CN104045704B - PEGization recombined human IFN-λ 1, Preparation Method And The Use - Google Patents
PEGization recombined human IFN-λ 1, Preparation Method And The Use Download PDFInfo
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- CN104045704B CN104045704B CN201310076833.1A CN201310076833A CN104045704B CN 104045704 B CN104045704 B CN 104045704B CN 201310076833 A CN201310076833 A CN 201310076833A CN 104045704 B CN104045704 B CN 104045704B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—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
- A61K47/59—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
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention provides a kind of PEGization recombined human IFN λ 1, Preparation Method And The Use.Specifically, present invention achieves the PEG pointed decoration to recombined human IFN λ 1, present invention also offers the thus obtained PEGization recombined human IFN λ 1 purposes in the medicine of preparation treatment chronic hepatitis B or hepatocarcinoma.The homogeneity of prepared according to the methods of the invention PEGization recombined human IFN λ 1 is good, and purity is more than 98%.PEGization recombined human IFN λ 1 can significantly inhibit the growth of tumor bearing nude mice Subcutaneous tumor, significantly reduces HBsAg content in nude mouse serum, shows preferable safety simultaneously.
Description
Technical field
The present invention relates to biological technical field.More specifically, relate to one and pass through recombinant technique
The interferon that obtains with chemical modification technology, and its production and use.Particularly relate to one
Plant the recombined human IFN-λ 1 of pegylation, its preparation method and treat chronic in preparation
Purposes in the medicine of hepatitis B or hepatocarcinoma.
Background technology
Interferon-λ s(IFN-λ s) it is a class new forms of interferon, classification is set to type iii interferon,
By IFN-λ 1(IL-29), IFN-λ 2(IL-28A) and IFN-λ 3(IL-28B) form.2003
Year by American scientist reported first this type interferon (Sheppard P. et al., 2003；
Kotenko1S.V. et al., 2003).
IFN-λ gene is positioned at No. 19 chromosomes, and containing multiple introns in gene, its gene is tied
Structure is quite similar with IL-10 family member, and differs greatly with IFN-α.On aminoacid forms,
The aminoacid of IFN-λ s also only 15-19% is identical with IFN-α.While it is true, IFN-λ s exists
Functionally quite similar with IFN-α, hepatitis B/hepatitis C virus can be suppressed
(HBV/HCV) duplication, it is also possible to suppress encephalomyocarditis virus (EMCV), people big and small
Cellular virus (CMV), lymphocytic choriomeningitis virus (LCMV), herpes simplex
The activity (Bobek M.D. et al., 2005) of the viruses such as virus (HSV-2).Except antiviral
Outside activity, in cytologic experiment, IFN-λ s can suppress neuroendocrine tumor, the esophageal carcinoma,
Colon cancer, pulmonary carcinoma and Burkitt ' s lymphoma cell and the propagation of melanoma cell, and
Useful mIFN-λ s suppresses mouse tumor cell by the immunologic mechanism of transfer host in Mice Body
As melanoma, fibrosarcoma and hepatoma cell proliferation report (Abushahba W. et al.,
2010).
The receptor of IFN-λ s belongs to II type cytokines receptor family, the allos two special by structure
Dimeric complexes forms.IL-28R α is aglucon binding subunit, determines receptor and is combined with IFN-λ
Specificity, IL-10 β R is auxiliary subunit.I type IFN(IFN-α/β with wide expression) it is subject to
Body is different, and the expression of IFN-λ receptor has cell-specific, is mainly expressed in surface epithelial cell,
The such as all cells in liver all expresses the receptor of IFN-α, and the receptor of IFN-λ is only liver
Cell is expressed.T lymphocyte, bone-marrow-derived lymphocyte, NK is included equally in peripheral blood lymphocyte
All wide expression IFN-α receptors such as cell, neutrophilic granulocyte and mononuclear cell, but it is in addition to B
The external hematopoietic cell of lymphocyte is not detected by expression (the Muir A.J. etc. of IFN-λ receptor
People, 2010).The diversity of this different IFN family receptors distribution will affect IFN in vivo
Effect, with I type IFN commonly used clinically at present toxic and side effects in treatment and tolerance
Property difference compare, IFN-λ s has potential advantage, it is possible to playing antiviral, antitumor and ginseng
With bioactive poison simultaneously effective reduced hemopoietic and central nervous system such as immunomodulating
Side effect.
But similar with I type IFN, IFN-λ s relative molecular mass less (about 20kD),
It is prone to by glomerular filtration；Internal instability, is easily easily degraded by proteases, and plasma half-life is short；
Treatment cycle is long, needs frequently injection, causes the interdependence of patient to reduce；As Heterologous System
The Recombinant protein biological preparation expressed there is also Immunogenicity.All these it is likely to
Weaken the clinical effectiveness of IFN-λ s.
Polyethylene Glycol (polyethylene glycol, PEG) be a kind of avirulence, non-immunogenicity,
The polymer of safety.With PEG chain covalent coupling modifying protein be from the seventies in last century with
Develop polypeptide and the technology of pharmaceutical grade protein of carrying the most in vivo.PEG modifies egg
White matter benefit in treatment includes:
-add protein molecular weight, reduce glomerular filtration rate thus extend blood plasma and partly decline
Phase；
-add protein physical and chemical stability, reduce the degraded of protease；
-reduce toxicity and limit the immunogenicity of protein；
-add the solubility of pharmaceutical grade protein；
-by improving pharmaceutical grade protein dynamic performance, compared with unmodified protein matter medicine, have
Enhance medicine activity (Veronese FM et al., 2005) in vivo to effect.To protein
PEGization method of modifying mainly use monomethoxyPEG molecule (mono-methoxy
PEG, mPEG) and protein surface reactive group covalent coupling, its main modification approach
It is that N-terminal amino or lysine side chain amino groups are carried out acylated modification, but this modification mode
Easily produce heterogeneity (i.e. isomer mixture).
Hepatocarcinoma is one of modal malignant tumor, worldwide in kinds of tumor mortality rate
Rank the 3rd.The whole world has more than 60 million new every year and increases liver cancer patient, the trouble of the most about 55%
Person is in China.It is the most important danger of induced hepatocellular carcinoma that chronic HBV (HBV) infects
Factor, the liver cancer patient in China about 80% have HBV infection history (Yang, J.D et al.,
2010；Parkin, D.M. et al., 2005).IFN-α for Anti-HBV activity treatment clinically by
It is considered effective, is also advantageous for the hepatocarcinoma preventing HBV infection relevant.But the most still have
IFN-α is treated nonreply by the Patients with Chronic HBV Infection of significant proportion, and patient Chang Yinwei goes out
The toxicity, side effect being now difficult to tolerate includes fatigue, heating, anorexia, depression and bone marrow depression
Deng and interrupt the treatment of IFN-α.IFN-λ s especially IFN-λ 1(IL-29) can be by liver
The IFN-λ receptor of cell surface specifically expressing excites antiviral activity, suppresses HBV virus replication,
Prompting IFN-λ 1 may be used for treating the relevant hepatocarcinoma of chronic HBV infection and HBV infection and suffers from
Person (Doyle SE. et al., 2006).
Summary of the invention
According to an aspect of the present invention, it is provided that a kind of PEGization recombined human IFN-λ 1(is also referred to as
PEG-rhIFN-λ 1).Its aminoacid composition and wild type human IFN-λ 1 close, and have with
The biological activity that wild type human IFN-λ 1 is identical.
The aminoacid sequence of above-mentioned PEGization recombined human IFN-λ 1 is as shown in SEQ ID No.1.
Being made up of 181 aminoacid, the 46th, its N end is glutamine.The half Guang ammonia of the 112nd
Acid residue (being called for short C112) is that PEGization is modified.
RhIFN-λ 1 provided by the present invention is the low-saccharification sudden change having lacked N-glycosylation site
Body IFN-λ 1, the 46th, its N end is glutamine, by Pichia sp. (Pichia pastoris)
System is expressed, its aminoacid sequence, special secretion expression's carrier, transgenic cell line and place
Main bacterium is disclosed in Publication No. CN10235192A Chinese patent application, the artificial China of application
Academy of Medical Sciences Institute of Basic Medical Sciences.
RhIFN-λ 1 provided by the present invention is produced from thermophilic methanol yeast-Pichi strain, finishes
Red yeast has many advantages of protokaryon and eukaryotic expression system concurrently, simultaneously can be by special letter
Number peptide guides and forms solubility secreting, expressing, makes recombinant exogenous protein form correct folding.This
The method of bright used Pichia anomala expression and purification has been disclosed in Authorization Notice No. CN
In 1962873B Chinese patent, patentee is Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.
In some embodiments, the PEG-rhIFN-λ 1 of the present invention, wherein said PEG are
Straight or branched structure.
In some embodiments, described PEG molecular weight is in the range of 20～40 kilodaltons；
Preferably 20 kilodaltons.
In some embodiments, described PEG is selected from maleimide-PEG
(mPEG-MAL), vinyl sulfone-PEG(mPEG-VS), two thiopyridines-PEG
(mPEG-Pyridyl disulfides) and iodoacetamide-PEG(mPEG-iodoacetamide).
In some specific embodiments, described PEG is mPEG-MAL.
According to a further aspect in the invention, additionally provide the preparation method of PEG-rhIFN-λ 1,
Including step:
Recombined human IFN-λ 1 is provided；
PEG is added in recombined human IFN-λ 1；
Coupling reaction is carried out 12-30 hour at 2-8 DEG C, then carries out 1-3 hour at 18-25 DEG C；
By cation-exchange chromatography, coupling product carried out purification for the first time；
Second time purification is carried out by gel permeation chromatography；
Results PEGization recombined human IFN-λ 1.
In a specific embodiment, coupling reaction is carried out 24 hours at 4 DEG C, then at 18-25
DEG C carry out 2 hours.
In some embodiments, described PEG is selected from maleimide-PEG
(mPEG-MAL), vinyl sulfone-PEG, two thiopyridines-PEG and iodoacetamide-PEG.
In a specific embodiment, PEG is mPEG-MAL.
In some embodiments, described PEG molecular weight is 20～40 kilodaltons.One
In individual specific embodiment, PEG be molecular weight be 20 kilodaltons.
In a specific embodiment, the molecule mol ratio of recombined human IFN-λ 1 and PEG is
L:5.
Analyzed prepared by said method by series connection level Four bar time of-flight mass spectrometer
PEG-rhIFN-λ 1, compares with the rhIFN-λ 1 modified without PEG, and result shows
The conjugation sites of mPEG-MAL is positioned on the C112 of rhIFN-λ 1, is pointed decoration.
In accordance with a further aspect of the present invention, it is provided that PEG-rhIFN-λ 1 is chronic in preparation treatment
Purposes in the medicine of hepatitis B or hepatocarcinoma.
In some embodiments, PEG-rhIFN-λ 1 is administered alone or with co-administered.One
In a little embodiments, PEG-rhIFN-λ 1 be administered alone or with other medicines such as chemotherapeutic drugs
Conjunction is used.Chemotherapeutics is such as but not limited to doxorubicin, oxaliplatin, 5-FU or Asia
Calcium Folinate-SF.In some specific embodiments, PEG-rhIFN-λ 1 is administered alone.At another
In a little specific embodiments, PEG-rhIFN-λ 1 is co-administered with doxorubicin.
In a particular embodiment, the PEG-rhIFN-λ 1 of the present invention is B-mode at continuous release
The tumor bearing nude mice that the hepatoma cell strain Hep3B of hepatitis virus surface antigen (HBsAg) is built moves
Object model significantly inhibits the growth of tumor bearing nude mice Subcutaneous tumor, significantly reduces in nude mouse serum
HBsAg content.
Accompanying drawing explanation
Fig. 1: PEG modification reaction mixture isolated and purified.
A:CM Sepharose F.F. cation-exchange chromatography graphy figure；Peak 1.
mPEG-MAL-rhIFN-λ1；Peak 2.rhIFN-λ 1；
B:SDS-PAGE schemes, M. protein markers；1 road .PEG reactant mixture；
2 road .mPEG-MAL-rhIFN-λ 1(eluting peaks 1)；3 road .rhIFN-λ 1(eluting peaks 2).
The SDS-PAGE testing result of Fig. 2: PEG-rhIFN-λ 1.
A:SDS-PAGE coomassie brilliant blue staining；M. molecular weight protein marker；1 road.
rhIFN-λ1；Shown in 2 road .mPEG-MAL-rhIFN-λ 1(arrows).
B:SDS-PAGE barium-iodine staining；M. molecular weight protein marker；1 road .rhIFN-
λ1；Shown in 2 road .mPEG-MAL-rhIFN-λ 1(arrows).
Fig. 3: ISRE luciferase reporter gene experiment detection PEG-rhIFN-λ's 1 is external
Activity.
The vitro stability of Fig. 4: PEG-rhIFN-λ 1 and activity.
A1:BCA method measures the content of albumen, rhIFN-λ 1(◇ after 37 DEG C hatch),
PEG-rhIFN-λ 1(△)；
Rear rhIFN-λ 1(◇ is hatched in A2: luciferase reporter gene experiment detection 37 DEG C) and
PEG-rhIFN-λ 1(△) activity；
B1:ELISA detection rhIFN-λ 1(◇ after jointly hatching with rat blood serum) and
PEG-rhIFN-λ 1() content；
B2: luciferase reporter gene experiment detection rhIFN-after jointly hatching with rat blood serum
λ 1(◇) and PEG-rhIFN-λ 1() activity.
Fig. 5: PEG-rhIFN-λ 1 and rhIFN-λ 1 Pharmacokinetics in Rat curve.
Fig. 6: PEG-rhIFN-λ 1 is for the hepatic carcinoma growth curve of tumor bearing nude mice treatment, * P
＜ 0.05；* P ＜ 0.01.
Fig. 7: PEG-rhIFN-λ 1 is for the serum HBsAg-ELISA of hepatocarcinoma tumor bearing nude mice treatment
Testing result, * * P ＜ 0.01.
The Mass Spectrometric Identification of Fig. 8: PEGization decorating site.
A:PEG-rhIFN-λ 1, sequence IQPQPTAGPRPR；
B:rhIFN-λ 1, sequence C IQPQPTAGPRPR.
Detailed description of the invention
Below in conjunction with specific embodiment, embodiments of the present invention and concrete operating process are carried out
Detailed elaboration, it is to be understood that protection scope of the present invention is not limited to following embodiment.Under
State method therefor in embodiment and be conventional method if no special instructions.The use of test kit according to
The description that manufacturer or supplier are provided is carried out.
Embodiment 1, the acquisition of rhIFN-λ 1
Prepare the secretion expression's carrier used by low-saccharification mutant rhIFN-λ 1, transgenic cell
System, Host Strains, fermentation expression and method for purifying proteins are all in Publication No. CN10235192A
Chinese patent application has a detailed description.
Embodiment 2, the preparation method of PEG-rhIFN-λ 1
RhIFN-λ 1 albumen 25mM Tris-HCl pH8.0's embodiment 1 obtained is slow
Rushing liquid dialysis equilibrium, rhIFN-λ 1 concentration adjusts to 1mg/ml；
In the ratio that molecule mol ratio is l:5 of rhIFN-λ 1 and PEG, add PEG powder (20
KD straight chain mPEG-MAL, is provided by Beijing Kai Zheng biological engineering Development Co., Ltd)；
It is gently mixed and makes powder dissolve；
After 4 DEG C of reaction 24h, then room temperature (18-25 DEG C) reaction 2h obtains coupling reaction product；
Above-mentioned coupling reaction product is carried out cation-exchange chromatography: by above-mentioned coupling reaction product
Add buffer A (25mM NaAC, the pH4.5) dilution of 5 times of volumes；It is splined on afterwards
(GE Healthcare Life Sciences carries the cation-exchange chromatography post of buffered liquid A balance
The CM Sepharose F.F of confession) on；Eluting uses (25mM from buffer A to buffer B
NaAC, 0.5M NaCl, pH4.5) linear elution (0～100%)；Collect each eluting peak；
The eluting peak of above-mentioned collection is carried out gel permeation chromatography: buffer is 50mM PBS,
150mM NaCl, pH7.0, chromatographic column is Superdex75, Hiload16/60 prepacked column (GE
Healthcare Life Sciences Products)；
The PEG-rhIFN-λ 1 of results purification.
Embodiment 3, Homogeneity Test
Above-mentioned cation-exchange chromatography separate result as it is shown in figure 1, through PEG modify rhIFN-
λ 1 homogeneity good (Fig. 1, B, swimming lane 1), this shows that the preparation method of the present invention not only carries
For single modification mode, and the rhIFN-λ 1(of unmodified can be schemed by cation-exchange chromatography
1, B, swimming lane 3) PEG-rhIFN-λ 1(Fig. 1 mono-modified with 20kD mPEG-MAL,
B, swimming lane 2) separate (Fig. 1, A) well.
Embodiment 4, purity check
Purified product embodiment 2 obtained, after SDS-PAGE electrophoresis, is examined respectively
The dyeing of Maas light blue and barium-iodine staining detection.
Can directly detect PEG with barium-iodine staining dyeing, method is: SDS-PAGE electrophoresis is tied
Shu Hou, by the soak 30min in the barium chloride solution of 5% after distilled water drip washing, uses
Distilled water barium chloride is washed off after by soak 30min in 0.1mM iodine solution, the most again
Decolour with distilled water.
Testing result is shown in Fig. 2, and wherein the A of Fig. 2 is coomassie brilliant blue staining, and swimming lane 1 is for dividing
Son amount is about the rhIFN-λ of 20kD；Swimming lane 2 is modified for 20kD mPEG-MAL
PEG-rhIFN-λ1；The B of Fig. 2 is barium-iodine staining, it is seen that only PEG-rhIFN-λ 1 can
To develop the color (Fig. 2, B, swimming lane 2)；Testing result shows, PEG-rhIFN-after purification
λ 1 purity is more than 98%.
Embodiment 5, the qualification in PEGization site
The PEG-rhIFN-λ 1 that embodiment 2 purification is obtained and the rhIFN-λ 1 of unmodified, warp
After trypsin degradation, carry out level Four bar time of-flight mass spectrometer analysis of connecting, collect spectrogram,
With all polypeptide fragments produced through trypsin degradation by wild type IFN-λ 1 in data base
Theoretical value compares, PEG modify and unmodified two samples in, all capture containing C15,
The polypeptide fragment of C49, C145 and C171, two samples do not have difference.Analysis trypsin drops
Solution collection of illustrative plates shows, the polypeptide piece segment length containing C112, when molecular weight is more than series connection level Four bar flight
Between the scope that can catch of mass spectrograph, point out the analysis result after only trypsin degradation still can not
Clearly judge that PEG is that pointed decoration is on C112 site.
To this end, the rhIFN-λ 1 of PEG-rhIFN-λ 1 and unmodified is again through proteinase K digestion,
Row series connection level Four bar time of-flight mass spectrometer is analyzed, and catches in rhIFN-λ 1 sample of unmodified
To the polypeptide fragment containing C15, C49 and C112, wherein contain the polypeptide fragment ammonia of C112
Base acid sequence is " CIQPQPTAGPRPR ", and in PEG-rhIFN-λ 1 sample except
Capture outside the polypeptide fragment containing C15, C49, also capture the polypeptide piece adjacent with C112
Section, its aminoacid sequence is " IQPQPTAGPRPR ".Comprehensive through trypsin and protease
K degrade respectively after mass spectrometry results, in rhIFN-λ 1 sample of unmodified, contain
5 polypeptide fragments of Cys are all caught in, and in PEG-rhIFN-λ 1 sample, only catch
Arrive other 4 in addition to the C112 polypeptide fragments containing Cys, shown owing to C112 position has
The modification of 20kD mPEG-MAL, molecular weight is far longer than series connection level Four bar flight time mass spectrum
The scope that instrument can capture, therefore can confirm that mPEG-MAL pointed decoration is in C112 position
Upper (result is shown in Fig. 8).
A total of 5 Cys residues in the aminoacid sequence of rhIFN-λ 1, but mPEG-MAL
Modification be located only within rhIFN-λ 1 aminoterminal and rise on the 112nd cysteine residues, this shows
The modification homogeneity of the PEG-rhIFN-λ 1 of the present invention is high, homogeneity is good, is that a kind of fixed point is repaiied
Decorations.
Embodiment 6, the external activity of PEG-rhIFN-λ 1
Use pISRE-luciferase reporter gene to survey the method for living and measure the external of PEG-rhIFN-λ 1
Activity.
PISRE-luciferase reporter gene surveys the principle of the method for living: interferon is special with cell surface
Property receptor combine after, enabling signal cascade reaction, cause interference with element the stimulated gene factor (ISGF,
IFN-stimulated gene factor) enter nucleus and cis element IFN-stimulated response element
(ISRE, IFN-stimulated response element) interacts, thus regulator gene
Transcribe, the final antiviral producing interferon mediation, anti-tumour cell proliferative and immunomodulating
Biological effect (Williams BR, 1991).
The present embodiment is assembled into luciferase reporter plasmid pISRE-TA-luc with ISRE,
After being stimulated by detection interferon, the expression of the luciferase reporter gene of ISRE regulation and control can
To reflect the biological activity of interferon.
Concrete grammar is as follows: collect logarithmic (log) phase HepG2 cell (hepatoma cell strain), adjusts thin
Born of the same parents' suspension concentration, with 2 × 105Individual/hole inoculating cell to 6 orifice plates, 37 DEG C, 5%CO2, incubate
Educate overnight；PISRE-TA-luc(5ug is transfected with lipofectamine2000) and pRL-SV40
(0.1ug, internal reference) plasmid, 37 DEG C, 5%CO2, hatch 24 hours, be replaced with serum-free
Hungry 2～4 hours of culture medium；Add rhIFN-λ 1, PEG-rhIFN-λ 1 the most respectively
Each 100ng/ml, collects cell after continuing to cultivate 24 hours, examines with luciferase reporter gene
Test agent box (Promega Products) is in TD20/20 light-emitting appearance readings respectively, record inspection
Survey result, using the ratio of each time point Luciferase/Renilla as the table of ISRE reporter gene
Reach level.
The result of PEG-rhIFN-λ 1 external activity experiment is shown in that Fig. 3, PEG still retain after modifying
The activity of 28%, illustrates that rhIFN-λ 1 is really by PEGization.
Embodiment 7, the vitro stability of PEG-rhIFN-λ 1
Thermal stability analysis in aqueous solution: by rhIFN-λ 1, PEG-rhIFN-λ 1 desalination to 20mM
In PBS, pH7.2 solution, the concentration of two albumen is all adjusted to 400 μ g/ml, is placed in 37
DEG C, in 0h, 4h, 8h, 24h, 48h, 72h sample, and 10,000rpm are centrifuged 20min
After take supernatant BCA method (test kit is Pierce Products) detection residual protein content；
Utilize pISRE-luciferase reporter gene to survey the residual activity of method analyzing proteins of living, result simultaneously
Represent with residual protein content and activity percent.
PEG-rhIFN-λ 1 thermal stability results in aqueous is shown in Fig. 4, A1 and A2, not
Through PEG modify rhIFN-λ 1 at 37 DEG C of 8h after residual protein content drop to 40%, 24
After h, protein content drops to 20%, and PEG-rhIFN-λ 1 contains at whole observation period residual protein
Amount does not significantly reduce with former protein content, stills remain in 90%(Fig. 4, A1 after 3 days).
And the biological activity of ISRE luciferase reporter gene reflection remains to be retained in more than 75%, and not
The biological activity retained through the rhIFN-λ 1 of PEG modification is then less than 30%(Fig. 4, A2).
Above-mentioned experimental result illustrates that PEG modifies recombiant protein stability enhancing after rhIFN-λ 1 really,
The biological activity retention time extends.
Thermal stability analysis in serum: by rhIFN-λ 1, PEG-rhIFN-λ 1 desalination to 20mM
In PBS, pH7.2 solution, two albumen are adjusted to same concentrations, add isopyknic SD
Rat blood serum, is placed in 37 DEG C, and in 0h, 4h, 8h, 12h, 24h, 48h, 72h sample,
10,000rpm takes supernatant IL-29ELISA test kit after being centrifuged 20min (ebioscience is public
Department's product) detection by quantitative residual protein content；Utilize pISRE-luciferase reporter gene simultaneously
Surveying the residual activity of method analyzing proteins of living, result represents with residual protein content and activity percent.
PEG-rhIFN-λ 1 thermal stability results such as shown in Fig. 4, B1 and B2 in serum, nothing
Opinion is residual protein content or ISRE reporter gene activity all has a certain degree of decline, but
Compared with the rhIFN-λ 1 of unmodified, after 3 days, residual protein content and ISRE reporter gene are biological
Activity is all positively retained at more than 40%, far above non-PEGization rhIFN-λ 1, show PEG and
The coupling of rhIFN-λ 1 significantly increases the heat stability of rhIFN-λ 1 and is delayed egg in serum
The white enzyme degraded to rhIFN-λ 1.
Embodiment 8, PEG-rhIFN-λ 1 pharmacokinetics in rats are studied
Experiment uses SD rat, female, body weight 270-290g, random packet, often group 3,
Cage is divided to feed, animal fasting 12h before being administered；List is given respectively with the dosage of 200 μ g/kg body weight
Secondary subcutaneous injection rhIFN-λ 1 and PEG-rhIFN-λ 1；Tail venous blood sampling after administration, takes the blood time
Be respectively as follows: rhIFN-λ 1 injection after 0,5min, 10min, 0.5h, 1h, 2h, 3h, 4h,
12h、24h；PEG-rhIFN-λ 1 injection after 0,0.5h, 1h, 2h, 4h, 8h, 12
h、24h、48h、72h、96h、120h、144h；Blood sample through 5,000rpm, 10min
Take blood plasma fractions after Li Xin, put-70 DEG C of preservations, use ELISA(IFN-lambda1Platinum
ELISA, ebioscience company) detect the content of IFN-λ 1 in blood plasma.Soft with Kinetica
Part carries out curve fitting, and calculates various pharmacokinetic parameter.Detection data acquisition Excel software
Statistics, statistical result represents with average ± standard deviation (x+SD).Result is shown in Fig. 5 and Biao 1:
The pharmacokinetic parameter of table 1.rhIFN-λ 1 and PEG-rhIFN-λ 1
| PK parameter | RhIFN-λ 1(x ± SD) | PEG-rhIFN-λ (x ± SD) |
| AUC(ng h/ml) | 160.72±25.36 | 2082.16±203.44 |
| CMaximum(ng/ml) | 54.05±4.21 | 69.18±4.58 |
| TMaximum(h) | 0.50±0.04 | 7.35±2.10 |
| t1/2(h) | 1.46±0.25 | 15.70±0.77 |
| CL(ml/h/kg) | 417.49±65.18 | 31.91±3.26 |
Note: AUC: area under curve；CMaximum: maximum peak concentration；TMaximum: peak time；t1/2:
Eliminate the half-life；CL: elimination factor.
Result shows: through kinetica software comparision of goodness of fit, rhIFN-λ 1 and PEG-rhIFN-
λ 1 all meets a compartment model of first order absorption at SD rat physiological disposition.With without PEG
The rhIFN-λ 1 modified compares, and after PEG-rhIFN-λ 1 subcutaneous injection, the maximum peak in blood plasma is dense
Degree (CMaximum) improve, peak time (TMaximum) and eliminate half-life (t1/2) significantly extend,
It is respectively 1.28 times of rhIFN-λ 1,14.7 times and 10.75 times；Area under the drug-time curve is notable
Increasing (for 12.9 times of rhIFN-λ 1), elimination factor (CL) reduces 92.4%.These
Data show, rhIFN-λ 1 can improve rhIFN-λ 1 at body effectively through the modification of PEG
Interior absorption and distribution, it is possible to extend its biological half-life significantly, improve rhIFN-λ 1 at body
Interior bioavailability.
Embodiment 9, PEG-rhIFN-λ 1 are to the safety of hepatocarcinoma Xenograft Tumor Models under fell
Property and pharmacodynamic evaluation research
Experiment uses human hepatoma cell strain Hep3B, and this cell strain has HBV gene group to integrate,
Sustainable generation HBsAg, can be as cell model (the Chen HC. observing Anti-HBV drugs curative effect
Et al., 1997).
Experimental technique: BALB/c nude mice dorsal sc inoculation Hep3B cell, sets up people's hepatocarcinoma
Hep3B cell strain xenograft tumours animal model, dorsal sc inoculation 5 on the right side of every Mus
×106Hep3B cell adds Matrigel glue, and 0.1ml/ is only.Treat tumor average volume about 232mm3
Time, according to tumor size random packet.Experiment packet is shown in Table 2:
Table 2. experimental design
Note: administration volume: 10 μ l/g body weight；NS: normal saline；Qd: be administered once a day；
Q4d: every four days are administered once；Q7d: Per-Hop behavior is once.
In experimentation, routine monitoring includes tumor growth and the treatment shadow to animal normal behaviour
Ringing, the activeness of laboratory animal, ingest and drinking-water situation, body weight change situation (measures weekly 2
Secondary), eyes, by hair and other abnormal conditions.Enter according to the weight of animals change and death condition
Row safety evaluatio, according to the Relative tumor rate of increase (T/C(%)) and during tumor growth delay
Between (T-C) carry out therapeutic evaluation.
Gross tumor volume rate of increase T/C(%): in point sometime, treatment group and matched group tumor
The percent value of volume.T and C is respectively treatment group and matched group at a certain particular point in time
Gross tumor volume average.
Computing formula is as follows: T/C(%)=TTV/CTV* 100%(TTV: treatment group tumors body
Long-pending average；CTV: negative control group gross tumor volume average).
Tumor T-C time delay: refer to that (this experiment is 1200mm to tumor growth to certain volume3)
Time, the natural law that treatment group postpones than matched group.T is that the mean tumour volume for the treatment of group reaches specific
Required natural law during value；C is that matched group mean tumor volume reaches required natural law during same sample value.T-C value
The biggest, time delay is the longest, illustrates that drug effect is the best；Vice versa.
The situation of each treatment group and the change of control group mice tumor volume growth is shown in Fig. 6.Result shows
Showing, the rhIFN-λ 1(modified without PEG is administered every day) hepatocarcinoma subcutaneous to tumor bearing nude mice is swollen
Tumor growth has certain inhibitory action, but inhibitory action is more weak.At the end of administration, T/C(%)
Value is respectively 79.3%(3a group) and 79.1%(4a group), with negative control group mouse tumor
Volume is compared, and difference does not has significance.
Compared with non-PEGization rhIFN-λ 1 effect, PEG-rhIFN-λ 1(administration number of times reduces),
But the inhibitory action of hepatic carcinoma subcutaneous to tumor bearing nude mice growth is dose-dependent effect.It is administered knot
Shu Shi, 0.1mg/kg and 2.5mg/kg group T/C(%) value is 85.31%(3b group respectively) and
54.84%(4b group), wherein 2.5mg/kg(4b group) gross tumor volume and negative control group phase
Ratio has the difference (P=0.002) of highly significant, and gross tumor volume reaches 1200mm3Time
Between be delayed 13 days than negative control group.
PEG-rhIFN-λ 1 share than the effect being used alone chemotherapeutic with chemotherapeutic doxorubicin
Good, at the end of administration, 0.1mg/kg and 2.5mg/kg PEG-rhIFN-λ 1 respectively with chemotherapeutic
The T/C(% of combination group) value is 65.59%(5 group) and 64.29%(6 group), it is used alone
The T/C(% of chemotherapeutic group) value is 74.81%(2 group), PEG-rhIFN-λ 1 and chemotherapeutic
All significantly less than negative control group, (P value is respectively 0.016 He to the two groups of gross tumor volumes share
0.012), gross tumor volume reaches 1200mm3Time be delayed 7.5 respectively than negative control group
It and 6.5 days (table 3).
Table 3. each treatment group tumors volume T/C(%) value and T-C time delay
Note: a. mean+/-standard error；B. relative tumour volume is compared to negative control group.
The PEG-rhIFN-λ 1 safety results to Hep3B people's Hepatic neoplasm model: during experiment,
Negative control group mice weight loss after tumor inoculation 21 days, more than 10%, illustrates this animal mould
Type has obvious malignant tumor matter feature.Each administration group body weight compares without significant difference with negative control group,
And drug toxicity reaction does not occurs；Negative control group and each administration group inoculate the 36th day eventually in cell
Only experiment, mice has carried out euthanasia and has dissected, and main organs is no abnormal, shows mice
Better tolerance to rhIFN-λ 1 and PEG-rhIFN-λ 1, drug safety is good.Do not rule together
After treatment group and matched group are administered, body weight change is shown in Table 4.
Table 4. each treatment group and matched group body weight change situation
Note: a. mean+/-standard error.
Hepatoma carcinoma cell Hep3B xenotransplantation under fell is swollen by embodiment 10, PEG-rhIFN-λ 1
The detection of serum HBsAg in tumor model
Owing to human hepatoma cell strain Hep3B has HBV gene group to integrate, sustainable generation
HBsAg, carries out HBsAg to be detected from tumor bearing nude mice serum during xenograft tumours experiment
(Knowles BB. et al., 1980).When zoopery terminates, collection mice serum ,-80
DEG C preserve, use HBsAg-ELISA detection kit (Shanghai China of section biological engineering share is limited
Company) analyze the HBsAg content in mice serum.
Result is shown in 1 group of mice serum of rhIFN-λ that Fig. 7,2.5mg/kg modify without PEG
The content of HBsAg, compared with negative control group, only have dropped 4.7%, and rhIFN-λ 1 is through PEG
After modification, can effectively strengthen Anti-HBV activity effect, suppress Hep3B emiocytosis HBsAg,
In 1 group of mice serum of 0.1mg/kg and 2.5mg/kg PEG-rhIFN-λ, the content of HBsAg is respectively
Have dropped 28.5% and 62.5%, wherein the content of 2.5mg/kg group HBsAg in serum is with cloudy
Property matched group is compared the difference of very significant (P=0.000), shows provided by the present invention
Novel PEG-rhIFN-λ 1 has the effect of the anti HBV infecting of affirmative.
List of references
Abushahba W.,et al.(2010)Antitumor activity of Type I and Type III
interferons in BNL hepatoma model.Cancer Immunol Immunother.
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Bobek M.D.,Boyd B.S.&Chisari F.V.(2005)Lambda interferon
inhibits hepatitis B and C virus replication.J Virol.79(6):3851-3854；
Chen HC.,et al.(1997)Suppressive effects of destruxin B on hepatitis
B virus surface antigen gene expression in human hepatoma cells.Antiviral
Res.34(3):137-144；
Doyle SE.,et al.(2006)Interleukin-29Uses a Type1Interferon-Like
Program to Promote Antiviral Responses in Human Hepatocytes.
Hepatology.44:896-906;
Knowles BB.,et al(1980)Human hepatocellular carcinoma cell lines
secrete the major plasma proteins and hepatitis B surface antigen.
Science.25;209(4455):497-499；
Kotenko1S.V.,et al.(2003)IFN-λs mediate antiviral protection
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Muir A.J.,et al.(2010)Phase1b study of pegylated interferon lambda
1with or without ribavirin in patients with chronic genotype1hepatitis C
virus infection.Hepatology.52(3):822-32；
Parkin,D.M.,et al.(2005)Global cancer statistics,2002.CA Cancer J.
Clin.55,74–108；
Sheppard P.,et al.(2003)IL-28,IL-29and their class II cytokine
receptor IL-28R.Nat Immunol.4(1),63-68；
Veronese FM,Pasut G.(2005)PEGylation,successful approach to
drug delivery.Drug Discov Today.10(21):1451-1458；
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Claims (13)
1. a PEGization recombined human IFN-λ 1, its aminoacid sequence such as SEQ ID NO.1 institute
Showing, wherein the cysteine residues of the 112nd is that PEGization is modified.
PEGization recombined human IFN-λ 1 the most according to claim 1, wherein said PEG
For straight chain PEG or side chain PEG.
PEGization recombined human IFN-λ 1 the most according to claim 1, wherein said PEG
Molecular weight is 20 to 40 kilodaltons.
PEGization recombined human IFN-λ 1 the most according to claim 3, wherein said PEG
Molecular weight is 20 kilodaltons.
PEGization recombined human IFN-λ 1 the most according to claim 1, wherein said PEG
It is selected from: maleimide-PEG, vinyl sulfone-PEG, two thiopyridines-PEG and iodoacetamide
-PEG。
PEGization recombined human IFN-λ 1 the most according to claim 5, wherein said PEG
For maleimide-PEG.
7. a preparation method of PEGization recombined human IFN-λ 1, it includes step:
With Tris-HCl buffer dialysis equilibrium recombined human IFN-λ 1 egg that 25mM pH is 8.0
In vain, recombined human IFN-λ 1 concentration is adjusted to 1mg/ml；
In the ratio that molecule mol ratio is l:5 of recombined human IFN-λ 1 and PEG, to recombined human
IFN-λ 1 adds PEG；
Coupling reaction is carried out 24 hours at 4 DEG C, then carries out 2 hours at 18-25 DEG C；
By cation-exchange chromatography, coupling product carried out purification for the first time；
Second time purification is carried out by gel permeation chromatography；
PEGization recombined human IFN-λ 1 described in results.
Preparation method the most according to claim 7, wherein said PEG is selected from: horse
Come acid imide-PEG, vinyl sulfone-PEG, two thiopyridines-PEG and iodoacetamide-PEG.
Preparation method the most according to claim 7, wherein said PEG is maleoyl
Imines-PEG.
Preparation method the most according to claim 7, wherein said PEG molecular weight is 20
To 40 kilodaltons.
11. preparation methoies according to claim 10, wherein said PEG molecular weight is
20 kilodaltons.
PEGization recombined human IFN-λ 1 according to any one of 12. claim 1-6 controls in preparation
Treat the purposes in the medicine of chronic hepatitis B or hepatocarcinoma.
13. purposes according to claim 12, wherein said PEGization recombined human IFN-λ 1
It is administered alone or co-administered.
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| WO2007013944A2 (en) * | 2005-07-20 | 2007-02-01 | Zymogenetics, Inc. | Use of truncated cysteine il28 and il29 mutants to treat cancers and autoimmune disorders |
| WO2007041713A1 (en) * | 2005-10-04 | 2007-04-12 | Zymogenetics, Inc. | Production and purification of il-29 |
| WO2009149377A1 (en) * | 2008-06-05 | 2009-12-10 | Zymogenetics, Llc | Use of pegylated type iii interferons for the treatment of hepatitis c |
| CN102351952A (en) * | 2011-10-19 | 2012-02-15 | 中国医学科学院基础医学研究所 | Low-saccharification mutant interferon lambda1 as well as expression and purification methods and application thereof |
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| WO2007013944A2 (en) * | 2005-07-20 | 2007-02-01 | Zymogenetics, Inc. | Use of truncated cysteine il28 and il29 mutants to treat cancers and autoimmune disorders |
| WO2007041713A1 (en) * | 2005-10-04 | 2007-04-12 | Zymogenetics, Inc. | Production and purification of il-29 |
| WO2009149377A1 (en) * | 2008-06-05 | 2009-12-10 | Zymogenetics, Llc | Use of pegylated type iii interferons for the treatment of hepatitis c |
| CN102351952A (en) * | 2011-10-19 | 2012-02-15 | 中国医学科学院基础医学研究所 | Low-saccharification mutant interferon lambda1 as well as expression and purification methods and application thereof |
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