CN103923209B - A kind of Lambda interferon mutant and polyethyleneglycol derivative - Google Patents
A kind of Lambda interferon mutant and polyethyleneglycol derivative Download PDFInfo
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- CN103923209B CN103923209B CN201410200460.9A CN201410200460A CN103923209B CN 103923209 B CN103923209 B CN 103923209B CN 201410200460 A CN201410200460 A CN 201410200460A CN 103923209 B CN103923209 B CN 103923209B
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Abstract
The present invention relates to a kind of Lambda interferon mutant, the 62nd of interferon lambda1 leucine is sported serine, obtain a kind of novel mutant interferon Lambda, described mutant interferon Lambda is better stability before having sudden change relatively, the invention still further relates to polyethyleneglycol derivative of this Lambda interferon mutant and application thereof。
Description
Technical field
The present invention relates to IFN-λ 1 mutant, its polyethyleneglycol derivative and preparation method thereof and the application that the polyethyleneglycol derivative comprising described IFN-λ 1 mutant or this mutant is in having prevention or the treatment medicine such as viral disease, tumor。
Background technology
Interferon is the familial cytokine that a class is important, has the antiviral of wide spectrum, cell proliferation and immunoregulation effect。
So far, having identified the interferon of 6 kinds of forms, they are divided into three big group。So-called " I type " interferon includes interferon-ALPHA, interferon beta, interferon ω, interferon δ, interferon-tau。At present, a subclass of interferon gamma and interferon-ALPHA is only II type interferon。Type iii interferon is a recently discovered type cytokines family, including 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 recently discovered new protein family, described protein families and I type interferon sequence homology, and have the homology of gene order with IL-10。This new family is described later in detail in common all of PCT application WO02/086087 and Sheppard etc., NatureImmunol.4:63-68,2003 (being all incorporated herein reference in full)。Functionally, IL-28 and IL-29 is similar to I type interferon, all can antiviral state in inducing cell, with I type interferon the difference is that, they do not demonstrate the antiproliferative activity of some B cell system anti-。
200 amino acid polypeptides of wild type IL-29 (interferon lambda 1) gene code, this protein maturation aminoacid sequence is shown in SEQIDNO:1, and the corresponding polynucleotide encoding IL-29 polypeptide region as herein described, territory, motif, residue and sequence are shown in SEQIDNO:2。The spiral prediction of IL-29 is as follows: spiral A has amino acid residue 30 (Ser) defined to 44 (Leu)；Spiral B is defined to 65 (Val) by amino acid residue 57 (Asn)；Spiral C has amino acid residue 70 (Val) defined 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 application WO02/086087WO02/02627), further analyses and prediction based on the multiple ratio IL-29 to carrying out, amino acid residue the 49th and 145, and the cysteine of the 112nd and 171 will form intramolecular disulfide bond, the cysteine of the 15th is free, it is possible to form intermolecular disulfide bond。
No matter being I type interferon, II type interferon or type iii interferon, as pharmaceutical grade protein, due to poor stability, plasma clearance height, Half-life in vivo is short, is easily generated antigen-antibody etc., restriction very big in clinical treatment。Technique for gene engineering makes synthesis human recombination protein on a large scale be possibly realized, engineered protein is by changing certain or certain the several aminoacid in wild protein sequence, to obtain the recombiant protein relatively more stable, specific activity is higher, and reducing its immunogenicity, this to some extent solves instability or the Immunogenicity that heterologous protein causes。
Despite this, plasma clearance also cannot be overcome fast and the shortcoming such as bioavailability is low, the result that these shortcomings cause is: needs frequent injection of interferon can be only achieved effective plasma level and treats concentration；And, all can cause the larger fluctuation of blood drug level after per injection, form peak value and the valley of drug level。Thus it is likely to the risk adding medical expense and administration inconvenience and untoward reaction。Therefore, the curative effect adopting various drug delivery techniques to improve pharmaceutical grade protein it is attempted to。
The bioavailability of protein drug is generally by the restriction of plasma half-life, and proteasome degradation is sensitive, and the technology hindering the application polyethylene glycol modified protein on clinical treatment is a kind of new technique for improving protein drug Internal pharmacokinetics character that development in recent years is got up。It is to be bonded on protein molecule surface by the peg molecule (PEG) of activation, thus affecting the space structure of protein, ultimately result in 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, plasma clearance reduction etc.。
Summary of the invention
IFN-λ 1 there is many places aminoacid be available for suddenling change, but the change of these aminoacid sequences is contemplated that the folding of IFN-λ 1 and space structure is unaffected and at least obtains the mutant that can keep being equivalent to wild type IFN-λ 1 activity。It is an object of the invention to provide and a kind of carry out, in the 62nd amino acids, a kind of novel mutant IFN-λ 1 obtained that suddenlys change, the Leu of the 62nd is sported Ser, this mutant is relative to wild type IFN-λ 1, and activity is higher, stability is better。
The present invention provides the method for preparation 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 degeneration and renaturation, obtained solubility IFN-λ 1 mutant。
Required protein is from soluble cell matter part, solubility Periplasmic fractions, the soluble part of culture medium and inclusion body reclaim and purification, any method of recovery and protein purification from culture medium, Cytoplasm, 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, method that chromatography (includes molecular exclusion chromatography) etc.。Reclaim the appropriate method with purification desired protein and depend in part on characteristic and the purpose purposes of protein。
Being adopted the technical scheme that utilize protein engineering techniques by realizing this purpose, the aminoacid sequence shown in SEQIDNO:1 being carried out rite-directed mutagenesis, the Leu of 62 are sported Ser, concrete grammar is:
1) external site-directed mutagenesis technique is utilized to obtain the recombination of coding destination protein；
2) this recombination is inserted in expression vector obtain codified recombiant protein recombiant plasmid, described expression vector includes but not limited to pET-23b；
3) recombinant plasmid transformed competent escherichia coli cell is obtained can the engineering bacteria of stably express destination protein, described competent escherichia coli cell includes but not limited to BL21 (DE3)；
4) fermentation separate the destination protein that purification is expressed with occlusion body form。Expression product inclusion bodies exists, expression accounts for more than the 40% of bacterial protein, the purification of recombiant protein adopts Blue dye affinity chromatography, copper ion affinity chromatograph, CM weak cation exchange chromatography three step purifying process successively, it is possible to obtain highly purified restructuring destination protein IFN-λ 1 mutant。
The individual purpose of the present invention is to provide polyethyleneglycol derivative of IFN-λ 1 mutant as above and preparation method thereof, Polyethylene Glycol (PEG) reagent and the free cysteine residues in IFN-λ 1 is made to react acquisition specificity conjugate by thiol reactant, 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 method described in the similar WO9412219 (Cox and McDermott) of PEGization method of protein and WO9422466 (Cox and Russell), and improve 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 containing free cysteine or protein contacted with excessive " sulfhydrylation PEG " reacts, and in reaction condition, 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 SDS-PAGE change measuring molecular weight can detect the PEGization of protein, it is considered that producing a large amount of single PEGization product and not producing the minimum flow of the PEG of two PEGization products is best (it is generally acknowledged 80% be transformed into list PEGization product be good)。
Cysteine of the present invention includes any suitable polymer with " peg moiety " that form " PEGization " protein, for instance, linear or branched polyhydric alcohol。Preferred polyhydric alcohol is Polyethylene Glycol, and it is the synthesis polymer of ethylene oxide unit composition, can change ethylene oxide unit will pass through size exclusion chromatography acquisition apparent molecular weight and range of about 3,000-70, the PEGization protein variants of 000Da。The size of peg moiety directly affects its circulating half-life。Therefore, can by changing the size of peg moiety or structure process the protein variants with different circulating half-life so as to for concrete treatment use or preferred dosage。
" mercaptolation PEG agent " refers to the PEG derivant that any mercapto alcohol groups with cysteine residues is reacted as used in this application, these reactive PEG end groups modified for cysteine residues, it includes but not limited to that two sulfur are to pyridine, vinyl sulfone(RemzaolHuo Xingranliaohuoxingjituan), maleimide, iodoacetamide。PEG end group reply free sulfhydryl groups has specificity, it is achieved single PEGization。But react when not injury protein matter。With the PEG agent that its list-methoxylation form uses, only one of which 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, it is preferred to mercapto-reagent be that mPEG-bis-sulfur is to pyridine (mPEG-OPSS) or mPEG-maleimide (mPEG-MAL)。It should be noted that the reaction of above-mentioned two PEG agent and IFN-λ is site-specific, be dissociating at IFN-λ 1 respectively15Cys and other naturally occurring Cys Residue positions owing to being easily formed intramolecular disulfide bond, discord thiol-reactive PEG agent reaction。
In the present invention, the method additionally providing PEG-IFN-λ 1 conjugate of a kind of purification gained of the present invention, in general, through such as dialysis, ultrafiltration, molecular-exclusion chromatography, ion-exchange chromatography, affinity chromatograph, reversed phase chromatography or hydrophobic chromatography, never purification PEGization protein conjugate in PEGization protein and unreacted PEG, it is used as other purification process, such as biphase organic extraction 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 bigger molecular weight), carrying out amino acid sequencing subsequently can complete。In amino acid sequencing process, the aminoacid of PEG coupling occurs with blank。
Term " chromatography method " or " chromatography " refer to any technology for separate mixture components, flowing through implant (fixing phase) by mixture puts on solvent (mobile phase), the separation principle being easily separated chromatography is based on the different physical characteristics of fixing phase and mobile phase。
A further object of the present invention is in that the application providing a kind of IFN-λ 1 mutant of the present invention and polyethylene glycol conjugation thing thereof to have in treatment or prophylaxis of viral diseases or in tumor in preparation, virus refers to the IFN any virus being resistant to, such as hepatitis virus, papillomavirus, herpes simplex virus, HIV, Epstein-Barr virus, coronavirus and/or influenza virus etc., preferably hepatitis virus, such as HBV or HCV；Antibacterial infects and cancer (such as, myeloma, lymphatic cancer, hepatocarcinoma, breast carcinoma, melanoma, leukemia etc.)。PEG-IFN-λ conjugate of the present invention is given to hepatocellular carcinoma H22, myocarditis virus EMCV in embodiments of the invention
For therapeutic use, technical staff is readily determined suitable dosage, administration frequency and route of administration。The factor making these decisions includes, but not limited to the characteristic of protein used, the disease of required treatment, potential patient's contingency, the age of patient, body weight and individual reaction etc.。
PEG-IFN-λ 1 conjugate of the present invention or the compositions containing polyhydric alcohol-IFN-λ 1 conjugate, in conjunction with the pharmaceutical carrier being suitable for or excipient, for treating various diseases, 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 intestinal (such as, oral), locally, suppository, suction and parenteral route, it is preferred to parenteral administration, such as subcutaneous, muscle or intravenous injection。
When preparing the compositions of oral liquid dosage forms (such as, suspensoid, tincture, colloid or solution), it is possible to adopt typical drug media, such as water, ethylene glycol, glycerol, oil, ethanol, flavour enhancer, preservative, coloring agent etc.。Similarly, when preparing oral dosage form (such as tablet, capsule etc.), such as starch, saccharide, diluent, granulating agent, lubricant, binding agent, disintegrating agent etc. can be adopted。
Medicine composition for parenteral administration can be prepared as the injection type including effective ingredient and suitable carrier。For parenteral administration, carrier generally includes aseptic injection water, it is possible to includes some other available cosolvent or preservative component, also can prepare injection-type suspensoid in addition, in this case, by liquid-carrier suitable for employing, suspending agent etc.。Know in the art for the carrier of parenteral administration and include, water, saline solution, ringer's solution and/or glucose, this carrier can comprise a small amount of excipient to keep stability and the isotonicity of medicament, and these solution can be prepared according to usual way。
For topical, PEG-IFN-λ 1 conjugate of the present invention can be prepared by gentle hydrated matrix, such as ointment or emulsifiable paste, be suitable for ointment base such as vaseline, lanoline and water in oil emulsion such as EucerinTM, it is available from Beiersdorf (Cincinnati, Ohio)；Cold cream (USP)；PurposeCreamTM, available from Johnson&Johnson (NewBrunswick, NewJersey) etc.。
PEG-IFN-λ 1 conjugate of the present invention is generally administered with the form of unit dose, the compound of the present invention is generally with every day, weekly and monthly dosed administration, from about 0.01 μ g/kg body weight to about 50mg/kg body weight, it is preferably from about 0.1 μ g/kg body weight to about 25mg/kg body weight, more preferably from about 1 μ g/kg body weight to about 5mg/kg body weight。For the average weight of 75kg, dosage can between every day 10 μ g and 1mg, more preferably between 20 μ g to 200 μ g。To modified PEG-IFN-λ, its dosage period can extend, for instance, 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, depend on a number of factors according to the specified quantitative that the medicine of the present invention forms, include but not limited to desired biological activity, the state of patient and toleration etc. to medicine。
Accompanying drawing explanation
Fig. 1: represent the engineering bacteria body protein abduction delivering SDS-PAGE collection of illustrative plates of interferon 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: represent the SDS-PAGE collection of illustrative plates of the interferon lambda1L62S mutant after purification。1 swimming lane is molecular weight of albumen Marker；2 swimming lanes are the interferon lambda1L62S mutant protein after purification；
Fig. 3: represent the SDS-PAGE collection of illustrative plates of the interferon lambda1L62S mutant modified of the PEG after purification。1 swimming lane is molecular weight of albumen Marker；2 swimming lanes are the interferon lambda1L62S mutant protein modified of the PEG after purification。
Detailed description of the invention
The expression engineering bacteria of embodiment 1:IFN-λ 1L62S mutant builds and sequence confirms
Extracting the plasmid of IFN-λ 1 as template, 62 leucines (CTG) are sported serine (TCT), first run PCR includes two reaction systems:
Reaction system 1 primer is:
P1:5 '-CCATATGGGTCCGGTGCCGACCTCGAAACCG-3 '
P2:5 '-ACGGCCGCGCGGACGCGGGCC-3 '
Expand 62 sites and DNA sequence upstream thereof。
Reaction system 2 primer is:
P3:5 '-GGCCCGCGTCCGCGCGGCCGT-3 '
P4:5 '-CCTCGAGTTATTATTAGGTCGATTCCGG-3 '
Expand 62 sites and DNA sequence downstream thereof。
PCR condition is: 94 DEG C of denaturation 5min, 94 DEG C of degeneration 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 with the product of first round PCR for template, carries out pcr amplification with P1, P2 for primer。Reaction condition is: 94 DEG C of denaturation 5min, 94 DEG C of degeneration 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 EcoRI/NdeI endonuclease reaction respectively, 37 DEG C of reaction 1h, reclaim linearized vector and purpose fragment (about 600bp)。The endonuclease bamhi of PCR primer and carrier is attached by T4DNA ligase: 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 overnight。
Coupled reaction liquid is directly converted BL21 (DE3) competent cell, converts above-mentioned connection product, be coated with ammonia benzyl flat board, 37 DEG C of incubated overnight。
Picking list bacterium colony is as template, and primer P1, P2 for above-mentioned design expand, and agarose gel electrophoresis detects, and positive colony band occurs at about 600bp, is consistent with expection situation, preliminary explanation construction of recombinant plasmid success。For further confirming that its sequence, carry out full-automatic sequencing with T7 universal primer by ABI377 sequenator。
The expression of embodiment 2:IFN-λ 1L62S mutant and purification
Engineering bacteria is inoculated in the LB culture medium containing ampicillin with the ratio of 1:100,37 DEG C of shaken cultivation to bacterium solution OD600 be 0.4-0.6 time, add final concentration of 0.5mMIPTG induction, after continuing cultivation 4h, collect thalline (Fig. 1 is shown in by the SDS-PAGE collection of illustrative plates of thalline)。The IFN-λ 1 of expression when e. coli bl21 (DE3) is as host accounts for the 30-50% of bacterial protein, mainly exists with inclusion bodies。
Fermentation thalli being washed thalline 3 times with TE solution (m:V=1:10), then carries out high-pressure homogenization and crush, 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), 8000rpm is centrifuged 15min, collects broken bacterial sediment。Take broken bacterial sediment and be placed in beaker, add inclusion body cleaning mixture (10mMTris-HCl+1mMEDTA+0.5%Triton-X100, pH6.5, m:V=1:10), on magnetic stirrer, stir 30min, wash 3～5 times。Inclusion body inclusion body lysate cracks (7M guanidine hydrochloride+50mMTris-HCl+10mMDTT, pH6.5, m:V=1:10), is stirred at room temperature, overnight。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 making final concentration of protein is 0.2mg/ml, is stirred at room temperature, overnight。
Renaturation solution 8000rpm is centrifuged 5min, collects supernatant。Utilizing ultrafiltration cup, ultrafilter membrane aperture is 30kDa, balances ultrafilter membrane with A liquid (20mMPBpH7.0), then by 1L supernatant concentration 10 times。Concentrated solution adds 2 times of buffer and continues ultrafiltration, final collection sample solution 50ml。Concentrated solution is loaded to the BlueSepharoseFF chromatographic column fully balanced with A liquid, rinses with the A liquid of two column volumes, then carry out eluting with B liquid (20mMPB+2MNaClpH7.0), collect eluting 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 loading after the filtrate that BlueSepharoseFF chromatography obtains dilutes 5 times with BufferA。5 column volumes are balanced respectively with BufferA, BufferB (50mmol/LTris (pH7.5)+0.5mol/LNaCL), steady to baseline。BufferC (50mmol/LGly (pH3.0)+0.3mol/LNaCL) eluting。(pillar is regenerated BufferD by 50mmol/LEDTA (pH8.0)。The eluting peak component of ChelatingSepharoseFastFlow affinity chromatograph 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 eluting peak component。After above purification flow process, IFN-λ 1 purity finally obtained (SDS-PAGE figure is shown in Fig. 2) more than 95%。
The PEG coupling of embodiment 3:IFN-λ 1L62S mutant
The ratio of the mPEG-MAL 1/10-1/5 in molar ratio of protein I FN-the λ 1 and molecular weight 20kDa of purification is mixed, in 25mMTris-HCl buffer, after putting 4 DEG C of reaction 10h, regulates reaction system pH to less than 5.0 to terminate reaction with acetum。The coupling degree of SDS-PAGE detection reaction。
The purification of embodiment 4IFN-λ 1L62S mutant derivative
Modified outcome is carried out SPSepharoseHP purification, balance liquid is 25mM sodium acetate buffer (pH5.5) eluent 25mM sodium acetate buffer, containing 1MNaCl, pH5.5, carry out linear elution from 0-100%BufferB (referring to embodiment 2)。This step chromatography purpose is to remove many modified proteins and unmodified protein, the purity of IFN-λ 1 mutant that the single PEG finally obtained modifies more than 95% (see Fig. 3)。
Embodiment 5: adopt reporter gene method to measure the cell in vitro biologic activity of interferon IFN-λ 1, mutant IFN-λ 1 and mutant IFN-λ 1 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。Taking cultured cells and discard culture fluid, after washing 1 time with PBS, digestion and collection cell, be configured to every 1ml containing 3.5 × 10 with mensuration culture fluid5～4.5 × 105The cell suspension of individual cell。The standard solution prepared and need testing solution being moved into and can be used in 96 orifice plates that cell is cultivated with chemiluminescence microplate reader reading, every hole adds 100 μ l, then by above-mentioned cell suspension inoculation in same 96 orifice plates, and every hole 100 μ l。In 37 DEG C, cultivate 19～23 hours under 5% carbon dioxide conditions。Supernatant in careful exhaustion 96 orifice plates, adds cell pyrolysis liquid and luciferase substrate, is measured by chemiluminescence microplate reader by Luciferase Assay Reagent box description, 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。
By IFN-λ 1, IFN-λ 1 mutant and IFN-λ 1mPEG-MAL modified outcome, the dialysis of IFN-λ 1 mutant mPEG-MAL modified outcome to 25mMpH5.0 hac buffer containing 2mMZnCl2With in 100mMNaCl。Detecting above-mentioned purity of protein respectively by SDS-PAGE electrophoresis and size-exclusion HPLC method, the stability of IFN-λ 1 mutant is remarkably reinforced, and the purity result of Peg-IFN alpha-2b Lambda mutant is all more than 97%, and activity change is little。
125 DEG C of stability test results of table 1IFN-λ
Table 25 DEG C of stability test results of 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 it can be seen that IFN-λ 1 mutant is after L62S suddenlys change, before its ED50 value is less than sudden change, activity is better；25 DEG C of stability test results prove that sudden change rear stability is improved significantly。
Embodiment 6: pharmacokinetics in rats is studied
24 female SPF SD rats, body weight 270-290g/ is only, it is randomly divided into 4 groups, respectively interferon Lambda group, interferon Lambda mutant group and interferon LambdamPEG-MAL modified outcome group, interferon Lambda mutant mPEG-MAL modified outcome group, often group 6。Animal fasting 12h before administration, can't help water。Dosed administration and interferon sample by setting are degerming through diafiltration。Single subcutaneous injection is administered, and dosage is 200 μ g/kg body weight。After the administration of interferon Lambda group, interferon Lambda mutant group 0,5,15,30,60,120,240,720 and 1440min tail vein blood, after the administration of interferon LambdamPEG-MAL modified outcome group, interferon 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 off density takes 0.5ml。The blood sample taked, through 5000rpm, centrifugal 8min, separated plasma, puts-70 ° of preservations。Use HumanIL-29 (IFN-lambda1) the ELISA kit detection of eBioscience company。Kinetica software analysis result homogeneously should be higher than that not mutated IFN-λ 1 in Table, IFN-λ 1 mutant and the Cmax of polyethyleneglycol derivative, AUC。
Table 5: pharmacokinetic parameter
Claims (10)
1. IFN-λ 1 mutant, it is characterised in that the aminoacid sequence of described mutant is that the Leu of the 62nd of aminoacid sequence shown in SEQIDNO:1 the is sported Ser。
2. a nucleotide sequence, it is characterised in that coding IFN-λ 1 mutant described in claim 1。
3. an expression vector, it is characterised in that described expression vector contains the nucleotide sequence described in claim 2。
4. cultured cells, has wherein had been introduced into the expression vector described in claim 3, and described cell is expressed by the polypeptide of this region of DNA section coding。
5. the polyethyleneglycol derivative of IFN-λ 1 mutant, it is characterised in that described polyethyleneglycol modified dose be connected to IFN-λ 1 mutant described in claim 1 the Cys of the 15th on。
6. the polyethyleneglycol derivative of IFN-λ 1 mutant described in claim 5, wherein said polyethyleneglycol modified dose is mercapto-polyglycol dressing agent, is have selected from two sulfur polyethyleneglycol derivative to the functional group of pyridine, vinyl sulfone(RemzaolHuo Xingranliaohuoxingjituan), maleimide or iodoacetamide。
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 claim 5 for one kind, the method of the polyethyleneglycol derivative of IFN-λ 1 mutant described in any one claim in 6 or 7, it is characterized in that comprising the steps: with thiol-reactive polyethylene glycol reagents and the reaction of described IFN-λ 1 mutant, described IFN-λ 1 mutant has single free cysteine residues, the locus specificity covalent bond of described polyethylene glycol reagents and described IFN-λ 1 mutant, form covalent thioether key, obtain Polyethylene Glycol-IFN-λ 1 mutant conjugate, reclaim the purpose Polyethylene Glycol-IFN-λ 1 mutant conjugate that purification produces, obtain。
9. a Pharmaceutical composition, it is characterized in that, said composition includes in claim 1 to 2 IFN-λ 1 mutant described in any one claim or the polyethyleneglycol derivative of IFN-λ 1 mutant described in any one claim in claim 5-7, and acceptable carrier or excipient on materia medica。
10. IFN-λ 1 mutant described in claim 1, or the polyethyleneglycol derivative of IFN-λ 1 variant described in any one claim in claim 5-7, or the compositions described in claim 9 preparation have treatment inflammation, viral infection, antibacterial infect and cancer drug in purposes。
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| JP6987741B2 (en) * | 2015-07-24 | 2022-01-05 | 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 Physiologically Active Polypeptide Bond |
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Patent Citations (2)
| 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 |
| WO2010059984A1 (en) * | 2008-11-20 | 2010-05-27 | Zymogenetics, Llc | Il-29 mutants and uses thereof |
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