibody may be, for example, a cytotoxic agent that inhibits the presence of an endothelin receptor, either in vitro or in vivo. can be used in assays to detect

[0111] The antibodies may also be prepared by any conventional technique. For example, the antibodies may be prepared by Can they be purified from cells that naturally express them (e.g., antibodies can be purified from the cells that produce them)? The nucleotide sequences can be purified from lysosomes (e.g., purified from lysosomes) or synthesized using any technique known in the art. It can be produced in a recombinant expression system. s,Hybridomas:A New Dimension in Biologic al Analyses,Kennet et al.(eds.),Plenum P ress, (1980); and Antibodies: A Laboratory Ma nual, Harlow and Land (eds.), Cold Spring H See Arbor Laboratory Press (1988). These are explained below in the section on nucleic acids.

[0112] Antibodies may be prepared and screened for desired properties by any conventional technique. Several techniques are available for the detection of polyclonal antibodies of interest (e.g., anti-endothelin receptor antibodies). Isolation of nucleic acids encoding peptide chains (or parts thereof) and nucleic acids by recombinant DNA techniques The nucleic acid may be fused to another related nucleic acid or may be linked to one or more amino acids. to add, delete, or substitute (e.g., by induced mutagenesis or other conventional techniques) (Thus) can be modified.

[0113] Improving the affinity of antibodies of the present invention containing one or more of the above-described CDRs If necessary, maintenance of CDRs (Yang et al., 1995, J. Mol. Biol.,254:392-403), chain shuffling (Marks et al .,1992,Bio / Technology,10:779-783), Escherichia coli (E. coli) (Low et al., 1996, J. Mol. Biol. l., 250:350-368), DNA rearrangement (Patten et al., 199 7, Curr. Opin. Biotechnol., 8:724-733), phage Display (Thompson et al., 1996, J. Mol. Biol., 2 56:7-88) and further PCR techniques (Crameri et al., 1998, Several affinity maturation protocols are available, including (Nature, 391:288-291) All of these affinity maturation methods are described in detail in Vaughan et al., 1999. 8, Nature Biotechnology, 16:535-539 do.

[0114] In one embodiment, the antibodies described herein are directed to anti-endothelin receptor fragments. The fragment may consist entirely of antibody-derived sequences or may contain additional sequences. Examples of antigen-binding fragments include Fab, F(ab')2, single-chain antibodies, and double-chain antibodies. Examples include bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and domain antibodies. An example is Lunde et al.,2002,Biochem.Soc.Trans.3 It is provided at 0:500-06.

[0115] Single-chain antibodies are made by cleaving the polypeptide chains together using an amino acid bridge (a short peptide linker) to produce a single polypeptide chain. By linking heavy and light chain variable domain (Fv region) fragments via Such single-chain Fvs (scFvs) can be formed by combining two variable domain polypeptides. The fusion DNA encodes a peptide linker between the DNAs encoding the VL and VH domains. The resulting polypeptide folds back on itself to form an antigen-binding moiety. They can form monomers or they can be separated by a flexible linker between the two variable domains. Depending on the length, polymers (e.g., dimers, trimers, or tetramers) can be formed (Kor tt et al., 1997, Prot. Eng. 10:423; and Kortt e (T al., 2001, Biomol. Eng. 18:95-108). Various VL and By combining VH-containing polypeptides, polymers that bind to various phenotypes can be cFv can be formed (Kriangkum et al., 2001, Biomol. Eng.18:31-40). Techniques developed for the production of single-chain antibodies include: U.S. Patent No. 4,946,778; Bird, 1988, Science 242 :423;Huston et al.,1988,PNAS USA 85:5879 -5883;Ward et al.,1989,Nature 334:544-54 6;de Graaf et al.,2002,Methods Mol.Biol. 178:379-87. Single chain antibodies derived from the antibodies described herein, including scFvs comprising the combination L1H1 are included within the scope of this specification.

[0116] Antigen-binding fragments derived from antibodies can also be prepared by isolating the antibody protein according to conventional methods. proteolytic hydrolysis, e.g., obtained by pepsin or papain digestion of intact antibodies; For example, antibody fragments include the SS fragment called F(ab')2. This fragment can be produced by enzymatic cleavage of antibodies with pepsin, resulting in This is further cleaved using a sulfhydryl reducing agent to give the 3.5S Fab' monovalent fragment. An optional scheme involves a cleavage reaction with a sulfhydryl protecting group to give a disulfide. Further, enzymatic cleavage using papain results in the cleavage of two These methods directly produce monovalent Fab and Fc fragments of, for example, See, for example, Goldenberg et al., U.S. Pat. No. 4,331,647; Nisonof fet et al.,1960,Arch.Biochem.Biophys.89: 230;Porter,1959,Biochem.J.73:119;Edelman et al.,Methods in Enzymology 1:422(Acad emic Press 1967); and Andrews and Titus, J.A. .in Current Protocols in Immunology(Coli John Wiley & Sons, 2003 ),pages 2.8.1-2.8.10 and 2.10A.1-2.10A.5 The heavy chains are prepared to form monovalent heavy and light chain fragments (Fd). Other methods for cleaving antibodies, such as by cleaving the fragments, or other enzymatic methods , chemical, or genetic techniques can also be used to generate fragments that are recognized by intact antibodies. Any antibody may be used as long as it binds to the antigen.

[0117] Another form of antibody fragment contains one or more complementarity-determining regions (CDRs) of an antibody. CDRs are peptides containing the CDRs. Such polynucleotides can be obtained by, for example, cloning antibody-producing cells as a template. The variable region was synthesized using the polymerase chain reaction (PCR) with mRNA from the cells. can be prepared (e.g., Larrick et al., 1991, Methods: A Companion to Methods in Enzymology 2:106 ;Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies”,in Monoclonal An tibodies:Production,Engineering and Clin ical Application, Ritter et al. (eds.), pag e 166(Cambridge University Press 1995);W ard et al., “Genetic Manipulation and Exp. Antibodies”,in Monoclonal Ant ibodies:Principles and Applications,Birc h et al., (eds.), page 137 (Wiley-Liss, Inc. 1995). Antibody fragments comprise at least one amino acid sequence of an antibody described herein. Thus, for example, a V region domain may comprise a monomer and V H or V L domains, which may be independently at least 1×1 as described below. 0 -7 It can bind to the endothelin receptor with an affinity of less than or equal to M.

[0118] The variable region domain may be any naturally occurring variable domain or a genetically engineered form thereof. Genetically engineered forms are those produced by recombinant DNA techniques. Such genetically engineered forms include, for example, specific Insertions, deletions, or changes in or to the amino acid sequence of the antibody A specific example is one that is generated from a single CDR from an antibody. and optionally one or more framework amino acids. The antibody comprises a selected variable region domain and the remainder of the variable region domain from another antibody.

[0119] The variable region domains are those that, at the C-terminal amino acid, have at least one other antibody domain or Thus, for example, a fragment of a nucleotide sequence present in a variable region domain may be covalently linked to the fragment. Existing V H The domain is immunoglobulin C H1 linked to the domain or its fragment Similarly, V L The domain is C K It can be linked to a domain or a fragment thereof. For example, the antibody can be a Fab fragment, in which the antigen-binding domain is , and at their C-termini, C H1 and a combination covalently linked to the Cκ domain Matched V H and V L Contains domains. C H1 The domain is extended with additional amino acids For example, the hinge region or hinge region domain as found in the Fab' fragment antibody C H2 and C H3 domains, and more domains. You can.

[0120] Antibody Derivatives and Variants The nucleotide sequences L1 and H1 encoding the amino acid sequence A-1 are, for example, random Mutagenesis or site-directed mutagenesis (e.g., oligonucleotide-directed mutagenesis) site-directed mutagenesis) compared to the non-mutated polynucleotide , modified polynucleotides containing one or more specific nucleotide substitutions, deletions, or insertions Examples of techniques for making such modifications can be found in Walder et al., 1986, Gene 42:133; Bauer et al., 1985, Gene 37:73;Craik,1985,BioTechniques,3:12-19;S mith et al.,1981,Genetic Engineering:Pri Principles and Methods, Plenum Press; and U.S. Patent Nos. 4,518,584 and 4,737,462. These and other methods are well-suited to providing antibodies with desirable properties, such as endogenous cleavage, compared to underivatized antibodies. Increased affinity, avidity, or specificity for the doserine receptor , having improved in vivo or in vitro activity or stability, or reduced in vivo side effects , for example, can be used to generate derivatives of anti-endothelin receptor antibodies.

[0121] Other derivatives of anti-endothelin receptor antibodies in the art are directed against other proteins or together with the polypeptide, for example, the N-terminus or C-terminus of the anti-endothelin receptor antibody polypeptide. Anti-enzyme expression is achieved by expression of a recombinant fusion protein containing a heterologous polypeptide fused to its terminus. and covalent or aggregative conjugates of endosin receptor antibodies or fragments thereof. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide, e.g., an enzyme The fusion protein may be the parent α-factor leader peptide or an epitope tag peptide. Containing antibodies may contain a poly-His group added to facilitate purification or identification of the antibody (e.g., poly-His). Antibodies may also contain peptides that are capable of binding to the nucleotides of the nucleotides described by Hopp et al., 1988, Bio / T Technology 6:1204, and U.S. Pat. No. 5,011,912 The FLAG peptide can be linked to a FLAG peptide, such that the FLAG peptide is highly antigenic. It provides an epitope that is reversibly bound by a specific monoclonal antibody (mAb). This allows for rapid assay and easy purification of the expressed recombinant protein. A method useful for preparing a fusion protein in which the LAG peptide is fused to a given polypeptide. The reagents are commercially available (Sigma-Aldrich, St. Louis, MO). In embodiments, the oligomer containing one or more antibodies is an endothelin receptor antagonist. The oligomers may be used as antibiotics or higher oligomers. in the form of interdigitated or non-covalently linked dimers, trimers, or higher oligomers. The use of oligomers containing two or more antibodies is contemplated, an example being homodimers. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterodimers, and heterodimers. Examples include heterotetramers.

[0122] One embodiment is directed to the antibody binding via covalent or non-covalent interactions between peptide moieties fused to the antibody. Such peptides are also directed to oligomers comprising multiple antibodies linked together. It may be a linker (spacer) or a peptide with properties that promote oligomerization. Certain polypeptides derived from leucine zippers and antibodies are described in more detail below. As described above, it is a peptide that can promote antibody oligomerization.

[0123] In certain embodiments, the oligomer comprises two to four antibodies. , in any of the forms described above, e.g., mutants or fragments, Preferably, the oligomer comprises an antibody having an endothelin receptor.

[0124] In one embodiment, the oligomer is prepared using polypeptides derived from immunoglobulins. The antibodies are prepared by fusion with various portions of antibody-derived polypeptides (including the Fc domain). The preparation of fusion proteins containing certain heterologous polypeptides is possible, for example, by the use of Ashkenazi e t al.,1991,PNAS USA 88:10535;Byrn et al. , 1990, Nature 344:677; and Hollenbaugh et al. .,Construction of Immunoglobulin Fusion Proteins,Current Protocols in Immunology ,Suppl.4, pages 10.19.1-10.19.11. One embodiment herein is directed to an endothelin-binding fragment of an anti-endothelin receptor antibody. The present invention includes two fusion proteins produced by fusing a fusion protein (Fc) to the Fc region of an antibody. Regarding dimers, dimers can be prepared by the following methods: for example, by coating a fusion protein The gene fusion to be transduced is inserted into an appropriate expression vector and the recombinant expression vector is used to express the gene. The transformed fusion gene is expressed, and antibody molecules similar to the expressed fusion protein are synthesized. The Fc fragments can be generated by allowing the fragments to stand, immediately followed by the formation of interchain disulfide bonds between the Fc portions. , forming a dimer.

[0125] As used herein, the term "Fc polypeptide" refers to the Fc region of an antibody. This includes polypeptides in the form of native proteins and muteins derived from the Also included are truncated forms of such polypeptides containing the hinge region that facilitates Fc binding. The fusion protein containing the fragment (and oligomers formed therefrom) is a protein A or protein B complex. It offers the advantage of easy purification by affinity chromatography on a protein G column. Provide.

[0126] PCT Application WO 93 / 10151 (incorporated herein by reference) Suitable Fc polypeptides described in the literature are derived from the N-terminal hinge region of a human IgG1 antibody. Another useful Fc polypeptide is a single-chain polypeptide extending from the C-terminus of the Fc region to the native C-terminus of the Fc region. The tides are described in U.S. Pat. No. 5,457,035 and Baum et al., 1994 , EMBO J. 13:3992-4001. The amino acid sequence of this mutant protein is such that amino acid 19 is changed from Leu to Ala. The amino acid 20 is changed from Leu to Glu, and the amino acid 22 is changed from Gly to The amino acid sequence of the amino acid sequence of WO 93 / 10151 is the same as that of the amino acid sequence of WO 93 / 10151 except that the amino acid sequence of ... The muteins are identical to those of the native Fc sequences shown. In another embodiment, the heavy chain and / or heavy chain of the anti-endothelin receptor antibody exhibits reduced affinity. Or the light chain may be replaced with the variable portion of the heavy and / or light chain.

[0127] Alternatively, the oligomer may be a nucleotide with or without a peptide linker (spacer peptide). A fusion protein containing multiple antibodies. Suitable peptide linkers include those described in U.S. Pat. Nos. 4,751,180 and 4,935,233. There is.

[0128] Another method for preparing oligomeric antibodies involves the use of leucine zippers. 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 240:175 9), and has since been found in a variety of different proteins. Among these are natural peptides or their derivatives that can dimerize or trimerize. Examples of leucine zipper domains suitable for producing mer proteins are described in PCT application WO 02 / 04999. Pulmonary surfactant protein D (SPD) is described in Publication No. 94 / 10308. ) is a leucine zipper derived from Hoppe et al., 1994, FEBS L Etc. 344:191, which is incorporated herein by reference. Use of a modified leucine zipper that allows stable trimerization of heterologous proteins fused to it However, Fanslow et al., 1994, Semin. Immunol. 6:26 In one method, an anti-enantiomer fused to a leucine zipper peptide is used. Recombinant fusion proteins containing antibody fragments or derivatives thereof are preferred. and a soluble oligomeric anti-endothelin receptor antibody fragment expressed in a suitable host cell. or a derivative thereof is recovered from the culture supernatant.

[0129] In another embodiment, the antibody derivative comprises at least one of the CDRs disclosed herein. For example, one or more CDRs may comprise a known antibody framework region (e.g., IgG1, IgG2, etc.) or a suitable vehicle to increase its half-life. Suitable vehicles include, but are not limited to, Fc, albumin, and thiamin. These and other suitable vehicles are known in the art. Such CDR-conjugated peptides may be in the form of a monomer, a dimer, a tetramer, or In one embodiment, the one or more water-soluble polymers may be one or The binding agent is attached at multiple specific positions, for example, at the amino terminus of the binding agent. Conductors include, but are not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. For example, U.S. Patent No. 4,640,835, U.S. Patent No. 4,496,689, U.S. Patent No. 4,301,144, U.S. Patent No. 4,670,417, U.S. See U.S. Patent Nos. 4,791,192 and 4,179,337 In some embodiments, the derivative is monomethoxy-polyethyleneglycol. cellulose, dextran, cellulose, or other carbohydrate-based polymers, poly(N-vinyl alcohol), hydroxypyrrolidone)-polyethylene glycol, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, and mixtures of such polymers In certain embodiments, the one or more water-soluble polymers include one or more In one embodiment, PEG is attached randomly to multiple side chains. These methods may serve to improve the therapeutic efficacy of the drug combination. No. 6,133,426, which is incorporated herein by reference for all purposes. It's listed in the fine print.

[0130] As long as the antibody retains its binding specificity, the antibodies described herein may be used in combination with at least one antibody. It will be understood that modifications to the antibody structure may have amino acid substitutions. These modifications are within the scope of the invention and are intended to improve the ability of the antibody to bind to the endothelin receptor. Conservative amino acid substitutions may be included which do not disrupt the sequence and may be conservative or non-conservative. The substitutions are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These amino acid residues may include natural amino acid residues. Conservative amino acid substitutions also include substitutions of amino acid residues at that position. The natural acid residues are introduced by non-natural residues so that there is little or no effect on the polarity or charge of the acid residues. Non-conservative substitutions may involve substituting a natural amino acid residue for an amino acid or amino acid analog. Members of a class of organisms can be differentiated by different physical properties (e.g., size, polarity, hydrophobicity, and This may involve replacing a member of a class with a member from another class that has a different charge.

[0131] Furthermore, one skilled in the art can easily create tested sequences containing one amino acid substitution at each desired amino acid residue. Mutants can be generated that are capable of inhibiting the activity of the polypeptides. Mutants can be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about preferred variants. For example, changes to specific amino acid residues may result in disruption or undesirable degradation. or otherwise found to result in inappropriate activity, variants containing such changes are , can be avoided. In other words, based on the information gathered from such routine experiments, Those skilled in the art will recognize that further substitutions should be avoided, either alone or in combination with other mutations. Certain amino acids can be easily determined.

[0132] Those skilled in the art will be able to identify suitable variants of the polypeptides described herein using well-known techniques. In some embodiments, one skilled in the art can determine the amount of ATP that is not important for activity. By targeting specific regions, one can identify suitable areas of the molecule that can be altered without destroying activity. In some embodiments, residues or conserved molecules among similar polypeptides may be identified. In some embodiments, the portion of the Important regions may be modified without destroying biological activity or adversely affecting polypeptide structure. Furthermore, those skilled in the art can easily make conservative amino acid substitutions without any need for modification of the amino acid sequence, without regard to whether the amino acid sequence is important for activity or structure. Structure-function studies that identify residues in similar polypeptides can be considered. In light of such comparisons, the presence of nucleotides that are important for activity or structure in similar proteins is important. Predicting the importance of a particular amino acid residue in a protein Those skilled in the art can substitute chemically similar amino acid residues for such predicted important amino acid residues. The amino acid substitutions can be selected.

[0133] Those skilled in the art will recognize the three-dimensional structure and amino acid sequence associated with that structure in similar polypeptides. In view of such information, one skilled in the art can determine its three-dimensional structure. In some embodiments, the alignment of amino acid residues of an antibody with respect to Therefore, one skilled in the art can make radical changes to amino acid residues predicted to be on the surface of the protein. It may be possible to choose not to have such residues present in the nucleotide sequence, since such residues may not be involved in important interactions with other molecules. Several scientific publications have been devoted to the prediction of secondary structure. . Moult,1996,Curr.Op.Biotech.7:422-427;Ch ou et al.,1974,Biochemistry 13:222-245;C hou et al., 1974, Biochemistry 113:211-222 ;Chou et al.,1978,Adv.Enzymol.Relat.Area s Mol.Biol.47:45-148;Chou et al.,1979,An n. Rev. Biochem. 47:251-276 and Chou et al., Bi See Ophys. J. 26:367-384. Computer programs are now available to assist in this. Two polypeptides or proteins that share identity or a similarity of greater than 40% are considered to have a high level of similarity. The recent growth of the Protein Data Bank (PDB) has Improved secondary structure, including the potential number of folds within the structure of a polypeptide or protein. Provided predictability. Holm et al., 1999, Nucl. Acid. Re See s.27:244-247. There are a critical number of folds, and once a critical number of structures have been determined, structure prediction becomes remarkably accurate. It has been suggested that this may be the case (Brenner et al., 1997, Curr. O p. Struct. Biol. 7:369-376). Further methods for predicting secondary structure. For example, "threading" (Jones, 1997, Cur r. Opin. Struct. Biol. 7:377-87; and Sippl et al. l., 1996, Structure 4:15-19); "Profile Analysis" (Bo wie et al.,1991,Science 253:164-170;Grib skov et al.,1990,Meth.Enzym.183:146-159; and Gribskov et al., 1987, PNAS USA 84:4355- 4358, and "evolutionary linkage" (H see Olm, supra (1999), and Brenner, supra (1997). In certain embodiments, antibody variants include glycosylation variants, where glycosylation The number and / or type of cosylation sites are altered compared to the amino acid sequence of the parent polypeptide. In one embodiment, the variant may have more or fewer Ns than the native protein. -linked glycosylation sites. Alternatively, removal of such sequences by substitution can be achieved by removing the existing Removal of N-linked carbohydrate chains. Rearrangement of N-linked carbohydrate chains is also provided, where one or multiple N-linked carbohydrate chain sites (typically those occurring in nature) have been deleted, resulting in one or multiple new N-linked sites are generated. Additional preferred antibody mutants include cysteine-binding domains. and amino acid variants, wherein one or more cysteine ​​residues are , deleted or substituted with another amino acid (e.g., serine). The antibody is refolded (e.g., after isolation of soluble inclusion bodies) into a biologically active conformation. Cysteine ​​variants can be useful when the protein must be folded. Proteins have fewer cysteine ​​residues, and interactions typically arise from unpaired cysteines. It has an even number of cysteines to minimize

[0134] Desired amino acid substitutions (whether conservative or non-conservative) are made in the presence of a nucleotide sequence in which such substitutions are desired. This can be determined by one of skill in the art as desired. In certain embodiments, amino acid substitutions are To identify important residues of a human endothelin receptor antibody or to identify the human endothelin receptor These antibodies can be used to increase or decrease the affinity of antibodies to endothelin receptors. According to an embodiment, preferred amino acid substitutions are those that (1) reduce susceptibility to proteolysis. (2) reduce susceptibility to oxidation; and (3) form protein complexes. (4) modifying the binding affinity of such polypeptides; and / or These compounds provide or modify other physicochemical or functional properties of the peptide. According to embodiments, single or multiple amino acid substitutions (in some embodiments, conservative amino acid substitutions) are The substitutions may be made within the native sequence (in some embodiments, within the domains that form intermolecular contacts). In one embodiment, the conservative amino acid sequence is Substitutions typically cannot substantially alter the structural characteristics of the parent sequence (e.g., The amino acid substitution should not disrupt the helix present in the parent sequence or otherwise characterize the parent sequence. (It should not interfere with other types of secondary structure present in the protein.) Examples of secondary and tertiary structures of polypeptides are given in Proteins, Structures and Molecular Principles,Creighton, ed. WHFreeman and Company,(1984);Introduct ion to Protein Structure, Branden and Too ze, eds., Garl and Publishing, (1991); and Tho rnton et al., 1991, Nature 354:105, each of which is incorporated herein by reference.

[0135] In certain embodiments, the antibodies described herein may be linked to a polymer, lipid, or other moiety. The antigen-binding agent can be chemically bonded to the present invention, which is incorporated into a biocompatible framework structure. It may contain at least one of the CDRs described herein. Framework structure is the distribution of antigens in localized surface regions (e.g., CDRs, variable regions, etc.). a conformationally stable molecule capable of presenting one or more amino acid sequences that bind to Polypeptides or polypeptides sufficient to form a structural support, or framework, or scaffold. Such structures may be derived from natural polypeptides or polypeptide "folds" (structures). The amino acid sequence may be a structural motif (a structural motif) or a sequence of amino acids compared to the native polypeptide or fold. These scaffolds may have one or more modifications such as additions, deletions or substitutions. of any species (or more than one species), such as mammals, other vertebrates, invertebrates, bacteria, or viruses Typically, the biocompatible framework structure is derived from an immunomodulatory polypeptide. They are based on protein scaffolds or frameworks other than globulin domains. Nectin, ankyrin, lipocalin, neocarzinostatin, cytochrome b, CP1 Finger protein, PST1, coiled-coil protein, LACI-D1, Z-do Those protein scaffolds based on the main and tendamistat domains can be used ( For example, Nygren and Uhlen, 1997, Current Opinio n in Structural Biology 7:463-469).

[0136] Furthermore, those skilled in the art will appreciate that suitable binding agents may be selected from the heavy chain CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR19, CDR11, CDR12, CDR13, C DR2, and CDR3, and one or more of light chain CDR1, CDR2, and CDR3 It will be appreciated that the term "antibody" includes portions of these antibodies, such as heavy chain CDR1, CDR2, At least one of the light chain CDR1, CDR2 and CDR3 regions is unsubstituted in the antibody. As long as the binding specificity of the CDR is maintained, at least one amino acid substitution may be made. The non-CDR portion can be a non-protein molecule, where the binding agent is a non-CDR molecule of the present invention for human ETA. cross-blocking the binding of the antibodies disclosed herein and / or receptor-mediated endothelial cell proliferation. The non-CDR portion of the antibody can be a non-protein molecule. The antibody binds to the human ETA peptide in a competitive binding assay. and / or exhibit a binding type similar to that of at least one of the endothelins The non-CDR portions of the antibody may be composed of amino acids, where the antibody is a recombinant recombinant. The recombinant binding protein is a recombinant protein or synthetic peptide, and the ... cross-blocking the binding of the antibodies disclosed herein and / or in vitro or in vivo Neutralizes endothelin activity. The non-CDR portion of the antibody may be composed of amino acids, The antibody is a recombinant antibody, and the recombinant antibody binds to human ET in a competitive binding assay. exhibiting a binding type similar to that of at least one of antibodies A-1 / A-2 to the A peptide, and / or neutralize endothelin signaling.

[0137] nucleic acid In one aspect, provided herein is an isolated nucleic acid molecule. The nucleic acid molecule can be, for example, , all or a portion of an antibody, e.g., an antibody described herein, or a fragment thereof, a derivative Polynucleotides encoding one or both strands of the derivative, mutein, or variant a polynucleotide sufficient for use as a hybridization probe; Identifying, analyzing, mutating, or amplifying polynucleotides encoding polypeptides PCR primers or sequencing primers for detecting the expression of polynucleotides The nucleic acid may be of any length and may be an antisense nucleic acid or a complementary sequence thereof. The nucleic acid may be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 5 0, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400 , 450, 500, 750, 1000, 1500, 3000, 5000 or more The nucleic acid sequence may be of any nucleotide length and / or may contain one or more additional sequences, e.g., regulatory sequences. The nucleic acid may be single-stranded and / or may be part of a larger nucleic acid, e.g., a vector. or double-stranded, and may contain RNA and / or DNA nucleotides, and artificial variants thereof (e.g. For example, peptide nucleic acids.

[0138] Encoding an antibody polypeptide (e.g., heavy or light chain, variable domain only, or full length) Nucleic acids corresponding to the ETA antigen can be isolated from B cells of mice immunized with the ETA antigen. The antibodies may be isolated by conventional methods such as polymerase chain reaction (PCR).

[0139] Nucleic acid sequences encoding the heavy and light chain variable regions are shown above. Those skilled in the art will appreciate that the genetic code Due to degeneracy, each of the polypeptide sequences disclosed herein can be synthesized from a large number of other nucleic acid sequences. It will be understood that the antibodies described herein may be encoded by Each degenerate nucleotide sequence is provided herein.

[0140] Under certain hybridization conditions, other nucleic acids (e.g., either A-1 / A-2) Further provided herein are nucleic acids that hybridize to the nucleic acid comprising the nucleotide sequence of Methods for hybridizing nucleic acids are well known in the art. For example, Current Protocols in Molecular Biology gy, John Wiley & Sons, NY (1989), 6.3.1-6. See 3.6. As defined herein, for example, moderately stringent Gentle hybridization conditions were 5x sodium chloride / sodium citrate ( SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), approximately 50% formaldehyde Prewash containing amide hybridization buffer, 6x SSC ng) solution, 55°C hybridization temperature (or 42°C hybridization temperature) Other similar hybridization methods, such as solutions containing approximately 50% formamide at room temperature, ion solution), and elution conditions of 60°C in 0.5x SSC and 0.1% SDS are used. Stringent hybridization conditions were hybridization in 6x SSC at 45°C. The cells are soybean-soaked and subsequently washed one or more times with 0.1×SSC and 0.2% SDS at 68°C. Furthermore, those skilled in the art have at least 65%, 70%, 75%, 80%, 85%, 90% Nucleic acids containing nucleotide sequences that are 95%, 98% or 99% similar to each other are typically The stringency of hybridization should be adjusted so that the target protein remains hybridized to the target protein. Manipulating hybridization and / or washing conditions to increase or decrease The basic parameters that influence the choice of hybridization conditions and Guidance for devising suitable conditions can be found, for example, in Sambrook, Fritsch and d Maniatis 1989,Molecular Cloning: A Labo ratory Manual,Cold Spring Harbor Laborat ory Press, chapters 9 and 11; and Current Pr otocols in Molecular Biology,1995,Ausube l et al.,Eds.,John Wiley & Sons,Inc.,sec. Sections 2.10 and 6.3-6.4 are described, for example, in the length of DNA. The mutation can be readily determined by one skilled in the art based on the length and / or base composition. can be introduced into the nucleic acid by mutation, thereby allowing the polypeptide it encodes (e.g. Mutations result in changes in the amino acid sequence of the target protein (antigen binding protein). In one embodiment, one or more specific Certain amino acid residues are changed using, for example, site-directed mutagenesis protocols. In another embodiment, one or more randomly selected residues are, e.g., randomly However it is created, the mutant polypeptide is altered using a mutagenesis protocol. The polypeptides can be expressed and screened for desired properties.

[0141] A mutation may occur in the nucleus without significantly altering the biological activity of the polypeptide it encodes. For example, nucleotides that result in amino acid substitutions at non-essential amino acid residues can be introduced into the In one embodiment, L-1 to L-2 and H-1 to H-2 are The nucleotide sequences provided herein, or fragments, variants, or variants thereof, The amino acid sequences encoded by the amino acid sequences L-1 to L-2 shown in the present specification are also suitable. and has two or more different residues compared to those encoded by H-1 to H-2. The sequence is modified to include one or more deletions or substitutions of amino acid residues to produce a sequence In another embodiment, mutagenesis is performed on a sequence having two or more different residues. One or more of L-1 to L-2 and H-1 to H-2 shown herein Alternatively, one or more mutations may be inserted into the nucleic acid. The polypeptides that they encode can be introduced into the host cell to enhance their biological activity (e.g., binding to ETA). For example, mutations can selectively alter biological activity quantitatively or qualitatively. Examples of quantitative changes include increasing, decreasing, or eliminating activity. An example of a qualitative change includes changing the antigen specificity of an antibody.

[0142] In another embodiment, a primer or hybrid for the detection of a nucleic acid sequence herein Nucleic acid molecules suitable for use as dimerization probes are provided herein. The nucleic acid molecules herein are those encoding the full-length polypeptides herein. Only a portion of a nucleic acid sequence, for example, a fragment that can be used as a probe or primer or an active portion (e.g., ETA-binding portion) of the polypeptide herein. The nucleic acid sequences may be used to detect nucleic acids. Detecting nucleic acids or similar nucleic acids, e.g., transcripts encoding the polypeptides herein. The probe may contain a label group, such as a radioisotope, a fluorescent compound, an enzyme, or Such probes can be used to identify cells expressing the polypeptide. can be identified.

[0143] In another embodiment, a nucleic acid encoding a polypeptide or a portion thereof as described herein is provided. Provided herein are vectors containing the vectors. Examples of vectors include, but are not limited to, , plasmids, viral vectors, non-episomal mammalian vectors and expression vectors, e.g. For example, a recombinant expression vector is exemplified. It may include the nucleic acids herein in a form suitable for expression of the nucleic acid in a host cell. An expression vector is a host cell used for expression that is operably linked to a nucleic acid sequence to be previously expressed. The control sequence may include one or more regulatory sequences that are screened based on the host cell. Those that direct the constitutive expression of nucleotide sequences in many types of host cells (e.g., SV40 early gene enhancer, Rous sarcoma virus promoter and cytomegalovirus rus promoter), which directs expression of a nucleotide sequence only in a specific host cell (See, e.g., tissue-specific regulatory sequences, Voss et al., 1986, Trends B iochem.Sci.11:287, and Maniatis et al., 1987 , Science 236:1237, the disclosures of each of which are incorporated herein by reference in their entirety. (incorporated herein by reference), and in response to specific treatments or conditions, the nucleotide sequence those that direct inducible expression of genes (e.g., metallothionein promoters in mammalian cells) and tet-responsive and / or serotype-specific phenotypes in both prokaryotic and eukaryotic cell systems. The design of the expression vector includes a streptomycin-responsive promoter (see above). The choice of host cell to be transformed and the level of expression of the desired protein may depend on such factors as: It should be understood by those skilled in the art that the expression vectors herein are fusion proteins encoded by the nucleic acids described herein. Proteins or peptides may be produced, including proteins or peptides.

[0144] In another aspect, provided herein is a host cell into which the expression vector can be introduced. The cell can be any prokaryotic or eukaryotic cell. Prokaryotic host cells are Gram-negative. or Gram-positive bacteria, such as E. coli or Bacillus sp. 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 and their derivatives such as Veggie CHO and related cells grown in serum-free medium. system (Rasmussen et al., 1998, Cytotechnology 2 8:31) or the DHFR-deficient CHO strain DXB-11 (Urlaub et al. al., 1980, PNAS USA 77:4216-20). CHO cell lines are CHO-K1(ATCC#CCL-61), EM9(ATCC# CR L-1861), and UV20 (ATCC# CRL-1862). The cells were the monkey kidney COS-7 cell line (ATCC# CRL-1651) (Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCCCCL-163), AM-1 / D cells (US Patent No. 6,210,9 24), HeLa cells, BHK (ATCC CRL-10) cell line, The CV1 / EBNA cell line (ATCC 94444) derived from the African green monkey kidney cell line CV1 L-70)(McMahan et al.,1991,EMBO J.10:2821 ), human embryonic kidney cells such as 293, 293EBNA, or MSR293, human epidermal A 431 cells, human C010220 cells, other transformed primate cell lines, normal diploid Cell lines derived from in vitro culture of cells, primary tissues, and primary explants, including HL-60 and U937 , HaK, or Jurkat cells. Suitable cloning and expression vectors for use in the present invention are described in Pouwels et al. ing Vectors:A Laboratory Manual,Elsevier , 1985).

[0145] Vector DNA can be introduced into prokaryotes by conventional transformation or transfection techniques. or eukaryotic cells. For stable transfection of mammalian cells Depending on the expression vector and transfection technique used, a small percentage of cells Only these components are known to be capable of integrating foreign DNA into their genome. Selection (e.g., for resistance to antibiotics) to identify and screen for A gene encoding a pharmacological marker is generally introduced into the host cell along with the gene of interest. Preferred selectable markers are G418, hygromycin and methotrexate. These include those that confer resistance to drugs such as steroids. Integrating cells can be identified by drug screening, among other methods ( For example, cells that have integrated a selectable gene will survive, while other cells will die. (This is the case.)

[0146] The transformed cells may be cultured under conditions that promote expression of the polypeptide, The peptide can be recovered by conventional protein purification methods. One such purification method is The polypeptides contemplated for use herein are described in the Examples below. The antibody is a substantially homogeneous recombinant mammalian anti-endonuclease antibody that is substantially free of contaminating endogenous substances. Includes serine receptor antibody polypeptides.

[0147] Antibody activity In one embodiment, the antibodies described herein specifically bind to an endothelin receptor. and inhibit signal transduction, resulting in therapeutic biological effects, e.g., pulmonary artery disease in animal models. In another embodiment, the antibodies described herein are human endogenous antibodies. The antibody is a mouse antibody or a humanized antibody that can specifically bind to the doserin receptor. These include antagonistic or neutralizing antibodies that can reduce or neutralize endothelin signaling.

[0148] In one embodiment, the antibodies described herein bind to the human endothelin receptor ETA. When binding, approximately 0.01nM to 1000nM, 0.1nM to 500nM, 0.5nM K of 200 nM, 1 nM to 200 nM, or 10 nM to 100 nM d It has a different real value. In embodiments, the antibodies described herein bind to the human endothelin receptor ETA. When d In another embodiment, the The antibodies described herein have a binding affinity of approximately 10 nM to 100 nM to the human endothelin receptor ETA. K of M d In another embodiment, the antibodies described herein have a human endogenous When binding to serine receptor ETA, approximately 1 nM, 2 nM, 5 nM, 10 nM, 20 nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, or 100 nM K d It has a value.

[0149] In one embodiment, the antibodies described herein inhibit human endothelin signaling. When reducing, approximately 0.01nM to 500nM, 0.1nM to 200nM, 0.5nM to 2 IC of 00nM, 1nM to 200nM, or 10nM to 100nM 50 It has value. In embodiments, the antibodies described herein reduce human endothelin signaling. When used in combination with HCl, the IC value is approximately 1nM to 200nM. 50 In another embodiment, the present invention The antibodies described herein reduce human endothelin signaling with a potency of approximately 10 nM to 1 IC of 00nM 50 In another embodiment, the antibodies described herein have a value of Attenuating human endothelin signaling was observed at approximately 1 nM, 2 nM, 5 nM, 10 nM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, or 100 nM IC 50 It has a value.

[0150] In one embodiment, the ETA antibody has one or more of the following properties: a. Binds to the human endothelin receptor ETA as well as or better than the reference antibody Good K d to provide; b. In inhibiting the activation of the human endothelin receptor ETA by endothelin, see IC as good as or better than antibody 50 to provide; and c. Cross-compete for binding with a reference antibody to the human endothelin receptor ETA.

[0151] In another embodiment, the ETA antibodies described herein exhibit one or more of the following properties: The antibodies have the following numbers: a. The same or similar binding to the reference ETA antibody in binding to the human endothelin receptor ETA Better K d to provide; b. In inhibiting the activation of the human endothelin receptor ETA by endothelin, see IC as good as or better than ETA antibody 50 to provide; and c. Cross-compete for binding with a reference ETA antibody to the human endothelin receptor ETA and.

[0152] In one embodiment, the reference antibody comprises the light chain variable domain amino acid sequence SEQ ID NO: 138 and the heavy chain variable domain amino acid sequence SEQ ID NO: 139. In another embodiment, the amino acid sequence of the variable chain domain is SEQ ID NO: 166. The antibody is monoclonal antibody A-1, A-2, A-7, A-9, or A-12. As used herein, the term "substantially similar" refers to the IC of an antibody described herein. 50 or K d The values ​​are equivalent to those of the reference antibody or are about 200%, 18 0%, 160%, 150%, 140%, 120%, 110%, 100%, 99%, 98% , 97%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, or 50% In one embodiment, the reference antibody is, for example, a heavy and light chain combination L1 In another embodiment, the reference antibody is an ETA antibody. In one embodiment, the ETA antibodies described herein are directed against human endothelial cells. It can specifically bind to phospho-receptors and reduce pulmonary arterial hypertension in animal models. In one embodiment, pulmonary arterial hypertension is reduced by 2% compared to untreated animals. In another embodiment, pulmonary arterial hypertension is reduced by about 5% compared to untreated animals. In another embodiment, pulmonary arterial hypertension is reduced by about 10% compared to untreated animals. In another embodiment, pulmonary arterial hypertension is reduced by about 15% compared to untreated animals. In another embodiment, pulmonary arterial hypertension is reduced by about 20% compared to untreated animals. In the treated animals, pulmonary arterial hypertension is reduced by approximately 25% compared to untreated animals. The amount of symptom relief is controlled by the dose. The dose is that which reduces pulmonary arterial hypertension to the normal range.

[0153] TGF-β Trap In one embodiment, the TGF-β Trap described herein inhibits the function of TGF-β. In another embodiment, the TGF-α protein described herein is a protein capable of inhibiting the function of the TGF-α protein. -β Trap comprises the amino acid sequence: SEQ ID NO: 207. The amino acid sequence of TGF-β Trap described in the specification is SEQ ID NO:207.

[0154] Peptide Linker (Linker) In one embodiment, the peptide linker (linker) described herein is: each independently selected from one of SEQ ID NO: 208, SEQ ID NO: 209, and SEQ ID NO: 210; The amino acid sequence includes an amino acid sequence selected from the group consisting of:

[0155] In one embodiment, the amino acid sequence of the peptide linker described herein is In another embodiment, the amino acid sequence of the peptide linker described herein is The amino acid sequence is SEQ ID NO: 209. In another embodiment, the peptides described herein The amino acid sequence of the peptide linker is SEQ ID NO:210.

[0156] Fusion protein of ETA antibody and TGF-β Trap In one embodiment, a combination of an ETA antibody and a TGF-β Trap as provided herein The fusion protein of the present invention comprises an ETA antibody and a TGF-β Trap fragment described herein. Includes segments.

[0157] In another embodiment, the ETA antibody and TGF-β Tr as provided herein are The fusion protein of ap can be used with the ETA antibody, 1, 2, 3, 4, 5, 6, Contains 7 or 8 TGF-β Traps and the same number of peptide linkers (linkers); The conjugated protein is composed of the amino terminus of TGF-β Trap and the light or heavy chain of the ETA antibody. The carboxyl terminus is linked via a peptide linker sequence, or the fusion protein is The carboxyl terminus of F-β Trap is paired with the amino terminus of the light or heavy chain of the ETA antibody. They are linked via a peptide linker sequence.

[0158] In another embodiment, the ETA antibody and TGF-β Tr as provided herein are The fusion protein of ap can comprise an ETA antibody as described herein, one, two, three or four TGs. The fusion protein contains F-β Trap and an equal number of peptide linkers (linkers); The amino terminus of TGF-β Trap is connected to the carboxyl terminus of the light or heavy chain of the ETA antibody. , linked via a peptide linker sequence, or the fusion protein is TGF-β Trap The carboxyl terminus of the ETA antibody is connected to the amino terminus of the light chain or heavy chain of the ETA antibody via a peptide linker sequence. Linking via columns.

[0159] In another embodiment, the ETA antibody and TGF-β Tr as provided herein are The fusion protein of ap comprises an ETA antibody described herein, one or two TGF-β The fusion protein contains TGF-Trap and an equal number of peptide linkers (linkers); The amino terminus of β Trap is linked to the carboxyl terminus of the light or heavy chain of the ETA antibody. The fusion protein is linked via a linker sequence or a carbohydrate of TGF-β Trap. The xyl terminus is connected to the amino terminus of the light or heavy chain of the ETA antibody via a peptide linker sequence. and connect them.

[0160] In another embodiment, the ETA antibody and TGF-β Tr as provided herein are The fusion protein of ap contains the ETA antibody described herein, two TGF-β Trap p and two peptide linkers (linkers); the fusion protein The amino terminus of rap is connected to the carboxyl terminus of the light or heavy chain of the ETA antibody and the peptide phosphorylase. The fusion protein is linked via the Car sequence to the carboxyl group of TGF-β Trap. The end is connected to the amino terminus of the light or heavy chain of the ETA antibody via a peptide linker sequence. do.

[0161] In one embodiment, a combination of an ETA antibody and a TGF-β Trap as provided herein The fusion protein of p is composed of the amino terminus of TGF-β Trap and the light or heavy chain of the ETA antibody. The carboxyl terminus of the chain is connected via a peptide linker sequence. The fusion protein of the ETA antibody and TGF-β Trap provided herein The amino terminus of TGF-β Trap was linked to the carboxyl terminus of the light chain of the ETA antibody. In another embodiment, the peptide linker sequences provided herein are linked via a peptide linker sequence. The fusion protein of ETA antibody and TGF-β Trap is The amino terminus is connected to the carboxyl terminus of the heavy chain of the ETA antibody via a peptide linker sequence. To connect.

[0162] In one embodiment, a combination of an ETA antibody and a TGF-β Trap as provided herein The fusion protein of p has the amino acid sequences: SEQ ID NO: 162, SEQ ID NO: 190, SEQ ID NO: 20 7, and SEQ ID NO: 210.

[0163] In one embodiment, the fusion protein of ETA antibody and TGF-β Trap is The present invention provides a method for the production of an ETA antibody, a TGF-β Trap, and a peptide linker. - The sequence is as follows: (3) The amino terminus of TGF-β Trap is linked to ETA via a peptide linker sequence. Linked to the carboxyl terminus of the antibody heavy / light chain: N'-R-linker-TGF-β Trap-C'; and (4) The carboxyl terminus of TGF-β Trap is linked to the N'-TGF-β Trap-Lys is linked to the amino terminus of the light or heavy chain of the ETA antibody. Binker-R-C' fused to one of the following to form a fusion protein; where N' represents the amino terminus of the polypeptide chain and C' represents the carboxyl terminus of the polypeptide chain. TGF-β Trap represents the carboxyl terminus of the TGF-β Trap fragment. wherein R is the amino acid sequence of the light chain or heavy chain of the ETA antibody, and the linker is a peptide linker. Represents a car.

[0164] In one embodiment, a combination of an ETA antibody and a TGF-β Trap as provided herein The fusion protein of p comprises the amino acid sequence: SEQ ID NO: 211 or SEQ ID NO: 212. In embodiments, the ETA antibody and TGF-β Trap provided herein The fusion protein comprises the amino acid sequences: SEQ ID NO:211 and SEQ ID NO:212.

[0165] Biological activity of the fusion protein of ETA antibody and TGF-β Trap The biological activity of the fusion protein of ETA antibody and TGF-β Trap was The biological activity of Trap and the biological activity of ETA antibodies are included. , effectively inhibiting the increase in vascular pressure caused by endothelin, and This may reduce the symptoms of rheumatoid arthritis and improve the patient's exercise capacity and hemodynamics. The biological activity of the fusion protein of ETA antibody and TGF-β Trap is Binds to and blocks the cellular stress response induced by pulmonary artery hypertension in vivo. The biological activity of the therapeutic effects on hypertension, pulmonary hypertension and other related symptoms of pulmonary fibrosis. The above-mentioned cellular stress responses include, but are not limited to, pulmonary artery These include a decrease in aortic pressure, a decrease in aortic pressure, and associated changes in cardiovascular and pulmonary vascular remodeling. By combining the biological activities of TGF-β Trap and ETA antibodies, The TGF-β Trap fusion proteins described herein can be used to detect TGF-β and E The fusion protein can treat various conditions and diseases associated with TA. Since it exerts its biological effect by acting on β- and / or ETA, The TGF-β Trap fusion protein described in the document is used to "reduce TGF-β stimulation" " or "reduced ETA stimulation" to treat subjects with conditions and diseases that respond favorably to These subjects may be those who are in need of reduced TGF-β stimulation or those who are in need of ET Subjects who need to reduce A stimulation are called "Pulmonary arterial hypertension, pulmonary hypertension, pulmonary fibrosis" These include vascular disease and other related conditions of cardiovascular fibrosis.

[0166] In one embodiment, the biology of the fusion protein of ETA antibody or TGF-β Trap The alteration of the activity of ETA is achieved by the use of ETA antibodies or TGF-β inhibitors that inhibit ETA and block TGF-β in vitro. To quantify the function of the GF-βTrap fusion protein, calcium flux assays and It is detected by reporter gene detection methods.

[0167] Pharmaceutical Composition In one embodiment, a combination of an ETA antibody and a TGF-β Trap as provided herein and one or more pharmaceutically acceptable carriers, Provided herein.

[0168] In one embodiment, the pharmaceutical compositions described herein are administered intravenously or subcutaneously. can be.

[0169] Treatment method In one embodiment, the present invention provides a method for treating, preventing, or ameliorating pulmonary arterial hypertension and pulmonary arterial hypertension-related disorders. and a combination of an ETA antibody and a TGF-β T cell line as described herein in the preparation of a medicament for improving Uses of rap fusion proteins are provided herein.

[0170] In another embodiment, the method of treating, preventing, or ameliorating pulmonary hypertension and pulmonary hypertension-related disorders is and a TGF-β Trap antibody as described herein in the preparation of a medicament for The use of the fusion protein of is provided herein.

[0171] In another embodiment, to treat, prevent, or ameliorate pulmonary fibrosis and pulmonary fibrosis-related diseases. The fusion of the ETA antibody and TGF-β Trap described herein in the preparation of a medicament for Uses of the fusion proteins are provided herein.

[0172] In another embodiment, the method comprises treating, preventing, or ameliorating cardiovascular fibrosis and cardiovascular fibrosis-related diseases. and a combination of an ETA antibody and a TGF-β Tr as described herein in the preparation of a medicament for the treatment of Uses of fusion proteins of ap are provided herein.

[0173] In a further embodiment, pulmonary arterial hypertension, pulmonary hypertension, pulmonary fibrosis or cardiovascular fibrosis. In the preparation of a medicament for simultaneously treating, preventing, or ameliorating two or more diseases among The use of the fusion protein of ETA antibody and TGF-β Trap described in the present specification Provided in the specification.

[0174] As used herein, the term "subject" refers to a mammal, including a human, and the term "patient" refers to a The term is used synonymously with the term.

[0175] The term "treatment" refers to the alleviation of at least one symptom or other aspect of a disease, or a decrease in the severity of the disease. The combination of ETA antibodies and TGF-β Trap provided herein includes reduction in the severity of the disease. To be an effective therapeutic agent, the fusion protein may provide a complete cure or alleviate all symptoms or symptoms of a disease. As recognized in the relevant art, a drug as a therapeutic agent does not necessarily eradicate the symptoms. Although a drug may be used to reduce the severity of a given condition, it must be effective to treat the disease to be considered an effective therapeutic agent. It is not necessary to eliminate all signs of the disease. Similarly, prophylactic treatments may be effective prophylactically. It does not need to be completely effective in preventing the onset of symptoms in order to be effective. and (e.g., by reducing the number or severity of the symptoms or by achieving another therapeutic effect). or by producing another beneficial effect), or It is sufficient to reduce the likelihood of the condition occurring or worsening in the body. Embodiments include the measurement of sustained improvement above baseline levels of indicators reflecting the severity of a particular disease. The fusion protein of ETA antibody and TGF-β Trap was administered in an amount and for a time sufficient to induce the effect. The present invention relates to a method comprising administering to a patient a compound ...

[0176] Pharmaceutical compositions of the fusion protein of ETA antibody and TGF-β Trap include, but are not limited to, However, they may be administered by any suitable technique, including parenteral, topical, or by inhalation. When injected, the pharmaceutical composition can be administered, for example, intraarticularly, intravenously, intramuscularly, intralesionally, intraperitoneally, or intraperitoneally. By the subcutaneous route, it can be administered as a bolus or continuous infusion. Localized administration at the site of disease or injury, such as sustained release administration of implants Inhalation administration can be achieved by, for example, nasal or oral inhalation, spray, Other alternatives include inhaling antibodies in aerosol form. Examples include oral formulations including lozenges.

[0177] A composition containing one or more other ingredients, such as a physiologically acceptable carrier, excipient, or diluent. The fusion tag of ETA antibody and TGF-β Trap is provided herein in the form of a composition. It is advantageous to administer a protein containing the composition. The composition may optionally contain one or more bioactive compounds as described below. In various specific embodiments, the composition may further comprise a physiologically active agent. The antibody (such as a murine antibody or a humanized antibody) provided herein and one or more of TGF-β. The fusion protein may contain one, two, three, four, five, or six physiologically active agents other than the multiple fusion proteins. nothing.

[0178] In one embodiment, the pharmaceutical composition comprises a murine antibody or a human antibody provided herein. a fusion protein of a fused antibody and TGF-β Trap, and one selected from the following: or a plurality of substances: a substance having a pH suitable for the fusion protein of the antibody and TGF-β Trap Buffers, antioxidants such as ascorbic acid, low molecular weight polypeptides (less than 10 amino acids) Proteins, amino acids, carbohydrates such as dextrin, E Contains complexing agents such as DTA, glutathione, stabilizers and excipients. Therefore, preservatives may also be added. The composition may be frozen using a suitable excipient solution as a diluent. The appropriate ingredients may be formulated into a dry powder. Further examples of ingredients that may be used in pharmaceutical formulations are found in Remington's Pharmaceutical Sciences,16th edition(198 0) and 20th edition (2000). The blishing company provides kits for use by physicians, and the kits The present invention relates to a method for producing a fusion protein comprising administering to a subject an antibody or antibodies provided herein and one or more fusion proteins of an antibody or antibodies provided herein and TGF-β Trap. Proteins, and tags or other instructions for treating any of the diseases described herein. In one embodiment, the kit comprises one or more tube-shaped compositions in the form of the compositions described above. A sterile preparation of one or more fusion proteins of an antibody and TGF-β Trap in a bottle. .

[0179] The dosage and frequency of administration will depend on the following factors: the route of administration, the particular antibody and TGF used. -β Trap fusion protein, the nature and severity of the disease being treated, whether the condition is acute or chronic The dosage may vary depending on the dosage and the size and general condition of the patient. The amount of hydroxybenzoate can be determined by methods well known in the art, for example, by dose escalation studies in clinical trials.

[0180] The antibody and TGF-β Trap fusion proteins provided herein are The dose may be administered once or several times at regular intervals over a period of time. In this study, the fusion protein of mouse antibody or humanized antibody and TGF-β Trap was found to be effective at least in at least once a month or more over a period of time, e.g., over a period of 1, 2, or 3 months, or For treating chronic conditions, long-term treatment is usually most effective. However, to treat acute symptoms, short-term administration, e.g., for 1 to 6 weeks, is recommended. Generally, human antibodies are administered at a baseline concentration for a selected indication or endpoint. The drug is administered until the patient shows medically relevant improvement above the standard dose.

[0181] An example of a treatment regimen provided herein is for the treatment of pulmonary arterial hypertension, pulmonary hypertension, pulmonary fibrosis, For treating symptoms of cardiovascular disease and cardiovascular fibrosis, at appropriate doses, A single subcutaneous injection of a fusion protein of antibody and TGF-β Trap was administered. The GF-β Trap fusion protein may be administered in a manner that is consistent with the patient's symptoms, for example, until the desired result is achieved. It may be administered once a week or once a month until symptoms subside. Treatment may be resumed as needed. Alternatively, a maintenance dose may be administered.

[0182] The patient's pulmonary artery pressure was measured using a fusion antibody and TGF-β Trap to detect changes in pressure. The patient's condition may be monitored before, during, and / or after treatment with the fusion protein. For diseases such as pulmonary artery disease, changes in pulmonary artery pressure may vary depending on factors such as disease progression. The pressure can be determined using known techniques.

[0183] Certain embodiments of the methods and compositions provided herein include, for example, antibodies and T a fusion protein of GF-β Trap and one or more endothelin antagonists; Two or more fusion proteins of an antibody and TGF-β Trap provided herein or a fusion protein of the antibody and TGF-β Trap provided herein, In a further embodiment, the use of one or more other endothelin antagonists is included. Therefore, the fusion proteins of the antibodies and TGF-β Trap provided herein can be used alone. It may be administered alone or in combination with other drugs to treat the condition from which the patient is suffering. Examples of these drugs include protein and non-protein drugs. If administered, the dosage should be adjusted accordingly, as is well known in the art. "Combined administration" Combination therapy is not limited to simultaneous administration, but includes at least one and administering to the patient other therapeutic agents containing antigens and proteins during the course of treatment. This also includes treatment regimens in which both drugs are administered once.

[0184] In another embodiment, for treating the symptoms of pulmonary arterial hypertension, pulmonary hypertension, and pulmonary fibrosis. The method of claim 1, wherein the antibody provided herein is for the treatment of a disease related to the above-mentioned disease. A mixture of fusion proteins of TGF-β Trap and TGF-β Trap and a pharmaceutically acceptable excipient is used. The method for preparing the medicament is described above. That's right.

[0185] Fusion protein of human ETA and antibody capable of specifically binding to TGF-β Trap Related compositions, kits and methods are further provided herein. A polynucleotide encoding all or part of a fusion protein of an antibody and TGF-β Trap. Nucleic acid sequences containing nucleotide sequences and derivatives and fragments thereof, e.g., human ETA binding All antibodies, antibody fragments, antibody derivatives, and fusion proteins of TGF-β Trap Nucleic acid sequences encoding the nucleic acid sequences or portions thereof are provided. Vectors containing the nucleic acid sequences, as well as nucleic acids and Further provided herein are cells comprising the vector. For example, a fusion protein of a human ETA-binding antibody and TGF-β Trap can be prepared, identified, or The present invention relates to a method for isolating a fusion protein of a human ETA-binding antibody and TGF-β Trap. Methods for determining whether a protein can still bind to ETA, human ETA binding The TGF-β fusion protein, which blocks TGF-β, is a fusion protein that blocks TGF-β. Methods for determining signal transduction and for measuring biological activity in vivo include: A fusion protein of human ETA-binding antibody and TGF-β Trap is administered to model animals. The present invention includes a method for:

[0186] To further illustrate the technical solutions described herein, the following specific examples are provided. Used.

[0187] Unless otherwise specified herein, all raw materials, equipment, etc. used are commercially available. These are well known or commonly used in the art. All methods therein are conventional in the art unless otherwise specified. [Example]

[0188] 1. Cloning and subcloning of antibody genes Antibody-secreting hybridoma cells were collected and purified using the QIAGEN mRNA extraction kit. Extract the mRNA from the hybridoma cells according to the manufacturer's protocol. The extracted mRNA is then reverse transcribed into cDNA. The reverse transcription primer is a mouse light chain and a primer specific to the heavy chain constant region, and the heavy chain reverse transcription primer was (5' -TTTGGRGGGAAGATGAAGAC-3' (SEQ ID NO: 199), and The reverse transcription primer was (5'-TTAACACTCTCCCCTGTTGAA-3')( SEQ ID NO: 200) and (5'-TTAACACTCATTCCTGTTGAA-3') ( The RT-PCR reaction conditions were as follows: 25°C, 5 50°C for 60 minutes; 70°C for 15 minutes. The reverse transcribed cDNA was diluted to 500 μL. Dilute with 0.1 mM TE and filter in ultrafiltration centrifuge tubes (Amicon Ultra-0.5). Add to PBS and centrifuge at 2000 g for 10 minutes; remove the filtrate and add 500 μL of 0. Add 1 mM TE and centrifuge at 2000 g for 10 min; remove the filtrate and prepare The tube was inverted into a new centrifuge tube and centrifuged at 2000 g for 10 minutes. Obtain purified cDNA; take 10 μL of purified cDNA as template and 4 μL of 5x Tailing Buffer (Promega, commercially available), 4 μL of dATP ( 1 mM) and 10 U of terminal transferase (Promega, commercially available) Mix thoroughly and incubate at 37°C for 5 minutes, then at 65°C for 5 minutes; then add polyA The tailed cDNA was used as a template to generate the antibody light and heavy chain variable regions by PCR. The upstream primers were all OligodT, and the heavy chain downstream primers were all OligodT. The dimer was (5'-TGGACAGGGATCCAGAGTTCC-3') (SEQ ID NO: 2 02) and (5'-TGGACAGGGCTCCATAGTTCC-3') (SEQ ID NO: 2 03), and the light chain downstream primer was (5'-ACTCGTCCTTGGTCAACG TG-3') (SEQ ID NO: 204). The PCR reaction conditions were as follows: 5°C, 5 min; 95°C, 30 sec, 56°C, 30 sec, 72°C, 1 min, 40 cycles; 72°C, 7 min. The PCR product was transferred to the PMD 18-T vector (Takara Bio, commercially available) The CDR sequences of the cloned antibodies are shown in Table 1 and Table 2. Shown in Figure 2.

[0189] PCR primers were designed based on the sequenced DNA sequences of the antibodies and therefore Complete light and heavy chain signal peptides and variable domains and mouse IgG1 constant region is ligated into the expression vector pTM5.

[0190] 2. Preparation of Expression Plasmids for Antibody Fusion Protein Genes The human TGF-β Trap (hTGF-β Trap) gene sequence was The Nhe1 restriction enzyme site is fused to the C-terminus of the heavy chain of the anti-ETA antibody by PCR. A Not1 restriction enzyme site was added to the 5' end of the heavy chain variable region of the fusion protein, and a Not1 restriction enzyme site was added to the PC The R primer adds the complete heavy chain and hTGF-β to the 3' end of the fusion protein. The Trap fusion protein gene was loaded into the expression vector pTM5; An Nhe1 restriction enzyme site was added to the 5' end of the light chain variable region, and a Bsiw1 restriction enzyme site was added to the 5' end of the light chain variable region. The complete light chain variable region sequence, along with its 3' end, is then added to the expression vector loaded with the light chain constant region. The vector is then ligated into pTM5.

[0191] 3. Transient Expression of Antibody Fusion Proteins 5×10 5 HEK293 or CHO expression cell lines were inoculated into shake flasks at a concentration of 1000 / mL. Seed and culture at 1 x 10 cells / well for 24 h at 37 °C and 5% CO. 6 Achieve a density of 1000p / mL The cells were then incubated under rotation until transfection was complete, and then used for transfection. During the transfection process, polyethyleneimine (PEI) was used as the transfection medium. and DNA (the amount of DNA is 1 × 10 6 0.5 μg per cell, where antibody The light chain to heavy chain ratio of the PEI is 3:2, where the preferred ratio of PEI to DNA is The ratio is 3:1. After 15 minutes of static incubation, After receiving the PEI and DNA mixture, the cells are incubated at 37°C for 24 h. The cells were incubated for 1 hour at 37°C and 5% CO2 under rotation, and then 0.5% tryptone was added. Add the necessary additives for expression to the cell culture solution, and finally, the cell supernatant is used for expression. After completion of incubation (>96 hours), the cells are harvested for antibody purification and isolation.

[0192] 4. Purification and isolation of antibody fusion proteins The collected cell supernatant was centrifuged at high speed (8000 rpm, 15 min) to separate the cells and cells. The vesicle debris is removed and then filtered and clarified using a 0.45 μm filter membrane. The purified supernatant is used for purification. The purification process is completed by chromatography. The supernatant was first run through a protein G affinity chromatography column, Meanwhile, the antibodies contained in the supernatant are purified by a protein G affinity chromatography column. The ligand binds to the ATP and is retained in the column. Washing the chromatography column with elution buffer to release the antibody bound to it The collected antibody eluate, which has a low pH value, was immediately neutralized with 1 M Tris-HCl. The resulting antibody fusion protein eluate was then permeated for 16 hours to protect the antibody from activation. After precipitation, the buffer system is replaced with PBS.

[0193] 5. Calcium flux assays demonstrate ETA antibodies and TGF-β T block ETA in vitro Detecting the biological activity of RAP fusion proteins CHO-DHFR cells co-expressing hETA-aequorin were cultured at 25,000 cells per well. Inoculate 100 cells into a black 96-well cell culture plate and incubate overnight at 37°C. The cell supernatant was removed and 50 μl of the substrate coelenterandin h (Promega, commercially available) was added. Add 50 μl of purified antibody or fusion protein and incubate in the dark for 2 hours. Add the ligated protein and incubate for 30 minutes. on the SpectraMax L microplate reader from Incular Devices Endothelin-1 is perfused through the endothelin-1 membrane. An immediate change in calcium flux is detected within 40 seconds. The peak time and peak value are recorded. Both the TGF-β and TGF-β Trap fusion proteins were mediated by human ETA. Intracellular Ca 2+ Change can be effectively inhibited.

[0194] 6. Reporter gene experiments demonstrate that TGF-β signaling pathway is blocked in vitro Detecting the fusion protein of antibody A and TGF-β Trap 4T1 cells stably expressing SBE-luciferase were cultured at 34,000 cells per well. The cells were seeded into a 96-well cell culture plate and cultured overnight at 37°C. ng / ml TGF-β1 and serially diluted ETA antibody and TGF-β Trap The fusion proteins are pre-incubated in equal volumes for 30 minutes at room temperature. During the incubation period, the medium supernatant was removed from the 96-well cell culture plate and Wash the cell surface twice with serum-free medium and aspirate the remaining liquid. The reaction solution was added to a 96-well cell culture plate, and the mixture was incubated at 37°C for 6 hours. After incubation, add 100 μL of Bright Glo Chemiluminescent Substrate ( Finally, the cell lysate was transferred to a white 96-well plate and incubated for 1 hour. Fluorescence intensity was measured using a SpectraMax L microplate reader (Molecular Figure 2 shows the activity of ETA antibody and TGF-β1 receptor. Detecting a TGF-β Trap fusion protein that blocks TGF-β1 from activation 1 shows the inhibition curves of reporter gene experiments.

[0195] 7. Antibody fusion protein in bleomycin-induced pulmonary fibrosis in C57BL / 6 mice In vivo activity of proteins Bleomycin (BLM) was administered intratracheally to normal C57BL / 6 mouse lung tissue. A pulmonary fibrosis model (BPF) was induced and used to evaluate the efficacy of pulmonary fibrosis in the treatment of this model. Evaluate the effect of intraperitoneal injection of 15F3-(G4S)4-TGF-β Trap in mice The animals were anesthetized with 2% isoflurane, fixed to the laboratory table in a supine position, and the indwelling tube was quickly inserted into their trachea. A 100 μL microsyringe is inserted into the body with a specific amount of saline solution according to the body weight. The patient was placed in a vein to withdraw fluid (normal control group) or bleomycin solution (2.5 mg / kg). The indwelling needle is inserted into the annular cavity of the cartilage, and the medical solution is injected at a dose of 50 μL / 20 g. After injection, the mouse is quickly placed upright and turned over, and the medical solution is The fluid is distributed evenly within the lungs. The animals are randomly divided according to their body weight into a normal group and 12 mice in the mouse group, 20 mice in the model group, and 15F3-(G4S)4-TGF-β Trap low-dose group There were 14 animals in each of the low-dose and high-dose groups. The ap or blank formulation was administered according to the protocol one day after modeling, and then administered for 3 weeks. The efficacy of the drug is evaluated based on indicators such as animal survival and lung lesions. As shown in Figure 3, in the model group, Masson staining of lung lesions revealed In this case, most of the alveolar structures disappear, the alveolar septa expand, and a large amount of collagen fibers accumulate. Tracheal administration of bleomycin successfully induced pulmonary fibrosis in normal C57BL / 6 mice. Low and high doses of h15F3-(G4S)4-TGF-β Trap were shown to be The groups showed significant improvements in the above pulmonary fibrosis indices, demonstrating a clear dose-effect relationship. Furthermore, as shown in Figure 4, h15F3-(G4S)4-TGF-β Trap treatment The drug was able to extend the survival time of mice, indicating that it slowed down the process of pulmonary fibrosis. This indicates that it can be inhibited.

[0196] The above embodiments are only preferred solutions of the present invention, which should not be construed as limiting the present invention. It is not to be construed in any way as a limitation on the scope of the technical solutions defined in the claims. Other variations and modifications are possible without departing from the scope of the present invention.

Claims

[Claim 1] The novel products, methods and processes substantially as herein described.

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