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WO2023051412A1 - 一种β-NGF融合蛋白及其制备方法和用途 - Google Patents

一种β-NGF融合蛋白及其制备方法和用途 Download PDF

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WO2023051412A1
WO2023051412A1 PCT/CN2022/120976 CN2022120976W WO2023051412A1 WO 2023051412 A1 WO2023051412 A1 WO 2023051412A1 CN 2022120976 W CN2022120976 W CN 2022120976W WO 2023051412 A1 WO2023051412 A1 WO 2023051412A1
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ngf
fusion protein
seq
ngf fusion
protein according
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PCT/CN2022/120976
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French (fr)
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肖建国
黄浩旻
朱祯平
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三生国健药业（上海）股份有限公司
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    • 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
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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

Definitions

  • the invention belongs to the field of biopharmaceuticals. Specifically, the invention relates to a ⁇ -NGF fusion protein molecule, and also relates to a preparation method and application of the ⁇ -NGF fusion protein molecule.
  • Nerve Growth Factor is the first neurotrophic factor discovered by humans. It was first isolated in 1956 by Italian neuroscientist Rita Levi-Montalcini and American biochemist Stanley Cohen; Cohen also accidentally discovered another A growth factor that can promote the growth, proliferation and differentiation of epidermal cells, so the factor is named epidermal growth factor (Epidermal Growth Factor, EGF). For this, Levi-Montalcini and Cohen were jointly awarded the Nobel Prize in Physiology or Medicine in 1986.
  • Nerve Growth Factor is the earliest neurotrophic factor discovered, the most thoroughly studied at present, and a nerve cell growth regulator with dual biological functions of neuron nutrition and neurite growth. Differentiation, growth, regeneration and expression of functional properties are all important regulators.
  • the human NGF gene is located on the short arm of chromosome 1, zone 2, zone 2, and the short arm of chromosome 1, zone 2, zone 1, subband 1, and consists of 6 exons and 5 introns.
  • NGF is initially in a 7S, 130 kDa complex composed of three protein subunits, which consists of three proteins: ⁇ -NGF, ⁇ -NGF and ⁇ -NGF (2:1:2 ratio, Figure 1).
  • This form of NGF is also known as proNGF (NGF precursor).
  • the ⁇ subunit of the complex belongs to heterogeneous acid protein, and its pI is about 4.3. Its main function is to prevent the hydrolysis of the ⁇ subunit by the ⁇ subunit, thereby protecting it.
  • the ⁇ subunit has arginine ester peptidase activity, and its main function is related to the synthesis of NGF, which can activate the inactive ⁇ subunit.
  • the nerve growth factor that is often said in the art usually refers to the 2.5S, 26kDa ⁇ subunit (ie ⁇ -NGF) of the protein, which is the only component of the biologically active 7S NGF complex (ie acts as a signaling molecule).
  • ⁇ -NGF which is a dimer formed by two peptide chains containing 118 amino acids through non-covalent bonds.
  • the mature NGF chain contains 6 cysteine residues, which can produce 3 pairs of disulfide bonds in the chain. Disulfide bonds are very important for maintaining the biological activity of NGF. Once the disulfide bonds are destroyed, the biological activity of NGF will be greatly reduced or even lost.
  • NGF is an endogenous protein involved in neuronal differentiation and maintenance, and this endogenous protein brings strong support to the integrity of the cornea through multiple mechanisms. NGF acts directly on corneal cells to stimulate cell growth and survival. In addition, NGF is known to bind to receptors on the lacrimal gland (Figure 2), promoting tear production, which provides lubrication and natural protection to the eye from pathogens. This protein has been shown to support corneal innervation, a nerve function that is lost in neurotrophic keratitis.
  • Oxervate TM (cenegermin, recombinant human nerve growth factor, rhNGF), an eye drop originally developed by Italian pharmaceutical manufacturer Domoutheastern, is a treatment for adult patients with moderate to severe neurotrophic keratitis (Neurotrophic keratitis, NK) Orphan drug, which is currently on the market in the European Union, the United States and China.
  • NK neurotrophic keratitis
  • its indication for the treatment of dry eye disease is under research, and the phase II clinical results are good. But it needs to be instilled every 2 hours, which is very inconvenient to use.
  • Mouse nerve growth factor has been on the market in China for many years for the treatment of peripheral nerve injury. T1/2( ⁇ ) is 4.83 hours, injected once a day. However, 10% of the amino acids in mouse nerve growth factor are different from those of human, and there is a risk of potential immunogenicity.
  • the original manufacturer In order to prepare recombinant human nerve growth factor, the original manufacturer first expresses proNGF inclusion bodies, then refolds, purifies the proNGF refolding solution, and digests and purifies recombinant human nerve growth factor.
  • this method requires enzyme digestion and purification after enzyme digestion, with many steps and cumbersome process.
  • the NGF heat source prepared by this method is not easy to remove and has a short half-life.
  • Chinese patent CN108300736B provides a method for CHO cells to prepare rhNGF-FC, but one rhNGF-FC molecule contains only one NGF active molecule, the expression level is 18 mg/L, and the N-terminus is not uniform, requiring furin digestion.
  • the inventors of the present application intend to develop a ⁇ -NGF fusion protein with good activity, high yield, long half-life and uniform N-terminus.
  • the present invention provides a ⁇ -NGF fusion protein molecule, the ⁇ -NGF fusion protein molecule contains two ⁇ -NGF active molecules, has good activity, high yield, long half-life and uniform N-terminus.
  • the first object of the present invention is to provide a ⁇ -NGF fusion protein, comprising:
  • the second object of the present invention is to provide an isolated nucleic acid molecule encoding said ⁇ -NGF fusion protein.
  • the third object of the present invention is to provide an expression vector containing the nucleic acid molecule.
  • the fourth object of the present invention is to provide a host cell containing the expression vector.
  • the fifth object of the present invention is to provide a method for preparing the ⁇ -NGF fusion protein.
  • the sixth object of the present invention is to provide a pharmaceutical composition containing the ⁇ -NGF fusion protein.
  • the seventh object of the present invention is to provide the ⁇ -NGF fusion protein or the pharmaceutical composition for use with another therapeutic agent.
  • the eighth object of the present invention is to provide the use of the ⁇ -NGF fusion protein or the pharmaceutical composition.
  • the ninth object of the present invention is to provide a method for treating diseases, comprising the step of: administering the ⁇ -NGF fusion protein of the present invention, or the pharmaceutical composition of the present invention to a subject in need.
  • a ⁇ -NGF fusion protein comprising:
  • the light chain and the heavy chain are linked by a non-covalent bond.
  • the ⁇ -NGF is human ⁇ -NGF.
  • the ⁇ -NGF comprises the amino acid sequence shown in SEQ ID NO:1.
  • the ⁇ -NGF and the Fc fragment are connected by a linker.
  • the heavy chain consists of ⁇ -NGF, a linker and an Fc fragment sequentially from the N-terminus to the C-terminus.
  • the Fc fragment is derived from IgM, IgG, IgA, IgD or IgE.
  • the Fc fragment is derived from IgG.
  • the IgG is derived from mammals; preferably from mice, cynomolgus monkeys or humans; preferably from humans.
  • the Fc fragment is selected from IgG1, IgG2, IgG3 or IgG4; preferably IgG4.
  • the Fc fragment comprises a human IgG hinge region, CH2 and CH3 regions.
  • the Fc fragment comprises a human IgG4 hinge region, CH2 and CH3 regions.
  • the Fc fragment comprises the amino acid sequence shown in SEQ ID NO: 2.
  • the Fc fragment comprises an Fc variant
  • the Fc variant comprises one or more amino acid substitutions, deletions and/or insertions.
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:4.
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 1.
  • the ⁇ -NGF fusion protein comprises the amino acid sequence shown in SEQ ID NO: 4 and/or SEQ ID NO: 1.
  • an isolated nucleic acid molecule comprising a nucleotide sequence encoding the ⁇ -NGF fusion protein according to the first aspect of the present invention.
  • the nucleic acid molecule comprises a light chain nucleotide sequence as shown in SEQ ID NO: 8 and a heavy chain nucleotide sequence as shown in SEQ ID NO: 9.
  • the nucleic acid molecule comprises a light chain nucleotide sequence as shown in SEQ ID NO: 11 and a heavy chain nucleotide sequence as shown in SEQ ID NO: 12.
  • the nucleic acid molecule comprises a light chain nucleotide sequence as shown in SEQ ID NO: 15 and a heavy chain nucleotide sequence as shown in SEQ ID NO: 16.
  • an expression vector comprises the nucleic acid molecule described in the second aspect of the present invention.
  • the expression vector is selected from pCGS3, pcDNA3.4, pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), pDHFR or pTT5; preferably pCGS3.
  • a host cell comprising the expression vector described in the third aspect of the present invention.
  • the host cell is selected from prokaryotic expression cells or eukaryotic expression cells.
  • the host cell is selected from COS, CHO, NSO, sf9, sf21, DH5 ⁇ , BL21 (DE3), TG1, CHO-K1, 293E or Expi293F cells; preferably selected from CHO cells, 293E or Expi293F cells; more preferably CHO cells.
  • a method for preparing a ⁇ -NGF fusion protein comprising:
  • step (b) isolating and purifying the ⁇ -NGF fusion protein described in step (a).
  • a pharmaceutical composition which comprises the ⁇ -NGF fusion protein as described in the first aspect of the present invention and a pharmaceutically acceptable carrier or adjuvant.
  • a ⁇ -NGF fusion protein as described in the first aspect of the present invention or a pharmaceutical composition as described in the sixth aspect of the present invention for preparing and treating neurotrophic keratitis Or the use of peripheral nerve injury drugs.
  • the ninth aspect of the present invention provides a method for treating diseases, comprising the step of: administering the ⁇ -NGF fusion protein as described in the first aspect of the present invention to a subject in need, or as described in the sixth aspect of the present invention pharmaceutical composition.
  • the present invention provides a ⁇ -NGF fusion protein molecule, which contains two ⁇ -NGF active molecules, has good activity, high yield, long half-life and uniform N-terminus.
  • Figure 1 shows the structure of NGF.
  • FIG. 2 shows a schematic diagram of the binding of NGF to NGF receptor (NGFR).
  • Fig. 3 shows the structure of ⁇ -NGF fusion protein.
  • Fig. 4 shows the SDS-PAGE identification diagram of the ⁇ -NGF fusion protein obtained by separation and purification.
  • Figure 5 shows the detection of the in vitro biological activity of the ⁇ -NGF fusion protein by ELISA.
  • Figure 6 shows the in vitro biological activity of ⁇ -NGF fusion protein detected by TF-1 cell method.
  • Figures 7A-7C show the spectrum detection diagrams of ⁇ -NGF fusion proteins, and Figures 7B-7C are partial enlarged views of Figure 7A.
  • fusion protein has a structure as shown in Figure 3, which refers to a ⁇ -NGF fusion protein comprising (a) a light chain comprising ⁇ -NGF; and (b) a heavy chain, the The heavy chain comprises ⁇ -NGF and Fc fragments.
  • the present invention also provides a method for preparing a ⁇ -NGF fusion protein, that is, a method for constructing the ⁇ -NGF fusion protein of the present invention.
  • the method is as described in the fifth aspect of the present invention.
  • the ⁇ -NGF fusion protein obtained by the construction method of the present invention contains two ⁇ -NGF active molecules, has good activity, high yield, long half-life and uniform N-terminus.
  • the terms "Fc fragment” and “immunoglobulin Fc fragment” can be used interchangeably, and both refer to a fragment containing an immunoglobulin hinge region, an immunoglobulin heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3). ) and does not contain immunoglobulin heavy chain variable region and light chain variable region, heavy chain constant region 1 (CH1) and light chain constant region (CL) protein.
  • the immunoglobulin Fc fragment of the present invention may comprise part or all of the heavy chain constant region 1 (CH1) and/or the light chain constant region (CL) in addition to the heavy chain variable region and the light chain variable region.
  • the IgG Fc fragment may be a fragment having a deletion in a relatively long part of the CH2 and/or CH3 amino acid sequence as long as it has a physiological function substantially similar to or better than that of the natural protein. That is, the immunoglobulin Fc fragment of the present invention may comprise 1) CH1 domain, CH2 domain, CH3 domain and CH4 domain, 2) CH1 domain and CH2 domain, 3) CH1 domain and CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains and an immunoglobulin hinge region (or part of a hinge region), or 6) the individual domains of the heavy and light chain constant regions dimer.
  • the Fc fragments of the present invention include native amino acid sequences and variants thereof.
  • Amino acid sequence derivatives are sequences that differ from the native amino acid sequence by deletion, insertion, non-conservative or conservative substitution, or combinations thereof, of one or more amino acid residues.
  • the Fc fragment can be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, acetylation, amidation, etc., if desired.
  • the aforementioned Fc derivatives are derivatives that have the same biological activity as the Fc fragment of the present invention or have improved structural stability (such as resistance to heat, pH, etc.).
  • these Fc fragments are available in native form isolated from humans and other animals including cattle, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, or may be recombinant or derivatives thereof, Obtained from transformed animal cells or microorganisms.
  • they can be obtained from natural immunoglobulins by isolating intact immunoglobulins from the human or animal body and treating them with proteolytic enzymes.
  • Papain digests native immunoglobulins into Fab and Fc fragments, while pepsin treatment results in the production of pF'c and F(ab')2 fragments.
  • These fragments can be subjected to eg size exclusion chromatography to isolate Fc or pF'c.
  • the human Fc fragment is a recombinant immunoglobulin Fc fragment obtained from microorganisms.
  • the immunoglobulin Fc fragment of the present invention may be in a form having natural sugar chains, increased sugar chains compared with the natural form, or reduced sugar chains compared with the natural form, or may be in a deglycosylated form.
  • the increase, decrease or removal of immunoglobulin Fc sugar chains can be accomplished by common methods in the art, such as chemical methods, enzymatic methods and genetic engineering methods using microorganisms.
  • the immunoglobulin Fc fragment may be from humans or other animals, including cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, preferably humans.
  • the immunoglobulin Fc fragment may be an Fc fragment derived from IgG, IgA, IgD, IgE, and IgM, or an Fc fragment prepared by combining or hybridizing them.
  • the Fc fragment is from IgG or IgM, which are one of the most abundant proteins in human blood, most preferably from IgG (which is known to prolong the half-life of ligand-binding proteins).
  • linker refers to a short linker sequence between ⁇ -NGF and Fc fragment, preferably a flexible linker.
  • suitable linkers include single glycine (Gly) or serine (Ser) residues, the identity and sequence of the amino acid residues in the linker may vary with the type of secondary structural elements desired to be achieved in the linker.
  • the present invention provides a nucleic acid molecule encoding the above-mentioned light chain.
  • the ⁇ -NGF gene was obtained by adding the precursor pro sequence, signal peptide signal 1 sequence, Kozak sequence and codon optimization to obtain the light chain gene and related element sequences (SEQ ID NO: 8).
  • the present invention also provides a nucleic acid molecule encoding the aforementioned heavy chain ( ⁇ -NGF+(G4S)4+Fc).
  • ⁇ -NGF+(G4S)4+Fc a nucleic acid molecule encoding the aforementioned heavy chain
  • the ⁇ -NGF+(G4S)4+Fc gene was added to the precursor pro sequence, signal peptide signal 2 sequence, Kozak sequence and codon optimization to obtain the heavy chain gene and related element sequences (SEQ ID NO: 9).
  • the preparation method of the nucleic acid molecule of the present invention is a conventional preparation method in the art, preferably including the following preparation methods: obtain the nucleic acid molecule encoding the above-mentioned polypeptide through gene cloning techniques such as PCR method, or obtain the encoded polypeptide by artificial full-sequence synthesis.
  • a nucleic acid molecule of the above-mentioned polypeptide obtained by gene cloning techniques such as PCR method, or obtain the encoded polypeptide by artificial full-sequence synthesis.
  • the term "expression vector” contains any of the nucleic acid molecules described above, which is a conventional expression vector in the art, and refers to the expression vector containing appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences , enhancer sequence, marker gene and/or sequence, and other appropriate sequence expression vectors.
  • the expression vector can be a virus or a plasmid, such as a suitable phage or phagemid.
  • a suitable phage or phagemid for more technical details, please refer to, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Many known techniques and protocols for nucleic acid manipulation are found in Current Protocols in Molecular Biology, 2nd Edition, edited by Ausubel et al.
  • the expression vector of the present invention is preferably selected from pCGS3, pcDNA3.4, pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), pDHFR and pTT5, preferably pCGS3.
  • the term "host cell” contains the above-mentioned expression vector, which is a variety of conventional host cells in the art, as long as the above-mentioned recombinant expression vector can stably replicate itself, and the nucleotides carried can be effectively expressed That's it.
  • said host cells include prokaryotic expression cells and eukaryotic expression cells, preferably including: COS, CHO (Chinese Hamster Ovary, Chinese Hamster Ovary), NSO, sf9, sf21, DH5 ⁇ , BL21 (DE3) or TG1, more preferably The ground is E.coli TG1, BL21(DE3) cells or CHO-K1 cells.
  • the preferred recombinant expression transformants of the present invention can be obtained by transforming the aforementioned expression vectors into host cells.
  • the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electroporation method.
  • the host cells of the present invention are eukaryotic cells, preferably selected from CHO cells, 293E and Expi293F cells.
  • the invention provides a kind of preparation method, described preparation method comprises the following steps:
  • step (b) isolating and purifying the ⁇ -NGF fusion protein described in step (a).
  • nucleotide molecules encoding the light chain as shown in SEQ ID NO: 8 and the nucleotide molecules encoding the heavy chain as shown in SEQ ID NO: 9 are respectively inserted into different multiple cloning sites of pCGS3 to construct Expression vector; secreted and expressed in CHO cells; the supernatant was collected by centrifugation and purified by Protein A affinity chromatography to obtain highly pure ⁇ -NGF fusion protein.
  • the method for cultivating host cells and the method for isolating and purifying the ⁇ -NGF fusion protein described in the present invention are conventional methods in the art.
  • specific operation methods please refer to the corresponding cell culture technical manual and protein separation and purification technical manual.
  • the ⁇ -NGF fusion protein can be purified into a substantially uniform substance, such as a single band on non-reducing SDS-PAGE electrophoresis.
  • the purified ⁇ -NGF fusion protein molecule of the present invention has biological activity and can be used as a medicine for treating diseases.
  • the term "pharmaceutical composition” means that the ⁇ -NGF fusion protein of the present invention and a pharmaceutically acceptable carrier or adjuvant together form a pharmaceutical preparation composition so as to exert a more stable curative effect. These preparations can guarantee the efficacy of the present invention.
  • the effective amount of the ⁇ -NGF fusion protein of the present invention or its pharmaceutical composition is administered to a subject, the amount or dose will produce the expected effect in the treated individual, and the expected effect includes the improvement of the individual's disease.
  • the subjects include but are not limited to mammals, such as humans, non-human primates, rats, and mice.
  • the ⁇ -NGF fusion protein of the present invention or its pharmaceutical composition can also be used together with other therapeutic agents.
  • “Pharmaceutically acceptable” means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
  • the "pharmaceutically acceptable carrier or excipient” should be compatible with the active ingredient, that is, it can be blended with it without greatly reducing the effect of the drug under normal circumstances.
  • Specific examples of some substances that can be used as pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, Ethyl cellulose and methyl cellulose; Gum tragacanth powder; Malt; Gelatin; Talc; Solid lubricants such as stearic acid and magnesium stearate; Calcium sulfate; Vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oils, corn oil, and cocoa butter; polyols, such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such
  • the pharmaceutical dosage form is not particularly limited, and can be made into dosage forms such as injections, oral liquids, tablets, capsules, dripping pills, sprays, etc., and can be prepared by conventional methods.
  • the choice of drug dosage form should match the mode of administration.
  • the present invention also provides a ⁇ -NGF fusion protein as described in the first aspect of the present invention or a pharmaceutical composition as described in the sixth aspect of the present invention for the preparation of drugs for treating neurotrophic keratitis or peripheral nerve injury use.
  • the neurotrophic keratitis or peripheral nerve injury is diagnosed and determined according to existing clinical diagnosis principles or clinicians' experience.
  • the neurotrophic keratitis is selected from moderate to severe neurotrophic keratitis.
  • the present invention also provides a method for treating diseases, comprising the step of: administering the ⁇ -NGF fusion protein as described in the first aspect of the present invention, or the pharmaceutical composition as described in the sixth aspect of the present invention to a subject in need.
  • the disease is selected from central and peripheral neurological diseases, ophthalmic diseases, endocrine diseases, pediatric diseases or orthopedic diseases.
  • the diseases of the central and peripheral nerves are selected from neurotoxicity, stroke, Alzheimer's disease, Parkinson's disease, and central nerve injury or peripheral nerve injury.
  • the ophthalmic disease is selected from any one or more of the following: acute and persistent optic nerve, retina, corneal damage; optic neuritis; neurotrophic keratitis; allergic conjunctivitis; retinal detachment; amblyopia; optic atrophy; glaucoma; cornea ulcer.
  • the endocrine disease is selected from diabetic peripheral neuritis and diabetic retinopathy.
  • the pediatric disease is selected from children with cerebral palsy, children with hypoxic-ischemic encephalopathy or sequelae of encephalitis.
  • the orthopedic disease is selected from limb nerve injury, sciatic nerve injury, sciatica, multiple neuritis, intervertebral disc pain, nerve rupture and degenerative disease.
  • Subjects in need include but are not limited to animals, preferably mammals; said mammals are preferably rodents, artiodactyls, perissodactyla, lagomorphs, primates and the like. Such mammals include, for example, humans, non-human primates (e.g., monkeys), mice, pigs, cows, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, or other non-human mammals; Mammals include, for example, non-mammalian vertebrates, such as birds (eg, chickens or ducks) or fish, and non-mammalian invertebrates.
  • the object of need may be a human being.
  • Treatment includes preventing or alleviating a condition, reducing the rate at which a condition occurs or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or symptoms, produce complete or partial reversal of a state, cure a state, or a combination of the above.
  • the dosage When administered to a subject, the dosage varies according to the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. The results of animal experiments and various situations can be referred to. The total dosage cannot exceed a certain range.
  • the methods described herein can further comprise administering one or more additional therapies (eg, one or more additional therapeutic agents and/or one or more treatment regimens) in combination with other compounds.
  • the one or more additional therapies may include, but are not limited to: surgery, chemotherapy, and combinations thereof.
  • the examples do not include detailed descriptions of conventional methods, such as those for expressing and purifying known amino acid sequences to obtain polypeptide fragments, for constructing vectors and plasmids, for inserting genes encoding proteins into vectors and plasmids, or for inserting plasmids into Methods of introducing into host cells, etc.
  • Such methods are well known to those of ordinary skill in the art and are described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual 2nd edition, Cold spring Harbor Laboratory Press.
  • Microplate reader SpectraMax 190, wavelength 450nm
  • the light and heavy chains were expressed separately using different expression cassettes.
  • the nucleotide sequence encoding ⁇ -NGF in the light chain is shown in SEQ ID NO: 15, and the nucleotide sequence encoding ⁇ -NGF+Fc in the heavy chain is shown in SEQ ID NO: 16.
  • the precursor sequence Pro SEQ ID NO: 5
  • Signal peptide signal 1 SEQ ID NO: 6
  • signal peptide signal 2 SEQ ID NO: 7
  • the nucleotide sequence encoding the light chain signal 1+ProNGF amino acid was optimized with CHO preferred codons to obtain the optimized nucleotide sequence (SEQ ID NO: 8); the nucleotide encoding the heavy chain signal2+ProNGF+Fc amino acid
  • the sequence was optimized with CHO preferred codons to obtain the optimized nucleotide sequence (SEQ ID NO: 9).
  • a Kozak sequence: GCCACC (SEQ ID NO: 10) was added at the 5' end of the nucleotide (SEQ ID NO: 8) encoding the light chain signal 1+ProNGF amino acid.
  • the GCCACC (SEQ ID NO: 10) Add an endonuclease HindIII site at the 5' end, and add an endonuclease Xhol site at the 3' end of SEQ ID NO: 8 to obtain the light chain gene signal 1+ProNGF and related element sequences (SEQ ID NO: 11 ).
  • a Kozak sequence: GCCACC (SEQ ID NO: 10) was added to the 5' end of the nucleotide (SEQ ID NO: 9) encoding the heavy chain signal 2+ProNGF+Fc amino acid.
  • CHO cells were transfected with the linearized plasmid ⁇ -NGF Fusion-pCGS3, and the cell pool was pressurized and screened first, then single clones were picked, and finally each single clone was pressurized and expanded to continue screening.
  • the protein A probe was used to measure the expression level of ⁇ -NGF fusion protein, and the cell line with the highest expression level was selected for subsequent experiments, and the highest expression level was 1.164g/L.
  • the ⁇ -NGF fusion protein recombinant cell line obtained in Example 2 was fed-batch cultured.
  • the cell line with the highest expression was recovered from cryopreservation and passaged every 3-4 days. After the cells have recovered from viability, they are plated in Gibco ExpiCHO Medium. After a minimum of 3 passages, the cells are fully adapted to the medium. Cells with a doubling time of less than 24 hours during culture can be considered fully adapted by default. After full adaptation, cells were then inoculated into 30 mL volume shake flasks at 0.5 x 10 6 /mL. On the 3rd day of culture, feeding was started. During the cultivation process, glucose solution of 250g/L was used to control the glucose above 3g/L.
  • Purifier150 chromatography system was used for affinity chromatography, and the column was MabSelect TM SuRe TM 1 mL.
  • the operation of the instrument was carried out according to the operating instructions, the A1 pump was Buffer A (PBS, pH7.4), and the B1 pump was Buffer B (50mM citric acid buffer, pH2.5).
  • Buffer A was washed at a flow rate of 1mL/min for 10CV to maintain the Protein A gel environment suitable for the combination of ⁇ -NGF fusion protein and Protein A.
  • Sample loading The clarified and filtered fermentation broth passes through the Protein A gel at a rate of 1mL/min, so that the ⁇ -NGF fusion protein can specifically bind to Protein A.
  • Buffer A was flushed at a flow rate of 1mL/min until the absorbance at 280nm was lower than 0.01.
  • NEF antigen ⁇ -NGF
  • SEQ ID NO: 1 Coating antigen ⁇ -NGF (NGF, SEQ ID NO: 1), 100 ⁇ L per well, at a concentration of 80 nM, and incubate overnight at 4°C; coating ⁇ -NGF fusion protein ( ⁇ -NGF Fusion), 100 ⁇ L per well , at a concentration of 80 nM, and incubated overnight at 4°C.
  • Blocking 200 ⁇ L of blocking solution (PBST+1% BSA) was added to each well, and blocked at room temperature for 2 hours.
  • Washing wash the plate 3 times with washing buffer PBST, and pat dry for later use.
  • Diluted antibody Dilute the primary antibody in a 96-well cell culture plate (the diluent is the blocking solution): the initial concentration is 1 ⁇ g/mL, dilute by 2 times from left to right, and dilute 10 gradients, the 11th and Column 12 was set as a blank control without primary antibody. For each sample, 2 replicate wells were set for each gradient.
  • Washing wash the plate 3 times with washing buffer PBST, and pat dry for later use.
  • Washing wash the plate 3 times with washing buffer PBST, and pat dry for later use.
  • Color development add 100 ⁇ L of freshly prepared color development solution (TMB) to each well, and incubate at room temperature in the dark for 10 minutes.
  • TMB color development solution
  • Termination Add 70 ⁇ L of stop solution (2M H 2 SO 4 ) to each well, mix well, and immediately read on a microplate reader, and the detection wavelength is 450 nm.
  • the processed data map is shown in Figure 5.
  • the EC50 of the antigen ⁇ -NGF is 0.534nM
  • the EC50 of the ⁇ -NGF fusion protein is 0.524nM, indicating that the ⁇ -NGF fusion protein has biological activity.
  • the TF-1 cell method is based on the granulocyte-macrophage colony-stimulating factor (GM-CSF) of the TF-1 cell line is highly dependent and uses NGF to bind to the NGF high-affinity receptor TrkA on the surface of TF-1 cells to induce TF-1
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • TrkA NGF high-affinity receptor
  • TF-1 cells in the logarithmic growth phase were washed twice with 1640 medium preheated at 37°C, and centrifuged at 300-500g for 5 minutes;
  • Count TF-1 cells suspend to appropriate density with 1640 medium containing 10% FBS, inoculate into 96-well plate, 10000 cells/150 ⁇ L/well;
  • the EC50 of the antigen ⁇ -NGF is 0.045nM
  • the EC50 of the ⁇ -NGF fusion protein is 0.292nM, which shows that the ⁇ -NGF fusion protein has biological activity.
  • the mass spectrometry conditions are as follows:
  • the liquid phase part of the system is configured as: BSM binary high-pressure mixing pump, SM sample manager, TUV ultraviolet detector; the mass spectrometry part is configured as: ESI source, Q-TOF detector.
  • Masslynx V4.1 and BiopharmaLynx analysis software were used for data processing and analysis.
  • the MS data are collected in the Resolution mode in the continuum mode; the LockSpray acquisition mode is: real-time acquisition without calibration.
  • Calibration solution real-time calibration (LockSpray) solution: 2ng/ ⁇ L LE solution;
  • Calibration solution for mass axis 2 ⁇ g/ ⁇ L sodium iodide solution.
  • N-glycoamidase excises the sugar chain connected to the asparagine residue (Asn), and then performs mass spectrometry detection.
  • test results are shown in Figures 7A-7C and Table 3. It can be seen that the N-terminus of the ⁇ -NGF fusion protein of the present invention is uniform, which is completely consistent with the design.

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Abstract

一种β-NGF融合蛋白及其制备方法和用途。具体地，涉及一种β-NGF融合蛋白，包含：(a)轻链，所述轻链包含β-NGF；和(b)重链，所述重链包含β-NGF和Fc片段。通过构建的表达载体和转染宿主细胞可以表达出有生物活性的β-NGF融合蛋白，制备过程不需要酶切，且获得的融合蛋白含有两个β-NGF活性分子，活性好，产量高，半衰期长，N端均一。

Description

一种β-NGF融合蛋白及其制备方法和用途 技术领域
本发明属于生物制药领域，具体地，本发明涉及一种β-NGF融合蛋白分子，本发明还涉及所述β-NGF融合蛋白分子的制备方法及用途。
背景技术
神经生长因子(Nerve Growth Factor,NGF)是人类发现的第一个神经营养因子，首先是由意大利神经科学家Rita Levi-Montalcini和美国生物化学家Stanley Cohen于1956年分离成功；Cohen还意外发现了另一种能促进表皮细胞生长、增殖和分化的生长因子，因而将该因子命名为表皮生长因子(Epidermal Growth Factor,EGF)。为此，Levi-Montalcini和Cohen于1986年共同获得了诺贝尔生理学或医学奖。
神经生长因子(NGF)是神经营养因子中最早被发现、目前研究最为透彻、具有神经元营养和促突起生长双重生物学功能的一种神经细胞生长调节因子，它对中枢及周围神经元发育、分化、生长、再生和功能特性表达均具有重要调控作用。
人NGF基因位于1号染色体短臂2区2带及1号染色体短臂2区1带1号亚带上，由6个外显子及5个内含子组成。
NGF最初是在3种蛋白质亚基组成的7S、130kDa复合物中，该复合物由α-NGF、β-NGF和γ-NGF(2：1：2比例，图1)三个蛋白组成。这种形式的NGF也称为proNGF(NGF前体)。该复合物的α亚单位属于非匀质性酸蛋白，其pI约为4.3，它的作用主要是阻止γ亚单位对β亚单位的水解，从而对其起到保护作用。γ亚单位具有精氨酸酯肽酶活性，其主要作用与NGF的合成有关，可以将无活性的β亚基激活。
本领域常说的神经生长因子通常是指蛋白质的2.5S、26kDa的β亚基(即β-NGF)，是生物活性的7S NGF复合物的唯一组分(即充当信号分子)。β-NGF，它是由两条含有118个氨基酸的肽链通过非共价键形成的二聚体。成熟的NGF链内含有6个半胱氨酸残基，可以产生3对链内二硫键，二硫键对维持NGF的生物学活性十分重要，一旦二硫键被破坏，NGF的生物学活性会大大降低甚至是丧失。
NGF是一种参与神经元分化和维护的内源性蛋白质，这种内源性蛋白质通过多种机制为眼角膜完整性带来有力的支持。NGF直接作用于角膜细胞来刺激细胞的生长与存活。此外，已知NGF能结合泪腺上的受体(图2)，促进泪液分泌，为眼睛提供润滑和自然保护，免受病原体伤害。这种蛋白质经证实能够支持角膜神经支配，即神经营养性角膜炎当中已经损失 的一种神经功能。
Oxervate TM(cenegermin，重组人神经生长因子，rhNGF)是意大利制药商Dompé原研的一款滴眼液，是一种用于中度至重度神经营养性角膜炎(neurotrophic keratitis，NK)成人患者的治疗的孤儿药，目前已在欧盟、美国和中国上市。另外，其用于治疗干眼病的适应症在研，二期临床结果良好。但是需要每2小时滴眼一次，使用很不方便。
鼠神经生长因子已经在中国上市多年，用于治疗外周神经损伤。T1/2(β)为4.83小时，每天注射一次。但鼠神经生长因子有10％氨基酸与人的不一样，有潜在免疫原性的风险。
为了制备重组人神经生长因子，原研厂商首先表达proNGF包涵体，然后再变复性，纯化proNGF复性液，酶切纯化得到重组人神经生长因子。然而，该方法需要酶切，酶切后还需要纯化，步骤较多，过程较为繁琐。这种方法制备的NGF热源不容易去除，半衰期短。
中国专利CN108300736B提供了一种CHO细胞制备rhNGF-FC的方法，但其一个rhNGF-FC分子只含有一个NGF活性分子，表达量为18mg/L，况且N端不均一，需要furin酶切。
鉴于现有技术中存在的上述问题，本申请的发明人拟开发一种活性好，产量高，半衰期长，N端均一的β-NGF融合蛋白。
发明内容
针对现有技术的不足，本发明提供了一种β-NGF融合蛋白分子，所述β-NGF融合蛋白分子含有两个β-NGF活性分子，活性好，产量高，半衰期长，N端均一。
因此，本发明的第一个目的在于提供一种β-NGF融合蛋白，包含：
(a)轻链，所述轻链包含β-NGF；和
(b)重链，所述重链包含β-NGF和Fc片段。
本发明第二个目的在于提供编码所述β-NGF融合蛋白的分离的核酸分子。
本发明的第三个目的在于提供含有所述核酸分子的表达载体。
本发明的第四个目的在于提供含有所述表达载体的宿主细胞。
本发明第五个目的在于提供所述β-NGF融合蛋白的制备方法。
本发明第六个目的在于提供含有所述β-NGF融合蛋白的药物组合物。
本发明第七个目的在于提供所述β-NGF融合蛋白或所述药物组合物与另外的治疗剂使用。
本发明的第八个目的在于提供所述β-NGF融合蛋白或所述药物组合物的用途。
本发明的第九个目的在于提供一种治疗疾病的方法，包括步骤：给需要的对象施用如本发明所述的β-NGF融合蛋白，或本发明所述的药物组合物。
为了实现上述目的，本发明采用了如下技术方案：
在本发明的第一方面，提供一种β-NGF融合蛋白，包含：
(a)轻链，所述轻链包含β-NGF；和
(b)重链，所述重链包含β-NGF和Fc片段。
在另一优选例中，所述轻链和重链通过非共价键连接。
在另一优选例中，所述β-NGF为人β-NGF。
在另一优选例中，所述β-NGF包含如SEQ ID NO：1所示的氨基酸序列。
在另一优选例中，所述β-NGF和所述Fc片段通过连接子连接。
在另一优选例中，所述重链从N端到C端依次为β-NGF、连接子和Fc片段。
在另一优选例中，所述连接子包含通式(GGGGS)n，n为整数，n＝1～10；较佳地n＝4～10；优选地n＝4，氨基酸序列如SEQ ID NO：3所示。
在另一优选例中，所述Fc片段来源于IgM、IgG、IgA、IgD或IgE。
在另一优选例中，所述Fc片段来源于IgG。
在另一优选例中，所述IgG来源于哺乳动物；较佳地来源于鼠、食蟹猴或人；优选来源于人。
在另一优选例中，所述Fc片段选自IgG1、IgG2、IgG3或IgG4；较佳地选自IgG4。
在另一优选例中，所述Fc片段包含人IgG铰链区、CH2和CH3区。
在另一优选例中，所述Fc片段包含人IgG4铰链区、CH2和CH3区。
在另一优选例中，所述Fc片段包含如SEQ ID NO：2所示氨基酸序列。
在另一优选例中，所述Fc片段包含Fc变体，所述Fc变体包含一个或多个氨基酸的取代、缺失和/或插入。
在另一优选例中，所述重链包含如SEQ ID NO：4所示的氨基酸序列。
在另一优选例中，所述轻链包含如SEQ ID NO：1所示的氨基酸序列。
在另一优选例中，所述β-NGF融合蛋白包含如SEQ ID NO：4和/或SEQ ID NO：1所示的氨基酸序列。
在本发明的第二方面，提供了一种分离的核酸分子，所述核酸分子包含编码如本发明的第一方面所述的β-NGF融合蛋白的核苷酸序列。
在另一优选例中，所述核酸分子包含如SEQ ID NO：8所示轻链核苷酸序列和如SEQ ID  NO：9所示重链核苷酸序列。
在另一优选例中，所述核酸分子包含如SEQ ID NO：11所示轻链核苷酸序列和如SEQ ID NO：12所示重链核苷酸序列。
在另一优选例中，所述核酸分子包含如SEQ ID NO：15所示轻链核苷酸序列和如SEQ ID NO：16所示重链核苷酸序列。
在本发明的第三方面，提供了一种表达载体，所述的表达载体包含本发明的第二方面所述的核酸分子。
在另一优选例中，所述表达载体选自pCGS3、pcDNA3.4、pDR1、pcDNA3.1(+)、pcDNA3.1/ZEO(+)、pDHFR或pTT5；较佳地选自pCGS3。
在本发明的第四方面，提供了一种宿主细胞，所述宿主细胞包含本发明的第三方面所述的表达载体。
在另一优选例中，所述宿主细胞选自原核表达细胞或真核表达细胞。
在另一优选例中，所述宿主细胞为选自COS、CHO、NS0、sf9、sf21、DH5α、BL21(DE3)、TG1、CHO-K1、293E或Expi293F细胞；较佳地选自CHO细胞、293E或Expi293F细胞；更佳地为CHO细胞。
在本发明的第五方面，提供了一种β-NGF融合蛋白的制备方法，其特征在于，所述方法包括：
(a)在表达条件下，培养上述的宿主细胞，从而表达β-NGF融合蛋白；
(b)分离并纯化步骤(a)所述的β-NGF融合蛋白。
在本发明的第六方面，提供了一种药物组合物，所述药物组合物包含如本发明的第一方面所述的β-NGF融合蛋白和药学上可接受的载体或辅料。
在本发明的第七方面，提供了如本发明的第一方面所述的β-NGF融合蛋白或本发明第六方面所述的药物组合物，其与另外的治疗剂分别、依次或同时使用。
在本发明的第八方面，提供了一种如本发明的第一方面所述的β-NGF融合蛋白或如本发明的第六方面所述的药物组合物用于制备治疗神经营养性角膜炎或者外周神经损伤药物的用途。
本发明的第九方面，提供了一种治疗疾病的方法，包括步骤：给需要的对象施用如本发明的第一方面所述的β-NGF融合蛋白，或如本发明的第六方面所述的药物组合物。
本发明的有益效果：本发明提供了一种β-NGF融合蛋白分子，该β-NGF融合蛋白分子含有两个β-NGF活性分子，活性好，产量高，半衰期长，N端均一。
附图说明
图1表示NGF的结构。
图2表示NGF与NGF受体(NGFR)结合示意图。
图3表示β-NGF融合蛋白结构。
图4表示分离纯化获得的β-NGF融合蛋白SDS-PAGE鉴定图。
图5表示ELISA法检测β-NGF融合蛋白体外生物活性。
图6表示TF-1细胞法检测β-NGF融合蛋白体外生物活性。
图7A-7C表示β-NGF融合蛋白质谱检测图，图7B-7C为图7A的局部放大图。
具体实施方式
本发明中，术语“融合蛋白”结构如图3所示，是指一种β-NGF融合蛋白，包含(a)轻链，所述轻链包含β-NGF；和(b)重链，所述重链包含β-NGF和Fc片段。
应理解，本发明还提供一种β-NGF融合蛋白的制备方法，即构建本发明β-NGF融合蛋白的方法。所述方法如本发明第五方面所述。通过本发明的构建方法获得的β-NGF融合蛋白，含有两个β-NGF活性分子，活性好，产量高，半衰期长，N端均一。
本发明中，术语“Fc片段”和“免疫球蛋白Fc片段”可以互换使用，均指含有免疫球蛋白铰链区、免疫球蛋白重链恒定区2(CH2)和重链恒定区3(CH3)且不含免疫球蛋白重链可变区和轻链可变区、重链恒定区1(CH1)和轻链恒定区(CL)的蛋白质。此外，除了重链可变区和轻链可变区之外，本发明的免疫球蛋白Fc片段可能包含重链恒定区1(CH1)和/或轻链恒定区(CL)的一部分或全部。同样，只要它具有与天然蛋白质基本上相似或优于天然蛋白质的生理学功能，IgG Fc片段就可以是在CH2和/或CH3氨基酸序列的相对较长部分中有缺失的片段。即，本发明的免疫球蛋白Fc片段可能包含1)CH1结构域、CH2结构域、CH3结构域和CH4结构域，2)CH1结构域和CH2结构域，3)CH1结构域和CH3结构域，4)CH2结构域和CH3结构域，5)一个或多个结构域和免疫球蛋白铰链区(或铰链区的一部分)的组合，或6)重链恒定区和轻链恒定区各个结构域的二聚体。
本发明的Fc片段包括天然氨基酸序列和其变体。氨基酸序列衍生物是由于一个或多个氨基酸残基的缺失、插入、非保守性或保守性取代或它们的联合而不同于天然氨基酸序列的序列。
此外，如果需要，Fc片段可以通过磷酸化、硫酸化、丙烯酸化、糖基化、甲基化、法呢 基化、乙酰化、酰胺化等等作用被修饰。
上述Fc衍生物是具有与本发明Fc片段同样的生物学活性或提高了结构稳定性(例如抗热、pH等等)的衍生物。
此外，这些Fc片段可获自从人和包括牛、山羊、猪、小鼠、兔子、仓鼠、大鼠和豚鼠在内的其它动物中分离的天然形式，或可以是重组体或它们的衍生物，获自被转化的动物细胞或微生物。在此，它们可通过从人或动物体中分离完整的免疫球蛋白并用蛋白水解酶加以处理而从天然免疫球蛋白获得。木瓜蛋白酶将天然的免疫球蛋白消化成Fab和Fc片段，而胃蛋白酶处理则导致了pF′c和F(ab′)2片段的产生。可对这些片段进行例如大小排阻层析，以分离Fc或pF′c。
优选的，人源Fc片段是获自微生物的重组免疫球蛋白Fc片段。
此外，本发明的免疫球蛋白Fc片段可以是具有天然糖链、与天然形式相比糖链增加或与天然形式相比糖链减少的形式，或者可以是去糖基化的形式。免疫球蛋白Fc糖链的增加、减少或去除可以通过本领域的通用方法完成，诸如化学法、酶促法和利用微生物的遗传工程方法。
另一方面，免疫球蛋白Fc片段可来自人类或其它动物，包括牛、山羊、猪、小鼠、兔子、仓鼠、大鼠和豚鼠，优选人类。此外，免疫球蛋白Fc片段可以是来自IgG、IgA、IgD、IgE和IgM的Fc片段，或者通过它们的组合或杂合制备而来的Fc片段。优选Fc片段来自IgG或IgM，它们是人类血液中最丰富的蛋白质之一，最优选来自IgG(已知它能延长配体结合蛋白质的半衰期)。
本发明中，术语“连接子”是指β-NGF和Fc片段之间的短的连接子序列，优选为柔性接头。合适的连接子实例包括单甘氨酸(Gly)或丝氨酸(Ser)残基，接头中氨基酸残基的标识和序列可随着连接子中需要实现的次级结构要素的类型而变化。
本发明提供了一种核酸分子，所述核酸分子编码上述的轻链。其中将β-NGF基因，通过加入前体pro序列、信号肽signal 1序列、Kozak序列以及密码子优化后获得了轻链基因和相关元件序列(SEQ ID NO：8)。
本发明还提供了一种核酸分子，所述核酸分子编码上述的重链(β-NGF+(G4S)4+Fc)。其中将β-NGF+(G4S)4+Fc基因，通过加入前体pro序列、信号肽signal 2序列、Kozak序列以及密码子优化后获得了重链基因和相关元件序列(SEQ ID NO：9)。
本发明核酸分子的制备方法为本领域常规的制备方法，较佳地包括以下制备方法：通过基因克隆技术例如PCR方法等，获得编码上述多肽的核酸分子，或者通过人工全序列合成的 方法得到编码上述多肽的核酸分子。
本发明中，术语“表达载体”含有上述任一所述的核酸分子，为本领域常规的表达载体，是指包含适当的调控序列，例如启动子序列、终止子序列、多腺苷酰化序列、增强子序列、标记基因和/或序列以及其他适当的序列的表达载体。所述表达载体可以是病毒或质粒，如适当的噬菌体或者噬菌粒，更多技术细节请参见例如Sambrook等，Molecular Cloning：A Laboratory Manual，第二版，Cold Spring Harbor Laboratory Press，1989。许多用于核酸操作的已知技术和方案请参见Current Protocols in Molecular Biology，第二版，Ausubel等编著。
本发明所述表达载体较佳地为选自pCGS3，pcDNA3.4，pDR1，pcDNA3.1(+)，pcDNA3.1/ZEO(+)，pDHFR和pTT5，优选自pCGS3。
本发明中，术语“宿主细胞”含有上述表达载体，为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核苷酸可被有效表达即可。其中所述宿主细胞包括原核表达细胞和真核表达细胞，较佳地包括：COS、CHO(中国仓鼠卵巢，Chinese Hamster Ovary)、NS0、sf9、sf21、DH5α、BL21(DE3)或TG1，更佳地为E.coli TG1、BL21(DE3)细胞或者CHO-K1细胞。将前述表达载体转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
作为优选的方案，本发明所述宿主细胞是真核细胞，优选自CHO细胞，293E和Expi293F细胞。
本发明提供了一种制备方法，所述制备方法包括以下步骤：
(a)在表达条件下，培养上述的宿主细胞，从而表达β-NGF融合蛋白；
(b)分离并纯化步骤(a)所述的β-NGF融合蛋白。
更具体地，用如SEQ ID NO：8所示编码轻链的核苷酸分子和用如SEQ ID NO：9所示编码重链的核苷酸分子分别插入pCGS3不同的多克隆位点，构建表达载体；在CHO细胞中分泌表达；离心分离收集上清液，用Protein A亲和层析纯化，得到高纯度的β-NGF融合蛋白。
本发明所述的宿主细胞的培养方法、所述β-NGF融合蛋白的分离和纯化方法为本领域常规方法，具体操作方法请参考相应的细胞培养技术手册以及蛋白分离纯化技术手册。利用上述方法，可以将β-NGF融合蛋白纯化为基本均一的物质，例如在非还原SDS-PAGE电泳上为单一条带。
本发明所述纯化后β-NGF融合蛋白分子具有生物活性，可以作为治疗疾病的药物。
本发明中，术语“药物组合物”是指本发明的β-NGF融合蛋白和药学上可以接受的载体 或辅料一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明公开的β-NGF融合蛋白的氨基酸核心序列的构象完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。有效量的本发明的β-NGF融合蛋白或其药物组合物施用受试者后，在治疗的个体中产生预期效果的量或剂量，该预期效果包括个体病症的改善。其中受试者包括但不限于哺乳动物，例如人、非人灵长类动物、大鼠和小鼠等。此外，本发明的β-NGF融合蛋白或其药物组合物还可与其他治疗剂一起使用。
“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。
“药学上可接受的载体或辅料”应当与所述有效成分相容，即能与其共混而不会在通常情况下大幅度降低药物的效果。可作为药学上可接受的载体或辅料的一些物质的具体例子是糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如Tween；润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；磷酸盐缓冲液等。这些物质根据需要用于帮助配方的稳定性或有助于提高活性或它的生物有效性或在口服的情况下产生可接受的口感或气味。
本发明中，除非特别说明，药物剂型并无特别限定，可以被制成针剂、口服液、片剂、胶囊、滴丸、喷剂等剂型，可通过常规方法进行制备。药物剂型的选择应与给药方式相匹配。
本发明还提供一种如本发明的第一方面所述的β-NGF融合蛋白或如本发明的第六方面所述的药物组合物用于制备治疗神经营养性角膜炎或者外周神经损伤药物的用途。
所述神经营养性角膜炎或者外周神经损伤依据现有临床诊断原则或临床医生经验诊断确定。
所述神经营养性角膜炎选自中度至重度神经营养性角膜炎。
本发明还提供一种治疗疾病的方法，包括步骤：给需要的对象施用如本发明的第一方面所述的β-NGF融合蛋白，或如本发明的第六方面所述的药物组合物。
所述疾病选自中枢及周围神经疾病、眼科疾病、内分泌疾病、儿科疾病或骨科疾病。
所述中枢及周围神经疾病选自神经中毒、脑卒中、阿尔茨海默病、帕金森氏病以及中枢神经损伤或外周神经损伤。
所述眼科疾病选自以下任一项或多项：急性和迁延性视神经、视网膜、角膜损伤；视神 经炎；神经营养性角膜炎；过敏性结膜炎；视网膜脱离；弱视；视神经萎缩；青光眼；角膜溃疡。
所述内分泌疾病选自糖尿病末梢神经炎、糖尿病视网膜病。
所述儿科疾病选自小儿脑性瘫痪、小儿缺血缺氧性脑病或脑炎后遗症。
所述骨科疾病选自四肢神经损伤、坐骨神经损伤、坐骨神经痛、多发性神经炎、椎间盘痛、神经断裂及退行性病变。
“需要的对象”包括但不限于动物，优选为哺乳动物；所述哺乳动物优选为啮齿目动物、偶蹄目动物、奇蹄目动物、兔形目动物、灵长目动物等。所述哺乳动物包括例如人、非人灵长类动物(例如猴)、小鼠、猪、牛、山羊、兔、大鼠、豚鼠、仓鼠、马、猴、绵羊或其他非人哺乳动物；非哺乳动物，包括例如非哺乳动物脊椎动物，例如鸟(例如鸡或鸭)或鱼，以及非哺乳动物无脊椎动物。需要的对象可以是人类。
“治疗”包括预防或减轻某种状态，降低某种状态发生或发展的速度，减少发展出某种状态的风险，预防或延迟与某种状态相关的症状发展，减少或终止与某种状态相关的症状，产生某种状态的完全或部分的逆转，治愈某种状态，或以上的组合。
在向受试者施用时，给药剂量因病人的年龄和体重，疾病特性和严重性，以及给药途径而异，可以参考动物实验的结果和种种情况，总给药量不能超过一定范围。
在一些实施方式中，本文描述的方法可以进一步包括与其他化合物联合给予一种或多种另外的疗法(例如，一种或多种另外的治疗剂和/或一种或多种治疗方案)。一种或多种另外的疗法可以包括但不限于：手术，化学疗法，以及它们的组合。
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外， 根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
实施例不包括对传统方法的详细描述，如那些将已知氨基酸序列表达纯化获得多肽片段的方法，用于构建载体和质粒的方法，将编码蛋白的基因插入到载体和质粒的方法或将质粒引入宿主细胞的方法等。这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais,T.(1989)Molecular Cloning:A Laboratory Manual 2nd edition,Cold spring Harbor Laboratory Press.
以下实施例中使用的检测方法说明如下：
1、SDS-PAGE检测：
检测系统：Mini protein Tetra system
检测条件：140V恒压45-55min
2、紫外检测：
仪器型号：Nanodrop one(Thermo)
消光系数：1.42
3、Protein A亲和层析
层析柱：XK16/20(GE)
填料：MabSelect TM SuRe TM(GE)
层析系统：AKTA Pure150(GE)
操作系统：unicorn 7.0(GE)
流速；1.0mL/min
4、ELISA检测及处理
酶标仪：SpectraMax 190，波长450nm
处理软件：GraphPad Prism 9
以下实施例中使用的实验材料和实验试剂，如果没有特殊说明，均为常规商购获得。
实施例1.β-NGF融合蛋白表达载体的构建
1.β-NGF融合蛋白氨基酸的设计，以及其基因和相关元件序列的设计
将轻链和重链用不同的表达框分别表达。编码轻链中β-NGF的核苷酸序列如SEQ ID NO：15所示，编码重链中β-NGF+Fc的核苷酸序列如SEQ ID NO：16所示。为了便于表达折叠，分别在轻链和重链β-NGF的N端增加前体序列Pro(SEQ ID NO：5)；为了使融合蛋白分泌 到发酵液上清中，在轻链和重链NGF前体序列Pro的N端分别增加信号肽signal 1(SEQ ID NO：6)和信号肽signal 2(SEQ ID NO：7)。编码轻链signal 1+ProNGF氨基酸的核苷酸序列用CHO偏好的密码子优化，得到优化后的核苷酸序列(SEQ ID NO：8)；编码重链signal2+ProNGF+Fc氨基酸的核苷酸序列用CHO偏好的密码子优化，得到优化后的核苷酸序列(SEQ ID NO：9)。为了提高表达量，在编码轻链signal 1+ProNGF氨基酸的核苷酸(SEQ ID NO：8)5’端添加Kozak序列：GCCACC(SEQ ID NO：10)，为了便于克隆，在GCCACC(SEQ ID NO：10)5’端添加内切酶HindIII位点，在SEQ ID NO：8的3’端添加内切酶Xhol位点得到轻链基因signal 1+ProNGF和相关元件序列(SEQ ID NO：11)。同理为了提高表达量，在编码重链signal 2+ProNGF+Fc氨基酸的核苷酸(SEQ ID NO：9)5’端添加Kozak序列：GCCACC(SEQ ID NO：10)，为了便于克隆，在GCCACC(SEQ ID NO：10)5’端添加内切酶BstB I位点，在SEQ ID NO：9的3’端添加内切酶Pac I位点得到重链基因signal2+ProNGF+Fc和相关元件序列(SEQ ID NO：12)。
2.β-NGF融合蛋白表达载体的制备
全基因合成重链基因signal 2+ProNGF+Fc和相关元件序列(SEQ ID NO：12)和轻链基因signal 1+ProNGF和相关元件序列(SEQ ID NO：11)，分别插入pCGS3不同的表达框，构建β-NGF Fusion-pCGS3和质粒菌。大量提取质粒β-NGF Fusion-pCGS3，用内切酶Pvu1(NEB公司)线性化，0.22μm无菌过滤除菌备用。
实施例2.β-NGF融合蛋白CHO稳转株的构建
用线性化的质粒β-NGF Fusion-pCGS3转染CHO细胞，先对细胞池进行加压筛选，随后挑取单克隆，最后针对每个单克隆进行加压扩大培养继续筛选。用Protein A探针测β-NGF融合蛋白的表达量，挑选表达量最高的细胞株进行后续试验，表达量最高为1.164g/L。
实施例3.β-NGF融合蛋白表达纯化
1.β-NGF融合蛋白表达
将实施例2中获得的β-NGF融合蛋白重组细胞株进行流加培养。将表达最高的细胞株从冻存中复苏，每3-4天传代。细胞从生长活力恢复后，将细胞接种至Gibco ExpiCHO培养基中。最少经过3次传代，直到细胞完全地适应培养基。细胞在培养过程中倍增时间小于24小时可默认为已完全适应。在完全适应后，随后将细胞以0.5×10 6/mL接种至30mL体积的摇瓶 中。培养的第3天开始流加补料。培养过程中使用250g/L的葡萄糖溶液将葡萄糖控制在3g/L以上。
2.Protein A亲和层析分离纯化β-NGF融合蛋白
使用Purifier150层析系统来进行亲和层析，色谱柱为MabSelect TM SuRe TM 1mL。仪器操作按操作指南进行，A1泵为Buffer A(PBS，pH7.4)，B1泵为Buffer B(50mM柠檬酸缓冲液，pH2.5)。
清洗：以0.1M NaOH，流速1mL/min，冲洗Protein A凝胶柱5个柱体积(CV)，再用超纯水以流速1mL/min清洗5CV，去除Protein A凝胶上的杂质残留。
平衡：Buffer A以1mL/min流速冲洗10CV，以维持Protein A凝胶环境适合β-NGF融合蛋白与Protein A的结合。
上样：澄清过滤后的发酵液以1mL/min的速率通过Protein A凝胶，使β-NGF融合蛋白与Protein A能特异性的结合。
冲平：Buffer A以1mL/min流速冲洗至280nm吸收值低于0.01。
洗脱：以1mL/min流速，Buffer B冲洗Protein A凝胶，收集洗脱峰。
中和：洗脱峰收集样品用1.5mol/L Tris-HCl，pH9.0调pH至5.6。SDS-PAGE检测分析纯化得到的β-NGF融合蛋白。其结构示意图如图3所示，SDS-PAGE如图4所示。
实施例4.β-NGF融合蛋白ELISA检测
1、包被抗原β-NGF(NGF，SEQ ID NO：1)，每孔100μL，浓度为80nM，，于4℃孵育过夜；包被β-NGF融合蛋白(β-NGF Fusion)，每孔100μL，浓度为80nM，于4℃孵育过夜。
2、洗涤：取出包被好抗原的Elisa板，用洗涤缓冲液PBST洗板3次，拍干Elisa板等待下一步封闭。
3、封闭：每孔加入200μL封闭液(PBST+1％BSA)，于室温封闭2h。
4、洗涤：用洗涤缓冲液PBST洗板3次，拍干备用。
5、加入一抗Fulranumab(轻链氨基酸序列如SEQ ID NO：13所示，重链氨基酸序列如SEQ ID NO：14所示；参考IMGT数据库)
(1)稀释抗体：在96孔细胞培养板中进行一抗稀释(稀释液为封闭液)：初始浓度为1μg/mL，从左至右依次以2倍稀释，稀释10个梯度，第11和12列设置为不加一抗的空白对照。每个样品，每个梯度设置2个复孔。
(2)每孔加入100μL稀释好的一抗，于室温封闭2h。
6、洗涤：用洗涤缓冲液PBST洗板3次，拍干备用。
7、加入二抗：按1：1000的比例用封闭液(PBST+1％BSA)稀释Fab HRP(苏州博特龙)，每孔加入100μL稀释好的二抗，于室温避光孵育1h。
8、洗涤：用洗涤缓冲液PBST洗板3次，拍干备用。
9、显色：每孔加入100μL新鲜配制的显色液(TMB)，于室温避光孵育10分钟。
10、终止：每孔加入70μL终止液(2M H 2SO 4)，混匀后，立即到酶标仪上读数，检测波长为450nm。
处理数据图谱如图5所示。抗原β-NGF的EC50为0.534nM，β-NGF融合蛋白的EC50为0.524nM，说明β-NGF融合蛋白具有生物活性。
实施例5.TF-1细胞法体外生物活性分析
TF-1细胞法是基于TF-l细胞系的粒细胞巨噬细胞集落刺激因子(GM-CSF)高度依赖性利用NGF与TF-1细胞表面的NGF高亲和力受体TrkA结合后诱导TF-1细胞增殖的特点，建立的一种新的NGF活性测定方法。该方法目前已成为检测重组人神经生长因子(rhNGF)WHO参考品的标准方法。
材料：1640培养基，Gibco，货号：22400-105；96孔平底细胞培养板，Corning，货号CLS3599；CCK-8试剂盒，Dojindo，货号CK04；胎牛血清，GIBCO，货号10099-141；TF-1细胞，ATCC，CRL-2003 TM
1.处于对数生长期的TF-1细胞用37℃预热的1640培养基洗2遍，300-500g离心5分钟；
2.计数TF-1细胞，用含10％FBS的1640培养基悬浮到适当密度，接种到96孔板中，10000个/150μL/孔；
3.在96孔板中用1640培养基梯度稀释β-NGF(NGF)样品或者β-NGF融合蛋白(β-NGF Fusion)样品，100nM起始，以三倍连续稀释9个梯度；将稀释好的样品加入96孔细胞培养板中，50μL/孔；96孔周围用200μL/孔的蒸馏水补齐；
4.在37℃、5％CO 2条件的孵箱中孵育培养3天；
5.在96孔细胞培养板每孔加入20μL CCK-8溶液，在37℃孵箱中继续培养8h；
6.混匀后酶标仪读OD450值，GraphPad Prism6进行数据分析，作图并计算EC50。
生物活性如图6所示，抗原β-NGF的EC50为0.045nM，β-NGF融合蛋白的EC50为0.292nM，由此可知，β-NGF融合蛋白具有生物活性。
实施例6.质谱检测
质谱条件如下：
Waters公司UPLC-XEVO G2 Q-TOF液质联用系统。系统液相部分配置为：BSM二元高压混合泵，SM样品管理器，TUV紫外检测器；质谱部分配置为：ESI源，Q-TOF检测器。数据处理分析采用Masslynx V4.1及BiopharmaLynx分析软件(Version：1.2)。
液相条件
色谱柱：Mass PREPTM Micro Desalting Column 2.1×5mm(完整蛋白分子量分析)，柱温：80℃；
流动相A：0.1％FA-H 2O
流动相B：0.1％FA-CAN
Seal Wash溶液：10％IPA
质谱清洗液：50％ACN
质谱IntelliStart阀清洗液：50％MeOH
进样体积：10μL
样品室温度：10℃
梯度洗脱条件：
表1.梯度洗脱条件
Figure PCTCN2022120976-appb-000001
Figure PCTCN2022120976-appb-000002
质谱条件
MS数据均采用轮廓图(continuum)模式，在Resolution模式下采集；LockSpray采集模式为：实时采集并不应用校准。
校准溶液：实时校准(LockSpray)溶液：2ng/μL LE溶液；
质量轴的校正溶液：2μg/μL碘化钠溶液。
质谱参数：
表2.质谱参数
Figure PCTCN2022120976-appb-000003
脱N糖分子量：N-糖酰胺酶(PNGase)切除连接在天冬酰胺残基(Asn)上的糖链，再进行质谱检测。
表3.β-NGF融合蛋白脱N-糖完整分子量
Figure PCTCN2022120976-appb-000004
检测结果如图7A-7C和表3所示，由此可知，本发明的β-NGF融合蛋白N端均一，与设计完全一致。
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法的变化，在不脱离本发明的范围和精神的前提下对本 领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。

Claims (21)

  1. 一种β-NGF融合蛋白，其特征在于，包含：
    (a)轻链，所述轻链包含β-NGF；和
    (b)重链，所述重链包含β-NGF和Fc片段。
  2. 如权利要求1所述的β-NGF融合蛋白，其特征在于，所述β-NGF为人β-NGF；优选地，所述β-NGF包含如SEQ ID NO：1所示氨基酸序列。
  3. 如权利要求1所述的β-NGF融合蛋白，其特征在于，所述β-NGF和所述Fc片段通过连接子连接。
  4. 如权利要求3所述的β-NGF融合蛋白，其特征在于，所述重链从N端到C端依次为β-NGF、连接子和Fc片段。
  5. 如权利要求3或4所述的β-NGF融合蛋白，其特征在于，所述连接子包含通式(GGGGS)n，n为整数，n＝1～10；较佳地n＝4～10；优选地n＝4，氨基酸序列如SEQ ID NO：3所示。
  6. 如权利要求1所述的β-NGF融合蛋白，其特征在于，所述Fc片段来源于IgM、IgG、IgA、IgD或IgE。
  7. 如权利要求6所述的β-NGF融合蛋白，其特征在于，所述IgG来源于哺乳动物；较佳地来源于鼠、食蟹猴或人；优选来源于人。
  8. 如权利要求6所述的β-NGF融合蛋白，其特征在于，所述Fc片段选自IgG1、IgG2、IgG3或IgG4；较佳地选自IgG4。
  9. 如权利要求6所述的β-NGF融合蛋白，其特征在于，所述Fc片段包含人IgG铰链区、CH2和CH3区；较佳地，包含人IgG4铰链区、CH2和CH3区；优选地，包含如SEQ ID NO：2所示氨基酸序列。
  10. 如权利要求1所述的β-NGF融合蛋白，其特征在于，所述Fc片段包含Fc变体，所述Fc变体包含一个或多个氨基酸的取代、缺失和/或插入。
  11. 如权利要求1所述的β-NGF融合蛋白，其特征在于，所述轻链包含如SEQ ID NO：1所示的氨基酸序列；和/或所述重链包含如SEQ ID NO：4所示的氨基酸序列。
  12. 一种分离的核酸分子，其特征在于，所述核酸分子包含编码如权利要求1-11中任一项所述的β-NGF融合蛋白的核苷酸序列。
  13. 如权利要求12所述的核酸分子，其特征在于，还包括如下特征中的一项或多项：
    (1)所述核酸分子包含如SEQ ID NO：8所示轻链核苷酸序列和如SEQ ID NO：9所示重链核苷酸序列；
    (2)所述核酸分子包含如SEQ ID NO：11所示轻链核苷酸序列和如SEQ ID NO：12所 示重链核苷酸序列；
    (3)所述核酸分子包含如SEQ ID NO：15所示轻链核苷酸序列和如SEQ ID NO：16所示重链核苷酸序列。
  14. 一种表达载体，其特征在于，所述的表达载体包含如权利要求12或13所述的核酸分子。
  15. 一种宿主细胞，其特征在于，所述宿主细胞包含如权利要求14所述的表达载体。
  16. 如权利要求1-11中任一项所述的β-NGF融合蛋白的制备方法，其特征在于，所述方法包括：
    (a)在表达条件下，培养如权利要求15所述的宿主细胞，从而表达β-NGF融合蛋白；
    (b)分离并纯化步骤(a)所述的β-NGF融合蛋白。
  17. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1-11中任一项所述的β-NGF融合蛋白和药学上可接受的载体或辅料。
  18. 如权利要求1-11中任一项所述的β-NGF融合蛋白或权利要求17所述的药物组合物，其与另外的治疗剂分别、依次或同时使用。
  19. 如权利要求1-11中任一项所述的β-NGF融合蛋白或权利要求18所述的药物组合物用于制备治疗神经营养性角膜炎或者外周神经损伤药物的用途。
  20. 一种治疗疾病的方法，其特征在于，包括步骤：给需要的对象施用如权利要求1-11中任一项所述的β-NGF融合蛋白，或如权利要求17所述的药物组合物。
  21. 如权利要求20所述的方法，其特征在于，所述疾病选自中枢及周围神经疾病、眼科疾病、内分泌疾病、儿科疾病或骨科疾病；优选的，所述疾病为神经营养性角膜炎或者外周神经损伤。
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