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WO2018032787A1 - Protéine de fusion d'hormone de croissance humaine hautement glycosylée, son procédé de fabrication et son application - Google Patents

Protéine de fusion d'hormone de croissance humaine hautement glycosylée, son procédé de fabrication et son application Download PDF

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WO2018032787A1
WO2018032787A1 PCT/CN2017/079873 CN2017079873W WO2018032787A1 WO 2018032787 A1 WO2018032787 A1 WO 2018032787A1 CN 2017079873 W CN2017079873 W CN 2017079873W WO 2018032787 A1 WO2018032787 A1 WO 2018032787A1
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Prior art keywords
fusion protein
human
ctp
hgh
protein according
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PCT/CN2017/079873
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English (en)
Chinese (zh)
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李强
陈思
郑云程
李子瑞
马心鲁
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安源医药科技（上海）有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—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
    • A61K38/27—Growth hormone [GH], i.e. somatotropin
    • 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
    • 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
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to the field of fusion proteins, and more particularly to a highly glycosylated human growth hormone fusion protein, a preparation method thereof and use thereof.
  • hGH Human growth hormone
  • hGH Human growth hormone
  • Its physiological function is mainly to promote substance metabolism and growth.
  • hGH mediates action on cartilage tissue by growth-promoting factors or insulin-like growth factors, increasing bone length, thereby promoting linear growth of the human body.
  • More functions of hGH have been discovered in recent years, including promoting skeletal muscle and cardiomyocyte growth, promoting protein synthesis, regulating immune system function, and enhancing immune defense capabilities.
  • the large-scale clinical application of hGH has also expanded from the prevention and treatment of childhood growth hormone deficiency in children with short stature to burns, acute pancreatitis, and aging in the elderly. Its clinical indications are still expanding.
  • PEG polyethylene glycol
  • U93/15199 glycosylation modification
  • the PEG-modified protein can prevent hydrolysis without causing serious side effects.
  • the coupling of PEG to the target protein belongs to non-specific covalent binding. This random coupling hinders the interaction between the target protein and its receptor to varying degrees. In vivo activity is reduced.
  • Increased glycosylation can increase the molecular weight of the target protein, especially for proteins that are at the renal filtration threshold ( ⁇ 30KD), which can reduce the sensitivity of proteins to protease hydrolysis.
  • Korean Patent Application Publication No. KR10-2013-0029713 discloses a method of attempting to increase the half-life in vivo by introducing at least one mutation into the ⁇ -1 antitrypsin to add an N-glycosyl group.
  • the attachment and addition of a sugar chain can cause the physiologically active protein to be inactivated, and the selection site of the site of the physiologically active protein additionally attached to the sugar chain is also very narrow, thereby introducing an additional glycosylation site to the protein for glycosylation.
  • Engineering technology has not been widely used.
  • An Fc fusion protein is a novel recombinant protein produced by fusion of a biologically active functional protein molecule (a soluble ligand, a receptor or other biologically active substance requiring an extended half-life) with an Fc fragment by techniques such as genetic engineering.
  • the fusion protein not only retains the biological activity of the functional protein molecule, but also prolongs the half-life of the fusion protein and enhances its stability.
  • the interchain disulfide bond of the Fc fragment facilitates the formation of a dimer of the fusion molecule, thereby enhancing ligand binding.
  • the ability to enhance biological activity; the introduction of an Fc fragment also facilitates increased expression levels of the fusion molecule in mammalian cells.
  • manufacture of a fusion protein containing an Fc region linked to a human IgG protein will help to prolong the circulating half-life of the drug and/or increase its biological activity.
  • Patent CN102875683 discloses a long-acting recombinant human growth hormone Fc fusion protein, which adds a flexible peptide linker to hGH and human IgG Fc variants to reduce steric hindrance effects, with the expectation of prolonging serum half-life, biological activity Increased, thereby improving pharmacokinetics and efficacy.
  • the active site of hGH is mainly at the C-terminus, other proteins fused at the C-terminus greatly affect the activity and function of hGH.
  • the CN102875683 patent shows that the dimerized hGH-L-vFc has a molar activity of 1.2 nM. The active phase is still less than ideal for recombinant hGH.
  • the present invention aims to find a new fusion method to further increase the serum half-life and in vivo activity of the fusion protein.
  • CTP is a short peptide derived from the carboxy terminus of the ⁇ -subunit of human chorionic gonadotropin (hCG).
  • hCG human chorionic gonadotropin
  • reproductive-related peptide hormones FSH
  • FSH follicle stimulating hormone
  • LH luteinizing hormone
  • TSH thyrotropin
  • hCG chorionic gonadotropin
  • CTP contains 37 amino acid residues with four O-glycosylation sites.
  • the terminal is a sialic acid residue.
  • CTP can increase the glycosylation level of the protein and increase the activity of the target protein, while the negatively charged, highly sialylated CTP can resist the clearance of the kidney, thereby prolonging the half-life of the protein in vivo.
  • U.S. Patent No. 13,195,931 discloses a method for ligating the growth hormone half-life by only about 6-fold by attaching the chorionic gonadotropin carboxy-terminal peptide (CTP) to hGH, but does not mention several hGH/CTP chimeras.
  • the present inventors have creatively used a CTP polypeptide having a plurality of O-glycosyl sites as part of a linker peptide for ligation of hGH and Fc fragments, rather than acting as a fusion ligand due to its natural glycosylation site
  • the half-life of the fusion protein can be further prolonged, the bioavailability is improved, and the steric hindrance effect of the fusion ligand Fc on hGH is greatly reduced, thereby maintaining high biological activity.
  • hGH-L-vFc Chinese Patent No. CN102875683
  • a recombinant hGH fusion protein comprising human growth hormone (hGH), a flexible peptide linker (L), at least one human chorionic gonadotropin from N-terminus to C-terminus is provided Hormone ⁇ -subunit carboxy terminal rigid peptide (CTP) and human immunoglobulin Fc fragment; wherein the Fc fragment is preferably a human IgG Fc variant (denoted as vFc).
  • the flexible peptide linker is preferably non-immunogenic and produces a sufficient distance between hGH and Fc to minimize steric effects between each other.
  • a flexible peptide linker comprising two or more of the following amino acids: Gly (G), Ser (S), Ala (A) and Thr (T) is used.
  • the flexible peptide linker comprises G and S residues. The length of the linker peptide is very important for the activity of the fusion protein.
  • the flexible peptide linker amino acid composition has the structural formula of (GS) a (GGS) b (GGGS) c (GGGGS) d , wherein a, b, c and d are greater than or equal to 0 An integer, and a+b+c+d ⁇ 1.
  • the peptide linker is selected from the group consisting of:
  • CTP carboxy terminal peptide rigid peptide
  • the CTP rigid peptide comprises SEQ ID NO: 1 or a truncated sequence thereof.
  • the CTP rigid peptide comprises at least 2 glycosylation sites; for example, in a preferred embodiment of the invention, the CTP comprises 2 glycosylation sites, exemplarily, the CTP comprises SEQ ID NO: 10 amino acids at the 1N terminus, ie SSSS*KAPPPS* (* represents a glycosylation site); or the CTP comprises 14 amino acids at the C-terminus of SEQ ID NO: 1 , ie S*RLPGPS*DTPILPQ; In one embodiment, the CTP comprises three glycosylation sites, exemplarily, the CTP comprises 16 amino acids of the N-terminus of SEQ ID NO: 1 , ie SSSS*KAPPPS*LPSPS*R; In one embodiment, the CTP comprises four glycosylation sites, exemplarily, the CTP sequence comprises 28, 29, 30, 31, 32 or 33 amino acids and begins with human chorionic gonadotropin beta subunit The 113th, 114th, 115th, 116th, 117th or 118
  • the CTP rigid units provided herein are at least 70% homologous to the native CTP amino acid sequence; in other embodiments, the CTP rigid units provided herein are at least 80% homologous to the native CTP amino acid sequence; In other embodiments, the CTP rigid units provided herein are at least 90% homologous to the native CTP amino acid sequence; in other embodiments, the CTP rigid sheets provided by the present invention The element is at least 95% homologous to the native CTP amino acid sequence.
  • the CTP rigid peptide may preferably comprise the following sequence elements:
  • the fusion protein of the present invention may further comprise two or more of the above CTP rigid peptide sequence units.
  • the CTP comprises two CTP 3 units: SSSSKAPPPSSSSSKAPPPS (CTP 3- CTP 3 , Or expressed as (CTP 3 ) 2 ).
  • the extended half-life portion is preferably a self-immunoglobulin IgG, IgM, IgA Fc fragment; more preferably an Fc fragment from human IgG1, IgG2, IgG3 or IgG4 and variants thereof; further, the human IgG Fc variant comprises in the wild At least one amino acid modification in a human IgG Fc, and the variant has reduced effector function (ADCC and/or CDC effect) and/or enhanced binding affinity to the neonatal receptor FcRn. Further, the human IgG Fc variant may be selected from the group consisting of:
  • vFc ⁇ 1 human IgG1 hinge region, CH2 and CH3 region containing the Leu234Val, Leu235Ala and Pro331Ser mutations (such as the amino acid sequence shown in SEQ ID NO: 4);
  • vFc ⁇ 2-1 human IgG2 hinge region, CH2 and CH3 region containing the Pro331Ser mutation (such as the amino acid sequence shown in SEQ ID NO: 5);
  • vFc ⁇ 2-2 human IgG2 hinge region, CH2 and CH3 regions containing the Pro331Ser, Thr250Gln and Met428Leu mutations (such as the amino acid sequence shown in SEQ ID NO: 6);
  • vFc ⁇ 4 human IgG4 hinge region, CH2 and CH3 region containing the Ser228Pro and Leu235Ala mutations (such as the amino acid sequence shown in SEQ ID NO: 7).
  • amino acid sequence of the hGH-L-CTP-vFc fusion protein is set forth in SEQ ID NO: 2.
  • a DNA molecule encoding the recombinant hGH-L-CTP-vFc fusion protein of the first aspect of the invention is provided, preferably wherein the DNA sequence has the sequence shown in SEQ ID NO: Nucleotide sequence.
  • a vector comprising the DNA sequence of the second aspect of the invention is provided.
  • a host cell comprising a third party of the present invention is provided.
  • the vector is surfaced or transfected with the above vector.
  • the host cell is a CHO derived cell line DXB-11.
  • the host cell produces a recombinant hGH-L-CTP-vFc fusion protein according to the first aspect of the invention in more than 30 ⁇ g/million cells per 24 hours in its growth medium.
  • a method for the preparation of the recombinant hGH-L-CTP-vFc fusion protein of the first aspect of the invention comprising:
  • step (c) culturing step (b) the selected cell strain to express the fusion protein
  • the CHO-derived cell line in step (a) is DXB-11.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent, and an effective amount of said hGH-L-CTP- vFc fusion protein.
  • the present invention provides the hGH-L-CTP-vFc fusion protein for treating growth and development disorders caused by insufficient secretion of endogenous growth hormone, and the short and chronic renal function caused by Turner syndrome. Depletion, Prader-Willi syndrome or idiopathic short stature.
  • the present invention provides the use of the fusion protein for the preparation of a medicament for the treatment of osteoporosis, in particular for slowing bone loss due to aging.
  • the fusion protein structure of the present invention has the following characteristics:
  • the fusion ligand human IgG Fc variant used in the fusion protein is non-lytic, which reduces the effector function triggered by binding to Fc ⁇ Rs and Clq;
  • the fusion protein prepared by the invention has good stability in fermentation, purification process and storage process
  • CTP rigid peptide can not only further prolong its half-life in vivo, but also increase the spatial distance between fusion molecules by blocking the glycosylation side chain, and promote hGH and Fc segments.
  • the fusion protein has reduced immunogenicity, which reduces the risk of inducing neutralizing antibody production
  • hGH-L-CTP-vFc Compared with PEG-rhGH, hGH-L-CTP-vFc has improved bioavailability, low fluctuation of blood concentration, faster onset, and extended half-life of circulating in vivo, which can reduce the frequency of injection and improve patient compliance.
  • Sex
  • hGH-L-CTP-vFc Compared with recombinant hGH, hGH-L-CTP-vFc has a simple purification step and high purification efficiency.
  • the Fc element is derived from the constant region Fc fragment of immunoglobulin IgG, which plays an important role in eradicating the immune defense of pathogens.
  • the effector function of Fc-mediated IgG is exerted through two mechanisms: (1) binding to cell surface Fc receptors (Fc ⁇ Rs), digestion of pathogens by phagocytosis or cleavage or killer cells via antibody-dependent cellular cytotoxicity (ADCC) pathway , or (2) binding to C1q of the first complement component C1, eliciting a complement-dependent cytotoxicity (CDC) pathway, thereby lysing the pathogen.
  • Fc ⁇ Rs cell surface Fc receptors
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the Fc region of the fusion protein preferably does not have adverse effector functions and thus does not dissolve. Or remove these target cells. Therefore, the Fc region of hGH-Fc must be insoluble, and the Fc region is preferably inactive in binding to Fc ⁇ Rs and Clq to trigger effector functions.
  • IgG1 and IgG3 efficiently bind to Fc ⁇ Rs, and the binding affinity of IgG4 to Fc ⁇ Rs is low, and the binding of IgG2 to Fc ⁇ Rs is too low to be determined, so human IgG2 has almost no ADCC effect.
  • human IgG1 and IgG3 can also efficiently bind to C1q to activate the complement cascade.
  • Human IgG2 binds relatively weakly to C1q, whereas IgG4 does not bind to C1q (Jefferis R et al, Immunol Rev, 1998, 163: 59-76), so the human IgG2 CDC effect is also weak.
  • the plasma half-life of IgG depends on its binding to FcRn, which typically binds at pH 6.0 and dissociates at pH 7.4 (plasma pH). By studying the binding sites of the two, the site of binding to FcRn on IgG was engineered to increase the binding ability at pH 6.0. Mutations in some residues of the human Fc ⁇ domain important for binding to FcRn have been shown to increase serum half-life. In particular, in human Fc ⁇ 1, when three residues were replaced by the other 19 conventional amino acids, some point mutations showed increased FcRn binding affinity (Hinton et al, J Biol Chem, 279(8): 6213-6216, 2004). Hinton et al.
  • the antibody has improved pharmacokinetic parameters compared to wild-type antibodies, increased in vivo exposure, reduced clearance, and improved subcutaneous bioavailability (Datta-Mannan A et al. MAbs. Taylor & Francis, 2012, 4(2): 267- 273.).
  • CTP is a short peptide derived from the carboxy terminus of the ⁇ -subunit of human chorionic gonadotropin (hCG).
  • hCG human chorionic gonadotropin
  • reproductive-related peptide hormones follicle stimulating hormone (FSH), luteinizing hormone (LH), thyrotropin (TSH), and chorionic gonadotropin (hCG) contain the same alpha-subunit and their respective specific beta - Yaki.
  • FSH follicle stimulating hormone
  • LH luteinizing hormone
  • TSH thyrotropin
  • hCG chorionic gonadotropin
  • the half-life of hCG is significantly prolonged, mainly due to the unique carboxy terminal peptide (CTP) on its ⁇ -subunit (Fares FA et al, Proc Natl Acad Sci USA, 1992, 89: 4304-4308). ).
  • CTP contains 37 amino acid residues with four O-glycosylation sites and a terminal sialic acid residue. Negatively charged, highly sialylated CTP is resistant to the clearance of the kidneys, thereby prolonging the half-life of the protein in the body.
  • the inventors creatively add at least one CTP polypeptide after a suitable length of flexible linker peptide such that the half-life of the fusion protein is further extended and the bioavailability is increased.
  • a CTP peptide between hGH and an Fc variant corresponds to the addition of a rigid linker peptide.
  • This aspect ensures that the N-terminally fused hGH does not affect the binding site of the Fc variant and FcRn, thereby affecting the half-life; in addition, the Fc Protein A binding site is important for the purification step in the preparation process, and the CTP is guaranteed to N- The end-fused hGH also does not "cover" its binding site to Protein A.
  • the addition of CTP also allows the Fc fragment of about 25 kD size to not interfere with the correct folding of the N-terminally fused hGH, thereby causing a decrease or loss of its biological activity/function.
  • the correct three-dimensional conformation does not affect each other's biological activity.
  • the fusion proteins of the invention are typically prepared by biosynthetic methods. According to the nucleotide sequence of the present invention, one skilled in the art can conveniently prepare the nucleic acid of the present invention by various known methods. These methods are, for example but not limited to, PCR, DNA synthesis, etc. For specific methods, see J. Sambrook, Molecular Cloning Experiment Guide. As an embodiment of the present invention, the nucleic acid sequence of the present invention can be constructed by a method of segmentally synthesizing a nucleotide sequence and performing overlap extension PCR.
  • the invention also provides an expression vector comprising a sequence encoding a fusion protein of the invention and an expression control sequence operably linked thereto.
  • operably linked or “operably linked” is meant a condition in which portions of a linear DNA sequence are capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, if a promoter controls the transcription of a sequence, then it is operably linked to the coding sequence.
  • the expression vector may be a commercially available vector such as, but not limited to, pcDNA3, pIRES, pDR, pUC18 or the like which can be used for expression of a eukaryotic cell system.
  • pcDNA3, pIRES, pDR, pUC18 or the like which can be used for expression of a eukaryotic cell system.
  • One skilled in the art can select a suitable expression vector based on the host cell.
  • the skilled person can prepare the present invention by inserting the coding sequence of the fusion protein of the present invention into a suitable restriction site by restriction enzyme cleavage and splicing according to a conventional method. Recombinant expression vector.
  • the invention also provides a host cell expressing a fusion protein of the invention comprising a coding sequence for a fusion protein of the invention.
  • the host cell is preferably a eukaryotic cell such as, but not limited to, CHO, COS Cells, 293 cells, RSF cells, etc.
  • the cell is a CHO cell which can express the fusion protein of the present invention well, and a fusion protein having good binding activity and good stability can be obtained.
  • the present invention also provides a method for producing a fusion protein of the present invention using recombinant DNA, the steps of which include:
  • Introduction of the coding sequence into a host cell can employ a variety of known techniques in the art such as, but not limited to, calcium phosphate precipitation, protoplast fusion, lipofection, electroporation, microinjection, reverse transcription, phage Transduction method, alkali metal ion method.
  • the fusion protein obtained as described above can be purified to a substantially uniform property, such as a single band on SDS-PAGE electrophoresis.
  • a commercially available ultrafiltration membrane can be used to separate the protein, for example, from Millipore, Pellicon, etc., and the expression supernatant is first concentrated.
  • the concentrate may be further purified by gel chromatography or by ion exchange chromatography. For example, anion exchange chromatography (DEAE, etc.) or cation exchange chromatography.
  • the gel matrix may be a matrix commonly used for protein purification such as agarose, dextran, polyamide, and the like.
  • the Q- or SP- group is a preferred ion exchange group.
  • the purified product may be further purified by hydroxyapatite adsorption chromatography, metal chelate chromatography, hydrophobic interaction chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). All of the above purification steps can utilize different combinations to ultimately achieve a substantially uniform protein purity.
  • the expressed fusion protein can be purified using an affinity chromatography column containing a specific antibody, receptor or ligand of the fusion protein.
  • the fusion polypeptide bound to the affinity column can be eluted using conventional methods such as high salt buffer, pH change, and the like.
  • the amino terminus or carboxy terminus of the fusion protein may also contain one or more polypeptide fragments as a protein tag. Any suitable label can be used in the present invention.
  • the tags may be FLAG, HA, HAl, c-Myc, 6-His or 8-His, etc. These tags can be used to purify the fusion protein.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount (e.g., 0.000001 to 90% by weight; preferably 0.1 to 50% by weight; more preferably 5 to 40% by weight) of the fusion protein of the present invention, and pharmaceutically acceptable Accepted carrier.
  • an effective amount of a fusion protein of the invention can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably, the pH is from about 6 to about 8.
  • the term "effective amount” or “effective amount” refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • a “pharmaceutically acceptable” ingredient is one which is suitable for use in humans and/or mammals without excessive adverse side effects (such as toxicity, irritation, and allergies), i.e., materials having a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • Pharmaceutically acceptable carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the pharmaceutical preparation of the present invention can also be formulated into a sustained release preparation.
  • the effective amount of the fusion protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, pharmacokinetic parameters of the fusion protein such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, body weight of the patient, immune status of the patient, administration Ways, etc.
  • a satisfactory effect can be obtained.
  • several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
  • Figure 1 shows the nucleotide sequence and deduced amino acid sequence of the fusion protein of the Spe I-EcoR I fragment in the pAPG-3 expression vector.
  • the position of the peptide linker in the mature protein is indicated by a dotted line.
  • the bold nucleotides and corresponding amino acid variants are underlined.
  • Figure 2 shows the ability of hGH fusion protein to stimulate proliferation of Nb2-11 cells.
  • Figure 3 shows the growth curves of each group after administration of the hGH fusion protein.
  • Figure 4 shows the histological observation of the tibia of rats after administration of hGH fusion protein.
  • A model group
  • B TL group
  • C TM group
  • D TH group
  • E SL group
  • F SM group
  • G SH group.
  • Example 1 Construction of an expression plasmid encoding a hGH fusion protein
  • the gene sequences encoding the hGH leader peptide and mature hGH as well as the different lengths of flexible peptide linkers, different lengths of CTP rigid peptides and different IgG Fc variants are artificially optimized CHO cell-preferred codons obtained by chemical synthesis.
  • the fusion gene after sequencing verification was digested with SpeI and EcoRI, and then inserted into the corresponding restriction sites of the expression plasmid PXY1A1 which was modified with PCDNA3.1 as a template to obtain a fusion gene expression plasmid pAPG.
  • the PXY1A1 plasmid includes, but is not limited to, the following important expression components: 1) human cytomegalovirus early promoter and mammalian cells with high exogenous expression of the desired enhancer; 2) dual screening marker with kanamycin resistance in bacteria sexuality, G418 resistance in mammalian cells; 3) Murine dihydrofolate reductase (DHFR) gene expression cassette, when the host cell is DHFR gene-deficient, methotrexate (MTX) can amplify the fusion gene And the DHFR gene (see U.S. Patent 4,399,216).
  • DHFR Murine dihydrofolate reductase
  • the present invention constructs a series of hGH-Fc fusion proteins having flexible peptide linkers of different lengths, CTP compositions, and also different positions, as well as IgG Fc variant (vFc) elements of several different subtypes.
  • the nucleotide sequence of APG-3 and the translated amino acid sequence are shown in Figure 1.
  • the series of expression plasmids obtained in Example 1 were transfected into 3 ⁇ 10 7 CHO cells in a 30 ml shake flask using DNAFect LT reagent (ATGCell), and the transfected cells were grown in serum-free growth medium for 5 days, according to The concentration of the fusion protein in the supernatant was determined by the corresponding ELISA method, and its in vitro biological activity was determined by the method described in Example 5. The ELISA results showed that the transient expression levels of several plasmids under these conditions were similar, but their activities showed large differences (Table 2).
  • APG-1 we defined the molar activity of APG-1 as 100%.
  • the activity of APG-2 was 80.9% of APG-1, indicating that the elongation of the peptide linker alone did not effectively improve the activity of the fusion protein, i.e., the steric hindrance effect of each other could not be attenuated with the extension of the peptide linker.
  • Even an excessively long peptide linker not only does not increase the activity of the fusion protein, but instead causes the protein to fold in error and is secreted as an inactive multimer.
  • APG-4 in which CTP was placed at the Fc C-terminus was only 61.3% of APG-3 in which CTP was placed at the Fc N-terminus, and the activity of APG-4 was similar to that of APG-1 without CTP.
  • the activity of APG-3 is higher than that of APG-4, and the activities of APG-5, APG-6 and APG-7 are much higher than other fusion proteins.
  • the above results confirmed that CTP is critical for the activity of the fusion protein, and that CTP is placed at the N-terminus of the Fc to increase the activity of the fusion protein.
  • the recombinant expression plasmid is transfected into a mammalian host cell line to express the hGH-L-CTP-vFc fusion protein.
  • a preferred host cell line is a DHFR enzyme deficient CHO-cell (see U.S. Patent 4,818,679).
  • a preferred method of transfection is electroporation, and other methods can be used, including calcium phosphate co-sedimentation, lipofection, and protoplast fusion.
  • the transfected fusion protein gene was co-amplified with the DHFR gene in growth medium containing increasing concentrations of MTX. Subclones with limiting dilution were able to grow transfectants in up to 6 [mu]M MTX medium.
  • the secreted rate was determined for further analysis of the subcloned cell lines. A cell line having a secretion rate level of more than about 30 (preferably about 50) ⁇ g/10 6 (i.e., millions) of cells per 24 hours is adapted to suspension culture using serum-free growth medium.
  • the fusion protein is then purified using conditioned medium.
  • the conditioned medium containing the fusion protein was titrated to pH 7-8 with 1 N NaOH and then filtered through a 0.45 micron nitrocellulose filter.
  • the filtrate was loaded onto a phosphate buffered saline (PBS) equilibrated Protein A column. After the fusion protein is bound to the Protein A column, the effluent components are discarded.
  • the column was washed with PBS until the OD value at 280 nm was less than 0.01.
  • the bound fusion protein was then eluted with 0.1 M citrate buffer pH 3.75.
  • the eluate was neutralized with 0.4 volume of 1 M K 2 HPO 4 and the fractions containing the purified protein were combined and dialyzed against PBS. It was then filtered through a 0.22 micron nitrocellulose filter and stored at 4 °C. Protein products were identified and analyzed by SDS-PAGE under non-reducing conditions.
  • the fusion protein was quantified by BCA protein analysis using BSA as a standard.
  • Example 5 In vitro biological activity analysis of fusion protein
  • the in vitro biological activity of the recombinant hGH-L-CTP-Fc fusion protein was examined using Nb2-11 cell proliferation.
  • Mouse lymphoma cell Nb2-11 (US ATCC cell bank) was cultured normally in Fischer's medium containing 10% fetal bovine serum. Using a serum-free medium, the fusion protein was diluted 3-fold at 1000 ng/ml, and 8 samples of different concentrations were obtained, 100 ⁇ l per well, added to a 96-well plate, and the medium was a negative control.
  • the cells in the log phase growth phase were taken, and the cells were washed once with serum-free medium to adjust the density to 3 ⁇ 10 6 cells per ml, and 100 ⁇ l per well was added to the above 96-well plate.
  • the cells were cultured for 48 hours at 37 ° C in a 5% CO 2 incubator, and cell proliferation was measured using a CCK-8 kit (Cell Counting Kit, purchased from Shanghai Shengsheng Biotechnology Co., Ltd.).
  • the absorbance at 450 nm was measured with a microplate reader, and the OD reading was plotted against the concentration of the fusion protein, and the resulting dose response curve was used to calculate the biological activity of the fusion protein.
  • Figure 2 shows the ability of hGH fusion protein to stimulate proliferation of Nb2 cells.
  • Table 3 EC50 values (half maximal effective concentration, EC 50 ) different fusion proteins. Since the amino acid at the C-terminus of growth hormone is closely related to its function, Fc directly linked to the C segment of hGH affects its biological activity. After the addition of the linker peptide to hGH and Fc, the activity of the hGH fusion protein is increased. As can be seen from the results, the activity of APG-3 was nearly doubled compared with APG-1.
  • CTP exerts its own function on the one hand, and on the other hand, as a rigid linker peptide between Fc and hGH, CTP is coupled with a flexible peptide, which allows the fusion protein to fold into a more favorable three-dimensional structure, ensuring hGH Biological activity.
  • PEG-rhGH (Changchun Jinsai Pharmaceutical Co., Ltd.) high, medium and low doses.
  • the groups were 27 IU/kg/7d (SH), 9 IU/kg/7d (SM) and 3 IU/kg/7d (SL), respectively.
  • the high, medium and low doses of APG-3 were 27 IU/kg/7d (TH). , 9 IU / kg / 7d (TM), 3 IU / kg / 7d (TL).
  • Different doses of APG-3 fusion protein and PEG-rhGH were administered once a week, ie, d1, d7, d14, d21, and d28, for 5 weeks, for a total of 5 times, and the model group was given a vehicle.
  • Each rat was weighed at the same time every day after administration, and the weight gain of the rats was calculated every day, and the experiment was terminated on the 35th day (d35), and the rats were weighed.
  • the pituitary rats were 7 weeks old when they started to be administered. They were adult rats once a week. After 5 weeks, the rats were 12 weeks old and were adult rats. After the growth period, the growth was about to stagnate. .
  • the body weight of the model group did not change, and there was no increase, while the body weight of APG-3 and the positive control drug PEG-rhGH increased significantly (P ⁇ 0.01).
  • the effect relationship indicates that APG-3, like PEG-rhGH, increases body growth and increases protein synthesis.
  • the ⁇ bw of each dose group showed a significant difference; on the 35th day after the administration, APG-3 had a very significant effect on the weight gain of the rat, and the high dose APG-3 induced the pituitary.
  • the weight gain ( ⁇ bw value) of the resected rats was about 1.5 times higher than that of the high dose PEG-rhGH group; while the high dose PEG-rhGH induced weight gain was slightly better than the medium dose APG-3, and the growth trend of the growth curve from d26 Smooth. This may Because APG-3 should have a longer in vivo half-life than PEG-rhGH, this in vivo accumulation effect makes the APG-3 group have a steeper growth curve after the third administration.
  • the rats were subjected to a dark avoidance test.
  • the rat escaping instrument uses a plastic box that is divided into two chambers, light and dark. There is an arched small door between the light and dark boxes. The bottom of the two boxes are copper grids. The bottom of the chamber has a copper grid and can be energized. The voltage is 36V.
  • the rats were first placed in a bright room illuminated by strong light, and they were found to find the passage between the light and dark chambers and enter the dark room. The rats were repeatedly trained until they could be found within 60s.
  • the dark room is energized to give 36V, 50Hz AC electrical stimulation once, lasting about 5s, let the rats rest for 30s and then repeat the above process until In the Ming room, he did not dare to enter the darkroom for more than 300s.
  • the rats were repeatedly trained in the darkness reaction box, and the rats were shocked and fled to the bright room.
  • the rats were first placed in a clear box, adapted to 5 min, and the small door was opened. The time when the rat entered the dark box from the bright box was the darkness incubation period. The rats entered the dark box, and the bottom of the dark box was stimulated by alternating current.
  • Rats Then escaped into the dark room, within 5 minutes, the number of times the rats were shocked by entering the black box was the number of shuttles.
  • the darkness meter automatically records the number of times the rat enters the darkroom within 5 minutes, which is the number of shuttles, and the time of entering the darkroom for the first time, which is the darkness incubation period, and statistical analysis (measured by the pharmacological laboratory, the instrument used is the pharmacology room of Beijing Drug Testing Institute). ).
  • Avoiding darkness avoidance experiments is a common method for screening nootropics. It is established by using the animal's good darkness (light and dark) and fear and memory of aversive stimuli (such as electric shock). One of the methods of memory performance in rats. Judging criteria are that the longer the animal avoids dark latency, the less the number of shuttles, and the stronger the animal's ability to learn and remember. It can be seen from Table 5 that the model group has the shortest latency, the most frequent shuttle times, and the worst learning and memory ability.
  • the APG-3 dose group has a long time to avoid darkness (prolonged by 290% ⁇ 1220%), the APG-3 high- and medium-dose group and the PEG-rhGH middle-dose group and the model group were significantly different (P ⁇ 0.05) (the SH group has a large gap within the group, there is no statistical significance)
  • the number of shuttles in each dose group of APG-3 was significantly reduced, and the difference was significant compared with the model group (P ⁇ 0.05).
  • the results showed that APG-3, like the positive drug PEG-rhGH, had a certain effect on improving the cognitive function and puzzle of pituitary rats.
  • IGF-1 is an important growth factor on the growth axis. It has a variety of physiological functions. In addition to regulating the growth of the body, it has a mitotic effect on many cell lines, including promoting osteoblast proliferation and differentiation, and increasing osteoblast activity. And the amount that prevents osteoblast apoptosis. Growth hormone is regulated by growth of IGF-1. At present, more studies suggest that changes in serum IGF-1 levels are relatively reliable and sensitive indicators for determining the efficacy of GH therapy.
  • IGY-1 insulin-like factor
  • the length of the tail root to the tip of each rat ie the length of the tail (cm) was measured by a measuring tape.
  • the sacrificed rats were harvested from the liver, the attached tissues were removed, washed with physiological saline, and the filter paper was blotted to dry the surrounding blood (g).
  • the sacrificed rats were harvested and the left humerus was removed.
  • the attached muscles and connective tissues were removed. Neutral 10% formaldehyde was stored.
  • the tissue samples were excised from the median sagittal plane at the top of the proximal humerus and harvested to a thickness of 3 mm.
  • the paraffin was embedded and sectioned. After staining with hematoxylin eosin (HE), the neutral resin was sealed, observed by light microscopy and photomicrographed.
  • HOLOGIC dual energy X The line bone densitometer (Hologic, USA) was used to determine the bone mineral density (BMD) and bone mineral content (BMC) of the rat femur using special software for small animals. At the time of measurement, 7 rats in each group were measured together, and the BMD and BMC values of the central and distal ends of the femur were separately measured and measured.
  • Bone mineral density is the mineral mass contained in the unit bone area in the bone tissue of the body, and is one of the important factors affecting bone strength. It is a commonly used index for evaluating bone strength.
  • APG-3 and PEG-rhGH have the same effect of increasing bone mineral content, can promote bone calcification, increase bone density, promote bone cell proliferation, increase its activity, and strengthen bone mineralization process.
  • the bone mass is increased; therefore, it is clinically expected to be used for the treatment of osteoporosis, especially for relieving bone loss due to aging.
  • the tibia specimen was subjected to conventional decalcification and embedding, and a 4 ⁇ m thick tissue section was prepared, and the longitudinal section was taken for HE staining.
  • the sections were subjected to pathological observation and microphotographing ( ⁇ 2), and the image pro insight 8.0 software was used for bone morphology measurement. Sections of 7 rats in each group, each section was taken for central field of view measurement, calculation of bone area (Tissear Area, T.Ar); Trabecular Bone Area (Tb. Ar), trabecular circumference ( Trabecular Perimeter, Tb. Pm), Trabecular thickness (Tb.
  • the pathological sections of the left tibia of the rats were observed by morphological observation, micrograph and data analysis.
  • the conclusions were as follows: except for the largest separation of the trabecular bone in the model group, the other parameters were the lowest; the pathological observation was The trabecular bone of the model group is slender and pale, with poor structural integrity, with distortion and fracture. The trabecular bone is short and the growth is not good. The size of the medullary cavity is different, and the hematopoietic cells in the cavity are reduced. The trabecular thickness of each dose group increased, and the APG-3 medium dose group increased (P ⁇ 0.01). The other parameters of APG-3 increased significantly (P ⁇ 0.01 or P ⁇ 0.05). As shown in Fig.
  • the pathological morphology was observed as TH group, TM group and SH group.
  • the trabecular bone was relatively thick, full and dark, and the trabecular bone structure was intact, without distortion or fracture, and the growth was good. Smaller, the number of red blood cells in the lumen is larger, and the trabecular bone structure of the TL group is better than the model group. The greater the separation of the trabecular bone, the greater the distance between the trabecular bone, the bone The poorer the structure, the increased bone resorption, and osteoporosis may occur.
  • model group>SL>TL>SM>TM>SH>TH suggesting that APG-3 and PEG-rhGH can improve bone quality loose.
  • the elimination phase half-life of PEG-rhGH drug on SD rats excised from the pituitary gland was 9.89 h, and the peak time was about 24 h.
  • the T 1/2 of the high-dose and medium-dose groups of the fusion protein were 11 h and 12 h, respectively, and did not change substantially with the change in dose.
  • the APG-3 fusion protein has a longer half-life.
  • the combined Fc variant effectively prolongs the half-life, and on the other hand introduces a CTP rigid structure.
  • the negatively charged, highly sialylated CTP is resistant to the kidney. Its scavenging effect further extends the half-life of the fusion protein.
  • the peak time of the fusion protein drug was about 8 hours, and the APG-3 fusion protein was more effective than the 24-hour peak of PEG-rhGH.
  • APG-3 exhibits superior performance in terms of biological activity, bioavailability, and pharmacokinetics compared to PEG-rhGH.

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Abstract

L'invention concerne une protéine de fusion d'hormone de croissance humaine hautement glycosylée, comprenant, de l'extrémité N-terminale à l'extrémité C-terminale, une hormone de croissance humaine (hGH), un lieur peptidique flexible (L), et un peptide rigide carboxy-terminal (CTP) de sous-unité bêta de la gonadotrophine chorionique humaine (CTP), et un fragment Fc d'une immunoglobuline humaine. L'invention concerne en outre un procédé de fabrication hautement efficace de la protéine de fusion. La protéine de fusion présente une efficacité pharmaceutique améliorée in vivo et une demi-vie de circulation étendue in vivo par comparaison avec une hGH recombinante, réduisant fortement la fréquence d'administration et améliorant la biodisponibilité d'un médicament. La protéine de fusion présente également un processus de fabrication simple et très efficace.
PCT/CN2017/079873 2016-08-19 2017-04-10 Protéine de fusion d'hormone de croissance humaine hautement glycosylée, son procédé de fabrication et son application WO2018032787A1 (fr)

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WO2018032638A1 (fr) 2016-08-19 2018-02-22 安源医药科技（上海）有限公司 Peptide de liaison pour la construction d'une protéine de fusion
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