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CN103923209A - Lambda interferon mutant and polyethylene glycol derivative - Google Patents

Lambda interferon mutant and polyethylene glycol derivative Download PDF

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CN103923209A
CN103923209A CN201410200460.9A CN201410200460A CN103923209A CN 103923209 A CN103923209 A CN 103923209A CN 201410200460 A CN201410200460 A CN 201410200460A CN 103923209 A CN103923209 A CN 103923209A
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mutant
ifn
interferon
protein
polyethyleneglycol
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CN103923209B (en
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田硕
杨璐
潘海
陈全民
李尧
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Hangzhou Xianweida Biotechnology Co ltd
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BEIJING KAWIN TECHNOLOGY Co Ltd
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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • 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
    • A61K47/56—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
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    • A61K47/60—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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Abstract

The invention relates to a Lambda interferon mutant. The 62nd leucine of an interferon lambda1 mutates into serine, and then the novel mutant interferon Lambda is obtained. Compared with the interferon lambda1, the mutant interferon Lambda has higher stability. The invention further relates to a polyethylene glycol derivative of the Lambda interferon mutant and the purpose of the polyethylene glycol derivative.

Description

A kind of Lambda interferon mutant and polyethyleneglycol derivative
Technical field
The present invention relates to IFN-λ 1 mutant, its polyethyleneglycol derivative and preparation method thereof, and the polyethyleneglycol derivative that comprises described IFN-λ 1 mutant or this mutant is in the application having aspect the medicine such as prevention or treatment virus disease, tumour.
Background technology
Interferon, rabbit is the important familial cytokine of a class, has antiviral, inhibition of cell proliferation and the immunoregulation effect of wide spectrum.
So far, identified the Interferon, rabbit of 6 kinds of forms, they are divided into three large group.So-called " I type " Interferon, rabbit comprises interferon alpha, interferon beta, Interferon, rabbit ω, Interferon, rabbit δ, interferon-tau.At present, interferon-gamma and interferon alpha subclass is only II type Interferon, rabbit.Type iii interferon is a type cytokines family of recent findings, comprises interferon lambda 1, λ 2 and λ 3, also referred to as IL-28A, IL-28B and IL-29
The new protein family that IL-28A, IL-28B and IL-29 comprise recent findings, described protein families and I type Interferon, rabbit have sequence homology, and have the homology of gene order with IL-10.This new family is at common all PCT application WO02/086087 and Sheppard etc., and Nature Immunol.4:63-68, is described later in detail in 2003 (including in full reference herein in).Say from function, IL-28 and IL-29 are similar to I type Interferon, rabbit, all antiviral states in energy inducing cell, and different from I type Interferon, rabbit, they do not demonstrate the antiproliferative activity that resists some B clone.
200 amino acid polypeptides of wild-type IL-29 (interferon lambda 1) genes encoding, this protein maturation aminoacid sequence is presented in SEQ ID NO:1, and the corresponding polynucleotide of encode IL-29 polypeptide as herein described region, territory, motif, residue and sequence are presented in SEQ ID NO:2.The spiral prediction of IL-29 is as follows: spiral A has amino-acid residue 30 (Ser) to define to 44 (Leu); Spiral B is defined to 65 (Val) by amino-acid residue 57 (Asn); Spiral C has amino-acid residue 70 (Val) to define to 85 (Ala); Spiral D is defined to 111 (Gln) by amino-acid residue 92 (Lys); Spiral E is defined to 139 (Lys) by amino-acid residue 118 (Thr); And spiral F is defined to 170 (Leu) by amino-acid residue 144 (Gly).
Known IL-29 molecule has 5 cysteine residues (PCT applies for WO02/086087WO02/02627), further analyses and prediction based on multiple ratio to the IL-29 carrying out, amino-acid residue the 49th and 145, and the 112nd and the halfcystine of 171 will form intramolecular disulfide bond, the halfcystine of the 15th dissociates, and can form intermolecular disulfide bond.
No matter be I type Interferon, rabbit, II type Interferon, rabbit or type iii interferon, as pharmaceutical grade protein, because poor stability, plasma clearance are high, Half-life in vivo is short, easily produces antigen-antibody etc., very large restriction in clinical treatment.Genetic engineering technique makes to synthesize on a large scale human recombination protein becomes possibility, engineered protein is by changing certain or certain the several amino acid in wild protein sequence, to obtain the recombinant protein relatively more stable, specific activity is higher, and reducing its immunogenicity, this has solved the unstable or Immunogenicity that heterologous protein causes to a certain extent.
Even now, also cannot overcome the shortcomings such as the fast and bioavailability of plasma clearance is low, and the result that these shortcomings cause is: need frequent injection of interferon just can reach effective plasma level and treat concentration; And, after per injection, all can cause the larger fluctuation of Plasma Concentration, form peak value and the valley of drug level.So just may increase the risk of medical expense and administration inconvenience and untoward reaction.Therefore, people attempt to adopt various useful for drug delivery technology to improve the curative effect of pharmaceutical grade protein.
The bioavailability of protein drug is subject to the restriction of plasma half-life conventionally, and to proteasome degradation sensitivity, the technology that hinders the application polyethylene glycol modified protein on clinical treatment is a kind of new technology for improvement of pharmacokinetics character in protein drug body that development in recent years is got up.It is that the peg molecule (PEG) of activation is bonded on protein molecule surface, thereby affect the space structure of protein, finally cause the change of the various biochemical properties of protein, as: chemical stability increases, opposing protease hydrolysis ability improves, immunogenicity and toxicity reduce disappearance, and Half-life in vivo extends, and plasma clearance reduces etc.
Summary of the invention
On IFN-λ 1, have the many places amino acid can be for suddenling change, but the change of these aminoacid sequences should consider the folding of IFN-λ 1 and space structure is unaffected, and at least obtain can keeping being equivalent to the mutant of wild-type IFN-λ 1 activity.The object of this invention is to provide and a kind ofly at the 62nd amino acids a kind of novel mutant IFN-λ 1 obtaining that suddenlys change, the Leu of the 62nd is sported to Ser, this mutant is with respect to wild-type IFN-λ 1, and activity is stronger, stability is better.
The invention provides the method for preparing solubility IFN-λ 1 mutant, the method is by first building design IFN-λ 1 mutant primer, build and obtain mutant gene, gained gene is inserted into expression vector, the carrier of gained is transferred in host e. coli, expressed accordingly engineering bacteria, then carried out fermentation and abduction delivering, inclusion body denature and renature, obtained solubility IFN-λ 1 mutant.
Required protein is from soluble cell matter part, in the soluble fractions of solubility pericentral siphon part, substratum and inclusion body, reclaim and purifying, can use from substratum, tenuigenin, pericentral siphon part and inclusion body and to reclaim and any method of protein purification, these reclaim and purification process be known or those skilled in the art easily definite, include, but are not limited to, centrifugal, filter dialysis, the methods such as chromatography (comprising molecular exclusion chromatography).The appropriate method of recovery and purifying desired protein depends in part on characteristic and the object purposes of protein.
Be to utilize protein engineering technology for realizing the technical scheme that this object takes, the aminoacid sequence shown in SEQ ID NO:1 is carried out to rite-directed mutagenesis, the Leu of 62 is sported to Ser, concrete grammar is:
1) utilize external site-directed mutagenesis technique to obtain the recombination of coding target protein;
2) this recombination is inserted into the recombinant plasmid that obtains codified recombinant protein in expression vector, described expression vector includes but not limited to pET-23b;
3) recombinant plasmid transformed competent escherichia coli cell is obtained can stably express target protein engineering bacteria, described competent escherichia coli cell includes but not limited to BL21 (DE3);
4) target protein that fermentation separation and purification are expressed with occlusion body form.Expression product inclusion body form exists, expression amount accounts for the more than 40% of bacterial protein, the purifying of recombinant protein adopts Blue dye affinity chromatography, cupric ion affinity chromatography, CM weak cation exchange chromatography three step purifying process successively, can obtain highly purified restructuring target protein IFN-λ 1 mutant.
Of the present invention object is to provide polyethyleneglycol derivative of IFN-λ 1 mutant as above and preparation method thereof, by thiol reactant, polyoxyethylene glycol (PEG) reagent is reacted with the free cysteine residues in IFN-λ 1 and obtain specificity conjugate, in products therefrom, PEG realizes at free cysteine pointed decoration, PEG-IFN-λ 1 mutant obtaining.More the IFN-λ 1 of PEGization does not compare, and the PEG-IFN-λ 1 of gained has stability raising, reduced immunogenicity.
In general, the method described in the similar WO9412219 of PEGization method of protein (Cox and McDermott) and WO9422466 (Cox and Russell), and improvement slightly, quote these two sections of documents herein for reference.(general PEG: the mol ratio of protein or polypeptide is 1:1,5:1,10:1 and 50:1) stirring that polypeptide or protein containing free cysteine are contacted with excessive " sulfhydrylation PEG " is reacted, and in reaction conditions, preferred temperature is 4 DEG C to 37 DEG C; Preferred pH scope can from 6.5 to 9.5, but more preferably 7.5 to 8.5.Use the change of SDS-PAGE determining molecular weight can detect the PEGization of protein, it is generally acknowledged a large amount of single PEGization products of generation and the minimum quantity that do not produce the PEG of two PEGization products is best (it is generally acknowledged 80% be transformed into single PEGization product be good).
Halfcystine of the present invention comprises any suitable polymer with " peg moiety " that form " PEGization " protein, for example, and linear or branched polyvalent alcohol.Preferred polyvalent alcohol is polyoxyethylene glycol, and it is the synthetic polymer of ethylene oxide unit composition, can change ethylene oxide unit to obtain apparent molecular weight scope by size exclusion chromatography and be approximately 3,000-70, the PEGization protein variants of 000Da.The size of peg moiety directly affects its circulating half-life.Therefore, can process the protein variants with different circulating half-lifes and make it for concrete treatment application or preferred dosage by changing the size of peg moiety or structure.
" mercaptolation PEG agent " refers to any PEG derivative reacting with the mercapto alcohol groups of cysteine residues as used in this application, the reactive PEG end group that these are modified for cysteine residues, it includes but not limited to that two sulphur are to pyridine, vinyl sulfone(RemzaolHuo Xingranliaohuoxingjituan), maleimide, iodo-acid amide.PEG end group reply free sulfhydryl groups has specificity, realizes single PEGization.But react under the condition of injury protein matter not.The PEG agent using with its list-methoxylation form, wherein only an end can be coupled.
Term " single PEGization " is defined as by single peg moiety and is covalently attached to the protein that the specific site of protein is modified." single PEGization " method can be used any method known to those skilled in the art, includes, but not limited to method cited in the embodiment of the present invention.
In the present invention, preferred mercapto-reagent is that mPEG-bis-sulphur are to pyridine (mPEG-OPSS) or mPEG-maleimide (mPEG-MAL).It should be noted that above-mentioned two PEG agents are site-specific with reacting of IFN-λ, is respectively dissociating at IFN-λ 1 15cys and other naturally occurring Cys residue sites are owing to easily forming intramolecular disulfide bond, the reaction of discord thiol-reactive PEG agent.
In the present invention, the method of PEG-IFN-λ 1 conjugate of a kind of purifying gained of the present invention is also provided, in general, process is such as dialysis, ultrafiltration, molecular-exclusion chromatography, ion exchange chromatography, affinity chromatography, reverse-phase chromatography or hydrophobic chromatography, never purifying PEGization protein conjugate in PEGization protein and unreacted PEG, also can use other purification process, extraction as organic in two-phase or salt precipitation method.
Can test to confirm that peg moiety is attached on protein in correct site, through chemical cracking or this protein of proteolytic digestion, with size-exclusion, ion-exchange or reversed phase chromatography purifying PEG polypeptide (it has larger molecular weight), carrying out subsequently amino acid sequencing can complete.In amino acid sequencing process, the amino acid of PEG coupling occurs with blank.
Term " chromatography method " or " chromatography " refer to any technology that is used for separating mixture component, flow through weighting material (stationary phase) by mixture being put on to solvent (moving phase), the separation principle that separates chromatography is the different physical propertys based on stationary phase and moving phase.
Another object of the present invention is to provide a kind of IFN-λ 1 mutant of the present invention and polyethylene glycol conjugation thing thereof to have aspect treatment or prophylaxis of viral diseases or the application of tumour aspect in preparation, virus refers to any virus that IFN can resist, such as hepatitis virus, papillomavirus, herpes simplex virus, HIV, Epstein-Barr virus, coronavirus and/or influenza virus etc., preferably hepatitis virus, as HBV or HCV; Bacterium infects and cancer (for example, myelomatosis, lymphatic cancer, liver cancer, mammary cancer, melanoma, leukemia etc.).As provided PEG-IFN-λ conjugate of the present invention in embodiments of the invention to liver cancer cell HepG2, myocarditis virus EMCV
For therepic use, technician can easily determine suitable dosage, administration frequency and route of administration.The factor of making these decisions includes, but not limited to the characteristic of protein used, the illness of required treatment, potential patient's complaisance, patient's age, body weight and individual reaction etc.
PEG-IFN-λ 1 conjugate of the present invention or containing the composition of polyvalent alcohol-IFN-λ 1 conjugate, in conjunction with applicable pharmaceutical carrier or vehicle, for treating various illnesss, specific support used in these pharmaceutical compositions can be taked various forms, and it depends on desirable administration type.Suitable route of administration includes, but not limited to enteron aisle (for example, oral), part, suppository, suction and parenteral route, is preferably parenteral administration, as subcutaneous, muscle or intravenous injection.
For example, in the time preparing the composition of liquid oral formulation (, suspensoid, tincture, colloid or solution), can adopt typical drug media, as water, ethylene glycol, glycerol, oil, ethanol, odorant, sanitas, tinting material etc.Similarly, in the time preparing oral dosage form (as tablet, capsule etc.), can adopt such as starch, carbohydrate, thinner, granulating agent, lubricant, tackiness agent, disintegrating agent etc.
Can be prepared into the injection type that comprises effective constituent and suitable carrier for the ingredients of parenteral administration.For parenteral administration, carrier generally includes aseptic injection water, also can comprise some other available solubility promoter or preservative component, also can prepare in addition injection-type suspensoid, in this case, will adopt suitable liquid vehicle, suspension agent etc.For the carrier of parenteral administration the art be know and comprise, water, salts solution, ringer's solution and/or glucose, this carrier can comprise a small amount of vehicle to keep stability and the isotonicity of medicament, and these solution can be prepared according to usual method.
For topical, PEG-IFN-λ 1 conjugate of the present invention can be prepared by gentle hydrated matrix, as ointment or emulsifiable paste, be suitable for ointment base if Vaseline, lanolin and water-in-oil emulsion are as Eucerin tM, it can be available from Beiersdorf (Cincinnati, Ohio); Cold cream (USP); Purpose Cream tM, can be available from Johnson & Johnson (New Brunswick, New Jersey) etc.
PEG-IFN-λ 1 conjugate of the present invention is generally with the form administration of unitary dose, compound of the present invention is conventionally with every day, dosed administration weekly and monthly, from approximately 0.01 μ g/kg body weight to about 50mg/kg body weight, be preferably from approximately 0.1 μ g/kg body weight to about 25mg/kg body weight, more preferably from approximately 1 μ g/kg body weight to about 5mg/kg body weight.For the mean body weight of 75kg, dosage can be between every days 10 μ g and 1mg, and more preferably 20 μ g are between 200 μ g.To modified PEG-IFN-λ, its administration cycle can extend, for example, and weekly or every two weeks dosage regimens.For example, everyone every weekly dose can be extremely approximately 500 μ g of 10 μ g, and in some specific embodiment, everyone every weekly dose can be extremely approximately 250 μ g of approximately 50 μ g, and at some, in other embodiment, everyone every weekly dose can be approximately 100 to approximately 200 μ g.As clear where the person skilled in the art, depend on a number of factors according to the specified quantitative of ingredients of the present invention, include but not limited to desirable biological activity, patient's state and the tolerance to medicine etc.
Brief description of the drawings
Fig. 1: the engineering bacteria body protein abduction delivering SDS-PAGE collection of illustrative plates that represents Interferon, rabbit lambda1L62S mutant.1 swimming lane is molecular weight of albumen Marker; 2 swimming lanes are abduction delivering 0h; 3 swimming lanes are abduction delivering 1h; 4 swimming lanes are abduction delivering 2h; 5 swimming lanes are abduction delivering 3h;
Fig. 2: the SDS-PAGE collection of illustrative plates that represents the Interferon, rabbit lambda1L62S mutant after purifying.1 swimming lane is molecular weight of albumen Marker; 2 swimming lanes are the Interferon, rabbit lambda1L62S mutant protein after purifying;
Fig. 3: the SDS-PAGE collection of illustrative plates that represents the Interferon, rabbit lambda1L62S mutant of the PEG modification after purifying.1 swimming lane is molecular weight of albumen Marker; 2 swimming lanes are the Interferon, rabbit lambda1L62S mutant protein that the PEG after purifying modifies.
Embodiment
The expression engineering bacteria of embodiment 1:IFN-λ 1L62S mutant builds and sequence is confirmed
Extract the plasmid of IFN-λ 1 as template, 62 leucines (CTG) are sported to Serine (TCT), first run PCR comprises two reaction systems:
Reaction system 1 primer is:
P1：5’-CCATATGGGTCCGGTGCCGACCTCGAAACCG-3’
P2：5’-ACGGCCGCGCGGACGCGGGCC-3’
62 sites and upstream DNA sequence dna thereof increase.
Reaction system 2 primers are:
P3：5’-GGC?CCG?CGT?CCG?CGC?GGC?CGT-3’
P4：5’-C?CTCGAGTTATTATTAGGTCGATTCCGG-3’
62 sites and downstream DNA sequence thereof increase.
PCR condition is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 53 DEG C annealing 30s, 72 DEG C extend 1min, circulate 30 times, finally again 72 DEG C extension 5min.
Second takes turns PCR taking the product of first round PCR as template, carries out pcr amplification taking P1, P2 as primer.Reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 53 DEG C annealing 30s, 72 DEG C extend 1min, circulate 30 times, finally again 72 DEG C extension 5min.
PET-23b carrier and pcr amplification product are carried out respectively to EcoRI/NdeI endonuclease reaction, and 37 DEG C of reaction 1h, reclaim linearized vector and object fragment (about 600bp).T4DNA ligase enzyme is connected the endonuclease bamhi of PCR product and carrier: 10 × T4DNALigase Buffer5 μ L, and PCR double digestion reclaims product 15 μ L, and plasmid double digestion reclaims product 30 μ L, T4DNA Ligase2 μ L, ddH 2o adds to 50 μ L, and 16 DEG C are spent the night.
Ligation liquid is directly transformed to BL21 (DE3) competent cell, transform above-mentioned connection product, coating ammonia benzyl flat board, 37 DEG C of incubated overnight.
Picking list bacterium colony, as template, increases for primer P1, the P2 of above-mentioned design, and agarose gel electrophoresis detects, and positive colony occurs conforming to band with expection situation in 600bp left and right, and construction of recombinant plasmid success is tentatively described.For further confirming its sequence, carry out full-automatic sequencing with T7 universal primer by ABI377 sequenator.
Expression and the purifying of embodiment 2:IFN-λ 1L62S mutant
Engineering bacteria is inoculated in the LB substratum containing penbritin with the ratio of 1:100, when 37 DEG C of shaking culture are 0.4-0.6 to bacterium liquid OD600, adding final concentration is 0.5mM IPTG induction, continues to collect thalline (Fig. 1 is shown in by the SDS-PAGE collection of illustrative plates of thalline) after cultivation 4h.The IFN-λ 1 of the expression of e. coli bl21 (DE3) during as host accounts for the 30-50% of bacterial protein, mainly exists with inclusion body form.
The thalline that will ferment washs thalline 3 times with TE solution (m:V=1:10), then carries out high-pressure homogenization fragmentation, and broken condition is: 35Mpa high-pressure homogenization.After homogenate, microscopy breaks bacterium rate.In the time of bacterial cell disruption rate approximately 95% (approximately broken bacterium 2～3 times), the centrifugal 15min of 8000rpm, collects broken bacterial sediment.Get broken bacterial sediment and be placed in beaker, add inclusion body washings (10mMTris-HCl+1mM EDTA+0.5%Triton-X100, pH6.5, m:V=1:10), on magnetic stirrer, stir 30min, wash 3～5 times.Inclusion body inclusion body lysate cracking (7M Guanidinium hydrochloride+50mM Tris-HCl+10mM DTT, pH6.5, m:V=1:10), stirring at room temperature, spends the night.The albumen of cracking adds at a slow speed in renaturation solution (100mM Tris-HCl, 0.5M Arginine, 0.5%PEG3350 (m:V), 2mM GSH:0.5mM GSSG, pH8.5), and making final concentration of protein is 0.2mg/ml, and stirring at room temperature, spends the night.
The centrifugal 5min of renaturation solution 8000rpm, collects supernatant.Utilize ultrafiltration cup, ultra-filtration membrane aperture is 30kDa, with A liquid (20mMPB pH7.0) balance ultra-filtration membrane, then by 10 times of 1L supernatant concentration.Concentrated solution adds 2 times of damping fluids to continue ultrafiltration, finally collects sample solution 50ml.Concentrated solution is loaded to the Blue Sepharose FF chromatography column by the abundant balance of A liquid, with the A liquid flushing of two column volumes, then uses B liquid (20mM PB+2M NaCl pH7.0) to carry out wash-out, collect elution peak component; Chelating Sepharose Fast Flow filler dress post, 0.1mol CuSO4 upper prop, Buffer A (50mmol/L Tris (pH0.5)+50mmol/LNaCL+0.1mmol/L CuSO4) balance, until detector baseline is steady.Cross filtrate that Blue Sepharose FF chromatography obtains by loading after 5 times of Buffer A dilutions.Use respectively 5 column volumes of Buffer A, Buffer B (50mmol/L Tris (pH7.5)+0.5mol/L NaCL) balance, steady to baseline.Buffer C (50mmol/L Gly (pH3.0)+0.3mol/L NaCL) wash-out.(50mmol/L EDTA's BufferD (pH8.0) regenerates to pillar.The elution peak component of Chelating Sepharose Fast Flow affinity chromatography is loaded to the fully good CM Sepharose FF cation-exchange chromatography post of balance of C liquid (25mmol/L sodium-acetate buffer pH4.5), with D liquid (25mmol/L sodium-acetate buffer+0.3mol/L NaCl pH4.5) flushing, collect elution peak component.After above purifying flow process, IFN-λ 1 purity finally obtaining (SDS-PAGE figure is shown in Fig. 2) more than 95%.
The PEG coupling of embodiment 3:IFN-λ 1L62S mutant
By the mPEG-MAL of the protein I FN-λ 1 of purifying and molecular weight 20kDa in molar ratio the ratio of 1/10-1/5 mix, in 25mM Tris-HCl damping fluid, put after 4 DEG C of reaction 10h, regulate reaction system pH to 5.0 following with termination reaction with acetum.The coupling degree of SDS-PAGE detection reaction.
The purifying of embodiment 4IFN-λ 1L62S mutant derivative
Modified outcome is carried out to SP Sepharose HP purifying, balance liquid is 25mM sodium acetate buffer (pH5.5) elutriant 25mM sodium acetate buffer, containing 1M NaCl, pH5.5, carries out linear elution from 0-100%Buffer B (referring to embodiment 2).This step chromatography object is to remove many modified proteins and unmodified protein, and the purity of IFN-λ 1 mutant that the single PEG finally obtaining modifies (is shown in Fig. 3) more than 95%.
Embodiment 5: adopt reporter gene method to measure Interferon, rabbit IFN-λ 1, the cell in vitro biologic activity of mutant IFN-λ 1 and mutant IFN-λ 1 polyethyleneglycol derivative.
Make HEK293-ISRE-Luc cell adherent growth in complete culture solution.Went down to posterity by 1: 4,2～3 times weekly, in complete culture solution, grow.Get cultured cells and discard nutrient solution, wash 1 time with PBS and digest afterwards and collecting cell, be mixed with every 1ml with mensuration nutrient solution and contain 3.5 × 10 5～4.5 × 10 5the cell suspension of individual cell.The standard solution that preparation is completed and need testing solution move into and can be used in 96 orifice plates of cell cultures and chemoluminescence microplate reader reading, and every hole adds 100 μ l, then by above-mentioned cell suspension inoculation in same 96 orifice plates, every hole 100 μ l.Under 37 DEG C, 5% carbon dioxide conditions, cultivate 19～23 hours.Supernatant liquor in careful exhaustion 96 orifice plates, adds cell pyrolysis liquid and luciferase substrate by luciferase detection kit specification sheets, measures by chemoluminescence microplate reader, records its ED50 value, records measurement result.
25 DEG C of stability test results of embodiment 6:IFN-λ 1, IFN-λ 1 mutant and IFN-λ 1mPEG-MAL modified outcome, IFN-λ 1 mutant mPEG-MAL modified outcome.
IFN-λ 1, IFN-λ 1 mutant and IFN-λ 1mPEG-MAL modified outcome, IFN-λ 1 mutant mPEG-MAL modified outcome are dialysed to 25mM pH5.0 hac buffer containing 2mM ZnCl 2in 100mM NaCl.Detect respectively above-mentioned purity of protein by SDS-PAGE electrophoresis and size-exclusion HPLC method, the stability of IFN-λ 1 mutant obviously strengthens, and the purity result of polyoxyethylene glycol Interferon, rabbit Lambda mutant is all more than 97%, and activity change is little.
125 DEG C of stability test results of table 1IFN-λ
25 DEG C of stability test results of table 2IFN-λ 1 mutant
25 DEG C of stability test results of table 3IFN-λ 1mPEG-MAL modified outcome
25 DEG C of stability test results of table 4IFN-λ 1 mutant mPEG-MAL modified outcome
From upper table result, IFN-λ 1 mutant is after L62S sudden change, and its ED50 value is less than before sudden change, active better; 25 DEG C of stability test results prove that sudden change rear stability is improved significantly.
Embodiment 6: pharmacokinetics in rats research
24 female SPF SD rats, body weight 270-290g/ only, be divided at random 4 groups, be respectively Interferon, rabbit Lambda group, Interferon, rabbit Lambda mutant group and Interferon, rabbit Lambda mPEG-MAL modified outcome group, Interferon, rabbit Lambda mutant mPEG-MAL modified outcome group, 6 every group.Animal fasting 12h before administration, can't help water.By set dosed administration and Interferon, rabbit sample through diafiltration degerming.Single subcutaneous injection administration, dosage is 200 μ g/kg body weight.After Interferon, rabbit Lambda group, the administration of Interferon, rabbit Lambda mutant group 0,5,15,30,60,120,240,720 and 1440min tail vein blood, after Interferon, rabbit Lambda mPEG-MAL modified outcome group, the administration of Interferon, rabbit Lambda mutant mPEG-MAL modified outcome group 0,0.5,1,2,4,8,12,24,48,72,96,120,144h tail vein blood.The 1ml that once takes a blood sample, blood sampling point of density is got 0.5ml.The blood sample of taking is through 5000rpm, centrifugal 8min, and separated plasma, puts-70 ° of preservations.Use Human IL-29 (IFN-lambda1) the ELISA test kit of eBioscience company to detect.Kinetica software analysis the results are shown in Table, and Cmax, the AUC value of IFN-λ 1 mutant and polyethyleneglycol derivative thereof are all corresponding to the IFN-λ 1 without sudden change.
Table 5: pharmacokinetic parameter

Claims (10)

1. IFN-λ 1 mutant, is characterized in that the Leu of the 62nd of the aminoacid sequence of described mutant sports Ser.
2. a nucleotide sequence, IFN-λ 1 mutant claimed in claim 1 is characterized in that encoding.
3. an expression vector, is characterized in that described expression vector contains nucleotide sequence claimed in claim 2.
4. cultured cells, has wherein imported expression vector claimed in claim 3, and described cell expressing is by the polypeptide of this DNA section coding.
5. a polyethyleneglycol derivative for IFN-λ 1 mutant, it is characterized in that described polyethyleneglycol modified dose be connected in described IFN-λ 1 mutant the Cys of the 15th on.
6. the polyethyleneglycol derivative of IFN-λ 1 mutant claimed in claim 5, wherein said polyethyleneglycol modified dose is mercapto-polyglycol modifier, is to have the polyol derivative that is selected from the functional group of two sulphur to pyridine, vinyl sulfone(RemzaolHuo Xingranliaohuoxingjituan), maleimide or iodo-acid amide.
7. the polyethyleneglycol derivative of IFN-λ 1 mutant described in claim 5 or 6, the molecular weight of wherein said polyethyleneglycol modified dose is 10-40kDa.
8. prepare in claim 5,6 or 7 method of the polyethyleneglycol derivative of IFN-λ 1 mutant described in any claim for one kind, it is characterized in that comprising the steps: with thiol-reactive polyvalent alcohol agent and the reaction of described polypeptide, described polypeptide has single free cysteine residues, the locus specificity covalent attachment of described polyvalent alcohol reagent and described polypeptide, form covalency thioether bond, obtain polyvalent alcohol-polypeptide conjugate, reclaim object polyvalent alcohol-polypeptide conjugate that purifying produces, to obtain final product.
9. a medicinal compositions, it is characterized in that, said composition comprises in claim 1 to 2 in IFN-λ 1 mutant described in any claim or claim 5-7 the polyethyleneglycol derivative of IFN-λ 1 mutant described in any claim, and acceptable carrier or vehicle on pharmacology.
10. IFN-λ 1 mutant described in claim 1 or 2, or the polyethyleneglycol derivative of IFN-λ 1 variant described in any claim in claim 5-7, or composition claimed in claim 9 preparation have that treatment inflammation, virus infection, bacterium infect and cancer drug in purposes.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105037523A (en) * 2015-06-25 2015-11-11 北京三元基因工程有限公司 Interferon mutant and polyethylene glycol derivative thereof
JP2018523646A (en) * 2015-07-24 2018-08-23 ハンミ ファーマシューティカル カンパニー リミテッド Method for producing bioactive polypeptide conjugate
CN108707194A (en) * 2018-04-12 2018-10-26 中国农业科学院生物技术研究所 7 interferon mutants of pig ω and its preparation method and application
CN108823138A (en) * 2018-07-06 2018-11-16 中国科学院微生物研究所 One plant of engineering bacteria and its preparing the application in fused interferon
WO2021238302A1 (en) * 2020-05-27 2021-12-02 杭州先为达生物科技有限公司 Interleukin 29 mutant protein

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007012033A2 (en) * 2005-07-20 2007-01-25 Zymogenetics, Inc. Il28 and il29 truncated cysteine mutants and antiviral methods of using same
US20070020227A1 (en) * 2005-07-20 2007-01-25 Sheppard Paul O Use of truncated cysteine IL28 and IL29 mutants to treat cancers and autoimmune disorders
WO2010059984A1 (en) * 2008-11-20 2010-05-27 Zymogenetics, Llc Il-29 mutants and uses thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007012033A2 (en) * 2005-07-20 2007-01-25 Zymogenetics, Inc. Il28 and il29 truncated cysteine mutants and antiviral methods of using same
US20070020227A1 (en) * 2005-07-20 2007-01-25 Sheppard Paul O Use of truncated cysteine IL28 and IL29 mutants to treat cancers and autoimmune disorders
WO2010059984A1 (en) * 2008-11-20 2010-05-27 Zymogenetics, Llc Il-29 mutants and uses thereof

Cited By (8)

* Cited by examiner, † Cited by third party
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CN105037523A (en) * 2015-06-25 2015-11-11 北京三元基因工程有限公司 Interferon mutant and polyethylene glycol derivative thereof
CN105037523B (en) * 2015-06-25 2019-02-19 北京三元基因药业股份有限公司 Interferon mutant and its polyethyleneglycol derivative
JP2018523646A (en) * 2015-07-24 2018-08-23 ハンミ ファーマシューティカル カンパニー リミテッド Method for producing bioactive polypeptide conjugate
CN108707194A (en) * 2018-04-12 2018-10-26 中国农业科学院生物技术研究所 7 interferon mutants of pig ω and its preparation method and application
CN108707194B (en) * 2018-04-12 2020-09-29 中国农业科学院生物技术研究所 Porcine ω7 interferon mutant and preparation method and application thereof
CN108823138A (en) * 2018-07-06 2018-11-16 中国科学院微生物研究所 One plant of engineering bacteria and its preparing the application in fused interferon
CN108823138B (en) * 2018-07-06 2020-05-01 中国科学院微生物研究所 Engineering bacterium and application thereof in preparation of fusion interferon
WO2021238302A1 (en) * 2020-05-27 2021-12-02 杭州先为达生物科技有限公司 Interleukin 29 mutant protein

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