CN1547590A - 人源化抗-LT-β-R抗体 - Google Patents
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- CN1547590A CN1547590A CNA018205917A CN01820591A CN1547590A CN 1547590 A CN1547590 A CN 1547590A CN A018205917 A CNA018205917 A CN A018205917A CN 01820591 A CN01820591 A CN 01820591A CN 1547590 A CN1547590 A CN 1547590A
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Abstract
本发明提供了抗LT-β-R的人源化抗体以及它们的应用方法。
Description
相关申请
这是2001年6月21日提交的美国申请60/299,987的部分继续申请,而后者是2001年3月13日提交的美国申请60/275,289的部分继续申请,而后者是2000年10月13日提交的美国申请60/240,285的部分继续申请。上述每一专利申请都全文引入本文作为参考。
技术领域
本发明主要涉及特异性针对淋巴毒素β受体(LT-β-R)的人源化抗体。
背景技术
淋巴毒素β受体(本文称为LT-β-R)是肿瘤坏死因子家族的一员,已知其在免疫系统的发育、以及包括滤泡树突状细胞在内的免疫系统多种细胞和多种基质细胞的功能维持方面发挥作用(Matsumoto等,Immunol.Rev.156:137(1997)。LT-β-R的已知配体包括LTα1/β2以及另一种称为LIGHT的配体(Mauri等Immunity 8:21(1998))。有文献表明,LT-β-R的活化包括一些癌细胞系的体内凋亡性死亡(PCT/US96/01386)。用激动型LT-β-R激活剂,如特异性人源化抗LT-β-R抗体进行的治疗,将因此可用于治疗或减缓受试者(如,人)中瘤形成(neoplasia)的进展、严重程度或影响。
发明简述
本发明提供了人源化抗淋巴毒素β受体(LT-β-R)的抗体以及应用这些抗体治疗或缓解受试者(如,人)中瘤形成的进展、严重程度或影响的方法。
具体地,本发明涉及特异性结合LT-β-R(如人LT-β-R)的人源化抗体。这种抗体包含由SEQ ID NO:1中氨基酸残基24-34,50-56和89-97所限定的轻链互补决定区,和/或由SEQ ID NO:2中氨基酸残基31-35,50-66和99-109限定的重链互补决定区,并且在其轻链中包含下列的至少1种(例如,1,2,3,4,或5种)残基:K3,W41,I46,Q69和Y71;或在其重链中包含下列至少1种(例如1,2,3,4,或5种)残基:F37,T40,A49,M89和V93(按照Kabat编号原则进行编号)。
本发明的人源化抗体可包含由SEQ ID NO:8的氨基酸残基1-107所限定的轻链可变区序列和/或由SEQ ID NO:16的氨基酸残基1-120所限定的重链可变区序列。所述人源化抗体还可包含与由细胞系E46.4(ATCC专利保藏物PTA-3357)或细胞系E77.4(ATCC专利保藏物3765)产生的抗体相同的重链和/或轻链多肽序列。
在另一实施方案中,本发明的人源化抗体充分保留了亲本抗体的结合特性。在一个实施方案中,本发明的人源化抗体以一定的功能性亲和力与LT-β-R结合,所述亲和力是,例如,约1pM-约10pM,或者,约10pM-约20pM,或者,约20pM-约30pM,或者,约30pM-约40pM,或者,约40pM-约50pM,或者,约50pM-约60pM,或者,约60pM-约70pM,或者,约70pM-约80pM,以及,或者,约80pM-约90pM,所述功能性亲和力按照实施例8所述用FACS进行测定。
在另一实施方案中,本发明的人源化抗体与免疫毒素(例如,蓖麻毒蛋白A链和假单胞菌毒素)相连。本发明的人源化抗体还可与化疗药物(例如,阿霉素(Adriamycin),5FU,长春花碱,放线菌素D,依托泊苷(Etoposide),顺铂(Cisplatin),氨甲蝶呤和阿霉素(Doxorubicin))或与放射性同位素相连。本发明还涉及一种联合疗法,其中例如,本发明的与免疫毒素相连的人源化抗体和本发明的与化疗药物相连的人源化抗体联合使用。本发明还涉及一种适于对哺乳动物(即人)给药的组合物,所述哺乳动物具有过度表达LTβR的肿瘤,所述组合物包含:a)人源化抗LTβR抗体,单独或与免疫毒素或与化疗药物相连,和b)细胞毒因子,每种均以通过对哺乳动物给药而有效减小肿瘤体积的量存在。细胞毒因子可以包含例如,TNF-α,TNF-β,IL-1,INF-γ,IL-2。或者,细胞毒因子可以是一种化疗药物。该化疗药物可以包含例如,阿霉素,5FU,长春花碱,放线菌素D,依托泊苷,顺铂,氨甲蝶呤和阿霉素。
本发明的抗体可以是,例如,任何同种型和亚型的完整抗体(即具有两条全长轻链和两条全长重链)(例如IgM,IgD,IgG1,IgG2,IgG3,IgG4,IgE,IgA1和IgA2);或者,它可以是完整抗体的抗原结合片段(例如,Fab,F(ab’)2,和Fv)。
本发明涉及的还有,一种组合物,该组合物包含可药用载体;一种分离的核酸,该核酸包含编码SEQ ID NO:8的序列;一种分离的核酸,该核酸包含编码SEQ IDNO:16的序列;一种分离的核酸,该核酸包含编码抗体轻链的序列,该抗体由细胞系E46.4(ATCC专利保藏物PTA-3357)或细胞系E77.4(ATCC专利保藏物3765)产生;一种分离的核酸,该核酸包含编码抗体重链的序列,该抗体由细胞系E46.4(ATCC专利保藏物PTA-3357)或细胞系E77.4(ATCC专利保藏物3765)产生;一种分离的核酸,该核酸包含编码SEQ ID NO:8的1-107残基的序列;和一种分离的核酸,该核酸包含编码SEQ ID NO:16的1-120残基的序列。
本发明中涉及的还有,产生人源化抗LTβR抗体的细胞系的细胞,所述细胞系包括,例如,细胞系E46.4(ATCC专利保藏物PTA-3357)和细胞系E77.4 (ATCC专利保藏物3765)。在一个实施方案中,所述细胞系产生约250mg/L-约300mg/L所述抗体,或者,所述细胞系产生约300mg/L-约350mg/L所述抗体,或者,所述细胞系产生约350mg/L-约400mg/L所述抗体,或者,所述细胞系产生约400mg/L-约450mg/L所述抗体,或者,所述细胞系产生约450mg/L-约500mg/L所述抗体,或者,所述细胞系产生约500mg/L-约550 mg/L所述抗体,或者,所述细胞系产生约550mg/L-约600mg/L所述抗体。所述细胞系产生的抗体的浓度可以是补料分批培养10天后所测定的收获物的滴度。
本发明还提供了一种治疗或缓解受试者(例如,人)中瘤形成的进展、严重程度或影响的方法,该方法包括以有效量的本发明抗体对受试者给药。组合物的有效量可以以一或多剂量给药。在另一实施方案中,本发明提供了一种治疗或缓解受试者(例如,人)中瘤形成的进展、严重程度或影响的方法,该方法包括以有效量的本发明抗体和细胞毒素因子对受试者给药。细胞毒素因子可包括例如,TNF-α,TNF-β,IL-1,INF-γ,IL-2。或者,细胞毒素因子可以是化疗药物。化疗药物可包括例如,阿霉素,5FU,长春花碱,放线菌素D,依托泊苷,顺铂,氨甲蝶呤和阿霉素。
附图说明
图1显示对于WiDr细胞的细胞毒活性。mCBE11(鼠)(钻石形),huCBE11#2(人源化抗LT-β-R抗体,含第二种轻链(VL#2)和第二种重链(VH#2))(圆形),huCBE11#4(人源化抗LT-β-R抗体,含第三种轻链(VL#3)和第四种重链(VH#4))(星形)。
图2显示IL-8对A375细胞的激动作用(agonism)。mCBE11(钻石形),huCBE11#2(人源化抗LT-β-R抗体,含第二种轻链(VL#2)和第二种重链(VH#2))(圆形),huCBE11#4(人源化抗LT-β-R抗体,含第三种轻链(VL#3)和第四种重链(VH#4))(星形)。
图3显示肿瘤体积相对于给药天数。mCBE11(三角形),huCBE11#4(人源化抗LT-β-R抗体,含第三种轻链(VL#3)和第四种重链(VH#4))(圆形),未经治疗(方形)。
图4显示动物存活百分比相对于肿瘤注射后天数。mCBE11(三角形),huCBE11#4(人源化抗LT-β-R抗体,含第三种轻链和第四种重链)(圆形),未经处理(方形)。
图5显示肿瘤体积相对于注射后天数。HuCBE11#4(人源化抗LT-β-R抗体,含第三种轻链(VL#3)和第四种重链(VH#4))(圆形)和与未经处理的对照(方形)。
图6显示肿瘤的预生长体积相对于处理后的天数。对照(方形);huCBE11#4(人源化抗LT-β-R抗体,含第三种轻链(VL#3)和第四种重链(VH#4))于不同剂量:500ug(圆形),100ug(三角形),和20ug(钻石形);mCBE11(十字形)。
图7显示带有预生长肿瘤的动物的存活百分比相对于处理后的天数。对照(方形);huCBE11#4(人源化抗LT-β-R抗体,含第三种轻链(VL#3)和第四种重链(VH#4))于不同剂量:500ug(圆形),100ug(三角形),和20ug(钻石形);mCBE11(十字形)。
图8显示平均荧光强度相对于huCBE11#4浓度的对数(1og)。
发明详述
序列识别号
在说明书中提到的核苷酸和氨基酸序列给出下列序列识别号:
SEQ ID NO:1-mCBE11重链可变区的氨基酸序列。
SEQ ID NO:2-mCBE11轻链可变区的氨基酸序列。
SEQ ID NO:3-人源化CBE11轻链可变区(version 1-VL#1)核酸序列。
SEQ ID NO:4-人源化CBE11轻链可变区(version 1-VL#1)氨基酸序列。
SEQ ID NO:5-人源化CBE11轻链可变区(version 2-VL#2)核酸序列。
SEQ ID NO:6-人源化CBE11轻链可变区(version 2-VL#2)氨基酸序列。
SEQ ID NO:7-人源化CBE11轻链可变区(version 3-VL#3)核酸序列。
SEQ ID NO:8-人源化CBE11轻链可变区(version 3-VL#3)氨基酸序列。
SEQ ID NO:9-人源化CBE11重链可变区(version 1-VH#1)核酸序列。
SEQ ID NO:10-人源化CBE11轻链可变区(version 1-VH#1)氨基酸序列。
SEQ ID NO:11-人源化CBE11重链可变区(version 2-VH#2)核酸序列。
SEQ ID NO:12-人源化CBE11重链可变区(version 2-VH#2)氨基酸序列。
SEQ ID NO:13-人源化CBE11重链可变区(version 3-VH#3)核酸序列。
SEQ ID NO:14-人源化CBE11重链可变区(version 3-VH#3)氨基酸序列。
SEQ ID NO:15-人源化CBE11重链可变区(version 4-VH#4)核酸序列。
SEQ ID NO:16-人源化CBE11重链可变区(version 4-VH#4)氨基酸序列。
SEQ ID NO:17-FR1引物,用于引入Bsu36I位点。
SEQ ID NO:18-FR2引物,用于引入NciI和HpaII位点。
SEQ ID NO:19-FR3引物,用于引入Bsu36I和PstI位点。
SEQ ID NO:20-FR2引物,用于引入SmaI位点。
SEQ ID NO:21-FR3引物,用于引入PvuI位点。
SEQ ID NO:22-FR2引物,用于引入SmaI和HhaI位点。
SEQ ID NO:23-FR3引物,用于引入PvuII和FspI位点。
SEQ ID NO:24-FR1引物,用于引入Hinfl和NsiI位点。
SEQ ID NO:25-FR2引物,用于引入HaeII和HhaI位点。
SEQ ID NO:26-FR3引物,用于引入Bsu36I,DdeI和PstI位点。
SEQ ID NO:27-FR1引物,用于引入EcoRV位点。
SEQ ID NO:28-FR3引物,用于引入RsaI位点。
SEQ ID NO:29-FR1引物,用于引入EcoRV位点。
SEQ ID NO:30-FR2引物,用于引入HindIII位点。
SEQ ID NO:31-FR3引物,用于引入RsaI位点。
SEQ ID NO:32-完整huCBE11轻链(version3),包含恒定区。
SEQ ID NO:33-完整huCBE11重链(version4),包含恒定区。
定义
本文中术语人源化抗体是指衍生自非人抗体(通常衍生自鼠)的抗体,其保留或充分保留了亲本抗体的抗原结合特性,但在人体内具有更小的致免疫性。
本文中术语互补决定区(CDR)是指多个氨基酸序列,它们共同限定了天然免疫球蛋白结合位点的天然Fv区的结合亲和力和特异性,如Kabat等描绘(1991)。
本文中术语框架区(FR),是指位于CDR之间的氨基酸序列。抗体的这些部分起到使CDR保持适当方位(从而使CDR可以结合抗原)的作用。
本文中术语恒定区(CR),是指抗体分子中赋予效应功能的部分。在本发明中,鼠的恒定区被人的恒定区取代。本发明中嵌合抗体或人源化抗体的恒定区衍生于人的免疫球蛋白。重链恒定区可选自5种同种型之一:α,δ,ε,γ或μ。另外,不同亚类的重链(例如重链的IgG亚类)负责不同的效应功能并因此,通过挑选所需重链恒定区,可以产生具有所需效应功能的抗体。优选的恒定区是γ1(IgG1),γ3(IgG3)和γ4(IgG4)。更优选的是γ1(IgG1)同种型的Fc区。轻链恒定区可以具有κ或λ型,优选具有κ型。
本文中术语嵌合抗体是指包含源自于两种不同抗体(通常为不同种类)的序列的抗体。最通常地,嵌合抗体包含人和鼠的抗体片段,一般包含人的恒定区和鼠的可变区。
本文中术语致免疫性是指靶向蛋白或治疗性半分子给药至受体时激发免疫应答(体液免疫应答或细胞免疫应答)的能力。本发明涉及人源化抗体的致免疫性。
致免疫性降低的人源化抗体是指相对于亲本抗体(例如,鼠抗体)致免疫性降低的人源化抗体。
充分保留了亲本抗体的结合特性的人源化抗体是指,保留了特异性结合抗原的能力的人源化抗体,所述抗原是由产生这类人源化抗体的亲本抗体所识别的。优选这种人源化抗体显示与亲本抗体相同或基本相同的抗原结合亲和力和亲合力。理想地,该抗体的亲和力不低于亲本抗体亲和力的10%,更优选不低于约30%,最优选不低于50%。分析抗原结合亲和力的方法在本领域众所周知,包括半量最大结合试验,竞争试验,和Scatchard分析。在本申请中描述了适当的抗原结合试验。
本发明涉及结合人LT-β-R的人源化单克隆抗体和它们作为治疗药物的用途。本发明还涉及了编码所述人源化抗体的核酸序列,和它们在重组宿主细胞中的表达。更具体的,本发明涉及源自与人LT-β-R特异性结合的鼠CBE11的人源化抗体。
鼠CBE11(mCBE11)是从用人LT-β-R-Ig融合蛋白免疫的小鼠中分离的鼠IgG1、κ抗体(Browning等,J.Immunol.154:33(1995))。mCBE11能在体外和体内激活LT-β-R(PCT/US96/01386),对它的分离和抗肿瘤特性已有报道(Browning等J.Exp.Med.183:867(1996)。产生mCBE11的杂交细胞系已经由本发明的申请人根据布达佩斯条约的规定,保藏在美国典型培养物保藏中心(ATCC),保藏号为HB 11793(PCT/US96/01386)。申请人还显示,LT-β受体与不同的激动型抗LT-β-R抗体的交联可激活LT-β受体(即它能模拟天然配体的效果)(PCT/US96/01386)。受体的激活在表达LT-β受体的各种体内肿瘤模型中抑制肿瘤的生长。LT-β受体在多种癌细胞上表达,包括例如非小细胞肺癌细胞(NSCLC),结直肠癌细胞(CRC),乳腺癌细胞,以及前列腺,胃(gastric),皮肤,胃部(stomach),食道和膀胱癌细胞。可被激动型LT-β-R抗体抑制的肿瘤的非限定性实例包括以下实体瘤:HT29结肠腺癌,HT3宫颈癌,A375黑素瘤,MDA-231乳腺癌和原发性结肠癌。因此,激动型LT-β-R抗体具有使得它可用于治疗疾病的特性,所述治疗是指需要激活LT-β-R和/或调节LT-β-R/LT-β-R配体相互作用的那些治疗,包括例如治疗或缓解受试者(例如,人)瘤形成的恶化,严重性或影响。
本发明描述了mCBE11单克隆抗体的人源化,包括充分保留该mCBE11单克隆抗体的结合特性所必需的建模分析以及回复突变。
小鼠可变区的建模分析
CDR包含最有可能结合抗原的残基,且必须保留在再成形抗体中。CDR由Kabat等的序列定义,参见Sequence of Proteins of Immunological Interest,5thEdition,The United States Department of Health和Human Services,TheUnited States Government Printing Offce,1991。CDR分为多个规范的类别(Chothia et al,1989 Nature,342,877-883),在这些类别中,关键残基很大程上决定了CDR环的结构性构象。这些残基几乎总是保留在再成形抗体中。mCBE11的轻链可变区的多肽序列如下所示,CDR以下划线表示,残基位置编号根据Kabat编号系统指定:
1 DIKMTQSPSS MYASLGERVT ITC
KAGQDIK
SYLSWYQQKP
41 WKSPKILIY
Y
ATRLADGVPS RFSGSGSGQD YSLTISSLES
81 DDTATYYC
LQ
HGESPWTFGG GTKLEIK
(SEQ ID NO:1)
mCBE11的重链可变区的多肽序列如下所示,CDR以下划线表示,残基位置编号根据Kabat编号系统指定:
1 EVQLVESGGG LVKPGGSLKL SCAASGFTFS
DYYMYWFRQT
41 PEKRLEWVA
T
ISDGGSYTYY
PDSVKGRFTI SRDNAKNNLY
81 LQMSSLKSED TAMYYCVR
EE
NGNFYYFDYW GQGTTVTVSS
(SEQ ID NO:2)
mCBE11的轻链可变区和重链可变区使用FASTA程序,与小鼠和人各亚组的共有序列作比较(Johnson,G.,Wu,T.T.Kabat Database和itsapplications:future directions Nucleic Acid Research,29,205-206,2001;Wu和Kabat,J.Exp.Med.132:211-250(1970))。mCBE11轻链可变区是小鼠亚组V的一个成员,在110个氨基酸的重叠部分中具有74%的同一性,mCBE11重链可变区是小鼠亚组IIId的一个成员,在132个氨基酸的重叠部分中具有79%的同一性。轻链可变区与人亚组I相对应,在113个氨基酸的重叠部分具有66%的同一性。重链可变区与人亚组III相对应,在131个氨基酸的重叠部分具有71%的同一性。
本发明的CDR分为多个规范的类别。L1环划归类2(11个残基的环),L2划归类1(7个残基)和L3划归类1(9个残基)。H1环划归类1(5个残基),H2环划归类3(17个残基)。H3环不属于规范的类别。
轻链可变区和重链可变区交接处的残基已被定义(Chothia et al,1985 J.Mol.Biol.,186,651-663)。它们通常保留在再成形抗体中。在mCBE11中这些残基中的数个通常位于交接处,即VL中的S34,I46,L89,H91和VH中的Y35,F37,V93,E95。
非常规框架残基通过分析1999年9月版的Kabat数据库[NCBI,NIH]中小鼠和人的所有可变链序列来确定。据信,mCBE11-特异性差异如果接近结合位点则可能指示那些能增强结合活性的体细胞突变。更远离结合位点的非常规小鼠残基,还有非常规的人的残基,如果在再成形抗体中产生致免疫性表位,则被除去。在mCBE11中发现的非常规框架残基是轻链的K3,M11,Y12,W41,Q69,S72,D81,T83和重链的F37,T40,E42,A49,N77。这其中大多数残基未能保留在人源化CBE11抗体中,一部分非常规框架残基得以保留,包括,如重链的F37和A49。
对可变区结构建模
将本发明的轻链和重链在非-丰余(non-redundant)数据库中进行对比排列,以便确定用于构建mCBE11轻链和重链的三维模型的结构框架。利用BLAST发现,轻链与鼠单克隆抗体5g9(1AHW)有93%的序列同一性,重链与鼠IGGA2 Fab片段(Fab 17/9)(1IFH)有81%的序列同一性。利用分子建模软件包Sybyl(Tripos Inc.),分别用5g9的轻链和IGGA2 Fab片段的重链构建轻链和重链的三维结构。所建模型的结构完整性在控制台(console)进行评估,发现比较合理。
对再成形的可变区的设计
用同源性匹配选择“接受”mCBE11 CDR的人受体框架区。用软件程序BLAST在Kabat数据库和非丰余数据库,NCBI,ENTRZ(The NationalInstitutes of Health)中进行搜索。对人的受体框架区的选择是基于mCBE11框架区与人的框架区(除了来源于以前人源化的抗体的框架区以外)之间的序列同一性。
最终选择的人的框架区中,轻链可变区部分(VL)来自抗人肿瘤坏死因子α的抗体TNF-A1′CL(kabat ID 004770)(Griffths等,1993 EMBO J.12:725-734)(人κ亚组I),重链可变区部分(VH)来自特异性不明的抗体FLA-IgG′CL(kabat id 040003)(Malisan等,1996 Blood 87:717-724)(人亚组III)。人VL和VH的框架区与鼠的序列相比有15和11个残基的差异。
入框架区的回复突变
单克隆抗体人源化过程中最无法预测的步骤是,对亲本抗体(本例中为鼠源抗体)中需要予以保留以便充分保留亲本抗体的结合特性、同时又使所得抗体的潜在致免疫性最小化的关键性框架残基的鉴定。尤其重要的是保留靠近结合位点的规范残基、界面包装残基和非常规鼠残基。还要考虑‘Vernier区'中的残基(形成一个能留置CDR的平台)(Foote & Winter,1992 J.Mol.Biol.224,487-499)和那些靠近CDR H3的残基。向亲本抗体回复的突变(即,从人的框架残基向鼠的残基的突变)在后文中都称为回复突变。
制备三种再成形的可变轻链(hu-CBE11 VL)和四种再成形的可变重链(hu-CBE11 VH)。第一种包含回复得最多的突变,第三种包含回复得最少的突变(即,最为“人源化”),只有第四种hu-CBE11 VH例外。本发明涉及源自mCBE11的人源化抗体,它以任意组合方式具有选自下列(即,VL#1,VL#2或VL#3)的可变轻链和选自下列(即,VH#1,VH#2,VH#2,或VH#4)的可变重链。
(A)轻链:
3Q(谷氨酰胺)->K(赖氨酸)它保留在第一种形式中,因为此前的再成形实验(例如Kolbinger等,1993 Prot.Eng.,6,971-980)显示它可能对于抗原结合或CDR构象很重要。
41G(甘氨酸)->W(色氨酸)它保留在第一种和第二种形式中。
46L(亮氨酸)->I(异亮氨酸)它保留在第一和第二种形式中,因为它既是界面残基,又位于vernier区中。而且它还是在鼠序列中出现9次,和在人的序列中出现1次的非常规残基。它看来会影响可变链的包装并且可能与CDR接触。
69T(苏氨酸)->Q(谷氨酰胺)该残基位于vernier之中并可能影响CDR构象。从较短的T变为较长的Q还可能意味着它能与抗原接触。Q在小鼠中非常规地出现了58次,在人类中仅出现2次。它保留在第一种形式中。
71F(苯丙氨酸)->Y(酪氨酸)该残基为一个规范位点,它保留在所有形式中。它在人的序列中仅出现25次,这相对来讲不太常规。
(B)重链
37V(缬氨酸)->F(苯丙氨酸)它保留在第一、二和第四种形式中。该位置的F不是常规的,它在小鼠中仅出现15次,在人类中出现18次。它还是一种界面残基。
40A(丙氨酸)->T(苏氨酸) 它保留在第一种形式中。此前有5次人源化实验对该位置进行了突变,但都不是从A变为T。一例是将BrE-3表面(veneering)的A变为S(Couto等,1994 Hybridoma,13,215-219),导致结合亲和力增强,但原因不明。在该例中,重链也是人的亚组III。
49S(丝氨酸)->A(丙氨酸) 该残基位于CDR之下、vernier区中,并且它保留在所有形式中。
89V(缬氨酸)->M(甲硫氨酸)它在第一种形式得以保留。有多项实验对该位置进行了回复突变。
93A(丙氨酸)->V(缬氨酸)该位置既是界面残基又位于vernier区之中。它被保留在第一和第二种形式中。
所制备的可变轻链和可变重链的每种不同形式,它们的氨基酸和核酸序列如下:
再成形的可变轻链
再成形的CBE11可变轻链-第1种轻链(VL#1)(质粒pAND066)
1 GATATTAAGATGACCCAGTCTCCATCATCCTTGTCTGCATCGGTGGGAGACAGGGTCACT 60
D I K M T Q S P S S L S A S V G D R V T
aa3
61 ATCACTTGCAAGGCGGGTCAGGACATTAAAAGCTATTTAAGCTGGTACCAGCAGAAACCA 120
I T C K A G Q D I K S Y L S W Y Q Q K P
121 TGGAAAGCGCCTAAGATCCTGATCTATTATGCAACAAGGTTGGCAGATGGGGTCCCATCA 180
W K A P K I L I Y Y A T R L A D G V P S
aa41 aa46
181 AGATTCAGTGGCAGTGGATCTGGGCAAGATTATACTCTAACCATCAGCAGCCTGCAGCCT 240
R F S G S G S G Q D Y T L T I S S L Q P
aa69 aa71
241 GAGGATTTCGCAACTTATTACTGTCTACAGCATGGTGAGAGCCCGTGGACGTTCGGTGGA 300
E D F A T Y Y C L Q H G E S P W T F G G
301 GGCACCAAGCTGGAGATCAAA 321
G T K L E I K
SEQ ID NO:3-表示上述再成形V#1的核酸序列。
SEQ ID NO:4-表示上述再成形VL#1的氨基酸序列。
再成形的CBE11可变轻链-第2种轻链(VI#2)(质粒pAND070)
1 GATATCCAGATGACCCAGTCTCCATCATCCTTGTCTGCATCGGTGGGAGACAGGGTCACT 60
D I Q M T Q S P S S L S A S V G D R V T
61 ATCACTTGCAAGGCGGGTCAGGACATTAAAAGCTATTTAAGCTGGTACCAGCAGAAACCA 120
I T C K A G Q D I K S Y L S W Y Q Q K P
121 TGGAAAGCGCCTAAGATCCTGATCTATTATGCAACAAGGTTGGCAGATGGGGTCCCATCA 180
W K A P K I L I Y Y A T R L A D G V P S
aa41 aa46
181 AGATTCAGTGGCAGTGGATCTGGTACAGATTATACTCTAACCATCAGCAGCCTGCAGCCT 240
R F S G S G S G T D Y T L T I S S L Q P
aa71
241 GAGGATTTCGCAACTTATTACTGTCTACAGCATGGTGAGAGCCCGTGGACGTTCGGTGGA 300
E D F A T Y Y C L Q H G E S P W T F G G
301 GGCACCAAGCTGGAGATCAAA 321
G T K L E I K
SEQ ID NO:5-表示上述再成形VL#2的核酸序列。
SEQ ID NO:6-表示上述再成形VL#2的氨基酸序列。
再成形的CBE11可变轻链-第3种轻链(VL#3)(质粒pAND074)
1 GATATCCAGATGACCCAGTCTCCATCATCCTTGTCTGCATCGGTGGGAGACAGGGTCACT 60
D I Q M T Q S P S S L S A S V G D R V T
61 ATCACTTGCAAGGCGGGTCAGGACATTAAAAGCTATTTAAGCTGGTACCAGCAGAAACCA 120
I T C K A G Q D I K S Y L S W Y Q Q K P
121 GGGAAAGCGCCTAAGCCTCTGATCTATTATGCAACAAGGTTGGCAGATGGGGTCCCATCA 180
G K A P K L L I Y Y A T R L A D G V P S
181 AGATTCAGTGGCAGTGGATCTGGTACAGATTATACTCTAACCATCAGCAGCCTGCAGCCT 240
R F S G S G S G T D Y T L T I S S L Q P
aa71
241 GAGGATTTCGCAACTTATTACTGTCTACAGCATGGTGAGAGCCCGTGGACGTTCGGTGGA 300
E D F A T Y Y C L Q H G E S P W T F G G
301 GGCACCAAGCTGGAGATCAAA 321
G T K L E I K
SEQ ID NO:7-表示上述再成形VL#3的核酸序列。
SEQ ID NO:8-表示上述再成形VL#3的氨基酸序列。
再成形的可变重链:
再成形的CBE11可变重链-第1种重链(VH#1)(质粒pAND067)
1 GAGGTACAACTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAGGCTC 60
E V Q L V E S G G G L V K P G G S L R L
61 TCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGTTTCGCCAGACT 120
S C A A S G F T F S D Y Y M Y W F R Q T
aa37 aa40
121 CCGGGAAAGGGGCTGGAGTGGGTCGCAACCATTAGTGATGGTGGTAGTTACACCTACTAT 180
P G K G L E W V A T I S D G G S Y T Y Y
aa49
181 CCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAGCCTCTAC 240
P D S V K G R F T I S R D N A K N S L Y
241 CTGCAGATGAGCAGCCTGAGGGCTGAGGACACAGCCATGTATTACTGTGTAAGAGAGGAG 300
L Q M S S L R A E D T A M Y Y C V R E E
aa89 aa93
301 AATGGTAACTTTTACTACTTTGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 360
N G N F Y Y F D Y W G Q G T T V T V S S
SEQ ID NO:9-表示上述再成形VH#1的核酸序列。
SEQ ID NO:10-表示上述再成形VH#1的氨基酸序列。
再成形的CBE11可变重链-第2种重链(VH#2)(质粒pAND071)
1 GAGGTACAACTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAGGCTC 60
E V Q L V E S G G G L V K P G G S L R L
61 TCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGTTTCGCCAGGCC 120
S C A A S G F T F S D Y Y M Y W F R Q A
aa37
121 CCGGGAAAGGGGCTGGAGTGGGTCGCAACCATTAGTGATGGTGGTAGTTACACCTACTAT 180
P G K G L E W V A T I S D G G S Y T Y Y
aa49
181 CCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAGCCTCTAC 240
P D S V K G R F T I S R D N A K N S L Y
241 CTGCAGATGAGCAGCCTGAGGGCTGAGGACACAGCTGTGTATTACTGTGTAAGAGAGGAG 300
L Q M S S L R A E D T A V Y Y C V R E E
aa93
301 AATGGTAACTTTTACTACTTTGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 360
N G N F Y Y F D Y W G Q G T T V T V S S
SEQ ID NO:11-表示上述再成形VH#2的核酸序列。
SEQ ID NO:12-表示上述再成形VH#2的氨基酸序列。
再成形的CBE11可变重链-第3种重链(VH#3)(质粒pAND075)
1 GAGGTACAACTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAGGCTC 60
E V Q L V E S G G G L V K P G G S L R L
61 TCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTGCGCCAGGCC 120
S C A A S G F T F S D Y Y M Y W V R Q A
121 CCGGGAAAGGGGCTGGAGTGGGTCGCAACCATTAGTGATGGTGGTAGTTACACCTACTAT 180
P G K G L E W V A T I S D G G S Y T Y Y
aa49
181 CCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAGCCTCTAC 240
P D S V K G R F T I S R D N A K N S L Y
241 CTGCAGATGAGCAGCCTGAGGGCTGAGGACACAGCTGTGTATTACTGCGCAAGAGAGGAG 300
L Q M S S L R A E D T A V Y Y C A R E E
301 AATGGTAACTTTTACTACTTTGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 360
N G N F Y Y F D Y W G Q G T T V T V S S
SEQ ID NO:13-表示上述再成形VH#3的核酸序列。
SEQ ID NO:14-表示上述再成形VH#3的氨基酸序列。
再成形的CBE11可变重链-第4种重链(VH#4)(质粒pAND090)
1 GAGGTACAACTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAGGCTC 60
E V Q L V E S G G G L V K P G G S L R L
61 TCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGTTTCGCCAGGCC 120
S C A A S G F T F S D Y Y M Y W F R Q A
aa37
121 CCGGGAAAGGGGCTGGAGTGGGTCGCAACCATTAGTGATGGTGGTAGTTACACCTACTAT 180
P G K G L E W V A T I S D G G S Y T Y Y
aa49
181 CCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAGCCTCTAC 240
P D S V K G R F T I S R D N A K N S L Y
241 CTGCAGATGAGCAGCCTGAGGGCTGAGGACACAGCTGTGTATTACTGCGCAAGAGAGGAG 300
L Q M S S L R A E D T A V Y Y C A R E E
301 AATGGTAACTTTTACTACTTTGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 360
N G N F Y Y F D Y W G Q G T T V T V S S
SEQ ID NO:15-表示上述再成形VH#4的核酸序列。
SEQ ID NO:16-表示上述再成形VH#4的氨基酸序列。
制备由不同类型的轻链和重链组成的抗体并用于进一步的研究。例如,制备由再成形的huCBE11第3种可变轻链(VL#3)和再成形的huCBE11第4种可变重链(VH#4)组成的抗体,命名为huCBE11#4或hCBE11。将产生所述抗体的细胞系:E46.4和E77.4保藏在A.T.C.C.(ATCC专利保藏物编号分别为PTA-3357和3765)。
本发明还涉及再成形VH和VL序列的等价体和变体,即那些包含一或多个不实质影响LT-β-R结合的保守氨基酸取代者。含有这些人源化可变重链和轻链的序列的人源化LT-β-R抗体可通过实施例中描述的重组方法获得。
应用
本发明的人源化抗LT-β-R抗体可用于治疗那些当LT-β-R激活时具有疗效的疾病,如用于治疗、预防或减少瘤形成的进展、严重性或影响。
本发明一个实施方案涉及治疗哺乳动物(即,人类)中与不良细胞增生有关的疾病的方法,包括给与该哺乳动物治疗有效量的含本发明人源化LT-β-R抗体的组合物。
本发明另一实施方案涉及治疗哺乳动物(即,人类)中过表达LT-β-R的实体瘤(即,癌症)的方法,包括给与该哺乳动物有效量的、与LT-β-R结合从而减小肿瘤体积的人源化LT-β-R抗体。细胞增生受LT-β-R调节的那些癌症的实例可通过测量表达在肿瘤组织文库中的LT-β-R和/或LT-β-R配体(即,LTα1β2或LIGHT)信息的体外水平来进行筛选。高表达LT-β-R和/或LT-β-R配体(即,LTα1β2或LIGHT)信息的肿瘤组织文库将是候选者。本发明涉及的肿瘤类型包括实体瘤,它包括但不限于,非小细胞肺癌(NSCLC),结直肠癌(CRC),乳腺癌,以及前列腺、胃(gastric)、皮肤、胃部(stomach)、食道和膀胱等部位的癌症。
本发明用于治疗与不良细胞增生有关的疾病(尤其肿瘤)的人源化抗体优选抑制肿瘤细胞的生长,其判定标准可以是:例如,肿瘤体积减小约10%,20%,30%或40%以上,最优选减小约50%以上。人源化抗体通过筛选获得(例见实施例3)。例如,本发明所用人源化抗体可根据肿瘤体积相对于未处理的癌症细胞的减小来选择(例如,减小约10%,20%,30%,40%或50%以上)。
本发明还提供了包含本发明人源化抗体和可药用赋形剂的药用组合物。适当的载体,例如,以及它们的配制剂参见
Remington’Pharmaceutical Sciences,第16版,1980,Mack Publishing Co.,编者Oslo等。通常在配制剂中使用适当量的可药用盐来造成等渗。载体的实例有缓冲剂如盐水、Ringer溶液和葡萄糖溶液。溶液的pH优选约5-8,更优选约7.4-7.8。其它载体包括持续释放制剂,如由固体疏水性聚合物组成的半透性基质,为成形的颗粒,例如脂质体、膜或微粒体。本领域技术人员很清楚,根据给药途径和所给的药用组合物的浓度不同,还有更优选的载体。
给药可通过注射(如静脉内、腹腔内、皮下、肌肉内)或通过诸如灌注等确保有效运送至血流的其它方法来实现。
本发明的人源化抗体可以以有效剂量给与以便治疗具体的临床疾病。针对具体的应用,本领域技术人员完全可以根据,例如患者的体重和状态、治疗所要达到的程度以及患者对治疗的耐受程度,来确定优选的药物配制剂和进行有效治疗所需的剂量方案。例如,有效剂量可以是约0.05-100mg/kg体重/日。更优选约0.05mg,0.1mg,0.2mg,0.3mg,0.4mg,0.5mg,0.6mg,0.7mg,0.8mg,0.9mg,1mg,2mg,3mg,4mg,5mg,6mg,7mg,8mg,9mg,10mg,15mg,20mg,或25mg/kg体重/日。或者,约0.05-100mg,更优选约0.05mg,0.1mg,0.2mg,0.3mg,0.4mg,0.5mg,0.6mg,0.7mg,0.8mg,0.9mg,1mg,2mg,3mg,4mg,5mg,6mg,7mg,8mg,9mg,10mg,15mg,20mg,或25mg/kg体重/周。或者,约0.05-100mg,更优选约0.05mg,0.1mg,0.2mg,0.3mg,0.4mg,0.5mg,0.6mg,0.7mg,0.8mg,0.9mg,1mg,2mg,3mg,4mg,5mg,6mg,7mg,8mg,9mg,10mg,15mg,20mg,或25mg/kg体重/2周。或者约0.05-100mg,更优选约0.05mg,0.1mg,0.2mg,0.3mg,0.4mg,0.5mg,0.6mg,0.7mg,0.8mg,0.9mg,1mg,2mg,3mg,4mg,5mg,6mg,7mg,8mg,9mg,10mg,15mg,20mg,或25mg/kg体重/3周。或者约0.05-100mg,更优选约0.05mg,0.1mg,0.2mg,0.3mg,0.4mg,0.5mg,0.6mg,0.7mg,0.8mg,0.9mg,1mg,2mg,3mg,4mg,5mg,6mg,7mg,8mg,9mg,10mg,15mg,20mg,或25mg/kg体重/4周。
除非特别说明,本发明的实施可应用本领域内的细胞生物学,细胞培养,分子生物学,微生物学,重组DNA,蛋白质化学以及免疫学的常规技术。这类技术都有文献记载。例如,可参见Molecular Cloning:A LaboratoryManual,2nd edition.(Sambrook,Fritsch和Maniatis,eds.),Cold Spring HarborLaboratory Press,1989;DNA Cloning,Volumes I和II(D.N.Glover,ed),1985;Oligonucleotide Synthesis,(M.J.Gait,ed.),1984;U.S.Patent No.4,683,195(Mullis等,);Nucleic Acid Hybridization(B.D.Hames和S.J.Higgins,eds.),1984;Transcription和Translation(B.D.Hames和S.J.Higgins,eds.),1984;Culture of Animal Cells(R.I.Freshney,ed).Alan R.Liss,Inc.,1987;Immobilized Cells和Enzymes,IRL Press,1986;A Practical Guide toMolecular Cloning(B.Perbal),1984;Metbods in Enzymology,Volumes 154和155(Wu等,eds),Academic Press,New York;Gene Transfer Vectors forMammalian Cells(J.H.Miller和M.P.Calos,eds.),1987,Cold Spring HarborLaboratory;Immunochemical Methods in Cell和Molecular Biology(Mayer和Walker,eds.),Academic Press,London,1987;Handbook of ExperimentImmunology,Volumes I-IV(D.M.Weir和C.C.Blackwell,eds.),1986;Manipulating the Mouse Embryo,Cold Spring Harbor Laboratory Press,1986.
以下实施例旨在举例说明本发明,不应理解为对本发明的限制。
实施例:
实施例1
构建并表达chCBE11
用编码鼠CBE11重链和轻链可变区的cDNA来构建鼠-人嵌合体(chCBE11)的表达载体,其中muCBE11可变区与人的IgG1和κ恒定区相连。为了构建重链嵌合体,将来自CBE11重链亚克隆pEAG970的0.36kbPstI-BstEII片段,亚克隆至来自5a8(它是此前由Biogen所鉴定的CD4-特异性mAb的分子克隆形式)重链质粒pLCB7经磷酸酶处理的2.82kbPstI-BstEII载体片段中,从而在muCBE11重链可变区添加鼠重链信号序列和剪接供体位点。所得质粒为pEAG979,其中的重链成熟N-末端与经Edman降解得到的纯化的标准CBE11的重链N-末端序列有两个残基的差异,因为它在PCR过程中是通过引物来确定的。为了校正重链N-末端,利用Amersham Pharmacia Biotech USE诱变试剂盒,按照厂家建议对pEAG979进行唯一位点消除(USE)诱变。突变的质粒通过筛选所导入的AvaII,PstI,和RsaI改变来鉴定。所得质粒为pEAG981,其中的重链序列通过DNA测序来证实。将来自pEAG981的0.44kb NotI-HindIII重链可变区片段和来自pEAG964的1.21kb HindIII-NotI片段(含人IgG1恒定区),亚克隆至pCEP4(Invitrogen)EBV表达载体所衍生的质粒pCH269的NotI位点,得到质粒pEAG983。
为了构建轻链嵌合体,将来自pMDR985的0.11kb NotI-EcoRV片段和来自CBE11轻链可变区质粒pEAG967的0.37kb EcoRV-BamHI片段,亚克隆至Stratagene公司提供的pBluescriptIISK+克隆载体经磷酸酶处理的2.94kb NotI-BamHI载体片段,以便添加鼠轻链信号序列和5’NotI位点,所得质粒为pEAG978。利用Amersham Pharmacia Biotech USE诱变试剂盒,按照厂家建议对该质粒进行USE诱变,所用的诱变引物编码V3K取代,该引物与标准CBE11轻链的N-末端匹配,并可在轻链可变区的3’末端引入一个BglII位点。突变的质粒通过筛选所引入的BglII,EcoRV,和MseI位点改变来鉴定。所得质粒为pEAG980,其中的轻链序列通过DNA测序来证实。将来自pEAG980的0.41kb NotI-BglII轻链可变区片段和来自pEAG963的0.68kbBclI-NotI片段(含有人κ轻链恒定区),亚克隆至pCEP4(Invitrogen)EBV表达载体所衍生的质粒pCH269的NotI位点,得到质粒pEAG982。
用表达载体(chCBE11重链载体pEAG983和chCBE11轻链载体pEAG982)共转染293-EBNA细胞,检验转染的细胞的抗体分泌和抗体特异性(用被空载体转染的细胞以及被ch5c8转染的细胞作为对照,ch5c8是此前由Biogen所鉴定的CD154-特异性mAb的分子克隆形式)。全细胞裂解物以及经过细胞培养后的培养基用蛋白A进行免疫沉淀,对沉淀物的Western印迹分析(用抗人重链和轻链的抗体来显示)表明,被chCBE11-转染的细胞能够合成并有效分泌重链和轻链,其水平与被ch5c8-转染的细胞相似。用培养过转染细胞的培养基对表达LT-β-R的HT-29细胞进行染色,随后的FACS表明,chCBE11抗体被结合并产生类似于muCBE11的染色模式,而培养假转染细胞的培养基,以及培养被ch5c8转染的细胞的培养基都没能对HT-29细胞上的LT-β-R实现染色。将瞬时转染产生的嵌合CBE11纯化,用表达LT-β-R的A375黑素瘤细胞证实其诱导IL-8的分泌,还证实它能在裸鼠中抑制WiDRr腺癌细胞的生长。
实施例2
构建并表达huCBE11
如上述设计再成形的可变区,以制备人源化CBE11(huCBE11)。根据同源性匹配来选择人的受体框架:轻链选人κ亚组I mAb TNF-A1(Griiffths等,1993),重链选人亚组III mAb FLA-IgG(Malisan等,1996)。每种可变轻链共设计了三种形式,再成形的可变重链设计了四种形式。一般第1种形式包含向鼠供体序列回复得最多的突变,而最后一种形式包含回复得最少的突变(即,最为“人源化”)。
用Amersham Pharmacia Biotech USE诱变试剂盒按照厂家建议经唯一位点消除法(USE)制备huCBE11可变区,其利用了chCBE11可变区质粒作为起始模板。引起框架(FR)变化的诱变引物如下。使用人的受体框架的cDNA序列(轻链为Kabat数据库#004770,重链为Kabat#040003),其中引入了静默突变以便产生有利于鉴定突变质粒的限制性位点变化。突变的质粒通过检查所引入的限制性位点变化来鉴定。所得质粒中的可变区cDNA序列通过DNA测序证实。
VH#1以pEAG981为模板,并使用以下引物:FR1引物5’GCC TGGAGG GTC CCT GAG GCT CTC CTG TGC AGC CTC 3’(SEQ ID NO:17),它引入了一个Bsu36I位点;FR2引物5’GTT TCG CCA GAC TCC GGG AAAGGG GCT GGA GTG GGT CGC AAC 3’(SEQ ID NO:18),它引入了NciI和HpaII位点;FR3引物5’CAG AGA CAA TGC CAA GAA CAG CCT CTA CCTGCA GAT GAG CAG CCT GAG GGC TGA GGA CAC AGC CAT G 3’(SEQID NO:19),它引入了Bsu36I和PstI位点、并除去了一个RsaI位点。所得VH#1质粒为pAND067。
VH#2以pAND067为模板,并使用以下引物:FR2引物5’CAT GTA TTGGTT TCG CCA GGC CCC GGG AAA GGG GCT GG 3’(SEQ ID NO:20),它引入了一个SmaI位点;FR3引物5’GGG CTG AGG ACA CAG CTG TGT ATTACT GTG TAA GAG 3’(SEQ ID NO:21),它引入了一个PVuII位点。所得VH#2质粒为pAND071。
VH#3以质粒pAND067为模板,并使用以下引物:FR2引物5’GTG ACTATT ACA TGT ATT GGG TGC GCC AGG CCC CGG GAA AGG GGC TGGAG 3’(SEQ ID NO:22),它引入了SmaI和HhaI位点;FR3引物5’GAG GGCTGA GGA CAC AGC TGT GTA TTA CTG CGC AAG AGA GGA GAA TGGTAA C 3’(SEQ ID NO:23),它引入了PvuII和FspI位点。所得VH#3质粒为pAND075。
huCBE11重链的表达载体如下制备:将来自pAND067,pAND071,或pAND075的0.44kb NotI-HindIII重链可变区片段,和来自pEAG964的1.21kb HindIII-NotI片段(含人IgG1恒定区),亚克隆至pCEP4 EBV表达载体所衍生的质粒pCH269中的NotI位点,从而得到重链表达载体pAND069(VH#1),pAND073(VH#2),和pAND077(VH#3)。
VL#1以质粒pEAG980为模板,并使用以下引物:FR1引物5’CTT GCAAGT GAT AGT GAC CCT GTC TCC CAC CGA TGC AGA CAA GGA TGATGG AGA CTG GGT CAT C 3’(SEQ ID NO:24),它除去了HinfI和NsiI位点;FR2引物5’CAT AAT AGA TCA GGA TCT TAG GCG CTT TCC ATG GTTTCT GCT G 3’(SEQ ID NO:25),它引入了HaeII和HhaI位点;FR3引物5’GTA GAC AGT AAT AAG TTG CGA AAT CCT CAG GCT GCA GGC TGCTGA TGG TTA GAG TAT AAT CTT GCC CAG ATC 3’(SEQ ID NO:26),它引入了Bsu36I,DdeI,和PstI位点。所得VL#1质粒为pAND066。
VL#2以质粒pAND066为模板,并使用以下引物:FR1引物5’GAT GGAGAC TGG GTC ATC TGG ATA TCA CCT CTG GCA CCT G 3’(SEQ IDNO:27),它引入了一个EcoRV位点;FR3引物5’GAT GGT TAG AGT ATAATC TGT ACC AGA TCC ACT GCC ACT G 3’(SEQ ID NO:28),它引入了一个RsaI位点。所得VL#2质粒为pAND070。
VL#3以质粒pAND066为模板,并使用以下引物:FR1引物5’GAT GGAGAC TGG GTC ATC TGG ATA TCA CCT CTG GCA CCT G 3’(SEQ IDNO:29),它引入了一个EcoRV位点;FR2引物5’CAA CCT TGT TGC ATAATA GAT CAG AAG CTT AGG CGC TTT CCC TGG TTT CTG CTG GTA CC3’(SEQ ID NO:30),它引入了一个HindIII位点,它还除去了NcoI和StyI位点;FR3引物5’GAT GGT TAG AGT ATA ATC TGT ACC AGA TCC ACTGCC ACT G 3’(SEQ ID NO:31),它引入了一个RsaI位点。所得VL#3质粒为pAND074。
huCBE11轻链的表达载体如下制备:将来自pAND066,pAND070,或pAND074的0.41kb NotI-BglII轻链可变区片段,和来自pEAG963的0.68kbBclI-NotI片段(含人κ轻链恒定区),亚克隆至pCEP4 EBV表达载体所衍生的质粒pCH269中的NotI位点,从而得到轻链表达载体pAND068(VL#1),pAND072(VL#2),和pAND076(VL#3)。
将表达载体共转染至293-EBNA细胞中,检验转染的细胞的抗体分泌和抗体特异性(用被空载体转染的细胞作为对照)。全细胞裂解物以及经过细胞培养后的培养基用蛋白A进行免疫沉淀,对沉淀物的Western印迹分析(用抗人重链和轻链的抗体来显示)表明,被huCBE11-转染的细胞能够合成并有效分泌重链和轻链,其水平与被chCBE11-转染的细胞相似。用培养过转染细胞的培养基对表达LT-β-R的HT-29细胞进行染色,随后的FACS表明,相对于chCBE11而言,huCBE11#3mAb的结合不及huCBE11#1和huCBE11#2这两种mAb,见下表1,其中huCBE11#1(VL#1+VH#1);huCBE#2(VL#2+VH#2)和huCBE11#3(VL#3+VH#3)。混合和匹配共转染提示:结合减弱可能是VH#3所致,它有两个框架残基不同于VH#2:FR2 F37V和FR3V93A。为了检测这些变化各自造成的影响,构建了新的重链表达载体。质粒pAND089是VH#2的F37V变体,它如下制备:将来自pAND075的311bp NotI-PstI片段,来自pAND071的126bp PstI-HindIII片段和来自pEAG964的1.21kb HindIII-NotI片段,亚克隆至pCEP4 EBV表达载体所衍生的质粒pCH269的NotI位点。质粒pAND090是VH#2的V93A变体,它如下制备:将来自pAND071的311bp NotI-PstI片段,来自pAND075的126bp PstI-HindIII片段和来自pEAG964的1.21kb HindIII-NotI片段,亚克隆至pCEP4 EBV表达载体所衍生的质粒pCH269的NotI位点。将这些H2/H3嵌合重链与VL#2或VL#3一起共转染293-EBNA细胞。FACS分析表明,V93AH2变体当与VL#3配合时恢复了LT-β-R结合(见表1)。pAND076和pAND090配对被称为huCBE11#4(见表1)。
用不同形式的chCBE11和huCBE11(#1-4)对293-EBNA细胞进行的共转染按比例递增,收获经过培养的培养基。将抗体在蛋白A-Sepharose上纯化。分析纯化后mAb的活性。
表1.chCBE11和huCBE11对HT-29细胞的FACS染色
轻链 重链
相对MFI
ChCBE11 pEAG982 pEAG983 1.00
HuCBE11#1 pAND068 pAND069 1.00
HuCBE11#2 pAND072 pAND073 1.00
HuCBE11#3 pAND076 pAND077 0.62
L2/H3 pAND072 pAND077 0.42
L3/H2 pAND076 pAND073 1.00
L2/F37VH2 pAND072 pAND089 0.65
L2/V93AH2 pAND072 pAND090 0.75
L3/F37VH2 pAND076 pAND089 0.80
HuCBE11#4 pAND076 pAND090 1.00
用培养了瞬时转染的细胞的培养基通过在冰上静置30min而对HT-29细胞进行染色,将细胞用FACS缓冲液(PBS,含5%FBS和0.05%叠氮钠)洗两次,用偶联了PE抗人IgG(H+L)(Jackson ImmunoResearch Laboratories,Inc.)在冰上的FACS缓冲液中染色30min,细胞用FACS缓冲液洗两次,然后重悬于FACS缓冲液中以备进一步分析。相对MFI是指chCBE11观测值经标准化以后的平均MFI。所显示的数据代表两轮独立的转染的平均值。
实施例3
IL-8对A375细胞的激动作用(Agonism)
分析纯化mAb的活性。在A375人黑素瘤细胞上进行的IL-8释放试验的结果见图1,它测量了抗LT-β-R抗体与表达在A375人黑素瘤细胞表面的LT-β-R结合所致的IL-8释放的量。将105/ml的A375细胞加入96孔板,板中含有可溶性抗体、或捕获在包被了山羊抗人IgG Fc(JacksonImmunoResearch Laboratories)的孔中的抗体。将培养平板保温过夜。收获培养后的培养基,通过ELISA分析IL-8。
实施例4
对WiDr细胞的细胞毒活性
用WiDr结肠癌细胞以及可溶性抗LT-β-R抗体在包被了抗人IgG Fc的孔中进行细胞毒试验,结果证实,抗LT-β-R抗体在癌细胞中增强了细胞毒活性,见图2。在有80单位/ml huIFN-γ存在的条件下,将6×104/ml的WiDr细胞加入96孔板,板中含有可溶性抗体、或捕获在包被了山羊抗人IgG Fc(Jackson ImmunoResearch Laboratories)的孔中的抗体。将培养板保温5天。加入MTT,维持4小时,所得沉淀通过与10% SDS的10mM HCl溶液保温过夜而溶解,在微滴板读取仪上读取O.D.值。
实施例5
制备由再成形的huCBE11第3种可变轻链(VL#3)和再成形的huCBE11第4种可变重链(VH#4)组成的抗体,命名为huCBE11#4或hCBE11。将产生所述抗体的细胞系保藏在A.T.C.C.(ATCC专利保藏物编号为PTA-3357)。轻链和重链各自的全长多肽序列如下(包括恒定区):
成熟huCBE11第3种轻链的序列(SEQ ID NO:32):
1 DIQMTQSPSS LSASVGDRVT ITC
KAGQDIK
SYLSWYQQKP GKAPKLLIY
Y
51
ATRLADGVPS RFSGSGSGTD YTLTISSLQP EDFATYYC
LQ
HGESPWTFGG
101 GTKLEIK [RTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV
151 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG
201 LSSPVTKSFN RGEC]
CDR用下划线标示,回复突变F71Y以粗体标示,恒定区用括号标示。
成熟huCBE11第4种重链的序列(SEQ ID NO:33):
V37F S49A
1 EVQLVESGGG LVKPGGSLRL SCAASGFTF
S
DYYMYWFRQA PGKGLEWVA
T
51
ISDGGSYTYY PDSVKGRFTI SRDNAKNSLY LQMSSLRAED TAVYYCAR
EE
101
NGNFYYFDYW GQGTTVTVSS[ASTKGPSVFP LAPSSKSTSG GTAALGCLVK
151 DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT
201 YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP
251 KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN
301 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ
351 VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV
401 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG]
CDR用下划线标示,回复突变V37F和S49A以粗体标示,恒定区用括号标示。
6周龄裸鼠腹腔注射100ug抗LFA3抗体(1E6),100ug抗LTBR抗体(huCBE11#4)或不接受注射(对照)。然后给它们皮下注射1×106WIDR结肠腺癌细胞。经huCBE11#4-处理的小鼠每周再用100ug抗体处理,mCBE11小鼠仅在第14天进行再处理。每周测量肿瘤大小并计算肿瘤球体积(图3)。当肿瘤体积达到2.0cm3(直径16mm)时,处死小鼠,将它们的死亡标注在存活曲线中(图4)。
实施例6
6周龄裸鼠腹腔注射100ug抗LTBR抗体(huCBE11#4)或不接受注射(对照)。然后给它们皮下注射1×106WIDR结肠腺癌细胞。经huCBE11#4-处理的小鼠每周再用100ug huCBE11#4处理。每周测量肿瘤大小并计算肿瘤球体积。所示肿瘤体积代表10只对照小鼠和8只huCBE11#4-处理鼠的均值(图5)。
每周用huCBE11#4处理显著抑制了经皮下方式植入裸鼠的WIDR肿瘤的生长速率。经抗体连续处理21天的小鼠,在停止处理后的2周继续表现出肿瘤生长速率的减小。
实施例7
huCBE11#4 使已经长成的WIDR肿瘤的生长减缓并使携有WIDR肿瘤的裸鼠的存活延长
106 WIDR细胞已在裸鼠皮下生长10天。给小鼠皮下注射PBS或huCBE11#4(每周)或mCBE11(隔周)。测量肿瘤的宽度和长度,以此计算肿瘤重量。将肿瘤超过2000mg的小鼠处死,处死当时的肿瘤重量被纳入统计学平均值的计算中。错误柱代表标准误。用以下公式计算肿瘤重量:(宽×宽×长)/2=肿瘤重量的mg数。结果见图6,可看出huCBE11#4能在体内减缓已生成的肿瘤的生长。
此外,如上述使肿瘤生长并进行处理,测量小鼠的存活百分比。结果见图7,可看出huCBE11#4能使体内已有肿瘤生成的小鼠延长存活期。
实施例8
测量抗体亲和力
将HT-29细胞在添加了L-谷氨酰胺、非必需氨基酸、丙酮酸钠和10%胎牛血清的DMEM中培养。细胞用PBS洗一次,通过与添加了20mM EDTA的PBS室温保温5分钟而从平板中取出。将细胞以1000rpm(110×g)离心5分钟,然后以1×107细胞/mL重悬在PBS中。
将HuCBE11#4抗LTβR抗体和作为阴性对照的人源化抗CD40L用PBS稀释,按照1∶4稀释12个梯度,使终浓度范围为2.37pM-10μM。将100μL细胞悬液和100μL抗体稀释液一起添加至96孔微滴板的V形底的孔中。使抗体和细胞在4℃保温2小时。将平板在4℃以1000rpm(110×g)离心10分钟。弃去上清,细胞沉淀用冷PBS洗6次。
山羊-抗人IgG-藻红蛋白偶联物(Jackson Immunoresearch)用PBS进行1∶100稀释,每孔加200μL。使细胞与此种第二抗体在4℃保温1小时,如上述离心,用冷PBS洗一次。然后将细胞转移至聚苯乙烯试管中。在FACSCalibur仪(Beckton Dickinson)上测量荧光强度。
在Delta图中,将抗CD40L非特异性结合对照的染色的平均荧光强度值对抗体浓度作图。所述值成一条直线,将每一抗体浓度的非特异性结合理论值从huCBE11#4稀释系列的每一数值中减去。
然后,将这些特异性荧光强度值对huCBE11#4浓度的对数值作图。所得曲线为钟型,对称状,它反映了高浓度时抗体结合的自我抑制。该曲线的左半部分可用一种4参数等式找出所述抗体的功能性亲和力。从所得曲线可知60pM huCBE11#4与HT-29结合的EC50值。
本领域技术人员显然明了,在不背离本发明精神或超出本发明范围的前提下,可以对本发明的多肽、组合物和方法进行各种修改和变动。因此,本发明包括了落在所附权利要求范围内的对本发明进行的修改和变动以及它们的等效形式。
序 列 表
<110>比奥根公司(BIOGEN,INC.)
艾伦·加伯(GARBER,Ellen)
保罗·莱恩(LYNE,Paul)
乔斯·W·塞尔丹哈(SALDHANA,Jose W.)
<120>人源化抗-LT-β-R抗体
<130>A100 PCT
<150>60/240,285
<151>2000-10-13
<150>60/275,289
<151>2001-03-13
<150>60/299,987
<151>2001-06-21
<160>33
<170>FastSEQ for Windows Version 4.0
<210>1
<211>107
<212>PRT
<213>鼠
<400>1
Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Thr Ile Thr Cys Lys Ala Gly Gln Asp Ile Lys Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Trp Lys Ser Pro Lys Ile Leu Ile
35 40 45
Tyr Tyr Ala Thr Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser
65 70 75 80
Asp Asp Thr Ala Thr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210>2
<211>120
<212>PRT
<213>鼠
<400>2
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Tyr Met Tyr Trp Phe Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Val Arg Glu Glu Asn Gly Asn Phe Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210>3
<211>321
<212>DNA
<213>人(Homo Sapien)
<400>3
gatattaaga tgacccagtc tccatcatcc ttgtctgcat cggtgggaga cagggtcact 60
atcacttgca aggcgggtca ggacattaaa agctatttaa gctggtacca gcagaaacca 120
tggaaagcgc ctaagatcct gatctattat gcaacaaggt tggcagatgg ggtcccatca 180
agattcagtg gcagtggatc tgggcaagat tatactctaa ccatcagcag cctgcagcct 240
gaggatttcg caacttatta ctgtctacag catggtgaga gcccgtggac gttcggtgga 300
ggcaccaagc tggagatcaa a 321
<210>4
<211>107
<212>PRT
<213>人(Homo Sapien)
<400>4
Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Gly Gln Asp Ile Lys Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Trp Lys Ala Pro Lys Ile Leu Ile
35 40 45
Tyr Tyr Ala Thr Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gln Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210>5
<211>321
<212>DNA
<213>人(Homo Sapien)
<400>5
gatatccaga tgacccagtc tccatcatcc ttgtctgcat cggtgggaga cagggtcact 60
atcacttgca aggcgggtca ggacattaaa agctatttaa gctggtacca gcagaaacca 120
tggaaagcgc ctaagatcct gatctattat gcaacaaggt tggcagatgg ggtcccatca 180
agattcagtg gcagtggatc tggtacagat tatactctaa ccatcagcag cctgcagcct 240
gaggatttcg caacttatta ctgtctacag catggtgaga gcccgtggac gttcggtgga 300
ggcaccaagc tggagatcaa a 321
<210>6
<211>107
<212>PRT
<213>人(Homo Sapien)
<400>6
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Gly Gln Asp Ile Lys Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Trp Lys Ala Pro Lys Ile Leu Ile
35 40 45
Tyr Tyr Ala Thr Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210>7
<211>321
<212>DNA
<213>人(Homo Sapien)
<400>7
gatatccaga tgacccagtc tccatcatcc ttgtctgcat cggtgggaga cagggtcact 60
atcacttgca aggcgggtca ggacattaaa agctatttaa gctggtacca gcagaaacca 120
gggaaagcgc ctaagcctct gatctattat gcaacaaggt tggcagatgg ggtcccatca 180
agattcagtg gcagtggatc tggtacagat tatactctaa ccatcagcag cctgcagcct 240
gaggatttcg caacttatta ctgtctacag catggtgaga gcccgtggac gttcggtgga 300
ggcaccaagc tggagatcaa a 321
<210>8
<211>107
<212>PRT
<213>人(Homo Sapien)
<400>8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Gly Gln Asp Ile Lys Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Ala Thr Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210>9
<211>360
<212>DNA
<213>人(Homo Sapien)
<400>9
gaggtacaac tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaggctc 60
tcctgtgcag cctctggatt cactttcagt gactattaca tgtattggtt tcgccagact 120
ccgggaaagg ggctggagtg ggtcgcaacc attagtgatg gtggtagtta cacctactat 180
ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa cagcctctac 240
ctgcagatga gcagcctgag ggctgaggac acagccatgt attactgtgt aagagaggag 300
aatggtaact tttactactt tgactactgg ggccaaggga ccacggtcac cgtctcctca 360
<210>10
<211>120
<212>PRT
<213>人(Homo Sapien)
<400>10
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Tyr Met Tyr Trp Phe Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Val Arg Glu Glu Asn Gly Asn Phe Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210>11
<211>360
<212>DNA
<213>人(Homo Sapien)
<400>11
gaggtacaac tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaggctc 60
tcctgtgcag cctctggatt cactttcagt gactattaca tgtattggtt tcgccaggcc 120
ccgggaaagg ggctggagtg ggtcgcaacc attagtgatg gtggtagtta cacctactat 180
ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa cagcctctac 240
ctgcagatga gcagcctgag ggctgaggac acagctgtgt attactgtgt aagagaggag 300
aatggtaact tttactactt tgactactgg ggccaaggga ccacggtcac cgtctcctca 360
<210>12
<211>120
<212>PRT
<213>人(Homo Sapien)
<400>12
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Tyr Met Tyr Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Arg Glu Glu Asn Gly Asn Phe Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210>13
<211>360
<212>DNA
<213>人(Homo Sapien)
<400>13
gaggtacaac tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaggctc 60
tcctgtgcag cctctggatt cactttcagt gactattaca tgtattggtt tcgccaggcc 120
ccgggaaagg ggctggagtg ggtcgcaacc attagtgatg gtggtagtta cacctactat 180
ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa cagcctctac 240
ctgcagatga gcagcctgag ggctgaggac acagctgtgt attactgtgt aagagaggag 300
aatggtaact tttactactt tgactactgg ggccaaggga ccacggtcac cgtctcctca 360
<210>14
<211>120
<212>PRT
<213>人(Homo Sapien)
<400>14
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Glu Asn Gly Asn Phe Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210>15
<211>360
<212>DNA
<213>人(Homo Sapien)
<400>15
gaggtacaac tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaggctc 60
tcctgtgcag cctctggatt cactttcagt gactattaca tgtattggtt tcgccaggcc 120
ccgggaaagg ggctggagtg ggtcgcaacc attagtgatg gtggtagtta cacctactat 180
ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa cagcctctac 240
ctgcagatga gcagcctgag ggctgaggac acagctgtgt attactgcgc aagagaggag 300
aatggtaact tttactactt tgactactgg ggccaaggga ccacggtcac cgtctcctca 360
<210>16
<211>120
<212>PRT
<213>人(Homo Sapien)
<400>16
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Tyr Met Tyr Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Glu Asn Gly Asn Phe Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210>17
<211>36
<212>DNA
<213>人(Homo Sapien)
<400>17
gcctggaggg tccctgaggc tctcctgtgc agcctc 36
<210>18
<211>42
<212>DNA
<213>人(Homo Sapien)
<400>18
gtttcgccag actccgggaa aggggctgga gtgggtcgca ac 42
<210>19
<211>67
<212>DNA
<213>人(Homo Sapien)
<400>19
cagagacaat gccaagaaca gcctctacct gcagatgagc agcctgaggg ctgaggacac 60
agccatg 67
<210>20
<211>38
<212>DNA
<213>人(Homo Sapien)
<400>20
catgtattgg tttcgccagg ccccgggaaa ggggctgg 38
<210>21
<211>36
<212>DNA
<213>人(Homo Sapien)
<400>21
gggctgagga cacagctgtg tattactgtg taagag 36
<210>22
<211>50
<212>DNA
<213>人(Homo Sapien)
<400>22
gtgactatta catgtattgg gtgcgccagg ccccgggaaa ggggctggag 50
<210>23
<211>52
<212>DNA
<213>人(Homo Sapien)
<400>23
gagggctgag gacacagctg tgtattactg cgcaagagag gagaatggta ac 52
<210>24
<211>64
<212>DNA
<213>人(Homo Sapien)
<400>24
cttgcaagtg atagtgaccc tgtctcccac cgatgcagac aaggatgatg gagactgggt 60
catc 64
<210>25
<211>43
<212>DNA
<213>人(Homo Sapien)
<400>25
cataatagat caggatctta ggcgctttcc atggtttctg ctg 43
<210>26
<211>72
<212>DNA
<213>人(Homo Sapien)
<400>26
gtagacagta ataagttgcg aaatcctcag gctgcaggct gctgatggtt agagtataat 60
cttgcccaga tc 72
<210>27
<211>40
<212>DNA
<213>人(Homo Sapien)
<400>27
gatggagact gggtcatctg gatatcacct ctggcacctg 40
<210>28
<211>40
<212>DNA
<213>人(Homo Sapien)
<400>28
gatggttaga gtataatctg taccagatcc actgccactg 40
<210>29
<211>40
<212>DNA
<213>人(Homo Sapien)
<400>29
gatggagact gggtcatctg gatatcacct ctggcacctg 40
<210>30
<211>59
<212>DNA
<21 3>人(Homo Sapien)
<400>30
caaccttgtt gcataataga tcagaagctt aggcgctttc cctggtttct gctggtacc 59
<210>31
<211>40
<212>DNA
<213>人(Homo Sapien)
<400>31
gatggttaga gtataatctg taccagatcc actgccactg 40
<210>32
<211>663
<212>PRT
<213>人(Homo Sapien)
<400>32
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Gly Gln Asp Ile Lys Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Ala Thr Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly
210 215 220
Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
225 230 235 240
Phe Thr Phe Ser Asp Tyr Tyr Met Tyr Trp Phe Arg Gln Ala Pro Gly
245 250 255
Lys Gly Leu Glu Trp Val Ala Thr Ile Ser Asp Gly Gly Ser Tyr Thr
260 265 270
Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
275 280 285
Ala Lys Asn Ser Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp
290 295 300
Thr Ala Val Tyr Tyr Cys Ala Arg Glu Glu Asn Gly Asn Phe Tyr Tyr
305 310 315 320
Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser
325 330 335
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
340 345 350
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
355 360 365
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
370 375 380
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
385 390 395 400
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
405 410 415
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
420 425 430
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
435 440 445
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
450 455 460
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
465 470 475 480
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
485 490 495
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
500 505 510
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
515 520 525
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
530 535 540
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
545 550 555 560
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
565 570 575
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
580 585 590
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
595 600 605
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
610 615 620
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
625 630 635 640
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
645 650 655
Ser Leu Ser Leu Ser Pro Gly
660
<210>33
<211>4852
<212>PRT
<213>人(Homo Sapien)
<400>33
Pro Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu
1 5 10 15
Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Gly Gln Asp Ile Lys Ser
20 25 30
Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Trp Lys Ser Pro Lys Ile Leu
35 40 45
Ile Tyr Tyr Ala Thr Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu
65 70 75 80
Ser Asp Asp Thr Ala Thr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro
85 90 95
Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Pro Glu Val Gln
100 105 110
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys
115 120 125
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr Met Tyr
130 135 140
Trp Phe Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr Ile
145 150 155 160
Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg
165 170 175
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr Leu Gln Met
180 185 190
Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg Glu
195 200 205
Glu Asn Gly Asn Phe Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr
210 215 220
Val Thr Val Ser Ser Asp Gly Ala Thr Ala Thr Thr Ala Ala Gly Ala
225 230 235 240
Thr Gly Ala Cys Cys Cys Ala Gly Thr Cys Thr Cys Cys Ala Thr Cys
245 250 255
Ala Thr Cys Cys Thr Thr Gly Thr Cys Thr Gly Cys Ala Thr Cys Gly
260 265 270
Gly Thr Gly Gly Gly Ala Gly Ala Cys Ala Gly Gly Gly Thr Cys Ala
275 280 285
Cys Thr Ala Thr Cys Ala Cys Thr Thr Gly Cys Ala Ala Gly Gly Cys
290 295 300
Gly Gly Gly Thr Cys Ala Gly Gly Ala Cys Ala Thr Thr Ala Ala Ala
305 310 315 320
Ala Gly Cys Thr Ala Thr Thr Thr Ala Ala Gly Cys Thr Gly Gly Thr
325 330 335
Ala Cys Cys Ala Gly Cys Ala Gly Ala Ala Ala Cys Cys Ala Thr Gly
340 345 350
Gly Ala Ala Ala Gly Cys Gly Cys Cys Thr Ala Ala Gly Ala Thr Cys
355 360 365
Cys Thr Gly Ala Thr Cys Thr Ala Thr Thr Ala Thr Gly Cys Ala Ala
370 375 380
Cys Ala Ala Gly Gly Thr Thr Gly Gly Cys Ala Gly Ala Thr Gly Gly
385 390 395 400
Gly Gly Thr Cys Cys Cys Ala Thr Cys Ala Ala Gly Ala Thr Thr Cys
405 410 415
Ala Gly Thr Gly Gly Cys Ala Gly Thr Gly Gly Ala Thr Cys Thr Gly
420 425 430
Gly Gly Cys Ala Ala Gly Ala Thr Thr Ala Thr Ala Cys Thr Cys Thr
435 440 445
Ala Ala Cys Cys Ala Thr Cys Ala Gly Cys Ala Gly Cys Cys Thr Gly
450 455 460
Cys Ala Gly Cys Cys Thr Gly Ala Gly Gly Ala Thr Thr Thr Cys Gly
465 470 475 480
Cys Ala Ala Cys Thr Thr Ala Thr Thr Ala Cys Thr Gly Thr Cys Thr
485 490 495
Ala Cys Ala Gly Cys Ala Thr Gly Gly Thr Gly Ala Gly Ala Gly Cys
500 505 510
Cys Cys Gly Thr Gly Gly Ala Cys Gly Thr Thr Cys Gly Gly Thr Gly
515 520 525
Gly Ala Gly Gly Cys Ala Cys Cys Ala Ala Gly Cys Thr Gly Gly Ala
530 535 540
Gly Ala Thr Cys Ala Ala Ala Pro Asp Ile Lys Met Thr Gln Ser Pro
545 550 555 560
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys
565 570 575
Ala Gly Gln Asp Ile Lys Ser Tyr Leu Ser Trp Tyr Gln Gln Lys Pro
580 585 590
Trp Lys Ala Pro Lys Ile Leu Ile Tyr Tyr Ala Thr Arg Leu Ala Asp
595 600 605
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Thr
610 615 620
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
625 630 635 640
Leu Gln His Gly Glu Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu
645 650 655
Glu Ile Lys Asp Gly Ala Thr Ala Thr Cys Cys Ala Gly Ala Thr Gly
660 665 670
Ala Cys Cys Cys Ala Gly Thr Cys Thr Cys Cys Ala Thr Cys Ala Thr
675 680 685
Cys Cys Thr Thr Gly Thr Cys Thr Gly Cys Ala Thr Cys Gly Gly Thr
690 695 700
Gly Gly Gly Ala Gly Ala Cys Ala Gly Gly Gly Thr Cys Ala Cys Thr
705 710 715 720
Ala Thr Cys Ala Cys Thr Thr Gly Cys Ala Ala Gly Gly Cys Gly Gly
725 730 735
Gly Thr Cys Ala Gly Gly Ala Cys Ala Thr Thr Ala Ala Ala Ala Gly
740 745 750
Cys Thr Ala Thr Thr Thr Ala Ala Gly Cys Thr Gly Gly Thr Ala Cys
755 760 765
Cys Ala Gly Cys Ala Gly Ala Ala Ala Cys Cys Ala Thr Gly Gly Ala
770 775 780
Ala Ala Gly Cys Gly Cys Cys Thr Ala Ala Gly Ala Thr Cys Cys Thr
785 790 795 800
Gly Ala Thr Cys Thr Ala Thr Thr Ala Thr Gly Cys Ala Ala Cys Ala
805 810 815
Ala Gly Gly Thr Thr Gly Gly Cys Ala Gly Ala Thr Gly Gly Gly Gly
820 825 830
Thr Cys Cys Cys Ala Thr Cys Ala Ala Gly Ala Thr Thr Cys Ala Gly
835 840 845
Thr Gly Gly Cys Ala Gly Thr Gly Gly Ala Thr Cys Thr Gly Gly Thr
850 855 860
Ala Cys Ala Gly Ala Thr Thr Ala Thr Ala Cys Thr Cys Thr Ala Ala
865 870 875 880
Cys Cys Ala Thr Cys Ala Gly Cys Ala Gly Cys Cys Thr Gly Cys Ala
885 890 895
Gly Cys Cys Thr Gly Ala Gly Gly Ala Thr Thr Thr Cys Gly Cys Ala
900 905 910
Ala Cys Thr Thr Ala Thr Thr Ala Cys Thr Gly Thr Cys Thr Ala Cys
915 920 925
Ala Gly Cys Ala Thr Gly Gly Thr Gly Ala Gly Ala Gly Cys Cys Cys
930 935 940
Gly Thr Gly Gly Ala Cys Gly Thr Thr Cys Gly Gly Thr Gly Gly Ala
945 950 955 960
Gly Gly Cys Ala Cys Cys Ala Ala Gly Cys Thr Gly Gly Ala Gly Ala
965 970 975
Thr Cys Ala Ala Ala Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
980 985 990
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Gly
995 1000 1005
Gln Asp Ile Lys Ser Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Trp Lys
1010 1015 1020
Ala Pro Lys Ile Leu Ile Tyr Tyr Ala Thr Arg Leu Ala Asp Gly Val
1025 1030 1035 1040
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr
1045 1050 1055
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln
1060 1065 1070
His Gly Glu Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
1075 1080 1085
Lys Asp Gly Ala Thr Ala Thr Cys Cys Ala Gly Ala Thr Gly Ala Cys
1090 1095 1100
Cys Cys Ala Gly Thr Cys Thr Cys Cys Ala Thr Cys Ala Thr Cys Cys
1105 1110 1115 1120
Thr Thr Gly Thr Cys Thr Gly Cys Ala Thr Cys Gly Gly Thr Gly Gly
1125 1130 1135
Gly Ala Gly Ala Cys Ala Gly Gly Gly Thr Cys Ala Cys Thr Ala Thr
1140 1145 1150
Cys Ala Cys Thr Thr Gly Cys Ala Ala Gly Gly Cys Gly Gly Gly Thr
1155 1160 1165
Cys Ala Gly Gly Ala Cys Ala Thr Thr Ala Ala Ala Ala Gly Cys Thr
1170 1175 1180
Ala Thr Thr Thr Ala Ala Gly Cys Thr Gly Gly Thr Ala Cys Cys Ala
1185 1190 1195 1200
Gly Cys Ala Gly Ala Ala Ala Cys Cys Ala Gly Gly Gly Ala Ala Ala
1205 1210 1215
Gly Cys Gly Cys Cys Thr Ala Ala Gly Cys Cys Thr Cys Thr Gly Ala
1220 1225 1230
Thr Cys Thr Ala Thr Thr Ala Thr Gly Cys Ala Ala Cys Ala Ala Gly
1235 1240 1245
Gly Thr Thr Gly Gly Cys Ala Gly Ala Thr Gly Gly Gly Gly Thr Cys
1250 1255 1260
Cys Cys Ala Thr Cys Ala Ala Gly Ala Thr Thr Cys Ala Gly Thr Gly
1265 1270 1275 1280
Gly Cys Ala Gly Thr Gly Gly Ala Thr Cys Thr Gly Gly Thr Ala Cys
1285 1290 1295
Ala Gly Ala Thr Thr Ala Thr Ala Cys Thr Cys Thr Ala Ala Cys Cys
1300 1305 1310
Ala Thr Cys Ala Gly Cys Ala Gly Cys Cys Thr Gly Cys Ala Gly Cys
1315 1320 1325
Cys Thr Gly Ala Gly Gly Ala Thr Thr Thr Cys Gly Cys Ala Ala Cys
1330 1335 1340
Thr Thr Ala Thr Thr Ala Cys Thr Gly Thr Cys Thr Ala Cys Ala Gly
1345 1350 1355 1360
Cys Ala Thr Gly Gly Thr Gly Ala Gly Ala Gly Cys Cys Cys Gly Thr
1365 1370 1375
Gly Gly Ala Cys Gly Thr Thr Cys Gly Gly Thr Gly Gly Ala Gly Gly
1380 1385 1390
Cys Ala Cys Cys Ala Ala Gly Cys Thr Gly Gly Ala Gly Ala Thr Cys
1395 1400 1405
Ala Ala Ala Pro AsD Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
1410 1415 1420
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Gly Gln Asp
1425 1430 1435 1440
Ile Lys Ser Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
1445 1450 1455
Lys Leu Leu Ile Tyr Tyr Ala Thr Arg Leu Ala Asp Gly Val Pro Ser
1460 1465 1470
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser
1475 1480 1485
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Gly
1490 1495 1500
Glu Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Asp
1505 1510 1515 1520
Gly Ala Gly Gly Thr Ala Cys Ala Ala Cys Thr Gly Gly Thr Gly Gly
1525 1530 1535
Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr
1540 1545 1550
Ala Gly Thr Gly Ala Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly
1555 1560 1565
Thr Cys Cys Cys Thr Gly Ala Gly Gly Cys Thr Cys Thr Cys Cys Thr
1570 1575 1580
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr
1585 1590 1595 1600
Cys Ala Cys Thr Thr Thr Cys Ala Gly Thr Gly Ala Cys Thr Ala Thr
1605 1610 1615
Thr Ala Cys Ala Thr Gly Thr Ala Thr Thr Gly Gly Thr Thr Thr Cys
1620 1625 1630
Gly Cys Cys Ala Gly Ala Cys Thr Cys Cys Gly Gly Gly Ala Ala Ala
1635 1640 1645
Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly Gly Gly Thr Cys
1650 1655 1660
Gly Cys Ala Ala Cys Cys Ala Thr Thr Ala Gly Thr Gly Ala Thr Gly
1665 1670 1675 1680
Gly Thr Gly Gly Thr Ala Gly Thr Thr Ala Cys Ala Cys Cys Thr Ala
1685 1690 1695
Cys Thr Ala Thr Cys Cys Ala Gly Ala Cys Ala Gly Thr Gly Thr Gly
1700 1705 1710
Ala Ala Gly Gly Gly Gly Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala
1715 1720 1725
Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Gly Cys
1730 1735 1740
Cys Ala Ala Gly Ala Ala Cys Ala Gly Cys Cys Thr Cys Thr Ala Cys
1745 1750 1755 1760
Cys Thr Gly Cys Ala Gly Ala Thr Gly Ala Gly Cys Ala Gly Cys Cys
1765 1770 1775
Thr Gly Ala Gly Gly Gly Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys
1780 1785 1790
Ala Gly Cys Cys Ala Thr Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr
1795 1800 1805
Gly Thr Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Ala Ala Thr Gly
1810 1815 1820
Gly Thr Ala Ala Cys Thr Thr Thr Thr Ala Cys Thr Ala Cys Thr Thr
1825 1830 1835 1840
Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly Gly Cys Cys Ala Ala
1845 1850 1855
Gly Gly Gly Ala Cys Cys Ala Cys Gly Gly Thr Cys Ala Cys Cys Gly
1860 1865 1870
Thr Cys Thr Cys Cys Thr Cys Ala Pro Glu Val Gln Leu Val Glu Ser
1875 1880 1885
Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
1890 1895 1900
Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr Met Tyr Trp Phe Arg Gln
1905 1910 1915 1920
Thr Pro Gly Lys Gly Leu Glu Trp Val Ala Thr Ile Ser Asp Gly Gly
1925 1930 1935
Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
1940 1945 1950
Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Ser Ser Leu Arg
1955 1960 1965
Ala Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg Glu Glu Asn Gly Asn
1970 1975 1980
Phe Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser
1985 1990 1995 2000
Ser Asp Gly Ala Gly Gly Thr Ala Cys Ala Ala Cys Thr Gly Gly Thr
2005 2010 2015
Gly Gly Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys
2020 2025 2030
Thr Thr Ala Gly Thr Gly Ala Ala Gly Cys Cys Thr Gly Gly Ala Gly
2035 2040 2045
Gly Gly Thr Cys Cys Cys Thr Gly Ala Gly Gly Cys Thr Cys Thr Cys
2050 2055 2060
Cys Thr Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala
2065 2070 2075 2080
Thr Thr Cys Ala Cys Thr Thr Thr Cys Ala Gly Thr Gly Ala Cys Thr
2085 2090 2095
Ala Thr Thr Ala Cys Ala Thr Gly Thr Ala Thr Thr Gly Gly Thr Thr
2100 2105 2110
Thr Cys Gly Cys Cys Ala Gly Gly Cys Cys Cys Cys Gly Gly Gly Ala
2115 2120 2125
Ala Ala Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly Gly Gly
2130 2135 2140
Thr Cys Gly Cys Ala Ala Cys Cys Ala Thr Thr Ala Gly Thr Gly Ala
2145 2150 2155 2160
Thr Gly Gly Thr Gly Gly Thr Ala Gly Thr Thr Ala Cys Ala Cys Cys
2165 2170 2175
Thr Ala Cys Thr Ala Thr Cys Cys Ala Gly Ala Cys Ala Gly Thr Gly
2180 2185 2190
Thr Gly Ala Ala Gly Gly Gly Gly Cys Gly Ala Thr Thr Cys Ala Cys
2195 2200 2205
Cys Ala Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr
2210 2215 2220
Gly Cys Cys Ala Ala Gly Ala Ala Cys Ala Gly Cys Cys Thr Cys Thr
2225 2230 2235 2240
Ala Cys Cys Thr Gly Cys Ala Gly Ala Thr Gly Ala Gly Cys Ala Gly
2245 2250 2255
Cys Cys Thr Gly Ala Gly Gly Gly Cys Thr Gly Ala Gly Gly Ala Cys
2260 2265 2270
Ala Cys Ala Gly Cys Thr Gly Thr Gly Thr Ala Thr Thr Ala Cys Thr
2275 2280 2285
Gly Thr Gly Thr Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Ala Ala
2290 2295 2300
Thr Gly Gly Thr Ala Ala Cys Thr Thr Thr Thr Ala Cys Thr Ala Cys
2305 2310 2315 2320
Thr Thr Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly Gly Cys Cys
2325 2330 2335
Ala Ala Gly Gly Gly Ala Cys Cys Ala Cys Gly Gly Thr Cys Ala Cys
2340 2345 2350
Cys Gly Thr Cys Thr Cys Cys Thr Cys Ala Pro Glu Val Gln Leu Val
2355 2360 2365
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
2370 2375 2380
Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr Met Tyr Trp Phe
2385 2390 2395 2400
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Thr Ile Ser Asp
2405 2410 2415
Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr
2420 2425 2430
Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Ser Ser
2435 2440 2445
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg Glu Glu Asn
2450 2455 2460
Gly Asn Phe Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr
2465 2470 2475 2480
Val Ser Ser Asp Gly Ala Gly Gly Thr Ala Cys Ala Ala Cys Thr Gly
2485 2490 2495
Gly Thr Gly Gly Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly
2500 2505 2510
Gly Cys Thr Thr Ala Gly Thr Gly Ala Ala Gly Cys Cys Thr Gly Gly
2515 2520 2525
Ala Gly Gly Gly Thr Cys Cys Cys Thr Gly Ala Gly Gly Cys Thr Cys
2530 2535 2540
Thr Cys Cys Thr Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly
2545 2550 2555 2560
Gly Ala Thr Thr Cys Ala Cys Thr Thr Thr Cys Ala Gly Thr Gly Ala
2565 2570 2575
Cys Thr Ala Thr Thr Ala Cys Ala Thr Gly Thr Ala Thr Thr Gly Gly
2580 2585 2590
Thr Thr Thr Cys Gly Cys Cys Ala Gly Gly Cys Cys Cys Cys Gly Gly
2595 2600 2605
Gly Ala Ala Ala Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly
2610 2615 2620
Gly Gly Thr Cys Gly Cys Ala Ala Cys Cys Ala Thr Thr Ala Gly Thr
2625 2630 2635 2640
Gly Ala Thr Gly Gly Thr Gly Gly Thr Ala Gly Thr Thr Ala Cys Ala
2645 2650 2655
Cys Cys Thr Ala Cys Thr Ala Thr Cys Cys Ala Gly Ala Cys Ala Gly
2660 2665 2670
Thr Gly Thr Gly Ala Ala Gly Gly Gly Gly Cys Gly Ala Thr Thr Cys
2675 2680 2685
Ala Cys Cys Ala Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Ala
2690 2695 2700
Ala Thr Gly Cys Cys Ala Ala Gly Ala Ala Cys Ala Gly Cys Cys Thr
2705 2710 2715 2720
Cys Thr Ala Cys Cys Thr Gly Cys Ala Gly Ala Thr Gly Ala Gly Cys
2725 2730 2735
Ala Gly Cys Cys Thr Gly Ala Gly Gly Gly Cys Thr Gly Ala Gly Gly
2740 2745 2750
Ala Cys Ala Cys Ala Gly Cys Thr Gly Thr Gly Thr Ala Thr Thr Ala
2755 2760 2765
Cys Thr Gly Thr Gly Thr Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly
2770 2775 2780
Ala Ala Thr Gly Gly Thr Ala Ala Cys Thr Thr Thr Thr Ala Cys Thr
2785 2790 2795 2800
Ala Cys Thr Thr Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly Gly
2805 2810 2815
Cys Cys Ala Ala Gly Gly Gly Ala Cys Cys Ala Cys Gly Gly Thr Cys
2820 2825 2830
Ala Cys Cys Gly Thr Cys Thr Cys Cys Thr Cys Ala Pro Glu Val Gln
2835 2840 2845
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg
2850 2855 2860
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr Met Tyr
2865 2870 2875 2880
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Thr Ile
2885 2890 2895
Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg
2900 2905 2910
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met
2915 2920 2925
Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu
2930 2935 2940
Glu Asn Gly Asn Phe Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr
2945 2950 2955 2960
Val Thr Val Ser Ser Asp Gly Ala Gly Gly Thr Ala Cys Ala Ala Cys
2965 2970 2975
Thr Gly Gly Thr Gly Gly Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly
2980 2985 2990
Ala Gly Gly Cys Thr Thr Ala Gly Thr Gly Ala Ala Gly Cys Cys Thr
2995 3000 3005
Gly Gly Ala Gly Gly Gly Thr Cys Cys Cys Thr Gly Ala Gly Gly Cys
3010 3015 3020
Thr Cys Thr Cys Cys Thr Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys
3025 3030 3035 3040
Thr Gly Gly Ala Thr Thr Cys Ala Cys Thr Thr Thr Cys Ala Gly Thr
3045 3050 3055
Gly Ala Cys Thr Ala Thr Thr Ala Cys Ala Thr Gly Thr Ala Thr Thr
3060 3065 3070
Gly Gly Thr Thr Thr Cys Gly Cys Cys Ala Gly Gly Cys Cys Cys Cys
3075 3080 3085
Gly Gly Gly Ala Ala Ala Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly
3090 3095 3100
Thr Gly Gly Gly Thr Cys Gly Cys Ala Ala Cys Cys Ala Thr Thr Ala
3105 3110 3115 3120
Gly Thr Gly Ala Thr Gly Gly Thr Gly Gly Thr Ala Gly Thr Thr Ala
3125 3130 3135
Cys Ala Cys Cys Thr Ala Cys Thr Ala Thr Cys Cys Ala Gly Ala Cys
3140 3145 3150
Ala Gly Thr Gly Thr Gly Ala Ala Gly Gly Gly Gly Cys Gly Ala Thr
3155 3160 3165
Thr Cys Ala Cys Cys Ala Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala
3170 3175 3180
Cys Ala Ala Thr Gly Cys Cys Ala Ala Gly Ala Ala Cys Ala Gly Cys
3185 3190 3195 3200
Cys Thr Cys Thr Ala Cys Cys Thr Gly Cys Ala Gly Ala Thr Gly Ala
3205 3210 3215
Gly Cys Ala Gly Cys Cys Thr Gly Ala Gly Gly Gly Cys Thr Gly Ala
3220 3225 3230
Gly Gly Ala Cys Ala Cys Ala Gly Cys Thr Gly Thr Gly Thr Ala Thr
3235 3240 3245
Thr Ala Cys Thr Gly Cys Gly Cys Ala Ala Gly Ala Gly Ala Gly Gly
3250 3255 3260
Ala Gly Ala Ala Thr Gly Gly Thr Ala Ala Cys Thr Thr Thr Thr Ala
3265 3270 3275 3280
Cys Thr Ala Cys Thr Thr Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly
3285 3290 3295
Gly Gly Cys Cys Ala Ala Gly Gly Gly Ala Cys Cys Ala Cys Gly Gly
3300 3305 3310
Thr Cys Ala Cys Cys Gly Thr Cys Thr Cys Cys Thr Cys Ala Pro Glu
3315 3320 3325
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser
3330 3335 3340
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr
3345 3350 3355 3360
Met Tyr Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala
3365 3370 3375
Thr Ile Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys
3380 3385 3390
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu
3395 3400 3405
Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
3410 3415 3420
Arg Glu Glu Asn Gly Asn Phe Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly
3425 3430 3435 3440
Thr Thr Val Thr Val Ser Ser Asp Gly Cys Cys Thr Gly Gly Ala Gly
3445 3450 3455
Gly Gly Thr Cys Cys Cys Thr Gly Ala Gly Gly Cys Thr Cys Thr Cys
3460 3465 3470
Cys Thr Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Ser Glu Gln Asp
3475 3480 3485
Gly Thr Thr Thr Cys Gly Cys Cys Ala Gly Ala Cys Thr Cys Cys Gly
3490 3495 3500
Gly Gly Ala Ala Ala Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr
3505 3510 3515 3520
Gly Gly Gly Thr Cys Gly Cys Ala Ala Cys Ser Glu Gln Asp Cys Ala
3525 3530 3535
Gly Ala Gly Ala Cys Ala Ala Thr Gly Cys Cys Ala Ala Gly Ala Ala
3540 3545 3550
Cys Ala Gly Cys Cys Thr Cys Thr Ala Cys Cys Thr Gly Cys Ala Gly
3555 3560 3565
Ala Thr Gly Ala Gly Cys Ala Gly Cys Cys Thr Gly Ala Gly Gly Gly
3570 3575 3580
Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys Ala Gly Cys Cys Ala Thr
3585 3590 3595 3600
Gly Asp Cys Ala Thr Gly Thr Ala Thr Thr Gly Gly Thr Thr Thr Cys
3605 3610 3615
Gly Cys Cys Ala Gly Gly Cys Cys Cys Cys Gly Gly Gly Ala Ala Ala
3620 3625 3630
Gly Gly Gly Gly Cys Thr Gly Gly Asp Gly Gly Gly Cys Thr Gly Ala
3635 3640 3645
Gly Gly Ala Cys Ala Cys Ala Gly Cys Thr Gly Thr Gly Thr Ala Thr
3650 3655 3660
Thr Ala Cys Thr Gly Thr Gly Thr Ala Ala Gly Ala Gly Asp Gly Thr
3665 3670 3675 3680
Gly Ala Cys Thr Ala Thr Thr Ala Cys Ala Thr Gly Thr Ala Thr Thr
3685 3690 3695
Gly Gly Gly Thr Gly Cys Gly Cys Cys Ala Gly Gly Cys Cys Cys Cys
3700 3705 3710
Gly Gly Gly Ala Ala Ala Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly
3715 3720 3725
Asp Gly Ala Gly Gly Gly Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys
3730 3735 3740
Ala Gly Cys Thr Gly Thr Gly Thr Ala Thr Thr Ala Cys Thr Gly Cys
3745 3750 3755 3760
Gly Cys Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Ala Ala Thr Gly
3765 3770 3775
Gly Thr Ala Ala Cys Asp Cys Thr Thr Gly Cys Ala Ala Gly Thr Gly
3780 3785 3790
Ala Thr Ala Gly Thr Gly Ala Cys Cys Cys Thr Gly Thr Cys Thr Cys
3795 3800 3805
Cys Cys Ala Cys Cys Gly Ala Thr Gly Cys Ala Gly Ala Cys Ala Ala
3810 3815 3820
Gly Gly Ala Thr Gly Ala Thr Gly Gly Ala Gly Ala Cys Thr Gly Gly
3825 3830 3835 3840
Gly Thr Cys Ala Thr Cys Asp Cys Ala Thr Ala Ala Thr Ala Gly Ala
3845 3850 3855
Thr Cys Ala Gly Gly Ala Thr Cys Thr Thr Ala Gly Gly Cys Gly Cys
3860 3865 3870
Thr Thr Thr Cys Cys Ala Thr Gly Gly Thr Thr Thr Cys Thr Gly Cys
3875 3880 3885
Thr Gly Asp Gly Thr Ala Gly Ala Cys Ala Gly Thr Ala Ala Thr Ala
3890 3895 3900
Ala Gly Thr Thr Gly Cys Gly Ala Ala Ala Thr Cys Cys Thr Cys Ala
3905 3910 3915 3920
Gly Gly Cys Thr Gly Cys Ala Gly Gly Cys Thr Gly Cys Thr Gly Ala
3925 3930 3935
Thr Gly Gly Thr Thr Ala Gly Ala Gly Thr Ala Thr Ala Ala Thr Cys
3940 3945 3950
Thr Thr Gly Cys Cys Cys Ala Gly Ala Thr Cys Asp Gly Ala Thr Gly
3955 3960 3965
Gly Ala Gly Ala Cys Thr Gly Gly Gly Thr Cys Ala Thr Cys Thr Gly
3970 3975 3980
Gly Ala Thr Ala Thr Cys Ala Cys Cys Thr Cys Thr Gly Gly Cys Ala
3985 3990 3995 4000
Cys Cys Thr Gly Asp Gly Ala Thr Gly Gly Thr Thr Ala Gly Ala Gly
4005 4010 4015
Thr Ala Thr Ala Ala Thr Cys Thr Gly Thr Ala Cys Cys Ala Gly Ala
4020 4025 4030
Thr Cys Cys Ala Cys Thr Gly Cys Cys Ala Cys Thr Gly Asp Gly Ala
4035 4040 4045
Thr Gly Gly Ala Gly Ala Cys Thr Gly Gly Gly Thr Cys Ala Thr Cys
4050 4055 4060
Thr Gly Gly Ala Thr Ala Thr Cys Ala Cys Cys Thr Cys Thr Gly Gly
4065 4070 4075 4080
Cys Ala Cys Cys Thr Gly Asp Cys Ala Ala Cys Cys Thr Thr Gly Thr
4085 4090 4095
Thr Gly Cys Ala Thr Ala Ala Thr Ala Gly Ala Thr Cys Ala Gly Ala
4100 4105 4110
Ala Gly Cys Thr Thr Ala Gly Gly Cys Gly Cys Thr Thr Thr Cys Cys
4115 4120 4125
Cys Thr Gly Gly Thr Thr Thr Cys Thr Gly Cys Thr Gly Gly Thr Ala
4130 4135 4140
Cys Cys Asp Gly Ala Thr Gly Gly Thr Thr Ala Gly Ala Gly Thr Ala
4145 4150 4155 4160
Thr Ala Ala Thr Cys Thr Gly Thr Ala Cys Cys Ala Gly Ala Thr Cys
4165 4170 4175
Cys Ala Cys Thr Gly Cys Cys Ala Cys Thr Gly Pro Asp Ile Gln Met
4180 4185 4190
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
4195 4200 4205
Ile Thr Cys Lys Ala Gly Gln Asp Ile Lys Ser Tyr Leu Ser Trp Tyr
4210 4215 4220
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Ala Thr
4225 4230 4235 4240
Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
4245 4250 4255
Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
4260 4265 4270
Thr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Trp Thr Phe Gly Gly
4275 4280 4285
Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe
4290 4295 4300
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
4305 4310 4315 4320
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
4325 4330 4335
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
4340 4345 4350
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr
4355 4360 4365
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
4370 4375 4380
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
4385 4390 4395 4400
Glu Cys Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys
4405 4410 4415
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
4420 4425 4430
Ser Asp Tyr Tyr Met Tyr Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu
4435 4440 4445
Glu Trp Val Ala Thr Ile Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro
4450 4455 4460
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
4465 4470 4475 4480
Ser Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val
4485 4490 4495
Tyr Tyr Cys Ala Arg Glu Glu Asn Gly Asn Phe Tyr Tyr Phe Asp Tyr
4500 4505 4510
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
4515 4520 4525
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
4530 4535 4540
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
4545 4550 4555 4560
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
4565 4570 4575
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
4580 4585 4590
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
4595 4600 4605
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
4610 4615 4620
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
4625 4630 4635 4640
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
4645 4650 4655
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
4660 4665 4670
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
4675 4680 4685
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
4690 4695 4700
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
4705 4710 4715 4720
Asn Gly Lys G1u Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
4725 4730 4735
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
4740 4745 4750
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
4755 4760 4765
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
4770 4775 4780
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
4785 4790 4795 4800
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
4805 4810 4815
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
4820 4825 4830
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
4835 4840 4845
Leu Ser Pro Gly
4850
Claims (17)
1.人源化抗淋巴毒素β受体(LT-β-R)的抗体,其轻链互补决定区由SEQID NO:1中氨基酸残基24-34,50-56和89-97限定,其重链互补决定区由SEQ ID NO:2中氨基酸残基31-35,50-66和99-109限定,并且该抗体在其轻链中包含下列的至少1种残基:K3,W41,I46,Q69和Y71;或在其重链中包含下列的至少1种残基:F37,T40,A49,M89和V93(按照Kabat编号原则进行编号)。
2.人源化抗淋巴毒素β受体(LT-β-R)的抗体,其轻链互补决定区由SEQID NO:1中氨基酸残基24-34,50-56和89-97限定,其重链互补决定区由SEQ ID NO:2中氨基酸残基31-35,50-66和99-109限定,并且该抗体在其轻链中包含Y71残基(按照Kabat编号原则进行编号)。
3.人源化抗淋巴毒素β受体(LT-β-R)的抗体,其轻链互补决定区由SEQID NO:1中氨基酸残基24-34,50-56和89-97限定,其重链互补决定区由SEQ ID NO:2中氨基酸残基31-35,50-66和99-109限定,并且该抗体在其重链中包含F37和A49残基(按照Kabat编号原则进行编号)。
4.权利要求1的抗体,其中该抗体包含由SEQ ID NO:8中氨基酸残基1至107限定的轻链可变区序列。
5.权利要求1的抗体,其中该抗体包含由SEQ ID NO:16中氨基酸残基1至120限定的重链可变区序列。
6.权利要求4的抗体,其中该抗体还包含由SEQ ID NO:16中氨基酸残基1至120限定的重链可变区序列。
7.一种抗体,它包含与由细胞系E46.4(ATCC专利保藏物PTA-3357)或细胞系E77.4(ATCC专利保藏物3765)产生的抗体相同的重链和轻链多肽序列。
8.权利要求1-7任一项的抗体,其中该抗体充分保留了亲本抗体的结合特性。
9.权利要求1-7任一项的抗体,其中该抗体还与免疫毒素相连。
10.权利要求1-7任一项的抗体,其中该抗体还与化疗药物相连。
11.一种组合物,它包含权利要求1-7任一项的抗体和可药用载体。
12.一种治疗或缓解人的瘤形成的进展、严重程度或影响的方法,该方法包含给药权利要求11的组合物。
13.一种分离的核酸,它包含抗体轻链的编码序列,该抗体由细胞系E46.4(ATCC专利保藏物PTA-3357)或细胞系E77.4(ATCC专利保藏物3765)产生。
14.一种分离的核酸,它包含抗体重链的编码序列,该抗体由细胞系E46.4(ATCC专利保藏物PTA-3357)或细胞系E77.4(ATCC专利保藏物3765)产生。
15.一种分离的核酸,它包含编码SEQ ID NO:8中残基1至107的序列。
16.一种分离的核酸,它包含编码SEQ ID NO:16中残基1至120的序列。
17.细胞系E46.4(ATCC专利保藏物PTA-3357)或细胞系E77.4(ATCC专利保藏物3765)的细胞。
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US24028500P | 2000-10-13 | 2000-10-13 | |
US60/240,285 | 2000-10-13 | ||
US27528901P | 2001-03-13 | 2001-03-13 | |
US60/275,289 | 2001-03-13 | ||
US29998701P | 2001-06-21 | 2001-06-21 | |
US60/299,987 | 2001-06-21 |
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CNA018205917A Pending CN1547590A (zh) | 2000-10-13 | 2001-10-12 | 人源化抗-LT-β-R抗体 |
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US (2) | US7429644B2 (zh) |
EP (1) | EP1326897A2 (zh) |
JP (1) | JP2004532608A (zh) |
KR (1) | KR20030041164A (zh) |
CN (1) | CN1547590A (zh) |
AR (1) | AR035352A1 (zh) |
AU (2) | AU2002211747B2 (zh) |
BG (1) | BG107802A (zh) |
BR (1) | BR0114646A (zh) |
CA (1) | CA2425809A1 (zh) |
CZ (1) | CZ20031307A3 (zh) |
EA (1) | EA006945B1 (zh) |
EE (1) | EE200300179A (zh) |
GE (1) | GEP20063752B (zh) |
HU (1) | HUP0302573A2 (zh) |
IL (1) | IL155340A0 (zh) |
IS (1) | IS6779A (zh) |
MX (1) | MXPA03003144A (zh) |
NO (1) | NO20031642L (zh) |
NZ (1) | NZ525793A (zh) |
PL (1) | PL366307A1 (zh) |
SK (1) | SK5672003A3 (zh) |
TR (2) | TR200602095T2 (zh) |
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MXPA03003144A (es) | 2004-12-06 |
IL155340A0 (en) | 2003-11-23 |
KR20030041164A (ko) | 2003-05-23 |
GEP20063752B (en) | 2006-02-27 |
EP1326897A2 (en) | 2003-07-16 |
TR200300478T2 (tr) | 2004-10-21 |
EA006945B1 (ru) | 2006-06-30 |
BR0114646A (pt) | 2006-02-21 |
NO20031642L (no) | 2003-06-13 |
AU2002211747B2 (en) | 2007-11-29 |
AU1174702A (en) | 2002-04-22 |
NZ525793A (en) | 2008-04-30 |
US7429644B2 (en) | 2008-09-30 |
NO20031642D0 (no) | 2003-04-10 |
SK5672003A3 (en) | 2003-10-07 |
PL366307A1 (en) | 2005-01-24 |
WO2002030986A2 (en) | 2002-04-18 |
TR200602095T2 (tr) | 2007-02-21 |
CA2425809A1 (en) | 2002-04-18 |
CZ20031307A3 (cs) | 2003-10-15 |
US20040058394A1 (en) | 2004-03-25 |
AR035352A1 (es) | 2004-05-12 |
WO2002030986A3 (en) | 2003-03-13 |
US20090092601A1 (en) | 2009-04-09 |
YU28503A (sh) | 2006-05-25 |
HUP0302573A2 (hu) | 2003-10-28 |
IS6779A (is) | 2003-04-11 |
JP2004532608A (ja) | 2004-10-28 |
BG107802A (bg) | 2004-01-30 |
EA200300464A1 (ru) | 2003-10-30 |
EE200300179A (et) | 2003-08-15 |
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