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WO2024131534A1 - Protéine de fusion fc hétérodimère et son utilisation - Google Patents

Protéine de fusion fc hétérodimère et son utilisation Download PDF

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Publication number
WO2024131534A1
WO2024131534A1 PCT/CN2023/136888 CN2023136888W WO2024131534A1 WO 2024131534 A1 WO2024131534 A1 WO 2024131534A1 CN 2023136888 W CN2023136888 W CN 2023136888W WO 2024131534 A1 WO2024131534 A1 WO 2024131534A1
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Prior art keywords
peptide segment
seq
amino acid
terminus
acid sequence
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English (en)
Chinese (zh)
Inventor
邱玉信
秦锁富
张乡陵
胡俊宏
童童
潘志福
陈晓娟
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Shenzhen Kexing Pharmaceutical Co Ltd
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Shenzhen Kexing Pharmaceutical Co Ltd
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Publication of WO2024131534A1 publication Critical patent/WO2024131534A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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
    • A61K38/27—Growth hormone [GH], i.e. somatotropin
    • 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
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • 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
    • 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 invention belongs to the field of biopharmaceutical technology. Specifically, the present invention relates to a heterodimer Fc fusion protein and its use. More specifically, the present invention relates to an Fc mutant, fusion protein, nucleic acid molecule, expression vector and pharmaceutical composition and its use.
  • Growth hormone deficiency is a recognized clinical syndrome associated with many metabolic abnormalities, including abnormal body composition, decreased physical fitness, altered lipid metabolism, decreased bone mass, increased insulin resistance, and decreased quality of life. Most metabolic abnormalities associated with growth hormone deficiency can be reversed by recombinant human growth hormone (rhGH) replacement.
  • rhGH human growth hormone
  • Traditional treatment for growth hormone deficiency involves daily subcutaneous injections of rhGH. This dosing regimen of daily injections of rhGH places a huge burden on patients and their families. Data show that 66%-77% of adult and pediatric GHD patients do not comply with daily injections during the course of treatment, and compliance is relatively poorer with long courses of treatment. Poor treatment compliance will affect the overall treatment effect. Long-acting rhGH preparations can reduce the number of injections, improve patient treatment compliance, and help improve the efficacy of GH treatment.
  • Fc fusion protein long-acting growth hormone There are three main technologies for long-acting growth hormone: sustained-release technology, PEG chemical modification technology and fusion protein technology. Fusion protein technologies currently reported include XTEN fusion protein long-acting growth hormone, CTP fusion protein long-acting growth hormone, Albumin fusion protein long-acting growth hormone and Fc fusion protein long-acting growth hormone. Compared with other fusion protein long-acting growth hormones, one of the advantages of Fc fusion protein long-acting growth hormone is simple process and convenient purification. Fc fusion protein long-acting growth hormone products under development include Genexine/Tianjian Bio's GX-H9, Yifan Bio's F-899 and Anke Bio's AK2017.
  • Fc fusion protein long-acting growth hormones are homodimers. Due to steric hindrance, the homodimer form of GH protein will interfere with its binding with the corresponding receptor and affect its activity. For this reason, some Fc fusion protein long-acting growth hormones (such as GX-H9) are made by screening a flexible and long IgD hinge region as the connecting peptide between the GH molecule and the Fc protein. The purpose is to reduce the effect of steric hindrance of the homodimer form of GH protein on the activity of growth hormone. However, the effect of reducing the steric hindrance effect by optimizing the connecting peptide is limited.
  • HM-0560A long-acting growth hormones
  • HM-0560A Other long-acting growth hormones
  • HM-0560A are firstly Produce and purify an Fc homodimer skeleton, and then use chemical methods to cross-link a monomer rhGH molecule on one arm of the Fc homodimer skeleton through a PEG long chain.
  • the Fc structure of this monomer drug avoids the spatial hindrance effect of the dimer form of drug protein, and to a certain extent reduces the effect of steric hindrance on the activity of growth hormone.
  • this method of cross-linking through PEG has complex cross-linking and subsequent purification processes, which increases the production cost of the product.
  • the present invention proposes an Fc mutant.
  • the Fc mutant comprises: a first peptide segment, the first peptide segment has mutations at the following sites compared to the Fc fragment of wild-type IgG1: positions 366 and 354, and at least one of positions 351, 368, 392, 405 and 409; or positions 366 and 394; or positions 370, 349, 392 and 409; a second peptide segment, the second peptide segment has mutations at the following sites compared to the Fc fragment of wild-type IgG1: at least one of positions 349, 351, 354, 356, 357, 366, 368, 394, 399 and 407; wherein the first peptide segment and the second peptide segment are connected.
  • the Fc mutant of the present invention has higher thermal stability, and the fusion protein prepared by using the mutant has higher purity and better biological activity.
  • amino acid sequence of the wild-type IgG1 in this article can be found in the Uniprot database number: P01857.
  • the first peptide segment and the second peptide segment are linked via a disulfide bond.
  • the present invention provides a fusion protein.
  • the fusion protein includes: the aforementioned Fc mutant; and a third peptide segment, the third peptide segment includes a first biologically active molecule or a functional region thereof, and the third peptide segment is connected to the first peptide segment or the second peptide segment.
  • the fusion protein of the present invention has the advantages of high purity and good biological activity.
  • the present invention provides a nucleic acid molecule.
  • the nucleic acid molecule encodes the aforementioned The Fc mutant or the aforementioned fusion protein.
  • the present invention proposes an expression vector.
  • the expression vector carries the aforementioned nucleic acid molecule.
  • 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 come directly from the vector itself, or they can be exogenous, that is, they are not from the vector itself.
  • the nucleic acid molecule can be operably connected to the control element.
  • operably connected means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected functions of regulating the transcription and translation of the exogenous gene.
  • Commonly used vectors can be, for example, plasmids, bacteriophages, etc.
  • the present invention provides a recombinant cell.
  • the recombinant cell comprises: carrying the aforementioned nucleic acid molecule; or, expressing the aforementioned Fc mutant or the aforementioned fusion protein.
  • the recombinant cell can effectively express the aforementioned Fc mutant or fusion protein in the cell under suitable conditions.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises: the aforementioned fusion protein.
  • the pharmaceutical composition according to an embodiment of the present invention can be used to prevent and treat diseases.
  • the present invention proposes a use of the aforementioned fusion protein, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, and the aforementioned pharmaceutical composition in the preparation of a drug for treating and/or preventing a disease.
  • FIG1 is a schematic diagram of the overall structure of the heterodimeric Fc fusion protein in Example 3 of the present invention.
  • FIG2 is the SDS-PAGE detection result of different heterodimeric Fc fusion proteins in Example 3 of the present invention.
  • FIG3 is a schematic diagram of the overall structure of different recombinant long-acting human growth hormone Fc heterodimer fusion proteins in Example 4 of the present invention.
  • FIG4 is the SEC-HPLC detection result of GB08_A_01 fusion protein in Example 4 of the present invention.
  • FIG5 is the SEC-HPLC test result of GB08_A_03 fusion protein in Example 4 of the present invention.
  • FIG6 is the SEC-HPLC test result of GB08_A_04 fusion protein in Example 4 of the present invention.
  • FIG7 is the SEC-HPLC detection result of GB08_A_05 fusion protein in Example 4 of the present invention.
  • FIG8 is the SEC-HPLC test result of GB08_A_06 fusion protein in Example 4 of the present invention.
  • FIG9 is the SEC-HPLC test result of GB08_A_10 fusion protein in Example 4 of the present invention.
  • FIG10 is the SEC-HPLC detection result of JB09 fusion protein in Example 4 of the present invention.
  • FIG11 is the CE-SDS detection result of GB08_A_01 fusion protein in Example 4 of the present invention.
  • FIG12 is the CE-SDS detection result of GB08_A_03 fusion protein in Example 4 of the present invention.
  • FIG13 is the CE-SDS detection result of GB08_A_04 fusion protein in Example 4 of the present invention.
  • FIG14 is the CE-SDS detection result of GB08_A_05 fusion protein in Example 4 of the present invention.
  • FIG15 is the CE-SDS detection result of GB08_A_06 fusion protein in Example 4 of the present invention.
  • FIG16 is the CE-SDS detection result of GB08_A_10 fusion protein in Example 4 of the present invention.
  • FIG17 is the CE-SDS detection result of the JB09 fusion protein in Example 4 of the present invention.
  • FIG. 18 is the in vitro activity data of each fusion protein in Example 5 of the present invention.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality” is two or more.
  • the terms “optionally”, “optional” or “optionally” generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
  • variant or “mutant” generally refers to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.
  • position 336 refers to position 336 according to the Eu numbering numbering rules
  • T366W refers to the replacement of threonine at position 336 according to the Eu numbering numbering rules by tryptophan
  • S354C refers to the replacement of serine at position 354 according to the Eu numbering numbering rules by cysteine
  • K409R/D/E refers to the replacement of lysine at position 409 according to the Eu numbering numbering rules by arginine, aspartic acid or glutamic acid
  • L368R/A refers to the replacement of leucine at position 368 according to the Eu numbering numbering rules by arginine or alanine
  • Y407V/A/T refers to the replacement of tyrosine at position 407 according to the Eu numbering numbering rules by valine, alanine or
  • (GGGGS) n means n GGGGS connected together, for example, “(GGGGS) 5 ” means GGGGSGGGGSGGGGSGGGGSGGGGS.
  • fusion protein generally refers to a protein obtained by fusion of two or more proteins or polypeptides.
  • the genes or nucleic acid molecules encoding the two or more proteins or polypeptides can be connected to each other to form a fusion gene or a fused nucleic acid molecule, which can encode the fusion protein.
  • the translation of the fusion gene produces a single polypeptide, which has the properties of at least one or even each of the two or more proteins or polypeptides before fusion.
  • Recombinant fusion proteins are artificially created by recombinant DNA technology for biological research or treatment.
  • Recombinant fusion proteins are proteins created by genetic engineering of fusion genes.
  • the present invention relates to recombinant fusion proteins, and the terms fusion proteins and recombinant fusion proteins are used in the same meaning herein.
  • the fusion proteins described herein generally include at least two domains (A and C), and optionally include a third component, a joint between the two domains.
  • the generation of recombinant fusion proteins is known in the art, and generally involves removing the stop codon from the cDNA sequence encoding the first protein or polypeptide, and then attaching the cDNA sequence of the second protein in a frame-compliant manner by connection or overlap extension PCR. The DNA sequence will then be expressed by the cell as a single protein.
  • the protein may be engineered to include the entire sequence of either original protein or polypeptide, or only a portion thereof.
  • the fusion protein of the present invention is usually prepared by a biosynthetic method.
  • a person skilled in the art can easily prepare the encoding nucleic acid of the present invention by various known methods. These methods include, but are not limited to, PCR, artificial DNA synthesis, etc. For specific methods, see J. Sambrook, "Molecular Cloning Laboratory Guide”.
  • the encoding nucleic acid sequence of the present invention can be constructed by synthesizing the nucleotide sequence in segments and then performing overlap extension PCR.
  • identity is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences determined by conventional methods.
  • Computer programs using these algorithms are also available, and include but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2; or GAP, BESTFIT, FASTA, and TFASTA are available; and CLUSTAL in the PC/Gene program.
  • the term "at least 90% identity” refers to at least 90%, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to each reference sequence.
  • the term "expression vector” generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and/or between host cells.
  • the expression vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and/or translation of DNA or RNA.
  • the expression vector also includes vectors with multiple of the above functions.
  • the expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.
  • the term "recombinant cell” generally refers to a cell with unique stable genetic traits obtained by modifying or recombining the genetic material of a host cell using genetic engineering technology or cell fusion technology.
  • the term “transformed” or “transfected” as used herein refers to the introduction of nucleic acids (e.g., vectors) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the present invention and can be used for the expression and/or secretion of target proteins.
  • suitable host cells include immortalized hybridoma cells, NS/0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (human amniotic fluid-derived cells), and CoS cells.
  • the term "pharmaceutical composition” generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and thoroughly combining the active compound with a liquid carrier, a finely divided solid carrier, or both.
  • the term "pharmaceutically acceptable excipient” may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients incompatible with the compound of the present invention, such as any adverse biological effect produced or any other component of the pharmaceutically acceptable composition that interacts in a harmful manner, their use is also within the scope of the present invention.
  • the term "administration" refers to the introduction of a predetermined amount of material into a patient by a certain suitable method.
  • Fusion protein of the present invention can be administered by any common approach, as long as it can reach the expected tissue.
  • the various modes of administration are expected, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, local, nasal, pulmonary, rectal and smearing, but the present invention is not limited to these exemplified modes of administration.
  • compositions of the present invention are administered by injection.
  • growth hormone abnormality-related diseases generally refers to diseases caused by abnormal growth hormone, such as growth hormone deficiency or related diseases caused by alienation, including but not limited to, childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner Syndrome, Prader Willi Syndrome, intrauterine growth retardation, idiopathic short stature, renal failure, chemotherapy treatment and alienation during AIDS treatment.
  • Growth hormone deficiency may include congenital or acquired deficiency. Regarding congenital defects, growth hormone deficiency may occur when the pituitary gland does not develop a disorder of growth hormone secretion. Acquired growth hormone deficiency may occur due to brain tissue damage caused by hypoxia due to difficulty in delivery.
  • growth hormone deficiency causes include pituitary damage caused by radiation used to treat brain tumors or postnatal tuberculous meningitis.
  • Growth hormone deficiency exhibits symptoms such as growth retardation and short stature, and congenital growth hormone deficiency exhibits low glucose symptoms, starting from newborns.
  • children exhibit symptoms such as increased anxiety and reduced vitality.
  • treatment refers to the use of drugs to obtain the desired pharmacological and/or physiological effects.
  • the effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing a disease and/or the adverse effects caused by the disease.
  • Treatment covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease.
  • Treatment covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit an individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.
  • the present invention provides an Fc mutant, a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell and a pharmaceutical composition and uses thereof, which will be described in detail below.
  • the present invention proposes an Fc mutant.
  • the Fc mutant comprises: a first peptide segment, the first peptide segment has mutations at the following sites compared to the Fc fragment of wild-type IgG1: positions 366 and 354, and at least one of positions 351, 368, 392, 405 and 409; or positions 366 and 394; or positions 370, 349, 392 and 409; a second peptide segment, the second peptide segment has mutations at the following sites compared to the Fc fragment of wild-type IgG1: at least one of positions 349, 351, 354, 356, 357, 366, 368, 394, 399 and 407; wherein the first peptide segment and the second peptide segment are connected.
  • the Fc mutant of the present invention has higher thermal stability, and the fusion protein prepared by using the mutant has higher purity and better biological activity.
  • an "Fc fragment” has two identical peptide chains, each of which is a CH2-CH3 and optionally a hinge region.
  • the mutation sites of the first peptide segment and the second peptide segment are relative to one of the peptide chains.
  • the Fc mutant of the present invention is a heterodimer.
  • the first peptide segment has mutations at the following positions compared to the Fc fragment of wild-type IgG1: 1) positions 351, 354, 366 and 368, and optionally positions 405 and/or 409; or 2) positions 354, 356 and 357; 3) 366 and 409, and optionally 392; or 3) 370, 349, 392 and 409; or 4) 366 and 394.
  • the inventors have found through experiments that the above mutation combination can improve the thermal stability and purity of the Fc mutant, and in particular, the use of the Fc mutant can further improve the purity of the heterodimeric Fc fusion protein.
  • the first peptide segment has mutations at the following positions compared to the Fc fragment of wild-type IgG1: 1) position 351, position 354, position 366 and position 368; or 2) position 351, position 354, position 366, position 368 and position 405; or 3) position 351, position 354, position 366, position 368 and position 409; or 4) position 354, position 366 and position 409; or 5) position 354, position 366, position 392 and position 409; or 6) position 370, position 349, position 392 and position 409; or 7) position 366 and position 394.
  • the second peptide segment has mutations at the following sites compared to the Fc fragment of wild-type IgG1: 1) 349, 351, 366, 368 and 407; or 2) 349, 399 and 407, and optionally 356 and/or 368; or 3) 354, 356, 357 and 399; or 4) 366, 368, 394 and 407.
  • the above mutation combination can improve the thermal stability and purity of the Fc mutant, and in particular, the use of the Fc mutant can further improve the purity of the heterodimeric Fc fusion protein.
  • the second peptide segment has mutations at the following positions compared to the Fc fragment of wild-type IgG1: 1) position 349, position 366, position 368, position 407 and position 351; or 2) position 349, position 399 and position 407; or 3) position 349, position 368, position 399 and position 407; or 4) position 349, position 356, position 399 and position 407; or 5) position 366, position 368, position 394 and position 407; or 6) position 354, position 356, position 357 and position 399.
  • the first peptide segment and/or the second peptide segment further has mutations at the following sites compared to the Fc fragment of wild-type IgG1: at least one of positions 234, 235 and 322.
  • the combination of the above mutations can eliminate the ADCC effect and CDC effect caused by the binding of the Fc fragment to receptors such as Fc ⁇ RIIIa and complement C1q.
  • the first peptide segment has mutations at the following positions compared to the Fc fragment of wild-type IgG1: 1) positions 234, 235, 322, 351, 354, 366 and 368; or 2) positions 234, 235, 322, 351, 354, 366, 368 and 405; or 3) positions 234, 235, 322, 351, 354, 366, 368 and 405.
  • the first peptide segment has mutations at the following positions compared to the Fc fragment of wild-type IgG1: 1) position 234, 235, 322, 351, 354, 366 and 368; or 2) position 234, 235, 322, 351, 354, 366, 368 and 405; or 3) position 234, 235, 322, 351, 354, 366, 368 and 409; or 4) position 234, 235, 322, 354, 366, 392 and 409.
  • the second peptide segment has mutations at the following positions compared to the Fc fragment of wild-type IgG1: 1) positions 234, 235, 322, 349, 366, 368, 407 and 351; or 2) positions 234, 235, 322, 349, 399 and 407; or 3) positions 234, 235, 322, 349, 399 and 407.
  • the second peptide segment has mutations at the following positions compared to the Fc fragment of wild-type IgG1: 1) position 234, position 235, position 322, position 349, position 351, position 366, position 368 and position 407; or 2) position 234, position 235, position 322, position 349, position 368, position 399 and position 407.
  • the first peptide segment has mutations at the following sites compared to the Fc fragment of wild-type IgG1: T366W/Y and S354C, and at least one of L351D, L368E, K392D/E, F405L and K409R/D/E; or K370D, Y349C, K392D/E and K409R/D/E; or T366W/Y and T394C; the second peptide segment, the second peptide segment has mutations at the following sites compared to the Fc fragment of wild-type IgG1: Y349C, L351K, S354C, D356K/R, E357K, T366S/K, L368R/A, T394C, At least one of D399K/R and Y407V/A/T.
  • the above mutation combination can further improve the thermal stability and purity of the Fc mutant, and in particular, the above mutation combination can further improve the thermal
  • the first peptide segment has mutations at the following sites compared to the Fc fragment of wild-type IgG1: 1) L351D, S354C, T366W/Y and L368E, and optionally at least one of F405L and K409R/D/E; or 2) S354C, T366W/Y and K409R/D/E, and optionally K392D/E; or 3) K370D, Y349C, K392D/E and K409R/D/E; or 4) T366W/Y and T394C.
  • the above mutation combination can further improve the thermal stability and purity of the Fc mutant, and in particular, the use of the Fc mutant can further improve the purity of the heterodimeric Fc fusion protein.
  • the first peptide segment has mutations at the following sites compared to the Fc fragment of wild-type IgG1: 1) L351D, S354C, T366W/Y and L368E; or 2) L351D, S354C, T366W/Y, L368E and F405L; or 3) L351D, S354C, T366W/Y, L368E and K409R/D/E; or 4) S354C, T366W/Y and K409R/D/E; or 5) S354C, T366W/Y, K392E/D and K409R/D/E.
  • the second peptide segment has mutations at the following sites compared to the Fc fragment of wild-type IgG1: 1) Y349C, L351K, T366S/K, L368R/A and Y407V/A/T; or 2) Y349C, D399K/R and Y407V/A/T, and optionally D356K/R and/or L368R/A; or 3) S354C, D356K/R, E357K and D399K/R; or 4) T366S/K, L368R/A, T394C and Y407V/A/T.
  • the above mutation combination can further improve the thermal stability and purity of the Fc mutant, and in particular, the use of the Fc mutant can further improve the purity of the heterodimeric Fc fusion protein.
  • the second peptide segment has mutations at the following sites compared to the Fc fragment of wild-type IgG1: 1) Y349C, L351K, T366S/K, L368R/A and Y407V/A/T; or 2) Y349C, D399K/R and Y407V/A/T; or 3) Y349C, L368R/A, D399K/R and Y407V/A/T; or 4) Y349C, D356K/R, D399K/R and Y407V/A/T.
  • the first peptide segment and/or the second peptide segment further has mutations at the following sites compared to the Fc fragment of wild-type IgG1: at least one of L234A, L235A and K322A.
  • the combination of the above mutations can further eliminate the ADCC effect and CDC effect caused by the binding of the Fc fragment to the FcR.
  • the first peptide segment has mutations at the following sites compared to the Fc fragment of wild-type IgG1: 1) L234A, L235A, K322A, L351D, S354C, T366W/Y and L368E; or 2) L234A, L235A, K322A, L351D, S354C, T366W/Y, L368E and F405L; or 3) L234A, L235A, K322A, L351D, S354C, T366W/Y, L368E and K409 R/D/E; or 4) L234A, L235A, K322A, S354C, T366W/Y and K409R/D/E; or 5) L234A, L235A, K322A, S354C, T366W/Y, K392E/D and K409R/D/E; or 6) L234A, L234A, L
  • the first peptide segment has mutations at the following sites compared to the Fc fragment of wild-type IgG1: 1) L234A, L235A, K322A, L351D, S354C, T366W/Y and L368E; or 2) L234A, L235A, K322A, L351D, S354C, T366W/Y, L368E and F405L; or 3) L234A, L235A, K322A, L351D, S354C, T366W/Y, L368E and K409R/D/E; or 4) L234A, L235A, K322A, S354C, T366W/Y, K392D/E and K409R/D/E.
  • the second peptide segment has mutations at the following sites compared to the Fc fragment of wild-type IgG1: 1) L234A, L235A, K322A, Y349C, L351K, T366S/K, L368R/A and Y407V/A/T; or 2) L234A, L235A, K322A, Y349C, D399K/R and Y407V/A/T; or 3) L234A, L235A, K322A, Y349C, L368R/A /A, D399K/R and Y407V/A/T; or 4) L234A, L235A, K322A, Y349C, D356K/R, D399K/R and Y407V/A/T; or 5) L234A, L235A, K322A, S354C, D356K/R, E357K and
  • the second peptide segment has mutations at the following sites compared to the Fc fragment of wild-type IgG1:
  • the first peptide segment includes an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 4, 45 to 48; or the second peptide segment includes an amino acid sequence as shown in any one of SEQ ID NOs: 5 to 6, 49 to 50.
  • the first peptide segment includes the amino acid sequence shown in SEQ ID NO:1 and the second peptide segment includes the amino acid sequence shown in SEQ ID NO:5; or, the first peptide segment includes the amino acid sequence shown in SEQ ID NO:3 and the second peptide segment includes the amino acid sequence shown in SEQ ID NO:5; or, the first peptide segment includes the amino acid sequence shown in SEQ ID NO:4 and the second peptide segment includes the amino acid sequence shown in SEQ ID NO:5; or, the first peptide segment includes the amino acid sequence shown in SEQ ID NO:2 and the second peptide segment includes the amino acid sequence shown in SEQ ID NO:6; Alternatively, the first peptide segment includes the amino acid sequence shown in SEQ ID NO:45 and the second peptide segment includes the amino acid sequence shown in SEQ ID NO:49; alternatively, the first peptide segment includes the amino acid sequence shown in SEQ ID NO:47 and the second peptide segment includes the amino acid sequence shown in SEQ ID NO:5; or,
  • the first peptide segment includes an amino acid sequence as shown in SEQ ID NO: 1 and the second peptide segment includes an amino acid sequence as shown in SEQ ID NO: 5.
  • the inventors have found through experiments that the fusion protein obtained by fusing the Fc mutant with a biologically active molecule (especially growth hormone) has a higher purity and better biological activity.
  • the Fc mutant further includes a first hinge region fragment and/or a second hinge region fragment.
  • the C-terminus of the first hinge region fragment is connected to the N-terminus of the first peptide segment; and/or, the C-terminus of the second hinge region fragment is connected to the N-terminus of the second peptide segment.
  • the first hinge region fragment and the second hinge region fragment are the same or different.
  • the first hinge region fragment and the second hinge region fragment both include the amino acid sequence shown in SEQ ID NO:7.
  • the first peptide segment and the second peptide segment are connected via an interchain disulfide bond.
  • the present invention provides a fusion protein.
  • the fusion protein includes: the aforementioned Fc mutant; and a third peptide segment, the third peptide segment includes a first biologically active molecule or a functional region thereof, and the third peptide segment is connected to the first peptide segment or the second peptide segment.
  • the fusion protein of the present invention has the advantages of high purity and good biological activity.
  • the N-terminus of the third peptide segment is connected to the C-terminus of the first peptide segment; or, the N-terminus of the third peptide segment is connected to the C-terminus of the second peptide segment; or, the C-terminus of the third peptide segment is connected to the N-terminus of the first peptide segment or the first hinge region fragment; or, the C-terminus of the third peptide segment is connected to the N-terminus of the second peptide segment or the second hinge region fragment.
  • the prepared fusion protein has the advantages of high purity.
  • the fusion protein further includes: a fourth peptide segment, the fourth peptide segment includes a second biologically active molecule or a functional region thereof, and the fourth peptide segment is connected to the first peptide segment or the second peptide segment.
  • the N-terminus of the third peptide segment is connected to the C-terminus of the first peptide segment, and the C-terminus of the fourth peptide segment is connected to the N-terminus of the first peptide segment or the first hinge region fragment; or, the N-terminus of the third peptide segment is connected to the C-terminus of the first peptide segment, and the C-terminus of the fourth peptide segment is connected to the N-terminus of the second peptide segment or the second hinge region fragment; or, the third peptide segment
  • the N-terminus of the third peptide segment is connected to the C-terminus of the second peptide segment, and the C-terminus of the fourth peptide segment is connected to the N-terminus of the first peptide segment or the first hinge region fragment; or, the N-terminus of the third peptide segment is connected to the C-terminus of the second peptide segment, and the C-terminus of the fourth peptide segment is connected to the
  • the first bioactive molecule and the second bioactive molecule are independently selected from at least one of growth hormone, IFN ⁇ , IFN ⁇ , Epo, coagulation factor FIX and complement factor.
  • first bioactive molecule and the second bioactive molecule of the present invention may be the same or different.
  • the first bioactive molecule and the second bioactive molecule are both growth hormones.
  • the third peptide segment and the fourth peptide segment each independently include growth hormone, growth hormone analogs, growth hormone functional regions or growth hormone analog functional regions, preferably human growth hormone or human growth hormone functional regions.
  • the third peptide segment and the fourth peptide segment both include an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 90% identity thereto.
  • the fusion protein further includes a first connecting peptide and/or a second connecting peptide.
  • the N-terminus of the third peptide segment is connected to the C-terminus of the first connecting peptide
  • the N-terminus of the first connecting peptide is connected to the C-terminus of the first peptide segment
  • the C-terminus of the fourth peptide segment is connected to the N-terminus of the second connecting peptide
  • the C-terminus of the second connecting peptide is connected to the N-terminus of the first peptide segment or the first hinge region fragment
  • the N-terminus of the third peptide segment is connected to the C-terminus of the first connecting peptide
  • the N-terminus of the first connecting peptide is connected to the C-terminus of the first peptide segment
  • the C-terminus of the fourth peptide segment is connected to the N-terminus of the second connecting peptide
  • the C-terminus of the second connecting peptide is connected to the N-terminus of the second peptide segment or the second hinge region fragment.
  • the N-terminus of the third peptide segment is connected to the C-terminus of the first connecting peptide
  • the N-terminus of the first connecting peptide is connected to the C-terminus of the second peptide segment
  • the C-terminus of the fourth peptide segment is connected to the N-terminus of the second connecting peptide
  • the C-terminus of the second connecting peptide is connected to the N-terminus of the first peptide segment or the first hinge region fragment
  • the N-terminus of the third peptide segment is connected to the C-terminus of the first connecting peptide
  • the N-terminus of the first connecting peptide is connected to the C-terminus of the second peptide segment
  • the C-terminus of the fourth peptide segment is connected to the N-terminus of the second connecting peptide
  • the C-terminus of the second connecting peptide is connected to the N-terminus of the second peptide segment or the second hinge region fragment.
  • the in vitro activity of the fusion protein IFN ⁇ (monomer)-Fc (dimer) is 3 times higher than that of IFN ⁇ (dimer)-Fc (dimer), and the in vivo half-life is extended by 2.4 times; the in vivo half-life of the fusion protein Epo (monomer)-Fc (dimer) is extended by 1.5 times than that of Epo (dimer)-Fc (dimer); the in vitro activity of the fusion protein IFNa (monomer)-Fc (dimer) is 10 times higher than that of IFNa (dimer)-Fc (dimer); the in vivo half-life of the fusion protein FIX (monomer)-Fc (dimer) is more biologically active than FIX (dimer)-Fc (dimer).
  • the inventor unexpectedly discovered that when the third peptide segment and the fourth peptide segment both include the amino acid sequence (rhGH sequence) shown in SEQ ID NO: 8, since the C-terminus of the human growth hormone amino acid sequence is an a-helical structure, there are more amino acid residues that interact with the GHR receptor, and the C182-C189 disulfide bond is also located at the C-terminus, while the N-terminus of the growth hormone amino acid sequence is a loop region, and the spatial conformation has a certain flexibility.
  • rhGH sequence amino acid sequence shown in SEQ ID NO: 8
  • the C-terminus of GH is connected to the fusion protein Fc molecule, which will cause a certain steric hindrance, thereby affecting the activity of GH; while the N-terminus of GH is connected to the fusion protein Fc molecule, the C-terminus of GH can be released, and the steric hindrance of GH binding to GHR will be relatively smaller, thereby having a relatively small effect on the activity of GH. Therefore, by adopting the above-mentioned connection method, the N-terminus and C-terminus of the Fc mutant are respectively bound to only one rhGH, which can avoid the steric hindrance of the monomer drug binding to its receptor.
  • the rhGH at the C-terminus of the Fc mutant is connected to the Fc mutant through the N-terminal Loop region of rhGH, which can further improve the biological activity of the fusion protein drug. Therefore, the fusion protein can reduce the steric hindrance of the mutual binding of rhGH and GHR and has better biological activity in vivo and in vitro.
  • the first connecting peptide and the second connecting peptide each independently include at least one selected from the following amino acid sequences: an amino acid sequence as shown in (GGGGS) n , wherein n is any integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; or, the amino acid sequence shown in SEQ ID NO:9.
  • the first connecting peptide and the second connecting peptide each independently include an amino acid sequence selected from SEQ ID NO: 9 and SEQ ID NO: 10.
  • the first connecting peptide and the second connecting peptide both include an amino acid sequence as shown in SEQ ID NO: 10.
  • the fusion protein has better biological activity in vivo and in vitro.
  • the fusion protein comprises the amino acid sequence shown in SEQ ID NO:11 and the amino acid sequence shown in SEQ ID NO:12; or, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:13 and the amino acid sequence shown in SEQ ID NO:14; or, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:15 and the amino acid sequence shown in SEQ ID NO:16; or, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:17 and the amino acid sequence shown in SEQ ID NO:18; or, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:21 and the amino acid sequence shown in SEQ ID NO:31; or, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:32 and the amino acid sequence shown in SEQ ID NO:51; or, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:52 and the amino acid sequence shown in SEQ ID NO:53; or, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:11 and the amino acid sequence shown
  • the fusion protein comprises the amino acid sequence shown in SEQ ID NO:57; or the fusion protein comprises the amino acid sequence shown in SEQ ID NO:58 and the amino acid sequence shown in SEQ ID NO:59; or the fusion protein comprises the amino acid sequence shown in SEQ ID NO:60 and the amino acid sequence shown in SEQ ID NO:61; or the fusion protein comprises the amino acid sequence shown in SEQ ID NO:62 and the amino acid sequence shown in SEQ ID NO:63; or the fusion protein comprises the amino acid sequence shown in SEQ ID NO:64 and the amino acid sequence shown in SEQ ID NO:65; or the fusion protein comprises the amino acid sequence shown in SEQ ID NO:66 and the amino acid sequence shown in SEQ ID NO:67; or the fusion protein comprises the amino acid sequence shown in SEQ ID NO:68 and the amino acid sequence shown in SEQ ID NO:69; or the fusion protein comprises the amino acid sequence shown in SEQ ID NO:43 and the amino acid sequence shown in SEQ ID NO:44.
  • the present invention provides a nucleic acid molecule.
  • the nucleic acid molecule encodes the aforementioned Fc mutant or the aforementioned fusion protein.
  • the nucleic acid molecule encodes the aforementioned Fc mutant or the aforementioned fusion protein.
  • the nucleic acid molecule is DNA.
  • nucleic acid molecules mentioned in the present invention actually include any one or both of the complementary double strands.
  • the nucleic acid sequence in the present invention includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.
  • the present invention proposes an expression vector.
  • the expression vector carries the aforementioned nucleic acid molecule.
  • 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 come directly from the vector itself, or they can be exogenous, that is, they are not from the vector itself.
  • the nucleic acid molecule can be operably connected to the control element.
  • operably connected means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected functions of regulating the transcription and translation of the exogenous gene.
  • Commonly used vectors can be, for example, plasmids, bacteriophages, etc.
  • the expression vector is a eukaryotic expression vector.
  • the expression vector is a plasmid expression vector.
  • the present invention provides a recombinant cell.
  • the recombinant cell comprises: carrying the aforementioned nucleic acid molecule; or, expressing the aforementioned Fc mutant or the aforementioned fusion protein.
  • the recombinant cell can effectively express the aforementioned Fc mutant or fusion protein in the cell under suitable conditions.
  • suitable conditions refer to conditions suitable for the expression of the Fc mutant or fusion protein of the present invention. It is easy for those skilled in the art to understand that the conditions suitable for the expression of the Fc mutant or fusion protein include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions” are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the Fc mutant or fusion protein according to the specific environment of the laboratory.
  • the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.
  • the recombinant cell is a eukaryotic cell.
  • the recombinant cell is a mammalian cell.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises: the aforementioned fusion protein.
  • the pharmaceutical composition according to an embodiment of the present invention can be used to prevent and treat diseases, such as treating and/or preventing diseases related to abnormal growth hormone.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
  • the dosage form of the pharmaceutical composition is an injection.
  • the administration route of the pharmaceutical composition includes subcutaneous injection or intravenous injection.
  • the present invention proposes a use of the aforementioned fusion protein, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, and the aforementioned pharmaceutical composition in the preparation of a drug for treating and/or preventing a disease.
  • the medicine is used for treating and/or preventing diseases related to abnormal growth hormone.
  • the abnormal growth hormone-related diseases include childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, diseases caused by alienation during chemotherapy and AIDS treatment, and intrauterine growth retardation.
  • the present invention provides a method for preventing and/or treating diseases related to abnormal growth hormone.
  • the method comprises: administering a pharmaceutically acceptable amount of the aforementioned fusion protein or the aforementioned pharmaceutical composition to a subject, wherein the third peptide segment or the fourth peptide segment of the fusion protein comprises growth hormone.
  • the method of the present invention can effectively prevent and/or treat diseases related to abnormal growth hormone.
  • the effective amount of the fusion protein or pharmaceutical composition of the present invention may vary depending on the mode of administration and the severity of the disease to be treated.
  • the selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials).
  • the factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, several divided doses may be administered daily, or the dose may be reduced proportionally, depending on the urgency of the treatment situation.
  • the fusion protein or pharmaceutical composition of the present invention can be incorporated into a drug suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • parenteral administration e.g., intravenous, subcutaneous, intraperitoneal, intramuscular
  • these drugs can be prepared in various forms.
  • liquid, semisolid and solid dosage forms including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders.
  • Typical drugs are in the form of injection solutions or infusion solutions.
  • the aforementioned fusion protein or pharmaceutical composition can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
  • Feed is commercially available SMS 293-SUPI; Buffer A is 20mM PB+100mM NaCl; Buffer B is 30mM HAC/NaAC.
  • MOE software was used to predict all possible interchain disulfide bond formation sites in the wild-type homodimer Fc molecule (crystal structure: 2WAH), KiH heterodimer Fc molecule (crystal structure: 4NQS), ZW1 heterodimer Fc molecule (crystal structure: BSW) and DD-KK heterodimer Fc molecule (crystal structure: 5DK2), and the dStability values were ranked from low to high.
  • the crystal structure information of 2WAH, 4NQS, BSW and 5DK2 is shown in the following table:
  • the structure of the mutant is considered to be stable.
  • the above disulfide bond prediction results show that the dStability values of the S354C-Y349C intermolecular disulfide bond in the wild-type Fc structure and several Fc heterodimer structures are all ⁇ -0.5 kcal/mol, that is, the disulfide bond formed by the mutation of these two sites to cysteine may be stable in various Fc mutants. Therefore, the heterodimer Fc mutant containing the S354C-Y349C intermolecular disulfide bond was further screened.
  • the inventors used MOE software to perform multi-site amino acid combination virtual mutations on the crystal structure of the wild-type homodimer Fc molecule (crystal structure: 2WAH), and determined the optimal Fc site-directed mutation combination based on the mutation energy value. Among them, the thermal stability prediction results of the Fc mutants of each mutation combination are shown in the following table:
  • the structure of the mutant is considered to be stable.
  • the inventors found that P006, P009, P011, P014, P016, P017, P021, P026, P029, P030 and P033 have good thermal stability.
  • the multiple Fc mutants obtained in Example 2 were fused with human growth hormone (SEQ ID NO: 8) to obtain fusion proteins, wherein the hinge region amino acid sequence is DKTHTCP (SEQ ID NO: 7), the connecting peptide sequence is GGGGSGGGGSGGGGS (SEQ ID NO: 20) flexible linker, and the overall structure description and schematic diagram are shown in Figure 1, wherein the A chain: rhGH-(G4S) 3 -Hinge-CH2-CH3, the B chain: Hinge-CH2-CH3.
  • the heterodimer Fc mutant is composed of two different polypeptide chains, the A chain and the B chain, with a theoretical molecular weight of about 73.7KDa, and the rhGH molecule is connected to the A chain of the Fc heterodimer through the connecting peptide sequence.
  • the inventors added signal peptide sequences to the N-termini of all A-chain and B-chain molecules, respectively, and then sent out the genes for optimization (the optimized host cells were 293F cells) and synthesis.
  • the Kozak sequence was GCCGCCACC (SEQ ID NO: 22), and the stop codon was TGATAA (SEQ ID NO: 23) double terminator.
  • Plasmid extraction and preparation The above expression vectors were amplified using DH5a host cells, and then the plasmids were extracted using Tiangen's extraction kit to obtain the corresponding plasmids.
  • 293F cell line was used as host cell, and the multiple plasmids obtained above were transfected into host cells using chemical transfection reagent PEI.
  • the specific operation was as follows:
  • the 293F cells are diluted to (0.3-0.35) ⁇ 10 6 cells/mL, and SMM293-TII cell culture medium preheated at 37°C is added.
  • the cells are cultured in a shake flask for 3 days.
  • the cells are counted and the cell density is adjusted to (3-4) ⁇ 10 6 cells/ml with SMM293-TII cell culture medium to obtain a cell culture fluid.
  • transfection reagent dilute DNA (plasmid obtained in step 2.3) to 5 mL (final DNA concentration is 1 ⁇ g/mL) with Expi293 TM expression medium, mix well and let stand for 5 minutes to obtain DNA dilution solution; dilute transfection reagent to 5 mL (final PEI concentration is 2.5 ⁇ g/mL) with Expi293 TM expression medium, mix well and let stand for 5 minutes to obtain transfection reagent dilution solution.
  • transfection reagent diluent to the DNA diluent, mix well and place for 10 minutes, then add to the cell culture medium and culture in a constant temperature shaker at 37°C and 100rpm (relative humidity is 80%, 8% CO2 ); add Feed (35mL/1L) 20-24h after transfection, and then add Feed every other day for 6 days. Collect the cell culture supernatant from the 6th day after transfection.
  • the cell culture supernatant obtained in step 2 was divided into sterile centrifuge bottles, centrifuged at 4°C and 1000 rpm for 5 min, and the supernatant was transferred to another sterile centrifuge bottle, and centrifuged at 4°C and 10000 rpm for 5 min; the culture supernatant was transferred to a sterile centrifuge bottle, and 2.5 ml of fully resuspended Protein A gel solution was added to each bottle of supernatant, and the mixture was incubated at 37°C for 1 hour after being fully mixed to obtain a mixture. After 1 hour, the target protein was purified and recovered using a gravity column.
  • the mixture was first transferred to a gravity column, and the Protein A gel was washed with 10 times the column volume of Buffer A buffer until the liquid in the mixture was drained. After the Buffer A buffer was drained, the protein eluate was collected 3 times, and 4 mL of Buffer B eluate with a pH value of 3.5 was added each time. After incubation at room temperature for 5 min, the eluate was eluted and collected in a clean 15 mL centrifuge tube. The protein concentration was determined using a micro-UV instrument to measure the absorbance of protein A280, and then converted into the corresponding protein concentration.
  • GB08_A_01 "GB08-A-01” and "GB08-A-001" are synonymous with GB08_A_03, GB08_A_04, GB08_A_05, GB08_A_06 and GB08_A_010.
  • Example 4 Design and expression verification of recombinant long-acting human growth hormone Fc heterodimer fusion protein
  • the Fc mutants 1 to 4 obtained above were prepared into fusion proteins with human growth hormone (SEQ ID NO:8), wherein the fusion proteins prepared using mutant 1 were GB08_A_01 (also known as GB08_P_26), GB08_A_03, GB08_A_04 (also known as GB08_KB_3), GB08_A_05, GB08_A_06 and GB08_A_010, and the fusion proteins prepared using mutant 2 were P029-04 (including an A chain with an amino acid sequence as shown in SEQ ID NO:52 and a B chain with an amino acid sequence as shown in SEQ ID NO:53, which has the same structure as GB08_A_04 in Table 1, except that the mutants are different), P029-05 (including an A chain with an amino acid sequence as shown in SEQ ID NO:54 and a B chain with an amino acid sequence as shown in SEQ ID NO:55), and GB08_A_06 (including an A chain with an amino acid sequence as shown in SEQ
  • the fusion proteins prepared by mutant 3 are P030-04 (comprising an A chain as shown in SEQ ID NO: 60 and a B chain as shown in SEQ ID NO: 61, the same structure as GB08_A_04 in Table 1, the only difference being the mutant), P030-05 (comprising an A chain as shown in SEQ ID NO: 79 and an B chain as shown in SEQ ID NO: 80, the same structure as GB08_A_05 in Table 1, the only difference being the mutant), P029-06 (comprising an A chain as shown in SEQ ID NO: 21 and an B chain as shown in SEQ ID NO: 31, the same structure as GB08_A_06 in Table 1, the only difference being the mutant), and P029-010 (comprising an A chain as shown in SEQ ID NO: 32 and an B chain as shown in SEQ ID NO: 51, the same structure as GB08_A_010 in Table 1, the only difference being the mutant).
  • the fusion proteins prepared by mutant 4 are P033-04 (comprising an A chain as shown in SEQ ID NO: 68 and an amino acid sequence as shown in SEQ ID NO: 70, which are the same as the structure of GB08_A_05 in Table 1, except that the mutants are different), P030-06 (comprising an A chain as shown in SEQ ID NO: 56 and an amino acid sequence as shown in SEQ ID NO: 57, which are the same as the structure of GB08_A_06 in Table 1, except that the mutants are different), and P030-010 (comprising an A chain as shown in SEQ ID NO: 58 and an amino acid sequence as shown in SEQ ID NO: 59, which are the same as the structure of GB08_A_010 in Table 1, except that the mutants are different).
  • the B chain has a sequence as shown in SEQ ID NO:69, which is the same as the structure of GB08_A_04 in Table 1, and the only difference is that the mutant is different), P033-05 (including the A chain with an amino acid sequence as shown in SEQ ID NO:43 and the B chain with an amino acid sequence as shown in SEQ ID NO:44, which is the same as the structure of GB08_A_05 in Table 1, and the only difference is that the mutant is different), P033-06 (including the A chain with an amino acid sequence as shown in SEQ ID NO:64 and the B chain with an amino acid sequence as shown in SEQ ID NO:65, which is the same as the structure of GB08_A_06 in Table 1, and the only difference is that the mutant is different) and P033-010 (including the A chain with an amino acid sequence as shown in SEQ ID NO:66 and the B chain with an amino acid sequence as shown in SEQ ID NO:67, which is the same as the structure of GB08_A_010 in Table 1, and the only difference
  • the nucleotide sequences of the A and B chains of P029-04, the A and B chains of P029-05, the A and B chains of P029-06, the A and B chains of P029-010, the A and B chains of P030-04, the A and B chains of P030-05, the A and B chains of P030-06, the A and B chains of P030-010, the A and B chains of P033-04, the A and B chains of P033-05, the A and B chains of P033-06, and the A and B chains of P033-010 can be obtained according to their amino acid sequences using conventional methods or conventional software (for example, the online program Vectorbuilder (website: https://www.vectorbuilder.cn/tool/codon-optimization.html), the GeneOptimizer online program, etc.).
  • the hinge region amino acid sequence is DKTHTCP
  • the connecting peptide sequence is (G 4 S) 5 (SEQ ID NO: 10) flexible linker or a_Helix[LEA(EAAAK) 4 ALEA(EAAAK) 4 ALE](SEQ ID NO: 9) rigid linker
  • the specific preparation method is shown in Example 3
  • the difference is that the connecting peptide is different and/or the human growth hormone connection method is different, see Figure 3 and Table 1 for details.
  • the amino acid sequences of the A chain and B chain of JB09 are shown in SEQ ID NO: 41
  • the preparation method thereof is shown in Example 3
  • the structure and connection method thereof are shown in Figure 3 and Table 1.
  • Figure A in Figures 4 to 10 is a SEC-HPLC spectrum (the horizontal axis is time, the vertical axis is the absorbance unit AU), and Figure B is the information such as the peak time and peak area corresponding to each peak in the SEC-HPLC spectrum, and Figure A in Figures 11 to 17 is a CE-SDS spectrum (the horizontal axis is time, the vertical axis is the absorbance unit AU), and Figure B is the information such as the peak time and peak area corresponding to each peak in the CE-SDS spectrum.
  • SEQ ID NO:26 is used to encode the A chain of GB08_A_01 (the amino acid sequence is SEQ ID NO:25), and SEQ ID NO:28 is used to encode the B chain of GB08_A_01 (the amino acid sequence is SEQ ID NO:27):
  • SEQ ID NO:30 is used to encode the A chain of GB08_A_03 (amino acid sequence is SEQ ID NO:29), the B chain of GB08_A_03
  • the amino acid sequence and nucleotide sequence of GB08_A_01 are the same as those of the B chain of GB08_A_01, specifically referring to the amino acid sequence shown in SEQ ID NO:27 and the nucleotide sequence shown in SEQ ID NO:28:
  • SEQ ID NO:33 is used to encode the A chain of GB08_A_04 (amino acid sequence is SEQ ID NO:15), and SEQ ID NO:34 is used to encode the B chain of GB08_A_04 (amino acid sequence is SEQ ID NO:16):
  • SEQ ID NO:35 is used to encode the A chain of GB08_A_05 (amino acid sequence is SEQ ID NO:17), and SEQ ID NO:36 is used to encode the B chain of GB08_A_05 (amino acid sequence is SEQ ID NO:18):
  • SEQ ID NO:37 is used to encode the A chain of GB08_A_06 (amino acid sequence is SEQ ID NO:11), and SEQ ID NO:38 is used to encode the B chain of GB08_A_06 (amino acid sequence is SEQ ID NO:12):
  • SEQ ID NO:39 is used to encode the A chain of GB08_A_010 (amino acid sequence is SEQ ID NO:13), and SEQ ID NO:40 is used to encode the B chain of GB08_A_010 (amino acid sequence is SEQ ID NO:14):
  • SEQ ID NO:42 is used to encode the A chain of JB09 (amino acid sequence is SEQ ID NO:41).
  • JB09 is a homodimer, and its A chain and B chain are identical:
  • Table 1 Types of Fc mutants, linker peptides and hinge regions of each fusion protein
  • the inventors used the fusion proteins of GB08_A_01, GB08_A_03, GB08_A_04, GB08_A_05, GB08_A_06 and GB08_A_010 prepared in Example 4 as candidates, and used the above-mentioned JB09 as a control group.
  • the specific experimental operation was as follows:

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Abstract

L'invention concerne un mutant Fc et une protéine de fusion Fc hétérodimère. Le mutant Fc comprend : un premier segment peptidique, par comparaison avec un fragment Fc d'IgG1 de type sauvage, le premier segment peptidique ayant les mutations suivantes : positions 366 et 354, et au moins l'une des positions 351, 368, 392, 405 et/ou 409 ; et un second segment peptidique, par rapport au fragment Fc d'IgG1 de type sauvage, le second segment peptidique ayant les mutations suivantes : au moins l'une des positions 349, 351, 366, 368, 399 et/ou 407, le premier segment peptidique et le second segment peptidique étant liés. La protéine de fusion comprend le mutant Fc décrit ci-dessus.
PCT/CN2023/136888 2022-12-19 2023-12-06 Protéine de fusion fc hétérodimère et son utilisation Ceased WO2024131534A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102875683A (zh) * 2011-07-11 2013-01-16 旭华（上海）生物研发中心有限公司 长效重组人生长激素的Fc融合蛋白
US20180127478A1 (en) * 2016-09-16 2018-05-10 Wei-Chiang Shen SINGLE CHAIN Fc-DIMER-HUMAN GROWTH HORMONE FUSION PROTEIN FOR IMPROVED DRUG DELIVERY
US20200317787A1 (en) * 2017-12-26 2020-10-08 Nanjingjinsirui Science & Technology Biology Corp. Fusion protein dimer using antibody fc region as backbone and use thereof
US20220017634A1 (en) * 2018-08-16 2022-01-20 Denali Therapeutics Inc. Engineered bispecific proteins
WO2022143912A1 (fr) * 2020-12-31 2022-07-07 信达生物制药(苏州)有限公司 Protéine contenant un anticorps hétérodimère fc et son procédé de préparation
CN114874333A (zh) * 2021-10-18 2022-08-09 深圳科兴药业有限公司 一种生长激素融合蛋白及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102875683A (zh) * 2011-07-11 2013-01-16 旭华（上海）生物研发中心有限公司 长效重组人生长激素的Fc融合蛋白
US20180127478A1 (en) * 2016-09-16 2018-05-10 Wei-Chiang Shen SINGLE CHAIN Fc-DIMER-HUMAN GROWTH HORMONE FUSION PROTEIN FOR IMPROVED DRUG DELIVERY
US20200317787A1 (en) * 2017-12-26 2020-10-08 Nanjingjinsirui Science & Technology Biology Corp. Fusion protein dimer using antibody fc region as backbone and use thereof
US20220017634A1 (en) * 2018-08-16 2022-01-20 Denali Therapeutics Inc. Engineered bispecific proteins
WO2022143912A1 (fr) * 2020-12-31 2022-07-07 信达生物制药(苏州)有限公司 Protéine contenant un anticorps hétérodimère fc et son procédé de préparation
CN114874333A (zh) * 2021-10-18 2022-08-09 深圳科兴药业有限公司 一种生长激素融合蛋白及其应用

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