CN1200148A - 在细胞培养中增强干扰素生产的方法 - Google Patents
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Abstract
本发明描述了在动物细胞培养物中强化干扰素生产的方法。这些方法基于干扰素生产的某些诱导剂的细胞水平,尤其是双链RNA依赖性激酶(dsRNA-PKR,或PKR)的细胞水平的操作。在超量生产PKR的细胞培养中,诱导干扰素合成至高水平,并且无需传统的病毒诱导干扰素而可收获显著量的干扰素。
Description
本发明涉及在细胞培养中增强干扰素生产的方法。
根据其基本序列可将干扰素(IFN)分成两大类。I型干扰素,IFN-α和IFN-β,是由含IFN-α基因族和所组成的无内含子的基因超家族和单个IFN-β基因所编码的。II型干扰素,或IFN-γ,仅由单型构成并且只限于淋巴细胞(T细胞和自然杀伤细胞)。I型干扰素介导各种生物学过程,包括:诱导抗病毒活性,调节细胞生长和分化,并调节免疫功能(Sen,G.C.和Lengyel,P.(1992)生物化学杂志(J.Biol.Chem.),267,5017-5020;Pestka,S.和Langer,J.A.(1987)生物化学年鉴(Ann.Rev.Biochem.),56,727-777)。一般在病毒感染后测定包括IFN-α和IFN-β基因族的I型IFN的诱导表达。许多研究已鉴定和I型IFN表达调节相关的启动子元件和转录因子(Du,W.,Thanos,D.和Maniatis,T.(1993)细胞(Cell),74,887-898;Matsuyama,T.,Kimura,T.,Kitagawa,M.,Pfeffer,K.,Kawakami,T.,Watanabe,N.,Kundig,T.M.,Amakawa,R.,Kishihara,k.,Wakeham,A.,Potter,J.,Furlonger,C.L.,Narendran,A.,Suzuki,H.,Ohashi,P.S.,Paige,C.J.,Taniguchi,T.和Mak,T.W.(1993)细胞(Cell),75,83-97;Tanaka,N和Taniguchi,T.(1992)免疫学进展(Adv.Immurol.),52,263-81)。然而,还不清楚是什么生化信号表示病毒感染了细胞和有关的信号传输机理(一个最近的有关干扰素系统的综述,见Jaramillo等,癌症研究(Cancer Investigation),1995,13:327-337)。
IFN属于一类阴性生长因子,其具有抑制包括正常的和转化的表型的广谱的细胞种类的生长的能力。已发现IFN治疗对人类恶性肿瘤如:长波济肉瘤,慢性骨髓性白血病,非何杰金淋巴瘤和多毛细胞白血病以及传染性疾病如乳头瘤病毒(生殖道疣)及乙肝和丙肝是有效的(综述于Gutterman的美国国家科学院院刊(Proc.Natl.Acad.Sci.),91:1198-1205,1994)。最近已批准将由遗传工程细菌生产的IFN-β用于治疗多发性硬化,其是一种仅在美国就影响了至少25,000个病人的较常见神经病。
目前,治疗用途的IFN生产有两大来源,源自人白细胞或白细胞系的天然IFN和在细菌细胞中生产的重组IFN。由于天然IFN由IFN的整个补体所组成并且有合适的结构,所以认为它更优选,但其生产费用昂贵且费时。细菌生产的重组IFN则较便宜且能更有效率地生产,但研究表明细菌生产的蛋白有较高的排斥率,尤其是在长期使用后。例如,以前的医学研究表明由抗体生成所反映的排斥发生率在细菌生产的IFN中与天然的IFN-α的仅1.2%相比高达20-38%(Antonelli等,传染病杂志(J.Inf.Disease),163:882-885 1991;Quesad等,临床肿瘤学杂志(J.Clin.Oncology)3:1522-1528,1985)。因此,一个强化天然IFN生产以使之降低成本的方法是有用的。
IFN的生物学活性是通过结合于其能识别的受体并随后通过信号传导而导致诱导IFN-刺激基因(ISGs)而诱发。已鉴定出一些ISGs并检测了其生物学活性。研究的最充分的ISGs的例子包括双链RNA依赖性激酶(dsRNA-PKR,或者就是PKR,以前称为p68激酶),2'-5'-连接的寡腺等酸(2-5A)合成酶,和Mx蛋白(TaylorJL,Grossberg SE.病毒研究(Virus Research),1990 15:1-26.;Williams BRG.欧洲生物化学杂志(Eur.J.Biochem.),1991200:1-11.)。
唯一鉴定出具有激酶活性的dsRNA-结合蛋白是PKR(RNA依赖性蛋白激酶的缩写)。PKR是一种Ser/Thr激酶,其酶活性需要dsRNA结合和随后的自身磷酸化(Galabru,J.和Hovanessian,A.(1987)生物化学杂志(J.Biol.Chem.),262,15538-15544;Meurs,E.,Chony,K.,Galabru,J.,Thomas,N.S.,Kerr,I:M.,Williams,B.R.G.和Hovanessian,A-G.(1990)细胞,62,379-390)。PKR在字面上也指dsRNA-激活的蛋白激酶,P1/elF2激酶,DAI或dsI(dsRNA-激活的抵制剂),以及p68(人)或p65(鼠)激酶。已描述有类似的酶存在于兔网织红细胞,各种鼠组织,以及人外周血单核细胞中(Farrel等)(1977)细胞11,187-200;Levin等,(1987)美国国家科学院院刊,75,1121-1125;Hovanessian(1980)生物化学(Biochimie),62,775-778;Krust等(1982)病毒学(Virology)120,240-246;Butffet-Janvresse等(1986)干扰素研究杂志(J.Interferon Res.),6,85-96)。研究得最充分的PKR在体内的底物是真核起始因子-2的α-亚基(eIF-2a),其一旦磷酸化将最终导致抑制细胞和病毒蛋白合成(Hershey,J.W.B.(1991)生物化学年鉴,60,717-755)。已建议将PKR的这种特殊功能作为引起介导IFN-α和IFN-β的抗病毒和抗增质活性的一个机理。PKR的另一个生物学功能是推断的作为信号转导蛋白的作用。Kumar等人证实PKR可以磷酸化IκBα从而导致核因子κB(NF-κB)的释放和激活(Kumar,A,Haque,J.,Lacoste,J.,Hiscott,J,和Williams,B.R.G.(1994)美国国家科学院院刊91,6288-6292)。鉴于已完全鉴定的NF-κB位点在IFN-β启动子中,这可能代表一个PKR介导IFN-β转录的dsRNA活化的机理(Visvanathan,K.V.和Goodbourne,S.(1989)欧洲分子生物学学会杂志(EMBO J.)8,1129-1138)。
本发明人惊讶地发现某些ISG的操纵表达可便于其应用于干扰素生产。他们发现PKR蛋白的超表达诱导IFN-α和IFN-β干扰素的超量生产,从而用于强化动物细胞培养中的干扰素生产。
目前,有两种大量生产干扰素的方法:在细菌或哺乳动物细胞中生产重组IFN或于用病毒或其它IFN诱导剂刺激后从人白细胞中产生天然IFN。美国专利第5,376,567和第4,966,843号描述了在中国仓鼠卵巢细胞中生产重组人干扰素;美国专利第5,196,323号描述了在E.coli细胞中生产重组人IFN-α。许多专利描述了用各种方法从人白细胞中生产干扰素;例如,美国专利第4,745,053号描述了一个用病毒诱导剂从人全血中生产干扰素的方法,美国专利第4,680,261号描述了一个用抗坏血酸衍生物或无机钒化合物在哺乳动物细胞培养物中诱生干扰素的方法,而美国专利第4,548,900号描述了一个用多元醇在引发阶段诱导干扰素的方法。目前的干扰素生产方法的主要缺点是由于其它诱导剂不能产生足够高水平的用于商业目的的干扰素,所以一般用病毒作为IFN诱导剂。病毒必需要在使用前从该干扰素除去,从而增加了该生产方法的时间和成本。此外,使用病毒作为诱导剂最终导致干扰素生产细胞死亡,从而不可能循环和再利用该细胞。
本发明通过提高一个无需使用病毒诱导剂而能生产高水平的干扰素的干扰素生产系统。虽然病毒诱导剂可用于本发明的系统,其它无需在IFN使用前除去的诱导剂也能以商业可接受的水平产生IFN。
本发明的一个目的是提供一个无需加入病毒作为诱导剂而在动物细胞培养物中强化干扰素生产的方法。
一般地,本目的是通过提供一个动物细胞培养物,其表达干扰素基因的水平比正常表达的要有实质性提高而实现。这可以通过操作那些能在体内调节干扰素水平的因子的表达水平而影响,这些因子包括干扰素转录调节因子(例如IRF1),干扰素受体,及干扰素刺激基因产物(如PKR和2-5A合成酶)。
因此,增加的IFN生产和本发明的其它目的在这里明显是通过在动物细胞培养中进行干扰素生产而完成的,而这些细胞培养中的干扰素调节蛋白,尤其是双链RNA依赖性激酶(PKR)和2'-5'寡腺等酸合成酶(2-5A合成酶)的活性水平比正常水平有显著增加。
附图简述
图1.pCMV-PKR和pMT-PKR的示意图。
1(A)是示由源于人巨细胞病毒(CMV)立即早期的基因的启动子序列控制的正向人PKR cDNA的pCMV-PKR的图。
1(B)是示由可被镉诱导的金属硫蛋白II启动子控制的正向人PKR cDMA的pMT-PKR图。
图2在U937-PKR+细胞和对照U937-neo细胞中诱导干扰素。U937-PKR+细胞和U937-neo细胞(0.5×106/m)培养于补充有5%胎牛血清的RPMI 1640培养基。用或不用10nM PMA预处理(“引发”)20小时,随后用10μg/ml Poly r(I):Poly r(C)或用0.1 TCID50/细胞EMCV诱导20小时。诱导24小时后,收集培养物之清液并测定IFN-α。空白条表示U937-neo细胞,阴影条表示U937-PKR+细胞。实验组表示各种处理:1-未处理;2-单独用Poly r(I):Poly r(C)诱导;3-单独用EMCV诱导;单独用4-PMA引发;5-PMA引发和Poly r(I):Poly r(C)诱导;6-PMA引发和脑心肌炎病毒(EMCV)诱导。
本发明部分地源自本发明人的发现,即细胞培养物中干扰素生产水平可以通过控制某些通常在体内调节干扰素表达的蛋白质的表达或活性而调节。这些因子包括干扰素特异性转录因子(尤其是IRF1),干扰素受体,及某些干扰素刺激的基因的产物(也称为干扰素介导的抗病毒反应),尤其是PKR,任何这些因子的表达或活性增强将导致比正常水平更高的干扰素基因表达。而这可产生的结果之一是可以以更高的效率和更低的成本进行干扰素生产。本说明书的下文中将以PKR为例,但可以理解的是这里所述的其它干扰素增强因子可以代替PKR。
通过在动物细胞中增加PKR蛋白水平(并因此的PKR活性),可以增加干扰素生产。因此,那些表达出更高的组成水平的PKR或其中的PKR表达可被诱导到更高水平的动物细胞培养物可用于生产干扰素。本发明的方法解决了与细胞培养生产干扰素相关的常见问题,如收获率低和污染有用于诱导干扰素表达的病毒。因此,可以得到无需用病毒诱导的高收率的干扰素蛋白(虽然可用病毒)来以更高产量生产INF)。
该方法基于利用PKR-超量生产细胞作为干扰素源,但任何已知的或最近发现的干扰素生产方法均可使用。除了使用了常用的非病毒性的干扰素诱导剂外,无需特别的干扰素生产方法。特别地,本方法包括(a)在足以超量产生PKR的条件下培养能超量生产PKR或其类似物或同系物的动物细胞培养物,以及(b)适当地处理该细胞培养物以诱导干扰素,尤其是IFN-β和IFN-α基因的表达。一般地,在这些步骤后进行所生产的干扰素的纯化。在某些情况下,超量生产的并非PKR本身,而是PKR类似物,即意谓着能介导干扰素转录的dsRNA活化的非天然的蛋白激酶(通常通过编码一种特定的PKR的基因的遗传操作而获自选定的动物细胞系)。用于生产干扰素的细胞培养物可以超量生产源自任何用于生产干扰素的动物细胞素的PKR,如常见于兔网织红细胞,各种鼠组织,或人外周血单核细胞的PKR。通常,特定的细胞系的天然PKR(相对于外源性PKR)用于超量表达。鼠p65激酶和人p68激酶分别优选地和更优选地超量生产于相应的鼠或人细胞培养。
可以通过包括许多本领域周知的方法分离PKR基因能超量生产的动物细胞培养物。该方法包括选择表达较高水平PKR的细胞,用含一个PKR基因(或cDNA)的载体在一个组成型启动子(如CMV,RSV或SV40启动子)的控制下转染,或者用一个含PKR基因或cDMA在可诱导的启动子(如,热激基因,金属硫蛋白启动子或者糖皮质素启动子)的控制下转染。如前述进行转染并选择出超量生产PKR的转染子。PKR的超量生产意味着比PKR活性的正常水平更高。正常指在所用细胞系的正常条件下(见,例如,建议的培养条件如市售细胞的目录所提供,特别是获自美国典型培养物保藏中心,Rockville,Maryland,USA的ATCC品系目录,或者若非市售,则源自描述所用的细胞系科技出版物),而在各种优选的实施方案中是指在其它相同的条件下于亲本细胞中生产INF,由亲本细胞选择出生产细胞系或通过遗传工程产生出生产细胞系。比正常水平更高优选地意谓着正常PKR水平的至少150%,更优选地至少200或300%,最优选地至少500%。根据分离或制备该培养物的特定方法,PKR-超量表达细胞培养物可能是PKR超量生产所必需或可诱导PKR超量表达。优选地可诱发该PKR-超量表达细胞培养物超量生产PKR以便调节可诱导IFN的PKR水平。明显地,若可诱导PKR超量生产细胞培养物的超量表达,可在诱导条件下检测PKR的超量生产活性。PKR活性可通过任何本领域已知的方法或者下列实施例所述的方法测定。
任何已知的细胞培养物可作为制备PKR-超量生产细胞培养物的亲本株。任何能正常地生产干扰素的细胞都适于作为亲本细胞,尤其是衍生于成纤维细胞或包括B细胞和单核细胞的免疫细胞的细胞。特别合适的细胞培养物是前-单核U937细胞,Namalwa(淋巴母细胞B)细胞和MRC-5(人成纤维细胞)细胞。合适细胞还有:WI-38细胞,Flow1000细胞,Flow4000细胞,FS-4和FS-7细胞,MG-63细胞,CCRF-SB细胞和CCRF-CEM细胞。
在PKR-超量生产细胞培养物生产干扰素可通过本领域周知的方法进行。一般地,将该干扰素生产细胞在任何合适的培养基中培养,用诱导剂诱导干扰素的表达(以及用一个PKR基因诱导剂,在PKR中于可诱导的启动子控制下提供),并在诱导干扰素生产后培养。然后,分离该干扰素。该细胞可在诱导前加入启动剂而被启动。干扰素有多种且在本领域是周知的,而几乎任何已知的干扰素诱导剂均可用于本发明。典型的诱导剂包括Poly(I):Poly(C),仙台病毒,新城疫病毒,伴刀豆球蛋白A,衣原体,立克次氏体,促细胞分裂剂和脂多糖。优选地,该诱导剂为非病毒诱导剂。更优选地,该诱导剂为Poly(I):Poly(C)或Poly r(I):Poly r(C)。由于细胞不遭受暴露于病毒所致的有害损伤并因此可以循环再利用从而可以使生产成本较低,因而该诱导剂优选地为非病毒性的。典型的启动剂包括因豆蔻酸乙酸佛波醇酯(PMA),钙离子载体和干扰素。
用本领域周知的常用技术纯化IFN。这些技术包括抗体-亲和柱层析,离子交换层析,蛋白沉淀和离心(美国专利第5,391,713);用CM-琼脂糖凝胶,con A-琼脂糖凝胶,和苯基-琼脂糖凝胶(美国专利第4,658,017)的色谱,用玻璃吸着剂柱的三步色谱(美国专利第4,732,683);免疫亲和色谱,反相HPLC,阳离子交换柱,和凝胶过滤(美国专利第4,765,903号);盐酸胍作为溶剂和HIC柱色谱(美国专利第4,845,032)。
制剂中IFN-α水平可通过滴定L929(鼠成纤维细胞)抗“国家过敏性和传染性疾病研究所”的G-023-901-527标准品而测定其抗病毒活性(Lau等人,临床研究杂志,82:1415-1421 1988)。在该技术中,将5×104L929细胞接种到96孔微量滴定板的每一孔,然后,用两倍系列的培养物上清液的稀释液培养16小时。随后,用EMCV或仙台病毒(0.1或0.01TCID/细胞)攻击。通过显微镜检并通过用溶于5%乙醇液的0.1%结晶紫染细胞而测定病毒-诱导的致细胞病变效果。该IFN滴定度定义为能保护50%的细胞免于病毒-诱导的致细胞病变效果的培养物上清液的最高稀释度的倒数。
上述步骤的是体的例子如下列实施例如述。然而,对本领域的普通技术人员来说明显的是,可以进行许多修改,而除了特别指出外,实施例只是为了说明的目的而非对本发明进行限制而提供。
实施例实施例1:超量表达结构性PKR的细胞培养制备
通过将源于pBS-8.6R的PKR cDNA插入到pRC/CMV载体的Hind III位点而从pRC/CMV中制备质粒pCMV-PKR以便使编码PKR的序列的表达在CMV启动子的控制之下。该pRC/CMV质粒(Invitrogen)一般用于真核表达。该载体提供了如下的适于PKR转录的特征:i)源于人CMV(巨细胞病毒)立即早期基因的用于高水平转录的启动子序列;ii)源于牛生长激素(BGH)基因的多腺苷酸信号和转录终止序列以增强RMA稳定性;iii)游离型复制和简单载体拯救的SV40起点;iv)位于多重克隆位点侧翼的T7和Sp6RNA启动子用于在体外转录有义和反义RNA;以及V)用于筛选和E.coli中维持的氨苄青霉素抗性基因(AMP)和CoIE1起点。该载体还包括一个G418抗性标记(NEO)以便于在转染到真核细胞后筛选和鉴定该质粒。该pCMV-PKR结构如图1A所示。
在一台置于500μF,250V的基因脉冲仪(BioRad)上,用10mg的每种质粒电穿孔5×106处于指数生长期的生长可含DEAE-葡聚糖(50mg/ml)的无血清RPMT-1640的U937细胞而收致细胞病变稳定的转染体。用400ug/ml遗传霉素(GIBCO-BRL)筛选3周得到稳定的转染体集群。随后,通过有限稀释克隆得到克隆系。在含10%胎牛血清(完全培养基)和遗传霉素的RPMI-1640中培养细胞系。筛选出一个代表性转染细胞系并称为“U937-PKR+”。检测U937-PKR+中生产的PKR水平并发现比正常(亲本)水平增加了大约五倍。作为对照,用pRC/CMV转染U937细胞,并分离出一个代表性的转染细胞系,称为“U937-neo”。在有或无启动剂和/或诱导剂存在下测定该转染细胞系的.IFN生产。
在补充有胎牛血清的RPMI 1640培养基中培养U937-PKR+细胞和U937-neo细胞。用有或无豆蔻酰佛波醇乙酯(PMA)(10nM)预处理该细胞20小时并随后用或不用Poly r(I):r(C)(10ug/ml)刺激20小时。用Poly r(I):r(C)刺激24小时后,收集培养上清液用于IFN检测。
如图2所示,两种转染细胞系在无启动剂或诱导剂存在下均不能自发地产生IFN。响应于Polyr(I):Polyr(C),对照U937-neo细胞在有或无PMA启动(空白柱2和5)下均表现出最低的IFN-α合成。相反,U937-PKR+细胞在有或无PMA启动下响应于Polyr(I):Polyr(C)诱导而表现出增加的IFN生产,并且当用Polyr(I):Polyr(C)和PMA(阴影柱2和5)同时处理时可达到4000U/ml的水平,在无PMA启动时,EMCV在两个转染细胞系(超过1000U/ml)均诱导出高水平IFN,而在U937-PKR+细胞(柱3)中则只诱导出稍高水平的,在无EMCV或Polyr(I):Polyr(C)诱导剂时,U937-PKR+细胞响应于只有PMA启动(柱4)而产出比U937-neo细胞的更高的IFN水平。用PMA启动和EMCV诱导,两个细胞系均产生出超过4000U/ml IFN(柱6)。总之,这些结果表明用PMA启动后,以Polyr(I):Polyr(C)作为诱导剂时在IFN生产方面该U937-PKR+细胞在同对照细胞有以病毒作为诱导剂时一样有效。因此,当使用PKR-超量表达细胞时,可以避免使用活病毒进行IFN生产。
用Polyr(I):Polyr(C)诱导后,用台盼蓝排斥实验检测U937-PKR+细胞生存力。我们发现有超过95%的细胞存活。相反,使用EMCV作为诱导剂导致了全部细胞的死亡。因此,U937-PKR+细胞可以循环利用来进行连续的IFN生产。实施例2:可诱导超量表达PKR的细胞培养的制备
用镉-可诱导性金属硫蛋白II启动子代替pCMV-PKR中PKR克隆位点上游的CMV启动子而制备质粒pMT-PKR(Hewison等,免疫学杂志(J.Immunol.),153:5709-5719 1994)。pMT-PKR的结构示于图1B。如实施例1中对pCMV-PKR转染子所述分离pMT-PKR对U937细胞的稳定的转染体。用锌或镉离子可诱导MT II启动子。在加入20μM氯化镉诱导该转染的PKR基因后,测定该转染体中PKR活性水平。
本说明书中涉及的所有出版物和专利申请均以每个出版物或专利申请单独引入的相同的程度而以参考文献的形式并入本发明。
现已充分描述了本发明,很明显地,对本领域的普通技术人员来说,许多改变和修改均可在未脱离所述的发明的精神和范围的条件下进行。
Claims (16)
1.一种在动物细胞培养物中生产干扰素的方法,包括:
(a)在足以超量生产PKR的条件下培养动物细胞培养物,并且
(b)处理该培养物以诱导干扰素生产。
2.一种在动物细胞培养物中生产干扰素的方法,包括:
(a)在足以超量生产PKR类似物的条件下培养动物细胞培养物,并且
(b)处理该培养物以诱导干扰素生产。
3.权利要求2的方法,其中所述类似物可获自鼠组织。
4.权利要求3的方法,其中所述类似物是鼠p65激酶。
5.权利要求2的方法,其中所述类似物可获自兔网织红细胞。
6.权利要求2的方法,其中所述类似物可获自人外周血单核细胞。
7.权利要求1的方法,其中所述PKR是人p68激酶。
8.权利要求1的方法,其中所述干扰素生产用一种非病毒性诱导剂诱导。
9.权利要求8的方法,其中所述非病毒性诱导剂是Poly(I):Poly(C)或Polyr(I):Polyr(C)。
10.权利要求1的方法,其中所述步骤(a)进一步包括用启动剂处理。
11.权利要求1的方法,其进一步包括所生产的干扰素的纯化步骤。
12.权利要求1的方法,其中所述动物细胞培养物源于能正常地生产干扰素的亲本细胞。
13.权利要求12的方法,其中所述PKR超量生产是通过用一个包括在启动子控制下的编码PKR的DNA的载体转染该亲本细胞而实现。
14.权利要求13的方法,其中的启动子是诱导型启动子。
15.权利要求14的方法,其中的诱导启动子是金属硫蛋白启动子。
16.权利要求12的方法,其中的亲本细胞是U937细胞。
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- 1996-08-21 US US08/700,198 patent/US5840565A/en not_active Expired - Lifetime
- 1996-08-22 AT AT96929066T patent/ATE289348T1/de active
- 1996-08-22 WO PCT/US1996/013745 patent/WO1997008292A1/en active IP Right Grant
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- 1996-08-22 AU AU68576/96A patent/AU6857696A/en not_active Abandoned
- 1996-08-22 WO PCT/US1996/013627 patent/WO1997008324A1/en not_active Application Discontinuation
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MX9801423A (es) | 1998-05-31 |
US20010001290A1 (en) | 2001-05-17 |
JP3432519B2 (ja) | 2003-08-04 |
CA2229405A1 (en) | 1997-03-06 |
US20040157310A1 (en) | 2004-08-12 |
AU707130B2 (en) | 1999-07-01 |
CA2229163A1 (en) | 1997-03-06 |
US7132271B2 (en) | 2006-11-07 |
US5840565A (en) | 1998-11-24 |
EP0846160A4 (en) | 2002-01-02 |
CN1161469C (zh) | 2004-08-11 |
CA2229163C (en) | 2011-04-05 |
EP0846160A1 (en) | 1998-06-10 |
US6673591B2 (en) | 2004-01-06 |
EP0846174A1 (en) | 1998-06-10 |
EP0846160B1 (en) | 2005-02-16 |
US6686190B2 (en) | 2004-02-03 |
US20010001709A1 (en) | 2001-05-24 |
ATE289348T1 (de) | 2005-03-15 |
AU6860896A (en) | 1997-03-19 |
BR9610550A (pt) | 1999-07-06 |
JP4267693B2 (ja) | 2009-05-27 |
DE69634360T2 (de) | 2005-12-29 |
US6489144B1 (en) | 2002-12-03 |
EP0846174A4 (en) | 2003-02-05 |
CA2229405C (en) | 2004-03-02 |
JPH11511324A (ja) | 1999-10-05 |
WO1997008324A1 (en) | 1997-03-06 |
US6159712A (en) | 2000-12-12 |
JPH11514344A (ja) | 1999-12-07 |
DE69634360D1 (de) | 2005-03-24 |
WO1997008292A1 (en) | 1997-03-06 |
AU6857696A (en) | 1997-03-19 |
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