CN108473591A - 位点特异性her2抗体药物缀合物 - Google Patents
位点特异性her2抗体药物缀合物 Download PDFInfo
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- CN108473591A CN108473591A CN201680072749.4A CN201680072749A CN108473591A CN 108473591 A CN108473591 A CN 108473591A CN 201680072749 A CN201680072749 A CN 201680072749A CN 108473591 A CN108473591 A CN 108473591A
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Abstract
本发明提供了位点特异性HER2抗体药物缀合物以及其制备方法和其应用。
Description
技术领域
本发明系关于位点特异性HER2抗体药物缀合物。本发明另关于将该抗体药物缀合物用于治疗癌之方法。
先前技术
跨膜受体酪氨酸激酶ErbB家族成员为细胞生长、细胞分化、细胞迁移、与细胞凋亡的重要介质。该受体家族包括四个不同成员,其包括表皮生长因子受体(EGFR或ErbB1)、HER2(ErbB2或p185)、HER3(ErbB3)及HER4(ErbB4或tyro2)。
HER2起初被认定是来自经化学治疗的大鼠之神经胚细胞瘤的转型基因产物。HER2过度表达被证实在体外(Di Fiore et al.,1987,Science 237(4811):178-82;Hudziak etal.,1987,PNAS 84(20):7159-63;Chazin et al.,1992,Oncogene 7(9):1859-66)及在动物模型中(Guy et al.,1992,PNAS 89(22):10578-82)都是致瘤的。引起受体过度表达之编码HER2的基因扩增出现于乳癌与卵巢癌且与预后不良有关联(Slamon et al.,1987,Science 235(4785):177-82;Slamon et al.,1989,Science 244:707-12;Anbazhagan etal.,1991,Annals Oncology 2(1):47-53;Andrulis et al.,1998,J Clinical Oncology16(4):1340-9)。HER2过度表达(通常但不一定是由于基因扩增)也在其它癌型观测到,该其它癌型包括胃癌、子宫内膜癌、非小细胞肺癌、结肠癌、胰腺癌、膀胱癌、肾癌、前列腺癌及子宫颈癌(Scholl et al.,2001,Annals Oncology 12(Suppl.1):S81-7;Menard et al.,2001,Ann Oncol 12(Suppl.1):S15-9;Martin et al.,2014,Future Oncology 10:1469-86)。
(曲妥单抗(trastuzumab))为结合至HER2的胞外功能域之人源化单克隆抗体(Carter et al.1992,PNAS89:4285-9及美国专利第5,821,337号)。用于治疗肿瘤过度表达HER2蛋白质的转移性乳癌患者在1998年9月25日得到食品药物管理局的上市许可。尽管是治疗接受过大规模先前抗癌疗法之HER2过度表达性乳癌患者的一个突破性进展,然而有部分患者对治疗没反应、反应很小或变成有抗性。
(曲妥单抗-DM1或T-DM1)为由通过稳定的硫醚连接子MCC(4-[N-顺丁烯二酰亚胺甲基]环己烷-1-羧酸酯)缀合至类美登素药剂(maytansinoid agent)DM1之曲妥单抗组成的抗体药物缀合物(Lewis et al.,2008,Cancer Res.68:9280-90;Krop etal.,2010,J Clin Oncol.28:2698-2704;美国专利第8,337,856号)。用于治疗先前经与紫杉烷药剂治疗过且变成难治之患者中HER2阳性转移性乳癌在2013年2月22日得到食品药物管理局的上市许可。类似长期疗法对一部分HER2过度表达性乳癌患者并没成功。
因此,临床上很需要针对对与/或治疗没反应、反应很小或变成有抗性的患HER2过度表达性肿瘤或其它与HER2过度表达有关连之疾病的患者发展另外的HER2导向癌疗法。
发明内容
本发明提供位点特异性HER2抗体药物缀合物及其在治疗HER2过度表达性癌的用途。ADC使疗法能靶向递输给癌细胞且能提供更具选择性之疗法而减少已知的脱靶效应造成的毒性。
本发明之位点特异性HER2ADC一般如式Ab-(L-D)所示,其中Ab为结合至HER2的抗体或其抗原结合片段;及L-D是连接子-药物部分,其中L是连接子,及D是药物。
本发明之ADC的抗体(Ab)可为任何HER2结合抗体。在本发明之某些实施方式中,该Ab结合至和曲妥单抗相同的HER2上的表位。在本发明之其它实施方式中,该Ab具有和曲妥单抗相同的重链与轻链CDR。在本发明之具体实施方式中,该Ab具有和曲妥单抗相同的重链可变区(VH)与轻链可变区(VL)。
本发明之HER2ADC以位点特异方式缀合至药物。为了符合此类型的缀合,必须将抗体衍生化以提供工程化在一或多个特异位点的反应性半胱氨酸残基或酰基供体谷氨酰胺残基(工程化在一或多个特异位点或附接肽标签)。该修饰应是在不瓦解抗体之抗原结合力的位点上。在较佳实施方式中,在抗体之重链恒定区与/或轻链恒定区进行一或多个修饰。
在本发明之某些实施方式中,位点特异性HER2ADC可以使用包含曲妥单抗的重链可变区CDR与轻链可变区CDR及表1中揭示之重链恒定区与轻链恒定区的任何组合之抗体,条件是当该重链恒定区是SEQ ID No:5时,则该轻链恒定区不是SEQ ID No:11。在这样的实施方式中,该重链恒定区可选自SEQ ID No:17、5、13、21、23、25、27、29、31、33、35、37或39中任一者,而该轻链恒定区可选自SEQ ID No:41、11或43中任一者,条件是该组合不是SEQ IDNo:5与SEQ ID No:11。
在一个具体实施方式中,被用来构成该位点特异性HER2ADC之抗体包含附接至SEQID No:17的重链恒定区及SEQ ID No:41的轻链恒定区之具有SEQ ID No:2至4的CDR之VH区及具有SEQ ID No:8至10的CDR之VL区。在另一个具体实施方式中,被用来构成该位点特异性HER2ADC的抗体包含附接至SEQ ID No:13的重链恒定区及SEQ ID No:43的轻链恒定区之具有SEQ ID No:2至4的CDR之VH区及具有SEQ ID No:8至10的CDR之VL区。
在其它具体实施方式中,本发明之ADC可以使用包含表1中揭示之重链与轻链的任何组合之抗体,条件是若该重链是SEQ ID No:6,则该轻链恒定区不是SEQ ID No:12。在这样的实施方式中,该重链可选自SEQ ID No:18、6、14、22、24、26、28、30、32、34、36、38或40中任一者,而该轻链可选自SEQ ID No:42、12或44中任一者,条件是该组合不是SEQ ID No:6与SEQ ID No:12。
在一个具体实施方式中,本发明之ADC可以使用包含SEQ ID No:18的重链与SEQID No:42的轻链。在另一个具体实施方式中,本发明之ADC可以使用包含SEQ ID No:14的重链与SEQ ID No:44的轻链。
本发明中所揭示之位点特异性HER2ADC中任一者可以用可用于治疗癌的治疗剂-药物(D)制得。在一个具体实施方式中,该治疗剂是抗有丝分裂剂。在另一个具体实施方式中,本发明之ADC中的抗有丝分裂剂药物成分是耳抑素(auristatin)(例如0101、8261、6121、8254、6780与0131)。在一个更具体实施方式中,本发明之ADC中的耳抑素药物成分是2-甲基丙氨酰基-N-[(3R,4S,5S)-3-甲氧基-1-{(2S)-2-[(1R,2R)-1-甲氧基-2-甲基-3-氧代-3-{[(1S)-2-苯基-1-(1,3-噻唑-2-基)乙基]氨基}丙基]吡咯烷-1-基}-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺(valinamide)(也通称为0101)。较佳地,本发明之ADC的药物成分是可透膜的。
本发明中所揭示之位点特异性HER2ADC中任一者可以用可切割的或不可切割的连接子(L)制得。较佳地,该连接子是可切割的。可切割之连接子包括但不限于vc、AcLysvc及m(H20)c-vc。更佳地,该连接子是vc或AcLysvc。
在本发明之一个特定实施方式中,如式Ab-(L-D)之位点特异性HER2ADC包含(a)抗体Ab,其包含SEQ ID No:18的重链与SEQ ID No:42的轻链;及(b)连接子-药物部分L-D,其中L是连接子,及D是药物,其中该连接子是vc且其中该药物是0101。
在本发明之另一个特定实施方式中,如式Ab-(L-D)之位点特异性HER2ADC包含(a)抗体Ab,其包含SEQ ID No:14的重链与SEQ ID No:44的轻链;及(b)连接子-药物部分L-D,其中L是连接子,及D是药物,其中该连接子是AcLysvc且其中该药物是0101。
本发明之另一个方面包括本发明中所揭示之抗体药物缀合物及用于制备、合成与缀合本发明中所揭示之抗体药物缀合物的中间物之构成方法、制备方法、合成方法、缀合方法及纯化方法。
本发明另外提供药学组合物,其包含本发明中所揭示之位点特异性HER2ADC及药学上可接受的载剂。
本发明设想到编码位点特异性HER2ADC之抗体部分的核酸。本发明也设想到另外的包含该核酸之载体与宿主细胞。
本发明也提供位点特异性HER2ADC在治疗HER2表达性癌的用法。欲用本发明之位点特异性HER2ADC治疗的HER2表达性癌可以表达高、中或低水平的HER2。在某些实施方式中,该欲治疗之癌对用曲妥单抗和/或曲妥单抗emtansine(T-DM1)单独或合并紫杉烷之治疗有抗性、是该治疗难治的、和/或在该治疗后复发。欲治疗之癌包括但不限于乳癌、卵巢癌、肺癌、胃癌、食道癌、结肠直肠癌、尿路上皮癌、胰腺癌、唾液腺癌及脑癌或上述癌之转移。在一个更具体实施方式中,该乳癌是雌激素与黄体激素受体阴性乳癌或三阴性乳癌(TNBC)。在另一个实施方式中,该肺癌是非小细胞肺癌(NSCLC)。
通过重新探讨整个申请案会了解本发明之上述方面及其它方面。
附图说明
图1A至1B绘示(A)T(κK183C+K290C)-vc0101ADC及(B)T(LCQ05+K222R)-AcLysvc0101ADC。各黑圆圈代表缀合至单克隆抗体之连接子/载荷物(payload)。显示各种ADC之一种这样的连接子/载荷物结构。藉以发生缀合之抗体上的氨基酸残基提供加底线实体。
图2A至2E绘示来自疏水性交互作用层析术(HIC)之选定ADC光谱,其显示在将曲妥单抗衍生的抗体缀合至不同连接子载荷物后在滞留时间方面之变化。
图3A至3B绘示结合至HER2之ADC的图。(A)直接结合至HER2阳性BT474细胞及(B)与PE标记的曲妥单抗竞争性结合至BT474细胞。这些结果指示在这些ADC中的抗体结合性质不随缀合方法而改变。
图4绘示曲妥单抗衍生之ADC的ADCC活性。
图5绘示一些曲妥单抗衍生之ADC对一些具有不同HER2表达水平的细胞株的以nM载荷物浓度表示之体外细胞毒性数据(IC50)。
图6绘示一些曲妥单抗衍生之ADC对一些具有不同HER2表达水平的细胞株的以ng/ml抗体浓度表示之体外细胞毒性数据(IC50)。
图7A至7I绘示9种曲妥单抗衍生之ADC对N87异种移植物的以肿瘤体积对时间表示之抗肿瘤活性。(A)T(κK183C+K290C)-vc0101;(B)T(κK183C)-vc0101;(C)T(K290C)-vc0101;(D)T(LCQ05+K222R)-AcLysvc0101;(E)T(K290C+K334C)-vc0101;(F)T(K334C+K392C)-vc0101;(G)T(N297Q+K222R)-AcLysvc0101;(H)T-vc0101;(I)T-DM1。N87胃癌细胞表达高水平的HER2。
图8A至8E绘示6种曲妥单抗衍生之ADC对HCC1954异种移植物的以肿瘤体积对时间表示之抗肿瘤活性。(A)T(LCQ05+K222R)-AcLysvc0101;(B)T(K290C+K334C)-vc0101;(C)T(K334C+K392C)-vc0101;(D)T(N297Q+K222R)-AcLysvc0101;(E)T-DM1。HCC1954肺癌细胞表达高水平的HER2。
图9A至9G绘示7种曲妥单抗衍生之ADC对JIMT-1异种移植物的以肿瘤体积对时间表示之抗肿瘤活性。(A)T(κK183C+K290C)-vc0101;(B)T(LCQ05+K222R)-AcLysvc0101;(C)T(K290C+K334C)-vc0101;(D)T(K334C+K392C)-vc0101;(E)T(N297Q+K222R)-AcLysvc0101;(F)T-vc0101;(G)T-DM1。JIMT-1乳癌细胞表达中/低水平的HER2。
图10A至10D绘示5种曲妥单抗衍生之ADC对MDA-MB-361(DYT2)异种移植物的以肿瘤体积对时间表示之抗肿瘤活性。(A)T(LCQ05+K222R)-AcLysvc0101;(B)T(N297Q+K222R)-AcLysvc0101;(C)T-vc0101;(D)T-DM1。MDA-MB-361(DYT2)乳癌细胞表达中/低水平的HER2。
图11A至11E绘示5种曲妥单抗衍生之ADC对PDX-144580患者衍生之异种移植物的以肿瘤体积对时间表示之抗肿瘤活性。(A)T(κK183C+K290C)-vc0101;(B)T(LCQ05+K222R)-AcLysvc0101;(C)T(N297Q+K222R)-AcLysvc0101;(D)T-vc0101;(E)T-DM1。PDX-144580患者衍生之细胞是TNBC PDX模型。
图12A至12D绘示4种曲妥单抗衍生之ADC对PDX-37622患者衍生之异种移植物的以肿瘤体积对时间表示之抗肿瘤活性。(A)T(κK183C+K290C)-vc0101;(B)T(N297Q+K222R)-AcLysvc0101;(C)T(K297C+K334C)-vc0101;(D)T-DM1。PDX-37622患者衍生之细胞是表达中水平的HER2之NSCLC PDX模型。
图13A至13B绘示经(A)T-DM1或(B)T-vc0101治疗且行磷酸化组织蛋白H3与IgG抗体染色之N87肿瘤异种移植物的免疫组织细胞化学法。
图14绘示一些曲妥单抗衍生之ADC与游离载荷物对体外T-DM1抗药性细胞(N87-TM1与N87-TM2)或T-DM1感药性亲代细胞(N87细胞)之以nM载荷物浓度及ng/ml抗体浓度表示的体外细胞毒性数据(IC50)。
图15A至15G绘示7种曲妥单抗衍生之ADC对T-DM1感药性(N87细胞)与抗药性(N87-TM1与N87-TM2)胃癌细胞的抗肿瘤活性。(A)T-DM1;(B)T-mc8261;(C)T(297Q+K222R)-AcLysvc0101;(D)T(LCQ05+K222R)-AcLysvc0101;(E)T(K290C+K334C)-vc0101;(F)T(K334C+K392C)-vc0101;(G)T(κK183C+K290C)-vc0101。
图16A至16B绘示显示T-DM1感药性(N87细胞)与抗药性(N87-TM1与N87-TM2)胃癌细胞的(A)MRP1药物排出泵蛋白质表达与(B)MDR1药物排出泵蛋白质表达之蛋白印迹法。
图17A至17B绘示T-DM1感药性(N87细胞)与抗药性(N87-TM1与N87-TM2)胃癌细胞的HER2表达及与曲妥单抗之结合。(A)显示HER2蛋白质表达的蛋白印迹法与(B)和细胞表面HER2之结合。
图18A至18D绘示T-DM1感药性(N87细胞)与抗药性(N87-TM1与N87-TM2)胃癌细胞之蛋白质表达水平的特性分析。(A)523蛋白质的蛋白质表达水平变化;(B)显示IGF2R、LAMP1和CTSB蛋白质表达之蛋白印迹法;(C)显示CAV1蛋白质表达的蛋白印迹法;(D)植入N87细胞(左)与N87-TM2细胞(右)后在体内产生之肿瘤的CAV1蛋白质表达的IHC。
图19A至19C绘示植入下列后在体内产生之肿瘤对曲妥单抗与各种曲妥单抗衍生之ADC的感药性:(A)T-DM1感药性N87亲代细胞;(B)T-DM1抗药性N87-TM1细胞;(C)T-DM1抗药性N87-TM2细胞。
图20A至20F绘示植入T-DM1感药性N87亲代细胞与T-DM1抗药性N87-TM2细胞或N87-TM1细胞后在体内产生之肿瘤对曲妥单抗与各种曲妥单抗衍生之ADC的感药性。(A)在曲妥单抗或二种曲妥单抗衍生之ADC存在下N87肿瘤体积对时间的图;(B)在曲妥单抗或二种曲妥单抗衍生之ADC存在下N87-TM2肿瘤体积对时间的图;(C)在曲妥单抗或二种曲妥单抗衍生之ADC存在下N87细胞肿瘤体积加倍的时间;(D)在曲妥单抗或二种曲妥单抗衍生之ADC存在下N87-TM2细胞肿瘤体积加倍的时间;(E)在7种不同曲妥单抗衍生之ADC存在下N87-TM2肿瘤体积对时间的图;(F)在第14日加入曲妥单抗衍生之ADC的N87-TM1肿瘤体积对时间的图。
图21A至21E绘示在体内产生之T-DM1抗药性细胞的产生与特性分析。(A)在植入体内初期N87胃癌细胞对T-DM1有感药性。(B)随时间过去,经植入的N87细胞变成对T-DM1有抗药性,但对下列仍有感药性:(C)T-vc0101;(D)T(N297Q+K222R)-AcLysvc0101及(E)T(κK183+K290C)-vc0101。
图22A至22D绘示4种曲妥单抗衍生之ADC对在体内产生之T-DM1抗药性细胞(N87-TDM)和T-DM1感药性亲代N87细胞的以肿瘤体积对时间表示之体外细胞毒性。(A)T-DM1;(B)T(κK183+K290C)-vc0101;(C)T(LCQ05+K222R)-AcLysvc0101;(D)T(N297Q+K222R)-AcLysvc0101。
图23A至23B绘示在体内产生之T-DM1抗药性细胞(N87-TDM1,来自小鼠2、17与18)和T-DM1感药性亲代N87细胞的HER2蛋白质表达水平比较。(A)FACS分析法与(B)蛋白印迹分析法。没有观测到HER2蛋白质表达的明显差异。
图24A至24D绘示在N87-TDM1(小鼠2、17与17)中的T-DM1抗药性不是由于药物排出泵。(A)显示MDR1蛋白质表达之蛋白印迹法。在游离药物(B)0101;(C)多柔比星;(D)T-DM1存在下的T-DM1抗药性细胞(N87-TDM1)与T-DM1感药性N87亲代细胞之体外细胞毒性。
图25A至25B绘示(A)在对马来猴投药后之总Ab与曲妥单抗ADC(T-vc0101)或T(κK183C+K290C)位点特异性ADC和(B)在对马来猴投药后之曲妥单抗(T-vc0101)的ADC被分析物或不同位点特异性ADC的浓度对时间特性及药物动力学/毒物动力学。
图26绘示由疏水性交互作用层析术(HIC)得到之相对滞留值对在大鼠中的暴露量(AUC)。X轴代表由HIC得到之相对滞留时间;而Y轴代表在大鼠中的药物动力学剂量标准化之暴露量(“曲线下面积”,AUC,用于抗体,从0至336小时,除以10mg/kg的药物剂量)。符号形状表示大概的载药量(DAR):菱形=DAR2;圆形=DAR4。箭头指示T(κK183C+K290C)-vc0101。
图27绘示使用T-vc0101常规缀合物ADC与T(κK183C+K290C)-vc0101位点特异性ADC的毒性研究。在5mg/kg下的T-vc0101诱发严重的嗜中性粒细胞减少症,而在9mg/kg下的T(κK183C+K290C)-vc0101造成嗜中性粒细胞最小的下降。
图28A至28C绘示下列之晶体结构:(A)T(K290C+K334C)-vc0101;(B)T(K290C+K392C)-vc0101;与(C)T(K334C+K392C)-vc0101。
图29绘示在使用N87细胞株之异种移植物模型上的体内效力。在3mpk的被试验的ADC全部显示功效。
图30绘示在PDX-GA0044患者衍生之异种移植物上的曲妥单抗与二种曲妥单抗衍生之ADC的抗癌活性,以肿瘤体积对时间作图。动物被下列处理:媒剂(空心菱形),曲妥单抗(空心三角形),T-DM1(空心圆形),或T(κK183C+K290C)-vc0101(实心圆形与实心菱形)。PDX-GA0044患者衍生之细胞是表达中水平的HER2的胃PDX模型。
发明详述
本发明提供位点特异性HER2抗体药物缀合物(ADC),使用HER2抗体、连接子、与药物载荷物、及编码用于制造ADC之抗体的核酸来制备该缀合物之方法。本发明之ADC可用于制备与制造能用来治疗HER2表达性癌的组合物(例如医药品)。
ADC由通过连接子的使用缀合至药物载荷物之抗体成分组成。常规ADC缀合策略依靠把药物载荷物通过在抗体重链与/或轻链上的内生性赖氨酸或半胱氨酸随机地缀合至该抗体。因此,这样的ADC是显出不同药物:抗体比(DAR)之物种的异质混合物。反之,本发明中所揭示之ADC是在抗体重链与/或轻链上的特定工程化之残基上把药物载荷物缀合至抗体的位点特异性ADC。所以,该位点特异性ADC是由具定义药物:抗体比(DAR)的物种组成之ADC的同质总体。因此,该位点特异性ADC显示导致该缀合物之改进的药性、体内分布性与安全性。本发明之ADC包括缀合至一或多个连接子/载荷物部分的本发明之抗体。
本发明提供如式Ab-(L-D)之抗体药物缀合物,其中(a)Ab是结合至HER2的抗体,或其抗原结合片段,及(b)L-D是连接子-药物部分,其中L是连接子,及D是药物。
本发明也包含如式Ab-(L-D)p之抗体药物缀合物,其中(a)Ab是结合至HER2的抗体,或其抗原结合片段,(b)L-D是连接子-药物部分,其中L是连接子,及D是药物,和(c)p是附接至该抗体之连接子/药物部分的数量。对位点特异性ADC而言,p是整数,由于该ADC的均质。在某些实施方式中,p是4。在其它实施方式中,p是3。在其它实施方式中,p是2。在其它实施方式中,p是1。在其它实施方式中,p大于4。
本发明中所用之用语“HER2”乃指属于EGFR家族的跨膜酪氨酸激酶受体。HER2也称为ErbB2、p185与CD340。此受体家族包括四个成员(EGFR/HER1、HER2、HER3与HER4),其通过刺激生长因子讯息传递路径(例如PI3K-AKT-mTOR路径)而产生作用。HER2的放大与/或过度表达和多种人恶性肿瘤有关连。野生型人HER2蛋白质的描述参见,例如,Semba et al.,1985,PNAS 82:6497-6501及Yamamoto et al.,1986,Nature 319:230-4和GenbankAccession Number X03363。
本发明中所用之用语“抗体(Ab)”乃指能通过至少一个位于免疫球蛋白分子的可变区之抗原识别位点来识别与结合至特异靶或抗原的免疫球蛋白分子(例如多肽)。该用语可以包含任何类型之抗体,其包括但不限于:单克隆抗体、完整抗体的抗原结合片段(其保留特异性结合至给定抗原(即Fab、Fab'、F(ab')2、Fd、Fv、Fc等)之能力)及其突变体的抗体。
天然抗体(native antibody或naturally-occurring antibody)与天然免疫球蛋白一般是由二个相同轻(L)链与二个相同重(H)链组成之约150,000道尔顿的异质四聚体糖蛋白。每一轻链通过一个共价二硫键连接至重链,而在不同免疫球蛋白同型之重链中的二硫键数目不同。每一重链与轻链也具有规律间隔之链间二硫键。每一重链在一端具有可变区(VH)后面接着一些恒定区。每一轻链在一端具有可变区(VL)而另一端有恒定区;该轻链恒定区与该重链的第一恒定区密切结合,及该轻链可变区与该重链可变区密切结合。用语“可变的”乃指可变区的某些部分在抗体序列中广泛不同。
本发明中所用之抗体特异性结合至HER2。在一个具体实施方式中,该HER2抗体结合至和曲妥单抗相同之在HER2上的表位。在一个更具体实施方式中,该HER2抗体具有和曲妥单抗相同的可变区CDR。在又一个更具体实施方式中,该HER2抗体具有和曲妥单抗相同之可变区(即VH与VL)。
本发明中所用之用语“连接子(L)”描述该抗体直接或间接连接至该药物载荷物。可以各式各样方式将连接子附接至抗体,该方式例如通过表面赖氨酸、与氧化碳水化合物还原偶合、还原链间二硫键释出半胱氨酸残基、在特异位点工程化反应性半胱氨酸残基、和在转谷氨酰胺酶与胺存在下工程化多肽使含有酰基供体谷氨酰胺之标签或内生性谷氨酰胺变成反应性的。本发明使用位点特异方法将抗体连接至药物载荷物。在一个实施方式中,缀合通过被工程化到抗体恒定区中之半胱氨酸残基而发生。在另一个实施方式中,缀合通过酰基供体谷氨酰胺残基而发生,该酰基供体谷氨酰胺a)通过肽标签被加到抗体恒定区,b)被工程化到抗体恒定区中,或c)通过工程化周围之残基而变成可接近的/反应性的。连接子可为可切割的(即,在细胞内条件下容易切割)或不可切割的。在某些实施方式中,该连接子是可切割之连接子。
本发明中所用之用语“药物(D)”乃指可用来治疗癌的任何治疗剂。该药物具有生物活性或可检测之活性,例如细胞毒性剂、化学治疗剂、细胞抑制剂、与免疫调节剂。在较佳实施方式中,治疗剂对肿瘤有细胞毒效应,包括耗减、消除、与/或杀死肿瘤细胞。用语药物(drug)、载荷物(payload)、与药物载荷物(drug payload)可交换地使用。在一个具体实施方式中,该药物是抗有丝分裂剂。在一个更具体实施方式中,该药物是耳抑素。在又一个更具体实施方式中,该药物是2-甲基丙氨酰基-N-[(3R,4S,5S)-3-甲氧基-1-{(2S)-2-[(1R,2R)-1-甲氧基-2-甲基-3-氧代-3-{[(1S)-2-苯基-1-(1,3-噻唑-2-基)乙基]氨基}丙基]吡咯烷-1-基}-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺(也称为0101)。在某些具体实施方式中,该药物较佳为可透膜的。
本发明中所用之用语“L-D”乃指由连接至连接子(L)的药物(D)产生的连接子-药物部分。
与本发明有关之另外的科技用语,除非另外指出,否则应当具有该领域之具有普通技能者一般理解的意义。此外,除非上下文另外要求,否则单数词应当包括复数,及复数词应当包括单数。一般来说,本发明中所述之细胞与组织培养、分子生物学、免疫学、微生物学、遗传学与、蛋白质与核酸化学、和杂交相关命名法与技术是该领域中众所周知且常用的。
I.HER2抗体
就制备本发明之位点特异性HER2ADC而言,该抗体可为特异性结合至HER2的胞外功能域之任何抗体。在一个实施方式中,该抗体用来使该ADC结合至和曲妥单抗相同之HER2的表位,与/或使该ADC与曲妥单抗竞争HER2结合。在另一个实施方式中,该抗体用来使该ADC具有和曲妥单抗相同之重链可变区CDR与轻链可变区CDR。在又一个实施方式中,该抗体用来使该ADC具有和曲妥单抗相同之重链可变区与轻链可变区。
本发明中所用之与抗体有关的用语“竞争”表示第一抗体(或其抗原结合片段)以足够类似于第二抗体(或其抗原结合片段)结合方式结合至表位,使得在第二抗体存在下第一抗体与其同源表位之结合比在无第二抗体存在下第一抗体的结合可检测出地减少。在第一抗体存在下第二抗体与其表位之结合也可检测出地减少可为但不必是非此即彼的案例。也就是说,在无第二抗体抑制第一抗体与其表位之结合的情况下,第一抗体能抑制第二抗体与其表位之结合。然而,在各抗体在相同、更大或更小的程度上可检测出地抑制其它抗体与其同源表位或配体之结合的情况下,认为该抗体彼此“交叉竞争”其各自表位之结合。本发明包含竞争性抗体与交叉竞争性抗体。不论这样的竞争或交叉竞争发生之机制(例如,立体阻碍、构象改变、或结合至共同表位、或其部分),根据本发明中提供的明示说明,本领域技术人员会理解本发明中所揭示之方法包含且能使用这样的竞争性抗体与/或交叉竞争性抗体。
曲妥单抗(商品名称)是一种结合至HER2之胞外功能域的人源化单克隆抗体。其可变区之氨基酸序列的揭示参见美国专利第5,821,337号(VH是SEQ ID No:42及VL是SEQ ID No:41)和下文的表1(分别是SEQ ID No:1与7)。重链可变区CDR之氨基酸序列是SEQ ID No:2至4,而轻链CDR之氨基酸序列是SEQ ID No:6至10(下文的表1)。完整的重链与轻链之氨基酸序列分别是SEQ ID No:6与12。
T-DM1(商品名称)是一种由通过稳定的硫醚连接子MCC(4-[N-顺丁烯二酰亚胺甲基]环己烷-1-羧酸酯)缀合至类美登素药剂DM1之曲妥单抗组成的抗体药物缀合物(美国专利第8,337,856号)。此ADC之抗体成分同于曲妥单抗。使用常规缀合(位点特异缀合以外的)技术把载荷物缀合至曲妥单抗,使得该ADC是具有不同量的彼此缀合之DM1的物种之异质总体。该DM1载荷物通过抑制微管蛋白聚合来抑制在细胞有丝分裂过程中的微管形成以抑制细胞增生(Remillard et al.,1975,Science 189:1002-5)。获准于治疗先前经与紫杉烷药剂治疗过且变成难治之患者中HER2阳性转移性乳癌。在实施例节中所述的实验中所用之T-DM1系使用公开可利用的信息而自制。
以位点特异方式把本发明之ADC缀合至载荷物。为符合此类型缀合,必须将抗体衍生化来提供工程化在一或多个特异位点的反应性半胱氨酸残基,和在转谷氨酰胺酶与胺存在下工程化多肽使含有酰基供体谷氨酰胺之标签或内生性谷氨酰胺变成反应性的。可利用该领域中已知之任何方法把氨基酸修饰,及许多这样的方法是本领域技术人员众所周知且为标准。例如,但不限于,可能使用任何众所周知的以PCR为基础之技术来实现氨基酸取代、删除与插入。可能利用定点突变法来氨基酸取代(参见,例如,Zoller and Smith,1982,Nucl.Acids Res.10:6487-6500;及Kunkel,1985,PNAS 82:488)。
在需要保留抗原结合之应用上,这样的修饰应当是在不扰乱抗体之抗原结合能力的位点上。在较佳实施方式中,该一或多个修饰是在重链恒定区与/或轻链恒定区完成。
本发明中所用之抗体的用语“恒定区”乃指抗体轻链之恒定区或抗体重链的恒定区,以单一或组合形式。用来制造本发明之ADC的抗体之恒定区可能由下列中任一者的恒定区衍生:IgA、IgD、IgE、IgG、IgM、或其任何同型及亚类和突变型。
恒定区不直接涉及将抗体结合至抗原,但显出不同效应功能,例如Fc受体(FcR)结合、抗体参与抗体依赖性细胞毒性(ADCC)、调理作用、引发补体依赖性细胞毒性、及肥胖细胞去颗粒作用。该领域中熟悉之用语“Fc区”用于定义免疫球蛋白重链的C末端区。该“Fc区”可为天然序列Fc区或变异体Fc区。尽管免疫球蛋白重链之Fc区的定义可能改变,然而人IgG重链Fc区之定义一般是从第Cys226位或从第Pro230位氨基酸残基延伸至其羧基末端。在Fc区中的残基编号是根据Kabat之EU Index(Kabat et al.,Sequences of Proteins ofImmunological Interest,5th Ed.Public Health Service,National Institutes ofHealth,Bethesda,Md.,1991)。免疫球蛋白Fc区一般具有二个恒定区:CH2与CH3。
抗体使用二个不同轻链恒定区:CLκ与CLλ。CLκ具有已知的多型基因座CLκ-V/A45与CLκ-L/V83(使用Kabat编号系统,如Kabat et al.(1991,NIH Publication 91-3242,National Technical Information Service,Springfield,VA)明示,所以κ位与λ位全都根据Kabat系统来编号),因此考虑到多型性Km(1):CLκ-V45/L83;Km(1,2):CLκ-A45/L83;与Km(3):CLκ-A45/V83。本发明之多肽、抗体与ADC能具有抗体成分,其具有这些轻链恒定区中任一者。
为了明确起见,除非另外具体指定,否则抗体之人IgG重链恒定区中的氨基酸残基系根据Edelman et al.,1969,Proc.Natl.Acad.Sci.USA 63(1):78-85之EU Index,如Kabat et al.,1991中所述(在本发明中称为“Kabat之EU index”),来编号。典型上,该Fc区包含人IgG1恒定区的从约第236位至约第447位氨基酸残基。C编号间的相似处能在(例如)IGMT数据库中找到。轻链恒定区之氨基酸残基系根据Kabat et al.,1991来编号。抗体恒定区氨基酸残基的编号也在国际专利公布WO 2013/093809中显示。Kabat之EU index用于IgG重链恒定区的唯一例外是实施例中所述之残基A114。A114乃指Kabat编号,而对应的EUindex号是118。这是由于此位点之位点特异缀合的最初公开使用Kabat编号并且把此位点称为A114C,且自此以后在该领域中广泛地以“114”位点来使用。参见Junutula et al.,Nature Biotechnology 26,925-932(2008)。为了和在该领域中此位点的习惯用法一致,在实施例中使用“A114”、“A114C”、“C114”或“114C”。
能将编码用于制造本发明之ADC的抗体之重链与轻链的核酸克隆到用于表达或增殖之载体上。可把编码关注抗体之序列保留于宿主细胞内的载体中,然后把该宿主细胞扩增与冷冻以便未来之用。
本发明中所用之用语“载体”乃指能输送,且较佳地表达宿主细胞内一或多种关注基因或序列的构建体。载体实例包括但不限于:病毒载体、裸DNA或RNA表达载体、质粒载体、黏质粒载体或噬菌体载体、阳离子型缩合剂关连性DNA或RNA表达载体、包覆于脂质体中的DNA或RNA表达载体、与某些真核细胞(例如生产细胞)。
本发明中所用之用语“宿主细胞”包括可为或已为用于混入多核苷酸嵌入物的载体之接受体的个别细胞或细胞培养物。宿主细胞包括单一宿主细胞之后代,且由于自然突变、意外突变、或人为突变,该后代可能不必和原始母细胞完全相同(在型态上或在基因体DNA互补上)。宿主细胞包括受核酸或本发明之载体体内转染的细胞。
表1提供用来构建本发明之位点特异性ADC的人源化HER2抗体的氨基酸(蛋白质)序列与相关核酸(DNA)序列。所示之CDR利用Kabat编号方案来定义。
表1中所示之抗体重链与轻链具有曲妥单抗重链可变区(VH)与轻链可变区(VL)。重链恒定区与轻链恒定区由曲妥单抗衍生化且含有一或多个修饰以在制造本发明之ADC时得以位点特异缀合。
将位点特异缀合之抗体恒定区中的氨基酸序列修饰加底线且加粗。由曲妥单抗衍生化之抗体的命名法是T(曲妥单抗)后面接着小括号,在小括号中修饰之氨基酸位点的两侧是野生型残基之单字母氨基酸码和现于衍生化抗体中的位点之残基的单字母氨基酸码。此命名法之二个例外是“κK183C”,其表示轻(κ)链上的第183位已经由赖氨酸修饰成半胱氨酸,及“LCQ05”,其表示被附接至轻链恒定区之C末端的含有谷氨酰胺的8个氨基酸标签。
表1中所示之修饰中的一者不是用于缀合。在重链上的第222位之残基(使用Kabat编号方案的EU index)可被改变而导致更均质的抗体与载荷物缀合物,更佳之在抗体与载荷物间的分子间交联,与/或链间交联明显减少。
表1:人源化HER2抗体的序列
在某些实施方式中,本发明之ADC能使用包含下列的抗体:曲妥单抗之重链可变区CDR与轻链可变区CDR(SEQ ID No:2至4的VH CDR与SEQ ID No:8至10的VL CDR)及表1中所揭示之重链恒定区与轻链恒定区的任何组合,条件是当该重链恒定区是SEQ ID No:5时,则该轻链恒定区不是SEQ ID No:11(由于此组合再产生野生型曲妥单抗而因此不会考虑到位点特异缀合)。在这样的实施方式中,该重链恒定区可以选自SEQ ID No:17、5、13、21、23、25、27、29、31、33、35、37或39中任一者,而该轻链恒定区可以选自SEQ ID No:41、11或43中任一者,条件是该组合不是SEQ ID No:5及SEQ ID No:11,如上所述。
在更具体实施方式中,本发明之ADC能使用包含下列的抗体:曲妥单抗之重链可变区CDR与轻链可变区CDR(SEQ ID No:2至4的VH CDR与SEQ ID No:8至10的VL CDR)及选自下列的重链恒定区与轻链恒定区组合:
(a)重链恒定区是SEQ ID No:17及轻链恒定区是SEQ ID No:41;
(b)重链恒定区是SEQ ID No:5及轻链恒定区是SEQ ID No:41;
(c)重链恒定区是SEQ ID No:17及轻链恒定区是SEQ ID No:11;
(d)重链恒定区是SEQ ID No:21及轻链恒定区是SEQ ID No:11;
(e)重链恒定区是SEQ ID No:23及轻链恒定区是SEQ ID No:11;
(f)重链恒定区是SEQ ID No:25及轻链恒定区是SEQ ID No:11;
(g)重链恒定区是SEQ ID No:27及轻链恒定区是SEQ ID No:11;
(h)重链恒定区是SEQ ID No:23及轻链恒定区是SEQ ID No:41;
(i)重链恒定区是SEQ ID No:25及轻链恒定区是SEQ ID No:41;
(j)重链恒定区是SEQ ID No:27及轻链恒定区是SEQ ID No:41;
(k)重链恒定区是SEQ ID No:29及轻链恒定区是SEQ ID No:11;
(l)重链恒定区是SEQ ID No:31及轻链恒定区是SEQ ID No:11;
(m)重链恒定区是SEQ ID No:33及轻链恒定区是SEQ ID No:43;
(n)重链恒定区是SEQ ID No:35及轻链恒定区是SEQ ID No:11;
(o)重链恒定区是SEQ ID No:37及轻链恒定区是SEQ ID No:11;
(p)重链恒定区是SEQ ID No:39及轻链恒定区是SEQ ID No:11;或
(q)重链恒定区是SEQ ID No:13及轻链恒定区是SEQ ID No:43。
在又一个更具体实施方式中,本发明之ADC包含具有下列的抗体:SEQ ID No:2至4之VH CDR与SEQ ID No:8至10的VL CDR和SEQ ID No:17之重链恒定区及SEQ ID No:41的轻链恒定区。
在另一个更具体实施方式中,本发明之ADC包含具有下列的抗体:SEQ ID No:2至4之VH CDR与SEQ ID No:8至10的VL CDR和SEQ ID No:13之重链恒定区及SEQ ID No:43的轻链恒定区。
在其它实施方式中,本发明之ADC能使用包含下列的抗体:表1中所揭示之重链与轻链的任何组合,条件是若该重链是SEQ ID No:6,则该轻链不是SEQ ID No:12(由于此组合再产生野生型曲妥单抗而因此不会考虑到位点特异缀合)。在这样的实施方式中,该重链可以选自SEQ ID No:18、6、14、22、24、26、28、30、32、34、36、38或40中任一者,而该轻链可以选自SEQ ID No:42、12或44中任一者,条件是该组合不是SEQ ID No:6及SEQ ID No:12,如上所述。
在更具体实施方式中,本发明之ADC能使用包含选自下列的重链与轻链组合之抗体:
(a)重链是SEQ ID No:18及轻链是SEQ ID No:42;
(b)重链是SEQ ID No:6及轻链是SEQ ID No:42;
(c)重链是SEQ ID No:18及轻链是SEQ ID No:12;
(d)重链是SEQ ID No:22及轻链是SEQ ID No:12;
(e)重链是SEQ ID No:24及轻链是SEQ ID No:12;
(f)重链是SEQ ID No:26及轻链是SEQ ID No:12;
(g)重链是SEQ ID No:28及轻链是SEQ ID No:12;
(h)重链是SEQ ID No:24及轻链是SEQ ID No:42;
(i)重链是SEQ ID No:26及轻链是SEQ ID No:42;
(j)重链是SEQ ID No:28及轻链是SEQ ID No:42;
(k)重链是SEQ ID No:30及轻链是SEQ ID No:12;
(l)重链是SEQ ID No:32及轻链是SEQ ID No:12;
(m)重链是SEQ ID No:34及轻链是SEQ ID No:44;
(n)重链是SEQ ID No:36及轻链是SEQ ID No:12;
(o)重链是SEQ ID No:38及轻链是SEQ ID No:12;
(p)重链是SEQ ID No:40及轻链是SEQ ID No:12;或
(q)重链是SEQ ID No:14及轻链是SEQ ID No:44。
在又一个更具体实施方式中,本发明之ADC包含具有下列的抗体:SEQ ID No:18之重链及SEQ ID No:42的轻链。含有编码SEQ ID No:18之重链及SEQ ID No:42的轻链的核酸的质粒已于2015年11月17日保藏于美国菌种保存中心(ATCC,10801University Blvd.,Manassas,VA20110-2209),保藏编号分别是PTA-122672与PTA-122673。该保藏系根据国际承认用于专利程序的微生物保存布达佩斯条约之规定进行。这确保该保藏之存活性培养物从保藏日起算保持30年。根据布达佩斯条约的条款保藏于ATCC,且遵守Pfizer Inc.与ATCC间之协议,该协议确保在发布切合的美国专利或向公众公开任何美国或外国之专利申请案后(以先到者为准)立即向公众开放该保藏的培养物之后代的永久性与不受限制之可利用性,并且根据35U.S.C.Section 122及据此的Commissioner's rules(包括37C.F.R.Section 1.14,特别是886OG 638)确保由美国专利商标局局长决定之培养物的后代之可利用性的权利。
本申请案之受让人业已就下列达成协议:万一保藏的材料之培养物在合适条件下培养时死亡或丧失或被消灭,该材料在通知后将立即被替换成另一个相同材料。所保藏的材料之可利用性不应被理解成在违反任何政府根据其专利法授权所授予的权利之情况下实践本发明的许可。
在另一个更具体实施方式中,本发明之ADC包含具有SEQ ID No:14的重链及SEQID No:44之轻链的抗体。
在本发明之某些方面,本发明的ADC包括具有包含氨基酸序列之重链与/或轻链的抗体,该氨基酸序列和上文揭示之重链或轻链中任一者有至少90%、95%、98%、或99%相同。被改变的残基可为在抗体可变区或恒定区中。在某些实施方式中,和上文揭示之重链或轻链中任一者相比,有不超过1、2、3、4或5个残基被改变。在其它实施方式中,在可变区CDR中任一者中没有被改变的残基。
在有关氨基酸序列之论述中,用语“同一性”(或“%相同”)表示在符合最大一致时相同的二个序列中之残基数。有一些可用于测量氨基酸序列同一性的该领域熟悉之不同算法(即Basic Local Alignment Tool或)。除非另外具体指定,否则使用特定程序或算法的预设参数。
就制备ADC而言,本发明中所述之HER2抗体可为实质纯的,即至少50%纯(即不含污染物),更佳地,至少90%纯,更佳地,至少95%纯,又更佳地,至少98%纯,及最佳地,至少99%纯。
II.药物
可用于制备本发明之位点特异性HER2ADC的药物包括可用于治疗癌的任何治疗剂,其包括但不限于:细胞毒性剂、细胞抑制剂、免疫调节剂与化学治疗剂。细胞毒性效应乃指耗减、消除与/或杀死靶细胞(即肿瘤细胞)。细胞毒性剂乃指对细胞具有细胞毒性效应的药剂。细胞生长抑制效应乃指抑制细胞增生。细胞抑制剂乃指对细胞具有细胞生长抑制效应,藉此抑制特异细胞亚群(即肿瘤细胞)生长与/或增殖的药剂。免疫调节剂乃指通过直接或间接使另一种药剂更有效来产生细胞因子与/或抗体与/或调节T细胞功能来刺激免疫反应,藉此抑制或降低细胞亚群(即肿瘤细胞)生长的药剂。化学治疗剂乃指可用于治疗癌的化合物。药物也可能是药物衍生物,其中已经把药物官能化以能够和本发明之抗体缀合。
在某些实施方式中,该药物是可透膜药物。在这样的实施方式中,该载荷物(即药物)能引出旁观者效应,其中包围住最初把ADC内化之细胞的细胞被该载荷物杀死。这在该载荷物从抗体中释出(即通过可切割的连接子切割)及越过细胞膜和在扩散后立即诱发杀死周围的细胞时发生。
根据所揭示的方法,将该药物用于制备如式Ab-(L-D)之抗体药物缀合物,其中(a)Ab是结合至HER2的抗体,及(b)L-D是连接子-药物部分,其中L是连接子,及D是药物。
药物对抗体比(DAR)或载药量指示每一抗体缀合之药物(D)分子的数量。本发明之抗体药物缀合物使用位点特异缀合,使得ADC组合物中实质有具有1的DAR的ADC同质总体。在某些实施方式中,该DAR是1。在某些实施方式中,该DAR是2。在其它实施方式中,该DAR是3。在其它实施方式中,该DAR是4。在其它实施方式中,该DAR大于4。
使用常规缀合(位点特异缀合以外的)导致不同种ADC之异质总体,各种ADC具有不同的个别DAR。以此方式制得之ADC组合物包括多个抗体,每一抗体缀合至特定数量的药物分子。因此,该组合物具有平均DAR。T-DM1在赖氨酸残基上使用常规缀合且具有约4的平均DAR,该广大分布包括载荷0、1、2、3、4、5、6、7或8个药物分子之ADC(Kim etal.,2014,Bioconj Chem 25(7):1223-32)。
多个ADC之组合物、批料、与/或配制物可经由平均DAR鉴定特性。DAR与平均DAR能利用各种常规方式测得,该方式例如UV光谱分析、质谱分析、ELISA检定、放射线测定方法、疏水性交互作用层析术(HIC)、电泳法与HPLC。
在本发明之实施方式中,HER2ADC可能具有1的DAR、2的DAR、3的DAR、4的DAR、5的DAR、6的DAR、7的DAR、8的DAR、9的DAR、10的DAR、11的DAR、12的DAR、或大于12的DAR。在本发明之实施方式中,HER2ADC可能具有1个药物分子、或2个药物分子、或3个药物分子、或4个药物分子、或5个药物分子、或6个药物分子、或7个药物分子、或8个药物分子、或9个药物分子、或10个药物分子、或11个药物分子、或12个药物分子、或大于12个药物分子。
在本发明之实施方式中,HER2ADC可能具有在约2至约4范围内的平均DAR、或在约3至约5范围内之平均DAR、或在约4至约6范围内的平均DAR、或在约5至约7范围内之平均DAR、或在约6至约8范围内的平均DAR、或在约7至约9范围内之平均DAR、或在约8至约10范围内的平均DAR、或在约9至约11范围内之平均DAR、或在约10至约12范围内的平均DAR等。在某些实施方式中,HER2ADC之组合物、批料、与/或配制物可能具有约1的平均DAR、或约2之平均DAR、或约3的平均DAR、或约4之平均DAR、或约5的平均DAR、或约6之平均DAR、或约7的平均DAR、或约8之平均DAR、或约9的平均DAR、或约10之平均DAR、或约11的平均DAR、或约12之平均DAR、或大于12的平均DAR。如上述平均DAR范围,用语“约”表示+/-0.5%。
HER2ADC之组合物、批料、与/或配制物可经由较佳的平均DAR范围鉴定特性,例如在约3至约5范围内之平均DAR、在约3至约4范围内的平均DAR、或在约4至约5范围内之平均DAR。此外,HER2ADC之组合物、批料、与/或配制物可经由较佳的平均DAR范围鉴定特性,例如在3至5范围内之平均DAR、在3至4范围内的平均DAR、或在4至5范围内之平均DAR。
在本发明之某些实施方式中,HER2ADC之组合物、批料、与/或配制物可经由下列鉴定特性:约1.0的平均DAR、或1.0之平均DAR、或1.1的平均DAR、或1.2之平均DAR、或1.3的平均DAR、或1.4之平均DAR、或1.5的平均DAR、或1.6之平均DAR、或1.7的平均DAR、或1.8之平均DAR、或1.9的平均DAR。在另一个实施方式中,HER2ADC之组合物、批料、与/或配制物可经由下列鉴定特性:约2.0的平均DAR、或2.0之平均DAR、或2.1的平均DAR、或2.2之平均DAR、或2.3的平均DAR、或2.4之平均DAR、或2.5的平均DAR、或2.6之平均DAR、或2.7的平均DAR、或2.8之平均DAR、或2.9的平均DAR。在另一个实施方式中,HER2ADC之组合物、批料、与/或配制物可经由下列鉴定特性:约3.0的平均DAR、或3.0之平均DAR、或3.1的平均DAR、或3.2之平均DAR、或3.3的平均DAR、或3.4之平均DAR、或3.5的平均DAR、或3.6之平均DAR、或3.7的平均DAR、或3.8之平均DAR、或3.9的平均DAR。在另一个实施方式中,HER2ADC之组合物、批料、与/或配制物可经由下列鉴定特性:约4.0的平均DAR、或4.0之平均DAR、或4.1的平均DAR、或4.2之平均DAR、或4.3的平均DAR、或4.4之平均DAR、或4.5的平均DAR、或4.6之平均DAR、或4.7的平均DAR、或4.8之平均DAR、或4.9的平均DAR、或5.0之平均DAR。
在另一个实施方式中,HER2ADC之组合物、批料、与/或配制物可经由下列鉴定特性:12或以下的平均DAR、11或以下之平均DAR、10或以下的平均DAR、9或以下之平均DAR、8或以下的平均DAR、7或以下之平均DAR、6或以下的平均DAR、5或以下之平均DAR、4或以下的平均DAR、3或以下之平均DAR、2或以下的平均DAR、或1或以下之平均DAR。
在其它实施方式中,HER2ADC之组合物、批料、与/或配制物可经由下列鉴定特性:11.5或以下的平均DAR、10.5或以下之平均DAR、9.5或以下的平均DAR、8.5或以下之平均DAR、7.5或以下的平均DAR、6.5或以下之平均DAR、5.5或以下的平均DAR、4.5或以下之平均DAR、3.5或以下的平均DAR、2.5或以下之平均DAR、1.5或以下的平均DAR。
在本发明之某些实施方式中,本发明中所揭示之通过半胱氨酸残基的常规缀合方法与纯化条件提供具有在约3至5范围内,较佳为约4之最佳化平均DAR的HER2ADC之组合物、批料、与/或配制物。
在本发明之某些实施方式中,本发明中所揭示的通过工程化之半胱氨酸残基的位点特异缀合方法与纯化条件提供具有在约3至5范围内,较佳为约4之最佳化平均DAR的HER2ADC之组合物、批料、与/或配制物。
在本发明之某些实施方式中,通过本发明中所揭示的以转谷氨酰胺酶为基础之缀合的位点特异缀合方法与纯化条件提供具有在约1至3范围内,较佳为约2之最佳化平均DAR的HER2ADC之组合物、批料、与/或配制物。
本发明也包含如式Ab-(L-D)p之抗体药物缀合物,其中(a)Ab是结合至HER2的抗体,或其抗原结合片段,(b)L-D是连接子-药物部分,其中L是连接子,及D是药物,和(c)p是附接至该抗体之连接子/药物部分的数量。对位点特异性ADC而言,p是整数,由于该ADC的均质。在某些实施方式中,p是4。在其它实施方式中,p是3。在其它实施方式中,p是2。在其它实施方式中,p是1。在其它实施方式中,p大于4。
在一个实施方式中,本发明之ADC的药物成分是抗有丝分裂剂。在一个具体实施方式中,该抗有丝分裂剂是耳抑素(例如0101、8261、6121、8254、6780与0131;参见下表2)。在一个更具体实施方式中,该耳抑素药物是2-甲基丙氨酰基-N-[(3R,4S,5S)-3-甲氧基-1-{(2S)-2-[(1R,2R)-1-甲氧基-2-甲基-3-氧代-3-{[(1S)-2-苯基-1-(1,3-噻唑-2-基)乙基]氨基}丙基]吡咯烷-1-基}-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺(也通称为0101)。
耳抑素通过抑制微管蛋白聚合来抑制在细胞有丝分裂过程中的微管形成以抑制细胞增生。PCT国际公布WO2013/072813(将其全部内容并入本案)揭示可用于制造本发明之ADC的耳抑素及提供制造该耳抑素的方法。
表2:药物
在本发明之某些实施方式中,该细胞毒性剂能使用脂质粒或生物兼容性聚合物制得。能把本发明中所述之HER2抗体缀合至该生物兼容性聚合物以增加血清半衰期与生物活性,与/或延长体内半衰期。生物兼容性聚合物实例包括水溶性聚合物,例如聚乙二醇(PEG)或其衍生物及含有两性离子之生物兼容性聚合物(例如含有磷酸胆碱的聚合物)。
III.连接子
本发明之位点特异性HER2ADC系使用连接子把药物连接至或缀合至HER2抗体而制得。连接子是可用于连接药物与抗体以形成抗体药物缀合物(ADC)的双官能性化合物。这样的缀合物使药物得以选择性输送到肿瘤细胞。合适连接子包括,例如,可切割的连接子与不可切割的连接子。可切割之连接子典型地在细胞内条件下容易切割。经缀合的药物从抗体中切割之主要机制包括在溶酶体(腙类、缩醛类、与类顺式乌头酸酯的酰胺类)的酸性pH下水解、通过溶酶体酶类(组织蛋白酶与其它溶酶体酶)使肽切割、及二硫键还原。由于这些不同切割机制,把药物连接至抗体的机制也大幅变化并且能使用任何合适连接子。
合适可切割的连接子包括但不限于可被细胞内蛋白酶切割的肽连接子,该细胞内蛋白酶例如溶酶体蛋白酶或核内体蛋白酶,例如顺丁烯二酰亚胺基己酰基-缬氨酸-瓜氨酸-对氨酸苄氧基羰基(vc)、N~2~-乙酰基-L-赖氨酰基-L-缬氨酰基-L-瓜氨酸-对氨酸苄氧基羰基-N,N'-二甲基氨基乙基-CO-(AcLysvc)及m(H20)c-vc(下表3)。在具体实施方式中,该连接子是可切割的连接子,使得一旦该连接子切割,该载荷物便能诱发旁观者效应。该旁观者效应是在可透膜之药物从抗体中释出(即通过可切割的连接子切割)及越过细胞膜和在扩散后立即诱发杀死包围住最初把ADC内化之细胞的细胞时发生。
合适不可切割的连接子包括但不限于顺丁烯二酰亚胺基己酰基(mc)、顺丁烯二酰亚胺-(聚乙二醇)6(MalPeg6)、Mal-PEG2C2、Mal-PEG3C2及m(H20)c(下表3)。
其它合适连接子包括在特异pH或在pH范围下可水解的连接子,例如腙连接子。另外之合适可切割的连接子包括二硫键连接子。连接子可能与抗体共价键结到该抗体在细胞内一定被降解而释出药物的程度,例如mc连接子等。
在本发明之特定实施方式中,在本发明之位点特异性HER2ADC中的连接子是可切割的且可为vc或AcLysvc。
许多缀合至抗体之治疗剂(药物)具有少的(若有的话)水溶性,而该少的水溶性能限制该缀合物之载药量,由于该缀合物的聚集作用。一个克服这问题之方法是把增溶基加到连接子中。可使用由PEG与二肽组成的连接子制成之缀合物,其包括具有附接至该抗体的PEG-二酸、硫醇-酸、或顺丁烯二酰亚胺-酸,二肽间隔子,及结合至蒽环类抗生素或双联霉素(duocarmycin)类似物之胺的酰胺键之缀合物。另一个实例是用结合至细胞毒性剂的含有PEG之二硫化物连接子与结合至抗体的酰胺制得之缀合物。混入PEG基团的方法可能有益于克服聚集作用及限制载药量。
表3:连接子
连接子通过分子之左侧附接至单克隆抗体及通过分子的右侧附接至药物,如表3中所示。
IV.制备位点特异性HER2ADC之方法
本发明也提供制备本发明之抗体药物缀合物的方法。例如,制造本发明中所揭示之位点特异性HER2ADC的方法可以包括:(a)将该连接子连接至该药物;(b)将该连接子药物部分缀合至该抗体;及(c)纯化该抗体药物缀合物。
本发明之HER2ADC使用位点特异方法来把该HER2抗体缀合至该药物载荷物。
在一个实施方式中,该位点特异缀合通过一或多个被工程化到抗体恒定区中之半胱氨酸残基而发生。制备用于通过半胱氨酸残基的位点特异缀合之HER2抗体的方法可以按照PCT公布WO2013/093809(将其全部内容并入本案中作为参考)中所述地来进行。以下位点(对IgG1恒定区使用Kabat编号方案的EU Index及对κ链恒定区使用Kabat编号方案)中的一或多者可被改变成半胱氨酸,且从而成为用于缀合之位点:a)在重链恒定区上的第114、246、249、265、267、270、276、278、283、290、292、293、294、300、302、303、314、315、318、320、327、332、333、334、336、345、347、354、355、358、360、362、370、373、375、376、378、380、382、386、388、390、392、393、401、404、411、413、414、416、418、419、421、428、431、432、437、438、439、443、与444位残基,与/或b)在κ链恒定区上的第111、149、183、188、207、与210位残基。
在一个具体实施方式中,该一或多个可被改变成半胱氨酸之位点(使用Kabat编号方案的EU Index)a)在重链恒定区上的是第290、334、392与/或443位点,与/或b)在轻链恒定区上的是第183位点(Kabat编号)。
在一个更具体实施方式中,在重链恒定区上的第290位点及在轻链恒定区上的第183位点被改变成用于缀合之半胱氨酸。
在另一个实施方式中,该位点特异缀合通过一或多个被工程化到抗体恒定区中之酰基供体谷氨酰胺残基而发生。制备用于通过谷氨酰胺残基的位点特异缀合之HER2抗体的方法可以按照PCT公布WO2012/059882(将其全部内容并入本案中作为参考)中所述地来进行。可以三种不同方式工程化抗体以表达用于位点特异缀合的谷氨酰胺残基。
可以将含有谷氨酰胺残基之短肽标签混入轻链与/或重链的一些不同位点(即在N末端、C末端内)。在第一个实施方式中,可以把含有谷氨酰胺残基之短肽标签附接至重链与/或轻链的C末端。可以附接以下含有谷氨酰胺之标签中的一或多者以成为用于药物缀合之酰基供体:GGLLQGPP(SEQ ID NO:81)、GGLLQGG(SEQ ID NO:82)、LLQGA(SEQ ID NO:83)、GGLLQGA(SEQ ID NO:84)、LLQG(SEQ ID NO:85)、LLQGPG(SEQ ID NO:86)、LLQGPA(SEQ IDNO:87)、LLQGP(SEQ ID NO:88)、LLQP(SEQ ID NO:89)、LLQPGK(SEQ ID NO:90)、LLQGAPGK(SEQ ID NO:91)、LLQGAPG(SEQ ID NO:92)、LLQGAP(SEQ ID NO:93)、LLQX1X2X3X4X5,其中X1是G或P,其中X2是A、G、P、或不存在,其中X3是A、G、K、P、或不存在,其中X4是G、K、或不存在,及其中X5是K或不存在(SEQ ID NO:94),或LLQX1X2X3X4X5,其中X1是任何天然氨基酸及其中X2、X3、X4与X5是任何天然氨基酸或不存在(SEQ ID NO:95)。
在一个具体实施方式中,GGLLQGPP(SEQ ID NO:81)被附接至轻链的C末端。
在第二个实施方式中,可以利用定点突变把重链与/或轻链上的残基改变成谷氨酰胺残基。在一个具体实施方式中,可以把重链上的第297位残基(使用Kabat编号方案之EUIndex)改变成谷氨酰胺(Q)从而成为用于缀合的位点。
在第三个实施方式中,可以改变重链或轻链上的残基导致该位点之无糖基化,使得一或多个内生性谷氨酰胺变成缀合可接近的/反应性的。在一个具体实施方式中,可以把重链上的第297位残基(使用Kabat编号方案之EU Index)改变成丙氨酸(A)。在这样的情况下,重链上的第295位谷氨酰胺(Q)能用于缀合。
可利用反应变量之变型凭经验求出用于形成缀合物的最佳化反应条件,该反应变量例如温度、pH、连接子-载荷物部分投入、及添加剂浓度。该领域之本领域技术人员无庸过度实验即可求出适用于缀合其它药物的条件。通过工程化之半胱氨酸残基的位点特异缀合在下文实施例5A中被举例说明。通过谷氨酰胺残基的位点特异缀合在下文实施例5B中被举例说明。
可将药物缀合至聚乙二醇(PEG),其包括直链或支链聚乙二醇聚合物与单体,以进一步增加每一抗体药物缀合物之药物分子数量。PEG单体如式-(CH2CH2O)-所示。可把药物与/或肽类似物直接或间接地结合至PEG,即通过合适间隔基团,例如糖类。PEG-抗体药物组合物也可包括另外的亲油性与/或亲水性部分以促进药物稳定性及输送到体内靶位点。含有PEG之组合物的代表性制法可在美国专利第6,461,603号、第6,309,633号、与第5,648,095号中找到。
在缀合后,可利用常规方法由未缀合之反应物与/或缀合物聚集物中分离出缀合物及纯化。这可以包括以下方法:粒径排阻层析术(SEC)、超过滤/渗滤法、离子交换层析术(IEC)、层析聚焦法(CF)、HPLC、FPLC、或Sephacryl S-200层析术。该分离也可利用疏水性交互作用层析术(HIC)实现。合适HIC介质包括Phenyl Sepharose 6 Fast Flow层析介质、Butyl Sepharose 4 Fast Flow层析介质、Octyl Sepharose 4 Fast Flow层析介质、Toyopearl Ether-650M层析介质、Macro-Prep methyl HIC层析介质或Macro-Prep t-Butyl HIC层析介质。
下表4显示用来产生本发明中明示之实施例节中的数据之HER2ADC。表4(第1至17列)中所示的位点特异性HER2ADC是本发明之位点特异性ADC的实施例。
可以使用位点特异技术通过上文III节中所揭示之任何连接子把上文I节中所揭示的任何HER2抗体缀合至上文II节中所揭示之任何药物,以制造本发明之位点特异性HER2ADC。在较佳实施方式中,该连接子是可切割的(例如vc或AcLysvc)。在其它较佳实施方式中,该药物是耳抑素(例如0101)。
在本发明的一个特定实施方式中,如式Ab-(L-D)之位点特异性HER2ADC包含(a)抗体Ab,其包含SEQ ID No:18的重链与SEQ ID No:42的轻链;及(b)连接子-药物部分L-D,其中L是连接子,及D是药物,其中该连接子是vc且其中该药物是0101。这样的ADC之示意图参见图1A。
在本发明的另一个特定实施方式中,如式Ab-(L-D)之位点特异性HER2ADC包含(a)抗体Ab,其包含SEQ ID No:14的重链与SEQ ID No:44的轻链;及(b)连接子-药物部分L-D,其中L是连接子,及D是药物,其中该连接子是AcLysvc且其中该药物是0101。这样的ADC之示意图参见图1B。
在本发明的另一个特定实施方式中,如式Ab-(L-D)之位点特异性HER2ADC包含(a)抗体Ab,其包含SEQ ID No:24的重链与SEQ ID No:42的轻链;及(b)连接子-药物部分L-D,其中L是连接子,及D是药物,其中该连接子是vc且其中该药物是0101。
在本发明的另一个特定实施方式中,如式Ab-(L-D)之位点特异性HER2ADC包含(a)抗体Ab,其包含SEQ ID No:26的重链与SEQ ID No:42的轻链;及(b)连接子-药物部分L-D,其中L是连接子,及D是药物,其中该连接子是vc且其中该药物是0101。
在本发明的另一个特定实施方式中,如式Ab-(L-D)之位点特异性HER2ADC包含(a)抗体Ab,其包含SEQ ID No:28的重链与SEQ ID No:42的轻链;及(b)连接子-药物部分L-D,其中L是连接子,及D是药物,其中该连接子是vc且其中该药物是0101。
在本发明的另一个特定实施方式中,如式Ab-(L-D)之位点特异性HER2ADC包含(a)抗体Ab,其包含SEQ ID No:30的重链与SEQ ID No:12的轻链;及(b)连接子-药物部分L-D,其中L是连接子,及D是药物,其中该连接子是vc且其中该药物是0101。
在本发明的另一个特定实施方式中,如式Ab-(L-D)之位点特异性HER2ADC包含(a)抗体Ab,其包含SEQ ID No:32的重链与SEQ ID No:12的轻链;及(b)连接子-药物部分L-D,其中L是连接子,及D是药物,其中该连接子是vc且其中该药物是0101。
在本发明的另一个特定实施方式中,如式Ab-(L-D)之位点特异性HER2ADC包含(a)抗体Ab,其包含SEQ ID No:34的重链与SEQ ID No:44的轻链;及(b)连接子-药物部分L-D,其中L是连接子,及D是药物,其中该连接子是AcLysvc且其中该药物是0101。
在本发明的另一个特定实施方式中,如式Ab-(L-D)之位点特异性HER2ADC包含(a)抗体Ab,其包含SEQ ID No:36的重链与SEQ ID No:12的轻链;及(b)连接子-药物部分L-D,其中L是连接子,及D是药物,其中该连接子是AcLysvc且其中该药物是0101。
在本发明的另一个特定实施方式中,如式Ab-(L-D)之位点特异性HER2ADC包含(a)抗体Ab,其包含SEQ ID No:38的重链与SEQ ID No:12的轻链;及(b)连接子-药物部分L-D,其中L是连接子,及D是药物,其中该连接子是vc且其中该药物是0101。
在本发明的另一个特定实施方式中,如式Ab-(L-D)之位点特异性HER2ADC包含(a)抗体Ab,其包含SEQ ID No:40的重链与SEQ ID No:12的轻链;及(b)连接子-药物部分L-D,其中L是连接子,及D是药物,其中该连接子是vc且其中该药物是0101。
表4:HER2ADCs
1C=可切割的;N=不可切割的
V.位点特异性HER2抗体药物缀合物之用途
本发明之抗体药物缀合物可用于治疗HER2表达性癌的治疗方法。在本发明之某些方面中,本发明提供抑制具有HER2表达性肿瘤的个体中肿瘤生长或进展之方法,其包括对有其需要的个体施用有效量之具有一或多种本发明中所述的ADC之组合物(即药学组合物)。在本发明的其它方面中,本发明提供抑制个体中HER2表达性癌细胞转移之方法,其包括对有其需要的个体施用有效量之具有一或多种本发明中所述的ADC之组合物(即药学组合物)。在本发明的其它方面中,本发明提供诱发个体中HER2表达性癌消退之方法,其包括对有其需要的个体施用有效量之具有一或多种本发明中所述的ADC之组合物(即药学组合物)。在其它方面中,本发明提供用于上述方法中使用的包含一或多种本发明中所述之ADC的药学组合物。在其它方面中,本发明提供一或多种本发明中所述之ADC或包含本发明中所述的ADC之药学组合物的用途,其系用于制造上述方法中使用之医药品。
和对照组或基准值相比,本发明中所揭示之治疗方法的所欲结果通常是可以计量之值。本发明中所用的相对用语例如“改进”、“增加”或“降低”指示相对于对照组之计量值,例如在开始本发明中所述的治疗前相同个体中的计量值,或在没有本发明中所述之治疗的情况下对照个体(或多重对照个体)中的计量值。代表性对照个体是患有和受治疗个体相同形式之癌的个体,两者年龄大约相同(以保证受治疗个体与对照个体的疾患期别可相比拟)。
对疗法作出反应的变化或改进一般达统计上显著性。本发明中所用之用语“显著性”或“显著的”系关于在二或多个实体间有不均的现象之机率的统计分析。为确定关系是否是“显著的”或具有“显著性”,数据之统计处理可为“P值”。低于使用者定义截止点之P值被视为显著的。小于或等于0.1、小于0.5、小于0.01、小于0.005、或小于0.001之P值可被视为显著的。
V.A.癌
本发明之ADC可用于治疗HER2表达性癌。在一个实施方式中,该HER2表达性癌是实体瘤。在一个更具体实施方式中,HER2表达性实体瘤包括但不限于:乳癌(例如雌激素与黄体激素受体阴性、三阴性乳癌)、卵巢癌、肺癌(例如非小细胞肺癌(包括腺癌、鳞状细胞癌与大细胞癌)和小细胞肺癌)、胃癌、食道癌、结肠直肠癌、尿路上皮癌(例如微乳突状尿路上皮癌与典型尿路上皮癌)、胰腺癌、唾液腺癌(例如黏液上皮样癌、腺样囊状癌与终末管腺癌)及脑癌或上述癌的转移(即HER2+乳癌的肺转移)(Martin et al.,2014,Future Oncol.10(8):1469-86)。
在一个更具体实施方式中,HER2表达性实体瘤包括但不限于:乳癌、卵巢癌、肺癌与胃癌。
在另一个实施方式中,该乳癌是雌激素受体与黄体激素受体阴性的。在一个更具体实施方式中,该乳癌是三阴性乳癌(TNBC)。
在另一个实施方式中,该肺癌是非小细胞肺癌(NSCLC)。
在本发明的一个方面中,本发明中所揭示之ADC可用于治疗先前未经治疗剂治疗过的HER2表达性癌(即,作为第一线治疗)。
在本发明的另一个方面中,本发明中所揭示之ADC可用于治疗对用另一治疗剂治疗有抗性、是该治疗难治的、和/或在该治疗后复发的HER2表达性癌(即,作为第二线治疗)。在一个实施方式中,该先前治疗是曲妥单抗(曲妥单抗或)单独或合并另一治疗剂(即紫杉烷,例如紫杉醇(paclitaxel)、多西他赛(docetaxel)、卡巴他赛(cabazitaxel)等)。在另一个实施方式中,该先前治疗是曲妥单抗emtansine(T-DM1或)单独或合并另一治疗剂(即紫杉烷,例如紫杉醇、多西他赛、卡巴他赛等)。
在本发明的另一个实施方式中,本发明中所揭示之ADC可用于治疗对用多于一种的其它治疗剂治疗有抗性、是该治疗难治的、和/或在该治疗后复发的HER2表达性癌(即,作为第三线治疗或第四线治疗等)。
本发明之ADC可用于治疗表达高水平的HER2之癌(即IHC 3+)、表达中水平的HER2之癌(即2+IHC或2+/3+IHC)、或表达低水平的HER2之癌(即IHC 1+、IHC 2+或IHC 1+/2+)(参见IVB节HER2检测方法)。这和曲妥单抗与T-DM1相反,该曲妥单抗与T-DM1对表达低或中水平的HER2之癌无效(Burris et al.,2011,J Clinical Oncology 29(4):398-405)。
本发明之ADC可用于治疗大部分癌细胞表达相似量的HER2之同源性癌。另外,本发明之ADC可用于治疗有表达不同水平的HER2之不同肿瘤细胞群体的实质性异源癌。
V.B.HER2检测方法
关于评估肿瘤上的HER2表达水平的最佳方式的方面已被论述且临床意义已被概述(Sauter et al.,2009,J Clin Oncol.27:1323-33;Wolff et al.,2007,J ClinicalOncology 25:118-45;Wolff et al.,2013,J Clinical Oncology 31:3997-4014)。现在可以利用下列来评估HER2状态:免疫组织化学法(IHC)、荧光原位杂交法(FISH)、与色质原位杂交法(CISH)。
IHC识别在细胞膜上的HER2蛋白质表达。结果通常使用范围从0+(无表达)到3+(高度表达)的半定量计分系统来表示。显示无水平(0+)或低水平(1+)的表达的肿瘤被认为是HER2阴性;相反地,显示高水平(3+)的表达的肿瘤应当被认为是HER2阳性。此方法具经济效益且容易取得,但受困于低灵敏度与不同观测者间的高差异(Gancberg et al.,2002,Breast Cancer Res Treat.74:113-20)。
使用IHC之HER2检测有四种FDA许可的商用试剂盒:HercepTestTM(Dako DenmarkA/S产品);Pathway(Ventana Medical Systems,Inc.产品);Insite HER2/NEU kit(Biogenex Laboratories,Inc.产品)和Bond Oracle HER2 IHC System(LeicaBiosystems产品)。这些都是高度标准化之半定量检测法,其将HER2表达水平分成0(每一细胞<20,000个受体,无可看见的表达)、1+(每一细胞~100,000个受体,一部分细胞膜染色,<10%的过度表达HER-2之细胞)、2+(每一细胞~500,000个受体,轻度到中度细胞膜完全染色,>10%的过度表达HER-2之细胞)、和3+(每一细胞~2,000,000个受体,高度细胞膜完全染色,>10%的过度表达HER-2之细胞)。忽略细胞质表达的存在。
FISH用DNA探针检测HER2基因扩增且比IHC更特异及灵敏(Owens et al.,2004,Clin Breast Cancer.5:63-69;Press et al.,2005,Clin Cancer Res.11:6598-6607;Vogel et al.,2002,J Clinical Oncology 20(3):719-726)。FISH提供每染色体17中节的HER2基因副本数量之定量结果。结果为HER2信号数对染色体17中节信号数比率。小于1.8的比率被认为在正常限制内。1.8至2.0之比率是可疑的且需要进一步试验。大于2.0的比率是HER2基因序列扩增。
使用FISH之HER2检测有四种FDA许可的商用试剂盒:HER2FISH Pharm DxTM kit(Dako Denmark产品);Pathvysion HER2 DNA Probe Kit(Abbott Molecular Inc.产品);Inform HER2/NEU和Inform HER2 Dual ISH DNA Probe Cocktail(二者都是VentanaMedical Systems,Inc.产品)。
另一种评估HER2基因扩增之方法是CISH。CISH非常类似于FISH,但使用在标准亮视野显微镜可看见的常规过氧化酶或碱性磷酸酶方法。使用CISH之HER2检测有二种FDA许可的商用试剂盒:HER2 CISH PharmDx Kit(Dako Denmark A/S产品)与Spot-Light HER2CISH Kit(Life Technologies,Inc.产品)。
常将利用FISH或CISH检测之基因扩增及利用IHC的蛋白质表达二者作为最初试验来评估HER2状态。该二种方法间有良好关系(Jacobs et al.,1999,J Clinical Oncology17(7):1974-82)。然而在肿瘤被分成可疑的(即IHC 2+,或1.8至2.2的FISH/CISH比,或每核4至6个信号的平均HER2基因副本数量)情况下,常见的方法是用另一种方法检验该肿瘤(Wolff et al.,2007,J Clinical Oncology 25:118-45)。
因此,具有如免疫组织化学法(IHC)所测得之3+水平和/或≥2.0的荧光原位杂交(FISH)放大率的肿瘤被认为是高HER2表达。具有如免疫组织化学法(IHC)所测得之2+水平和/或<2.0的荧光原位杂交(FISH)放大率的肿瘤被认为是中HER2表达。具有如免疫组织化学法(IHC)所测得之1+水平和/或<2.0的荧光原位杂交(FISH)放大率的肿瘤被认为是低HER2表达。
在一个实施方式中,利用IHC测得HER2水平。在一个更具体实施方式中,使用DakoHercptestTM检定来进行IHC。
在另一个实施方式中,利用FISH测得HER2水平。在一个更具体实施方式中,使用Dako HER2 FISH Pharm DxTM检定来进行FISH。
代表性肿瘤样品包括含有肿瘤细胞的任何生物样品或临床样品,例如组织样品、活体组织切片、血液样品、血浆样品、唾液样品、尿液样品等。
VI.配制物
本发明提供药学组合物,其包括本发明中所揭示之位点特异性HER2抗体药物缀合物与药学上可接受的载剂中任一者。此外,该药学组合物可以包括多于一种的本发明中所揭示之位点特异性HER2ADC。
本发明中所用之组合物可以进一步包括药学上可接受的载剂、赋形剂、或稳定剂(Remington:The Science and practice of Pharmacy 21st Ed.,2005,LippincottWilliams and Wilkins,Ed.K.E.Hoover),其呈冷冻干燥配制物或水溶液形式。可接受之载剂、赋形剂、或稳定剂在使用的剂量与浓度下对接受者无毒性,且可能包括缓冲剂(例如磷酸盐、柠檬酸盐、与其它有机酸类)、抗氧化剂(包括抗坏血酸与甲硫氨酸)、防腐剂(例如氯化十八基二甲基苄基铵、氯化六甲双铵、氯化烷基二甲基苄基铵、氯化苄乙氧铵、酚、丁醇或苯甲醇、羟苯甲酸烷基酯(例如羟苯甲酸甲酯或羟苯甲酸丙酯)、儿茶酚、间苯二酚、3-戊醇、与间甲酚)、低分子量(小于约10个残基)多肽类、蛋白质(例如血清白蛋白、明胶、或免疫球蛋白)、亲水性聚合物(例如聚乙烯吡咯啶酮)、氨基酸类(例如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸、或赖氨酸)、单醣类、双醣类、及其它碳水化合物类(包括葡萄糖、甘露糖、或葡聚糖)、螯合剂(例如EDTA)、糖类(例如蔗糖、甘露糖醇、海藻糖、或山梨糖醇)、成盐相对离子(例如钠)、金属复合物(例如Zn-蛋白质复合物)、与/或非离子性界面活性剂(例如TWEENTM、PLURONICSTM、或聚乙二醇(PEG))。本发明中所用的“药学上可接受之盐”乃指药学上可接受的分子或巨分子之有机盐或无机盐。药学上可接受之赋形剂在本发明中进一步说明。
一或多种位点特异性HER2ADC的各种配制物可用于施用,该配制物包括但不限于包含一或多种药学上可接受之赋形剂的配制物。药学上可接受之赋形剂是该领域中熟悉且为促进药理学有效物质之施用的相对惰性物质。例如,赋形剂能赋予形态或坚硬度,或作为稀释剂。合适赋形剂包括但不限于稳定剂、润湿剂与乳化剂、用于改变容积渗透浓度的盐类、包封剂、缓冲剂、与皮肤穿透增强剂。赋形剂与用于非肠道与经肠道之药物传输的配制物参见Remington,The Science and Practice of Pharmacy 20th Ed.Mack Publishing,2000。
在本发明之某些方面中,这些药剂可针对注射施用(例如腹膜内注射、静脉内注射、皮下注射、肌内注射等)而配制。因此,这些药剂可和药学上可接受之媒剂(例如盐溶液、林格氏液、葡萄糖溶液等)混合。特定给药方案(即剂量、时机与重复)将取决于特定个体及其病史。
把具有所欲纯度ADC和随意的药学上可接受之载剂、赋形剂或稳定剂混合来制得用于储存的根据本发明使用之位点特异性HER2 ADC的治疗用配制物(Remington,TheScience and Practice of Pharmacy 21st Ed.Mack Publishing,2005),其呈冷冻干燥配制物或水溶液形式。可接受之载剂、赋形剂、或稳定剂在使用的剂量与浓度下对接受者无毒性,且可能包括缓冲剂(例如磷酸盐、柠檬酸盐、与其它有机酸类)、盐类(例如氯化钠)、抗氧化剂(包括抗坏血酸与甲硫氨酸)、防腐剂(例如氯化十八基二甲基苄基铵、氯化六甲双铵、氯化烷基二甲基苄基铵、氯化苄乙氧铵、酚、丁醇或苯甲醇、羟苯甲酸烷基酯(例如羟苯甲酸甲酯或羟苯甲酸丙酯)、儿茶酚、间苯二酚、3-戊醇、与间甲酚)、低分子量(小于约10个残基)多肽类、蛋白质(例如血清白蛋白、明胶、或免疫球蛋白)、亲水性聚合物(例如聚乙烯吡咯啶酮)、氨基酸类(例如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸、或赖氨酸)、单醣类、双醣类、及其它碳水化合物类(包括葡萄糖、甘露糖、或糊精)、螯合剂(例如EDTA)、糖类(例如蔗糖、甘露糖醇、海藻糖、或山梨糖醇)、成盐相对离子(例如钠)、金属复合物(例如Zn-蛋白质复合物)、与/或非离子性界面活性剂(例如TWEENTM、PLURONICSTM、或聚乙二醇(PEG))。
含有位点特异性HER2ADC的脂质体可以利用该领域中熟悉之方法制得,该方法的描述参见(例如):Eppstein,et al.,1985,PNAS 82:3688-92;Hwang,et al.,1908,PNAS77:4030-4;和美国专利第4,485,045号与第4,544,545号。具有增强循环时间之脂质体在美国专利第5,013,556号中揭示。特别有用的脂质体可以通过逆相蒸发法用包括卵磷脂、胆固醇与PEG衍生之磷脂酰乙醇胺(PEG-PE)的脂质组合物产生。通过定义孔径之滤器来挤压脂质体产生具有所欲直径的脂质体。
活性成分也可包埋于所制得之微囊剂中,例如利用凝聚技术或界面聚合(例如羟甲基纤维素微囊剂或明胶微囊剂及聚甲基丙烯酸甲酯微囊剂),包埋于胶态药物输送系统(例如脂质体、白蛋白微球体、微乳液、纳米粒子与纳米囊剂)或包埋于粗乳液。这样的技术之揭示参见Remington,The Science and Practice of Pharmacy 21st Ed.MackPublishing,2005。
可制得持续释放式制剂。合适持续释放式制剂实例包括含有抗体之固体疏水性聚合物的半透性基质,该基质呈成形对象(例如膜或微囊剂)形式。持续释放式基质实例包括聚酯、水凝胶(例如聚(2-羟乙基-甲基丙烯酸酯)、或聚乙烯醇)、聚交酯(美国专利第3,773,919号)、L-谷氨酸与7乙基-L-谷氨酸酯共聚物、不可降解的乙烯-乙酸乙烯酯、可降解的乳酸-羟基乙酸共聚物,例如LUPRON DEPOTTM(由乳酸-羟基乙酸共聚物与leuprolide acetate组成之可注射的微球体)、蔗糖乙酸异丁酸酯、聚-D-(-)-3-羟基丁酸。
体内施用用的配制物必须灭菌。这利用(例如)通过过滤灭菌薄膜来过滤容易第实现。治疗用位点特异性HER2ADC组合物一般放进具无菌入口的容器中,例如静脉溶液袋或具有可由皮下注射针刺穿的塞子的管形瓶中。
合适界面活性剂包括,特别是,非离子性药剂,例如聚氧乙烯山梨糖醇酐(例如TWEENTM 20、40、60、80或85)及其它山梨糖醇酐(例如SpanTM 20、40、60、80或85)。具界面活性剂之组合物会便利地包括在0.05至5%间,且可为在0.1至2.5%间的界面活性剂。应了解可视需要加入其它成分,例如甘露糖醇或其它药学上可接受的媒剂。
合适乳液可使用市售脂肪乳液,例如INTRALIPIDTM、LIPOSYNTM、INFONUTROLTM、LIPOFUNDINTM与LIPIPHYSANTM。可将活性成分溶于预混乳液组合物,或可溶于油(例如大豆油、红花油、棉籽油、芝麻油、玉米油或杏仁油)及与磷脂(例如卵磷脂、大豆磷脂或大豆卵磷脂)与水混合后形成的乳液。应了解可加入其它成分,例如甘油或葡萄糖,来调整乳液的渗性。合适乳液典型地会含有至多20%,例如在5至20%间的油。脂肪乳液可包括在0.1至1.0μm间,特别是在0.1至0.5μm间的脂肪滴,及具有在5.5至8.0范围内的pH。乳液组合物可利用把位点特异性HER2ADC与INTRALIPIDTM或其组分(大豆油、卵磷脂、甘油与水)混合来制得。
本发明也提供用于即用方法之试剂盒。本发明之试剂盒包括一或多个装有一或多种本发明中所述之位点特异性HER2ADC的容器,及根据本发明中所述之方法中任一者用的指示说明书。一般来说,这些指示说明书包括把用于上述治疗处理的位点特异性HER2ADC的施用说明。
关于本发明中所述之位点特异性HER2ADC的用途之指示说明一般包括用于意图治疗的剂量、给药方案、与施用途径之信息。容器可为单位剂量、散装包装(例如多剂量包装)、或亚单位剂量包装。本发明之试剂盒中提供的指示说明书典型地是在标签或包装说明书上的书面式指示说明(例如试剂盒中所包括的纸张),但也可提供机器可读式指示说明(例如在磁性或光学储存碟上载的指示说明)。
本发明之试剂盒在合适的包装中。合适的包装包括但不限于:管型瓶、瓶、大口瓶、软质包装(例如密封Mylar袋或塑料袋)等。也包含和特殊装置(例如输注装置(例如袖珍泵))并用的包装。试剂盒可能具有无菌入口(例如容器可能是静脉溶液袋或具有可由皮下注射针刺穿的塞子的管形瓶)。组合物中的至少一种活性药剂是位点特异性HER2ADC。容器可能进一步包括第二药学活性药剂。
试剂盒可能随意地提供额外的组分,例如缓冲液与解说用数据。通常,试剂盒包括容器与在该容器上或和该容器一起的标签或包装说明书。
VII.投药与施用
对体内施用而言,提供或施用有效剂量之位点特异性HER2ADC。本发明中所用之措辞“有效剂量”或“有效量”乃指要直接或间接地达到任何一或多个有益或所欲之治疗结果必需的药物、化合物、或药学组合物的量。例如当对患癌个体施用时,有效剂量包括足够引出抗癌活性的量,该抗癌活性包括癌细胞溶解、抑制癌细胞增生、诱发癌细胞凋亡、减少癌细胞抗原、延迟肿瘤生长、与/或抑制转移。肿瘤缩小被普遍接受为效力之临床替代指标。另一个被普遍接受的效力之指标是无疾病进展存活期。
有效剂量可以一或多次施用来施用。药物、化合物、或药学组合物之有效剂量可能连同或可能不连同另一种药物、化合物、或药学组合物来达到。因此,有效剂量可能被认为是施用一或多种治疗剂,及单一药剂可能被考虑以有效量被给予,若(连同一或多种其它药剂)可能达到或达到满意的结果。
可通过合适途径对个体施用位点特异性HER2ADC。该领域之本领域技术人员应当理解本发明中所述之实施例无意限制而是要介绍可用之技术。因此,在本发明之某些实施方式中,根据已知方法对个体施用位点特异性HER2ADC,该已知方法例如静脉内施用(例如推注或在一段期间内连续输注)、肌内施用、腹膜内施用、脑脊髓内施用、颅内施用、透皮施用、皮下施用、关节内施用、舌下施用、滑液膜内施用、通过吹入、脑脊髓膜内施用、口服、通过吸入、或局部施用。施用可为全身施用,例如静脉内施用或局部施用。市售用于液体配制物的气雾器(包括喷射气雾器与超音波气雾器可用于施用。液体配制物可被直接雾化,及冷冻干燥的粉末可在重新配制后被雾化。或者,可使用氟碳化合物配制物与计量吸入器把位点特异性HER2ADC喷雾化,或以冷冻干燥粉末及磨细粉末形式吸入。
在本发明之某些实施方式中,通过位点特异或靶向局部输送技术施用位点特异性HER2ADC。位点特异或靶向局部输送技术实例包括:各种位点特异性HER2ADC可植入式储库源或局部输药导管(例如输注导管、留置导管、针型导管、合成植入物、外膜套、引流管及支架或其它可植入式装置、位点特异性载剂、直接注射、或直接施用。见,例如,PCT国际公布WO2000/53211与美国专利第5,981,568号。
为了本发明之目的,位点特异性HER2ADC的合适剂量将取决于:所用之特定ADC(或其组合物),要治疗之症状的类型与严重程度,药剂是否以治疗目的施用,先前疗法,患者病史及对药剂的反应,患者之所施用的药物廓清率,及主治医师的裁量。临床医师可施用位点特异性HER2ADC直到剂量达到或超出所欲之结果为止。剂量与/或次数可以随疗程而变动,但也可能保持固定。经验上的考虑(例如半衰期)通常有助于剂量的决定。例如,和人类免疫系统兼容的抗体(例如人源化抗体或纯人类抗体)可用来延长抗体的半衰期及防止抗体被宿主免疫系统攻击。在疗程中可决定与调整给药次数,通常,但不必根据症状的治疗与/或抑制与/或改善,例如抑制肿瘤或延迟生长等。或者,位点特异性HER2ADC的持续释放式配制物可能合适。用于达到持续释放的各种配制物与装置是该领域中已知的。
通常,位点特异性HER2ADC施用的初始候选剂量可为约2mg/kg。为了本发明之目的,典型每日剂量范围可能从约3μg/kg至30μg/kg至300μg/kg至3mg/kg,至30mg/kg,至100mg/kg或更多中任一者。例如,可能使用约1mg/kg,约2.5mg/kg,约5mg/kg,约10mg/kg,与约25mg/kg的剂量。对在几日或更长的时间内重复施用而言,视疾患而定,持续治疗直到所欲之症状的抑制发生,或直到达到足够的治疗水平,例如,足够抑制或延迟肿瘤生长/进展或癌细胞转移。示范性给药方案包括约2mg/kg的初始剂量,接着保持每周约1mg/kg的位点特异性HER2ADC的剂量,或接着保持每隔一周约1mg/kg的剂量。其它示范性给药方案包括施用递增的剂量(例如1mg/kg的初始剂量与逐步增加为每周或更长的期间内一或多次较高剂量)。视医师希望达到的药物动力学衰减模式而定,也可能使用其它给药方案。例如,在本发明之某些方面中,包含1周从1次至4次。在其它方面中,包含1个月1次,或每隔1个月1次,或每3个月1次,及每周1次,每2周1次与每3周1次施用。此疗法之进展可利用常规技术与检定容易地监控。给药方案(包括所用之特定位点特异性HER2ADC)可以随时间而变动。
VIII.组合疗法
在本发明之某些实施方式中,本发明中所述之方法进一步包括用另一形式的疗法治疗个体的步骤。在某些实施方式中,该另一形式的疗法是附加抗癌疗法,其包括但不限于化学疗法、放射线疗法、外科手术、激素疗法、与/或附加免疫疗法。
本发明所揭示之位点特异性HER2ADC可以初步治疗形式被施用,或可被施用用于治疗对常规疗法无反应的癌。此外,位点特异性HER2ADC可与其它疗法(例如切除、放射线、额外的抗癌药剂等)并用,藉此引出加成或增强的治疗效应与/或降低某些抗癌药剂之细胞毒性。本发明之位点特异性HER2ADC可和额外的药剂共同施用或共同配制、或可针对和额外的药剂以任何顺序连续施用而配制。
本发明之位点特异性HER2ADC可和其它治疗剂并用,该其它治疗剂包括但不限于治疗用抗体、ADC、免疫调节剂、细胞毒性剂、与细胞抑制剂。细胞毒性效应乃指耗减、消除、与/或杀死靶细胞(即癌细胞)。细胞毒性剂乃指对细胞具有细胞毒性效应与/或细胞生长抑制效应的药剂。细胞生长抑制效应乃指抑制细胞增生。细胞抑制剂乃指对细胞具有细胞生长抑制效应,藉此抑制特异细胞亚群(即癌细胞)生长与/或增殖的药剂。免疫调节剂乃指通过产生细胞因子与/或抗体与/或调节T细胞功能来刺激免疫反应,藉此直接或间接允许另一种更有效之药剂来抑制或降低细胞亚群(即癌细胞)生长的药剂。
就组合疗法而言,位点特异性HER2ADC与/或一或多种额外的治疗剂在适合进行意图之疗法的任何时段内被施用。因此,单一药剂可被实质上合并施用(即单一配制物或在数分钟或数小时内)或被以任何顺序连续施用。例如,单一药剂治疗可彼此在约1年内(例如在约10、8、6、4、或2个月内、或在4、3、2、或1周内、或在5、4、3、2、或1日内)被施用。
本发明所揭示之组合疗法可引出协同增加治疗效应,即大于其个别效应或治疗结果的总和的效应。例如协同增加治疗效应可为大于单一药剂所引出之治疗效应,或给定组合的单一药剂所引出之治疗效应总和的至少约2倍、或至少约5倍、或至少约10倍、或至少约20倍、或至少约50倍、或至少约100倍之效应。协同增加治疗效应也可为比单一药剂所引出的治疗效应,或给定组合之单一药剂所引出的治疗效应总和增加至少10%、或至少20%、或至少30%、或至少40%、或至少50%、或至少60%、或至少70%、或至少80%、或至少90%、或至少100%、或更多之治疗效应。协同增加治疗效应也是在将治疗剂并用时允许降低给药的效应。
[实施例]
以下实施例被提供仅作介绍用,并且无意以任何方式限制本发明之范围。当然,除本发明中所显示及描述者外,本发明的各式各样修改为该领域之本领域技术人员从前面的描述中显而易知的并且属于后附申请专利范围之范围内。
实施例1:用于位点特异缀合之曲妥单抗衍生的抗体之制备
A.通过半胱氨酸缀合
一般按PCT公布WO2013/093809(将其全部内容并入本案)中所述地进行通过半胱氨酸残基来制备用于位点特异缀合之曲妥单抗衍生物的方法。将在轻链(第183位,使用Kabat编号方案)或重链(第290、334、392与/或443位,使用Kabat编号方案的EU index)上的一或多个残基利用定点突变法改变成半胱氨酸(C)残基。
B.通过转谷氨酰胺酶缀合
一般按PCT公布WO2012/059882(将其全部内容并入本案)中所述地进行通过谷氨酰胺残基来制备用于位点特异缀合之曲妥单抗衍生物的方法。以三种不同方式工程化曲妥单抗以表达用于缀合的谷氨酰胺残基。
就第一种方法而言,把含有谷氨酰胺残基的8个氨基酸残基标签(LCQ05)附接至轻链之C末端(即SEQ ID No:81)。
就第二种方法而言,将重链上的残基(第297位,使用Kabat编号方案的EU index)利用定点突变法由天冬酰胺(N)改变成谷氨酰胺(Q)残基。
就第三种方法而言,将重链上之残基(第297位,使用Kabat编号方案的EU index)由天冬酰胺(N)改变成丙氨酸(A)。这导致第297位的无糖基化及第295位之可接近的/反应性的内生性谷氨酰胺。
另外,某些曲妥单抗衍生物具有缀合没用到的改变。将重链上之第222位残基(使用Kabat编号方案的EU Index)由赖氨酸(K)改变成精氨酸(R)残基。我们发现K222R取代导致更均质的抗体与载荷物缀合物,更佳之在抗体与载荷物间的分子间交联,与/或和在抗体轻链C末端上的谷氨酰胺标签之链间交联明显减少。
实施例2:表达曲妥单抗衍生的抗体之稳定性转染的细胞之制造
A.半胱氨酸突变体
为了测定能在细胞中稳定地表达且大量制造的单与双半胱氨酸工程化之曲妥单抗衍生的抗体变异体,用编码九种曲妥单抗衍生之抗体变异体(T(κK183C)、T(K290C)、T(K334C)、T(K382C)、T(κK183C+K290C)、T(κK183C+K382C)、T(K290C+K334C)、T(K334C+K392C)、与T(K290C+K392C))的DNA转染CHO细胞,及使用该领域中众所周知的标准方法分离稳定之大量生产库。用使用标准方法编码此双半胱氨酸工程化之抗体变异体的重链DNA与轻链DNA瞬时共转染HEK-293细胞(ATCC Accession#CRL-1573)。使用二管柱法(即Protein-A亲和捕获管柱后面接着TMAE管柱)或三管柱法(即Protein-A亲和捕获管柱后面接着TMAE管柱再接着CHA-TI管柱)从浓缩CHO库起始原料中分离这些曲妥单抗变异体。使用这些纯化法,利用分析粒径排阻层析测得全部半胱氨酸工程化之曲妥单抗衍生的抗体变异体制剂都含有>97%之关注峰(peak-of-interest)(POI)(表5)。表5中所示之这些结果表明在Protein A树脂洗提十种曲妥单抗衍生的半胱氨酸变异体后都检测到可接受水平之高分子量(HMW)聚集物种,及此不理想的HMW物种可使用粒径排阻层析术移除。另外,该数据表明工程化之半胱氨酸残基的存在不改变人IgG1恒定区中的Protein A结合位点。
表5:衍生的半胱氨酸抗体变体的制备
ND=未测定
实施例3:曲妥单抗衍生的抗体之完整性
进行该工程化之半胱氨酸与转谷氨酰胺酶变异体的分子评估来评估相对于曲妥单抗野生型抗体之关键生物物理学性质,以保证该变异体会适合于标准抗体制造平台方法。
A.半胱氨酸突变体
为测定经纯化之通过稳定的CHO表达所制得之工程化的半胱氨酸抗体变异体制剂之完整性,使用非还原毛细管凝胶电泳术(Caliper LabChip GXII:Perkin ElmerWaltham,MA)求出峰的纯度。结果显示工程化之半胱氨酸抗体变异体T(κK183C+K290C)与T(K290C+K334C)含有低水平的片段与高分子量物种(HMMS),类似于曲妥单抗野生型抗体。反之,T(K334C+K392C)含有相对于所评估的其它双工程化之半胱氨酸变异体而言高水平的片段抗体峰(表6)。这些结果表明工程化之半胱氨酸的特异组合物能影响用于位点特异缀合之抗体的完整性。
表6:非还原电泳图计算的峰的纯度百分数
抗体 | 主峰(%) | 片段(%) | HMMS(%) |
曲妥单抗WT | 95 | 5 | 0 |
T(KK183C+K290C) | 95.78 | 4.18 | 0.04 |
T(K290C+K334C) | 94.6 | 5.2 | 0.2 |
T(K334C+K392C) | 80.7 | 19.3 | 0 |
实施例4:载荷物药物化合物之产生
根据PCT公布WO2013/072813(将其全部内容并入本案)中所述之方法制得耳抑素药物化合物0101、0131、8261、6121、8254与6780。在公开的申请案中,利用表7中所示的编号系统来标示该耳抑素化合物。
表7
耳抑素药物化合物 | WO2013/072813中指定 |
0101 | #54 |
0131 | #118 |
8261 | #69 |
6121 | #117 |
8254 | #70 |
6780 | #112 |
根据PCT公布WO2013/072813,根据以下步骤制得药物化合物0101。
步骤1.合成N-[(9H-芴-9-基甲氧基)羰基]-2-甲基丙氨酰基-N-[(3R,4S,5S)-3-甲氧基-1-{(2S)-2-[(1R,2R)-1-甲氧基-2-甲基-3-氧代-3-{[(1S)-2-苯基-1-(1,3-噻唑-2-基)乙基]氨基}丙基]吡咯烷-1-基}-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺(#53)。根据通用步骤D,由#32(2.05g,2.83mmol,1eq.)与二氯甲烷(2.05mL,0.1M)和N,N-二甲基甲酰胺(3mL)溶液、胺#19(2.5g,3.4mmol,1.2eq.)、HATU(1.29g,3.38mmol,1.2eq.)与三乙胺(1.57mL,11.3mmol,4eq.)合成粗制所欲材料,利用硅胶层析纯化(溶剂梯度:0%至55%丙酮之庚烷溶液),产生固体状#53(2.42g,74%)。LC-MS:m/z 965.7[M+H+],987.6[M+Na+],滞留时间=1.04分钟;HPLC(Protocol A):m/z 965.4[M+H+],滞留时间=11.344分钟(纯度>97%);1H NMR(400MHz,DMSO-d6),被推定为旋转异构物之混合物,特性信号:δ7.86-7.91(m,2H),[7.77(d,J=3.3Hz)与7.79(d,J=3.2Hz),总共1H],7.67-7.74(m,2H),[7.63(d,J=3.2Hz)与7.65(d,J=3.2Hz),总共1H],7.38-7.44(m,2H),7.30-7.36(m,2H),7.11-7.30(m,5H),[5.39(ddd,J=11.4,8.4,4.1Hz)与5.52(ddd,J=11.7,8.8,4.2Hz),总共1H],[4.49(dd,J=8.6,7.6Hz)与4.59(dd,J=8.6,6.8Hz),总共1H],3.13,3.17,3.18与3.24(4s,总共6H),2.90与3.00(2br s,总共3H),1.31与1.36(2br s,总共6H),[1.05(d,J=6.7Hz)与1.09(d,J=6.7Hz),总共3H]。
步骤2.合成2-甲基丙氨酰基-N-[(3R,4S,5S)-3-甲氧基-1-{(2S)-2-[(1R,2R)-1-甲氧基-2-甲基-3-氧代-3-{[(1S)-2-苯基-1-(1,3-噻唑-2-基)乙基]氨基}丙基]吡咯烷-1-基}-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺(#54或0101)。根据通用步骤A,由#53(701mg,0.726mmol)与二氯甲烷(10mL,0.07M)合成粗制所欲材料,利用硅胶层析纯化(溶剂梯度:0%至10%甲醇之二氯甲烷溶液)。用二乙醚与庚烷稀释残留物,以真空浓缩,产生白色固体状#54(或0101)(406mg,75%)。LC-MS:m/z 743.6[M+H+],滞留时间=0.70分钟;HPLC(Protocol A):m/z 743.4[M+H+],滞留时间=6.903分钟,(纯度>97%);1H NMR(400MHz,DMSO-d6),被推定为旋转异构物之混合物,特性信号:δ[8.64(br d,J=8.5Hz)与8.86(brd,J=8.7Hz),总共1H],[8.04(br d,J=9.3Hz)与8.08(br d,J=9.3Hz),总共1H],[7.77(d,J=3.3Hz)与7.80(d,J=3.2Hz),总共1H],[7.63(d,J=3.3Hz与7.66(d,J=3.2Hz),总共1H],7.13-7.31(m,5H),[5.39(ddd,J=11,8.5,4Hz)与5.53(ddd,J=12,9,4Hz),总共1H],[4.49(dd,J=9,8Hz)与4.60(dd,J=9,7Hz),总共1H],3.16,3.20,3.21与3.25(4s,总共6H),2.93与3.02(2br s,总共3H),1.21(s,3H),1.13与1.13(2s,总共3H),[1.05(d,J=6.7Hz)与1.10(d,J=6.7Hz),总共3H],0.73-0.80(m,3H)。
根据PCT公布WO 2013/072813中所揭示之方法自制药物化合物MMAD、MMAE与MMAF。
通过美国专利第5,208,020号中概述之步骤由购得的美登醇(maytansinol)自制药物化合物DM1。
实施例5:曲妥单抗衍生之抗体的生物缀合
将本发明之曲妥单抗衍生的抗体通过连接子缀合至载荷物以产生ADC。所用之缀合法是位点特异缀合法(即,通过特定半胱氨酸残基或特定谷氨酰胺残基)或常规缀合法。
A.半胱氨酸位点特异性
通过下述半胱氨酸位点特异方法来缀合表8之ADC。
表8
将500mM三(2-羧乙)膦盐酸盐(TCEP)溶液(50至100摩尔当量)加到抗体(5mg)中,使得在含有20mM EDTA之PBS中的最终抗体浓度为5至15mg/mL。使反应物在37℃下静置2.5小时,使用凝胶过滤管柱(PD-10脱盐管柱,GE Healthcare)把抗体之缓冲液交换成含有5mMEDTA的PBS。用新制50mM的DHA之1:1PBS/EtOH溶液(最终DHA浓度=1mM至4mM)处理所产生之在含有5mM EDTA的PBS中之抗体(5至10mg/mL)并且使混合物在4℃下静置过夜。
把该抗体/DHA混合物之缓冲液交换成含有5mM EDTA的PBS(用磷酸把平衡缓冲液之pH调整成~7.0)及用50kD MW截止自旋浓缩装置来浓缩。用5至7摩尔当量的10mM之顺丁烯二酰亚胺载荷物的DMA溶液处理所产生之在含有5mM EDTA的PBS中之抗体(抗体浓度~5至10mg/mL)。静置1.5至2.5小时后,将材料之缓冲液交换(PD-10)。利用SEC纯化(根据需要)以移除任何聚集的材料且保留游离载荷物。
B.转谷氨酰胺酶位点特异性
通过下述转谷氨酰胺酶位点特异方法来缀合表9之ADC。
表9
在转酰胺基反应中,在抗体中的谷氨酰胺具有酰基供体作用,而含有胺之化合物具有酰基受体(胺供体)作用。将浓度33μM的经纯化之HER2抗体在2%(w/v)茂原链轮丝菌(Streptoverticillium mobaraense)转谷氨酰胺酶(ACTIVATM,Ajinomoto,Japan)与150-mM氯化钠和Tris HCl缓冲液(pH范围7.5至8)和0.31mM还原型谷胱甘肽(除非指明)存在下,培养于10至25M过量之酰基受体(范围在33至83.3μM间的AcLysvc-0101)中。针对个别酰基供体来调整反应条件,其中T(LCQ05+K222R)在pH8.0与无还原型谷胱甘肽下培养于10M过量之酰基受体中,T(N297Q+K222R)与T(N297Q)在pH 7.5下培养于20M过量的酰基受体中,和T(N297A+K222R+LCQ05)在pH7.5下培养于25M过量之酰基受体中。在37℃下培养16至20个小时后,把抗体在MabSelect resin或Butyl Sepharose High Performance(GEHealthcare,Piscataway,NJ)上用该领域之本领域技术人员熟悉的标准层析方法纯化,该标准层析方法例如来自GE Healthcare的商用亲和层析术与疏水性交互作用层析术。
C.常规缀合方法
通过下述常规缀合方法来缀合表10与11之ADC。
表10
表11
将抗体透析到Dulbecco氏磷酸盐缓冲盐水(DPBS,Lonza)中。把经透析之抗体用含有5mM乙二胺四乙酸(2,2’,2”,2’”-(ethane-1,2-diyldinitrilo)tetraacetic acid)(EDTA)的PBS(pH 7)稀释成15mg/mL。用2至3当量之三(2-羧乙)膦盐酸盐(TCEP,5mM之蒸馏水溶液)处理所产生的抗体并且在37℃下静置1至2个小时。在冷却至室温后立即加入二甲基乙酰胺(DMA)到10%(v/v)总有机物。将混合物用8至10当量的适当连接子-载荷物处理成为10mM之以DMA为溶剂的母液。使反应在室温下静置1至2个小时,然后使用GE HealthcareSephadex G-25M缓冲液交换管柱根据制造商指示把缓冲液交换成DPBS(pH 7.4)。
利用粒径排阻层析术(SEC)使用GE AKTA Explorer系统(具有GE Superdex200管柱与PBS(pH 7.4)洗提液)来纯化保持死循环的材料(表10之ADC)。把最终样品浓缩成~5mg/mL蛋白质,用过滤方法灭菌,及用下文概述的质谱分析条件来检验载药。
立即使用超过滤装置(50kD MW截止)把用于丁二酰亚胺环水解的材料(表11之ADC)的缓冲液交换成50mM硼酸盐缓冲液(pH 9.2)。将所产生之溶液加热到45℃维持48个小时。冷却所产生之溶液,把缓冲液交换成PBS,及利用SEC(如下所述)纯化,以移除任何聚集的材料。将最终样品浓缩成~5mg/mL蛋白质,用过滤方法灭菌,及用下文概述的质谱分析条件来检验载药。
D.T-DM1缀合方法
曲妥单抗-类美登素缀合物(T-DM1)结构类似于曲妥单抗emtansineT-DM1由通过双官能性连接子硫代丁二酰亚胺基4-(N-顺丁烯二酰亚胺基甲基)环己烷-1-羧酸酯(sulfo-SMCC)与DM1类美登素共价键结的曲妥单抗抗体组成。首先在25℃下于50mM磷酸钾,2mM EDTA,pH 6.8,在10:1反应化学计量下,把sulfo-SMCC缀合至抗体上的游离胺1个小时,然后从经缀合之抗体中把未结合的连接子脱盐。然后在25℃下于50mM磷酸钾,50mM NaCl,2mM EDTA,pH 6.8,在10:1反应化学计量下,把此抗体-MCC中间物缀合至该MCC连接子抗体上的游离顺丁烯二酰亚胺基末端之DM1硫化物。然后用L-半胱氨酸将剩余的未反应之顺丁烯二酰亚胺封端,及通过Superdex200管柱把ADC分级以除去非单体物种(Chari et al.,1992,Cancer Res 52:127-31)。
实施例6:ADC之纯化
一般使用下述粒径排阻层析术(SEC)来将ADC纯化及特性分析。使用各式各样方法来测定到指定缀合位点上的载药,该方法包括如下文更完全地描述之质谱分析(MS)、逆相HPLC、与疏水性交互作用层析术(HIC)。这三种分析方法之组合提供各式各样证实与定量到抗体上的载荷物剂量之方式,藉此精确测定各缀合物的DAR。
A.制备型SEC
一般使用SEC层析术来纯化ADC以移除蛋白质聚集体及移除反应混合物中剩下的微量载荷物-连接子,该SEC层析术使用Akta Explorer FPLC系统与Waters Superdex20010/300GL管柱。有时ADC在SEC纯化前并没有聚集体与小分子,因此不进行制备级SEC。所用之洗提液是流率1mL/分钟的PBS。在这些条件下,从非聚集材料(在室温下洗提约15分钟)容易地分离出聚集材料(在室温下洗提约10分钟)。疏水性载荷物-连接子组合物常导致SEC峰“右偏移”。在不希望受任何特定理论所束缚情况下,此SEC峰偏移可能由于该连接子-载荷物与固定相的疏水性交互作用。在某些情况下,此右偏移使经缀合之蛋白质得以从未缀合的蛋白质部分地析出。
B.分析型SEC
在Agilent 1100HPLC上使用PBS作为洗提液进行分析型SEC以评估ADC之纯度与单体状态。在220与280nm下检测洗提液。当管柱为TSKGel G3000SW管柱(7.8x300mm,型号R874803P)时,所用之移动相为流率0.9mL/分钟与洗提30分钟的PBS。当管柱为BiosepSEC3000管柱(7.8x300mm)时,所用之移动相为流率1.0mL/分钟与洗提25分钟的PBS。
实施例7:ADC之特性分析
A.质谱分析(MS)
将约20μl的样品(约1mg/ml的ADC之PBS溶液)与20μl的20mM二硫苏糖醇(DTT)混合来制得用于LCMS分析之样品。把混合物在室温下静置5分钟,然后将样品注入安装AgilentPoroshell 300SB-C8(2.1x75mm)管柱的Agilent 110HPLC系统中。把系统温度设定于60℃。使用从20%至45%乙腈之水溶液(具有0.1%甲酸改性剂)梯度洗提5分钟。利用UV(220nM)与Waters Micromass ZQ质谱仪(电喷雾电离;锥电压:20V;离子源温度:120℃;去溶剂化温度:350℃)来检测洗提液。使用MaxEnt1 within MassLynx 4.1软件包根据制造商指示来反褶积含有多电荷物种的原质谱。
B.每一抗体载药量之MS测定
制造ADC之抗体的总载荷物量称为药物对抗体比或DAR。计算出每一所制得之ADC的DAR(表12)。
将整个洗提窗域(通常5分钟)的质谱合并到单一总质谱(即代表整个样品的MS的质谱)中。把ADC样品的MS结果直接和对应的相同无载药对照组抗体的MS作比较。这能识别载药/无载药重链(HC)峰及载药/无载药轻链(LC)峰。可使用各种峰的比率来建立以下列方程式(方程式1)为基础的载药量。计算基于下面的假设,即载药链和无载药链相同离子化(已被确定为通常有效的假设)。
进行下列计算来建立DAR:
方程式1:
载药量=2*[LC1/(LC1+LC0)]+2*[HC1/(HC0+HC1+HC2)]+4*[HC2/(HC0+HC1+HC2)]
其中指示变量是下列的相对丰富度:LC0=无载药轻链,LC1=单载药轻链,HC0=无载药重链,HC1=单载药重链,及HC2=双载药重链。该领域之具有普通技能者可理解本发明包含此计算放大到包含更高载药物种(例如LC2、LC3、HC3、HC4、HC5等)。
下列方程式2被用于估计到工程化之半胱氨酸残基上的载药量。对工程化之Fc突变体而言,到轻链(LC)上的载药量通过定义被认为是非特异性载药量。此外,假设仅LC有载药是HC-LC二硫键非故意还原之结果(即该抗体被“过度还原”)。已知把大量过量的顺丁烯二酰亚胺用于共轭反应(通常对单突变体而言是约5当量及对双突变体而言是约10当量),假设到轻链上的任何非特异性载药量伴有对应量之到重链上的任何非特异性载药量(即另“一半”之断裂的HC-LC二硫键)。基于这些假设,使用下列方程式(方程式2)来估计到蛋白质上的非特异性载药量:
方程式2:
非特异性载药量=4*[LC1/(LC1+LC0)]
其中指示变量是下列的相对丰富度:LC0=无载药轻链,LC1=单载药轻链。
表12:ADC的药物抗体比(DAR)
ADC | DAR |
T(κK183C)-vc0101 | 2 |
T(K290C)-vc0101 | 2 |
T(K334C)-vc0101 | 2 |
T(K392C)-vc0101 | 2 |
T(κK183C+K290C)-vc0101 | 4 |
T(κK183C+K334C)-vc0101 | 4 |
T(κK183C+K392C)-vc0101 | 4 |
T(K290C+K334C)-vc0101 | 4 |
T(K290C+K392C)-vc0101 | 4 |
T(K334C+K392C)-vc0101 | 4 |
T(N297Q)-AcLysvc0101 | 4 |
T(N297Q+K222R)-AcLysvc0101 | 4 |
T(N297A+K222R+LCQ05)-AcLysvc0101 | 4 |
T(LCQ05+K222R)-AcLysvc0101 | 2 |
T-mc8261 | 4.2 |
T-m(H20)c8261 | 3.6 |
T-MalPeg8261 | 3.1 |
T-vc8261 | 4.3 |
T-mc6121 | 3.5 |
T-MalPeg6121 | 3.6 |
T-mc0101 | 4.8 |
T-vc0101 | 4.2 |
T-vc8254 | 4 |
T-vc6780 | 4.2 |
T-vc0131 | 4.5 |
T-MalPegMMAD | 4.4 |
T-vcMMAE | 3.8 |
T-DM1 | 4.2 |
C.用蛋白质水解来建立载药之位点
对半胱氨酸突变之ADC而言,到抗体上的亲电子载荷物之任何非特异性载药量被推定发生在“链间”,也称为“内部”半胱氨酸残基(即,其典型上是HC-HC或HC-LC二硫键之一部分)。为了区别到Fc区中的工程化之半胱氨酸上的载亲电子剂量对到内部半胱氨酸残基(典型上另外形成在HC-HC或HC-LC间之S-S键)上的载亲电子剂量,用已知把抗体之Fab区与Fc区间切断的蛋白酶来处理缀合物。一种这样之蛋白酶是半胱氨酸蛋白酶IdeS,作为由Genovis销售,及其说明参见von Pawel-Rammingen et al.,2002,EMBO J.21:1607。
简单地说,按照制造商建议之条件,用蛋白酶处理ADC,及把样品在37℃下培养30分钟。将约20μl的样品(在PBS中约1mg/ml)与20μl的20mM二硫苏糖醇(DTT)混合来制得用于LCMS分析之样品,及把混合物在室温下静置5分钟。这样处理人IgG1产生三种抗体片段,大小全都是在约23至26kD范围内:LC片段,其包含典型地形成LC-HC链间二硫键之内部半胱氨酸;N末端的HC片段,其包含三个内部半胱氨酸(其中一个典型地形成LC-HC二硫键,而其余二个半胱氨酸在抗体之绞链区中发现且典型地形成在抗体的二重链间的HC-HC二硫键);和C末端之HC片段,其不含由本发明中所揭示之构建体中的突变导入的以外之反应性半胱氨酸。如上所述利用MS来分析样品。以和上述相同的方式进行载药量计算以定量LC、N末端之HC、与C末端的HC之载药量。在C末端的HC上的载药量被认为是“特异性”载药量,而在LC与N末端之HC上的载药量被认为是“非特异性”载药量。
为了交叉检查载药量计算,也使用下文更加详述的其他方法(逆相高效液相层析[rpHPLC基和疏水性交互作用层析[HIC]基方法])评估ADC亚组的载药量。
D.逆相HPLC分析
将约20μl的样品(在PBS中约1mg/ml)与20μl的20mM二硫苏糖醇(DTT)混合来制得用于逆相HPLC分析之样品。把混合物在室温下静置5分钟,然后将样品注入安装AgilentPoroshell 300SB-C8(2.1x75mm)管柱的Agilent 1100HPLC系统中。把系统温度设定于60℃,利用UV(220nM与280nM)来检测洗提液。使用从20%至45%乙腈之水溶液(具有0.1%TFA改性剂)梯度洗提20分钟:T=0分钟:25%乙腈;T=2分钟:25%乙腈;T=19分钟:45%乙腈;及T=20分钟:25%乙腈。使用这些条件把抗体的HC与LC基线分离。此分析结果指示LC仍有大量未被修饰(除含有T(κK183C)与T(LCQ05)的抗体外),而HC被修饰(数据未显示)。
E.疏水性交互作用层析(HIC)
将样品用PBS稀释成约1mg/ml来制得用于HIC分析之化合物。自动注射15μl到具TSK-GEL Butyl NPR管柱(4.6 x 3.5mm,2.5μm孔径;Tosoh Biosciences part#14947)的Agilent 1200 HPLC上来分析样品。该系统包括具恒温装置、管柱加热器与UV检测器之自动取样器。
按照下列来使用梯度洗提法:
移动相A:1.5M硫酸铵、50mM磷酸氢二钾(pH7);移动相B:20%异丙醇、50mM磷酸氢二钾(pH 7);T=0分钟,100%A;T=12分钟,0%A。
滞留时间参见表13。选定谱参见图2A至2E。使用位点特异缀合(T(κK183C+K290C)-vc0101、T(K334C+K392C)-vc0101与T(LCQ05+K222R)-AcLysvc0101)的ADC(图1A至1C)显示一个主峰,而使用常规缀合(T-vc0101与T-DM1)的ADC(图2D至2E)显示差别载药之缀合物的混合物。
表13:疏水性相互作用层析(HIC)的ADC滞留时间
ADC | RT(min) | RRT |
T-vc0101 | 8.8±0.1 | 1.68 |
T(κK183C)-vc0101 | 7.2±0.1 | 1.40 |
T(K334C)-vc0101 | ND | |
T(K392C)-vc0101 | 6.7±0.1 | 1.29 |
T(L443C)-vc0101 | 10.1±0.1 | 1.98 |
T(κK183C+K290C)-vc0101 | 9.0±0.0 | 1.77 |
T(κK183C+K334C)-vc0101 | ND | |
T(κK183C+K392C)-vc0101 | 7.7±0.1 | 1.54 |
T(κK183C+L443C)-vc0101 | 10.6 | 2.04 |
T(K290C+K334C)-vc0101 | 6.3±0.0 | 1.21 |
T(K290C+K392C)-vc0101 | 7.8±0.0 | 1.54 |
T(K334C+K392C)-vc0101 | 6.0±0.3 | 1.18 |
T(K392C+L443C)-vc0101 | 10.8±0.0 | 2.08 |
T(LCQ05+K222R)-AcLys-vc0101 | 6.5 | 1.27 |
T(N297A+K222R+LCQ05)-AcLys-vc0101 | 6.3±0.1 | 1.24 |
ND=未测定
RT=HIC上的滞留时间(min)
RRT=平均相对滞留时间,计算方式为将ADC的RT除以基准未缀合的野生型曲妥单抗的RT(具有5.0-5.2分钟的典型滞留时间)
F.热稳定性
使用微差扫描热量分析(DCS)来测定工程化之半胱氨酸与转谷氨酰胺酶抗体变异体、及对应的Aur-06380101位点特异性缀合物之热稳定性。对此分析法而言,将在PBS-CMF(pH 7.2)中配制的样品分配到具Autosampler(GE Healthcare Bio-Sciences,Piscataway,NJ)之MicroCal VP-Capillary DSC的样品盘中,在5℃下平衡5分钟,然后在100℃/小时速率下扫描到110℃为止。选定16秒之过滤周期。把原数据作基线校正,及将蛋白质浓度标准化。使用Origin Software 7.0(OriginLab Corporation,Northampton,MA)用合适的转变数把数据拟合MN2-State Model。
全部单与双半胱氨酸工程化之抗体变异体、及含有工程化之LCQ05酰基供体谷氨酰胺的标签之抗体显出出色的热稳定性,如由第一熔化转变(Tm1)>65℃测得(表14)。
使用位点特异缀合方法缀合至0101之曲妥单抗衍生的单克隆抗体也被评估且表明也具有优秀热稳定性(表15)。然而,T(K392C+L443C)-vc0101ADC的Tm1最受到载荷物缀合之影响,由于其相对于未缀合的抗体是-4.35℃。
将这些结果合在一起显示工程化之半胱氨酸的抗体变异体与含有酰基供体谷氨酰胺之标签的抗体变异体皆是热稳定的,及显示通过vc连接子之0101位点特异缀合产生具有出色热稳定性的缀合物。此外,观测到T(K392C+L443C)-vc0101比未缀合之抗体更低的热稳定性指示通过vc连接子把0101缀合至工程化之半胱氨酸残基的某些组合物能对ADC之稳定性产生影响。
表14:工程化曲妥单抗衍生的变体的热稳定性
抗体 | Tm1(℃) | Tm2(℃) | Tm3(℃) |
T(κK183C) | 72.17±0.029 | 80.78±0.37 | 82.81±0.055 |
T(L443C) | 72.02±0.06 | 80.98±1.10 | 82.96±0.11 |
T(LCQ05) | 72.22±0.027 | 81.16±0.19 | 82.88±0.033 |
T(κK183C+K290C) | 75.4 | 81.1 | 82.9 |
T(κK183C+K392C) | 75 | 81 | 83 |
T(κK183C+L443C) | 72.24±0.05 | 80.89±0.89 | 82.87±0.16 |
T(K290C+K334C) | 75.0±0.14 | 83.0±0.1 | 81.1±0.4 |
T(K334C+K392C) | 75.3±0.25 | 82.7±0.53 | 81.0±2.9 |
T(K290C+K392C) | 77 | 81 | 83 |
T(K392C+L443C) | 73.95±0.29 | 80.54±0.70 | 82.81±0.17 |
表15:位点特异性构建体缀合至耳抑素0101的热稳定性
位点特异性缀合物 | Tm1(℃) | Tm2(℃) | Tm3(℃) | Tm1SSC–Tm1Ab |
T(κK183C)-vc0101 | 70.16±0.03 | 80.45±0.12 | 82.04±0.03 | -2.01 |
T(L443C)-vc0101 | 72.34±0.10 | 80.20±0.59 | 82.44±0.10 | 0.32 |
T(κK183C+L443C)-vc0101 | 70.11±0.02 | 78.89±0.59 | 81.38±0.10 | -2.13 |
T(K392C+L443C)-vc0101 | 69.60±0.35 | 79.21±0.43 | 82.10±0.05 | -4.35 |
实施例8:结合至HER2之ADC
A.直接结合
使BT474细胞(HTB-20)受胰蛋白酶作用,离心而甩到底部及在新培样基中再悬浮。然后将该细胞在4℃下培养于ADC或未缀合之曲妥单抗的起始浓度1μg/ml之一连串梯度稀释液中1个小时。然后把该细胞用冰冷PBS清洗2次,及培养于抗人Alexafluor 488二级抗体(Cat#A-11013,Life technologies)中30分钟。然后把该细胞清洗2次及然后在PBS中再悬浮。使用Accuri流式细胞仪(BD Biosciences San Jose,CA)读取平均荧光强度。
表16:ADC结合至HER2
ADC/Ab | EC50 |
曲妥单抗 | 0.37 |
T(κK183C+K392C)-vc0101 | 0.56 |
T(κK183C+K290C)-vc0101 | 0.47 |
T(K290C+K392C)-vc0101 | 0.32 |
T-DM1(Kadcyla) | 0.40 |
T(LCQ05+K222R)-AcLysvc0101 | 0.37 |
T(N297Q+K222R)-AcLysvc0101 | 0.36 |
EC50=给出半最大结合的抗体或ADC的浓度
如图3A与表16中所示,在直接结合上,ADC T(LCQ05+K222R)-AcLysvc0101、T(N297Q+K222R)-AcLysvc0101、T(κK183C+K290C)-vc0101、T(κK183C+K392C)-vc0101、T(K290C+K392C)-vc0101具有和T-DM1与曲妥单抗类似的亲和力。这指示修饰本发明之ADC中的抗体及加入连接子-载荷物不显著地影响结合。
B.利用FACS竞争性结合
使BT474细胞受胰蛋白酶作用,离心而甩到底部及在新培样基中再悬浮。然后将该细胞在4℃下培养于ADC或未缀合之曲妥单抗和1μg/mL的曲妥单抗-PE(1:1PE标记之曲妥单抗,向eBiosciences(San Diego,CA)订制)的一连串梯度稀释液中1个小时。然后把该细胞清洗2次及然后在PBS中再悬浮。使用Accuri流式细胞仪(BD Biosciences San Jose,CA)读取平均荧光强度。
如图3B中所示,在竞争性结合至PE标记之曲妥单抗上,ADC T(LCQ05+K222R)-AcLysvc0101、T(N297Q+K222R)-AcLysvc0101、T(κK183C+K290C)-vc0101、T(κK183C+K392C)-vc0101、T(K290C+K392C)-vc0101具有和T-DM1与曲妥单抗类似的亲和力。这指示修饰本发明之ADC中的抗体及加入连接子-载荷物不显著地影响结合。
实施例9:结合至人FcRn之ADC
该领域中相信FcRn以pH依赖性方式与IgG(不拘亚型)相互作用及通过阻止抗体进入使抗体降解之溶酶体区而保护抗体免于降解。因此,考虑选择反应性半胱氨酸导入野生型IgG-Fc区中的位点是为了避免改变FcRn结合性质与包含工程化之半胱氨酸的抗体之半衰期。
进行分析来测定曲妥单抗衍生的单克隆抗体及其个别ADC结合至人FcRn之稳态亲和力(KD)。技术利用在曲妥单抗衍生的单克隆抗体或其个别ADC结合至被固定在传感器表面层上的人FcRn蛋白质后该表面层之折射率变化。利用从该表面折射的雷射光之表面电浆共振(SPR)来检测结合。通过使用BirA试剂(Catalog#:BIRA500,Avidity,LLC,Aurora,Colorado)且被固定在链霉抗生物素蛋白(SA)传感器基片上的工程化之Avi-标签把人FcRn特异生物素化,使在传感器上的人FcRn蛋白质能均匀定位。接着,将各种浓度之曲妥单抗衍生的单克隆抗体或其个别ADC,或20mM MES(2-(N-吗啉基)乙磺酸)(pH 6.0)与150mM NaCl、3mM EDTA(乙二胺四乙酸)、0.5%Surfactant P20(MES-EP)注射到基片表面上。在注射周期间使用HBS-EP+0.05%Surfactant P20(GEHealthcare,Piscataway,NJ)(pH 7.4)使该表面再生。测定曲妥单抗衍生之单克隆抗体或其个别ADC的稳态亲和力,并将其与野生型曲妥单抗抗体(在IgG1 Fc区中不含半胱氨酸,无TGase工程化之标签或载荷物的位点特异缀合)作比较。
这些数据显示工程化之半胱氨酸残基混入在本发明之指示位点上的IgG-Fc区不改变对FcRn之亲和力(表17)。
表17:位点特异性缀合物结合人FcRn的稳定状态亲和力
ND=未测定
实施例10:结合至Fcγ受体之ADC
评估使用位点特异缀合法之ADC对人Fcγ受体的结合以了解是否缀合至载荷物改变了能对抗体相关官能性(例如抗体依赖性之细胞媒介的细胞毒性(ADCC))产生影响之结合。FcγIIIa(CD16)在NK细胞及巨噬细胞上被表达,及其通过抗体结合与靶表达细胞的共同结合诱发ADCC。使用分析来测定曲妥单抗衍生之单克隆抗体及其个别ADC对Fc-γ受体IIa(CD32a)、IIb(CD32b)、IIIa(CD16)与FcγRI(CD64)的结合。
对此表面电浆共振(SPR)检定而言,将重组人表皮生长因子受体2(Her2/neu)胞外功能域蛋白质(Sino Biological Inc.,Beijing,P.R.China)固定于CM5基片(GEHealthcare,Piscataway,NJ)上,及捕获曲妥单抗衍生之单克隆抗体或其个别ADC的~300至400个反应单位(RU)。将T-DM1纳入本评估中作为阳性对照组,由于其保留比得上未缀合之曲妥单抗抗体的在缀合至Fcγ受体后的结合性质。然后,在测定表面与结合期间注入各种浓度之Fcγ受体FcγIIa(CD32a)、FcγIIb(CD32b)、FcγIIIa(CD16a)与FcγRI(CD64)。
FcγRs IIa、IIb与IIIa显出快速开/关速率,所以使感应图谱拟合稳态模型而得到Kd值。FcγRI显出较慢的开/关速率,所以使数据拟合动态模型而得到Kd值。
载荷物缀合至工程化之第290与334位半胱氨酸与其未缀合之对应抗体和T-DM1相比,显出FcγR亲和力中度丧失,尤其是对CD16a、CD32a与CD64的亲和力(表18)。然而,用T(K290C+K334C)-vc0101与T(K334C+K392C)-vc0101观测到同步缀合至第290、334与392位点导致对CD16a、CD32a与CD32b的亲和力实质丧失,但未丧失对CD64之亲和力(表18)。有趣地,T(κK183C+K290C)-vc0101显出对此研究中所评估的一切FcγR之可比拟的结合,尽管包含在K290C位点之载荷物(表18)。如预期,转谷氨酰胺酶媒介的缀合之T(N297Q+K222R)-AcLysvc0101未结合至所评估的Fcγ受体中任一者,由于含有酰基供体谷氨酰胺之卷标的位置消除了N-糖基化。相反地,T(LCQ05+K222R)-AcLysvc0101保留完全结合至Fcγ受体,由于含有谷氨酰胺的标签被工程化于人κ轻链恒定区中。
综合言之,这些结果表明缀合的载荷物之位置能影响ADC结合至FcγR,及可能影响缀合物的抗体官能性。
表18:位点特异性缀合物对Fcγ受体结合至CD16a、CD32a、CD32b和CD64的结合亲和性
ND=未测定,NB=未结合
实施例11:ADCC活性
在ADCC检定中,将Her2表达性细胞株BT474与SKBR3作为靶细胞,而把NK-92细胞(来自50岁男性白种人的外周血液单核细胞细胞衍生的介白素-2依赖型自然杀手细胞株,Conkwest)或人类外周血液单核细胞(PBMC)(由从健康捐血人新抽的血分离,#179)作为效应细胞。
将1X104细胞/100μl/孔之靶细胞(BT474或SKBR3)放进96孔盘中,及在37℃/5%CO2下于RPMI1640培养基中培养过夜。次日,移除培养基及替换成60μl检定缓冲液(含有10mMHEPES的RPMI1640培养基)、20μl之1μg/ml抗体或ADC,然后在每一孔中加入20μl 1 X 105(对SKBR3而言)或5x105(对BT474而言)PBMC悬浮液或2.5 X 105NK92细胞来达到50:1的效应细胞对靶细胞比(对BT474而言)或25:1(对用于PBMC的SKBR3而言),10:1(对NK92而言)。所有样品一式三份地进行。
将检定盘在37℃/5%CO2下培养6个小时,然后平衡至室温。使用CytoTox-OneTM试剂在560nm的激发波长及590nm的发射波长下测量细胞切割释出的LDH。就阳性对照组而言,在对照孔中加入8μL的Triton来产生最大LDH释出。图4中所示之比细胞毒性是用下式计算出:
“实验”对应于在上述任一条件下测定的信号
“效应自发性”对应于在仅PBMC存在下测定的信号
“目标自发性”对应于在仅目标细胞存在下测定的信号
“目标最大值”对应于在仅去污剂溶解目标细胞存在下测定的信号
图4显示曲妥单抗、T-DM1、与vc0101 ADC缀合物之实测ADCC活性。数据符合曲妥单抗与T-DM1被报告的ADCC活性。由于N297Q之突变体位于糖基化位点,T(N297Q+K222R)-AcLysvc0101不会具有ADCC活性,这也在检定中被确认。对单突变体(K183C、K290C、K334C、K392C,包括LCQ05)ADC而言,保留ADCC活性。出乎意外地,对双突变体(K183C+K290C,K183C+K392C,K183C+K334C K290C+K392C,K290C+K334C,K334C+K392C)ADC而言,全部保留ADCC活性,除二个和K334C位点有关的双突变体ADC(K290C+K334C与K334C+K392C)外。
实施例12:体外细胞毒性检定
按照实施例3中指示制得抗体药物缀合物。将细胞在低密度下接种于96孔盘中,然后在后续日子用ADC与未缀合之载荷物一式二份地以10个浓度的3倍系列稀释来处理。把细胞在加湿的37℃/5%CO2培养箱培养4日。将孔盘用96AQueous One MTSSolution(Promega,Madison,WI)培养1.5小时来收获孔盘及在Victor plate reader(Perkin-Elmer,Waltham,MA)上于波长490nm下测量吸光度。使用具XLfit的四参数逻辑模型(IDBS,Bridgewater,NJ)计算出IC50值及在图5中被报告为nM载荷物浓度和在图6中被报告为ng/ml抗体浓度。IC50被显示为在小括号中的独立测定之数字+/-标准偏差。
和基准ADC、T-DM1(Kadcyla)相比,含有vc-0101或AcLysv-0101连接子-载荷物之ADC高效对抗Her2阳性细胞模型及选择性对抗Her2阴性细胞模型。
用位点特异缀合至曲妥单抗所制得之ADC显示对抗Her2细胞模型的高效力及选择性。尤其,在中或低的Her2表达性细胞模型中,几种曲妥单抗-vc0101ADC比T-DM1更有效。例如,在MDA-MB-175-VII细胞(具1+Her2表达)中T(κK183C+K290C)-vc0101之体外细胞毒性IC50是351ng/ml,比T-DM1的3626ng/ml的十分之一更低。对具2++水平之Her2表达的细胞(例如MDA-MB-361-DYT2细胞与MDA-MB-453细胞)-而言,(κK183C+K290C)-vc0101之IC50是12至20ng/ml,T-DM1的IC50是38至40ng/ml。
实施例13:异种移植物模型
本发明之曲妥单抗衍生的ADC在下列中测试:N87胃癌异种移植物模型、37622肺癌异种移植物模型、与一些乳癌异种移植物模型(即,HCC 1954、JIMT-1、MDA-MB-361(DYT2)与144580(PDX)模型)。对下述之每一模型而言,在第1日给予第一剂。每周至少测量一次肿瘤及其体积用下式计算出:肿瘤体积(mm3)=0.5x(肿瘤宽度2)(肿瘤长度)。计算出每一治疗组的平均肿瘤体积(±S.E.M.),该治疗组包括最多8至10只与最少6至8只的动物。
A.N87胃腺异种移植物
在免疫缺陷小鼠中测验曲妥单抗衍生的ADC之效应,该免疫缺陷小鼠体内有由具有高水平HER2表达的N87细胞株(ATCC CRL-5822)建立之人肿瘤异种移植物生长。在母裸小鼠(Nu/Nu,Charles River Lab,Wilmington,MA)皮下植入7.5x106N87细胞与50%Matrigel(BD Biosciences)来产生异种移植物。当肿瘤体积达到250至450mm3时,将肿瘤分期以保证在不同治疗组中的肿瘤块一致。N87胃腺模型接受每4日给一次药,共给4次(Q4dx4)的静脉内施用PBS媒剂、曲妥单抗ADC(在0.3、1与3mg/kg下)、或T-DM1(1、3与10mg/kg)(图7)。
数据表明曲妥单抗衍生之ADC以剂量依赖性方式抑制N87胃腺异种移植物的生长(图7A至7H)。
如图7I中所示,在1与3mg/kg下的T-DM1使肿瘤生长延迟,而在10mg/kg下使肿瘤完全消退。然而,在1与3mg/kg下的T(κK183C+K290C)-vc0101使肿瘤完全消退,而在0.3mg/kg下使肿瘤一部分消退(图7A)。数据显示在此模型中,T(κK183C+K290C)-vc0101明显比T-DM1更有效(~10倍)。
具DAR4之ADC的体内效力类似于183+290(图6E、6F与6G)。此外,评估单突变体DAR2ADC(图7B、7C与7D)。一般而言,这些ADC比DAR4ADC效力差但比T-DM1更有效。在DAR2ADC中,以体内效力数据为基础,LCQ05似乎是最有效的ADC。
B.HCC1954乳腺异种移植物
HCC1954(ATCC#CRL-2338)是一种高HER2表达性乳癌细胞株。在母SHO小鼠(Charles River,Wilmington,MA)皮下植入5x106HCC1954细胞与50%Matrigel(BDBiosciences)来产生异种移植物。当肿瘤体积达到200至250mm3时,将肿瘤分期以保证在不同治疗组中的肿瘤块一致。HCC1954乳腺模型接受Q4dx4的静脉内施用PBS媒剂、曲妥单抗衍生之ADC及阴性对照组ADC(图8A至8E)。
数据表明曲妥单抗ADC以剂量依赖性方式抑制HCC1954乳腺异种移植物的生长。比较1mg/kg剂量,vc0101缀合物比T-DM1更有效。比较3mg/kg剂量,载荷DAR4之ADC(图8B、8C与8D)比载荷DAR2之ADC(图8A)更有效。此外,和媒剂对照组相比,在1mg/kg下的阴性对照组ADC对肿瘤生长影响很小(图8D)。然而,T(N297Q+K222R)-AcLysvc0101使肿瘤完全消退指示靶特异性。
C.JIMT-1乳腺异种移植物
JIMT-1是一种中/低Her2表达性乳癌细胞株且固有地对曲妥单抗有抗药性。在母裸(Nu/Nu)小鼠皮下植入5x106JIMT-1细胞(DSMZ#ACC-589)与50%Matrigel(BDBiosciences)来产生异种移植物。当肿瘤体积达到200至250mm3时,将肿瘤分期以保证在不同治疗组中的肿瘤块一致。JIMT-1乳腺模型接受Q4dx4的静脉内施用PBS媒剂、T-DM1(图9G)、使用位点特异缀合的曲妥单抗衍生之ADC(图9A至9E)、使用常规缀合的曲妥单抗衍生之ADC(图9F)、及阴性对照组huNeg-8.8ADC。
数据表明被试验之vc0101缀合物以剂量依赖性方式造成肿瘤减少。这些ADC在1mg/kg剂量下能造成肿瘤消退。然而,在此中/低Her2表达性乳癌模型中T-DM1无效,即使在6mg/kg下。
D.MDA-MB-361(DYT2)乳腺异种移植物
MDA-MB-361(DYT2)是一种中/低Her2表达性乳癌细胞株。在100cGy/分钟下对母裸(Nu/Nu)小鼠照射4分钟,及在3日后在皮下植入1.0x107MDA-MB-361(DYT2)细胞(ATCC#HTB-27)与50%Matrigel(BD Biosciences)来产生异种移植物。当肿瘤体积达到300至400mm3时,将肿瘤分期以保证在不同治疗组中的肿瘤块一致。DYT2乳腺模型接受Q4dx4的静脉内施用PBS媒剂、使用位点特异缀合与常规缀合的曲妥单抗衍生之ADC、T-DM1、及阴性对照组ADC(图10A至10D)。
数据表明曲妥单抗ADC以剂量依赖性方式抑制DYT2乳腺异种移植物的生长。尽管DYT2是中/低Her2表达性细胞株,然而对微管抑制剂比其它中/低Her2表达性细胞株更有感药性。
E.144580患者衍生之乳癌异种移植物
在免疫缺陷小鼠中测验曲妥单抗衍生的ADC之效应,该免疫缺陷小鼠体内有由根据合适同意程序所得到的新切除的144580乳腺肿瘤的片段建立的人肿瘤异种移植物生长。在采用新鲜活体组织切片时144580的肿瘤特性分析是三阴性(ER-、PR-、与HER2-)乳癌肿瘤。将144580患者衍生之乳癌异种移植物片段以从动物到动物方式植入母裸(Nu/Nu)小鼠皮下。当肿瘤体积达到150至300mm3时,将肿瘤分期以保证在不同治疗组中的肿瘤块一致。该144580乳腺模型接受每4日给一次药,共给4次(Q4dx4)的静脉内施用PBS媒剂、使用位点特异缀合的曲妥单抗ADC、使用常规缀合的曲妥单抗衍生之ADC、及阴性对照组ADC(图11A至11E)。
在此HER2-(由临床定义)PDX模型中,T-DM1在全部试验剂量(1、5、3与6mg/kg)下是无效的(图10E)。对DAR4vc0101ADC(图11A、11C与11D)而言,在3mg/kg下能造成肿瘤消退(在图11C中甚至还在1mg/kg下)。在3mg/kg下,DAR2 vc0101 ADC(图11B)比DAR4 ADC没效。然而,在6mg/kg下,DAR 2 vc0101 ADC有效,不像T-DM1。
F.37622患者衍生之非小细胞肺癌异种移植物
在根据合适同意程序所得到的37622患者衍生之非小细胞肺癌异种移植物模型中试验几种ADC。将37622患者衍生之异种移植物片段以从动物到动物方式植入母裸(Nu/Nu)小鼠皮下。当肿瘤体积达到150至300mm3时,将肿瘤分期以保证在不同治疗组中的肿瘤块一致。该37622PDX模型接受每4日给一次药,共给4次(Q4dx4)的静脉内施用PBS媒剂、使用位点特异缀合的曲妥单抗衍生之ADC、T-DM1、及阴性对照组ADC(图12A至12D)。
Her2表达利用经修饰的Hercept试验来特性分析及被归类为2+,比在细胞株中看到的更不均匀。以连接子-载荷物形式缀合vc0101的ADC(图12A至12C)在1and 3mg/kg下有效造成肿瘤消退。然而,T-DM1仅在10mg/kg下提供某些治疗效益(图12D)。比较从10mg/kg(T-DM1)到1mg/kg(vc0101ADC),vc0101ADC似乎比T-DM1够有效了10倍。对异源性肿瘤来说,旁观者效应可能对效力很重要。
G.GA0044患者衍生之胃癌异种移植物
在根据合适同意程序所得到的患者衍生之胃癌异种移植物模型(GA0044)中试验曲妥单抗与抗HER2ADC。将GA0044患者衍生之异种移植物片段以从动物到动物方式植入母裸(Nu/Nu)小鼠皮下。当肿瘤体积达到150至300mm3时,将肿瘤分期以保证在不同治疗组中的肿瘤块一致。该GA0044PDX模型接受每4日给一次药,共给4次(Q4dx4)的静脉内施用PBS媒剂、曲妥单抗、T-DM1、或使用位点特异缀合至vc0101的曲妥单抗衍生之ADC(图30)。
在GA0044中HER2表达利用经修饰的Hercept试验来特性分析及被归类为2+,具有不均匀的分布。以载荷物形式缀合vc0101(即T(κK183C+K290C)-vc0101)的ADC在1and 3mg/kg剂量下有效且造成肿瘤完全消退。和经媒剂治疗的肿瘤相比,曲妥单抗与T-DM1没有在肿瘤生长上显示明显差异。在此具有异源性靶(即HER2)表达的肿瘤中,旁观者效应可能对效力很重要。
H.在N87胃腺异种移植物中T-vc0101ADC之旁观者效应的示范
T-DM1 ADC之释出的代谢产物已经证实为经赖氨酸封端之mcc-DM1连接子载荷物(即Lys-mcc-DM1),其为不可透膜的化合物(Kovtun et al.,2006,Cancer Res 66:3214-21;Xie et al.,2004,J Pharmacol Exp Ther 310:844)。然而,由T-vc0101ADC释出的代谢产物是耳抑素0101,一种比Lys-mcc-DM1更可透膜之化合物。释出的ADC载荷物杀死附近细胞之能力就是旁观者效应。由于可透膜载荷物释出,T-vc0101能引出强的旁观者效应,而T-DM1不能。图13显示N87细胞株异种移植物肿瘤的免疫组织化学,该肿瘤接受单次剂量的在6mg/kg下的T-DM1(图13A)或在3mg/kg下的T-vc0101(图13B),然后收获及在甲醛中浸泡96个小时。将肿瘤切片进行染色,以人IgG为标记来检测结合至肿瘤细胞的ADC,及以磷酸化组织蛋白H3(pHH3)为标记来检测有丝分裂细胞,作为二种ADC之载荷物的假定作用机制之读出。
在二种情况下在肿瘤外围检出ADC。在经T-DM1治疗之肿瘤(图13A)中,pHH3阳性肿瘤细胞大多数位于ADC附近。然而,在经T-vc0101治疗的肿瘤(图13B)中,pHH3阳性肿瘤细胞大多数延伸到ADC位置以外及在肿瘤内部。这些数据表明具有可切割之连接子与可透膜的载荷物之ADC能引出强的体内旁观者效应。
实施例14:体外T-DM1抗药性模型
A.体外T-DM1抗药性细胞之产生
将N87细胞通入二个烧瓶中及每一烧瓶用相同抗药性产生操作程序来处理使能产生生物复制品。把细胞暴露于5个周期之在约IC80浓度(10nM载荷物浓度)下的T-DM1缀合物3日,接着在没有处理下恢复约4至11日。在10nM之T-DM1缀合物下5个周期后,把细胞以相似方式暴露于另外6个周期的100nM之T-DM1。本步骤意图模拟在典型地被用于临床细胞毒性治疗剂之最大耐受剂量下的长期且多周期(开/关)投药,接着恢复期。由N87衍生之亲代细胞称为N87,而长期暴露于T-DM1的细胞称为N87-TM。N87-TM细胞在4个月内发展出中至高水平之抗药性。在~3至4个月的处理周期后,当持续暴露于药物后抗药性水平不再增加时,药物选择压力消除。在之后的约3至6个月在所培养之细胞株中的反应与表达型保持稳定。后来,偶尔观测到细胞毒性检定所测得之抗药性表达型的数量降低,在此种情况下,将先前经冷冻保存之T-DM1抗药性细胞解冻用于另外的研究。所有被报告之特性分析都是在T-DM1选择压力消除至少2至8周后才进行以保证细胞稳定。在模型开发后约1至2年内从各种解冻之经冷冻保存的由单一选择衍生之总体采集数据以保证结果的一致性。选定胃癌细胞株N87用于对曲妥单抗-类美登素抗体药物缀合物(T-DM1)之抗药性研究,对个别细胞株在约IC80(~10nM载荷物浓度)剂量下进行处理周期。亲代N87细胞对该缀合物固有地具感药性(IC50=1.7nM载荷物浓度;62ng/ml抗体浓度)(图14)。将二个亲代N87细胞总体暴露于处理周期,及在只暴露于100nM之T-DM1下约4个月的周期后,和亲代细胞相比,这二个总体(以后命名为N87-TM-1与N87-TM-2)的ADC难治性分别变成114倍与146倍(图14与15A)。有趣地是观测到对该对应之未缀合的无类美登素之药物DM1有极小的交叉抗药性(~2.2-2.5X)(图14)。
B.细胞毒性研究
按照实施例3中指示制得ADC。未缀合之美登素类似物(DM1)与耳抑素类似物制造商为Pfizer Worldwide Medicinal Chemistry(Groton,CT)。其它标准照护化学治疗剂购自Sigma(St.Louis,MO)。将细胞在低密度下接种于96孔盘中,然后在后续日子用ADC与未缀合之载荷物一式二份地以10个浓度的3倍系列稀释来处理。把细胞在加湿的37℃/5%CO2培养箱培养4日。将孔盘用96AQueous One MTS Solution(Promega,Madison,WI)培养1.5小时来收获孔盘及在Victor plate reader(Perkin-Elmer,Waltham,MA)上于波长490nm下测量吸光度。使用具XLfit的四参数逻辑模型(IDBS,Bridgewater,NJ)计算出IC50值。
测得对其他曲妥单抗衍生之ADC的交叉抗药性特性。观测到对许多由用抗微管蛋白作用机制来输送载荷物之不可切割的连接子组成之曲妥单抗衍生的ADC之显著交叉抗药性(图14)。例如,在N87-TM对N87-亲代细胞中,观测到对T-mc8261(图14与15B)与T-MalPeg8261(图14)的效力降低>330倍及>272倍,其分别代表以耳抑素为基础之载荷物通过不可切割的顺丁烯二酰亚胺基己酰基或Mal-PEG连接子连接至曲妥单抗。在对抗T-mcMalPegMMAD的N87-TM细胞中观测到超过235倍之抗药性,该抗T-mcMalPegMMAD N87-TM细胞是另一种具输送单甲基尾海兔素(monomethyl dolastatin)(MMAD)的不同之不可切割的连接子之曲妥单抗ADC(图14)。
明显地,观测到当通过可切割之连接子输送时,N87-TM细胞株保留对载荷物的敏感性,即使这些药物在功能上抑制类似的靶(即微管解聚合)。克服抗药性之ADC实施例包括但不限于:T(N297Q+K222R)-AcLysvc0101(图14与图15C)、T(LCQ05+K222R)-AcLysvc0101(图14与图15D)、T(K290C+K334C)-vc0101(图10与图11E)、T(K334C+K392C)-vc0101(图14与图15F)及T(κK183C+K290C)-vc0101(图14与图15G)。这些代表输送耳抑素类似物0101的以曲妥单抗为基础之ADC,但该载荷物被vc连接子蛋白溶解性切割而在细胞内释出。
为了测定这些ADC抗药性癌细胞是否大体上对其他疗法具抗药性,用一组具不同作用机制的标准照护化学治疗剂来治疗N87-TM细胞模型。通常,微管与DNA功能的小分子抑制剂保留有效对抗N87-TM抗药性细胞株的能力(图14)。尽管这些细胞被制成对输送微管解聚合剂(美登素)类似物的ADC有抗药性,仍然观测到对几种微管蛋白解聚合剂或聚合剂有最低的或没有交叉抗药性。相似地,二种细胞株保留对干扰DNA功能的药剂有感药性,该药剂包括拓朴异构酶抑制剂、抗代谢药、及烷基化剂/交联剂。通常,N87-TM细胞不是大范围的细胞毒性剂难治的,排除一般生长或与抗药性极相似的细胞周期缺陷。
二个N87-TM总体也保留对对应未缀合之药物的感药性(即DM1与0101;图14)。因此,当时,N87-TM细胞是曲妥单抗-类美登素缀合物难治的,其显出对通过不可切割之连接子输送的其它以微管为基础之ADC的交叉抗药性,但保留对未缀合之微管抑制剂与其它化学治疗剂的感药性。
测得MDR1与MRP1药物排出泵之蛋白质表达水平,以确定在N87-TM细胞中的T-DM1抗药性的分子机制。这是由于小分子微管蛋白抑制剂是MDR1与MRP1药物排出泵的已知基质(Thomas and Coley,2003,Cancer Control 10(2):159-165)。我们测得来自亲代N87与N87-TM抗药性细胞之总溶胞产物的这二种蛋白质之蛋白质表达水平(图16)。免疫印迹分析表明N87-TM抗药性细胞不显著地过度表达MDR1(图16A)或MRP1(图16B)蛋白质。综合言之,这些数据和缺乏在N87-TM细胞中的药物排出泵之已知基质(例如紫杉醇、多柔比星(doxorubicin))的交叉抗药性表明药物排出泵过度表达不是在N87-TM细胞中的T-DM1抗药性之分子机制。
由于ADC之作用机制需要结合至特异抗原,抗原耗减或降低的抗体结合可能引起在N87-TM细胞中的T-DM1抗药性。将来自亲代N87与N87-TM抗药性细胞之总溶胞产物的HER2蛋白质表达水平相比较来求出在N87-TM细胞中T-DM1的抗原是否已经显著地耗减(图17A)。免疫印迹分析表明和亲代N87细胞相比,N87-TM细胞不具有显著地降低之HER2蛋白质表达量。
测得结合至N87-TM细胞之细胞表面HER2抗原的抗体量。在使用荧光活化细胞分选法之细胞表面结合研究中,N87-TM细胞确实具有~50%减少的结合至细胞表面抗原之曲妥单抗(图17B)。由于在癌细胞株中N87细胞是HER2蛋白质的高表达者(Fujimoto-Ouchi etal.,2007,Cancer Chemother Pharmacol 59(6):795-805),在这些细胞中HER2抗体结合降低~50%可能不代表在N87-TM细胞中的T-DM1抗药性的主要机制。支持此解释的证据是N87-TM抗药性细胞保留对其他具不同连接子与载荷物的HER2结合曲妥单抗衍生之ADC的感药性(图14)。
通过蛋白质体方法作出亲代N87与N87-TM抗药性细胞模型之特性以便大范围识别可能引起T-DM1抗药性的膜蛋白质表达水平变化,来以不偏差的方法求出T-DM1抗药性之潜在机制。观测到在二个细胞株模型间的523蛋白质表达水平之显著变化(图18A)。对被预测相对于N87细胞来说在N87-TM细胞中表达不足的蛋白质(IGF2R、LAMP1、CTSB)(图18B)与过度表达蛋白质(CAV1)(图18C)进行N87与N87-TM之总溶胞产物的免疫印迹分析,来证实这些被预测之蛋白质变化的选择。在NSG小鼠皮下植入N87与N87-TM-2细胞来产生体内肿瘤,以评估在体内观测到的蛋白质变化是否和在体外看到的蛋白质变化极相似。和N87肿瘤相比,N87-TM-2肿瘤保留CAV1蛋白质之过度表达(图18D)。尽管已预测在二个模型中在小鼠基质中的CAV1染色,然而上皮之CAV1染色仅在N87-TM-2模型看到。
C.体内效力研究
将亲代N87细胞与N87-TM-2细胞增殖及注入取自The Jackson Laboratory(BarHarbor,ME)的母非肥胖糖尿病型重症联合(NSG)免疫缺陷型小鼠(NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ)侧腹来测定在细胞培养中观测到之抗药性是否在体内重演。把N87或N87-TM细胞悬浮液注入右侧腹皮下(每一注射有7.5x106细胞和50%Matrigel)。当肿瘤达到~0.3g(~250mm3)时,将小鼠随机分派至研究组。在第0日静脉内施用T-DM1缀合物或媒剂之盐溶液及每4日给一次药,共给4次(Q4Dx4)。每周一次测量肿瘤且将肿瘤块计算为:体积=(宽度x宽度x长度)/2。进行事件发生时间分析(肿瘤倍增)及利用对数等级(Mantel-Cox)检定来评估重要性。在这些研究中,在所有治疗组中都没观测到小鼠体重减轻。
用下列药剂治疗小鼠:(1)媒剂对照组PBS;(2)在13mg/kg下,接着在4.5mg/kg下的曲妥单抗抗体;(3)在6mg/kg下之T-DM1;(4)在10mg/kg下的T-DM1;(5)在10mg/kg下之T-DM1,然后在3mg/kg下的T(N297Q+K222R)-AcLysvc0101;(6)在3mg/kg下之T(N297Q+K222R)-AcLysvc0101。检测肿瘤大小及结果参见图20。N87(图19与图20A)肿瘤及N87-TM-2(图19与图20B)肿瘤显示类似于体外细胞毒性检定中看到的ADC效力特性(图19与图20B),其中N87-TM抗药性细胞是T-DM1难治的,但仍对具可切割之连接子的曲妥单抗衍生之ADC有反应。事实上,将T-DM1难治的且生长到约1克之肿瘤传换成用T(N297Q+K222R)-AcLysvc0101的疗法且有效地消退(图20B)。在此研究之事件发生时间分析中,在6与10mg/kg下的T-DM1在N87模型中在至少60日内防止>50%之小鼠肿瘤倍增,但T-DM1在N87-TM-2模型中却不能做到(图20C与图20D)。施用在3mg/kg下的T(N297Q+K222R)-AcLysvc0101在研究期间内(~80日)防止在小鼠中N87与N87-TM肿瘤的任何肿瘤倍增(图20C与图20D)。
在另一个研究中,克服体外T-DM1抗药性的所有具可切割之连接子的ADC在此对T-DM1无反应的N87-TM2肿瘤模型中仍然有效(图19与图20E)。
然后评估T(κK183+K290C)-vc0101ADC是否能抑制T-DM1难治之肿瘤的生长。尽管用媒剂或T-DM1治疗之N87-TM肿瘤生长,然而在第14日转换成T(κK183C+K290C)-vc0101疗法的肿瘤立即消退(图20F)。
实施例15:体内T-DM1抗药性模型
A.体内T-DM1抗药性细胞之产生
一切动物研究都是由Pfizer Pearl River Institutional Animal Care andUse Committee根据明确指导方针而核准。把7.5x 106 N87细胞和50%Matrigel(BDBiosciences)植入母裸小鼠皮下来产生异种移植物。当平均肿瘤体积达到~300mm3时,将小鼠随机分派成二组:1)媒剂对照组(n=10)及2)T-DM1治疗组(n=20)。在第0日静脉内施用T-DM1ADC(6.5mg/kg)或媒剂(PBS)之盐溶液,然后每周施用6.5mg/kg到30周为止。每周二次或每周一次测量肿瘤且将肿瘤块计算为:体积=(宽度x宽度x长度)/2。在这些研究中,在所有治疗组中都没观测到小鼠体重减轻。
当个别肿瘤体积达到~600mm3(在随机分派时的原本大小加倍)时,小鼠被认为是T-DM1治疗难治的或是在T-DM1治疗后复发。和对照组相比,大多数肿瘤刚开始时对T-DM1治疗有反应,如图21A中所示。更具体地说,20只小鼠中有17只刚开始时对T-DM1治疗有反应,但大量肿瘤(20只中有13只)在T-DM1治疗后复发。随时间过去,被植入之N87肿瘤细胞变成T-DM1抗药性(图21B)。收集刚开始时对T-DM1治疗无反应的3颗肿瘤用于HER2表达测定,IHC指示HER2表达没变化。其余之10颗复发的肿瘤如下所述。
将4颗刚开始时对T-DM1治疗有反应然后复发之肿瘤在第77日转换成每周一次在2.6mg/kg下的T-vc0101治疗(小鼠1与16),在第91日转换(小鼠19),在第140日转换(小鼠6)。如图19C中所示,体内产生之T-DM1抗药性肿瘤对T-vc0101有反应指示后天性T-DM1抗药性肿瘤对vc0101ADC治疗有感药性。
另外3颗刚开始时对T-DM1治疗有反应然后复发之肿瘤在第110日转换成每周一次在2.6mg/kg下的T(N297Q+K222R)-AcLysvc0101治疗(小鼠4、13与18)。如图21D中所示,体内产生之T-DM1抗药性肿瘤对T(N297Q+K222R)-AcLysvc0101有反应指示后天性T-DM1抗药性肿瘤对vc0101ADC治疗有感药性。进行后续试验来评估T(κK183C+K290C)-vc0101,得到类似的结果指示体内产生之T-DM1抗药性肿瘤对T(κK183C+K290C)-vc0101治疗有感药性,如图21E中所示。
总而言之,一切具有后续治疗之T-DM1难治性肿瘤都对vc0101ADC治疗有感药性(7只中有7只)指示可以用可切割的vc0101缀合物治疗体内产生之T-DM1抗药性肿瘤。
将另外3颗刚开始时对T-DM1治疗有反应然后复发之肿瘤(小鼠7、17与2,如图21B中所示)切除用于体外特性分析。该被切除的肿瘤在体外培养2至5个月后,对这些细胞评估T-DM1抗药性及进行体外特性分析(见以下的本实施例的B与C节)。
B.细胞毒性研究
将在T-DM1治疗后复发且经体外培养之细胞(如本实施例的A节中所述)接种于96孔盘中及在后续日子用ADC或未缀合之载荷物以4倍系列稀释来处理。把细胞在加湿的37℃/5%CO2培养箱培养96个小时。把CellTiter Glo Solution(Promega,Madison,WI)加到该盘中及在Victor plate reader(Perkin-Elmer,Waltham,MA)上于波长490nm下测量吸光度。使用具XLfit的四参数逻辑模型(IDBS,Bridgewater,NJ)计算出IC50值。
细胞毒性筛选结果汇总于表19与20中。该细胞和亲代相比较时具有T-DM1抗药性(图22A),但对可切割的vc0101缀合物T-vc0101(数据未显示)、T(κK183C+K290C)-vc0101(图22B)、T(LCQ05+K222R)-AcLysvc0101(图22C)、T(N297Q+K222R)-AcLysvc0101(图22D)有感药性(表19)。出乎意外地,T-DM1抗药性细胞对亲代载荷物DM1及0101载荷物有感药性(表20)。
表19:抗性细胞对ADC的敏感性
每个细胞系显示的IC50值
表20:抗性细胞系对游离载荷物的敏感性
细胞系 | DM1-Sme | Aur-0101 | 多柔比星 |
N87 | 10 | 0.5 | 48 |
N87-T-DM1_Ms2 | 23 | 0.40 | 46 |
N87-T-DM1_Ms7 | 20 | 0.60 | 79 |
N87-T-DM1_Ms17 | 27 | 0.28 | 34 |
C.利用FACS与蛋白印迹法之Her2表达
对在T-DM1治疗后复发且经体外培养之细胞(如本实施例的A节中所述)进行Her2表达特性分析。对FACS分析而言,使细胞受胰蛋白酶作用,离心而甩到底部及在新培样基中再悬浮。然后将该细胞在4℃下培养于5μg/mL的曲妥单抗-PE(1:1PE标记之曲妥单抗,向eBiosciences(San Diego,CA)订制)中1个小时。然后把该细胞清洗2次及然后在PBS中再悬浮。使用Accuri流式细胞仪(BD Biosciences San Jose,CA)读取平均荧光强度。
对蛋白印迹法分析而言,把细胞在冰上使用RIPA lysis buffer(具蛋白酶抑制剂与磷酸酶抑制剂)来溶解15分钟,然后在4℃下于微量离心机中在最大速度下涡漩震荡与甩到底部。采集上清液,加入4X样品缓冲液与还原剂(标准化每一样品中的总蛋白质)。将样品在4至12%的Bis tris gel上运行及转移到硝化纤维素薄膜上。把薄膜封闭1个小时及在4℃下培养于HER2抗体(Cell Signalling,1:1000)中过夜。然后将薄膜在1X TBST中清洗3次及培养于抗-小鼠HRP抗体(Cell Signalling,1:5000)中1个小时及清洗3次和探测。
在T-DM1治疗后复发之肿瘤的HER2表达水平类似于对照组肿瘤(无T-DM1治疗),评估方法系利用FACS(图23A)及蛋白印迹法(图23B)。
D.T-DM1抗样性不是由于药物排出泵
利用蛋白印迹法得知该细胞系不表达MDR1(图24A)及该细胞对MDR-1基质游离药物0101无抗药性(图24B)。观测到对多柔比星无抗药性(图24C)指示抗药机制不是通过MRP1。然而,该细胞对游离DM1仍有抗药性(图24D)。
实施例16:药物动力学(PK)
在对马来猴施用5或6mg/kg剂量之静脉推注后测定常规的或位点特异性vc0101抗体药物缀合物之暴露量。使用(LBA)配体结合分析测量总抗体(总Ab;缀合之mAb与未缀合之mAb的计量)与ADC(缀合至至少一个药物分子之mAb)浓度。在所有情况下,ADC是用vc0101制得,除T(LCQ05)是用AcLysvc0101制得外。使用常规缀合(非位点特异缀合)由曲妥单抗制得ADC。
在对马来猴施用总Ab与曲妥单抗ADC(T-vc0101)(5mg/kg)或T(κK183C+K290C)位点特异性ADC(6mg/kg)后之浓度对时间特性和药物动力学/毒物动力学(图25A与表21)。和常规的缀合物相比,T(κK183C+K290C)位点特异性ADC之暴露量与稳定性都增加。
在对马来猴施用曲妥单抗(T-vc0101)(5mg/kg)或T(κK183C+K290C)、T(LCQ05)、T(K334C+K392C)、T(K290C+K334C)、T(K290C+K392C)与T(κK183C+K392C)位点特异性ADC(6mg/kg)后ADC被分析物的浓度对时间特性和药物动力学/毒物动力学(图25B与表21)。和使用常规缀合法之曲妥单抗ADC相比,几种位点特异性ADC(T(LCQ05)、T(κK183C+K290C)、T(K290C+K392C)与T(κK183C+K392C))的暴露量更高。然而,其余二种位点特异性ADC(T(K290C+K334C)与T(K334C+K392C))之暴露量没比曲妥单抗ADC更高,这指示并非全位点点特异性ADC都具有优于使用常规缀合法所制得之曲妥单抗ADC的药物动力学性质。
表21:药物动力学
实施例17:利用疏水性交互作用层析术的相对滞留值对大鼠中暴露量(AUC)。
疏水性是可以利用疏水性交互作用层析术(HIC)评估的蛋白质之物理性质,而蛋白质样品的滞留时间根据其相对疏水性而不同。通过计算相对滞留时间(RRT)可以将ADC与其个别抗体作比较,该相对滞留时间是ADC之HIC滞留时间除以个别抗体的HIC滞留时间之比值。高疏水性ADC具有较高的RRT,且高疏水性ADC也可能更具有药物动力学可靠度,尤其是较低之曲线下面积(AUC或暴露量)。当把具各种位点突变的ADC之HIC值和其在大鼠中的实测AUC相比较时,观测到在图26中的分布。
具RRT≥1.9之ADC显示较低的AUC值,而具较低RRT之ADC倾向于具有较高的AUC,尽管不是直接的关系。观测到ADC T(κK183C+K290C)-vc0101具有相对高之RRT(1.77的平均值),因此预测具有相对低之AUC。出乎意外地,观测到的ADC相对高,因此从疏水性数据不太能预测此ADC之暴露量。
实施例18:毒性研究
在二个独立探究之毒性研究中,将公母共10只的马来猴分成5个剂量组(1/性别/剂量)及每3周IV投药一次(第1、22与43个研究日)。在第46个研究日(第3次投药后第3日)使马来猴安乐死,及采集操作程序规定的血液与组织样品。在生存时及验尸后进行临床观测、临床病理学、宏观病理学与微观病理学评估。对解剖病理学评估而言,以主观的、相对的、研究特异性为基础来记录组织病理学发现之严重程度。
在3与5mg/kg下的马来猴探究毒性研究中,在第一次投药后第11日,T-vc0101引起短暂而明显(390/μl)到严重(40/μl至不能检测)之嗜中性粒细胞减少症。和在9mg/kg下相反,被施用T(κK183C+K290C)-vc0101的马来猴都没有嗜中性粒细胞减少症,在试验之任何时间点都是大于500/μl的良好嗜中性粒细胞计数(图27)。事实上,和媒剂对照组相比,被施用T(κK183C+K290C)-vc0101之马来猴在第11与14日显示平均的嗜中性粒细胞计数(>1000/μL)。
在3与5mg/kg下的骨髓中,显微镜观测到被施用T-vc0101之马来猴具有化合物相关的增加之M/E比。增加的骨髓/类红血球(M/E)比由减少的类红血球前驱物和主要成熟颗粒球增加组成。相反地,在6与9mg/kg下,只有被施用6mg/kg的T(κK183C+K290C)-vc0101之公马来猴具有最小至中度的增加之成熟颗粒球细胞构成(数据未显示)。
因此,显微镜观测之血液数据清楚地指示以位点特异性突变技术为基础的ADC缀合物T(κK183C+K290C)-vc0101清楚地改进T-vc0101诱发的骨髓毒性与嗜中性粒细胞减少症。
实施例19:ADC晶体结构
得到T(K290C+K334C)-vc0101、T(K290C+K392C)-vc0101与T(K334C+K392C)-vc0101之晶体结构。选择这些特定ADC作晶体照相术是由于缀合至K290C+K334C与K334C+K392C双半胱氨酸变异体,而不是K290C+K392C,彻底破坏ADCC活性。
使用木瓜酵素使ADC切割来制得用于晶体照相术的经缀合之Fc区。使用下列相同条件得到三个经缀合之IgG1-Fc区的相同型态的结晶:100mM柠檬酸钠(pH 5.0)+100mMMgCl2+15%PEG 4K。
保藏于PDB中的野生型人IgG1-Fc结构相对地相似,表明CH2-CH2结构域通过经Asn297键联之多糖(碳水化合物或多糖末端)彼此接触,及表明CH3-CH3结构域形成在结构间相对固定的稳定界面。Fc结构以“闭合”或“开放”构象存在及去糖基化之Fc结构采取“开放”结构构象从而表明该多糖末端把CH2结构域固定在一起。此外,未缀合的Phe241Ala-IgG1Fc突变体(Yu et al.“Engineering Hydrophobic Protein-Carbohydrateinteractions to fine-tune monoclonal antibodies”.JACS 2013)被公开之结构显示一个部份无序的CH2结构域,由于此突变体导致CH2-多糖界面与CH2-CH2界面不稳定,乃因为芳香族Phe残基不能使碳水化合物稳定。
人IgG Fc区之“CH2”结构域(也称为“Cγ2”结构域)通常从约第231位氨基酸延伸至约第340位氨基酸。该CH2结构域在与另一区不紧密配对方面是唯一的。相反,二个N-键联之分支碳水化合物链置于完整天然IgG分子的二个CH2结构域间。推测该碳水化合物可能提供区-区配对之代替物及帮助使该CH2结构域稳定(Burton et al.,1985,Molec.Immunol.22:161-206)。
“CH3结构域”包含C末端残基延伸至Fc中的CH2结构域(即从约第341位氨基酸残基到IgG之约第447位氨基酸残基)。
T(K290C+K334C)-vc0101与T(K290C+K392C)-vc0101Fc区之已解释的结构相似,表明该Fc二聚体含有一个CH2与二个高度有序之CH3(像野生型Fc)。然而,其也含有附接多糖的无序之CH2(图28A与图28B)。一个CH2结构域的较高程度之去稳定化乃归因于缀合位点紧密接近多糖末端。考虑到0101载荷物几何形状,在K290、K334、K392位点中任一者上的缀合能扰乱多糖从CH2界面离开之总体轨迹,使多糖与CH2结构自身去稳定化,结果产生CH2-CH2界面(图28C)。相对于WT-Fc、Phe241Ala-Fc或去糖基化的Fc,较高程度之不均匀性有助于这些0101位点特异缀合双半胱氨酸-Fc-变异体。当工程化之半胱氨酸-变异体位点对应到WT-Fc与FcYR型IIb复合物的结构时,显示在C334之缀合可以直接干扰对FcYRIIb的结合(图28C)。此由突变或缀合引起之在CH2定位时的不均匀性可以导致FcRIIb结合明显减少。因此,这些结果表明构象不均匀性或0101缀合至在IgG1-Fc内的工程化之半胱氨酸的某些组合物或可能二者,可以影响含有K334位点之双半胱氨酸变异体的ADCC活性。
实施例20:不同缀合位点导致不同的ADC性质
A.用于合成半胱氨酸突变之ADC的通用步骤
混入一或多个工程化之半胱氨酸残基的曲妥单抗溶液(如表22中所示)是在50mM磷酸盐缓冲液(pH 7.4)中制得。加入PBS、EDTA(0.5M原液)、与TCEP(0.5M原液),使得最终蛋白质浓度是10mg/mL,最终EDTA浓度是20mM,及最终TCEP浓度是约6.6mM(100摩尔当量)。使反应在室温下静置48个小时,然后使用GE PD-10Sephadex G25管柱根据制造商指示把缓冲液交换成PBS。用50当量之脱氢抗坏血酸(在1:1EtOH/水中的50mM原液)处理所产生之溶液。使抗体在4℃下静置过夜,接着使用GE PD-10Sephadex G25管柱根据制造商指示把缓冲液交换成PBS。对某些突变体使用上述步骤之轻微的变型。
将所制得之抗体在含有10%(v/v)DMA的PBS中稀释成~2.5mg/mL,及用vc0101(10摩尔当量)处理成为在DMA中的10mM原液。在室温下经过2小时后,把缓冲液交换成PBS(如上述)及利用粒径排阻层析术在Superdex200管柱上纯化。将单体馏份浓缩及用过滤方法灭菌,而产生最终ADC。参见下表22的产物特性分析。
表22:ADC性质总结
B.用于缀合实施例之通用分析方法
LCMS:管柱=Waters BEH300-C4,2.1 x 100mm(P/N=186004496);仪器=AcquityUPLC(具SQD2质谱检测器);流率=0.7mL/min;温度=80℃;缓冲液A=水+0.1%甲酸;缓冲液B=乙腈+0.1%甲酸。梯度:在2分钟内从3%B进行至95%B,在95%B下保持0.75分钟,然后在3%B下再平衡。在即将注射前用TCEP或DTT把样品还原。利用LCMS(400至2000道尔顿)检测洗出液,及使用MaxEnt1把蛋白质峰反折积。DAR为重量平均载药量。
SEC:管柱=Superdex200(5/150GL);移动相:磷酸盐缓冲液(含有2%乙腈)(pH7.4);流率=0.25mL/min;温度=环境温度;仪器=Agilent 1100 HPLC。
HIC:管柱=TSKGel Butyl NPR,4.6mm x 3.5cm(P/N=S0557-835);缓冲液A=1.5M硫酸铵(含有10mM磷酸盐)(pH 7);缓冲液B=10mM磷酸盐(pH 7)+20%异丙醇;流率=0.8mL/min;温度=环境温度;梯度=在12分钟内从0%B进行至100%B,在100%B下保持2分钟,然后在100%A下再平衡;仪器:Agilent 1100 HPLC。
C.位点特异性vc0101缀合物之疏水性的测定
利用疏水性交互作用层析术(上述方法)评估表22之ADC,以测定各种缀合物的相对疏水性。ADC疏水性已被报告和总抗体暴露量有关系。
和未修饰之抗体相比,第334、375、与392位点缀合物显出最小的滞留时间偏移,而第421、443、与347位点缀合物显出最大的滞留时间偏移。将ADC之滞留时间除以未修饰之抗体的滞留时间计算出各ADC之相对疏水性,从而求出“相对滞留时间”或“RRT”。~1的RRT指示ADC具有和未修饰之抗体大约相同的疏水性。每个ADC的RRT显示在表22中。
D.位点特异性vc0101缀合物的ADC之血浆中稳定性
将ADC样品(~1.5mg/mL)在小鼠、大鼠或人血浆中稀释而产生在血浆中50μg/mLADC的最终溶液。把样品在37℃下于5%CO2下培养,在三个时间点(0、24小时与72小时)采取等分样品。将各时间点之来自血浆培养的ADC样品(25μL)在37℃下用IgG0去糖基化1个小时。在去糖基化后,加入捕获抗体(用于小鼠与大鼠血浆的1mg/mL生物素化之山羊抗人IgG1Fcγ特异性片段,或用于人血浆的1mg/mL生物素化之抗曲妥单抗抗体)及在37℃下加热混合物1个小时,接着在室温下轻轻地搅拌1个小时。将Dynabead MyOne Streptavidin T1磁珠加到样品中及在室温下轻轻地搅拌下培育1个小时。然后用200μL PBS+0.05%Tween-20,200μL PBS与HPLC等级水清洗试样板。用55μL之2%(v/v)甲酸(FA)洗提结合的ADC。把50μL之各等分样品移到新板中,接着加入5μL的200mM TCEP。
用外加nanoAcquity UPLC(Waters)(使用BEH300C4,1.7μm,0.3×100mm iKey管柱)之Xevo G2 Q-TOF质谱仪来进行完整蛋白质分析。移动相A(MPA)由0.1%(v/v)FA之水溶液组成,及移动相B(MPB)由0.1%(v/v)FA之乙腈溶液组成。使用从5%至90%的MPB之线性梯度在流率0.3μL/分钟下洗提,在7分钟内达到层析分离。把LC管柱温度设定于85℃。用MassLynx software version 4.1进行数据获取。质量获取范围从700Da至2400Da。使用Biopharmalynx version 1.33进行包括反折积的数据分析。
随着时间检测载药与丁二酰亚胺开环(一个+18道尔顿峰)。载药数据为和0小时DAR相比的%DAR丧失。开环数据为和在72小时时存在之全部物种相比的已开环物种的%。几个位点突变体导致很稳定之ADC(334C、421C、与443C),而某些位点丧失大量的连接子-载荷物(380C与114C)。开环率在位点间有相当大之不同。几个位点(例如392C、183C、与334C)导致很少的开环,而其它位点(例如421C、388C、与347C)导致快速且自发之开环。
导致快速且自发之开环的位点可能可用于产生具有降低的疏水性与/或增加之PK暴露量的缀合物。此一发现和环之稳定性与血浆中稳定性相互关联之普遍理解相反。因此在某些方面中,当使用具高疏水性的连接子-载荷物时在位点421C、388C、与347C中之一或多者上的缀合会特别有优势。在某些方面中,高疏水性是1.5或更多之相对滞留时间(RRT)值(利用HIC测得)。在某些方面中,高疏水性是1.7或更多的RRT值。在某些方面中,高疏水性是1.8或更多之RRT值。在某些方面中,高疏水性是1.9或更多的RRT值。在某些方面中,高疏水性是2.0或更多之RRT值。
表23:各种ADC的血浆稳定性
ADC | DAR损失%@72-h | 琥珀酰胺水解%@72-h |
T(K334C)-vc0101 | 0% | 18% |
T(N421C)-vc0101 | 0% | 100 |
T(L443C)-vc0101 | 0% | 40% |
T(K388C)-vc0101 | -1.3% | 100% |
T(K392C)-vc0101 | 3.0% | 0% |
T(K290C)-vc0101 | 9.5% | 21% |
T(Q347C)-vc0101 | 10% | 66% |
T(κK183C)-vc0101 | 11% | 29% |
T(S375C)-vc0101 | 12% | 46% |
T(A114C)-vc0101 | 20% | 33% |
T(E380C)-vc0101 | 49% | 29% |
E.位点特异性vc0101缀合物之谷胱甘肽中稳定性
将ADC样品在谷胱甘肽水溶液中稀释而产生在磷酸盐缓冲液(pH 7.4)中0.5mM之最终GSH浓度与~0.1mg/mL的最终蛋白质浓度。然后把样品在37℃下培养及在三个时间点取出等分来测定DAR(T-0、T-3日、T-6日)。将各时间点之等分样品用TCEP处理及利用LC-MS根据实施例20.A中所述之方法来分析。
随着时间检测载药与丁二酰亚胺开环(一个+18道尔顿峰)。载药数据为和0小时DAR相比的%DAR丧失(表24)。开环数据为和在72小时时存在之全部物种相比之已开环物种的%。几个位点突变体导致很稳定之ADC(334C、421C、与443C),而某些位点丧失大量的连接子-载荷物(380C与114C)。开环率在位点间有相当大之不同。几个位点(例如392C、183C、与334C)导致很少的开环,而其它位点(例如421C、388C、与347C)导致大量开环。此检定之结果和血浆中稳定性结果(实施例20.D)十分相互关联,表明硫醇媒介的早期解离是血浆中载荷物损失的主要途径。结合来说,这些结果表明特定位点(例如334、443、290、与392)可能特别地可用于通过硫醇媒介之早期解离而容易丧失的载荷物-连接子的缀合。这样之载荷物-连接子包括使用常见的mc与vc键联(例如vc-101、vc-MMAE、mc-MMAF等)之载荷物-连接子。
表24:各种vc0101位点特异性缀合物的谷胱甘肽稳定性
ADC | DAR损失%@72-h | 琥珀酰胺水解%@72-h |
T(A114C)-vc0101 | 12% | 41% |
T(κK183C)-vc0101 | 7% | 17% |
T(K334C)-vc0101 | 4% | 26% |
T(Q347C)-vc0101 | 10% | 71% |
T(S375C)-vc0101 | 18% | 47% |
T(E380C)-vc0101 | 79% | 50% |
T(K388C)-vc0101 | 19% | 100% |
T(K392C)-vc0101 | 0% | 17% |
T(N421C)-vc0101 | 0% | 80% |
T(L443C)-vc0101 | 12% | 41% |
T(K290C)-vc0101 | 17% | 33% |
F.小鼠中选择位点特异性vc0101缀合物之药代动力学评价
由Charles River Laboratories取得不带肿瘤之母无胸腺(裸)小鼠(6至8周龄)。所有使用到小鼠的步骤都是由Institutional Animal Care and Use Committee根据明确指导方针而核准。根据抗体成分,对小鼠(n=3或4)静脉内施用一次3mg/kg之ADC。在投药后的0.083、6、24、48、96、168与336小时通过尾静脉对每只小鼠采集血液样品。利用LBA测得总抗体(Tab)与ADC浓度,其中把绵羊抗人IgG抗体用于捕获,将山羊抗人IgG抗体用于检测Tab,或把抗载荷物抗体用于检测ADC。使用Watson LIMS version 7.4(Thermo)分析每只小鼠之血浆中浓度数据。暴露量根据位点而不同。用290C与443C突变体制成的ADC显出最低暴露量,而用183C与392C位点制成之ADC显出最高暴露量。就许多应用而言,具高暴露量的位点可能较佳,由于这将导致治疗剂持续时间增加。然而,就某些应用而言,较佳为使用具低暴露量的缀合物(例如290C与443C)。特别地,较低暴露量(即,较低PK)之应用可能包括但不限于用于脑、CNS、及眼。适应症包括癌,尤其是脑癌、CNS癌、及眼癌。
表25:各种位点特异性vc0101 ADC的PK暴露
ADC | tAb AUC(0-最后)(mg*h/mL) | ADC AUC(0-最后)(mg*h/mL) |
T(κK183C)-vc0101 | 7150 | 5980 |
T(K290C)-vc0101 | 4240 | 3480 |
T(K334C)-vc0101 | 5130 | 4500 |
T(Q347C)-vc0101 | 5080 | 4070 |
T(K388C)-vc0101 | 6100 | 3680 |
T(K392C)-vc0101 | 6400 | 6010 |
T(L443C)-vc0101 | 4430 | 4500 |
G.位点特异性vc0101缀合物之组织蛋白酶切割
在37℃下使用6mM二硫苏糖醇(DTT)之150mM乙酸钠溶液(pH 5.2)把组织蛋白酶B活化15分钟。然后将50ng活化的组织蛋白酶B与20uL之1mg/mL的ADC在2mM DTT、50mM乙酸钠(pH 5.2)之最终浓度下混合。在37℃下培育20分钟、1个小时、2个小时与4个小时后,使用10uM E-64半胱氨酸蛋白酶抑制剂之250mM硼酸盐缓冲液(pH 8.5)终止反应。在检定后,使用TCEP将样品还原及利用LC/MS使用实施例21.A中所述之条件来分析。数据显示连接子切割速率大大地取决于缀合的位点。特定位点很快地被切割(例如443C、388C、与290C),而其它位点很慢地被切割(例如334C、375C、与392C)。在某些方面中,缀合至提供自身来使切割变慢之位点可能有利。在其它方面中,较佳为快速切割。例如,可能较佳为快速释放载荷物来降低花费在核内体的时间。在另外的方面中,快速载荷物切割能有利于使载荷物能穿透,其中缀合之分子可能不能这样做,例如某些实体瘤。在另外的方面中,快速切割能使药物能输送至没表达抗体之抗原的相邻细胞,从而能治疗(例如)异源性肿瘤。
表26:各种位点特异性vc0101ADC的连接子切割动力学
H.位点特异性vc0101缀合物之热稳定性
将ADC在含有10mM EDTA之PBS(pH 7.4)中稀释成0.2mg/mL。把ADC放入密封的管形瓶中及加热到45℃。每增加1周取出等分样品(10μL)利用粒径排阻层析术(SEC)来评估随着时间而形成之高分子量物种(HMWS)与低分子量物种(LMWS)的水平。SEC条件在实施例21.A中概述。在这些条件下,在约3.6分钟洗提出单体。洗提到单体峰左侧的任何蛋白质材料被视为HMWS,及洗提到单体峰右侧之任何蛋白质材料被视为LMWS。结果参见下表27。选定ADC显示极高的热稳定性(例如183C、375C、与334C),而其它ADC显示显著的分解(例如443C与392C+443C)。
表27:各种位点特异性vc0101 ADC的热稳定性
I.各种vc0101位点突变体之效力
在使用N87细胞株之靶表达性异种移植物模型中进行抗体药物缀合物的体内效力研究。把在50%基质胶中的约750万个肿瘤细胞植入6至8周龄的裸小鼠皮下到肿瘤大小达到在250与350mm3间为止。通过尾静脉推注来投药。每4日(在第1、5、9、与13日)一次为小鼠注射10、3、或1mg/kg之抗体药物缀合物,共注射4次。每周为所有小鼠检测体重变化。在前50日每周2次而之后每周1次用卡尺测量肿瘤大小,并且用下式计算:肿瘤体积=(长度x宽度2)/2。在肿瘤体积达到2500mm3前将小鼠人道处死。通常在治疗一周后观测到肿瘤大小减少。在中止治疗后持续为小鼠检测肿瘤复发(到治疗后100日为止)。来自3mpk投药组之数据参见图29。由388C与347C突变体产生的ADC显出比由334C、183C、392C与443C突变体产生之ADC稍低的效价。
序列表
<110> 辉瑞公司
<120> 位点特异性HER2抗体药物缀合物
<130> PC72091
<150> US 62/260,854
<151> 2015-11-30
<150> US 62/289,744
<151> 2016-02-01
<150> US 62/409,105
<151> 2016-10-17
<150> US 62/289,727
<151> 2016-02-01
<160> 95
<170> PatentIn version 3.5
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
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Asp Thr Tyr Ile His
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<212> PRT
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Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
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<212> PRT
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Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
1 5 10
<210> 5
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<223> Synthetic peptide sequence
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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly
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<212> PRT
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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 Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
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<212> PRT
<213> Artificial Sequence
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<223> Synthetic peptide sequence
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Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala
1 5 10
<210> 9
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
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Ser Ala Ser Phe Leu Tyr Ser
1 5
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<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
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Gln Gln His Tyr Thr Thr Pro Pro Thr
1 5
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Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
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Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
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<212> PRT
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<223> Synthetic peptide sequence
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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 Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val 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
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Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
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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
210
<210> 13
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<212> PRT
<213> Artificial Sequence
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<223> Synthetic peptide sequence
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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Arg Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 14
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Arg Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 15
<211> 329
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 15
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Cys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
<210> 16
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 16
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Cys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly
<210> 17
<211> 329
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 17
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Cys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
<210> 18
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 18
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Cys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly
<210> 19
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 19
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 20
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 20
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 21
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 21
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 22
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 22
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 23
<211> 329
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 23
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Cys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
<210> 24
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 24
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Cys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly
<210> 25
<211> 329
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 25
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Cys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
<210> 26
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 26
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Cys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly
<210> 27
<211> 329
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 27
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Cys Ser Pro Gly
325
<210> 28
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 28
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Cys Ser Pro
435 440 445
Gly
<210> 29
<211> 329
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 29
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Cys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Cys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
<210> 30
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 30
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Cys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Cys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly
<210> 31
<211> 329
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 31
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Cys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Cys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
<210> 32
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 32
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Cys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Cys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly
<210> 33
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 33
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Arg Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 34
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 34
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Arg Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 35
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 35
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Arg Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 36
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 36
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Arg Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 37
<211> 329
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 37
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Cys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Cys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
<210> 38
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 38
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Cys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Cys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly
<210> 39
<211> 329
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 39
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Cys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Cys Ser Pro Gly
325
<210> 40
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 40
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Cys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Cys Ser Pro
435 440 445
Gly
<210> 41
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 41
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Cys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 42
<211> 213
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 42
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 Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Cys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys
210
<210> 43
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 43
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Leu Leu Gln
100 105 110
Gly Pro Pro
115
<210> 44
<211> 222
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 44
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 Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val 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 Gly Gly Leu Leu Gln Gly Pro Pro
210 215 220
<210> 45
<211> 360
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 45
gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg 60
tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca 120
cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta caccagatac 180
gccgactccg tgaagggccg gttcaccatc tccgccgaca cctccaagaa caccgcctac 240
ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgctc cagatgggga 300
ggcgacggct tctacgccat ggactactgg ggccagggca ccctggtcac cgtgtctagc 360
<210> 46
<211> 1347
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 46
gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg 60
tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca 120
cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta caccagatac 180
gccgactccg tgaagggccg gttcaccatc tccgccgaca cctccaagaa caccgcctac 240
ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgctc cagatgggga 300
ggcgacggct tctacgccat ggactactgg ggccagggca ccctggtcac cgtgtctagc 360
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc cccgggt 1347
<210> 47
<211> 321
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 47
gacatccaga tgacccagtc cccctccagc ctgtccgcct ctgtgggcga cagagtgacc 60
atcacctgtc gggcctccca ggacgtgaac accgccgtgg cctggtatca gcagaagccc 120
ggcaaggccc ccaagctgct gatctactcc gcctccttcc tgtactccgg cgtgccctcc 180
cggttctccg gctccagatc tggcaccgac tttaccctga ccatctccag cctgcagccc 240
gaggacttcg ccacctacta ctgccagcag cactacacca ccccccccac ctttggccag 300
ggcaccaagg tggaaatcaa g 321
<210> 48
<211> 642
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 48
gacatccaga tgacccagtc cccctccagc ctgtccgcct ctgtgggcga cagagtgacc 60
atcacctgtc gggcctccca ggacgtgaac accgccgtgg cctggtatca gcagaagccc 120
ggcaaggccc ccaagctgct gatctactcc gcctccttcc tgtactccgg cgtgccctcc 180
cggttctccg gctccagatc tggcaccgac tttaccctga ccatctccag cctgcagccc 240
gaggacttcg ccacctacta ctgccagcag cactacacca ccccccccac ctttggccag 300
ggcaccaagg tggaaatcaa gcggaccgtg gccgctccct ccgtgttcat cttcccaccc 360
tccgacgagc agctgaagtc cggcaccgcc tccgtcgtgt gcctgctgaa caacttctac 420
ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 480
gaatccgtca ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 540
ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600
ctgtccagcc ccgtgaccaa gtccttcaac cggggcgagt gc 642
<210> 49
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 49
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtagtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg accgtactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggaaaa 990
<210> 50
<211> 1350
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 50
gaggtgcagc tggtggagtc cggcggcggc ctggttcagc ccggcggatc actgaggctc 60
tcctgtgccg ccagcggctt caacatcaag gacacataca tccactgggt tcgccaggct 120
cctggcaagg gactggagtg ggtcgctagg atctacccca ccaatgggta caccaggtac 180
gccgactccg tgaaggggcg gttcacaatc tcagccgata ctagcaaaaa tacagcctac 240
ttgcagatga actccctgag agcagaggat accgccgtgt actattgctc tcgctggggc 300
ggcgacggct tctacgctat ggattattgg ggccagggaa ccttggtcac cgtctcctca 360
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtagtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg accgtactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc tccgggaaaa 1350
<210> 51
<211> 987
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 51
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccatgcccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc cccgggt 987
<210> 52
<211> 1347
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 52
gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg 60
tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca 120
cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta caccagatac 180
gccgactccg tgaagggccg gttcaccatc tccgccgaca cctccaagaa caccgcctac 240
ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgctc cagatgggga 300
ggcgacggct tctacgccat ggactactgg ggccagggca ccctggtcac cgtgtctagc 360
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccatgcccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc cccgggt 1347
<210> 53
<211> 987
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 53
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacatgcc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc cccgggt 987
<210> 54
<211> 1347
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 54
gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg 60
tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca 120
cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta caccagatac 180
gccgactccg tgaagggccg gttcaccatc tccgccgaca cctccaagaa caccgcctac 240
ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgctc cagatgggga 300
ggcgacggct tctacgccat ggactactgg ggccagggca ccctggtcac cgtgtctagc 360
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacatgcc cgcgggagga gcagtacaac 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc cccgggt 1347
<210> 55
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 55
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtagtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacgcc 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggaaaa 990
<210> 56
<211> 1350
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 56
gaggtgcagc tggtggagtc cggcggcggc ctggttcagc ccggcggatc actgaggctc 60
tcctgtgccg ccagcggctt caacatcaag gacacataca tccactgggt tcgccaggct 120
cctggcaagg gactggagtg ggtcgctagg atctacccca ccaatgggta caccaggtac 180
gccgactccg tgaaggggcg gttcacaatc tcagccgata ctagcaaaaa tacagcctac 240
ttgcagatga actccctgag agcagaggat accgccgtgt actattgctc tcgctggggc 300
ggcgacggct tctacgctat ggattattgg ggccagggaa ccttggtcac cgtctcctca 360
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtagtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacgcc 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc tccgggaaaa 1350
<210> 57
<211> 1055
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 57
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtagtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtaccaa 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggaaaa gccgccagcg gcttcaacat caaggacaca 1020
tacatccact gggttcgcca ggctcctggc aaggg 1055
<210> 58
<211> 1415
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 58
gaggtgcagc tggtggagtc cggcggcggc ctggttcagc ccggcggatc actgaggctc 60
tcctgtgccg ccagcggctt caacatcaag gacacataca tccactgggt tcgccaggct 120
cctggcaagg gactggagtg ggtcgctagg atctacccca ccaatgggta caccaggtac 180
gccgactccg tgaaggggcg gttcacaatc tcagccgata ctagcaaaaa tacagcctac 240
ttgcagatga actccctgag agcagaggat accgccgtgt actattgctc tcgctggggc 300
ggcgacggct tctacgctat ggattattgg ggccagggaa ccttggtcac cgtctcctca 360
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtagtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtaccaa 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc tccgggaaaa gccgccagcg gcttcaacat caaggacaca 1380
tacatccact gggttcgcca ggctcctggc aaggg 1415
<210> 59
<211> 987
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 59
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagtg caccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc cccgggt 987
<210> 60
<211> 1347
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 60
gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg 60
tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca 120
cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta caccagatac 180
gccgactccg tgaagggccg gttcaccatc tccgccgaca cctccaagaa caccgcctac 240
ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgctc cagatgggga 300
ggcgacggct tctacgccat ggactactgg ggccagggca ccctggtcac cgtgtctagc 360
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagtg caccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc cccgggt 1347
<210> 61
<211> 987
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 61
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact actgcaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc cccgggt 987
<210> 62
<211> 1347
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 62
gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg 60
tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca 120
cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta caccagatac 180
gccgactccg tgaagggccg gttcaccatc tccgccgaca cctccaagaa caccgcctac 240
ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgctc cagatgggga 300
ggcgacggct tctacgccat ggactactgg ggccagggca ccttggtcac cgtgtctagc 360
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact actgcaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc cccgggt 1347
<210> 63
<211> 987
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 63
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctcctgctc cccgggt 987
<210> 64
<211> 1347
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 64
gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg 60
tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca 120
cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta caccagatac 180
gccgactccg tgaagggccg gttcaccatc tccgccgaca cctccaagaa caccgcctac 240
ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgctc cagatgggga 300
ggcgacggct tctacgccat ggactactgg ggccagggca ccctggtcac cgtgtctagc 360
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctcctgctc cccgggt 1347
<210> 65
<211> 987
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 65
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacatgcc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagtg caccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc cccgggt 987
<210> 66
<211> 1347
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 66
gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg 60
tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca 120
cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta caccagatac 180
gccgactccg tgaagggccg gttcaccatc tccgccgaca cctccaagaa caccgcctac 240
ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgctc cagatgggga 300
ggcgacggct tctacgccat ggactactgg ggccagggca ccctggtcac cgtgtctagc 360
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacatgcc cgcgggagga gcagtacaac 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagtg caccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc cccgggt 1347
<210> 67
<211> 987
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 67
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacatgcc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact actgcaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc cccgggt 987
<210> 68
<211> 1347
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 68
gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg 60
tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca 120
cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta caccagatac 180
gccgactccg tgaagggccg gttcaccatc tccgccgaca cctccaagaa caccgcctac 240
ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgctc cagatgggga 300
ggcgacggct tctacgccat ggactactgg ggccagggca ccctggtcac cgtgtctagc 360
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacatgcc cgcgggagga gcagtacaac 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact actgcaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc cccgggt 1347
<210> 69
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 69
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtagtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg accgtactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacgcc 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggaaaa 990
<210> 70
<211> 1350
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 70
gaggtgcagc tggtggagtc cggcggcggc ctggttcagc ccggcggatc actgaggctc 60
tcctgtgccg ccagcggctt caacatcaag gacacataca tccactgggt tcgccaggct 120
cctggcaagg gactggagtg ggtcgctagg atctacccca ccaatgggta caccaggtac 180
gccgactccg tgaaggggcg gttcacaatc tcagccgata ctagcaaaaa tacagcctac 240
ttgcagatga actccctgag agcagaggat accgccgtgt actattgctc tcgctggggc 300
ggcgacggct tctacgctat ggattattgg ggccagggaa ccttggtcac cgtctcctca 360
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtagtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg accgtactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacgcc 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc tccgggaaaa 1350
<210> 71
<211> 1055
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 71
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtagtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg accgtactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtaccaa 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggaaaa gccgccagcg gcttcaacat caaggacaca 1020
tacatccact gggttcgcca ggctcctggc aaggg 1055
<210> 72
<211> 1415
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 72
gaggtgcagc tggtggagtc cggcggcggc ctggttcagc ccggcggatc actgaggctc 60
tcctgtgccg ccagcggctt caacatcaag gacacataca tccactgggt tcgccaggct 120
cctggcaagg gactggagtg ggtcgctagg atctacccca ccaatgggta caccaggtac 180
gccgactccg tgaaggggcg gttcacaatc tcagccgata ctagcaaaaa tacagcctac 240
ttgcagatga actccctgag agcagaggat accgccgtgt actattgctc tcgctggggc 300
ggcgacggct tctacgctat ggattattgg ggccagggaa ccttggtcac cgtctcctca 360
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtagtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg accgtactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtaccaa 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc tccgggaaaa gccgccagcg gcttcaacat caaggacaca 1380
tacatccact gggttcgcca ggctcctggc aaggg 1415
<210> 73
<211> 987
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 73
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagtg caccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact actgcaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc cccgggt 987
<210> 74
<211> 1347
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 74
gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg 60
tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca 120
cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta caccagatac 180
gccgactccg tgaagggccg gttcaccatc tccgccgaca cctccaagaa caccgcctac 240
ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgctc cagatgggga 300
ggcgacggct tctacgccat ggactactgg ggccagggca ccctggtcac cgtgtctagc 360
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagtg caccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact actgcaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc cccgggt 1347
<210> 75
<211> 987
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 75
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact actgcaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctcctgctc cccgggt 987
<210> 76
<211> 1347
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 76
gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg 60
tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca 120
cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta caccagatac 180
gccgactccg tgaagggccg gttcaccatc tccgccgaca cctccaagaa caccgcctac 240
ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgctc cagatgggga 300
ggcgacggct tctacgccat ggactactgg ggccagggca ccctggtcac cgtgtctagc 360
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact actgcaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctcctgctc cccgggt 1347
<210> 77
<211> 321
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 77
cggaccgtgg ccgctccctc cgtgttcatc ttcccaccct ccgacgagca gctgaagtcc 60
ggcaccgcct ccgtcgtgtg cctgctgaac aacttctacc cccgcgaggc caaggtgcag 120
tggaaggtgg acaacgccct gcagtccggc aactcccagg aatccgtcac cgagcaggac 180
tccaaggaca gcacctactc cctgtcctcc accctgaccc tgtcctgcgc cgactacgag 240
aagcacaagg tgtacgcctg cgaagtgacc caccagggcc tgtccagccc cgtgaccaag 300
tccttcaacc ggggcgagtg c 321
<210> 78
<211> 642
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 78
gacatccaga tgacccagtc cccctccagc ctgtccgcct ctgtgggcga cagagtgacc 60
atcacctgtc gggcctccca ggacgtgaac accgccgtgg cctggtatca gcagaagccc 120
ggcaaggccc ccaagctgct gatctactcc gcctccttcc tgtactccgg cgtgccctcc 180
cggttctccg gctccagatc tggcaccgac tttaccctga ccatctccag cctgcagccc 240
gaggacttcg ccacctacta ctgccagcag cactacacca ccccccccac ctttggccag 300
ggcaccaagg tggaaatcaa gcggaccgtg gccgctccct ccgtgttcat cttcccaccc 360
tccgacgagc agctgaagtc cggcaccgcc tccgtcgtgt gcctgctgaa caacttctac 420
ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 480
gaatccgtca ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 540
ctgtcctgcg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600
ctgtccagcc ccgtgaccaa gtccttcaac cggggcgagt gc 642
<210> 79
<211> 321
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 79
cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60
ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120
tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180
agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240
aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300
agcttcaaca ggggagagtg t 321
<210> 80
<211> 666
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic nucleotide sequence
<400> 80
gatatccaga tgacacagtc cccctccagc ctctccgcta gtgtcggaga tagagtgaca 60
attacatgtc gggcaagcca ggacgtcaat accgccgtgg cctggtatca gcagaagcca 120
ggaaaggccc caaaactcct gatctactcc gcctccttcc tgtactcagg ggtcccttca 180
cgcttctccg gttcccggag cggcaccgac ttcactctga ctatctcaag cttgcagccc 240
gaggacttcg ccacatacta ttgccagcag cactatacca ccccccctac cttcggtcag 300
ggaactaagg tggaaattaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360
tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gtggtggcct gcttcagggc 660
ccacca 666
<210> 81
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 81
Gly Gly Leu Leu Gln Gly Pro Pro
1 5
<210> 82
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 82
Gly Gly Leu Leu Gln Gly Gly
1 5
<210> 83
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 83
Leu Leu Gln Gly Ala
1 5
<210> 84
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 84
Gly Gly Leu Leu Gln Gly Ala
1 5
<210> 85
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 85
Leu Leu Gln Gly
1
<210> 86
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 86
Leu Leu Gln Gly Pro Gly
1 5
<210> 87
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 87
Leu Leu Gln Gly Pro Ala
1 5
<210> 88
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 88
Leu Leu Gln Gly Pro
1 5
<210> 89
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 89
Leu Leu Gln Pro
1
<210> 90
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 90
Leu Leu Gln Pro Gly Lys
1 5
<210> 91
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 91
Leu Leu Gln Gly Ala Pro Gly Lys
1 5
<210> 92
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 92
Leu Leu Gln Gly Ala Pro Gly
1 5
<210> 93
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<400> 93
Leu Leu Gln Gly Ala Pro
1 5
<210> 94
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa is G or P
<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa is A, G, P, or absent
<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa is A, G, K, P, or absent
<220>
<221> MISC_FEATURE
<222> (7)..(7)
<223> Xaa is G, K or absent
<220>
<221> MISC_FEATURE
<222> (8)..(8)
<223> Xaa is K or absent
<400> 94
Leu Leu Gln Xaa Xaa Xaa Xaa Xaa
1 5
<210> 95
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide sequence
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa is any naturally occurring amino acid
<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa is any naturally occurring amino acid or absent
<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa is any naturally occurring amino acid or absent
<220>
<221> MISC_FEATURE
<222> (7)..(7)
<223> Xaa is any naturally occurring amino acid or absent
<220>
<221> MISC_FEATURE
<222> (8)..(8)
<223> Xaa is any naturally occurring amino acid or absent
<400> 95
Leu Leu Gln Xaa Xaa Xaa Xaa Xaa
1 5
Claims (57)
1.一种如下式的抗体药物缀合物:
Ab-(L-D),
其中:
(a)Ab是结合至HER2的抗体及包含
(1)重链可变区,其包括含SEQ ID No:2、3和4的三个CDR;
(2)SEQ ID No:17、5、13、21、23、25、27、29、31、33、35、37或39中任一者的重链恒定区;
(3)轻链可变区,其包括含SEQ ID No:8、9和10的三个CDR;
(4)SEQ ID No:41、11或43中任一者的轻链恒定区;及
(b)L-D是连接子-药物部分,其中L是连接子,及D是药物,
条件是当所述重链恒定区是SEQ ID No:5时,所述轻链恒定区不是SEQ ID No:11。
2.如权利要求第1项的抗体药物缀合物,其中
(a)所述重链恒定区是SEQ ID No:17及所述轻链恒定区是SEQ ID No:41;
(b)所述重链恒定区是SEQ ID No:5及所述轻链恒定区是SEQ ID No:41;
(c)所述重链恒定区是SEQ ID No:17及所述轻链恒定区是SEQ ID No:11;
(d)所述重链恒定区是SEQ ID No:21及所述轻链恒定区是SEQ ID No:11;
(e)所述重链恒定区是SEQ ID No:23及所述轻链恒定区是SEQ ID No:11;
(f)所述重链恒定区是SEQ ID No:25及所述轻链恒定区是SEQ ID No:11;
(g)所述重链恒定区是SEQ ID No:27及所述轻链恒定区是SEQ ID No:11;
(h)所述重链恒定区是SEQ ID No:23及所述轻链恒定区是SEQ ID No:41;
(i)所述重链恒定区是SEQ ID No:25及所述轻链恒定区是SEQ ID No:41;
(j)所述重链恒定区是SEQ ID No:27及所述轻链恒定区是SEQ ID No:41;
(k)所述重链恒定区是SEQ ID No:29及所述轻链恒定区是SEQ ID No:11;
(l)所述重链恒定区是SEQ ID No:31及所述轻链恒定区是SEQ ID No:11;
(m)所述重链恒定区是SEQ ID No:33及所述轻链恒定区是SEQ ID No:43;
(n)所述重链恒定区是SEQ ID No:35及所述轻链恒定区是SEQ ID No:11;
(o)所述重链恒定区是SEQ ID No:37及所述轻链恒定区是SEQ ID No:11;
(p)所述重链恒定区是SEQ ID No:39及所述轻链恒定区是SEQ ID No:11;或
(q)所述重链恒定区是SEQ ID No:13及所述轻链恒定区是SEQ ID No:43。
3.如权利要求第1项的抗体药物缀合物,其中
(a)所述重链包含SEQ ID No:18、6、14、22、24、26、28、30、32、34、36、38或40中任一者;及
(b)所述轻链包含SEQ ID No:42、12或44中任一者,
条件是当所述重链是SEQ ID No:6时,所述轻链不是SEQ ID No:12。
4.如权利要求第3项的抗体药物缀合物,其中
(a)所述重链是SEQ ID No:18及所述轻链是SEQ ID No:42;
(b)所述重链是SEQ ID No:6及所述轻链是SEQ ID No:42;
(c)所述重链是SEQ ID No:18及所述轻链是SEQ ID No:12;
(d)所述重链是SEQ ID No:22及所述轻链是SEQ ID No:12;
(e)所述重链是SEQ ID No:24及所述轻链是SEQ ID No:12;
(f)所述重链是SEQ ID No:26及所述轻链是SEQ ID No:12;
(g)所述重链是SEQ ID No:28及所述轻链是SEQ ID No:12;
(h)所述重链是SEQ ID No:24及所述轻链是SEQ ID No:42;
(i)所述重链是SEQ ID No:26及所述轻链是SEQ ID No:42;
(j)所述重链是SEQ ID No:28及所述轻链是SEQ ID No:42;
(k)所述重链是SEQ ID No:30及所述轻链是SEQ ID No:12;
(l)所述重链是SEQ ID No:32及所述轻链是SEQ ID No:12;
(m)所述重链是SEQ ID No:34及所述轻链是SEQ ID No:44;
(n)所述重链是SEQ ID No:36及所述轻链是SEQ ID No:12;
(o)所述重链是SEQ ID No:38及所述轻链是SEQ ID No:12;
(p)所述重链是SEQ ID No:40及所述轻链是SEQ ID No:12;或
(q)所述重链是SEQ ID No:14及所述轻链是SEQ ID No:44。
5.如权利要求第1至4项中任一项的抗体药物缀合物,其中所述连接子系选自由下列所组成的群组:vc、AcLysvc、mc、MalPeg6、m(H20)c及m(H20)cvc。
6.如权利要求第5项的抗体药物缀合物,其中所述连接子是可切割的。
7.如权利要求第6项的抗体药物缀合物,其中所述连接子是vc或AcLysvc。
8.如权利要求第1至7项中任一项的抗体药物缀合物,其中所述药物是可透膜的。
9.如权利要求第1至8项中任一项的抗体药物缀合物,其中所述药物是耳抑素。
10.如权利要求第1至9项中任一项的抗体药物缀合物,其中所述耳抑素选自由下列所组成的群组:
2-甲基丙氨酰基-N-[(3R,4S,5S)-3-甲氧基-1-{(2S)-2-[(1R,2R)-1-甲氧基-2-甲基-3-氧代-3-{[(1S)-2-苯基-1-(1,3-噻唑-2-基)乙基]氨基}丙基]吡咯烷-1-基}-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺;
2-甲基丙氨酰基-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-羧基-2-苯基乙基]氨基}-1-甲氧基-2-甲基-3-氧代丙基]吡咯烷-1-基}-3-甲氧基-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺;
2-甲基-L-脯氨酰基-N-[(3R,4S,5S)-3-甲氧基-1-{(2S)-2-[(1R,2R)-1-甲氧基-3-{[(2S)-1-甲氧基-1-氧代-3-苯基丙-2-基]氨基}-2-甲基-3-氧代丙基]吡咯烷-1-基}-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺,三氟乙酸盐;
2-甲基丙氨酰基-N-[(3R,4S,5S)-3-甲氧基-1-{(2S)-2-[(1R,2R)-1-甲氧基-3-{[(2S)-1-甲氧基-1-氧代-3-苯基丙-2-基]氨基}-2-甲基-3-氧代丙基]吡咯烷-1-基}-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺;
2-甲基丙氨酰基-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-羟基-1-苯基丙-2-基]氨基}-1-甲氧基-2-甲基-3-氧代丙基]吡咯烷-1-基}-3-甲氧基-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺;
2-甲基-L-脯氨酰基-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-羧基-2-苯基乙基]氨基}-1-甲氧基-2-甲基-3-氧代丙基]吡咯烷-1-基}-3-甲氧基-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺,三氟乙酸盐;
N-甲基-L-缬氨酰基-N-[(3R,4S,5S)-3-甲氧基-1-{(2S)-2-[(1R,2R)-1-甲氧基-2-甲基-3-氧代-3-{[(1S)-2-苯基-1-(1,3-噻唑-2-基)乙基]氨基}丙基]吡咯烷-1-基}-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺;
N-甲基-L-缬氨酰基-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-羟基-1-苯基丙-2-基]氨基}-1-甲氧基-2-甲基-3-氧代丙基]吡咯烷-1-基}-3-甲氧基-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺;及
N-甲基-L-缬氨酰基-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-羧基-2-苯基乙基]氨基}-1-甲氧基-2-甲基-3-氧代丙基]吡咯烷-1-基}-3-甲氧基-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺,
或其药学上可接受的盐或溶剂化物。
11.如权利要求第10项的抗体药物缀合物,其中所述耳抑素是2-甲基丙氨酰基-N-[(3R,4S,5S)-3-甲氧基-1-{(2S)-2-[(1R,2R)-1-甲氧基-2-甲基-3-氧代-3-{[(1S)-2-苯基-1-(1,3-噻唑-2-基)乙基]氨基}丙基]吡咯烷-1-基}-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺或其药学上可接受的盐或溶剂化物。
12.一种如下式的抗体药物缀合物:
Ab-(L-D),
其中:
(a)Ab是结合至HER2的抗体及包括含有SEQ ID No:18的重链及含有SEQ ID No:42的轻链;及
(b)L-D是连接子-药物部分,其中L是vc连接子及D是耳抑素2-甲基丙氨酰基-N-[(3R,4S,5S)-3-甲氧基-1-{(2S)-2-[(1R,2R)-1-甲氧基-2-甲基-3-氧代-3-{[(1S)-2-苯基-1-(1,3-噻唑-2-基)乙基]氨基}丙基]吡咯烷-1-基}-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺或其药学上可接受的盐或溶剂化物。
13.一种如下式的抗体药物缀合物:
Ab-(L-D),
其中:
(a)Ab是结合至HER2的抗体及包括含有SEQ ID No:14的重链及含有SEQ ID No:44的轻链;及
(b)L-D是连接子-药物部分,其中L是AcLysvc连接子及D是耳抑素2-甲基丙氨酰基-N-[(3R,4S,5S)-3-甲氧基-1-{(2S)-2-[(1R,2R)-1-甲氧基-2-甲基-3-氧代-3-{[(1S)-2-苯基-1-(1,3-噻唑-2-基)乙基]氨基}丙基]吡咯烷-1-基}-5-甲基-1-氧代庚-4-基]-N-甲基-L-缬氨酰胺或其药学上可接受的盐或溶剂化物。
14.一种药学组合物,其包含如权利要求第1至13项中任一项的抗体药物缀合物及药学上可接受的载剂。
15.一种组合物,其包含多个如权利要求第1至12项中任一项的抗体药物缀合物,及可选存在地药学载剂,其中所述组合物具有4的DAR。
16.一种组合物,其包含多个如权利要求第1至11与13项中任一项的抗体药物缀合物,及可选存在地药学载剂,其中所述组合物具有2的DAR。
17.一种核酸,其编码如权利要求第1至4项中任一项的Ab的重链或轻链。
18.一种核酸,其为编码重链的SEQ ID No:54、46、50、54、58、60、62、64、60、62、64、66、68、70、72、74或76中任一者,或为编码轻链的SEQ ID No:78、48或80中任一者,条件是当SEQID No:46编码所述重链时,所述轻链不被SEQ ID No:48编码。
19.一种编码抗体的重链或抗体的轻链的核酸,其中
(a)SEQ ID No:54编码所述重链及SEQ ID No:78编码所述轻链;
(b)SEQ ID No:46编码所述重链及SEQ ID No:78编码所述轻链;
(c)SEQ ID No:54编码所述重链及SEQ ID No:48编码所述轻链;
(d)SEQ ID No:58编码所述重链及SEQ ID No:48编码所述轻链;
(e)SEQ ID No:60编码所述重链及SEQ ID No:48编码所述轻链;
(f)SEQ ID No:62编码所述重链及SEQ ID No:48编码所述轻链;
(g)SEQ ID No:64编码所述重链及SEQ ID No:78编码所述轻链;
(h)SEQ ID No:60编码所述重链及SEQ ID No:78编码所述轻链;
(i)SEQ ID No:62编码所述重链及SEQ ID No:78编码所述轻链;
(j)SEQ ID No:64编码所述重链及SEQ ID No:78编码所述轻链;
(k)SEQ ID No:66编码所述重链及SEQ ID No:48编码所述轻链;
(l)SEQ ID No:68编码所述重链及SEQ ID No:48编码所述轻链;
(m)SEQ ID No:70编码所述重链及SEQ ID No:80编码所述轻链;
(n)SEQ ID No:72编码所述重链及SEQ ID No:48编码所述轻链;
(o)SEQ ID No:74编码所述重链及SEQ ID No:48编码所述轻链;
(p)SEQ ID No:76编码所述重链及SEQ ID No:48编码所述轻链;或
(q)SEQ ID No:50编码所述重链及SEQ ID No:80编码所述轻链。
20.一种载体,其包含如权利要求第17至19项中任一项的核酸。
21.一种宿主细胞,其包含如权利要求第17至19项中任一项的核酸。
22.一种制造如权利要求第1至16项中任一项的抗体药物缀合物的方法,所述方法包含:
(a)将所述连接子连接至所述药物;
(b)将所述连接子药物部分缀合至所述抗体;及
(c)纯化所述抗体药物缀合物。
23.如权利要求第22项的方法,其中所述缀合对所述抗体上的一或多个工程化的半胱氨酸和/或谷氨酰胺残基具有位点特异性。
24.一种治疗HER2表达性癌的方法,其包含对有其需要的个体施用治疗有效量的包含如权利要求第1至13项中任一项的抗体药物缀合物的组合物。
25.如权利要求第24项的方法,其中所述癌是实体瘤。
26.如权利要求第25项的方法,其中所述实体瘤选自由下列所组成的群组:乳癌、卵巢癌、肺癌、胃癌、食道癌、结肠直肠癌、尿路上皮癌、胰腺癌、唾液腺癌及脑癌。
27.如权利要求第26项的方法,其中所述实体瘤系选自由下列所组成的群组:乳癌、卵巢癌、肺癌及胃癌。
28.如权利要求第27项的方法,其中所述乳癌是雌激素与黄体激素受体阴性或三阴性乳癌(TNBC)。
29.如权利要求第27项的方法,其中所述肺癌是非小细胞肺癌(NSLC)。
30.如权利要求第24至29项中任一项的方法,其中所述个体事先已接受过曲妥单抗和/或曲妥单抗emtansine单独治疗或合并另一治疗剂治疗。
31.如权利要求第30项的方法,其中所述治疗剂是紫杉烷。
32.如权利要求第30项的方法,其中所述癌对用曲妥单抗和/或曲妥单抗emtansine单独或合并另一治疗剂治疗有抗性、是所述治疗难治的、和/或在所述治疗后复发。
33.如权利要求第32项的方法,其中所述治疗剂是紫杉烷。
34.如权利要求第24至33项中任一项的方法,其中所述癌表达高水平的HER2。
35.如权利要求第34项的方法,其中所述癌表达如免疫组织化学法(IHC)所测得的3+水平和/或荧光原位杂交(FISH)放大率≥2.0的HER2。
36.如权利要求第24至33项中任一项的方法,其中所述癌表达中度水平的HER2。
37.如权利要求第36项的方法,其中所述癌表达如免疫组织化学法(IHC)所测得的2+水平和/或荧光原位杂交(FISH)放大率<2.0的HER2。
38.如权利要求第24至33项中任一项的方法,其中所述癌表达低水平的HER2。
39.如权利要求第38项的方法,其中所述癌表达如免疫组织化学法(IHC)所测得的1+水平和/或荧光原位杂交(FISH)放大率<2.0的HER2。
40.如权利要求第35、37与39项中任一项的方法,其中使用Dako HercptestTM检定进行IHC,及使用Dako HER2 FISH Pharm DxTM检定进行FISH。
41.一种如权利要求第1至13项中任一项的抗体药物缀合物在制备用于治疗个体中HER2表达性癌的医药品中的用途。
42.如权利要求第41项的用途,其中所述癌是实体瘤。
43.如权利要求第42项的用途,其中所述实体瘤选自由下列所组成的群组:乳癌、卵巢癌、肺癌、胃癌、食道癌、结肠直肠癌、尿路上皮癌、胰腺癌、唾液腺癌及脑癌。
44.如权利要求第43项的用途,其中所述实体瘤选自由下列所组成的群组:乳癌、卵巢癌、肺癌及胃癌。
45.如权利要求第44项的用途,其中所述乳癌是雌激素与黄体激素受体阴性或三阴性乳癌(TNBC)。
46.如权利要求第44项的用途,其中所述肺癌是非小细胞肺癌(NSLC)。
47.如权利要求第42至46项中任一项的用途,其中所述个体事先已接受过曲妥单抗和/或曲妥单抗emtansine单独治疗或合并另一治疗剂治疗。
48.如权利要求第47项的用途,其中所述治疗剂是紫杉烷。
49.如权利要求第47项的用途,其中所述癌对用曲妥单抗和/或曲妥单抗emtansine单独治疗或合并另一治疗剂的治疗是有抗性的、是所述治疗难治的、和/或在所述治疗后复发。
50.如权利要求第49项的用途,其中所述治疗剂是紫杉烷。
51.如权利要求第42至50项中任一项的用途,其中所述癌表达高水平的HER2。
52.如权利要求第51项的用途,其中所述癌表达如免疫组织化学法(IHC)所测得的3+水平和/或荧光原位杂交(FISH)放大率≥2.0的HER2。
53.如权利要求第42至50项中任一项的用途,其中所述癌表达中度水平的HER2。
54.如权利要求第53项的用途,其中所述癌表达如免疫组织化学法(IHC)所测得的2+水平和/或荧光原位杂交(FISH)放大率<2.0的HER2。
55.如权利要求第42至50项中任一项的用途,其中所述癌表达低水平的HER2。
56.如权利要求第55项的用途,其中所述癌表达如免疫组织化学法(IHC)所测得的1+水平和/或荧光原位杂交(FISH)放大率<2.0的HER2。
57.如权利要求第52、54与56项中任一项的用途,其中使用Dako HercptestTM检定进行IHC,及使用Dako HER2 FISH Pharm DxTM检定进行FISH。
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