CN101849006A - 改良的抗体分子 - Google Patents
改良的抗体分子 Download PDFInfo
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Abstract
本发明提供包含优于TOCILIZUMAB的第2代分子的药物组合物以及上述药物组合物的制备方法,所述优于TOCILIZUMAB的第2代分子是指通过改变人源化抗IL-6受体IgG1抗体、即TOCILIZUMAB的可变区和恒定区的氨基酸序列,使抗原中和能力增强、同时提高药物动力学,从而减少给药频率、持续发挥治疗效果,并且使免疫原性、安全性、理化性质(稳定性和均匀性)得到改善。
Description
技术领域
本发明涉及含有抗IL-6受体抗体作为有效成分的药物组合物及其制备方法等。
背景技术
抗体在血浆中的稳定性高、副作用也少,因此作为药品而受到关注。其中,有多种IgG型抗体药物已上市,目前还有多种抗体药物正在开发中(非专利文献1、非专利文献2)。IL-6是与各种自身免疫疾病、炎症性疾病、恶性肿瘤等有关的细胞因子(非专利文献3),作为人源化抗IL-6受体IgG1抗体的TOCILIZUMAB(トシリズマブ)与IL-6受体进行特异性结合。由于TOCILIZUMAB中和IL-6的生物学作用,因此认为TOCILIZUMAB可以用作类风湿性关节炎等与IL-6有关的疾病的治疗药(专利文献1、2、3、非专利文献4),在日本TOCILIZUMAB被承认作为卡斯尔曼病(Castleman’s disease)和类风湿性关节炎的治疗药(非专利文献5)。
TOCILIZUMAB这样的人源化抗体是第1代抗体药物,将第1代抗体药物改良使药效、便利性、成本得到改善的第2代抗体药物目前正在开发中。作为可适用于第2代抗体药物的技术,人们开发了各种技术,有人报道了提高效应子功能、抗原结合能力、药物动力学、稳定性的技术或降低免疫原性风险的技术等。作为增强药效或者减少给药量的方法,有人报道了通过取代IgG抗体Fc区的氨基酸来增强抗体依赖性细胞毒性(ADCC活性)或补体依赖性细胞毒性(CDC活性)的技术(非专利文献6)。另外,作为增强抗原结合能力、抗原中和能力的技术,有人报道了亲和力成熟技术(非专利文献7),通过向可变区的互补性决定区(CDR;complementarity determining region)等的氨基酸中导入突变,可以增强与抗原的结合活性。通过增强抗原结合能力,可以提高在体外的生物活性、或者减少给药量,还可以进一步提高在体内的药效(非专利文献8)。目前,发挥优于抗RSV抗体的第一代药物、即帕利珠单抗(Palivizumab)的效果的莫维珠单抗(Motavizumab)(利用亲和力成熟技术制作)的临床试验正在进行(非专利文献9)。在抗IL-6受体抗体中,有人报道了比TOCILIZUMAB的亲和力强的、亲和力为约0.05nM的抗体(专利文献4),但具有强于0.05nM的亲和力的人抗体或人源化抗体或嵌合抗体未见报道。
目前的抗体药物所面临的问题有:给药蛋白量非常多所导致的高制造成本。就人源化抗IL-6受体IgG1抗体、即TOCILIZUMAB而言,给药量也设定为8mg/kg/月左右的静脉内注射(非专利文献4)。关于给药方式,当为慢性自身免疫疾病时,优选皮下给药制剂。通常皮下给药制剂必需是高浓度制剂,当为IgG型抗体制剂时,从稳定性等方面考虑,通常认为100mg/mL左右的制剂是极限(非专利文献10)。为了能够发挥持续的治疗效果,通过延长抗体的血浆中半衰期来减少给药蛋白量、赋予高稳定性,从而可以以长给药间隔进行皮下给药,可以提供成本低且便利性高的第2代抗体药物。
在抗体的药物动力学方面,FcRn参与较多,关于抗体同种型间的血浆中半衰期的不同,已知IgG1和IgG2的血浆中半衰期最优异,而IgG3和IgG4比它们差(非专利文献11)。作为进一步提高血浆中半衰期优异的IgG1和IgG2抗体的血浆中半衰期的方法,有人报道了增强与FcRn的结合的恒定区的氨基酸取代(非专利文献12、13)。另外,从免疫原性的角度考虑,与恒定区相比,更希望通过可变区的氨基酸取代来进一步提高血浆中半衰期(专利文献5),但迄今为止未见通过修饰可变区来提高IL-6受体抗体的血浆中半衰期的报道。
开发生物药品时,最重要的一个问题是免疫原性。通常,小鼠抗体通过人源化而使免疫原性降低。通过在人源化时的模板构架中使用种系(ジヤ一ムライン,germline)序列,可以进一步降低免疫原性的风险(非专利文献14)。但即使是作为完全人抗TNF抗体的阿达木单抗(Adalimumab),也显现高频率(13~17%)免疫原性,在显现出免疫原性的患者中发现治疗效果下降(非专利文献15、16)。即使是人抗体,也有可能在CDR中存在T细胞表位,CDR上的T细胞表位有可能成为免疫原性的原因。有人报道了在计算机芯片上或者在体外预测T细胞表位的方法(非专利文献17、18),认为通过除去采用上述方法预测的T细胞表位,可以降低免疫原性风险(非专利文献19)。
作为人源化抗IL-6受体IgG1抗体的TOCILIZUMAB是将小鼠PM1抗体人源化得到的IgG1抗体。H链、L链分别使用人NEW、人REI的序列作为模板构架进行CDR嫁接,但作为对保持活性重要的氨基酸,有5个氨基酸作为小鼠序列残留在构架中(非专利文献20)。迄今为止尚无在不使作为人源化抗体的TOCILIZUMAB的构架中残留的小鼠序列活性降低的情况下进行完全人源化的报道。另外,TOCILIZUMAB的CDR序列是小鼠序列,与阿达木单抗一样,在CDR中有可能存在T细胞表位,不能否定免疫原性的风险。在TOCILIZUMAB的临床试验中,在8mg/kg的药效用量时没有确认到抗TOCILIZUMAB抗体的出现,但在4mg/kg和2mg/kg时确认到了该抗体(专利文献6)。由此认为:在TOCILIZUMAB的免疫原性上还存在改善的空间。但迄今为止尚无有关通过氨基酸取代使免疫原性风险得到改善的TOCILIZUMAB的报道。
TOCILIZUMAB的同种型是IgG1,但同种型的不同即为恒定区序列的不同,认为恒定区的序列对效应子功能、药物动力学、理化性质等有很大影响,所以恒定区序列的选择对抗体药物的开发极为重要(非专利文献11)。近年来,人们非常重视抗体药物的安全性,认为在TGN1412的I期临床试验中发现的重大副作用的原因之一是抗体的Fc部分与Fcγ受体的相互作用(效应子功能)(非专利文献21)。在以中和抗原的生物学作用为目的的抗体药物中,不必与对ADCC等的效应子功能重要的Fcγ受体结合,从副作用方面考虑,认为可能也宁可不优选与Fcγ受体的结合。作为降低与Fcγ受体结合的方法,有将IgG抗体的同种型由IgG1变为IgG2或IgG4的方法(非专利文献22),但从与Fcγ受体I的结合和药物动力学方面考虑,与IgG4相比更优选IgG2(非专利文献11)。TOCILIZUMAB的同种型是IgG1、即IL-6受体中和抗体,所以在不需要ADCC等的效应子功能、而考虑副作用的可能性时,认为同种型还有可能优选IgG2。
另一方面,在开发抗体作为药品时,其蛋白质的理化性质、其中均匀性和稳定性极为重要,有报道称:IgG2同种型其来自铰链区的二硫键的异质性(heterogeneity)非常多(非专利文献23)。在维持来源于此的目标物质/相关物质的异质性的制造间差的同时作为药品进行大量制造并不容易,考虑到成本增加的关系,优选尽可能是单一物质。并且作为抗体H链C末端序列的异质性,有人报道了:由C末端氨基酸的赖氨酸残基的缺失、和C末端的2个氨基酸的甘氨酸、赖氨酸的缺失引起的C末端羧基的酰胺化(非专利文献24),开发IgG2同种型的抗体作为药品时,希望在维持高稳定性的同时降低上述异质性。为了制作便利性优异的、稳定的高浓度皮下给药制剂,希望不仅稳定性高、而且血浆中半衰期也较TOCILIZUMAB的同种型、即IgG1优异。但迄今为止尚无与IgG2同种型的恒定区的抗体有关的异质性下降、具有高稳定性、具有优于IgG1同种型的恒定区抗体的血浆中半衰期的恒定区序列的报道。
本发明的在先技术文献如下。
专利文献1:WO92/19759
专利文献2:WO96/11020
专利文献3:WO96/12503
专利文献4:WO2007/143168
专利文献5:WO2007/114319
专利文献6:WO2004/096273
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发明内容
发明所要解决的课题
本发明鉴于上述状况而设,其目的在于提供:包含优于TOCILIZUMAB的第2代分子的药物组合物(以下,在本说明书中有时还记作“药剂”或“制剂”)、以及上述药物组合物的制备方法,所述优于TOCILIZUMAB的第2代分子是指通过修饰人源化抗IL-6受体IgG1抗体、即TOCILIZUMAB的可变区和恒定区的氨基酸序列,使抗原中和能力增强、同时提高了药物动力学,从而减少了给药频率、持续发挥治疗效果,且免疫原性、安全性、理化性质(稳定性和均匀性)得到改善。
解决课题的方法
本发明人等为了研制优于TOCILIZUMAB的第2代分子进行了深入研究,通过修饰第1代人源化抗IL-6受体IgG1抗体、即TOCILIZUMAB的可变区和恒定区的氨基酸序列,使药效增强、同时提高药物动力学,从而减少给药频率、持续发挥治疗效果,且免疫原性、安全性、理化性质(稳定性和均匀性)得到改善。其结果,本发明人等在TOCILIZUMAB的可变区中发现了多个使与抗原的结合能力(亲和力)提高的CDR突变,利用其组合成功地大幅提高了亲和力。另外,本发明人等通过导入使可变区序列的等电点降低的修饰,成功提高了药物动力学。本发明人等通过对与作为抗原的IL-6受体的结合赋予pH依赖性,可以利用1分子抗体多次中和抗原,成功提高了药物动力学。本发明人等通过将残留在TOCILIZUMAB的构架中的、来自小鼠的序列完全人源化,降低了可变区中通过在计算机芯片上预测的T细胞表位肽的数目,从而成功降低了免疫原性风险。并且,本发明人等在TOCILIZUMAB的恒定区中成功发现了新的恒定区序列,所述新的恒定区序列为了提高安全性,使与Fcγ受体的结合低于IgG1,使药物动力学较IgG1得到改善,并且在不降低稳定性的情况下降低来源于IgG2的铰链区的二硫键的异质性和来源于H链C末端的异质性。通过适当组合上述CDR区氨基酸序列的修饰、可变区氨基酸序列的修饰、恒定区氨基酸序列的修饰,成功研制了优于TOCILIZUMAB的第2代分子。
本发明涉及包含人源化抗IL-6受体IgG抗体的药物组合物、以及上述药物组合物的制备方法,所述人源化抗IL-6受体IgG抗体是通过修饰人源化抗IL-6受体IgG1抗体、即TOCILIZUMAB的可变区和恒定区的氨基酸序列,具有更优异的、与抗原(IL-6受体)结合的能力、具有更优异的药物动力学、更优异的安全性、免疫原性风险、理化性质(稳定性、均匀性)。更具体而言,本发明提供下述[1]~[11]。
[1]下述(a)~(f)中任一项所述的多肽:
(a)多肽,该多肽包含具有SEQ ID NO:1(VH4-M73的CDR1)的序列的CDR1、具有SEQ ID NO:2(VH4-M73的CDR2)的序列的CDR2和具有SEQ ID NO:3(VH4-M73的CDR3)的序列的CDR3;
(b)多肽,该多肽包含具有SEQ ID NO:4(VH3-M73的CDR1)的序列的CDR1、具有SEQ ID NO:5(VH3-M73的CDR2)的序列的CDR2和具有SEQ ID NO:6(VH3-M73的CDR3)的序列的CDR3;
(c)多肽,该多肽包含具有SEQ ID NO:7(VH5-M83的CDR1)的序列的CDR1、具有SEQ ID NO:8(VH5-M83的CDR2)的序列的CDR2和具有SEQ ID NO:9(VH5-M83的CDR3)的序列的CDR3;
(d)多肽,该多肽包含具有SEQ ID NO:10(VL1的CDR1)的序列的CDR1、具有SEQ ID NO:11(VL1的CDR2)的序列的CDR2和具有SEQ ID NO:12(VL1的CDR3)的序列的CDR3;
(e)多肽,该多肽包含具有SEQ ID NO:13(VL3的CDR1)的序列的CDR1、具有SEQ ID NO:14(VL3的CDR2)的序列的CDR2和具有SEQ ID NO:15(VL3的CDR3)的序列的CDR3;
(f)多肽,该多肽包含具有SEQ ID NO:16(VL5的CDR1)的序列的CDR1、具有SEQ ID NO:17(VL5的CDR2)的序列的CDR2和具有SEQ ID NO:18(VL5的CDR3)的序列的CDR3。
[2]下述(a)~(c)中任一项所述的抗体:
(a)抗体,该抗体包含重链可变区及轻链可变区,所述重链可变区包含具有SEQ ID NO:1(VH4-M73的CDR1)的序列的CDR1、具有SEQ ID NO:2(VH4-M73的CDR2)的序列的CDR2和具有SEQ ID NO:3(VH4-M73的CDR3)的序列的CDR3;
所述轻链可变区包含具有SEQ ID NO:10(VL1的CDR1)的序列的CDR1、具有SEQ ID NO:11(VL1的CDR2)的序列的CDR2和具有SEQ ID NO:12(VL1的CDR3)的序列的CDR3;
(b)抗体,该抗体包含重链可变区及轻链可变区,所述重链可变区包含具有SEQ ID NO:4(VH3-M73的CDR1)的序列的CDR1、具有SEQ ID NO:5(VH3-M73的CDR2)的序列的CDR2和具有SEQ ID NO:6(VH3-M73的CDR3)的序列的CDR3;
所述轻链可变区包含具有SEQ ID NO:13(VL3的CDR1)的序列的CDR1、具有SEQ ID NO:14(VL3的CDR2)的序列的CDR2和具有SEQ ID NO:15(VL3的CDR3)的序列的CDR3;
(c)抗体,该抗体包含重链可变区及轻链可变区,所述重链可变区包含具有SEQ ID NO:7(VH5-M83的CDR1)的序列的CDR1、具有SEQ ID NO:8(VH5-M83的CDR2)的序列的CDR2和具有SEQ ID NO:9(VH5-M83的CDR3)的序列的CDR3;
所述轻链可变区包含具有SEQ ID NO:16(VL5的CDR1)的序列的CDR1、具有SEQ ID NO:17(VL5的CDR2)的序列的CDR2和具有SEQ ID NO:18(VL5的CDR3)的序列的CDR3。
[3]下述(a)~(f)中任一项所述的可变区:
(a)重链可变区,其中具有SEQ ID NO:19(VH4-M73的可变区)的序列;
(b)重链可变区,其中具有SEQ ID NO:20(VH3-M73的可变区)的序列;
(c)重链可变区,其中具有SEQ ID NO:21(VH5-M83的可变区)的序列;
(d)轻链可变区,其中具有SEQ ID NO:22(VL1的可变区)的序列;
(e)轻链可变区,其中具有SEQ ID NO:23(VL3的可变区)的序列;
(f)轻链可变区,其中具有SEQ ID NO:24(VL5的可变区)的序列。
[4]下述(a)~(c)中任一项所述的抗体:
(a)抗体,该抗体包含重链可变区和轻链可变区,所述重链可变区具有SEQ ID NO:19(VH4-M73的可变区)的序列,所述轻链可变区具有SEQ ID NO:22(VL1的可变区)的序列;
(b)抗体,该抗体包含重链可变区和轻链可变区,所述重链可变区具有SEQ ID NO:20(VH3-M73的可变区)的序列,所述轻链可变区具有SEQ ID NO:23(VL3的可变区)的序列;
(c)抗体,该抗体包含重链可变区和轻链可变区,所述重链可变区具有SEQ ID NO:21(VH5-M83的可变区)的序列,所述轻链可变区具有SEQ ID NO:24(VL5的可变区)的序列。
[5]下述(a)~(f)中任一项所述的重链或轻链:
(a)重链,其中具有SEQ ID NO:25(VH4-M73)的序列;
(b)重链,其中具有SEQ ID NO:26(VH3-M73)的序列;
(c)重链,其中具有SEQ ID NO:27(VH5-M83)的序列;
(d)轻链,其中具有SEQ ID NO:28(VL1)的序列;
(e)轻链,其中具有SEQ ID NO:29(VL3)的序列;
(f)轻链,其中具有SEQ ID NO:30(VL5)的序列。
[6]下述(a)~(c)中任一项所述的抗体:
(a)抗体,该抗体包含重链和轻链,所述重链具有SEQ ID NO:25(VH4-M73)的序列,所述轻链具有SEQ ID NO:28(VL1)的序列;
(b)抗体,该抗体包含重链和轻链,所述重链具有SEQ ID NO:26(VH3-M73)的序列,所述轻链具有SEQ ID NO:29(VL3)的序列;
(c)抗体,该抗体包含重链和轻链,所述重链具有SEQ ID NO:27(VH5-M83)的序列,所述轻链具有SEQ ID NO:30(VL5)的序列。
[7]基因,该基因编码[1]~[6]中任一项所述的多肽。
[8]载体,该载体包含[7]所述的基因。
[9]宿主细胞,该宿主细胞保有[8]所述的载体。
[10]通过培养[9]的宿主细胞来制备[1]~[6]中任一项所述的多肽的方法。
[11]药物组合物,其中含有[1]~[6]中任一项所述的多肽或按照[10]所述的方法制备的多肽。
发明效果
根据本发明得到的人源化抗IL-6受体IgG抗体通过增强药效、提高药物动力学,可以减少给药频率,可以持续发挥治疗效果。
附图说明
图1是显示汇总了提高TOCILIZUMAB与IL-6受体的亲和性的突变位点的列表的图。HCDR2的TOCILIZUMAB序列见SEQ ID NO:81,HCDR2的突变后序列(上段)见SEQ ID NO:82,HCDR2的突变后序列(下段)见SEQ ID NO:83,HCDR3的TOCILIZUMAB序列见SEQID NO:84,HCDR3的突变后序列(上段)见SEQ ID NO:85,HCDR3的突变后序列(下段)见SEQ ID NO:86,LCDR1的TOCILIZUMAB序列见SEQ ID NO:87,LCDR1的突变后序列(上段)见SEQ ID NO:88,LCDR1的突变后序列(下段)见SEQ ID NO:89,LCDR3的TOCILIZUMAB序列见SEQ ID NO:90,LCDR3的突变后序列(上段)见SEQ ID NO:91,LCDR3的突变后序列(下段)见SEQ ID NO:92。
图2是显示TOCILIZUMAB和RDC-23在BaF/gp130中的中和活性的图。
图3是显示汇总了在不大幅降低TOCILIZUMAB与IL-6受体的结合的情况下可以降低可变区的等电点的突变位点的列表的图。图中的星号是为了形成人序列虽然不影响等电点但发生了突变的位点。HFR1的TOCILIZUMAB序列见SEQ ID NO:93,HFR1的突变后序列见SEQ ID NO:94,HCDR1的TOCILIZUMAB序列见SEQ ID NO:95,HCDR1的突变后序列见SEQ ID NO:96,HFR2的TOCILIZUMAB序列见SEQ ID NO:97,HFR2的突变后序列见SEQ ID NO:98,HCDR2的TOCILIZUMAB序列见SEQ ID NO:81,HCDR2的突变后序列见SEQ ID NO:99,HFR4的TOCILIZUMAB序列见SEQ ID NO:100,HFR4的突变后序列见SEQ ID NO:101,LFR1的TOCILIZUMAB序列见SEQ ID NO:102,LFR1的突变后序列见SEQ ID NO:103,LCDR1的TOCILIZUMAB序列见SEQ ID NO:87,LCDR1的突变后序列见SEQ ID NO:104,LFR2的TOCILIZUMAB序列见SEQ ID NO:105,LFR2的突变后序列见SEQ ID NO:106,LCDR2的TOCILIZUMAB序列见SEQ ID NO:107,LCDR2的突变后序列见SEQ ID NO:108,109,LFR3的TOCILIZUMAB序列见SEQ ID NO:110,LFR3的突变后序列见SEQ ID NO:111,LFR4的TOCILIZUMAB序列见SEQ IDNO:112,LFR4的突变后序列见SEQ ID NO:113。
图4是显示TOCILIZUMAB和H53/L28在BaF/gp130中的中和活性的图。
图5是显示将TOCILIZUMAB和H53/L28对小鼠进行静脉内给药后血浆中浓度变化的曲线图。
图6是显示将TOCILIZUMAB和H53/L28对小鼠进行皮下给药后血浆中浓度变化的曲线图。
图7是显示IgG分子通过在核内体内从膜型抗原上解离而再次与新抗原结合的模式图。
图8是显示汇总了可以对TOCILIZUMAB与IL-6受体的结合赋予pH依赖性(在pH7.4时结合、在pH5.8时解离)的突变位点的列表的图。HFR1的TOCILIZUMAB序列见SEQ ID NO:93,HFR1的突变后序列见SEQ ID NO:114,HCDR1的TOCILIZUMAB序列见SEQ IDNO:95,HCDR1的突变后序列见SEQ ID NO:115,LCDR1的TOCILIZUMAB序列见SEQ ID NO:87,LCDR1的突变后序列见SEQID NO:116,LCDR2的TOCILIZUMAB序列见SEQ ID NO:107,LCDR2的突变后序列见SEQ ID NO:117。
图9是显示TOCILIZUMAB和H3pI/L73在BaF/gp130中的中和活性的图。
图10是显示将TOCILIZUMAB和H3pI/L73对食蟹猴(カニクイザル)进行静脉内给药后血浆中浓度变化的曲线图。
图11是显示将TOCILIZUMAB和H3pI/L73对人IL-6受体转基因小鼠进行静脉内给药后血浆中浓度变化的曲线图。
图12是显示利用阳离子交换色谱评价TOCILIZUMAB、TOCILIZUMABΔK和TOCILIZUMABΔGK的来自C末端的异质性的结果的图。
图13是显示利用阳离子交换色谱评价TOCILIZUMAB-IgG1、TOCILIZUMAB-IgG2和TOCILIZUMAB-SKSC的来自二硫键的异质性的结果的图。
图14是显示利用差示扫描量热法(DSC)测定的TOCILIZUMAB-IgG1、TOCILIZUMAB-IgG2和TOCILIZUMAB-SKSC的变性曲线和各Fab结构域的Tm值的图。
图15是显示将TOCILIZUMAB-IgG1、TOCILIZUMAB-M44、TOCILIZUMAB-M58和TOCILIZUMAB-M73对人FcRn转基因小鼠进行静脉内给药后血浆中浓度变化的曲线图。
图16是显示TOCILIZUMAB、对照和Fv5-M83在BaF/gp130中的中和活性的图。
图17是显示TOCILIZUMAB、Fv3-M73和Fv4-M73在BaF/gp130中的中和活性的图。
图18是显示将TOCILIZUMAB、对照、Fv3-M73、Fv4-M73和Fv5-M83对食蟹猴进行静脉内给药后血浆中浓度变化的曲线图。
图19是显示将TOCILIZUMAB、对照、Fv3-M73、Fv4-M73和Fv5-M83对食蟹猴进行静脉内给药后CRP浓度变化的曲线图。
图20是显示将TOCILIZUMAB、对照、Fv3-M73、Fv4-M73和Fv5-M83对食蟹猴进行静脉内给药后非结合型可溶型IL-6受体率的变化的曲线图。
图21是显示TOCILIZUMAB和Fv4-M73对来自人RA患者的滑膜细胞的MCP-1产生的抑制作用的图。
图22是显示TOCILIZUMAB和Fv4-M73对来自人RA患者的滑膜细胞的VEGF产生的抑制作用的图。
实施发明的最佳方式
本发明提供下述(a)~(f)中任一项所述的多肽。
(a)多肽,该多肽包含具有SEQ ID NO:1(VH4-M73的CDR1)的序列的CDR1、具有SEQ ID NO:2(VH4-M73的CDR2)的序列的CDR2和具有SEQ ID NO:3(VH4-M73的CDR3)的序列的CDR3;
(b)多肽,该多肽包含具有SEQ ID NO:4(VH3-M73的CDR1)的序列的CDR1、具有SEQ ID NO:5(VH3-M73的CDR2)的序列的CDR2和具有SEQ ID NO:6(VH3-M73的CDR3)的序列的CDR3;
(c)多肽,该多肽包含具有SEQ ID NO:7(VH5-M83的CDR1)的序列的CDR1、具有SEQ ID NO:8(VH5-M83的CDR2)的序列的CDR2和具有SEQ ID NO:9(VH5-M83的CDR3)的序列的CDR3;
(d)多肽,该多肽包含具有SEQ ID NO:10(VL1的CDR1)的序列的CDR1、具有SEQ ID NO:11(VL1的CDR2)的序列的CDR2和具有SEQ ID NO:12(VL1的CDR3)的序列的CDR3;
(e)多肽,该多肽包含具有SEQ ID NO:13(VL3的CDR1)的序列的CDR1、具有SEQ ID NO:14(VL3的CDR2)的序列的CDR2和具有SEQ ID NO:15(VL3的CDR3)的序列的CDR3;
(f)多肽,该多肽包含具有SEQ ID NO:16(VL5的CDR1)的序列的CDR1、具有SEQ ID NO:17(VL5的CDR2)的序列的CDR2和具有SEQ ID NO:18(VL5的CDR3)的序列的CDR3。
在本发明中,对多肽没有特别限定,但优选为与人IL-6受体具有结合活性的抗原结合物质。抗原结合物质的优选例子有:抗体的重链可变区(VH)、抗体的轻链可变区(VL)、抗体的重链、抗体的轻链、抗体等。
在上述(a)~(f)的多肽中,(a)~(c)的多肽的优选例子有:抗体的重链可变区,(d)~(f)的多肽的优选例子有:抗体的轻链可变区。
上述可变区可以用作抗人IL-6受体抗体的一部分。使用了上述可变区的抗人IL-6受体抗体具有优异的结合活性、优异的药物动力学、优异的安全性·免疫原性、和/或优异的理化性质。在本发明中,优异的药物动力学或药物动力学的提高是指对机体内给予抗体时,由血浆中浓度变化算出的药物动力学参数之一、即“清除率(Clearance,CL)”的减少、“浓度曲线下面积(Area Under Curve、AUC)”的增大、“平均滞留时间(Mean Residence Time)”的增大、“血浆中半衰期(t1/2)”的增大中的任一者。在本发明中,对优异的理化性质或理化性质的提高没有特别限定,意思是指稳定性的提高、异质性的降低等。
与CDR连接的人抗体构架区(framework region;FR)选择CDR形成良好的抗原结合部位的构架区。对本发明的可变区中所用的FR没有特别限定,可以使用任意的FR,但优选使用来自人的FR。来自人的FR可以使用具有天然序列的FR,也可以根据需要取代、缺失、添加和/或插入具有天然序列的构架区的1个或多个氨基酸等,使CDR形成适当的抗原结合部位。例如,通过测定、评价使用进行了氨基酸取代的FR的抗体与抗原的结合活性,可以选择具有所期望性质的突变FR序列(Sato,K.等人,Cancer Res.(1993)53,851-856)。
在上述CDR序列中,可以取代、缺失、添加和/或插入1个或多个氨基酸等。优选取代、缺失、添加和/或插入1个或多个氨基酸后的CDR序列在结合活性、中和活性、稳定性、免疫原性和/或药物动力学方面与修饰前的CDR序列具有相同的活性。对取代、缺失、添加和/或插入的氨基酸数目没有特别限定,但每一个CDR中优选3个氨基酸以内,进一步优选2个氨基酸以内,更优选为1个氨基酸。
作为将1个或多个氨基酸残基取代成其他目标氨基酸的方法,例如有位点特异性诱变法(Hashimoto-Gotoh,T,Mizuno,T,Ogasahara,Y,and Nakagawa,M.(1995).An oligodeoxyribonucleotide-directed dualamber method for site-directed mutagenesis(寡脱氧核糖核苷酸定向双琥珀色法位点定向诱变).Gene 152,271-275、Zoller,MJ,and Smith,M.(1983)Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors(DNA片段克隆到M13载体的寡聚核苷酸定向诱变).Methods Enzymol.100,468-500、Kramer,W,Drutsa,V,Jansen,HW,Kramer,B,Pflugfelder,M,and Fritz,HJ(1984)The gapped duplex DNA approach tooligonucleotide-directed mutation construction(带缺口的双链体DNA接近于寡聚核苷酸定向突变结构).Nucleic Acids Res.12,9441-9456、Kramer W,and Fritz HJ(1987)Oligonucleotide-directed construction of mutationsvia gapped duplex DNA Methods(经由带缺口的双链体DNA法突变的寡聚核苷酸定向结构).Enzymol.154,350-367、Kunkel,TA(1985)Rapid andefficient site-specific mutagenesis without phenotypic selection(无表型筛选的迅速及高效的位点特异性诱变法).Proc Natl Acad Sci U S A.82,488-492)。利用该方法可以将抗体所期望的氨基酸取代成其他目标氨基酸。另外,通过使用构架改组(Mol Immunol.2007 Apr;44(11):3049-60)和CDR修复(US2006/0122377)等文库技术,还可以将构架和CDR中的氨基酸取代成其他适当的氨基酸。
本发明还提供下述(a)~(c)中任一项所述的抗体。
(a)抗体,该抗体包含重链可变区及轻链可变区,所述重链可变区包含具有SEQ ID NO:1(VH4-M73的CDR1)的序列的CDR1、具有SEQ ID NO:2(VH4-M73的CDR2)的序列的CDR2和具有SEQ ID NO:3(VH4-M73的CDR3)的序列的CDR3,所述轻链可变区包含具有SEQID NO:10(VL1的CDR1)的序列的CDR1、具有SEQ ID NO:11(VL1的CDR2)的序列的CDR2和具有SEQ ID NO:12(VL1的CDR3)的序列的CDR3;
(b)抗体,该抗体包含重链可变区及轻链可变区,所述重链可变区包含具有SEQ ID NO:4(VH3-M73的CDR1)的序列的CDR1、具有SEQ ID NO:5(VH3-M73的CDR2)的序列的CDR2和具有SEQ ID NO:6(VH3-M73的CDR3)的序列的CDR3,所述轻链可变区包含具有SEQID NO:13(VL3的CDR1)的序列的CDR1、具有SEQ ID NO:14(VL3的CDR2)的序列的CDR2和具有SEQ ID NO:15(VL3的CDR3)的序列的CDR3;
(c)抗体,该抗体包含重链可变区及轻链可变区,所述重链可变区包含具有SEQ ID NO:7(VH5-M83的CDR1)的序列的CDR1、具有SEQ ID NO:8(VH5-M83的CDR2)的序列的CDR2和具有SEQ ID NO:9(VH5-M83的CDR3)的序列的CDR3,所述轻链可变区包含具有SEQID NO:16(VL5的CDR1)的序列的CDR1、具有SEQ ID NO:17(VL5的CDR2)的序列的CDR2和具有SEQ ID NO:18(VL5的CDR3)的序列的CDR3。
上述抗体可以用作具有优异的结合活性、优异的药物动力学、优异的安全性·免疫原性、和/或优异的理化性质的抗人IL-6受体抗体。
本发明的与CDR连接的人抗体的构架区选择CDR形成良好的抗原结合部位的构架区。对本发明的可变区中使用的FR没有特别限定,可以使用任何的FR,但优选使用来自人的FR。来自人的FR可以使用具有天然序列的FR,根据需要可以取代、缺失、添加和/或插入具有天然序列的构架区的1个或多个氨基酸等,使CDR形成适当的抗原结合部位。例如,通过测定、评价使用了进行了氨基酸取代的FR的抗体与抗原的结合活性,可以选择具有所期望性质的突变FR序列(Sato,K.等人,Cancer Res.(1993)53,851-856)。
对本发明的抗体中使用的恒定区没有特别限定,可以使用任何的恒定区。本发明的抗体中使用的恒定区的优选例子有:来自人的恒定区(来自IgG1、IgG2、IgG3、IgG4、Cκ、Cλ的恒定区等)。来自人的恒定区中可以取代、缺失、添加和/或插入1个或多个氨基酸。例如,作为来自人的恒定区的优选例子,当为重链恒定区时,例如有:具有SEQ ID NO:31(VH4-M73的恒定区)的氨基酸序列的恒定区、具有SEQ ID NO:32(VH3-M73的恒定区)的氨基酸序列的恒定区、具有SEQ ID NO:33(VH5-M83的恒定区)的氨基酸序列的恒定区;当为轻链恒定区时,例如有:具有SEQ ID NO:34(VL1)的氨基酸序列的恒定区、具有SEQ ID NO:35(VL3)的氨基酸序列的恒定区、具有SEQ IDNO:36(VL5)的氨基酸序列的恒定区。
在上述CDR序列中可以取代、缺失、添加和/或插入1个或多个氨基酸等。优选取代、缺失、添加和/或插入1个或多个氨基酸后的CDR序列在结合活性、中和活性、稳定性、免疫原性和/或药物动力学方面与修饰前的CDR序列具有同等的活性。对取代、缺失、添加和/或插入的氨基酸数目没有特别限定,每个CDR优选3个氨基酸以内,进一步优选2个氨基酸以内、更优选为1个氨基酸。
氨基酸的取代、缺失、添加和/或插入等还可以通过上述方法来进行。
本发明还提供下述(a)~(f)中任一项所述的可变区。
(a)重链可变区,其中具有SEQ ID NO:19(VH4-M73的可变区)的序列;
(b)重链可变区,其中具有SEQ ID NO:20(VH3-M73的可变区)的序列;
(c)重链可变区,其中具有SEQ ID NO:21(VH5-M83的可变区)的序列;
(d)轻链可变区,其中具有SEQ ID NO:22(VL1的可变区)的序列;
(e)轻链可变区,其中具有SEQ ID NO:23(VL3的可变区)的序列;
(f)轻链可变区,其中具有SEQ ID NO:24(VL5的可变区)的序列。
上述可变区可以用作抗人IL-6受体抗体的一部分。使用了上述可变区的抗人IL-6受体抗体具有优异的结合活性、优异的药物动力学、优异的安全性·免疫原性、和/或优异的理化性质。
上述可变区中可以取代、缺失、添加和/或插入1个或多个(例如5个氨基酸以内、优选3个氨基酸以内)氨基酸。作为将1个或多个氨基酸残基取代成其他目标氨基酸的方法,例如有上述方法。
本发明还提供包含上述可变区的多肽。
本发明还提供下述(a)~(c)中任一项所述的抗体。
(a)抗体,该抗体包含重链可变区和轻链可变区,所述重链可变区具有SEQ ID NO:19(VH4-M73的可变区)的序列,所述轻链可变区具有SEQ ID NO:22(VL1的可变区)的序列;
(b)抗体,该抗体包含重链可变区和轻链可变区,所述重链可变区具有SEQ ID NO:20(VH3-M73的可变区)的序列,所述轻链可变区具有SEQ ID NO:23(VL3的可变区)的序列;
(c)抗体,该抗体包含重链可变区和轻链可变区,所述重链可变区具有SEQ ID NO:21(VH5-M83的可变区)的序列,所述轻链可变区具有SEQ ID NO:24(VL5的可变区)的序列。
上述可变区可以用作抗人IL-6受体抗体的一部分。使用了上述可变区的抗人IL-6受体抗体具有优异的结合活性、优异的药物动力学、优异的安全性·免疫原性、和/或优异的理化性质。
上述可变区中可以取代、缺失、添加和/或插入1个或多个(例如5个氨基酸以内、优选3个氨基酸以内)氨基酸。作为将1个或多个氨基酸残基取代成其他目标氨基酸的方法,例如有上述方法。
此外,对本发明的抗体中使用的恒定区没有特别限定,可以使用任何的恒定区。本发明的抗体中使用的恒定区的优选例子有:来自人的恒定区(来自IgG1、IgG2、IgG3、IgG4、κ链、λ链的恒定区等)。来自人的恒定区中可以取代、缺失、添加和/或插入1个或多个氨基酸。例如,作为来自人的恒定区的优选例子,当为重链恒定区时,例如有:具有SEQ ID NO:31(VH4-M73的恒定区)的氨基酸序列的恒定区、具有SEQ ID NO:32(VH3-M73的恒定区)的氨基酸序列的恒定区、具有SEQ ID NO:33(VH5-M83的恒定区)的氨基酸序列的恒定区;当为轻链恒定区时,例如有:具有SEQ ID NO:34(VL1)的氨基酸序列的恒定区、具有SEQ ID NO:35(VL3)的氨基酸序列的恒定区、具有SEQ IDNO:36(VL5)的氨基酸序列的恒定区。
本发明还提供下述(a)~(f)中任一项所述的重链或轻链。
(a)重链,该重链具有SEQ ID NO:25(VH4-M73)的序列;
(b)重链,该重链具有SEQ ID NO:26(VH3-M73)的序列;
(c)重链,该重链具有SEQ ID NO:27(VH5-M83)的序列;
(d)轻链,该轻链具有SEQ ID NO:28(VL1)的序列;
(e)轻链,该轻链具有SEQ ID NO:29(VL3)的序列;
(f)轻链,该轻链具有SEQ ID NO:30(VL5)的序列。
上述重链或轻链可以用作抗人IL-6受体抗体的一部分。使用了上述重链或轻链的抗人IL-6受体抗体具有优异的结合活性、优异的药物动力学、优异的安全性·免疫原性、和/或优异的理化性质。
上述重链或轻链中可以取代、缺失、添加和/或插入1个或多个(例如10个氨基酸以内、优选5个氨基酸以内、进一步优选3个氨基酸以内)氨基酸。作为将1个或多个氨基酸残基取代成其他目标氨基酸的方法,例如可以使用上述方法。
1个或多个氨基酸的取代、缺失、添加和/或插入可以在可变区中进行,也可以在恒定区中进行,或者在可变区和恒定区两区中进行。
本发明还提供下述(a)~(c)中任一项所述的抗体。
(a)抗体,该抗体包含重链和轻链,所述重链具有SEQ ID NO:25(VH4-M73)的序列,所述轻链具有SEQ ID NO:28(VL1)的序列;
(b)抗体,该抗体包含重链和轻链,所述重链具有SEQ ID NO:26(VH3-M73)的序列,所述轻链具有SEQ ID NO:29(VL3)的序列;
(c)抗体,该抗体包含重链和轻链,所述重链具有SEQ ID NO:27(VH5-M83)的序列,所述轻链具有SEQ ID NO:30(VL5)的序列。
上述抗体是具有优异的结合活性、优异的药物动力学、优异的安全性·免疫原性、和/或优异的理化性质的抗人IL-6受体抗体。
上述抗体中可以取代、缺失、添加和/或插入1个或多个(例如20个氨基酸以内、优选10个氨基酸以内、进一步优选5个氨基酸以内)氨基酸。作为将1个或多个氨基酸残基取代成其他目标氨基酸的方法,例如有上述方法。
1个或多个氨基酸的取代、缺失、添加和/或插入可以在可变区中进行,也可以在恒定区中进行,或者在可变区和恒定区两区中进行。
本发明的抗体优选为人源化(humanized)抗体。
人源化抗体也称重构(reshaped)人抗体,其是将来自人以外的哺乳动物的CDR嫁接到人抗体的CDR中得到的抗体,其一般的基因重组手法也已知(参照欧州专利申请公开号EP125023号公报、WO96/02576号公报)。
具体而言,例如使用制作成在CDR和FR的末端区均具有重叠部分的多个寡核苷酸作为引物,利用PCR法合成设计成连接目标CDR和目标构架区(FR)的DNA序列(参照WO98/13388号公报中记载的方法)。将得到的DNA与编码人抗体恒定区或人抗体恒定区变体的DNA连接,之后插入到表达载体中,再将其导入宿主中,产生人源化抗体而得到(参照欧州专利申请公开号EP239400、国际专利申请公开号WO96/02576)。
与CDR连接的人抗体的构架区选择CDR形成良好的抗原结合部位的构架区。根据需要,可以对抗体可变区中的构架区的氨基酸进行取代、缺失、添加和/或插入等。
在人源化抗体的恒定区中,可以使用人抗体恒定区或者在人抗体恒定区中取代、缺失、添加和/或插入1个或多个氨基酸的人抗体恒定区变体。
例如,在H链中可以使用Cγ1、Cγ2、Cγ3、Cγ4、Cμ、Cδ、Cα1、Cα2、Cε,在L链中可以使用Cκ、Cλ。Cκ的氨基酸序列见SEQ ID NO:38,编码该氨基酸序列的核苷酸序列见SEQ ID NO:37。Cγ1的氨基酸序列见SEQ ID NO:40,编码该氨基酸序列的核苷酸序列见SEQ IDNO:39。Cγ2的氨基酸序列见SEQ ID NO:42,编码该氨基酸序列的核苷酸序列见SEQ ID NO:41。Cγ4的氨基酸序列见SEQ ID NO:44,编码该氨基酸序列的核苷酸序列见SEQ ID NO:43。
另外,为了改善抗体或其产生的稳定性,可以对人抗体C区进行修饰。人源化时所用的人抗体可以是IgG、IgM、IgA、IgE、IgD等任何的同种型的人抗体,但在本发明中优选使用IgG。作为IgG,可以使用IgG1、IgG2、IgG3、IgG4等。
需要说明的是,制作人源化抗体后,可以将可变区(例如CDR、FR)或恒定区中的氨基酸用其他氨基酸取代、缺失、添加和/或插入等,本发明的人源化抗体中还包含上述进行了氨基酸取代等的人源化抗体。
在本发明的抗体中,只要与IL-6受体具有结合活性和/或中和活性即可,不仅包含IgG所代表的二价抗体,还包含一价抗体或IgM所代表的多价抗体。本发明的多价抗体中包含具有完全相同的抗原结合部位的多价抗体或具有一部分不同或完全不同的抗原结合部位的多价抗体。本发明的抗体并不限于抗体的全长分子,只要与IL-6受体蛋白结合即可,可以是小分子抗体或其修饰物。
小分子抗体是包含全长抗体(whole antibody,例如whole IgG等)的一部分发生缺失的抗体片段的抗体,只要与IL-6受体具有结合活性和/或中和活性即可,没有特别限定。在本发明中,小分子抗体只要包含全长抗体的一部分即可,没有特别限定,优选包含VH或VL,特别优选为包含VH和VL两者的小分子抗体。另外,本发明的小分子抗体的其他优选例子有:包含抗体的CDR的小分子抗体。小分子抗体所含的CDR可以包含抗体的全部6个CDR,也可以包含部分CDR。
本发明中的小分子抗体优选比全长抗体的分子量小,例如有时还会形成二聚体、三聚体、四聚体等多聚物等,其分子量有时比全长抗体的分子量大。
抗体片段的具体例子有:例如Fab、Fab’、F(ab’)2、Fv等。小分子抗体的具体例子有:例如Fab、Fab’、F(ab’)2、Fv、scFv(单链Fv)、双抗体、sc(Fv)2(单链(Fv)2)等。这些抗体的多聚物(例如二聚体、三聚体、四聚体、聚合物)也包含在本发明的小分子抗体内。
抗体片段例如可以通过用酶处理抗体生成抗体片段而得到。作为生成抗体片段的酶,例如木瓜蛋白酶、胃蛋白酶或者纤溶酶等是公知的。或者,构建编码上述抗体片段的基因,将其导入表达载体中,之后可以使之在适当的宿主细胞中表达(例如参照Co,M.S.等人,J.Immunol.(1994)152,2968-2976;Better,M.& Horwitz,A.H.Methodsin Enzymology(1989)178,476-496;Pluckthun,A.& Skerra,A.Methodsin Enzymology(1989)178,476-496;Lamoyi,E.,Methods inEnzymology(1989)121,652-663;Rousseaux,J.等人,Methods inEnzymology(1989)121,663-669;Bird,R.E.等人,TIBTECH(1991)9,132-137)。
消化酶切断抗体片段的特定位置,产生下述特定结构的抗体片段。对于利用上述酶得到的抗体片段,当应用基因工程学方法时,可以使抗体的任意部分缺失。
使用上述消化酶时得到的抗体片段如下。
木瓜蛋白酶消化:F(ab)2或Fab
胃蛋白酶消化:F(ab’)2或Fab’
纤溶酶消化:Facb
本发明中的小分子抗体只要与IL-6受体具有结合活性和/或中和活性即可,可以包含缺失了任意区的抗体片段。
双抗体是指通过基因融合构建的二价(bivalent)抗体片段(Holliger P等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)、EP404,097号、WO93/11161号等)。双抗体是由两条多肽链构成的二聚体。通常,构成二聚体的多肽链各自在相同链中VL和VH经由接头进行结合。双抗体中的接头通常短至VL和VH彼此无法结合。具体而言,构成接头的氨基酸残基例如为5个残基左右。因此,编码在同一多肽链上的VL和VH无法形成单链可变区片段,而是与其他单链可变区片段形成二聚体。其结果,双抗体具有2个抗原结合部位。
scFv抗体是将VH和VL通过接头等结合形成单链多肽的抗体(Huston,J.S.等人,Proc.Natl.Acad.Sci.U.S.A.(1988)85,5879-5883;Pluckthun“The Pharmacology of Monoclonal Antibodies”第113卷,Resenburg和Moore编,Springer Verlag,New York,第269-315页,(1994))。scFv中的H链V区和L链V区可以来自本说明书所记载的任一种抗体。对连接V区的肽接头没有特别限定。例如可以使用包含3~25个残基左右的任意单链肽作为接头。具体而言,例如可以使用后述的肽接头等。
两链的V区例如可以按照上述PCR法进行连接。为了利用PCR法连接V区,首先在下述DNA中,利用编码全部或所期望的部分氨基酸序列的DNA作为模板。
编码抗体的H链或H链V区的DNA序列、以及
编码抗体的L链或L链V区的DNA序列
通过使用了具有与应扩增的DNA两端序列对应的序列的一对引物的PCR法,分别扩增编码H链和L链的V区的DNA。之后,准备编码肽接头部分的DNA。编码肽接头的DNA也可以利用PCR来合成。此时,可以在利用的引物的5’侧添加能够与另外合成的各V区的扩增产物连接的核苷酸序列。之后,利用[H链V区DNA]-[肽接头DNA]-[L链V区DNA]的各DNA和装配PCR用引物进行PCR反应。
装配PCR用引物包含在[H链V区DNA]的5’侧退火的引物和在[L链V区DNA]的3’侧退火的引物的组合。即,装配PCR用引物是指能够扩增编码应合成的scFv的全长序列的DNA的引物组。另一方面,在[肽接头DNA]中添加能够与各V区DNA连接的核苷酸序列。其结果,上述DNA被连接起来,再通过装配PCR用引物最终以扩增产物的形式生成全长scFv。若暂且制作编码scFv的DNA,则按照常规方法可以取得含有这些DNA的表达载体和通过该表达载体进行转化的重组细胞。其结果,通过培养所得的重组细胞,使编码该scFv的DNA表达,可以取得该scFv。
对所结合的VH和VL的顺序没有特别限定,可以按照任意顺序进行排列,例如有下述排列。
[VH]接头[VL]
[VL]接头[VH]
sc(Fv)2是将2个VH和2个VL通过接头等结合形成单链的小分子抗体(Hudson等人,J Immunol.Methods 1999;231:177-189)。sc(Fv)2例如可以通过用接头连接scFv来制作。
另外,优选2个VH和2个VL以单链多肽的N末端侧为基点按照VH、VL、VH、VL([VH]接头[VL]接头[VH]接头[VL])的顺序进行排列为特征的抗体,2个VH和2个VL的顺序并非特别限于上述排列,可以按照任意顺序进行排列。例如有下述排列。
[VL]接头[VH]接头[VH]接头[VL]
[VH]接头[VL]接头[VL]接头[VH]
[VH]接头[VH]接头[VL]接头[VL]
[VL]接头[VL]接头[VH]接头[VH]
[VL]接头[VH]接头[VL]接头[VH]
可以对小分子抗体中VH或VL的氨基酸序列进行取代、缺失、添加和/或插入。并且,当使VH与VL缔合时,只要具有抗原结合活性即可,可以使一部分缺失,还可以添加其他多肽。另外,可以将可变区嵌合化或人源化。
在本发明中,连接抗体可变区的接头可以使用能通过基因工程导入的任意的肽接头或合成化合物接头、例如Protein Engineering,9(3),299-305,1996中公开的接头。
在本发明中,优选的接头为肽接头。对肽接头的长度没有特别限定,本领域技术人员可以根据目的适当选择,但通常为1~100氨基酸、优选3~50氨基酸、进一步优选5~30氨基酸、特别优选为12~18氨基酸(例如15氨基酸)。
作为肽接头的氨基酸序列,例如有下述序列。
Ser
Gly·Ser
Gly·Gly·Ser
Ser·Gly·Gly
Gly·Gly·Gly·Ser(SEQ ID NO:45)
Ser·Gly·Gly·Gly(SEQ ID NO:46)
Gly·Gly·Gly·Gly·Ser(SEQ ID NO:47)
Ser·Gly·Gly·Gly·Gly(SEQ ID NO:48)
Gly·Gly·Gly·Gly·Gly·Ser(SEQ ID NO:49)
Ser·Gly·Gly·Gly·Gly·Gly(SEQ ID NO:50)
Gly·Gly·Gly·Gly·Gly·Gly·Ser(SEQ ID NO:51)
Ser·Gly·Gly·Gly·Gly·Gly·Gly(SEQ ID NO:52)
(Gly·Gly·Gly·Gly·Ser(SEQ ID NO:47))n
(Ser·Gly·Gly·Gly·Gly(SEQ ID NO:48))n
[n为1以上的整数]等。
关于肽接头的氨基酸序列,本领域技术人员根据目的可以适当选择。例如,决定上述肽接头长度的n通常为1~5,优选1~3,更优选为1或2。
合成化合物接头(化学交联剂)是通常用于肽交联的交联剂,例如有:N-羟基琥珀酰亚胺(NHS)、辛二酸二琥珀酰亚胺酯(DSS)、辛二酸双(磺基琥珀酰亚胺酯)(BS3)、二硫代双(丙酸琥珀酰亚胺酯)(DSP)、二硫代双(丙酸磺基琥珀酰亚胺酯)(DTSSP)、乙二醇双(琥珀酸琥珀酰亚胺酯)(EGS)、乙二醇双(琥珀酸磺基琥珀酰亚胺酯)(Sulfo-EGS)、二琥珀酰亚氨基酒石酸盐(DST)、二磺基琥珀酰亚氨基酒石酸盐(Sulfo-DST)、双[2-(琥珀酰亚氨氧基羰基氧基)乙基]砜(BSOCOES)、双[2-(磺基琥珀酰亚氨氧基羰基氧基)乙基]砜(Sulfo-BSOCOES)等,上述交联剂市场上可以买到。
在连接4个抗体可变区时,通常需要3个接头。这些接头可以使用相同的也可以使用不同的。
本发明的抗体还包括:在本发明抗体的氨基酸序列中添加有1个或多个氨基酸残基的抗体。上述抗体与其他肽或蛋白融合的融合蛋白也包括在内。制作融合蛋白的方法可以采用本领域技术人员所公知的方法,例如连接编码本发明抗体的多核苷酸与编码其它肽或多肽的多核苷酸使构架一致,之后将其导入表达载体中,使之在宿主中表达即可。作为用于与本发明抗体融合的其他肽或多肽,例如有FLAG(Hopp,T.P.等人,Bio Technology(1988)6,1204-1210)、包含6个His(组氨酸)残基的6×His、10×His、流感血凝素(HA)、人c-myc片段、VSV-GP片段、p18HIV片段、T7-tag、HSV-tag、E-tag、SV40T抗原片段、lcktag、α-微管蛋白片段、B-tag、蛋白C片段等公知的肽。另外,作为用于与本发明抗体融合的其他多肽,例如有:GST(谷胱甘肽-S-转移酶)、HA(流感血凝素)、免疫球蛋白恒定区、β-半乳糖苷酶、MBP(麦芽糖结合蛋白)等。使编码市售的上述肽或多肽的多核苷酸与编码本发明抗体的多核苷酸融合,并使由此制备的融合多核苷酸表达,从而可以制备融合多肽。
本发明的抗体可以是与聚乙二醇(PEG)或透明质酸等高分子物质、放射性物质、荧光物质、发光物质、酶、毒素等各种分子结合的缀合抗体。上述缀合抗体可以通过对所得抗体实施化学修饰而得到。需要说明的是,抗体的修饰方法在该领域已经确立(例如US5057313、US5156840)。本发明中的“抗体”还包含上述缀合抗体。
本发明的抗体还包含糖链发生修饰的抗体。
并且,本发明所使用的抗体可以是双重特异性抗体(双特异性抗体,bispecific antibody)。双重特异性抗体是指在同一抗体分子内具有识别不同表位的可变区的抗体。本发明中,双重特异性抗体可以是识别IL-6受体分子上的不同表位的双重特异性抗体,还可以是其中一个抗原结合部位识别IL-6受体、另一个抗原结合部位识别其他物质的双重特异性抗体。作为包含识别IL-6受体的本发明抗体的双重特异性抗体的另一个抗原结合部位所结合的抗原,例如有:IL-6,TNFα,TNFR1,TNFR2,CD80,CD86,CD28,CD20,CD19,IL-1α,IL-β,IL-1R,RANKL,RANK,IL-17,IL-17R,IL-23,IL-23R,IL-15,IL-15R,BlyS,淋巴细胞毒素α,淋巴细胞毒素β,LIGHT配体,LIGHT,VLA-4,CD25,IL-12,IL-12R,CD40,CD40L,BAFF,CD52,CD22,IL-32,IL-21,IL-21R,GM-CSF,GM-CSFR,M-CSF,M-CSFR,IFN-α,VEGF,VEGFR,EGF,EGFR,CCR5,APRIL,APRILR等。
用于制备双重特异性抗体的方法是公知的。例如,使识别抗原不同的2种抗体结合,可以制作双重特异性抗体。进行结合的抗体可以是分别具有H链和L链的1/2分子,也可以是仅包含H链的1/4分子。或者,使产生不同单克隆抗体的杂交瘤融合,也可以制作双重特异性抗体产生融合细胞。还可以利用基因工程学方法制作双重特异性抗体。
如后所述,本发明的抗体根据产生抗体的细胞或宿主或者纯化方法而在氨基酸序列、分子量、等电点、或是否具有糖链、或构象等方面有所不同。但所得抗体只要与本发明的抗体具有同等的功能,即包含在本发明中。例如,使本发明的抗体在原核细胞、例如大肠杆菌中表达时,在原抗体(original antibody)的氨基酸序列的N末端添加甲硫氨酸残基。本发明的抗体也包括这样的抗体。
本发明的抗IL-6受体抗体等多肽可以利用本领域技术人员所公知的方法进行制备。
例如,根据所得抗IL-6受体抗体的序列,使用本领域技术人员所公知的基因重组技术,可以制作抗IL-6受体抗体。具体而言,根据识别IL-6受体的抗体的序列,构建编码抗体的多核苷酸,将其导入表达载体中,之后使之在适当的宿主细胞中表达即可(例如参照Co,M.S.等人,J.Immunol.(1994)152,2968-2976;Better,M.and Horwitz,A.H.,Methods Enzymol.(1989)178,476-496;Pluckthun,A.and Skerra,A.,Methods Enzymol.(1989)178,497-515;Lamoyi,E.,Methods Enzymol.(1986)121,652-663;Rousseaux,J.等人,Methods Enzymol.(1986)121,663-669;Bird,R.E.and Walker,B.W.,Trends Biotechnol.(1991)9,132-137)。
因此,本发明提供制备本发明的多肽、或由编码本发明多肽的基因编码的多肽的方法,该方法包括培养宿主细胞的步骤,所述宿主细胞包含导入有编码本发明多肽的多核苷酸的载体。
更具体而言,提供本发明的多肽的制备方法,该方法包括以下步骤。
(a)培养宿主细胞的步骤,所述宿主细胞包含导入有编码本发明多肽的基因的载体;
(b)取得由该基因编码的多肽的步骤。
载体的例子有:M13系载体、pUC系载体、pBR322、pBluescript、pCR-Script等。为了亚克隆、切除cDNA时,除上述载体外,例如还有pGEM-T、pDIRECT、pT7等。为了生产本发明的抗体而使用载体时,表达载体特别有用。作为表达载体,例如为了在大肠杆菌中表达时,载体除了具有在大肠杆菌中扩增的特征外,还必须持有可以在JM109、DH5α、HB101、XL1-Blue等大肠杆菌宿主中高效率地表达的启动子、例如lacZ启动子(Ward等人,Nature(1989)341,544-546;FASEB J.(1992)6,2422-2427)、araB启动子(Better等人,Science(1988)240,1041-1043)或T7启动子等。作为这样的载体,除上述载体外,还有pGEX-5X-1(Pharmacia制)、“QIAexpress system”(Quiagen制)、pEGFP或pET(此时宿主优选表达T7 RNA聚合酶的BL21)等。
在表达质粒的载体中可以包含用于抗体分泌的信号序列。作为用于抗体分泌的信号序列,当向大肠杆菌的周质中产生时,可以使用pelB信号序列(Lei,S.P.等人,J.Bacteriol.(1987)169,4379)。例如可以利用氯化钙法、电穿孔法将载体导入宿主细胞中。
除大肠杆菌外,作为用于制备本发明抗体的载体,例如还有:来源于哺乳动物的表达载体(例如pcDNA3(Invitrogen社制)或pEF-BOS(Nucleic Acids.Res.1990,18(17),第5322页)、pEF、pCDM8);来源于昆虫细胞的表达载体(例如“Bac-to-BAC杆状病毒表达系统”(Gibco-BRL制)、pBacPAK8);来源于植物的表达载体(例如pMH1、pMH2);来源于动物病毒的表达载体(例如pHSV、pMV、pAdexLcw);来源于反转录病毒的表达载体(例如pZIPneo);来源于酵母的表达载体(例如“Pichia Expression试剂盒”(Invitrogen制)、pNV11、SP-Q01);来源于枯草杆菌的表达载体(例如pPL608、pKTH50)。
为了在CHO细胞、COS细胞、NIH3T3细胞等动物细胞中表达时,表达质粒的载体必须具有在细胞内表达所必需的启动子、例如SV40启动子(Mulligan等人,Nature(1979)277,108)、MMLV-LTR启动子、EF1α启动子(Mizushima等人,Nucleic Acids Res.(1990)18,5322)、CMV启动子等,进一步优选具有用于选择转化细胞的基因(例如可用药物(新霉素、G418等)判别的耐药性基因)。作为具有上述特性的载体,例如有:pMAM、pDR2、pBK-RSV、pBK-CMV、pOPRSV、pOP13等。
并且,为了稳定表达基因且扩增细胞内的基因拷贝数时,可以列举以下方法,即向缺乏核酸合成路径的CHO细胞中导入填补该路径的具有DHFR基因的载体(例如pSV2-dhfr(“Molecular Cloning第二版”Cold Spring Harbor Laboratory Press,(1989))等),再用甲氨蝶呤(MTX)扩增该载体的方法。另外,为了瞬时表达基因,可以列举以下方法,即使用染色体上具有表达SV40 T抗原的基因的COS细胞,用持有SV40的复制起点的载体(pcD等)进行转化的方法。复制起点可以使用来源于多瘤病毒、腺病毒、牛乳头状瘤病毒(BPV)等的起点。为了在宿主细胞系中扩增基因拷贝数,表达载体可以进一步含有氨基糖苷转移酶(APH)基因、胸苷激酶(TK)基因、大肠杆菌黄嘌呤鸟嘌呤磷酸核糖转移酶(Ecogpt)基因、二氢叶酸还原酶(dhfr)基因等作为选择标记。
如此操作而得到的本发明抗体,可以将其从宿主细胞内或细胞外(培养基等)分离,并纯化成实质上纯粹且均匀的抗体。抗体的分离、纯化可以使用通常抗体的纯化中所使用的分离、纯化方法,但没有任何限制。例如,可以适当选择、组合层析柱、过滤器、超滤、盐析、溶剂沉淀、溶剂提取、蒸馏、免疫沉淀、SDS-聚丙烯酰胺凝胶电泳、等电点电泳法、透析、重结晶等来分离、纯化抗体。
层析例如有:亲合层析、离子交换层析、疏水层析、凝胶过滤、反相层析、吸附层析等(Strategies for Protein Purification andCharacterization(蛋白质纯化与鉴定实验指南):A Laboratory CourseManual.Ed Daniel R.Marshark等,Cold Spring Harbor Laboratory Press,1996)。上述层析可以使用液相层析例如HPLC、FPLC等来进行。用于亲合层析的柱有:蛋白A柱、蛋白G柱。蛋白A柱例如有:HyperD,POROS,Sepharose FF(GE Amersham Biosciences)等。本发明还包含利用上述纯化方法被高度纯化的抗体。
所得抗体与IL-6受体的结合活性可以利用本领域技术人员所公知的方法进行测定。例如,作为测定抗体的抗原结合活性的方法,可以采用ELISA(酶联免疫吸附测定法)、EIA(酶免疫测定法)、RIA(放射免疫测定法)或荧光抗体法。例如,采用酶免疫测定法时,在包被有抗原的平板上加入含有抗体的试样、例如抗体产生细胞的培养上清或纯化抗体。添加用碱性磷酸酶等酶标记的二次抗体并温育平板,清洗后加入对硝基苯磷酸等酶底物,之后测定吸光度,从而可以评价抗原结合活性。
药物组合物
本发明还提供含有上述多肽作为有效成分的药物组合物。本发明的药物组合物可用于与IL-6相关的类风湿性关节炎等疾病。即,本发明还提供以上述抗体作为有效成分的类风湿性关节炎等疾病的治疗剂。作为本发明的对象疾病的优选例子有:类风湿性关节炎、幼年特发性关节炎、全身型幼年特发性关节炎、卡斯尔曼病、系统性红斑狼疮(SLE)、狼疮肾炎、克罗恩氏病(Crohn’s disease)、淋巴瘤、溃疡性大肠炎、贫血、血管炎、川崎病、Still病、淀粉样变性病、多发性硬化症、移植、老年性黄斑变性、强直性脊椎炎、干癣、干癣性关节炎、慢性阻塞性肺病(COPD)、IgA肾病、变形性关节症、哮喘、糖尿病性肾病、GVHD、子宫内膜症、肝炎(NASH)、心肌梗塞、动脉硬化、败血症、骨质疏松症、糖尿病、多发性骨髓瘤、前列腺癌、肾癌、B细胞性非霍奇金淋巴瘤、胰癌、肺癌、食道癌、大肠癌、癌恶液质、癌神经浸润、心肌梗塞、近视性脉络膜新生血管、特发性脉络膜新生血管、葡萄膜炎、慢性甲状腺炎、延迟性过敏症、接触性皮炎、特应性皮炎、间皮瘤、多发性肌炎、皮肌炎、全葡萄膜炎、前葡萄膜炎、中间葡萄膜炎、巩膜炎、角膜炎、眼眶炎症、视神经炎、糖尿病视网膜病变、增生性玻璃体视网膜病变、干眼、术后炎症等,但并不限于这些。
“含有抗IL-6受体抗体作为有效成分”是指含有抗IL-6受体抗体作为至少一种活性成分,对其含有率没有限定。本发明的药物组合物除含有上述多肽外,可以一并含有其他有效成分。
需要说明的是,本发明的药物组合物不仅用于治疗目的,还可用于预防目的。
本发明的多肽可以按照常规方法制成制剂(例如,Remington’sPharmaceutical Science,latest edition,Mark Publishing Company,Easton,U.S.A)。并且,根据需要,可以同时含有药物上可接受的载体和/或添加剂。例如可以含有:表面活性剂(PEG、Tween等)、赋形剂、抗氧剂(抗坏血酸等)、着色剂、着香剂、保存剂、稳定剂、缓冲剂(磷酸、枸橼酸、其他有机酸等)、螯合剂(EDTA等)、悬浮剂、等渗剂、粘合剂、崩解剂、润滑剂、助流剂、矫味剂等。但本发明的炎症性疾病的预防或治疗剂并不限于这些,可以适当含有其他常用的载体。具体有:轻质硅酸酐、乳糖、结晶纤维素、甘露醇、淀粉、羧甲基纤维素钙、羧甲基纤维素钠、羟丙基纤维素、羟丙基甲基纤维素、聚乙烯缩醛二乙基氨基乙酸酯、聚乙烯吡咯烷酮、明胶、中链脂肪酸三甘油酯、聚氧乙烯氢化蓖麻油60、蔗糖、羧甲基纤维素、玉米淀粉、无机碱等。此外,可以含有其他小分子的多肽、血清白蛋白、明胶和免疫球蛋白等蛋白、以及氨基酸。制成注射用水溶液时,例如可以将抗IL-6受体抗体溶于含有生理盐水、葡萄糖或其他辅剂的等渗溶液中。作为辅剂,例如有:D-山梨醇、D-倭勒米糖、D-甘露醇、氯化钠,还可以与适当的助溶剂、例如醇(乙醇等)、多元醇(丙二醇、PEG等)、非离子性表面活性剂(聚山梨酯80、HCO-50)等结合使用。
此外,根据需要,可以将多肽封入微囊(羟甲基纤维素、明胶、聚甲基丙烯酸甲酯]等的微囊)中、或者制成胶体给药系统(脂质体、白蛋白微球、微乳、纳米微粒和纳米囊等)(参照Remington’s PharmaceuticalScience第16版,Oslo Ed.(1980)等)。并且,将药物制成缓释制剂的方法也已公知,可应用于多肽(Langer等人,J.Biomed.Mater.Res.(1981)15:167-277;Langer,Chem.Tech.(1982)12:98-105;美国专利第3,773,919号;欧州专利申请公开(EP)第58,481号;Sidman等人,Biopolymers(1983)22:547-56;EP第133,988号)。并且,通过在本剂中添加或者混合透明质酸酶,还可以增加皮下给药的液量(例如WO2004/078140等)。
本发明的药物组合物可以口服给药,也可以胃肠外给药,但优选胃肠外给药。具体而言,对患者进行注射给药和经皮给药。注射剂型的例子有:例如可以通过静脉内注射、肌肉内注射或皮下注射等进行全身或局部给药。可以对治疗部位或其周边进行局部注入,特别是肌肉内注射。经皮给药剂型的例子有:例如软膏剂、凝胶剂、霜剂、湿布剂和贴剂等,可以进行全身或局部给药。此外,可以根据患者的年龄、症状选择适当的给药方法。给药量例如可以是:每次每1kg体重在0.0001mg~100mg的范围内选择活性成分。或者,例如对病人给药时,每名患者以0.001~1000mg/kg体重的范围选择活性成分,每次的给药量例如优选含有0.01~50mg/kg体重左右的量的本发明抗体。但本发明的药物组合物并不限于上述给药量。
需要说明的是,本发明中记载的氨基酸序列中所含的氨基酸有时在翻译后会经受修饰(例如N末端的谷氨酰胺通过焦谷氨酰化(pyroglutamylation)修饰成焦谷氨酸,这是本领域技术人员所熟知的修饰),即使是按照这种方式将氨基酸翻译后进行修饰,当然也包含在本发明所记载的氨基酸序列中。
进行结合的糖链的结构可以是任何结构。EU编号的第297位糖链可以是任何的糖链结构(优选岩藻糖基化的糖链),也可以不结合糖链(例如可以在大肠杆菌中生产、或者进行修饰使糖链不结合在EU编号的第297位上)。
需要说明的是,本说明书中引用的所有在先技术文献均作为参照而纳入本说明书中。
实施例
以下,通过实施例具体说明本发明,但本发明并不限于这些实施例。
[实施例1]提高TOCILIZUMAB与IL-6受体的亲和性的可变区突变位点的鉴定
为了提高TOCILIZUMAB(H链WT-IgG1/SEQ ID NO:53、L链WT-κ/SEQ ID NO:54)与IL-6受体的亲和性,制作在CDR序列中导入有突变的文库进行研究。筛选在CDR中导入有突变的文库,结果发现了提高与IL-6受体的亲和性的突变,将其汇总在图1中。组合有这些突变的高亲和性TOCILIZUMAB的例子有:RDC-23(H链RDC23H-IgG1/SEQ ID NO:55、L链RDC-23L-κ/SEQ ID NO:56)。将RDC-23的与可溶型IL-6受体的亲和性和BaF/gp130所产生的生物活性、与TOCILIZUMAB进行比较(方法参照参考例)。
亲和性的测定结果见表1。BaF/gp130所产生的生物活性(IL-6终浓度为30ng/mL)的测定结果见图2。结果发现:与TOCILIZUMAB相比,RDC-23的亲和性提高约60倍,作为BaF/gp130的100%抑制浓度,提高约100倍活性。
[表1]
[实施例2]提高由TOCILIZUMAB的等电点降低引起的药物动力学的突变的鉴定
为了提高TOCILIZUMAB的药物动力学,研究在不使与IL-6受体的结合大幅下降的情况下可以降低可变区的等电点的突变位点。筛选由TOCILIZUMAB的立体结构模型推测的可变区突变位点,结果发现了在不使与IL-6受体的结合大幅下降的情况下可以降低可变区的等电点的突变位点,将它们汇总在图3中。组合有这些突变的等电点降低TOCILIZUMAB的例子有:H53/L28(H链H53-IgG1/SEQ ID NO:57、L链L28-κ/SEQ ID NO:58)。将H53/L28的与可溶型IL-6受体的亲和性、BaF/gp130所产生的生物活性、等电点和在小鼠体内的药物动力学、与TOCILIZUMAB进行比较(方法参照参考例)。
亲和性的测定结果见表2。BaF/gp130所产生的生物活性(IL-6终浓度为30ng/mL)的测定结果见图4。与TOCILIZUMAB相比,H53/L28的亲和性提高约6倍,作为BaF/gp130的100%抑制浓度,活性提高数倍左右。
[表2]
利用本领域技术人员公知的等电点电泳测定等电点,结果TOCILIZUMAB的等电点为约9.3,H53/L28的等电点为约6.5~6.7,H53/L28与TOCILIZUMAB相比等电点下降约2.7。通过GENETYX(GENETYX CORPORATION)计算可变区VH/VL的理论等电点时,TOCILIZUMAB的理论等电点为9.20,H53/L28的理论等电点为4.52,H53/L28与TOCILIZUMAB相比等电点下降约4.7。
为了评价等电点降低了的修饰抗体H53/L28的药物动力学,比较TOCILIZUMAB和H53/L28在正常小鼠体内的药物动力学。将TOCILIZUMAB和H53/L28以1mg/kg对小鼠(C57BL/6J、日本CharlesRiver)进行静脉内(IV)和皮下(SC)单次给药,评价血浆中的浓度变化。TOCILIZUMAB和H53/L28静脉内给药后的血浆中浓度变化见图5,皮下给药后的血浆中浓度变化见图6,通过WinNonlin(Pharsight社制)得到的药物动力学参数(清除率(CL)、半衰期(T1/2))见表3。H53/L28静脉内给药后的血浆中半衰期(T1/2)延长至TOCILIZUMAB的约1.3倍,清除率下降约1.7倍。H53/L28皮下给药后的T1/2延长至TOCILIZUMAB的约2倍,清除率下降约2.1倍。结果发现:通过如此地利用氨基酸取代来降低TOCILIZUMAB的等电点,可以大幅提高药物动力学。
[表3]
[实施例3]降低TOCILIZUMAB的免疫原性的突变位点的鉴定降低由存在于可变区的T细胞表位产生的免疫原性风险的突变位点的 鉴定
使用TEPITOPE(Methods.2004 Dec;34(4):468-75)分析存在于TOCILIZUMAB的可变区序列中的T细胞表位。其结果,预测L链CDR2中存在多个与HLA结合的T细胞表位(存在免疫原性风险高的序列)。因此,在TEPITOPE分析中,研究了降低L链CDR2的免疫原性风险但不降低稳定性、结合活性、中和活性的氨基酸取代。
筛选的结果发现:通过象下述那样将TOCILIZUMAB的L链CDR2(SEQ ID NO:59)的L51(Kabat编号、Kabat EA等人.1991.Sequences of Proteins of Immunological Interest.NIH)的苏氨酸取代成甘氨酸、将L53的精氨酸取代成谷氨酸(SEQ ID NO:60),可以在不降低稳定性、结合活性、中和活性的情况下降低免疫原性风险。
TOCILIZUMAB L链CDR2(SEQ ID NO:59)
除去了T细胞表位的TOCILIZUMAB L链CDR2(SEQ ID NO:60)
[实施例4]TOCILIZUMAB的可变区构架序列的完全人源化所引起的免疫原性风险的降低
在TOCILIZUMAB的可变区序列中,H链FR1的H27,H28,H29,H30和H链FR3的H71(Kabat编号、Kabat EA等人.1991.Sequencesof Proteins of Immunological Interest.NIH)为了在人源化过程中维持结合活性,在构架序列中残留有小鼠序列(Cancer Res.1993 Feb 15;53(4):851-6)。由于残留的小鼠序列是提高免疫原性风险的原因,所以为了进一步降低TOCILIZUMAB的免疫原性风险,研究将构架序列完全人源化。
结果发现:通过将TOCILIZUMAB的H链FR1(SEQ ID NO:61)取代成下述的人源化H链FR1-A(SEQ ID NO:62)、将TOCILIZUMAB的H链FR3(SEQ ID NO:63)取代成下述的人源化H链FR3(SEQ IDNO:64),可以在不降低稳定性、结合活性、中和活性的情况下将TOCILIZUMAB的整个构架完全人源化。
TOCILIZUMAB H链FR1(SEQ ID NO:61)
人源化H链FR1-A(SEQ ID NO:62)(来自种系IMGT hVH_4_)
TOCILIZUMAB H链FR3(SEQ ID NO:63)
人源化H链FR3(SEQ ID NO:64)(来自Mol.Immunol.2007,44(4):412-422)
[实施例5]用于提高TOCILIZUMAB与IL-6受体的pH依赖性结合的药物动力学的突变位点的鉴定
作为提高TOCILIZUMAB的药物动力学的方法之一,考虑改良分子使1分子的TOCILIZUMAB反复结合、中和多个IL-6受体的方法。认为:TOCILIZUMAB与膜型IL-6受体结合后,保持与膜型IL-6受体结合的状态不变,通过内化整合到细胞内的核内体中,之后TOCILIZUMAB在与膜型IL-6受体结合的状态下直接移至溶酶体中,同时被溶酶体分解。即,认为通常1分子的TOCILIZUMAB与1分子~2分子的膜型IL-6受体(一价~二价)结合,内化后被溶酶体分解,所以1分子的TOCILIZUMAB只能结合、中和1分子~2分子的膜型IL-6受体。
因此,如图7所示,认为只要能够制作出在中性条件下TOCILIZUMAB的结合得到维持而在酸性条件下TOCILIZUMAB的结合大幅降低的pH依赖性结合的TOCILIZUMAB,pH依赖性结合的TOCILIZUMAB在核内体内从作为抗原的膜型IL-6受体上解离,与存在于核内体内的FcRn结合,从而可以返回到血浆中,返回到血浆中的pH依赖性结合的TOCILIZUMAB可以再次与膜型IL-6受体结合。认为通过反复进行上述在血浆中的结合和在核内体内的解离,1分子的TOCILIZUMAB可以反复结合、中和多个分子的IL-6受体,由此认为:与TOCILIZUMAB相比,pH依赖性结合的TOCILIZUMAB的药物动力学得到提高。
为了使TOCILIZUMAB在核内体内的酸性条件下从IL-6受体上解离,与中性条件下相比必需大幅降低酸性条件下的结合。由于TOCILIZUMAB在细胞表面必需与IL-6受体强有力结合以将其中和,所以在细胞表面的pH7.4下抗体必需与IL-6受体进行和TOCILIZUMAB相比为同等程度以上的结合。有报道称核内体内的pH通常为pH5.5~pH6.0(Nat Rev Mol Cell Biol.2004 Feb;5(2):121-32),由此认为:只要是改良成在pH5.5~pH6.0下与IL-6受体弱结合的pH依赖性结合的TOCILIZUMAB,在核内体内的酸性条件下即可从IL-6受体上解离。即,认为只要是改良成在细胞表面的pH7.4下与IL-6受体强有力结合、在核内体内的pH5.5~pH6.0下与IL-6受体弱结合的pH依赖性结合的TOCILIZUMAB,即可以以1分子与多个IL-6受体结合、中和,由此提高药物动力学。
为了对TOCILIZUMAB与IL-6受体的结合赋予pH依赖性,考虑下述方法:将pKa存在于6.0~6.5左右、在中性条件下(pH7.4)与酸性条件下(pH5.5~pH6.0)之间质子的解离状态发生变化的组氨酸残基导入TOCILIZUMAB的可变区中。因此,筛选由TOCILIZUMAB的立体结构模型推测的可变区的组氨酸导入位点。另外,制作设计成TOCILIZUMAB的被选择的可变区序列随机置换成组氨酸的文库,进行筛选。以在pH7.4下与IL-6受体结合、在pH5.5~pH5.8下从IL-6受体上解离或亲和性降低为指标进行筛选。
结果发现了可以赋予TOCILIZUMAB与IL-6受体显示pH依赖性结合(pH7.4时结合、pH5.8时解离的性质)的突变位点,将它们汇总在图8中。图8的H27的酪氨酸被取代成组氨酸,这并不是CDR的突变,而是H链的FR1的突变,但如Eur.J.Immunol.1992.22:1719-1728所述,H27为组氨酸时的序列以人序列(SEQ ID NO:65)的形式存在,所以通过使用下述的构架,可以与实施例4一并进行完全人源化。
人源化H链FR1-B(SEQ ID NO:65)
组合有上述突变的pH依赖性结合的TOCILIZUMAB的例子有:H3pI/L73(H链H3pI-IgG1/SEQ ID NO:66、L链L73-κ/SEQ ID NO:67)。将H3pI/L73的在pH7.4下与可溶型IL-6受体的亲和性、在pH7.4和pH5.8下从膜型IL-6受体上的解离速度、BaF/gp130所产生的生物活性以及在食蟹猴和人IL-6受体转基因小鼠体内的药物动力学、与TOCILIZUMAB进行比较(方法参照参考例)。
在pH7.4下与可溶型IL-6受体的亲和性的测定结果见表4。BaF/gp130所产生的生物活性(IL-6终浓度为30ng/mL)的测定结果见图9。显示出H3pI/L73与TOCILIZUMAB相比具有大致相同的、在pH7.4下与可溶型IL-6受体的亲和性和BaF/gp130的活性。
[表4]
TOCILIZUMAB和H3pI/L73在pH7.4和pH5.8下从膜型IL-6受体上的解离速度的测定结果见表5。显示出:与TOCILIZUMAB相比,H3pI/L73在pH5.8时的解离速度变快,从膜型IL-6受体上的解离速度的pH依赖性提高约2.6倍。
[表5]
将TOCILIZUMAB和H3pI/L73以1mg/kg对食蟹猴进行静脉内单次给药,评价血浆中浓度变化。TOCILIZUMAB和H3pI/L73静脉内给药后的血浆中浓度变化见图10。其结果,与TOCILIZUMAB相比,H3pI/L73在食蟹猴体内的药物动力学大幅改善。
将TOCILIZUMAB和H3pI/L73以25mg/kg对人IL-6受体转基因小鼠(hIL-6R tg小鼠、Proc Natl Acad Sci U S A.1995 May 23;92(11):4862-6)进行静脉内单次给药,评价血浆中浓度变化。TOCILIZUMAB和H3pI/L73静脉内给药后的血浆中浓度变化见图11。其结果,与TOCILIZUMAB相比,H3pI/L73在人IL-6受体转基因小鼠体内的药物动力学大幅改善。
与TOCILIZUMAB相比,pH依赖性结合的TOCILIZUMAB、即H3pI/L73在食蟹猴和人IL-6受体转基因小鼠体内的药物动力学大幅改善,由此认为:通过赋予在pH7.4时与抗原结合、在pH5.8时从抗原上解离的性质,1分子的H3pI/L73可以和多个IL-6受体进行结合、中和。另外,认为通过对与IL-6受体的结合赋予比H3pI/L73更强的pH依赖性,可以进一步改善药物动力学。
[实施例6]TOCILIZUMAB的恒定区的最优化
TOCILIZUMAB H链C末端的异质性的降低
作为IgG抗体H链C末端序列的异质性,有人报道了由C末端氨基酸的赖氨酸残基的缺失、以及C末端的甘氨酸、赖氨酸这2个氨基酸均缺失引起的C末端羧基的酰胺化(Anal Biochem.2007 Jan 1;360(1):75-83)。即使在TOCILIZUMAB中,其主要成分也是存在于核苷酸序列上的C末端氨基酸的赖氨酸通过翻译后修饰发生缺失的序列,但残留有赖氨酸的副成分、和甘氨酸、赖氨酸均缺失所引起的C末端羧基的酰胺化的副成分也作为异质性而存在。在维持目标物质/相关物质的异质性的制造间差的同时以药品的形式大量制造,这并不容易,而且成本也增加,所以希望尽可能是单一物质,在开发抗体作为药品时,希望降低上述异质性。因此,以药品形式进行开发时,希望H链C末端的异质性不存在。
为了降低C末端氨基酸的异质性而对C末端氨基酸进行修饰。结果发现,通过使TOCILIZUMAB H链恒定区的C末端的赖氨酸和甘氨酸预先在核苷酸序列上缺失,可以避免来自C末端的异质性。利用阳离子交换色谱评价TOCILIZUMAB、C末端缺失赖氨酸的TOCILIZUMAB(TOCILIZUMABΔK、H链WT-IgG1ΔK/SEQ ID NO:68、L链WT-κ/SEQ ID NO:54)和C末端缺失赖氨酸和甘氨酸的TOCILIZUMAB(TOCILIZUMABΔGK、H链WT-IgG1ΔGK/SEQ IDNO:69、L链WT-κ/SEQ ID NO:54)的异质性。柱使用ProPac WCX-10,4×250mm(Dionex),流动相A使用25mmol/L MES/NaOH,pH6.1;流动相B使用25mmol/L MES/NaOH,250mmol/L NaCl,pH6.1,采用适当的流量和梯度进行实施。利用阳离子交换色谱进行评价的结果见图12。其结果,通过预先使不仅H链恒定区C末端的赖氨酸、H链恒定区C末端的赖氨酸和甘氨酸也均在核苷酸序列上缺失,首次发现可以降低C末端氨基酸的异质性。在人抗体恒定区IgG1、IgG2、IgG4中,C末端序列均为:EU编号(参照Sequences of proteins of immunologicalinterest,NIH Publication No.91-3242)第447位变为赖氨酸、第446位变为甘氨酸,由此认为:本研究中发现的、降低C末氨基酸的异质性的方法也可用于IgG2恒定区和IgG4恒定区或它们的变体。
TOCILIZUMAB的IgG2同种型中来自二硫键的异质性的下降
TOCILIZUMAB的同种型是IgG1,但由于TOCILIZUMAB是中和抗体,所以在考虑免疫原性和副作用时,认为与Fcγ受体的结合有可能并不优选。作为降低与Fcγ受体的结合的方法,考虑将IgG抗体的同种型从IgG1变成IgG2或IgG4的方法(Ann Hematol.1998 Jun;76(6):231-48),从与Fcγ受体I的结合和药物动力学的角度考虑,与IgG4相比,更优选IgG2(Nat Biotechnol.2007 Dec;25(12):1369-72)。另一方面,开发抗体作为药品时,其蛋白的理化性质、其中均匀性和稳定性极其重要,关于IgG2同种型,有报道称其来自铰链区的二硫键的异质性非常多(J Biol Chem.2008 Jun 6;283(23):16206-15)。在维持来自其的目标物质/相关物质的异质性的制造间差的同时以药品的形式大量制造,这并不容易,而且成本增加,所以希望尽可能是单一物质。因此,开发IgG2同种型的抗体作为药品时,希望在不降低稳定性的情况下使来自二硫键的异质性降低。
为了降低IgG2同种型的异质性,对各种变体进行了研究,结果发现:在IgG2恒定区序列中,通过WT-SKSC(SEQ ID NO:70)可以在不降低稳定性的情况下降低异质性,所述WT-SKSC是指将存在于H链的CH1结构域的EU编号第131位的半胱氨酸和第133位的精氨酸分别取代成丝氨酸和赖氨酸、并且将存在于H链的上铰链(upper hinge)的EU编号第219位的半胱氨酸取代成丝氨酸的恒定区。制备TOCILIZUMAB-IgG1(H链WT-IgG1/SEQ ID NO:53、L链WT-κ/SEQ ID NO:54)、TOCILIZUMAB-IgG2(H链WT-IgG2/SEQ IDNO:71、L链WT-κ/SEQ ID NO:54)和TOCILIZUMAB-SKSC(H链WT-SKSC/SEQ ID NO:70、L链WT-κ/SEQ ID NO:54),评价异质性和稳定性。异质性的评价通过阳离子交换色谱来进行。柱使用ProPacWCX-10(Dionex),流动相A使用20mM乙酸钠,pH5.0;流动相B使用20mM乙酸钠、1M NaCl、pH5.0;采用适当的流量和梯度来实施。利用阳离子交换色谱进行评价的结果见图13。稳定性评价通过使用差示扫描量热法(DSC)(VP-DSC、Microcal社制)进行的热变性中间温度(Tm值)的评价来实施。在20mM乙酸钠、150mM NaCl、pH6.0的条件下,DSC测定结果和Fab结构域的Tm值见图14。
结果发现:与TOCILIZUMAB-IgG1相比,TOCILIZUMAB-IgG2的异质性显著增大,但通过形成TOCILIZUMAB-SKSC,可以大幅降低异质性。与TOCILIZUMAB-IgG1相比,TOCILIZUMAB-IgG2在DSC中的Fab结构域的热变性峰中确认到认为是由杂成分产生的、稳定性低即Tm低的肩峰(Fab*)成分,但通过形成TOCILIZUMAB-SKSC,认为是由杂成分产生的Tm值低的肩峰消失,显示出与TOCILIZUMAB-IgG1和TOCILIZUMAB-IgG2的Fab结构域同等的约94℃的Tm值,由此发现:TOCILIZUMAB-SKSC具有高稳定性。
提高TOCILIZUMAB的药物动力学的恒定区突变位点的鉴定
如上所述,发现由作为TOCILIZUMAB的同种型的IgG1可以降低C末端的异质性、以及可以在降低与Fcγ受体的结合性且维持高稳定性的状态下降低IgG2同工型的恒定区的抗体的异质性,但关于药物动力学,也希望是比TOCILIZUMAB的同种型-IgG1具有优异的恒定区。
为了找到比IgG1同种型的恒定区的抗体具有优异的血浆中半衰期的恒定区,对于具有高稳定性、与IgG2同种型恒定区的抗体有关的上述异质性已降低的TOCILIZUMAB-SKSC,为了提高其药物动力学,筛选突变位点,结果发现了:将WT-SKSC的EU编号第137位的谷氨酸取代成甘氨酸、将第138位的丝氨酸取代成甘氨酸、将第268位的组氨酸取代成谷氨酰胺、将第355位的精氨酸取代成谷氨酰胺、将第419位的谷氨酰胺取代成谷氨酸、除此以外为了降低H链C末端的异质性而使第446位的甘氨酸和第447位的赖氨酸缺失的WT-M58(SEQ ID NO:72(氨基酸序列))。另一方面,制作了将IgG1的第434位的天冬酰胺取代成丙氨酸的WT-M44(SEQ ID NO:73(氨基酸序列))。并且,为了降低M44的H链C末端的异质性,制作了使M44的第446位的甘氨酸和第447位的赖氨酸缺失的WT-M83(SEQ ID NO:74(氨基酸序列))。制作了将WT-M58的第434位的天冬酰胺取代成丙氨酸的WT-M73(SEQ ID NO:75(氨基酸序列))。
制备TOCILIZUMAB-M44(H链WT-M44/SEQ ID NO:73、L链WT-κ/SEQ ID NO:54)、TOCILIZUMAB-M58(H链WT-M58/SEQ IDNO:72、L链WT-κ/SEQ ID NO:54)和TOCILIZUMAB-M73(H链WT-M73/SEQ ID NO:75、L链WT-κ/SEQ ID NO:54),评价与人FcRn的亲和性和在人FcRn转基因小鼠体内的药物动力学(方法参照参考例)。
利用Biacore评价TOCILIZUMAB-IgG1、TOCILIZUMAB-M44、TOCILIZUMAB-M58和TOCILIZUMAB-M73与人FcRn的结合性,结果见表6,TOCILIZUMAB-M44、TOCILIZUMAB-M58和TOCILIZUMAB-M73的结合性分别比TOCILIZUMAB-IgG1强约2.7倍、约1.4倍和约3.8倍左右。
[表6]
评价TOCILIZUMAB-IgG1、TOCILIZUMAB-M44、TOCILIZU-MAB-M58和TOCILIZUMAB-M73在人FcRn转基因小鼠体内的药物动力学,结果见图15。如图15所示,发现与TOCILIZUMAB-IgG1相比,TOCILIZUMAB-M44、TOCILIZUMAB-M58和TOCILIZU-MAB-M73的药物动力学均有提高。该药物动力学的提高效果与和人FcRn的结合能力相关。其中,TOCILIZUMAB-M73与TOCILIZUMAB-IgG1相比,28天后的血浆中浓度改善约16倍,由此认为:即使在人体内,具有M73的恒定区的抗体与具有IgG1的恒定区的抗体相比药物动力学也大幅提高。
[实施例7]PK/PD已改善的完全人源化IL-6受体抗体的制作
制作组合有多个上述实施例中发现的TOCILIZUMAB的可变区和恒定区的突变的TOCILIZUMAB变体,进行各种筛选,结果发现了:作为完全人源化IL-6受体抗体的Fv3-M73(H链VH4-M73/SEQ IDNO:25、L链VL1-κ/SEQ ID NO:28)、Fv4-M73(H链VH3-M73/SEQID NO:26、L链VL3-κ/SEQ ID NO:29)、Fv5-M83(H链VH5-M83/SEQ ID NO:27、L链VL5-κ/SEQ ID NO:30)。
将制作的Fv3-M73、Fv4-M73和Fv5-M83与IL-6受体的亲和性、与TOCILIZUMAB进行比较(方法参照参考例)。测定上述抗体在pH7.4下与可溶型IL-6受体的亲和性,结果见表7。另外,将BaF/gp130的中和活性与TOCILIZUMAB和对照(参考例的公知的高亲和性高IL-6受体抗体、US2007/0280945中的VQ8F11-21 hIgG1)进行比较(方法参照参考例)。测定上述抗体的BaF/gp130所产生的生物活性,结果见图16(IL-6终浓度为300ng/mL:TOCILIZUMAB、对照、Fv5-M83)和图17(IL-6终浓度为30ng/mL:TOCILIZUMAB、Fv3-M73、Fv4-M73)。如表7所示,Fv3-M73、Fv4-M73与TOCILIZUMAB相比,具有2~3倍左右的强亲和性,Fv5-M83与TOCILIZUMAB相比,显示出100倍左右的强亲和性(由于在Fv5-M83中亲和性难以测定,所以使用将恒定区形成IgG1的Fv5-IgG1(H链VH5-IgG1/SEQ ID NO:76、L链VL5-κ/SEQ ID NO:30)来测定亲和性,认为恒定区通常不影响亲和性)。如图17所示,Fv3-M73、Fv4-M73与TOCILIZUMAB相比,显示出稍强的活性,如图16所示,Fv5-M83与TOCILIZUMAB相比,在50%抑制浓度下具有100倍以上的极强的活性,且与对照(公知的高亲和性高IL-6受体抗体)相比,在50%抑制浓度下也显示出约10倍左右的高中和活性。
[表7]
测定TOCILIZUMAB、Fv3-M73和Fv4-M73在pH7.4和pH5.8下从膜型IL-6受体上的解离速度,结果见表8(方法参照参考例)。结果显示:Fv3-M73和Fv4-M73与TOCILIZUMAB相比,自膜型IL-6受体上的解离速度的pH依赖性分别提高约11倍和10倍。与实施例5中的H3pI/L73相比,解离速度的pH依赖性大幅提高,由此认为:与H3pI/L73相比,Fv3-M73和Fv4-M73的药物动力学大幅提高。
[表8]
利用本领域技术人员公知的方法,通过等电点电泳测定TOCILIZUMAB、对照、Fv3-M73、Fv4-M73和Fv5-M83的等电点,结果TOCILIZUMAB的等电点为约9.3、对照的等电点为约8.4~8.5、Fv3-M73的等电点为约5.7~5.8、Fv4-M73的等电点为约5.6~5.7、Fv5-M83的等电点为5.4~5.5,与TOCILIZUMAB和对照相比,所有抗体的等电点均大幅下降。通过GENETYX(GENETYXCORPORATION)计算可变区VH/VL的理论等电点时,TOCILIZUMAB的理论等电点为9.20,对照的理论等电点为7.79,Fv3-M73的理论等电点为5.49、Fv4-M73的理论等电点为5.01、Fv5-M83的理论等电点为4.27,与TOCILIZUMAB和对照相比,所有抗体的等电点均大幅下降。实施例2显示:通过降低等电点,药物动力学得到改善,由此认为:与TOCILIZUMAB和对照相比,Fv3-M73、Fv4-M73和Fv5-M83的药物动力学有所提高。
利用TEPITOPE(Methods.2004 Dec;34(4):468-75)分析存在于TOCILIZUMAB、Fv3-M73、Fv4-M73和Fv5-M83的可变区序列中的T细胞表位。其结果,如实施例3所示,预测TOCILIZUMAB的多个序列存在与HLA结合的T细胞表位,但Fv3-M73、Fv4-M73和Fv5-M83的、预测结合在T细胞表位上的序列大幅减少。另外,Fv3-M73、Fv4-M73和Fv5-M83被完全人源化,在构架中没有残留小鼠序列。由此表明:Fv3-M73、Fv4-M73和Fv5-M83与TOCILIZUMAB相比,免疫原性风险有可能大幅降低。
[实施例8]完全人源化IL-6受体抗体在猴中的PK/PD试验
将TOCILIZUMAB、对照、Fv3-M73、Fv4-M73和Fv5-M83以1mg/kg对食蟹猴进行静脉内单次给药,评价血浆中浓度变化(方法参照参考例)。TOCILIZUMAB、Fv3-M73、Fv4-M73和Fv5-M83静脉内给药后的血浆中浓度变化见图18。其结果,Fv3-M73、Fv4-M73和Fv5-M83与TOCILIZUMAB和对照相比,在食蟹猴体内的药物动力学均大幅改善。其中,Fv3-M73和Fv4-M73的药物动力学与TOCILIZUMAB相比大幅改善。
为了评价食蟹猴膜型IL-6受体以某种程度被中和的药效,在抗体给药第6天~第18天(TOCILIZUMAB则为第3天~第10天)将食蟹猴IL-6以5μg/kg的量在腰背部连续进行皮下给药,测定24小时后各个体的CRP浓度(方法参照参考例)。各抗体给药时的CRP浓度变化见图19。为了评价食蟹猴可溶型IL-6受体以某种程度被中和的药效,测定食蟹猴血浆中的非结合型的食蟹猴可溶型IL-6受体浓度,计算非结合型的可溶型IL-6受体率(方法参照参考例)。各抗体给药时的非结合型的可溶型IL-6受体率的变化见图20。
Fv3-M73、Fv4-M73和Fv5-M83与TOCILIZUMAB和对照(公知的高亲和性抗IL-6受体抗体)相比,均更持续地中和食蟹猴膜型IL-6受体,长期抑制CRP的增加。Fv3-M73、Fv4-M73和Fv5-M83与TOCILIZUMAB和对照相比,均更持续地中和食蟹猴可溶型IL-6受体,长期抑制非结合型的食蟹猴可溶型IL-6受体的增加。由此发现:对于膜型IL-6受体和可溶型IL-6受体的中和的持续性,Fv3-M73、Fv4-M73和Fv5-M83均较TOCILIZUMAB和对照优异。其中,Fv3-M73和Fv4-M73的中和持续性极其优异。另一方面,与Fv3-M73和Fv4-M73相比,Fv5-M83将CRP和非结合型的食蟹猴可溶型IL-6受体抑制在低水平,由此认为:Fv5-M83比Fv3-M73、Fv4-M73和对照(公知的高亲和性抗IL-6受体抗体)更强效地中和膜型IL-6受体和可溶型IL-6受体。认为这是:与对照相比,Fv5-M83与IL-6受体的亲和性强、且在BaF/gp130中的生物活性强在食蟹猴体内得到反映的结果。
由此认为:与TOCILIZUMAB和对照相比,Fv3-M73和Fv4-M73作为抗IL-6受体中和抗体,其作用的持续性极其优异,可以大幅降低给药频率和给药量;Fv5-M83作为抗IL-6受体中和抗体,作用强度极其优异,作用的持续性也优异。因此,认为Fv3-M73、Fv4-M73和Fv5-M83可以作为IL-6拮抗剂用于药品。
[实施例9]
已知单核细胞趋化蛋白(MCP)-1参与单核细胞、T细胞、NK细胞、嗜碱细胞的细胞侵袭。有报道称:MCP-1在RA患者的滑膜组织、滑液中高度表达(J Clin Invest.1992 Sep;90(3):772-9),认为其与RA的病态有关(Inflamm Allergy Drug Targets.2008 Mar;7(1):53-66)。
已知VEGF是强效的血管新生因子,由RA患者滑膜中的巨噬细胞、成纤维细胞、滑膜细胞等产生(J Rheumatol.1995 Sep;22(9):1624-30)。另外,RA患者血清中的VEGF水平与疾病活动性或放射进展(radiographic progression)相关(Arthritis Rheum.2003 Jun;48(6):1521-9.、Arthritis Rheum.2001 Sep;44(9):2055-64),通过使用抗IL-6R抗体TOCILIZUMAB治疗RA患者,血清中的VEGF水平降低,由此认为:VEGF在RA的病态中也担负着重要的作用(Mod Rheumatol.2009;19(1):12-9.、Mediators Inflamm.2008;2008:129873)。
因此,利用下述的方法研究TOCILIZUMAB和Fv4-M73能否抑制由sIL-6R和IL-6刺激产生的、来自人RA患者的滑膜细胞的MCP-1和VEGF产生。
将来自人RA患者的滑膜细胞(TOYOBO)加入到含5%FCS的IMDM培养基中以2×104/0.05mL/孔接种在96孔平板中,在37℃、5%CO2的培养箱中静置90分钟。添加0.05mL浓度已适当稀释的TOCILIZUMAB和Fv4-M73,静置15分钟,之后添加0.05mL可溶型IL-6受体(SR344:按照参考例的方法进行制备),再静置30分钟,之后进一步添加0.05mL IL-6(TORAY)(可溶型IL-6受体和IL-6的终浓度均为50ng/mL)。培养2天后回收培养上清,使用ELISA试剂盒(Biosource和Pierce Biotechnology)测定培养上清中MCP-1和VEGF的浓度。结果见图21和图22。TOCILIZUMAB和Fv4-M73浓度依赖性地抑制了由可溶型IL-6受体和IL-6刺激产生的、来自人RA患者的滑膜细胞的MCP-1和VEGF产生。
由此可知:Fv4-M73作为抗IL-6受体中和抗体,其作用(与IL-6受体结合,阻断膜型IL-6受体和可溶型IL-6受体的信号)的持续性与TOCILIZUMAB相比极其优异,与TOCILIZUMAB相比,可以大幅减少给药频率和给药量,并且,Fv4-M73抑制来自人RA患者的滑膜细胞的MCP-1和VEGF产生,由此表明:Fv4-M73对RA是极有用的治疗药。
[参考例]
重组可溶型人IL-6受体的制备
作为抗原的人IL-6受体的重组可溶型人IL-6受体如下制备。制作J.Biochem.108,673-676(1990)中报道的、包含N末端侧第1位~第344位氨基酸序列的可溶型人IL-6受体(Yamasaki等人,Science1988;241:825-828(GenBank # X12830))的CHO细胞恒定表达株(定常発現株)。使用Blue Sepharose 6 FF柱色谱、固定有SR344特异性抗体的柱的亲和色谱、凝胶过滤柱色谱这3种柱色谱,从由SR344表达CHO细胞得到的培养上清中纯化可溶型人IL-6受体。以作为主峰洗脱的组分作为最终纯品。
重组可溶型食蟹猴IL-6受体(cIL-6R)的制备
根据所公开的恒河猴IL-6受体基因序列(Birney等人,Ensembl2006,Nucleic Acids Res.2006 Jan 1:34(Database issue):D556-61)制作寡DNA引物,以由食蟹猴胰脏制备的cDNA为模板,使用引物通过PCR法制备编码食蟹猴IL-6受体基因全长的DNA片段。将得到的DNA片段插入哺乳动物细胞表达载体中,使用其制作CHO恒定表达株(cyno.sIL-6R产生CHO细胞)。将cyno.sIL-6R产生CHO细胞的培养液用HisTrap柱(GE Healthcare Bioscience)进行纯化,之后用AmiconUltra-15 Ultracel-10k(Millipore)进行浓缩,再用Superdex200pg16/60凝胶过滤柱(GE Healthcare Bioscience)进一步纯化,得到可溶型食蟹猴IL-6受体(以下记作“cIL-6R”)的最终纯品。
重组食蟹猴IL-6(cIL-6)的制备
食蟹猴IL-6如下制备。作成编码在SWISSPROT Accession No.P79341中注册的212个氨基酸的核苷酸序列,克隆到哺乳动物细胞表达载体中,再将其导入CHO细胞中,从而制作恒定表达细胞株(cyno,IL-6产生CHO细胞)。将cyno.IL-6产生CHO细胞的培养液用SP-Sepharose/FF柱(GE Healthcare Bioscience)进行纯化,之后使用Amicon Ultra-15 Ultracel-5k(Millipore)进行浓缩,再用Superdex75pg26/60凝胶过滤柱(GE Healthcare Bioscience)进一步纯化,用Amicon Ultra-15 Ultracel-5k(Millipore)进行浓缩,得到食蟹猴IL-6(以下记作“cIL-6”)的最终纯品。
公知高亲和性抗IL-6受体抗体的制作
作为公知的高亲和性抗IL-6受体抗体,为了使US 2007/0280945A1中记载的高亲和性抗IL-6受体抗体、即VQ8F11-21 hIgG1(US2007/0280945A1,H链氨基酸序列:SEQ ID NO:77、L链氨基酸序列:SEQ ID NO:78)表达,构建哺乳动物细胞表达用载体。利用组合有合成寡DNA的PCR法(assembly PCR,装配PCR)制作抗体可变区,恒定区使用IgG1。通过装配PCR法使抗体可变区与恒定区结合,插入哺乳动物表达用载体中,制作目标H链表达载体和L链表达载体。所得表达载体的核苷酸序列通过本领域技术人员公知的方法来确定。使用制作的表达载体进行表达和纯化。表达和纯化按照实施例1中记载的方法来进行,得到高亲和性抗IL-6受体抗体(以后记作“对照”)。
TOCILIZUMAB突变体的制作、表达和纯化
关于TOCILIZUMAB的突变体,使用QuikChange位点定向诱变试剂盒(Stratagene),按照附录说明书记载的方法制作突变体,将得到的质粒片段插入哺乳动物细胞表达载体中,制作目标H链表达载体和L链表达载体。所得表达载体的核苷酸序列按照本领域技术人员公知的方法来确定。抗体的表达采用下述的方法来进行。将来自人胚肾癌细胞的HEK293H株(Invitrogen)悬浮在含10%胎牛血清(Invitrogen)的DMEM培养基(Invitrogen)中,以5~6×105个/mL的细胞密度在粘附细胞用培养皿(直径为10cm,CORNING)的各皿中分别接种10mL,在37℃、5%CO2的培养箱内培养一昼夜,之后吸除培养基,添加6.9mLCHO-S-SFM-II(Invitrogen)培养基。利用脂质转染法将制备的质粒导入细胞中。回收所得的培养上清,之后在室温下以约2000g的离心力离心5分钟以除去细胞,再通过0.22μm的滤器MILLEX(R)-GV(Millipore)进行灭菌,得到培养上清。使用rProtein A SepharoseTM Fast Flow(Amersham Biosciences),按照本领域技术人员公知的方法从所得的培养上清中纯化抗体。至于纯化抗体浓度,使用分光光度计测定280nm处的吸光度。由所得的值通过PACE法算出吸光系数,使用该吸光系数算出抗体浓度(Protein Science 1995;4:2411-2423)。
人gp130表达BaF3细胞株的建立
为了得到显示IL-6依赖增殖性的细胞株,如下建立表达人gp130的BaF3细胞株。
通过PCR扩增全长人gp130 cDNA(Hibi等人、Cell 1990;63:1149-1157(GenBank # NM_002184)),除去pCHOI(Hirata等人、FEBSLetter 1994;356:244-248)的DHFR基因表达部位,再克隆到插入有Zeocin耐性基因表达部位的表达载体pCOS2Zeo中,构建pCOS2Zeo/gp130。通过PCR扩增全长人IL-6R cDNA,克隆到pcDNA3.1(+)(Invitrogen)中,构建hIL-6R/pcDNA3.1(+)。
将10μg的pCOS2Zeo/gp130混合在悬浮于PBS中的BaF3细胞(0.8×107细胞)中,使用Gene Pulser(Bio-Rad)以0.33kV、950μFD的容量施加脉冲。将通过电穿孔处理导入有基因的BaF3细胞在含有0.2ng/mL的小鼠白介素-3(Peprotech)、10%胎牛血清(以下记作“FBS”、HyClone)的RPMI1640培养基(Invitrogen)中培养一昼夜,加入含100ng/mL的人白介素-6(R&D systems)、100ng/mL的人白介素-6可溶性受体(R&D systems)和10%FBS的RPMI1640培养基进行筛选,建立人gp130表达BaF3细胞株(以下记作“BaF3/gp130”)。由于该BaF/gp130在人白介素-6(R&D systems)和可溶型人IL-6受体的存在下增殖,所以可用于评价抗IL-6受体抗体的增殖抑制活性(即IL-6受体中和活性)。
评价人gp130表达BaF3细胞(BaF/gp130)的生物活性
使用显示IL-6/IL-6受体依赖性增殖的BaF3/gp130评价IL-6受体中和活性。将BaF3/gp130用含10%FBS的RPMI1640培养基清洗3次,之后悬浮于含600ng/mL~60ng/mL的人白介素-6(TORAY)(终浓度为300ng/mL~30ng/mL)、适量的可溶型人IL-6受体和10%FBS的RPMI1640培养基中,使达到5×104细胞/mL的浓度,向96孔平板(CORNING)的各孔中分别注入50μL。接下来,将已纯化的抗体用含10%FBS的RPMI1640稀释,向各孔中分别混合50μL。在37℃、5%CO2的条件下培养3天,按20μL/孔加入经PBS稀释2倍的WST-8试剂(Cell Counting Kit-8、株式会社同仁化学研究所),之后立即使用SUNRISE CLASSIC(TECAN)测定450nm处的吸光度(参比波长为620nm)。培养2小时后,再次测定450nm的吸光度(参比波长为620nm),以2个小时的吸光度变化为指标,评价IL-6受体中和活性。
利用Biacore进行的与可溶型人IL-6受体的结合评价
使用Biacore T100(GE Healthcare)进行抗原抗体反应的速度论的分析。通过胺耦合法将适量的蛋白A或蛋白A/G或抗IgG(γ-链特异性)F(ab’)2固定在传感器芯片上,接下来在pH7.4下使目标抗体结合,再将在pH7.4下制备成各种浓度的可溶型IL-6受体以分析物的形式流动,测定抗体与可溶型人IL-6受体的相互作用。所有测定均在37℃下进行。由测定中得到的传感图算出动力学参数、即结合速度常数ka(1/Ms)和解离速度常数kd(1/s),根据该值算出KD(M)。使用BiacoreT100评估软件(GE Healthcare)计算各参数。
利用Biacore进行的自膜型IL-6受体的pH依赖性解离评价
使用Biacore T100(GE Healthcare)观测在pH5.8、pH7.4下与膜型IL-6受体的抗原抗体反应。通过评价与固定在传感器芯片上的可溶型人IL-6受体的结合,评价与膜型IL-6受体的结合。按照本领域技术人员公知的方法将SR344生物素化,利用链亲合素与生物素的亲和性经由链亲合素将生物素化可溶型人IL-6受体固定在传感器芯片上。所有测定均在37℃下进行,流动相的缓冲液为10mM MES pH5.8、150mM NaCl、0.05%Tween20,在pH7.4的条件下向其中注入pH依赖性结合克隆,使其与可溶型人IL-6受体结合,之后(注入样品缓冲液为10mM MES pH7.4、150mM NaCl、0.05%Tween20),在流动相的pH5.8下观测各克隆的pH依赖性解离。使用Biacore T100评估软件(GEHealthcare),仅对样品浓度为0.25μg/mL、在10mM MES pH7.4、150mMNaCl、0.05%Tween20中结合、在10mM MES pH5.8、150mM NaCl、0.05%Tween20中解离时的pH5.8下的解离相进行拟合,从而算出pH5.8时的解离速度常数(kd(1/s))。同样,使用Biacore T100评估软件(GE Healthcare),仅对样品浓度为0.5μg/mL、在10mM MES pH7.4、150mM NaCl、0.05%Tween20中结合、在10mM MES pH7.4、150mMNaCl、0.05%Tween20中解离时的pH7.4下的解离相进行拟合,从而算出pH7.4时的解离速度常数(kd(1/s))。
与人FcRn的结合评价
FcRn是FcRn与β2-微球蛋白的复合体。根据公开的人FcRn基因序列(J.Exp.Med.180(6),2377-2381(1994)),制作寡DNA引物。以人cDNA(Human Placenta Marathon-Ready cDNA,Clontech)为模板,使用制作的引物,通过PCR法调整编码基因全长的DNA片段。以所得DNA片段为模板,利用PCR法扩增编码包含信号区的胞外区(Met1-Leu290)的DNA片段,将其插入哺乳动物细胞表达载体中(人FcRn氨基酸序列/SEQ ID NO:79)。同样,根据所公开的人β2-微球蛋白基因序列(Proc.Natl.Acad.Sci.U.S.A.99(26),16899-16903(2002))制作寡DNA引物。以人cDNA(Hu-Placenta Marathon-Ready cDNA,CLONTECH)为模板,使用制作的引物,利用PCR法制备编码基因全长的DNA片段。以所得DNA片段为模板,利用PCR法扩增编码包含信号区的β2-微球蛋白全长(Met1-Met119)的DNA片段,将其插入哺乳动物细胞表达载体中(人β2-微球蛋白氨基酸序列/SEQ ID NO:80)。
可溶型人FcRn的表达按以下程序进行。将制备的人FcRn和人β2-微球蛋白的质粒通过使用了10%胎牛血清(Invitrogen)的脂质转染法导入来自人胚肾癌细胞的HEK293H株(Invitrogen)的细胞中。回收所得的培养上清,之后使用IgG Sepharose 6 Fast Flow(AmershamBiosciences),按照(J Immunol.2002 Nov 1;169(9):5171-80)的方法进行纯化。之后通过HiTrap Q HP(GE Healthcare)进行纯化。
小鼠血浆中抗体浓度的测定
小鼠血浆中抗体浓度的测定通过ELISA法、利用本领域技术人员公知的方法进行测定。
猴PK/PD试验的血浆中抗体浓度、CRP浓度、非结合型可溶型IL-6
受体的测定
食蟹猴血浆中浓度的测定通过ELISA法、利用本领域技术人员公知的方法进行测定。
利用Cias R CRP(关东化学株式会社),使用自动分析装置(TBA-120FR、东芝Medical Systems株式会社)测定CRP浓度。
食蟹猴血浆中的非结合型的可溶型食蟹猴IL-6受体浓度如下测定。将30μL食蟹猴的血浆放在0.22μm的滤杯(Millipore)中,添加适量的已干燥的rProtein A Sepharose Fast Flow(GE Healthcare)树脂,使存在于血浆中的所有IgG型抗体(食蟹猴IgG、抗人IL-6受体抗体和抗人IL-6受体抗体-可溶型食蟹猴IL-6受体复合体)吸附在蛋白A上。之后,用高速离心机进行旋转下降(spin-down),回收通过的溶液。由于通过的溶液中不含与蛋白A结合的抗人IL-6受体抗体-可溶型食蟹猴IL-6受体复合体,所以通过测定通过蛋白A的溶液中的可溶型食蟹猴IL-6受体浓度,可以测定非结合型的可溶型IL-6受体浓度。至于可溶型食蟹猴IL-6受体浓度,以上述制作的可溶型食蟹猴IL-6受体(cIL-6R)作为标准品,利用测定人IL-6受体浓度的本领域技术人员公知的方法进行测定。非结合型的可溶型IL-6受体率通过下式计算。
(抗体给予后的非结合型的可溶性IL-6受体浓度÷抗体给予前的可溶性IL-6受体浓度)×100
序列表
<110>中外制药株式会社
<120>改良的抗体分子
<130>C1-A0805Y2P
<150>JP 2008-248213
<151>2008-09-26
<150>JP 2009-60806
<151>2009-03-13
<150>JP 2009-67925
<151>2009-03-19
<160>117
<170>PatentIn version 3.4
<210>1
<211>6
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>1
His Asp His Ala Trp Ser
1 5
<210>2
<211>16
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>2
Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu Gln Gly
1 5 10 15
<210>3
<211>10
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>3
Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr
1 5 10
<210>4
<211>6
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>4
His Asp His Ala Trp Ser
1 5
<210>5
<211>16
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>5
Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Gln Gly
1 5 10 15
<210>6
<211>10
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>6
Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr
1 5 10
<210>7
<211>6
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>7
Asp Asp His Ala Val Ser
1 5
<210>8
<211>16
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>8
Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu Gln Asp
1 5 10 15
<210>9
<211>10
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>9
Leu Leu Ala Arg Ala Thr Ala Met Asp Val
1 5 10
<210>10
<211>11
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>10
Gln Ala Ser Arg Asp Ile Ser Ser His Leu Asn
1 5 10
<210>11
<211>7
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>11
Tyr Gly Ser His Leu Leu Ser
1 5
<210>12
<211>9
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>12
Gly Gln Gly Asn Arg Leu Pro Tyr Thr
1 5
<210>13
<211>11
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>13
Gln Ala Ser Thr Asp Ile Ser Ser His Leu Asn
1 5 10
<210>14
<211>7
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>14
Tyr Gly Ser His Leu Leu Ser
1 5
<210>15
<211>9
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>15
Gly Gln Gly Asn Arg Leu Pro Tyr Thr
1 5
<210>16
<211>11
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>16
Gln Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn
1 5 10
<210>17
<211>7
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>17
Tyr Gly Ser Glu Leu Glu Ser
1 5
<210>18
<211>9
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>18
Gly Gln Gly Asn Arg Leu Pro Tyr Thr
1 5
<210>19
<211>119
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>19
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp
35 40 45
Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu
50 55 60
Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210>20
<211>119
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>20
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp
35 40 45
Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210>21
<211>119
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>21
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp
20 25 30
His Ala Val Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp
35 40 45
Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu
50 55 60
Gln Asp Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Leu Ala Arg Ala Thr Ala Met Asp Val Trp Gly Glu Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210>22
<211>107
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>22
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Arg Asp Ile Ser Ser His
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile
35 40 45
Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu
100 105
<210>23
<211>107
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>23
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Thr Asp Ile Ser Ser His
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile
35 40 45
Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu
100 105
<210>24
<211>107
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>24
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile
35 40 45
Tyr Tyr Gly Ser Glu Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu
100 105
<210>25
<211>443
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>25
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp
35 40 45
Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu
50 55 60
Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu
210 215 220
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
290 295 300
Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440
<210>26
<211>443
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>26
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp
35 40 45
Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu
210 215 220
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
290 295 300
Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440
<210>27
<211>447
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>27
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp
20 25 30
His Ala Val Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp
35 40 45
Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu
50 55 60
Gln Asp Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Leu Ala Arg Ala Thr Ala Met Asp Val Trp Gly Glu Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
<210>28
<211>214
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>28
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Arg Asp Ile Ser Ser His
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile
35 40 45
Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu 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
210
<210>29
<211>214
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>29
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Thr Asp Ile Ser Ser His
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile
35 40 45
Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu 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
210
<210>30
<211>214
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>30
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile
35 40 45
Tyr Tyr Gly Ser Glu Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu 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
210
<210>31
<211>324
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<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 Asn Phe Gly Thr Gln Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
115 120 125
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
130 135 140
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
145 150 155 160
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
165 170 175
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
195 200 205
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
210 215 220
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
225 230 235 240
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
260 265 270
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
275 280 285
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
290 295 300
Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu
305 310 315 320
Ser Leu Ser Pro
<210>32
<211>324
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>32
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 Asn Phe Gly Thr Gln Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
115 120 125
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
130 135 140
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
145 150 155 160
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
165 170 175
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
195 200 205
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
210 215 220
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
225 230 235 240
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
260 265 270
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
275 280 285
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
290 295 300
Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu
305 310 315 320
Ser Leu Ser Pro
<210>33
<211>328
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<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 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 Asp Glu
225 230 235 240
Leu 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 Ala His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro
325
<210>34
<211>107
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>34
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
100 105
<210>35
<211>107
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>35
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
100 105
<210>36
<211>107
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>36
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
100 105
<210>37
<211>327
<212>DNA
<213>Homo sapiens
<400>37
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 ttgataa 327
<210>38
<211>107
<212>PRT
<213>Homo sapiens
<400>38
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
100 105
<210>39
<211>990
<212>DNA
<213>Homo sapiens
<400>39
gctagcacca 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 ccgggatgag 720
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggtaaa 990
<210>40
<211>330
<212>PRT
<213>Homo sapiens
<400>40
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 Asp Glu
225 230 235 240
Leu 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>41
<211>984
<212>DNA
<213>Homo sapiens
<400>41
gctagcacca agggcccatc ggtcttcccc ctggcgccct cctccaagag cacctccgag 60
agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgctctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg cacccagacc 240
tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc 300
aaatcttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc 360
ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc 420
gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc 480
gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt 540
gtggtcagcg tcctcaccgt cgtgcaccag gactggctga acggcaagga gtacaagtgc 600
aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg 660
cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 720
caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 780
gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct ggactccgac 840
ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 900
gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacaca gaagagcctc 960
tccctgtctc cgggtaaatg ataa 984
<210>42
<211>326
<212>PRT
<213>Homo sapiens
<400>42
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Glu Ser 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 Asn Phe Gly Thr Gln Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
115 120 125
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
130 135 140
Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
145 150 155 160
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
165 170 175
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
195 200 205
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
210 215 220
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
225 230 235 240
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
260 265 270
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
275 280 285
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
290 295 300
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
305 310 315 320
Ser Leu Ser Pro Gly Lys
325
<210>43
<211>995
<212>DNA
<213>Homo sapiens
<400>43
gctagcacca agggcccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag 60
agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 240
tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 300
aaatatggtc ccccatgccc accatgccca gcacctgagt tcctgggggg accatcagtc 360
ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 420
tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 480
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 540
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 600
tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 660
gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 720
aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 780
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 840
gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 900
aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 960
ctctccctgt ctctgggtta atgataagcg gccgc 995
<210>44
<211>326
<212>PRT
<213>Homo sapiens
<400>44
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser 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 Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly
325
<210>45
<211>4
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>45
Gly Gly Gly Ser
1
<210>46
<211>4
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>46
Ser Gly Gly Gly
1
<210>47
<211>5
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>47
Gly Gly Gly Gly Ser
1 5
<210>48
<211>5
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>48
Ser Gly Gly Gly Gly
1 5
<210>49
<211>6
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>49
Gly Gly Gly Gly Gly Ser
1 5
<210>50
<211>6
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>50
Ser Gly Gly Gly Gly Gly
1 5
<210>51
<211>7
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>51
Gly Gly Gly Gly Gly Gly Ser
1 5
<210>52
<211>7
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>52
Ser Gly Gly Gly Gly Gly Gly
1 5
<210>53
<211>449
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>53
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210>54
<211>214
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>54
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 Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
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
210
<210>55
<211>449
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>55
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Leu Ala Arg Ile Thr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210>56
<211>214
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>56
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 Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr
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
210
<210>57
<211>449
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>57
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210>58
<211>214
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>58
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile
35 40 45
Tyr Tyr Gly Ser Glu Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu 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
210
<210>59
<211>7
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>59
Tyr Thr Ser Arg Leu His Ser
1 5
<210>60
<211>7
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>60
Tyr Gly Ser Glu Leu His Ser
1 5
<210>61
<211>30
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>61
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr
20 25 30
<210>62
<211>30
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>62
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser
20 25 30
<210>63
<211>32
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>63
Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg
1 5 10 15
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210>64
<211>32
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>64
Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210>65
<211>30
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>65
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser
20 25 30
<210>66
<211>449
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>66
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp
20 25 30
His Ala His Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210>67
<211>214
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>67
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln His Ile Ser Ser His
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile
35 40 45
Tyr Tyr Gly Ser His Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu 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
210
<210>68
<211>448
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>68
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210>69
<211>447
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>69
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
<210>70
<211>445
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>70
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Glu Ser Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu
210 215 220
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
290 295 300
Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210>71
<211>445
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>71
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu
210 215 220
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
290 295 300
Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210>72
<211>443
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>72
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu
210 215 220
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
290 295 300
Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440
<210>73
<211>449
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>73
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210>74
<211>447
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>74
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
<210>75
<211>443
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>75
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu
210 215 220
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
290 295 300
Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440
<210>76
<211>449
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>76
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp
20 25 30
His Ala Val Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp
35 40 45
Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu
50 55 60
Gln Asp Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Leu Ala Arg Ala Thr Ala Met Asp Val Trp Gly Glu Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210>77
<211>446
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>77
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Phe Thr Phe Asp Asp Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Ser Trp Asn Ser Gly Arg Ile Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Ser Leu Phe
65 70 75 80
Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Lys Gly Arg Asp Ser Phe Asp Ile Trp Gly Gln Gly Thr Met Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210>78
<211>214
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>78
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Ser Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210>79
<211>267
<212>PRT
<213>Homo sapiens
<400>79
Ala Glu Ser His Leu Ser Leu Leu Tyr His Leu Thr Ala Val Ser Ser
1 5 10 15
Pro Ala Pro Gly Thr Pro Ala Phe Trp Val Ser Gly Trp Leu Gly Pro
20 25 30
Gln Gln Tyr Leu Ser Tyr Asn Ser Leu Arg Gly Glu Ala Glu Pro Cys
35 40 45
Gly Ala Trp Val Trp Glu Asn Gln Val Ser Trp Tyr Trp Glu Lys Glu
50 55 60
Thr Thr Asp Leu Arg Ile Lys Glu Lys Leu Phe Leu Glu Ala Phe Lys
65 70 75 80
Ala Leu Gly Gly Lys Gly Pro Tyr Thr Leu Gln Gly Leu Leu Gly Cys
85 90 95
Glu Leu Gly Pro Asp Asn Thr Ser Val Pro Thr Ala Lys Phe Ala Leu
100 105 110
Asn Gly Glu Glu Phe Met Asn Phe Asp Leu Lys Gln Gly Thr Trp Gly
115 120 125
Gly Asp Trp Pro Glu Ala Leu Ala Ile Ser Gln Arg Trp Gln Gln Gln
130 135 140
Asp Lys Ala Ala Asn Lys Glu Leu Thr Phe Leu Leu Phe Ser Cys Pro
145 150 155 160
His Arg Leu Arg Glu His Leu Glu Arg Gly Arg Gly Asn Leu Glu Trp
165 170 175
Lys Glu Pro Pro Ser Met Arg Leu Lys Ala Arg Pro Ser Ser Pro Gly
180 185 190
Phe Ser Val Leu Thr Cys Ser Ala Phe Ser Phe Tyr Pro Pro Glu Leu
195 200 205
Gln Leu Arg Phe Leu Arg Asn Gly Leu Ala Ala Gly Thr Gly Gln Gly
210 215 220
Asp Phe Gly Pro Asn Ser Asp Gly Ser Phe His Ala Ser Ser Ser Leu
225 230 235 240
Thr Val Lys Ser Gly Asp Glu His His Tyr Cys Cys Ile Val Gln His
245 250 255
Ala Gly Leu Ala Gln Pro Leu Arg Val Glu Leu
260 265
<210>80
<211>99
<212>PRT
<213>Homo sapiens
<400>80
Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu
1 5 10 15
Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro
20 25 30
Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys
35 40 45
Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu
50 55 60
Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys
65 70 75 80
Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp
85 90 95
Arg Asp Met
<210>81
<211>16
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>81
Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu Lys Ser
1 5 10 15
<210>82
<211>16
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>82
Phe Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu Lys Ser
1 5 10 15
<210>83
<211>16
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>83
Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys Ser
1 5 10 15
<210>84
<211>10
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>84
Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr
1 5 10
<210>85
<211>10
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>85
Leu Leu Ala Arg Thr Thr Ala Met Asp Tyr
1 5 10
<210>86
<211>10
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>86
Ser Leu Ala Arg Ala Thr Ala Met Asp Tyr
1 5 10
<210>87
<211>11
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>87
Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn
1 5 10
<210>88
<211>11
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>88
Arg Ala Ser Thr Asp Ile Ser Ser Tyr Leu Asn
1 5 10
<210>89
<211>11
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>89
Arg Ala Ser Arg Asp Ile Ser Ser Tyr Leu Asn
1 5 10
<210>90
<211>9
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>90
Gln Gln Gly Asn Thr Leu Pro Tyr Thr
1 5
<210>91
<211>9
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>91
Gly Gln Gly Asn Thr Leu Pro Tyr Thr
1 5
<210>92
<211>9
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>92
Gln Gln Gly Asn Arg Leu Pro Tyr Thr
1 5
<210>93
<211>30
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>93
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr
20 25 30
<210>94
<211>30
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>94
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser
20 25 30
<210>95
<211>6
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>95
Ser Asp His Ala Trp Ser
1 5
<210>96
<211>6
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>96
Asp Asp His Ala Trp Ser
1 5
<210>97
<211>14
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>97
Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile Gly
1 5 10
<210>98
<211>14
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>98
Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp Ile Gly
1 5 10
<210>99
<211>16
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>99
Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu Gln Asp
1 5 10 15
<210>100
<211>11
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>100
Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser
1 5 10
<210>101
<211>11
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>101
Trp Gly Glu Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210>102
<211>23
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>102
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
20
<210>103
<211>23
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>103
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Ser Val Thr Ile Thr Cys
20
<210>104
<211>11
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>104
Gln Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn
1 5 10
<210>105
<211>15
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>105
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
1 5 10 15
<210>106
<211>15
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>106
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile Tyr
1 5 10 15
<210>107
<211>7
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>107
Tyr Thr Ser Arg Leu His Ser
1 5
<210>108
<211>7
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>108
Tyr Thr Ser Glu Leu Glu Ser
1 5
<210>109
<211>7
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>109
Tyr Thr Ser Arg Leu Leu Ser
1 5
<210>110
<211>32
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>110
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys
20 25 30
<210>111
<211>32
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>111
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Phe Thr Ile Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
20 25 30
<210>112
<211>10
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>112
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
1 5 10
<210>113
<211>10
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>113
Phe Gly Gln Gly Thr Lys Val Glu Ile Glu
1 5 10
<210>114
<211>30
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>114
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly His Ser Ile Thr
20 25 30
<210>115
<211>6
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>115
His Asp His Ala Trp Ser
1 5
<210>116
<211>11
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>116
Arg Ala Ser Gln Asp Ile Ser Ser His Leu Asn
1 5 10
<210>117
<211>7
<212>PRT
<213>人工序列
<220>
<223>人工合成多肽序列
<400>117
Tyr Thr Ser His Leu His Ser
1 5
Claims (11)
1.下述(a)~(f)中任一项所述的多肽:
(a)多肽,该多肽包含具有SEQ ID NO:1的序列的CDR1、具有SEQ ID NO:2的序列的CDR2和具有SEQ ID NO:3的序列的CDR3,其中,SEQ ID NO:1为VH4-M73的CDR1、SEQ ID NO:2为VH4-M73的CDR2、SEQ ID NO:3为VH4-M73的CDR3;
(b)多肽,该多肽包含具有SEQ ID NO:4的序列的CDR1、具有SEQ ID NO:5的序列的CDR2和具有SEQ ID NO:6的序列的CDR3,其中,SEQ ID NO:4为VH3-M73的CDR1、SEQ ID NO:5为VH3-M73的CDR2、SEQ ID NO:6为VH3-M73的CDR3;
(c)多肽,该多肽包含具有SEQ ID NO:7的序列的CDR1、具有SEQ ID NO:8的序列的CDR2和具有SEQ ID NO:9的序列的CDR3,其中,SEQ ID NO:7为VH5-M83的CDR1、SEQ ID NO:8为VH5-M83的CDR2、SEQ ID NO:9为VH5-M83的CDR3;
(d)多肽,该多肽包含具有SEQ ID NO:10的序列的CDR1、具有SEQ ID NO:11的序列的CDR2和具有SEQ ID NO:12的序列的CDR3,其中,SEQ ID NO:10为VL1的CDR1、SEQ ID NO:11为VL1的CDR2、SEQ ID NO:12为VL1的CDR3;
(e)多肽,该多肽包含具有SEQ ID NO:13的序列的CDR1、具有SEQ ID NO:14的序列的CDR2和具有SEQ ID NO:15的序列的CDR3,其中,SEQ ID NO:13为VL3的CDR1、SEQ ID NO:14为VL3的CDR2、SEQ ID NO:15为VL3的CDR3;
(f)多肽,该多肽包含具有SEQ ID NO:16的序列的CDR1、具有SEQ ID NO:17的序列的CDR2和具有SEQ ID NO:18的序列的CDR3,其中,SEQ ID NO:16为VL5的CDR1、SEQ ID NO:17为VL5的CDR2、SEQ ID NO:18为VL5的CDR3。
2.下述(a)~(c)中任一项所述的抗体:
(a)抗体,该抗体包含重链可变区及轻链可变区,所述重链可变区包含具有SEQ ID NO:1的序列的CDR1、具有SEQ ID NO:2的序列的CDR2和具有SEQ ID NO:3的序列的CDR3,其中,SEQ ID NO:1为VH4-M73的CDR1、SEQ ID NO:2为VH4-M73的CDR2、SEQ ID NO:3为VH4-M73的CDR3;
所述轻链可变区包含具有SEQ ID NO:10的序列的CDR1、具有SEQ ID NO:11的序列的CDR2和具有SEQ ID NO:12的序列的CDR3,其中,SEQ ID NO:10为VL1的CDR1、SEQ ID NO:11为VL1的CDR2、SEQ ID NO:12为VL1的CDR3;
(b)抗体,该抗体包含重链可变区及轻链可变区,所述重链可变区包含具有SEQ ID NO:4的序列的CDR1、具有SEQ ID NO:5的序列的CDR2和具有SEQ ID NO:6的序列的CDR3,其中,SEQ ID NO:4为VH3-M73的CDR1、SEQ ID NO:5为VH3-M73的CDR2、SEQ ID NO:6为VH3-M73的CDR3;
所述轻链可变区包含具有SEQ ID NO:13的序列的CDR1、具有SEQ ID NO:14的序列的CDR2和具有SEQ ID NO:15的序列的CDR3,其中,SEQ ID NO:13为VL3的CDR1、SEQ ID NO:14为VL3的CDR2、SEQ ID NO:15为VL3的CDR3;
(c)抗体,该抗体包含重链可变区及轻链可变区,所述重链可变区包含具有SEQ ID NO:7的序列的CDR1、具有SEQ ID NO:8的序列的CDR2和具有SEQ ID NO:9的序列的CDR3,其中,SEQ ID NO:7为VH5-M83的CDR1、SEQ ID NO:8为VH5-M83的CDR2、SEQ ID NO:9为VH5-M83的CDR3;
所述轻链可变区包含具有SEQ ID NO:16的序列的CDR1、具有SEQ ID NO:17的序列的CDR2和具有SEQ ID NO:18的序列的CDR3,其中,SEQ ID NO:16为VL5的CDR1、SEQ ID NO:17为VL5的CDR2、SEQ ID NO:18为VL5的CDR3。
3.下述(a)~(f)中任一项所述的可变区:
(a)重链可变区,其中具有SEQ ID NO:19的序列,其中SEQ ID NO:19为VH4-M73的可变区;
(b)重链可变区,其中具有SEQ ID NO:20的序列,其中SEQ IDNO:20为VH3-M73的可变区;
(c)重链可变区,其中具有SEQ ID NO:21的序列,其中SEQ ID NO:21为VH5-M83的可变区;
(d)轻链可变区,其中具有SEQ ID NO:22的序列,其中SEQ IDNO:22为VL1的可变区;
(e)轻链可变区,其中具有SEQ ID NO:23的序列,其中SEQ ID NO:23为VL3的可变区;
(f)轻链可变区,其中具有SEQ ID NO:24的序列,其中SEQ ID NO:24为VL5的可变区。
4.下述(a)~(c)中任一项所述的抗体:
(a)抗体,该抗体包含重链可变区和轻链可变区,所述重链可变区具有SEQ ID NO:19的序列,所述轻链可变区具有SEQ ID NO:22的序列,其中,SEQ ID NO:19为VH4-M73的可变区、SEQ ID NO:22为VL1的可变区;
(b)抗体,该抗体包含重链可变区和轻链可变区,所述重链可变区具有SEQ ID NO:20的序列,所述轻链可变区具有SEQ ID NO:23的序列,其中,SEQ ID NO:20为VH3-M73的可变区、SEQ ID NO:23为VL3的可变区;
(c)抗体,该抗体包含重链可变区和轻链可变区,所述重链可变区具有SEQ ID NO:21的序列,所述轻链可变区具有SEQ ID NO:24的序列,其中,SEQ ID NO:21为VH5-M83的可变区、SEQ ID NO:24为VL5的可变区。
5.下述(a)~(f)中任一项所述的重链或轻链:
(a)重链,该重链具有SEQ ID NO:25的序列,其中SEQ ID NO:25为VH4-M73;
(b)重链,该重链具有SEQ ID NO:26的序列,其中SEQ ID NO:26为VH3-M73;
(c)重链,该重链具有SEQ ID NO:27的序列,其中SEQ ID NO:27为VH5-M83;
(d)轻链,该轻链具有SEQ ID NO:28的序列,其中SEQ ID NO:28为VL1;
(e)轻链,该轻链具有SEQ ID NO:29的序列,其中SEQ ID NO:29为VL3;
(f)轻链,该轻链具有SEQ ID NO:30的序列,其中SEQ ID NO:30为VL5。
6.下述(a)~(c)中任一项所述的抗体:
(a)抗体,该抗体包含重链和轻链,所述重链具有SEQ ID NO:25的序列,所述轻链具有SEQ ID NO:28的序列,其中,SEQ ID NO:25为VH4-M73、SEQ ID NO:28为VL1;
(b)抗体,该抗体包含重链和轻链,所述重链具有SEQ ID NO:26的序列,所述轻链具有SEQ ID NO:29的序列,其中,SEQ ID NO:26为VH3-M73、SEQ ID NO:29为VL3;
(c)抗体,该抗体包含重链和轻链,所述重链具有SEQ ID NO:27的序列,所述轻链具有SEQ ID NO:30的序列,其中,SEQ ID NO:27为VH5-M83、SEQ ID NO:30为VL5。
7.基因,该基因编码权利要求1~6中任一项所述的多肽。
8.载体,该载体包含权利要求7所述的基因。
9.宿主细胞,该宿主细胞保有权利要求8所述的载体。
10.通过培养权利要求9的宿主细胞来制备权利要求1~6中任一项所述的多肽的方法。
11.药物组合物,其中含有权利要求1~6中任一项所述的多肽或按照权利要求10所述的方法制备的多肽。
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- 2021-11-12 HU HUS2100050C patent/HUS2100050I1/hu unknown
- 2021-11-17 LT LTPA2021013C patent/LTC2330193I2/lt unknown
- 2021-12-08 NL NL301152C patent/NL301152I2/nl unknown
- 2021-12-13 FI FIC20210049C patent/FIC20210049I1/fi unknown
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2022
- 2022-06-01 US US17/829,641 patent/US20220306755A1/en not_active Abandoned
- 2022-09-08 AR ARP220102433A patent/AR127010A2/es unknown
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2023
- 2023-01-12 US US18/096,066 patent/US20230159648A1/en not_active Abandoned
- 2023-09-11 US US18/464,407 patent/US20240010738A1/en active Pending
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