WO2020156467A1 - 人α干扰素受体结合相关位点突变体及其用途 - Google Patents

人α干扰素受体结合相关位点突变体及其用途 Download PDF

Info

Publication number
WO2020156467A1
WO2020156467A1 PCT/CN2020/073903 CN2020073903W WO2020156467A1 WO 2020156467 A1 WO2020156467 A1 WO 2020156467A1 CN 2020073903 W CN2020073903 W CN 2020073903W WO 2020156467 A1 WO2020156467 A1 WO 2020156467A1
Authority
WO
WIPO (PCT)
Prior art keywords
ifn
eifk
human
receptor binding
interferon
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2020/073903
Other languages
English (en)
French (fr)
Inventor
袁正宏
陈捷亮
李亚明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fudan University
Original Assignee
Fudan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fudan University filed Critical Fudan University
Priority to US19/133,302 priority Critical patent/US20260092098A1/en
Publication of WO2020156467A1 publication Critical patent/WO2020156467A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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]
    • C07K14/56—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7156—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the technical field of medicine and bioengineering, and relates to human alpha interferon receptor binding-related site mutants and uses thereof, and particularly relates to alpha interferon mutants prepared by modifying human alpha interferon and purifying and preparing anti-interferon mutants.
  • Use in a hepatitis B virus preparation which reduces or eliminates viral surface antigen (HBsAg) and DNA in hepatocytes infected by hepatitis B virus.
  • Hepatitis B virus is an important pathogen that seriously endangers human health. According to relevant statistics, there are approximately 240 million HBV carriers in the world, of which nearly 80 million are chronically infected with HBV in China; although there is currently a hepatitis B vaccine that can prevent HBV infection, there are still hundreds of thousands of new chronic hepatitis B infections every year At the same time, hundreds of thousands of people die from liver disease caused by chronic hepatitis B each year.
  • hepatitis B surface antigen (HBsAg) conversion and lasting cccDNA silence, or even complete cure, that is, viral genomic DNA (cccDNA) clearance is still a difficult problem in this technical field.
  • HBsAg hepatitis B surface antigen
  • cccDNA viral genomic DNA
  • Interferon is a type of cytokine with direct antiviral effect and immunomodulatory effect. It was first discovered and named in 1957. It plays a key role in the host's antiviral immune response. There are more than a dozen types of IFNs that have been identified. According to the different receptors they bind, IFNs are roughly divided into two types: I and II: Type I interferons mainly include IFN- ⁇ and IFN- ⁇ (IFN- ⁇ is generally classified as III Type II interferon), Type II interferon is mainly IFN- ⁇ .
  • interferon activates the transduction of the downstream JAK-STAT signaling pathway through IFNAR that specifically binds to the cell surface and induces the transcription and expression of interferon-stimulating genes (ISGs) to exert antiviral effects; currently known to include IFN- ⁇ 2 in Within, 13 human IFN- ⁇ subtypes have been identified one after another, and their coding genes are all located on human chromosome 9.
  • ISGs interferon-stimulating genes
  • Type IFN- ⁇ activates the downstream classical or alternative signaling pathways in different ways and extent; at the same time, different viruses and different cells have different sensitivities to IFN subtypes; it has been found through research on the affinity of interferon and its receptor , Interferon usually has high binding power to IFNAR2, but low binding power to IFNAR1, and the amino acid positions related to the interferon receptor binding in the interferon amino acid sequence have been basically resolved.
  • each IFN- ⁇ subtype inhibits HBV secretion into the supernatant of viral antigens and intracellular Regression analysis of HBV RNA levels revealed that the inhibitory effect of different subtypes of IFN- ⁇ on HBV is positively correlated with their affinity for IFNAR1 but not IFNAR2; further analysis of IFN- ⁇ by comparing the amino acid sequences of IFN- ⁇ 2 and IFN- ⁇ 14 It is found that there are 4 amino acid positions difference between the two amino acid positions that bind to IFNAR1.
  • IFN- ⁇ 2 4 amino acid positions on IFN- ⁇ 2 are mutated to the corresponding IFN- ⁇ 14 amino acids, and then the IFN- ⁇ 2 mutant
  • the antiviral function and signal pathway activation were tested, and it was found that it had antiviral effects and signal pathway activation effects similar to IFN- ⁇ 14; on the one hand, the research results enriched the antiviral mechanism of alpha interferon from scientific knowledge.
  • Recognition on the other hand, provides a theoretical and technical basis for the development of new treatment methods for chronic hepatitis B based on interferon receptor binding-related site mutants.
  • the inventor of the present application intends to provide human alpha interferon receptor binding-related site mutants and their uses.
  • the alpha interferon mutants prepared by modifying human alpha interferon and purification and their applications Use in preparing anti-hepatitis B virus preparations.
  • the purpose of the present invention is to provide human alpha interferon receptor binding-related site mutants based on the status and foundation of the prior art
  • Another object of the present invention is to provide the use of the human alpha interferon receptor binding related site mutants. By mutating specific amino acids at the interaction site of human IFN- ⁇ 2 and interferon receptor 1 subunit, the direct anti-HBV effect is improved.
  • the research of the present invention shows that the interferon mutant obtained by mutating individual specific amino acid sites has stronger anti-HBV effect and lower working concentration than the current clinical use of IFN- ⁇ 2, and can reduce or eliminate the virus surface in hepatocytes infected by hepatitis B virus.
  • Antigen (HBsAg) and DNA are included in the genome of the interferon mutant obtained by mutating individual specific amino acid sites.
  • the present invention provides new ideas and theoretical technical support for the development of novel preparations for the treatment of chronic hepatitis B based on specific alpha interferon mutants.
  • IFN- ⁇ 14 has the most significant inhibitory effect on HBV replication at the same concentration; and the 13 subtypes and interference of IFN- ⁇ reported in research
  • the binding and dissociation constants of the two subunits of the receptor are different, and the regression analysis of the viral antigens of each IFN- ⁇ subtype inhibiting the secretion of HBV into the supernatant and the intracellular HBV RNA level shows that the different subtypes of IFN-
  • the effect of ⁇ inhibiting HBV is positively correlated with its affinity for IFNAR1 but not IFNAR2, and comparing the amino acid sequences of IFN- ⁇ 2 and IFN- ⁇ 14 and analyzing the binding amino acid positions of IFN- ⁇ and IFNAR1, it is found that there are 4 amino acid positions between the two There are differences.
  • the 4 amino acid positions on IFN- ⁇ 2 were mutated to the corresponding IFN- ⁇ 14 amino acids, and the IFN- ⁇ 2 mutant was tested for antiviral function and signal pathway activation, and it was found that it had similar IFN - ⁇ 14 antiviral effect and signal pathway activation effect, etc. research basis; the present invention constructs prokaryotically expressed human IFN- ⁇ and its mutant plasmids, and obtains biologically active human IFN- ⁇ through the method of prokaryotic expression and purification of proteins Subtype and corresponding mutant recombinant protein.
  • the amino acid sequence of IFN- ⁇ 14 with stronger anti-HBV effect is compared with clinical Using the IFN- ⁇ 2 amino acid sequence, it was found that 4 amino acid positions related to the binding of IFNAR1 were different in the two subtypes of IFN- ⁇ ; for this, the present invention changed the aspartic acid at position 82 of human IFN- ⁇ 2 Mutation to glutamic acid, mutation of threonine at position 86 to isoleucine, mutation of tyrosine at position 89 to phenylalanine, and mutation of arginine at position 120 to lysine to obtain an improved affinity for IFNAR1 Interferon mutant IFN- ⁇ 2-EIFK.
  • amino acid sequence of the related IFN- ⁇ 2 recombinant protein is obtained from the human genome, and the sequence is SEQ ID NO.1;
  • the IFN- ⁇ 14 sequence compared with IFN- ⁇ 2 in amino acid sequence is SEQ ID NO.3;
  • the sequence of IFN- ⁇ 2-EIFK, which mutates the four IFNAR1 receptor binding-related amino acid positions of IFN- ⁇ 2, is SEQ ID NO.2.
  • the human IFN- ⁇ 2 gene coding sequence (SEQ ID No. 1), IFN- ⁇ 2-EIFK (SEQ ID NO. 2) interferon mutant sequence (as shown in Figure 1A), and IFN- ⁇ 14 sequence (SEQ ID No. .3) Clone into a prokaryotic expression vector, obtain the interferon after prokaryotic expression of the recombinant protein, and then concentrate the protein to remove the endotoxin in the interferon, obtain purified interferon with different dilutions, aliquot, and store in- 80°C.
  • the present invention carries out the purification of interferon by prokaryotic expression system and its purity evaluation; and the comparison experiment of human IFN- ⁇ 2 and IFN- ⁇ 2-EIFK in inhibiting HBV antigen and DNA level in HBV infection replication model, and human IFN- Comparison of ⁇ 2 and IFN- ⁇ 2-EIFK on the activation of the classic JAK-STAT1/STAT2 pathway, and comparison of the difference between human IFN- ⁇ 2 and IFN- ⁇ 2-EIFK in inducing some interferon-stimulating genes.
  • the present invention shows that the interferon mutant IFN- ⁇ 2-EIFK is compared with human IFN- ⁇ 2 through immunoblotting, interferon stimulating response element (ISRE) fluorescence reporter system and quantitative PCR experimental data. It has a stronger activating effect on the classic interferon pathway JAK-STAT pathway, and a stronger activating effect on ISRE, which can induce higher levels of ISGs. Among them, ISGs related to the anti-HBV effect of interferon are compared with the prior art. Subgroups have a higher inducing effect.
  • ISRE interferon stimulating response element
  • the human alpha interferon receptor binding related site mutants of the present invention have been tested and tested, and the results show that the interferon mutant IFN- ⁇ 2-EIFK has the ability to inhibit HBV surface antigen, e antigen and viral DNA content.
  • the activity of IFN- ⁇ 2 is superior to that of the currently used IFN- ⁇ 2, and there is no cytotoxic effect; under the similar antiviral effect, the working concentration of IFN- ⁇ 2-EIFK is more than 10 times lower than that of IFN- ⁇ 2.
  • the IFN- ⁇ mutant can be used to prepare new drugs for the treatment of chronic hepatitis B.
  • A human IFN- ⁇ 2, IFN- ⁇ 14 and IFN- ⁇ 2-EIFK mutant sequence alignment diagram
  • B human IFN- ⁇ 2 and IFN- ⁇ 2-EIFK purified interferon Coomassie brilliant blue results
  • FIG. 1 Comparison of human IFN- ⁇ 2 and IFN- ⁇ 2-EIFK in the inhibition of HBV antigen and DNA levels in the HBV infection replication model. Among them, A, IFN- ⁇ 2 and IFN- ⁇ 2-EIFK two interferons Antiviral effect in HepG2-NTCP cells; Antiviral effect of B, IFN- ⁇ 2 and IFN- ⁇ 2-EIFK in PHH cells;
  • FIG. 3 Comparison of human IFN- ⁇ 2 and IFN- ⁇ 2-EIFK on the activation of the classic JAK-STAT1/STAT2 pathway. Among them, A IFN- ⁇ 2 and IFN- ⁇ 2-EIFK stimulate the difference in phosphorylation levels of STAT1 and STAT2 ; B, the difference in activation of ISRE between IFN- ⁇ 2 and IFN- ⁇ 2-EIFK;
  • the human IFN- ⁇ 2 gene coding sequence (SEQ ID No. 1), IFN- ⁇ 2-EIFK (SEQ ID NO. 2) interferon mutant sequence (as shown in Figure 1A), and IFN- ⁇ 14 sequence (SEQ ID No. .3) Cloning into a prokaryotic expression vector, and then performing prokaryotic expression of the recombinant protein;
  • the centrifuged cells are resuspended in buffer A (20mM phosphate buffer, 0.5M sodium chloride, 20mM imidazole), and the volume of the resuspension is 1/50-1/100 of the amount of bacteria; Suspend the clumps, blow them away, transfer to a 2ml centrifuge tube, ultrasonic on ice: ultrasonic breaker high, break for 10s, cool for 10s, cycle 6 times; this step is repeated 3 times. Centrifuge at 10000g for 25min at 4°C and collect the supernatant. Dilute the collected bacterial lysate supernatant with buffer A to 1/20 of the shake volume, and filter the diluted sample. First pass through a 0.45 ⁇ m filter membrane, and then through a 0.22 ⁇ m filter membrane. Set 4°C for standby;
  • Triton X-114 was used to remove endotoxin in interferon. Interferon and 10% Triton X-114 were mixed at a ratio of 9:1, and magnetically stirred at 4°C for 60 minutes, and mixed well. 30°C metal bath, shaking at 1000 rpm, 20 min; take it out and invert and mix, and then 30°C metal bath, shaking at 1000 rpm, 20 min. Centrifuge at 14000g for 15min at .25°C, carefully remove the upper aqueous phase into the newly unpacked tube, repeat the above steps again;
  • the purified human IFN- ⁇ 2 and IFN- ⁇ 2-EIFK recombinant proteins are treated with HepG2-NTCP ( Figure 2A) or PHH ( Figure 2B) cells infected with hepatitis B virus particles.
  • the production of hepatitis B virus e antigen (HBeAg) and DNA is different. Degree of inhibition:
  • HepG2-NTCP cell culture DMEM culture medium (Gibco company, plus 10% fetal bovine serum, 100U/ml penicillin, 100mg/ml streptomycin) is used for normal culture at 37°C under 5% CO2 saturated water vapor environment Constant temperature culture.
  • the infection medium ordinary medium + 2.5% DMSO
  • PHH cell culture purchased from Shanghai Ruide Biotechnology and cultured in a special commercial medium;
  • the hepatitis B virus used for infection is purified by our laboratory. After collecting the HepAD38 supernatant, use the PEG8000 precipitation method to concentrate the supernatant about 100 times, and infect at a concentration of 200 copies/cell during infection;
  • the above examples illustrate that the present invention prepares wild-type and mutant interferons through cloning and purification, and compares the effects of human IFN- ⁇ 2 and IFN- ⁇ 2-EIFK mutants in anti-HBV.
  • the experimental results show that amino acids related to IFNAR1 are mutated Site, IFN- ⁇ 2-EIFK obtained by changing the affinity of IFN- ⁇ 2 and IFNAR1 has a more significant anti-HBV effect at the level of inhibiting HBV antigen and DNA. This effect is related to its ability to stimulate higher levels of STAT1 and STAT2 phosphorylation, activate higher levels of ISRE, and induce higher levels of antiviral-related ISGs.
  • This method of mutating IFN- ⁇ interferon receptor binding related sites will provide new ideas for the development of new anti-viral interferons, and the development of the new interferons has good application and development prospects.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Virology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供一种人α干扰素受体结合相关位点突变体IFN-α2-EIFK，所述突变体较IFN-α2具有更强的抗乙肝病毒活性，且在抗病毒浓度下无细胞毒效应，可制备抗乙肝病毒药物。

Description

人α干扰素受体结合相关位点突变体及其用途 技术领域
本发明属医药和生物工程学技术领域，涉及人α干扰素受体结合相关位点突变体及其用途，尤其涉及通过改造人类α干扰素及纯化制备的α干扰素突变体及其在制备抗乙型肝炎病毒制剂中的用途，所述制剂降低或清除乙肝病毒感染肝细胞中病毒表面抗原(HBsAg)及DNA。
背景技术
乙型肝炎病毒(HBV)是严重危害人类健康的重要病原体。据有关统计，全球大约有2.4亿HBV携带者，其中我国慢性HBV感染者近8000万人；虽然目前已有可预防HBV感染的乙肝疫苗，但是，每年仍有几十万的新发乙肝慢性感染病例，同时，每年有几十万人死于慢乙肝导致的肝脏疾病。由于特异性治疗手段的缺乏，如何达到慢乙肝功能性治愈，即乙肝表面抗原(HBsAg)转阴和cccDNA持久沉默，乃至完全治愈即病毒基因组DNA(cccDNA)清除，仍是本技术领域中的难题，其中，如，若干患者为控制病毒感染及治疗疾患需长期乃至终身服用核苷(酸)类抗病毒药物，造成严重的经济负担及降低了生活质量。
干扰素(Interferon,IFN)是一类具有直接抗病毒效应、免疫调节作用的细胞因子，于1957年最初被发现并命名，在宿主抗病毒免疫应答过程中发挥着关键作用。目前已鉴定的IFNs有十几种，根据所结合受体的不同，IFNs大致被分为I和II两型：I型干扰素主要包括IFN-α和IFN-β(IFN-λ一般归为III型干扰素)，II型干扰素则主要是IFN-γ，其中，随着基因工程技术的出现及发展，重组人IFN-α于上世纪被克隆及生产应用于治疗病毒性肝炎等疾病；相较另一大类用于治疗慢乙肝的药物—核苷(酸)类似物，研究显示，IFN-α及其PEG化产物(PEG-IFN-α)除了具有直接抗病毒作用外还兼具免疫调节作用，因此具有相对更高的HBsAg转阴及持续应答率更高的优势；然而，临床统计表明，IFN-α治疗慢乙肝的效率仍较低，如，长效干扰素治疗48个月HBe抗原(HBeAg)阳性患者仅约三成可出现HBeAg转阴，而HBsAg的转阴率则更低于5％，为此，业内共识，亟待优化提升干扰素抗乙肝病毒效率和应答率。
研究显示，干扰素通过特异性结合到细胞表面的IFNAR启动下游JAK-STAT信号通路的转导进而诱导干扰素刺激基因(ISGs)的转录表达而发挥抗病毒作用；目前已知包括IFN-α2在内，有13种人类IFN-α亚型被陆续鉴定，它们的编码基因均位于人9号染色体，各亚型α干扰素间具有较多相似的结构域，但有30％左右序列为非保守；部分报道表明，虽然不同亚型IFN-α均通过与I型干扰素受体的两个亚基IFNAR1和IFNAR2结合而发挥作用，但由于与两受体亚基的结合亲和力各异，各亚型IFN-α激活下游经典或旁路信号通路的方式及程度存在差异；同时，不同病毒及不同细胞对于IFN亚型的敏感性也存在不同；有通过对干扰素及其受体亲和力的研究发现，干扰素通常对IFNAR2的结合力高，而对IFNAR1的结合力低，而干扰素氨基酸序列中与干扰素受体结合相关的氨基酸位点已基本被解析。
前期研究显示，HBV对不同IFN-α亚型的敏感性不同，其中同等作用浓度下IFN-α14抑制HBV复制的效果最为显著。另有研究报道IFN-α的13种亚型与干扰素受体两亚基的结合解离常数不同，据此对各IFN-α亚型抑制HBV分泌到上清中的病毒抗原及细胞内的HBV RNA水平进行回归性分析，发现不同亚型的IFN-α抑制HBV的效应与其对IFNAR1的亲和力而非IFNAR2呈正相关；进一步通过比对IFN-α2和IFN-α14的氨基酸序列，分析IFN-α与IFNAR1结合的氨基酸位点，发现两者间有4个氨基酸位点存在差异，将IFN-α2上这4个氨基酸位点突变为相应的IFN-α14的氨基酸，进而对该IFN-α2突变体进行抗病毒功能和信号通路激活上的检测，发现其具有了类似IFN-α14的抗病毒效果和信号通路激活效应；该研究结果一方面从科学认知上丰富了对α干扰素抗病毒机制的认识，另一方面为开发基于干扰素受体结合相关位点突变体的新型治疗慢乙肝手段提供了理论和技术基础。
基于现有技术的现状与基础，本申请的发明人拟提供人α干扰素受体结合相关位点突变体及其用途，通过改造人类α干扰素及纯化制备的α干扰素突变体及其在制备抗乙型肝炎病毒制剂中的用途。
发明内容
本发明目的是基于现有技术的现状与基础，提供人α干扰素受体结合相关位 点突变体，
本发明的再一目的在于提供所述人α干扰素受体结合相关位点突变体的用途。通过突变特定人IFN-α2与干扰素受体1亚基相互作用位点的氨基酸，提升其直接抗HBV效应。
本发明研究显示，突变个别特定氨基酸位点得到的干扰素突变体相较目前临床所使用的IFN-α2抗HBV效应更强而工作浓度更低，能降低或清除乙肝病毒感染肝细胞中病毒表面抗原(HBsAg)及DNA。
本发明为开发基于特定α干扰素突变体的新型治疗慢乙肝的制剂提供了新的思路和理论技术支撑。
基于本申请前期研究，HBV对不同IFN-α亚型的敏感性不同，其中同等作用浓度下IFN-α14抑制HBV复制的效果最为显著；和有研究报道的IFN-α的13种亚型与干扰素受体两亚基的结合解离常数不同，及对各IFN-α亚型抑制HBV分泌到上清中的病毒抗原及细胞内的HBV RNA水平进行回归性分析显示，不同亚型的IFN-α抑制HBV的效应与其对IFNAR1的亲和力而非IFNAR2呈正相关，以及比对IFN-α2和IFN-α14的氨基酸序列和分析IFN-α与IFNAR1结合的氨基酸位点发现两者间有4个氨基酸位点存在差异，将IFN-α2上这4个氨基酸位点突变为相应的IFN-α14的氨基酸，对该IFN-α2突变体进行抗病毒功能和信号通路激活上的检测，发现其具有了类似IFN-α14的抗病毒效果和信号通路激活效应，等研究基础；本发明通过构建原核表达的人IFN-α及其突变质粒，通过原核表达纯化蛋白的方法制得有生物学活性的人IFN-α亚型及相应突变体重组蛋白。
具体的，本发明中，在关于不同亚型IFN-α抗HBV差异及该差异与干扰素-IFNAR1亲和力呈正相关的研究基础上，通过比较抗HBV效应更强的IFN-α14的氨基酸序列与临床使用的IFN-α2氨基酸序列，发现4个与IFNAR1结合相关的氨基酸位点在两种亚型的IFN-α中存在差异；针对此，本发明将人IFN-α2的第82位天冬氨酸突变为谷氨酸，第86位苏氨酸突变为异亮氨酸，第89位酪氨酸突变为苯丙胺酸，及第120位精氨酸突变为赖氨酸，获得一个对IFNAR1亲和力提升的干扰素突变体IFN-α2-EIFK。继而，在HBV感染细胞模型HepG2-NTCP 和原代肝细胞(primary human hepatocytes，PHH)中，对人IFN-α及相应突变体的抗HBV效果进行了比较，检测人IFN-α2和IFN-α2-EIFK对HBeAg，HBsAg及HBV DNA的抑制效果，结果显示，所述的干扰素突变体IFN-α2-EIFK具有类似IFN-α14的强效抗HBV效果，与同等浓度IFN-α2相比较，其对病毒HBs与HBe抗原和病毒DNA的抑制效果强2-10倍，并且在工作浓度下均无细胞毒性。
本发明中，相关IFN-α2重组蛋白的氨基酸序列获取自人基因组，序列为SEQ ID NO.1；
本发明中，与IFN-α2进行氨基酸序列对比的IFN-α14序列为SEQ ID NO.3；
本发明中，对IFN-α2的4个IFNAR1受体结合相关氨基酸位点进行突变的IFN-α2-EIFK的序列为SEQ ID NO.2。
本发明中通过下述方法制备与纯化人IFN-α2和IFN-α2-EIFK：
将人IFN-α2基因编码序列(SEQ ID No.1)，IFN-α2-EIFK(SEQ ID NO.2)干扰素突变体序列(如图1A所示)，及IFN-α14序列(SEQ ID No.3)克隆到原核表达载体上，进行重组蛋白原核表达后获得干扰素，再进行蛋白浓缩，去除干扰素中的内毒素，获得纯化后的稀释不同倍数的干扰素，分装，保存于-80℃。
本发明进行了原核表达系统纯化干扰素及其纯度评价；以及人IFN-α2和IFN-α2-EIFK两种干扰素在HBV感染复制模型中抑制HBV抗原及DNA水平的比较实验，以及人IFN-α2和IFN-α2-EIFK两种干扰素对经典JAK-STAT1/STAT2通路激活效应比较，和人IFN-α2和IFN-α2-EIFK两种干扰素诱生部分干扰素刺激基因的差异比较。
本发明在HepG2-NTCP细胞中，通过免疫印迹、干扰素刺激反应原件(ISRE)荧光报告系统及定量PCR实验数据表明，相较人IFN-α2，所述的干扰素突变体IFN-α2-EIFK对干扰素经典通路JAK-STAT通路具有更强的激活效应，对ISRE具有更强的活化效应，可诱生更高水平的ISGs，其中，较现有技术的与干扰素抗HBV疗效相关的ISGs亚群有更高的诱生效应。
本发明的人α干扰素受体结合相关位点突变体，经试验及检测，结果表明，所述的干扰素突变体IFN-α2-EIFK在抑制HBV表面抗原、e抗原和病毒DNA含量上具有优于当前所使用IFN-α2的活性，且无细胞毒效应；类似抗病毒效应下， 所述IFN-α2-EIFK比IFN-α2工作浓度低10倍以上。进一步，所述的IFN-α突变体可用于制备治疗慢性乙肝新型药物。
为便于理解，以下将通过具体的附图对本发明所述IFN-α2-EIFK突变体相较IFN-α2具有更优抗HBV活性进行详细地描述。需指出的是，该附图仅是为了说明，显然本领域的普通技术人员可根据本文说明，在本发明的范围内对本发明做出个别位点和流程等的修改，这些修改也纳入本发明的范围内。
附图说明
图1.原核表达系统纯化干扰素及其纯度评价；
其中，A，人IFN-α2、IFN-α14和IFN-α2-EIFK突变体序列比对示意图；B，人IFN-α2和IFN-α2-EIFK纯化干扰素的考马斯亮蓝结果；
图2.人IFN-α2和IFN-α2-EIFK两种干扰素在HBV感染复制模型中，抑制HBV抗原及DNA水平的比较，其中，A，IFN-α2和IFN-α2-EIFK两种干扰素在HepG2-NTCP细胞中的抗病毒效应；B，IFN-α2和IFN-α2-EIFK两种干扰素在PHH细胞中的抗病毒效应；
图3.人IFN-α2和IFN-α2-EIFK两种干扰素对经典JAK-STAT1/STAT2通路激活效应比较，其中，A IFN-α2和IFN-α2-EIFK刺激STAT1和STAT2磷酸化水平的差异；B，IFN-α2和IFN-α2-EIFK活化ISRE的差异；
图4.人IFN-α2和IFN-α2-EIFK两种干扰素诱生部分干扰素刺激基因的差异比较。
具体实施方式
实施例1人IFN-α2和IFN-α2-EIFK的制备与纯化
将人IFN-α2基因编码序列(SEQ ID No.1)，IFN-α2-EIFK(SEQ ID NO.2)干扰素突变体序列(如图1A所示)，及IFN-α14序列(SEQ ID No.3)克隆到原核表达载体上，随后进行重组蛋白原核表达；
(1)将重组质粒转化入E.coli BL-21，涂布于含干扰素的固体LB培养基，37℃待16h后，挑取单菌落到3-4ml LB重进行小摇，过夜。以1:100的比例将菌接入大摇，待OD600读数在0.5-0.6之间时，IPTG终浓度10μM进行诱导，温度16℃表达20小时，蛋白诱导表达完成后，将菌液加入到50ml离心管，5000g， 4℃离心10min，弃上清；
(2)离心后的菌体用缓冲液A(20mM磷酸盐缓冲液，0.5M氯化钠，20mM咪唑)重悬，重悬液的体积是菌液量的1/50-1/100；重悬菌块，将其充分吹散后，转移至2ml离心管，冰上超声：超声破碎仪high档，破碎10s，冷却10s，循环6次；该步骤重复3次。10000g，4℃离心25min，收集上清。将收集到的菌体裂解液上清用缓冲液A稀释到摇菌体积的1/20，对稀释好的样品进行过滤，首先过0.45μm的滤膜，然后再过0.22μm的滤膜，样品置4℃备用；
(3)使用GE公司AKTA avant机器配合GE Histrap HP(货号：17524701)进行亲和纯化。随后使用GE公司AKRA avant机器配合GE Hitrap Q HP(货号：17115401)进行离子交换；
(4)收集后的样品进行考马斯亮蓝染色，对干扰素纯度进行鉴定(如图1B所示)；
(5)将纯化好的干扰素用超滤管进行蛋白浓缩，并将干扰素的缓冲液置换为PBS；
(6)为排除内毒素对实验结果的影响，随后用Triton X-114对干扰素中的内毒素进行去除。干扰素与10％Triton X-114以9:1的比例进行混合，4℃磁力搅拌60min，充分混匀。30℃金属浴，1000rpm振荡，20min；拿出来颠倒混匀一下，再30℃金属浴，1000rpm振荡，20min。.25℃，14000g离心15min，小心移出上层水相到新拆封的tube管中，以上步骤再重复一次；
(7)将上述干扰素转移至预处理过的透析袋中，透析袋外侧缓冲液为预冷过的PBS，透析过夜；
(8)将纯化完成后的干扰素稀释不同的倍数，进行BCA定量。全部完成后，将干扰素分装，保存在-80℃冰箱。
实施例2 HBV感染系统
将纯化的人IFN-α2和IFN-α2-EIFK重组蛋白处理感染有乙肝病毒颗粒的HepG2-NTCP(图2A)或PHH(图2B)细胞，乙肝病毒e抗原(HBeAg)和DNA的产生受不同程度抑制：
(1)HepG2-NTCP细胞的培养：普通培养采用DMEM培养液(Gibco公司，加10％ 胎牛血清、100U/ml青霉素、100mg/ml链霉素)在5％CO2饱和水蒸气环境下37℃恒温培养。进行乙肝病毒感染实验时，感染用培养基：普通培养基+2.5％DMSO，PHH细胞的培养：购自上海瑞德生物并采用专用商业培养基培养；
(2)感染所用的乙型肝炎病毒，由本室纯化。将HepAD38上清收集后，采用PEG8000沉淀的方法浓缩上清约100倍，感染时以200copies/cell的浓度进行感染；
(3)细胞感染3天后，向各组细胞分别加入人IFN-α2或IFN-α2-EIFK突变体干扰素(HepG2-NTCP系统中干扰素浓度为0.2或1ng/ml，PHH系统中干扰素浓度为0.04或0.2ng/ml)，每孵育72小时进行换液及再处理；
(4)感染后第9天，收集细胞上清ELISA检测病毒抗原标志物HBeAg，用特异性HBV引物qPCR检测上清中病毒DNA的产生情况；
(5)在两种HBV感染细胞模型中，结果均显示IFN-α2-EIFK具有优于IFN-α2的抗HBV抗原及病毒DNA产生的效果(如图2所示)；
(6)Western Blot检测结果进一步显示，IFN-α2-EIFK相较IFN-α2可更有力地激活经典JAK-STAT1/STAT2通路，即更高地刺激STAT1和STAT2的磷酸化水平；还可以更高水平的活化干扰素刺激原件ISRE(如图3所示)；
(7)进一步检测IFN-α2和IFN-α2-EIFK处理HepG2-NTCP细胞6小时后细胞内ISGs诱生情况，结果显示IFN-α2-EIFK相较IFN-α2，可诱生更高幅度的ISGs(如图4所示)，分析认为这可能与其具有更好的抗病毒效果相关。
上述实例说明本发明通过克隆及纯化制备野生型和突变体干扰素，比较了人IFN-α2和IFN-α2-EIFK突变体在抗HBV中的效应，实验结果表明，通过突变与IFNAR1相关的氨基酸位点，改变IFN-α2与IFNAR1的亲和力所得到的IFN-α2-EIFK在抑制HBV抗原和DNA水平上具有更为显著的抗HBV效应。这种效应与其可刺激更高水平的STAT1和STAT2磷酸化，活化更高水平的ISRE以及诱生更高水平的抗病毒相关ISGs相关。这种突变IFN-α干扰素受体结合相关位点的方法，将为开发新的用于抗病毒的干扰素提供新的思路，所述新型干扰素的开发具有良好的应用和发展前景。
Figure PCTCN2020073903-appb-000001

Claims (6)

  1. 人α干扰素受体结合相关位点突变体，其特征在于，通过将人IFN-α2的第82位天冬氨酸突变为谷氨酸，第86位苏氨酸突变为异亮氨酸，第89位酪氨酸突变为苯丙胺酸，及第120位精氨酸突变为赖氨酸，获得IFN-α2受体结合相关突变体IFN-α2-EIFK；
    所述的IFN-α2重组蛋白的氨基酸序列获取自人基因组，序列为SEQ ID NO.1；
    所述的IFN-α2-EIFK的序列为SEQ ID NO.2。
  2. 按权利要求1所述的人α干扰素受体结合相关位点突变体，其特征在于，所述的IFN-α2受体结合相关突变体在用于制备治疗慢性乙肝病毒感染药物中的应用。
  3. 按权利要求2所述的人α干扰素受体结合相关位点突变体，其特征在于，所述的IFN-α2-EIFK在制备降低和清除乙肝病毒表面抗原HBsAg，HBeAg及病毒基因组DNA制剂中的应用。
  4. 按权利要求3所述的人α干扰素受体结合相关位点突变体，其特征在于，所述的IFN-α2-EIFK抑制感染乙肝病毒的肝细胞中病毒表面抗原HBsAg，e抗原HBeAg病毒蛋白及病毒DNA产生的效应比IFN-α2强。
  5. 按权利要求2所述的人α干扰素受体结合相关位点突变体，其特征在于，所述的IFN-α2-EIFK在于相同工作浓度下，抗乙肝病毒活性较IFN-α2显著；类似抗病毒效应下，IFN-α2-EIFK较IFN-α2工作浓度低10倍以上。
  6. 按权利要求2所述的人α干扰素受体结合相关位点突变体，其特征在于，所述的IFN-α2-EIFK突变体重组蛋白显著降低及清除感染乙肝病毒的肝细胞中的病毒表面抗原(HBsAg)，e抗原(HBeAg)病毒蛋白及病毒DNA的产生同时激活JAK-STAT1/STAT2和ISRE的活化、诱生高水平的抗病毒分子ISGs。
PCT/CN2020/073903 2019-01-30 2020-01-22 人α干扰素受体结合相关位点突变体及其用途 Ceased WO2020156467A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US19/133,302 US20260092098A1 (en) 2019-01-30 2020-01-22 Human a interferon receptor binding-related site mutant and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910090516.2A CN111499718B (zh) 2019-01-30 2019-01-30 人α干扰素受体结合相关位点突变体及其用途
CN201910090516.2 2019-01-30

Publications (1)

Publication Number Publication Date
WO2020156467A1 true WO2020156467A1 (zh) 2020-08-06

Family

ID=71841893

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/073903 Ceased WO2020156467A1 (zh) 2019-01-30 2020-01-22 人α干扰素受体结合相关位点突变体及其用途

Country Status (3)

Country Link
US (1) US20260092098A1 (zh)
CN (1) CN111499718B (zh)
WO (1) WO2020156467A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021214054A1 (en) * 2020-04-21 2021-10-28 Universität Duisburg-Essen Interferon alpha 2 variants and uses thereof
WO2024040247A1 (en) 2022-08-18 2024-02-22 Regeneron Pharmaceuticals, Inc. Interferon proproteins and uses thereof
WO2024040249A1 (en) 2022-08-18 2024-02-22 Regeneron Pharmaceuticals, Inc. Interferon receptor agonists and uses thereof
WO2024238415A1 (en) 2023-05-12 2024-11-21 Regeneron Pharmaceuticals, Inc. Interferon receptor antagonists and uses thereof

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113440603B (zh) * 2020-03-26 2022-07-08 复旦大学 人α干扰素亚型及受体结合相关位点突变体在制备防治新型冠状病毒感染药物中的用途
CN113009156B (zh) * 2021-03-22 2022-08-12 华南农业大学 一种利用绿色荧光蛋白报告基因检测犬IFN-α生物学活性的方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000006735A1 (en) * 1998-07-28 2000-02-10 The United States Of America, Represented By The Secretary, The Department Of Health & Human Services Interferon alpha hybrids
WO2014194100A1 (en) * 2013-05-29 2014-12-04 The Regents Of The University Of California Anti-cspg4 fusions with interferon for the treatment of malignancy
CN104470536A (zh) * 2012-03-03 2015-03-25 免疫基因公司 工程化的抗体-干扰素突变体融合分子
CN105658669A (zh) * 2013-07-19 2016-06-08 弗拉芒区生物技术研究所 细胞因子拮抗剂的靶向
CN109071632A (zh) * 2016-02-05 2018-12-21 奥里尼斯生物科学公司 靶向性治疗剂及其用途

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1511849A (zh) * 2002-12-30 2004-07-14 北京三元基因工程有限公司 新型α干扰素突变体及其制备方法
KR20050107435A (ko) * 2003-02-18 2005-11-11 메르크 파텐트 게엠베하 개선된 특성의 인터페론 알파 뮤테인의 융합 단백질
EP1888633A4 (en) * 2005-05-18 2010-01-27 Maxygen Inc UNFOLDED INTERFERON ALPHA POLYPEPTIDE
CN104274828B (zh) * 2013-07-04 2017-11-17 复旦大学 IFN‑α处理细胞所分泌外体在制备抑制肝炎病毒的药物中的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000006735A1 (en) * 1998-07-28 2000-02-10 The United States Of America, Represented By The Secretary, The Department Of Health & Human Services Interferon alpha hybrids
CN104470536A (zh) * 2012-03-03 2015-03-25 免疫基因公司 工程化的抗体-干扰素突变体融合分子
WO2014194100A1 (en) * 2013-05-29 2014-12-04 The Regents Of The University Of California Anti-cspg4 fusions with interferon for the treatment of malignancy
CN105658669A (zh) * 2013-07-19 2016-06-08 弗拉芒区生物技术研究所 细胞因子拮抗剂的靶向
CN109071632A (zh) * 2016-02-05 2018-12-21 奥里尼斯生物科学公司 靶向性治疗剂及其用途

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021214054A1 (en) * 2020-04-21 2021-10-28 Universität Duisburg-Essen Interferon alpha 2 variants and uses thereof
WO2024040247A1 (en) 2022-08-18 2024-02-22 Regeneron Pharmaceuticals, Inc. Interferon proproteins and uses thereof
WO2024040249A1 (en) 2022-08-18 2024-02-22 Regeneron Pharmaceuticals, Inc. Interferon receptor agonists and uses thereof
WO2024238415A1 (en) 2023-05-12 2024-11-21 Regeneron Pharmaceuticals, Inc. Interferon receptor antagonists and uses thereof

Also Published As

Publication number Publication date
US20260092098A1 (en) 2026-04-02
CN111499718A (zh) 2020-08-07
CN111499718B (zh) 2022-06-14

Similar Documents

Publication Publication Date Title
WO2020156467A1 (zh) 人α干扰素受体结合相关位点突变体及其用途
AU2021301953B2 (en) Construction and application of fusion protein vaccine platform
JP2025041775A5 (zh)
CN113876938A (zh) 融合蛋白疫苗平台的构建与应用
Temonen et al. Effect of interferon-α and cell differentiation on Puumala virus infection in human monocyte/macrophages
JP2023526317A5 (zh)
US20150174206A1 (en) Uses of interferons with altered spatial structure
CN102268093B (zh) 一种肝靶向肽与人干扰素a2b融合蛋白及其制备方法和应用
CN109381698A (zh) 人α干扰素亚型在制备抗乙型肝炎病毒药物中的用途
CN102757492A (zh) 全人乙肝表面蛋白单克隆抗体及其在制备预防hbv感染药物中的用途
CN113121672A (zh) 猫干扰素γ的可溶性原核表达和纯化方法及应用
WO2019029081A1 (zh) 干扰素κ在制备抗囊膜病毒药物方面的应用
Langer et al. Purification, bacterial expression, and biological activities of the human interferons
EP1663110A2 (en) Uses of interferons with altered spatial structure
CN113440603B (zh) 人α干扰素亚型及受体结合相关位点突变体在制备防治新型冠状病毒感染药物中的用途
CN116333166B (zh) 一种靶向HIV gp120蛋白的重组干扰素及其应用
CN102199217B (zh) 乙型肝炎病毒多表位融合蛋白及其制备方法和应用
CN110204613B (zh) 一种针对胡宁病毒的中和抗体、其制备方法及其应用
CN116854803B (zh) IFN-a2位点突变融合蛋白及其在制备抗呼吸道病毒免疫制剂中的应用
Wang et al. Association of interleukin 27 expression and p28 gene polymorphism with chronic hepatitis B virus infection
JP2023522423A (ja) インターフェロンアルファ２バリアント及びその使用
Ningrum Human interferon Alpha2a as anti Hepatitis B and C
CN108721646B (zh) 一种抑制病毒感染的方法及抗病毒药物
CN115920006B (zh) Abcf1或其激动剂在制备抗dna病毒制剂中的应用
Zuyun et al. Molecular characterization of ATG5 in Larimichthys crocea and its role in promoting virus proliferation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20749571

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 30/11/2021)

122 Ep: pct application non-entry in european phase

Ref document number: 20749571

Country of ref document: EP

Kind code of ref document: A1