CN105085658A - Interleukin 29 mutant and polyethylene glycol derivative - Google Patents
Interleukin 29 mutant and polyethylene glycol derivative Download PDFInfo
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- CN105085658A CN105085658A CN201410203564.5A CN201410203564A CN105085658A CN 105085658 A CN105085658 A CN 105085658A CN 201410203564 A CN201410203564 A CN 201410203564A CN 105085658 A CN105085658 A CN 105085658A
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Abstract
The invention relates to an interleukin 29 mutant. The aspartic acid at position 167 of an interleukin 29 mutates transforms into serine so that a novel interleukin 29 mutant is obtained with higher stability than the interleukin 29 before mutation. The invention also relates to uses of the mutant and of a polyethylene glycol derivative thereof.
Description
Technical field
The present invention relates to the mutant of interleukin II 9, its polyethyleneglycol derivative and preparation method thereof, and comprise the application of polyethylene glycol conjugation thing in prevention or treatment virus disease, tumour etc. of described interleukin II 9 mutant or this mutant.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, a subclass of interferon-gamma and interferon alpha is only II type Interferon, rabbit.Type iii interferon is the type cytokines family found recently, comprises interferon lambda 1, λ 2 and λ 3, also referred to as IL-28A, IL-28B and IL-29
IL-28A, IL-28B and IL-29 comprise the new protein family found recently, described protein families and I type Interferon, rabbit sequence homology, and have the homology of gene order with IL-10.This new family, at jointly all PCT application WO02/086087 and Sheppard etc., NatureImmunol.4:63-68, is described later in detail in 2003 (all including reference herein in full).Functionally, IL-28 and IL-29 is similar to I type Interferon, rabbit, all can antiviral state in inducing cell, with I type Interferon, rabbit unlike, they do not demonstrate the antiproliferative activity of some B cell system anti-.
Wild-type IL-29 (interferon lambda 1) genes encoding 200 amino acid polypeptides, this protein maturation aminoacid sequence is presented in SEQIDNO:1, and the corresponding polynucleotide of IL-29 polypeptide region as herein described of encoding, territory, motif, residue and sequence are presented in SEQIDNO: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 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 defined to 111 (Gln) by amino-acid residue 92 (Lys); Spiral E defined to 139 (Lys) by amino-acid residue 118 (Thr); And spiral F defined to 170 (Leu) by amino-acid residue 144 (Gly).
Known IL-29 molecule has 5 cysteine residues (PCT application WO02/086087WO02/02627), based on the further analyses and prediction of multiple ratio to the IL-29 carried 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 is free, 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., restriction very large in clinical treatment.Genetic engineering technique makes to synthesize human recombination protein on a large scale 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 to some extent solves instability or Immunogenicity that heterologous protein causes.
Even now, also cannot overcome the shortcomings such as the fast and bioavailability of plasma clearance is low, the result that these shortcomings cause is: need frequent injection of interferon just can reach effective plasma level and treat concentration; And, all can cause the larger fluctuation of Plasma Concentration after per injection, form peak value and the valley of drug level.So just may add the risk of medical expense and administration inconvenience and untoward reaction.Therefore, people attempt to adopt various drug delivery techniques to improve the curative effect of pharmaceutical grade protein.
The bioavailability of protein drug is usually by the restriction of plasma half-life, and responsive to proteasome degradation, hinder the technology of the application polyethylene glycol modified protein on clinical treatment to be a kind of new technology for improvement of protein drug Internal pharmacokinetics character that development in recent years is got up.It is that the peg molecule (PEG) of activation is bonded to protein molecule on the surface, thus affect the space structure of protein, finally cause the change of the various biochemical property of protein, as: chemical stability increases, opposing protease hydrolysis ability improves, immunogenicity and toxicity reduce disappearance, prolonged half-life in vivo, and plasma clearance reduces etc.
Summary of the invention
IFN-λ 1 has many places amino acid can for suddenling change, but the change of these aminoacid sequences should consider the folding of IFN-λ 1 and space structure unaffected and at least obtain the mutant that can keep being equivalent to wild-type IFN-λ 1 activity.The object of this invention is to provide and a kind ofly carry out in the 167th amino acids a kind of novel mutant IFN-λ 1 obtained that suddenlys change, the Asp of the 167th is sported Ser, this mutant is relative to wild-type IFN-λ 1, and activity is stronger, stability is better.
The invention provides the method 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 engineering bacteria accordingly, 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, reclaim and purifying in the soluble fractions of solubility Periplasmic fractions, substratum and inclusion body, any method of recovery and protein purification from substratum, tenuigenin, Periplasmic fractions and inclusion body can be used, these reclaim and purification process is known or those skilled in the art easily determines, 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.
For the technical scheme realizing taking utilizes protein engineering techniques, carry out rite-directed mutagenesis to the aminoacid sequence shown in SEQIDNO:1, the Asp of 167 is sported Ser, and concrete grammar is:
1) external site-directed mutagenesis technique is utilized to obtain the recombination of coding target protein;
2) this recombination is inserted in expression vector the recombinant plasmid obtaining codified recombinant protein, described expression vector includes but not limited to pET-23b;
3) recombinant plasmid transformed competent escherichia coli cell is obtained the engineering bacteria that Absorbable organic halogens expresses target protein, described competent escherichia coli cell includes but not limited to BL21 (DE3);
4) the engineering bacteria target protein of expressing with occlusion body form.Expression product inclusion bodies exists, expression amount accounts for 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, the free cysteine residues in polyoxyethylene glycol (PEG) reagent and IFN-λ 1 is reacted and obtain specificity conjugate, in products therefrom, PEG realizes at free cysteine pointed decoration, PEG-IFN-λ 1 mutant obtained.The IFN-λ 1 of more non-PEGization compares, and the PEG-IFN-λ 1 of gained has stability raising, reduced immunogenicity.
In general, the similar WO9412219 of PEGization method of protein (Cox and McDermott) and the method described in WO9422466 (Cox and Russell), and improve slightly, quote these two sections of documents herein for reference.Contact with excessive " sulfhydrylation PEG " containing the polypeptide of free cysteine or protein (general PEG: the mol ratio of protein or polypeptide is 1:1,5:1,10:1 and 50:1) is stirred and reacted, and in reaction conditions, preferred temperature is 4 DEG C to 37 DEG C; Preferred pH scope from 6.5 to 9.5, but can be 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 product 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, such as, and linear or branched polyvalent alcohol.Preferred polyvalent alcohol is polyoxyethylene glycol, and it is the synthesis polymer of ethylene oxide unit composition, can change ethylene oxide unit to obtain apparent molecular weight scope by size exclusion chromatography being approximately 3,000-70, the PEGization protein variants of 000Da.The size of peg moiety directly affects its circulating half-life.Therefore, size or structure by changing peg moiety are processed the protein variants with different circulating half-life and are made it for concrete treatment use or preferred dosage.
" mercaptolation PEG agent " refers to any PEG derivative reacted with the mercapto alcohol groups of cysteine residues as used in this application, these reactive PEG end groups 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 not injury protein matter.With the PEG agent that its list-methoxylation form uses, wherein only an end can be coupled.
Term " single PEGization " is defined through single peg moiety and is covalently attached to the specific site of protein and the protein modified." single PEGization " method can use 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 is to pyridine (mPEG-OPSS) or mPEG-maleimide (mPEG-MAL).The reaction that it should be noted that above-mentioned two PEG agents and IFN-λ is site-specific, is free at IFN-λ 1 respectively
15cys and other naturally occurring Cys Residue positions owing to easily forming intramolecular disulfide bond, the reaction of discord thiol-reactive PEG agent.
In the present invention, additionally provide the method for PEG-IFN-λ 1 conjugate of a kind of purifying gained of the present invention, in general, through 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 other purification process can be used, extraction as organic in two-phase or salt precipitation method.
Can carry out testing 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 reverse phase chromatography PEG polypeptide (it has larger molecular weight), carrying out amino acid sequencing subsequently 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 being used for separate mixture components, flow through weighting material (stationary phase) by mixture being put on solvent (moving phase), carrying out being separated the separation principle of chromatography is 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 the application in treatment or prophylaxis of viral diseases or in tumour 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; Bacteriological infection and cancer (such as, myelomatosis, lymphatic cancer, liver cancer, mammary cancer, melanoma, leukemia etc.).As given PEG-IFN-λ conjugate of the present invention in embodiments of the invention to hepatocellular carcinoma H22, myocarditis virus EMCV
For therepic use, technician can easily determine suitable dosage, administration frequency and route of administration.Make these factors determined and include, but not limited to the characteristic of protein used, the illness of required treatment, potential patient's complaisance, age of patient, body weight and individual reaction etc.
PEG-IFN-λ 1 conjugate of the present invention or the composition containing polyvalent alcohol-IFN-λ 1 conjugate, in conjunction with the pharmaceutical carrier be applicable to or vehicle, for the various illness for the treatment of, specific support used in these pharmaceutical compositions can take various forms, and it depends on desired administration fashion.Suitable route of administration include, but not limited to enteron aisle (such as, oral), locally, suppository, suction and parenteral route, be preferably parenteral administration, as subcutaneous, muscle or intravenous injection.
When preparing the composition of oral liquid dosage forms (such as, suspensoid, tincture, colloid or solution), typical drug media can be adopted, as water, ethylene glycol, glycerol, oil, ethanol, odorant, sanitas, tinting material etc.Similarly, when preparing oral dosage form (as tablet, capsule etc.), such as starch, carbohydrate, thinner, granulating agent, lubricant, tackiness agent, disintegrating agent etc. can be adopted.
Medicine composition for parenteral administration can be prepared into the injection type comprising effective constituent and suitable carrier.In order to parenteral administration, carrier generally includes aseptic injection water, also can comprise some other available solubility promoter or preservative component, also can prepare injection-type suspensoid in addition, in this case, will adopt suitable liquid vehicle, suspension agent etc.For parenteral administration carrier 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.
In order to topical, PEG-IFN-λ 1 conjugate of the present invention can be prepared by the hydrated matrix of gentleness, as ointment or emulsifiable paste, be suitable for ointment base if Vaseline, lanolin and water-in-oil emulsion are as Eucerin
tM, it can available from Beiersdorf (Cincinnati, Ohio); Cold cream (USP); PurposeCream
tM, can available from Johnson & Johnson (NewBrunswick, NewJersey) 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 usually with every day, weekly and monthly dosed administration, from about 0.01 μ g/kg body weight to about 50mg/kg body weight, be preferably from about 0.1 μ g/kg body weight to about 25mg/kg body weight, be more preferably from about 1 μ g/kg body weight to about 5mg/kg body weight.For the mean body weight of 75kg, dosage between every day 10 μ g and 1mg, can be more preferably between 20 μ g to 200 μ g.To modified PEG-IFN-λ, its dosage period can extend, such as, weekly or every two weeks dosage regimens.Such as, everyone every weekly dose can be 10 μ g to about 500 μ g, and in some specific embodiment, everyone every weekly dose can be about 50 μ g to about 250 μ g, and at some in other embodiment, everyone every weekly dose can be about 100 to about 200 μ g.As clear where the person skilled in the art, the specified quantitative that medicine according to the present invention forms depends on a number of factors, and includes but not limited to desired biological activity, the state of patient and the tolerance etc. to medicine.
Accompanying drawing explanation
Fig. 1: the engineering bacteria body protein abduction delivering SDS-PAGE collection of illustrative plates representing Interferon, rabbit lambda1D167S 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 representing the Interferon, rabbit lambda1D167S mutant that the Interferon, rabbit lambda1D167S mutant after purifying and PEG modify.1 swimming lane is molecular weight of albumen Marker; 2 swimming lanes are the Interferon, rabbit lambda1D167S mutant protein after purifying; 3 swimming lanes are the Interferon, rabbit lambda1D167S mutant protein that the PEG after purifying modifies.
Embodiment
Embodiment 1: the expression engineering bacteria of Interferon, rabbit lambda1D167S mutant builds and confirms with sequence
Extract the plasmid of Interferon, rabbit lambda1 as template, 167 aspartic acids (GAC) sport Serine (TCT), and first run PCR comprises two reaction systems
Reaction system primer is:
Upstream primer: 5 '-CCATATGGGTCCGGTGCCGACCTCGAAACCG-3 ',
Downstream primer:
5’-CCTCGAGTTATTATTAGGTCGATTCCGGGTGGGTCGAGGTACGCAGGCACAGATTGCCGTC-3’；
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.
PET-23b carrier and pcr amplification product are carried out NdeI/XhoI endonuclease reaction respectively, and 37 DEG C of reaction 1h, reclaim linearized vector and object fragment (about 600bp).T4DNA ligase enzyme is connected with the endonuclease bamhi of carrier PCR primer: 10 × T4DNALigaseBuffer5 μ L, PCR double digestion reclaims product 15 μ L, and plasmid double digestion reclaims product 30 μ L, T4DNALigase2 μ L, ddH2O adds to 50 μ L, and 16 DEG C are spent the night.
Ligation liquid is directly transformed BL21 (DE3) competent cell, transform above-mentioned connection product, coating ammonia benzyl is dull and stereotyped, 37 DEG C of incubated overnight.
Picking list bacterium colony is as template, and primer P1, P2 for above-mentioned design increase, and agarose gel electrophoresis detects, and positive colony occurs band at about 600bp, conforms to expection situation, the success of preliminary explanation construction of recombinant plasmid.For confirming its sequence further, carry out full-automatic sequencing with T7 universal primer by ABI377 sequenator.
Embodiment 2: the expression and purification of Interferon, rabbit lambda1D167S mutant
Be inoculated in the LB substratum containing penbritin by engineering bacteria 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 0.5mMIPTG induction, collects thalline after continuing to cultivate 4h.E. coli bl21 (DE3) accounts for the 30-50% of bacterial protein as the IFN-lambda of expression during host, mainly exists with inclusion bodies.
Fermentation thalli is washed thalline 3 times with TE solution (m:V=1:10), then carries out high-pressure homogenization fragmentation, broken condition is: 35Mpa high-pressure homogenization.After homogenate, microscopy breaks bacterium rate.When bacterial cell disruption rate about 95% (about broken bacterium 2 ~ 3 times), the centrifugal 15min of 8000rpm, collects broken bacterial sediment (SDS-PAGE figure is shown in Fig. 1).Get broken bacterial sediment and be placed in beaker, add inclusion body washings (10mMTris-HCl+1mMEDTA+0.5%Triton-X100, pH6.5, m:V=1:10), on magnetic stirrer, stir 30min, wash 3 ~ 5 times.Inclusion body is with the cracking of inclusion body lysate (7M Guanidinium hydrochloride+50mMTris-HCl+10mMDTT, pH6.5, m:V=1:10), and stirring at room temperature, spends the night.The albumen of cracking adds at a slow speed in renaturation solution (100mMTris-HCl, 0.5MArginine, 0.5%PEG3350 (m:V), 2mMGSH:0.5mMGSSG, pH8.5), and make final concentration of protein be 0.2mg/ml, 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, balances ultra-filtration membrane with A liquid (20mMPBpH7.0), then by 1L supernatant concentration 10 times.Concentrated solution adds 2 times of damping fluids and continues ultrafiltration, final collection sample solution 50ml.Concentrated solution is loaded to the BlueSepharoseFF chromatography column fully balanced with A liquid, rinses with the A liquid of two column volumes, then use B liquid (20mMPB+2MNaClpH7.0) to carry out wash-out, collect elution peak component; ChelatingSepharoseFastFlow filler dress post, 0.1molCuSO4 upper prop, BufferA (50mmol/LTris (pH0.5)+50mmol/LNaCL+0.1mmol/LCuSO4) balances, until detector baseline is steady.Cross filtrate that BlueSepharoseFF chromatography obtains dilute 5 times with BufferA after loading.BufferA, BufferB (50mmol/LTris (pH7.5)+0.5mol/LNaCL) is used to balance 5 column volumes respectively, steady to baseline.BufferC (50mmol/LGly (pH3.0)+0.3mol/LNaCL) wash-out.(50mmol/LEDTA (pH8.0) regenerates pillar BufferD.The elution peak component of ChelatingSepharoseFastFlow affinity chromatography is loaded to the CMSepharoseFF cation-exchange chromatography post fully balanced with C liquid (25mmol/L sodium-acetate buffer pH4.5), rinse with D liquid (25mmol/L sodium-acetate buffer+0.3mol/LNaClpH4.5), collect elution peak component.After above purifying flow process, the purity (see accompanying drawing 2) more than 95% of the IFN-lambda1 mutant finally obtained.
Embodiment 3: the PEG coupling of Interferon, rabbit lambda1D167S mutant
The ratio of the mPEG-MAL of the protein I FN-lambda of purifying and molecular weight 20kDa 1/10-1/5 is in molar ratio mixed, in 25mMTris-HCl damping fluid, after putting 4 DEG C of reaction 10h, regulates below reaction system pH to 5.0 with termination reaction with acetum.The coupling degree of SDS-PAGE detection reaction.
Embodiment 4: the purifying of Interferon, rabbit lambda1D167S mutant derivative
Modified outcome is carried out SPSepharoseHP purifying, balance liquid is 25mM sodium acetate buffer (pH5.5) elutriant 25mM sodium acetate buffer, containing 1MNaCl, pH5.5, carry out linear elution from 0-100%BufferB (shown in embodiment 2 BufferB).This step chromatography object is the many modified proteins of removing and unmodified protein, the purity (see accompanying drawing 3) more than 95% of the IFN-lambda1 mutant that the single PEG finally obtained modifies.
Embodiment 5: adopt reporter gene method to measure the cell in vitro biologic activity of IFN-lambda1, IFN-lambda1 polyethyleneglycol derivative, mutant IFN-lambda1 and mutant IFN-lambda1 polyethyleneglycol derivative.
Make HEK293-ISRE-Luc cell adherent growth in complete culture solution.Go down to posterity by 1: 4,2 ~ 3 times weekly, grow in complete culture solution.Get cultured cells and discard nutrient solution, wash 1 digestion and collecting cell afterwards with PBS, be mixed with the cell suspension of every 1ml containing 3.5 × 105 ~ 4.5 × 105 cells with mensuration nutrient solution.The standard solution prepared and need testing solution being moved into 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.In 37 DEG C, cultivate 19 ~ 23 hours under 5% carbon dioxide conditions.Supernatant liquor in careful exhaustion 96 orifice plate, adds cell pyrolysis liquid and luciferase substrate by Luciferase Assay Reagent box specification sheets, measures by chemoluminescence microplate reader, records its ED50 value, record measurement result.
Embodiment 6: 25 DEG C of stability test results of Interferon, rabbit Lambda1, Interferon, rabbit Lambda1 mutant and Interferon, rabbit Lambda1mPEG-MAL modified outcome, Interferon, rabbit Lambda1 mutant mPEG-MAL modified outcome
Interferon, rabbit Lambda1, Interferon, rabbit Lambda1 mutant and Interferon, rabbit Lambda1mPEG-MAL modified outcome, the dialysis of Interferon, rabbit Lambda1 mutant mPEG-MAL modified outcome are contained in 2mMZnCl2 and 100mMNaCl to 25mMpH5.0 hac buffer.Detect above-mentioned purity of protein respectively by SDS-PAGE electrophoresis and size-exclusion HPLC method, the stability of Interferon, rabbit Lambda mutant obviously strengthens, and the purity result of Peg-IFN alpha-2b Lambda1 mutant is all more than 97%, and activity change is little.
Table 1 Interferon, rabbit Lambda125 DEG C of stability test result
Table 2 Interferon, rabbit Lambda1 mutant 25 DEG C of stability test results
25 DEG C of stability test results of table 3 Interferon, rabbit Lambda1mPEG-MAL modified outcome
25 DEG C of stability test results of table 4 Interferon, rabbit Lambda1 mutant mPEG-MAL modified outcome
From upper table result, IFN-λ 1 mutant is after D167S sudden change, before its ED50 value is less than sudden change, active better; 25 DEG C of stability test results prove that sudden change rear stability is improved significantly.
Embodiment 7: pharmacokinetics in rats is studied
24 female SPF SD rats, body weight 270-290g/ only, be divided into 4 groups at random, be respectively Interferon, rabbit Lambda1 group, Interferon, rabbit Lambda1 mutant group and Interferon, rabbit Lambda1mPEG-MAL modified outcome group, Interferon, rabbit Lambda1 mutant mPEG-MAL modified outcome group, often organize 6.Before administration, animal fasting 12h, can't help water.Dosed administration and the Interferon, rabbit sample of pressing setting are degerming through diafiltration.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 LambdamPEG-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.Once take a blood sample 1ml, and blood sampling point of density gets 0.5ml.The blood sample taked is through 5000rpm, centrifugal 8min, and separated plasma, puts-70 ° of preservations.HumanIL-29 (IFN-lambda1) ELISA kit of eBioscience company is used to detect.Kinetica software analysis the results are shown in Table, and under equal conditions, Cmax and AUC value all improves mutant.
Table 5: pharmacokinetic parameter
Claims (10)
1. IFN-λ 1 mutant, is characterized in that the Asp of the 167th of the aminoacid sequence of described mutant sports Ser.
2. a nucleotide sequence, is characterized in that IFN-λ 1 mutant according to claim 1 of encoding.
3. an expression vector, is characterized in that described expression vector contains nucleotide sequence according to claim 2.
4. cultured cells, has wherein imported expression vector according to claim 3, the polypeptide that described cell expressing is encoded by this region of DNA section.
5. a polyethyleneglycol derivative for IFN-λ 1 mutant, it is characterized in that described polyethyleneglycol modified dose be connected to described IFN-λ 1 mutant the Cys of the 15th on.
6. the polyethyleneglycol derivative of IFN-λ 1 mutant according to claim 5, wherein said polyethyleneglycol modified dose is mercapto-polyglycol modifier, is to have to be selected from the polyol derivative of two sulphur to the functional group of 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 the method for the polyethyleneglycol derivative of IFN-λ 1 mutant described in any claim in claim 5,6 or 7 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 covalent thioether key, 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 the polyethyleneglycol derivative of IFN-λ 1 mutant described in any claim in IFN-λ 1 mutant described in any claim in claim 1 to 2 or claim 5-7, 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 according to claim 9 has the purposes in treatment inflammation, virus infection, bacteriological infection and cancer drug in preparation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112694526A (en) * | 2020-05-27 | 2021-04-23 | 杭州先为达生物科技有限公司 | Interleukin 29 mutant protein |
| CN112870336A (en) * | 2021-02-24 | 2021-06-01 | 杭州先为达生物科技有限公司 | Interleukin 29 mutant protein preparation |
| 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)
| 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 |
| WO2007013944A2 (en) * | 2005-07-20 | 2007-02-01 | Zymogenetics, Inc. | 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 |
-
2014
- 2014-05-14 CN CN201410203564.5A patent/CN105085658B/en active Active
Patent Citations (3)
| 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 |
| WO2007013944A2 (en) * | 2005-07-20 | 2007-02-01 | Zymogenetics, Inc. | 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 |
Non-Patent Citations (1)
| Title |
|---|
| PAUL SHEPPARD等: "IL-28, IL-29 and their class II cytokine receptor IL-28R", 《NATURE IMMUNOLOGY》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112694526A (en) * | 2020-05-27 | 2021-04-23 | 杭州先为达生物科技有限公司 | Interleukin 29 mutant protein |
| CN113150107A (en) * | 2020-05-27 | 2021-07-23 | 杭州先为达生物科技有限公司 | Interleukin 29 mutant protein |
| 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 |
| CN112870336A (en) * | 2021-02-24 | 2021-06-01 | 杭州先为达生物科技有限公司 | Interleukin 29 mutant protein preparation |
| CN112870336B (en) * | 2021-02-24 | 2022-06-14 | 杭州先为达生物科技有限公司 | Interleukin 29 mutant protein preparation |
| WO2022178922A1 (en) * | 2021-02-24 | 2022-09-01 | 杭州先为达生物科技有限公司 | Interleukin-29 mutant protein preparation |
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