KR100511749B1 - Modified interferon-beta, and chemically modified conjugates thereof - Google Patents
Modified interferon-beta, and chemically modified conjugates thereof Download PDFInfo
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- KR100511749B1 KR100511749B1 KR10-2001-0068852A KR20010068852A KR100511749B1 KR 100511749 B1 KR100511749 B1 KR 100511749B1 KR 20010068852 A KR20010068852 A KR 20010068852A KR 100511749 B1 KR100511749 B1 KR 100511749B1
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- beta
- interferon
- leu
- peg
- asn
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- C—CHEMISTRY; METALLURGY
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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Abstract
본 발명은 야생형 인터페론-베타 (Interferon-β; IFN-β)의 80 번 아스파라진 잔기 및 CD 루프(loop) 중 108∼117 번 잔기의 아미노산이 하나 이상 시스테인 잔기로 치환되거나, 또는 인터페론-베타의 아미노산 서열에 시스테인 잔기가 추가로 첨가된 시스테인이 부가된 변이체(cysteine added variant) 인터페론-베타 및 이에 설프히드릴 반응성 생폴리머(biopolymer)가 결합된 배합체(conjugate)를 제공하고자 하는 것으로, 이와 같이 변형된 인터페론-베타에 PEG 유도체와 같은 생체고분자를 위치지정(site-directed) 공유결합시킴으로써, 항원유발성(immunogenicity)이 감소되고 천연 단백질과 동일한 생물학적 활성과 시험관내 활성을 가지며 전신 분포가 상당히 증가하여 향상된 약동학 프로필(pharmacokinetic profile)과 약리학 성질을 갖게 된다.The present invention is directed to the substitution of one or more cysteine residues at amino acids 80 and 117 in the CD loop and asparagine residues of wild type Interferon-beta (IFN-β). To provide a conjugate in which a cysteine added variant interferon-beta having an additional cysteine residue added to an amino acid sequence and a sulfhydryl reactive biopolymer is bound thereto. Site-directed covalent linkage of biopolymers, such as PEG derivatives, to modified interferon-beta results in reduced immunogenicity, the same biological and in vitro activity as native proteins, and significantly increased systemic distribution. This results in improved pharmacokinetic profile and pharmacological properties.
Description
본 발명은 인터페론-베타(Interferon-β)의 특정 아미노산 잔기에 시스테인(Cysteine) 잔기를 첨가(insertion)하거나, 야생형(wild-type) 아미노산 서열에 존재하는 특정 아미노산 잔기를 시스테인으로 치환하여 변형시킨 인터페론-베타 변이체, 및 이에 폴리에틸렌글리콜 등의 반응성을 갖는 생체고분자를 위치지정(site-directed) 공유결합시킴으로써 향상된 약동학 프로필(pharmacokinetic profile)과 약리학 성질을 갖도록 변형된 인터페론-베타 배합체(conjugates)에 관한 것이다.The present invention provides an interferon modified by inserting a cysteine residue into a specific amino acid residue of interferon-beta, or by replacing a specific amino acid residue present in a wild-type amino acid sequence with cysteine. Beta variants and interferon-beta conjugates modified to have improved pharmacokinetic profile and pharmacological properties by site-directed covalent bonding of reactive polymers such as polyethylene glycol will be.
약리학적으로 유용한 단백질들은 비경구(parenteral) 경로를 통해 투여될 때 항원성을 가질 수 있으며, 대체로 수용성이 낮고 체내 잔존기간이 짧다는 단점이 있어 이를 극복하고자 하는 연구가 수행되고 있다.Pharmacologically useful proteins may have antigenicity when administered via the parenteral route, and generally have a disadvantage in that they have low water solubility and short duration of life in the body.
데이비스(Frank F. Davis) 등의 미국특허 제4,179,337호에서는, 폴리에틸렌글리콜(polyethylene glycol, 이하 PEG로 약칭)와 결합된 단백질 및 효소 등을 치료제로 사용할 경우, PEG가 갖는 장점인 항원성의 감소, 수용성의 증가, 체내 잔류 기간 증가 등의 효과를 얻을 수 있음을 개시하고 있다.In US Patent No. 4,179,337 to Frank F. Davis et al., The use of proteins and enzymes combined with polyethylene glycol (abbreviated to PEG) as therapeutic agents reduces the antigenicity and water solubility of PEG. It is disclosed that the effect of the increase of, the increase in the residual period of the body and the like can be obtained.
이러한 데이비스 특허 이후, 단백질을 PEG 등의 생체고분자와 결합시킴으로써 생리활성 단백질의 단점을 극복하고자 하는 시도가 이루어지고 있는데, 예를 들어, 베로니즈 등(Veronese et al., Applied Biochem. and Biotech. 11: 141-152, 1985)은 리보뉴클레아제(ribonuclease)와 수퍼옥사이드 디스뮤타제(superoxide dismutase)를 PEG와 결합시킨 바 있다. 또한, 카터 등(Katre et al.)은 미국특허 제4,766,106호와 제4,917,888호에서 단백질에 PEG를 포함한 폴리머(polymer, 중합체)를 결합시켜 단백질의 수용성을 증가시킨 내용을 개시하고 있으며, 나이테키 등(Nitecki et al.)은 미국특허 제4,902,502호에서 PEG나 다른 중합체들을 재조합 단백질에 결합시킴으로써 항원성을 줄이고 체내 잔존 기간을 증가시켰다고 기술하고 있다.After this Davis patent, there is an attempt to overcome the disadvantages of the physiologically active protein achieved by coupling a protein and bio-polymers such as PEG, e.g., Vero needs such as (Veronese et al., Applied Biochem . And Biotech. 11 141-152, 1985) combined ribonuclease and superoxide dismutase with PEG. In addition, Carter et al. , U.S. Patent Nos. 4,766,106 and 4,917,888 discloses the binding of a polymer containing PEG to a protein to increase the water solubility of the protein. Nitecki et al. , In US Pat. No. 4,902,502, describe the binding of PEG or other polymers to recombinant proteins to reduce antigenicity and increase the duration of life in the body.
그러나, PEG와 단백질의 결합에는 이와 같은 장점 외에 결점도 존재한다. 즉, PEG는 대개 결합할 단백질의 하나 또는 그 이상의 자유 라이신(lysine, Lys) 잔기에 공유결합을 통해 결합하게 되는데, 이때 단백질의 표면 부위중 단백질의 활성도와 직접적인 관계가 있는 부위가 PEG와 결합할 경우, 그 부위는 더 이상 생물학적 기능을 수행할 수 없게 되므로 단백질의 활성도가 감소하게 된다. 또한, PEG와 라이신 잔기의 결합은 대개 무작위적으로 일어나게 되므로 많은 종류의 PEG-단백질 배합체들이 혼합물로 존재하게 되고, 따라서 원하는 배합체를 순수 분리하는 과정이 복잡하고 어려워지게 된다. 따라서, 이와 같은 시험관 내(in vitro) 생물활성(bioactivity)의 감소를 상쇄하기 위해서는 천연 단백질에 비해 체내(in vivo) 반감기를 상당히 증가시켜야 한다.However, in addition to these advantages, there are drawbacks to PEG and protein binding. That is, PEG usually binds covalently to one or more free lysine (lys) residues of the protein to be bound, where a site directly related to protein activity on the surface of the protein is bound to PEG. In that case, the site will no longer be able to perform biological functions, thereby reducing the activity of the protein. In addition, the binding of PEG and lysine residues usually occurs randomly, so that many kinds of PEG-protein combinations are present in the mixture, which makes the process of pure separation of the desired combination complicated and difficult. Thus, in order to offset this decrease in in vitro bioactivity, the in vivo half-life must be significantly increased compared to natural proteins.
PEG 폴리머를 위치지정(site-directed) 공유결합에 의해 단백질에 결합시키면 생물학적 활성의 감소를 방지하거나 최대한 줄이면서 혈장내 반감기는 수 배 증가시킬 수 있다. 특정한 단백질을 위치특이적(site-specific)이고 화학량적으로 변환시키는 것은, 시스테인 잔기나 시스테인을 포함한 작용기를 단백질에 도입하고 이와 반응성이 있는 활성화된 PEG 유도체를 티오에테르(thioether) 결합을 통해 선택적으로 결합시킴으로써 수행될 수 있다. 이 때, 새로이 도입되거나 치환된 시스테인 잔기에 PEG 분자를 결합하는 것은 단백질에 구조적인 무리를 주지 않는 것으로 예상되며, 설사 시험관내(in vitro) 활성의 감소가 있다 하더라도 이를 최소한으로 줄일 수 있을 것으로 예상된다.Binding of PEG polymers to proteins by site-directed covalent bonds can increase the plasma half-life several times while preventing or minimizing the reduction of biological activity. Site-specific and stoichiometric conversion of a particular protein involves the introduction of a cysteine residue or a functional group containing cysteine into the protein and the selective activation of a reactive PEG derivative that is reactive to the protein via a thioether bond. Can be performed by binding. At this time, the binding of PEG molecules to newly introduced or substituted cysteine residues is expected not to impose structural strain on the protein, and even if there is a decrease in in vitro activity, it is expected to be reduced to a minimum. do.
PEG가 결합된(pegylated) 유도체는 천연 단백질과 비교했을 때 상당히 증가된 반감기(half-life, t1/2)를 갖는데, 이와 같은 반감기의 증가에는 순환계로부터 단백질의 제거에 관련된 여러 과정들의 억제가 일부 관여하는 것으로 생각된다. 여기에서, 단백질의 제거에 관련된 여러 과정들에는 단백질 분해(proteolysis), 면역 복합체(immune complex) 형성, 그리고 신장 사구체를 통한 여과 등이 포함된다. PEG-단백질 배합체의 신장여과율은 이들의 전하(charge), 가변형성 (deformability), 그리고 특히 PEG 폴리머의 수화(hydration)에 기인하는 단백질 크기의 증가에 영향을 받을 수 있다. 일례로 야마오카(Yamaoka, J. Pharm. Sci., 83: 601-606, 1994) 등은 쥐 모델에서 분자량 6,000의 PEG 분자는 10 분 미만의 최종 반감기를 갖지만 분자량 20,000의 PEG 분자는 반감기가 약 3 시간이었음을 밝힌 바 있는데, 이는 PEG 폴리머의 반복단위[repeat unit, -(-CH2CH2O-)n-] 당 2.3∼2.8 개의 물 분자들이 결합한다는 사실을 반증하는 것이다.PEG-derived derivatives have a significantly increased half-life (t 1/2 ) compared to natural proteins, which increases the inhibition of the processes involved in the removal of proteins from the circulation. It seems to be involved in some. Here, several processes involved in protein removal include proteolysis, immune complex formation, and filtration through renal glomeruli. The stretch filtration rate of PEG-protein combinations can be influenced by the increase in protein size due to their charge, deformability, and especially the hydration of PEG polymers. For example, Yamaoka ( J. Pharm. Sci., 83 : 601-606, 1994) et al . Described that in a mouse model, PEG molecules with a molecular weight of 6,000 have a final half-life of less than 10 minutes, while PEG molecules with a molecular weight of 20,000 have a half-life of about 3 It has been shown that this is a testament to the fact that 2.3 to 2.8 water molecules bind per repeat unit (-(-CH 2 CH 2 O-) n- ) of the PEG polymer.
인터페론(interferon, IFN)은 핵이 존재하는 대부분의 세포로부터 유래하는 당단백질로, 바이러스의 복제를 억제함으로써 항 바이러스 성질을 가지며, 세포 증식을 억제하고 면역 반응을 조절한다. 인터페론은 세포 표면 세포막의 특정 수용체와 결합하여 일련의 세포내 반응을 개시하는데, 이러한 세포내 반응에는 특정 효소들의 활성화 유도, 세포 증식의 억제 등이 포함되고, 면역조절 반응으로서 대식세포(macrophage)의 식세포(phagocytosis) 활성의 증가, 표적 세포에 대한 임파구들의 세포 독성 증가, 그리고 바이러스에 감염된 세포들의 바이러스 증식 억제 등이 있다.Interferon (IFN) is a glycoprotein derived from most cells in which the nucleus is present. It has antiviral properties by inhibiting the replication of the virus, inhibits cell proliferation and modulates the immune response. Interferon binds to specific receptors on cell surface cell membranes and initiates a series of intracellular responses. These intracellular responses include the activation of specific enzymes, inhibition of cell proliferation, and the like. Increased phagocytosis activity, increased cytotoxicity of lymphocytes to target cells, and inhibition of virus proliferation of virus infected cells.
인간의 인터페론은 유래세포(cellular origin)와 항원성에 따라 크게 3 종류로 분류할 수 있는데, 인터페론-알파는 말초 혈액 백혈구나 림프 모세포에 의해 분비되고, 인터페론-베타는 섬유아세포(fibroblast), 그리고 인터페론-감마는 B 세포에 의해 분비된다. 특히, 인터페론-베타는 다발성 경화증(Multiple Sclerosis, MS)에 상당한 치료 효과를 보이므로 그 표면에 PEG 분자들을 결합시켜 치료효과를 증진시키려는 시도가 행해지고 있다. 만일 인터페론-베타가 수용체와 결합할 때 관여하지 않는 부위에 PEG 폴리머를 결합시킬 수 있다면 매우 유용한 인터페론-베타 배합체(conjugates)가 될 것이다.Human interferons can be classified into three types according to their cellular origin and antigenicity. Interferon-alpha is secreted by peripheral blood leukocytes or lymphoblasts, and interferon-beta is fibroblast and interferon. Gamma is secreted by B cells. In particular, since interferon-beta has a significant therapeutic effect on Multiple Sclerosis (MS), attempts have been made to enhance the therapeutic effect by binding PEG molecules to its surface. If interferon-beta is able to bind PEG polymers to sites that are not involved in binding to the receptor, it would be a very useful interferon-beta conjugate.
현재 미국에는 다발성 경화증 치료를 위한 2가지 종류의 인터페론-베타가 존재한다. 첫 번째 인터페론-베타-1a(Interferon-β-1a, IFN-β-1a)는 인간 인터페론 -베타 유전자를 포함한 포유류 세포주로부터 생산하는 166 개 아미노산 잔기로 구성된 당화(glycosylated) 단백질이다. 두 번째 인터페론-베타-1b(Interferon-β-1b, IFN-β-1b)는 인터페론-베타의 당이 결여된(non-glycosylated) 재조합 단백질로, 대장균(E. coli)으로부터 생산하는 165 개 아미노산 잔기로 구성되는데, 아미노산 1 번 메티오닌(Methionine) 잔기가 결실되어 있고, 17 번 시스테인(Cysteine) 잔기가 세린(Serine)으로 치환되어 있다.There are currently two types of interferon-beta for the treatment of multiple sclerosis in the United States. The first Interferon-beta-1a (IFN-β-1a) is a glycosylated protein consisting of 166 amino acid residues produced from a mammalian cell line containing human interferon-beta genes. The second interferon-beta-1b (IFN-β-1b) is a non-glycosylated recombinant protein of interferon-beta, a 165 amino acid produced from E. coli . It is composed of residues, amino acid No. 1 methionine residues are deleted, and Cysteine residues 17 are substituted with Serine.
런켈(Runkel, Pharm. Res., 15: 641, 1998) 등은 표준 항 바이러스 실험 (standard antiviral assay)에서 시험관내 생물학적 활성이 인터페론-베타-1a (20×107 IU/㎎)가 인터페론-베타-1b (2×107 IU/㎎) 보다 10 배 이상 높았다고 보고하였다. 활성에 영향을 주는 인터페론-베타-1a와 인터페론-베타-1b 간의 유일한 구조적 차이는 당화(glycosylation)였으며, 인터페론-베타-1b는 비당화로 인하여 응집 (aggregation)이 증가하고 열 변성에 대한 민감도가 증가하였다고 기술하고 있다. 이 외에도 우추미(Utsumi, Eur. J. Biochem., 181: 545, 1989; J. Interferon Res., 11: S160, 1991) 등은 인터페론-베타에 대한 N-결합 당(N-linked sugar)의 긍정적인 영향을 보고하고 있다. 즉, 탄수화물 사슬의 안정화 효과는 다발성 경화증 치료의 임상요법에서 알 수 있는데, 임상에서 당화된 인터페론-베타-1a(AvonexR)는 일 주일에 1 회 30 ㎍(microgram, mcg)의 근육내 주사를 필요로 한 반면, 비 당화된 인터페론-베타-1b(BetaseronR)는 이틀마다 250 ㎍, 즉 일 주일에 875 ㎍의 피하주사 용량을 필요로 하고 있다(Physicians Desk Reference, Ed. 51, pp 664, 656, 1997).Runkel, Pharm. Res., 15 : 641, 1998, et al ., Reported that interferon-beta-1a (20 × 10 7 IU / mg) in vitro in vitro activities was tested by standard antiviral assays. Reported at least 10-fold higher than −1b (2 × 10 7 IU / mg). The only structural difference between interferon-beta-1a and interferon-beta-1b that affects activity was glycosylation, and interferon-beta-1b has increased aggregation and sensitivity to thermal denaturation due to aglycosylation. It is said to increase. In addition, Utsumi, Eur. J. Biochem., 181 : 545, 1989; J. Interferon Res., 11 : S160, 1991, et al., Described the N-linked sugars for interferon-beta. The positive impact is reported. In other words, the stabilizing effect of the carbohydrate chain can be seen in the clinical therapy of multiple sclerosis treatment. Clinically glycated interferon-beta-1a (Avonex R ) is injected intramuscularly at 30 μg (microgram, mcg) once a week. On the other hand, unglycosylated interferon-beta-1b (Betaseron R ) requires a subcutaneous dose of 250 μg every other day, 875 μg a week (Physicians Desk Reference, Ed. 51, pp 664, 656, 1997).
한편, 다발성 경화증은 중추신경계 질환으로, 뇌와 척수에서 신경세포섬유들을 절연하며 신경전도를 촉진하는 수초(myelin)들의 감소를 수반한다. 즉, 신경세포섬유들이 신경전달을 관장하는데 다발성 경화증 환자들은 신경전달이 감소하거나 완전히 차단되어 기능이 감소하거나 상실하게 되는 것이다. 이러한 신경퇴행성 질환에 대한 다수의 제약품들이 개발되고 있는데, 이들의 치료효과는 이들 약품을 혈액-뇌 장벽(blood-brain barrier)을 지나서 중추신경계로 전달할 수 있는 운반물질들에 의존한다.Multiple sclerosis, on the other hand, is a central nervous system disease that involves the reduction of myelin, which insulates nerve cell fibers from the brain and spinal cord and promotes nerve conduction. In other words, neurofibrillary cells govern neurotransmission, and patients with multiple sclerosis have reduced or completely blocked neurotransmission, resulting in decreased or lost function. Many pharmaceuticals for these neurodegenerative diseases have been developed and their therapeutic effects depend on carriers that can deliver these drugs across the blood-brain barrier to the central nervous system.
프리든 등(Friden et al., Science, 259: 373, 1993)은 신경성장인자(Nerve Growth Factor, NGF) 같은 거대 단백질을 혈액-뇌 장벽을 건너서 효율적으로 전달할 수 있는 기술을 보고한 바 있다. 이 기술은 뇌 모세혈관의 내피세포들 표면에 존재하는 트랜스페린 수용체(transferrin receptor)를 인식하는 운반 항체(carrier antibody)를 NGF와 결합시키는 과정을 포함한다. 즉, 항체-NGF 배합체는 혈액-뇌 장벽을 건너서 세포내 이입-세포외 유출(endocytosis-exocytosis) 기작을 통해 신경세포들로 전달되고, 이후에 NGF는 항체와 연결된 이황화(disulfide) 결합의 환원을 통해서 캐리어 항체로부터 유리되는 것이다. 교감신경절(sympathetic ganglion) 신경세포 성장을 촉진하는데 항체-NGF 배합체가 NGF 단독투여의 경우 만큼 효율적이라는 보고가 있었다(Granholm et al., J. Pharmacol. Exp. Ther., 268: 448∼459, 1994). 트랜스페린 수용체 항체는 또한 항체-IFN-β 배합체 형태로 인터페론-베타 단백질을 뇌로 운반할 수 있다.Frieden et al ., Science, 259 : 373, 1993, have reported a technique for efficiently delivering large proteins such as the Nerve Growth Factor (NGF) across the blood-brain barrier. This technique involves binding NGF to a carrier antibody that recognizes a transferrin receptor on the surface of endothelial cells of brain capillaries. That is, the antibody-NGF combination is delivered to neurons via the endocytosis-exocytosis mechanism across the blood-brain barrier, whereupon NGF reduces the disulfide bonds associated with the antibody. Through the carrier antibody. It has been reported that antibody-NGF combinations are as effective as NGF alone in promoting sympathetic ganglion neuronal growth (Granholm et al. , J. Pharmacol. Exp. Ther., 268 : 448-459, 1994). ). The transferrin receptor antibody can also carry the interferon-beta protein to the brain in the form of an antibody-IFN-β combination.
인터페론-베타 단백질을 혈액-뇌 장벽을 가로질러 운반하는 다른 시스템들에는, 인터페론-베타와 트랜스페린 수용체 항체를 다른 이종이기능성 커플링 시약으로 접합하거나, 인터페론-베타를 운반작용을 하는 다른 시약들이나 뇌로 단백질을 지속전달(sustained delivery)하는 다른 다양한 수단들을 사용해서 접합시키는 것들이 포함된다(Bartus et al., Science, 281: 1161∼1162, 1998).Other systems that deliver interferon-beta proteins across the blood-brain barrier include conjugation of interferon-beta and transferrin receptor antibodies to other heterofunctional coupling reagents, or other reagents or brains that transport interferon-beta. Conjugation using various other means of sustained delivery of the protein (Bartus et al. , Science, 281 : 1161-1116, 1998).
본 발명의 목적은 인터페론-베타(Interferon-β)의 특정 아미노산 잔기에 시스테인(Cysteine) 잔기를 첨가(insertion)하거나, 야생형(wild-type) 아미노산 서열에 존재하는 특정 아미노산 잔기를 시스테인으로 치환하여 변형시킨 인터페론-베타 변이체를 제공하고자 하는 것이다.An object of the present invention is to modify by inserting a cysteine residue into a specific amino acid residue of Interferon-beta or by replacing a specific amino acid residue in a wild-type amino acid sequence with cysteine. To provide interferon-beta variants.
본 발명의 다른 목적은, 이러한 인터페론-베타 변이체에서 수용체와 결합할 때 관여하지 않는 부위에 생체고분자를 위치지정(site-directed) 공유결합시킴으로써, 항원유발성(immunogenicity)을 감소시키고 야생형이나 재조합 인터페론-베타 단백질의 치료효과에 필요한 투여량(dose)이나 투여횟수를 더욱 줄여 향상된 약동학 프로필(pharmacokinetic profile)과 약리학 성질을 갖도록 변형된 인터페론-베타 배합체(conjugates)를 제공하는 것이다.Another object of the present invention is to site-directed covalent attachment of biopolymers to sites not involved in binding to receptors in these interferon-beta variants, thereby reducing immunogenicity and reducing wild type or recombinant interferon. To further reduce the dose or frequency required for the therapeutic effect of beta protein, it provides interferon-beta conjugates modified to have improved pharmacokinetic profile and pharmacological properties.
상기 목적을 달성하기 위하여 본 발명에서는, 야생형 인터페론-베타 (Interferon-β; IFN-β)의 80 번 아스파라진 잔기 및 CD 루프(loop) 중 108∼117 번 잔기의 아미노산이 하나 이상 시스테인 잔기로 치환되거나, 또는 인터페론-베타의 아미노산 서열에 시스테인 잔기가 추가로 첨가된 시스테인이 부가된 변이체(cysteine added variant) 인터페론-베타를 제공한다.In order to achieve the above object, in the present invention, amino acids of residues 80 to 117 of CD 80 loop and asparagine residue of wild type Interferon-beta (IFN-β) are substituted with at least one cysteine residue. Or a cysteine added variant interferon-beta with an additional cysteine residue added to the amino acid sequence of the interferon-beta.
여기에서, 인터페론-베타는 인터페론-베타-1b 또는 아미노산 1 번 메티오닌 잔기가 제거된 인터페론-베타-1b일 수 있으며, 특히 인터페론-베타의 80 번 아스파라진 잔기가 시스테인 잔기로 치환되거나, 인터페론-베타의 CD 루프(loop) 중 108∼117 번 잔기의 아미노산이 하나 이상 시스테인 잔기로 치환된 것이 바람직하다.Wherein the interferon-beta may be interferon-beta-1b or interferon-beta-1b from which the amino acid no. 1 methionine residue has been removed, in particular the 80th asparagine residue of interferon-beta is substituted with a cysteine residue, or an interferon-beta Preferably, amino acids of residues 108-117 in the CD loop of are substituted with one or more cysteine residues.
상기 다른 목적을 달성하기 위하여 본 발명에서는 상기 시스테인이 부가된 변이체 인터페론-베타에 설프히드릴 반응성 생폴리머(biopolymer)가 결합된 배합체 (conjugate)를 제공한다.In order to achieve the above another object, the present invention provides a conjugate in which a sulfhydryl reactive biopolymer is bonded to the variant interferon-beta to which the cysteine is added.
여기에서, 생폴리머는 덱스트란과 같은 탄수화물 폴리머, 아미노산 폴리머 및 바이오틴 유도체의 적어도 하나일 수 있으며, 특히 폴리에틸렌글리콜 (polyethylene glycol; PEG) 유도체인 것이 바람직하다.Here, the biopolymer may be at least one of a carbohydrate polymer such as dextran, an amino acid polymer and a biotin derivative, and it is particularly preferable that it is a polyethylene glycol (PEG) derivative.
이들 PEG 유도체는 직선형(linear) 메톡시폴리에틸렌글리콜-말레이미드, 가지형(branched) 폴리에틸렌글리콜-말레이미드, 또는 펜던트(pendant)형 폴리에틸렌글리콜-말레이미드이거나, 교차결합을 위한 이기능성(bifunctional) 또는 다기능성(multifunctional) 그룹을 포함할 수 있으며, 분자량 범위가 5,000 내지 100,000인 것이 바람직하다.These PEG derivatives are linear methoxypolyethyleneglycol-maleimide, branched polyethyleneglycol-maleimide, or pendant polyethyleneglycol-maleimide, or are bifunctional for crosslinking or It may include multifunctional groups, and preferably has a molecular weight range of 5,000 to 100,000.
본 발명에서는, 인터페론-베타에서 수용체 결합에 관여하지 않는 특정 아미노산 잔기를 시스테인으로 치환하거나 시스테인 잔기를 첨가(insertion)하고, 이와 같이 부가된 시스테인의 자유 티올기(free thiol group, -SH group)에 반응성을 갖는 PEG 유도체 등의 생체고분자를 반응시켜 위치지정(site-directed) 공유결합된 배합체를 합성하였다. 이와 같이 합성된 인터페론-베타와 PEG 유도체의 배합체는 향상된 약동학 프로필(pharmacokinetic profile)과 약리학 성질을 가질 수 있다.In the present invention, in the interferon-beta, a specific amino acid residue not involved in receptor binding is substituted with cysteine or a cysteine residue is inserted into the free thiol group (-SH group) of cysteine added in this manner. Site-directed covalently bonded compounds were synthesized by reacting biopolymers such as PEG derivatives having reactivity. The combination of interferon-beta and PEG derivatives thus synthesized may have improved pharmacokinetic profile and pharmacological properties.
이하, 본 발명을 더욱 구체적으로 설명한다.Hereinafter, the present invention will be described in more detail.
인터페론-베타는 섬유아세포(fibroblast)에서 생산되며 다발성 경화증에 치료 효과가 있는 것으로 알려져 있다. 인터페론-베타-1a(IFN-β-1a)는 166 개 아미노산으로 구성된 당화(glycosylated) 단백질로 1차 서열과 탄수화물 함량으로 천연 인터페론-베타와 구조적으로 구별하기 어렵다. 인터페론-베타-1b(IFN-β-1b)는 인터페론-베타의 당이 결여된(non-glycosylated) 재조합 단백질로, 아미노산 1 번 잔기 메티오닌이 결실되어 있고, 17 번 잔기 시스테인이 세린으로 치환되어 있다.Interferon-beta is produced in fibroblasts and is known to have a therapeutic effect on multiple sclerosis. Interferon-beta-1a (IFN-β-1a) is a glycosylated protein consisting of 166 amino acids and is difficult to distinguish structurally from natural interferon-beta by primary sequence and carbohydrate content. Interferon-beta-1b (IFN-β-1b) is a non-glycosylated recombinant protein that lacks the amino acid residue 1 methionine and residue 17 cysteine is substituted for serine. .
본 발명에서는 인터페론-베타의 천연적으로 존재하는(naturally occurring) 아미노산 잔기들을 선택하여 하나 또는 그 이상을 시스테인 잔기로 치환시키거나 또는 아미노산 서열에 시스테인을 부가시키는 위치지정 돌연변이(site-directed mutagenesis) 기법을 사용한 후, 이 시스테인 잔기 또는 잔기들에 설프히드릴기 (sulfhydryl group, -SH)와 반응하는 PEG를 결합시켜 위치특이적으로 변형된 인터페론 유도체들을 합성하였다. 이러한 유도체들은 천연 단백질과 동일한 생물학적 활성과 시험관 내(in vitro) 활성을 가지며 동시에 전신 분포(systemic exposure)가 상당히 증가한다.In the present invention, a site-directed mutagenesis technique that selects naturally occurring amino acid residues of interferon-beta to replace one or more with cysteine residues or add cysteines to the amino acid sequence. After use, the cysteine residue or residues were combined with PEG reacting with a sulfhydryl group (-SH) to synthesize site-specifically modified interferon derivatives. These derivatives have the same biological and in vitro activity as natural proteins, while at the same time significantly increasing systemic exposure.
본 발명에서 바람직한 인터페론-베타의 시스테인 치환 잔기는 80 번째 아스파라진(Asparagine)으로서 탄수화물이 N-결합으로 연결되며 아미노산 108∼117 번 잔기들로 구성된 CD 루프 내에 위치한다.Preferred interferon-beta cysteine substitution residues in the present invention are the 80 th asparagine (Asparagine), which is carbohydrate linked by an N-link and is located in a CD loop consisting of amino acids 108-117 residues.
본 발명에서는 비 당화 인터페론-베타-1b의 탄수화물 부분(moiety)의 부재를 상쇄하고 안정성(stability), 활성, 체내분포, 용해도, 혈액내 반감기를 증가시키기 위해 아미노산 80 번 위치에 PEG 폴리머를 위치지정 공유 결합시키고 있는데, 이를 위해 재조합 인터페론-베타-1b를 변형시켰다. 아미노산 80 번 잔기는 야생형 인간 인터페론-베타에서 유일한 당 부가 위치이다. 야생형에 존재하는 다당류 사슬을 PEG 폴리머로 치환함으로써 당화 단백질과 그 수용체와의 결합이 촉진되도록 하는 기술은 인터류킨-2(IL-2)에 의해 입증되었다. 즉, 굿슨 등(Goodson et al., Bio/Technology, 8: 343∼346, 1990)은 IL-2에서 유일한 당 부가 위치인 아미노산 3 번 잔기에 분자량 6,000의 PEG 폴리머를 공유결합시킨 PEG 6000-Cys3-IL-2 배합체를 보고하고 있다. 크기 배제 크로마토그래피(size exclusion chromatography)로 결정된 이 배합체의 분자량 크기는 55 킬로달톤(KDa)이었는데, 이는 PEG 분자량 6 킬로달톤과 IL-2의 분자량 13 킬로달톤의 합계보다 큰 것으로 PEG 분자들의 광범위한 수화(hydration) 가능성을 설명하고 있다. PEG-IL-2 배합체는 완전한 생물학적 활성을 갖고 당화 형태 즉, 야생형보다 전신분포도(systemic exposure)가 4 배 증가한 것으로 보고되었다.In the present invention, PEG polymers are positioned at amino acid position 80 to counteract the absence of carbohydrate moieties of unglycosylated interferon-beta-1b and to increase stability, activity, body distribution, solubility, and blood half-life. Covalently binding, for which the recombinant interferon-beta-1b was modified. Amino acid residue 80 is the only sugar addition site in wild type human interferon-beta. Interleukin-2 (IL-2) demonstrated a technique that promotes the binding of glycosylated proteins to their receptors by substituting PEG polymers for the polysaccharide chains present in the wild type. In other words, Goodson et al. , Bio / Technology, 8: 343-346, 1990, covalently bonded PEG 6000-Cys3 to a PEG polymer having a molecular weight of 6,000 to amino acid residue 3, the only sugar addition site in IL-2. The -IL-2 blend is reported. The molecular weight of the compound, determined by size exclusion chromatography, was 55 kilodaltons (KDa), which is greater than the sum of 6 kilodaltons of PEG molecular weight and 13 kilodaltons of IL-2. The possibility of hydration is described. The PEG-IL-2 combination has been reported to have a complete biological activity and a four-fold increase in systemic exposure than the glycated form, ie wild type.
본 발명에서는 야생형 인터페론-베타와 비교하였을 때 하나의 시스테인 잔기가 더 부가된 비 당화 인터페론-베타 하이브리드(hybrid)를 제공한다. 우추미 등(Utzumi et al., J. Biochem., 101: 1199, 1987)은 아미노산 잔기 31 번과 141 번에 위치하는 2개의 보존된(conserved) 시스테인 잔기들이 이황화 결합(disulfide bond)를 형성하고 이것이 야생형 인터페론-베타의 활성구조를 유지하는데 필요하다는 것을 밝혔다. 즉, 아미노산 잔기 31 번과 141 번의 시스테인 잔기가 형성하는 이황화 결합이 인터페론-베타의 생물학적 활성을 평가하는 기준이 되므로, 본 발명에서는 부가적인 시스테인 코돈(codon)을 DNA 수준(level)에서 원하는 부위에 삽입(insertion)하거나 원하는 부위의 기존 잔기를 시스테인 잔기로 치환시켜 도입하였다.The present invention provides an unglycosylated interferon-beta hybrid with one more cysteine residue added as compared to the wild type interferon-beta. Utzumi et al. , J. Biochem., 101 : 1199, 1987, found that two conserved cysteine residues located at amino acid residues 31 and 141 form a disulfide bond, This is necessary to maintain the active structure of wild type interferon-beta. That is, since the disulfide bonds formed by amino acid residues 31 and 141 cysteine residues serve as a criterion for evaluating the biological activity of interferon-beta, in the present invention, an additional cysteine codon is added to a desired site at the DNA level. Either by insertion or by replacing existing residues of the desired site with cysteine residues.
이러한 돌연변이체들은 기존의 분자생물학 기술들을 사용하여 합성할 수 있다. 인터페론-베타의 위치지정 돌연변이는 포터 등(Porter et al., DNA, 5: 137-148, 1986)의 논문을 참고하였다.Such mutants can be synthesized using existing molecular biology techniques. For the positional mutations of interferon-beta, see Porter et al. , DNA, 5 : 137-148, 1986.
본 발명에 따라, 시험관내 돌연변이로 비 당화 인터페론-베타 단백질들을 합성하였다. 이들 단백질은 17 번 시스테인 잔기가 세린(serine)으로, 80 번 아스파라진 잔기가 시스테인으로 바뀌고, 1 번 메티오닌(methionine) 잔기가 존재하거나 또는 제거된 것을 포함한다.According to the present invention, non-glycosylated interferon-beta proteins were synthesized with in vitro mutations. These proteins include those where the cysteine residue 17 is serine, the asparagine residue 80 is cysteine, and the methionine residue 1 is present or removed.
서열 번호 1은 야생형 인간 인터페론-베타-1a의 뉴클레오티드 및 아미노산 서열을 나타낸 것이고, 서열 번호 3은 아미노산 17 번 시스테인 잔기가 세린으로 치환된 대장균 유래 재조합 인간 인터페론-베타의 뉴클레오티드 및 아미노산 서열을 나타낸 것이다. 또한, 서열 번호 5는 아미노산 17 번 시스테인 잔기가 세린으로 치환되고, 1 번 메티오닌 잔기가 결실(deletion)된 대장균 유래 재조합 인간 인터페론-베타의 뉴클레오티드 및 아미노산 서열을 나타낸 것이다.SEQ ID NO: 1 shows the nucleotide and amino acid sequence of wild type human interferon-beta-1a, and SEQ ID NO: 3 shows the nucleotide and amino acid sequence of E. coli derived recombinant human interferon-beta in which the cysteine residue of amino acid 17 is substituted with serine. SEQ ID NO: 5 shows the nucleotide and amino acid sequences of E. coli-derived recombinant human interferon-beta, in which the cysteine residue of amino acid 17 is substituted with serine and the methionine residue is deleted.
한편, 서열 번호 7은 본 발명에 따라 아미노산 17 번 시스테인 잔기가 세린으로 치환되고, 80 번 아스파라진(asparagine) 잔기가 PEG 유도체와 결합시키기 위해 시스테인으로 치환된 대장균 유래 재조합 인간 인터페론-베타의 뉴클레오티드 및 아미노산 서열을 나타낸 것이고, 서열 번호 9는 아미노산 1 번 메티오닌 잔기가 결실되고, 17 번 시스테인 잔기가 세린으로 치환되고, 80 번 아스파라진 (asparagine) 잔기가 PEG 유도체와 결합시키기 위해 시스테인으로 치환된 대장균 유래 재조합 인간 인터페론-베타의 뉴클레오티드 및 아미노산 서열을 나타낸 것이다.On the other hand, SEQ ID NO: 7 shows the nucleotides of E. coli-derived recombinant human interferon-beta in which the cysteine residue of amino acid 17 is substituted with serine and the asparagine residue 80 is substituted with cysteine to bind a PEG derivative according to the present invention. Amino acid sequence, SEQ ID NO: 9 is derived from E. coli, wherein amino acid No. 1 methionine residue is deleted, cysteine residue No. 17 is substituted with serine, and asparagine residue No. 80 is substituted with cysteine to bind a PEG derivative Nucleotide and amino acid sequences of recombinant human interferon-beta are shown.
이와 같이 변형된 인터페론-β 분자들에 충분한 양의 설프히드릴 반응성 분자(sulfhydryl reactive compound)를 반응시킴으로써 80 번 시스테인 잔기에 공유결합시킨다. 설프히드릴 반응성 분자의 양(amount)은 적어도 인터페론-베타와 동일한 당량(equimolar)으로 가해 주어야 하며, 시스테인의 자유 티올(free thiol; -SH) 그룹과의 완전한 반응을 유도하기 위해 과량 첨가하는 것이 바람직하다.The modified interferon-β molecules are covalently bound to the cysteine residue at 80 by reacting a sufficient amount of sulfhydryl reactive compound. The amount of the sulfhydryl reactive molecule should be added at least equal to the equivalent of interferon-beta, and excessive addition to induce a complete reaction with the free thiol (-SH) group of cysteine. desirable.
설프히드릴 반응성 분자들로서는, 알킬 말단을 갖는 폴리알킬렌 옥사이드 폴리머[alkyl terminated poly(alkylene oxide)]로 통칭되는 분자들이 사용될 수 있으며, 여기에는 PEG 호모폴리머(homopolymer), 알킬 캡드 폴리에틸렌 옥사이드 [alkyl capped poly(ethylene oxide)], 폴리알킬렌 옥사이드의 블록 코폴리머 (block copolymer)와 이들의 유도체들이 포함된다. 이들은 에스-피리딜(S-pyridyl) 그룹, 말레이미드(maleimide), 그리고 활성화된 에스터(ester) 그룹 같은 커플링 (coupling) 또는 활성화된 부분(moiety)들을 포함하고 있다.As the sulfhydryl reactive molecules, molecules collectively referred to as alkyl terminated poly (alkylene oxides) with alkyl ends can be used, including PEG homopolymers, alkyl capped polyethylene oxides [alkyl] capped poly (ethylene oxide)], block copolymers of polyalkylene oxides and derivatives thereof. These include coupling or activated moieties such as S-pyridyl groups, maleimide, and activated ester groups.
본 발명에서 변형된 인터페론-베타에 결합되는 설프히드릴 반응성 PEG 유도체들은 특정한 분자량을 가질 필요는 없으나 분자량 범위가 5,000 내지 100,000인 것이 바람직하고, 5,000∼60,000 범위의 것이 더욱 바람직하다. 이들 PEG 유도체들로는 선바이오(주)가 합성하거나 판매하는 제품을 이용할 수 있다.The sulfhydryl reactive PEG derivatives bound to the modified interferon-beta in the present invention do not need to have a specific molecular weight, but preferably have a molecular weight range of 5,000 to 100,000, more preferably 5,000 to 60,000. As these PEG derivatives, the product which Sun Bio Co., Ltd. synthesizes or sells can be used.
이들 PEG 유도체로는 직선형(linear) 구조를 갖는 것, 가지형(branched) 구조를 갖는 것, 교차결합에 유용한 이기능성(bifunctional) 그룹을 갖는 것, 그리고 PEG 뼈대에 말레이미도(maleimido) 그룹이 펜단트(pendant)형태로 결합된 폴리머들이 사용될 수 있다.These PEG derivatives include linear structures, branched structures, bifunctional groups useful for crosslinking, and maleimido groups in PEG skeletons. Polymers bound in pendant form may be used.
다음 화학식 1∼3은 3 종류의 직선형(linear) 말레이미도 모노메톡시 (maleimido monomethoxy) PEG의 구조를 나타낸 것이다.The following Chemical Formulas 1 to 3 show the structure of three types of linear maleimido monomethoxy PEG.
다음 화학식 4 및 5는 2 종류의 가지형 사슬(branched chain, BC) 말레이미도 모노메톡시 PEG의 구조를 나타낸 것이다.The following Formulas 4 and 5 show the structure of two branched chain (BC) maleimido monomethoxy PEG.
다음 화학식 6은 교차결합에 유용한 이기능성(bifunctional) 말레이미도 PEG의 구조를 나타낸 것이다.Formula 6 shows the structure of bifunctional maleimido PEG useful for crosslinking.
다음 화학식 7은 PEG 뼈대에 말레이미도(maleimido) 그룹이 펜단트(pendant)형태로 결합된 폴리머를 나타낸다.Formula 7 shows a polymer in which a maleimido group is bound to a PEG skeleton in the form of pendant.
여기에서, R1은 수소 또는 낮은 탄소수의 알킬(lower alkyl)이고, R2 및 R3는 CH2CH2 또는 CH2CHCH3일 수 있으나 서로 동일하지는 않고, R4는 말레이미도 (maleimido) 그룹이다.Wherein R 1 is hydrogen or lower alkyl, R 2 and R 3 are CH 2 CH 2 or CH 2 CHCH 3 , but not identical to each other, R 4 is a maleimido group.
이밖의 설프히드릴 반응성 분자들에는 덱스트란(dextran) 등과 같은 탄수화물에 근거한 폴리머, 아미노산 폴리머 및 바이오틴(biotin) 유도체들이 포함된다.Other sulfhydryl reactive molecules include carbohydrate based polymers, amino acid polymers and biotin derivatives, such as dextran.
대표적인 설프히드릴 커플링 시약은 말레이미도 모노메톡시(maleimido monomethoxy) PEG로써, 설프히드릴 반응성 분자들이 변형된 시스테인 잔기에 공유결합해서 티올 부분(thiol moiety)과 말레이미드 그룹(maleimide group) 간에 티오에테르(thioether) 결합이 형성된다.A representative sulfhydryl coupling reagent is maleimido monomethoxy PEG, in which sulfhydryl reactive molecules are covalently bonded to the modified cysteine residues to form a thiou moiety between the thiol moiety and the maleimide group. An ether bond is formed.
다음 반응식 1은 시스테인이 치환된 인터페론-베타-1b(이하, Cys80-IFN-β-1b로 약칭)에 직선형의 말레이미도 모노메톡시 PEG를 결합시켜 PEG-Cys80-IFN-β-1b를 합성하는 예를 도시한 것이다.Scheme 1 shows the synthesis of PEG-Cys80-IFN-β-1b by binding a linear maleimido monomethoxy PEG to a cysteine-substituted interferon-beta-1b (abbreviated to Cys80-IFN-β-1b). An example is shown.
Cys80-IFN-β-1b의 시스테인 잔기에만 선택적으로 균일하게 폴리머를 결합시키기 위해서는 말레이미도 모노메톡시 PEG 같은 설프히드릴에 특이적인 시약을 사용하는데, 다른 반응성 부위보다 시스테인 잔기에 훨씬 증가된 효율로 공유결합을 형성한다. Cys80-IFN-β-1b 단백질과 PEG-말레이미드의 결합 반응은 DTT (dithiothreitol), EDTA(ethylenediaminetetraacetate), 및 염산 구아니딘 (guanidine HCl)을 포함하는 NaH2PO4 완충액(buffer), pH 7.0(pH 6.0∼7.5 범위가 바람직하다) 조건에서 이루어진다. 반응은 4 ℃에서 수행되며, 그 과정 중에 SDS-아크릴아마이드(acrylamide) 젤로 PEG-Cys80-IFN-β-1b 배합체의 형성을 확인하는데, 확인 후 2 시간 더 반응시킨다.To selectively and uniformly bind the polymer only to the cysteine residues of Cys80-IFN-β-1b, reagents specific to sulfhydryls, such as maleimido monomethoxy PEG, are used, with much increased efficiency at cysteine residues than other reactive sites. To form a covalent bond. The coupling reaction of Cys80-IFN-β-1b protein with PEG-maleimide was performed by NaH 2 PO 4 buffer, pH 7.0 (pH), including DTT (dithiothreitol), EDTA (ethylenediaminetetraacetate), and guanidine hydrochloride Range from 6.0 to 7.5 is preferred). The reaction is carried out at 4 ℃, during the process to confirm the formation of the PEG-Cys80-IFN-β-1b compound with SDS-acrylamide gel, the reaction is further reacted for 2 hours.
다음 반응식 2는 시스테인이 치환된 Cys80-IFN-β-1b에 가지형 사슬의 말레이미도 모노메톡시 PEG를 결합시켜 PEG-Cys80-IFN-β-1b를 합성하는 예를 도시한 것이다.Scheme 2 shows an example of synthesizing PEG-Cys80-IFN-β-1b by binding a branched chain maleimido monomethoxy PEG to a cysteine substituted Cys80-IFN-β-1b.
PEG-말레이미드와 단백질과의 결합반응 예는 굿슨 등의 논문(Goodson et al., Bio/Technology, 8: 343-346, 1990) 및 쇼 등(Shaw et al.)의 미국특허 제5,166,322호에 기술되어 있다.Examples of binding reactions between PEG-maleimide and proteins are shown in Goodson et al. , Bio / Technology, 8 : 343-346, 1990 and US Pat. No. 5,166,322 to Shaw et al . Described.
다음 반응식 3은 시스테인이 치환된 Cys80-IFN-β-1b에 펜단트 PEG 분자(Ⅱ)를 결합시켜 PEG-Cys80-IFN-β-1b(Ⅲ)를 합성하는 예를 도식화한 것이다.Scheme 3 shows an example of synthesizing PEG-Cys80-IFN-β-1b (III) by binding a pendant PEG molecule (II) to Cys80-IFN-β-1b substituted with cysteine.
여기에서, 폴리-말레이미도(poly-maleimido) 시약(Ⅱ)은 PEG 뼈대에 2∼20 개의 펜단트 프로피오닉산(pendant propionic acid) 그룹을 도입시킨 멀티-카르복실화(multi-carboxylated) 산물(Ⅰ)로부터 제조한다. 폴리-말레이미도 시약(Ⅱ)을 시스테인 잔기가 변형된 Cys80-IFN-β-1b와 반응시켜 배합체 산물인 PEG-Cys80-IFN-β-1b(Ⅲ)을 얻는다.Here, poly-maleimido reagent (II) is a multi-carboxylated product (I) having introduced 2 to 20 pendant propionic acid groups in a PEG skeleton. From). The poly-maleimido reagent (II) is reacted with Cys80-IFN-β-1b modified cysteine residues to give the combination product PEG-Cys80-IFN-β-1b (III).
한편, 본 발명에 따른 인터페론-베타는 혈액-뇌 장벽을 가로질러 중추신경계로 효율적으로 전달하는 트랜스페린 수용체 항체와 결합하여 항체-IFN-β배합체 형태로 뇌로 운반될 수 있다. 항체-IFN-β 배합체는 항체의 라이신(lysine) 잔기에 존재하는 입실론 아미노(ε-amino) 잔기에 교차결합 가능한 이종이기능성 (heterobifunctional) 말레이미도 PEG를 커플링시킨 뒤, 인터페론-베타에 새로 도입된 시스테인 잔기와 말레이미드의 티올 반응 그룹(maleimide thiol reactive group) 사이에서 반응이 일어나는 단계별 과정을 통해 합성된다.On the other hand, the interferon-beta according to the present invention can be transported to the brain in the form of antibody-IFN-β conjugates by binding to transferrin receptor antibodies that efficiently deliver to the central nervous system across the blood-brain barrier. The antibody-IFN-β combination couples a crosslinkable heterobifunctional maleimido PEG to an epsilon amino residue present in the lysine residue of the antibody, followed by new interferon-beta. It is synthesized through a stepwise process in which a reaction takes place between the introduced cysteine residue and the maleimide thiol reactive group.
도 1은 트랜스페린 수용체 항체와 시스테인이 치환된 인터페론-베타-1b (Cys80-IFN-β-1b) 배합체의 결합 과정을 도식화한 것이다.Figure 1 illustrates the binding process of the transferrin receptor antibody and cysteine substituted interferon-beta-1b (Cys80-IFN-β-1b) combination.
또한, 트랜스페린 수용체 항체-IFN-β 배합체는 이황화 결합을 갖도록 합성될 수도 있다. 첫 단계에서 트랜스페린 수용체 항체의 라이신 잔기에 존재하는 입실론 아미노 잔기에 교차결합 가능한 이종이기능성 PEG 유도체(heterobifunctional PEG derivative)를 커플링시키고, 두 번째 단계로 인터페론-베타에 새로 도입된 시스테인 잔기의 설프히드릴 그룹과 오르쏘피리딜(orthopyridyl) 이황화 유도체의 축합(condensation)에 의한 이황화결합이 형성된다.In addition, the transferrin receptor antibody-IFN-β combination may be synthesized to have disulfide bonds. In the first step, a cross-linkable heterobifunctional PEG derivative is coupled to the epsilon amino residue present in the lysine residue of the transferrin receptor antibody, and in the second step, a sulfide of a newly introduced cysteine residue in interferon-beta. Disulfide bonds are formed by condensation of a drill group with an orthopyridyl disulfide derivative.
도 2는 이황화 링커(linker)를 포함하는 트랜스페린 수용체 항체와 시스테인이 치환된 인터페론-베타-1b(Cys80-IFN-β-1b) 배합체의 결합 과정을 도식화한 것이다.Figure 2 illustrates the binding process of a transferrin receptor antibody comprising a disulfide linker and a cysteine substituted interferon-beta-1b (Cys80-IFN-β-1b) combination.
이하, 실시예를 통해 본 발명을 더욱 상세히 설명한다. 단, 이들 실시예는 본 발명의 일부 실험방법과 조성을 나타낸 예시일 뿐, 본 발명의 범위가 이들만으로 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only examples showing some experimental methods and compositions of the present invention, but the scope of the present invention is not limited thereto.
[실시예 1]Example 1
시스테인이 치환된 인간 인터페론-베타의 클로닝(cloning)Cloning of Cysteine-Substituted Human Interferon-beta
인간 섬유아세포 인터페론(IFN-β) cDNA 서열의 클로닝과 대장균에서의 발현에 대해서는 더링크 등(Derynck et al., Nature, 285: 542, 1980), 괴달 등 (Goeddal et al., Nucleic Acids Res., 8: 4057, 1980), 타니구치 등(Taniguichi et al., Proc. Natl. Acad. Sci. U.S.A., 77: 5230, 1980), 그리고 셰퍼드 등(Shepard et al., Nature, 294: 563, 1981)에 의해 보고되어 있다.For cloning of human fibroblast interferon (IFN-β) cDNA sequences and expression in E. coli, Derinck et al. , Nature, 285 : 542, 1980, Goeddal et al., Nucleic Acids Res. , 8 : 4057, 1980), Taniguchi et al., Proc. Natl. Acad. Sci. USA, 77 : 5230, 1980, and Shepard et al. (Shepard et al., Nature, 294 : 563, 1981). Reported by.
IFN-β 서열에 특정 돌연변이를 유도하는 과정은, 먼저 야생형 IFN-β를 코딩하는 DNA 서열을 분리하거나 합성한 후, PCR(polymerase chain reaction, 연쇄중합반응) 돌연변이 그리고 플라스미드(plasmid) 수준에서 위치지정 돌연변이 등의 기술을 이용하여 특정 아미노산 코돈을 도입하고자 하는 코돈으로 대체함으로써 가능하다. IFN-β의 아미노산 17 번 시스테인 잔기를 세린으로 치환시키는 돌연변이는 런켈 등(Runkel et al., J. Biol. Chem., 273: 8003, 1998)이 야생형 IFN-β 유전자를 17 번 시스테인으로부터 세린으로 돌연변이가 유도된 5'-프라이머(primer)를 사용하여 PCR 증폭함으로써 제조하고 분리한 예가 있다.The process of inducing specific mutations in the IFN-β sequence first involves isolating or synthesizing the DNA sequence encoding the wild type IFN-β, followed by positioning at the polymerase chain reaction (PCR) mutation and the plasmid level. This can be accomplished by replacing specific amino acid codons with the codon to be introduced using mutations or other techniques. Mutations that replace the amino acid 17 cysteine residue of IFN-β with serine are described by Runkel et al. (J. Biol. Chem., 273 : 8003, 1998) from the wild type IFN-β gene from cysteine 17 to serine. There is an example prepared and isolated by PCR amplification using a 5'-primer induced mutation.
특정한 한 아미노산이 하나 이상의 코돈으로 코딩(coding)될 수 있는데 이를 리던던시(redundancy)라고 하며, 예를 들어 시스테인의 경우 TGT와 TGC의 2 개의 코돈으로부터 코딩될 수 있다. 즉, 특정한 돌연변이 단백질을 코딩하는 DNA 서열이 여러 개 존재할 수 있다는 것으로, 동일한 아미노산을 코딩하는 가능한 모든 서열들이 본 발명의 범위 내에 속할 수 있다.One particular amino acid may be encoded into one or more codons, which is called redundancy, for example cysteine from two codons of TGT and TGC. That is, because there may be multiple DNA sequences encoding a particular mutant protein, all possible sequences encoding the same amino acid may fall within the scope of the present invention.
[실시예 2]Example 2
Cys80-IFN-β-1b와 직선형 PEG-말레이미드(maleimide)의 배합(conjugation)Conjugation of Cys80-IFN-β-1b with Straight PEG-maleimide
돌연변이 Cys80-IFN-β-1b 단백질과 직선형 PEG-말레이미드(평균 분자량 5,000∼40,000) 폴리머를 DTT, EDTA, 구아니딘 염산을 포함하는 NaH2PO4, pH 7.0 완충액 중에서 반응시켜 배합체를 형성하였다. 반응은 4 ℃에서 수행하며, 반응 중 SDS-폴리아크릴아마이드 전기영동(SDS-polyacrylamide electrophoresis)으로 배합체(PEG-Cys80-IFN-β-1b)를 확인한 후 2 시간 더 반응시켰다.The mutant Cys80-IFN-β-1b protein and the linear PEG-maleimide (average molecular weight 5,000-40,000) polymer were reacted in NaH 2 PO 4 , pH 7.0 buffer containing DTT, EDTA, guanidine hydrochloric acid, to form a formulation. The reaction was carried out at 4 ℃, and after the reaction was confirmed by the SDS-polyacrylamide electrophoresis (SDS-polyacrylamide electrophoresis) compound (PEG-Cys80-IFN-β-1b) for 2 hours.
[실시예 3]Example 3
Cys80-IFN-β-1b와 가지형 사슬 PEG-말레이미드(maleimide)의 배합(conjugation)Conjugation of Cys80-IFN-β-1b with Branched Chain PEG-maleimide
돌연변이 Cys80-IFN-β-1b 단백질과 가지형 사슬 PEG-말레이미드(평균 분자량 10,000∼40,000) 폴리머를 실시예 2에 기재된 것과 동일한 조건으로 반응시켜 배합체를 형성하였다.The mutant Cys80-IFN-β-1b protein and the branched chain PEG-maleimide (average molecular weight 10,000-40,000) polymer were reacted under the same conditions as described in Example 2 to form a combination.
[실시예 4]Example 4
Cys80-IFN-β-1b와 펜단트형 PEG-말레이미드(maleimide)의 배합(conjugation)Conjugation of Cys80-IFN-β-1b with Pendant PEG-maleimide
돌연변이 Cys80-IFN-β-1b 단백질과 다수의 말레이미드 곁사슬(side chain)을 갖는 펜단트형 PEG-말레이미드(평균 분자량 10,000∼100,000) 폴리머를 실시예 2에 기재된 것과 동일한 조건으로 반응시켜 배합체를 형성하였다.The compound was reacted with a mutant Cys80-IFN-β-1b protein and a pendant PEG-maleimide (average molecular weight 10,000 to 100,000) polymer having a plurality of maleimide side chains under the same conditions as described in Example 2. Formed.
이상에서 살펴본 바와 같이, 인터페론-베타의 특정 아미노산 잔기에 시스테인 잔기를 첨가하거나, 야생형 아미노산 서열에 존재하는 특정 아미노산 잔기를 시스테인으로 치환하여 변형시킨 본 발명의 인터페론-베타 변이체는, PEG 유도체와 같은 생체고분자를 위치지정(site-directed) 공유결합시킨 배합체를 제조함으로써, 항원유발성(immunogenicity)이 감소되고 천연 단백질과 동일한 생물학적 활성과 시험관내 활성을 가지며 전신 분포가 상당히 증가하여 향상된 약동학 프로필 (pharmacokinetic profile)과 약리학 성질을 갖게 된다.As described above, the interferon-beta variant of the present invention modified by adding a cysteine residue to a specific amino acid residue of interferon-beta or by replacing a specific amino acid residue present in a wild-type amino acid sequence with cysteine is a bio-material such as a PEG derivative. By preparing a combination of site-directed covalent polymers, the improved pharmacokinetic profile is reduced by immunogenicity, the same biological and in vitro activity as natural proteins, and a significant increase in systemic distribution. profile) and pharmacological properties.
도 1은 트랜스페린 수용체 항체와 시스테인이 치환된 인터페론-베타-1b (Cys80-IFN-β-1b) 배합체의 결합 과정을 도식화한 것,1 is a diagram illustrating the binding process of a transferrin receptor antibody and a cysteine-substituted interferon-beta-1b (Cys80-IFN-β-1b) combination,
도 2는 이황화 링커(linker)를 포함하는 트랜스페린 수용체 항체와 시스테인이 치환된 인터페론-베타-1b(Cys80-IFN-β-1b) 배합체의 결합 과정을 도식화한 것.Figure 2 illustrates the binding process of a transferrin receptor antibody comprising a disulfide linker and a cysteine substituted interferon-beta-1b (Cys80-IFN-β-1b) combination.
<110> SUNBIO INC. <120> MODIFIED INTERFERON-BETA, AND CHEMICALLY MODIFIED CONJUGATES THEREOF <130> ppba0582 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 507 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (7)..(504) <400> 1 ctttcc atg agc tac aac ttg ctt gga ttc cta caa aga agc agc aat 48 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn 1 5 10 ttt cag tgt cag aag ctc ctg tgg caa ttg aat ggg agg ctt gaa tat 96 Phe Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr 15 20 25 30 tgc ctc aag gac agg atg aac ttt gac atc cct gag gag att aag cag 144 Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln 35 40 45 ctg cag cag ttc cag aag gag gac gcc gca ttg acc atc tat gag atg 192 Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met 50 55 60 ctc cag aac atc ttt gct att ttc aga caa gat tca tct agc act ggc 240 Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly 65 70 75 tgg aat gag act att gtt gag aac ctc ctg gct aat gtc tat cat cag 288 Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln 80 85 90 ata aac cat ctg aag aca gtc ctg gaa gaa aaa ctg gag aaa gaa gat 336 Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp 95 100 105 110 ttt acc agg gga aaa ctc atg agc agt ctg cac ctg aaa aga tat tat 384 Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr 115 120 125 ggg agg att ctg cat tac ctg aag gcc aag gag tac agt cac tgt gcc 432 Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala 130 135 140 tgg acc ata gtc aga gtg gaa atc cta agg aac ttt tac ttc att aac 480 Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn 145 150 155 aga ctt aca ggt tac ctc cga aac tga 507 Arg Leu Thr Gly Tyr Leu Arg Asn 160 165 <210> 2 <211> 166 <212> PRT <213> Homo sapiens <400> 2 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 Thr Gly Tyr Leu Arg Asn 165 <210> 3 <211> 507 <212> DNA <213> Artificial Sequence <220> <223> genetically modified human interferon-beta gene <220> <221> CDS <222> (7)..(504) <400> 3 ctttcc atg agc tac aac ttg ctt gga ttc cta caa aga agc agc aat 48 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn 1 5 10 ttt cag agt cag aag ctc ctg tgg caa ttg aat ggg agg ctt gaa tat 96 Phe Gln Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr 15 20 25 30 tgc ctc aag gac agg atg aac ttt gac atc cct gag gag att aag cag 144 Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln 35 40 45 ctg cag cag ttc cag aag gag gac gcc gca ttg acc atc tat gag atg 192 Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met 50 55 60 ctc cag aac atc ttt gct att ttc aga caa gat tca tct agc act ggc 240 Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly 65 70 75 tgg aat gag act att gtt gag aac ctc ctg gct aat gtc tat cat cag 288 Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln 80 85 90 ata aac cat ctg aag aca gtc ctg gaa gaa aaa ctg gag aaa gaa gat 336 Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp 95 100 105 110 ttt acc agg gga aaa ctc atg agc agt ctg cac ctg aaa aga tat tat 384 Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr 115 120 125 ggg agg att ctg cat tac ctg aag gcc aag gag tac agt cac tgt gcc 432 Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala 130 135 140 tgg acc ata gtc aga gtg gaa atc cta agg aac ttt tac ttc att aac 480 Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn 145 150 155 aga ctt aca ggt tac ctc cga aac tga 507 Arg Leu Thr Gly Tyr Leu Arg Asn 160 165 <210> 4 <211> 166 <212> PRT <213> Artificial Sequence <400> 4 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 Thr Gly Tyr Leu Arg Asn 165 <210> 5 <211> 504 <212> DNA <213> Artificial Sequence <220> <223> genetically modified human interferon-beta gene <220> <221> CDS <222> (7)..(501) <400> 5 ctttcc agc tac aac ttg ctt gga ttc cta caa aga agc agc aat ttt 48 Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe 1 5 10 cag agt cag aag ctc ctg tgg caa ttg aat ggg agg ctt gaa tat tgc 96 Gln Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys 15 20 25 30 ctc aag gac agg atg aac ttt gac atc cct gag gag att aag cag ctg 144 Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu 35 40 45 cag cag ttc cag aag gag gac gcc gca ttg acc atc tat gag atg ctc 192 Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu 50 55 60 cag aac atc ttt gct att ttc aga caa gat tca tct agc act ggc tgg 240 Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp 65 70 75 aat gag act att gtt gag aac ctc ctg gct aat gtc tat cat cag ata 288 Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile 80 85 90 aac cat ctg aag aca gtc ctg gaa gaa aaa ctg gag aaa gaa gat ttt 336 Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe 95 100 105 110 acc agg gga aaa ctc atg agc agt ctg cac ctg aaa aga tat tat ggg 384 Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly 115 120 125 agg att ctg cat tac ctg aag gcc aag gag tac agt cac tgt gcc tgg 432 Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp 130 135 140 acc ata gtc aga gtg gaa atc cta agg aac ttt tac ttc att aac aga 480 Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg 145 150 155 ctt aca ggt tac ctc cga aac tga 504 Leu Thr Gly Tyr Leu Arg Asn 160 165 <210> 6 <211> 165 <212> PRT <213> Artificial Sequence <400> 6 Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln Ser 1 5 10 15 Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu Lys 20 25 30 Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln Gln 35 40 45 Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln Asn 50 55 60 Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn Glu 65 70 75 80 Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn His 85 90 95 Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr Arg 100 105 110 Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg Ile 115 120 125 Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr Ile 130 135 140 Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu Thr 145 150 155 160 Gly Tyr Leu Arg Asn 165 <210> 7 <211> 507 <212> DNA <213> Artificial Sequence <220> <223> genetically modified human interferon-beta gene <220> <221> CDS <222> (7)..(504) <400> 7 ctttcc atg agc tac aac ttg ctt gga ttc cta caa aga agc agc aat 48 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn 1 5 10 ttt cag agt cag aag ctc ctg tgg caa ttg aat ggg agg ctt gaa tat 96 Phe Gln Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr 15 20 25 30 tgc ctc aag gac agg atg aac ttt gac atc cct gag gag att aag cag 144 Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln 35 40 45 ctg cag cag ttc cag aag gag gac gcc gca ttg acc atc tat gag atg 192 Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met 50 55 60 ctc cag aac atc ttt gct att ttc aga caa gat tca tct agc act ggc 240 Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly 65 70 75 tgg tgc gag act att gtt gag aac ctc ctg gct aat gtc tat cat cag 288 Trp Cys Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln 80 85 90 ata aac cat ctg aag aca gtc ctg gaa gaa aaa ctg gag aaa gaa gat 336 Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp 95 100 105 110 ttt acc agg gga aaa ctc atg agc agt ctg cac ctg aaa aga tat tat 384 Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr 115 120 125 ggg agg att ctg cat tac ctg aag gcc aag gag tac agt cac tgt gcc 432 Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala 130 135 140 tgg acc ata gtc aga gtg gaa atc cta agg aac ttt tac ttc att aac 480 Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn 145 150 155 aga ctt aca ggt tac ctc cga aac tga 507 Arg Leu Thr Gly Tyr Leu Arg Asn 160 165 <210> 8 <211> 166 <212> PRT <213> Artificial Sequence <400> 8 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Cys 65 70 75 80 Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 Thr Gly Tyr Leu Arg Asn 165 <210> 9 <211> 504 <212> DNA <213> Artificial Sequence <220> <223> genetically modified human interferon-beta gene <220> <221> CDS <222> (7)..(501) <400> 9 ctttcc agc tac aac ttg ctt gga ttc cta caa aga agc agc aat ttt 48 Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe 1 5 10 cag agt cag aag ctc ctg tgg caa ttg aat ggg agg ctt gaa tat tgc 96 Gln Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys 15 20 25 30 ctc aag gac agg atg aac ttt gac atc cct gag gag att aag cag ctg 144 Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu 35 40 45 cag cag ttc cag aag gag gac gcc gca ttg acc atc tat gag atg ctc 192 Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu 50 55 60 cag aac atc ttt gct att ttc aga caa gat tca tct agc act ggc tgg 240 Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp 65 70 75 tgc gag act att gtt gag aac ctc ctg gct aat gtc tat cat cag ata 288 Cys Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile 80 85 90 aac cat ctg aag aca gtc ctg gaa gaa aaa ctg gag aaa gaa gat ttt 336 Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe 95 100 105 110 acc agg gga aaa ctc atg agc agt ctg cac ctg aaa aga tat tat ggg 384 Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly 115 120 125 agg att ctg cat tac ctg aag gcc aag gag tac agt cac tgt gcc tgg 432 Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp 130 135 140 acc ata gtc aga gtg gaa atc cta agg aac ttt tac ttc att aac aga 480 Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg 145 150 155 ctt aca ggt tac ctc cga aac tga 504 Leu Thr Gly Tyr Leu Arg Asn 160 165 <210> 10 <211> 165 <212> PRT <213> Artificial Sequence <400> 10 Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln Ser 1 5 10 15 Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu Lys 20 25 30 Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln Gln 35 40 45 Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln Asn 50 55 60 Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Cys Glu 65 70 75 80 Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn His 85 90 95 Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr Arg 100 105 110 Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg Ile 115 120 125 Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr Ile 130 135 140 Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu Thr 145 150 155 160 Gly Tyr Leu Arg Asn 165<110> SUNBIO INC. <120> MODIFIED INTERFERON-BETA, AND CHEMICALLY MODIFIED CONJUGATES THEREOF <130> ppba0582 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 507 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (7) .. (504) <400> 1 ctttcc atg agc tac aac ttg ctt gga ttc cta caa aga agc agc aat 48 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn 1 5 10 ttt cag tgt cag aag ctc ctg tgg caa ttg aat ggg agg ctt gaa tat 96 Phe Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr 15 20 25 30 tgc ctc aag gac agg atg aac ttt gac atc cct gag gag att aag cag 144 Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln 35 40 45 ctg cag cag ttc cag aag gag gac gcc gca ttg acc atc tat gag atg 192 Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met 50 55 60 ctc cag aac atc ttt gct att ttc aga caa gat tca tct agc act ggc 240 Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly 65 70 75 tgg aat gag act att gtt gag aac ctc ctg gct aat gtc tat cat cag 288 Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln 80 85 90 ata aac cat ctg aag aca gtc ctg gaa gaa aaa ctg gag aaa gaa gat 336 Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp 95 100 105 110 ttt acc agg gga aaa ctc atg agc agt ctg cac ctg aaa aga tat tat 384 Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr 115 120 125 ggg agg att ctg cat tac ctg aag gcc aag gag tac agt cac tgt gcc 432 Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala 130 135 140 tgg acc ata gtc aga gtg gaa atc cta agg aac ttt tac ttc att aac 480 Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn 145 150 155 aga ctt aca ggt tac ctc cga aac tga 507 Arg Leu Thr Gly Tyr Leu Arg Asn 160 165 <210> 2 <211> 166 <212> PRT <213> Homo sapiens <400> 2 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 Thr Gly Tyr Leu Arg Asn 165 <210> 3 <211> 507 <212> DNA <213> Artificial Sequence <220> <223> genetically modified human interferon-beta gene <220> <221> CDS (222) (7) .. (504) <400> 3 ctttcc atg agc tac aac ttg ctt gga ttc cta caa aga agc agc aat 48 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn 1 5 10 ttt cag agt cag aag ctc ctg tgg caa ttg aat ggg agg ctt gaa tat 96 Phe Gln Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr 15 20 25 30 tgc ctc aag gac agg atg aac ttt gac atc cct gag gag att aag cag 144 Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln 35 40 45 ctg cag cag ttc cag aag gag gac gcc gca ttg acc atc tat gag atg 192 Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met 50 55 60 ctc cag aac atc ttt gct att ttc aga caa gat tca tct agc act ggc 240 Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly 65 70 75 tgg aat gag act att gtt gag aac ctc ctg gct aat gtc tat cat cag 288 Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln 80 85 90 ata aac cat ctg aag aca gtc ctg gaa gaa aaa ctg gag aaa gaa gat 336 Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp 95 100 105 110 ttt acc agg gga aaa ctc atg agc agt ctg cac ctg aaa aga tat tat 384 Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr 115 120 125 ggg agg att ctg cat tac ctg aag gcc aag gag tac agt cac tgt gcc 432 Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala 130 135 140 tgg acc ata gtc aga gtg gaa atc cta agg aac ttt tac ttc att aac 480 Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn 145 150 155 aga ctt aca ggt tac ctc cga aac tga 507 Arg Leu Thr Gly Tyr Leu Arg Asn 160 165 <210> 4 <211> 166 <212> PRT <213> Artificial Sequence <400> 4 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 Thr Gly Tyr Leu Arg Asn 165 <210> 5 <211> 504 <212> DNA <213> Artificial Sequence <220> <223> genetically modified human interferon-beta gene <220> <221> CDS (222) (7) .. (501) <400> 5 ctttcc agc tac aac ttg ctt gga ttc cta caa aga agc agc aat ttt 48 Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe 1 5 10 cag agt cag aag ctc ctg tgg caa ttg aat ggg agg ctt gaa tat tgc 96 Gln Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys 15 20 25 30 ctc aag gac agg atg aac ttt gac atc cct gag gag att aag cag ctg 144 Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu 35 40 45 cag cag ttc cag aag gag gac gcc gca ttg acc atc tat gag atg ctc 192 Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu 50 55 60 cag aac atc ttt gct att ttc aga caa gat tca tct agc act ggc tgg 240 Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp 65 70 75 aat gag act att gtt gag aac ctc ctg gct aat gtc tat cat cag ata 288 Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile 80 85 90 aac cat ctg aag aca gtc ctg gaa gaa aaa ctg gag aaa gaa gat ttt 336 Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe 95 100 105 110 acc agg gga aaa ctc atg agc agt ctg cac ctg aaa aga tat tat ggg 384 Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly 115 120 125 agg att ctg cat tac ctg aag gcc aag gag tac agt cac tgt gcc tgg 432 Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp 130 135 140 acc ata gtc aga gtg gaa atc cta agg aac ttt tac ttc att aac aga 480 Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg 145 150 155 ctt aca ggt tac ctc cga aac tga 504 Leu Thr Gly Tyr Leu Arg Asn 160 165 <210> 6 <211> 165 <212> PRT <213> Artificial Sequence <400> 6 Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln Ser 1 5 10 15 Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu Lys 20 25 30 Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln Gln 35 40 45 Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln Asn 50 55 60 Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn Glu 65 70 75 80 Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn His 85 90 95 Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr Arg 100 105 110 Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg Ile 115 120 125 Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr Ile 130 135 140 Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu Thr 145 150 155 160 Gly Tyr Leu Arg Asn 165 <210> 7 <211> 507 <212> DNA <213> Artificial Sequence <220> <223> genetically modified human interferon-beta gene <220> <221> CDS (222) (7) .. (504) <400> 7 ctttcc atg agc tac aac ttg ctt gga ttc cta caa aga agc agc aat 48 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn 1 5 10 ttt cag agt cag aag ctc ctg tgg caa ttg aat ggg agg ctt gaa tat 96 Phe Gln Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr 15 20 25 30 tgc ctc aag gac agg atg aac ttt gac atc cct gag gag att aag cag 144 Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln 35 40 45 ctg cag cag ttc cag aag gag gac gcc gca ttg acc atc tat gag atg 192 Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met 50 55 60 ctc cag aac atc ttt gct att ttc aga caa gat tca tct agc act ggc 240 Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly 65 70 75 tgg tgc gag act att gtt gag aac ctc ctg gct aat gtc tat cat cag 288 Trp Cys Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln 80 85 90 ata aac cat ctg aag aca gtc ctg gaa gaa aaa ctg gag aaa gaa gat 336 Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp 95 100 105 110 ttt acc agg gga aaa ctc atg agc agt ctg cac ctg aaa aga tat tat 384 Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr 115 120 125 ggg agg att ctg cat tac ctg aag gcc aag gag tac agt cac tgt gcc 432 Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala 130 135 140 tgg acc ata gtc aga gtg gaa atc cta agg aac ttt tac ttc att aac 480 Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn 145 150 155 aga ctt aca ggt tac ctc cga aac tga 507 Arg Leu Thr Gly Tyr Leu Arg Asn 160 165 <210> 8 <211> 166 <212> PRT <213> Artificial Sequence <400> 8 Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Cys 65 70 75 80 Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 Thr Gly Tyr Leu Arg Asn 165 <210> 9 <211> 504 <212> DNA <213> Artificial Sequence <220> <223> genetically modified human interferon-beta gene <220> <221> CDS (222) (7) .. (501) <400> 9 ctttcc agc tac aac ttg ctt gga ttc cta caa aga agc agc aat ttt 48 Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe 1 5 10 cag agt cag aag ctc ctg tgg caa ttg aat ggg agg ctt gaa tat tgc 96 Gln Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys 15 20 25 30 ctc aag gac agg atg aac ttt gac atc cct gag gag att aag cag ctg 144 Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu 35 40 45 cag cag ttc cag aag gag gac gcc gca ttg acc atc tat gag atg ctc 192 Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu 50 55 60 cag aac atc ttt gct att ttc aga caa gat tca tct agc act ggc tgg 240 Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp 65 70 75 tgc gag act att gtt gag aac ctc ctg gct aat gtc tat cat cag ata 288 Cys Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile 80 85 90 aac cat ctg aag aca gtc ctg gaa gaa aaa ctg gag aaa gaa gat ttt 336 Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe 95 100 105 110 acc agg gga aaa ctc atg agc agt ctg cac ctg aaa aga tat tat ggg 384 Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly 115 120 125 agg att ctg cat tac ctg aag gcc aag gag tac agt cac tgt gcc tgg 432 Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp 130 135 140 acc ata gtc aga gtg gaa atc cta agg aac ttt tac ttc att aac aga 480 Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg 145 150 155 ctt aca ggt tac ctc cga aac tga 504 Leu Thr Gly Tyr Leu Arg Asn 160 165 <210> 10 <211> 165 <212> PRT <213> Artificial Sequence <400> 10 Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln Ser 1 5 10 15 Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu Lys 20 25 30 Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln Gln 35 40 45 Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln Asn 50 55 60 Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Cys Glu 65 70 75 80 Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn His 85 90 95 Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr Arg 100 105 110 Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg Ile 115 120 125 Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr Ile 130 135 140 Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu Thr 145 150 155 160 Gly Tyr Leu Arg Asn 165
Claims (16)
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| Application Number | Priority Date | Filing Date | Title |
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| KR10-2001-0068852A KR100511749B1 (en) | 2001-11-06 | 2001-11-06 | Modified interferon-beta, and chemically modified conjugates thereof |
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| KR10-2001-0068852A KR100511749B1 (en) | 2001-11-06 | 2001-11-06 | Modified interferon-beta, and chemically modified conjugates thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2016013911A1 (en) * | 2014-07-24 | 2016-01-28 | 에이비온 주식회사 | Pegylated interferon-beta variant |
| WO2016140528A1 (en) * | 2015-03-03 | 2016-09-09 | 에이비온 주식회사 | Human interferon-beta variant conjugated immunocytokine and method for preparing same |
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| KR100480430B1 (en) * | 2001-12-04 | 2005-04-06 | 선바이오(주) | Conjugates of interferon-beta and polyethylene glycol derivatives |
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| US9249407B2 (en) | 2006-02-03 | 2016-02-02 | Opko Biologics Ltd. | Long-acting coagulation factors and methods of producing same |
| WO2007092252A2 (en) * | 2006-02-03 | 2007-08-16 | Modigene Inc | Long-acting polypeptides and methods of producing same |
| US9663778B2 (en) | 2009-07-09 | 2017-05-30 | OPKO Biologies Ltd. | Long-acting coagulation factors and methods of producing same |
| US20150158926A1 (en) | 2013-10-21 | 2015-06-11 | Opko Biologics, Ltd. | Long-acting polypeptides and methods of producing and administering same |
| RU2017108448A (en) * | 2014-10-24 | 2018-11-27 | Политерикс Лимитед | CONJUGATES AND CONJUGATING REAGENTS |
| ES2893616T3 (en) | 2015-06-19 | 2022-02-09 | Opko Biologics Ltd | Long-acting coagulation factors and methods for their production |
| SG10202100189WA (en) | 2016-07-11 | 2021-02-25 | Opko Biologics Ltd | Long-acting coagulation factor vii and methods of producing same |
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| WO2016140528A1 (en) * | 2015-03-03 | 2016-09-09 | 에이비온 주식회사 | Human interferon-beta variant conjugated immunocytokine and method for preparing same |
| US10806799B2 (en) | 2015-03-03 | 2020-10-20 | Genopharm Inc. | Human interferon-beta variant conjugated immunocytokine and method for preparing same |
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| KR20030037598A (en) | 2003-05-14 |
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