WO2023065700A1 - A growth hormone fusion protein and itsuse thereof - Google Patents

A growth hormone fusion protein and itsuse thereof Download PDF

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Publication number
WO2023065700A1
WO2023065700A1 PCT/CN2022/100240 CN2022100240W WO2023065700A1 WO 2023065700 A1 WO2023065700 A1 WO 2023065700A1 CN 2022100240 W CN2022100240 W CN 2022100240W WO 2023065700 A1 WO2023065700 A1 WO 2023065700A1
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amino acid
growth hormone
wild
fusion protein
fragment
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PCT/CN2022/100240
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French (fr)
Inventor
Suofu QIN
Chengwei YAN
Yuxin QIU
Nining GUO
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Shenzhen Kexing Pharmaceutical Co., Ltd.
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Publication of WO2023065700A1 publication Critical patent/WO2023065700A1/en

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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
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • 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
    • A61K38/25—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/305—Growth hormone [GH], aka. somatotropin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2511/00—Cells for large scale production
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present disclosure relates to the field of biomedicine, in particular to a growth hormone fusion protein and its use thereof. More particularly, the present invention relates to an Fc mutant, a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition and use thereof, and a method of preventing and/or treating abnormal growth hormone related diseases.
  • Growth hormone deficiency is a recognized clinical syndrome associated with a number of metabolic abnormalities, including abnormal body composition, decreased physical fitness, altered lipid metabolism, decreased bone mass, increased insulin resistance, and reduced quality of life. Most of the metabolic abnormalities associated with growth hormone deficiency can be reversed by recombinant human growth hormone (rhGH) substitutes.
  • rhGH human growth hormone
  • Traditional treatment for growth hormone deficiency involves daily subcutaneous injections of rhGH. However, injections are too frequent and inconvenient for many patients, raising concerns about poor treatment compliance, which can lead to reduced efficacy. Long-acting rhGH preparations can not only reduce the number of injections, thereby improving the compliance, but also help to improve the efficacy of GH treatment.
  • Fusion proteins have been prepared using immunoglobulin Fc to increase the half-life of the fusion protein (Cheol Ryong Ku et al., Eur J Endocrinol. 2018 Sep; 179 (3) : 169-179; Wolfgang Glaesner et al., Diabetes Metab Res Rev. 2010 May; 26 (4) : 287-96. ) . Since the biological activity of growth hormone requires a certain spatial effect, the improper connection of Fc in the fusion protein will cause a space thymus effect. However, studies have found that the combination of GGGGSGGGSGGGGS or some fragments of Genexine GX-H9 with Fc has low binding force to human growth hormone receptors, low activity to promote cell proliferation, and low activity in vitro.
  • the aim of the present invention is to solve at least one of the technical problems of the prior art.
  • the present invention is based on the following findings of the present inventor:
  • the present disclosure provides an Fc mutant.
  • the Fc mutant comprises the first peptide chain
  • the first peptide chain 1) comprises mutantions at the following positions in comparision to a wild-type IgG4 Fc fragment: at least one of amino acid in positions 234, 235, 298, 299, and 300 ; or positions 231-239 ; or 2) comprises mutantions at the following positions in comparision to a wild-type IgG1 Fc fragment: positions 228-239, and/or, at least one of amino acid in positions 268, 296, 330 and 331.
  • the inventor carries out the above-mentioned mutations to an Fc fragment of IgG4 or IgG1 antibody respectively, and it is understood by those skilled in the art thatthe mutations at different position of amino acid sequence have different effects.
  • the Fc mutant according toan embodiment of the invention can effectively reduce the fragment content during the preparation process of the Fc fragment, and avoid the formation of semi-antibody, and eliminate or lowerthe ADCC and CDC effect.
  • the fusion protein prepared bythe Fc mutant in this application has strong in vitro and in vivo binding activity with human growth hormone receptor, can eliminate or lower ADCC and CDC effect, promote NB2-11 cell proliferation, and effectively treat or prevent abnormal growth hormone related diseases.
  • the Fc mutant can further comprise at least one of the following additional technical characteristics:
  • the Fc mutant further comprises a second peptide chain, a C-terminal of the second peptide chain is connected with an N-terminal of the first peptide chain; and the second peptide chain.
  • the Fc mutant further includes a second peptide chain whose C-terminal is connected to the N-terminal of the first peptide chain, and the second peptide chain has the following mutation sites: 1) comprises mutantions at the position 228 in comparision to the wild-type IgG4 hinge region; or 2) comprises a wild-type IgD fragment 1 or a IgD mutational fragment 1.
  • amino acid sequence of the wild-type IgG4 hinge region comprising:
  • the wild-type IgD fragment 1 comprises amino acid at positions 287-298 of the wild-type IgD.
  • the IgD mutational fragment 1 comprises mutantions at the following positions in comparisionto a wild-type IgD Fc fragment 1: at least one of amino acid at positions 291-296.
  • the Fc mutant further comprises a third peptide chain, a C-terminal of the third peptide chain is connected withan N-terminal of the second peptide chain.
  • the wild-type IgD fragment 2 comprises amino acid in positions 262-291 of the wild-type IgD.
  • the first peptide chain 1 comprises mutantions at the following positions in comparision to the wild-type IgG4 Fc fragment: positions 234, 235; or 298, 299, 300; or positions 231-239; or 2) comprises mutantions at the following positions in comparision to the wild-type IgG1 Fc fragment: positions 228-239, 268, 296, 330 and 331.
  • the second peptide chain 1) comprises mutantions at the following positions in comparision to the wild-type IgG4 hinge region: positions 228; or 2) comprises the wild-type IgD fragment 1 or the IgD mutational fragment 1.
  • the IgD mutational fragment 1 comprises mutantions at the following in comparision to the wild-type IgD Fc fragment 1: positions 291-296.
  • the first peptide chain 1) comprises mutantions at the following positions in comparision to the wild-type IgG4 Fc fragment: F234A and L235E; or F234V and L235A; or F234V and L235E; or S298N, T299A and Y300S ; or amino acid deletion at positons 231-239 ; or 2) comprises mutantions at the following positions in comparision to the wild-type IgG1 Fc fragment: amino acid deletion at positions 228-239, and H268Q, Y296F, A330S and P331S.
  • different sites are mutated with different effects. Therefore, the Fc mutant obtained by combining each mutation site have different effects.
  • F234V&L235E can eliminate ADCC effect
  • S298N&T299A&Y300S can eliminate ADCC effect ADCC and CDC effects.
  • the second peptide chain 1) comprises mutantions at the following positions in comparision to the wild-type IgG4 hinge region: S228P; or 2) comprises the wild-type IgD fragment 1 or the IgD mutational fragment 1.
  • S228P mutation can avoid the formation of half-antibodies in the IgG4 constant region.
  • the IgD mutational fragment 1 comprises mutantions at the following positions in comparision to the wild-type IgD fragment 1: P291C, S292H, H293P, T294R, Q295L, P296S.
  • amino acid sequence of the wild-type IgD fragment 2 comprising:
  • the first peptide chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 55, 56, 57, 58, 68 and 69.
  • the second peptide chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 59, 60 and 61.
  • the third peptide chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 62, 63, 64 and 65.
  • the wild-type IgG4, the wild-type IgG1 and the wild-type IgD are human antibodies.
  • the Fc mutant comprises: 1) an amino acid sequence as shown in any one of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54; or 2) an amino acid sequence with at least 90%identity with the amino acid sequence as shown in any one of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54.
  • the present disclosure provides a fusion protein.
  • the fusion protein comprising a first peptide, wherein the first peptide comprises a bioactive molecular functional region; a second peptide conncected with the first peptide, wherein the second peptide comprises an Fc mutant of the first aspect, wherein a N-terminal of the second peptide is connected to a C-terminal of the first peptide.
  • the fusion protein has strong in vitro and in vivo binding activity with human growth hormone receptor, can effectively eliminate or lower ADCC and CDC effect, promote NB2-11 cell proliferation, and effectively treat or prevent abnormal growth hormone related diseases.
  • the fusion protein can further comprise at least one of the following additional technical characteristics:
  • the first peptide comprises a growth hormone, a growth hormone analog, a growth hormone functional region or a growth hormone analog functional region.
  • the first peptide comprises the human growth hormone or the human growth hormone functional region.
  • the human growth hormone comprisies an amino acid sequence as shown in SEQ ID NO: 1 or at least 90%homologous thereof.
  • the fusion protein further comprisies a linker peptide.
  • an N-terminal of the linker peptide is connected with the C-terminal of the first peptide, and a C-terminal of the linker peptide is connected with the N-terminal of the second peptide.
  • the linker peptide comprises an amino acid sequence as shown in the SEQ ID NO: 46.
  • the linker peptide comprises mutantions at the following positions in comparision to the amino acid sequence as shown in the SEQ ID NO: 46: at least one S>T in any one GGGGS, increasing G mutation at N-terminal and increasing L mutation at C-terminal.
  • the linker peptide comprisies an amino acid sequence as shown in SEQ ID NO: 11.
  • the fusion protein comprises: 1) an amino acid sequence as shown in any one of SEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, 28; or 2) an amino acid sequence with at least 90%and 95%identity with the amino acid sequence as shown in any one of SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28.
  • the inventor unexpectedly found that the binding activity of the fusion protein obtained after the combination of the second peptide and the linker peptide with the human growth hormone receptor and the promoting effect of NB2-11 cell proliferation were significantly superior.
  • the present disclosure provides a nucleic acid molecule.
  • the nucleic acid molecule encoding the Fc mutant of the first aspect or the fusion protein of the second aspect.
  • the Fc mutant obtained according to the nucleic acid molecule of the embodiment of the present invention can effectively avoid to format of semi-antibodies and eliminate ADCC and CDC effects.
  • the fusion protein obtained in present invention has strong in vitro and in vivo binding activity with human growth hormone receptor and can effectively eliminate or lower ADCC and CDC effects and promote the proliferation of NB2-11 cells, and effectively treat or prevent abnormal growth hormone related diseases.
  • the nucleic acid molecule can further comprise at least one of the following additional technical characteristics:
  • the nucleic acid molecule is DNA.
  • the nucleic acid molecule comprises an nucleotide sequence as shown in any one ofSEQ ID NOs : 32, 33, 34, 35, 36, 37, 38, 39 or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20.
  • nucleic acid includes any one, or two, of a complementary double-strand.
  • only one strand is provided in most cases for convenience, but the disclosure includes the other one strand of the complementary double-strand.
  • the gene sequence in the present disclosure includes both the DNA form and the RNA form, wherein in the case that one form is disclosed, the other one is also disclosed.
  • the term "encoding" refers to the inherent properties of polynucleotides such as genes, cDNAs, or mRNAs in which specific nucleotide sequences are used as templates for the synthesis of other polymers and macromolecules in biological processes.
  • the polymers and macromolecules have a certain nucleotide sequence (e.g. rRNA, tRNA, and mRNA) or defined amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene, cDNA, or RNA encodes the protein.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence.
  • the present disclosure provides an expression vector.
  • the expression vector carriesthe nucleic acid molecule of first aspect.
  • the nucleic acid molecule can be directly or indirectly connected to the control element on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule.
  • these control elements can be directly originaed from the vector itself, but also can be exogenous, that is, not only from the vector itself.
  • the nucleic acid molecule can be operationally connected to the control element.
  • operable linkage refers to the connection of exogenous genes to the vector so that the control elements in the vector, such as transcription control sequence and translation control sequence, can play their expected functions of regulating transcription and translation of exogenous genes.
  • Common vectors such as plasmids, phages, etc.
  • the expression vector can further comprise at least one of the following additional technical characteristics:
  • the expression vector is an eukaryotic expression vector, a prokaryotic expression vector or a viral vector.
  • the viruses comprises lentiviruses.
  • the present disclosure provides an recombinant cell.
  • the recombinant cell carriesthe nucleic acid molecule of third aspect, or the expression vector of fourth aspect, or expressing the Fc mutant of any one of first aspect or the fusion protein of second aspect.
  • the recombinant can be used for in vitro expression and mass availability of the Fc mutants of first aspect or fusion proteins of second aspect.
  • the recombinant cell can further comprise at least one of the following additional technical characteristics:
  • the recombinant cell is obtainable by introducing the expression vector mentioned of the fourth aspect into a host cell.
  • the expression vector is introduced into the host cell by means of electrical transduction.
  • the recombinant cell isan eukaryotic cell.
  • the recombinant cell is amammalian cell.
  • the eukaryotic cell not comprises animal germ cell, oospermand embryonic stem cell.
  • the recombinant cell in the present invention are not subject to special restrictions and it can comprise a prokaryotic cell, the eukaryotic cell or a phage.
  • the prokaryotic cell can comprise Escherichia coli, Bacillus subtilis, Streptomyces or Exotic proteobacteria.
  • the eukaryotic cell can comprise pichia pastoris, Saccharomyces cerevisiae, Fission yeast, Fungi, such as Trichoderma, insect cell, such as Grass armyworm, plant cell, such as tobacco, mammalian cell such as BHK cell, CHO cell, COS cell, myeloma cell,
  • the recombinant cells are mammalian cellspreferably, comprising BHK cell, CHO cell, NSO cell or COS cell, and do not comprising animal germ cell, oosperm or embryonic stem cell.
  • suitable conditions refers to the conditions suitable for the expression of Fc mutants or fusion proteins.
  • the conditions suitable for the expression of Fc mutants or fusion proteins comprise but are not limited to suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. There are no special restrictions on “suitable conditions” and the skilled artisan can optimize the conditions for the expression of the Fc mutant or fusion protein according to the laboratory environment.
  • the present disclosure provides a composition.
  • the composition comprises the fusion protein ofthe second aspect, or the nucleic acid molecule ofthe third aspect, or the expression vector ofthe fourth aspect, or the recombinant cell ofthe fifith aspect.
  • the fusion protein and the fusion protein obtained after the expression of the nucleic acid molecule, or the expression vector or the recombinant cell can not only effectively bind with the human growth hormone receptor, but also effectively promote the proliferation of NB2-11 cell. Therefore, the fusion protein contained or expressed in the composition according to embodiments of the invention also can bind the human growth hormone receptor, promote the proliferation of NB2-11 cell, and effectively treat or prevent abnormal growth hormone related diseases.
  • the composition comprises an food composition, or a drug composition, etc.
  • the composition comprises combination that is separate in time and/or space, as long as it can act together to achieve the purpose of the invention.
  • the components contained in the composition may be applied to the subject as a whole or separately.
  • each component can be applied simultaneously or sequentially to the subject.
  • the present disclosure provides the use of the fusion proteins ofany one of the second aspect, the nucleic acid molecules ofthe third aspect, the expression vectors of the fourth aspect, the recombinant cell of the fifth aspect, or the compositions of the sixth aspect in the preparation of medicament for preventing or treating disease associated with abnormal growth hormone.
  • the medicament is used to treat or prevent abnormal growth hormone related diseases.
  • the usecan further comprise at least one of the following additional technical characteristics:
  • the disease associated with abnormal growth hormone comprises at least one of the following: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner's syndrome, Prader-Willi syndrome, kidney failure, disorders caused by alienated states during chemotherapy and AIDS treatment, and intrauterine growth retardation.
  • the present disclosure provides a drug.
  • the drug comprises fusion proteins of the second aspect, the nucleic acid molecules of the third aspect, the expression vector of the forth aspect, the recombinant cell of the fifth aspect, or the composition of the sixth aspect.
  • the fusion protein and the fusion protein obtained after the expression of the nucleic acid molecule, or the expression vector or the recombinant cell can not only effectively bind with the human growth hormone receptor, but also effectively promote the proliferation of NB2-11 cell. Therefore, the fusion protein contained or expressed in the composition according to embodiments of the invention also can bind the human growth hormone receptor, promote the proliferation of NB2-11 cell, and effectively treat or prevent the abnormal growth hormone related diseases.
  • the drugcan further comprise at least one of the following additional technical characteristics:
  • the drug can further comprise pharmaceutically acceptable carriers, for example, solvent, stabilizers, solid excipient, antioxidants, diluent, binder, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspension agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid adhesive, flow aid or lubricant, and so on, that are nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed, to fit the specific target dosage form.
  • pharmaceutically acceptable carriers for example, solvent, stabilizers, solid excipient, antioxidants, diluent, binder, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspension agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid adhesive, flow aid or lubricant, and so on, that are nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed, to fit the specific target dosage form.
  • the fusion protein of the present disclosure can be incorporated into a drug suitable for parenteral administration (e.g. intravenously, subcutaneously, intraperitoneally, intramuscularly) .
  • parenteral administration e.g. intravenously, subcutaneously, intraperitoneally, intramuscularly
  • these drugs can be prepared in various forms, such as liquid, semi-solid and solid dosage forms, comprising but not limited to liquid solutions (e.g., injection and infusion solutions) , dispersants or suspensions, tablets, pills, powders, liposomes and suppositories. Drugs typically take the form of an injectable or infusion solution.
  • the fusion protein can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
  • the drug of the present disclosure also can be suitable for oral administration.
  • the drug are used (e.g., administered to a subject in need of treatment, such as a human individual) by oral administration.
  • the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions.
  • Active component (s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.
  • inactive ingredients examples include red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink.
  • Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract.
  • Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
  • Dosages and desired concentration of the drug of the present disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan.
  • Administration of a drug of the present disclosure can be continuous or intermittent, depending, for example, on the recipient’s physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. It is within the scope of the present disclosure that dosages may be administered by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • the drug can further comprise at least one of the following additional technical features:
  • the present disclosure provides a method of preventing or treating diseases associated with abnormal growth hormone.
  • the method comprises applying to subjects at least one of the following: 1) the fusion protein of the second aspect; 2) the nucleic acid molecule of the third aspect; 3) The expression vector of the fourth aspect; 4) The recombinant cells of the fifth aspect; 5) the composition of the sixth aspect; or 6) the drug of the eigth aspect.
  • the fusion protein and the fusion protein obtained after the expression of the nucleic acid molecule, or the expression vector or the recombinant cell can not only effectively bind with the human growth hormone receptor, but also effectively promote the proliferation of NB2-11 cell. Therefore, the fusion protein contained or expressed in the composition according to embodiments of the invention also can bind the human growth hormone receptor, promote the proliferation of NB2-11 cell, and effectively treat or prevent the abnormal growth hormone related diseases.
  • the method can further comprise at least one of the following additional technical characteristics:
  • the fusion protein is adminstered by subcutaneous injection or intravenous injection.
  • the abnormal growth hormone related diseases comprises at least one of the following: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner's syndrome, Prader-Willi syndrome, kidney failure, disorders caused by alienated states during chemotherapy and AIDS treatment, and intrauterine growth retardation.
  • the present disclosure provides the fusion protein of the fusion protein of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the recombinant cells of the fifth aspect, the composition of the sixth aspect, or the drug of the eigth aspect, for use in preventing or treating a diseaseassociated withabnormal growth hormone.
  • the fusion protein and the fusion protein obtained after the expression of the nucleic acid molecule, or the expression vector or the recombinant cell can not only effectively bind with the human growth hormone receptor, but also effectively promote the proliferation of NB2-11 cell. Therefore, the fusion protein contained or expressed in the composition according to embodiments of the invention also can bind the human growth hormone receptor, promote the proliferation of NB2-11 cell, and effectively treat or prevent the abnormal growth hormone related diseases.
  • the use can further comprise at least one of the following additional technical characteristics:
  • the present disclosure provides the fusion protein of the fusion protein of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the recombinant cells of the fifth aspect, the composition of the sixth aspect, or the drug of eigth aspect, for use in preventing or treating a diseaseassociated withabnormal growth hormone comprises at least one of the following:
  • childhood growth hormone deficiency idiopathic short stature, adult growth hormone deficiency, Turner's syndrome, Prader-Willi syndrome, kidney failure, disorders caused by alienated states during chemotherapy and AIDS treatment, and intrauterine growth retardation.
  • FIG. 1 is a schematic diagram of fusion protein in one embodiment of the present invention.
  • FIG. 2 is the result graph of ADCC effect induced by fusion protein 9#.
  • the fusion protein of the invention is generally prepared by a biosynthesis method.
  • the nucleotide sequence of the present invention the one skilled in the art to which this invention belongs can easily obtain the coding nucleic acid of the present invention by various known methods. These methods comprise but are not limited to: PCR, artificial DNA synthesis, etc. For specific methods, please refer to Joseph. Sambrook ⁇ Molecular cloning: a laboratory manual ⁇ .
  • the coding nucleic acid sequence of the present invention can be constructed by the method of segmented synthesis of nucleotide sequence followed byoverlap extension PCR.
  • the term “comprise” , “contain” or “include” is an open expression, it means comprising the contents disclosed herein, but don’ t exclude other contents.
  • the terms “optionally” or “optional” generally means that the subsequently described event or condition can occur, but may not must, and that the description includes the circumstances in which the event or condition occurs and the circumstances in which the event or condition does not occur.
  • room temperature refers to the ambient temperature, which may be 20°C-30°C. In some embodiments, the room temperature may be 22°C-28°C; In some embodiments, 24°C-26°C; In some embodiments, 25°C.
  • fusion protein refers to the product obtained by the fusion of antibody fragment with other bioactive protein using genetic engineering technology.
  • mutant generally refers to any naturally occurring or engineered molecule that contains one or more nucleotide or amino acid mutations.
  • antibody “Fc fragment” includes the CH2 and CH3 regions of the antibody, for example, the wild-type IgG4 Fc fragment comprises the CH2 and CH3 regions of the wild-type IgG1 antibody, and the wild-type IgG1 Fc fragment comprises the CH2 and CH3 regions of the wild-type IgG1 antibody.
  • “semi-antibody” refers to the two antibody fragments formed after the disulfide bond of the antibody Fc fragment is broken.
  • the Fc fragment of the wild-type human IgG4 antibody forms the semi-antibody fragments containing the CH2 and CH3 regions of the wild-type human IgG4 antibody
  • the Fc fragment of wild-type human IgG1 antibody forms the semi-antibody fragmentscontaining fragments of the CH2 and CH3 regions of wild-type human IgG1 antibody.
  • nucleotide generally is ribonucleotides, deoxynucleotides, or any type of nucleotide, and theirmodified forms, and their combinations.
  • host cell used in present invention refers to prokaryotic or eukaryotic cell that can be introduced into recombinant expression vectors.
  • transformed or “transfected” in present invention refers to the introduction of nucleic acids (such as vectors) into cells through various techniques known in the art.
  • the term “identity” is used to describe the relative to the reference sequence of amino acid sequencesor nucleic acid sequence, determines the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequencesby conventional methods, see, for example, Ausubel et al., eds. (1995) , Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York) ; and the ALIGN program (Dayhoff (1978) , Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C. ) .
  • ALIGN ALIGN or Megalign (DNASTAR) software
  • WU-BLAST-2 Altschul et al., Meth. Enzym., 266: 460-480 (1996)
  • GAP BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA
  • CLUSTAL CLUSTAL of PC/Gene program provided by Intelligenetics, Mountain View, California.
  • the amino acid number of the Fc fragment of IgG4 or IgG1 is numbered according to the EU numbering system.
  • the "S228P" refers to the serine at position 228 of the EU numbering system replacedby proline.
  • the amino acid of IgD fragment is numbered according to the reference: Takahashi, Nobuhiro, Et al. "Sequence of Complete Amino Acid of the ⁇ Heavy Chain of Human Immunoglobulin D. " Proceedings of the National Academy of Sciences of the United States of America, Vol. 79, No. 9, 1982, pp. 2850 --54, http: //www. jstor. org/stable/11720. Accessed 12 Apr. 2022.
  • the present disclosure provides a fusion protein, the general structure of the fusion protein is expressed as X-L-Y, wherein X is the first bioactive molecule; L is absent or linker peptide; Y is the second bioactive molecule; -is the peptide bond; and the X or Y is selected from the protein or protein domain, polypeptide, antibody or antibody fragment.
  • the fusion protein can further comprise at least one of the following additional technical characteristics:
  • the X is human growth hormone.
  • the human growth hormone comprisies an amino acid sequence as shown in SEQ ID NO: 1 or at least 80-99%homologous thereof or at least part of SEQ ID NO: 1.
  • the Y is an Fc fragment or a variant thereof.
  • the Fc fragment comprisies an amino acid sequence as shown in SEQ ID NO: 29, SEQ ID NO: 30 or at least 80-99%homologous thereof, or at least part of its sequence; or the amino acid sequence of the Fc fragment variant comprises selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7or at least 80-99%homologous thereof, or part of its sequence.
  • the amino acid sequence of the Fc fragment variant 1 comprising:
  • amino acid sequence of the Fc fragment variant 2 amino acid sequences comprising:
  • amino acid sequence of the Fc fragment variant 3 amino acid sequences comprising:
  • amino acid sequence of the Fc fragment variant 4 amino acid sequences comprising:
  • amino acid sequences of the Fc fragment variant 5 amino acid sequences comprising:
  • amino acid sequence of the Fc fragment variant 6 amino acid sequences comprising:
  • amino acid sequence of the wild-type IgG4 fragment comprising:
  • amino acid sequence of the wild-type IgG1 Fc fragment comprising:
  • the amino acid sequenceof the fusion protein comprisesselected fromSEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28 or any amino acid sequence or at least 80-99%homologous thereof , or part of its sequence.
  • the amino acid sequence of the fusion protein 8# (bioactive molecular functional region -Fc mutant 1) comprising:
  • the amino acid sequence of the fusion protein 9# (bioactive molecular functional region -Fc mutant 2) comprising:
  • the amino acid sequence of the fusion protein 10# (bioactive molecular functional region -Fc mutant 4) comprising :
  • the amino acid sequence of the fusion protein 11# (bioactive molecular functional region - (G 4 S) 3 -FC mutant 5) comprising:
  • the amino acid sequence of the fusion protein 12# (bioactive molecular functional region -GS linker peptied mutant -Fc mutant 3) comprising:
  • the amino acid sequence of the fusion protein 13# (bioactive molecular functional region -Fc mutant 6) comprising:
  • the amino acid sequence of the fusion protein 14# (bioactive molecular functional region - (G4S) 3-FC mutant 7) comprising :
  • the amino acid sequence of the fusion protein 15# (bioactive molecular functional region - (G 4 S) 3 -FC mutant 8) comprising:
  • the linker peptide comprises one or more amino acids selected from glycine, serine, alanine and threonine.
  • the linker peptide comprises the amino acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 or at least 80-99%homologous thereof , or part of its sequence.
  • the amino acid sequence of the Linker1 (the third peptide 1) comprising:
  • the amino acid sequence of the Linker 2 (the third peptide 2) comprising:
  • the amino acid sequence of the Linker3 (the third peptide 3) comprising:
  • the amino acid sequence of the Linker4 comprising:
  • the amino acid sequence of the Linker5 (the third peptide 4) comprising:
  • the present disclosure provides a nucleotide sequenceaccording to an embodiment of the present invention, the nucleotide sequenceencodes thelinker peited previously described.
  • the present disclosure provides a nucleotide sequenceaccording to an embodiment of the present invention, the nucleotide sequenceencodes the the fusion protein previously described.
  • the present disclosure provides an vector.
  • the vector carriesthe nucleic acid molecule encodeding the the fusion protein or linker pepited.
  • the vector can further comprise at least one of the following additional technical characteristics:
  • the expression vector is an expression vector.
  • the term "vector” usually refers to a nucleic acid molecule capable of self-replicating in a suitable host by transferring the inserted nucleic acid molecule to and/or between host cells.
  • the vector can comprise a vector primarily used for inserting DNA or RNA into cells, a vector primarily used for DNA or RNA replication, and an expression vector primarily used for DNA or RNA transcription and/or translation.
  • the vector also includes a vector with a variety of thefunctions.
  • the vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the vector can produce the desired expression product by culturing an appropriate host cell containing the vector.
  • the present invention presents an engineered cell characterized by either the engineered cell containing the fusion protein previously described or the engineered cell containing the nucleotide sequence or expression vector previously described.
  • engineered cell refers to a cell that is modified or recombined with the genetic material of the host cell by means of genetic engineering techniques or cell fusion techniques to obtain a unique character with stable inheritance.
  • the present invention presents a pharmaceutical composition containing the fusion protein previously described.
  • the pharmaceutical composition may further include at least one of the following additional technical features:
  • drug combination is used for oral and intravenous use, such as pushing injection or continuous infusion for a period of time, through the subcutaneous, intramuscular, inside arteries, peritoneal, lungs, brain within the spinal cord, intra-articular, within the synovial membrane and its sheath, damage, or suction path such as nasal, usually by intravenous or subcutaneous application of drug combination.
  • the dosage forms of the drug composition are tablets, capsules, sprays, injections, freeze-dried powder injections or pre-filled needle injections.
  • the present invention proposes a preparation method of the fusion protein described above, which includes: (a) culture of the engineered cells described above to obtain a culture medium containing the fusion protein; (b) the fusion protein is isolated from the culture medium.
  • the present invention proposes the use of the aforementioned fusion protein or drug composition in the preparation of drugs for the treatment or prevention of diseases lacking human growth hormone.
  • treatment is used to refer to the achievement of a desired pharmacological and/or physiological effect.
  • the effects may be preventive in terms of complete or partial prevention of the disease or its symptoms, and/or therapeutic in terms of partial or complete cure of the disease and/or adverse effects resulting from the disease.
  • the term “treatment” is used in this context to cover diseases of mammals and, in particular, of humans, including: (a) prevention of disease or illness in susceptible but undiagnosed individuals; (b) suppression of disease, for example by blocking its progression; or (c) alleviating the disease, for example by reducing symptoms associated with the disease.
  • treatment as used herein includes any administration of a drug or compound to an individual to treat, cure, alleviate, improve, mitigate, or inhibit an individual's disease, including but not limited to the administration of a drug containing the compound to an individual in need.
  • the present invention has at least one of the following beneficial effects:
  • the long-acting human growth hormone fusion protein of the invention has low EC50 value and strong binding activity with human GHR, especially the binding activity effect of the human growth hormone fusion protein 8#, 9#, 10#and 15#is significantly better than that of Genexine GX-H9 and GH+G/SLinker +IGG4-1.
  • the long-acting human growth hormone fusion protein of the invention has a good proliferation effect on NB2-11 cells; the human growth hormone fusion protein 8#, 9#, 10#, 12#had better proliferative activity than Genexine GX-H9 and GH+G/SLinker + IGG4-1.
  • the long-acting human growth hormone fusion protein reporter cells of the present invention have good luciferase activity.
  • the human growth hormone fusion proteins 8#, 9#, 10#and 12# have better luciferase activity than Genexine GX-H9 and GH+G/SLinker + IGG4-1.
  • the third peptide chains are provided by the invention can improve the binding activity of the human growth hormone fusion protein containing the third peptide chain to human GHR, the proliferation activity of NB2-11 cells and the luciferase expression activity of reporter cells.
  • the SEQ ID NO: 54 is provided by the invention is conducive to improving the binding activity of human growth hormone fusion protein containing the second bioactive molecule of SEQ ID NO: 54 amino acid sequence to human GHR.
  • the third peptide chain is provided by the invention, as shown in SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, is conducive to improving the proliferation activity of the human growth hormone fusion protein containing the third peptide chain against NB2-11 cells.
  • the second bioactive molecule is provided by the invention, as shown in SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 50, is conducive to improving the proliferation activity of the human growth hormone fusion protein containing the second bioactive molecule against NB2-11 cells..
  • the third peptide chain is provided by the invention, as shown in SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, is conducive to improving the luciferase expression activity of the human growth hormone fusion protein.
  • the second bioactive molecule is provided by the invention, as shown in SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 50, is conducive to improving the luciferase expression activity of the human growth hormone fusion protein containing the second bioactive molecule.
  • the fusion proteins are provided by the invention has a longer half-life in vivo.
  • the protein disclosed herein may be prepared by methods described herein, wherein the peotein are as defined for above, except where further noted.
  • the following non-limiting schemes and examples are presented to further exemplify the invention.
  • Example 1 The preparing of the expression vector of human growth hormone fusion protein
  • the human growth hormone (SEQ ID NO: 1) was linked to a variety of antibody Fc mutants (any one of SEQ ID NO: 47-54, the second peptide chain was indicated in bold letters in the sequence) to construct fusion proteins.
  • Some fusion proteins had linker peptides, and the specific structure of the fusion proteins with linker peptides was shown in Figure 1.
  • the linker peptide is GGGGSGGGGSGGGGS (SEQ ID NO: 45) or obtained by mutation
  • the antibody Fc mutant is obtained by mutation of wild-type IgG4 Fc fragment (SEQ ID NO: 71) or IgG1 Fc fragment (SEQ ID NO: 30) .
  • the mutation modes include point mutation, the addition of wild-type IgG4 and/or IgD antibody fragments or their corresponding mutants, etc.
  • the specific experimental operations are as follows:
  • nucleotide sequence encoding human growth hormone SEQ ID NO: 31
  • nucleotide sequence encoding linker peptide SEQ ID NO: 14
  • nucleotide sequence encoding multiple antibody Fc mutants SEQ ID NO: 32-39
  • nucleotide sequences encoding fusion protein SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 and SEQ ID NO: 20 were respectively synthesized by total gene synthesis and molecular cloning techniques and were cloned into the expression vector pCDNA3.4 (purchased from Thermo Fisher) , and the fusion protein expression vector of each nucleotide sequence was obtained.
  • the amino acid sequence of the Human Growth hormone (hGH) is as follows:
  • amino acid sequence of the first peptide chain 1 (conprising F234V and L235E mutations compared to wild-type IgG4 Fc fragment) is as follows:
  • the amino acid sequence of the first peptide chain 2 (comprising S298N, T299A and Y300S mutations compared to wild-type IgG4 Fc fragment) is as follows:
  • amino acid sequence of the first peptide chain 3 (with amino acid deletion at position 231-239 compared to the wild-type IgG4 Fc fragment) is as follows:
  • the amino acid sequence of the first peptide chain 4 (comprising amino acid deletion at position 228-239, H268Q, Y296F, A330S, and P331S mutations compared to the Fc fragment of wild-type IgG1) is as follows:
  • amino acid sequence of first peptide chain 5 (F234A and L235E mutations compared to the Fc fragment of wild-type IgG4 antibody) is as follows:
  • amino acid sequence of first peptide chain 6 (F234V and L235A mutations compared to the Fc fragment of wild-type IgG4 antibody) is as follows:
  • amino acid sequence of the second peptide chain 1 is as fpllows:
  • the amino acid sequence of the second peptide chain 2 (IgD mutational fragment 1) is as follows:
  • the amino acid sequence of the second peptide chain 3 (wild-type IgD fragment 1) is as follows:
  • the amino acid sequence of the third peptide chain 1 is as follows:
  • the amino acid sequence ofthe third peptide chain 2 is as follows:
  • the amino acid sequence of the third peptide 3 is as follows:
  • the amino acid sequence of the third peptide 4 is as follows:
  • the amino acid sequence of the Fc mutant 1 (first peptide chain 5-second peptide chain 1-third peptide chain 1) is as follows:
  • the amino acid sequence of the Fc mutant 2 (first peptide chain 6-second peptide chain 1-third peptide chain 2) is as follows :
  • the amino acid sequence of Fc mutant 3 (first peptide chain 5-second peptide chain 1) is as follows:
  • the amino acid sequence of the Fc mutant 4 (peptide-1 -peptide-1 -peptide-3) is as follows:
  • the amino acid sequence of the Fc mutant 5 (first peptide chain 2-second peptide chain 1) is as follows:
  • amino acid sequence of the Fc mutant 6 (firstpeptide chain 2-secondpeptide chain 1-third peptide chain 4) amino acid sequence is as follows:
  • the amino acid sequence of theFc mutant 7 (first peptide chain 3-second peptide chain 2) :
  • the amino acid sequence of the Fc mutant 8 (first peptide chain 4-second peptide chain 3) is as follows:
  • the amino acid sequence of the Linker pepited (4) is as follows:
  • hGH human growth hormone
  • the nucleotide acid sequence encoding the Fc mutant 1 is as follows:
  • the nucleotide acid sequence encoding the Fc mutant 2 is as follows:
  • the nucleotide acid sequence encoding the Fc mutant 3 is as follows:
  • the nucleotide acid sequence encoding the Fc mutant 4 is as follows:
  • the nucleotide acid sequence encoding the Fc mutant 5 is as follows:
  • the nucleotide acid sequence encoding the Fc mutant 6 is as follows:
  • the nucleotide acid sequence encoding the Fc mutant 7 is as follows:
  • the nucleotide acid sequence encoding the Fc mutant 8 is as follows:
  • the nucleotide acid sequence encoding the third peptide chain 1 is as follows:
  • the nucleotide acid sequence encoding the third peptide chain 2 is as follows:
  • the nucleotide acid sequence encoding the third peptide chain 3 is as follows:
  • the nucleotide acid sequence encoding the third peptide chain 4 is as follows:
  • the nucleotide acid sequence encoding the Linker4 is as follows:
  • the nucleotide sequence encoding the fusion protein 8# is as follows:
  • the nucleotide sequence encoding the fusion protein 9# is as follows:
  • the nucleotide sequence encoding the fusion protein 10# is as follows:
  • the nucleotide sequence encoding the fusion protein 11# is as follows:
  • the nucleotide sequence encoding the fusion protein 12# is as follows:
  • the nucleotide sequence encoding the fusion protein 13# is as follows:
  • the nucleotide sequence encoding the fusion protein 14# is as follows:
  • the nucleotide sequence encoding the fusion protein 15# is as follows:
  • amino acid sequence of the Fc fragment of wild-type IgG4 is as follows:
  • Example 2 The expression of human growth hormone fusion protein
  • the multiple plasmids obtained from example 1 were transfected into Expi CHO-S (Gibco, A29133) host cells, and the long-acting human growth hormone fusion protein was transient expressed by chemical transfection reagent Polyplus-Fectopro (Polyplus, 116-010) . Its corresponding amino acid sequence is SEQ ID NO: 21-28.
  • the specific experimental operations are as follows:
  • Expi CHO-Scells were subcultured one day before transient transfection, and the cell density was adjusted to about 3 ⁇ 10 6 cells/mL by medium (CD FortiCHO TM medium) . Then, the cell culture flask was put back to the shaker (37°C, 8%CO 2 ) for further culture.
  • transfection complex 16 sterile cell culture bottles were taken, 8 labeled “DNA” and 8 labeled “FectoPRO” , and transfection reagent Polyplus-fectopro was added into the "FectoPRO” flask and mixed; 60mL OPti-MEM solution and fusion protein expression vector containing each nucleotide sequence of example 1was added into ench "DNA” flask and mixed. And obtained the diluent of fusion protein expression vector.
  • diluent of fusion protein expression vector of each nucleotide sequence was added into the "FectoPRO” flask and mixed, and was incubated at room temperature for 10 minutes, then was added to the cell solution and shaken, and the cell culture flask was returned to the shaker to continue culture and transfection.
  • step 3 After the cell culture medium obtained in step 3) was transfected for 18-22 hours, an appropriate amount of OPM-CHO ProFeed was added, and the biochemical indicators of the cell solution were measured. According to biochemical indicators, the glucose was supplied to 6g/L. Titer was measured 4 days after transfection, and the sugar and culture solution were supplied every other day. The CHO cell fermentation broth could be obtained when the cell viability is less than 80%, and the supernatant can be harvested for purification.
  • the CHO cell fermentation liquid obtained in example 2 was centrifuged in two stages (first stage: 3,000 ⁇ g, 30 min; Second stage: 12,000 ⁇ g, 20 min) , the supernatant was collected and filtered through a 0.2 ⁇ m filter, and set aside.
  • Protein A affinity chromatography The aqueous solution containing 20 mM phosphate and 150 mM NaCl at pH 7.2 was used to equilibrate the chromatographycolumnfor at least 3 columnvolumes, and the filtered clarified filtrate was loaded and remained on the chromatography column for 5 minutes. After loading the sample, the aqueous solution containing 20 mM phosphate and 150 mM NaCl at pH 7.2 was used to equilibrate the chromatographycolumnfor at least 1 column volume, followed by 50 mM pH 4.5 Acetic acid (HAc) buffer, 50 mM pH 4.0 HAc buffer, and 50 mM pH 3.5 HAcbuffer were used to elute the target protein respectively.
  • HAc Acetic acid
  • Expi Cho-Scells respectively containing nucleotide sequence SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28 were fermented and chromatographed to obtain fusion protein 8#, fusion protein 9#, fusion protein 10#, fusion protein 11#, fusion protein 12#, fusion protein 13#, fusion protein 14#and fusion protein 15#.
  • Example 4 Determination of receptor-binding activity of human growth hormone fusion protein in vitro
  • in vitro receptor Human growth hormone receptor, Human GHR binding activity of the purified fusion protein obtained from example 3 was detected.
  • the Genexine GX-H9 SEQ ID NO: 70
  • GH+G/SLinker + IgG 4 -1 SEQ ID NO: 43
  • the specific experimental procedures were as follows:
  • HGHR was diluted to prepare 0.5 ⁇ g/mL coating solution, which was added to enzyme plate at 100 ⁇ L/well, and coated at 2-8 °C for more than 12 hours.
  • the plate residue was discarded, 300 ⁇ L of1%BSA-PBST (phosphate Tween buffer containing 1%bovine serum albumin) was added into each well, and blocked for 1 hour at 37°C.
  • 300 ⁇ L of PBST (Phosphate Tween Buffer) was added to each wellfor 3 washes.
  • the recombinant long-acting human growth hormone fusion protein and Genexine GX-H9 were diluted to 5 ⁇ g/ml, and then diluted 5 times to 8 gradient concentrations, addedto 100 ⁇ L/well to ELISA plate.
  • the ELISA plate comprising the long-acting human growth hormone fusion protein or Genexine GX-H9 was incubated at 37°C for 1 hour, then 300 ⁇ L of PBST was added to each wellfor 3 washes, and the Goat anti-human IgG Fc-HRP that dilute 10000 foldwith 1%BSA-PBST was added, 100 ⁇ L/well of a sample. After incubation at 37°C for 1 hour, 300 ⁇ L of PBST was added to each wellfor 3 washes, and patted dry. TMB Substrate was added, 100 ⁇ L per well. After 5 minutes of reaction at room temperature, 2M H 2 SO 4 aqueous solution was added to stop the reaction, 100 ⁇ L/well. The ELISA plate that stopped the reaction was put on a microplate reader, the absorbance OD 450 value was read at a wavelength of 450nm, and the EC 50 of each fusion protein's binding activity to hGHR was calculated.
  • the specific experimental results were shown in Table 2.
  • the long-acting Human growth hormone fusion protein had low EC 50 value of the invention and had strong binding activity with hGHR.
  • the Human growth hormone fusion proteins 8#, 9#, 10#and 15#encoded by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 20 respectinely showed significantly better binding activity to Human GHR than Genexine Gx-h9 and GH+G/SLinker + IgG 4 -1;
  • the human growth hormone fusion protein 15# had the highest binding activity to human GHR, followed by the human growth hormone fusion protein 9#.
  • Example 5 Effects of human growth hormone fusion protein on proliferation of NB2-11 cells
  • Example 3 the purified fusion protein obtained in Example 3 was used for the experiment, and the above-mentioned Genexine GX-H9 (commercially available product) and GH+G/SLinker + IGG4-1 were used as the control group.
  • the specific experimental procedures were as follows:
  • Cells were collected and suspended in medium (PRMI 1640 medium containing 1%FBS and 50 ⁇ M ⁇ -mercaptoethanol) at a concentration of 1 ⁇ 10 5 cells/mL. Add each 50 ⁇ L cell sample to each well in the 96-well cell culture plate. The cells were cultured in 50 ⁇ L assay medium containing various concentrations of recombinant long-acting human growth hormone fusion protein, Genexine GX-H9 and GH+G/Slinker+IgG4-1 (%) from 0.051 ng/mL to 3000 ng/mL, respectively.
  • medium PRMI 1640 medium containing 1%FBS and 50 ⁇ M ⁇ -mercaptoethanol
  • the Cell plates were incubated at 37°C in a 5%CO 2 moist incubator for 96 h and 50 ⁇ L cellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571) was added to each well. After 10 minutes, the chemiluminescence signal was detected by a microplate reader. The bioactivity of recombinant long-acting human growth hormone fusion protein was determined by dose-response curve.
  • the specific experimental results were shown in Table 3, wherein, (1) The long-acting human growth hormone fusion protein of the invention had a good proliferation effect on NB2-11 cells;
  • the human growth hormone fusion protein 8#, 9#, 10#and 12#encoded by the sequence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17 had significantly better proliferative effect than Genexine GX-H9 and GH+G/Slinker + IgG4-1;
  • the human growth hormone fusion protein 10# had the highest proliferation activity againstNB2-11 cells, followed by the human growth hormone fusion protein 8#, and the human growth hormone fusion protein 9#again.
  • the third peptide chain encoded by the sequence SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 could enhance the proliferation activity of human growth hormone fusion protein against NB2-11 cells.
  • the second bioactive molecule encoded by SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 50 could improve the proliferation activity of the human growth hormone fusion protein against NB2-11 cells.
  • Example 6 Biological activity of human growth hormone fusion protein on reporter cells
  • the purified human growth hormone fusion protein obtained in Example 3 was subjected to this experiment, and the Genexine GX-H9 (commercially available product) and GH+G/SLinker + IGG4-1 were used as the control group.
  • the specific experimental procedures were as follows.
  • Reporter cells at the logarithmic growth stage (293-GHR/STAT5 cell line) were adjust to 4 ⁇ 10 4 cells/well, 50 ⁇ L/well after trypsin digestion (CoSTAR, 3917) , and were incubated overnight in a 96-well whiteboard at 37 °C in a 5%CO 2 incubator. Thereporter cells were added with gradient dilution recombinant long-acting human growth hormone fusion protein at the initial concentration of 100 nM, 5 times gradient dilution at 10 gradient concentrations. The dilution was added to the cells at the volume of 50 ⁇ L/well. The 96-well whiteboard was incubated at 37 °C in a 5%CO 2 incubator for 6 hours.
  • the 96-well white board and nano-Glo Luciferase Assay kit (Promega, N112B) were taken out and balanced to room temperature, the reaction substrate was added to the sample wells at a volume of 50 ⁇ L/well and placed at room temperature for 10 minutes.
  • the instrument (Promega, GM2000) recorded the luminescence signal value, with the protein concentration as the X-axis and the luminescence signal value as the Y-axis.
  • the EC50 value was calculated by using GraphPad Prisim 5 as four-parameter fitting.
  • the recombinant long-acting human growth hormone fusion protein reporter cells of the present invention had good luciferase activity.
  • the human growth hormone fusion proteins 8#, 9#, 10#, 12#comprisedan nucleotide sequence as shown in sequence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17 respectively could promote luciferase expression activity of reporter cells better than Genexine GX-H9 and GH+G/S would + IgG4-1;
  • the human growth hormone fusion protein 9# had the highest activity of promoting luciferase expression in reporter cells, followed by human growth hormone fusion protein 8#and the human growth hormone fusion protein 10#.
  • the third peptide chain encoded by the sequence SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 could improve the luciferase expression activity of the human growth hormone fusion protein.
  • the second bioactive molecule encoded by the sequence SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 50 was beneficial to increase the luciferase expression activity of reporter cells containing the human growth hormone fusion protein with the second bioactive molecule.
  • Example 7 Detection of ADCC effect of Human growth hormone fusion protein induced effector cell Jurkate-CD16A-Luc on target cell Cho-K1-PD-L1/GHR
  • Target cells CHO-K1-PD-L1 /GHR (Shanghai Kabe Biomedical Technology Co., LTD., SBTCL015) treatment: The cells were passaged 2 days before the experiment, and the supernatant was discarded. After washing the cells with 5 mL of PBS, 1 mL of 0.25%Trypsin-EDTA was added.
  • the cho-K1-PD-L1 /GHR density of target cells was adjusted to 1.83 ⁇ 10 5 cells /mL in analytical medium 1 (40mL F12 medium+ 0.4ml FBS) , and the target cells were plinked overnight at 50 ⁇ L/well.
  • analytical medium 1 40mL F12 medium+ 0.4ml FBS
  • SBT123 Molecule 9#and Genexine GX-H9
  • positive control M7824 and negative control Acterma were first diluted to 0.1mg/mL and 24 ⁇ g/mL in analysis Medium 2 (RPMI Medium 1640) . Then dilute 9 gradients by 3 times, 25 ⁇ L/well was taken into cell plates, mixed well, and incubated at 37°C for 45min, then removed the cell plates.
  • the density of effector cells Jurkat-CD16A-Luc was adjusted to 1.47 ⁇ 10 6 cells /mL in analysis Medium 2 (RPMI Medium 1640) , and 25 ⁇ L/well was added to the cell plate, mixed well, and incubated at 37°Cfor 5.5h. After the cell plate was taken out at room temperature and balanced, 100 ⁇ L/well of one-GLo Luciferase Assay System (Promega, E6120) was added to the cell plate and pipetted blow 10 times, mixed well. The chemiluminescence signal was detected by a microplate analyzer after 10min of light-resistant pyrolysis at room temperature.
  • Example 8 The CDC effect of the Human growth hormone fusion protein indyced human serum complement on target cells Cho-K1-PD-L1 /GHR.
  • Target cells CHO-K1-PD-L1 /GHR (Shanghai Kabe Biomedical Technology Co., LTD., SBTCL015) treatment: The cells were passaged 2 days before the experiment, and the supernatant was discarded. After washing the cells with 5 mL of PBS, 1 mL of 0.25%Trypsin-EDTA was added.
  • the density of target cells CHO-K1-PD-L1 /GHR was adjusted to 2.0 ⁇ 10 5 cells /mL in differentiation medium (99%F12 medium + 1%FBS) , and the target cells were plated overnight at 50 ⁇ L/well.
  • the test samples (9#andGenexine GX-H9) , positive control Avelumab (PD-1 antibody, Shanghai Jiabei Biomedical Technology Co., Ltd.

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Abstract

Provided herein are a growth hormone fusion protein and its use thereof. The fusion protein comprises an Fc mutant, wherein the Fc mutant comprises the first peptide chain, wherein the first peptide chain 1) comprises mutantions at the following positions in comparision to a wild-type IgG4 Fc fragment: at least one of amino acid in positions 234, 235, 298, 299, and 300; Or positions 231-239; Or 2) comprising mutantions at the following positions in comparision to a wild-type IgG1 Fc fragment: positions 228-239, and/or, at least one of amino acid in positions 268, 296, 330 and 331.

Description

A GROWTH HORMONE FUSION PROTEIN AND ITSUSE THEREOF
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefits of Chinese Patent Application 202111208636.1, filed October18, 2021, and Chinese Patent Application 202210459224.3, filed April 27, 2022, which are incorporated herein by reference in their entirety.
FIELD
The present disclosure relates to the field of biomedicine, in particular to a growth hormone fusion protein and its use thereof. More particularly, the present invention relates to an Fc mutant, a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition and use thereof, and a method of preventing and/or treating abnormal growth hormone related diseases.
BACKGROUND
Growth hormone deficiency is a recognized clinical syndrome associated with a number of metabolic abnormalities, including abnormal body composition, decreased physical fitness, altered lipid metabolism, decreased bone mass, increased insulin resistance, and reduced quality of life. Most of the metabolic abnormalities associated with growth hormone deficiency can be reversed by recombinant human growth hormone (rhGH) substitutes. Traditional treatment for growth hormone deficiency involves daily subcutaneous injections of rhGH. However, injections are too frequent and inconvenient for many patients, raising concerns about poor treatment compliance, which can lead to reduced efficacy. Long-acting rhGH preparations can not only reduce the number of injections, thereby improving the compliance, but also help to improve the efficacy of GH treatment.
Fusion proteins have been prepared using immunoglobulin Fc to increase the half-life of the fusion protein (Cheol Ryong Ku et al., Eur J Endocrinol. 2018 Sep; 179 (3) : 169-179; Wolfgang Glaesner et al., Diabetes Metab Res Rev. 2010 May; 26 (4) : 287-96. ) . Since the biological activity of growth hormone requires a certain spatial effect, the improper connection of Fc in the fusion protein will cause a space thymus effect. However, studies have found that the combination of GGGGSGGGSGGGGS or some fragments of Genexine GX-H9 with Fc has low binding force to human growth hormone receptors, low activity to promote cell proliferation, and low activity in vitro.
Therefore, there is still an urgent need for a fusion protein with high binding activity to human growth hormone receptors and can promote T cell proliferation.
SUMMARY
The aim of the present invention is to solve at least one of the technical problems of the prior art. The present invention is based on the following findings of the present inventor:
In the first aspect, the present disclosure provides an Fc mutant. According to an embodiment of the present invention, wherein the Fc mutant comprises the first peptide chain, wherein the first peptide chain 1)  comprises mutantions at the following positions in comparision to a wild-type IgG4 Fc fragment: at least one of amino acid in positions 234, 235, 298, 299, and 300 ; or positions 231-239 ; or 2) comprises mutantions at the following positions in comparision to a wild-type IgG1 Fc fragment: positions 228-239, and/or, at least one of amino acid in positions 268, 296, 330 and 331. In the research and development process, the inventor carries out the above-mentioned mutations to an Fc fragment of IgG4 or IgG1 antibody respectively, and it is understood by those skilled in the art thatthe mutations at different position of amino acid sequence have different effects. The Fc mutant according toan embodiment of the invention can effectively reduce the fragment content during the preparation process of the Fc fragment, and avoid the formation of semi-antibody, and eliminate or lowerthe ADCC and CDC effect. More imporantly, the fusion protein prepared bythe Fc mutant in this application has strong in vitro and in vivo binding activity with human growth hormone receptor, can eliminate or lower ADCC and CDC effect, promote NB2-11 cell proliferation, and effectively treat or prevent abnormal growth hormone related diseases.
According to embodiment of the present invention, the Fc mutant can further comprise at least one of the following additional technical characteristics:
According to an embodiment of the present invention, the Fc mutant further comprises a second peptide chain, a C-terminal of the second peptide chain is connected with an N-terminal of the first peptide chain; and the second peptide chain.
According to an embodiment of the present invention, the Fc mutant further includes a second peptide chain whose C-terminal is connected to the N-terminal of the first peptide chain, and the second peptide chain has the following mutation sites: 1) comprises mutantions at the position 228 in comparision to the wild-type IgG4 hinge region; or 2) comprises a wild-type IgD fragment 1 or a IgD mutational fragment 1.
The amino acid sequence of the wild-type IgG4 hinge region comprising:
Figure PCTCN2022100240-appb-000001
According to an embodiment of the present invention, the wild-type IgD fragment 1 comprises amino acid at positions 287-298 of the wild-type IgD.
According to an embodiment of the present invention, the IgD mutational fragment 1 comprises mutantions at the following positions in comparisionto a wild-type IgD Fc fragment 1: at least one of amino acid at positions 291-296.
According to an embodiment of the present invention, the Fc mutant further comprises a third peptide chain, a C-terminal of the third peptide chain is connected withan N-terminal of the second peptide chain.
According to an embodiment of the present invention, the third peptide chaincomprises mutantions at the following positions in comparision to a wild-type IgD fragment 2: at least one of the amino acid at positions: 263, 264, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 280, 281, 283, 284, 285, 286, 287, 288, 289, 290, and 291.
According to an embodiment of the present invention, the wild-type IgD fragment 2 comprises amino acid in positions 262-291 of the wild-type IgD.
According to an embodiment of the present invention, the first peptide chain 1) comprises mutantions at the following positions in comparision to the wild-type IgG4 Fc fragment: positions 234, 235; or 298, 299, 300; or positions 231-239; or 2) comprises mutantions at the following positions in comparision to the wild-type IgG1 Fc fragment: positions 228-239, 268, 296, 330 and 331.
According to an embodiment of the present invention, the second peptide chain 1) comprises mutantions at the following positions in comparision to the wild-type IgG4 hinge region: positions 228; or 2) comprises the wild-type IgD fragment 1 or the IgD mutational fragment 1.
According to an embodiment of the present invention, the IgD mutational fragment 1 comprises mutantions at the following in comparision to the wild-type IgD Fc fragment 1: positions 291-296.
According to an embodiment of the present invention, the third peptide chaincomprises mutantions at the following positions in comparision to a wild-type IgD fragment 2: 1) 266, 267, 268, 277, 278, 280, 283, 285, 286 and 287; or 2) 266, 277, 278, 280, 283, 285, 286 and 287; or 3) 266, 269, 270, 271, 273, 274, 275, 277, 278, 281 and 283; or 4) 263, 264, 266, 269, 270, 272, 274, 276, 284, and 287-291.
According to an embodiment of the present invention, the first peptide chain 1) comprises mutantions at the following positions in comparision to the wild-type IgG4 Fc fragment: F234A and L235E; or F234V and L235A; or F234V and L235E; or S298N, T299A and Y300S ; or amino acid deletion at positons 231-239 ; or 2) comprises mutantions at the following positions in comparision to the wild-type IgG1 Fc fragment: amino acid deletion at positions 228-239, and H268Q, Y296F, A330S and P331S. According to the specific embodiment of the present invention, different sites are mutated with different effects. Therefore, the Fc mutant obtained by combining each mutation site have different effects. For example, F234V&L235E can eliminate ADCC effect, S298N&T299A&Y300S can eliminate ADCC effect ADCC and CDC effects.
According to an embodiment of the present invention, the second peptide chain 1) comprises mutantions at the following positions in comparision to the wild-type IgG4 hinge region: S228P; or 2) comprises the wild-type IgD fragment 1 or the IgD mutational fragment 1. Wherein, the S228P mutation can avoid the formation of half-antibodies in the IgG4 constant region.
According to an embodiment of the present invention, the IgD mutational fragment 1 comprises mutantions at the following positions in comparision to the wild-type IgD fragment 1: P291C, S292H, H293P, T294R, Q295L, P296S.
According to an embodiment of the present invention, the third peptide chaincomprises mutantions at the following positions in comparision to a wild-type IgD fragment 2: 1) R266E, G267R, G268E, K277E, E278K, Q280E, R283E, T285E, K286G and T287E; or 2) R266K, K277E, E278K, Q280N, R283E, T285E, K286R and T287E; or 3) R266K, E269D, E270D, K271E, K273E, E274D, K275E, K277E, E278K, E281Q and R283G; or 4) N263Q, T264S, R266K, E269D, E270D, K272R, E274D, E276D, E284D, and amino acid deletion at positons 287-291.
The amino acid sequence of the wild-type IgD fragment 2 comprising:
Figure PCTCN2022100240-appb-000002
According to an embodiment of the present invention, the first peptide chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 55, 56, 57, 58, 68 and 69.
According to an embodiment of the present invention, the second peptide chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 59, 60 and 61.
According to an embodiment of the present invention, the third peptide chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 62, 63, 64 and 65.
According to an embodiment of the present invention, the wild-type IgG4, the wild-type IgG1 and the wild-type IgD are human antibodies.
According to an embodiment of the present invention, the Fc mutant comprises: 1) an amino acid sequence as shown in any one of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54; or 2) an amino acid sequence with at least 90%identity with the amino acid sequence as shown in any one of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54.
In the second aspect of the invention, the present disclosure provides a fusion protein. According to an embodiment of the present invention, the fusion protein comprisinga first peptide, wherein the first peptide comprises a bioactive molecular functional region; a second peptide conncected with the first peptide, wherein the second peptide comprises an Fc mutant of the first aspect, wherein a N-terminal of the second peptide is connected to a C-terminal of the first peptide. According to an embodiment of the present invention, the fusion protein has strong in vitro and in vivo binding activity with human growth hormone receptor, can effectively eliminate or lower ADCC and CDC effect, promote NB2-11 cell proliferation, and effectively treat or prevent abnormal growth hormone related diseases.
According to embodiment of the present invention, the fusion protein can further comprise at least one of the following additional technical characteristics:
According to an embodiment of the present invention, the first peptide comprises a growth hormone, a growth hormone analog, a growth hormone functional region or a growth hormone analog functional region.
According to an embodiment of the present invention, the first peptide comprises the human growth hormone or the human growth hormone functional region.
According to an embodiment of the present invention, the human growth hormone comprisies an amino acid sequence as shown in SEQ ID NO: 1 or at least 90%homologous thereof.
According to an embodiment of the present invention, the fusion protein further comprisies a linker peptide.
According to an embodiment of the present invention, an N-terminal of the linker peptide is connected with the C-terminal of the first peptide, and a C-terminal of the linker peptide is connected with the N-terminal of the second peptide.
According to an embodiment of the present invention, the linker peptide comprises an amino acid sequence as shown in the SEQ ID NO: 46.
According to an embodiment of the present invention, the linker peptide comprises mutantions at the following positions in comparision to the amino acid sequence as shown in the SEQ ID NO: 46: at least one S>T in any one GGGGS, increasing G mutation at N-terminal and increasing L mutation at C-terminal.
(G 4S)  n (SEQ ID NO: 46) , where “n” is an integer greater than 0. The “n” preferred value is 2～6.
According to an embodiment of the present invention, the linker peptide comprisies an amino acid sequence as shown in SEQ ID NO: 11.
According to an embodiment of the present invention, wherein the fusion protein comprises: 1) an amino acid sequence as shown in any one of SEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, 28; or 2) an amino acid sequence with at least 90%and 95%identity with the amino acid sequence as shown in any one of SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28. In this invention, the inventor unexpectedly found that the binding activity of the fusion protein obtained after the combination of the second peptide and the linker peptide with the human growth hormone receptor and the promoting effect of NB2-11 cell proliferation were significantly superior.
In the third aspect of the invention, the present disclosure provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid moleculeencoding the Fc mutant of the first aspect or the fusion protein of the second aspect. The Fc mutant obtained according to the nucleic acid molecule of the embodiment of the present invention can effectively avoid to format of semi-antibodies and eliminate ADCC and CDC effects. The fusion protein obtained in present invention has strong in vitro and in vivo binding activity with human growth hormone receptor and can effectively eliminate or lower ADCC and CDC effects and promote the proliferation of NB2-11 cells, and effectively treat or prevent abnormal growth hormone related diseases.
According to embodiments of the present invention, the nucleic acid molecule can further comprise at least one of the following additional technical characteristics:
According to an embodiment of thepresent invention, the nucleic acid molecule is DNA.
According to an embodiment of thepresent invention, the nucleic acid molecule comprisesan nucleotide sequence as shown in any one ofSEQ ID NOs : 32, 33, 34, 35, 36, 37, 38, 39 or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20.
It should be noted that the nucleic acid, as mentioned in the description and claims of the present disclosure, includes any one, or two, of a complementary double-strand. In the description and claims of the present disclosure, only one strand is provided in most cases for convenience, but the disclosure includes the other one strand of the complementary double-strand. For example, when referring to SEQ ID NOs : 32, 33, 34, 35, 36, 37, 38, 39 or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, they include their complementary sequences. It would be also understood that one strand can be determined using the other one strand of the complementary double-strand, vice versa.
The gene sequence in the present disclosure includes both the DNA form and the RNA form, wherein in the case that one form is disclosed, the other one is also disclosed.
The term "encoding" refers to the inherent properties of polynucleotides such as genes, cDNAs, or mRNAs in which specific nucleotide sequences are used as templates for the synthesis of other polymers and macromolecules in biological processes. The polymers and macromolecules have a certain nucleotide sequence (e.g. rRNA, tRNA, and mRNA) or defined amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene, cDNA, or RNA encodes the protein. The coding strand and its nucleotide sequence are identical to the mRNA sequence and are usually provided in the sequence listing, while the non-coding strand used as a template for the transcription of a gene or cDNA can be referred to as a coding protein or other products of the gene or cDNA. Unless otherwise specified, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence.
In the fourth aspect of the invention, the present disclosure provides an expression vector. According to an embodiment of thepresent invention, the expression vectorcarriesthe nucleic acid molecule of first aspect. When the nucleic acid molecule is connected to the carrier, the nucleic acid molecule can be directly or indirectly connected to the control element on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly originaed from the vector itself, but also can be exogenous, that is, not only from the vector itself. Of course, the nucleic acid molecule can be operationally connected to the control element. In this invention, "operable linkage" refers to the connection of exogenous genes to the vector so that the control elements in the vector, such as transcription control sequence and translation control sequence, can play their expected functions of regulating transcription and translation of exogenous genes. Common vectors such as plasmids, phages, etc. After the expression vectors of some specific embodiments of the present invention are introduced into appropriate receptor cells, the expression of the Fc mutant or fusion protein mentioned above can be effectively realized under the mediation of the regulatory system, and then the Fc mutant or fusion protein can be obtained in large quantities in vitro.
According to embodiments of the present invention, the expression vector can further comprise at least one of the following additional technical characteristics:
According to an embodiment of thepresent invention, the expression vector is an eukaryotic expression vector, a prokaryotic expression vector or a viral vector.
According to an embodiment of thepresent invention, the viruses comprises lentiviruses.
In the fifth aspect of the invention, the present disclosure provides an recombinant cell. According to an embodiment of the present invention, the recombinant cellcarriesthe nucleic acid molecule of third aspect, or the expression vector of fourth aspect, or expressing the Fc mutant of any one of first aspect or the fusion protein of second aspect. According to an embodiment of the present invention, the recombinant can be used for in vitro expression and mass availability of the Fc mutants of first aspect or fusion proteins of second aspect.
According to embodiments of the present invention, the recombinant cell can further comprise at least one of the following additional technical characteristics:
According to an embodiment of thepresent invention, the recombinant cell is obtainable by introducing the expression vector mentioned of the fourth aspect into a host cell.
According to an embodiment of the present invention, the expression vector is introduced into the host cell by means of electrical transduction.
According to an embodiment of the present invention, the recombinant cell isan eukaryotic cell.
According to an embodiment of the presentinvention, the recombinant cell is amammalian cell.
According to embodiments of the present invention, the eukaryotic cell not comprises animal germ cell, oospermand embryonic stem cell.
It should be noted that the recombinant cell in the present invention are not subject to special restrictions and it can comprisea prokaryotic cell, the eukaryotic cell or a phage. The prokaryotic cell can comprise Escherichia coli, Bacillus subtilis, Streptomyces or Exotic proteobacteria. The eukaryotic cell can comprise pichia pastoris, Saccharomyces cerevisiae, Fission yeast, Fungi, such as Trichoderma, insect cell, such as Grass armyworm, plant cell, such as tobacco, mammalian cell such as BHK cell, CHO cell, COS cell, myeloma cell, In some embodiments, the recombinant cells are mammalian cellspreferably, comprising BHK cell, CHO cell, NSO cell or COS cell, and do not comprising animal germ cell, oosperm or embryonic stem cell.
It should be noted that the "suitable conditions" in this invention refers to the conditions suitable for the expression of Fc mutants or fusion proteins. The skilled artisan will understand thatthe conditions suitable for the expression of Fc mutants or fusion proteins comprise but are not limited to suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. There are no special restrictions on "suitable conditions" and the skilled artisan can optimize the conditions for the expression of the Fc mutant or fusion protein according to the laboratory environment.
In the sixth aspect of the invention, the present disclosure provides a composition. According to an embodiment of the present invention, the compositioncomprises the fusion protein ofthe second aspect, or the nucleic acid molecule ofthe third aspect, or the expression vector ofthe fourth aspect, or the recombinant cell ofthe fifith aspect. As mentioned above, the fusion protein and the fusion protein obtained after the expression of the nucleic acid molecule, or the expression vector or the recombinant cell can not only effectively bind with the human growth hormone receptor, but also effectively promote the proliferation of NB2-11 cell. Therefore, the fusion protein contained or expressed in the composition according to embodiments of the invention also can bind the human growth hormone receptor, promote the proliferation of NB2-11 cell, and effectively treat or prevent abnormal growth hormone related diseases.
The skilled artisan can understand that the composition comprises an food composition, or a drug composition, etc. The composition comprises combination that is separate in time and/or space, as long as it can act together to achieve the purpose of the invention. For example, the components contained in the  composition may be applied to the subject as a whole or separately. When the components contained in the composition are applied separately to the subject, each component can be applied simultaneously or sequentially to the subject.
In the seventh aspect of the invention, the present disclosure provides the use of the fusion proteins ofany one of the second aspect, the nucleic acid molecules ofthe third aspect, the expression vectors of the fourth aspect, the recombinant cell of the fifth aspect, or the compositions of the sixth aspect in the preparation of medicament for preventing or treating disease associated with abnormal growth hormone. According to an embodiment of the present invention, the medicament is used to treat or prevent abnormal growth hormone related diseases.
According to embodiments of the present invention, the usecan further comprise at least one of the following additional technical characteristics:
According to an embodiment of thepresent invention, the disease associated with abnormal growth hormone comprises at least one of the following: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner's syndrome, Prader-Willi syndrome, kidney failure, disorders caused by alienated states during chemotherapy and AIDS treatment, and intrauterine growth retardation.
In the eighth aspect of the invention, the present disclosure provides a drug. According to an embodiment of the present invention, the drug comprises fusion proteins of the second aspect, the nucleic acid molecules of the third aspect, the expression vector of the forth aspect, the recombinant cell of the fifth aspect, or the composition of the sixth aspect. As mentioned above, the fusion protein and the fusion protein obtained after the expression of the nucleic acid molecule, or the expression vector or the recombinant cell can not only effectively bind with the human growth hormone receptor, but also effectively promote the proliferation of NB2-11 cell. Therefore, the fusion protein contained or expressed in the composition according to embodiments of the invention also can bind the human growth hormone receptor, promote the proliferation of NB2-11 cell, and effectively treat or prevent the abnormal growth hormone related diseases.
According to embodiments of the present invention, the drugcan further comprise at least one of the following additional technical characteristics:
According to an embodiment of thepresent invention, the drug can further comprise pharmaceutically acceptable carriers, for example, solvent, stabilizers, solid excipient, antioxidants, diluent, binder, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspension agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid adhesive, flow aid or lubricant, and so on, that are nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed, to fit the specific target dosage form. In addition to the extent to which any conventional excipients are incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other component of a pharmaceutically acceptable composition in a harmful manner, their uses are also considered in the scope of the present invention.
For example, the fusion protein of the present disclosure can be incorporated into a drug suitable for parenteral administration (e.g. intravenously, subcutaneously, intraperitoneally, intramuscularly) . These drugs  can be prepared in various forms, such as liquid, semi-solid and solid dosage forms, comprising but not limited to liquid solutions (e.g., injection and infusion solutions) , dispersants or suspensions, tablets, pills, powders, liposomes and suppositories. Drugs typically take the form of an injectable or infusion solution. The fusion protein can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
The drug of the present disclosure also can be suitable for oral administration. In some embodiments, the drug are used (e.g., administered to a subject in need of treatment, such as a human individual) by oral administration. For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Active component (s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate. Examples of additional inactive ingredients that may be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
Dosages and desired concentration of the drug of the present disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan.
Administration of a drug of the present disclosure can be continuous or intermittent, depending, for example, on the recipient’s physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. It is within the scope of the present disclosure that dosages may be administered by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
According to embodiments of the invention, the drug can further comprise at least one of the following additional technical features:
In the ninth aspect of the invention, the present disclosure provides a method of preventing or treating diseases associated with abnormal growth hormone. According to an embodiment of the present invention, the methodcomprises applying to subjects at least one of the following: 1) the fusion protein of the second aspect; 2) the nucleic acid molecule of the third aspect; 3) The expression vector of the fourth aspect; 4) The recombinant cells of the fifth aspect; 5) the composition of the sixth aspect; or 6) the drug of the eigth aspect. As mentioned above, the fusion protein and the fusion protein obtained after the expression of the  nucleic acid molecule, or the expression vector or the recombinant cell can not only effectively bind with the human growth hormone receptor, but also effectively promote the proliferation of NB2-11 cell. Therefore, the fusion protein contained or expressed in the composition according to embodiments of the invention also can bind the human growth hormone receptor, promote the proliferation of NB2-11 cell, and effectively treat or prevent the abnormal growth hormone related diseases.
According to embodiments of the present invention, the method can further comprise at least one of the following additional technical characteristics:
According to an embodiment of thepresent invention, the fusion protein is adminstered by subcutaneous injection or intravenous injection.
According to an embodiment of thepresent invention, the abnormal growth hormone related diseases comprises at least one of the following: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner's syndrome, Prader-Willi syndrome, kidney failure, disorders caused by alienated states during chemotherapy and AIDS treatment, and intrauterine growth retardation.
In the tenthaspect of the invention, the present disclosure provides the fusion protein of the fusion protein of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the recombinant cells of the fifth aspect, the composition of the sixth aspect, or the drug of the eigth aspect, for use in preventing or treating a diseaseassociated withabnormal growth hormone. As mentioned above, the fusion protein and the fusion protein obtained after the expression of the nucleic acid molecule, or the expression vector or the recombinant cell can not only effectively bind with the human growth hormone receptor, but also effectively promote the proliferation of NB2-11 cell. Therefore, the fusion protein contained or expressed in the composition according to embodiments of the invention also can bind the human growth hormone receptor, promote the proliferation of NB2-11 cell, and effectively treat or prevent the abnormal growth hormone related diseases.
According to embodiments of the present invention, the use can further comprise at least one of the following additional technical characteristics:
According to an embodiment of thepresent invention, the present disclosure provides the fusion protein of the fusion protein of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the recombinant cells of the fifth aspect, the composition of the sixth aspect, or the drug of eigth aspect, for use in preventing or treating a diseaseassociated withabnormal growth hormone comprises at least one of the following:
childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner's syndrome, Prader-Willi syndrome, kidney failure, disorders caused by alienated states during chemotherapy and AIDS treatment, and intrauterine growth retardation.
BRIEF DESCRIPTION OF THE FIGURES
The present application can be best understood by reference to the following description taken in conjunction with the accompanying figures included in the specification.
FIG. 1 is a schematic diagram of fusion protein in one embodiment of the present invention.
FIG. 2 is the result graph of ADCC effect induced by fusion protein 9#.
DESCRIPTION OF THE DISCLOSURE
The present invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described herein. In the event that one or more of the incorporated literature, patents, and similar materials differ from or contradict this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.
It is further appreciated that certain features of the present invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the present invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one skilled in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entirety. Although many methods and materials similar or equivalent to those described herein could be used in the practice or test of the present invention, the preferred methods, equipment and materials are described in the present invention.
The fusion protein of the invention is generally prepared by a biosynthesis method. According to the nucleotide sequence of the present invention, the one skilled in the art to which this invention belongs can easily obtain the coding nucleic acid of the present invention by various known methods. These methods comprise but are not limited to: PCR, artificial DNA synthesis, etc. For specific methods, please refer to Joseph. Sambrook 《Molecular cloning: a laboratory manual》. In some implementation of the present invention, the coding nucleic acid sequence of the present invention can be constructed by the method of segmented synthesis of nucleotide sequence followed byoverlap extension PCR.
In present invention, the term “comprise” , “contain” or “include” is an open expression, it means comprising the contents disclosed herein, but don’ t exclude other contents.
In present invention, the terms “optionally” or “optional” generally means that the subsequently described event or condition can occur, but may not must, and that the description includes the circumstances in which the event or condition occurs and the circumstances in which the event or condition does not occur.
The term “room temperature” refers to the ambient temperature, which may be 20℃-30℃. In some embodiments, the room temperature may be 22℃-28℃; In some embodiments, 24℃-26℃; In some embodiments, 25℃.
In present invention, the term "fusion protein" refers to the product obtained by the fusion of antibody fragment with other bioactive protein using genetic engineering technology.
In present invention, the term "mutant" generally refers to any naturally occurring or engineered molecule that contains one or more nucleotide or amino acid mutations.
In present invention, the term antibody "Fc fragment" includes the CH2 and CH3 regions of the antibody, for example, the wild-type IgG4 Fc fragment comprises the CH2 and CH3 regions of the wild-type IgG1 antibody, and the wild-type IgG1 Fc fragment comprises the CH2 and CH3 regions of the wild-type IgG1 antibody.
In present invention, "semi-antibody" refers to the two antibody fragments formed after the disulfide bond of the antibody Fc fragment is broken. For example, the Fc fragment of the wild-type human IgG4 antibody forms the semi-antibody fragments containing the CH2 and CH3 regions of the wild-type human IgG4 antibody, the Fc fragment of wild-type human IgG1 antibody forms the semi-antibody fragmentscontaining fragments of the CH2 and CH3 regions of wild-type human IgG1 antibody.
In this application, the term "nucleotide" generally is ribonucleotides, deoxynucleotides, or any type of nucleotide, and theirmodified forms, and their combinations.
The term "host cell" used in present invention refers to prokaryotic or eukaryotic cell that can be introduced into recombinant expression vectors. The term "transformed" or "transfected" in present invention refers to the introduction of nucleic acids (such as vectors) into cells through various techniques known in the art.
In present invention, the term "identity" , "homologous" or "similar" are used to describe the relative to the reference sequence of amino acid sequencesor nucleic acid sequence, determines the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequencesby conventional methods, see, for example, Ausubel et al., eds. (1995) , Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York) ; and the ALIGN program (Dayhoff (1978) , Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C. ) . There are many algorithms for aligning sequences and determining sequence identity, including, the homology alignment algorithm ofNeedleman et al. ( (1970) J. Mol. Biol. 48: 443) ; the local homology algorithmof Smith et al. ( (1981) Adv. Appl. Math. 2: 482) ; the similarity search method of Pearson et al. ( (1988) Proc. Natl. Acad. Sci. 85: 2444) ; the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997) ) ; and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403-410) . Using computer programs for these algorithms are also available, and the computer programsinclude, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266: 460-480 (1996) ) ; or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and the CLUSTAL of PC/Gene program provided by Intelligenetics, Mountain View, California..
In present invention, the amino acid number of the Fc fragment of IgG4 or IgG1 is numbered according to the EU numbering system. For example, the "S228P" refers to the serine at position 228 of the EU numbering system replacedby proline.
In present invention, the amino acid of IgD fragment is numbered according to the reference: Takahashi, Nobuhiro, Et al. "Sequence of Complete Amino Acid of the δ Heavy Chain of Human Immunoglobulin D. " Proceedings of the National Academy of Sciences of the United States of America, Vol. 79, No. 9, 1982, pp. 2850 --54, http: //www. jstor. org/stable/11720. Accessed 12 Apr. 2022.
In one aspect, the present disclosure provides a fusion protein, the general structure of the fusion protein is expressed as X-L-Y, wherein X is the first bioactive molecule; L is absent or linker peptide; Y is the second bioactive molecule; -is the peptide bond; and the X or Y is selected from the protein or protein domain, polypeptide, antibody or antibody fragment.
According to some specificembodiments of the present invention, the fusion protein can further comprise at least one of the following additional technical characteristics:
According to an embodiment of the present invention, the X is human growth hormone.
According to an embodiment of the present invention, the human growth hormone comprisies an amino acid sequence as shown in SEQ ID NO: 1 or at least 80-99%homologous thereof or at least part of SEQ ID NO: 1.
Figure PCTCN2022100240-appb-000003
According to an embodiment of the present invention, the Y is an Fc fragment or a variant thereof.
According to an embodiment of the present invention, the Fc fragment comprisies an amino acid sequence as shown in SEQ ID NO: 29, SEQ ID NO: 30 or at least 80-99%homologous thereof, or at least part of its sequence; or the amino acid sequence of the Fc fragment variant comprises selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7or at least 80-99%homologous thereof, or part of its sequence.
The amino acid sequence of the Fc fragment variant 1 comprising:
Figure PCTCN2022100240-appb-000004
The amino acid sequence of the Fc fragment variant 2 amino acid sequences comprising:
Figure PCTCN2022100240-appb-000005
The amino acid sequence of the Fc fragment variant 3 amino acid sequences comprising:
Figure PCTCN2022100240-appb-000006
The amino acid sequence of the Fc fragment variant 4 amino acid sequences comprising:
Figure PCTCN2022100240-appb-000007
The amino acid sequences of the Fc fragment variant 5 amino acid sequences comprising:
Figure PCTCN2022100240-appb-000008
The amino acid sequence of the Fc fragment variant 6 amino acid sequences comprising:
Figure PCTCN2022100240-appb-000009
The amino acid sequence of the wild-type IgG4 fragment comprising:
Figure PCTCN2022100240-appb-000010
The amino acid sequence of the wild-type IgG1 Fc fragment comprising:
Figure PCTCN2022100240-appb-000011
According to an embodiment of thepresent invention, the amino acid sequenceof the fusion protein comprisesselected fromSEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28 or any amino acid sequence or at least 80-99%homologous thereof , or part of its sequence.
The amino acid sequence of the fusion protein 8# (bioactive molecular functional region -Fc mutant 1) comprising:
Figure PCTCN2022100240-appb-000012
Figure PCTCN2022100240-appb-000013
The amino acid sequence of the fusion protein 9# (bioactive molecular functional region -Fc mutant 2) comprising:
Figure PCTCN2022100240-appb-000014
The amino acid sequence of the fusion protein 10# (bioactive molecular functional region -Fc mutant 4) comprising :
Figure PCTCN2022100240-appb-000015
The amino acid sequence of the fusion protein 11# (bioactive molecular functional region - (G 4S)  3-FC mutant 5) comprising:
Figure PCTCN2022100240-appb-000016
The amino acid sequence of the fusion protein 12# (bioactive molecular functional region -GS linker peptied mutant -Fc mutant 3) comprising:
Figure PCTCN2022100240-appb-000017
Figure PCTCN2022100240-appb-000018
The amino acid sequence of the fusion protein 13# (bioactive molecular functional region -Fc mutant 6) comprising:
Figure PCTCN2022100240-appb-000019
The amino acid sequence of the fusion protein 14# (bioactive molecular functional region - (G4S) 3-FC mutant 7) comprising :
Figure PCTCN2022100240-appb-000020
The amino acid sequence of the fusion protein 15# (bioactive molecular functional region - (G 4S)  3-FC mutant 8) comprising:
Figure PCTCN2022100240-appb-000021
According to an embodiment of the present invention, the linker peptide comprises one or more amino acids selected from glycine, serine, alanine and threonine.
According to an embodiment of the present invention, the linker peptide comprises the amino acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 or at least 80-99%homologous thereof , or part of its sequence.
The amino acid sequence of the Linker1 (the third peptide 1) comprising:
Figure PCTCN2022100240-appb-000022
The amino acid sequence of the Linker 2 (the third peptide 2) comprising:
Figure PCTCN2022100240-appb-000023
The amino acid sequence of the Linker3 (the third peptide 3) comprising:
Figure PCTCN2022100240-appb-000024
The amino acid sequence of the Linker4 comprising:
Figure PCTCN2022100240-appb-000025
The amino acid sequence of the Linker5 (the third peptide 4) comprising:
Figure PCTCN2022100240-appb-000026
In another aspect, the present disclosure provides a nucleotide sequenceaccording to an embodiment of the present invention, the nucleotide sequenceencodes thelinker peited previously described.
In another aspect, the present disclosure provides a nucleotide sequenceaccording to an embodiment of the present invention, the nucleotide sequenceencodes the the fusion protein previously described.
In one aspect, the present disclosure provides an vector. According to an embodiment of thepresent invention, the vectorcarriesthe nucleic acid molecule encodeding the the fusion protein or linker pepited.
According to embodiments of the present invention, the vector can further comprise at least one of the following additional technical characteristics:
According to an embodiment of thepresent invention, the expression vectoris an expression vector.
In this application, the term "vector" usually refers to a nucleic acid molecule capable of self-replicating in a suitable host by transferring the inserted nucleic acid molecule to and/or between host cells. The vector can comprise a vector primarily used for inserting DNA or RNA into cells, a vector primarily used for DNA or RNA replication, and an expression vector primarily used for DNA or RNA transcription and/or translation. The vector also includes a vector with a variety of thefunctions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce the desired expression product by culturing an appropriate host cell containing the vector.
In some embodiments, the present invention presents an engineered cell characterized by either the engineered cell containing the fusion protein previously described or the engineered cell containing the nucleotide sequence or expression vector previously described.
In this application, the term "engineered cell" refers to a cell that is modified or recombined with the genetic material of the host cell by means of genetic engineering techniques or cell fusion techniques to obtain a unique character with stable inheritance.
In some embodiments, the present invention presents a pharmaceutical composition containing the fusion protein previously described.
Depending on some specific embodiments of the invention, the pharmaceutical composition may further include at least one of the following additional technical features:
According to some concrete implementing scheme of the present invention, as described in drug combination is used for oral and intravenous use, such as pushing injection or continuous infusion for a period of time, through the subcutaneous, intramuscular, inside arteries, peritoneal, lungs, brain within the spinal cord, intra-articular, within the synovial membrane and its sheath, damage, or suction path such as nasal, usually by intravenous or subcutaneous application of drug combination.
According to some specific embodiments of the invention, the dosage forms of the drug composition are tablets, capsules, sprays, injections, freeze-dried powder injections or pre-filled needle injections.
In some embodiments, the present invention proposes a preparation method of the fusion protein described above, which includes: (a) culture of the engineered cells described above to obtain a culture medium containing the fusion protein; (b) the fusion protein is isolated from the culture medium.
In some embodiments, the present invention proposes the use of the aforementioned fusion protein or drug composition in the preparation of drugs for the treatment or prevention of diseases lacking human growth hormone.
In this application, the term "treatment" is used to refer to the achievement of a desired pharmacological and/or physiological effect. The effects may be preventive in terms of complete or partial prevention of the disease or its symptoms, and/or therapeutic in terms of partial or complete cure of the disease and/or adverse effects resulting from the disease. The term "treatment" is used in this context to cover diseases of mammals and, in particular, of humans, including: (a) prevention of disease or illness in susceptible but undiagnosed individuals; (b) suppression of disease, for example by blocking its progression; or (c) alleviating the disease, for example by reducing symptoms associated with the disease. The term "treatment" as used herein includes any administration of a drug or compound to an individual to treat, cure, alleviate, improve, mitigate, or inhibit an individual's disease, including but not limited to the administration of a drug containing the compound to an individual in need.
The name and number of each sequence in presentinvention are shown in Table 1.
Table 1:
Figure PCTCN2022100240-appb-000027
Figure PCTCN2022100240-appb-000028
Figure PCTCN2022100240-appb-000029
Figure PCTCN2022100240-appb-000030
Compared with prior art, the present invention has at least one of the following beneficial effects:
(1) The long-acting human growth hormone fusion protein of the invention has low EC50 value and strong binding activity with human GHR, especially the binding activity effect of the human growth hormone fusion protein 8#, 9#, 10#and 15#is significantly better than that of Genexine GX-H9 and GH+G/SLinker +IGG4-1.
(2) The long-acting human growth hormone fusion protein of the invention has a good proliferation effect on NB2-11 cells; the human growth hormone fusion protein 8#, 9#, 10#, 12#had better proliferative activity than Genexine GX-H9 and GH+G/SLinker + IGG4-1.
(3) The long-acting human growth hormone fusion protein reporter cells of the present invention have good luciferase activity. Among them, the human growth hormone fusion proteins 8#, 9#, 10#and 12#have better luciferase activity than Genexine GX-H9 and GH+G/SLinker + IGG4-1.
(4) The third peptide chains are provided by the invention can improve the binding activity of the human growth hormone fusion protein containing the third peptide chain to human GHR, the proliferation activity of NB2-11 cells and the luciferase expression activity of reporter cells.
(5) The SEQ ID NO: 54 is provided by the invention is conducive to improving the binding activity of human growth hormone fusion protein containing the second bioactive molecule of SEQ ID NO: 54 amino acid sequence to human GHR.
(6) The third peptide chain is provided by the invention, as shown in SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, is conducive to improving the proliferation activity of the human growth hormone fusion protein containing the third peptide chain against NB2-11 cells.
(7) The second bioactive molecule is provided by the invention, as shown in SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 50, is conducive to improving the proliferation activity of the human growth hormone fusion protein containing the second bioactive molecule against NB2-11 cells..
(8) The third peptide chain is provided by the invention, as shown in SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, is conducive to improving the luciferase expression activity of the human growth hormone fusion protein.
(9) The second bioactive molecule is provided by the invention, as shown in SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 50, is conducive to improving the luciferase expression activity of the human growth hormone fusion protein containing the second bioactive molecule.
(10) The fusion proteins are provided by the invention has a longer half-life in vivo.
The scheme of the invention is explained below in combination with embodiments. Those skilled in the field will understand that the following embodiments are intended only to illustrate the invention and should not be regarded as limiting the scope of the invention. If the specific technology or conditions are not specified in the embodiment, the technology or conditions described in the literature in the field or the product specification shall be followed. Reagents or instruments used without manufacturer are conventional products that can be purchased in the market.
EXAMPLES
The following Examples are merely illustrative and is not meant to limit any aspects of the present disclosure in any way.
Generally, the protein disclosed herein may be prepared by methods described herein, wherein the peotein are as defined for above, except where further noted. The following non-limiting schemes and examples are presented to further exemplify the invention.
Professionals skilled in the art will recognize that the chemical reactions described may be readily adapted to prepare a number of other compounds disclosed herein, and alternative methods for preparing the compounds disclosed herein are deemed to be within the scope disclosed herein. For example, the synthesis of non-exemplified compounds according to the present invention may be successfully performed by modifications apparent to those skilled in the art, e.g., by appropriately protecting interfering groups, by utilizing other suitable reagents known in the art other than those described, and/or by making routine modifications of reaction conditions. Alternatively, the known reaction conditions or the reaction disclosed in  the present invention will be recognized as having applicability for preparing other compounds disclosed herein.
Example 1 The preparing of the expression vector of human growth hormone fusion protein
In this application, the human growth hormone (SEQ ID NO: 1) was linked to a variety of antibody Fc mutants (any one of SEQ ID NO: 47-54, the second peptide chain was indicated in bold letters in the sequence) to construct fusion proteins. Some fusion proteins had linker peptides, and the specific structure of the fusion proteins with linker peptides was shown in Figure 1. The linker peptide is GGGGSGGGGSGGGGS (SEQ ID NO: 45) or obtained by mutation, and the antibody Fc mutant is obtained by mutation of wild-type IgG4 Fc fragment (SEQ ID NO: 71) or IgG1 Fc fragment (SEQ ID NO: 30) . The mutation modes include point mutation, the addition of wild-type IgG4 and/or IgD antibody fragments or their corresponding mutants, etc. The specific experimental operations are as follows:
Combining the nucleotide sequence encoding human growth hormone (SEQ ID NO: 31) , the nucleotide sequence encoding linker peptide, and the nucleotide sequence encoding multiple antibody Fc mutants (SEQ ID NO: 32-39) , the nucleotide sequences encoding fusion protein SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 and SEQ ID NO: 20 were respectively synthesized by total gene synthesis and molecular cloning techniques and were cloned into the expression vector pCDNA3.4 (purchased from Thermo Fisher) , and the fusion protein expression vector of each nucleotide sequence was obtained.
The amino acid sequence of the Human Growth hormone (hGH) is as follows:
Figure PCTCN2022100240-appb-000031
The amino acid sequence of the first peptide chain 1 (conprising F234V and L235E mutations compared to wild-type IgG4 Fc fragment) is as follows:
Figure PCTCN2022100240-appb-000032
The amino acid sequence of the first peptide chain 2 (comprising S298N, T299A and Y300S mutations compared to wild-type IgG4 Fc fragment) is as follows:
Figure PCTCN2022100240-appb-000033
The amino acid sequence of the first peptide chain 3 (with amino acid deletion at position 231-239 compared to the wild-type IgG4 Fc fragment) is as follows:
Figure PCTCN2022100240-appb-000034
The amino acid sequence of the first peptide chain 4 (comprising amino acid deletion at position 228-239, H268Q, Y296F, A330S, and P331S mutations compared to the Fc fragment of wild-type IgG1) is as follows:
Figure PCTCN2022100240-appb-000035
The amino acid sequence of first peptide chain 5 (F234A and L235E mutations compared to the Fc fragment of wild-type IgG4 antibody) is as follows:
Figure PCTCN2022100240-appb-000036
The amino acid sequence of first peptide chain 6 (F234V and L235A mutations compared to the Fc fragment of wild-type IgG4 antibody) is as follows:
Figure PCTCN2022100240-appb-000037
The amino acid sequence of the second peptide chain 1 is as fpllows:
Figure PCTCN2022100240-appb-000038
The amino acid sequence of the second peptide chain 2 (IgD mutational fragment 1) is as follows:
Figure PCTCN2022100240-appb-000039
The amino acid sequence of the second peptide chain 3 (wild-type IgD fragment 1) is as follows:
Figure PCTCN2022100240-appb-000040
The amino acid sequence of the third peptide chain 1 is as follows:
Figure PCTCN2022100240-appb-000041
The amino acid sequence ofthe third peptide chain 2 is as follows:
Figure PCTCN2022100240-appb-000042
The amino acid sequence of the third peptide 3 is as follows:
Figure PCTCN2022100240-appb-000043
The amino acid sequence of the third peptide 4 is as follows:
Figure PCTCN2022100240-appb-000044
The amino acid sequence of the Fc mutant 1 (first peptide chain 5-second peptide chain 1-third peptide chain 1) is as follows:
Figure PCTCN2022100240-appb-000045
The amino acid sequence of the Fc mutant 2 (first peptide chain 6-second peptide chain 1-third peptide chain 2) is as follows :
Figure PCTCN2022100240-appb-000046
The amino acid sequence of Fc mutant 3 (first peptide chain 5-second peptide chain 1) is as follows:
Figure PCTCN2022100240-appb-000047
The amino acid sequence of the Fc mutant 4 (peptide-1 -peptide-1 -peptide-3) is as follows:
Figure PCTCN2022100240-appb-000048
The amino acid sequence of the Fc mutant 5 (first peptide chain 2-second peptide chain 1) is as follows:
Figure PCTCN2022100240-appb-000049
The amino acid sequence of the Fc mutant 6 (firstpeptide chain 2-secondpeptide chain 1-third peptide chain 4) amino acid sequence is as follows:
Figure PCTCN2022100240-appb-000050
Figure PCTCN2022100240-appb-000051
The amino acid sequence of theFc mutant 7 (first peptide chain 3-second peptide chain 2) :
Figure PCTCN2022100240-appb-000052
The amino acid sequence of the Fc mutant 8 (first peptide chain 4-second peptide chain 3) is as follows:
Figure PCTCN2022100240-appb-000053
The amino acid sequence of the Linker pepited (4) is as follows:
Figure PCTCN2022100240-appb-000054
The nucleotide sequence encoding human growth hormone (hGH) is as follows:
Figure PCTCN2022100240-appb-000055
The nucleotide acid sequence encoding the Fc mutant 1is as follows:
Figure PCTCN2022100240-appb-000056
Figure PCTCN2022100240-appb-000057
The nucleotide acid sequence encoding the Fc mutant 2 is as follows:
Figure PCTCN2022100240-appb-000058
The nucleotide acid sequence encoding the Fc mutant 3 is as follows:
Figure PCTCN2022100240-appb-000059
The nucleotide acid sequence encoding the Fc mutant 4 is as follows:
Figure PCTCN2022100240-appb-000060
Figure PCTCN2022100240-appb-000061
The nucleotide acid sequence encoding the Fc mutant 5 is as follows:
Figure PCTCN2022100240-appb-000062
The nucleotide acid sequence encoding the Fc mutant 6 is as follows:
Figure PCTCN2022100240-appb-000063
The nucleotide acid sequence encoding the Fc mutant 7 is as follows:
Figure PCTCN2022100240-appb-000064
Figure PCTCN2022100240-appb-000065
The nucleotide acid sequence encoding the Fc mutant 8 is as follows:
Figure PCTCN2022100240-appb-000066
The nucleotide acid sequence encoding the third peptide chain 1 is as follows:
Figure PCTCN2022100240-appb-000067
The nucleotide acid sequence encoding the third peptide chain 2 is as follows:
Figure PCTCN2022100240-appb-000068
The nucleotide acid sequence encoding the third peptide chain 3 is as follows:
Figure PCTCN2022100240-appb-000069
The nucleotide acid sequence encoding the third peptide chain 4 is as follows:
Figure PCTCN2022100240-appb-000070
The nucleotide acid sequence encoding the Linker4 is as follows:
Figure PCTCN2022100240-appb-000071
The nucleotide sequence encoding the fusion protein 8#is as follows:
Figure PCTCN2022100240-appb-000072
Figure PCTCN2022100240-appb-000073
The nucleotide sequence encoding the fusion protein 9#is as follows:
Figure PCTCN2022100240-appb-000074
The nucleotide sequence encoding the fusion protein 10#is as follows:
Figure PCTCN2022100240-appb-000075
Figure PCTCN2022100240-appb-000076
The nucleotide sequence encoding the fusion protein 11#is as follows:
Figure PCTCN2022100240-appb-000077
The nucleotide sequence encoding the fusion protein 12#is as follows:
Figure PCTCN2022100240-appb-000078
The nucleotide sequence encoding the fusion protein 13#is as follows:
Figure PCTCN2022100240-appb-000079
Figure PCTCN2022100240-appb-000080
The nucleotide sequence encoding the fusion protein 14#is as follows:
Figure PCTCN2022100240-appb-000081
The nucleotide sequence encoding the fusion protein 15#is as follows:
Figure PCTCN2022100240-appb-000082
Figure PCTCN2022100240-appb-000083
The amino acid sequence of the Fc fragment of wild-type IgG4 is as follows:
Figure PCTCN2022100240-appb-000084
Example 2 The expression of human growth hormone fusion protein
In this embodiment, the multiple plasmids obtained from example 1 were transfected into Expi CHO-S (Gibco, A29133) host cells, and the long-acting human growth hormone fusion protein was transient expressed by chemical transfection reagent Polyplus-Fectopro (Polyplus, 116-010) . Its corresponding amino acid sequence is SEQ ID NO: 21-28. The specific experimental operations are as follows:
(1) Expi CHO-Scells were subcultured one day before transient transfection, and the cell density was adjusted to about 3×10 6 cells/mL by medium (CD FortiCHO TM medium) . Then, the cell culture flask was put back to the shaker (37℃, 8%CO 2) for further culture.
(2) On the day of transient transfection : Expi CHO-Scell fluid was taken for cell counting, and the cell density was adjusted to about 6×10 6cells/mL by medium. Then 1L cell fluid with a density of 6×10 6 cells/mL was required for each fusion protein expression vector to be transfected.
(3) Preparation of transfection complex: 16 sterile cell culture bottles were taken, 8 labeled "DNA" and 8 labeled "FectoPRO" , and transfection reagent Polyplus-fectopro was added into the "FectoPRO" flask and mixed; 60mL OPti-MEM solution and fusion protein expression vector containing each nucleotide sequence of example 1was added into ench "DNA" flask and mixed. And obtained the diluent of fusion protein expression vector. And the diluent of fusion protein expression vector of each nucleotide sequence was added into the "FectoPRO" flask and mixed, and was incubated at room temperature for 10 minutes, then was added to the cell solution and shaken, and the cell culture flask was returned to the shaker to continue culture and transfection.
(4) After the cell culture medium obtained in step 3) was transfected for 18-22 hours, an appropriate amount of OPM-CHO ProFeed was added, and the biochemical indicators of the cell solution were measured. According to biochemical indicators, the glucose was supplied to 6g/L. Titer was measured 4 days after transfection, and the sugar and culture solution were supplied every other day. The CHO cell fermentation broth could be obtained when the cell viability is less than 80%, and the supernatant can be harvested for purification.
Example 3: Purification of human growth hormone fusion protein
In this embodiment, the CHO cell fermentation liquid obtained in example 2 was centrifuged in two stages (first stage: 3,000×g, 30 min; Second stage: 12,000×g, 20 min) , the supernatant was collected and filtered through a 0.2μm filter, and set aside.
Protein A affinity chromatography: The aqueous solution containing 20 mM phosphate and 150 mM NaCl at pH 7.2 was used to equilibrate the chromatographycolumnfor at least 3 columnvolumes, and the filtered clarified filtrate was loaded and remained on the chromatography column for 5 minutes. After loading the sample, the aqueous solution containing 20 mM phosphate and 150 mM NaCl at pH 7.2 was used to equilibrate the chromatographycolumnfor at least 1 column volume, followed by 50 mM pH 4.5 Acetic acid (HAc) buffer, 50 mM pH 4.0 HAc buffer, and 50 mM pH 3.5 HAcbuffer were used to elute the target protein respectively. When an absorption peak appeared, the peak range was 50 mAU-peak-50 mAU, and the desired SEC purity (> 98.0) fusion protein is obtained. A variety of Expi Cho-Scells respectively containing nucleotide sequence SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28 were fermented and chromatographed to obtain fusion protein 8#, fusion protein 9#, fusion protein 10#, fusion protein 11#, fusion protein 12#, fusion protein 13#, fusion protein 14#and fusion protein 15#.
Example 4: Determination of receptor-binding activity of human growth hormone fusion protein in vitro
In this embodiment, in vitro receptor (Human growth hormone receptor, Human GHR) binding activity of the purified fusion protein obtained from example 3 was detected. The Genexine GX-H9 (SEQ ID NO: 70) and GH+G/SLinker + IgG 4-1 (SEQ ID NO: 43) were used as the control group. The specific experimental procedures were as follows:
HGHR was diluted to prepare 0.5μg/mL coating solution, which was added to enzyme plate at 100μL/well, and coated at 2-8 ℃ for more than 12 hours. The plate residue was discarded, 300 μL of1%BSA-PBST (phosphate Tween buffer containing 1%bovine serum albumin) was added into each well, and blocked for 1 hour at 37℃. 300 μL of PBST (Phosphate Tween Buffer) was added to each wellfor 3 washes. The recombinant long-acting human growth hormone fusion protein and Genexine GX-H9 were diluted to 5 μg/ml, and then diluted 5 times to 8 gradient concentrations, addedto 100 μL/well to ELISA plate. The ELISA plate comprising the long-acting human growth hormone fusion protein or Genexine GX-H9 was incubated at 37℃ for 1 hour, then 300 μL of PBST was added to each wellfor 3 washes, and the Goat anti-human IgG Fc-HRP that dilute 10000 foldwith 1%BSA-PBST was added, 100 μL/well of a sample. After incubation at 37℃ for 1 hour, 300μL of PBST was added to each wellfor 3 washes, and patted dry. TMB Substrate was added, 100μL per well. After 5 minutes of reaction at room temperature, 2M H 2SO 4 aqueous solution was added to stop the reaction, 100 μL/well. The ELISA plate that stopped the reaction was put on a microplate reader, the absorbance OD 450 value was read at a wavelength of 450nm, and the EC 50 of each fusion protein's binding activity to hGHR was calculated.
Figure PCTCN2022100240-appb-000085
Figure PCTCN2022100240-appb-000086
The specific experimental results were shown in Table 2. (1) The long-acting Human growth hormone fusion protein had low EC 50 value of the invention and had strong binding activity with hGHR. Especially, the Human growth hormone fusion proteins 8#, 9#, 10#and 15#encoded by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 20 respectinely showed significantly better binding activity to Human GHR than Genexine Gx-h9 and GH+G/SLinker + IgG 4-1; Among them, the human growth hormone fusion protein 15#had the highest binding activity to human GHR, followed by the human growth hormone fusion protein 9#.
(2) The human growth hormone fusion protein 15#contained the second bioactive molecule encoded by SEQ ID NO: 54, indicated that the SEQ ID NO: 54 can improve the binding activity of human growth hormone fusion protein to hGHR.
Table 2:
Figure PCTCN2022100240-appb-000087
Figure PCTCN2022100240-appb-000088
Example 5: Effects of human growth hormone fusion protein on proliferation of NB2-11 cells
In this embodiment, the purified fusion protein obtained in Example 3 was used for the experiment, and the above-mentioned Genexine GX-H9 (commercially available product) and GH+G/SLinker + IGG4-1 were used as the control group. The specific experimental procedures were as follows:
Cells were collected and suspended in medium (PRMI 1640 medium containing 1%FBS and 50μM β-mercaptoethanol) at a concentration of 1×10 5 cells/mL. Add each 50μL cell sample to each well in the 96-well cell culture plate. The cells were cultured in 50μL assay medium containing various concentrations of recombinant long-acting human growth hormone fusion protein, Genexine GX-H9 and GH+G/Slinker+IgG4-1 (%) from 0.051 ng/mL to 3000 ng/mL, respectively. The Cell plates were incubated at 37℃ in a 5%CO 2 moist incubator for 96 h and 50μL cellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571) was added to each well. After 10 minutes, the chemiluminescence signal was detected by a microplate reader. The bioactivity of recombinant long-acting human growth hormone fusion protein was determined by dose-response curve.
The specific experimental results were shown in Table 3, wherein, (1) The long-acting human growth hormone fusion protein of the invention had a good proliferation effect on NB2-11 cells; The human growth hormone fusion protein 8#, 9#, 10#and 12#encoded by the sequence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17 had significantly better proliferative effect than Genexine GX-H9 and GH+G/Slinker + IgG4-1; Among them, the human growth hormone fusion protein 10#had the highest proliferation activity againstNB2-11 cells, followed by the human growth hormone fusion protein 8#, and the human growth hormone fusion protein 9#again.
(2) The third peptide chain encoded by the sequence SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 could enhance the proliferation activity of human growth hormone fusion protein against NB2-11 cells.
(3) The second bioactive molecule encoded by SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 50 could improve the proliferation activity of the human growth hormone fusion protein against NB2-11 cells.
Table 3:
Figure PCTCN2022100240-appb-000089
Figure PCTCN2022100240-appb-000090
Example 6: Biological activity of human growth hormone fusion protein on reporter cells
In this embodiment, the purified human growth hormone fusion protein obtained in Example 3 was subjected to this experiment, and the Genexine GX-H9 (commercially available product) and GH+G/SLinker + IGG4-1 were used as the control group. The specific experimental procedures were as follows.
Reporter cells at the logarithmic growth stage (293-GHR/STAT5 cell line) were adjust to 4×10 4 cells/well, 50 μL/well after trypsin digestion (CoSTAR, 3917) , and were incubated overnight in a 96-well whiteboard at 37 ℃ in a 5%CO 2 incubator. Thereporter cells were added with gradient dilution recombinant long-acting human growth hormone fusion protein at the initial concentration of 100 nM, 5 times gradient dilution at 10 gradient concentrations. The dilution was added to the cells at the volume of 50 μL/well. The 96-well whiteboard was incubated at 37 ℃ in a 5%CO 2 incubator for 6 hours. After incubation, the 96-well white board and nano-Glo Luciferase Assay kit (Promega, N112B) were taken out and balanced to room temperature, the reaction substrate was added to the sample wells at a volume of 50 μL/well and placed at room temperature for 10 minutes. The instrument (Promega, GM2000) recorded the luminescence signal value, with the protein concentration as the X-axis and the luminescence signal value as the Y-axis. The EC50 value was calculated by using GraphPad Prisim 5 as four-parameter fitting.
Specific experimental results were shown in Table 4, wherein:
(1) The recombinant long-acting human growth hormone fusion protein reporter cells of the present invention had good luciferase activity. the human growth hormone fusion proteins 8#, 9#, 10#, 12#comprisedan nucleotide sequence as shown in sequence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17 respectively, could promote luciferase expression activity of reporter cells better than Genexine GX-H9 and GH+G/S Would + IgG4-1; Among them, the human growth hormone fusion protein 9#had the highest activity of promoting luciferase expression in reporter cells, followed by human growth hormone fusion protein 8#and the human growth hormone fusion protein 10#.
(2) The third peptide chain encoded by the sequence SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 could improve the luciferase expression activity of the human growth hormone fusion protein.
(3) The second bioactive molecule encoded by the sequence SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 50 was beneficial to increase the luciferase expression activity of reporter cells containing the human growth hormone fusion protein with the second bioactive molecule.
Table 4:
Figure PCTCN2022100240-appb-000091
Example 7: Detection of ADCC effect of Human growth hormone fusion protein induced effector cell Jurkate-CD16A-Luc on target cell Cho-K1-PD-L1/GHR
7.1 methods
Sample information is shown in Table 5.
Table 5:
Sample name concentration (mg/mL)
M7824 2.05
9#fusion protein GH-IgG4-2) 5.04
5(Genexine GX-H9) 1.33
Acterma 20
7.2 cell processing
Target cells CHO-K1-PD-L1 /GHR (Shanghai Kabe Biomedical Technology Co., LTD., SBTCL015) treatment: The cells were passaged 2 days before the experiment, and the supernatant was discarded. After washing the cells with 5 mL of PBS, 1 mL of 0.25%Trypsin-EDTA was added. After digestion in a 37℃ incubator for 1 min, 6 mL of medium 1 (450 mL of F12 medium + 50 mL of FBS) was added to terminate digestion, and resuspend to single cell suspension, 5mL of cell suspension was discarded, and 8mL of medium 1 (450mL of F12 medium + 50mL of fetal bovine serum (FBS) ) was added, then 120μL of G418 was added and mixed well, the G418 work concentrationwas 600 μg/mL. The cells were put into the incubator to continue culturing.
Effector cells Jurkat-CD16A-LUC treatment: The cells were passaged 2 days before the experiment, and the supernatant was discarded, and the cell suspension was mixed by pipetting. The 5mL cell suspension was taken into the new T75 culture flask. Then 15mL Medium 2 (450mLRPMI Medium 1640+50mL FBS) was added, then 320μL G418 was added. The work concentration of G418 was 800μg/mL, 60μL Hygromycin B was added and mixed, the work concentration of Hygromycin B was 150μg/mL. The cells were put into the incubator to continue culturing..
7.3. Experimental process
On the first day, the cho-K1-PD-L1 /GHR density of target cells was adjusted to 1.83×10 5 cells /mL in analytical medium 1 (40mL F12 medium+ 0.4ml FBS) , and the target cells were plinked overnight at 50μL/well. On the second day, SBT123 (Molecule 9#and Genexine GX-H9) , positive control M7824 and negative control Acterma were first diluted to 0.1mg/mL and 24μg/mL in analysis Medium 2 (RPMI Medium 1640) . Then dilute 9 gradients by 3 times, 25μL/well was taken into cell plates, mixed well, and incubated at 37℃ for 45min, then removed the cell plates. The density of effector cells Jurkat-CD16A-Luc was adjusted to 1.47×10 6 cells /mL in analysis Medium 2 (RPMI Medium 1640) , and 25μL/well was added to the cell plate, mixed well, and incubated at 37℃for 5.5h. After the cell plate was taken out at room temperature and balanced, 100 μL/well of one-GLo Luciferase Assay System (Promega, E6120) was added to the cell plate and pipetted blow 10 times, mixed well. The chemiluminescence signal was detected by a microplate analyzer after 10min of light-resistant pyrolysis at room temperature. Results As shown in Figure 2, after the positive control PD-L1 antibody M7824, negative control IL-6R antibody Acterma and SBT123 were added into the ADCC  experiment, the positive control M7824 induced effector cell Jurkate-CD16A-Luc to have a significant ADCC effect on target cell Cho-K1-PD-L1 /GHR, while the negative control Acterma had no ADCC effect, and SBT123 had no ADCC effect.
Example 8: The CDC effect of the Human growth hormone fusion protein indyced human serum complement on target cells Cho-K1-PD-L1 /GHR.
8.1 methods
Sample information is shown in Table 6:
Table 6:
Figure PCTCN2022100240-appb-000092
(2) Cell processing
Target cells CHO-K1-PD-L1 /GHR (Shanghai Kabe Biomedical Technology Co., LTD., SBTCL015) treatment: The cells were passaged 2 days before the experiment, and the supernatant was discarded. After washing the cells with 5 mL of PBS, 1 mL of 0.25%Trypsin-EDTA was added. After digestion in a 37℃incubator for 1 min, 6 mL of medium 1 (450 mL of F12 medium + 50 mL of FBS) was added to terminate digestion, and resuspend to single cell suspension, 5mL of cell suspension was discarded, and 8mL of medium 1 (450mL of F12 medium + 50mL of fetal bovine serum (FBS) ) was added, then 120μL of G418 was added and mixed, G418 work concentration was 600 μg/mL. The cells were put into the incubator to continue culturing.
(3) Experimental process
On the first day, the density of target cells CHO-K1-PD-L1 /GHR was adjusted to 2.0×10 5 cells /mL in differentiation medium (99%F12 medium + 1%FBS) , and the target cells were plated overnight at 50μL/well. On the second day, the test samples (9#andGenexine GX-H9) , positive control Avelumab (PD-1 antibody, Shanghai Jiabei Biomedical Technology Co., Ltd. ) and negative control Acterma (Roche) were first diluted to 0.1mg/mL in differentiation medium (99%F12 medium + 1%FBS) , and were second diluted to 15μg/mL in differentiation medium (99%F12 medium + 1%FBS) , and then diluted by 3 times to 7 gradients, 50μL/well was added to the cell plate, and mixed well. After incubating at 37℃ for 30min, the cell plate was removed. 30%complement (containing 70%differentiation medium +30%complement) was added, 50μL/well, mixed. And it was placed in a 37℃ incubator for 6 hours of co-incubation. After the cell plate was removed and equilibrated at room temperature, 50 μL/well CellTiter-GLo Luciferase Assay System was added to the cell  plate and pipetted blow 10 times, mixed well. After lysing at room temperature for 10 min in the dark, the chemiluminescence signal was detected by a microplate analyzer.
The results showed that after adding positive control PD-L1 antibody Avelumab, negative control IL-6R antibody Acterma and SBT123 in the CDC experiment, the positive control Avelumab induced human serum complement to have obvious CDC effect on target cell CHO-K1-PD-L1/GHR, the negative control Acterma had no CDC effect, and the SBT123 had no CDC effect.
Reference throughout this specification to "an embodiment, " "some embodiments, " "one embodiment" , "another example, " "an example, " "a specific examples, " or "some examples, " means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as "in some embodiments, " "in one embodiment" , "in an embodiment" , "in another example, " in an example, " "in a specific examples, " or "in some examples, " in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and cannot be understood as a limitation of the present invention, and ordinary technicians in the field can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims (23)

  1. An Fc mutant comprisinga first peptide chain, wherein the first peptide chain 1) comprises mutantions at the following positions in comparisionto a wild-type IgG4 Fc fragment: at least one of amino acid in positions 234, 235, 298, 299, and 300; or positions 231-239; or 2) comprisesmutantions at the following positions in comparisiontoa wild-type IgG1 Fc fragment: positions 228-239, and/or, at least one of amino acid in positions 268, 296, 330 and 331.
  2. The Fc mutant of claim 1, whereinthe Fc mutant further comprises a second peptide chain, a C-terminal of the second peptide chain is connected withan N-terminal of the first peptide chain; and the second peptide chain:
    1) comprises mutantions at the position228 in comparision to the wild-type IgG4hinge region; or
    2) comprises a wild-type IgDfragment 1 or a IgD mutational fragment 1;
    optionally, the wild-type IgD fragment 1 comprises amino acid at positions 287-298 of the wild-type IgD;
    optionally, the IgD mutational fragment 1 comprises mutantions at the following positions in comparisionto a wild-type IgD Fc fragment 1: at least one of amino acid at positions 291-296;
    optionally, the Fc mutant further comprises a third peptide chain, a C-terminal of the third peptide chain is connected withan N-terminal of the second peptide chain;
    optionally, the third peptide chaincomprises mutantions at the following positions in comparision to a wild-type IgD fragment 2: at least one of the amino acid at positions: 263, 264, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 280, 281, 283, 284, 285, 286, 287, 288, 289, 290, and 291;
    optionally, the wild-type IgD fragment 2 comprises amino acid in positions 262-291 of the wild-type IgD.
  3. The Fc mutant of claim 1 or 2, wherein the first peptide chain 1) comprises mutantions at the following positions in comparision to the wild-type IgG4 Fc fragment: positions 234, 235; or 298, 299, 300 ; or positions 231-239; or 2) comprises mutantions at the following positions in comparision to the wild-type IgG1 Fc fragment: positions 228-239, 268, 296, 330 and 331;
    optionally, the second peptide chain 1) comprises mutantions at the following positions in comparision to the wild-type IgG4 hinge region: positions 228; or 2) comprises the wild-type IgD fragment 1 or the IgD mutational fragment 1;
    optionally, the IgD mutational fragment 1 comprises mutantions at the following in comparision to the wild-type IgD Fc fragment 1: positions 291-296;
    optionally, the third peptide chaincomprises mutantions at the following positions in comparision to a wild-type IgD fragment 2: 1) 266, 267, 268, 277, 278, 280, 283, 285, 286 and 287; or 2) 266, 277, 278, 280, 283, 285, 286 and 287; or 3) 266, 269, 270, 271, 273, 274, 275, 277, 278, 281 and 283; or 4) 263, 264, 266, 269, 270, 272, 274, 276, 284, and 287-291.
  4. The Fc mutant of claim 1 or 2, wherein the first peptide chain 1) comprises mutantions at the following positions in comparision to thewild-type IgG4 Fc fragment: F234A and L235E; or F234V and L235A; or F234V and L235E; or S298N, T299A and Y300S; or amino acid deletion at positons 231-239; or 2) comprises  mutantions at the following positions in comparision to the wild-type IgG1 Fc fragment: amino acid deletion at positions 228-239, and H268Q, Y296F, A330S and P331S;
    optionally, the second peptide chain 1) comprises mutantions at the following positions in comparision to the wild-type IgG4 hinge region: S228P; or 2) comprises the wild-type IgD fragment 1 or the IgD mutational fragment 1;
    optionally, the IgD mutational fragment 1 comprises mutantions at the following positions in comparision to the wild-type IgD fragment 1: P291C, S292H, H293P, T294R, Q295L, P296S;
    optionally, the third peptide chaincomprises mutantions at the following positions in comparision to the wild-type IgD fragment 2: 1) R266E, G267R, G268E, K277E, E278K, Q280E, R283E, T285E, K286G and T287E; Or 2) R266K, K277E, E278K, Q280N, R283E, T285E, K286R and T287E; Or 3) R266K, E269D, E270D, K271E, K273E, E274D, K275E, K277E, E278K, E281Q and R283G; Or 4) N263Q, T264S, R266K, E269D, E270D, K272R, E274D, E276D, E284D, and amino acid deletion at positons 287-291.
  5. The Fc mutant of claim 1 or 2, wherein the first peptide chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 55, 56, 57, 58, 68 and 69;
    optionally, the second peptide chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 59, 60 and 61;
    optionally, the third peptide chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 62, 63, 64 and 65;
    optionally, the wild-type IgG4, the wild-type IgG1 and the wild-type IgD are human antibodies.
  6. The Fc mutant of any one of claims 1 to 5, wherein comprises:
    1) an amino acid sequence as shown in any one of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54; or
    2) an amino acid sequence with at least 90%identity with the amino acid sequence as shown in any one of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54.
  7. A fusion protein comprising:
    a first peptide, wherein the first peptidecomprisesa bioactive molecular functional region;
    a second peptide conncected with the first peptide, wherein the second peptidecomprises an Fc mutant according to any one of claims 1-6, wherein aN-terminal of the second peptide is connected to a C-terminal of the first peptide.
  8. The fusion protein of claim7, wherein the first peptide comprises a growth hormone, a growth hormone analog, a growth hormone functional region or a growth hormone analog functional region;
    optionally, the first peptide comprises the human growth hormone or the human growth hormone functional region;
    optionally, the human growth hormone comprisies an amino acid sequence as shown in SEQ ID NO: 1 or at least 90%homologous thereof;
    optionally, the fusion protein further comprisies a linker peptide;
    optionally, an N-terminal of the linker peptide is connected with the C-terminal of the first peptide, and a C-terminal of the linker peptide is connected with the N-terminal of the second peptide;
    optionally, the linker peptide comprisesan amino acid sequence as shown in the SEQ ID NO: 46;
    optionally, the linker peptide comprises mutantions at the following positions in comparision to the amino acid sequence as shown in the SEQ ID NO: 46:
    at least one S>T in any one GGGGS;
    increasing G at N-terminal; and
    increasing L mutation at C-terminal;
    optionally, the linker peptide comprisies an amino acid sequence as shown in SEQ ID NO: 11.
  9. The fusion protein of claim 7, wherein the fusion protein comprises:
    1) an amino acid sequence as shown in any one of SEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, 28; or
    2) an amino acid sequence with at least 90%and 95%identity with the amino acid sequence as shown in any one of SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28.
  10. A nucleic acid molecule encoding the Fc mutant of any one of claims 1-6 or the fusion protein of any one of claims 7-9.
  11. The nucleic acid molecule of claim 10, whereinthe nucleic acid molecule is DNA;
    optionally, the nucleic acid molecule comprisesan nucleotide sequence as shown in any one of SEQ ID NOs : 32, 33, 34, 35, 36, 37, 38, 39 or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20.
  12. An expression vector carrying the nucleic acid molecule of claim 10 or 11.
  13. The expression vector of claim 12, whereinthe expression vector is an eukaryotic expression vector, a prokaryotic expression vector or a viral vector;
    optionally, the viruses comprises lentiviruses.
  14. A recombinant cell carrying the nucleic acid molecule of claim 10 or 11, or the expression vector of any one of claim 12 or 13, or expressing the Fc mutant of any one of claims 1-6 or the fusion protein of any one of claims 7-9.
  15. The recombinant cell of claim 14, wherein the recombinant cell is obtainable by introducing the expression vector of any one of claims 12 -13 into a host cell;
    optionally, the expression vector is introduced into the host cell by means of electrical transduction;
    optionally, the recombinant cell isan eukaryotic cell;
    optionally, the recombinant cell isa mammalian cell.
  16. A compositioncomprising the fusion protein of any one of claims 7-9, the nucleic acid molecule of claim 10 or 11, the expression vector of claim 12 or 13, or the recombinant cell of claim 14 or 15.
  17. Use of the fusion proteins ofany one of claims 7-9, the nucleic acid molecules ofany one of claims 10-11, the expression vectors of any one of claims 12-13, the recombinant cell of any one of claims 14-15, or the composition of claim 16 in the preparation of medicament for preventing or treating disease associated with abnormal growth hormone;
    optionally, the disease associated with abnormal growth hormone comprises at least one of the following: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner's syndrome, Prader-Willi syndrome, kidney failure, disorders caused by alienated states during chemotherapy  and AIDS treatment, and intrauterine growth retardation.
  18. A drug comprising the fusion protein ofany one of claims 7-9, the nucleic acid molecule of any one of claims 10-11, the expression vector of any one of claims 12-13, the recombinant cell of claim 14 or 15, or the composition of claim 17.
  19. A method of preventing or treating abnormal growth hormone related diseases, wherein the method comprises applying to a subject at least one of the following:
    1) the fusion protein of any one of claims 7-9;
    2) the nucleic acid molecule of any one of claims 10-11;
    3) the expression vector of any one of claims 12-13;
    4) the recombinant cells of any one of claims 14-15;
    5) the composition of claim 16; and
    6) the drug of claim 18.
  20. The method of claim 19, whereinthe fusion protein is adminstered by subcutaneous injection or intravenous injection.
  21. The method of claim 19, wherein the abnormal growth hormone related diseases comprise at least one of the following: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner's syndrome, Prader-Willi syndrome, kidney failure, disorders caused by alienated states during chemotherapy and AIDS treatment, and intrauterine growth retardation.
  22. The fusion protein of any one of claims 7-9, the nucleic acid molecule of any one of claims 10-11, the expression vector of any one of claims 12-13, the recombinant cells of any one of claims 14-15, the composition of claim 16, or the drug of claim 18, for use in preventing or treating a diseaseassociated withabnormal growth hormone.
  23. The fusion protein of any one of claims 7-9, the nucleic acid molecule of any one of claims 10-11, the expression vector of any one of claims 12-13, the recombinant cells of any one of claims 14-15, the composition of claim 16, or the drug of claim 18, for use in preventing or treating a diseaseassociated withabnormal growth hormone comprises at least one of the following:
    childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner's syndrome, Prader-Willi syndrome, kidney failure, disorders caused by alienated states during chemotherapy and AIDS treatment, and intrauterine growth retardation.
PCT/CN2022/100240 2021-10-18 2022-06-21 A growth hormone fusion protein and itsuse thereof WO2023065700A1 (en)

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