WO2020248967A1

WO2020248967A1 - Fusion protein of eta antibody and bnp, and pharmaceutical composition and application of fusion protein

Info

Publication number: WO2020248967A1
Application number: PCT/CN2020/095062
Authority: WO
Inventors: 药晨江; 张�成; 章华; 汪笑峰; 景书谦
Original assignee: 鸿运华宁（杭州）生物医药有限公司
Priority date: 2019-06-10
Filing date: 2020-06-09
Publication date: 2020-12-17
Also published as: CN112062857A

Abstract

The present invention provides a fusion protein of an ETA antibody and BNP. The present invention also provides a pharmaceutical composition of the fusion protein of the ETA antibody and BNP. The present invention further provides a method for treating, preventing, or improving one or more of symptoms of pulmonary arterial hypertension, pulmonary hypertension, or heart failure by using the fusion protein of the ETA antibody and BNP.

Description

Fusion protein of ETA antibody and BNP, and its pharmaceutical composition and application

Technical field

This article provides a fusion protein of ETA antibody and BNP. This article also provides a pharmaceutical composition of ETA antibody and BNP fusion protein. This document further provides methods for treating, preventing or ameliorating one or more symptoms of pulmonary hypertension, pulmonary hypertension, or heart failure by fusion protein of ETA antibody and BNP.

Background technique

Pulmonary Arterial Hypertension (PAH) is a rare and progressive disease characterized by a marked increase in pulmonary arterial blood pressure. Pulmonary hypertension has become an important disease threatening human health. According to data, the incidence of various types of pulmonary hypertension worldwide is about 2.4 to 7.6 per million, and the prevalence is about 15 to 26 per million population. The 3rd most common cardiovascular disease after ischemic heart disease and hypertension. The cause of pulmonary hypertension is not yet fully understood. Because of its insidious onset, most patients are already in the Ⅲ-Ⅳ grade of pulmonary hypertension and cardiac function. The accompanying symptoms of pulmonary hypertension usually include shortness of breath (especially during exercise), chest pain, intermittent fainting, etc. In addition, as the condition continues, the continuous high pressure of the pulmonary artery will make the right ventricle continue to fail to supply blood to the lungs. , Will eventually lead to right ventricular failure. Heart failure is the most common cause of death in patients with pulmonary hypertension.

There is no cure for pulmonary hypertension, and drug therapy is the first choice for maintenance treatment of pulmonary hypertension. The drugs approved by the FDA for the treatment of pulmonary hypertension are vasodilators, which can be divided into calcium channel blockers, prostacyclin receptor agonists, and phosphodiesterase type 5 (PDE5) inhibitors according to their mechanism. , Endothelin receptor inhibitors, etc.

Pulmonary hypertension is caused by vasoconstriction in the lungs or the blood vessels associated with the lungs (vasoconstriction). After the heart's blood supply to the lungs is insufficient, the heart's blood supply pressure to the lungs is compensatory. Local occlusion caused by vascular tightening, remodeling, stiffness or thrombosis, and then the resistance of blood vessels to pulmonary blood circulation increases (Simonneau et al., 2004, J. Am. Coll. Cardiol. 43: 5S-12S; Barst et al., 2004, J. Am.Coll.Cardiol.43:40S–47S).

Endothelin receptor (Endthelin Receptor A, ETA, or the ET _A R) can effectively block angiogenesis inhibitors pressure induced by endothelin to increase remission of symptoms of pulmonary hypertension, improve exercise capacity, and hemodynamic patient (Serasli Et al., 2010, Recent Pat. Cardiovasc. Drug Discov. 5: 184-95).

Brain Natriuretic Peptide (BNP), mainly expressed in the ventricles, is a natural hormone synthesized by cardiomyocytes, and it is also present in brain tissue. Blood BNP increases during ventricular dysfunction and protects the body from volume overload by maintaining renal function and sodium balance. Studies on patients with pulmonary hypertension have shown that blood BNP rises when right heart dysfunction occurs. In addition to being a biomarker of cardiovascular disease, BNP is also a treatment option for acute heart failure (ADHF). "Nesiritide" is a recombinant human B-type natriuretic peptide, which was approved by the FDA in 2001 to treat acute heart failure. BNP binds to guanylate cyclase receptors on vascular smooth muscle and endothelial cells, causing the level of second messenger cyclic guanosine monophosphate (cGMP) in the cell to increase, which triggers a series of physiological effects:( 1) It has endothelium-independent vasodilation activity, dilates arteries and veins, reduces systemic vascular resistance, filling pressure and pulmonary capillary wedge pressure, and reduces cardiac pre- and post-load; (2) Increases glomerular filtration rate and produces sodium excretion Diuretic effect, reduce body fluid load, increase cardiac output, and comprehensively improve cardiac function; (3) Inhibit the activation of renin-angiotensin-aldosterone system (RAAS) in the body and inhibit the reflex heart rate caused by vasodilator effect Increase, avoid the occurrence of arrhythmia; (4) Inhibit the growth of vascular endothelial cells, smooth muscle cells and fibroblasts, and inhibit cardiac hypertrophy.

This article provides a fusion protein of ETA antibody and BNP. On the one hand, the fusion protein of ETA antibody and BNP can inhibit the combined effect of ETA signaling pathway and BNP signaling pathway in reducing pulmonary vascular resistance and peripheral vascular resistance, reducing body fluid load, alleviating pulmonary hypertension and cardiac remodeling of heart failure, comprehensively Improve heart function. On the other hand, the fusion protein of ETA antibody and BNP can prolong the half-life of BNP, thereby achieving the purpose of prolonging the effective time of the drug and reducing the side effects of the drug.

Summary of the invention

This article provides a fusion protein of ETA antibody and BNP. This article also provides methods for treating, preventing or improving one or more symptoms of pulmonary hypertension, pulmonary hypertension, and heart failure.

This article provides a fusion protein of an ETA antibody and BNP, and its structure is characterized in that the fusion protein comprises an ETA antibody and one or more BNPs.

This article provides a fusion protein of ETA antibody and BNP, and its structure is characterized in that: the fusion protein contains one ETA antibody and one, two, three, four, five, six, seven, or eight A BNP; the fusion protein connects the amino terminus of a BNP to the carboxy terminus of the ETA antibody light chain or heavy chain, or the fusion protein connects the carboxy terminus of a BNP to the ETA antibody light or heavy chain The amino terminal is connected.

This article provides a fusion protein of ETA antibody and BNP. Its structure is characterized in that: the fusion protein contains one ETA antibody and one, two, three or four BNP; the fusion protein combines the amino terminus of a BNP with The carboxyl terminal of the light chain or heavy chain of the ETA antibody is connected, or the fusion protein connects the carboxyl terminal of a BNP with the amino terminal of the light or heavy chain of the ETA antibody.

This article provides a fusion protein of ETA antibody and BNP. Its structure is characterized in that: the fusion protein contains an ETA antibody and two BNPs; the fusion protein combines the amino terminus of a BNP with the light chain or heavy chain of the ETA antibody. The carboxyl terminal of the chain is connected, or the fusion protein connects the carboxyl terminal of a BNP with the amino terminal of the ETA antibody light chain or heavy chain.

This article provides a fusion protein of ETA antibody and BNP, its structure is characterized in that: the fusion protein comprises an ETA antibody and a BNP; the fusion protein combines the amino terminus of the BNP with the light chain of the ETA antibody or The carboxyl terminal of the heavy chain is connected, or the fusion protein connects the carboxyl terminal of the BNP with the amino terminal of the ETA antibody light chain or heavy chain.

This article provides a fusion protein of ETA antibody and BNP, and its structure is characterized in that: the fusion protein contains one ETA antibody and one, two, three, four, five, six, seven, or eight A BNP and a peptide linker (Linker); the fusion protein connects the amino terminal of a BNP with the carboxyl terminal of the ETA antibody light chain or heavy chain through a peptide linker sequence (Linker), or the fusion protein is connected through a peptide linker The sequence (Linker) connects the carboxyl end of a BNP with the amino end of the light chain or heavy chain of the ETA antibody.

This article provides a fusion protein of ETA antibody and BNP, its structure is characterized in that: the fusion protein contains one ETA antibody, and one, two, three or four BNP and peptide linker (Linker); A peptide linker sequence (Linker) connects the amino terminal of a BNP to the carboxyl terminal of the light or heavy chain of the ETA antibody, or the fusion protein connects the carboxyl terminal of a BNP to the carboxyl terminal of the ETA antibody via a peptide linker sequence (Linker). The ETA antibody light chain or the amino terminal of the heavy chain is attached.

This article provides a fusion protein of ETA antibody and BNP. Its structure is characterized in that: the fusion protein includes an ETA antibody, two BNPs and two peptide linkers (Linker); the fusion protein passes through a peptide linker sequence (Linker) Connect the amino terminus of a BNP to the carboxy terminus of the ETA antibody light or heavy chain, or the fusion protein connects the carboxy terminus of a BNP to the ETA antibody light or heavy chain through a peptide linker sequence (Linker). The amino end of the chain is connected.

This article provides a fusion protein of ETA antibody and BNP. Its structure is characterized in that: the fusion protein comprises an ETA antibody, a BNP and a peptide linker (Linker); the fusion protein passes through the peptide linker sequence (Linker) Connect the amino terminus of the BNP to the carboxy terminus of the light or heavy chain of the ETA antibody, or the fusion protein connects the carboxy terminus of the BNP to the carboxy terminus of the ETA antibody via the peptide linker sequence (Linker). ETA antibody light chain or heavy chain amino terminal connection.

This article provides a fusion protein of ETA antibody and BNP, its structure is characterized in that the ETA antibody, BNP and peptide linker sequence are fused by one of the following methods to form the fusion protein:

(1) Connect the amino terminus of a BNP and the carboxy terminus of an ETA antibody heavy chain/light chain through a peptide linker sequence (Linker): N'-R-Linker-BNP-C'; and

(2) Connect the carboxyl end of a BNP to the amino end of an ETA antibody light chain or heavy chain through a peptide linker sequence (Linker): N'-BNP-Linker-R-C';

Among them: N'represents the amino terminal of the polypeptide chain, C'represents the carboxyl terminal of the polypeptide chain, BNP represents a BNP, R is the amino acid sequence of the light or heavy chain of an ETA antibody, and Linker represents a peptide linker.

Provided herein is a polynucleotide encoding a fusion protein of an ETA antibody and BNP described herein.

Provided herein is a vector comprising a polynucleotide encoding a fusion protein of an ETA antibody and BNP described herein.

Provided herein is a host cell comprising a vector as described herein.

Provided herein is a pharmaceutical composition comprising a fusion protein of an ETA antibody and BNP described herein, and a pharmaceutically acceptable carrier.

Provided herein is the use of a fusion protein of an ETA antibody and BNP described herein in the preparation of a medicament for the treatment, prevention or amelioration of pulmonary hypertension and pulmonary hypertension-related disorders.

Provided herein is the use of a fusion protein of an ETA antibody and BNP described herein in the preparation of a medicament for weight loss or treatment, prevention or amelioration of heart failure and heart failure-related disorders.

Provided herein is the use of a fusion protein of an ETA antibody and BNP described herein in the preparation of a medicament for simultaneous treatment, prevention or amelioration of two or more diseases of pulmonary hypertension, pulmonary hypertension or heart failure.

Provided herein is a method for treating, preventing or ameliorating one or more symptoms of pulmonary hypertension and pulmonary hypertension-related disorders, which comprises administering to a subject a therapeutically effective amount of a fusion protein of an ETA antibody and BNP described herein.

Provided herein is a method for treating, preventing or ameliorating one or more symptoms of heart failure and heart failure-related disorders, which comprises administering to a subject a therapeutically effective amount of a fusion protein of an ETA antibody and BNP described herein.

Provided herein are methods for treating, preventing or ameliorating two or more diseases of pulmonary hypertension, pulmonary hypertension or heart failure, which include administering to a subject a therapeutically effective amount of a fusion protein of an ETA antibody and BNP described herein.

Description of the drawings:

[Corrected according to Rule 26 07.07.2020]
Figure 1: Shows the fusion protein h15F3-(G ₄ S) ₂ -BNP of ETA antibody and BNP (which includes SEQ ID NO: 162, SEQ ID NO: 190, SEQ ID NO: 218, and SEQ ID NO: 205) , H15F3-BNP (3-29) (which includes SEQ ID NO: 162, SEQ ID NO: 190, and SEQ ID NO: 210) and h15F3-BNP (9-32) (which includes SEQ ID NO: 162, SEQ ID NO: 190, and SEQ ID NO: 209) inhibit the result of human ETA-mediated Ca ²⁺ changes.

[Corrected according to Rule 26 07.07.2020]
Figure 2: Shows that the fusion protein h15F3-(G ₄ S) ₂ -BNP, h15F3-BNP(3-29) and h15F3-BNP(9-32) of ETA antibody and BNP reduced the normal size 15 minutes after administration The effect of rat arterial pressure.

[Corrected according to Rule 26 07.07.2020]
Figure 3: Shows the effect of different doses of h15F3-(G ₄ S) ₂ -BNP, a fusion protein of ETA antibody and BNP, in reducing right ventricular pressure in MCT rats.

[Corrected according to Rule 26 07.07.2020]
Figure 4: Shows that the fusion protein h15F3-(G ₄ S) ₂ -BNP, h15F3-BNP(3-29) and h15F3-BNP(9-32) of ETA antibody and BNP reduced the arteries of healthy monkeys 10 minutes after administration The effect of pressure.

[Corrected according to Rule 26 07.07.2020]
Figure 5: Shows that the fusion proteins h15F3-(G ₄ S) ₂ -BNP, h15F3-BNP(3-29) and h15F3-BNP(9-32) of ETA antibody and BNP reduced the arteries of healthy monkeys 60 minutes after administration The effect of pressure.

[Corrected according to Rule 26 07.07.2020]
Figure 6: Shows the pharmacokinetic results of the fusion protein h15F3-(G ₄ S) ₂ -BNP of ETA antibody and BNP in healthy monkeys.

[Corrected according to Rule 26 07.07.2020]
Figure 7: shows the pharmacokinetic results of the fusion protein h15F3-BNP (9-32) of ETA antibody and BNP in healthy monkeys.

Specific implementation definition

Unless otherwise defined herein, scientific and technical terms related to this document shall have the meaning understood by those of ordinary skill in the art. Generally, the nomenclature and technology related to pharmacology, biology, biochemistry, cell and tissue culture, biology, molecular biology, immunology, microbiology, genetics and protein nucleic acid chemistry and hybridization described herein are based Well-known and frequently used in the field.

Standard one-letter or three-letter abbreviations are used herein to indicate polynucleotide and polypeptide sequences. Unless otherwise specified, the amino terminus of the polypeptide sequence is on the left and their carboxy terminus is on the right, the 5'end of the upstream strand of single-stranded nucleic acid sequences and double-stranded nucleic acid sequences is on the left and their 3'ends are on the right. The specific part of the polypeptide can be represented by the number of amino acid residues, for example, amino acids 80 to 130, or represented by the actual residues at the position, for example, Lys80 to Lys130. The specific polypeptide or polynucleotide sequence can also be described by explaining the difference between it and the reference sequence.

The terms "peptide", "polypeptide", and "protein" all refer to a molecule comprising two or more amino acids connected to each other by a peptide bond. These terms encompass, for example, natural and artificial proteins, protein fragments, and polypeptide analogs of protein sequences (such as muteins, variants, and fusion proteins), and proteins that are post-transcriptionally or otherwise covalently or non-covalently modified. Peptides, polypeptides or proteins can be monomers or multimers.

The term "polypeptide fragment" refers to a polypeptide having amino-terminal and/or carboxy-terminal deletions compared to the corresponding full-length protein. The fragment length can be, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 80, 90, 100, 150, or 200 amino acids. The fragment length can be, for example, up to 1000, 750, 500, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11 or 10 amino acids. The fragment may further include one or more additional amino acids at one or both ends, for example, an amino acid sequence from a different natural protein (for example, Fc or leucine zipper domain) or an artificial amino acid sequence (for example, an artificial linker sequence).

The polypeptide herein includes polypeptides modified for any reason and by any method, for example, to: (1) reduce proteolytic sensitivity, (2) reduce oxidation sensitivity, (3) change the affinity for forming protein complexes, ( 4) Change the binding affinity and (4) confer or modify other physicochemical or functional properties. Analogs include mutant proteins of polypeptides. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in the native sequence (e.g., the portion of the polypeptide outside the domain that forms the intramolecular contact). "Conservative amino acid substitutions" are those that do not significantly change the structural characteristics of the parent sequence (for example, the replacement amino acid should not disrupt the helix appearing in the parent sequence or interfere with other secondary structure types that confer characteristics to the parent sequence or are necessary for its function).

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

A "derivative" of a polypeptide is a chemically modified polypeptide, for example, by binding to other chemical moieties such as polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.

Unless otherwise specified, the term "antibody" includes its derivatives, variants, fragments and muteins other than an antibody comprising two full-length heavy chains and two full-length light chains, examples of which are shown below.

The term "antibody" is a protein comprising a scaffold or framework part that binds to an antigen and optionally allows the antigen binding part to adopt a conformation that promotes the binding of the antibody to the antigen. Examples of antibodies include whole antibodies, antibody fragments (e.g., antigen-binding portions of antibodies), antibody derivatives, and antibody analogs. The antibody may comprise, for example, an alternative protein scaffold or an artificial scaffold with grafted CDRs or CDRs derivatives. The scaffold includes, but is not limited to, an antibody-derived scaffold that is introduced, for example, to stabilize the three-dimensional structure of the antibody, and a fully synthetic scaffold that contains, for example, a biocompatible polymer. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function and Bioinformatics 53: 121-129; Roque et al., 2004, Biotechnol. Prog. 20: 639-654. In addition, peptide-mimicking antibodies ("PAMs") and antibody-mimicking scaffolds can be used, which utilize fibronectin like scaffolds.

The antibody may have, for example, the structure of natural immunoglobulin. "Immunoglobulin" is a tetrameric molecule. In natural immunoglobulins, each tetramer is composed of two identical polypeptide chain pairs, each pair having a "light" (about 25kDa) and a "heavy" chain (about 50-70kDa). The amino terminus of each chain includes a variable domain of about 100 to 110 or more amino acids, which is mainly related to antigen recognition. The carboxy terminal part of each chain defines the constant region mainly related to effector action. Human light chains are divided into kappa and lambda light chains. Heavy chains are classified into μ, δ, α, or ε, and the isotype of the antigen is determined, such as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 amino acids. See, Fundamental Immunology Ch.7 (Edited by Paul, 2nd Edition, Raven Press, 1989) (the entire contents are in reference form and used herein for any purpose). The variable region of each light/heavy chain pair forms an antibody binding site, so that a complete immunoglobulin has two binding sites.

The natural immunoglobulin chain shows the same basic structure of relatively conserved framework regions (FR) connected by three hypervariable regions, which are also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both the light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The allocation of amino acids in each domain is consistent with the definition in Sequences of Proteins of Immunological Interest, 5th edition, US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991.

Unless otherwise specified, "antibody" refers to an intact immunoglobulin or an antigen-binding portion thereof that can compete with an intact antibody for specific binding. The antigen binding portion can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. The antigen-binding portion includes, in particular, Fab, Fab', F(ab') ₂ , Fv, domain antibodies (dAbs), fragments including complementarity determining regions (CDRs), single chain antibodies (scFv), chimeric antibodies, Diabodies, triabodies, tetrabodies, and polypeptides containing at least a portion of immunoglobulin sufficient to confer specific antigen binding to the polypeptide.

Fab fragments having a V _L, a monovalent fragment V _H, C _L and C _H1 domains; F (ab ') ₂ fragment is a bivalent fragment having two Fab fragments linked by the hinge region disulfide bonds; Fd fragments having a V _H or V _L domains; a dAb fragment having V _H domain, V _L domain, or a V _H or V _L domain antigen binding fragment (U.S. Patent No. US6,846,634, US6,696,245, U.S. Patent application Publication No. US2005/0202512, US2004/0202995, US2004/0038291, US2004/0009507, US2003/0039958, Ward et al., 1989, Nature 341:544-546.).

A single-chain antibody (scFv) is an antibody in which the V _L buckle V _H region is connected by a linker (for example, a synthetic amino acid residue sequence) to form a continuous protein, wherein the linker is long enough to allow the protein chain to fold back to itself and form Monovalent antigen binding sites (see, for example, Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-83).

Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises V _H domains and V _L, connected by a linker, the linker is short so as to allow pairing the two domains on the same chain, Therefore, each domain is allowed to pair with a complementary domain on another polypeptide chain (see, for example, Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., 1994, Structure 2 :1121-23). If the two polypeptide chains of the diabody are the same, the diabody paired with them will have the same antigen binding site. Polypeptide chains with different sequences can be used to prepare diabodies with different antigen binding sites. Similarly, tri-chain antibodies and tetra-chain antibodies are antibodies containing three and four polypeptide chains, respectively, and form three and four antigen binding sites, respectively, which may be the same or different.

The method described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th edition, US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991 can be used to identify the complementarity determining region of a given antibody (CDRs) and framework regions (FR). One or more CDRs can be incorporated covalently or non-covalently into the molecule to make it an antibody. Antibodies can incorporate CDR(s) into larger polypeptide chains. CDR(s) can be covalently linked to another peptide chain, or non-covalently incorporated into CDR(s). CDRs allow antibodies to specifically bind to specific related antigens.

Antibodies can have one or more binding sites. If there is more than one binding site, the binding site may be the same or different from the other. For example, natural human immunoglobulins usually have two identical binding sites, while "bispecific" or "bifunctional" antibodies have two different binding sites.

The term "murine antibody" includes all antibodies that have one or more variable and constant regions derived from mouse immunoglobulin sequences.

The term "humanized antibody" is an antibody made by transplanting the complementarity determining region sequence of a mouse antibody molecule into the variable region framework of a human antibody.

The term "antigen-binding domain", "antigen-binding region" or "antigen-binding site" refers to an antibody that contains amino acid residues (or other parts) that interact with an antigen and contributes to the specificity and affinity of the antibody for the antigen. section. For an antibody that specifically binds to its antigen, this will include at least part of at least one of its CDR domains.

The term "epitope" is a part of a molecule that binds to an antibody (eg, through an antibody). The epitope may comprise a non-contiguous part of the molecule (for example, in a polypeptide, non-contiguous amino acid residues in the primary sequence of the polypeptide are close enough to each other in the tertiary and quaternary structure of the polypeptide to be bound by an antibody) .

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

The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably throughout the text and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), the use of nucleotides DNA or RNA analogs and hybrids thereof produced by analogs (for example, peptide nucleic acids and non-natural nucleotide analogs). The nucleic acid molecule can be single-stranded or double-stranded. In one embodiment, the nucleic acid molecule herein comprises a continuous open reading frame encoding the antibody or fragment, derivative, mutein, or variant thereof provided herein.

If their sequences can be arranged in anti-parallel, two single-stranded polynucleotides are "complementary" to each other, so that each nucleotide in one polynucleotide is complementary to the other polynucleotide In contrast to nucleotides, no gaps are introduced and there are no unpaired nucleotides at the 5'or 3'end of each sequence. If two polynucleotides can hybridize to each other under moderately stringent conditions, then one polynucleotide is "complementary" to the other polynucleotide. Therefore, one polynucleotide can be complementary to another polynucleotide, but not its complementary sequence.

The term "vector" is a nucleic acid that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid", which refers to a linear or circular double-stranded DNA molecule to which additional nucleic acid segments can be attached. Another type of vector is a viral vector (e.g., replication defective retrovirus, adenovirus, and adenovirus-associated virus), in which additional DNA segments can be introduced into the viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors containing bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the host cell's genome when introduced into the host cell and thus replicate with the host genome.

An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

If the regulatory sequence affects the expression of the nucleotide sequence (eg, expression level, time, or location), then the nucleotide sequence is "operably linked" to the regulatory sequence. A "regulatory sequence" is a nucleic acid that can affect the expression (eg, expression level, time, or site) of a nucleic acid to which it is operably linked. Regulatory genes, for example, act directly on the regulated nucleic acid or through the action of one or more other molecules (for example, polynucleotides that bind to regulatory sequences and/or nucleic acids). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology, Volume 185, Academic Press, San Diego, CA and Baron, etc., 1995, Nucleic Acids Res. 23:3605-06.

The term "host cell" is a cell used to express nucleic acid, such as the nucleic acid provided herein. The host cell may be a prokaryote, such as Escherichia coli, or it may be a eukaryote, such as a single-celled eukaryote (e.g., yeast or other fungi), plant cells (e.g., tobacco or tomato plant cells), animal cells (e.g., Human cells, monkey cells, hamster cells, rat cells, mouse cells or insect cells) or hybridomas. Generally, the host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell. The phrase "recombinant host cell" can be used to describe a host cell that is transformed or transfected with the nucleic acid to be expressed. The host cell may also be a cell that contains the nucleic acid but is not expressed at the desired level, unless a regulatory sequence is introduced into the host cell so that it is operably linked to the nucleic acid. It should be understood that the term host cell refers not only to a specific subject cell but also to the progeny or possible progeny of that cell. Due to, for example, mutations or environmental influences, certain modifications will occur in subsequent generations, the offspring may in fact be different from the parent cell but still fall within the scope of the term used herein.

Endothelin receptor

Endothelin receptor (ETA) belongs to the A subfamily of the 7-transmembrane receptor family, which is coupled to one or more intracellular signaling pathways through a heterotrimeric guanine nucleotide binding protein (G protein) (Jelinek et al., 1993, Science 259:1614-1616, Segre et al., 1993, Trends Endocrinol. Metab. 4:309-314). As used herein, "endothelin receptors" and "ETA" or "ET _A R" may be used interchangeably.

In one embodiment, the antibodies described herein can be selected to bind to membrane-bound endothelin receptors expressed on cells and inhibit or block endothelin signaling through the endothelin receptors. In one embodiment, the antibodies described herein specifically bind to human endothelin receptors. In a further embodiment, antibodies that bind to human endothelin receptors can also bind to endothelin receptors of other species, such as rats. The examples below provide for the generation of murine antibodies that bind to human membrane-bound endothelin receptors, and in further embodiments bind to endothelin receptors of other species.

The polynucleotide and polypeptide sequences of endothelin receptors of several species are known. SEQ ID NO: 1-SEQ ID NO: 6 shows the sequences of human, monkey and rat. The sequence data comes from the GeneBank database of the National Center for Biotechnology Information.

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); accession number: AAB20278.

Cynomolgus monkey polynucleotide (SEQ ID NO: 3); accession number: JV635771.

Cynomolgus monkey amino acid (SEQ ID NO: 4); accession number: AFJ71111.

Rat (Rattus norvegicus) polynucleotide (SEQ ID NO: 5); accession number: M60786.

Rat (Rattus norvegicus) amino acid (SEQ ID NO: 6); accession number: AAA41114.

Endothelin receptor A (ETA) antibody

In one embodiment, the ETA antibody described herein comprises one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

a. Light chain CDR1 amino acid sequence: SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30;

b. Light chain CDR2 amino acid sequence: SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48;

c. Light chain CDR3 amino acid sequence: SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 220;

d. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and SEQ ID NO: 90;

e. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114; and

f. Heavy chain CDR3 amino acid sequence: 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.

Table 1 lists the amino acid sequences of the light chain CDRs of the ETA antibodies described herein and their corresponding polynucleotide coding sequences. Table 2 lists the amino acid sequences of the heavy chain CDRs of the ETA antibodies described herein and their corresponding polynucleotide coding sequences.

Table 1: Amino acid sequences of light chain CDRs and their polynucleotide coding sequences

Table 2: Amino acid sequences of heavy chain CDRs and their polynucleotide coding sequences

In one embodiment, the antibody described herein comprises a sequence that differs from the CDR amino acid sequences in Table 1 and Table 2 by 5, 4, 3, 2, or 1 single amino acid addition, substitution and/or deletion. In another embodiment, the antibody described herein comprises a sequence that differs from the CDR amino acid sequences in Table 1 and Table 2 by 4, 3, 2, or 1 single amino acid addition, substitution and/or deletion. In another embodiment, the antibody described herein comprises a sequence that differs from the CDR amino acid sequences in Table 1 and Table 2 by 3, 2, or 1 single amino acid addition, substitution and/or deletion. In another embodiment, the antibodies described herein comprise sequences that differ from the CDR amino acid sequences in Table 1 and Table 2 by 2 or 1 single amino acid additions, substitutions, and/or deletions, respectively. In another embodiment, the antibody described herein contains a sequence that differs from the CDR amino acid sequences in Table 1 and Table 2 by one single amino acid addition, substitution, and/or deletion.

In another embodiment, the ETA antibody (ETA-1 antibody) described herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

a. Light chain CDR1 amino acid sequence: SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30; and

b. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and SEQ ID NO: 90.

In one aspect, the ETA-1 antibody further comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

a. Light chain CDR2 amino acid sequence: SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48; and

b. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114.

On the other hand, the ETA-1 antibody also contains one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

a. Light chain CDR3 amino acid sequence: SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 220; and

b. Heavy chain CDR3 amino acid sequence: 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.

In another embodiment, the ETA antibody (ETA-2 antibody) described herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

In one aspect, the ETA-2 antibody further comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

On the other hand, the ETA-2 antibody also contains one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

In another embodiment, the ETA antibody (ETA-3 antibody) described herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

In one aspect, the ETA-3 antibody further comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

On the other hand, the ETA-3 antibody also contains one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

In one embodiment, the ETA antibody described herein comprises: a. A light chain CDR1 amino acid sequence independently selected from the following: SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30;

b. A light chain CDR2 amino acid sequence independently selected from the following: SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO:42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48;

c. A light chain CDR3 amino acid sequence independently selected from the following: SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, and SEQ ID NO: 68;

d. A heavy chain CDR1 amino acid sequence independently selected from the following: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and SEQ ID NO: 90;

e. A heavy chain CDR2 amino acid sequence independently selected from the following: SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114; and

f. A heavy chain CDR3 amino acid sequence independently selected from the following: 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.

In one embodiment, the ETA antibody described herein comprises a light chain CDR3 amino acid sequence independently selected from the following: SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO :56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 220. In another embodiment, the ETA antibody described herein comprises a heavy chain CDR3 amino acid sequence independently selected from the following: 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.

In another embodiment, the ETA antibody described herein comprises a combination of light chain and heavy chain CDR3 amino acid sequences independently selected from the following: SEQ ID NO: 50 and SEQ ID NO: 116, SEQ ID NO :50 and SEQ ID NO: 220, 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.

In one embodiment, the ETA antibody described herein comprises:

(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: 220;

Amino acid sequence of heavy chain CDR1: SEQ ID NO: 70;

Heavy chain CDR2 amino acid sequence: SEQ ID NO: 92; and

Amino acid sequence of heavy chain CDR3: 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;

Amino acid sequence of light chain CDR3: SEQ ID NO: 52;

Amino acid sequence of heavy chain CDR1: SEQ ID NO: 72;

Amino acid sequence of heavy chain CDR2: SEQ ID NO: 94; and

Amino acid sequence of heavy chain CDR3: 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;

Amino acid sequence of light chain CDR3: SEQ ID NO: 54;

Amino acid sequence of heavy chain CDR1: SEQ ID NO: 74;

Heavy chain CDR2 amino acid sequence: SEQ ID NO: 96; and

Amino acid sequence of heavy chain CDR3: 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;

Amino acid sequence of heavy chain CDR1: SEQ ID NO: 76;

Heavy chain CDR2 amino acid sequence: SEQ ID NO: 98; and

Amino acid sequence of heavy chain CDR3: 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

Amino acid sequence of heavy chain CDR3: 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;

Amino acid sequence of heavy chain CDR2: SEQ ID NO: 102; and

Amino acid sequence of heavy chain CDR3: SEQ ID NO: 126;

(g) Light chain CDR1 amino acid sequence: SEQ ID NO: 20 or 22;

Light chain CDR2 amino acid sequence: SEQ ID NO: 44;

Light chain CDR3 amino acid sequence: SEQ ID NO: 62;

Amino acid sequence of heavy chain CDR1: SEQ ID NO: 82;

Heavy chain CDR2 amino acid sequence: SEQ ID NO: 104 or 106; and

Amino acid sequence of heavy chain CDR3: 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

Amino acid sequence of heavy chain CDR3: 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;

Amino acid sequence of heavy chain CDR1: SEQ ID NO: 86;

Heavy chain CDR2 amino acid sequence: SEQ ID NO: 110; and

Amino acid sequence of heavy chain CDR3: 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;

Amino acid sequence of heavy chain CDR1: 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;

Amino acid sequence of heavy chain CDR1: 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.

In another embodiment, the ETA antibody described herein comprises:

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;

Amino acid sequence of heavy chain CDR1: 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.

In another embodiment, the ETA antibody described herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

a. Light chain variable domain amino acid sequence: 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: 148(L6), SEQ ID NO: 150(L7), SEQ ID NO: 152(L8), SEQ ID NO: 154(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 at least 80%, at least 85%, and at least 90%, or at least 95% identical amino acid sequence; and

b. Heavy chain variable domain amino acid sequence: 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: 176 (H6), SEQ ID NO: 178 (H7), SEQ ID NO: 180 (H8), SEQ ID NO: 182 (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%, and at least 90%, or at least 95% identical amino acid sequence.

In another embodiment, the polynucleotide coding sequence of the ETA antibody described herein comprises one or two polynucleotide sequences, wherein each polynucleotide sequence is independently selected from the following Polynucleotide sequence:

a. Polynucleotide coding sequence of the light chain variable domain: 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 a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to it; and

b. Polynucleotide coding sequence of the heavy chain variable domain: 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 a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical thereto.

In another embodiment, the ETA antibody described herein comprises:

a. A light chain variable domain amino acid sequence independently selected from the following: 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: 148 (L6), SEQ ID NO: 150 (L7), SEQ ID NO: 152 (L8), SEQ ID NO: 154 (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 related An amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical; and

b. A heavy chain variable domain amino acid sequence independently selected from the following: 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: 176 (H6), SEQ ID NO: 178 (H7), SEQ ID NO: 180 (H8), SEQ ID NO: 182 (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 its Have at least 80%, at least 85%, at least 90%, or at least 95% identical amino acid sequence.

In another embodiment, the ETA antibody described herein comprises:

a. A light chain variable domain amino acid sequence independently selected from the following: 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: 148 (L6), SEQ ID NO: 150 (L7), SEQ ID NO: 152 (L8), SEQ ID NO: 154 (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

b. A heavy chain variable domain amino acid sequence independently selected from the following: 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: 176 (H6), SEQ ID NO: 178 (H7), SEQ ID NO: 180 (H8), SEQ ID NO: 182 (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).

In another embodiment, the ETA antibody described herein comprises a combination of light chain and heavy chain variable domain amino acid sequences independently selected from the following: 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: 172 (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: 152 and SEQ ID NO: 180 (L8H8), SEQ ID NO: 154 and SEQ ID NO: 182 (L9H9), SEQ ID NO: 156 and SEQ ID NO: 184 (L10H10), SEQ ID NO: 158 and SEQ ID NO: 186 ( L11H11), SEQ ID NO: 160 and SEQ ID NO: 188 (L12H12), SEQ ID NO: 162 and SEQ ID NO: 190 (L13H13), and SEQ ID NO: 164 and SEQ ID NO: 192 (L14H14). In another embodiment, the ETA antibody described herein comprises a combination of light chain and heavy chain variable domain amino acid sequences: SEQ ID NO: 162 and SEQ ID NO: 190 (L13H13).

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

In another embodiment, the ETA antibody described herein comprises a light chain variable region selected from L1-L14 or a heavy chain variable region selected from H1-H14 and fragments, derivatives, muteins, or variants thereof Of antibodies.

In another embodiment, the ETA antibody described herein comprises a combination of light chain and heavy chain CDR3 amino acid sequences independently selected from the following: 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: 180, SEQ ID NO: 154 and SEQ ID NO: 182, SEQ ID NO: 156 and SEQ ID NO: 184, SEQ ID NO: 158 And SEQ ID NO: 186, SEQ ID NO: 160 and SEQ ID NO: 188, SEQ ID NO: 162 and SEQ ID NO: 190, and SEQ ID NO: 164 and SEQ ID NO: 192.

In one embodiment, the ETA antibody described herein comprises the amino acid sequence of the light chain variable domain of SEQ ID NO: 138 or the amino acid sequence of the heavy chain variable domain of SEQ ID NO: 166. In another embodiment, the ETA antibody described herein comprises a combination of the amino acid sequence of the light chain variable domain of SEQ ID NO: 138 and the amino acid sequence of the heavy chain variable domain of SEQ ID NO: 166. In another embodiment, the ETA antibody described herein further comprises a constant amino acid sequence, wherein each constant amino acid sequence is independently selected from the following amino acid sequences: a. Light chain constant amino acid sequence: SEQ ID NO: 194 And SEQ ID NO: 196; and b. Heavy chain constant amino acid sequence: SEQ ID NO: 198 and SEQ ID NO: 221.

In another embodiment, the ETA antibody described herein further comprises a constant amino acid sequence, wherein each constant amino acid sequence is independently selected from a combination of the light chain and heavy chain constant amino acid sequences listed below:

a. The combination of the light chain constant amino acid sequence SEQ ID NO: 194 and the heavy chain constant amino acid sequence SEQ ID NO: 198;

b. The combination of the light chain constant amino acid sequence SEQ ID NO: 194 and the heavy chain constant amino acid sequence SEQ ID NO: 221;

c. The combination of the light chain constant amino acid sequence SEQ ID NO: 196 and the heavy chain constant amino acid sequence SEQ ID NO: 198;

d. The combination of the light chain constant amino acid sequence SEQ ID NO: 196 and the heavy chain constant amino acid sequence SEQ ID NO: 221.

In another embodiment, the amino acid sequences of the light chain and the heavy chain of the ETA antibody described herein are SEQ ID NO: 222 and SEQ ID NO: 236, respectively.

In one embodiment, the antibodies described herein comprise the amino acid sequences of the light chain and heavy chain CDRs and FRs (framework) listed herein. In one embodiment, the antibody comprises the light chain CDR1 sequence listed herein. In another embodiment, the antibody comprises the light chain CDR2 sequence listed herein. In another embodiment, the antibody comprises the light chain CDR3 sequence listed herein. In another embodiment, the antibody comprises the heavy chain CDR1 sequence listed herein. In another embodiment, the antibody comprises the heavy chain CDR2 sequence listed herein. In another embodiment, the antibody comprises the heavy chain CDR3 sequence listed herein. In another embodiment, the antibody comprises the light chain FR1 sequence listed herein. In another embodiment, the antibody comprises the light chain FR2 sequence listed herein. In another embodiment, the antibody comprises the FR3 sequence of the light chain listed herein. In another embodiment, the antibody comprises the light chain FR4 sequence listed herein. In another embodiment, the antibody comprises the heavy chain FR1 sequence listed herein. In another embodiment, the antibody comprises the FR2 sequence of the heavy chain listed herein. In another embodiment, the antibody comprises the heavy chain FR3 thick array listed herein. In another embodiment, the antibody comprises the heavy chain FR4 sequence listed herein.

In one embodiment, the CDR3 sequence of the antibody differs from the combination of the light and heavy chain CDR3 amino acid sequence SEQ ID NO: 50 and SEQ ID NO: 116 listed herein by no more than 6, 5, 4, 3, 2, or 1 Single amino acid addition, substitution and/or deletion. In another embodiment, the light chain CDR3 sequence of the antibody does not differ from the light chain CDR3 amino acid sequence SEQ ID NO: 50 listed herein by more than 6, 5, 4, 3, 2, or 1 single amino acid addition, substitution and / Or missing. In another embodiment, the light chain CDR3 sequence of the antibody does not differ from the light chain CDR3 amino acid sequence SEQ ID NO: 50 listed herein by more than 6, 5, 4, 3, 2, or 1 single amino acid addition, substitution and / Or missing, and the heavy chain CDR3 sequence of the antibody differs from the heavy chain CDR3 amino acid sequence SEQ ID NO: 116 or SEQ ID NO: 118 listed herein by no more than 6, 5, 4, 3, 2, or 1 single Amino acid additions, substitutions and/or deletions. In another embodiment, the antibody further comprises 1, 2, 3, 4, 5, or 6 combinations of light and heavy chain CDR sequences listed herein. In another embodiment, the antibody further comprises a combination of 1, 2, 3, 4, 5, or 6 CDR light and heavy chain sequences, each of which is uniquely linked to the light and heavy chain CDR3 amino acid sequence SEQ ID NO: 50 listed herein. The combination of SEQ ID NO: 116 differs by no more than 6, 5, 4, 3, 2, or 1 single amino acid at most. In another embodiment, the antibody comprises the CDRs of the light chain variable region and the CDRs of the heavy chain variable region listed herein. In another embodiment, the antibody comprises a combination of 1, 2, 3, 4, 5, and/or 6 CDR light and heavy chain sequences listed herein.

In one embodiment, the antibody (eg, antibody or antibody fragment) comprises the L1 light chain variable domain sequence listed herein. In one embodiment, the light chain variable domain comprises the same light chain variable domain sequence as L1, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3. , 2, or 1 amino acid difference amino acid sequence, wherein each difference in the sequence is independently a deletion, insertion or substitution of an amino acid residue. In another embodiment, the light chain variable domain comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, The amino acid sequence is at least 97%, or at least 99% identical. In another embodiment, the light chain variable domain polynucleotide coding sequence comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90% of the L1 polynucleotide coding sequence. %, at least 95%, at least 97%, or at least 99% identical nucleotide coding sequence. In another embodiment, the light chain variable domain polynucleotide coding sequence comprises a polynucleus that hybridizes to the complementary sequence of the polynucleotide coding sequence of the light chain variable domain of L1 under moderate conditions Nucleotide sequence. In another embodiment, the polynucleotide coding sequence of the light chain variable domain comprises a polymer that hybridizes to the complementary sequence of the polynucleotide coding sequence of the light chain variable domain of L1 under stringent conditions. Nucleotide sequence.

In one embodiment, the antibody (eg, antibody or antibody fragment) comprises the H1 heavy chain variable domain sequence listed herein. In another embodiment, the variable domain comprises a heavy chain variable domain sequence selected from H1. There are 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 , 2, or 1 amino acid difference amino acid sequence, wherein each difference in the sequence is independently a deletion, insertion or substitution of an amino acid residue. In another embodiment, the heavy chain variable domain comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, H1, and the sequence of the heavy chain variable domain. The amino acid sequence is at least 97%, or at least 99% identical. In another embodiment, the heavy chain variable domain polynucleotide coding sequence comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90% of the H1 polynucleotide coding sequence. %, at least 95%, at least 97%, or at least 99% identical nucleotide coding sequence. In another embodiment, the heavy chain variable domain polynucleotide coding sequence comprises a polynucleotide that hybridizes to the complementary sequence of the polynucleotide coding sequence of the heavy chain variable domain of H1 under moderately stringent conditions. Nucleotide sequence. In one embodiment, the heavy chain variable domain polynucleotide coding sequence comprises a polynucleotide that hybridizes to the complementary sequence of the polynucleotide coding sequence of the heavy chain variable domain of H1 under stringent conditions Acid sequence.

In one embodiment, the antibodies described herein include antibodies comprising the combination L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, or L14H14, or the desired phenotype (For example, IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE and IgD), or Fab or F(ab') ₂ fragments thereof.

In one embodiment, the antibodies described herein include antibodies comprising the combination L1H1, or antibodies with class switching (eg, IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE, and IgD), or Fab or F ( ab') ₂ fragments.

The antibodies (e.g., antibodies, antibody fragments, and antibody derivatives) described herein may comprise any of the constant regions known in the art. The light chain constant region may be, for example, a kappa or lambda type light chain constant region, such as a mouse kappa or lambda type light chain constant region. The heavy chain constant region can be, for example, an alpha, delta, epsilon, gamma, or mu type heavy chain constant region, such as a mouse alpha, delta, epsilon, gamma, or mu type heavy chain constant region. In one embodiment, the light or heavy chain constant region is a fragment, derivative, variant, or mutein of the natural constant region.

In one embodiment, the antibodies described herein further comprise constant light chain kappa or lambda domains or fragments of these. The light chain constant region sequences and their polynucleotide coding sequences are provided as follows: polynucleotide (κ), (SEQ ID NO: 193); amino acid (κ), (SEQ ID NO: 194); multiple Polynucleotide (λ), (SEQ ID NO: 195); Amino acid (λ), (SEQ ID NO: 196).

In another embodiment, the antibody described herein further comprises a heavy chain constant domain or fragment thereof. The heavy chain constant region sequence and its polynucleotide coding sequence are provided as follows: polynucleotide (IgG4), (SEQ ID NO: 197); amino acid (IgG4), (SEQ ID NO: 198).

In one embodiment, the ETA antibodies described herein are selected from murine antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, and double-chain antibodies , Tri-chain antibodies, tetra-chain antibodies, Fab fragments, F(ab') _x fragments, domain antibodies, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, and IgG4 antibodies. In another embodiment, the ETA antibody described herein is an ETA monoclonal antibody. In another embodiment, the ETA antibody described herein is a murine ETA antibody. The ETA antibody described herein is a humanized ETA antibody.

In one embodiment, the ETA antibody described herein is monoclonal antibody A-1 (which includes SEQ ID NO: 138 and SEQ ID NO: 166), A-7 (which includes SEQ ID NO: 150 and SEQ ID NO :178), A-8 (which includes SEQ ID NO: 152 and SEQ ID NO: 180), A-9 (which includes SEQ ID NO: 154 and SEQ ID NO: 182), A-10 (which includes SEQ ID NO: 156 and SEQ ID NO: 184), A-11 (which includes SEQ ID NO: 158 and SEQ ID NO: 186), A-12 (which includes SEQ ID NO: 160 and SEQ ID NO: 188), A -13 (which includes SEQ ID NO: 162 and SEQ ID NO: 190), or A-14 (which includes SEQ ID NO: 164 and SEQ ID NO: 192).

Antibodies and antibody fragments

In one embodiment, the antibodies described herein are complete antibodies (including polyclonal, monoclonal, chimeric, humanized, or human antibodies with full-length heavy and/or light chains). In another embodiment, the antibodies described herein are antibody fragments, such as F(ab') ₂ , Fab, Fab', Fv, Fc, or Fd fragments, and can be integrated into single domain antibodies, single chain antibodies , Maxibodies, minibodies, intrabodies, two-chain antibodies, three-chain antibodies, four-chain antibodies, v-NAR and bis-scFv (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology 23:1126-1136). In another embodiment, the antibodies described herein also include antibody polypeptides such as those disclosed in US Patent No. 6,703,199, including fibronectin polypeptide single antibodies. In another embodiment, the antibodies described herein also include other antibody polypeptides disclosed in U.S. Patent Application Publication No. US2005/0238646, which are single-chain polypeptides.

In one embodiment, nucleotide primers are used to amplify variable regions of genes expressing related monoclonal antibodies in hybridomas. These primers can be synthesized by those of ordinary skill in the art or purchased from commercial sources (see, for example, Stratagene, La Jolla, California). These manufacturers sell murine and human variable region primers including V _Ha , V _Hb , V _Hc , V _Hd , C _H1, primer C _L and V _L regions. These primers can be used to amplify the variable region of the heavy chain or the light chain, and then insert them into a vector such as IMMUNOZAPTMH or ILLLFFLUNOZAPTML (Stratagene), respectively. These vectors are then introduced into E. coli, yeast or mammal-based expression systems. These methods may be used to produce large amounts comprising V _H and V _L, domain fused single-chain protein (see Bird et al., 1988, Science242: 423-426).

Once cells producing antibodies according to this document are obtained using any of the above-mentioned immunization and other techniques, specific antibody genes can be cloned by isolating and amplifying DNA or mRNA from them according to standard methods described herein. To sequence the antibodies produced therefrom and identify the CDRs, the DNA encoding the CDRs can be manipulated as previously described to generate other antibodies according to the description herein.

The antibodies described herein are preferably used in the cell-based assays described herein and/or in the in vivo assays described herein to modulate endothelin signaling and/or cross-block the binding and/or via of one of the antibodies described herein. One of the antibodies described in this application is cross-blocked by binding ETA. Therefore the assay described herein can be used to identify such binding agents.

In some embodiments, the antibodies described in this application and/or cross-blocking the antibodies described herein are first identified in the cell-based and/or in vivo assays described herein that bind to and/or neutralize and/or cross-block the cells overexpressing ETA One of the antibodies described in this application is bound to an antibody cross-blocked by ETA to generate an antibody.

Those skilled in the art should understand that some proteins, such as antibodies, may undergo various post-transcriptional modifications. The type and extent of these modifications depend on the host cell line used to express the protein and the culture conditions. Such modifications include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. The frequent modification of the action of carboxypeptidase leads to the loss of carboxy-terminal basic residues (such as lysine or arginine) (as described in Harris, 1995, Journal of Chromatography 705:129-134).

An alternative method of producing murine monoclonal antibodies is to inject hybridoma cells into the peritoneal cavity of syngeneic mice, such as mice that have been treated (eg, pristane primary immunization) to promote the formation of ascites containing monoclonal antibodies. Monoclonal antibodies can be separated and purified by a variety of established techniques. Such separation techniques include affinity chromatography using protein A-Sepharose, size exclusion chromatography and ion exchange chromatography. See, for example, Coligan on pages 2.7.1-2.7.12 and 2.9.1-2.9. 3 pages; Baines et al., "Purification of Immunoglobulin G(IgG)," Methods in Molecular Biology, Volume 10, Pages 79-104 (The Humana Press, Inc., 1992). Monoclonal antibodies can be purified by affinity chromatography using appropriate ligands screened based on the specific properties of the antibody (eg, heavy chain or light chain isotype, binding specificity, etc.). Examples of suitable ligands immobilized on a solid carrier include protein A, protein G, anti-constant region (light or heavy chain) antibodies, anti-idiotypic antibodies, and TGF-p binding protein or fragments or variants thereof.

Molecular evolution of complementarity determining regions (CDRs) in the center of the antibody binding site can be used to isolate antibodies with increased affinity, such as antibodies with increased affinity for c-erbB-2, such as Schier et al., 1996, J. Mol. Biol. .263:551-567. Therefore, this type of technology can be used to prepare human endothelin receptor antibodies. For example, antibodies against human endothelin receptors can be used in in vitro or in vivo assays to detect the presence of endothelin receptors.

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

Antibodies can be prepared by any known technique and screened for desired properties. Some techniques involve isolating nucleic acids encoding the polypeptide chains (or parts thereof) of related antibodies (eg, anti-endothelin receptor antibodies) and manipulating the nucleic acids by recombinant DNA technology. The nucleic acid can be fused with another related nucleic acid or modified (for example by mutagenesis or other conventional techniques) to add, delete or replace one or more amino acid residues.

When it is necessary to improve the affinity of an antibody containing one or more of the above-mentioned CDRs according to this document, a variety of affinity maturation schemes can be adopted including maintaining the CDRs (Yang et al., 1995, J. Mol. Biol. 254:392-403), Strand replacement (Marks et al., 1992, Bio/Technology 10:779-783), using mutant strains of E. coli (Low et al., 1996, J. Mol. Biol. 250: 350-368) DNA rearrangement (Patten et al., 1997 , Curr. Opin. Biotechnol. 8: 724-733), phage display (Thompson et al., 1996, J. Mol. Biol. 256: 7-88) and other PCR techniques (Crameri et al., 1998, Nature 391: 28 8-- 291). All these affinity maturation methods are discussed in Vaughan et al., 1998, Nature Biotechnology 16:535-539.

In one embodiment, the antibody described herein is an anti-endothelin receptor fragment. 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, diabodies, tri-chain antibodies, quadru-chain antibodies, and domain antibodies. Other examples are provided in Lunde et al., 2002, Biochem. Soc. Trans. 30:500-06.

The heavy chain and light chain variable domains (Fv region) can be connected via an amino acid bridge (short peptide linker) to form a single chain antibody, thereby obtaining a single polypeptide chain. It has DNA encoding a peptide linker between DNAs by fusing two coding variable domain polypeptides (V _L and V _H) for preparing the single-chain Fvs (scFvs). The resulting polypeptides can fold back to themselves to form antigen-binding monomers, or they can form multimers (for example, dimers, trimers, or tetramers), depending on the length of the flexible linker between the two variable domains (Kortt et al., 1997, Prot. Eng. 10:423; Kortt et al. 2001, Biomol. Eng. 18:95-108). By combining different polypeptide comprising a V _L and V _H, scFvs binding member may be formed with a plurality of different phenotypes (Kriangkum et, 2001, Biomol.Eng.18: 31-40). The technologies developed for the production of single-chain antibodies include US 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 -546; those described in deGraaf et al., 2002, Methods Mol. Biol. 178:379-87. Single-chain antibodies derived from the antibodies described herein include, but are not limited to, scFvs comprising the variable domain combination L1H1, all of which are encompassed herein.

Antigen-binding fragments derived from antibodies can also be obtained by proteolysis of antibodies, for example, pepsin or papain digestion of intact antibodies according to traditional methods. For example, the antibody can be cleaved with pepsinase to provide an SS fragment called F(ab') ₂ to produce antibody fragments. This fragment can be further cleaved using a sulfhydryl reducing agent to produce a 3.5S Fab' monovalent fragment. Alternative schemes include the use of a sulfhydryl protecting group for the cleavage reaction to obtain the cleavage of the disulfide bond; in addition, the enzymatic cleavage of papain can be used to directly generate two monovalent Fab fragments and one Fc fragment. These methods are described in, for example, Goldenberg, U.S. Patent No. 4,331,647, Nisonoff et al., 1960, Arch. Biochem. Biophys. 89:230; Porter, 1959, Biochem. J. 73:119; Edelman et al., Methods in Enzymology 1:422 (Academic Press, 1967); and Andrews and Titus, JA Current Protocols in Immunology (Coligan et al. editors, John Wiley & Sons, 2003), pages 2.8, 1-2.8.10 and pages 2.10A.1-2.10A.5. Other methods of cleaving antibodies, such as preparing heavy chains to form monovalent heavy and light chain fragments (Fd), further cleaving the fragments or other enzymatic, chemical or genetic techniques may also be used, as long as the fragments bind to an antigen that can be recognized by the intact antibody.

Another form of antibody fragments are peptides that contain one or more antibody complementarity determining regions (CDRs). CDRs can be obtained by constructing polypeptides encoding relevant CDRs. For example, such polypeptides can be prepared by synthesizing variable regions using polymerase chain reaction using mRNA of antibody-producing cells as a template, see, for example, Larrick et al., 1991, Methods: A Companion to Methods in Enzymology 2:106; Courtenay-Luck "Genetic Manipulation of Monoclonal Antibodies," Monoclonal Antibodies: Production, Engineering and Clinical Application, edited by Ritter et al., page 166 (Cambridge University Press, 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," Monoclonal Antibodies: Principles and Applications, edited by Birch et al., page 137 (Wiley-Liss, Inc., 1995). The antibody fragment may further comprise at least one variable domain of the antibody described herein. Thus, for example, V region domain may be monomeric and a V _H or V _L domain, which may be as follows at least equal to 1x10 ^-7 M or less independent binding affinity endothelin receptor hereinbefore described.

The variable region domain can be any natural variable domain or its genetically engineered form. The form of genetic engineering refers to the variable region domain produced by recombinant DNA engineering technology. The genetic engineering form includes, for example, the production from the variable region of the specific antibody by inserting, deleting or changing the amino acid sequence of the specific antibody. Specific examples include variable region domains comprising only one CDR and optionally one or more framework amino acids from one antibody and the remaining part of the variable region domain from another antibody and assembled by genetic engineering.

The variable region domain may be covalently linked to at least one other antibody domain or fragment thereof at the C-terminal amino acid. Thus, for example, present in the variable domain V _H region domain may be linked to an immunoglobulin C _H1 domain or fragment thereof. Similarly, V _L domain may be attached to C _K domain or fragment thereof. In this manner, for example, the antibody may be a Fab fragment wherein the antigen binding domain comprises a V _H and V _L, United domains thereof are connected to the C-terminus and C _K C _Hl domain covalently. Other amino acids can be used to extend the C _H1 domain, for example to provide a hinge region or as Fab 'portions of the hinge domain fragment or provide other domains, such as an antibody C _H2 and C _H3 domains.

Derivatives and variants of antibodies

For example, the nucleotide sequence L1 and H1 encoding the amino acid sequence A-1 can be changed by random mutagenesis or by site-directed mutagenesis (e.g., oligonucleotide-induced site-directed mutagenesis) to produce a non-mutated polynucleoside. An acid phase is an altered polynucleotide that includes one or more specific nucleotide substitutions, deletions, or insertions. Examples of techniques used to produce such changes are described in Walder et al., 1986, Gene 42:133; Bauer et al., 1985, Gene 37:73; Craik, 1985, BioTechniques, 3:12-19; Smith et al., 1981, Genetic Engineering : Principles and Methods, Plenum Press; and US patent numbers US4,518,584 and US4,737,462. These and other methods can be used to produce, for example, anti-endothelial agents that have desirable properties, such as enhanced affinity, affinity, or specificity for endothelin receptors, enhanced in vivo or in vitro activity or stability, or reduced side effects in vivo compared to underivatized antibodies. Derivatives of receptor antibodies.

Other anti-endothelin receptor antibody derivatives in this field include covalent or aggregated conjugates of anti-endothelin receptor antibodies or fragments thereof and other proteins or polypeptides, for example, by expressing the N-terminal of the anti-endothelin receptor antibody polypeptide Or a recombinant fusion protein of a heterologous polypeptide fused to the C-terminal. For example, the binding peptide may be a heterologous signal (or guide) polypeptide, such as a yeast alpha factor leader peptide or a peptide such as an epitope tag. The antibody containing the fusion protein may contain peptides (e.g., polyhistidine) added to assist in the purification or identification of the antibody. Antibodies can also be linked to FLAG peptides as described in Hopp et al., 1988, Bio/Technology 6:1204 and US Patent No. 5,011,912. FLAG peptides have high antigenicity and provide epitopes that can be reversibly bound by specific monoclonal antibodies (mAbs), allowing rapid detection and convenient purification of expressed recombinant proteins. Commercially available (Sigma-Aldrich, St. Louis, MO) reagents for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide. In another embodiment, oligomers containing one or more antibodies are available As an endothelin receptor antagonist or higher oligomers. The oligomer may be in the form of covalently linked or non-covalently linked dimers, trimers or higher oligomers. Oligo species containing two or more antibodies can be used, one example of which is a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers and the like.

One embodiment is directed to oligomers comprising multiple antibodies, which are linked by covalent or non-covalent interactions between the peptide moieties fused to the antibody. Such peptides can be peptide linkers (spacers) or peptides with the property of promoting oligomerization. Leucine zippers and certain antibody-derived polypeptides are peptides that can promote antibody oligomerization, as described in detail below.

In a specific embodiment, the oligomer contains two to four antibodies. The oligomeric antibody can be in any form, as described above, such as variants or fragments. Preferably, the oligomer comprises an antibody having endothelin receptor binding activity.

In one embodiment, oligomers are prepared using polypeptides derived from immunoglobulins. The preparation of heterologous polypeptides containing some fusions to different parts of antibody-derived polypeptides (including Fc domains) has been described in, for example, Ashkenazi et 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 a dimer comprising two fusion proteins produced by fusing an endothelin binding fragment of an anti-endothelin receptor antibody with the Fc region of the antibody. The dimer can be prepared by the following methods: for example, inserting the gene fusion encoding the fusion protein into an appropriate expression vector, expressing the fusion gene in a host cell transformed with the recombinant expression vector and allowing the expressed fusion protein to assemble like an antibody molecule, The interchain disulfide bonds between the Fc parts form dimers.

The term "Fc polypeptide" as used herein includes native and mutein forms of polypeptides derived from the Fc region of antibodies. Also included are truncated forms of such polypeptides that include hinge regions that promote dimerization. The fusion protein containing the Fc portion (and the oligomer formed therefrom) provides the advantage of easy purification by affinity chromatography on a protein A or protein G column.

One suitable Fc polypeptide in PCT application W093/10151 (incorporated herein by reference) is a single chain polypeptide that extends from the N-terminal hinge region to the natural C-terminal end of the Fc region of a human IgG1 antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Patent No. 5,457,035 and Baum et al., 1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutant protein is identical to that of the natural Fc sequence shown in W093/10151, except that amino acid 19 is changed from leucine to alanine, amino acid 20 is changed from leucine to glutamine, and amino acid 22 is changed from glycine. Become alanine. This mutant protein exhibits reduced affinity for Fc receptors. In other embodiments, the heavy chain and/or light chain of the anti-endothelin receptor antibody may be substituted with the variable part of the antibody heavy chain and/or light chain.

Alternatively, the oligomer is a fusion protein containing multiple antibodies, with or without spacer peptides. These suitable linker peptides are described in US Patent Nos. US 4,751,180 and US 4,935,233.

Another method of making oligomeric antibodies involves the use of leucine zippers. Leucine zipper domains are peptides that promote the oligomerization of their existing proteins. Leucine zippers were originally found to exist in several DNA-binding proteins (Landschulz et al., 1988, Science 240:1759), and later found to exist in various proteins. Among the known leucine zippers are natural peptides or derivatives thereof that can be dimerized or trimerized. An example of a leucine zipper domain suitable for the production of soluble oligomeric proteins is described in PCT application WO94/10308, and a leucine zipper derived from lung surfactant protein D (SPD) is described in Hoppe Zheng, 1994, FEBS Letters 344 :191, incorporated in this article as a reference. The use of modified leucine zippers that allow stable trimerization of heterologous proteins fused to it is described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In one method, a recombinant fusion protein containing an anti-endothelin receptor antibody fragment or derivative fused to a leucine zipper peptide is expressed in an appropriate host cell, and the soluble oligomeric anti-endothelial is collected from the culture supernatant Receptor antibody fragments or derivatives thereof.

In another embodiment, the antibody derivative may comprise at least one of the CDRs disclosed herein. For example, one or more CDRs can be integrated into known antibody framework regions (IgG1, IgG2, etc.) or combined with an appropriate carrier to enhance its half-life. Suitable carriers include, but are not limited to, Fc, albumin, transferrin and similar substances. These and other suitable vectors are known in the art. The binding CDR peptide can be monomer, dimer, tetramer or other forms. In one embodiment, one or more water-soluble polymers are bound at one or more specific sites of the binding agent, for example at the amino terminus. In one example, the antibody derivative contains one or more water-soluble polymer attachments including but not limited to polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, for example, US Patent Nos. US4,640,835, US4,496,689, US4,301,144, US4,670,417, US4,791,192 and US4,179,337. In some embodiments, the derivative contains one or more monomethoxy groups. Ethylene glycol, dextran, cellulose or other carbohydrate-based polymers, poly(N-vinylpyrrolidone). Polyethylene glycol, polyoxyethylene polyol (such as glycerin) and polyvinyl alcohol, and mixtures of such polymers. In some embodiments, one or more water-soluble polymers are randomly associated with one or more side chains. In some embodiments. PEG can enhance the therapeutic effect of binding agents such as antibodies. Some such methods are described in, for example, U.S. Patent No. 6,133,426, which is incorporated herein by reference for any purpose.

It should be understood that the antibodies described herein may have at least one amino acid substitution, as long as the antibody retains binding specificity. Therefore, modification of antibody structure is included in the scope of this article. These may include amino acid substitutions that do not destroy the antibody's endothelin receptor binding ability, which may be conservative or non-conservative. Conservative amino acid substitutions may include unnatural amino acid residues, which are usually integrated by chemical peptide synthesis rather than synthesis by biological systems. These include peptidomimetic and other amino acid portions in reverse or inverted forms. Conservative amino acid substitutions can also involve replacing natural amino acid residues with unnatural residues so that the polarity or charge of the amino acid residues at the site has little or no effect. Non-conservative substitutions may involve the exchange of a member of one class of amino acids or amino acid analogs with a member of another class of amino acids having different physical properties (eg, volume, polarity, hydrophobicity, charge).

Moreover, those skilled in the art can generate test variants containing amino acid substitutions at each desired amino acid residue. Such variants can be screened using activity assays known to those skilled in the art. Such variants can be used to collect information about appropriate variants. For example, if it is found that a certain amino acid residue can cause activity destruction, undesired reduction, or inappropriate activity, variants with such changes can be avoided. In other words, based on the information collected from these routine experiments, those skilled in the art can easily determine which amino acids should be avoided for further substitution (alone or in combination with other mutations).

The skilled person can use known techniques to determine appropriate variants of the polypeptides listed herein. In some embodiments, those skilled in the art can identify appropriate regions of the molecule that will not destroy the activity after modification by targeting regions that are not important for activity. In some embodiments, residues or portions of molecules that are conserved among similar polypeptides can be identified. In some embodiments, even regions that are important for biological activity or structure can be conservatively replaced without destroying biological activity or adversely affecting the polypeptide structure. In addition, those skilled in the art can investigate structure and function studies to identify residues in similar polypeptides that are important for activity or structure. In view of this comparison, the importance of amino acid residues in a protein corresponding to amino acid residues important for activity or structure in similar proteins can be predicted. Those skilled in the art can select chemically similar amino acid substitutions for these predicted important amino acid residues.

Those skilled in the art can also analyze the three-dimensional structure and amino acid sequence related to the structure of similar polypeptides. In view of this type of information, those skilled in the art can predict the alignment of the amino acid residues of the antibody in terms of three-dimensional structure. In some embodiments, those skilled in the art may choose not to make significant changes to the predicted amino acid residues on the surface of the protein, because such residues may be involved in important interactions with other molecules. Many scientific publications are devoted to the prediction of secondary structure. See, Moult, 1996, Curr. Op. Biotech. 7:422-427, Chou et al., 1974, Biochemistry 13:222-245; Chou et al., 1974, Biochemistry 113:211-222; Chou et al., 1978, Adv. Enzymol Relat. Areas Mol. Biol. 47: 45-148; Chou et al., 1979, Ann. Rev. Biochem. 47: 251-276 and Chou et al., Biophys. J. 26: 367-384. In addition, computer programs are currently available to assist in predicting secondary structure. For example, two polypeptides or proteins with sequence identity greater than 30% or similarity greater than 40% usually have similar high-level structures. The recent growth of the protein structure database (PDB) has enhanced the predictability of secondary structure, including the number of potential folds in the structure of a polypeptide or protein. See, Holm et al., 1999, Nucl. Acid. Res. 27:244-247. It has been shown (Brenner et al., 1997, Curr. Op. Struct. Biol. 7: 369-376) that there are a limited number of folds in a given polypeptide or protein and once a critical number of structures are determined, the structure prediction will become significantly more accurate. Other methods for predicting secondary structure include "threading" (Jones, 1997, Curr. Opin. Struct. Biol., 7:377-87; Sippl et al., 1996, Structure 4:15-19; "Atlas analysis (profile analysis)" (Bowie et al., 1991, Science 253: 164-170; Gribskov et al., 1990, Meth. Enzym. 183: 146-159; Gribskov et al., 1987, PNAS USA 84: 4355-4358 and "Evolutionary Links ( evolutionary linkage)” (see Holm, supra (1999), and Brenner, supra (1997)). In some embodiments, antibody variants include glycosylation variants in which the amino acid sequence of the parent polypeptide changes the sugar The number and/or type of sylation sites. In some embodiments, the variant has a greater or lesser number of N-linked glycosylation sites than the native protein. Alternatively, substitutions that remove the sequence can remove Existing N-linked sugar chains. Rearrangements of N-linked sugar chains are also provided, in which one or more N-linked sugar chain sites (usually those that occur naturally) are removed and one or more new N-linked sugar chains are created. Linkage site. Other preferred antibody variants include cysteine variants in which one or more cysteine residues are deleted or replaced by another amino acid (such as serine) compared to the parent amino acid sequence. When the antibody must be folded Cysteine variants can be used in a biologically active conformation (for example, after isolation of soluble inclusion bodies). Cysteine variants usually have fewer cysteine residues than natural proteins, and usually have an even number of halves. Cystine to minimize the interaction caused by unpaired cysteine.

Those skilled in the art can determine the desired amino acid substitution (conservative or non-conservative) when such substitutions are required. In some embodiments, amino acid substitutions can be used to identify important residues of human endothelin receptor antibodies or to increase or decrease the affinity of the human endothelin receptor antibodies described herein. According to some embodiments, the preferred amino acid substitutions are as follows: (1) reduce proteolytic sensitivity, (2) reduce oxidation sensitivity, (3) change the binding affinity for forming protein complexes, (4) change the binding affinity and/or (4) Endow or modify other physicochemical or functional properties on this type of polypeptide. According to some embodiments, single or multiple amino acid substitutions (conservative amino acid substitutions in some embodiments) can be made in a naturally occurring sequence (in some embodiments, the part of the polypeptide outside the domain that forms intermolecular contacts) . In some embodiments, conservative amino acid substitutions generally do not substantially change the structural properties of the parent sequence (for example, the replacement amino acid should not break the helix present in the parent sequence or interfere with other types of secondary structure that characterize the parent sequence). Examples of the secondary and tertiary structures of polypeptides recognized in the art are described in Proteins, Structures and Molecular Principles, edited by Creighton, WH Freeman and Company (1984); Introduction to Protein Structure, edited by Brand and Tooze, edited by Garl and Publishing (1991); And Thornton et al., 1991, Nature 354:105, which is incorporated herein by reference.

In some embodiments, the antibodies described herein may be chemically bonded to multimers, lipids, or other moieties. The antigen binding reagent may comprise at least one CDRs described herein, which is incorporated into a biocompatible backbone structure. In one example, the biocompatible framework structure comprises a polypeptide or a portion thereof sufficient to form a conformationally stable structure support or a framework or scaffold, which can display one or more amino acid sequences (e.g., , CDRs, variable regions, etc.). This type of structure can be a naturally occurring polypeptide or polypeptide "folding" (structural motif), or it can have one or more modifications, such as amino acid addition, deletion or substitution, relative to the natural polypeptide or folding. These scaffolds can be derived from polypeptides of any species (or more than one species), for example, humans, other mammals, other vertebrates, invertebrates, bacteria, or viruses. The biosoluble backbone structure is usually based on a protein scaffold or backbone rather than an immunoglobulin domain. For example, can be used based on fibronectin, ankyrin, lipocalin (lipocalin), new tumor suppressor protein, cytochrome b, CP1 zinc finger protein, PST1, coiled coil, LACI-D1, Z domain and starch Aprotinin domain (see, for example, Nygren and Uhlen, 1997, Current Opinion in Structural Biology 7:463-469).

In addition, those skilled in the art can recognize that suitable binding agents include portions of these antibodies, such as one or more heavy chain CDR1, CDR2, CDR3, light chain CDR1, CDR2, and CDR3, as specifically disclosed herein. At least one of the heavy chain CDR1, CDR2, CDR3, CDR1, CDR2 and CDR3 regions have at least one amino acid substitution, as long as the antibody retains the binding specificity of the non-substituted CDR. The non-CDR portion of the antibody may be a non-protein molecule, wherein the binding agent cross-blocks the binding of the antibody disclosed herein to human ETA and/or inhibits endothelin signaling via the receptor. The non-CDR portion of the antibody can be a non-protein molecule, wherein the antibody exhibits a binding type similar to that of at least one of the antibodies A-1/A-2 in a competitive binding assay with human ETA peptide, and/or Neutralizes the activity of endothelin. The non-CDR portion of an antibody may be composed of amino acids, wherein the antibody is a recombinant binding protein or a synthetic peptide, and the recombinant binding protein cross-blocks the binding of the antibody disclosed herein to human ETA and/or neutralizes endothelin activity in vivo or in vitro. The non-CDR portion of the antibody may be composed of amino acids, where the antibody is a recombinant antibody, and the recombinant antibody shows a binding to human ETA peptide similar to that shown by at least one of the antibodies A-1/A-2 in a competitive binding assay Type, and/or neutralize endothelin signaling.

Nucleic Acid

In one aspect, provided herein is an isolated nucleic acid molecule. The nucleic acid molecule comprises, for example, a polynucleotide encoding all or part of an antibody, such as one or two chains of the antibody herein, or a fragment, derivative, mutein or variant thereof; a polymer sufficient for use as a hybridization probe Nucleotides; PCR primers or sequencing primers used to identify, analyze, mutate or amplify polynucleotides encoding polypeptides; antisense nucleic acids used to inhibit the expression of polynucleotides and their complementary sequences. The nucleic acid can be any length. For example, their length can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides, and/or contain one or more additional sequences, such as regulatory sequences, and/or be part of a larger nucleic acid such as a vector. The nucleic acid may be single-stranded or double-stranded and include RNA and/or DNA nucleotides and artificial variants thereof (for example, peptide nucleic acids).

A nucleic acid encoding an antibody polypeptide (e.g., heavy or light chain, variable domain only, or full length) can be isolated from mouse B cells immunized with ETA antigen. The nucleic acid can be isolated by a conventional method such as polymerase chain reaction (PCR).

The nucleic acid sequences encoding the variable regions of the heavy and light chains are shown above. A skilled artisan can understand that due to the degeneracy of the genetic code, each polypeptide sequence disclosed herein can be encoded by a greater number of other nucleic acid sequences. Provided herein is each degenerate nucleotide sequence encoding the antibody described herein.

Further provided herein are nucleic acids that hybridize to other nucleic acids (e.g., nucleic acids comprising any nucleotide sequence of A-1/A-2) under specific hybridization conditions. Methods of hybridizing nucleic acids are well known in the art. See, for example, Current Protocols in Molecular Biology, John Wiley&Son (1989), 6.3.1-6.3.6. As defined herein, for example, medium stringent conditions use a pre-wash solution containing 5x sodium chloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), about 50% formamide hybridization buffer, Hybridization temperature of 6xSSC and 55°C (or other similar hybridization solution, for example, containing 50% formamide, hybridization at 42°C), and the elution condition is 60°C, using 0.5xSSC, 0.1% SDS. Stringent hybridization conditions were hybridized in 6xSSC at 45°C, and then washed one or more times in 0.1xSSC, 0.2% SDS at 68°C. In addition, those skilled in the art can manipulate hybridization and/or washing conditions to increase or decrease the stringency of hybridization such that they include nucleosides that are at least 65, 70, 75, 80, 85, 90, 95, 98, or 99% homologous to each other. The nucleic acids of the acid sequence can usually still hybridize to each other. The basic parameters that affect the selection of hybridization conditions and guidelines for designing appropriate conditions are listed in, for example, Sambrook, Fritsch, and Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Chapters 9 and 11; Current Protocols in Molecular Biology, 1995 , Edited by Ausubel et al., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4) and can be easily determined by a person with ordinary skill in the art based on, for example, the length and/or base composition of DNA. Mutations can be used to introduce changes in the nucleic acid, thereby causing changes in the amino acid sequence of the encoded polypeptide (eg, antigen binding protein). Any technique known in the art can be used to introduce mutations. In one embodiment, one or more specific amino acid residues are altered using, for example, a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are changed using, for example, a random mutagenesis protocol. Regardless of how it is produced, the mutant polypeptide can be expressed and screened for desired properties.

Mutations can be introduced into the nucleic acid without significantly changing the biological activity of the encoded polypeptide. For example, nucleotide substitutions that cause amino acid substitutions at non-essential amino acid residues can be made. In one embodiment, the nucleotide sequences provided herein for L-1 to L-2 and H-1 to H-2 or fragments, variants or derivatives thereof are mutated such that their codes include L-1 to L-2 as shown herein. One or more of the amino acid residues of L-2 and H-1 to H-2 are deleted or replaced to become two or more residues with different sequences. In another embodiment, mutagenesis inserts an amino acid near one or more amino acid residues of L-1 to L-2 and H-1 to H-2 shown herein to make two or more different sequences. Residues. Alternatively, one or more mutations can be introduced into the nucleic acid to selectively change the biological activity of the encoded polypeptide (for example, binding to ETA). For example, the mutation can alter biological activity quantitatively or qualitatively. Examples of quantitative changes include increasing, decreasing or eliminating the activity. Examples of qualitative changes include changing the antigen specificity of antibodies.

In another aspect, provided herein are nucleic acid molecules suitable for use as primers or hybridization probes for detecting nucleic acid sequences herein. The nucleic acid molecule herein may only comprise a part of the nucleic acid sequence encoding the full-length polypeptide herein, for example, a fragment that can be used as a probe or primer or a fragment that encodes an active portion of the polypeptide (e.g., an ETA binding portion). Probes based on the nucleic acid sequence herein can be used to detect the nucleic acid or similar nucleic acids, such as transcripts encoding the polypeptides herein. The probe may contain a labeling group, such as a radioisotope, fluorescent compound, enzyme, or enzyme cofactor. Such probes can be used to identify cells expressing the polypeptide.

In another aspect, provided herein is a vector comprising a nucleic acid encoding a polypeptide herein or a portion thereof. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors. The recombinant expression vector herein may comprise the nucleic acid herein in a form suitable for expression of the nucleic acid in a host cell. The recombinant expression vector includes one or more regulatory sequences, which are screened based on the host cell used for expression, and are operably linked to the pre-expressed nucleic acid sequence. Regulatory sequences include guiding nucleotide sequences for constitutive expression in multiple types of host cells (for example, SV40 early gene enhancer, Rous's sarcoma virus promoter and cytomegalovirus promoter), guiding only in certain hosts Expression of nucleotide sequences in cells (for example, tissue-specific regulatory sequences, see Voss et al., 1986, Trends Biochem.Sci. 11:287, Maniatis et al., 1987, Science 236:1237, the complete content of which is incorporated herein by reference ) And inducible expression of the nucleotide sequence in response to specific treatments or conditions (e.g., the metalthionine promoter in mammalian cells and the tet-sesponsive response in both prokaryotic and eukaryotic systems Promoter and/or streptomycin responsive promoter (same as before)). Those skilled in the art should understand that the design of the expression vector depends on factors such as the selection of the host cell used for transformation, the desired protein expression level and other factors. The expression vector herein can be introduced into a host cell, thereby producing the protein or peptide encoded by the nucleic acid described herein, including fusion protein or peptide.

In another aspect, provided herein is a host cell into which the expression vector herein can be introduced. The host cell can be any prokaryotic or eukaryotic cell. Prokaryotic host cells include Gram-negative or Gram-positive organisms, such as Escherichia coli or Bacillus. More advanced eukaryotic cells include insect cells, yeast cells, and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include Chinese Hamster Ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines grown in serum-free media (see Rasmussen et al., 1998, Cytotechnology 28:31) or CHO The strain DXB-11 lacks DHFR (see Urlaub et al., 1980, PNAS USA 77:4216-20). Other CHO cell lines include CHO-K1 (ATCC#CCL-61), EM9 (ATCC#CRL-1861), and UV20 (ATCC#CRL-1862), and other host cells include the COS-7 line of monkey kidney cells (ATCC# CRL-1651) (see Gluzman et al., 1981, Cell23:175), L cells, C127 cells, 3T3 cells (ATCCCCL-163), AM-1/D cells (described in U.S. Patent No. US6,210,924), HeLa cells, BHK (ATCCCRL-10) cell line, CV1/EBNA cell line (ATCCCCL-70) derived from the African green monkey kidney cell line CV1 (see McMahan et al., 1991, EMBO J. 10: 2821), human embryonic kidney cells such as 293 , 293EBNA or MSR293, human epithelial A431 cells, human C010205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro culture of primary tissues, primary transplants, HL-60, U937, HaK or Jurkat cells. Suitable cloning and expression vectors for bacteria, fungi, yeast and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, 1985).

The vector DNA can be introduced into prokaryotic or eukaryotic cells by traditional transformation or transfection techniques. For stable mammalian transfection, depending on the expression vector and transfection technique used, it is known that only a small percentage of cells can incorporate foreign DNA into their genome. In order to identify and screen these integrants, a gene encoding a selectable marker (for example, antibiotic resistance) is usually introduced into the host cell together with the gene of interest. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. In other methods, drug screening can be used to identify stably transfected cells containing the introduced nucleic acid (for example, cells integrated with the screening gene can survive, while other cells die).

The transformed cells can be cultured under conditions that increase the expression of the polypeptide, and the polypeptide can be recovered by conventional protein purification methods. One such purification method is described in the Examples below. Polypeptides intended for use herein include substantially homologous recombinant mammalian anti-endothelin receptor antibody polypeptides, which are substantially free of contaminating endogenous materials.

Antibody activity

In one embodiment, the antibodies described herein specifically bind to endothelin receptors, inhibit signal transduction, and exhibit therapeutic biological effects, such as reducing pulmonary hypertension in animal models. In another embodiment, the antibodies described herein are murine antibodies or humanized antibodies that can specifically bind to human endothelin receptors. Such antibodies include antagonistic or neutralizing antibodies that can reduce or neutralize endothelin signaling.

In one embodiment, the K _d of the antibody described herein when binding to human endothelin receptor ETA is approximately 0.01 nM to 1000 nM, 0.1 nM to 500 nM, 0.5 nM to 200 nM, 1 nM to 200 nM, or 10 nM to 100 nM. In another embodiment, the antibody described herein has a K _{d of} approximately 1 nM to 200 nM when binding to the human endothelin receptor ETA. In another embodiment, the antibody described herein has a K _{d of} approximately 10 nM to 100 nM when binding to the human endothelin receptor ETA. In another embodiment, the K _d of the antibody described herein when binding to human endothelin receptor ETA is approximately 1 nM, 2 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, Or 100nM.

In one embodiment, antibodies described herein in reducing the IC ₅₀ values of human endothelin signaling about 0.01nM to 500nM, 0.1nM to 200nM, 0.5nM to 200nM, 1nM to 200 nM, or 10nM to 100nM. In another embodiment, antibodies described herein in reducing the IC ₅₀ values of human endothelin signaling about 1nM to 200nM. In another embodiment, antibodies described herein in reducing the IC ₅₀ values of human endothelin signaling 10nM to about 100nM. In another embodiment, antibodies described herein in reducing human endothelin signaling IC ₅₀ value of about 1nM, 2nM, 5nM, 10nM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, or 100nM.

In one embodiment, the ETA antibodies described herein have one or more of the properties listed below:

a. When binding to human endothelin receptor ETA, its K _d is the same as or better than the reference antibody;

b. When inhibiting endothelin activation of human endothelin receptor ETA, its IC ₅₀ is the same as or better than the reference antibody; and

c. The ETA antibody cross-competes binding with the reference antibody on the human endothelin receptor ETA.

In another embodiment, the ETA antibody described herein is an antibody having one or more of the following properties:

a. When binding to human endothelin receptor ETA, its K _d is the same as or better than a reference ETA antibody;

b. When inhibiting endothelin activation of human endothelin receptor ETA, its IC ₅₀ is the same as or better than a reference ETA antibody; and

c. The ETA antibody cross-competes binding with a reference ETA antibody on the human endothelin receptor ETA.

In one aspect, the reference antibody comprises a combination of the light chain variable domain amino acid sequence of SEQ ID NO: 138 and the heavy chain variable domain amino acid sequence of SEQ ID NO: 166. In another aspect, the reference antibody is monoclonal antibody A-1, A-2, A-7, A-9, or A-12. As used herein, the term "substantially similar" means that the IC ₅₀ or K _d of the reference antibody is comparable to or approximately 200%, 180%, 160%, 150%, 140% of the IC ₅₀ or K _d value of the reference antibody. , 120%, 110%, 100%, 99%, 98%, 97%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, or 50%. In one embodiment, the reference antibody includes, for example, an antibody having a heavy chain and light chain combination L1H1 or L2H2. In another embodiment, the reference antibody includes ETA antibody A-1. In one embodiment, the ETA antibodies described herein can specifically bind to human endothelin receptors and can reduce pulmonary hypertension in animal models. In one embodiment, pulmonary hypertension is reduced by 2% compared to untreated animals. In another embodiment, the pulmonary hypertension is reduced by about 5% compared to untreated animals. In another embodiment, the pulmonary hypertension is reduced by about 10% compared to untreated animals. In another embodiment, the pulmonary hypertension is reduced by about 15% compared to untreated animals. In another embodiment, pulmonary hypertension is reduced by about 20% compared to untreated animals, and in another embodiment, pulmonary hypertension is reduced by about 25% compared to untreated animals. The amount of reduction in pulmonary hypertension is controlled by the dose. For animal or human patients, the therapeutically effective dose is the dose that reduces the pulmonary hypertension to the normal range.

BNP

In one embodiment, the BNP described herein is a peptide that can activate the function of NPRA. In another embodiment, the BNP described herein is ten to one thousand times weaker than natural BNP (SEQ ID NO: 205) in activating the function of NPRA.

In one embodiment, the BNP described herein comprises an amino acid sequence each independently selected from one of the following: SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO: 216.

In one embodiment, the amino acid sequence of BNP described herein is: SEQ ID NO: 205. In another embodiment, the amino acid sequence of the BNP described herein is: SEQ ID NO: 206. In another embodiment, the amino acid sequence of BNP described herein is: SEQ ID NO: 207. In another embodiment, the amino acid sequence of the BNP described herein is: SEQ ID NO: 208. In another embodiment, the amino acid sequence of the BNP described herein is: SEQ ID NO: 209. In another embodiment, the amino acid sequence of BNP described herein is: SEQ ID NO: 210. In another embodiment, the amino acid sequence of the BNP described herein is: SEQ ID NO: 211. In another embodiment, the amino acid sequence of BNP described herein is: SEQ ID NO: 212. In another embodiment, the amino acid sequence of BNP described herein is: SEQ ID NO: 213. In another embodiment, the amino acid sequence of BNP described herein is: SEQ ID NO: 214. In another embodiment, the amino acid sequence of the BNP described herein is: SEQ ID NO: 215. In another embodiment, the amino acid sequence of the BNP described herein is: SEQ ID NO: 216.

Peptide Linker (Linker)

In one embodiment, the peptide linker described herein includes an amino acid sequence each independently selected from one of the following: SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO: 219.

In one embodiment, the amino acid sequence of the peptide linker described herein is: SEQ ID NO: 217. In another embodiment, the amino acid sequence of the peptide linker described herein is: SEQ ID NO:218. In another embodiment, the amino acid sequence of the peptide linker described herein is: SEQ ID NO: 219.

Fusion protein of ETA antibody and BNP

In one embodiment, the fusion protein of ETA antibody and BNP provided herein includes one ETA antibody described herein and one or more BNPs.

In one embodiment, the fusion protein of ETA antibody and BNP provided herein comprises one ETA antibody described herein and one, two, three, four, five, six, seven, or eight BNP. In another embodiment, the fusion protein of ETA antibody and BNP provided herein comprises one ETA antibody described herein and one BNP. In another embodiment, the fusion protein of ETA antibody and BNP provided herein includes one ETA antibody described herein and two BNPs. In another embodiment, the fusion protein of ETA antibody and BNP provided herein includes one ETA antibody described herein and three BNPs. In another embodiment, the fusion protein of ETA antibody and BNP provided herein includes one ETA antibody described herein and four BNPs. In another embodiment, the fusion protein of ETA antibody and BNP provided herein includes one ETA antibody described herein and five BNPs. In another embodiment, the fusion protein of ETA antibody and BNP provided herein includes one ETA antibody described herein and six BNPs. In another embodiment, the fusion protein of ETA antibody and BNP provided herein includes one ETA antibody described herein and seven BNPs. In another embodiment, the fusion protein of ETA antibody and BNP provided herein includes one ETA antibody described herein and eight BNPs.

In one embodiment, the fusion protein of ETA antibody and BNP provided herein includes one ETA antibody and one, two, three, four, five, six, seven, or eight BNP; the fusion The protein connects the amino terminal of a BNP to the carboxy terminal of the light chain or heavy chain of the ETA antibody described herein, or the fusion protein connects the carboxy terminal of a BNP to the amino terminal of the light chain or heavy chain of the ETA antibody described herein.端连接。 End connection.

In another embodiment, the fusion protein of ETA antibody and BNP provided herein includes one ETA antibody and one, two, three, or four BNP; the fusion protein combines the amino terminus of a BNP with the amino terminus of the BNP described herein. The carboxyl terminal of the light chain or heavy chain of the ETA antibody is connected, or the fusion protein connects the carboxyl terminal of a BNP with the amino terminal of the light chain or heavy chain of the ETA antibody described herein.

In another embodiment, the fusion protein of ETA antibody and BNP provided herein comprises one ETA antibody and one or two BNP; the fusion protein combines the amino terminus of a BNP with the light chain of the ETA antibody described herein or The carboxy terminus of the heavy chain is connected, or the fusion protein connects the carboxy terminus of a BNP to the amino terminus of the light or heavy chain of the ETA antibody described herein.

In another embodiment, the fusion protein of ETA antibody and BNP provided herein includes one ETA antibody and two BNPs; the fusion protein combines the amino terminus of a BNP with the light chain or heavy chain of the ETA antibody described herein. Or the fusion protein connects the carboxy terminal of a BNP with the amino terminal of the light chain or heavy chain of the ETA antibody described herein.

In one embodiment, the fusion protein of ETA antibody and BNP provided herein connects the amino terminal of a BNP to the carboxyl terminal of the light chain or heavy chain of the ETA antibody described herein. In another embodiment, the fusion protein of ETA antibody and BNP provided herein connects the amino terminal of a BNP to the carboxy terminal of the light chain of the ETA antibody described herein. In another embodiment, the fusion protein of ETA antibody and BNP provided herein connects the amino terminus of a BNP to the carboxy terminus of the heavy chain of the ETA antibody described herein.

In one embodiment, the fusion protein of ETA antibody and BNP provided herein includes the amino acid sequence: SEQ ID NO: 162 and SEQ ID NO: 190, and SEQ ID NO: 209 or SEQ ID NO: 210. In another embodiment, the fusion protein of ETA antibody and BNP provided herein comprises the amino acid sequence: SEQ ID NO: 162, SEQ ID NO: 190, and SEQ ID NO: 209. In another embodiment, the fusion protein of ETA antibody and BNP provided herein includes the amino acid sequence: SEQ ID NO: 162, SEQ ID NO: 190, and SEQ ID NO: 210.

In one embodiment, the fusion protein of ETA antibody and BNP provided herein includes one ETA antibody, and one, two, three, four, five, six, seven, or eight BNP and the same A number of peptide linkers (Linker); the fusion protein connects the amino terminal of a BNP with the carboxyl terminal of the light chain or heavy chain of the ETA antibody described herein through a peptide linker sequence, or the fusion protein connects The carboxy terminus of a BNP is connected to the amino terminus of the light or heavy chain of the ETA antibody described herein.

In another embodiment, the fusion protein of ETA antibody and BNP provided herein comprises one ETA antibody, and one, two, three, or four BNP and the same number of peptide linkers (Linker); the fusion protein The amino terminus of a BNP is connected to the carboxy terminus of the light or heavy chain of the ETA antibody described herein through a peptide linker sequence, or the fusion protein connects the carboxy terminus of a BNP to the ETA described herein through a peptide linker sequence. The amino terminus of the light or heavy chain of the antibody is connected.

In another embodiment, the fusion protein of ETA antibody and BNP provided herein comprises an ETA antibody, and one or two BNPs which have passed the same number of peptide linkers (Linker); the fusion protein combines a peptide linker sequence The amino terminus of a BNP is connected to the carboxy terminus of the light chain or heavy chain of the ETA antibody described herein, or the fusion protein connects the carboxy terminus of a BNP with the light chain or heavy chain of the ETA antibody described herein through a peptide linker sequence. The amino end of the chain is connected.

In another embodiment, the fusion protein of ETA antibody and BNP provided herein comprises an ETA antibody, two BNPs and two peptide linkers (Linker); the fusion protein connects the amino terminal of a BNP through a peptide linker sequence Connected to the carboxy terminus of the light chain or heavy chain of the ETA antibody described herein, or the fusion protein connects the carboxy terminus of a BNP to the amino terminus of the light chain or heavy chain of the ETA antibody described herein through a peptide linker sequence .

In one embodiment, the fusion protein of ETA antibody and BNP provided herein connects the amino terminus of a BNP to the carboxy terminus of the light chain or heavy chain of the ETA antibody through a peptide linker sequence. In another embodiment, the fusion protein of ETA antibody and BNP provided herein connects the amino terminus of a BNP to the carboxy terminus of the light chain of the ETA antibody through a peptide linker sequence. In another embodiment, the fusion protein of ETA antibody and BNP provided herein connects the amino terminus of a BNP to the carboxy terminus of the heavy chain of the ETA antibody through a peptide linker sequence.

In one embodiment, the fusion protein of ETA antibody and BNP provided herein includes the amino acid sequence: SEQ ID NO: 162, SEQ ID NO: 190, SEQ ID NO: 205, and SEQ ID NO: 218.

In one embodiment, the fusion protein of ETA antibody and BNP provided herein, wherein the ETA antibody, BNP and peptide linker sequence are fused by one of the following methods to form the fusion protein:

(3) Connect the amino terminal of a BNP to the carboxy terminal of the heavy chain/light chain of the ETA antibody through a peptide linker sequence: N'-R-Linker-BNP-C'; and

(4) Connect the carboxyl terminal of a BNP to the amino terminal of the light chain or heavy chain of the ETA antibody through a peptide linker sequence: N'-BNP-Linker-R-C';

Wherein: N'represents the amino terminal of the polypeptide chain, C'represents the carboxyl terminal of the polypeptide chain, BNP represents a BNP, R is the amino acid sequence of the light chain or heavy chain of the ETA antibody, and Linker represents a peptide linker.

In one embodiment, the fusion protein of ETA antibody and BNP provided herein contains two identical light chains, whose amino acid sequence is SEQ ID NO: 222; and two identical heavy chains, whose amino acid sequences are listed below One of the sequences: SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO :230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, and SEQ ID NO: 235, in which the amino terminus of a BNP and an ETA antibody heavy chain (SEQ ID NO :235) is connected to the carboxyl terminal of an ETA antibody heavy chain or via a peptide linker sequence.

Biological activity of the fusion protein of ETA antibody and BNP

The biological activity of the fusion protein of ETA antibody and BNP includes the biological activity of BNP and the activity of ETA antibody. Antibody inhibitors of ETA can effectively block the increase in vascular pressure caused by endothelin to relieve the symptoms of pulmonary hypertension and improve the patient's exercise capacity and hemodynamics. "BNP biological activity" means that the fusion protein of ETA antibody and BNP binds in the body and activates the NPRA receptor and causes cellular stress response, and exhibits the biological activity of therapeutic effects, such as pulmonary hypertension, pulmonary hypertension and heart failure. Related symptoms. The aforementioned cellular stress response includes but is not limited to lowering pulmonary artery pressure, lowering aortic pressure, and related changes in cardiac and pulmonary vascular remodeling. Combining the biological activities of BNP and ETA antibodies, the BNP fusion protein described herein can be used to treat a variety of diseases and disorders associated with NPRA and ETA. The fusion protein exerts its biological effects by acting on NPRA and/or ETA. Therefore, the BNP fusion protein described herein can be used to treat diseases and disorders that respond favorably to "increased NPRA stimulation" or "decreases ETA stimulation". Subject. These subjects are referred to as subjects who "need NPRA stimulation therapy" or "need to reduce ETA stimulation". Including pulmonary hypertension, pulmonary hypertension and other related symptoms of heart and pulmonary vascular remodeling.

In one embodiment, the changes in the biological activity of the ETA antibody or BNP fusion protein are detected by calcium flux experiments and direct cGMP detection methods to quantify the function of the ETA antibody or BNP fusion protein to inhibit ETA and activate NPRA in vitro.

Pharmaceutical composition

In one embodiment, a pharmaceutical composition is provided herein, which includes a fusion protein of an ETA antibody and BNP provided herein, and one or more pharmaceutically acceptable carriers.

In one embodiment, the pharmaceutical composition described herein is for intravenous or subcutaneous injection.

The term "carrier" as used herein includes a carrier, a pharmaceutical excipient, or a harmless stabilizer that exposes cells or mammals to it at the dosage and concentration used.

treatment method

In one embodiment, provided herein is the use of the fusion protein of ETA antibody and BNP described herein in the preparation of a medicament for the treatment, prevention or amelioration of pulmonary hypertension and pulmonary hypertension-related disorders.

In another embodiment, provided herein is the use of the fusion protein of ETA antibody and BNP described herein in the preparation of a medicament for the treatment, prevention or amelioration of pulmonary hypertension and pulmonary hypertension-related disorders.

In another embodiment, provided herein is the use of the fusion protein of the ETA antibody and BNP described herein in the preparation of a medicament for the treatment, prevention or amelioration of heart failure and heart failure-related disorders.

In a further embodiment, provided herein is the use of the fusion protein of the ETA antibody and BNP described herein in the preparation of a medicament for the simultaneous treatment, prevention or amelioration of two or more diseases of pulmonary hypertension, pulmonary hypertension or heart failure use.

As used herein, the term "subject" refers to mammals, including humans, and is used interchangeably with the term "patient".

The term "treatment" includes alleviating at least one symptom or other aspect of the condition, or alleviating the severity of the disease. The fusion protein of ETA antibody and BNP provided herein does not need to produce a complete cure effect or eradicate all symptoms or manifestations of the disease to constitute an effective therapeutic agent. As recognized in the related art, drugs as therapeutic agents can reduce the severity of a given disease state, but do not need to eliminate all manifestations of the disease to be considered as effective therapeutic agents. Similarly, prophylactic administration treatment does not need to be completely effective in preventing the appearance of symptoms to constitute an effective preventive agent. Only reducing the impact of the disease (for example, by reducing the number or severity of its symptoms, or by enhancing another therapeutic effect, or by producing another effective effect), or by reducing the likelihood of the occurrence or aggravation of the disease in the subject enough. One embodiment herein relates to a method comprising administering an ETA antibody and a BNP fusion protein to a patient in an amount and time sufficient to induce continuous improvement of an indicator of the severity of a specific disorder above the baseline level.

The fusion protein pharmaceutical composition of ETA antibody and BNP can be administered by any appropriate technique including but not limited to parenteral, topical or inhalation. In the case of injection, the pharmaceutical composition can be administered by, for example, intra-articular, intravenous, intramuscular, intra-injured, intraperitoneal or subcutaneous route, as a rapid injection or continuous infusion. For example, local administration at the site of the disease or injury may be considered, such as transdermal administration and sustained release administration of implants. Inhalation administration includes, for example, nasal or oral inhalation, spraying, inhalation of antibodies in aerosol form, and the like. Other options include oral formulations including tablets, syrups or lozenges.

It is advantageous to administer the fusion protein of ETA antibody and BNP provided herein in the form of a composition comprising one or more other components, such as a physiologically acceptable carrier, adjuvant or diluent. The composition may optionally additionally contain one or more physiologically active agents as described below. In various specific embodiments, the composition comprises one, two, three, four, five fusion proteins other than one or more of the antibodies provided herein (eg, murine antibodies or humanized antibodies) and BNP fusion proteins. One or six physiologically active agents.

In one embodiment, the pharmaceutical composition comprises the murine antibody or the fusion protein of humanized antibody and BNP provided herein and one or more substances selected from the group consisting of: a buffer with a pH suitable for the fusion protein of the antibody and BNP, Antioxidants such as ascorbic acid, low molecular weight polypeptides (such as polypeptides containing less than 10 amino acids), proteins, amino acids, sugars such as dextrin, complexes such as EDTA, glutathione, stabilizers and excipients. According to appropriate industry standards, preservatives may also be added. The composition can be formulated into a freeze-dried powder using a suitable excipient solution as a diluent. Appropriate components are non-toxic to recipients at the dosage and concentration used. For further examples of components that can be used in pharmaceutical prescriptions, see Remington's Pharmaceutical Sciences, 16th edition (1980) and 20th edition (2000). Mack Publishing Company provides kits for medical practitioners, which include one or more of the antibodies provided herein. Fusion proteins with BNP and tags or other instructions for treating any of the conditions discussed herein. In one embodiment, the kit includes a sterile preparation of a fusion protein of one or more antibodies and BNP in one or more vials in the form of the above composition.

The dosage and frequency of administration can be changed according to the following factors: the route of administration, the fusion protein of the specific antibody used and BNP, the nature and severity of the disease to be treated, whether the symptoms are acute or chronic, and the size and overall symptoms of the patient. The appropriate dose can be determined by methods well known in the art, for example, including dose scaling studies in clinical trials.

The fusion protein of the antibody and BNP provided herein can be administered one or more times at regular intervals, for example. In a specific embodiment, the murine antibody or the fusion protein of humanized antibody and BNP is administered once at least one month or longer, for example one, two or three months or even indefinite. For the treatment of chronic symptoms, long-term treatment is usually the most effective. However, for the treatment of acute symptoms, short-term administration, for example, from one week to six weeks is sufficient. Generally, human antibodies are administered until the patient shows a medically relevant improvement of the selected sign or indicator above the baseline level.

An example of the treatment regimen provided herein includes subcutaneous injection of a fusion protein of antibody and BNP at an appropriate dose once a week or longer to treat symptoms of pulmonary hypertension, pulmonary hypertension, and heart failure. The fusion protein of antibody and BNP can be administered weekly or monthly until the desired result is achieved, such as the patient's symptoms disappear. The treatment can be renewed as needed, or, optionally, a maintenance dose can be given.

The pulmonary artery pressure of the patient can be monitored before, during and/or after treatment with the fusion protein of antibody and BNP to detect any changes in pressure. For certain conditions, changes in pulmonary artery pressure can vary with factors such as disease progression. Pulmonary artery pressure can be measured by known techniques.

Specific embodiments of the methods and compositions provided herein involve the use of, for example, a fusion protein of an antibody and BNP and one or more endothelin antagonists, two or more fusion proteins of an antibody provided herein and BNP, or an antibody provided herein Fusion protein with BNP and one or more other endothelin antagonists. In a further embodiment, the fusion protein of the antibody and BNP provided herein is administered alone or in combination with other agents for treating distressing symptoms of the patient. Examples of these agents include protein and non-protein drugs. When multiple drugs are administered in combination, their dosage should be adjusted accordingly as is well known in the art. "Combined administration" combination therapy is not limited to simultaneous administration, but also includes a treatment regimen in which antigen and protein are administered at least once during a course of treatment involving the administration of at least one other therapeutic agent to the patient.

On the other hand, this article provides a method for preparing the treatment of pulmonary hypertension, pulmonary hypertension and heart failure symptoms, which comprises a mixture of the fusion protein of the antibody and BNP provided herein and pharmaceutically acceptable excipients for the treatment of related conditions of the above diseases. The preparation method of the medicament is as described above.

This document further provides compositions, kits and methods related to the fusion protein of antibodies that specifically bind to human ETA and BNP. Nucleic acid molecules and their derivatives and fragments are also provided, which comprise polynucleotides encoding all or part of a fusion protein of a polypeptide bound to ETA and BNP, such as encoding all or part of anti-ETA antibodies, antibody fragments, and antibody derivatives The nucleic acid of the fusion protein of the substance and BNP. This document further provides vectors and plasmids containing such nucleic acids, and cells and cell lines containing such nucleic acids and/or vectors and plasmids. The provided method includes, for example, a method of preparing, identifying or isolating a fusion protein of an antibody that binds to human ETA and BNP, such as a method of a fusion protein of an anti-ETA antibody and BNP, a method of determining whether the fusion protein of an antibody and BNP binds to ETA, And a method of administering a fusion protein of an antibody that binds to ETA and BNP to an animal model.

The technical scheme of this article is further explained by specific examples below.

In this article, unless otherwise specified, the raw materials and equipment used can be purchased from the market or commonly used in the field. The methods in the following examples, unless otherwise specified, are conventional methods in the art.

1. Cloning and subcloning of antibody genes

The hybridoma cells secreting antibodies are collected, and the mRNA of the hybridoma cells is extracted according to the operating procedures of QIAGEN's mRNA extraction kit. Then the extracted mRNA is reverse transcribed into cDNA. The reverse transcription primers are specific primers for the constant regions of the mouse light and heavy chains, and the heavy chain reverse transcription primers are (5'-TTTGGRGGGAAGATGAAGAC-3') (SEQ ID NO: 199 ), the light chain reverse transcription primers are (5'-TTAACACTCTCCCCTGTTGAA-3') (SEQ ID NO: 200) and (5'-TTAACACTCATTCCTGTTGAA-3') (SEQ ID NO: 201). The reaction conditions of RT-PCR are: 25°C for 5 minutes (minutes); 50°C for 60 minutes; 70°C for 15 minutes. Dilute the reverse-transcribed cDNA with 0.1mM TE to 500μL, add it to an ultrafiltration centrifuge tube (Amicon Ultra-0.5), centrifuge at 2000g for 10min; discard the filtrate, add 500μL of 0.1mM TE, centrifuge at 2000g for 10min; discard Filtrate, invert the preparation tube into a new centrifuge tube, centrifuge at 2000g for 10 minutes to obtain purified cDNA; take 10μL of purified cDNA as a template, add 4μL of 5xtailing buffer (Promega, commercially available), 4μL of dATP (1mM) ) And 10U terminal transferase (Promega, commercially available), mix well, incubate at 37°C for 5 minutes and then 65°C for 5 minutes; then use the cDNA with PolyA tail as template to amplify the light and heavy chain variable region genes of the antibody . The upstream primers are OligodT, the heavy chain downstream primers are (5'-TGGACAGGGATCCAGAGTTCC-3') (SEQ ID NO: 202) and (5'-TGGACAGGGCTCCATAGTTCC-3') (SEQ ID NO: 203), and the light chain downstream primers are (5'-ACTCGTCCTTGGTCAACGTG-3') (SEQ ID NO: 204). PCR reaction conditions: 95°C for 5 minutes; 95°C for 30 seconds (seconds), 56°C for 30 seconds, 72°C for 1 minute and 40 cycles; 72°C for 7 minutes; PCR products are connected to PMD 18-T vector (Takara Bio, commercially available) and sequenced. The CDR sequences of the cloned antibody are shown in Table 1 and Table 2.

Based on the DNA sequence of the antibody obtained by sequencing, PCR primers were designed to connect the complete light chain, heavy chain signal peptide and variable domain, and mouse IgG1 constant region to the expression vector pTM5.

2. Preparation of antibody fusion protein gene expression plasmid

The human BNP (hBNP) gene sequence and the anti-ETA antibody were fused to the C-terminus of the heavy chain antibody by overlapping PCR. The 5'end of the variable region of the heavy chain of the fusion protein is brought into the Nhe1 restriction site by PCR primers, and the 3'end of the fusion protein is brought into the Not1 restriction site, thereby loading the complete heavy chain and hBNP fusion protein gene into the expression vector pTM5; Similarly, bring the 5'end of the light chain variable region into the Nhe1 restriction site, and the 3'end into the Bsiw1 restriction site, so that the complete light chain variable region sequence and the light chain constant region have been loaded The expression vector pTM5 is connected.

3. Transient expression of antibody fusion protein

Inoculate 5×10 ⁵ /mL of the suspended HEK293 or CHO expressing cell line into a spinner flask, after 24h (hours) 37°C, 5% CO ₂ spin culture, the density reaches 1×10 ⁶ /mL and then used for transfection . Polyethylenimine (PEI) is used as the transfection medium in the transfection process, and it is mixed with DNA (the amount of DNA is 0.5μg per 1×10 ⁶ cells, where the specific gravity of the antibody light chain and heavy chain is 3:2), and PEI The preferred ratio of mixing with DNA is 3:1. The mixture of the two was added to the cell culture after 15 minutes of incubation. After receiving the mixture of PEI and DNA, the cells were cultured at 37°C with 5% CO ₂ rotation for 24 hours, and 0.5% tryptone was added to the cell culture medium as an additive for expression. Finally, the cells were collected after the expression was completed (above 96h) The supernatant is used for the purification and separation of antibodies.

4. Purification and separation of antibody fusion protein

The collected cell supernatant was centrifuged at a high speed (8000 rpm, 15 min) to remove cells and cell debris, and then filtered and clarified with a 0.45 μm filter membrane. The clarified supernatant was used for purification. The purification process is completed by a chromatograph. The supernatant first flows through the protein G affinity chromatography column. During this period, the antibody contained in the supernatant binds to the ligand of the protein G affinity chromatography column and is retained in the column, and then the pH value is lowered (less than or equal to 3.0). The elution buffer lavage the chromatography column to dissociate the antibody bound to the chromatography column, and the low pH value of the collected antibody eluate is quickly neutralized with 1M Tris-HCl to protect the antibody from inactivation. The obtained antibody fusion protein eluate was dialyzed for 16 hours and then replaced with a PBS buffer system.

5. Calcium flow experiment detects the fusion protein of anti-ETA antibody and BNP to block the biological activity of ETA in vitro

25000 cells per well were seeded with CHO-DHFR cells co-expressing hETA-Aequorin to a black 96-well cell culture plate and cultured overnight at 37°C. On the next day, the cell supernatant was removed, 50 μL of the substrate coelenterazine h (Promega, commercially available) was added and incubated for 2 hours in the dark, and then 50 μl of hybridoma cell supernatant or purified antibody was added and incubated for 30 minutes. The autosampler was used to infuse endothelin 1 on the SpectraMax L microplate reader of Molecular Devices, the instantaneous calcium flow change was detected within 40s, and the peak time and peak value were recorded. As shown in Figure 1, differently constructed fusion proteins of ETA antibodies and BNP showed different results in inhibiting the intracellular Ca ²⁺ changes mediated by human ETA.

6. The cGMP experiment detects the biological activity of the fusion protein of ETA antibody and BNP to activate NPRA in vitro

HEK 293 cells expressing human/mouse NPRA were inoculated with 5.5×10 ⁴ per well to 96-well cell culture plates, and placed in a 37°C, 5% CO ₂ incubator overnight. On the second day, remove the cell supernatant, add 90 μL/well of serially diluted ETA antibody and BNP fusion protein or mutant, incubate in a 37°C, 5% CO ₂ incubator for 30 minutes, and add 10 μL/well of 10% Triton X -100, pyrolysis at room temperature, mix well with a row gun. A cGMP kit (CisBio) was used to detect the cGMP produced in the experiment. Take the above 10μL/well cell lysate in a white 384-well plate, add 5μL/well of 1:20 diluted cGMP-d2, and finally add 5μL/well of 1:20 diluted Anti-cGMP-Eu3-cryptate, and incubate at room temperature for 1h. Read the time-resolved fluorescence signal ratio of 665nm/620nm on Envision 2103 microplate reader, and then use Prism5.0 to calculate the EC ₅₀ value. Table III shows the direct cGMP assay ETA antibody to BNP fusion proteins activate the human / rat NPRA signaling pathway EC _50, wherein h15F3 represents a humanized ETA antibody comprises SEQ ID NO: 162 and SEQ ID NO: 190; wherein BNP ( Such as BNP, BNP (1-28), BNP (1-29NSF), BNP (3-28), BNP (3-29NSF), BNP (3-32), BNP (7-29), BNP (7-32) ), BNP(9-29), and BNP(9-32)) directly or through a peptide linker (such as G ₄ S and (G ₄ S) ₂ ) and the carboxyl end of the heavy chain of h15F3 antibody Connected.

Table 3. cGMP experiment to detect the biological activity of the fusion protein of ETA antibody and BNP

7. The in vivo activity of a single tail vein administration on reducing aortic pressure in normal C57 mice

Animals are randomly divided into groups according to their body weight. The mice are fixed in a mouse bag with a mouse net, and then placed in an incubator. The pressure sensor is placed at the base of the tail. The tip of the sensor mark is in the same direction as the tip of the tail, and the tail is inserted. After the sensor, the mouse blood pressure was detected by the Softron intelligent non-invasive blood pressure monitor (BP-2010). Record 5 times in a row and take the average value as the basic value; inject normal saline or drugs into the tail vein according to the animal’s body weight, measure blood pressure at 15 minutes after injection, and record 5 times in a row to get the average value. Detection of h15F3-BNP(3-29), h15F3-(G ₄ S) _2- BNP and h15F3-BNP(9-32) by Softron intelligent non-invasive blood pressure monitor and mouse instrument before and after intravenous injection to normal C57BL/6 mice The effect of arterial pressure. The three test substances, h15F3-(G ₄ S) ₂ -BNP, h15F3-BNP (3-29) and h15F3-BNP (9-32), were all administered at a dose of 10 mg/kg. As shown in Figure 2, the results show that the h15F3-(G ₄ S) ₂ -BNP and h15F3-BNP(9-32) groups can reduce the blood pressure of the mice 15 minutes after administration.

8. The in vivo activity of monocrotaline-induced pulmonary hypertension in SD rats after repeated tail vein administration

Animals were randomly grouped according to their body weight. Model rats were injected with MCT solution at a dose of 50 mg/kg subcutaneously on the back of the neck, and rats in the normal control group were injected with an equal volume of normal saline. One day after modeling, the dose was 10 mg/kg according to body weight. The administration started by tail vein administration. After the administration, the rats were anesthetized by intraperitoneal injection of 25% urethane (4mL/kg), fixed on the operating table, and inserted into the external jugular vein with the umbilical catheter pre-connected to the physiological recorder, and entered through the superior vena cava and right atrium. Right ventricle. The animal was placed for 3 to 5 minutes, and after the blood pressure stabilized, the heart rate, right ventricular systolic and diastolic blood pressure were recorded. As shown in Figure 3, in the normal group, model group, h15F3-(G ₄ S) ₂ -BNP low and high dose groups, the right ventricular systolic blood pressure of each group was 38.46±2.56, 55.20±8.59, 47.95±2.52 and 45.79, respectively. ±7.36mmHg, compared with the model group, the statistical P values were 0.000, 0.038, and 0.034, respectively. The right ventricular systolic pressure inhibition rate of the drug group relative to the model group was 43.27% and 56.19%, respectively.

9. In vivo activity of a single intravenous injection on blood pressure in healthy rhesus monkeys

The experiment was commissioned by Prime Biotechnology Co., Ltd., and the study was carried out by a single intravenous injection of 2 mg/kg with h15F3-(G ₄ S) ₂ -BNP, h15F3-BNP(3-29) and h15F3-BNP( 9-32) Effect on blood pressure of healthy rhesus monkeys. The monkeys were randomly grouped by body weight, fasted for 14-16 hours before anesthesia, and anesthetized the animals with 10 mg/kg ketamine hydrochloride intramuscular injection. Observed the size of the animal's arm and selected the correct cuff. The cuff was tied to the left upper arm of the animal for blood pressure testing. A total of 4 tests were performed, 2 times before administration, 48-72h before administration, 10min after anesthesia and 1h after anesthesia; 10min after administration and 1h after administration; blood pressure measurement before and after administration should be ensured as much as possible At the same time of the day. Among them, blood pressure measurement is performed every 1 minute after anesthesia, and the test ends when the difference between SBP/DBP/MBP of the three results (no need to be continuous) is less than 10mmHg. Investigate the inhibition of blood pressure in animals after administration (△P=average blood pressure after administration-average blood pressure before administration). As shown in Figure 4 and Figure 5, the experimental results show that both the h15F3-(G ₄ S) ₂ -BNP and h15F3-BNP(9-32) groups can reduce the blood pressure of the monkeys 10 minutes after administration, and the h15F3-(G ₄ S) ₂ -BNP can also lower blood pressure 60 min after administration.

10. Pharmacokinetic experiment of the fusion protein of ETA antibody and BNP on healthy cynomolgus monkeys

A total of 6 male and female cynomolgus monkeys were given a single subcutaneous injection of the fusion protein of human ETA antibody and BNP at a dose of 2 mg/kg, 0h before administration, immediately after administration (within 2 minutes), and 0.5 after administration h, 2h, 8h, 24h, 2d (days), 3d, 4d, 7d each time for administration. Take 0.6 mL of whole blood from lateral limb vein, place it in a centrifuge tube, wait for it to coagulate naturally, and centrifuge to extract serum, ultra-low temperature Store (-80℃) until testing. The BNP part of the fusion protein of human ETA antibody and BNP in serum samples was quantified by ELISA method, and its half-life in cynomolgus monkey was determined by software analysis.

PK studies have shown that the half-life T _1/2 of the BNP portion of the two BNP fusion proteins are about 44.0 and 29.5 hours, respectively. The PK curves and parameters of each group of monkeys are shown in Figure 6 and Figure 7 and Table 4.

Table 4: Pharmacokinetic results of ETA antibody and BNP fusion protein

The above-mentioned embodiment is only a preferred solution of the present invention, and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

A fusion protein of ETA antibody and BNP, its structure is characterized in that: the fusion protein comprises an ETA antibody and one or more BNPs.
The fusion protein of claim 1, wherein the ETA antibody comprises one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

a. Light chain CDR1 amino acid sequence: SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30;

b. Light chain CDR2 amino acid sequence: SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48;

c. Light chain CDR3 amino acid sequence: SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 220;

d. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and SEQ ID NO: 90;

e. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114; and

f. Heavy chain CDR3 amino acid sequence: 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.
The fusion protein of claim 1 or 2, wherein the ETA antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

a. Light chain CDR1 amino acid sequence: SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30; and

b. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and SEQ ID NO: 90.
The fusion protein of any one of claims 1 to 3, wherein the ETA antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

a. Light chain CDR2 amino acid sequence: SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48; and

b. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114.
The fusion protein of any one of claims 1 to 4, wherein the ETA antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

a. Light chain CDR3 amino acid sequence: SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 220; and

b. Heavy chain CDR3 amino acid sequence: 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.
The fusion protein of any one of claims 1 to 5, wherein the ETA antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences: SEQ ID NO: 8. SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 220.
The fusion protein of any one of claims 1 to 6, wherein the ETA antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences: SEQ ID NO: 70, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, and SEQ ID NO: 136.
The fusion protein of any one of claims 1 to 7, wherein the ETA antibody comprises a combination of light chain and heavy chain CDR3 amino acid sequences independently selected from the following: SEQ ID NO: 50 and SEQ ID NO: 116, SEQ ID NO: 50 and SEQ ID NO: 220, 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.
The fusion protein of any one of claims 1 to 8, wherein the ETA antibody comprises

(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: 220;

Amino acid sequence of heavy chain CDR1: SEQ ID NO: 70;

Heavy chain CDR2 amino acid sequence: SEQ ID NO: 92; and

Amino acid sequence of heavy chain CDR3: 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;

Amino acid sequence of light chain CDR3: SEQ ID NO: 52;

Amino acid sequence of heavy chain CDR1: SEQ ID NO: 72;

Amino acid sequence of heavy chain CDR2: SEQ ID NO: 94; and

Amino acid sequence of heavy chain CDR3: 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;

Amino acid sequence of light chain CDR3: SEQ ID NO: 54;

Amino acid sequence of heavy chain CDR1: SEQ ID NO: 74;

Heavy chain CDR2 amino acid sequence: SEQ ID NO: 96; and

Amino acid sequence of heavy chain CDR3: 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;

Amino acid sequence of heavy chain CDR1: SEQ ID NO: 76;

Heavy chain CDR2 amino acid sequence: SEQ ID NO: 98; and

Amino acid sequence of heavy chain CDR3: 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

Amino acid sequence of heavy chain CDR3: 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;

Amino acid sequence of heavy chain CDR2: SEQ ID NO: 102; and

Amino acid sequence of heavy chain CDR3: SEQ ID NO: 126;

(g) Light chain CDR1 amino acid sequence: SEQ ID NO: 20 or 22;

Light chain CDR2 amino acid sequence: SEQ ID NO: 44;

Light chain CDR3 amino acid sequence: SEQ ID NO: 62;

Amino acid sequence of heavy chain CDR1: SEQ ID NO: 82;

Heavy chain CDR2 amino acid sequence: SEQ ID NO: 104 or 106; and

Amino acid sequence of heavy chain CDR3: 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

Amino acid sequence of heavy chain CDR3: 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;

Amino acid sequence of heavy chain CDR1: SEQ ID NO: 86;

Heavy chain CDR2 amino acid sequence: SEQ ID NO: 110; and

Amino acid sequence of heavy chain CDR3: 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;

Amino acid sequence of heavy chain CDR1: 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;

Amino acid sequence of heavy chain CDR1: 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.
The fusion protein of claim 9, wherein the ETA antibody comprises

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;

Amino acid sequence of heavy chain CDR1: 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.
The fusion protein of any one of claims 1 to 10, wherein the ETA antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

a. Light chain variable domain amino acid sequence: SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO : 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, and SEQ ID NO: 164; and

b. Heavy chain variable domain amino acid sequence: SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO :178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, and SEQ ID NO: 192.
The fusion protein of any one of claims 1 to 11, wherein the polynucleotide coding sequence of the ETA antibody comprises one or two polynucleotide sequences, wherein each polynucleotide sequence Are independently selected from the polynucleotide sequences listed below:

a. Light chain variable domain polynucleotide coding sequence: SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, and SEQ ID NO: 163 ；

b. Heavy chain variable domain polynucleotide coding sequence: 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 .
The fusion protein of any one of claims 1 to 12, wherein the ETA antibody comprises a combination independently selected from the following amino acid sequences: SEQ ID NO: 138 and SEQ ID NO: 166, SEQ ID NO: 140 and SEQ ID NO: 168, SEQ ID NO: 142 and SEQ ID NO: 170, SEQ ID NO: 144 and SEQ ID NO: 172, SEQ ID NO: 146 and SEQ ID NO: 174, SEQ ID NO :148 and SEQ ID NO: 176, SEQ ID NO: 150 and SEQ ID NO: 178, SEQ ID NO: 152 and SEQ ID NO: 180, SEQ ID NO: 154 and SEQ ID NO: 182, SEQ ID NO: 156 And SEQ ID NO: 184, SEQ ID NO: 158 and SEQ ID NO: 186, SEQ ID NO: 160 and SEQ ID NO: 188, SEQ ID NO: 162 and SEQ ID NO: 190, and SEQ ID NO: 164 and SEQ ID NO: 192.
The fusion protein of any one of claims 1 to 13, wherein the ETA antibody comprises an amino acid sequence independently selected from the following: SEQ ID NO: 138, SEQ ID NO: 150, SEQ ID NO :152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, and SEQ ID NO:164.
The fusion protein of any one of claims 1 to 14, wherein the ETA antibody comprises an amino acid sequence independently selected from the following: SEQ ID NO: 166, SEQ ID NO: 178, SEQ ID NO :180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, and SEQ ID NO: 192.
The fusion protein of any one of claims 1 to 15, wherein the ETA antibody comprises a combination of amino acid sequences independently selected from the light chain and heavy chain variable regions listed below: 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: 180, SEQ ID NO: 154 and SEQ ID NO: 182, SEQ ID NO: 156 and SEQ ID NO: 184, SEQ ID NO: 158 and SEQ ID NO: 186, SEQ ID NO: 160 and SEQ ID NO: 188, SEQ ID NO: 162 and SEQ ID NO: 190, and SEQ ID NO: 164 and SEQ ID NO: 192.
The fusion protein of claim 16, wherein the ETA antibody comprises the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 166.
The fusion protein of claim 16, wherein the ETA antibody comprises a combination of the amino acid sequence of SEQ ID NO: 138 and SEQ ID NO: 166.
The fusion protein of claim 16, wherein the ETA antibody comprises the amino acid sequence of SEQ ID NO: 162 or SEQ ID NO: 190.
The fusion protein of claim 16, wherein the ETA antibody comprises a combination of the amino acid sequence of SEQ ID NO: 162 and SEQ ID NO: 190.
The fusion protein of any one of claims 1 to 20, wherein the ETA antibody comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:

a. Light chain constant amino acid sequence: SEQ ID NO: 194 and SEQ ID NO: 196;

b. Heavy chain constant amino acid sequence: SEQ ID NO: 198 and SEQ ID NO: 221.
The fusion protein of claim 1, wherein the ETA antibody has one or more of the following properties:

a. When binding to human endothelin receptor ETA, its K d is the same as or better than a reference ETA antibody;

b. When inhibiting endothelin activation of human endothelin receptor ETA, its IC 50 is the same as or better than a reference ETA antibody; and

c. The ETA antibody cross-competes binding with a reference ETA antibody on the human endothelin receptor ETA.
The fusion protein of claim 22, wherein the ETA antibody cross-competes with the reference ETA antibody for binding on the human endothelin receptor ETA.
The fusion protein of claim 22 or 23, wherein the reference ETA antibody comprises the antibody of any one of claims 1-21.
The fusion protein of claim 24, wherein the reference ETA antibody comprises a light chain variable domain amino acid sequence SEQ ID NO: 138 and a heavy chain variable domain amino acid sequence SEQ ID NO: 166 combination or light chain The combination of the variable domain amino acid sequence SEQ ID NO: 162 and the heavy chain variable domain amino acid sequence SEQ ID NO: 190.
The fusion protein of any one of claims 1 to 25, wherein the ETA antibody comprises a murine ETA antibody or a humanized ETA antibody.
The fusion protein of any one of claims 1 to 26, wherein the ETA antibody comprises an ETA monoclonal antibody.
The fusion protein of any one of claims 1 to 27, wherein the IC 50 value of the ETA antibody for reducing human endothelin signaling is about 1 nM to 200 nM or 10 nM to 100 nM.
The fusion protein of any one of claims 1 to 28, wherein the fusion protein comprises one ETA antibody, and one, two, three, four, five, six, seven, or eight BNP; the fusion protein connects the amino terminal of a BNP to the carboxy terminal of the light or heavy chain of the ETA antibody, or the fusion protein connects the carboxy terminal of a BNP to the amino of the light or heavy chain of the ETA antibody端连接。 End connection.
The fusion protein of claim 29, wherein the fusion protein comprises one ETA antibody and one, two, three, or four BNPs.
The fusion protein of claim 29, wherein the fusion protein comprises one ETA antibody and one or two BNPs.
The fusion protein of claim 29, wherein the fusion protein comprises one ETA antibody and two BNPs.
The fusion protein of any one of claims 29 to 32, wherein the fusion protein connects the amino terminus of a BNP to the carboxy terminus of the light chain or heavy chain of the ETA antibody.
The fusion protein of any one of claims 29 to 33, wherein the fusion protein connects the amino terminal of a BNP to the carboxy terminal of the heavy chain of the ETA antibody.
The fusion protein of any one of claims 1 to 34, wherein the fusion protein comprises an amino acid sequence: SEQ ID NO: 162 and SEQ ID NO: 190, and SEQ ID NO: 209 or SEQ ID NO: 210.
The fusion protein of any one of claims 1 to 28, wherein the fusion protein comprises one ETA antibody, and one, two, three, four, five, six, seven, or eight BNP and peptide linker (Linker); the fusion protein connects the amino terminal of a BNP to the carboxyl terminal of the light chain or heavy chain of the ETA antibody through a peptide linker sequence, or the fusion protein connects a BNP through a peptide linker sequence The carboxyl terminal of the ETA antibody is connected to the amino terminal of the light chain or heavy chain of the ETA antibody.
The fusion protein of claim 36, wherein the fusion protein comprises an ETA antibody, and one, two, three, or four BNPs and a peptide linker (Linker).
The fusion protein of claim 36, wherein said fusion protein comprises an ETA antibody, two BNPs and two peptide linkers (Linker).
The fusion protein of claim 36, wherein said fusion protein comprises an ETA antibody, a BNP and a peptide linker (Linker).
The fusion protein of any one of claims 36 to 39, wherein the fusion protein connects the amino terminus of a BNP to the carboxy terminus of the light chain or heavy chain of the ETA antibody via a peptide linker sequence.
The fusion protein of any one of claims 36 to 40, wherein the fusion protein connects the amino terminal of a BNP to the carboxy terminal of the heavy chain of the ETA antibody via a peptide linker sequence.
The fusion protein of any one of claims 36 to 41, wherein the fusion protein comprises an amino acid sequence: SEQ ID NO: 162, SEQ ID NO: 190, SEQ ID NO: 205, and SEQ ID NO: 218.
The fusion protein of any one of claims 36 to 42, wherein the ETA antibody, BNP, and peptide linker sequence are fused by one of the following methods to form the fusion protein:

(1) Connect the amino terminal of a BNP and the carboxy terminal of the heavy chain/light chain of the ETA antibody through a peptide linker sequence: N'-R-Linker-BNP-C'; and

(2) Connect the carboxyl end of a BNP to the amino end of the light chain or heavy chain of the ETA antibody through a peptide linker sequence: N'-BNP-Linker-R-C';

Wherein: N'represents the amino terminal of the polypeptide chain, C'represents the carboxyl terminal of the polypeptide chain, BNP represents a BNP, R is the amino acid sequence of the light chain or heavy chain of the ETA antibody, and Linker represents a peptide linker.
The fusion protein of any one of claims 1 to 43, wherein each of the BNPs independently comprises an amino acid sequence selected from one of the following: SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO: 216.
The fusion protein of claim 44, wherein each of the BNPs is independently selected from one of the following amino acid sequences: SEQ ID NO: 205, SEQ ID NO: 209, and SEQ ID NO: 210.
The fusion protein of any one of claims 36 to 45, wherein the sequence of the peptide linker (Linker) each independently comprises one of the following amino acid sequences: SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO:219.
The fusion protein of claim 46, wherein the sequence of the peptide linker (Linker) is SEQ ID NO:218.
A polynucleotide encoding the fusion protein of any one of claims 1 to 47.
A vector comprising a polynucleotide described in claim 48.
A host cell comprising a vector described in claim 49.
A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 47 and a pharmaceutically acceptable carrier.
A use of the fusion protein according to any one of claims 1 to 47 in the preparation of a medicament for preventing, improving, or treating pulmonary hypertension.
A use of the fusion protein according to any one of claims 1 to 47 in the preparation of a medicament for preventing, ameliorating, or treating pulmonary hypertension.
A use of the fusion protein according to any one of claims 1 to 47 in the preparation of a medicine for preventing, improving, or treating heart failure.
A use of the fusion protein according to any one of claims 1 to 47 in the preparation of a medicament for preventing, improving, or treating two or more diseases of pulmonary hypertension, pulmonary hypertension or heart failure.
The use according to any one of claims 52 to 55, wherein the drug is for intravenous or subcutaneous injection.