CN104045704A - PEGylated recombinant human IFN-[lambda]1, preparation method and application thereof - Google Patents
PEGylated recombinant human IFN-[lambda]1, preparation method and application thereof Download PDFInfo
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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 PEGylated recombinant human IFN-[lambda]1, a preparation method and an application thereof. The invention particularly achieves a Site-directed PEG modification of the recombinant human IFN-[lambda]1 and also provides the application of the obtained PEGylated recombinant human IFN-[lambda]1 in preparation of medicines which are used for treating chronic hepatitis B or liver cancer. According to the preparation method, the prepared PEGylated recombinant human IFN-[lambda]1 has a good homogeneity and is greater than 98% in purity. The PEGylated recombinant human IFN-[lambda]1 can significantly inhibits the growth of a subcutaneous tumor of a nude mice bearing the tumor and reduces the content of HBsAg in nude mice serum. In addition, the PEGylated recombinant human IFN-[lambda]1 shows a relative good safety.
Description
Technical field
The present invention relates to biological technical field.More specifically, relate to a kind of Interferon, rabbit obtaining by recombinant technology and chemical modification technology, and its production and use.Relate in particular to a kind of recombinant human IFN-λ 1, its preparation method and the purposes in the medicine of preparation treatment chronic hepatitis B or liver cancer thereof of pegylation.
Background technology
Interferon, rabbit-λ s(IFN-λ is s) a class new forms of interferon, classification be decided to be type iii interferon, by IFN-λ 1(IL-29), IFN-λ 2(IL-28A) and IFN-λ 3(IL-28B) form.2003 by American scientist reported first this type Interferon, rabbit (people such as Sheppard P., 2003; Kotenko1S.V. wait people, 2003).
IFN-λ gene is positioned at karyomit(e) No. 19, contains multiple introns in gene, and its gene structure and IL-10 family member are quite similar, and differ greatly with IFN-α.On amino acid composition, IFN-λ s also only has the amino acid of 15-19% identical with IFN-α.However, IFN-λ s is quite similar with IFN-α in function, can suppress copying of hepatitis B/hepatitis C virus (HBV/HCV), can also suppress activity people such as (, 2005) Bobek M.D. of the virus such as encephalomyocarditis virus (EMCV), human cytomegalic inclusion disease virus (CMV), lymphocytic choriomeningitis virus (LCMV), hsv (HSV-2).Except antiviral activity, in cytologic experiment, IFN-λ s can suppress the propagation of neuroendocrine tumor, the esophageal carcinoma, colorectal carcinoma, lung cancer and Burkitt ' s lymphoma cell and melanoma cell, and existing in Mice Body, suppress mouse tumor cell as the report of melanoma, fibrosarcoma and hepatoma cell proliferation people such as (, 2010) Abushahba W. by transferring host's immunologic mechanism with mIFN-λ s.
The acceptor of IFN-λ s belongs to II type cytokines receptor family, and the heterodimer mixture special by structure forms.IL-28R α is aglucon binding subunit, has determined the specificity that acceptor is combined with IFN-λ, and IL-10 β R is auxiliary subunit.I type IFN(IFN-α/β from wide expression) acceptor is different, the expression of IFN-λ acceptor has cell-specific, mainly be expressed in surface epithelial cell, for example all cells in liver is all expressed the acceptor of IFN-α, and the acceptor of IFN-λ is only at liver cell expression.Comprise all wide expression IFN-α acceptors such as T lymphocyte, bone-marrow-derived lymphocyte, NK cell, neutrophil leucocyte and monocyte at peripheral blood lymphocyte equally, but except the expression (people such as Muir A.J., 2010) of IFN-λ acceptor not detected in the external hematopoietic cell of bone-marrow-derived lymphocyte.The otherness that this different I FN family receptors distributes will affect IFN effect in vivo, compared with toxic side effect with the I type IFN generally using clinically at present in treatment and tolerance are poor, IFN-λ s has potential advantage, can effectively reduce the toxic side effect to hematopoiesis and central nervous system in bioactive whiles such as bringing into play antiviral, antitumor and participation immunomodulatory.
But IFN is similar with I type, IFN-λ s relative molecular mass less (being about 20kD), is easy to by glomerular filtration; Unstable in body, easily by proteasome degradation, plasma half-life is short; Treatment cycle is long, need to frequently inject, and causes patient's interdependence to reduce; Also there is Immunogenicity in the recombinant protein class biotechnological formulation as allos system expression.All these likely weakens the clinical effectiveness of IFN-λ s.
Polyoxyethylene glycol (polyethylene glycol, PEG) is the polymkeric substance of a kind of nontoxicity, non-immunogenicity, safety.Since the seventies in last century, to have developed the technology of carrying the most in vivo polypeptide and pharmaceutical grade protein with PEG chain covalent coupling modifying protein.The benefit of PEG modifying protein in treatment comprises:
-increase protein molecular weight, extend plasma half-life thereby reduce glomerular filtration rate(GFR;
-increase protein physical and chemical stability, reduce the degraded of proteolytic enzyme;
-reduce toxicity and limited the immunogenicity of protein;
-increase the solubility of pharmaceutical grade protein;
-by improving pharmaceutical grade protein dynamic performance, compared with unmodified protein matter medicine, effectively strengthen medicine activity (people such as Veronese FM, 2005) in vivo.Mainly to adopt mono methoxy PEG molecule (mono-methoxy PEG to the PEGization modifying method of protein; mPEG) with protein surface reactive group covalent coupling; its main modification approach is that N-terminal amino or lysine side-chain amino are carried out to acidylate modification, but this modification mode easily produces heterogeneity (being isomer mixture).
Liver cancer is one of modal malignant tumour, worldwide in kinds of tumor mortality ratio, ranks the 3rd.The whole world has every year and exceedes the newly-increased liver cancer patients of 600,000 example, and wherein approximately 55% patient is in China.It is the most important Hazard Factor of induced hepatocellular carcinoma that chronic HBV (HBV) infects, and has HBV to infect history (Yang, the people such as J.D, 2010 at the liver cancer patient of China nearly 80%; Parkin, the people such as D.M., 2005).IFN-α is considered to effectively clinically for anti-HBV treatment, is also of value to prevention HBV and infects relevant liver cancer.But still have clinically the Patients with Chronic HBV Infection of significant proportion to IFN-α treatment nonreply, and patient Chang Yinwei occurs that the toxicity, side effect that is difficult to tolerance comprises fatigue, heating, apocleisis, dysthymia disorders and bone marrow depression etc. and interrupts the treatment of IFN-α.Especially IFN-λ 1(IL-29 of IFN-λ s) can excite antiviral activity by the IFN-λ acceptor at surface of hepatocytes specifically expressing, suppress HBV virus replication, prompting IFN-λ 1 can be used for the treatment of chronic HBV infection and HBV and infect relevant liver cancer patient people such as (, 2006) Doyle SE..
Summary of the invention
According to an aspect of the present invention, provide a kind of PEGization recombinant human IFN-λ 1(also to claim PEG-rhIFN-λ 1).Its amino acid composition is close with wild-type people IFN-λ 1, and has the biological activity identical with wild-type people IFN-λ 1.
The aminoacid sequence of above-mentioned PEGization recombinant human IFN-λ 1 is as shown in SEQ ID No.1.Be made up of 181 amino acid, its 46th, N end is glutamine.The cysteine residues (being called for short C112) of the 112nd is that PEGization is modified.
RhIFN-λ 1 provided by the present invention is for having lacked the low-saccharification mutant IFN-λ 1 of N-glycosylation site, its the 46th, N end is glutamine, expressed by pichia spp (Pichia pastoris) system, it is in CN10235192A Chinese patent application that its aminoacid sequence, special secretion expression's carrier, transgenic cell line and Host Strains are disclosed in publication number, and application people is Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.
RhIFN-λ 1 provided by the present invention produces from thermophilic methanol yeast-Pichi strain, pichia spp has many advantages of protokaryon and eukaryotic expression system concurrently, can form solubility secreting, expressing by special signal peptide guiding simultaneously, recombinant exogenous protein be formed correct folding.The method of Pichia anomala expression of the present invention and purifying has been disclosed in Granted publication CN1962873B Chinese patent, and patentee is Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.
In some embodiments, PEG-rhIFN-λ 1 of the present invention, wherein said PEG is straight or branched structure.
In some embodiments, described PEG molecular weight is within the scope of 20～40 kilodaltons; Preferably 20 kilodaltons.
In some embodiments, described PEG is selected from maleimide-PEG(mPEG-MAL), vinyl sulphone-PEG(mPEG-VS), two thiopyridines-PEG(mPEG-Pyridyl disulfides) and iodo-acid amide-PEG(mPEG-iodoacetamide).
In some concrete embodiments, described PEG is mPEG-MAL.
According to a further aspect in the invention, also provide the preparation method of PEG-rhIFN-λ 1, comprised step:
Recombinant human IFN-λ 1 is provided;
In recombinant human IFN-λ 1, add PEG;
Linked 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 reaction product is carried out to purifying for the first time;
Carry out purifying for the second time by gel permeation chromatography;
Results PEGization recombinant human IFN-λ 1.
In a concrete embodiment, linked reaction is carried out 24 hours at 4 DEG C, then carries out 2 hours at 18-25 DEG C.
In some embodiments, described PEG is selected from maleimide-PEG(mPEG-MAL), vinyl sulphone-PEG, two thiopyridines-PEG and iodo-acid amide-PEG.In a concrete embodiment, PEG is mPEG-MAL.
In some embodiments, described PEG molecular weight is 20～40 kilodaltons.In a concrete embodiment, PEG is that molecular weight is 20 kilodaltons.
In a concrete embodiment, recombinant human IFN-λ 1 is l:5 with the molecule mol ratio of PEG.
Analyze the prepared PEG-rhIFN-λ 1 of aforesaid method by series connection level Four bar time-of-flight mass spectrometer, compare with the rhIFN-λ 1 modifying without PEG, it is upper that result shows that the coupling site of mPEG-MAL is positioned at the C112 of rhIFN-λ 1, is pointed decoration.
In accordance with a further aspect of the present invention, provide the purposes of PEG-rhIFN-λ 1 in the medicine of preparation treatment chronic hepatitis B or liver cancer.
In some embodiments, PEG-rhIFN-λ 1 uses separately or with co-administered.In some embodiments, PEG-rhIFN-λ 1 uses separately or uses with for example chemotherapy drugs in combination of other medicines.Chemotherapeutics is such as but not limited to Dx, oxaliplatin, 5-FU or Calciumlevofolinate.In some concrete embodiments, PEG-rhIFN-λ 1 uses separately.In other concrete embodiments, PEG-rhIFN-λ 1 is co-administered with Dx.
In concrete embodiment, in the tumor bearing nude mice animal model that PEG-rhIFN-λ 1 of the present invention builds at the hepatoma cell strain Hep3B of continuous release hepatitis B virus surface antigen (HBsAg), significantly suppress the growth of tumor bearing nude mice Subcutaneous tumor, significantly reduce the HBsAg content in nude mice serum.
Brief description of the drawings
Fig. 1: the separation and purification of PEG modification reaction mixture.
A:CM Sepharose F.F. cation-exchange chromatography graphy figure; Peak 1.mPEG-MAL-rhIFN-λ 1; Peak 2.rhIFN-λ 1;
B:SDS-PAGE figure, M. molecular weight of albumen mark; 1 road .PEG reaction mixture; 2 road .mPEG-MAL-rhIFN-λ 1(elution peaks 1); 3 road .rhIFN-λ 1(elution peaks 2).
The SDS-PAGE detected result of Fig. 2: PEG-rhIFN-λ 1.
A:SDS-PAGE coomassie brilliant blue staining; M. molecular weight protein marker; 1 road .rhIFN-λ 1; Shown in 2 road .mPEG-MAL-rhIFN-λ 1(arrows).
B:SDS-PAGE barium-iodine staining; M. molecular weight protein marker; 1 road .rhIFN-λ 1; Shown in 2 road .mPEG-MAL-rhIFN-λ 1(arrows).
Fig. 3: the experiment of ISRE luciferase reporter gene detects the external activity of PEG-rhIFN-λ 1.
Fig. 4: the vitro stability of PEG-rhIFN-λ 1 and activity.
A1:BCA method is measured the content of albumen after 37 DEG C hatch, rhIFN-λ 1(◇), PEG-rhIFN-λ 1(△);
A2: luciferase reporter gene experiment detects 37 DEG C and hatches rear rhIFN-λ 1(◇) and PEG-rhIFN-λ 1(△) activity;
B1:ELISA detect after jointly hatching with rat blood serum rhIFN-λ 1(◇) and PEG-rhIFN-λ 1() content;
B2: luciferase reporter gene experiment detects rhIFN-λ 1(◇ after jointly hatching with rat blood serum) 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 liver cancer tumor growth curve of tumor bearing nude mice treatment, * P < 0.05; * P < 0.01.
Fig. 7: PEG-rhIFN-λ 1 is for serum HBsAg-ELISA detected result of liver cancer tumor bearing nude mice treatment, * * P < 0.01.
Fig. 8: the Mass Spectrometric Identification of PEGization decorating site.
A:PEG-rhIFN-λ 1, sequence IQPQPTAGPRPR;
B:rhIFN-λ 1, sequence C IQPQPTAGPRPR.
Embodiment
Below in conjunction with specific embodiment, embodiments of the present invention and concrete operating process are explained in detail, but are to be understood that protection scope of the present invention is not limited to following embodiment.In following embodiment, method therefor is ordinary method if no special instructions.The specification sheets that the use of test kit provides according to manufacturers or supplier carries out.
The acquisition of embodiment 1, rhIFN-λ 1
Preparing low-saccharification mutant rhIFN-λ 1 secretion expression's carrier, transgenic cell line, Host Strains, fermentation expression and method for purifying proteins used is all to have a detailed description in CN10235192A Chinese patent application at publication number.
The preparation method of embodiment 2, PEG-rhIFN-λ 1
The damping fluid dialysis equilibrium of the rhIFN-λ 1 albumen 25mM Tris-HCl pH8.0 that embodiment 1 is obtained, rhIFN-λ 1 concentration is adjusted to 1mg/ml;
In rhIFN-λ 1 and the ratio that the molecule mol ratio of PEG is l:5, add PEG powder (20kD straight chain mPEG-MAL, Kai Zheng biotechnology Development Co., Ltd provides by Beijing);
Stir and make powder dissolution gently;
After 4 DEG C of reaction 24h, then room temperature (18-25 DEG C) reaction 2h obtains linked reaction product;
Above-mentioned linked reaction product is carried out to cation-exchange chromatography: buffer A (25mM NaAC, the pH4.5) dilution that above-mentioned linked reaction product is added to 5 times of volumes; Be splined on afterwards on the cation-exchange chromatography post (the CM Sepharose F.F that GE Healthcare Life Sciences provides) through buffer A balance; Wash-out adopts the linear elution (0～100%) of from buffer A to buffer B (25mM NaAC, 0.5M NaCl, pH4.5); Collect each elution peak;
The elution peak of above-mentioned collection is carried out to gel permeation chromatography: damping fluid is 50mM PBS, 150mM NaCl, pH7.0, chromatography column is Superdex75, Hiload16/60 prepacked column (GE Healthcare Life Sciences company product);
The PEG-rhIFN-λ 1 of results purifying.
Embodiment 3, Homogeneity Test
The result that above-mentioned cation-exchange chromatography separates as shown in Figure 1, good (the Fig. 1 of rhIFN-λ 1 homogeneity modifying through PEG, B, swimming lane 1), this shows that preparation method of the present invention not only provides single modification mode, and cation-exchange chromatography can be by rhIFN-λ 1(Fig. 1 of unmodified, B, swimming lane 3) PEG-rhIFN-λ 1(Fig. 1 mono-modified with 20kD mPEG-MAL, B, swimming lane 2) separation (Fig. 1, A) well.
Embodiment 4, purity test
The purified product that embodiment 2 is obtained, after SDS-PAGE electrophoresis, carries out respectively coomassie brilliant blue staining and barium-iodine staining and detects.
With the dyeing of barium-iodine staining can direct-detection PEG, method is: after SDS-PAGE electrophoresis finishes, gel after distilled water drip washing is immersed in to 30min in 5% barium chloride solution, after with distilled water, bariumchloride being washed off, gel is immersed in to 30min in 0.1mM iodine solution, and then decolour with distilled water.
Detected result is shown in Fig. 2, and wherein the A of Fig. 2 is coomassie brilliant blue staining, and swimming lane 1 is about the rhIFN-λ of 20kD for molecular weight; The PEG-rhIFN-λ 1 that swimming lane 2 is modified for 20kD mPEG-MAL; The B of Fig. 2 is barium-iodine staining, only has as seen PEG-rhIFN-λ 1 can develop the color (Fig. 2, B, swimming lane 2); Detected result demonstration, PEG-rhIFN-λ 1 purity after purifying is greater than 98%.
The qualification in embodiment 5, PEGization site
The PEG-rhIFN-λ 1 that embodiment 2 purifying are obtained and the rhIFN-λ 1 of unmodified, after trypsin degradation, the analysis of the level Four of connecting bar time-of-flight mass spectrometer, collect spectrogram, theoretical value comparison with all polypeptide fragments that produced through trypsin degradation by wild-type IFN-λ 1 in database, in two samples of PEG modification and unmodified, all capture the polypeptide fragment that contains C15, C49, C145 and C171, two samples do not have difference.Analyzing trypsin degradation collection of illustrative plates shows, the polypeptide piece segment length of containing C112, molecular weight is greater than the scope that series connection level Four bar time-of-flight mass spectrometer can catch, and points out the analytical results after trypsin degradation only still can not clearly judge that PEG is that pointed decoration is on C112 site.
For this reason, the rhIFN-λ 1 of PEG-rhIFN-λ 1 and unmodified degrades through Proteinase K again, row series connection level Four bar time-of-flight mass spectrometer is analyzed, in the rhIFN-of unmodified λ 1 sample, capture the polypeptide fragment that contains C15, C49 and C112, the polypeptide fragment aminoacid sequence that wherein contains C112 is " CIQPQPTAGPRPR ", and in PEG-rhIFN-λ 1 sample except capturing the polypeptide fragment that contains C15, C49, also capture the polypeptide fragment adjacent with C112, its aminoacid sequence is " IQPQPTAGPRPR ".The comprehensively mass spectrometry results after trypsinase and Proteinase K are degraded respectively, in the rhIFN-of unmodified λ 1 sample, 5 polypeptide fragments that contain Cys are all caught in, and in PEG-rhIFN-λ 1 sample, other 4 polypeptide fragments that contain Cys except C112 are only captured, show to have due to C112 position the modification of 20kD mPEG-MAL, molecular weight is far longer than the scope that series connection level Four bar time-of-flight mass spectrometer can capture, and therefore can confirm that mPEG-MAL pointed decoration (the results are shown in Figure 8) on C112 position.
In the aminoacid sequence of rhIFN-λ 1, always have 5 Cys residues, but the modification of mPEG-MAL is only positioned at rhIFN-λ 1 aminoterminal to be risen on the 112nd cysteine residues, this shows that the modification homogeneity of PEG-rhIFN-λ 1 of the present invention is high, homogeneity good, is a kind of pointed decoration.
The external activity of embodiment 6, PEG-rhIFN-λ 1
Adopt pISRE-luciferase reporter gene to survey the external activity of the method for living mensuration PEG-rhIFN-λ 1.
PISRE-luciferase reporter gene is surveyed the ratio juris of living: after Interferon, rabbit is combined with cell surface specific receptors, initiating signal cascade reaction, cause the interferon-stimulated gene factor (ISGF, IFN-stimulated gene factor) enter nucleus and cis element IFN-stimulated response element (ISRE, IFN-stimulated response element) interact, thereby transcribing of regulatory gene, it is antiviral that final generation Interferon, rabbit mediates, anti-tumour cell proliferative and immunoregulatory biological effect (Williams BR, 1991).
In the present embodiment, be assembled into luciferase reporter gene plasmid pISRE-TA-luc with ISRE, stimulate the expression level of the luciferase reporter gene of rear ISRE regulation and control can reflect the biological activity of Interferon, rabbit by detecting Interferon, rabbit.
Concrete grammar is as follows: collect logarithmic phase HepG2 cell (hepatoma cell strain), adjust concentration of cell suspension, with 2 × 10
5individual/hole inoculating cell to 6 orifice plate, 37 DEG C, 5%CO
2, overnight incubation; With lipofectamine2000 transfection pISRE-TA-luc(5ug) and pRL-SV40(0.1ug, internal reference) plasmid, 37 DEG C, 5%CO
2, hatch 24 hours, be replaced with serum free medium hunger 2～4 hours; In substratum, add respectively rhIFN-λ 1, the each 100ng/ml of PEG-rhIFN-λ 1, continue to cultivate collecting cell after 24 hours, by luciferase reporter gene detection kit (Promega company product) in TD20/20 light-emitting appearance value of reading respectively, record detected result, the expression level using the ratio of each time point Luciferase/Renilla as ISRE reporter gene.
The results are shown in Figure after 3, PEG modifies of PEG-rhIFN-λ 1 external activity experiment still retains 28% activity, illustrates that rhIFN-λ 1 is really by PEGization.
The vitro stability of embodiment 7, PEG-rhIFN-λ 1
Thermal stability analysis in the aqueous solution: by rhIFN-λ 1, PEG-rhIFN-λ 1 desalination to 20mMPBS, in pH7.2 solution, the concentration of two albumen is all adjusted to 400 μ g/ml, be placed in 37 DEG C, in 0h, 4h, 8h, 24h, 48h, 72h sampling, gets supernatant BCA method (test kit is Pierce company product) and detects residual protein content after the centrifugal 20min of 10,000rpm; Utilize pISRE-luciferase reporter gene to survey the remaining activity of the method for living analyzing proteins, result represents with residual protein content and active percentage ratio simultaneously.
The thermostability of PEG-rhIFN-λ 1 in the aqueous solution the results are shown in Figure 4, A1 and A2, rhIFN-λ 1 residual protein content after 37 DEG C of 8h of modifying without PEG drops to 40%, after 24h, protein content drops to 20%, and PEG-rhIFN-λ 1 does not obviously reduce at whole observation period residual protein content and former protein content, after 3 days, still remain on 90%(Fig. 4, A1).And the biological activity of ISRE luciferase reporter gene reflection still can be retained in more than 75%, and the biological activity that the rhIFN-λ 1 modifying without PEG retains is less than 30%(Fig. 4, A2).Above-mentioned experimental result illustrates that PEG modifies the rear recombinant protein stability of rhIFN-λ 1 and strengthens really, and the biological activity hold-time extends.
Thermal stability analysis in serum: rhIFN-λ 1, PEG-rhIFN-λ 1 desalination, to 20mMPBS, in pH7.2 solution, are adjusted to same concentrations by two albumen, add isopyknic SD rat blood serum, be placed in 37 DEG C, in 0h, 4h, 8h, 12h, 24h, 48h, 72h sampling, gets supernatant IL-29ELISA test kit (ebioscience company product) detection by quantitative residual protein content after the centrifugal 20min of 10,000rpm; Utilize pISRE-luciferase reporter gene to survey the remaining activity of the method for living analyzing proteins, result represents with residual protein content and active percentage ratio simultaneously.
PEG-rhIFN-λ 1 in serum thermostability result as Fig. 4, shown in B1 and B2, no matter be that residual protein content or ISRE reporter gene activity all have decline to a certain degree, but compared with the rhIFN-λ 1 of unmodified, after 3 days, residual protein content and ISRE reporter gene biological activity all can remain on more than 40%, far above PEGization rhIFN-λ 1 not, show that the coupling of PEG and rhIFN-λ 1 has significantly increased the thermostability of rhIFN-λ 1 and postponed the degraded of proteolytic enzyme to rhIFN-λ 1 in serum.
Embodiment 8, PEG-rhIFN-λ 1 pharmacokinetics in rats research
Experiment adopts SD rat, female, body weight 270-290g, and random packet, 3 every group, point cage is fed, animal fasting 12h before administration; Give respectively single subcutaneous injection rhIFN-λ 1 and PEG-rhIFN-λ 1 with the dosage of 200 μ g/kg body weight; Tail venous blood sampling after administration, gets the blood time to be respectively: after rhIFN-λ 1-injection 0,5min, 10min, 0.5h, 1h, 2h, 3h, 4h, 12h, 24h; After PEG-rhIFN-λ 1-injection 0,0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h, 120h, 144h; Blood sample, through 5,000rpm, is got blood plasma part after 10min is centrifugal, puts-70 DEG C of preservations, uses ELISA(IFN-lambda1Platinum ELISA, ebioscience company) detect the content of IFN-λ 1 in blood plasma.Carry out curve fitting with Kinetica software, calculate various pharmacokinetic parameters.Detect data acquisition Excel software statistics, statistics represents with mean number ± standard deviation (x+SD).The results are shown in Figure 5 and table 1:
The pharmacokinetic parameter of table 1.rhIFN-λ 1 and PEG-rhIFN-λ 1
| PK parameter | rhIFN-λ1（x±SD） | PEG-rhIFN-λ（x±SD） |
| AUC(ng?h/ml) | 160.72±25.36 | 2082.16±203.44 |
| C Maximum(ng/ml) | 54.05±4.21 | 69.18±4.58 |
| T Maximum(h) | 0.50±0.04 | 7.35±2.10 |
| t 1/2(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; C
maximum: maximum peak concentration; T
maximum: peak time; t
1/2: eliminate the transformation period; CL: elimination factor.
Result shows: through kinetica software comparision of goodness of fit, rhIFN-λ 1 and PEG-rhIFN-λ 1 all meet at SD rat physiological disposition the compartment model that one-level absorbs.Compared with the rhIFN-λ 1 modifying without PEG, the maximum peak concentration (C after PEG-rhIFN-λ 1 subcutaneous injection in blood plasma
maximum) improve peak time (T
maximum) and eliminate transformation period (t
1/2) significant prolongation, be respectively 1.28 times, 14.7 times and 10.75 times of rhIFN-λ 1; Area under the drug-time curve significantly increases (for 12.9 times of rhIFN-λ 1), and elimination factor (CL) has reduced by 92.4%.These data show, rhIFN-λ 1 can improve rhIFN-λ 1 absorption and distribution in vivo effectively through the modification of PEG, can extend significantly its biological half-life, improve rhIFN-λ 1 bioavailability in vivo.
Embodiment 9, PEG-rhIFN-λ 1 security and the pharmacodynamic evaluation research to liver cancer xenotransplantation tumor model under fell
Experiment adopts human hepatoma cell strain-Hep3B, and this cell strain has HBV genome conformity, and sustainable generation HBsAg can be used as cell model people such as (, 1997) Chen HC. of observing anti-HBV curative effect of medication.
Experimental technique: BALB/c nude mice back subcutaneous vaccination Hep3B cell, set up Hep3B cells cell strain xenotransplantation animal model for tumour, every mouse right side back subcutaneous vaccination 5 × 10
6hep3B cell adds Matrigel glue, and 0.1ml/ only.Treat the about 232mm of tumor average volume
3time, according to tumor size random packet.Experiment is divided into groups in table 2:
Table 2. experimental design
Note: administration volume: 10 μ l/g body weight; NS: physiological saline; Qd: be administered once every day; Q4d: be administered once for every four days; Q7d: be administered once weekly.
In experimentation, routine monitoring comprises tumor growth and the impact for the treatment of on animal normal behaviour, and the reactivity of laboratory animal, ingests and drinking-water situation, body weight change situation (measuring weekly 2 times), eyes, quilt hair and other abnormal conditions.According to the weight of animals change and death condition carry out safety evaluation, according to relative tumor proliferation rate (T/C(%)) and the tumor growth delay time (T-C) carry out therapeutic evaluation.
Gross tumor volume proliferation rate T/C(%): at point sometime, the percent value for the treatment of group and control group gross tumor volume.T and C are respectively treatment group and the control group gross tumor volume average at a certain particular point in time.
Calculation formula is as follows: T/C(%)=T
tV/ C
tV* 100%(T
tV: treatment group gross tumor volume average; C
tV: negative control group gross tumor volume average).
Tumour T-C time of lag: refer to that (this experiment is 1200mm to tumor growth to certain volume
3) time, the number of days that treatment group postpones than control group.T is the mean tumour volume for the treatment of group required number of days while reaching particular value; C is the average knurl volume of control group required number of days while reaching same sample value.T-C value is larger, and time of lag is longer, illustrates that drug effect is better; Vice versa.
The situation of each treatment group and control group mice gross tumor volume growth change is shown in Fig. 6.Result show, without PEG modify rhIFN-λ 1(administration every day) the subcutaneous liver cancer tumor growth of tumor bearing nude mice is had to certain restraining effect, but restraining effect a little less than.When administration finishes, T/C(%) value is respectively 79.3%(3a group) and 79.1%(4a group), compared with negative control group mouse tumor volume, difference does not have significance.
Compared with PEGization rhIFN-λ 1 does not act on, PEG-rhIFN-λ 1(administration number of times reduces), but the restraining effect of the subcutaneous liver cancer tumor growth of tumor bearing nude mice is to dose-dependent effect.When administration finishes, 0.1mg/kg and 2.5mg/kg group T/C(%) value is respectively 85.31%(3b group) and 54.84%(4b group), wherein 2.5mg/kg(4b group) gross tumor volume has the difference (P=0.002) of highly significant compared with negative control group, and gross tumor volume reaches 1200mm
3time postponed 13 days than negative control group.
PEG-rhIFN-λ 1 and chemotherapeutic Dx share effective than independent use chemotherapeutic, when administration finishes, 0.1mg/kg and 2.5mg/kg PEG-rhIFN-λ 1 respectively with the T/C(% of chemotherapeutic combination group) value is 65.59%(5 group) and 64.29%(6 group), use separately the T/C(% of chemotherapeutic group) be worth for 74.81%(2 group), PEG-rhIFN-λ 1 is all significantly less than negative control group (P value is respectively 0.016 and 0.012) with two groups of gross tumor volumes that chemotherapeutic is share, and gross tumor volume reaches 1200mm
3time postponed respectively 7.5 days and 6.5 days (table 3) than negative control group.
The each treatment group gross tumor volume of table 3. T/C(%) value and T-C time of lag
Note: a. mean+/-standard error; B. relative tumour volume is compared to negative control group.
The safety results of PEG-rhIFN-λ 1 to Hep3B people's Hepatic neoplasm model: experimental session, negative control group mouse tumor inoculation after 21 days weight loss exceed 10%, illustrate that this animal model has obvious malignant tumor matter feature.Each administration group body weight and negative control group compare without significant difference, and do not occur drug toxicity reaction; Negative control group and each administration group stop experiment on the 36th day in cell inoculation, and mouse has carried out euthanasia and dissected, and main organs is no abnormal, shows the better tolerance of mouse to rhIFN-λ 1 and PEG-rhIFN-λ 1, and drug safety is good.After different treatment groups and control group administration, body weight change is in table 4.
The each treatment group of table 4. and control group body weight change situation
Note: a. mean+/-standard error.
Embodiment 10, PEG-rhIFN-λ 1 detection to serum HBsAg in liver cancer cell Hep3B xenotransplantation tumor model under fell
Because human hepatoma cell strain Hep3B has HBV genome conformity, sustainable generation HBsAg can detect the people such as HBsAg(Knowles BB., 1980 while carrying out xenotransplantation tumor experiment from tumor bearing nude mice serum).When experimentation on animals stops, collect mice serum ,-80 DEG C of preservations, adopt HBsAg-ELISA detection kit (Shanghai Kehua Bio-engineering Co., Ltd) to analyze the HBsAg content in mice serum.
The results are shown in Figure 7, in 1 group of mice serum of rhIFN-λ that 2.5mg/kg modifies without PEG, the content of HBsAg is compared with negative control group, only decline 4.7%, and rhIFN-λ 1 is after PEG modifies, can effectively strengthen anti-HBV effect, suppress Hep3B emiocytosis HBsAg, in 0.1mg/kg and 1 group of mice serum of 2.5mg/kg PEG-rhIFN-λ, the content of HBsAg has declined respectively 28.5% and 62.5%, wherein the content of 2.5mg/kg group HBsAg in serum has the difference (P=0.000) of very significant compared with negative control group, show that New PEG-rhIFN-λ 1 provided by the present invention has the effect of sure anti HBV infecting.
Reference
Abushahba?W.,et?al.(2010)Antitumor?activity?of?Type?I?and?Type?III?interferons?in?BNL?hepatoma?model.Cancer?Immunol?Immunother.59:1059–1071；
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?through?a?distinct?class?II?cytokine?receptor?complex.Nature?immunology.4(1):69-77；
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；
Williams?BR.(1991)Signal?transduction?and?transcriptional?regulation?of?interferon-alpha-stimulated?genes.J?Interferon?Res.11(4):207-213;
Yang,J.D.&Roberts,L.R.(2010)Hepatocellular?carcinoma:a?global?view.Nat.Rev.Gastroenterol.Hepatol.7,448–458。
Claims (10)
1. a PEGization recombinant human IFN-λ 1, its aminoacid sequence is as shown in SEQ ID NO.1, and the cysteine residues that it is characterized in that the 112nd is that PEGization is modified.
2. PEGization recombinant human IFN-λ 1 according to claim 1, wherein said PEG is straight chain PEG or side chain PEG.
3. PEGization recombinant human IFN-λ 1 according to claim 1, wherein said PEG molecular weight is 20～40 kilodaltons; Preferably 20 kilodaltons.
4. PEGization recombinant human IFN-λ 1 according to claim 1, wherein said PEG is selected from maleimide-PEG, vinyl sulphone-PEG, two thiopyridines-PEG and iodo-acid amide-PEG; Be preferably maleimide-PEG.
5. a preparation method of PEGization recombinant human IFN-λ 1, it comprises step:
Recombinant human IFN-λ 1 is provided;
In recombinant human IFN-λ 1, add PEG;
Linked 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 reaction product is carried out to purifying for the first time;
Carry out purifying for the second time by gel permeation chromatography;
Results PEGization recombinant human IFN-λ 1.
6. preparation method according to claim 5, wherein said PEG is selected from maleimide-PEG, vinyl sulphone-PEG, two thiopyridines-PEG and iodo-acid amide-PEG; Be preferably maleimide-PEG.
7. preparation method according to claim 5, wherein said PEG molecular weight is 20～40 kilodaltons; Preferably 20 kilodaltons.
8. preparation method according to claim 5, wherein said recombinant human IFN-λ 1 is l:5 with the molecule mol ratio of PEG.
The purposes of 9.PEGization recombinant human IFN-λ 1 in the medicine of preparation treatment chronic hepatitis B or liver cancer.
10. purposes according to claim 9, wherein said PEGization recombinant human IFN-λ 1 uses separately or is co-administered.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105037523A (en) * | 2015-06-25 | 2015-11-11 | 北京三元基因工程有限公司 | Interferon mutant and polyethylene glycol derivative thereof |
| CN111065405A (en) * | 2017-09-08 | 2020-04-24 | 科学未来管理有限责任公司 | Pegylated interferon lambda with high oral bioavailability and preparation method thereof |
| EP4005586A4 (en) * | 2019-07-22 | 2023-08-09 | Xiamen Amoytop Biotech Co., Ltd. | Method for treating diseases based on interferon |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2013
- 2013-03-11 CN CN201310076833.1A patent/CN104045704B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105037523A (en) * | 2015-06-25 | 2015-11-11 | 北京三元基因工程有限公司 | Interferon mutant and polyethylene glycol derivative thereof |
| CN105037523B (en) * | 2015-06-25 | 2019-02-19 | 北京三元基因药业股份有限公司 | Interferon mutant and its polyethyleneglycol derivative |
| CN111065405A (en) * | 2017-09-08 | 2020-04-24 | 科学未来管理有限责任公司 | Pegylated interferon lambda with high oral bioavailability and preparation method thereof |
| EP3679943A4 (en) * | 2017-09-08 | 2020-10-21 | Obshchestvo S Ogranichennoy Otvetstvennost Yu "Scientific Fututre Management" | Pegylated interferon lambda for oral administration, and method for producing same |
| EP4005586A4 (en) * | 2019-07-22 | 2023-08-09 | Xiamen Amoytop Biotech Co., Ltd. | Method for treating diseases based on interferon |
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