WO2020248967A1 - Protéine de fusion d'anticorps eta et de bnp, et composition pharmaceutique et application de protéine de fusion - Google Patents

Protéine de fusion d'anticorps eta et de bnp, et composition pharmaceutique et application de protéine de fusion Download PDF

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WO2020248967A1
WO2020248967A1 PCT/CN2020/095062 CN2020095062W WO2020248967A1 WO 2020248967 A1 WO2020248967 A1 WO 2020248967A1 CN 2020095062 W CN2020095062 W CN 2020095062W WO 2020248967 A1 WO2020248967 A1 WO 2020248967A1
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amino acid
acid sequence
fusion protein
heavy chain
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药晨江
张�成
章华
汪笑峰
景书谦
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鸿运华宁（杭州）生物医药有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

  • pulmonary hypertension pulmonary hypertension or heart failure
  • 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.
  • 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.
  • Figure 7 shows the pharmacokinetic results of the fusion protein h15F3-BNP (9-32) of ETA antibody and BNP in healthy monkeys.
  • 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.
  • murine antibody includes all antibodies that have one or more variable and constant regions derived from mouse immunoglobulin sequences.
  • regulatory sequence affects the expression of the nucleotide sequence (eg, expression level, time, or location)
  • 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).
  • Cynomolgus monkey polynucleotide (SEQ ID NO: 3); accession number: JV635771.
  • 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 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:
  • 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.
  • Light chain CDR3 amino acid sequence SEQ ID NO: 50 or SEQ ID NO: 220;
  • Amino acid sequence of heavy chain CDR3 SEQ ID NO: 118;
  • Heavy chain CDR1 amino acid sequence SEQ ID NO: 78;
  • Light chain CDR3 amino acid sequence SEQ ID NO: 62;
  • Light chain CDR3 amino acid sequence SEQ ID NO: 66;
  • Heavy chain CDR2 amino acid sequence SEQ ID NO: 112;
  • Heavy chain CDR3 amino acid sequence SEQ ID NO: 134; or
  • 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:
  • 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
  • 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
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the antibodies described herein comprise the amino acid sequences of the light chain and heavy chain CDRs and FRs (framework) listed herein.
  • the antibody comprises the light chain CDR1 sequence listed herein.
  • the antibody comprises the light chain CDR2 sequence listed herein.
  • the antibody comprises the light chain CDR3 sequence listed herein.
  • the antibody comprises the heavy chain CDR1 sequence listed herein.
  • the antibody comprises the heavy chain CDR2 sequence listed herein.
  • the antibody comprises the heavy chain CDR3 sequence listed herein.
  • the antibody comprises the light chain FR1 sequence listed herein.
  • the antibody comprises the light chain FR2 sequence listed herein.
  • 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.
  • the antibodies described herein are complete antibodies (including polyclonal, monoclonal, chimeric, humanized, or human antibodies with full-length heavy and/or light chains).
  • 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).
  • the antibodies described herein also include antibody polypeptides such as those disclosed in US Patent No. 6,703,199, including fibronectin polypeptide single antibodies.
  • the antibodies described herein also include other antibody polypeptides disclosed in U.S. Patent Application Publication No. US2005/0238646, which are single-chain polypeptides.
  • 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).
  • 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.
  • 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.
  • the antibody derivative may comprise at least one of the CDRs disclosed herein.
  • 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.
  • one or more water-soluble polymers are bound at one or more specific sites of the binding agent, for example at the amino terminus.
  • 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.
  • the variant has a greater or lesser number of N-linked glycosylation sites than the native protein.
  • 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.
  • 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.
  • 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.
  • 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.
  • a protein scaffold or backbone rather than an immunoglobulin domain.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • CHO Chinese Hamster Ovary
  • Veggie CHO and related cell lines grown in serum-free media see Rasmussen et al., 1998, Cytotechnology 28:31
  • CHO The strain DXB-11 lacks DHFR (see Urlaub et al., 1980, PNAS USA 77:4216-20).
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the ETA antibodies described herein have one or more of the properties listed below:
  • the ETA antibody cross-competes binding with the reference antibody on the human endothelin receptor ETA.
  • the ETA antibody described herein is an antibody having one or more of the following properties:
  • the ETA antibody cross-competes binding with a reference ETA antibody on the human endothelin receptor ETA.
  • 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.
  • the reference antibody is monoclonal antibody A-1, A-2, A-7, A-9, or A-12.
  • 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%.
  • 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.
  • 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.
  • 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. ⁇ End connection.
  • 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.
  • 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.
  • 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.
  • 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
  • Linker represents a peptide linker.
  • a pharmaceutical composition which includes a fusion protein of an ETA antibody and BNP provided herein, and one or more pharmaceutically acceptable carriers.
  • the pharmaceutical composition described herein is for intravenous or subcutaneous injection.
  • 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 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.
  • 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 are physiologically active agents.
  • 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.
  • 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 cGMP experiment detects the biological activity of the fusion protein of ETA antibody and BNP to activate NPRA in vitro
  • 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.

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Abstract

La présente invention concerne une protéine de fusion d'un anticorps ETA et de BNP. La présente invention concerne également une composition pharmaceutique de la protéine de fusion de l'anticorps ETA et de BNP. La présente invention concerne en outre un procédé de traitement, de prévention ou d'attéuation d'un ou de plusieurs symptômes de l'hypertension artérielle pulmonaire, de l'hypertension pulmonaire, ou de l'insuffisance cardiaque à l'aide de la protéine de fusion de l'anticorps ETA et de BNP.
PCT/CN2020/095062 2019-06-10 2020-06-09 Protéine de fusion d'anticorps eta et de bnp, et composition pharmaceutique et application de protéine de fusion WO2020248967A1 (fr)

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WO2009006732A1 (fr) * 2007-07-06 2009-01-15 Theratechnologies Inc. Protéines de fusion bifonctionnelles de l'hormone de stimulation des alpha-mélanocytes (alpha-msh) et de la protéine natriurétique auriculaire (pna), et leurs utilisations dans l'hypertension et la lésion rénale aiguë
CN105669863A (zh) * 2014-12-05 2016-06-15 杭州鸿运华宁生物医药工程有限公司 一种能与人内皮素受体特异性结合的抗体及其应用
CN107987162A (zh) * 2016-10-27 2018-05-04 鸿运华宁（杭州）生物医药有限公司 Etar抗体，其药物组合物及其应用

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US20190022165A1 (en) * 2016-03-02 2019-01-24 Stealth Biotherapeutics Corp Methods and compositions for the treatment and prevention of pulmonary arterial hypertension

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Publication number Priority date Publication date Assignee Title
WO2009006732A1 (fr) * 2007-07-06 2009-01-15 Theratechnologies Inc. Protéines de fusion bifonctionnelles de l'hormone de stimulation des alpha-mélanocytes (alpha-msh) et de la protéine natriurétique auriculaire (pna), et leurs utilisations dans l'hypertension et la lésion rénale aiguë
CN105669863A (zh) * 2014-12-05 2016-06-15 杭州鸿运华宁生物医药工程有限公司 一种能与人内皮素受体特异性结合的抗体及其应用
CN107987162A (zh) * 2016-10-27 2018-05-04 鸿运华宁（杭州）生物医药有限公司 Etar抗体，其药物组合物及其应用

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