CN104045704B - PEGylated recombinant human IFN-λ1, its preparation method and use - Google Patents

Abstract

The invention provides a PEGylated recombinant human IFN-λ1, its preparation method and application. Specifically, the present invention realizes the PEG site-directed modification of recombinant human IFN-λ1, and the present invention also provides the use of the thus obtained PEGylated recombinant human IFN-λ1 in the preparation of medicines for treating chronic hepatitis B or liver cancer. The PEGylated recombinant human IFN-λ1 prepared according to the method of the present invention has good uniformity and a purity greater than 98%. PEGylated recombinant human IFN‑λ1 can significantly inhibit the growth of subcutaneous tumors in tumor-bearing nude mice, significantly reduce the content of HBsAg in the serum of nude mice, and show good safety.

Description

PEGylated recombinant human IFN-λ1, its preparation method and use

technical field

The present invention relates to the field of biotechnology. More specifically, it relates to an interferon obtained through recombinant technology and chemical modification technology, and its preparation method and use. In particular, it relates to a pegylated modified recombinant human IFN-λ1, its preparation method and its use in the preparation of medicines for treating chronic hepatitis B or liver cancer.

Background technique

Interferon-λs (IFN-λs) is a new type of interferon, classified as type III interferon, composed of IFN-λ1 (IL-29), IFN-λ2 (IL-28A) and IFN-λ3 (IL-28B )composition. This type of interferon was first reported by American scientists in 2003 (Sheppard P. et al., 2003; Kotenko1S.V. et al., 2003).

The IFN-λ gene is located on chromosome 19 and contains multiple introns. Its gene structure is very similar to IL-10 family members, but far from IFN-α. In terms of amino acid composition, only 15-19% of the amino acids of IFN-λs are the same as IFN-α. Nevertheless, IFN-λs is functionally very similar to IFN-α, and can inhibit the replication of hepatitis B/hepatitis C virus (HBV/HCV), as well as encephalomyocarditis virus (EMCV), human cytomegalovirus (CMV ), lymphocytic choriomeningitis virus (LCMV), herpes simplex virus (HSV-2) and other viruses (Bobek M.D. et al., 2005). In addition to antiviral activity, in cytological experiments, IFN-λs can inhibit the proliferation of neuroendocrine tumors, esophageal cancer, colon cancer, lung cancer and Burkitt's lymphoma cells and melanoma cells, and mIFN-λs has been used in mice It has been reported to inhibit the proliferation of mouse tumor cells such as melanoma, fibrosarcoma and liver cancer cells by mobilizing the host's immune mechanism (Abushahba W. et al., 2010).

The receptor of IFN-λs belongs to the class Ⅱ cytokine receptor family and consists of structurally specific heterodimer complexes. IL-28Rα is a ligand-binding subunit that determines the specificity of the receptor binding to IFN-λ, and IL-10βR is an auxiliary subunit. Unlike the ubiquitously expressed type I IFN (IFN-α/β) receptors, the expression of IFN-λ receptors is cell-specific and mainly expressed on the surface of epithelial cells, such as in the liver where all cells express IFN-α receptor, while the IFN-λ receptor is only expressed in hepatocytes. IFN-α receptors are also widely expressed in peripheral blood lymphocytes, including T lymphocytes, B lymphocytes, NK cells, neutrophils, and monocytes, but IFN is not detected in hematopoietic cells except for B lymphocytes - Expression of lambda receptors (Muir A.J. et al., 2010). The difference in the distribution of different IFN family receptors will affect the effect of IFN in vivo. Compared with the toxic side effects and poor tolerance of the current clinically used type I IFN, IFN-λs has potential advantages. It can effectively reduce the toxic and side effects on hematopoiesis and central nervous system while exerting biological activities such as anti-virus, anti-tumor and participating in immune regulation.

However, similar to type I IFN, IFN-λs has a relatively small molecular weight (about 20kD) and is easily filtered by the glomeruli; it is unstable in the body, easily degraded by proteases, and has a short plasma half-life; the treatment cycle is long and requires frequent injections. It leads to a reduction in the dependence of patients; recombinant protein biologics expressed as heterologous systems also have immunogenicity problems. All of these have the potential to attenuate the clinical effects of IFN-λs.

Polyethylene glycol (PEG) is a non-toxic, non-immunogenic and safe polymer. Modification of proteins with covalent coupling of PEG chains is the most successful technology for delivering peptides and protein drugs in vivo since the 1970s. Therapeutic benefits of PEGylated proteins include:

-Increased protein molecular weight, reduced glomerular filtration rate and thus prolonged plasma half-life;

-Increase the physical and chemical stability of protein and reduce the degradation of protease;

- Reduced toxicity and limited immunogenicity of proteins;

- Increased solubility of protein drugs;

- Efficiently enhances drug activity in vivo by improving protein pharmacokinetic properties compared to unmodified protein drugs (Veronese FM et al., 2005). The PEGylation method of protein mainly adopts mono-methoxyPEG molecule (mono-methoxyPEG, mPEG) to covalently couple with protein surface reactive group, and its main modification way is to N-terminal amino group or lysine side Chain amino groups are modified by acylation, but this modification method is prone to heterogeneity (ie isomer mixture).

Liver cancer is one of the most common malignant tumors and ranks third in the mortality rate of common tumors worldwide. There are more than 600,000 new cases of liver cancer every year in the world, of which about 55% are in China. Chronic hepatitis B virus (HBV) infection is the most important risk factor for liver cancer. About 80% of liver cancer patients in my country have a history of HBV infection (Yang, J.D. et al., 2010; Parkin, D.M. et al., 2005). IFN-α is clinically considered to be effective in anti-HBV therapy and also beneficial in preventing HBV infection-related liver cancer. However, there are still a considerable proportion of patients with chronic HBV infection clinically unresponsive to IFN-α treatment, and patients often interrupt IFN-α due to intolerable toxicity and side effects including fatigue, fever, anorexia, depression and bone marrow suppression. Treatment. IFN-λs, especially IFN-λ1 (IL-29), can stimulate antiviral activity and inhibit HBV virus replication through the IFN-λ receptor specifically expressed on the surface of liver cells, suggesting that IFN-λ1 can be used to treat chronic HBV infection and HBV Infection-associated HCC patients (Doyle SE. et al., 2006).

Contents of the invention

According to one aspect of the present invention, a PEGylated recombinant human IFN-λ1 (also called PEG-rhIFN-λ1) is provided. Its amino acid composition is similar to wild-type human IFN-λ1, and has the same biological activity as wild-type human IFN-λ1.

The amino acid sequence of the above PEGylated recombinant human IFN-λ1 is shown in SEQ ID No.1. It consists of 181 amino acids, and the 46th position of its N-terminal is glutamine. The 112th cysteine residue (C112 for short) is PEGylated.

The rhIFN-λ1 provided by the present invention is a low-glycosylation mutant IFN-λ1 lacking the N-glycosylation site, the 46th position of its N-terminus is glutamine, and it is expressed by Pichia pastoris. The amino acid sequence, special secretory expression vector, transgenic cell line and host bacteria are all disclosed in the Chinese patent application with publication number CN10235192A, and the applicant is the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

The rhIFN-λ1 provided by the present invention is produced from the methanolophilic yeast-Pichia pastoris strain. Pichia pastoris has many advantages of both prokaryotic and eukaryotic expression systems. The source protein forms a correct fold. The method for expression and purification of Pichia pastoris used in the present invention has been disclosed in the Chinese Patent Authorized Notification No. CN1962873B, and the patentee is the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

In some embodiments, the PEG-rhIFN-λ1 of the present invention, wherein the PEG has a linear or branched chain structure.

In some embodiments, the molecular weight of the PEG is in the range of 20-40 kilodaltons; preferably 20 kilodaltons.

In some embodiments, the PEG is selected from maleimide-PEG (mPEG-MAL), vinylsulfone-PEG (mPEG-VS), dithiopyridine-PEG (mPEG-Pyridyl disulfides) and iodoacetamide -PEG (mPEG-iodoacetamide).

In some specific embodiments, the PEG is mPEG-MAL.

According to another aspect of the present invention, a preparation method of PEG-rhIFN-λ1 is also provided, comprising the steps of:

Provide recombinant human IFN-λ1;

Add PEG to recombinant human IFN-λ1;

The coupling reaction is carried out at 2-8°C for 12-30 hours, and then at 18-25°C for 1-3 hours;

The coupling reaction product is first purified by cation exchange chromatography;

Second purification by gel filtration chromatography;

PEGylated recombinant human IFN-λ1 was harvested.

In a specific embodiment, the coupling reaction is carried out at 4°C for 24 hours, followed by 2 hours at 18-25°C.

In some embodiments, the PEG is selected from maleimide-PEG (mPEG-MAL), vinylsulfone-PEG, dithiopyridine-PEG, and iodoacetamide-PEG. In a specific embodiment, PEG is mPEG-MAL.

In some embodiments, the molecular weight of the PEG is 20-40 kilodaltons. In a specific embodiment, PEG has a molecular weight of 20 kilodaltons.

In a specific embodiment, the molecular molar ratio of recombinant human IFN-λ1 to PEG is 1:5.

The PEG-rhIFN-λ1 prepared by the above method was analyzed by a tandem quadrupole time-of-flight mass spectrometer, and compared with rhIFN-λ1 without PEG modification, the results showed that the coupling site of mPEG-MAL was located on C112 of rhIFN-λ1 , is fixed-point modification.

According to yet another aspect of the present invention, the use of PEG-rhIFN-λ1 in the preparation of medicines for treating chronic hepatitis B or liver cancer is provided.

In some embodiments, PEG-rhIFN-λ1 is administered alone or in combination with PEG-rhIFN-λ1. In some embodiments, PEG-rhIFN-λ1 is administered alone or in combination with other drugs, such as chemotherapeutic drugs. Chemotherapy drugs such as but not limited to doxorubicin, oxaliplatin, 5-fluorouracil or calcium folinate. In some specific embodiments, PEG-rhIFN-λ1 is administered alone. In other specific embodiments, PEG-rhIFN-λ1 is administered in combination with doxorubicin.

In a specific embodiment, PEG-rhIFN-λ1 of the present invention significantly inhibits subcutaneous tumors in tumor-bearing nude mice in a tumor-bearing nude mouse animal model established by the liver cancer cell line Hep3B that continuously secretes hepatitis B virus surface antigen (HBsAg). growth, significantly reduced the HBsAg content in the serum of nude mice.

Description of drawings

Figure 1: Separation and purification of PEG modification reaction mixture.

A: CM Sepharose F.F. Cation exchange chromatography trace; peak 1. mPEG-MAL-rhIFN-λ1; peak 2. rhIFN-λ1;

B: SDS-PAGE image, M. Protein molecular weight marker; Lane 1. PEG reaction mixture; Lane 2. mPEG-MAL-rhIFN-λ1 (elution peak 1); Lane 3. rhIFN-λ1 (elution peak 2).

Figure 2: SDS-PAGE detection results of PEG-rhIFN-λ1.

A: SDS-PAGE Coomassie Brilliant Blue staining; M. Protein molecular weight markers; Lane 1. rhIFN-λ1; Lane 2. mPEG-MAL-rhIFN-λ1 (indicated by the arrow).

B: SDS-PAGE barium-iodine staining; M. protein molecular weight markers; lane 1. rhIFN-λ1; lane 2. mPEG-MAL-rhIFN-λ1 (arrow).

Figure 3: The in vitro activity of PEG-rhIFN-λ1 was detected by ISRE dual luciferase reporter gene assay.

Figure 4: In vitro stability and activity of PEG-rhIFN-λ1.

A1: Determination of protein content after incubation at 37°C by BCA method, rhIFN-λ1 (◇), PEG-rhIFN-λ1 (△);

A2: Luciferase reporter gene assay detects the activities of rhIFN-λ1 (◇) and PEG-rhIFN-λ1 (△) after incubation at 37°C;

B1: ELISA detection of the content of rhIFN-λ1 (◇) and PEG-rhIFN-λ1 (□) after co-incubating with rat serum;

B2: Luciferase reporter gene assay detects the activity of rhIFN-λ1 (◇) and PEG-rhIFN-λ1 (□) after co-incubating with rat serum.

Figure 5: Pharmacokinetic curves of PEG-rhIFN-λ1 and rhIFN-λ1 in rats.

Figure 6: The growth curve of liver cancer tumors treated with PEG-rhIFN-λ1 in nude mice bearing tumors, *P<0.05; **P<0.01.

Figure 7: Serum HBsAg-ELISA detection results of PEG-rhIFN-λ1 used in the treatment of liver cancer tumor-bearing nude mice, **P<0.01.

Figure 8: Mass Spectrometric Identification of PEGylation Modification Sites.

A: PEG-rhIFN-λ1, sequence IQPQPTAGPRPR;

B: rhIFN-λ1, sequence CIQPQPTAGPRPR.

detailed description

The implementation mode and specific operation process of the present invention will be described in detail below in conjunction with specific examples, but it should be understood that the scope of protection of the present invention is not limited to the following examples. The methods used in the following examples are conventional methods unless otherwise specified. The kits were used according to the instructions provided by the manufacturer or supplier.

Example 1, the acquisition of rhIFN-λ1

The secretory expression vectors, transgenic cell lines, host bacteria, fermentation expression and protein purification methods used to prepare the low glycation mutant rhIFN-λ1 are described in detail in the Chinese patent application with publication number CN10235192A.

Embodiment 2, the preparation method of PEG-rhIFN-λ1

The rhIFN-λ1 protein obtained in Example 1 was dialyzed with 25 mM Tris-HCl pH 8.0 buffer solution, and the rhIFN-λ1 concentration was adjusted to 1 mg/ml;

Add PEG powder (20kD linear mPEG-MAL, provided by Beijing Kaizheng Bioengineering Development Co., Ltd.) according to the molar ratio of rhIFN-λ1 and PEG molar ratio of 1:5;

Gently stir to dissolve the powder;

After reacting at 4°C for 24h, react at room temperature (18-25°C) for 2h to obtain the coupling reaction product;

Carry out cation exchange chromatography on the above coupling reaction product: add 5 times the volume of buffer A (25mM NaAC, pH4.5) to dilute the above coupling reaction product; then load the sample on cation exchange chromatography equilibrated with buffer A On the column (CM Sepharose F.F provided by GE Healthcare Life Sciences); elution was linear elution (0-100%) from buffer A to buffer B (25mM NaAC, 0.5M NaCl, pH4.5); off peak;

Perform gel filtration chromatography on the elution peaks collected above: the buffer is 50mM PBS, 150mM NaCl, pH7.0, the chromatographic column is Superdex75, Hiload16/60 prepacked column (product of GE Healthcare Life Sciences);

Purified PEG-rhIFN-λ1 was harvested.

Embodiment 3, homogeneity test

The above cation-exchange chromatography separation results are shown in Figure 1, and the PEG-modified rhIFN-λ1 has good uniformity (Figure 1, B, lane 1), which shows that the preparation method of the present invention not only provides a single modification method, but also cation Unmodified rhIFN-λ1 (Fig. 1, B, lane 3) was well separated from 20kD mPEG-MAL monomodified PEG-rhIFN-λ1 (Fig. 1, B, lane 2) by exchange chromatography (Fig. 1, B, lane 2). A).

Embodiment 4, purity test

After SDS-PAGE electrophoresis, the purified product obtained in Example 2 was subjected to Coomassie brilliant blue staining and barium-iodine staining detection respectively.

Staining with barium-iodine staining can directly detect PEG. The method is: after SDS-PAGE electrophoresis, soak the gel washed with distilled water in 5% barium chloride solution for 30 minutes, and wash off the barium chloride with distilled water. Afterwards, the gel was soaked in 0.1 mM iodine solution for 30 min, and then decolorized with distilled water.

The detection results are shown in Figure 2, where A in Figure 2 is Coomassie Brilliant Blue staining, lane 1 is rhIFN-λ with a molecular weight of about 20kD; lane 2 is PEG-rhIFN-λ1 modified with 20kD mPEG-MAL; B in Figure 2 is barium - Iodine staining, it can be seen that only PEG-rhIFN-λ1 can develop color (Figure 2, B, lane 2); the test results show that the purity of purified PEG-rhIFN-λ1 is greater than 98%.

Embodiment 5, the identification of PEGylation site

The PEG-rhIFN-λ1 and unmodified rhIFN-λ1 purified and obtained in Example 2 were degraded by trypsin, analyzed by a tandem quadrupole time-of-flight mass spectrometer, and the spectra were collected and compared with those obtained from wild-type IFN-λ1 in the database. Comparing the theoretical values of all polypeptide fragments produced by trypsin degradation, polypeptide fragments containing C15, C49, C145 and C171 were captured in both PEG-modified and unmodified samples, and there was no difference between the two samples. Analysis of the trypsin degradation pattern shows that the polypeptide fragment containing C112 is long and the molecular weight is larger than the range that can be captured by the tandem quadrupole time-of-flight mass spectrometer, suggesting that the analysis results after trypsin degradation alone cannot clearly determine that the PEG is modified at the C112 position Point.

Therefore, PEG-rhIFN-λ1 and unmodified rhIFN-λ1 were degraded by proteinase K and analyzed by tandem quadrupole time-of-flight mass spectrometer. The amino acid sequence of the polypeptide fragment containing C112 is "CIQPQPTAGPRPR". In the PEG-rhIFN-λ1 sample, in addition to capturing the polypeptide fragment containing C15 and C49, the polypeptide fragment adjacent to C112 was also captured. The sequence is "IQPQPTAGPRPR". Based on the results of mass spectrometry after degradation by trypsin and proteinase K, in the unmodified rhIFN-λ1 sample, all five polypeptide fragments containing Cys were captured, while in the PEG-rhIFN-λ1 sample, only Except for C112, the other four Cys-containing polypeptide fragments indicate that due to the modification of 20kD mPEG-MAL at the C112 position, the molecular weight is much larger than the range that can be captured by the tandem quadrupole time-of-flight mass spectrometer, so it can be confirmed that mPEG-MAL The fixed-point modification was at position C112 (results shown in Figure 8).

There are a total of 5 Cys residues in the amino acid sequence of rhIFN-λ1, but the modification of mPEG-MAL is only located at the 112th cysteine residue from the amino terminal of rhIFN-λ1, which shows that the PEG-rhIFN-λ1 of the present invention The modification has high uniformity and good homogeneity, and it is a fixed-point modification.

Example 6, In Vitro Activity of PEG-rhIFN-λ1

The in vitro activity of PEG-rhIFN-λ1 was determined by pISRE-luciferase reporter gene activity assay.

The principle of pISRE-luciferase reporter gene activity assay: After interferon binds to specific receptors on the cell surface, a signal cascade reaction is initiated, leading to the entry of interferon-stimulated gene factor (ISGF, IFN-stimulated gene factor) into the nucleus and cis-element Interferon-stimulated response element (ISRE, IFN-stimulated response element) interacts to regulate gene transcription, and finally produces interferon-mediated anti-virus, anti-tumor cell proliferation and immune regulation biological effects (Williams BR, 1991) .

In this example, ISRE was used to assemble luciferase reporter gene plasmid pISRE-TA-luc, and the biological activity of interferon could be reflected by detecting the expression level of luciferase reporter gene regulated by ISRE after interferon stimulation.

The specific method is as follows: Collect logarithmic phase HepG2 cells (liver cancer cell line), adjust the concentration of the cell suspension, inoculate the cells into a 6-well plate at 2×10 ⁵ cells/well, and incubate overnight at 37°C, 5% CO ₂ ; use lipofectamine2000 Transfect pISRE-TA-luc (5ug) and pRL-SV40 (0.1ug, internal reference) plasmids, incubate at 37°C, 5% CO ₂ for 24 hours, replace with serum-free medium and starve for 2-4 hours; Add 100 ng/ml each of rhIFN-λ1 and PEG-rhIFN-λ1, continue to culture for 24 hours, collect the cells, use the dual luciferase reporter gene detection kit (promega company product) to read the values on the TD20/20 luminometer respectively, and record For the detection results, the ratio of Luciferase/Renilla at each time point was used as the expression level of the ISRE reporter gene.

The results of the in vitro activity test of PEG-rhIFN-λ1 are shown in Figure 3. 28% of the activity remained after PEG modification, indicating that rhIFN-λ1 was indeed PEGylated.

Embodiment 7, the in vitro stability of PEG-rhIFN-λ1

Thermal stability analysis in aqueous solution: desalt rhIFN-λ1 and PEG-rhIFN-λ1 into 20mMPBS, pH 7.2 solution, adjust the concentration of both proteins to 400μg/ml, place at 37°C for 0h, 4h, Take samples at 8h, 24h, 48h, and 72h, centrifuge at 10,000rpm for 20min, then take the supernatant and use the BCA method (the kit is a product of Pierce Company) to detect the remaining protein content; at the same time, use the pISRE-luciferase reporter gene activity assay to analyze the remaining activity of the protein, and the results Expressed as remaining protein content and activity percentage.

The thermal stability results of PEG-rhIFN-λ1 in aqueous solution are shown in Figure 4, A1 and A2. The remaining protein content of rhIFN-λ1 without PEG modification decreased to 40% after 8h at 37°C, and the protein content decreased to 20% after 24h. , while the remaining protein content and original protein content of PEG-rhIFN-λ1 did not decrease significantly during the entire observation period, and remained at 90% after 3 days (Figure 4, A1). And the biological activity reflected by the ISRE luciferase reporter gene can still retain more than 75%, while the biological activity of rhIFN-λ1 without PEG modification is less than 30% (Figure 4, A2). The above experimental results indeed show that the stability of the recombinant protein is enhanced after PEG modification of rhIFN-λ1, and the retention time of biological activity is prolonged.

Thermal stability analysis in serum: desalt rhIFN-λ1 and PEG-rhIFN-λ1 into 20mMPBS, pH7.2 solution, adjust the two proteins to the same concentration, add an equal volume of SD rat serum, place at 37°C, Samples were taken at 0h, 4h, 8h, 12h, 24h, 48h, and 72h, centrifuged at 10,000rpm for 20min, and the supernatant was taken to quantitatively detect the remaining protein content with IL-29ELISA kit (product of ebioscience); at the same time, the pISRE-luciferase reporter gene was used to measure the activity The remaining activity of the protein was analyzed by the method, and the results were expressed as the remaining protein content and activity percentage.

The thermostability results of PEG-rhIFN-λ1 in serum are shown in Figure 4, B1 and B2, both the remaining protein content and ISRE reporter gene activity decreased to a certain extent, but compared with unmodified rhIFN-λ1, After 3 days, both the remaining protein content and the biological activity of the ISRE reporter gene remained above 40%, much higher than that of non-PEGylated rhIFN-λ1, indicating that the coupling of PEG to rhIFN-λ1 significantly increased the thermal stability of rhIFN-λ1 and delayed The degradation of rhIFN-λ1 by protease in serum was inhibited.

Example 8, PEG-rhIFN-λ1 rat pharmacokinetic study

The experiment used SD rats, female, weighing 270-290g, randomly divided into groups, 3 rats in each group, fed in separate cages, and the animals were fasted for 12 hours before administration; a single subcutaneous injection of rhIFN-λ1 and PEG-rhIFN-λ1; blood was taken from the tail vein after administration, and the blood sampling time was as follows: rhIFN-λ1—0, 5min, 10min, 0.5h, 1h, 2h, 3h, 4h, 12h, 24h after injection; PEG-rhIFN -λ1—0, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h, 120h, 144h after injection; after the blood sample was centrifuged at 5,000rpm for 10min, the plasma part was collected and stored at -70°C. The content of IFN-λ1 in plasma was detected by ELISA (IFN-lambda1 PlatinumELISA, ebioscience company). Kinetica software was used for curve fitting to calculate various pharmacokinetic parameters. The test data were counted by Excel software, and the statistical results were expressed as mean ± standard deviation (x+SD). The results are shown in Figure 5 and Table 1:

Table 1. Pharmacokinetic parameters of rhIFN-λ1 and PEG-rhIFN-λ1

PK parameters rhIFN-λ1 (x ± SD) PEG-rhIFN-λ (x ± SD) AUC(ng h/ml) 160.72±25.36 2082.16±203.44 C _max (ng/ml) 54.05±4.21 69.18±4.58 _Tmax (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 the curve; Cmax: _maximum peak concentration; Tmax: _peak time; t _1/2 : elimination half-life; CL: elimination rate.

The results showed that the goodness of fit of rhIFN-λ1 and PEG-rhIFN-λ1 in SD rats was consistent with the one-compartment model of first-order absorption. Compared with non-PEG-modified rhIFN-λ1, PEG-rhIFN-λ1 increased the _maximum peak concentration (Cmax) in plasma after subcutaneous injection, and the time to _peak (Tmax) and elimination half-life (t _1/2 ) were significantly prolonged , were 1.28, 14.7 and 10.75 times that of rhIFN-λ1, respectively; the area under the drug-time curve increased significantly (12.9 times that of rhIFN-λ1), and the elimination rate (CL) decreased by 92.4%. These data show that the modification of rhIFN-λ1 with PEG can effectively improve the absorption and distribution of rhIFN-λ1 in vivo, significantly prolong its biological half-life, and increase the bioavailability of rhIFN-λ1 in vivo.

Example 9. Safety and Pharmacodynamic Evaluation of PEG-rhIFN-λ1 on Human Subcutaneous Liver Cancer Xenograft Tumor Model

The experiment uses a human liver cancer cell line—Hep3B, which has integrated HBV genome and can continuously produce HBsAg. It can be used as a cell model to observe the efficacy of anti-HBV drugs (Chen HC. et al., 1997).

Experimental method: Hep3B cells were subcutaneously inoculated on the back of BALB/c nude mice to establish a xenograft tumor animal model of human liver cancer Hep3B cell line, and 5×10 ⁶ Hep3B cells plus Matrigel glue were subcutaneously inoculated on the right back of each mouse, 0.1ml/mouse. When the average volume of the tumor was about 232mm ³ , they were randomly divided into groups according to the size of the tumor. The experimental groups are shown in Table 2:

Table 2. Experimental design

Note: Dosing volume: 10 μl/g body weight; NS: normal saline; Qd: once a day; Q4d: once every four days; Q7d: once a week.

During the experiment, routine monitoring included tumor growth and the effect of treatment on the normal behavior of animals, the activity of experimental animals, food intake and drinking conditions, body weight changes (measured twice a week), eyes, coat and other abnormalities. The safety was evaluated according to the animal body weight change and death, and the efficacy was evaluated according to the relative tumor proliferation rate (T/C (%)) and tumor growth delay time (T-C).

Tumor volume proliferation rate T/C (%): At a certain time point, the percentage value of the tumor volume in the treatment group and the control group. T and C are the mean tumor volumes of the treatment group and the control group at a specific time point, respectively.

The calculation formula is as follows: T/C (%)=T _TV /C _TV *100% (T _TV : mean tumor volume in the treatment group; C _TV : mean tumor volume in the negative control group).

Tumor delay time TC: refers to the number of days that the treatment group is delayed compared with the control group when the tumor grows to a certain volume (1200mm ³ in this experiment). T is the number of days required for the average tumor volume of the treatment group to reach a certain value; C is the number of days required for the average tumor volume of the control group to reach the same value. The larger the TC value, the longer the delay time, indicating the better drug effect; and vice versa.

The changes in tumor volume growth of mice in each treatment group and control group are shown in FIG. 6 . The results showed that rhIFN-λ1 without PEG modification (daily administration) had a certain inhibitory effect on tumor growth of subcutaneous liver cancer in tumor-bearing nude mice, but the inhibitory effect was weak. At the end of administration, the T/C (%) values were 79.3% (group 3a) and 79.1% (group 4a), respectively, and the difference was not significant compared with the tumor volume of mice in the negative control group.

Compared with the effect of non-PEGylated rhIFN-λ1, PEG-rhIFN-λ1 (administration frequency decreased), but the inhibitory effect on tumor growth of subcutaneous liver cancer in tumor-bearing nude mice was dose-dependent. At the end of the administration, the T/C (%) values of the 0.1mg/kg and 2.5mg/kg groups were 85.31% (3b group) and 54.84% (4b group), and the 2.5mg/kg (4b group) tumor volume and Compared with the negative control group, there was a very significant difference (P=0.002), and the tumor volume reached 1200mm ³ was delayed by 13 days compared with the negative control group.

The combination of PEG-rhIFN-λ1 and chemotherapeutic drug doxorubicin has a better effect than that of chemotherapeutic drug alone. At the end of administration, the T/ The C (%) value was 65.59% (group 5) and 64.29% (group 6), the T/C (%) value of the chemotherapy drug group alone was 74.81% (group 2), and PEG-rhIFN-λ1 was used in combination with chemotherapy drugs The tumor volumes of the two groups were significantly smaller than those of the negative control group (P values were 0.016 and 0.012, respectively), and the time for the tumor volume to reach 1200 ^mm3 was delayed by 7.5 days and 6.5 days compared with the negative control group (Table 3).

Table 3. Tumor volume T/C (%) value and T-C delay time in each treatment group

Note: a. mean ± standard error; b. relative tumor volume compared to the negative control group.

The safety results of PEG-rhIFN-λ1 on the Hep3B human liver cancer tumor model: During the experiment, the mice in the negative control group lost more than 10% of their body weight 21 days after tumor inoculation, indicating that the animal model has obvious malignant tumor characteristics. There was no significant difference in the body weight of each administration group compared with the negative control group, and there was no drug toxicity reaction; the negative control group and each administration group terminated the experiment on the 36th day of cell inoculation, and the mice were euthanized and dissected, and the main organs were not damaged. Abnormalities were found, indicating that mice have good tolerance to rhIFN-λ1 and PEG-rhIFN-λ1, and the drug safety is good. The changes in body weight after administration in different treatment groups and control groups are shown in Table 4.

Table 4. Body weight changes in each treatment group and control group

Note: a. Mean ± standard error.

Example 10, PEG-rhIFN-λ1 detection of serum HBsAg in human subcutaneous liver cancer cell Hep3B xenograft tumor model

Since the human liver cancer cell line Hep3B has integrated HBV genome and can continuously produce HBsAg, HBsAg can be detected from the serum of tumor-bearing nude mice during xenograft tumor experiments (Knowles BB. et al., 1980). At the end of the animal experiment, the mouse serum was collected and stored at -80°C, and the HBsAg content in the mouse serum was analyzed using the HBsAg-ELISA detection kit (Shanghai Kehua Bioengineering Co., Ltd.).

The results are shown in Figure 7. Compared with the negative control group, the level of HBsAg in the serum of mice in the 2.5mg/kg rhIFN-λ1 group without PEG modification was only decreased by 4.7%. After rhIFN-λ1 was modified with PEG, it could effectively Enhance the anti-HBV effect, inhibit the secretion of HBsAg from Hep3B cells, the content of HBsAg in the serum of mice in the 0.1mg/kg and 2.5mg/kg PEG-rhIFN-λ1 groups decreased by 28.5% and 62.5% respectively, and the serum levels in the 2.5mg/kg group Compared with the negative control group, the HBsAg content has a very significant difference (P=0.000), indicating that the novel PEG-rhIFN-λ1 provided by the present invention has positive anti-HBV infection effect.

references

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 interferoninhibits 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 hepatitis cells. AntiviralRes. 34 (3): 137-144;

Doyle SE., et al. (2006) Interleukin-29 Uses a Type 1 Interferon-Like Program to Promote Antiviral Responses in Human Hepatocytes. Hepatology. 44:896-906;

Knowles BB., et al (1980) Human hepatocellular carcinoma cell linesecrete 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 lambda1with or without ribavirin in patients with chronic genotype1hepatitis Cvirus 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-29 and their class II cytokinereceptor IL-28R. Nat Immunol. 4 (1), 63-68;

Veronese FM, Pasut G. (2005) PEGylation, successful approach todrug 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 (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.