WO2015161832A1

WO2015161832A1 - Long-acting recombinant human interferon α2b-fc fusion protein

Info

Publication number: WO2015161832A1
Application number: PCT/CN2015/077504
Authority: WO
Inventors: 朱文臣; 朱成功; 朱鹿燕; 朱石桥; 王晓山; 刘宾; 任子甲; 朱松林; 孙乃超; 李强; 苏鸿声; 孟庆乐; 崔浩
Original assignee: 辅仁药业集团有限公司; 河南辅仁医药科技开发有限公司
Priority date: 2014-04-25
Filing date: 2015-04-27
Publication date: 2015-10-29
Also published as: WO2015161832A8

Abstract

Provided is a human interferon α2b-Fc fusion protein, wherein the C-terminal of the human interferon α2b is linked to the N-terminal of Fc by a peptidic linker. Provided is a nucleic acid sequence encoding the fusion protein and a eukaryotic expression system expressing the fusion protein. Also provided is a use of the fusion protein in the preparation of an antiviral or antitumour drug. The fusion protein has an improved antiviral activity and prolonged in vivo half-life.

Description

Long-acting recombinant human interferon α2b-Fc fusion protein

This application claims the priority of the Chinese Patent Application entitled "Long-term Recombinant Fc Fusion Human Interferon α2b and Its Preparation Method" filed on April 25, 2014 by the Chinese Patent Office, Application No. CN201410170133.3, the entire contents of which is hereby incorporated by reference. This application is incorporated by reference.

Technical field

The present invention relates to a human interferon (IFN) interferon α2b-Fc fusion protein, and more particularly to a long-acting recombinant human interferon α2b-Fc fusion protein comprising a peptide linker, belonging to the field of biological genetic engineering.

Background technique

Interferon is a defensive substance produced by human cells. After more than half a century of basic and clinical research, it has proven to be an important broad-spectrum antiviral and anti-tumor therapeutic. Among them, IFNα2b is widely used in clinical practice and is the most used interferon. The US FDA approved it for the treatment of chronic hepatitis B, hepatitis C, hairy cell leukemia, Kaposi sarcoma and pigmentoma associated with AIDS. Subacute sclerosing encephalitis and T cell leukemia and other diseases. In China, IFNα2b also belongs to the first batch of genetic engineering high-tech drugs officially approved by the state. After more than 20 years of market introduction, it has treated a variety of common diseases and frequently-occurring diseases in China, and has achieved great economic and social benefits.

Although IFNα2b is widely used in clinical practice, it has the following disadvantages: due to its relatively small molecular weight, it is easily filtered by glomerulus during circulation in the body; it is unstable in the body and is easily degraded by serum protease; the plasma half-life is short and the treatment period is long. The half-life after intramuscular injection is 2-4 hours, and is almost undetectable in serum after 24 hours. Therefore, frequent administration is required in clinical treatment, and patient compliance is low. When used at high doses, it often leads to such as Red blood cells, white blood cells and thrombocytopenia, a series of side effects such as flu-like symptoms and gastrointestinal disorders directly affect the application of interferon in therapy. Therefore, improving the performance of interferon is still one of the focuses of the medical research.

At present, the internationally developed techniques for long-acting interferon mainly include the following: First, chemical modification, mainly by covalently linking a polymer (such as PEG) to interferon, and reducing the kidney by increasing the relative molecular weight of the interferon. Filtration of the pellets prolongs the half-life of the interferon in the body. There are currently two FDA-approved PEGylated interferon drugs: PEG-Intron and Pegasys. The half-life of PEG-Intron is only extended to 40 hours. Although the half-life of Pegasys is extended to 65 hours, the antiviral activity in vitro is only 7×. 10 ⁶ IU / mg; The second is the site-directed mutagenesis technology, which changes the individual amino acids of interferon to reduce the digestion ability of proteases in the blood. The use of site-directed mutagenesis to alter the original gene sequence of a protein often leads to a large change in the structure of the protein, which may lead to the production of new antigenic determinants and increase the toxic side effects of protein drugs. The third is protein fusion technology, which fuses the drug protein gene with a specific protein gene by genetic recombination, and prolongs the half-life of interferon. Human immunoglobulin G (IgG) is one of the most abundant proteins in human blood, and their half-life in the body is about 20 days. The Fc fragment is the main reason for IgG to maintain a longer half-life in vivo, and the Fc fragment also contributes to protein stability. This is because the Fc fragment can bind to the neonatal Fc receptor (FcRn), preventing IgG from being degraded into the lysosome, and Fc can also increase the molecular weight of the protein and reduce glomerular filtration. Linking the Fc fragment of IgG to the active protein to form a fusion protein has been shown to prolong the half-life of the active protein.

Patent application CN1361793 discloses an Fc-IFNα fusion protein having a half-life of only 19.3 hours; US Pat. No. 5,723,125 discloses an IFN-Fc fusion protein comprising a peptide linker which is a Gly-Ser repeat unit, Although the half-life of the fusion protein was prolonged, the antiviral activity was only 2.2 × 10 ⁸ IU/μmol (about 5 × 10 ⁶ IU/mg after conversion). In summary, a small extension of the half-life does not effectively reduce the frequency of dosing, but at the expense of antiviral activity to extend the half-life, and the corresponding therapeutic effect is not achieved. Therefore, in recent years, people have long-term interferon Efforts have been made to find a balance between activity and long-term efficacy, and it is hoped that the activity will be reduced within an acceptable range while maximizing its half-life in vivo.

The long-acting interferons currently available in the market are expressed in the E. coli expression system. However, the prokaryotic expression system has defects such as incompatibility of the inclusion body protein and incomplete protein modification. Conversely, eukaryotic expression systems can overcome the above problems. Most of the expression products of eukaryotic expression systems can be secreted, easily obtained and purified; the expression products can be glycosylated, closer to the natural protein; the stability of the expression system is good, and the expression can be Regulation. Common eukaryotic expression systems are yeast expression systems, insect cell expression systems, and mammalian cell expression systems. Among them, mammalian cell expression systems, especially dihydrofolate reductase (DHFR) diploid-deficient Chinese hamster ovary (CHO) expression system, have more and more pharmaceutical proteins in CHO cells. Highly expressed, some of which have been placed on the market, such as EPO, GCSF, etc. The CHO expression system has many advantages: 1) it has an accurate post-transcriptional modification function, and the expressed glycosylated drug protein molecule is in the molecule. The structure, physical and chemical properties and biological functions are closest to the natural protein molecules; 2) CHO has extracellular secretory function, and rarely secretes its own endogenous protein, which facilitates the separation and purification of downstream products; 3) serum-free suspension culture , the expression level is high. However, there is no report on the expression of IFNα2b-Fc fusion protein in eukaryotic cells. Therefore, there is an urgent need to adopt eukaryotic expression systems to overcome various problems of prokaryotic expression systems.

Summary of the invention

The present invention provides, based on the above problems, an object of the present invention is to provide a novel long-acting recombinant human interferon α2b-Fc fusion protein which passes through a peptide linker of a specific length and an N-terminus of Fc at the C-terminus of human interferon α2b The fusion forms a fusion protein, and the expression of the fusion protein is carried out by using a eukaryotic cell expression system, thereby obtaining a high protein expression amount, and the anti-viral activity of the prepared fusion protein is improved, and the half-life of the fusion protein is also prolonged.

The present invention provides a long-acting recombinant human interferon α2b-Fc fusion protein having the structure of human interferon α2b-peptide linker-Fc, wherein the peptide linker has the following structure: X-(GGGS)p-( GGGGS)n-(GGGS)q, where X is GS or GGS, p=0-2, q=0-2, n=1-8.

In a preferred embodiment of the invention, the peptide linker has the structure: X-(GGGS)p-(GGGGS)n-(GGGS)q, wherein X is GS or GGS, p=0-1, q =0-1, n=1-6.

In a more preferred embodiment of the invention, the peptide linker has SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6. The structure of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The Fc fragments of the invention include Fc fragments of human IgGl, IgG2, IgG3 and IgG4 subtypes, preferably Fc of IgG2 and IgG4 subtypes, most preferably Fc of the IgG2 subtype. The Fc fragment of the present invention includes a natural amino acid sequence and a sequence derivative (mutant) thereof. Amino acid sequence derivatives are sequences that differ from the native amino acid sequence due to deletions, insertions, non-conservative or conservative substitutions of one or more amino acid residues, or combinations thereof. For example, in IgG Fc, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 that are known to be important for binding can be used as appropriate targets for modification. In proteins or polypeptides, amino acid substitutions that generally do not alter the activity of the protein or peptide are well known to those skilled in the art (H. Neurath, R. L. Hill, Protein, Academic Press, New York, 1979).

In a preferred embodiment of the invention, the long acting recombinant human interferon alpha 2b-Fc fusion protein may be present as a homodimer.

The invention also provides a nucleic acid sequence encoding the aforementioned long acting recombinant human interferon alpha 2b-Fc fusion protein.

The present invention also provides a cell expression system expressing a long-acting recombinant human interferon α2b-Fc fusion protein, wherein the cell expression system is a yeast expression system, an insect cell expression system or a mammalian cell expression system.

In a preferred embodiment of the invention, the dihydrofolate reductase (DHFR) diploid deficient CHO cell expression system is utilized to express the fusion protein of the invention.

The present invention also provides the use of the aforementioned long-acting recombinant human interferon α2b-Fc fusion protein for the preparation of an antiviral or antitumor drug.

Furthermore, the IFNα2b-Fc fusion protein prepared by the present invention is formed by linking the C-terminus of IFNα2b to the N-terminus of Fc via a peptide linker, and can also form a double-headed homodimeric Fc fusion protein, and the obtained fusion protein is similar. IgG but no CH1 region and light chain. Due to structural homology, the IFNα2b-Fc fusion protein exhibits in vivo pharmacokinetic properties comparable to isotype human IgG, resulting in an extended half-life of the IFNα2b-Fc fusion protein, thereby reducing the frequency of dosing and enabling patients There is better compliance; and a decrease in fluctuations in the concentration of IFNα2b-Fc fusion protein in serum means an improvement in its safety and tolerability. In addition, there are two IFNα2b fractions on each fusion protein molecule, both of which can perform their functions separately, resulting in higher molar activity (herein "molar activity" refers to the specific activity per unit mole of fusion protein. Increased activity of the fusion protein can reduce the dose administered.

Without being bound by any theory, the present inventors have surprisingly found that the in vivo half-life of the IFNα2b-Fc fusion protein can be extended to more than 90 hours, while the in vitro antiviral activity can reach 10 ⁷ -10 ⁸ IU/mg. In addition, the eukaryotic cell expression system provided by the invention can increase the stability of the protein and the expression of the protein, can make the fusion protein yield at least greater than 900 mg/L, and simplifies the purification step and reduces the production cost, and has a larger Practical significance and broad application prospects.

DRAWINGS

In order to more clearly illustrate the embodiments of the present invention and the prior art, the following description of the embodiments and the drawings used in the prior art will be briefly described. It is obvious that the drawings in the following description are only Some embodiments of the invention may also be used to obtain other figures from these figures without departing from the art.

1 is an electrophoresis pattern of the IFNα2b-16L-IgG2Fc fusion protein and the IFNα2b-32L-IgG2Fc fusion protein of the present invention by SDS-PAGE electrophoresis; the sample of the R point of the lane is a reduced fusion protein (ie, dimer opening), lane NR The sample at the point is a non-reduced fusion protein;

2 is a graph showing the growth cell density, cell viability, and concentration trend of secreted IFNα2b-16L-IgG2Fc fusion protein in a highly expressed cell line selected from cell shake flask culture;

Figure 3 is a graph showing the growth cell density, cell viability, and concentration trend of secreted IFNα2b-32L-IgG2Fc fusion protein in a highly expressed cell line selected from cell shake flask culture;

Figure 4 is a result of HPLC detection of the purity of the purified IFNα2b-16L-IgG2Fc fusion protein of the present invention;

Figure 5 is a pharmacokinetic curve of the IFNα2b-16L-IgG2Fc fusion protein and IFNα2b-32L-IgG2Fc fusion protein of the present invention in rats.

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings. It is apparent that the described embodiments are only a part of the embodiments of the invention, and not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention.

Example 1 Preparation of Human Interferon α2b-peptide Linker-Fc Fusion Protein

1) Construction of expression vector

The fusion protein codons containing the peptide linkers in Table 1 were separately synthesized: IFNα2b-Fc was optimized according to CHO cell preference codon, and restriction endonuclease sites SpeI and EcoRI were added at the 5' and 3' ends.

Table 1

The EMCV IRES fragment obtained by the artificial synthesis method and the mouse dihydrofolate reductase (DHFR) gene were inserted into the sites of EcoRI (5') and XhoI (3') of the mammalian expression vector PCDNA3 (Invitrogen). The PCDNA3-DHFR vector was obtained. The full-length DNA fragment of the optimized IFNα2b-Fc was transferred to the SpeI (5') and EcoRI (3') sites on the PCDNA3-DHFR vector to obtain a PCDNA3-DHFR-IFNα2b-Fc expression vector.

SEQ ID No. 10 is the amino acid sequence of IFNα2b-16L-IgG2Fc fusion protein (wherein 1-16 amino acids are signal peptides, which are cleaved after secretion), and SEQ ID No. 11 is a fusion protein encoding IFNα2b-16L-IgG2Fc a nucleic acid sequence (wherein 1-6 bp in the sequence is a SpeI cleavage site; 7-12 bp is a Kozak sequence; 13-60 bp is a signal peptide sequence; 61-558 bp is an IFN-α2b sequence; and 559-606 bp is a peptide linker sequence; 607-1284 bp is the IgG2 Fc sequence); SEQ ID No. 12 is the amino acid sequence of the IFNα2b-32L-IgG2 Fc fusion protein (wherein 1-16 amino acids are signal peptides, which are cleaved after secretion), and SEQ ID No. 13 is an encoding The nucleic acid sequence of the IFNα2b-32L-IgG2Fc fusion protein (wherein 1-6 bp in the sequence is a SpeI cleavage site; 7-12 bp is Kozak) Sequence; 13-60 bp is the signal peptide sequence; 61-558 bp is the IFN-α2b sequence; 559-654 bp is the peptide linker sequence; 655-1332 bp is the IgG2 Fc sequence Pro331Ser).

2) Screening of transfected and highly expressed cell lines stably expressing cell lines

The host cells were transfected by electroporation (CHO DG44): a Bio-Rad electrorotator was used, and a 4 mm electric rotor was used for the electrorotation, the voltage was set to 280 V and the shock time was 25 msec. Each shock was 1 × 10 ⁷ cells, 40 μg of plasmid, and the total volume was 0.7 ml. After electroporation, the cells were transferred to shake flasks containing 30 ml of growth medium. After 24 hours of culture, the medium was changed to a screening culture containing 50 nM MTX, and seeded in a 96-well plate at 1000 cells/well. The cells were cultured for about 2 weeks until the clonal confluence rate reached 80% or higher. The expression of the anti-human IFN-α2b antibody and the anti-human IgG antibody was analyzed by sandwich ELISA, and the clones with relatively high expression were screened and transferred to 24 Expanded culture in well plates, 6-well plates, T25 cell culture flasks, and cell culture shake flasks.

To increase fusion protein production, cells were cultured using a pressurized method of increasing MTX concentration. Thus, the DHFR gene and the fusion protein gene are co-amplified by inhibition of the DHFR gene by MTX. When the cells were adapted to 2 μM MTX, clones with high levels of expression of the fusion protein were screened by limiting dilution subcloning. Single cell clones stably and highly expressed were screened by cell proliferation and expression of the target protein of different clones.

3) Production of fusion protein

The culture medium components were optimized, and the selected high-yield cell strains were inoculated at 5×10 ⁵ /ml in a 100 ml volume shake flask, and after 3 to 4 days of culture, the fed stream was cultured for 14 days, and the target protein was obtained after fermentation.

Figure 1 shows the results of SDS-PAGE electrophoresis of the fusion protein IFNα2b-16L-IgG2Fc and the fusion protein IFNα2b-32L-IgG2Fc, wherein the R lane is a reduced fusion protein (ie, monomer) and the NR lane is a non-reduced fusion protein. (ie dimer).

It can be seen from Fig. 2 that the high-yielding cell line expressing the IFNα2b-16L-IgG2Fc fusion protein has a viable cell density of up to 1.6×10 ⁷ cells/ml, a cell viability of more than 90%, and a protein yield of greater than 900 mg. /L.

As can be seen from Fig. 3, the high-yielding cell line expressing the IFNα2b-32L-IgG2Fc fusion protein has a viable cell density of up to about 1.5×10 ⁷ cells/ml, a cell viability of more than 90%, and a production of the target protein of which is greater than 1500mg/L.

4) Collect the supernatant and purify the fusion protein

The cell culture medium containing the protein of interest was collected by centrifugation and filtered through a 0.22 μm nitrocellulose filter. The filtrate was loaded onto a column MabSelect ^TM protein A (GE Healthcare) phosphate buffered saline (PBS) equilibrated. After the fusion protein was bound to the Protein A column, the effluent fraction was discarded and the column was washed with PBS until the OD value at 280 nm was less than 0.01. The bound fusion protein was then eluted with 20 mM Tris, 20 mM CaCl ₂ , 300 mM NaCl, 900 mM arginine, 45% propylene glycol (v/v), 0.05% Tween 80 (v/v), pH 6.8 buffer. The fractions containing the purified protein were pooled and dialyzed against PBS. It was then filtered through a 0.22 μm nitrocellulose filter and immediately stored at -70 °C. Under reducing conditions, the purified protein migrates to about 40-50 kDa to give a fusion protein.

The HPLC results of the purified IFNα2b-16L-IgG2Fc fusion protein were shown in Figure 4. The HPLC detection conditions were as follows: column: Ferromen S2000; flow rate: 0.5 mL/min; mobile phase: 0.1 M phosphate buffer + 0.1 M NaCl (pH 7.0) solution; detection wavelength: 280 nm.

Example 2 Determination of in vitro antiviral activity of fusion protein

A human amniotic cell line (WISH cells) was prepared into a cell suspension of 2.5×10 ⁵ -3.5×10 ⁵ cells/ml in complete culture medium, and inoculated into a 96-well cell culture plate, 100 μl per well, and placed in CO _{2 .} Incubate for 4-6 hours in an incubator. The sample to be tested was diluted 1000 times with the assay medium. 150 μl of the assay medium was added to each well of a 96-well plate, and then 50 μl of the diluted sample and the positive control rHuIFN (purchased from Beijing Yuance Pharmaceutical Co., Ltd.) were added to the A row, and each of the three replicate wells was sequentially performed. 4x gradient dilution for a total of 8 dilutions. Inoculate the inoculated cell culture plate. The diluted sample and the positive control solution were sequentially transferred into the cell culture plate, 100 μl per well, and cultured in a CO ₂ incubator for 18-24 hours. The cell culture plate was taken out, the culture solution was discarded, and the diluted virus solution was added to 100 μl per well. It was placed in a CO ₂ incubator for about 24 hours. The 50% lesions of the microscopic positive control were in the D or E row, the supernatant was discarded, 50 μl of staining solution was added to each well, and left at room temperature for 30 minutes. Carefully wash away the staining solution and blot the residual moisture. 100 μl of decolorizing solution was added to each well and allowed to stand at room temperature for 3-5 minutes. Colorimetric on a microplate reader, the measurement wavelength was 570 nm, the reference wavelength was 630 nm, and then the result was processed by a computer program (four-parameter regression calculation method). The antiviral activity results of several fusion proteins are shown in Table 2.

Example 3 Pharmacokinetics experiment of fusion protein in rats

Commercially produced rHuIFN (purchased from Beijing Yuance Pharmaceutical Co., Ltd.) and purified IFNα2b-Fc of the present invention were injected into SD rats (injection dose of 5 μg/kg body weight) by subcutaneous injection to inject SD rats of the same volume of PBS. As a control (3 per group). After injection, blood samples were collected by tail-cutting blood at different time points (0, 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, 96, 120 hours), and heparin sodium anticoagulation will The collected blood samples were centrifuged at 3000 rpm for 10 min, plasma was collected, and stored at -70 ° C for cryopreservation. The blood sample was tested for the content of the fusion protein having the activity of the IFNα2b of the present invention in the serum by using an ELISA kit according to the specification, and a graph reflecting the change in the protein content was plotted based on the result. As shown in Fig. 4, rHuIFN reached the blood concentration after 4 hours. The peak value was 1 nmol/ml, and the elimination half-life was about 8.07 hours. The IFNα2b-16L-IgG2Fc fusion protein reached a peak plasma concentration of 1.15 nmol/ml at 24 hours, and the elimination half-life was 91.15 hours. The IFNα2b-32L-IgG2Fc fusion protein reached blood at 24 hours. The drug concentration peaked at 1.1 nmol/ml and the elimination half-life was 93.25 hours.

Table 2 shows the results of comparison of the in vitro antiviral activity and elimination half-life of a part of the fusion protein of the present invention with the long-acting interferon reported in the literature.

Table 2

It can be seen that the long-acting recombinant human interferon α2b-Fc of the present invention is due to the steric hindrance effect of the fusion protein. The fusion protein, although reduced in vitro, has antiviral activity comparable to PEG-Intron and higher than Pegasys; and the in vivo half-life of the long-acting recombinant human interferon α2b-Fc fusion protein of the present invention is long-lived with these two commercially available products. Interferon PEG-Intron has been greatly improved compared to Pegasys. The prolongation of the half-life means that the administration time can be extended and the number of administrations can be reduced when the same dose is administered.

The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalents, improvements, etc., which are made within the spirit and principles of the present invention, should be included in the present invention. Within the scope of protection.

Claims

A long-acting recombinant human interferon α2b-Fc fusion protein having the structure of human interferon α2b-peptide linker-Fc, wherein the peptide linker has the following structure: X-(GGGS)p-(GGGGS)n- (GGGS)q, where X is GS or GGS, p=0-2, q=0-2, n=1-8.
The long-acting recombinant human interferon α2b-Fc fusion protein according to claim 1, wherein the Fc is a mutant of human IgG2 Fc or human IgG2 Fc.
The long-acting recombinant human interferon α2b-Fc fusion protein according to claim 2, wherein the mutant of human IgG2 Fc comprises a Pro331Ser mutation.
The long-acting recombinant human interferon α2b-Fc fusion protein according to any of the preceding claims, wherein the peptide linker has the following structure: X-(GGGS)p-(GGGGS)n-(GGGS)q, Wherein X is GS or GGS, p=0-1, q=0-1, n=1-6.
The long-acting recombinant human interferon α2b-Fc fusion protein according to claim 4, wherein the peptide linker has the following structure: X-(GGGS)p-(GGGGS)n-(GGGS)q, wherein X is GGS, p=1, q=1, n=1.
The long-acting recombinant human interferon α2b-Fc fusion protein according to claim 4, wherein the peptide linker has the following structure: X-(GGGS)p-(GGGGS)n-(GGGS)q, wherein X is GS, p=0, q=0, n=6.
The long-acting recombinant human interferon α2b-Fc fusion protein according to any of the preceding claims, which may exist in the form of a homodimer.
A nucleic acid sequence encoding the long acting recombinant human interferon alpha 2b-Fc fusion protein of any of the preceding claims.
A cell expression system for expressing the long-acting recombinant human interferon α2b-Fc fusion protein according to any one of claims 1 to 7, wherein the cell expression system is a yeast expression system, an insect cell expression system or a mammalian cell Expression system.
The cell expression system for expressing a long-acting recombinant human interferon α2b-Fc fusion protein according to claim 9, wherein the cell expression system is dihydrofolate reductase (DHFR) diploid deficiency A trapped CHO cell expression system.
Use of the long acting recombinant human interferon alpha 2b-Fc fusion protein of any of claims 1-7 for the preparation of an antiviral or antitumor drug.