WO2012053691A1 - Pegylated interferon alpha isomer - Google Patents

Pegylated interferon alpha isomer Download PDF

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WO2012053691A1
WO2012053691A1 PCT/KR2010/008080 KR2010008080W WO2012053691A1 WO 2012053691 A1 WO2012053691 A1 WO 2012053691A1 KR 2010008080 W KR2010008080 W KR 2010008080W WO 2012053691 A1 WO2012053691 A1 WO 2012053691A1
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peg
interferon alpha
interferon
kda
reaction
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송은경
고동현
고충현
김연향
송관규
고형곤
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(주)알엔디바이오랩
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol

Definitions

  • an amino acid sequence containing cysteine is added to a human interferon alpha gene by using a genetic manipulation method, and an appropriate size for specifically binding to cysteine by purifying interferon alpha isoform from the culture medium of the strain into which the gene is introduced.
  • PEG-interferon alpha can be produced in which the interferon alpha protein's persistence as well as the interferon alpha protein's specific activity is enhanced.
  • Interferon alpha belongs to the interferon type 1, interferon beta, omega in addition to the interferon type 1 and the amino acid sequence between them is very similar.
  • Interferon has biological activities such as antiviral, cell proliferation inhibitory effect, immunologically modifying effect (Stark et al., 1998). Because of this property, interferon alpha-2 has been used for cancer and viral diseases since the late 1980s. In particular, it has been used as a major drug in the treatment of chronic hepatitis B and C. (Zeuzem et.al., 2000; Heathcote et.al., 2000; RJ Wills. 1990; Barouki et.al., 1987: Craxi A. and Licata A. 2003; Choueiri et.al., 2003; Perry MC and Jarvis B., 1998)
  • Hepatitis B is mainly distributed in Korea, Japan and China.
  • PEG is a high-molecular compound having a structure of HO-(-CH 2 -CH 2 O-) nH.
  • PEG is strongly hydrophilic and binds to a protein to increase solubility.
  • the use of PEG molecular weight when binding to proteins ranges from about 1,000 to 100,000 Da, and the toxicity of PEG in this range is known to be very low.
  • PEG which can react mainly with amine groups present in interferon
  • amine-reactive PEG can react with 10 to 11 lysines (depending on subtype) to interferon alpha and can also bind to N-terminal amino acids (Monkarsh et. Al. 1997).
  • these homologues may bind mono-, di- and multiple-PEG to interferon.
  • Amine-PEGylated interferon alpha 2 has a significantly lower (4-14 times lower activity) biological activity in vitro compared to non-PEG-bound interferon alpha 2, increasing the amount of protein required to treat patients. Will be used.
  • This decrease in biological activity is large when interferon alpha is modified with high molecular weight PEG (ie, 20-, 40-kDa PEG) (Monkarsh et. Al., 1997; Wang et. Al., 2000; Bailon et. al., 2001).
  • PEGs of this kind are sized PEGs useful for enhancing half-life. As the size of protein-bound PEG increases, the amount of excretion in the kidney decreases, and the distribution in the body changes, such as increased degradation in the liver (Yamaoka et.al., 1994).
  • Interferon alpha 2 contains some lysine at sites important for binding to receptors (Radhakrishnan et.al. 1996). Therefore, the binding of amine-reactive PEG to one or more major lysine sites leads to a decrease in activity.
  • Rosendahl et al. (2005) investigated the biological activity of amino acids at various sites of human interferon alpha 2b by investigating their biological activity, and also the maleimide to cysteine-modified proteins to bind PEG to specific sites of interferon alpha 2b. The combination of -PEG was investigated for their biological activity and their persistence in the body.
  • Site-specific PEG binding is the introduction of cysteine residues that do not participate in disulfide bonds into specific regions of the protein by causing positional mutations. Then, the PEG composition, which performs a cysteine specific reaction, is covalently bound to the newly introduced cysteine to perform modification. An important significance here is to select a site that does not have biological activity, replace the existing amino acid with cysteine, and attach PEG to this position.
  • the binding of PEG to interferon alpha via site-specific PEG binding not only produces structurally homogeneous products, but also PEG-interferons with higher specific activity than amine-PEGylated interferons.
  • proline-cysteine or cysteine was added to the C-terminus of human interferon alpha protein as a specific binding position of PEG, and cysteine-specific PEG was bound to this position to achieve inactivity and body persistence.
  • the addition of proline-cysteine to the C-terminus of the human interferon alpha protein was first attempted in the present invention, and the addition of cysteine has been studied by Rosendahl et al., But these are insoluble when producing interferon with the cysteine added to the C-terminus in E. coli. It is present as an inclusion body, which is a state of, and does not attach importance.
  • the problem is solved by efficiently converting the interferon present as an insoluble inclusion body into a water-soluble state.
  • a feature of the present invention is the study of the size of PEG, as well as the position where PEG binds. As the size of PEG molecular weight increases, the effect of increasing interferon alpha's persistence in the body increases, but the degree of inactivation shows the opposite result. Therefore, the present invention insists on the selection of the smallest PEG that can increase the persistence.
  • the inactivation is maintained in the range of 30% to 52.9%, thereby minimizing the inactivation of interferon alpha and maintaining the body's persistence. Confirmed.
  • PEG-bound interferon alpha proteins have increased persistence in the body, but their inactivation is low, so administration of large amounts is inevitable. Therefore, the present invention solves the improvement of PEG-interferon alpha inactivation in addition to the increased persistence of PEG-binding interferon alpha.
  • Figure 1 confirms the increase in molecular weight by binding PEG to the interferon alpha protein homologue in SDS-PAGE.
  • A) was developed by SDS-PAGE, and then the developed sample was fixed on polyacrylamide gel in 10% acetic acid solution and then stained with KI-I 2 solution. Color development occurred only at the site where PEG is present, and thus it can be seen that the increase in molecular weight is a result of the binding of PEG.
  • the CBB of B) is the result of destaining the KI-I 2 stain with distilled water, followed by re-staining with Coomassie Brilliant Blue. Samples in each column are indicated at the bottom of the figure.
  • Figure 2 examines the biological activity of PEG-interferon alpha.
  • Interferon alpha homologues (3C: PEG-30 kDa-IFNAC +, 3PC: PEG-30 kDa-IFNAPC) bound to chain PEG 30 kDa exhibited moderate activity.
  • the interferon alpha homologue to which Y-form PEG 40 kDa bound (4C: PEG-40 kDa-IFNAC + 4PC: PEG-40 kDa-IFNAPC) showed the lowest activity.
  • C + 30 represents PEG-30 kDa-IFNAC +
  • PC30 represents PEG-30 kDa-IFNAPC
  • C + 40 represents PEG-40 kDa-IFNAC +.
  • the best goal of the invention is to maintain high activity and increase the persistence of human interferon alpha homologue.
  • oligonucleotide DNA was synthesized for the entire gene sequence, and synthetic DNA of complementary base pairs for each synthetic DNA was added, and the whole gene was prepared by polymerase reaction.
  • Specific methods include F1 and R13, F2 and R12, F3 and R11, F4 and R10, F5 and R9, F6 and R8, F7 and R7, F8 and R6, F9 and R5, F10 and R4, F11 and R3, F12 and R2, and F13 and R1 synthetic DNAs were reacted in 50 ⁇ l polymerase reaction solution by 100 pmol in each reaction vessel to synthesize nucleotide sequences for the unsynthesized portion in each complementary sequence. Next, 1 ⁇ l of each of the synthesized reaction solutions was taken, and a new polymerase chain reaction (PCR) was performed.
  • PCR polymerase chain reaction
  • reaction A 1 ⁇ l of the F1 and R13 reaction solution + 1 ⁇ l of the F2 and R12 reaction solution were added with F1 and R12 as the primer
  • reaction B 1 ⁇ l of the F3 and R11 reaction solution + 1 ⁇ l of the F3 and R10 reaction solution
  • F3 and Add R10 1 ⁇ l of F5 and R9 reaction solution + 1 ⁇ l of F6 and R8 reaction solution, add F5 and R8 as primer
  • reaction C 1 ⁇ l of F7 and R7 reaction solution + F8 and R6 reaction solution 1 ⁇ l of F7 and R6 was added as primer
  • reaction E 1 ⁇ l of F9 and R5 reaction solution + 1 ⁇ l of F10 and R4 reaction solution was added F9 and R4 as primer
  • F11 and R3 reaction solution 1 ⁇ l of the F1 and R13 reaction solution + 1 ⁇ l of the F2 and R12 reaction solution were added with F1 and R12 as the primer
  • reaction B 1 ⁇ l of the F3 and R11 reaction solution +
  • the polymerase chain reaction was performed using F11 and R2 (reaction F) as primers in 1 ⁇ l + 1 ⁇ l of the reaction solution of F12 and R2.
  • concentration of the primer was 10 pmol
  • the polymerase chain reaction conditions were denatured at 94 ° C. for 5 minutes, and the reaction was repeated 25 times for 1 minute at 94 ° C., 1 minute at 55 ° C., and 1 minute at 75 ° C.
  • the last reaction was carried out at 75 ° C. for 5 minutes. After these reactions were performed, it was confirmed that the size of the DNA fragments was increased by 2% agarose gel electrophoresis, and 1 ⁇ l of each of the synthesized reaction solutions was performed to perform a new polymerase chain reaction.
  • reaction G 1 ⁇ l of A reaction solution + 1 ⁇ l of B reaction solution was added with F1 and R10 as primers
  • reaction H 1 ⁇ l of C reaction solution + 1 ⁇ l of D reaction solution with F5 and R6 as primers
  • reaction H 1 ⁇ l of E reaction solution + 1 ⁇ l of F reaction solution was added with F9 and R2 as primers
  • reaction I polymerase chain reaction was performed.
  • the size of the DNA fragment was increased again in agarose gel electrophoresis, and then polymerase chain reaction was performed again.
  • reaction J 1 ⁇ l of G reaction solution + 1 ⁇ l of H reaction solution was added with F1 and R6 as a primer
  • reaction K 1 ⁇ l of reaction solution I was added with 1 ⁇ l of reaction solution with F9 and R1 as primer
  • reaction K Polymerase chain reaction was performed. Polymerase chain reaction conditions were performed in the same manner as before. After confirming that the size of the DNA fragment was increased in the 1.5% agarose gel, 1 ⁇ l of the J reaction solution and 1 ⁇ l of the K reaction solution were used as primers to synthesize human interferon gene using F1 and R1 primers.
  • the synthesized human interferon alpha gene was prepared using the following primers, the Xba I at the N-terminus and the BamH I restriction enzyme cleavage site at the C-terminus.
  • the synthesized DNA fragment was purified, digested with restriction enzymes Xba I and BamH I, conjugated to pET28a (+) plasmid DNA digested with the same restriction enzyme, and transformed into Escherichia coli BL21 (DE3). It was.
  • restriction enzymes and T4 DNA ligase for DNA conjugation was performed according to the manufacturer's method.
  • the N-terminal and C-terminal portions of the pET28a (+) plasmid DNA have His-Tag sequences for the convenience of protein purification. However, in the present invention, since the amino acid sequence is not necessary, the N-terminal region was modified to remove it.
  • the C-terminal part was not modified because the target protein was not affected by this sequence because protein synthesis was terminated before His-Tag.
  • Plasmid DNA was isolated from the transformed Escherichia coli and subjected to sequencing using the T7 promoter primer and the T7 terminator primer. As a result, the human interferon gene was completely synthesized.
  • An allogene of interferon alpha was constructed to bind PEG to the added cysteine position by adding cysteine or proline-cysteine to the C-terminal portion of human interferon alpha.
  • the interferon production was performed by refolding the inclusion body insoluble state of human interferon alpha homologue, and PEG binding reaction was performed on the protein purified therefrom.
  • amplification was performed by polymerase chain reaction using N-Xba I primer and primer C + or N-Xba I primer and primer PC on the interferon gene prepared in 1) of Example 1
  • the DNA fragment was purified and then transformed to BL21 (DE3) by conjugation to pET28a (+) plasmid DNA digested with restriction enzymes Xba I and BamH I and then digested with the same restriction enzyme.
  • Both homologues also isolated plasmid DNA and confirmed that human interferon gene was completely synthesized by sequencing using T7 promoter primer and T7 terminator primer.
  • Seeds cultured overnight at 37 ° C. were inoculated in 2 ⁇ YT medium containing 50 ⁇ g / ml kanamycin and transformed Escherichia coli prepared in Example 1 to 5 ⁇ 2 YT medium containing 50 ⁇ g / ml kanamycin. After incubation for 2 hours, IPTG was added to the final 1 mM, followed by 5 hours of incubation to induce the expression of human interferon alpha and allogenes. In order to confirm this, samples were taken from the culture solution, centrifuged, and then subjected to SDS-PAGE sample solution, which was developed by SDS-PAGE, and then confirmed expression.
  • Inclusion bodies were recovered by centrifuging the cells in the culture (10,000 g, 30 minutes) and then crushing the cells with a homogenizer and centrifuging the crushed solution to remove the supernatant. The collected inclusion body was suspended in distilled water and then washed by centrifugation to recover the precipitate. Inclusion bodies were completely dissolved in 8 M urea and 50 mM glycine buffer (pH 11), followed by centrifugation to obtain supernatant. 50 mM glycine solution was added to the inclusion body solution so that the concentration of urea was 2 M, and then the pH was adjusted to 9.0 with sodium hydroxide solution.
  • the pH-adjusted lysate was slowly stirred at room temperature for 15 hours to perform refolding of the protein.
  • the protein reconstituted solution was adjusted to pH 5.5 and then centrifuged to remove the precipitate.
  • the supernatant was taken and adjusted to pH 3.0 with hydrochloric acid, and then concentrated to a protein concentration of 1-5 mg / ml using a filter that fractionated 10 kDa molecular weight.
  • the concentrated solution was purified using anion exchange resin.
  • the PEG used in the reaction was 40 mg branched maleimide PEG-40 kDa (NOF, Sunbright GL2-400MA, Japan) or 30 mg single chained maleimide PEG 30 kDa (Sunbio, PIMAL-30, Korea). After addition, it was made to react at room temperature for 1 hour. After the reaction for 1 hour, the reaction solution was diluted 10-fold with distilled water, the pH was adjusted to 3.0, and the sample was loaded on a column filled with Carboxy Methyl Sepharose resin equilibrated with 50 mM acetic acid buffer (pH 3.0).
  • the column loaded with the sample was washed with 50 mM acetic acid buffer (pH 4.75), and then 50 mM, 100 mM, 150 mM, 200 mM and 250 mM of NaCl were contained in 50 mM acetic acid buffer (pH 4.75), respectively.
  • the solution separated human interferon alpha PEG polymer. PEGylated-interferon was eluted with a peak at 50 mM NaCl, and as the concentration of NaCl was increased, human interferon homomer monomer and human interferon homomer dimer were not eluted.
  • the interferon alpha activity was investigated by measuring the cytopathic effect of interferon antiviral level using MDBK cell line and VSV. The method was used by modifying the method of Rubinstein et al. (Rubinstein S., Familletti P., Pestka S .: Convienience assay for interferons, J. Virol. (1981), 37, 755-758).
  • the inactivation of 30 kDa chain PEG to the interferon alpha homologue was 30% to 52.9% (30% to 46.9% for IFNAC +; 33.8% to 52.9% for IFNAPC).
  • the degree of inactivation when 40 kDa branched PEG is bound to the interferon alpha homologue ranges from 7.4% to 15.1% (7.4% to 11.7% for IFNAC +; 9.6% to 15.1% for IFNAPC). .
  • IFNA 2b The persistence of IFNA 2b showed a sharp decrease, whereas PEG-interferon alpha showed persistence in the body up to 168 hours.
  • PEG-40 kDa-IFNAC + was shown to persist at higher concentrations than PEG-30 kDa-IFNAC + and PEG-30 kDa-IFNAPC, but the persistence patterns were very similar.
  • PEG polyethylene glycol
  • human interferon homologue is a generic term for a substance having interferon activity.
  • homologue refers to a protein that has been isolated and purified by mutation of the human interferon alpha gene. It also contains interferon alpha bound by PEG.
  • Human interferon alpha PEG polymer refers to a protein which has a PEG bond to a human interferon alpha homologue, and is a human interferon alpha homolog in a broad sense.
  • the human interferon alpha homologues produced as a result of the present invention not only increased the persistence in the body while maintaining the intrinsic properties of interferon, but also have high inactivity than previously reported products, and thus have industrial applicability.

Abstract

The present invention relates to production of human interferon having an increased persistence of specific activity, through genetic manipulation by transformation of an amino acid sequence in a specific region of human interferon alpha, then binding an appropriate size PEG to the transformed amino acid region.

Description

페길레이션된 인터페론 알파 동종체PEGylated Interferon Alpha Homolog
본 발명은 사람 인터페론 알파 유전자에 대해 유전자 조작 방법을 사용하여 시스테인을 포함하는 아미노산 서열을 첨가하고, 이 유전자가 도입된 균주의 배양액으로부터 인터페론 알파 동종체를 정제하여 시스테인에 특이적으로 결합하는 적정한 크기의 PEG와 결합시킴으로써 인터페론 알파 단백질의 체내 지속성은 물론 인터페론 알파 단백질의 비활성(specific activity)이 증대된 PEG-인터페론 알파를 제작하는 것이다.According to the present invention, an amino acid sequence containing cysteine is added to a human interferon alpha gene by using a genetic manipulation method, and an appropriate size for specifically binding to cysteine by purifying interferon alpha isoform from the culture medium of the strain into which the gene is introduced. By combining with PEG, PEG-interferon alpha can be produced in which the interferon alpha protein's persistence as well as the interferon alpha protein's specific activity is enhanced.
인터페론 알파는 제1형 인터페론에 속하며, 제1형 인터페론에는 그외에도 인터페론 베타, 오메가가 존재하며 그들 사이의 아미노산 서열은 매우 유사하다.Interferon alpha belongs to the interferon type 1, interferon beta, omega in addition to the interferon type 1 and the amino acid sequence between them is very similar.
인터페론은 항바이러스, 세포 증식 억제효과, 면역학적 수정 효과 (immunomodulatory) 등의 생물학적 활성을 갖는다(Stark 등, 1998). 이러한 특성 때문에 인터페론 알파-2는 1980년대 후반부터 암치료제와 바이러스 질환의 치료를 위해 사용되어 왔다. 특히 만성 B형과 C형 간염 치료제에서는 주요 의약품으로 사용되어 왔다. (Zeuzem et.al.,2000; Heathcote et.al.,2000; R.J. Wills. 1990; Barouki et.al.,1987: Craxi A. and Licata A. 2003; Choueiri et.al.,2003; Perry M.C. and Jarvis B.,1998))Interferon has biological activities such as antiviral, cell proliferation inhibitory effect, immunologically modifying effect (Stark et al., 1998). Because of this property, interferon alpha-2 has been used for cancer and viral diseases since the late 1980s. In particular, it has been used as a major drug in the treatment of chronic hepatitis B and C. (Zeuzem et.al., 2000; Heathcote et.al., 2000; RJ Wills. 1990; Barouki et.al., 1987: Craxi A. and Licata A. 2003; Choueiri et.al., 2003; Perry MC and Jarvis B., 1998)
간염 바이러스의 감염 인구를 살펴보면 전 세계적으로 B형 간염의 경우 3억 5천만 명이, C형 간염의 경우는 1억 7천만 명이 환자로 추정되고 있다. B형 간염의 경우는 한국, 일본 및 중국에 주로 분포하고 있다.In the infected population of hepatitis virus, 350 million cases of hepatitis B and 170 million cases of hepatitis C are estimated worldwide. Hepatitis B is mainly distributed in Korea, Japan and China.
많은 종류의 자연적인 또는 유전자 재조합으로 생산된 단백질은 약리학적인 효능이 있지만, 치료제로서 주사하였을 경우에 항원성을 유발할 수도 있고, 수용성이 낮아서 주사제로서의 어려움이 있을 수 있으며, 또한 체내 잔류 기간이 짧아서 약리효과가 좋지 않을 수도 있다.Many types of natural or genetically produced proteins are pharmacologically effective, but they may cause antigenicity when injected as therapeutics, have low water solubility and may be difficult as injections, and also have a short duration of pharmacology. It may not work well.
이러한 어려움을 극복하기 위해서 단백질과 PEG를 결합시키는 방법이 이용되어 왔으며, 이는 US Pat 4,179,337에서 처음 발표되었다. 이 특허에 의하면 PEG와 결합한 단백질 및 효소 등을 치료제로 사용할 경우, PEG가 갖는 장점인 항원성의 감소, 수용성의 증가, 체내 잔류 기간의 증가 등에서 효과를 나타낸다고 보고 하였다. PEG는 HO-(-CH2-CH2O-)n-H의 구조를 갖는 고분자 화합물로서, 친수성이 강하여 단백질과 결합해서 용해도를 증가시키기도 한다. 단백질에 결합할 때의 PEG 분자량의 사용 범위는 1,000-100,000 Da 정도의 크기로서, 이 범위에 속하는 PEG의 독성은 매우 낮은 것으로 알려져 있다. US Pat 4,766,106과 US Pat 4,917,888에서는 단백질에 PEG를 포함한 중합체를 결합시켜 단백질의 수용성을 증가시켰다고 밝혔다. US Pat 4,902,502에서는 PEG나 다른 중합체들을 재조합 단백질에 결합시켜 항원성을 줄이고 체내 잔존기간을 증가시켰다고 보고하고 있다.To overcome this difficulty, a method of combining protein and PEG has been used, which was first published in US Pat 4,179,337. According to this patent, the use of PEG-binding proteins and enzymes as a therapeutic agent has been shown to be effective in reducing antigenicity, increasing water solubility, and increasing duration of the body, which are advantages of PEG. PEG is a high-molecular compound having a structure of HO-(-CH 2 -CH 2 O-) nH. PEG is strongly hydrophilic and binds to a protein to increase solubility. The use of PEG molecular weight when binding to proteins ranges from about 1,000 to 100,000 Da, and the toxicity of PEG in this range is known to be very low. US Pat 4,766,106 and US Pat 4,917,888 found that binding of a polymer containing PEG to the protein increased the water solubility of the protein. US Pat 4,902,502 reports that PEG or other polymers can be linked to recombinant proteins to reduce antigenicity and increase body survival.
1990년대 초부터 인터페론 투여 빈도를 줄이기 위해 반감기를 증가시키는 연구가 집중적으로 이루어졌다. 이들은 주로 인터페론 내에 존재하는 amine기와 반응할 수 있는 PEG를 사용하여 인터페론 알파-2의 혈액 내에서 반감기를 증대시킬 수 있음을 보여주었다. Amine-reactive PEG의 사용은 인터페론 알파에 10~11개(아형에 따라)의 라이신과 반응할 수 있으며, 또 N-말단의 아미노산에도 결합을 할 수 있다(Monkarsh et.al. 1997). 더욱이 이들 동종체들은 인터페론에 mono-, di-, multiple-PEG가 결합할 수도 있다. 호프만 라-로슈의 특허 US Pat 5,382,657에 따르면 결합한 PEG의 분자량이 낮을수록, PEG가 인터페론에 1개 결합한 것이 1개 이상의 PEG가 결합한 것보다 항바이러스 활성이 높았다고 보고하고 있다. 따라서 인터페론의 활성을 최대한 유지하기 위해서는 인터페론에 결합하는 PEG의 숫자를 최소한으로 하면서 PEG의 장점을 갖기 위해서는 PEG의 분자량이 큰 것을 선택할 것을 주장하고 있다.Since the early 1990s, research has been focused on increasing the half-life to reduce the frequency of interferon administration. They showed that PEG, which can react mainly with amine groups present in interferon, can increase half-life in the blood of interferon alpha-2. The use of amine-reactive PEG can react with 10 to 11 lysines (depending on subtype) to interferon alpha and can also bind to N-terminal amino acids (Monkarsh et. Al. 1997). Moreover, these homologues may bind mono-, di- and multiple-PEG to interferon. According to Hoffman La Roche's patent US Pat 5,382,657, the lower the molecular weight of the bound PEG, the higher the antiviral activity of one PEG bound to interferon than the one or more PEG bound. Therefore, in order to keep the activity of interferon to the maximum, the number of PEG bound to interferon is minimized, and in order to have the advantages of PEG, it is insisted to select a large molecular weight of PEG.
Amine-PEGylated 인터페론 알파 2는 PEG가 결합하지 않은 인터페론 알파 2와 비교하여 in vitro에서 상당히 낮은 정도의(4~14배 낮은 활성) 생물학적 활성을 갖기 때문에 환자를 치료하기 위해 요구되는 단백질의 양을 증가시켜 사용하게 된다. 이 생물학적 활성의 감소는 인터페론 알파가 큰 분자량의 PEG(즉, 20-, 40- kDa PEG)로 수식될 때 크게 나타난다(Monkarsh et.al.,1997; Wang et.al.,2000; Bailon et.al.,2001). 이러한 종류의 PEG들은 반감기를 증대시키는데 유용한 크기의 PEG들이다. 또 단백질에 결합한 PEG는 크기가 증가함에 따라 신장에서의 배출되는 양은 줄고, 간에서의 분해가 증가 되는 등의 신체 내에서의 분포가 변하게 된다(Yamaoka et.al., 1994).Amine-PEGylated interferon alpha 2 has a significantly lower (4-14 times lower activity) biological activity in vitro compared to non-PEG-bound interferon alpha 2, increasing the amount of protein required to treat patients. Will be used. This decrease in biological activity is large when interferon alpha is modified with high molecular weight PEG (ie, 20-, 40-kDa PEG) (Monkarsh et. Al., 1997; Wang et. Al., 2000; Bailon et. al., 2001). PEGs of this kind are sized PEGs useful for enhancing half-life. As the size of protein-bound PEG increases, the amount of excretion in the kidney decreases, and the distribution in the body changes, such as increased degradation in the liver (Yamaoka et.al., 1994).
인터페론 알파 2에는 수용체와 결합하는 데 중요한 부위에 일부 라이신이 포함되어 있다(Radhakrishnan et.al. 1996). 그러므로 하나 혹은 그 이상의 주요 라이신 위치에 amine-reactive PEG가 결합하게 되면 활성의 감소를 일으키게 된다.Interferon alpha 2 contains some lysine at sites important for binding to receptors (Radhakrishnan et.al. 1996). Therefore, the binding of amine-reactive PEG to one or more major lysine sites leads to a decrease in activity.
쉐링-푸라우사의 특허 US Pat 5,908,621, US Pat 5,985,263과 US Pat 5,951,974,에서 분자량 12,000 Da의 PEG를 34번째 아미노산인 히스티딘에만 1개 결합한 PEG 인터페론의 경우 29%의 항바이러스 효과를 나타낸다고 발표하였다. 이와 같은 결과를 볼 때 체내 지속성을 증대시키기 위해 PEG를 결합할 경우 PEG가 결합하는 인터페론의 아미노산 위치가 매우 중요함을 알 수 있다.Schering-Plauau's patents US Pat 5,908,621, US Pat 5,985,263 and US Pat 5,951,974, reported a 29% antiviral effect for PEG interferon in which only one PEG 34 molecular weight PEG was bound to histidine, the 34th amino acid. From these results, it can be seen that the amino acid position of the interferon to which PEG binds is very important when PEG is bound to increase the persistence in the body.
단백질의 특정 위치에 PEG를 결합시키려는 시도는 인터루킨-2에서 실시된 바 있다(Goodson RJ, and Katre NV, 1990).Attempts to bind PEG to specific positions of proteins have been made in Interleukin-2 (Goodson RJ, and Katre NV, 1990).
Rosendahl등은(2005) 사람 인터페론 알파 2b의 여러 부위의 아미노산을 치환 혹은 교체, 추가하여 이들의 생물학적 활성을 조사하였고, 또한 인터페론 알파의 특정 부위에 PEG를 결합할 수 있도록 시스테인으로 변형된 단백질에 maleimide-PEG를 결합하여 이들에 대한 생물학적 활성과 체내 지속성에 대한 조사를 수행하였다. Rosendahl et al. (2005) investigated the biological activity of amino acids at various sites of human interferon alpha 2b by investigating their biological activity, and also the maleimide to cysteine-modified proteins to bind PEG to specific sites of interferon alpha 2b. The combination of -PEG was investigated for their biological activity and their persistence in the body.
위치 특이적 PEG 결합은 이황화 결합에 참여하지 않는 시스테인 잔기를 위치 지정 돌연변이를 유발시켜 단백질의 특정부위에 도입하는 것이다. 그리고 나서 시스테인 특이적 반응을 하는 PEG 조성물을 사용하여 새로이 도입된 시스테인에 공유결합으로 결합시켜 변형을 실시하는 것이다. 여기에서의 중요한 의의는 생물학적 활성을 미치지 않는 부위를 선정하여 시스테인으로 기존의 아미노산을 교체하고, 이 위치에 PEG 결합을 시키는 것이다. 위치 특이적 PEG 결합으로 인터페론 알파에 PEG를 결합하게 되면, 구조적으로 동질 한 제품을 만들 수 있을 뿐만 아니라, amine-PEGylated 인터페론보다 높은 비활성을 갖는 PEG-인터페론을 만들 수 있다. 그 결과 기존의 PEG-인터페론과는 유사한 정도의 반감기를 유지할 수 있어 투여 빈도를 줄일 수 있을 뿐만 아니라, 더 나아가서는 적은 용량의 PEG-인터페론을 투여할 수도 있을 것으로 기대된다. 이는 PEG-인터페론의 투여 용량의 감소로 인터페론이 갖고 있는 고유의 부작용을 최소화할 수 있을 뿐만 아니라 경제적인 효과도 가질 것으로 기대된다.Site-specific PEG binding is the introduction of cysteine residues that do not participate in disulfide bonds into specific regions of the protein by causing positional mutations. Then, the PEG composition, which performs a cysteine specific reaction, is covalently bound to the newly introduced cysteine to perform modification. An important significance here is to select a site that does not have biological activity, replace the existing amino acid with cysteine, and attach PEG to this position. The binding of PEG to interferon alpha via site-specific PEG binding not only produces structurally homogeneous products, but also PEG-interferons with higher specific activity than amine-PEGylated interferons. As a result, the half-life similar to that of the existing PEG-interferon can be maintained to reduce the frequency of administration, and furthermore, it is expected that a small amount of PEG-interferon can be administered. This is expected to have an economic effect as well as to minimize the inherent side effects of interferon due to the reduced dose of PEG-interferon.
본 발명에서는 PEG의 특이적 결합 위치로서 사람 인터페론 알파 단백질의 C-말단에 프롤린-시스테인 또는 시스테인을 추가하여, 이 위치에 시스테인 특이적 PEG를 결합시켜 비활성과 체내 지속성의 증대를 이루고자 하였다. 사람 인터페론 알파 단백질의 C-말단에 프롤린-시스테인 추가는 본 발명에서 처음 시도된 것이며, 시스테인의 추가는 Rosendahl 등에 의해 연구된 바 있으나, 이들은 C-말단에 시스테인이 추가된 인터페론을 대장균에서 생산할 때 불용성의 상태인 봉입체(inclusion body)로 존재하여 중요성을 부여하지 않았다. 본 발명에서는 불용성 상태의 봉입체로 존재하는 인터페론을 수용성의 상태로 효율적으로 전환시켜 문제점을 해결하였다. 또한, 본 발명의 특징은 PEG가 결합하는 위치뿐만 아니라, PEG의 크기에 대한 연구이다. PEG 분자량의 크기가 증대함에 따라 인터페론 알파의 체내 지속성의 증대 효과가 증가하지만, 비활성 정도는 그 반대의 결과를 나타낸다. 따라서 본 발명에서는 지속성을 증대시킬 수 있는 최소 크기의 PEG 선정을 주장한다. 본 발명에서는 20,000 Da 이상 40,000 Da 미만의 분자량을 가진 PEG를 사용하였을 때 비활성이 30%~52.9%의 범위를 유지함으로써, 인터페론 알파의 비활성 감소를 최소화할 수 있으며, 체내 지속성도 유지시킬 수 있음을 확인하였다.In the present invention, proline-cysteine or cysteine was added to the C-terminus of human interferon alpha protein as a specific binding position of PEG, and cysteine-specific PEG was bound to this position to achieve inactivity and body persistence. The addition of proline-cysteine to the C-terminus of the human interferon alpha protein was first attempted in the present invention, and the addition of cysteine has been studied by Rosendahl et al., But these are insoluble when producing interferon with the cysteine added to the C-terminus in E. coli. It is present as an inclusion body, which is a state of, and does not attach importance. In the present invention, the problem is solved by efficiently converting the interferon present as an insoluble inclusion body into a water-soluble state. In addition, a feature of the present invention is the study of the size of PEG, as well as the position where PEG binds. As the size of PEG molecular weight increases, the effect of increasing interferon alpha's persistence in the body increases, but the degree of inactivation shows the opposite result. Therefore, the present invention insists on the selection of the smallest PEG that can increase the persistence. In the present invention, when the PEG having a molecular weight of 20,000 Da or more than 40,000 Da is used, the inactivation is maintained in the range of 30% to 52.9%, thereby minimizing the inactivation of interferon alpha and maintaining the body's persistence. Confirmed.
PEG가 결합한 인터페론 알파 단백질은 체내 지속성은 증가 되었으나 비활성이 낮기 때문에 치료를 위해서는 많은 양의 투여가 불가피하다. 따라서 본 발명에서는 PEG가 결합한 인터페론 알파의 지속성 증가와 더불어 PEG-인터페론 알파 비활성의 향상을 해결하였다.PEG-bound interferon alpha proteins have increased persistence in the body, but their inactivation is low, so administration of large amounts is inevitable. Therefore, the present invention solves the improvement of PEG-interferon alpha inactivation in addition to the increased persistence of PEG-binding interferon alpha.
사람 인터페론 알파 단백질 유전자의 특정 부위에 아미노산 변형을 일으켜서 위치 특이적으로 적정한 크기의 PEG가 결합하게 함으로써 체내 지속성은 물론 비활성까지도 향상시켰다.Amino acid modifications were made to specific regions of the human interferon alpha protein gene, which allowed PEG to bind to a site-specific, appropriately sized molecule, thereby enhancing both persistence and inactivity.
도면 1.은 인터페론 알파 단백질 동종체에 PEG를 결합시킴으로써 분자량이 증가한 것을 SDS-PAGE에서 확인한 것이다. A)는 SDS-PAGE로 전개한 다음, 전개된 시료를 10 % 초산용액에서 폴리 아크릴 아미드 젤에 고정한 다음 KI-I2용액으로 염색한 것이다. PEG가 존재하는 부위에서만 발색이 일어났으며, 이로써 분자량의 증가는 PEG가 결합한 결과임을 알 수 있다. B)의 CBB는 KI-I2염색을 증류수로 탈색을 시킨 다음에 Coomassie Brilliant Blue로 다시 염색한 후 탈색한 결과이다. 각 칼럼의 시료들은 도면 하단에 표시되어 있다.Figure 1 confirms the increase in molecular weight by binding PEG to the interferon alpha protein homologue in SDS-PAGE. A) was developed by SDS-PAGE, and then the developed sample was fixed on polyacrylamide gel in 10% acetic acid solution and then stained with KI-I 2 solution. Color development occurred only at the site where PEG is present, and thus it can be seen that the increase in molecular weight is a result of the binding of PEG. The CBB of B) is the result of destaining the KI-I 2 stain with distilled water, followed by re-staining with Coomassie Brilliant Blue. Samples in each column are indicated at the bottom of the figure.
도면 2.는 PEG-인터페론 알파의 생물학적 활성을 조사한 것이다.Figure 2 examines the biological activity of PEG-interferon alpha.
각 시료들에 대해서 연속적으로 희석한 시료를 96 well 플레이트에 넣고 일정시간 MDBK세포주에 접촉하도록 한 다음, VSV를 공격용 바이러스로 사용하여 생존 세포의 수를 마이크로플레이트 리더로 흡광도를 조사하여 항바이러스 효과를 조사한 것이다. PEG가 결합하지 않은 인터페론 알파 및 그 동종체(standard: 표준품, a2b: 본 발명에서 제작된 인터페론 알파, C+: IFNAC+, PC: IFNAPC) 들은 낮은 농도에서도 50% 활성(IC50)을 나타내고 있으며, 다음으로 사슬 형 PEG 30 kDa이 결합한 인터페론 알파 동종체(3C: PEG-30 kDa-IFNAC+, 3PC: PEG-30 kDa-IFNAPC)들이 중간 정도의 활성을 나타낸다. 마지막으로 Y 형태 PEG 40 kDa가 결합한 인터페론 알파 동종체의 (4C: PEG-40 kDa-IFNAC+ 4PC: PEG-40 kDa-IFNAPC) 활성이 가장 낮게 나타남을 보여준다.Continuously diluted samples were placed in 96 well plates for each sample, and then contacted with MDBK cell lines for a certain period of time. Then, VSV was used as an attack virus, and the number of viable cells was examined by using a microplate reader for antiviral effects. It was investigated. Interferon alpha and its isoforms that do not bind to PEG (standard: standard, a2b: interferon alpha, C +: IFNAC +, PC: IFNAPC) produced in the present invention exhibit 50% activity (IC 50 ) even at low concentrations. Interferon alpha homologues (3C: PEG-30 kDa-IFNAC +, 3PC: PEG-30 kDa-IFNAPC) bound to chain PEG 30 kDa exhibited moderate activity. Finally, the interferon alpha homologue to which Y-form PEG 40 kDa bound (4C: PEG-40 kDa-IFNAC + 4PC: PEG-40 kDa-IFNAPC) showed the lowest activity.
도면 3.은 PEG-인터페론 알파의 in vivo 지속성을 rat에서 조사한 것을 보여준다. Non-PEG는 IFNA 2b를 사용하였으며, C+30은 PEG-30 kDa-IFNAC+을 나타낸 것이며, PC30은 PEG-30 kDa-IFNAPC을 나타내며, C+40은 PEG-40 kDa-IFNAC+을 표시한 것이다. 이 결과에서 볼 때 IFNA 2b의 지속성은 급격히 감소 됨을 보여주는 반면 PEG-인터페론 알파는 168시간까지 체내 지속성이 있음을 보여 준다. PEG-40 kDa-IFNAC+가 PEG-30 kDa-IFNAC+과 PEG-30 kDa-IFNAPC 보다 조금 높은 농도로 지속 됨을 보여 주고 있으나, 지속성의 양상은 매우 유사함을 보여주고 있다.3 shows the in vivo persistence of PEG-interferon alpha in rats. Non-PEG used IFNA 2b, C + 30 represents PEG-30 kDa-IFNAC +, PC30 represents PEG-30 kDa-IFNAPC, and C + 40 represents PEG-40 kDa-IFNAC +. These results show that the persistence of IFNA 2b is dramatically reduced, while PEG-interferon alpha is sustained in the body up to 168 hours. Although PEG-40 kDa-IFNAC + is sustained at a slightly higher concentration than PEG-30 kDa-IFNAC + and PEG-30 kDa-IFNAPC, the persistence patterns are very similar.
발명에서 이루고자 하는 최선의 목표는 사람 인터페론 알파 동종체에 대한 높은 활성유지와 체내 지속성의 증대에 있다.The best goal of the invention is to maintain high activity and increase the persistence of human interferon alpha homologue.
실시예 1.Example 1.
1) 사람 인터페론 알파 유전자의 제작1) Construction of Human Interferon Alpha Gene
사람 인터페론 알파 유전자 제작을 위해 전체 유전자 염기서열에 대한 올리고뉴클레오티드 DNA를 합성하여 각각의 합성 DNA에 대한 상보적인 염기쌍의 합성 DNA를 넣고 중합효소 반응으로 전체 유전자를 제작하였다.For the production of human interferon alpha gene, oligonucleotide DNA was synthesized for the entire gene sequence, and synthetic DNA of complementary base pairs for each synthetic DNA was added, and the whole gene was prepared by polymerase reaction.
표 1
Name Sequence
F1 5' ATGTGCGATC TGCCGCAGAC CCAT 3'
F2 5' AGCCTGGGCA GCCGTCGTAC CCTGATGCTG CTGGCGCAGA 3'
F3 5' TGCGTCGTAT CAGCCTGTTT AGCTGCCTGA AAGATCGTCA 3'
F4 5' TGATTTTGGC TTTCCGCAGG AAGAATTTGG CAACCAGTTT 3'
F5 5' CAGAAAGCGG AAACCATCCC GGTGCTGCAT GAAATGATCC 3'
F6 5' AGCAGATCTT TAACCTGTTT AGCACCAAAG ATAGCAGCGC 3'
F7 5' GGCGTGGGAT GAAACCCTGC TGGATAAATT TTATACCGAA 3'
F8 5' CTGTATCAGC AGCTGAACGA TCTGGAAGCG TGCGTGATCC 3'
F9 5' AGGGCGTGGG CGTGACCGAA ACCCCGCTGA TGAAAGAAGA 3'
F10 5' TAGCATCCTG GCGGTGCGTA AATATTTTCA GCGTATCACC 3'
F11 5' CTGTATCTGA AAGAAAAAAA ATATAGCCCG TGCGCGTGGG 3'
F12 5' AAGTGGTGCG TGCGGAAATC ATGCGTAGCT TTAGCCTGAG 3'
F13 5' CACCAACCTG CAAGAAAGCC TGCGTAGCAA AGAATAGTAA 3'
R1 5' TTACTATTCT TTGCTACGCA GGCT 3'
R2 5' TTCTTGCAGG TTGGTGCTCA GGCTAAAGCT ACGCATGATT 3'
R3 5' TCCGCACGCA CCACTTCCCA CGCGCACGGG CTATATTTTT 3'
R4 5' TTTCTTTCAG ATACAGGGTG ATACGCTGAA AATATTTACG 3'
R5 5' CACCGCCAGG ATGCTATCTT CTTTCATCAG CGGGGTTTCG 3'
R6 5' GTCACGCCCA CGCCCTGGAT CACGCACGCT TCCAGATCGT 3'
R7 5' TCAGCTGCTG ATACAGTTCG GTATAAAATT TATCCAGCAG 3'
R8 5' GGTTTCATCC CACGCCGCGC TGCTATCTTT GGTGCTAAAC 3'
R9 5' AGGTTAAAGA TCTGCTGGAT CATTTCATGC AGCACCGGGA 3'
R10 5' TGGTTTCCGC TTTCTGAAAC TGGTTGCCAA ATTCTTCCTG 3'
R11 5' CGGAAAGCCA AAATCATGAC GATCTTTCAG GCAGCTAAAC 3'
R12 5' AGGCTGATAC GACGCATCTG CGCCAGCAGC ATCAGGGTAC 3'
R13 5' GACGGCTGCC CAGGCTATGG GTCTGCGGCA GATCGCACAT 3'
Table 1
Name Sequence
F1 5 'ATGTGCGATC TGCCGCAGAC CCAT 3'
F2 5 'AGCCTGGGCA GCCGTCGTAC CCTGATGCTG CTGGCGCAGA 3'
F3 5 'TGCGTCGTAT CAGCCTGTTT AGCTGCCTGA AAGATCGTCA 3'
F4 5 'TGATTTTGGC TTTCCGCAGG AAGAATTTGG CAACCAGTTT 3'
F5 5 'CAGAAAGCGG AAACCATCCC GGTGCTGCAT GAAATGATCC 3'
F6 5 'AGCAGATCTT TAACCTGTTT AGCACCAAAG ATAGCAGCGC 3'
F7 5 'GGCGTGGGAT GAAACCCTGC TGGATAAATT TTATACCGAA 3'
F8 5 'CTGTATCAGC AGCTGAACGA TCTGGAAGCG TGCGTGATCC 3'
F9 5 'AGGGCGTGGG CGTGACCGAA ACCCCGCTGA TGAAAGAAGA 3'
F10 5 'TAGCATCCTG GCGGTGCGTA AATATTTTCA GCGTATCACC 3'
F11 5 'CTGTATCTGA AAGAAAAAAA ATATAGCCCG TGCGCGTGGG 3'
F12 5 'AAGTGGTGCG TGCGGAAATC ATGCGTAGCT TTAGCCTGAG 3'
F13 5 'CACCAACCTG CAAGAAAGCC TGCGTAGCAA AGAATAGTAA 3'
R1 5 'TTACTATTCT TTGCTACGCA GGCT 3'
R2 5 'TTCTTGCAGG TTGGTGCTCA GGCTAAAGCT ACGCATGATT 3'
R3 5 'TCCGCACGCA CCACTTCCCA CGCGCACGGG CTATATTTTT 3'
R4 5 'TTTCTTTCAG ATACAGGGTG ATACGCTGAA AATATTTACG 3'
R5 5 'CACCGCCAGG ATGCTATCTT CTTTCATCAG CGGGGTTTCG 3'
R6 5 'GTCACGCCCA CGCCCTGGAT CACGCACGCT TCCAGATCGT 3'
R7 5 'TCAGCTGCTG ATACAGTTCG GTATAAAATT TATCCAGCAG 3'
R8 5 'GGTTTCATCC CACGCCGCGC TGCTATCTTT GGTGCTAAAC 3'
R9 5 'AGGTTAAAGA TCTGCTGGAT CATTTCATGC AGCACCGGGA 3'
R10 5 'TGGTTTCCGC TTTCTGAAAC TGGTTGCCAA ATTCTTCCTG 3'
R11 5 'CGGAAAGCCA AAATCATGAC GATCTTTCAG GCAGCTAAAC 3'
R12 5 'AGGCTGATAC GACGCATCTG CGCCAGCAGC ATCAGGGTAC 3'
R13 5 'GACGGCTGCC CAGGCTATGG GTCTGCGGCA GATCGCACAT 3'
구체적인 방법으로는 F1과 R13, F2와 R12, F3와 R11, F4와 R10, F5와 R9, F6와 R8, F7과 R7, F8과 R6, F9과 R5, F10과 R4, F11과 R3, F12와 R2, 그리고 F13과 R1 합성 DNA를 각각의 반응용기에 100 pmol씩 50 ㎕ 중합효소 반응액에 넣어 반응시켜 각 각의 상보적인 서열에서 미합성 부분에 대한 염기서열을 합성하였다. 다음으로, 합성이 이루어진 반응액을 각 각 1 ㎕를 취하여, 새로운 중합효소 연쇄반응(PCR: polymerase chain reaction)을 수행하였다. F1과 R13 반응액 1 ㎕ + F2와 R12 반응액 1 ㎕에 프라이머로는 F1과 R12를 넣고(반응A), F3와 R11 반응액 1 ㎕ + F4와 R10 반응액 1 ㎕에 프라이머로는 F3와 R10을 넣고(반응B), F5와 R9 반응액 1 ㎕ + F6와 R8 반응액 1 ㎕에 프라이머로는 F5와 R8를 넣고(반응C), F7과 R7 반응액 1 ㎕ + F8과 R6 반응액 1 ㎕에 프라이머로는 F7과 R6를 넣고(반응D), F9과 R5 반응액 1 ㎕ + F10과 R4반응액 1 ㎕에 프라이머로는 F9과 R4를 넣고(반응E), F11과 R3 반응액 1 ㎕ + F12와 R2 반응액 1 ㎕에 프라이머로는 F11과 R2(반응F)를 사용하여 중합효소 연쇄반응을 수행하였다. 이때 프라이머의 농도는 10 pmol로 하고, 중합효소 연쇄반응 조건은 94℃에서 5분간 변성시키고, 94 ℃에서 1분, 55 ℃에서 1분, 75 ℃에서 1분간의 반응을 25회 반복하여 수행하고, 마지막 반응은 75 ℃에서 5분간 실시하였다. 이들 반응이 수행한 다음, 2 % 아가로스 젤 전기영동에서 DNA 단편의 크기가 증가 되었음을 확인하고, 다시 합성이 이루어진 반응액을 각 각 1 ㎕를 취하여 새로운 중합효소 연쇄반응을 수행하였다. A 반응액 1 ㎕ + B 반응액 1 ㎕에 프라이머로는 F1과 R10을 넣고(반응G), C 반응액 1 ㎕ + D 반응액 1 ㎕에 프라이머로는 F5와 R6를 넣고(반응H), E 반응액 1 ㎕ + F 반응액 1 ㎕에 프라이머로는 F9과 R2를 넣고(반응I) 중합효소 연쇄반응을 수행하였다. 반응이 종료된 다음 다시 아가로스 젤 전기영동에서 DNA 단편의 크기가 증가하였음을 확인한 다음 다시 중합효소 연쇄반응을 수행하였다. G 반응액 1 ㎕ + H 반응액 1 ㎕에 프라이머로는 F1과 R6를 넣고(반응J), I 반응액 1 ㎕ + F13과 R1 반응액 1 ㎕에 프라이머로는 F9과 R1을 넣고(반응K) 중합효소 연쇄반응을 수행하였다. 중합효소 연쇄반응 조건은 앞에서와 동일하게 수행하였다. 1.5 % 아가로스 젤에서 DNA 단편의 크기가 증가 되었음을 확인한 다음 J 반응액 1 ㎕와 K 반응액 1 ㎕에 프라이머로는 F1과 R1 프라이머를 사용하여 사람 인터페론 유전자를 합성하였다.Specific methods include F1 and R13, F2 and R12, F3 and R11, F4 and R10, F5 and R9, F6 and R8, F7 and R7, F8 and R6, F9 and R5, F10 and R4, F11 and R3, F12 and R2, and F13 and R1 synthetic DNAs were reacted in 50 µl polymerase reaction solution by 100 pmol in each reaction vessel to synthesize nucleotide sequences for the unsynthesized portion in each complementary sequence. Next, 1 μl of each of the synthesized reaction solutions was taken, and a new polymerase chain reaction (PCR) was performed. 1 μl of the F1 and R13 reaction solution + 1 μl of the F2 and R12 reaction solution were added with F1 and R12 as the primer (reaction A), 1 μl of the F3 and R11 reaction solution + 1 μl of the F3 and R10 reaction solution, and F3 and Add R10 (reaction B), 1 μl of F5 and R9 reaction solution + 1 μl of F6 and R8 reaction solution, add F5 and R8 as primer (reaction C), 1 μl of F7 and R7 reaction solution + F8 and R6 reaction solution 1 μl of F7 and R6 was added as primer (reaction D), 1 μl of F9 and R5 reaction solution + 1 μl of F10 and R4 reaction solution was added F9 and R4 as primer (reaction E), and F11 and R3 reaction solution. The polymerase chain reaction was performed using F11 and R2 (reaction F) as primers in 1 µl + 1 µl of the reaction solution of F12 and R2. At this time, the concentration of the primer was 10 pmol, and the polymerase chain reaction conditions were denatured at 94 ° C. for 5 minutes, and the reaction was repeated 25 times for 1 minute at 94 ° C., 1 minute at 55 ° C., and 1 minute at 75 ° C. The last reaction was carried out at 75 ° C. for 5 minutes. After these reactions were performed, it was confirmed that the size of the DNA fragments was increased by 2% agarose gel electrophoresis, and 1 μl of each of the synthesized reaction solutions was performed to perform a new polymerase chain reaction. 1 μl of A reaction solution + 1 μl of B reaction solution was added with F1 and R10 as primers (reaction G), 1 μl of C reaction solution + 1 μl of D reaction solution with F5 and R6 as primers (reaction H), 1 μl of E reaction solution + 1 μl of F reaction solution was added with F9 and R2 as primers (reaction I), and polymerase chain reaction was performed. After completion of the reaction, the size of the DNA fragment was increased again in agarose gel electrophoresis, and then polymerase chain reaction was performed again. 1 μl of G reaction solution + 1 μl of H reaction solution was added with F1 and R6 as a primer (reaction J), and 1 μl of reaction solution I was added with 1 μl of reaction solution with F9 and R1 as primer (reaction K). ) Polymerase chain reaction was performed. Polymerase chain reaction conditions were performed in the same manner as before. After confirming that the size of the DNA fragment was increased in the 1.5% agarose gel, 1 μl of the J reaction solution and 1 μl of the K reaction solution were used as primers to synthesize human interferon gene using F1 and R1 primers.
합성된 사람 인터페론 알파 유전자를 다음의 프라이머를 사용하여 N-말단에 Xba I, C-말단에 BamH I 제한효소 절단부위를 제작하였다.The synthesized human interferon alpha gene was prepared using the following primers, the Xba I at the N-terminus and the BamH I restriction enzyme cleavage site at the C-terminus.
표 2
Name Sequence
N-Xba I 5' CGCCTCTAGA AATAATTTTG TTTAACTTTA AGAAGGAGAT ATACATATGT GCGATCTGCC G 3'
C-BamH I 5' GAATTCGGAT CCTTACTATT CTTTGCTACG CAGGCT 3'
TABLE 2
Name Sequence
N-Xba I 5 'CGCCTCTAGA AATAATTTTG TTTAACTTTA AGAAGGAGAT ATACATATGT GCGATCTGCC G 3'
C-BamH I 5 'GAATTCGGAT CCTTACTATT CTTTGCTACG CAGGCT 3'
중합효소 연쇄반응이 완료된 후 합성된 DNA 단편을 정제하여 제한 효소 Xba I과 BamH I으로 절단한 다음, 동일한 제한효소로 절단된 pET28a(+) 플라스미드 DNA에 접합하고, 대장균 BL21(DE3)에 형질 전환하였다. 제한 효소 및 DNA 접합을 위한 T4 DNA ligase의 사용은 제조사의 방법에 따라 실시하였다. pET28a(+) 플라스미드 DNA의 N-말단 부위와 C-말단 부위에는 단백질 정제의 편의를 위해 His-Tag 서열이 있다. 그러나 본 발명에서는 이 아미노산 서열이 필요하지 않기 때문에 이를 제거하기 위해 N-말단 부위를 변형하였다. C-말단 부위는 목적 단백질이 His-Tag 앞에서 단백질 합성이 종료되기 때문에 이 서열의 영향을 받지 않으므로 변형을 하지 않았다. 형질 전환된 대장균으로부터 플라스미드 DNA를 분리하여 T7 promoter 프라이머와 T7 terminator 프라이머를 사용한 염기서열 분석을 하였으며, 그 결과 사람 인터페론 유전자가 완전하게 합성되었음을 확인하였다.After completion of the polymerase chain reaction, the synthesized DNA fragment was purified, digested with restriction enzymes Xba I and BamH I, conjugated to pET28a (+) plasmid DNA digested with the same restriction enzyme, and transformed into Escherichia coli BL21 (DE3). It was. The use of restriction enzymes and T4 DNA ligase for DNA conjugation was performed according to the manufacturer's method. The N-terminal and C-terminal portions of the pET28a (+) plasmid DNA have His-Tag sequences for the convenience of protein purification. However, in the present invention, since the amino acid sequence is not necessary, the N-terminal region was modified to remove it. The C-terminal part was not modified because the target protein was not affected by this sequence because protein synthesis was terminated before His-Tag. Plasmid DNA was isolated from the transformed Escherichia coli and subjected to sequencing using the T7 promoter primer and the T7 terminator primer. As a result, the human interferon gene was completely synthesized.
1) 사람 인터페론 알파 동종체 유전자의 제작1) Construction of Human Interferon Alpha Homologous Gene
사람 인터페론 알파의 C-말단 부위에 시스테인 또는 프롤린-시스테인을 추가하여 추가된 시스테인 위치에 PEG를 결합시키기 위해 인터페론 알파의 동종체를 제작하였다. An allogene of interferon alpha was constructed to bind PEG to the added cysteine position by adding cysteine or proline-cysteine to the C-terminal portion of human interferon alpha.
본발명에서 C-말단에 시스테인 또는 프롤린-시스테인의 추가가 인터페론의 활성에 영향을 미치지 않음을 확인하였다. 본 발명에서는 사람 인터페론 알파 동종체를 불용성의 상태인 봉입체를 재구성(refolding)하여 인터페론 생산을 하였으며, 이로부터 분리 정제한 단백질에 대해 PEG 결합 반응을 수행하였다. 동종체의 제작 방법으로는 실시예 1의 1) 항에서 제작된 인터페론 유전자에 대해 N-Xba I 프라이머 와 프라이머 C+ 또는 N-Xba I 프라이머와 프라이머 PC를 사용하여 중합효소 연쇄반응으로 제작하여 증폭된 DNA 단편을 정제한 다음, 제한 효소 Xba I과 BamH I으로 절단한 다음 동일한 제한 효소로 절단한 pET28a(+) 플라스미드 DNA에 접합하여 BL21(DE3)에 형질 전환하였다.In the present invention it was confirmed that the addition of cysteine or proline-cysteine at the C-terminal does not affect the activity of interferon. In the present invention, the interferon production was performed by refolding the inclusion body insoluble state of human interferon alpha homologue, and PEG binding reaction was performed on the protein purified therefrom. As a method for producing homologues, amplification was performed by polymerase chain reaction using N-Xba I primer and primer C + or N-Xba I primer and primer PC on the interferon gene prepared in 1) of Example 1 The DNA fragment was purified and then transformed to BL21 (DE3) by conjugation to pET28a (+) plasmid DNA digested with restriction enzymes Xba I and BamH I and then digested with the same restriction enzyme.
표 3
Name Sequence
Primer C+ 5' GAATTCGGAT CCTTACTAGC ATTCTTTGCT ACGCAGGCTT TC 3'
Primer PC 5' GAATTCGGAT CCTTACTAGC ACGGTTCTTT GCTACGCAGG CTTTC 3'
TABLE 3
Name Sequence
Primer C + 5 'GAATTCGGAT CCTTACTAGC ATTCTTTGCT ACGCAGGCTT TC 3'
Primer PC 5 'GAATTCGGAT CCTTACTAGC ACGGTTCTTT GCTACGCAGG CTTTC 3'
두 동종체 역시 플라스미드 DNA를 분리하여 T7 promoter 프라이머와 T7 terminator 프라이머를 사용한 염기서열 분석에서 사람 인터페론 유전자가 완전하게 합성되었음을 확인하였다.Both homologues also isolated plasmid DNA and confirmed that human interferon gene was completely synthesized by sequencing using T7 promoter primer and T7 terminator primer.
실시예 2.Example 2.
사람 인터페론 알파 동종체의 발현 및 정제Expression and Purification of Human Interferon Alpha Homologs
실시예 1에서 제조된 형질 전환 대장균을 50 ㎍/ml 가나마이신이 포함된 2 X YT 배지에 접종하여 37 ℃에서 하룻밤 배양한 종균을 새로운 50 ㎍/ml 가나마이신이 포함된 2 X YT 배지에 5 % 접종한 다음, 2시간 배양한 후 IPTG가 최종 1 mM되게 첨가한 후 5시간 배양함으로써 사람 인터페론 알파 및 동종체의 발현을 유도하였다. 이를 확인하기 위해서 배양액으로부터 시료를 채취하여 원심분리 후, SDS-PAGE 시료용 용액에 처리여 SDS-PAGE로 전개한 다음 발현을 확인하였다.Seeds cultured overnight at 37 ° C. were inoculated in 2 × YT medium containing 50 μg / ml kanamycin and transformed Escherichia coli prepared in Example 1 to 5 × 2 YT medium containing 50 μg / ml kanamycin. After incubation for 2 hours, IPTG was added to the final 1 mM, followed by 5 hours of incubation to induce the expression of human interferon alpha and allogenes. In order to confirm this, samples were taken from the culture solution, centrifuged, and then subjected to SDS-PAGE sample solution, which was developed by SDS-PAGE, and then confirmed expression.
2X YT 배지를 사용하여 5L 발효조 배양액으로부터 사람 인터페론 알파 및 동종체를 정제하였다. 배양액에서 균체를 원심분리한(10,000 g, 30분) 다음 호모게나이저로 균체를 파쇄하고 파쇄액을 원심 분리하여 상등액을 제거함으로써 봉입체를 회수하였다. 회수한 봉입체를 증류수에 현탁한 후, 원심 분리하여 침전물을 회수함으로써 깨끗이 세척하였다. 봉입체를 8 M 요소(urea)와 50 mM 글리신 완충용액(pH 11)에 완전히 용해시킨 후, 원심 분리하여 상등액을 취하였다. 요소의 농도가 2 M이 되도록 봉입체 용해액에 50 mM의 글리신 용액을 첨가한 다음 수산화나트륨 용액으로 pH를 9.0이 되도록 조정하였다. pH가 조정된 용해액을 상온에서 15시간 동안 서서히 교반하여 단백질의 재구성(refolding)을 수행하였다. 단백질 재구성이 완료된 용액을 pH 5.5로 조정한 다음, 원심분리를 하여 침전물을 제거하였다. 상등액을 취하여 염산으로 pH를 3.0으로 조정한 후, 분자량 10 kDa를 분획하는 필터를 사용하여 단백질 농도가 1~5 mg/ml이 되게 농축하였다. 농축된 용액을 음이온 교환수지를 이용하여 정제하였다. Human interferon alpha and allogenes were purified from 5 L fermentor broth using 2X YT medium. Inclusion bodies were recovered by centrifuging the cells in the culture (10,000 g, 30 minutes) and then crushing the cells with a homogenizer and centrifuging the crushed solution to remove the supernatant. The collected inclusion body was suspended in distilled water and then washed by centrifugation to recover the precipitate. Inclusion bodies were completely dissolved in 8 M urea and 50 mM glycine buffer (pH 11), followed by centrifugation to obtain supernatant. 50 mM glycine solution was added to the inclusion body solution so that the concentration of urea was 2 M, and then the pH was adjusted to 9.0 with sodium hydroxide solution. The pH-adjusted lysate was slowly stirred at room temperature for 15 hours to perform refolding of the protein. The protein reconstituted solution was adjusted to pH 5.5 and then centrifuged to remove the precipitate. The supernatant was taken and adjusted to pH 3.0 with hydrochloric acid, and then concentrated to a protein concentration of 1-5 mg / ml using a filter that fractionated 10 kDa molecular weight. The concentrated solution was purified using anion exchange resin.
실시예 3. PEGylationExample 3. PEGylation
정제한 인터페론 알파 동종체 1 mg(약 50 nmol)의 단백질을 50 mM Tris(pH 8.5)에 TCEP-HCl[{Tris(2-carboxyethyl)-Phosphine} Hydrochloride, Thermo사] 1 umole되도록 넣고 실온에서 10분간 방치한 다음 PEG 반응을 수행하였다.1 mg (approximately 50 nmol) of purified interferon alpha isoform was added to 50 mM Tris (pH 8.5) in 1 umole of TCEP-HCl [{Tris (2-carboxyethyl) -Phosphine} Hydrochloride, Thermo]] at room temperature. After leaving for a minute, PEG reaction was performed.
반응에 사용된 PEG는 40 mg branched maleimide PEG-40 kDa(NOF사, Sunbright GL2-400MA, Japan) 또는 30 mg의 single chained maleimide PEG 30 kDa (Sunbio, PIMAL-30, Korea)이며, 각각의 PEG를 첨가한 후, 실온에서 1시간 반응시켰다. 1시간 반응 후, 증류수로 반응액을 10배 희석한 다음, pH를 3.0으로 조정한 다음, 50 mM 아세트산 완충용액(pH 3.0)으로 평형화시킨 Carboxy Methyl Sepharose 수지로 충진한 칼럼에 시료를 loading하였다. 시료가 loading된 컬럼을 50 mM 아세트산 완충용액 (pH 4.75)로 세척한 다음, 50 mM 아세트산 완충용액(pH 4.75)에 NaCl이 각 각 50 mM, 100 mM, 150 mM, 200 mM, 250 mM들어 있는 용액으로 사람 인터페론 알파 PEG 중합체를 분리하였다. PEGylated-인터페론은 50mM의 NaCl에서 peak로 용출되며, NaCl의 농도가 증가함에 따라 PEG 반응이 일어나지 않은 사람 인터페론 동종체 monomer, 사람 인터페론 동종체 dimer가 용출되었다.The PEG used in the reaction was 40 mg branched maleimide PEG-40 kDa (NOF, Sunbright GL2-400MA, Japan) or 30 mg single chained maleimide PEG 30 kDa (Sunbio, PIMAL-30, Korea). After addition, it was made to react at room temperature for 1 hour. After the reaction for 1 hour, the reaction solution was diluted 10-fold with distilled water, the pH was adjusted to 3.0, and the sample was loaded on a column filled with Carboxy Methyl Sepharose resin equilibrated with 50 mM acetic acid buffer (pH 3.0). The column loaded with the sample was washed with 50 mM acetic acid buffer (pH 4.75), and then 50 mM, 100 mM, 150 mM, 200 mM and 250 mM of NaCl were contained in 50 mM acetic acid buffer (pH 4.75), respectively. The solution separated human interferon alpha PEG polymer. PEGylated-interferon was eluted with a peak at 50 mM NaCl, and as the concentration of NaCl was increased, human interferon homomer monomer and human interferon homomer dimer were not eluted.
실시예 4. 인터페론 알파의 활성조사Example 4 Activity Study of Interferon Alpha
MDBK 세포주와 VSV를 이용하여 인터페론의 항바이러스 정도로 cytopathic 효과를 측정함으로써 인터페론 알파의 활성을 조사하였다. 방법은 Rubinstein 등의 방법을 변형하여 사용하였다. (Rubinstein S., Familletti P., Pestka S.: Convienience assay for interferons, J. Virol. (1981), 37, 755-758).The interferon alpha activity was investigated by measuring the cytopathic effect of interferon antiviral level using MDBK cell line and VSV. The method was used by modifying the method of Rubinstein et al. (Rubinstein S., Familletti P., Pestka S .: Convienience assay for interferons, J. Virol. (1981), 37, 755-758).
구체적으로는 96-well의 평판 플레이트의 각 홈에 MDBK 세포주를 약 6 X 104 cells/50 ㎕를 넣었다. 순차적으로 희석한 인터페론 시료를(50 ㎕) 각 홈에 넣고 37 ℃의 CO2가 공급되는 배양기에서 2시간 방치하였다. 희석된 VSV 50 ㎕를 각각의 홈에 넣고 다시 20시간 배양하였다. 배양액에 세포를 2 % Neutral Red(Sigma St. Louis, MO)를 넣고 마이크로플레이트 리더에서 540 nm파장으로 측정하였다.Specifically, about 6 × 10 4 cells / 50 μl of MDBK cell line was placed in each groove of a 96-well plate. Sequentially diluted interferon samples (50 µl) were placed in each groove and allowed to stand for 2 hours in an incubator supplied with CO 2 at 37 ° C. 50 [mu] l of diluted VSV was put in each groove and incubated again for 20 hours. 2% Neutral Red (Sigma St. Louis, Mo.) was added to the culture solution and measured at 540 nm in a microplate reader.
인터페론의 활성도는 VSV에 대한 50 % 방어 효과가 있는 인터페론의 농도(IC50)로 비교 분석하였으며 그 결과는 표4와 같다.The activity of interferon was compared and analyzed by the concentration of interferon (IC 50 ) having a 50% protective effect against VSV. The results are shown in Table 4.
표 4
Name IC50 (pg/ml) 비활성(Specific acivity) 비고
표준품(IFN-alpha-2a) 40.423 100 식품의약품 안전청으로부터 입수한 표준품: Code 01/003 3,290,000 IU/vial
IFN-alpha-2b 30.204 100 자체 제작한 인터페론 표준품
IFNAC+ 25.833 100 인터페론 동종체
IFNAPC 34.232 100 인터페론 동종체
PEG-30 kDa-IFNAC+ 86.048 46.9 35.1 30.0 39.7 30 kDa single chained PEG가 결합한 인터페론 중합체
PEG-40 kDa-IFNAC+ 346.70 11.7 8.7 7.4 9.8 40 kDa branched PEG가 결합한 인터페론 중합체
PEG-30 kDa-IFNAPC 76.430 52.9 39.5 33.8 44.8 30 kDa single chained PEG가 결합한 인터페론 중합체
PEG-40 kDa-IFNAPC 267.278 15.1 11.3 9.6 12.8 40 kDa branched PEG가 결합한 인터페론 중합체
Table 4
Name IC 50 (pg / ml) Specific acivity Remarks
Standard product (IFN-alpha-2a) 40.423 100 Standard obtained from KFDA: Code 01/003 3,290,000 IU / vial
IFN-alpha-2b 30.204 100 Self-made interferon standard
IFNAC + 25.833 100 Interferon homologue
IFNAPC 34.232 100 Interferon homologue
PEG-30 kDa-IFNAC + 86.048 46.9 35.1 30.0 39.7 Interferon polymer bound with 30 kDa single chained PEG
PEG-40 kDa-IFNAC + 346.70 11.7 8.7 7.4 9.8 Interferon polymer bound with 40 kDa branched PEG
PEG-30 kDa-IFNAPC 76.430 52.9 39.5 33.8 44.8 Interferon polymer bound with 30 kDa single chained PEG
PEG-40 kDa-IFNAPC 267.278 15.1 11.3 9.6 12.8 Interferon polymer bound with 40 kDa branched PEG
30 kDa의 사슬 형 PEG를 인터페론 알파 동종체에 결합하였을 때의 비활성 정도는 30 %~52.9 %(IFNAC+의 경우는 30 %~46.9 %; IFNAPC의 경우는 33.8 %~52.9 %)정도로 나타났으며. 40 kDa의 가지 형 PEG를 인터페론 알파 동종체에 결합하였을 때의 비활성 정도는 7.4 %~15.1 %(IFNAC+의 경우는 7.4 %~11.7 %; IFNAPC의 경우는 9.6 %~15.1 %)의 범위를 나타내고 있다.The inactivation of 30 kDa chain PEG to the interferon alpha homologue was 30% to 52.9% (30% to 46.9% for IFNAC +; 33.8% to 52.9% for IFNAPC). The degree of inactivation when 40 kDa branched PEG is bound to the interferon alpha homologue ranges from 7.4% to 15.1% (7.4% to 11.7% for IFNAC +; 9.6% to 15.1% for IFNAPC). .
이 결과로 볼 때, 사람 인터페론 알파의 활성에 영향을 미치지 않는 부위에 PEG를 결합하여도 PEG의 크기에 따라 비활성의 차이가 크게 나타남을 알 수 있다. 또한, IFNAC+와 IFNAPC의 인터페론 알파 동종체의 경우 본 실시예에서 IFNAPC가 비활성이 다소 높게 나고 있다.As a result, it can be seen that even if PEG is coupled to a site that does not affect the activity of human interferon alpha, the difference in inactivation is large depending on the size of PEG. In addition, in the case of the interferon alpha homologs of IFNAC + and IFNAPC, IFNAPC is more inactive in this example.
실시예 5.Example 5.
인터페론 알파의 체내 지속성 조사In Vitro Persistence of Interferon Alpha
표 5
Name
IFN-alpha-2b 자체 제작한 인터페론 표준품
PEG-30 kDa-IFNAC+ 30 kDa single chained PEG가 결합한 인터페론 중합체
PEG-40 kDa-IFNAC+ 40 kDa branched PEG가 결합한 인터페론 중합체
PEG-30 kDa-IFNAPC 30 kDa single chained PEG가 결합한 인터페론 중합체
Table 5
Name
IFN-alpha-2b Self-made interferon standard
PEG-30 kDa-IFNAC + Interferon polymer bound with 30 kDa single chained PEG
PEG-40 kDa-IFNAC + Interferon polymer bound with 40 kDa branched PEG
PEG-30 kDa-IFNAPC Interferon polymer bound with 30 kDa single chained PEG
PEG-인터페론 중합체의 체내 지속성을 조사하기 위해 각 시료에 대해서 3마리씩의 Sprague Dawley Rats에 각각 100 ㎍/Kg의 용량으로 꼬리정맥에 단 회 투여한 다음 시간별로(최대 168시간: 7일) 시료를 0.4ml씩 채취하여 ELISA(Human Interferon alpha(Hu-IFN-α) ELISA kit, PBL Biomedical Lab. 사)로 분석하였다.To investigate the in vivo persistence of PEG-interferon polymers, three Sprague Dawley Rats were administered to the tail vein at a dose of 100 μg / Kg each for each sample, followed by hourly (up to 168 hours: 7 days) samples. 0.4 ml each was analyzed by ELISA (Human Interferon alpha (Hu-IFN-α) ELISA kit, PBL Biomedical Lab.).
IFNA 2b의 지속성은 급격히 감소함을 보여주는 반면, PEG-인터페론 알파는 168시간까지 체내 지속성이 있음을 보여주었다. PEG-40 kDa-IFNAC+이 PEG-30 kDa-IFNAC+과 PEG-30 kDa-IFNAPC 보다 높은 농도로 지속함을 보여 주고 있으나, 지속성의 양상은 매우 유사함을 보여주고 있다.The persistence of IFNA 2b showed a sharp decrease, whereas PEG-interferon alpha showed persistence in the body up to 168 hours. PEG-40 kDa-IFNAC + was shown to persist at higher concentrations than PEG-30 kDa-IFNAC + and PEG-30 kDa-IFNAPC, but the persistence patterns were very similar.
PEG는 polyethylene glycol의 약자이며, 사람 인터페론 동종체는 인터페론의 활성을 가지는 물질을 총칭하며, 좁은 의미로서 동종체는 사람 인터페론 알파 유전자에 돌연변이를 유발한 다음 분리정제한 단백질을 말하고, 넓은 의미로는 PEG가 결합한 인터페론 알파까지도 포함하고 있다. 사람 인터페론 알파 PEG 중합체는 사람 인터페론 알파 동종체에 PEG 결합을 한 단백질을 말하며 넓은 의미로 사람 인터페론 알파 동종체이다.PEG stands for polyethylene glycol, and human interferon homologue is a generic term for a substance having interferon activity. In a narrow sense, homologue refers to a protein that has been isolated and purified by mutation of the human interferon alpha gene. It also contains interferon alpha bound by PEG. Human interferon alpha PEG polymer refers to a protein which has a PEG bond to a human interferon alpha homologue, and is a human interferon alpha homolog in a broad sense.
본 발명의 결과로 제작된 사람 인터페론 알파 동종체는 인터페론 고유의 성질은 유지하면서도 체내 지속성을 증가시켰을 뿐만 아니라, 기존에 보고된 제품들보다 비활성이 높아서 산업적으로 이용가능성이 있다.The human interferon alpha homologues produced as a result of the present invention not only increased the persistence in the body while maintaining the intrinsic properties of interferon, but also have high inactivity than previously reported products, and thus have industrial applicability.

Claims (3)

  1. 아미노산 서열의 변형을 통해서 특정 위치에 PEG결합이 가능한 사람 인터페론 알파의 중합체.A polymer of human interferon alpha capable of PEG bonds at specific positions through modification of the amino acid sequence.
  2. 1항에서 아미노산 서열의 변형은 C-말단에 프롤린-시스테인인 또는 시스테의 아미노산 서열을 추가한 사람 인터페론 알파의 동종체.The amino acid sequence of claim 1 is a homologue of human interferon alpha, wherein the amino acid sequence of proline-cysteine or cyste is added to the C-terminus.
  3. 1항에서 아미노산 서열이 추가된 동종체에 PEG중합체를 제작함에 있어서 PEG의 크기는 분자량 20,000 Da 이상 40,000 Da 미만의 PEG 사용.In the preparation of the PEG polymer in the homolog having the amino acid sequence added in paragraph 1, the PEG has a PEG molecular weight of 20,000 Da or more and less than 40,000 Da.
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Citations (2)

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US5766897A (en) * 1990-06-21 1998-06-16 Incyte Pharmaceuticals, Inc. Cysteine-pegylated proteins
US7270809B2 (en) * 1997-07-14 2007-09-18 Bolder Biotechnology, Inc. Cysteine variants of alpha interferon-2

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