CN1273588A - 用于治疗和预防多毒素梭状芽胞杆菌毒素导致的疾病的氨基酸序列 - Google Patents
用于治疗和预防多毒素梭状芽胞杆菌毒素导致的疾病的氨基酸序列 Download PDFInfo
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- CN1273588A CN1273588A CN98808996A CN98808996A CN1273588A CN 1273588 A CN1273588 A CN 1273588A CN 98808996 A CN98808996 A CN 98808996A CN 98808996 A CN98808996 A CN 98808996A CN 1273588 A CN1273588 A CN 1273588A
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Abstract
讲述识别多毒素梭状芽胞杆菌(Clostridium difficile)内毒素(A毒素)或细胞毒素(B毒素)配体片段的抗原决定区、移位片段和催化片段并且可中和这些毒素的单克隆抗体。以及单克隆抗体的生产和在治疗、预防相应毒素导致的疾病方面的应用。
Description
治疗和预防多毒素梭状芽胞杆菌(Clostridium difficile)毒素引起的疾病的氨基酸序列
本发明的内容是从产生抗体的细胞、特别是产生单克隆抗体的杂交瘤细胞获得可中和多毒素梭状芽胞杆菌产生的内毒素和/或细胞毒素的氨基酸序列(多肽)。此外,对人体化的针对多毒素梭状芽胞杆菌毒素的单克隆抗体以及单克隆抗体的多变区作了描述。最后,本文也讲了获得这些氨基酸序列(多肽)的途径以及单克隆抗体的具体应用。
众所周知,大环内酯抗菌素如克林达霉素(Clindamycin)可引起严重的肠道疾病,表现为腹泻,严重时甚至有致死的假膜性结肠炎(PMC)出现。起先,这些疾病被称为“克林达霉素相关的腹泻”,现今,人们知道几乎所有在临床上采用的抗菌素和细胞抑制剂都可以导致PMC的临床表现。
临床上,严重腹泻是PMC的突出症状,由于体液和电介质的严重损失而导致死亡。随轻重不同,可出现腹部疼痛、血性腹泻、高热和白细胞增多。目前的治疗是停用抗菌素并给予万古霉素以及平衡液体和电解质。
很长时间,人们都没有弄清楚假膜性结肠炎的病因。直到1977年,证实大便中存在有毒性作用,这种毒性作用对CHO-细胞(中国地鼠卵巢肿瘤细胞)有细胞毒作用。进一步的研究证实,假膜性结肠炎的原因是多毒素梭状芽胞杆菌及其毒素。
多毒素梭状芽胞杆菌是专性厌氧、革兰氏染色阳性杆菌。它可形成近末端卵圆芽胞。其生化特点是可酵解单糖如葡萄糖、N-乙酰氨基葡萄糖和N-乙酰神经氨酸,但不能酵解甘露糖、木糖和阿拉伯糖。由于缺乏神经氨酸酶、β-半乳糖甙酶或涎酸酶,多毒素梭状芽胞杆菌不能将这些单糖从胃肠粘蛋白的侧链上裂解开来。在正常人体中,由于其生化特性,梭状芽胞杆菌在肠道不能构成危害。采用抗菌素或细胞抑制剂时,肠道正常菌丛因多毒素梭状芽胞杆菌而失调,后果就是PMC。
多毒素梭状芽胞杆菌产生两种致病因子:内毒素(A毒素)和细胞毒素(B毒素)。这两种毒素的获得、提纯、特性以及它们在制造单克隆抗体方面的应用,在欧洲专利说明书153 519和209 273、美国专利说明书4 879 218和5 098 826以及国际专利登记WO91/18293中都有详细说明。
显然,抗体在防止多毒素梭状芽胞杆菌感染的后果中起着很重要的作用。在PMC患者身上,可以查到针对A毒素和B毒素抗体。给地鼠抗菌素以后,再将其感染可产生毒素的多毒素梭状芽胞杆菌,可以在它们身上观察到典型的PMC,并且地鼠因此而死亡。如果事先用毒素对地鼠进行免疫,则它们不会患病。而毒素可以用抗体进行中和,这些抗体针对C-末端重复配体片段、中央移位片段或针对N-末端催化片段而发挥效用(片段结构请参见文献〔1〕)。第一种情况防止了毒素和细胞受体的结合,后种遏制了毒素介导的糖酵解反应,而针对中央移位片段的抗体阻止毒素向细胞内侵入。
这样,中和A毒素和/或B毒素的抗体提供了一个治疗和/或预防多毒素梭状杆菌引起的疾病的可能性,其作用方式不是灭菌,而是遏制毒素的作用。相应疾病的治疗和预防是对因的。
以A毒素为例,DSM ACC 2322细胞系可产生TTC8抗体,此抗体不仅和A毒素结合,而且也可中和其生物作用。TTC8抗体和A毒素结合的位置已经确定是在重复配体片段内。抗体和毒素的结合防止了后者和细胞受体的结合。单克隆抗体TTC8的结合位置是在A毒素的2480-2539氨基酸之间,其抗原序列(很可能)是TINGKYYF。美国US4879218号专利说明中的单克隆抗体PCG-4是和A毒素的2098-2141氨基酸之间的蛋白片段结合。
DSM ACC 2321细胞系产生的单克隆抗体2CV同样是和B毒素的配体片段向结合。位置是在B毒素的2233-2366氨基酸之间的蛋白片段。单克隆抗体PCG-4和单克隆抗体TCC8都是小鼠抗体,它们虽不适于人体治疗,但很适合作为诊断的辅助工具。
下面是我们实验室生产的单克隆抗体及其特性:
针对目标 毒素a) 毒素b)
催化片段b) 1212 2612,2688,3110,
1502,1115
移位片段b) 2825,2836,5288 2703,2740,
2747,2754,
5288,2784,
2788
配体片段b) 2620,TTC8 2912,2914,
2916,2926,
2562 2CV
注释:a)抗体在免疫电泳识别列举区域的重组蛋白。
b)A毒素和(B)毒素的片段氨基酸位置:催化片段:1-879(1-877);移位片段:880-1848(878-1850);配体片段:1849-2681(1851-2360)。
所有上述讲到的毒素特异单克隆抗体是从小鼠获得的,除小鼠以外,在别的种属(人或动物)这些抗体不能用于治疗和预防疾病。小鼠外的种属会识别这些抗体,因而产生免疫反应,造成抗体失活。因为目前不能确定这些抗体的变区和多变区,所以不能做到通过改良而使其适用于其他种属,特别是人体。
本发明的目的是获得适用于人体和其他动物的多肽,这些多肽有中和毒素的特性,但没有过强的免疫成分。多肽中和毒素作用的基础是抗体变区和相应毒素的结合。结合主要是由多变区决定(CDR:complementarity determing region)。所以,关键在于鉴别和改良和毒素结合并中和毒素作用的区域(变区和多变区(CDR)),改良的目的在于避免免疫反应。这些多肽既可用于治疗也可用于预防多毒素梭状芽胞杆菌引起的疾病,既适于人体也适于动物。
就目前的科研结果来看,多毒素梭状杆菌的A毒素可导致典型的假膜性结肠炎(PMC),而针对A毒素的单克隆抗体TTC8在活体(小鼠)可以中和其毒性作用。单克隆抗体TTC8属于IgG2b,其和A毒素特异性识别是由重链和轻链的多变区介导的。也就是说,抗体的(或者多肽的)特定片段负责识别抗原。鉴别多变区可使我们将多肽结构整合到其他种属的抗体序列上,从而使这些抗体适合于治疗和预防多毒素梭状芽胞杆菌引起的疾病,并且消除了从小鼠身上获得的抗体会引起的副作用。
因此,问题在于查明核甙酸及其相应的氨基酸序列。以部分多肽的形式或适于人体的抗体形式(整合到人类抗体基因内)用来治疗及预防人体疾病。如前面提到的对产生TTC8抗体的DSM2322细胞系的有关序列确定。TTC8抗体通过遏制A毒素的配体片段而中和其生物活性。
在DSM ACC 2322细胞系,多变区和变区多肽片段是SEQ ID No 1-12和/及SEQ ID No 13-16。这些片段在TTC8单克隆抗体起对毒素的中和作用。确定起中和作用抗体的变区可使生产相应的多肽成为可能。通过和多毒素梭状芽胞杆菌的内毒素和/或细胞毒素结合,这些多肽可消除毒素的生物效应。如SEQ ID No 13和/或SEQ ID No 16序列可单独或者整合到免疫球蛋白内用来治疗及预防多毒素梭状芽胞杆菌引起的疾病。要用于人体,则整合到人体球蛋白;用于动物,则整合到动物球蛋白。
这些多肽的衍生物可通过氨基酸的切断、插入、增加或交换,也就是说通过对位基因变换而获得,这些衍生物可和本来的多肽一样通过和多毒素梭状芽胞杆菌的内毒素和/或细胞毒素相结合而消除其生物活性,因而同样可用来治疗及预防多毒素梭状芽胞杆菌引起的疾病。
从德国微生物及细胞株收集有限公司(DSMZ)的DSM ACC 2322细胞系分离的TTC8抗体多变区(CDRs=complenentarity deternimingregions)已被确定。其氨基酸序列如下:
重链的CDR:
CDR-1:-Asn-Tyr-Trp-Met-Asn-
(SEQ ID No 2)
CDR-2:-Arg-Ile-Tyr-Pro-Gly-Asp-Gly-Asp-Ala-His-Tyr-Asn-Gly-Lys-Phe-Lys-Gly-
(SEQ ID No 4)
CDR-3:-Gly-Gly-Asn-Tyr-Asp-Asn-Arg-Val-Phe-Asp-Tyr-
(SEQ ID No 6)
轻链的CDR:
CDR-4:-Lys-Ala-Ser-Gln-Asn-Val-Gly-Thr-Asn-Val-Ala-
(SEQ ID No 8)
CDR-5:-Ser-Pro-Ser-Tyr-Arg-Tyr-Ser-
(SEQ ID No 10)
CDR-6:-Gln-Gln-Tyr-Asn-Ser-Tyr-Pro-Leu-Thr-
(SEQ ID No 12)
这些序列是这样确定的:第一步是先将产生TTC8抗体的杂交瘤细胞的整个RNA准备好,第二步是将mRNA从整个的RNA分离开来。采用的是和小(dt)25链结合的聚苯乙烯珠(Beads)。
从净化的mRNA合成CDNA,然后通过多聚酶链式反应(PCR)得到单克隆抗体TTC8的VH基因和VL基因,它们编码TTC8抗体的轻链(VL)和重链(VH)。这里,要注意的是选择有合适限制切点的多聚体,以便于克隆化的进行。
对单克隆抗体TTC8的VH基因和VL基因在puc 19进行克隆化,然后排序。由此找到了核甙酸(SEQ ID No 13和15)和相应的氨基酸序列SEQ ID No 14和SEQ ID No 16。
通过和胚层基因V102的比较,可鉴定出多变区,从重链基因(SEQID No 13)看出,重链的CDR-1包括5个氨基酸,从序列的30位开始。重链的CDR-2从49位开始,包括17个氨基酸。多变区的CDR-3从98位开始,包括11个氨基酸。
和胚层基因相比,单克隆抗体TTC8的VH-基因有36个等位基因变换。三个变换位于PCR-多聚体的结合区,可能是多聚体的序列所至。几乎所有位于多变区的变换都导致氨基酸序列的变化,而大约位于框架区变换的一半不起作用。
轻链基因(SEQ ID No 15)的多变区确定如下:CDR-4从22位开始,包括11个氨基酸;CDR-5从48位开始,包括7个氨基酸;CDR-6从87位开始,包括9个氨基酸。和单克隆抗体A23相比,单克隆抗体TTC8的VL-基因只有9个变换,其中4个位于多聚体的结合区。其他5个在核甙酸序列中的变换有2个变换在蛋白质水平不起作用。导致氨基酸序列变化的变换有一个在CDR-4,另外两个在2和3结构区。
用同样的方法,可以确定单克隆抗体2CV多变区的序列,2CV抗体由DSMZ提供的DSM ACC 2321号杂交瘤细胞系产生,此抗体可以识别多毒素梭状芽胞杆菌的细胞毒素(B毒素)。
从小鼠获得的单克隆抗体用于人体治疗会导致免疫反应。为解决这个问题,可以将抗体加以改良以适于待治疗的种属(这里指人体),也就是说,可以将抗体人体化。用相应的人体抗体成分来取代小鼠单克隆抗体中的免疫成分的方法很多,比如欧洲专利说明书中众所周知的184187、171 496和173 494。
用这些方法获得的人体化的单克隆抗体比从小鼠获得的抗体更适于人体治疗。
这些将抗体人体化的方法同样也可用来改良TTC8单克隆抗体以及其他的中和A毒素和B毒素的单克隆抗体,使其成为人体化的单克隆抗体。这种人体化的单克隆抗体是由两种成分构成的杂合子:有中和作用的单克隆抗体TTC8的多变区(如TTC8的SEQ ID No 1-12)插入到人体免疫球蛋白的构造区。免疫球蛋白可以是IgG亚型(主要用于胃肠外)也可以是IgA亚型(主要用于口服)。和来源于DSM ACC 2322细胞系的TTC8抗体一样,其他针对多毒素梭状芽胞杆菌的单克隆抗体的多变区同样可以克隆化、编序而用于生产人体化的抗体。
另外一个发明是,通过等位变换改变人类免疫球蛋白的变区和/或常区内轻链和/或重链的氨基酸序列,其和多毒素梭状芽胞杆菌的A毒素和/或B毒素相结合的能力依然存在。
生产所发明的单克隆抗体的生物“构件”,除特别讲明的以外,都有商品出售,如细胞系、质体、启动基因和复制源等。如果没有特别强调,这些生物“构件”对本发明没有决定性的影响,因为可以用其他的合适成分来替代它们。病毒宿主细胞用来加强所发明的DNA序列,如埃希氏大肠杆菌或杆菌属。
人体化抗体的生产可用真核生物细胞如酵母细胞、真菌或CHO(中国地鼠卵巢细胞)。如采用植物,则可用单子叶或双子叶植物。
下面举例说明用植物生产抗体的情况,其他类似。
用植物生产抗体的例子见〔2〕和国际专利说明书WO91/06320。用异基因植物生产单克隆抗体时,通过控制植物的一个或两个启动基因,在植物转换介质内将单克隆抗体的基因或其重组衍生物基因或单链抗体基因克隆化。
最终的转换介质中存在所有的待转换到植物中的DNA序列,这些序列从介质中转换到植物中去。另外一个途径是,将重链和轻链的基因分开整合到植物转换介质中,分别转换到植物细胞中去,然后再组合成完整抗体。植物转换可用多种合适的途径进行,如借助于植物肥大病菌属、直接基因转换或粒子枪。
抗体的生产可有针对性的在细胞的不同隔室内进行。分离对信息肽编码的序列时,从细胞浆提取抗体。为提高抗体的表现力,在基因的5’-末端联结一段DNA-序列或保存已有的序列,此序列编码输送到内质网的信息多肽。为确定细胞室,可将对多肽序列编码的DNA序列融合到基因中,如通过KDEL-序列实现在内质网的滞留。
抗体基因整合到多基因植物可通过植物染色体组DNA的分离和接着的诺登杂交进行分析和证实。基因的转录可用诺登电泳证实。生物合成的抗体可借助于特异次极抗体、针对常区多克隆或单克隆抗体、针对添加的序列(所谓Tag-序列)的单克隆或多克隆抗体在植物提取液中得到证明。
适合用的植物既有单子叶植物如大麦、小麦,也有双子叶植物如土豆、芝麻、胡萝卜和豌豆。抗体的提取可在植物的不同器官如叶子、种子、块茎等。
从植物提取的抗体可通过色谱方法而纯化,而色谱法一般情况下用于纯化从杂交瘤获取的抗体。此外,含有Tag-序列的重组抗体可用专门的亲合色谱方法如金属螯合色谱进行纯化。
所发明的人体化的单克隆抗体可经常规途径给予病人,治疗或预防多毒素芽胞杆菌导致的疾病。一般情况下,所发明的抗体经胃肠外给药,但最好是口服。可以考虑将含有抗体的植物作为生的素食品直接给病人吃。至于剂量问题,要按病人的情况如体重、年龄和病情的不同而不同。剂量要由有经验的医生确定,正常情况下是0.1mg/kg到70mg/kg,每天一次或多次给药,连给数天。
例子
例1:产生TTC8单克隆抗体的杂交瘤细胞DSM ACC 2322全部RNA的获取
用CsCl-梯度纯化RNA时用胍硫氰酸盐缓冲液打开细胞,然后机械打碎,细胞碎片离心分离,然后将溶液浇到CsCl-垫上(5.7M)。经过离心,RNA成球形沉积到试管的底部。用加热过的手术刀将试管的底部切开后,用H2O溶解球状的RNA。通过沉积对溶液作一步地浓缩,最后使溶液的总量在100μl H2O,RNA的浓度在1.5-3μg/μl之间。
例2:mRNA的分离
按Dynal的方法用小(DT)25珠将mRNA杂交而从其他的RNA分离。珠子的结合力是每mg结合2μg RNA,mRNA占全部RNA量的1-5%,所以1mg的小珠表面结合有80μg的RNA,纯化按照Dynal公司的办法,但略有不同:结合的mRNA先用缓冲液清洗两次,缓冲液的成分是:10mMTris/HCl(pH值7.5)、0.15mM LiCL、1mM EDTA、1%SDS。然后再用盐成分较低的缓冲液洗两次〔5mM Tris/HCL(pH值7.5)、75mM LiCl、0.5mM EDTA〕。其他步骤和Dynal公司一样,添加SDS并用低盐缓冲液加洗一次可提高mRNA的纯度。
例3:cDNA的合成
采用MMLV-反转录酶(Moloney Murine Leukemia Virus)从纯化的mRNA合成cDNA,为提高杂交mRNA-小(dT)分子的含量,小(dT)12-18聚合物的量比需要的高出两倍,反应时三磷酸吡啶核甙酸的浓度为1mM,为防止溶液中RNA的分解,添加了人体胎盘核糖核酸酶抑制剂。所获的ssDNA用于增加单克隆抗体TTC8的多变区的PCR-反应。
例4:为增加cDNA的多聚酶链式反应
cDNA的增加按照欧洲专利说明书0 388 914中的方法进行。根据一系列的文献设计了用于增加小鼠单克隆抗体多变区的聚合物,如下:
重链:(画线部分表示添加的SalI切点)
VH5Prim:5’-AG
GTCGACCTGCAG(C/G)AGTC(A/T)GG-3’
VH3Prim:5’-ACGGTGACA
GTCGACCCTTGGCCCC-3’
轻链:(画线部分表示添加的SacI切点)
VL5Prim:5’-GACATT
GAGCTCACCCAGTCTCCA-3’
VL3Prim:5’-GTTT
GAGCTCCAGCTTGGTCCC-3’
获取多变区的聚合物最佳浓度是0.5μM,dNTP的最终浓度是400μM。
第二个重要的参数是PCR-温度,VH的反应顺序为:变性1分钟,92℃;恢复1.5分钟,50℃;伸长3分钟,72℃,如此循环30次。VL的反应顺序是:变性1分钟92℃;恢复1.5分钟60℃;伸长3分钟,72℃,如此循环30次。
例5:单克隆抗体TTC8的VH-基因和VL-基因的克隆化
PCR-产物用SalI(VH)或SacI(VL)切开并用琼脂净化。克隆化在pUC19内均匀切点进行。目的克隆是通过控制消化确定的pVHT8和pVLT8.10构造物,它们有VH或VL-序列,这些序列和pUC19的启动基因成正序。
例6:单克隆抗体TTC8多变区的编序
插入物pVHT8的大小是341bp,pVLT8.10克隆的大小是312bp。两种克隆都具有在整个插入物延伸的阅读系统。PCR-聚合物的序列和切点很容易被鉴别。
通过序列的比较可将VH-片段归到J558多基因族,它和胚层序列V102有极大程度的相似性。
单克隆抗体TTC8的VL-区基因和来源于胚层基因vk19d的抗体有94%的一致性,所以TTC8的VL-区也应归结到这一基因族。
例7:人体化的单克隆抗体的生产
为避免用来源于其他种族(下面成为非人类抗体)的抗体时在人体导致免疫反应,必须将抗体的免疫区用人体的相应区域来替代。为此,有两种途径:
1)小鼠/人体杂合抗体,其中决定抗原性的变区是非人类抗体,而常区则来源于人类抗体。
2)单克隆抗体的完全人体化。单克隆抗体变区的构件区域由相应的人体序列来替代,只是CDR-区域保留或通过点变换的形式保留。
有大量的文献对此有报道,而且也为本领域的专业人员所熟知。
说明1)杂合抗体是在适当的介质中,将人体抗体的常区和非人类抗体的变区相结合。可以生产一个完整的杂合抗体,也可生产杂合抗体的一部分,称之为Fab-残片〔3〕。生产杂合抗体的优点是方法较为简便,由于小鼠抗体被部分保留的缘故,其有较高的抗原性,但在经口服时不会成为问题。
说明2)非人体单克隆抗体的完全人体化时,首先通过序列比较和3维模型找到一个人体抗体,此人体抗体的结构和非人体单克隆抗体的结构相似。然后,通过应用五个和人体抗原(VL或VH)的变区结合的小核甙酸聚合物以及三个含有小鼠抗体的CDR-区域序列的核甙酸,借助于PCR-反应生产完全人体化的抗体变区的基因〔4〕。另外一个改良过的方法是,将数个有部分重复的对目的序列编码的小核甙酸合成基因〔方法参见4〕。两种抗体的构造区的氨基酸可通过有针对性的基因变换而一致化〔5〕。这样改良的抗体有其种族特异性的变化,而此变化可以通过反变换而得到平衡〔5〕。最后一种生产完全人体化抗体的方法比较复杂,但在人体几乎没有免疫反应〔6〕。
参考文献
〔1〕Eichel-Streiber,C.V.,P.Boquet,M.Sauerborn undM.Thelestam.1996.Large clostridial cytotoxins-a family ofglycosyltransferases modifying small GTP-binding proteins.Trends in Microbiology,14:375-382.
〔2〕Düring.K.(1988)″Wundinduzierte Expression undSekretion von von T4 Lysozym und Monoklonalen Antikoerpern inNicotiana tabacum.″Ph.D.Thesis,Universitaet Koeln
〔3〕Skerra A.(1994).A general vector,pASK 84,forcloning,bacterial production and single step purification ofantibody Fab fragments.Gene 141:79-84.
〔4〕Sato K.,M.Tsuchiya,J.Saldanha,Y.Koishihara,Y.Ohsugi,T.Kishimoto und M.M.Bendig(1994).Humanization ofa mouse anti-human Interleukin-6 receptor antibody comparingtwo methods for selecting human framework regions.Mol.Immun.31:371-381.
〔5〕Benhar I.,E.A.Padlan,S.H.Lee,B.Lee und I.Pastan(1994).Rapid humanization of the Fv of monoclonal antibody B3by using framework exchange of the recombinant immunotoxinB3(Fv)-PE38.Proc.Natl.Acad.Sci.USA 91:12051-12055.
〔6〕Stephens S.,S.Emtage,O.Vetterlein,L.Chaplin,C.Bebbington,A.Nesbitt.M.Sopwith,D.Athwal,C.Nowak undM.Bodmer(1995).Comprehensive pharmacokinetics of ahumanized antibody and analysis of residual anti-idiotypicresponses.Immunology 85:668-674.
序列记录(1)一般说明:
(i)申请人:
(A)姓名:克里斯托夫·冯·埃歇尔—史泰伯(Christoph vonEichel-Streiber)
(B)街道:宾根路15号
(C)城市:施韦珀豪森
(D)联邦州:莱菌兰—法耳茨
(E)国家:联邦德国
(F)邮政编码:55444
(G)电话号码:06724/3398
(H)传真号码:06724/941078
(ii)发明名称:治疗和预防多毒素梭状芽胞杆菌毒素导致的疾病的氨基酸序列
(iii)序列数量:16
(iv)计算机阅读版本:
(A)数据载体:软盘
(B)机型:IBM个人电脑兼容机
(C)操作系统:PC-DOS/MS-DOS
(D)软件:PatenIn Release #1.0,Version #1.30(EPA)
(vi)原始申请资料:
(A)申报号码:DE 19739685.2
(B)申报日期:1997年9月10日(2)SEQ ID No 1说明:
(i)序列标志:
(A)长度:15个碱基对
(B)种类:核甙酸
(C)链型:单链
(D)拓扑学特征:不明
(ii)分子种类:cDNA
(iii)假定的:不
(iv)ANTISENSE:不
(v)残片种类:重链CDR1
(vi)原始来源:D
(B)株:单克隆抗体TTC8
(H)细胞系:杂交瘤
(vii)直接来源:
(A)库:DSM ACC 2322
(ix)特征:
(A)名称/编号:CDS
(B)位置:1……15
(xi)序列描述:SEQ ID No 1:AAC TAC TGG ATG AACAsn Tyr Trp Met Asn1 5(2)SEQ ID No 2说明:
(i)序列特征:
(A)长度:5个氨基酸
(B)种类:氨基酸
(D)拓扑学:线型
(ii)分子的种类:重链CDR1蛋白序列
(xi)序列描述:SEQ ID No 2:Asn Tyr Trp Met Asn1 5(2)SEQ ID NO 3说明:
(i)序列特征:
(A)长度:51个碱基对。
(B)种类:核甙酸
(C)链型:单链
(D)拓扑学:不明
(ii)分子种类:cDNA
(iii)假定:不
(iv)ANTISENSE:不
(v)残片种类:重链CDR2
(vi)原始来源:
(B)株:单克隆抗体TTC8
(H)细胞系:杂交瘤
(vii)直接来源:
(A)库:DSM ACC 2322
(ix)特征:
(A)名称/编码:CDS
(B)位置:1……51
(xi)SEQ ID No 3描述:CGG ATT TAT CCT GGA GAT GGA GAT GCT CAC TAC AAT GCG AAG TTC AAGArg Ile Tyr Pro Gly Asp Gly Asp Ala His Tyr Asn Gly Lys Phe Lys
5 10 15GGC 5Gly(2)SEQ ID No 4的说明:
(i)序列特征:
(A)长度:17个氨基酸。
(B)种类:氨基酸
(D)拓扑学:线型
(ii)分子种类:CDR2——重链的蛋白序列
(xi)SEQ ID No 4的描述:Arg Ile Tyr Pro Gly Asp Gly Asp Ala His Tyr Asn Gly Lys Phe Lys1 5 10 15Gly(2)SEQ ID No 5的说明:
(i)序列特征:
(A)长度:33个碱基对
(B)种类:核甙酸
(C)链型:单链
(D)拓扑:不明
(ii)分子种类:cDNA
(iii)假定:没有
(v)残片的种类:重链CDR3
(vi)原始来源:
(B)株:单克隆抗体TTC8
(H)细胞系:杂交瘤
(vii)直接来源:
(A)库:DSM ACC 2322
(ix)特征:
(A)名称/编码:CDS
(B)位置:1……33
(xi)SEQ ID No 5的序列描述:GGG GGG AAT TAC GAC GAC AGG GTC TTT GAC TACGly Gly Asn Tyr Asp Asp Arg Val Phe Asp Tyr1 5 10(2)SEQ ID No 6说明:
(i)序列特征:
(A)长度:11个氨基酸。
(B)种类:氨基酸
(D)拓扑学:线型
(ii)分子种类:CDR3重链蛋白序列
(xi)SEQ ID No 6的序列描述:Gly Gly Asn Tyr Asp Asp Arg Val Phe Asp Tyr 1
5 10(2)SEQ ID No 7说明:
(i)序列特征:
(A)长度:33个碱基对
(B)种类:核甙酸
(C)链型:单链
(D)拓扑学:不明
(ii)分子种类:cDNA
(v)残片的种类:轻链多变区(CDR)
(vi)原始来源:
(B)株:单克隆抗体TTC8
(H)细胞系:杂交瘤
(vii)直接来源:
(A)库:DSM ACC 2322
(ix)特征:
(A)名称/编码:CDS
(B)位置:1……33
(xi)SEQ ID No 7的序列描述:AAG GCC AGT CAG AAT GTG GGT ACT AAT GTA GCCLys Ala Ser Gln Asn Val Gly Thr Asn Val Ala
5 10(2)SEQ ID No 8的说明:
(i)序列特征:
(A)长度:11个氨基酸。
(B)种类:氨基酸
(D)拓扑学:线型
(ii)分子的种类:轻链CDR1蛋白序列
(xi)SEQ ID No 8序列描述:Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala 1
5 10(2)SEQ ID No 9的说明:
(i)序列特征:
(A)长度:21个碱基对
(B)种类:核甙酸
(C)链型:单链
(D)拓扑:不明
(ii)分子类型:cDNA
(iii)假定:不是
(v)残片的种类:轻链CDR2
(vi)原始来源:
(B)株:单克隆抗体TTC8
(H)细胞系:杂交瘤
(vii)直接来源:
(A)库:DSM ACC 2322
(ix)特征:
(A)名称/编码:CDS
(B)位置:1……21
(xi)SEQ ID No 9的序列说明:TCG CCA TCC TAC CGG ATC AGTSer Pro Ser Tyr Arg Tyr Ser
5(2)SEQ ID No 10的说明:
(i)序列特征:
(A)长度:7个氨基酸。
(B)种类:氨基酸
(D)拓扑学:线型
(ii)分子的类型:轻链CDR2蛋白序列
(xi)SEQ ID No 10的序列描述:Ser Pro Ser Tyr Arg Tyr Ser1 5(2)SEQ ID No 11的说明:
(i)序列特征:
(A)长度:27个碱基对
(B)种类:核甙酸
(C)链型:单链
(D)拓扑:不明
(ii)分子的种类:cDNA
(iii)假定:没有
(iv)ANTISENSE:不
(v)残片的种类:轻链CDR3
(vi)原始来源:
(B)株:单克隆抗体TTC8
(H)细胞系:杂交瘤
(vii)直接来源:
(A)库:DSM ACC 2322
(ix)特征:
(A)名称/编码:CDS
(B)位置:1……27
(xi)SEQ ID No 11的描述:CAG CAA TAT AAT AGC TAT CCT CTT ACGGln Gln Tyr Asn Ser Tyr Pro Leu Thr(2)SEQ ID No 12的说明:
(i)序列特征:
(A)长度:9个氨基酸。
(B)种类:氨基酸
(D)拓扑:线型
(ii)分子的种类:轻链CDR3蛋白序列
(xi)SEQ ID No 12的序列描述:Gln Gln Tyr Asn Ser Tyr Pro Leu Thr1 5(2)SEQ ID No 13的说明:
(i)序列特征:
(A)长度:341个碱基对
(B)种类:核甙酸
(C)链型:单链
(D)拓扑:不明
(ii)分子的种类:cDNA
(iii)假定:不是
(v)残片的种类:重链多变区VH
(vi)原始来源:
(B)株:单克隆抗体TTC8
(H)细胞系:杂交瘤
(vii)直接来源:
(A)库:DSM ACC 2322
(ix)特征:
(A)名称/编码:CDS
(B)位置:1……341
(IX)特征:
(A)名称/编码:primer_bind
(B)位置:1……21
(D)其他说明:/label=VH5PRIM
(ix)特征:
(A)名称/编码:primer_bind
(B)位置:325……341
(D)其他说明:/label=VH3PRIM
(xi)SEQ ID No 13的序列描述:GTC GAC CTG CAG CAG TCT GGA CCT GAG CTG GTG AAG CCT GGG GCC TCAVal Asp Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser1 5 10 15GTG AAG ATT TCC TGC AAA GCT TCT GGC TAC GCA TTC AGT AAC TAC TGGVal Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr Trp
20 25 30ATG AAC TGG GTG AAG CAG AGG CCT GGA AAG GGT CTT GAG TGG ATT GGAMet Asn Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45CGG ATT TAT CCT GGA GAT GGA GAT GCT CAC TAC AAT GGG AAG TTC AAGArg Ile Tyr Pro Gly Asp Gly Asp Ala His Tyr Asn Gly Lys Phe Lys
50 55 60GGC AAG GCC ACA CTG ACT GCA GAC AAA TCC TCC AGC ACA GCC TAC ATGGly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met65 70 75 80CAA CTC AGC AGC CTG ACA TCT GAG GAC TCT GCG GTC TAC TTC TGT GCAGln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala
85 90 95AGA GGG GGG AAT TAC GAC GAC AGG GTC TTT GAC TAC TGG GGC CAA GGGArg Gly Gly Asn Tyr Asp Asp Arg Val Phe Asp Tyr Trp Gly Gln Gly
100 105 110TCG ACSer(2)SEQ ID No 14说明:
(i)序列特征:
(A)长度:113个氨基酸
(B)种类:氨基酸
(D)拓扑学:线型
(ii)分子的种类:重链的变区VH蛋白序列。
(xi)SEQ ID No 14的序列描述:Val Asp Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser1 5 10 15Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr Trp
20 25 30Met Asn Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45Arg Ile Tyr Pro Gly Asp Gly Asp Ala His Tyr Asn Gly Lys Phe Lys
50 55 60Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met65 70 75 80Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala
85 90 95Arg Gly Gly Asn Tyr Asp Asp Arg Val Phe Asp Tyr Trp Gly Gln Gly
100 105 110Ser(2)SEQ ID No 15的说明:
(i)序列特征:
(A)长度:312碱基对
(B)种类:核甙酸
(C)链型:单链
(D)拓扑:不明
(ii)分子的种类:cDNA
(iii)假设:不是
(iv)ANTISENSE:不是
(v)残片的种类:轻链的变区
(vi)原始来源:
(B)株:单克隆抗体TTC8
(H)细胞系:杂交瘤
(vii)直接来源:
(A)库:DSM ACC 2322
(ix)特征:
(A)名称/编码:primer_bind
(B)位置:1……18
(D)其他说明:/label=VL5Prim
(ix)特征:
(A)名称/编码:primer_bind
(B)位置:289……312
(D)其他说明:/label=VL3PRIM
(ix)特征:
(A)名称/编码:CDS
(B)位置:1…312
(xi)SEQ ID No 15的描述:GAG CTC ACC CAG TCT CCA AAA TTC ATG TCC ACA TCA GTA GGA GAC AGGGlu Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg1 5 10 15GTC AGC GTC ACC TGC AAG GCC AGT CAG AAT GTG GGT ACT AAT GTA GCCVal Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala
20 25 30TGG TAT CAA CAG AAA CCA GGG CAA TCT CCT AAA ACA CTG ATT TAC TCGTrp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Thr Leu Ile Tyr Ser
35 40 45CCA TCC TAC CGG TAC AGT GGA GTC CCT GAT CGC TTC ACA GGC AGT GGAPro Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly
50 55 60TCT GGG ACG GAT TTC ACT CTC ACC ATC AGC AAT GTG CAG TCT GTT GACSer Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Val Asp65 70 75 80TTG GCA GAG TAT TTC TGT CAG CAA TAT AAT AGT TAT CCT CTT ACG TTCLeu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu Thr Phe
85 90 95GGC TCG GGG ACC AAG CTG GAG CTCGly Ser Gly Thr Lys Leu Glu Leu
100(2)SEQ ID No 16的说明:
(i)序列特征:
(A)长度:104个氨基酸
(B)种类:氨基酸
(D)拓扑:线型
(ii)分子的种类:轻链的多变区VL蛋白序列。
(xi)SEQ ID No 16的描述:Glu Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg1 5 10 15Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala
20 25 30Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Thr Leu Ile Tyr Ser
35 40 45Pro Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly
50 55 60Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Val Asp65 70 75 80Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu Thr Phe
85 90 95Gly Ser Gly Thr Lys Leu Glu Leu
100
Claims (29)
1.完全或部分从可和多毒素梭状芽胞杆菌的A毒素和/或B毒素或者和多毒素梭状芽胞杆菌A毒素和/或B毒素的重组残片相结合的抗体多变区序列获得的氨基酸序列(多肽)。
2.权利要求1和多毒素梭状芽胞杆菌的A毒素和/或B毒素相结合而中和毒素的生物效应的氨基酸序列(多肽)。
3.按权利要求1和2中可以通过聚合物对VH5Prim/VH3Prim或VL5Prim/VL3Prim经多聚酶链式反应而获得的DNA-序列和由此得出的氨基酸序列(多肽)。
4.权利要求3中的可以通过和聚合物VH5Prim、VH3Prim、VL5Prim、VL3Prim其中的一个或几个相似的聚合物对而获得的DNA-序列以及相应的氨基酸序列(多肽)。
5.权利要求的1和4中的用多毒素芽胞梭状杆菌的A毒素和/或B毒素或者这些毒素的重整残片免疫以后,这些氨基酸序列确定编码抗体变区的基因氨基酸序列。
6.编码权利要求5中的氨基酸序列的DNA-序列。
7.权利要求1和2中的以单价或多价识别和A毒素和/或B毒素的一个或数个配体片段、移位片段或催化片段的抗原区的氨基酸序列。
8.编码7中的氨基酸序列的DNA-序列。
9.由权利要求1到8中的氨基酸序列通过点变换(氨基酸交换、插入、去掉或添加)而得的氨基酸序列。
10.权利要求1和2中的部分或完全含有DSM AC 2322细胞系的SEQID No1-12(CDR-序列)和/或SEQ ID No 13-16(属VL和/或VH的变区)氨基酸序列。
11.和权利要求10中提到的来源于DSM AC 2322细胞系变区一致的氨基酸序列。
12.权利要求1和2中的含有部分或完整的DSM AC 2321细胞系VL和/或VH变区序列的氨基酸序列。
13.和权利要求12中提到的来源于DSM AC 2321细胞系变区序列一致的氨基酸序列。
14.人体化的由DNA重组技术而得,含有整合到人体抗体机构区(FRs)的全部或单个来源于其他种属并且和多毒素梭状芽胞杆菌的内毒素和/或细胞毒素相结合的抗体轻链和/或重链的多变区(CDRs)的单克隆抗体。
15.权利要求14中提到的构造区(Frs)和人体抗体的gamma或alpha亚型一致的人体化抗体。
16.权利要求14和15中提到的人体化的通过改良和多毒素梭状芽胞杆菌的A毒素和/或B毒素相结合的抗体且改良的方法是保留或改善其结合力,通过在氨基酸内部点变换改变抗体轻链和重链的变区和常区而获得的单克隆抗体。
17.权利要求14到16中提到的部分或完全含有来源于DSM ACC2321或2322细胞系的单克隆抗体的多变区的人体化单克隆抗体。
18.权利要求14到17中提到的多变区(CDRs)有氨基酸序列ID No.1到No.12或经其点变换而得的序列的人体化单克隆抗体。
19.和权利要求14和18中提到的含有家畜的结构区的抗体相似的抗体。
20.权利要求1和2中提到的用生化方法通过多肽合成而得、用于预防和治疗人体和动物的氨基酸序列。
21.获得在真核生物及前核生物细胞内权利要求1和2中提到的用于预防和治疗的多肽的方法以及获得权利要求14到19中提到的种族适应的单克隆抗体的方法。
22.权利要求21中提到的用革兰氏阳性或革兰氏阴性的前核生物的方法。
23.权利要求21中提到的用酵母、真菌、和动植物真核生物的方法。
24.权利要求23中提到的用单子叶和双子叶植物的方法。
25.权利要求24中提到的反应在植物的确定器官内进行的方法。
26.权利要求23和24中提到的方法的具体应用包括经植物获得的氨基酸序列或抗体分离和纯化后或者作为生的素食品用来治疗和预防多毒素梭状芽胞杆菌内毒素或细胞毒素引起的疾病。
27.含有权利要求1到13中提到的多肽或含有权利要求14到19中提到的种族适应的单克隆抗体的药物的合成。
28.权利要求27中提到的胃肠外给药的药物合成。
29.权利要求27中提到的经口给药的药物合成。
Applications Claiming Priority (2)
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DE19739685.2 | 1997-09-10 | ||
DE19739685A DE19739685A1 (de) | 1997-09-10 | 1997-09-10 | Monoklonale Antikörper zur Therapie und Prophylaxe von Erkrankungen durch Clostridium difficile |
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CN1273588A true CN1273588A (zh) | 2000-11-15 |
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CN98808996A Pending CN1273588A (zh) | 1997-09-10 | 1998-09-10 | 用于治疗和预防多毒素梭状芽胞杆菌毒素导致的疾病的氨基酸序列 |
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US (2) | US6667035B1 (zh) |
EP (1) | EP0994904B1 (zh) |
JP (1) | JP4318398B2 (zh) |
CN (1) | CN1273588A (zh) |
AT (1) | ATE254139T1 (zh) |
AU (1) | AU9742698A (zh) |
BR (1) | BR9815367A (zh) |
CA (1) | CA2303202C (zh) |
DE (2) | DE19739685A1 (zh) |
ES (1) | ES2210832T3 (zh) |
WO (1) | WO1999012971A2 (zh) |
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CN107022532A (zh) * | 2011-04-22 | 2017-08-08 | 惠氏有限责任公司 | 涉及突变体难辨梭菌毒素的组合物及其方法 |
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DE19739685A1 (de) * | 1997-09-10 | 1999-03-11 | Eichel Streiber Christoph Von | Monoklonale Antikörper zur Therapie und Prophylaxe von Erkrankungen durch Clostridium difficile |
US20030054009A1 (en) * | 2001-02-09 | 2003-03-20 | Windle Henry J. | Clostridium difficile vaccine |
GB0130267D0 (en) * | 2001-12-19 | 2002-02-06 | Neutec Pharma Plc | Focussed antibody technology |
GB0205206D0 (en) * | 2002-03-06 | 2002-04-17 | Oxoid Ltd | Synthetic peptides |
GB0309126D0 (en) * | 2003-04-17 | 2003-05-28 | Neutec Pharma Plc | Clostridium difficile focussed antibodies |
JP4588763B2 (ja) * | 2004-02-06 | 2010-12-01 | ユニバーシティー オブ マサチューセッツ | クロストリジウム・ディフィシル(Clostridiumdifficile)毒素に対する抗体およびその使用 |
DE602004011660T2 (de) * | 2004-06-16 | 2009-01-29 | Straumann Holding Ag | Abdeckmembran |
GB0414886D0 (en) | 2004-07-02 | 2004-08-04 | Neutec Pharma Plc | Treatment of bacterial infections |
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CA2618796C (en) * | 2005-08-11 | 2018-01-02 | Arpi Matossian-Rogers | Peptides for treatment and diagnosis of autoimmune disease |
CA2680741A1 (en) | 2007-03-22 | 2009-01-15 | The Regents Of The University Of California | Therapeutic monoclonal antibodies that neutralize botulinum neurotoxins |
WO2009136297A2 (en) * | 2008-05-09 | 2009-11-12 | Recopharma Ab | Compositions and methods for inhibiting toxin a from clostridium difficile |
DE102008029688B4 (de) * | 2008-06-24 | 2016-06-23 | Biodics Gmbh | Verfahren zum Nachweis und zur Identifikation eines varianten C. difficile Stammes in einer Probe |
WO2010014854A2 (en) | 2008-07-31 | 2010-02-04 | The Regents Of The University Of California | Antibodies that neutralize botulinum neurotoxins |
US8889363B2 (en) | 2009-07-27 | 2014-11-18 | Biodics | Method for the detection and identification of a variant C. difficile strain in a sample |
US20120269814A1 (en) * | 2009-11-10 | 2012-10-25 | Amgen Inc. | Anti-c mpl antibodies |
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US8906635B2 (en) | 2011-02-28 | 2014-12-09 | Northshore University Healthsystem | Methods of diagnosing Clostridium difficile infection |
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EP2780351B1 (en) | 2011-11-18 | 2019-12-18 | National Research Council of Canada (NRC) | Clostridium difficile lipoteichoic acid and uses thereof |
SG10201610123UA (en) | 2012-03-02 | 2017-01-27 | Regeneron Pharma | Human antibodies to clostridium difficile toxins |
BR122016023101B1 (pt) | 2012-10-21 | 2022-03-22 | Pfizer Inc | Polipeptídeo, composição imunogênica que o compreende, bem como célula recombinante derivada de clostridium difficile |
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-
1997
- 1997-09-10 DE DE19739685A patent/DE19739685A1/de not_active Ceased
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1998
- 1998-09-10 CN CN98808996A patent/CN1273588A/zh active Pending
- 1998-09-10 CA CA2303202A patent/CA2303202C/en not_active Expired - Fee Related
- 1998-09-10 AT AT98951374T patent/ATE254139T1/de active
- 1998-09-10 WO PCT/EP1998/005759 patent/WO1999012971A2/de active IP Right Grant
- 1998-09-10 BR BR9815367-6A patent/BR9815367A/pt not_active Application Discontinuation
- 1998-09-10 EP EP98951374A patent/EP0994904B1/de not_active Expired - Lifetime
- 1998-09-10 ES ES98951374T patent/ES2210832T3/es not_active Expired - Lifetime
- 1998-09-10 JP JP2000510776A patent/JP4318398B2/ja not_active Expired - Fee Related
- 1998-09-10 AU AU97426/98A patent/AU9742698A/en not_active Abandoned
- 1998-09-10 US US09/508,413 patent/US6667035B1/en not_active Expired - Fee Related
- 1998-09-10 DE DE59810172T patent/DE59810172D1/de not_active Expired - Lifetime
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2003
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102482331A (zh) * | 2009-07-27 | 2012-05-30 | 碧奥迪克斯有限公司 | 用于检测和鉴别样品中变异艰难梭菌菌株的方法 |
CN102482331B (zh) * | 2009-07-27 | 2014-12-24 | 碧奥迪克斯有限公司 | 用于检测和鉴别样品中变异艰难梭菌菌株的方法 |
CN107022532A (zh) * | 2011-04-22 | 2017-08-08 | 惠氏有限责任公司 | 涉及突变体难辨梭菌毒素的组合物及其方法 |
CN107022532B (zh) * | 2011-04-22 | 2021-05-04 | 惠氏有限责任公司 | 涉及突变体难辨梭菌毒素的组合物及其方法 |
Also Published As
Publication number | Publication date |
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EP0994904B1 (de) | 2003-11-12 |
WO1999012971A3 (de) | 1999-07-22 |
US20040137601A1 (en) | 2004-07-15 |
CA2303202C (en) | 2010-04-27 |
ATE254139T1 (de) | 2003-11-15 |
AU9742698A (en) | 1999-03-29 |
JP2001515920A (ja) | 2001-09-25 |
BR9815367A (pt) | 2001-11-06 |
EP0994904A2 (de) | 2000-04-26 |
JP4318398B2 (ja) | 2009-08-19 |
ES2210832T3 (es) | 2004-07-01 |
US7151159B2 (en) | 2006-12-19 |
WO1999012971A2 (de) | 1999-03-18 |
CA2303202A1 (en) | 1999-03-18 |
DE19739685A1 (de) | 1999-03-11 |
US6667035B1 (en) | 2003-12-23 |
DE59810172D1 (de) | 2003-12-18 |
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