WO2019148848A1 - 重组突变体α1-抗胰蛋白酶及其制备和应用 - Google Patents

重组突变体α1-抗胰蛋白酶及其制备和应用 Download PDF

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WO2019148848A1
WO2019148848A1 PCT/CN2018/105011 CN2018105011W WO2019148848A1 WO 2019148848 A1 WO2019148848 A1 WO 2019148848A1 CN 2018105011 W CN2018105011 W CN 2018105011W WO 2019148848 A1 WO2019148848 A1 WO 2019148848A1
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aat
mutant
buffer
solution
tris
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French (fr)
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蔺新力
李兰芬
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北京大学
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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
    • A61K38/55—Protease inhibitors
    • 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
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin
    • 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
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a method for producing a recombinant mutant ⁇ 1-antitrypsin (AAT) polypeptide and its use in the medical field.
  • AAT ⁇ 1-antitrypsin
  • AAT ⁇ 1-antitrypsin
  • HLE Human leukocyte elastase
  • AAT acts as an important inhibitor of HLE proteolysis to prevent damage to the alveolar matrix.
  • AAT is a 52KD glycoprotein that is mainly synthesized in the liver, but is also synthesized in neutrophils, monocytes, and macrophages. AAT can be secreted into the plasma, but its main site of action is in the lung parenchyma [7].
  • AAT In addition to HLE, AAT also inhibits two other proteases that release neutrophils into the lungs, namely cathepsin G (CatG) and protease 3 (Pr3). CatG and Pr3 can also cause lung damage by breaking down elastin and other extracellular matrix proteins. AAT can prevent this damage.
  • CatG and Pr3 can also cause lung damage by breaking down elastin and other extracellular matrix proteins. AAT can prevent this damage.
  • HLE is considered to be the main enzyme responsible for lung injury [8].
  • the normal biological function of AAT is essential for human health and is therefore known as the guardian of vascular tissue [9].
  • PiZZZ carriers have hereditary emphysema and can be treated with AAT-enhanced therapy [10].
  • COPD chronic obstructive pulmonary disease
  • Native ⁇ 1-antitrypsin is a 52 kDa glycoprotein with anti-protease action and is a physiological inhibitor of neutrophil serine proteases such as neutrophil elastase, cathepsin G and protease 3.
  • the main function of AAT is to protect the lungs from damage caused by proteases when inflammation occurs.
  • Genetic or acquired defects in AAT can lead to serious diseases such as hereditary emphysema and COPD. Due to its biological function, natural AAT is less stable and is easily oxidized. However, in order for AAT to be used as a drug for clinical treatment, it is necessary to increase its stability, antioxidant activity and half-life in vivo.
  • the present invention we utilized a highly efficient inclusion body renaturation technique [28] to reconstitute and purify recombinant AAT from inclusion bodies. Furthermore, in order to overcome the problem of AAT oxidative and E. coli expression of non-glycosylated proteins, we designed anti-oxidant and more stable AAT mutants to meet clinical treatment requirements. In this invention, we designed and screened a series of AAT mutants and successfully obtained a novel anti-oxidant and more stable triple mutant AAT suitable for drug development. In order to further extend the half-life of the AAT protein drug in vivo, in the present invention, we also designed, prepared and purified the chemically modified AAT at a specific site according to the structure of the AAT. The novel, chemically modified mutants obtained by the present invention enable large-scale application of AAT protein drugs.
  • ⁇ 1-antitrypsin in the form of inclusion bodies in E. coli and established efficient renaturation and purification methods.
  • the mutant or chemically modified mutant is expected to be an effective new AAT drug for the treatment of hereditary emphysema and other forms of lung disease such as smoking lung, cystic fibrosis, pulmonary hypertension, pulmonary fibrosis, chronic obstruction. Sexual lung disease, etc.
  • the object of the present invention is to improve the thermal stability and oxidation resistance by establishing an efficient and cost-effective recombinant AAT expression system while rationally designing the mutant, and improving its half-life in vivo by chemical modification.
  • the use of recombinant AAT for clinical treatment is achieved.
  • Our E. coli expression and renaturation system enables high yields, high purity and low cost.
  • the primary problem is that AAT is prone to oxidation and is unstable under physiological conditions [31-33].
  • the present invention seeks to solve this problem by constructing and selecting more stable and antioxidant mutants, making it more suitable for drug development.
  • E. coli is not glycosylated.
  • the half-life of unglycosylated AAT is much shorter than that of natural glycosylated AAT.
  • the present invention designs chemical modifications of E. coli recombinant renaturation AAT, including pegylation modifications and fatty acid modification (eg, palmitic acid modification) to increase protein stability and in vivo half-life.
  • pegylation modifications include pegylation modifications and fatty acid modification (eg, palmitic acid modification) to increase protein stability and in vivo half-life.
  • fatty acid modification eg, palmitic acid modification
  • the second mutant designed by the present invention is directed against oxidation resistance. It is well known that AAT is very sensitive to oxidation due to its in vivo functional regulation requirements [31, 34]. It is known that components inhaled in cigarette smoke, such as hydrogen peroxide, cause oxidation of AAT. It is inferred that the reduction of active AAT in the lungs of smokers is the pathophysiological cause of lung disease in smokers. The most sensitive residues are Met351 and Met358 [31,34], which are exactly the P8 and P1 positions of the AAT binding site ( Figure 7).
  • the in vivo half-life of unglycosylated rAAT expressed by E. coli is much shorter than that of native glycosylated AAT.
  • the plasma half-life of glycated rat AAT was measured in rat serum to be 170 minutes, while the non-glycosylated form of the molecule was only 30 minutes [35].
  • Polyethylene glycol binding (PEGylation) is one of the most effective methods for prolonging half-life in vivo and reducing protein immune responses [36].
  • PEGylation is one of the most effective methods for prolonging half-life in vivo and reducing protein immune responses [36].
  • Cys232 is the only cysteine in AAT and is present as a monomeric molecule ( Figure 7) and can therefore be PEGylated.
  • the PEGylated AAT showed similar binding rates to wild-type AAT in inhibiting porcine trypsin (PPE) in vitro ( Figure 4).
  • AAT represents both wild-type AAT and the mutant AAT of the invention.
  • the present invention provides a novel ⁇ 1-antitrypsin mutant, and a method of producing and purifying the novel recombinant mutant.
  • the novel mutants are designed to be more suitable for medical applications, structurally more stable, and capable of anti-oxidation candidate protein drugs through protein engineering based on protein structure.
  • the invention also provides methods for E. coli expression, inclusion body refolding and purification of such novel mutants. Further, the present invention also provides a method for chemically modifying a purified candidate drug to prolong the half-life of the protein drug in the body to achieve better drug efficacy.
  • FIG. 1 Amino acid mutation sites of mature AAT protein sequences and mutants. The starting position of ⁇ 5AAT is shown and an additional starting Met is inserted in the E. coli expression vector. Underlined indicates the amino acid change site. The mutation numbers in this paper are based on mature full-length AAT [39]. The nucleotide changes of the three mutants were: F51L, TTT ⁇ CTG; M351V/M358V, ATG ⁇ GTG/ATG ⁇ GTG; and F51L/M351V/M358V, TTT ⁇ CTG/ATG ⁇ GTG/ATG ⁇ GTG.
  • Lanes 1-3 Purified wild-type AAT inclusion bodies, loading amounts of 1, 3, 5 ⁇ l, respectively; Lane 4: BSA labeling; Lane 5: MW standard; Lanes 6-8: Purified F51L mutant protein inclusion bodies , sample loading is 5 ⁇ l, 3 ⁇ l, 1 ⁇ l; lanes 9-19: expression of soluble and insoluble cell extracts; lanes 9, 11: wild type, soluble extract; lanes 10, 12: wild type, insoluble extract Lanes 13, 15: M351V/M358V, soluble extract; Lane 14, 16: M351V/M358V, insoluble extract; lane 17: MW standard; Lane 18: F51L/M351V/M358V, soluble extract; Lane 19: F51L/M351V/M358V, insoluble extract.
  • FIG. 3 SDS-PAGE (A) of purified recombinant AAT and mutein and chemical assay for PPE inhibitory activity (B, C).
  • A SDS-PAGE results of purified samples, Coomassie blue staining; Lane 1: wild-type AAT; Lanes 2-4: increased purity of sample loading (1, 2, 4, 6 ⁇ g, respectively); Lane 2: F51L; lane 3: M351V/M358V; lane 4: F51L/M351V/M358V; molecular weight marker (MW).
  • B Recombinant AAT (wild type) and commercially available natural AAT drug as a control The activity comparison results.
  • C Results of assay for activity of purified mutants.
  • FIG. 4 PEGylation, purification and properties of Cys232.
  • A Cation exchange chromatogram of PEGylated AAT.
  • B Non-reducing SDS-PAGE of the fraction passing through the Q XL column, the number corresponding to the tube number of the gradient elution fraction shown in A.
  • C MALDI-TOF mass spectrometry analysis of samples after PEGylation, the molecular weight of each indicated peak is depicted at the top of the corresponding peak.
  • D Purified PEGylated AAT showed normal inhibitory activity in blocking PPE.
  • FIG. 1 Comparison of the thermal stability of wild type and mutant AAT.
  • the graph shows the relationship between fluorescence count (Y-axis) and temperature (X-axis, °C).
  • Figure 6 Determination of antioxidant properties.
  • the production of the aminolysis activity at 405 nm was monitored using a SpectraMax 250 microplate reader (Molecular Devices) at 37 ° C for 10 seconds, and the detection process was 20 minutes.
  • the IC50 (Y-axis) of each H 2 O 2 -treated AAT or its mutant was determined using GraFit version 7 (Erithacus Software).
  • X-axis shows the H 2 O 2 and the molar ratio of AAT (H 2 O 2: AAT, from 4:1 to 400:1).
  • Figure 7 Three-dimensional structure of AAT from Lomas and its collaborators, showing sites of antioxidant mutations (Met351 and Met358, P8 and P1, respectively, active sites), and conserved mutations buried deep in the hydrophobic core of the molecule ( Phe51) and Cys PEGylation (Cys232, exposed to the surface but not impeding activity).
  • the structural model was simulated using COOT software. The entire protein structure is shown in the form of cartoon, and the wild type and mutant residues are displayed in the form of a stick. The image was generated by PyMOL software.
  • the invention provides methods of expressing and purifying the AAT mutant.
  • the invention describes the expression of a host with E. coli, this does not limit the scope of the expression host of the invention.
  • any host capable of achieving high expression of the recombinant protein can be used to express the mutant protein.
  • Such hosts include mammalian and cell expression hosts, plant and plant cell expression hosts, insect expression hosts, fungal expression hosts, and bacterial expression hosts.
  • any vector that can express a protein in such a host can be used for expression of the protein.
  • E. coli can be used as an expression host for recombinant proteins.
  • the expression host used can be BL21 (DE3).
  • pET-11 (Novagen) can be used as an expression vector for E. coli.
  • ⁇ 5AAT cleaving the first 5 amino acids of the mature AAT protein
  • ⁇ 10AAT cleaging the first 10 amino acids of the mature AAT
  • E. coli expression under a number of conditions was tested, and finally the wild type and selected mutant AAT expressed well in an E. coli expression host ( Figure 2), and the results were shown using appropriate growth medium and When expressed, all expression constructs can be expressed in high yields, mostly in the form of insoluble inclusion bodies.
  • Methods for E. coli expression and inclusion body purification have been published (X. Lin, Umetsu, T., The high ph and ph-shift refolding technology, Current Pharmaceutical Technology 11 (2010), no. 3, 293-299.).
  • the purified inclusion bodies are dissolved in a high concentration of lysis buffer, such as a high concentration of urea buffer, such as about 8 M urea lysis buffer.
  • the purified inclusion bodies are dissolved in a high concentration of guanidine hydrochloride buffer, such as about 6 M guanidine hydrochloride lysis buffer.
  • AAT inclusion bodies dissolved in urea or guanidine hydrochloride buffer can be further purified, for example, by chromatography. Purification techniques for inclusion bodies are well known to those skilled in the art.
  • the purified inclusion bodies in the lysis buffer can be renatured in a plurality of renaturation buffers of different pH and different compositions.
  • wild-type and mutant AAT are renatured in different renaturation buffers for optimal renaturation.
  • the wild type and mutant AAT are renatured in the same renaturation buffer.
  • the renaturation buffer comprises Tris as a buffer.
  • the renaturation buffer comprises glycerin, sucrose, or any combination thereof.
  • the renaturation buffer can comprise from about 5% to about 30% glycerol (v/v, the same below), from about 5% to about 40% sucrose, or about 10% glycerol and about 10% sucrose.
  • the renaturation buffer comprises PEG.
  • the molecular weight of the PEG is from about 200 to about 20,000 Daltons.
  • the molecular weight of the PEG is about 200 Daltons.
  • the molecular weight of the PEG is about 600 Daltons.
  • the renaturation buffer may further comprise a detergent such as Tween-20, Tween-80, sodium deoxycholate, sodium cholate, and trimethylamine oxide (TMSO).
  • the renaturation method comprises rapidly diluting, for example, diluting about 20 times the AAT polypeptide solution dissolved in the lysis buffer with a refolding buffer. In certain embodiments, the renaturation method comprises dialyzing a solution of the AAT polypeptide dissolved in the lysis buffer with a refolding buffer, for example, with about 20 volumes of renaturation buffer.
  • the refolding buffer is at a high pH, such as a pH of about 9 or a pH of about 10. In certain embodiments, the refolding buffer begins with a high pH and is adjusted to a neutral pH after renaturation, such as a pH of about 8 or a pH of about 7. In certain embodiments, the method further comprises, prior to diluting the solubilized AAT polypeptide with a refolding buffer, modulating A 280 of the solubilized AAT polypeptide original solution with a lysis buffer to between about 2.0 and about 10.0 (eg, about 2.0 to about 5.0).
  • a method of producing a renatured recombinant AAT polypeptide comprises: a) dissolving a denatured AAT polypeptide with a lysis buffer comprising about 8 M urea, about 0.1 M Tris, about 1 mM glycine. , about 1mM EDTA, about 100mM ⁇ - mercaptoethanol, about pH 10, the resulting solution was dissolved the AAT polypeptide; b) with wildtype or mutant AAT fibrinogen a body of a lysis buffer solution adjusted to about 280 dissolved 2.0.
  • the lysis buffer comprises about 8 M urea, about 0.1 M Tris, about 1 mM glycine, about 1 mM EDTA, about 10 mM ⁇ -mercaptoethanol, about 10 mM dithiothreitol (DTT), about 1 mM reduced glutathione (GSH). And its pH is about 10.
  • the renaturation buffer further comprises from about 0.005% to about 0.02% Tween-20.
  • a method of producing a renatured recombinant AAT wild-type and mutant polypeptide comprises: a) dissolving a denatured AAT polypeptide with a lysis buffer comprising about 8 M urea, about 0.1 M Tris, about 1 mM glycine, about 1 mM EDTA, about 10 mM ⁇ -mercaptoethanol, about 10 mM dithiothreitol (DTT), about 1 mM reduced glutathione (GSH), and its pH is about 9, resulting in The dissolved AAT polypeptide solution; b) the above-dissolved AAT polypeptide is rapidly diluted by adding the above-dissolved AAT polypeptide to about 20 volumes of a refolding buffer containing about 20 mM Tris and about 10%. Glycerol, pH about 9; and c) slowly lowering the pH of the diluted solubilized AAT polypeptide to about 7.6, thereby producing a renatured AAT polypeptide
  • a method of producing a renatured recombinant AAT wild-type and mutant polypeptide comprises: a) dissolving a denatured AAT polypeptide with a lysis buffer comprising about 8 M urea, about 0.1 M Tris, about 1 mM glycine, about 1 mM EDTA, about 10 mM ⁇ -mercaptoethanol, about 10 mM dithiothreitol (DTT), about 1 mM reduced glutathione (GSH), and its pH is about 8, resulting in The dissolved AAT polypeptide solution; b) the above-dissolved AAT polypeptide is rapidly diluted by adding the above-dissolved AAT polypeptide to about 20 volumes of a refolding buffer containing about 20 mM Tris and about 10%. Glycerol, pH about 8; and c) slowly lowering the pH of the diluted solubilized AAT polypeptide to about 7.6, thereby producing a renatured AAT polypeptide
  • a method of producing a renatured recombinant AAT wild-type and mutant polypeptide comprises: a) dissolving a denatured AAT polypeptide with a lysis buffer comprising about 8 M urea, about 0.1 M Tris, about 1 mM glycine, about 1 mM EDTA, about 10 mM ⁇ -mercaptoethanol, about 10 mM dithiothreitol (DTT), about 1 mM reduced glutathione (GSH), and its pH is about 7.6, thereby Producing a dissolved AAT polypeptide solution; b) rapidly diluting the above-dissolved AAT polypeptide by adding the above-dissolved AAT polypeptide to about 20 volumes of a refolding buffer comprising about 20 mM Tris and about 10 % glycerol, pH about 7.6, thereby producing a renatured AAT polypeptide.
  • a lysis buffer comprising about 8 M urea, about 0.1 M Tris, about 1
  • the method further comprises a method of concentrating the renatured AAT wild type and mutant polypeptide.
  • the renatured AAT polypeptide can be concentrated 10-200 fold using an ultrafiltration concentration method.
  • the invention also provides a method for purifying correctly reconstituted AAT wild-type and mutant polypeptides from incorrectly renatured or non-refolded AAT, the method comprising: a) incorrectly renaturation under the action of a salt
  • the non-refoldable AAT polypeptide binds to a hydrophobic interaction chromatography resin; and b) collects the correctly renatured AAT polypeptide that is not bound to the resin.
  • the salt is ammonium sulfate [(NH 4 ) 2 SO 4 ], sodium chloride (NaCl), or potassium chloride (KCl).
  • the concentration of ammonium sulfate used therein is from about 0.25 M to about 1.2 M.
  • the concentration of sodium chloride used therein is from about 1.0 M to about 3.5 M. In certain embodiments, the concentration of potassium chloride used therein is from about 1.0 M to about 3.5 M. In certain embodiments, a suitably renatured AAT polypeptide is derived from a bacterial inclusion body.
  • a method of producing a refolded recombinant AAT wild-type and mutant polypeptide comprises: a) dissolving a denatured AAT polypeptide with a lysis buffer comprising about 8 M urea, about 0.1 M. Tris, glycine about 1mM, about 1mM EDTA, about 100mM ⁇ - mercaptoethanol, about pH 9.0, thereby producing a solution dissolved AAT polypeptide; b) a is dissolved with a buffer adjusted AAT dissolved fibrinogen solution was about 2.0 to 280.
  • the lysis buffer comprises about 8 M urea, about 0.1 M Tris, about 1 mM glycine, about 1 mM EDTA, about 10 mM ⁇ -mercaptoethanol, about 10 mM dithiothreitol (DTT), about 1 mM reduced glutathione (GSH). And its pH is about 9.0.
  • the AAT purification step of the renatured wild-type and mutant comprises the first step of concentrating the renaturation solution by ultrafiltration followed by an SEC column (Superdex 200 or Sephacryl 300, GE). Healthcare) Separating renatured monomeric proteins from unrefolded or partially renatured AAT.
  • the second step further purifies the refolding protein using ion exchange or hydrophobic interaction column chromatography.
  • Figure 3A is a SDS-PAGE result of a purified AAT sample obtained by hydrophobic interaction chromatography. It is shown that most of the protein is in monomeric form and has a small amount of dimeric form in the absence of a reducing agent when detected by SDS-PAGE.
  • Non-reducing SDS-PAGE has been routinely used to distinguish between folded and unfolded proteins.
  • human AAT glycosylation from Aventis Behring LLC
  • PPE protein pancreatic elastase
  • AAT protein has a unique cysteine at position 232 ( Figure 7). This site (Cys232) or N-terminal site can be used for chemical modification of the localization of AAT, and experiments have shown that the modification does not affect AAT activity.
  • the pegylation modification is performed at the Cys232 site.
  • Purified rAAT can be PEGylated at the unique Cys232 position with reference to published methods [37]. The efficiency of PEGylation was in the range of 50-65% in several experiments. Molecular models indicate that this unique cysteine moiety is exposed to aqueous solvents and is not in the vicinity of the AAT domain that interacts with elastase (see Figure 7). After PEGylation, unreacted maleimide-PEG ( ⁇ 20K Da, Nektar Therapeutics) and unpolymerized B were separated from PEGylated AAT by anion exchange chromatography (Q-HiTrap, GE Healthcare). Glycolized AAT.
  • Figure 4 shows the results of SDS-PAGE and MALDI-TOF mass spectrometry of PEGylated rAAT polypeptide and its normal function in blocking PPE.
  • This experiment shows that PEGylated AAT can be conveniently separated from unpegylated AAT and free unreacted mPEG20 by salt gradient elution.
  • the success of the PEGylation reaction has been confirmed by SDS-PAGE and MALDI-TOF mass spectrometry.
  • the molecular weight of the AAT polypeptide (non-PEGylated) is 43996.34 Daltons
  • the molecular weight of the PEGylation reagent Mal-PEG 20 is 22063.92 Daltons.
  • the molecular weight of the successfully pegylated rAAT is 65324.02 Daltons, which closely matches the predicted molecular weight. This indicates that rAAT has been successfully PEGylated.
  • the "free" rAAT and Mal-PEG 20 mass may be produced during the ionization decomposition of the mass spectrometry process.
  • the Cys232 site can be fatty acid modified, one of which is a palmitoylation modification.
  • Palmitic acid is a cetane fatty acid, also known as palmitic acid.
  • palmitic acid has a strong binding ability to serum albumin.
  • palmitic acid-modified protein drugs can bind to serum albumin in the blood, greatly extending the half-life in vivo. Palmitic acid modified protein or peptide drugs have been successfully applied in the clinic, producing good results. Novo Nordisk's treatment of diabetes and obesity, Liraglutide, is a successful example.
  • the present invention provides palmitic acid modified wild-type and mutant AAT proteins at the Cys232 site.
  • the following formula is a modification in which glutamate is used as a link "bridge" to attach palmitic acid to wild-type or mutant AAT-Cys232.
  • thermostable F51L single mutant was a thermostable F51L single mutant
  • the second was an anti-oxidant M351V/M358V double mutant
  • the third was a thermostable and antioxidant F51L/M351V/M358V triple mutant.
  • F51L/M351V/M358V triple mutant was compared with wild-type and mutant proteins (mutants).
  • mutants we used a fluorescence-based thermal denaturation assay, as shown in Figure 5.
  • the SYPRO Orange dye is bonded to the hydrophobic surface. As the protein denatures with increasing temperature, its hydrophobic surface is exposed and bound to the dye, resulting in increased fluorescence. A further increase in temperature separates the dye and protein to produce a denaturation peak.
  • Figure 5 shows that the wild-type and M351V/M358V mutant proteins were heat-denatured at around 48 °C, while the denaturation temperatures of the F51L, F51L and F51L/M351V/M358V mutant proteins were all increased to about 54 °C. The results showed that both the single mutant and the triple mutant containing F51L greatly improved the thermal stability of AAT as designed and expected.
  • the invention also provides a composition (including a pharmaceutical composition) comprising a biologically active AAT polypeptide.
  • This composition may also contain a pharmaceutically acceptable excipient.
  • the AAT polypeptide can be in the form of a lyophilized formulation or a liquid formulation.
  • the pharmaceutically acceptable excipient is non-toxic to the user at the dosage and concentration used, and may contain buffers such as phosphates, citrates; salts such as sodium chloride; sugars such as sucrose; and/or polyethylene glycol. (PEG). See Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover.
  • AAT polypeptide preparations can be prepared for different routes of administration, such as intravenous or intravenous (IV) liquid or lyophilized preparations. And a dry powder preparation or a gasification preparation for deep lung administration.
  • IV intravenous
  • a dry powder preparation or a gasification preparation for deep lung administration will be apparent to those skilled in the art, see, for example, Drug Delivery to the Lung, Bisgaard H., O'Callaghan C and Smaldone GC, editors, New York; Marcel Dekker, 2002.
  • the AAT polypeptides of the invention can be produced by any of the methods described herein.
  • the AAT polypeptide is produced from a bacterial (eg, E. coli) inclusion body.
  • the AAT polypeptide is non-glycosylated.
  • the AAT polypeptide has a purity of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
  • the specific activity of the AAT polypeptide is not less than about 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75 per milligram of total protein, 0.8, 0.85, 0.9 or 0.95 mg of active AAT polypeptide.
  • the AAT polypeptide is a wild-type AAT protein.
  • the AAT polypeptide is a mutant AAT protein.
  • the AAT polypeptide is a high stability, antioxidant, triple mutant AAT mutant as described herein.
  • the AAT polypeptide is a chemically modified AAT protein as described above.
  • the invention also provides a therapeutic use kit comprising the AAT polypeptide.
  • the kit of the invention comprises one or more containers containing AAT polypeptides.
  • This container can be a small vial, bottle, jar, or flexible package.
  • AAT polypeptides can be packaged in disposable vials containing 500 mg or 1,000 mg of active AAT polypeptide per vial.
  • the vial can have a sterile access port (eg a stopper that can be pierced by a hypodermic needle).
  • packages that are combined with special devices, such as inhalers, nasal delivery devices (e.g., nebulizers), or input devices such as micropumps.
  • At least one active agent is an AAT polypeptide.
  • the kit may also further comprise an active ingredient of the second medicament.
  • Application instructions for the method according to the invention may also be included in the packaging container. Generally, these instructions include instructions for the use of AAT polypeptides to treat diseases in accordance with the methods described herein. This specification may further include instructions for treating a disease using an AAT polypeptide, for example, treating a disease associated with AAT deficiency.
  • the instructions generally include the dosage, time of use, and route of application for treating the disease.
  • the instructions provided by the kit of the present invention are generally written on the label or on the label (for example, on the paper contained in the kit), but the machine-readable instructions (such as instructions loaded on the magnetic sheet or on the disc) are also acceptable. .
  • the kit may also include a device for pulmonary administration of a dry powder or nebulizer.
  • Example 1 Plasmid construction and expression.
  • a DNA fragment encoding the ⁇ 5-AAT polypeptide (Fig. 1) was obtained by PCR amplification.
  • the ⁇ 5-AAT polypeptide lacks the 1-5 amino acid sequence shown in Figure 1 and artificially methionine is added at the starting position to facilitate expression in E. coli.
  • the poly(poly)nucleotide sequence encoding the ⁇ 5-AAT polypeptide in the DNA fragment has been optimized for optimal expression in E. coli.
  • the above PCR product was cloned into the pET11a plasmid.
  • the resulting vector was pET11- ⁇ 5-AAT.
  • Example 2 Expression of wild-type and F51L mutant proteins.
  • the E. coli expression clone was expanded and then inoculated into 1.0 L of LB medium containing 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 50 mg of ampicillin.
  • OD 600 0.6
  • IPTG was added to 0.5 mM at 37 ° C. Express for 3 hours.
  • Example 3 Expression of M351V/M358V and F51L/M351V/M358V muteins.
  • Example 4 Purification of inclusion bodies. Collect cells by centrifugation and then contain 1% The suspension was suspended in a buffer of 20 ml TN (150 mM NaCl, 50 mM Tris, pH 8.0). 10 mg of lysozyme was added thereto, and the cells were suspended at -20 ° C overnight. The lysate was then dissolved and 20 ⁇ l of 1 M magnesium sulfate and 100 ⁇ l of 0.01 mg/ml DNAase were added. The cells are agitated and incubated until the released DNA is completely dissolved. Then use 250ml with 1% The lysate was diluted in TN and stirred for 2-4 hours.
  • 20 ml TN 150 mM NaCl, 50 mM Tris, pH 8.0
  • 10 mg of lysozyme was added thereto, and the cells were suspended at -20 ° C overnight.
  • the lysate was then dissolved and 20 ⁇ l of 1 M magnesium sulfate and 100
  • the inclusion bodies were collected by centrifugation, and the inclusion bodies were purified by washing 5 times with TN buffer (100 mM Tris, 250 mM NaCl, pH 8.0) containing 1% Triton X-100.
  • Example 5 Refolding.
  • the above-mentioned dissolved inclusion bodies were rapidly diluted to a volume of 20 volumes of a buffer containing 20 mM Tris, 10% glycerol, pH 9, and the final OD280 after dilution was 0.1.
  • the pH was then slowly adjusted to pH 8.0. After dilution, the pH of the solution was adjusted to 7.6 with 1-4 days in 1 M HCl.
  • renaturation methods included high concentrations of glycerol (20%) in renaturation buffer, or glycerol replacement with 20% sucrose, or 10% sucrose and 10% glycerol.
  • Tween-20 (0.005%-0.01%) was also included in the refolding buffer. All of these conditions result in a correctly renatured (active) AAT polypeptide.
  • the wild type and mutant AAT polypeptide inclusion bodies expressed can also be successfully renatured by a fixed pH method.
  • the washed inclusion bodies were lysed in a lysis buffer containing high concentration of urea (8 M urea, 0.1 M Tris, 1 mM glycine, 1 mM EDTA, 100 mM ⁇ -mercaptoethanol ( ⁇ -ME), pH 10.5), dissolved in a high OD 280 (20 -40) and slowly agitate for 12 hours at 4 °C.
  • the dissolved sample was ultracentrifuged (30 minutes x 66,000 g) to clarify to remove insoluble impurities.
  • OD 280 of the dissolved inclusion bodies was adjusted to 2.0.
  • the above-mentioned dissolved inclusion bodies were rapidly diluted to a volume of 20 volumes of a buffer containing 20 mM Tris, 10% glycerol, pH 8.5, and the final OD280 after dilution was 0.1.
  • the diluted solution was stored at 20 ° C for 16 hours, followed by ultrafiltration concentration and buffer exchange.
  • Example 6 Purification.
  • the refolded AAT was concentrated to A280 > 20.0 using a tangential flow ultrafiltration system and loaded into a buffer pre-equilibrated with a buffer containing 20 mM Tris, 0.15 M NaCl, 0.4 M urea, 1 mM DTT, 10% glycerol, pH 7.6.
  • Superdex 200 column The active peak fractions were collected and dialyzed against a buffer containing 20 mM Tris, 5% glycerol, 3 M NaCl, 0.001% Tween, 20, 1 mM DTT, pH 7.6.
  • the dialyzed protein was loaded into a phenyl sepharose (hydrophobic) column equilibrated with dialysis buffer.
  • the effluent containing the purified product of interest was collected, concentrated and dialyzed (NaCl) with a buffer containing 20 mM Tris, 5% glycerol, 0.001% Tween 20, 1 mM DTT, pH 7.6.
  • Example 7 PEGylation.
  • the highly purified AAT was passed through a PD-10 (BioRad) column pre-equilibrated with 50 mM sodium phosphate pH 7.5, 200 mM NaCl to remove the DTT and adjust the pH to 7.5 according to product requirements. Since the reducing agent DTT interferes with the PEGylation reaction, a buffer exchange process is usually performed twice to ensure that no trace amounts of DTT are present. The AAT after buffer exchange by molar extinction.
  • Solid PEG-mal20 polyethylene glycol maleimide 20, Nektar, Huntsville, AL
  • AAT solution in a molar ratio of 5:1 to 10:1
  • the reaction was stopped by the addition of 20 mM DTT and incubated for an additional 5 minutes at 37 °C.
  • the pegylated AAT (Peg-AAT) was then dialyzed into 20 mM Tris 8.0, 50 mM NaCl, 1 mM DTT to remove excess salt, then loaded onto a 5 mL Q XL HiTrap column and eluted with a gradient of 0-1000 mM NaCl.
  • Example 8 Enzyme activity assay.
  • the AAT biological activity of the refolded rAAT wild type and mutant was determined by measuring the inhibitory activity against HLE or PPE in vitro using the substrate color reaction method.
  • PPE isolated from pig pancreas was purchased from Sigma-Aldrich (St. Louis, MO, Cat. #E7885); HLE isolated from human sputum was purchased from Molecular Innovations (Southfield, MI Cat# HNE).
  • AAT concentrations ranged from 0.3 nM to 14 nM, incubated with 1.4 nM fixed concentrations of HLE or PPE for 15 minutes at 37 ° C, then aliquots of the incubation with 1 mM elastase substrate N-succinyl-ala ( PPE chromogenic substrate, Sigma) or N-methoxysuccinyl- ⁇ -alanyl- ⁇ -alanyl-p-nitroaniline (HLE chromogenic substrate, Sigma) was mixed. The hydrolysis kinetics of the substrate was determined at 21 ° C, 405 nm using a Molecular Devices spectrophotometer (Spectramax Plus).
  • the initial rate of each reaction was determined and the percent activity relative to the control (no AAT or AAT polypeptide) was calculated.
  • the percent elastase activity is plotted against the stoichiometric molar ratio of the AAT polypeptide/elastase concentration used in the corresponding reaction.
  • concentration of each form of AAT polypeptide, PPE and HLE stock used in the experiment was determined in advance by a known extinction coefficient using the known extinction coefficient from the Swiss Bioinformatics Institute's ExPASY proteomics server computer. Software program ProtParam (http://www.expasy.ch).
  • the kinetics of the elastase cleavage of the substrate was monitored at 405 nm at 21 °C. The speed was compared to the control (elastase only) and plotted as a stoichiometric ratio of % control elastase activity (y-axis) to AAT:PPE (x-axis).
  • the PPE concentration used in the measurement was obtained by measuring the extinction coefficient of pure PPE in 6 M ⁇ , 50 mM NaPi, pH 6.5 according to the ProtParam algorithm (www.expasy.ch) of the Swiss Institute of Bioinformatics.
  • the concentration of AAT was determined by first using the "almost irreversible" fluorescent substrate MUGB (4-methylumbelliferyl-4-mercaptobenzoate hydrochloride) from Novagen (www.novagen.com). , Fluka) Accurately titrate the concentration of the trypsin active site in the trypsin (Sigma) stock solution. Then using the chromogenic substrate BAPNA (N-benzoyl L-arg-4 nitroaniline hydrochloride, Sigma), at 21 ° C and 405 nm, in the stoichiometric analysis of any of the AAT stock solution in the blocking pancreas The concentration of action of the protease functional site.
  • MUGB 1-methylumbelliferyl-4-mercaptobenzoate hydrochloride
  • thermostability assay was performed using a 96-well culture plate.
  • the reaction volume was 110 ⁇ l, and the buffer contained: 1 ⁇ PBS buffer, 10% (v/v) glycerol, 10% DMSO, 5 mM DTT, 50 ⁇ SYPRO Orange, and purified AAT or its mutant each 15 ⁇ M.
  • the reaction plates were incubated at 25 ° C for 30 min and then warmed to 70 ° C at 0.5 ° C intervals. Ex 490 mM for each temperature and 200 mS of fluorescence at Em 580 mM were measured. The fluorescence was plotted against temperature.
  • Example 10 Oxidation resistance. To test the antioxidant activity of AAT and its mutants, 50 ⁇ M of each purified AAT or mutant was incubated in PBS buffer containing 0 mM, 2 mM, 10 mM, 50 mM, 100 mM, 200 mM H 2 O 2 at 25 ° C, respectively. For 15 minutes, an equal amount of DTT was then added to reduce excess H 2 O 2 . The antioxidant properties of the treated AAT and mutants were determined by measuring the inhibitory activity against PPE.
  • thermostable mutation located at the hydrophobic core of alpha 1-antitrypsin suppresses the folding defect of the Z-type variant, J Biol Chem. , 8597-601.
  • Unglycosylated rat alpha 1 proteinase inhibitor has a six-fold shorter plasma half-life than the mature glycoprotein, BBRC.126,630 -635.

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Abstract

提供了一种α1-抗胰蛋白酶突变体，其是具有活性的F51L/M351V/M358V三突变体和/或其化学修饰体。还提供了制备所述α1-抗胰蛋白酶突变体的方法、包含所述α1-抗胰蛋白酶突变体的药物组合物或药剂盒、以及所述α1-抗胰蛋白酶突变体在制备治疗肺部疾病的药物中的应用。

Description

重组突变体α1-抗胰蛋白酶及其制备和应用 技术领域
本发明涉及到生产重组突变体α1-抗胰蛋白酶(AAT)多肽方法及在医疗领域的应用。
背景技术
早在1963年人们就发现遗传性肺气肿与α1-抗胰蛋白酶(AAT)有关[1]。20世纪70年代，研究发现了AAT的功能以及由遗传缺陷引起的肺部疾病的科学基础[2,3]。在生理上，嗜中性粒细胞是肺部侵入微生物病原体免疫应答的重要介质[4]，它可以畅通无阻地通过肺毛细血管。嗜中性粒细胞作为炎症反应的一部分，会释放大量的防御性分子，包括活性分子氧、阳离子肽、类花生酸和蛋白水解酶[5]。这些杀灭病原体的分子是人体防御的重要组成部分，然而这些防御性分子的不受约束的作用会导致严重的肺损伤。
人类白细胞弹性蛋白酶(HLE)作为正常炎症反应的一部分，是从嗜中性粒细胞的嗜苯胺蓝颗粒(azurophilic granules)释放的丝氨酸蛋白酶[6]。在正常稳态条件下，AAT作为HLE蛋白水解反应的重要抑制剂而防止肺泡基质的损伤。AAT是一种主要在肝脏中合成的52KD糖蛋白，但也在中性粒细胞、单核细胞和巨噬细胞中合成。AAT可被分泌到血浆中，但其主要作用部位是在肺实质[7]。除了HLE，AAT还抑制中性粒细胞释放到肺部的两种其他蛋白酶，即组织蛋白酶G(CatG)和蛋白酶3(Pr3)。CatG和Pr3也可以通过分解弹性蛋白和其他细胞外基质蛋白而导致肺损伤。AAT可以防止这种损害。不过HLE被认为是造成肺损伤的主要酶[8]。AAT的正常生物学功能对人体健康至关重要，因此被称为脉管组织的卫士[9]。
据估计，全世界具有AAT无效突变(PiZZ)的人群有253,404人[10]：欧洲119,594，美洲和加勒比地区91,490，非洲3824，亚洲32,154，澳大利亚4,126，新西兰2,216。PiZZ携带者患有遗传性肺气肿，可用AAT增强疗法治疗[10]。根据世界卫生组织的统计，在全球范围内，2015年慢性阻塞性肺病(COPD)造成约300万人死亡，在许多情况下COPD可以接受AAT治疗。然而市场上现有的从人血清中天然纯化的AAT药物
Figure PCTCN2018105011-appb-000001
Figure PCTCN2018105011-appb-000002
只能满足不到10％的AAT遗传缺陷人群的治疗[11]。由于AAT供应有限，尚未对其在其他呼吸系统疾病(包括吸烟导致的肺气肿、囊性纤维化、肺动脉高压、肺纤维化和COPD)中的有益作用进行充分测试[12-14]。全世界估计至少有1.16亿的AAT基因突变携带者(PiMS和PiMZ：M，正常遗传Pi类型；S，低于正常；Z，低于S)和340万个缺失等位基因组合(PiSS，PiSZ，和PiZZ)[11,15]。这类人群对吸烟和环境污染等因素更敏 感，更容易罹患COPD类疾病。
为了制备重组AAT，迄今已有许多利用不同表达系统来表达AAT的工作，包括细菌[16,17]、酵母[18]、植物培养[19-21]和转基因羊[22-25]。由于治疗遗传性肺气肿治疗需要大剂量(4-6g/周·患者)静脉注射[26]，或者每天高达250mg的雾化肺部给药[27])，所以生产AAT的基本要求是可扩大规模——每年需要百万公斤级的药用级别的AAT药物，以及成本控制——使病人负担得起。大肠杆菌表达法是生产重组蛋白质药物的最具成本效益的方法之一。然而，AAT在细菌中过度表达时倾向于形成不溶性包涵体，从而限制了可溶性表达的可伸缩性。
发明内容
天然α1-抗胰蛋白酶(AAT)是一个52kDa的糖蛋白，具有抗蛋白酶的作用，是嗜中性粒细胞弹性蛋白酶、组织蛋白酶G和蛋白酶3等嗜中性粒细胞丝氨酸蛋白酶的生理抑制剂。AAT的主要功能是保护肺部在炎症发生时不受由蛋白酶造成的损伤。AAT的遗传或获得性缺陷会导致遗传性肺气肿、COPD等严重疾病。由其生物功能所决定，天然AAT较不稳定并很容易被氧化。但为了使AAT能够作为药物用于临床治疗，则需要增加其稳定、抗氧化性和体内半衰期。
在本发明中，利用我们开发的高效包涵体复性技术[28]，我们从包涵体中复性并纯化了重组AAT。更进一步，为了克服AAT易于氧化和大肠杆菌表达非糖基化蛋白的稳定性问题，我们设计了抗氧化和更稳定的AAT突变体用以满足临床治疗要求。在此发明中，我们设计、筛选了一系列AAT突变体，并成功获得了一个新型的适合药物研发的抗氧化和更稳定的三突变体AAT。为了更加延长AAT蛋白药在体内的半衰期，在本发明中，我们还根据AAT的结构，设计、制备和纯化了在特定位点的化学修饰的AAT。本发明所得到的全新的，化学修饰的突变体使AAT蛋白药的大规模应用成为可能。
我们已经在大肠杆菌中表达了包涵体形式的α1-抗胰蛋白酶并建立了高效的复性及纯化方法。我们设计了一系列α1-抗胰蛋白酶的突变体，使其在热稳定性和抗氧化性方面得到很大的改善。此外，我们通过半胱氨酸-聚乙二醇化合成了活性α1-抗胰蛋白酶，显著延长了它的体内半衰期。该突变体或化学修饰的突变体有望成为有效的AAT新药，用于治疗遗传性肺气肿以及其他形式的肺部疾病，例如吸烟肺、囊性纤维化、肺动脉高压、肺纤维化、慢性阻塞性肺病等。
本发明的目标是通过建立一个有效且具有成本效益的重组AAT表达系统，同时合理设计 突变体使其在热稳定性和抗氧化性两方面都得到提高，并通过化学修饰改善其体内半衰期，最终实现将重组AAT用于临床治疗。我们建立的大肠杆菌表达及复性系统可以实现高产量、高纯度和低成本。尽管如此，重组野生型蛋白质的医学应用还有很大的障碍。首要问题是AAT易于氧化，在生理条件下不稳定[31-33]。本发明则试图通过构建和选择更稳定和抗氧化的突变体来解决这个问题，使其更适合于药物研发。另外，大肠杆菌表达的AAT是没有被糖基化的。而未糖基化的AAT体内半衰期比天然糖基化的AAT要短很多。为此本发明设计了大肠杆菌重组复性AAT的化学修饰，包括聚乙二醇化修饰和脂肪酸化修饰(例如棕榈酸化修饰)，以提高蛋白的稳定性和体内半衰期。聚乙二醇化修饰原理：阻碍免疫系统对蛋白的识别，阻碍蛋白酶对治疗蛋白药物的降解；棕榈酸化修饰原理：修饰后的多肽或蛋白进入体内后，因棕榈酸与血清中人血清蛋白的结合而延长了所修饰蛋白的体内半衰期。
为了得到治疗性更好的重组AAT蛋白药，我们基于AAT的晶体结构设计了多个突变体。其中的一个是F51L突变蛋白，以期获得更稳定的rAAT。如图7所示，野生型AAT的第51位苯丙氨酸残基位于分子的疏水核心中，远离活性位点。有文献报道Kwon及其合作者[32]经过一轮非特异性的化学诱变和选择，发现这个位置上的脂肪酸氨基酸取代能显着提高AAT的热稳定性且不会使其失活、聚集或与弹性蛋白酶缔合动力学发生改变。大肠杆菌表达的非糖基化AAT突变体在57℃下显示可降低AAT的热失活10倍以上，这使得在热稳定性方面，突变体的表现已类似于血浆中的糖基化AAT。在本发明中，我们通过晶体结构模拟，用亮氨酸取代了这个苯丙氨酸残基(F51L)(图7)。结果表明，这种替换使AAT的热稳定性显着增加(图5)。值得注意的是，已有证明AAT的热稳定性与蛋白质的生物周转率相关[33]。因此，热稳定性好的AAT对于药物研发来说是更理想的。
本发明所设计的第二个突变体是针对抗氧化性的。众所周知，AAT由于其体内功能调节要求而对氧化非常敏感[31,34]。已知吸入香烟烟雾中的成分，如过氧化氢等，均会使AAT发生氧化，有推论认为吸烟者肺中活性AAT的减少是吸烟者肺病的病理生理学原因。最敏感的残基是Met351和Met358[31,34]，它们恰好是AAT结合位点的P8和P1位置(图7)。当这些位点用其他脂肪族氨基酸，例如缬氨酸[31]进行保存性置换时，将产生明显抗过氧化氢氧化的分子，并且对AAT的缔合动力学或与靶嗜中性粒细胞弹性蛋白酶的结合并无明显影响[31,33]。我们的研究结果也显示双突变体M351V/M358V具有明显提高的抗氧化性(图6)。
我们的目标是构建一种全新的组合突变体F51L/M351V/M358V，以期同时实现稳定性和抗氧化性的提高。本领域技术人员应该清楚，蛋白质的任何一个氨基酸变化都可能导致蛋 白的不稳定和性质的变化，所以任何新的突变体，特别是多突变体能否表达，其表达水平、稳定性、纯化条件等都不是可以预期的。此外，从上述涉及AAT单突变体和双突变体的早期研究论文我们可以看出，因受表达纯化技术水平所限，它们都是停留在纯理论研究的阶段。我们的研究发现，无论是单突变体、双突变体还是三突变体，其全长AAT的表达量都很低，所得到的表达量可以用来做研究，但达不到新药研发所需要的应用水平。
经过大量的条件摸索和创新性探索，我们成功寻找到合适的高包涵体表达及高效率复性条件，得到了一个完全活性的三突变体，并且已通过实验验证了它的热稳定性和抗氧化性(图5、6)。在本发明的表达条件和复性方法下，不论是天然蛋白还是突变体(我们发现突变体与天然蛋白的表达和复性条件有所不同)，都达到了前所未有的表达水平。从应用方面来说，这是一个量变到质变的过程，从纯理论研究到可以研发制备重组新药的过程。
大肠杆菌表达的未糖基化rAAT的体内半衰期比天然糖基化的AAT短得多。例如，在大鼠血清中测得糖化大鼠AAT的血浆半衰期为170分钟，而该分子的非糖基化形式仅为30分钟[35]。聚乙二醇结合(聚乙二醇化)是延长体内半衰期和减少蛋白质免疫反应的最有效方法之一[36]。如图4所示，我们已经根据文献的方法[37,38]成功地将野生型rAAT的Cys232聚乙二醇化。Cys232是AAT中唯一的半胱氨酸，并以单体分子存在(图7)，因此可以被聚乙二醇化。聚乙二醇化的AAT表现出与野生型AAT在体外抑制猪胰蛋白酶(PPE)(图4)方面具有相似的结合率。
综上所述，我们在大肠杆菌中表达了包涵体形式的高产率重组AAT，对包涵体进行了纯化和复性，建立了药物研发的高效复性及纯化技术。另外，为提高热稳定性和抗氧化性，以及利用聚乙二醇化作用而进一步延长体内半衰期，我们制备了一种全新的AAT三突变体形式。由此获得的新的候选药物产物将具有比人血清天然AAT更好的医疗应用性能。
需要说明的是，本发明所引用的参考文件代表了所能检索到的所有文献；本发明书所描述的AAT多肽和AAT蛋白具有等同的含义。在某些描述中，AAT同时代表野生型AAT和本发明的突变体AAT。
本发明提供了一种新型的α1-抗胰蛋白酶突变体，及生产和纯化这种新型重组突变体的方法。新型突变体是通过以蛋白质结构为依据的蛋白工程改造，设计出更加适合于医药应用的，结构上更稳定，并且能够抗氧化的候选蛋白药。本发明亦提供针对这种新的突变体的大肠杆菌表达，包涵体复性和纯化的方法。进一步，本发明亦提供对所纯化的候选药进行化学修饰的方法，以延长蛋白药在体内的半衰期，达到更佳的药效。
附图说明
图1.成熟AAT蛋白序列和突变体的氨基酸突变位点。显示了△5AAT的起始位置，并且在大肠杆菌表达载体中插入了另外的起始Met。下划线表示氨基酸变化位点。文中突变数字基于成熟的全长AAT[39]。三种突变体的核苷酸变化为：F51L，TTT→CTG；M351V/M358V，ATG→GTG/ATG→GTG；和F51L/M351V/M358V，TTT→CTG/ATG→GTG/ATG→GTG。
图2.表达的野生型、F51L、M351V/M358V和F51L/M351V/M358V突变蛋白的SDS/PAGE。蛋白质用考马斯蓝染色显现。小规模表达实验。泳道1-3：纯化后的野生型AAT包涵体，上样量分别为1、3、5μl；泳道4：BSA标记；泳道5：MW标准；泳道6-8：纯化后的F51L突变蛋白包涵体，上样量分别为5μl、3μl、1μl；泳道9-19：可溶性和不溶性细胞提取物的表达鉴定；泳道9、11：野生型，可溶性提取物；泳道10、12：野生型，不溶性提取物；泳道13、15：M351V/M358V，可溶性提取物；泳道14、16：M351V/M358V，不溶性提取物；第17道：MW标准；泳道18：F51L/M351V/M358V，可溶性提取物；泳道19：F51L/M351V/M358V，不溶性提取物。
图3.纯化的重组AAT和突变蛋白的SDS-PAGE(A)及对PPE抑制活性的化学测定(B，C)。其中，A.纯化样品的SDS-PAGE结果，考马斯蓝染色；泳道1：野生型AAT；泳道2-4：增加上样量的纯度检测(分别为1、2、4、6μg)；第2道：F51L；3道：M351V/M358V；泳道4：F51L/M351V/M358V；分子量标记(MW)。B.重组AAT(野生型)与作为对照的市售天然AAT药物
Figure PCTCN2018105011-appb-000003
的活性比较结果。C.纯化的突变体的活性测定结果。
图4.Cys232的聚乙二醇化、纯化及性质。其中：A.聚乙二醇化的AAT的阳离子交换色谱图。B.经过Q XL柱的馏分的非还原性SDS-PAGE，数字对应于A中所示的梯度洗脱馏分的管号。C.聚乙二醇化反应后样品的MALDI-TOF质谱分析，每个指示的峰的分子量描绘在相应峰的顶部。D.纯化的聚乙二醇化的AAT显示在阻断PPE中具有正常的抑制活性。
图5.野生型和突变型AAT的热稳定性的比较。该图显示了荧光计数(Y轴)与温度(X轴，℃)的关系。
图6.抗氧化性测定。使用SpectraMax 250酶标仪(Molecular Devices)，37℃，10秒间隔，监测405nm处的氨基分解活性的产生，检测过程为20分钟。使用GraFit版本7(Erithacus Software)测定每个H 2O 2处理的AAT或其突变体的IC50(Y轴)。X轴显示H 2O 2和AAT的摩尔比(H 2O 2：AAT，从4∶1到400∶1)。
图7.来自Lomas及其合作者的AAT的三维结构，显示抗氧化突变的位点(分别为活性 位点的Met351和Met358，P8和P1)、埋在分子疏水核心深处的保守性突变(Phe51)和Cys聚乙二醇化(Cys232，暴露在表面但不妨碍活性)。以已解析的晶体结构PDB坐标1QLP为基础，利用COOT软件模拟出该结构模型。整个蛋白结构以cartoon形式显示，野生型及突变体残基以stick形式显示。图片由PyMOL软件生成。
具体实施方式
本发明提供表达和纯化所述AAT突变体的方法。虽然本发明所描述的是用大肠杆菌表达宿主，但这不限制本发明的表达宿主范围。在某些实施方面，任何能够达到重组蛋白高表达的宿主都可以用于表达所述的突变体蛋白。此类宿主包括哺乳动物和细胞表达宿主，植物和植物细胞表达宿主，昆虫表达宿主，真菌表达宿主，和细菌表达宿主。在另一方面，任何可以在此类宿主中表达蛋白的载体都可以用于所述蛋白的表达。
在某些实施例中，大肠杆菌可用于重组蛋白的表达宿主。在某些实施例中，所用的表达宿主可以是BL21(DE3)。
在某些实施例中，可以应用pET-11(Novagen)作为大肠杆菌的表达载体。首先，在载体中表达了全长野生型AAT(图1)。结果发现表达量非常低。参考已发表的文献[16]我们做了N-末端截取，表达了△5AAT(截掉了成熟AAT蛋白的前5个氨基酸)和△10AAT(截掉了成熟AAT的前10个氨基酸)，两种都有很好的表达。最终我们选择了表达水平和复性率都更好的△5AAT。之后我们根据已发表的AAT蛋白晶体结构设计了多个突变体(如图7所示)，以期设计出更好的药物候选物，达到设计更稳定和抗氧化的突变体的目的。从早期的筛选工作中，我们最终选择了三个突变体进行表达。第一种是稳定突变体F51L[29,30]，第二种是设计用来减少氧化作用而失活的双突变体M351V/M358V[29]，第三种是兼具稳定性和抗氧化性的组合突变体(F51L/M351V/M358V)。所有的突变体均使用标准PCR突变技术构建，并通过测序验证。图1显示了△5AAT蛋白质序列的起始位置以及特定的氨基酸取代位点。
在某些实施例中，测试了许多条件下的大肠杆菌表达，最终野生型和选定的突变体AAT在大肠杆菌表达宿主中表达良好(图2)，结果显示当使用适当的生长培养基和条件时，所有表达构建体都可以高产率表达，大部分以不溶性包涵体形式存在。大肠杆菌表达和包涵体纯化的方法已经发表(X.Lin,Umetsu,T.,The high ph and ph-shift refolding technology,Current Pharmaceutical Technology 11(2010),no.3,293-299.)。在某些实施例中，纯化的包涵体溶解于高浓度的溶解缓冲液中，例如高浓度的尿素缓冲液中，例如可为约8M的尿素溶解缓冲液。在某些实施例中，纯化的包涵体溶解于高浓度的盐酸胍缓冲液中，例如可为约6M的盐酸胍 溶解缓冲液。
在某些实施例中，溶解于尿素或盐酸胍缓冲液中的AAT包涵体可进一步纯化，例如用色谱法纯化。包涵体的纯化技术是本领域技术人员所熟知的。
纯化后的在溶解缓冲液中包涵体可在多种不同pH、不同成分的复性缓冲液中复性。在某些实施例中，为达到最佳复性效果，野生型和突变体AAT在不同的复性缓冲液中复性。在某些实施例中，野生型和突变体AAT在相同的复性缓冲液中复性。
在某些实施例中，复性缓冲液中包含Tris作为缓冲液。在某些实施例中，复性缓冲液包含甘油、蔗糖或其任何组合。例如，复性缓冲液可以包含约5％至约30％的甘油(v/v，下同)，约5％至约40％的蔗糖，或约10％的甘油和约10％的蔗糖。在某些实施例中，复性缓冲液包含PEG。在某些实施例中，PEG的分子量是约200至约20,000道尔顿。在某些实施例中，PEG的分子量是约200道尔顿。在某些实施例中，PEG的分子量是约600道尔顿。在某些实施例中，复性缓冲液可以进一步包含去污剂，例如吐温-20、吐温-80、脱氧胆酸钠、胆酸钠和氧化三甲胺(TMSO)。
在某些实施例中，复性方法包括利用复性缓冲液将溶解于溶解缓冲液中的AAT多肽溶液快速稀释，例如稀释约20倍。在某些实施例中，复性方法包括利用复性缓冲液将溶解于溶解缓冲液中的AAT多肽溶液透析，例如用约20倍体积的复性缓冲液进行透析。
在某些实施例中，复性缓冲液是高pH，例如pH约9或pH约10。在某些实施例中，复性缓冲液开始是高pH，在复性后调节为中性的pH，例如pH约8或pH约7。在某些实施例中，此方法进一步包含在用复性缓冲液稀释溶解的AAT多肽之前，用溶解缓冲液调解溶解的AAT多肽原溶液的A 280到约2.0至约10.0(例如约2.0至约5.0)。
在一个示例性的实施例中，生产复性的重组AAT多肽的方法包括：a)用溶解缓冲液溶解变性的AAT多肽，所述溶解缓冲液包含约8M尿素，约0.1M Tris，约1mM甘氨酸，约1mM EDTA，约100mMβ-巯基乙醇，约pH 10，由此产生溶解的AAT多肽溶液；b)用一个溶解缓冲液调节溶解的AAT野生型或突变体蛋白原溶液的A 280到约2.0。此溶解缓冲液包含约8M尿素，约0.1M Tris，约1mM甘氨酸，约1mM EDTA，约10mMβ-巯基乙醇,约10mM二硫苏糖醇(DTT)，约1mM还原型谷胱甘肽(GSH)，并且其pH约为10。c)通过将上述溶解的AAT多肽加至约20倍体积的复性缓冲液中的方法将上述溶解的AAT多肽快速稀释，此复性缓冲液包含约20mM Tris，pH约10及下述1)～5)中的任何一种：1)约5％至约30％甘油，2)约5％至约40％蔗糖，3)约20％甘油和约20％蔗糖，4)约10％ 甘油和约10％蔗糖，和5)约5％至约10％聚乙二醇(PEG)；和d)将稀释的溶解的AAT多肽的pH降低至约7.6，由此产生了复性的AAT多肽。在某些变更中，复性缓冲液进一步包含约0.005％至约0.02％吐温-20(Tween 20)。
在另一个示例性的实施例中，生产复性的重组AAT野生型和突变体多肽的方法包括：a)用溶解缓冲液溶解变性的AAT多肽，所述溶解缓冲液包含约8M尿素，约0.1M Tris，约1mM甘氨酸，约1mM EDTA，约10mMβ-巯基乙醇，约10mM二硫苏糖醇(DTT)，约1mM还原型谷胱甘肽(GSH)，并且其pH约为9，由此产生溶解的AAT多肽溶液；b)通过将上述溶解的AAT多肽加至约20倍体积的复性缓冲液中的方法将上述溶解的AAT多肽快速稀释，此复性缓冲液包含约20mM Tris和约10％甘油，pH约9；和c)将稀释的溶解的AAT多肽的pH缓慢降低至约7.6，由此产生了复性的AAT多肽。
在另一个示例性的实施例中，生产复性的重组AAT野生型和突变体多肽的方法包括：a)用溶解缓冲液溶解变性的AAT多肽，所述溶解缓冲液包含约8M尿素，约0.1M Tris，约1mM甘氨酸，约1mM EDTA，约10mMβ-巯基乙醇，约10mM二硫苏糖醇(DTT)，约1mM还原型谷胱甘肽(GSH)，并且其pH约为8，由此产生溶解的AAT多肽溶液；b)通过将上述溶解的AAT多肽加至约20倍体积的复性缓冲液中的方法将上述溶解的AAT多肽快速稀释，此复性缓冲液包含约20mM Tris和约10％甘油，pH约8；和c)将稀释的溶解的AAT多肽的pH缓慢降低至约7.6，由此产生了复性的AAT多肽。
在另一个示例性的实施例中，生产复性的重组AAT野生型和突变体多肽的方法包括：a)用溶解缓冲液溶解变性的AAT多肽，所述溶解缓冲液包含约8M尿素，约0.1M Tris，约1mM甘氨酸，约1mM EDTA，约10mM β-巯基乙醇，约10mM二硫苏糖醇(DTT)，约1mM还原型谷胱甘肽(GSH)，并且其pH约为7.6，由此产生溶解的AAT多肽溶液；b)通过将上述溶解的AAT多肽加至约20倍体积的复性缓冲液中的方法将上述溶解的AAT多肽快速稀释，此复性缓冲液包含约20mM Tris和约10％甘油，pH约7.6，由此产生了复性的AAT多肽。
在某些实施例中，本方法进一步包括浓缩复性的AAT野生型和突变体多肽的方法。例如，可以用超滤浓缩方法将复性的AAT多肽浓缩10-200倍。
本发明亦提供将正确复性的AAT野生型和突变体多肽从不正确复性的或非复性的AAT中纯化出的方法，此方法包括：a)在盐的作用下将不正确复性的或非复性的AAT多肽与疏水作用层析树脂结合；和b)收集没有同树脂结合的正确复性的AAT多肽。在某些实施例中，其中所述的盐是硫酸铵[(NH 4) 2SO 4]、氯化钠(NaCl)或氯化钾(KCl)。在某些实施例中， 其中所用硫酸铵的浓度约为0.25M至约为1.2M。在某些实施例中，其中所用氯化钠的浓度约为1.0M至约为3.5M。在某些实施例中，其中所用氯化钾的浓度约为1.0M至约为3.5M。在某些实施例中，适当复性的AAT多肽来源于细菌包涵体。
在一个例证性的执行方案中，生产复性的重组AAT野生型和突变体多肽的方法包括：a)用溶解缓冲液溶解变性的AAT多肽，所述溶解缓冲液包含约8M尿素，约0.1M Tris，约1mM甘氨酸，约1mM EDTA，约100mMβ-巯基乙醇，约pH 9.0，由此产生溶解的AAT多肽溶液；b)用一个溶解缓冲液调节溶解的AAT蛋白原溶液的A 280到约2.0。此溶解缓冲液包含约8M尿素，约0.1M Tris，约1mM甘氨酸，约1mM EDTA，约10mMβ-巯基乙醇，约10mM二硫苏糖醇(DTT)，约1mM还原型谷胱甘肽(GSH)，并且其pH约为9.0。c)通过将上述溶解的AAT多肽加至约20倍体积的复性缓冲液中的方法将上述溶解的AAT多肽快速稀释，此复性缓冲液包含约20mM Tris，pH约9.0，及下述1)～5)中的任何一种：1)约10％至约30％甘油，2)约10％至约50％蔗糖，3)约20％甘油和约20％蔗糖，4)约10％甘油和约10％蔗糖，和5)约5％至约10％聚乙二醇(PEG)；d)在20℃将稀释的溶解的AAT多肽溶液孵育至少16小时；e)进一步在4℃将稀释的溶解的AAT多肽溶液孵育约24至约72小时；f)用超滤法将稀释的溶解的AAT多肽溶液浓缩；和g)用分子筛色谱法将稀释的溶解的AAT多肽溶液交换成具有约20mM Tris，约0.2M NaCl，约10％甘油或约15％蔗糖，约1mM DTT，约pH 7.6，由此产生复性的AAT多肽。在某些实施例中，复性缓冲液和在步骤g)中的缓冲液进一步包含约0.005％的吐温-20(Tween 20)。
在另一个例证性的执行方案中，复性的野生型和突变体的AAT纯化步骤包括，第一步先将复性溶液超滤浓缩，然后经过一个SEC色谱柱(Superdex 200或Sephacryl 300，GE Healthcare)将复性的单体蛋白与未复性或部分复性的AAT分离开。第二步使用离子交换或疏水相互作用柱层析进一步纯化复性蛋白。图3A是通过疏水相互作用色谱获得的纯化AAT样品的SDS-PAGE结果。显示蛋白质大部分是单体形式，SDS-PAGE检测时，在没有还原剂的情况下具有微量的二聚体形式。非还原性SDS-PAGE已被常规用于区分折叠和未折叠的蛋白质。当与可用于临床治疗的人AAT(来自Aventis Behring LLC的糖基化
Figure PCTCN2018105011-appb-000004
数据未显示)比较时，纯化的重组AAT显示出几乎相同的抑制活性。在AAT∶PPE为约1.07∶1的化学计量比下，PPE(猪胰弹性蛋白酶)被完全抑制(图3B)，表明纯化的重组AAT是完全活性的。纯化的突变蛋白(突变体)示于图3C。突变蛋白的活性与野生型相当。
化学修饰
对蛋白药进行化学修饰是提高体内半衰期的常用方法。AAT蛋白在232位点上有一个唯一的半胱氨酸(图7)。此位点(Cys232)或N-端位点可用于对AAT进行定位的化学修饰，且实验证明修饰后不影响AAT活性。
在某些实施例中，在Cys232位点进行聚乙二醇化修饰。参照已发表的方法，纯化的rAAT在唯一的Cys232位置可以被聚乙二醇化[37]。聚乙二醇化的效率在多个实验中在50-65％的范围内。分子模型表明，这个唯一的半胱氨酸部分暴露于含水溶剂中，并且不在与弹性蛋白酶相互作用的AAT结构域附近(参见图7)。聚乙二醇化后，通过阴离子交换色谱(Q-HiTrap，GE Healthcare)从聚乙二醇化的AAT中分离去除未反应的马来酰亚胺-PEG(～20K Da，Nektar Therapeutics)和未聚乙二醇化的AAT。图4是聚乙二醇化rAAT多肽的SDS-PAGE及MALDI-TOF质谱结果及其在阻断PPE中的正常功能。该实验显示，聚乙二醇化的AAT可以通过盐梯度洗脱而方便地从未聚乙二醇化的AAT和游离的未反应的mPEG20中被分离出来。并且，聚乙二醇化反应的成功已经过SDS-PAGE和MALDI-TOF质谱法证实。例如(图4C)，AAT多肽(非聚乙二醇化)的分子量是43996.34道尔顿，而聚乙二醇化试剂Mal-PEG20的分子量是22063.92道尔顿。成功聚乙二醇化的rAAT的分子量为65324.02道尔顿，与预测的分子量密切相符。这表明rAAT已成功聚乙二醇化。在质谱过程的电离分解过程中可能产生“游离”rAAT和Mal-PEG20质量。
在某些实施例中，可以对Cys232位点进行脂肪酸化修饰，例证之一是棕榈酸化修饰。棕榈酸是一个十六烷脂肪酸，也叫软脂酸。在血液中，棕榈酸与血清白蛋白有较强的结合能力。利用这个特点，棕榈酸修饰的蛋白药可以在血液中与血清白蛋白结合，极大的延长了体内半衰期。棕榈酸修饰的蛋白或多肽药已经成功的应用于临床，产生良好的效果。其中诺和诺德公司(Novo Nordisk)的治疗糖尿病和肥胖症的利拉鲁肽(Liraglutide)是一个成功的例证。本发明提供对Cys232位点的棕榈酸修饰的野生型和突变体AAT蛋白。下式是一种修饰方式，利用谷氨酸作为链接“桥”，将棕榈酸连接到野生型或突变体的AAT-Cys232。
Figure PCTCN2018105011-appb-000005
其它化学链接方法亦可以应用。比如在特定的化学反应条件下，可以进行N-端化学修饰(Christopher D.Spicer& Benjamin G.Davis Nature Communications 5,Article number:4740(2014).“Selective chemical protein modification”)化学修饰的技术为本领域技术人员所熟知。
热稳定性
为了提高重组AAT的热稳定性和抗氧化性，我们构建了三种突变体。第一个是热稳定性的F51L单突变体，第二个是抗氧化的M351V/M358V双突变体，第三个是热稳定性和抗氧化性的F51L/M351V/M358V三突变体。为了比较野生型和突变型蛋白质(突变体)的热稳定性，我们使用了基于荧光的热变性测定法，如图5所示。在这种情况下，SYPRO Orange染料在结合到疏水表面。当蛋白质随温度升高而变性时，其疏水表面暴露并结合染料，导致荧光增加。温度的进一步增加使染料和蛋白质分离而产生变性峰值。图5显示野生型和M351V/M358V突变蛋白在48℃左右发生热变性，而含有F51L，F51L和F51L/M351V/M358V突变蛋白的变性温度均升高至54℃左右。结果显示，按照设计和预期，含有F51L的单突变体和三突变体都大大提高了AAT的热稳定性。
抗氧化性
除构建抗氧化突变体M351V/M358V外，还构建了F51L/M351V/M358V的组合突变体，预测抗氧化性和热稳定性均将有所提高。如上所述(图5)，三突变体更热稳定，在抗氧化实验中，结果显示两种含M351V/M358V的突变蛋白都更耐氧化。图6显示，当H 2O 2与AAT的分子比从4∶1增加到400∶1时，天然AAT和F51L开始失去抑制PPE的体外活性，但是含有M351V/M358V的突变蛋白的抗氧化性都可达到400∶1的比例。据文献报道，与热稳定突变体一样，突变蛋白的体外抗氧化性能可以转化为增强体内稳定性，因此具有“可药用性”[33]。
药物组合物、治疗应用和药剂盒
本发明也提供所述的包含有生物活性的AAT多肽组合物(包括药品组合物)。此组合物亦可以包含药用赋形剂。AAT多肽可以是冻干制剂或液体制剂的形式。药用赋形剂是在所用的药量和浓度下对使用方无毒，并可以包含缓冲剂如磷酸盐、柠檬酸盐；盐如氯化钠；糖如蔗糖；及/或聚乙二醇(PEG)。见Remington:The Science and Practice of Pharmacy 20th Ed.(2000)Lippincott Williams and Wilkins,Ed.K.E.Hoover.可以制备出不同给药途径的AAT多肽制剂，如为静脉注射(IV)的液体或冻干制剂，及为深肺给药的干粉制剂或气化制剂。这些试剂对本领域技术人员是显而易见的，参见文献例如：Drug Delivery to the Lung,Bisgaard H.,O'Callaghan C and Smaldone GC,editors,New York；Marcel Dekker,2002。
本发明所述AAT多肽可以用此处所描述的任何方法产生。在某些实施例中，AAT多肽从细菌(如大肠杆菌)包涵体产生。在某些实施例中，AAT多肽是非糖基化的。在某些实施例 中，AAT多肽具有至少约80％、85％、90％、95％、96％、97％、98％或99％的纯度。在某些实施例中，AAT多肽的比活力(例如用猪胰弹力蛋白酶抑制测定)每毫克总蛋白不少于约0.3、0.35、0.4、0.45、0.5、0.55、0.6、0.65、0.7、0.75、0.8、0.85、0.9或0.95毫克的活性AAT多肽。在某些实例中，AAT多肽是野生型AAT蛋白。在某些实例中，AAT多肽是突变体AAT蛋白。在某些实例中，AAT多肽是本发明所描述的高稳定性、抗氧化的三突变AAT突变体。在某些实例中，AAT多肽是化学修饰的上述所描述的AAT蛋白。
本发明也提供包含所述AAT多肽的治疗应用药剂盒。本发明的药剂盒包含一个或多个含有AAT多肽的容器。此容器可以是小药水瓶、瓶子、广口瓶，或灵活的包装。例如，AAT多肽可以用一次性应用的小药水瓶包装，每瓶含有500毫克或1,000毫克的活性AAT多肽。此药瓶可以有一个无菌通入口(例如一个可以被皮下注射针头刺穿的瓶塞)。另外可预期的是用特殊装置组合起来的包装，例如吸入器、鼻给药装置(例如喷雾器)或输入装置如微泵。至少一种活性药剂是AAT多肽。此药剂盒也可以进一步包括第二个药物的有效成分。包装容器中亦可以包含根据本发明所描述方法的应用说明书。一般地，这些说明书包括根据本发明所描述方法的用AAT多肽治疗疾病的应用方法说明。此说明书可以进一步包括应用AAT多肽治疗疾病的说明，例如，治疗与AAT缺乏有关的疾病。说明一般包括使用剂量、使用时间和治疗所述疾病的应用途径。本发明药剂盒提供的使用说明一般在标签上或说明书上(例如包含于试剂盒中的纸张上)书写说明，但机器可读的说明(例如装载于磁片上或光盘上的说明)亦可以接受。此药剂盒也可以包括干粉或喷雾器肺部给药的装置。
下列实施例提供例证但不限制本发明。
实施例1.质粒构造和表达。用PCR扩增法得到编码Δ5-AAT多肽(图1)的DNA片断。Δ5-AAT多肽缺乏图1显示的1-5氨基酸序列并在起始位置人工附加了蛋氨酸以促进在大肠杆菌表达。编码Δ5-AAT多肽的在DNA片断中的多(聚)核苷酸序列已经为了在大肠杆菌中最佳表达而进行了优化。为了蛋白表达，将上述PCR产物克隆于pET11a质粒。在PCR、连接和转化入BL21(DE3)菌株后，选择单克隆菌落扩增，并最终对所选择的载体进行DNA序列测定以保证得到正确的DNA序列。所得载体为pET11-Δ5-AAT。
实施例2.野生型和F51L突变体蛋白的表达。首先扩大培养大肠杆菌表达克隆，然后接种到1.0L含有10g胰蛋白胨，5g酵母提取物，10g NaCl和50mg氨苄青霉素的LB培养基中，当OD 600＝0.6时加入IPTG至0.5mM，37℃下表达3小时。
实施例3.M351V/M358V和F51L/M351V/M358V突变蛋白的表达。首先将大肠杆菌表达克隆在LB培养基中扩增，然后接种到1.0L含有胰蛋白胨12g，酵母提取物24g，甘油4ml，17mM KH 2PO 4的培养基中，加入72mM KH 2PO 4和50mg氨苄青霉素，当OD 600＝0.6时加入IPTG至0.5mM，37℃下诱导表达4小时。
实施例4.包涵体纯化。通过离心收集细胞，然后在含有1％
Figure PCTCN2018105011-appb-000006
的20ml TN(150mM NaCl,50mM Tris,pH 8.0)的缓冲液中悬浮。向其中加入10mg溶菌酶，并将细胞悬浮在-20℃冰冻过夜。然后将裂解物溶化并加入20μl 1M硫酸镁和100μl 0.01mg/ml DNAase。搅动细胞，并将其温育至释放的DNA完全溶解。其后用250ml含有1％
Figure PCTCN2018105011-appb-000007
的TN稀释裂解物并搅动混合2-4小时。通过离心收集包涵体，通过用含有1％Triton X-100的TN缓冲液(100mM Tris，250mM NaCl，pH8.0)洗涤5次来纯化包涵体。将纯化的包涵体溶解于8M尿素缓冲溶液(8M尿素，0.1M Tris，1mM甘氨酸，1mM EDTA，100mMβ-巯基乙醇，pH 10)中，4℃下缓慢搅拌约16小时。然后将溶解物离心除去不溶性碎片。使用相同的8M尿素缓冲液作为稀释剂将纯化的包涵体调整至最终A 280＝2.0。
实施例5.复性。将上述溶解的包涵体快速冲稀至20倍体积的含：20mM Tris，10％甘油，pH 9的缓冲液，其稀释后的最后OD 280为0.1。然后将pH缓慢的调至pH 8.0。稀释后用1M HCl将溶液的pH在2-4天内，逐步的调到7.6。
其他试验过的复性方法包括在复性缓冲液中用高浓度的甘油(20％)，或用20％蔗糖取代甘油，或同时用10％蔗糖和10％甘油。在某些实验中，吐温-20(0.005％-0.01％)也被包含在复性缓冲液中。所有这些条件都产生了正确复性的(有活性的)AAT多肽。
用固定pH法对表达的野生型和突变体型的AAT多肽包涵体亦可以成功复性。将洗涤的包涵体融于含高浓度尿素的溶解缓冲液(8M尿素，0.1M Tris，1mM glycine，1mM EDTA，100mMβ-巯基乙醇(β–ME)，pH 10.5)，溶解于高OD 280(20-40)，并在4℃缓慢的搅动12小时。溶解的样品超速离心(30分钟×66,000g)澄清以去除不溶解的杂质。然后用8M尿素，0.1M Tris，1mM甘油，1mM EDTA，10mMβ-巯基乙醇(β–ME)，10mM二硫苏糖醇(DTT)，1mM还原型谷胱甘肽(GSH)，pH 10.5缓冲液将溶解的包涵体的OD 280调至2.0。将上述溶解的包涵体快速冲稀至20倍体积的含20mM Tris，10％甘油，pH 8.5的缓冲液，其稀释后的 最后OD 280为0.1。将稀释的溶液于20℃保存16小时，然后再进行超滤浓缩和缓冲液交换。
实施例6.纯化。使用切向流超滤系统将重折叠的AAT浓缩至A 280>20.0并加载到用含有20mM Tris，0.15M NaCl，0.4M尿素，1mM DTT，10％甘油，pH7.6的缓冲液预平衡的Superdex 200柱。收集活性峰组分并用含有20mM Tris，5％甘油，3M NaCl，0.001％吐温，20,1mM DTT，pH 7.6的缓冲液透析。将透析后的蛋白加载到用透析缓冲液平衡的苯基琼脂糖凝胶(疏水)柱中。收集含有目的纯化物的流出液，浓缩并用含有20mM Tris，5％甘油，0.001％吐温20，1mM DTT，pH 7.6的缓冲液透析除盐(NaCl)。蛋白质的浓度通过在6M盐酸胍，20mM磷酸钠，pH 6.5中的摩尔消光系数测定，对于该特定蛋白，消光系数ε 280＝19060M -1cm -1。
实施例7.聚乙二醇化。将高度纯化的AAT通过一个用50mM磷酸钠pH7.5，200mM NaCl预平衡的PD-10(BioRad)柱来去除DTT并根据产品要求将pH调至7.5。由于还原剂DTT会干扰聚乙二醇化反应，通常要进行两次缓冲液交换过程，以确保没有微量的DTT存在。通过摩尔消光定量缓冲液交换后的AAT。将-20℃下氩气中储存的固体PEG-mal20(聚乙二醇马来酰亚胺20，Nektar，Huntsville，AL)以5∶1至10∶1的摩尔比添加至AAT的溶液中，并在37℃下温育30分钟。加入20mM DTT终止反应，37℃下再孵育5分钟。然后将聚乙二醇化的AAT(Peg-AAT)透析到20mM Tris 8.0，50mM NaCl，1mM DTT中以去除过量的盐，然后加载到5mL Q XL HiTrap柱，用0-1000mM NaCl进行梯度洗脱。
实施例8.酶活性测定。复性的rAAT野生型和突变体的AAT生物学活性，采用底物显色反应法在体外测定对HLE或PPE的抑制活性来测定。我们测试了rAAT的抑制活性，并与Calbiochem公司生产的市售人血浆AAT(San Diego，CA目录#17825)或由Aventis Behring LLC生产和销售的市售糖基化全长AAT进行了对比。从生猪胰腺中分离的PPE购自Sigma-Aldrich(St.Louis，MO，货号#E7885)；从人类痰分离的HLE购自Molecular Innovations(Southfield，MI Cat#HNE)。AAT的浓度范围为0.3nM至14nM，与1.4nM固定浓度的HLE或PPE一起在37℃孵育15分钟，然后将孵育物的等分试样与1mM的弹性蛋白酶底物N-琥珀酰-ala(PPE显色底物，Sigma)或N-甲氧基琥珀酰-α-丙氨酰-α-丙氨酰-对-硝基苯胺(HLE显色底物，Sigma)混合。使用Molecular Devices分光光度计(Spectramax Plus)在21℃，405nm 测定底物的水解动力学反应。确定每个反应的初始速度并计算相对于对照(无AAT或AAT多肽)的百分比活性。将弹性蛋白酶活性百分比相对于在相应反应中使用的AAT多肽/弹性蛋白酶浓度的化学计量摩尔比作图。实验中使用的每种形式的AAT多肽、PPE和HLE的储备物的精确浓度通过已知的消光系数事先测定，所用的已知消光系数来自瑞士生物信息学研究所的ExPASY蛋白质组学服务器的计算机软件程序ProtParam(http://www.expasy.ch)。
下面详述图3的实验过程。向含有不同浓度AAT的Ependorf管中分别加入固定浓度的PPE(80μg/mL)，37℃下在50mM Tris pH8.8，38mM NaCl，0.01％吐温20的反应体系中温育15分钟。将10μl等分试样一式四份移液到微量滴定板中，然后用多道移液器将100μl等分的1mM生色底物对-α-丙氨酸-pro-val-pNA在同一缓冲液中吸取到微板孔中。在21℃在405nm处监测底物的弹性蛋白酶裂解的动力学。将速度与对照(仅弹性蛋白酶)进行比较，并作为％对照弹性蛋白酶活性(y轴)对AAT∶PPE(x轴)的化学计量比作图。测定中使用的PPE浓度，根据瑞士生物信息学研究所ProtParam算法(www.expasy.ch)，在6M胍，50mM NaPi，pH6.5中测量纯PPE的消光系数而获得。AAT的浓度通过如下方法测定：首先用来自Novagen(www.novagen.com)的“几乎不可逆的”荧光底物MUGB(4-甲基伞形酮基-4-胍基苯甲酸盐盐酸盐，Fluka)精确地滴定胰蛋白酶(Sigma)贮液中胰蛋白酶活性位点的浓度。然后用显色底物BAPNA(N-苯甲酰基L-arg-4硝基苯胺盐酸盐，Sigma)，在21℃和405nm下，在化学计量分析中测定任一种AAT原液在阻断胰蛋白酶功能位点的作用浓度。已经确定AAT的任一种形式的作用浓度几乎与通过使用来自瑞士生物信息学研究所网站(www.expasy.ch)的ProtParam计算机算法使用消光系数在结构上测量所确定的功能浓度相同，表明纯化的重组AAT的活性几乎接近100％。
实施例9.热稳定性。热稳定性测定使用96孔培养板进行。反应体积为110μl，缓冲液含有：1×PBS缓冲液，10％(v/v)甘油，10％DMSO，5mM DTT，50×SYPRO Orange和纯化的AAT或其突变体各15μM。将反应培养板在25℃下孵育30min，然后以0.5℃间隔升温至70℃。测定每个温度的Ex 490mM，Em 580mM下的荧光200mS。用荧光计数对温度作图。
实施例10.抗氧化性。为了测试AAT及其突变体的抗氧化性，分别将50μM的每种纯化的AAT或突变体在含有0mM，2mM，10mM，50mM，100mM，200mM H 2O 2的PBS缓冲液 里25℃下孵育15分钟，然后加入等量的DTT来还原过量的H 2O 2。处理后的AAT和突变体的抗氧化性通过测定对PPE的抑制活性来测定。
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Figure PCTCN2018105011-appb-000008
Figure PCTCN2018105011-appb-000009

Claims (18)

  1. 一种α1-抗胰蛋白酶突变体，是具有活性的F51L/M351V/M358V三突变体和/或其化学修饰体。
  2. 如权利要求1所述的α1-抗胰蛋白酶突变体，其特征在于，其氨基酸序列如序列表中SEQ ID No：1所示，或者是序列表中SEQ ID No：1在N-末端截掉1～10个氨基酸残基后的序列。
  3. 如权利要求2所述的α1-抗胰蛋白酶突变体，其特征在于，所述在N-末端截掉1～10个氨基酸残基后的序列是指：序列表中SEQ ID No：1截掉第1-5位氨基酸残基后的序列，或序列表中SEQ ID No：1截掉第1-10位氨基酸残基后的序列。
  4. 如权利要求1所述的α1-抗胰蛋白酶三突变体，其特征在于，所述化学修饰体是在F51L/M351V/M358V三突变体的特定位点上进行了化学修饰。
  5. 如权利要求4所述的α1-抗胰蛋白酶三突变体，其特征在于，在其Cys232位点上或N-端位点上具有化学修饰。
  6. 如权利要求4所述的α1-抗胰蛋白酶三突变体，其特征在于，所述化学修饰是聚乙二醇化修饰或脂肪酸化修饰，所述脂肪酸化修饰包括棕榈酸化修饰。
  7. 权利要求1～6任一所述α1-抗胰蛋白酶突变体的制备方法，包括以下步骤：
    1)将所述突变体的编码基因构建到表达载体上，通过表达宿主表达该突变体蛋白；
    2)收集并纯化含有所述突变体蛋白的包涵体；
    3)用溶解缓冲液溶解包涵体，然后通过复性缓冲液使突变体蛋白复性；
    4)纯化出复性的突变体蛋白。
  8. 如权利要求7所述的制备方法，其特征在于，在步骤1)以大肠杆菌为表达宿主过表达所述突变体蛋白；在步骤3)中所述溶解缓冲液为高浓度的尿素缓冲液或盐酸胍缓冲液，所述复性缓冲液为包含甘油、蔗糖和/或聚乙二醇的Tris缓冲液。
  9. 如权利要求8所述的制备方法，其特征在于，所述复性缓冲液还包含去污剂，所述去污剂选自下列物质中的一种或多种：吐温-20、吐温-80、脱氧胆酸钠、胆酸钠和氧化三甲胺。
  10. 如权利要求7所述的制备方法，其特征在于，所述步骤3)通过下述方法一至方法四中的一种实现：
    方法一：a)用第一溶解缓冲液溶解包涵体，所述第一溶解缓冲液包含6-8M尿素，0.01-0.1M Tris，1mM甘氨酸，1mM EDTA，10-100mMβ-巯基乙醇，pH7-10，获得溶解的多肽 原溶液；b)用第二溶解缓冲液调节溶解的多肽原溶液的A 280到1.0-4.0，所述第二溶解缓冲液包含6-8M尿素，0.1-0.1M Tris，1mM甘氨酸，1mM EDTA，1-10mMβ-巯基乙醇，1-10mM二硫苏糖醇，1mM还原型谷胱甘肽，pH8-10；c)将上述b)所得溶液加至10-50倍体积的复性缓冲液中快速稀释，此复性缓冲液包含1-20mM Tris，pH 7-10及下述I)～V)中的任何一种：I)5％至30％甘油，II)5％至40％蔗糖，III)20％甘油和20％蔗糖，IV)10％甘油和10％蔗糖，V)5％至10％聚乙二醇；d)将稀释后溶液的pH降低至7.0-8.5，由此产生复性的所述突变体蛋白；
    方法二：a)用溶解缓冲液溶解包涵体，所述溶解缓冲液包含6-8M尿素，0.01-0.1M Tris，1mM甘氨酸，1mM EDTA，1-10mMβ-巯基乙醇，1-10mM二硫苏糖醇，1mM还原型谷胱甘肽，pH8-10，获得溶解的多肽溶液；b)将该多肽溶液加至10-50倍体积的复性缓冲液中快速稀释，此复性缓冲液包含1-20mM Tris和5-30％甘油，pH8-10；c)将稀释后溶液的pH缓慢降低至7.0-8.5，由此产生复性的所述突变体蛋白；
    方法三：a)用溶解缓冲液溶解包涵体，所述溶解缓冲液包含6-8M尿素，0.01-0.1M Tris，1mM甘氨酸，1mM EDTA，1-10mMβ-巯基乙醇，1-10mM二硫苏糖醇，1mM还原型谷胱甘肽，pH8，获得溶解的多肽溶液；b)将该多肽溶液加至10-50倍体积的复性缓冲液中快速稀释，此复性缓冲液包含1-20mM Tris和5-30％甘油，pH8；c)将稀释后溶液的pH缓慢降低至7.6，由此产生了复性的所述突变体蛋白；
    方法四：a)用溶解缓冲液溶解包涵体，所述溶解缓冲液包含6-8M尿素，0.01-0.1M Tris，1mM甘氨酸，1mM EDTA，1-10mMβ-巯基乙醇，1-10mM二硫苏糖醇，1mM还原型谷胱甘肽，pH7.6，获得溶解的多肽溶液；b)将该多肽溶液加至10-50倍体积的复性缓冲液中快速稀释，此复性缓冲液包含约20mM Tris和10％甘油，pH7.6，由此产生复性的所述突变体蛋白。
  11. 如权利要求7所述的制备方法，其特征在于，所述步骤3)包括：a)用第一溶解缓冲液溶解包涵体，所述溶解缓冲液包含6-8M尿素，0.01-0.1M Tris，1mM甘氨酸，1mM EDTA，10-100mMβ-巯基乙醇，pH9.0，获得溶解的多肽原溶液；b)用第二溶解缓冲液调节溶解的多肽原溶液的A 280到1-4，所述第二溶解缓冲液包含6-8M尿素，0.01-0.1M Tris，1mM甘氨酸，1mM EDTA，1-10mMβ-巯基乙醇，1-10mM二硫苏糖醇，1mM还原型谷胱甘肽，pH 9.0；c)将上述b)所得溶液加至10-50倍体积的复性缓冲液中快速稀释，此复性缓冲液包含1-20mM Tris，pH 9.0，及下述I)～V)中的任何一种：I)5％至30％甘油， II)5％至50％蔗糖，III)20％甘油和20％蔗糖，IV)10％甘油和10％蔗糖，V)5％至10％聚乙二醇；d)在20℃将稀释后溶液孵育至少16小时；e)进一步在4℃将稀释后溶液孵育24至72小时；f)用超滤法将稀释后溶液浓缩；g)用分子筛色谱法将浓缩后溶液交换成含有10-20mM Tris，0.1-0.2M NaCl，5-30％甘油或5-40％蔗糖，1mM DTT，pH 7.6的缓冲液，由此产生复性的所述突变体蛋白。
  12. 如权利要求11所述的制备方法，其特征在于，步骤g)中的缓冲液进一步包含0.005％的吐温-20。
  13. 如权利要求7所述的制备方法，其特征在于，步骤4)的纯化方法是在盐溶液的作用下将不正确复性的或非复性的突变体蛋白与疏水作用层析树脂结合，而收集没有同树脂结合的正确复性的突变体蛋白。
  14. 如权利要求13所述的制备方法，其特征在于，所述盐溶液是含硫酸铵、氯化钠或氯化钾的溶液，其中硫酸铵的浓度为0.25M至1.2M，氯化钠的浓度为1.0M至3.5M，氯化钾的浓度为1.0M至3.5M。
  15. 如权利要求7所述的制备方法，其特征在于，步骤4)的纯化包括：第一步先将复性溶液超滤浓缩，然后经过一个SEC色谱柱将复性的单体蛋白与未复性或部分复性的蛋白分离开；第二步使用离子交换或疏水相互作用柱层析进一步纯化复性蛋白。
  16. 如权利要求7所述的制备方法，其特征在于，步骤4)还进一步包括对纯化的复性突变体蛋白的半胱氨酸位点进行化学修饰。
  17. 权利要求1～6任一所述α1-抗胰蛋白酶突变体在制备治疗肺部疾病的药物中的应用。
  18. 一种药物组合物或药剂盒，包含权利要求1～6任一所述α1-抗胰蛋白酶突变体。
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