一组CTLA-4单克隆抗体及其医药用途的制作方法

本发明属于肿瘤治疗和分子免疫学领域，具体地，本发明涉及一组抗CTLA-4的单克隆抗体及其在肿瘤和免疫医学中的应用。技术背景细胞毒性T淋巴细胞相关抗原-4，是一种由CTLA-4基因编码的跨膜蛋白，该基因位于人类第2号染色体2q33。CTLA-4是免疫球蛋白超级家族的一员，由胞外V区，跨膜区和胞浆区组成。CTLA-4与T细胞表面的协同刺激分子受体CD28具有同源性，两者竞争结合其配体B7-1(CD80)或B7-2(CD86)，这类配体主要表达于抗原递呈细胞表面。与CD28相比，CTLA-4与CD80和CD86的结合亲和力更高，因此能够竞争和阻断CD28介导的激活作用。CTLA-4通常表达在抑制性T细胞(Tregs)和活化状态的T细胞表面上，与B7类分子结合后，抑制T细胞的激活，参与免疫反应的负调控,所以CTLA-4在免疫调节中起到非常重要的作用。T细胞的激活需要两个信号的刺激，第一个信号来自T细胞受体(TCR)特异性结合抗原递呈细胞(APC)表面的抗原肽-MHC复合物，第二个信号通路则需要共刺激分子(如CD28)的参与，当CD28和B7-1/B7-2(CD80/CD86)结合后可以进一步活化T细胞，促进其成熟和增殖。目前的研究表明，在免疫应答过程中，CTLA-4通过下列途径下调T细胞的功能：其一，CTLA-4通过其与CD80/CD86的高亲和力，竞争性阻断CD28与CD80/86共刺激信号的传递，进而抑制T细胞的增殖，减少IL-2的分泌。其二，通过降低CD80/CD86在抗原递呈细胞(APC)的表达水平或者通过反式内吞作用将CD80/CD86分子从抗原递呈细胞(APC)表面移除，从而减少了CD28参与的T细胞激活。其三，CTLA-4可以通过介导树突状细胞结合CD80/CD86并诱导色氨酸降解酶IDO的表达，抑制TCR信号。此外，CTLA-4还可以通过招募抑制性分子结合到免疫突触，诱导产生调节性细胞因子,从而抑制APC和TCR信号的传递。CTLA-4信号紊乱与疾病的发生和发展密切相关。CTLA-4基因敲除小鼠会出现严重的淋巴组织增生，并且在出生后四周内会因大量的组织浸润和器官破坏而死亡。在过敏性脑脊髓炎(EAE)实验模型中，CTLA-4抗体通过阻断CTLA-4对T细胞的负调控信号，进一步加剧自身免疫性疾病，增加死亡率。研究表明，CTLA-4通过负向调节免疫反应参与癌症的发生，阻断CTLA-4信号传导可以抑制肿瘤生长。体内实验表明，CTLA-4抗体可以阻断CTLA-4介导的免疫抑制信号而促进机体的免疫功能，可用于治疗肿瘤，如纤维肉瘤，结肠癌和前列腺癌等。CTLA-4阻断性抗体还可与多种治疗方案，如化学疗法、放射疗法、疫苗、以及其他抗体药物联合使用，进一步提高肿瘤的治疗效果。虽然目前已经有少量CTLA-4抗体药物上市但是其核心技术都掌握在国外厂商手中。因此开发新的具有自主知识产权的阻断CTLA-4与CD80/CD86结合的抗体及其人源化抗体是一个迫切的任务。技术实现要素：针对上述问题，本发明通过杂交瘤技术获得了一组在阻断CTLA-4与CD80/CD86的相互作用方面有优异效果的抗CTLA-4单克隆抗体，并成功的对其进行了人源化改造。所述抗体在制备阻断，调节CTLA-4水平相关药物以及治疗癌症相关药物方面有巨大的应用前景。本发明提供一组CTLA-4单克隆抗体或其抗原结合片段，所述重链的CDR1的氨基酸序列选自SEQIDNO：37、43、49中的一种；所述重链的CDR2的氨基酸序列选自SEQIDNO：38、44、50中的一种；所述重链的CDR3的氨基酸序列选自SEQIDNO：39、45、51中的一种；所述轻链的CDR1氨基酸序列选自SEQIDNO：40、46、52中的一种；所述轻链的CDR2的氨基酸序列选自SEQIDNO：41、47、53中的一种；所述轻链的CDR3的氨基酸序列选自SEQIDNO：42、48、54中的一种；其中所述抗原结合片段的重链和轻链包含分别跨越所述抗体的重链和轻链的CDR1到CDR3的氨基酸序列。进一步的，上述抗体或其抗原结合片段，所述重链可变区的氨基酸序列选自SEQIDNO：3、7、11、15、19、23、27、31、35中的一种；所述轻链可变区的氨基酸序列选自SEQIDNO：4、8、12、16、20、24、28、32、36中的一种。进一步的，上述抗体或其抗原结合片段，所述重链和轻链经过人源化；所述人源化重链可变区的氨基酸序列选自SEQIDNO：55、59、63中的一种；所述人源化轻链可变区的氨基酸序列选自SEQIDNO：56、60、64中的一种。进一步的，上述抗体或其抗原结合片段，所述人源化重链的全长氨基酸选自SEQIDNO：57、61、65中的一种；所述人源化轻链的全长氨基酸选自SEQIDNO：58、62、66中的一种。进一步的，本发明公开了编码上述氨基酸片段的DNA序列，所述序列包括SEQIDNO：1、2、5、6、9、10、13、14、17、18、21、22、25、26、29、30、33、34。进一步的，本发明公开了表达上述DNA序列的载体。进一步的，本发明公开了所述CTLA-4单克隆抗体或其抗原结合片段在制备如下药物中的用途：阻断CTLA-4与B7结合的药物、调节CTLA-4活性或CTLA-4水平的药物、解除CTLA-4对机体免疫抑制的药物、激活T淋巴细胞的药物或者提高T淋巴细胞中IL-2表达的药物。进一步的，本发明公开了一种单克隆抗体偶联物，包括单克隆抗体和偶联部分，其中，所述单克隆抗体为上述抗CTLA-4单克隆抗体或其抗原结合片段，所述偶联部分为选自放射性核素、药物、毒素、细胞因子、酶、荧光素、和生物素中的一种或多种。进一步的，本发明公开了上述单克隆抗体偶联物在制备如下药物中的用途：阻断CTLA-4与B7结合的药物，调节CTLA-4活性或CTLA-4水平的药物、解除CTLA-4对机体免疫抑制的药物、激活T淋巴细胞的药物或者提高T淋巴细胞中IL-2表达的药物。进一步的，本发明公开了上述单克隆抗体偶联物在制备预防或者治疗肿瘤的药物中的用途。本发明的进步性表现为：本专利采用哺乳动物细胞表达系统制备重组CTLA-4作为抗原免疫小鼠，将小鼠脾脏细胞与骨髓瘤细胞融合获得杂交瘤细胞。通过对大量杂交瘤细胞的多次克隆及筛选后，得到一些单克隆杂交瘤细胞株。这些杂交瘤细胞株能够产生和CTLA-4特异性结合的单克隆抗体，并且这些单克隆抗体能够有效阻断CTLA-4与CD80或CD86的结合，并能促进金黄色葡萄球菌B型肠毒素(SEB)刺激的外周血单核细胞分泌细胞因子IL-2。通过RT-PCR克隆编码抗体轻链和重链可变区的基因，采用互补决定簇嫁接方法构建人源化抗体。体外功能试验表明，这些人源化的CTLA-4抗体能特异性结合CTLA-4蛋白，高效的阻断CTLA-4与CD80或CD86的结合，并能促进T细胞分泌细胞因子IL-2。以上实验结果表明本发明所述的单克隆抗体或其抗原结合片段，或者包含本发明所述单克隆抗体或其抗原结合片段的单克隆抗体偶联物在制备阻断CTLA-4与B7结合的药物、调节CTLA-4活性或CTLA-4水平的药物、解除CTLA-4对机体免疫抑制的药物、激活T淋巴细胞的药物或者提高T淋巴细胞中IL-2表达的药物，以及在预防和治疗或者辅助治疗肿瘤的药物中有良好的应用前景。说明书附图图1为用ELSIA方法检测鼠源CTLA-4单克隆抗体与CTLA-4-hFc蛋白结合的EC50；图2为用ELSIA方法检测鼠源CTLA-4单克隆抗体阻断配体结合CTLA-4-hFc的IC50；图3为用FACS方法检测鼠源CTLA-4单克隆抗体结合293T-CTLA-4细胞的EC50；图4为用FACS方法检测鼠源CTLA-4单克隆抗体阻断配体结合293T-CTLA-4细胞的IC50；图5为用ELISA方法检测鼠源CTLA-4单克隆抗体对PBMC分泌IL-2的刺激作用；图6为用ELSIA方法检测人源化CTLA-4抗体与CTLA-4-hFc蛋白结合的EC50；图7为用ELSIA方法检测人源化CTLA-4抗体阻断配体结合CTLA-4-hFc的IC50；图8为用FACS方法检测人源化CTLA-4抗体结合293T-CTLA-4细胞的EC50；图9为用FACS方法检测人源化CTLA-4抗体阻断配体结合293T-CTLA-4细胞的IC50；图10为用ELISA方法检测人源化CTLA-4抗体对PBMC分泌IL-2的刺激作用。具体实施方式下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制；下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到；本发明所涉及的抗体的氨基酸和核酸序见序列表。实施例1鼠源抗体库的构建，包括以下步骤(1)将人的CTLA-4-ECD-mFc融合蛋白作为抗原，与等体积完全弗氏佐剂(Sigma，CatNo:F5581)充分乳化后，经皮下免疫6-8周龄Balb/c小鼠(购自昭衍(苏州)新药研究中心有限公司)，抗原免疫量为20μg/只。随后每隔2周，用相同剂量的抗原与不完全弗氏佐剂(Sigma，CatNo:F5506)充分乳化后，经皮下免疫小鼠三次。三次免疫后测定小鼠血清效价，融合前3天经腹腔进行加强免疫。以PEGHybri-Max(Sigma,CatNo:7181)作为融合剂，将小鼠脾脏细胞与SP2/0细胞按照4：1的比例混合。将融合后的细胞加入到96孔板中(1x105细胞/孔)，每孔含有0.1mL1XHAT(Invitrogen，CatNo:21060-017)培养基。在第3天加入0.1mLHT(Invitrogen，CatNo:11067-030)培养基，在第7天吸掉96孔板中的培养基，补加0.2mL新鲜的HT培养基。在第9天，收取上清液进行ELISA和FACS检测。(2)用CTLA-4-hFc包被96孔ELISA板(Corning，CatNo:9018)，置室温过夜，用洗涤缓冲液(PBS+0.05％Tween20)洗涤3次后，加入封闭缓冲液(PBS+1％BSA(Sigma,CatNo:V90093))孵育1小时；洗涤96孔板3次；加入杂交瘤上清液孵育1小时，洗涤3次；每孔加入100μL1:400倍稀释的羊抗鼠IgG二抗(Thermo,CatNo:31432)，室温孵育1小时后洗涤3次；每孔加入100μLTMB(北京百奥赛博，CatNo:ES-002)显色3分钟，再加入100μL/孔的终止液(2NH2SO4)终止反应，用TecanSpark酶标仪测定各样品的OD405信号。(3)取50μl在上述ELISA检测中呈阳性的杂交瘤上清与50μl293T-CTLA-4细胞混合(1x105细胞/孔)加入到U形底96孔板中孵育1小时，用FACS缓冲液(PBS+3％FCS)离心洗涤2次后加入1:400倍稀释的PE标记羊抗鼠(Biolegend，CatNo:405307)二抗，孵育30分钟，经FACS缓冲液洗涤后，用BDC6流式细胞仪检测293T-CTLA-4细胞的PE信号。(4)采用竞争ELISA筛选能够阻断CTLA-4-Fc与CD80-biotin结合的杂交瘤抗体。将CTLA-4-hFc包被在96孔ELISA板上过夜孵育，用洗涤缓冲液(PBS+0.05％Tween20)洗涤3次后，加入200μl封闭缓冲液孵育1小时；将杂交瘤上清或纯化的抗体与CTLA-4-hFc孵育1小时，再加入0.1μg/mL的CD80-biotin孵育1小时，洗涤后加入二抗AvidinHRP(Invitrogen，CatNo:18-4100-51)孵育30分钟，加入TMB显色，用100μL/孔的终止液(2NH2SO4)终止反应，TecanSpark酶标仪测定各样品的OD405信号。(5)采用竞争FACS筛选能够阻断CTLA-4-Fc与CD80-biotin结合的杂交瘤抗体。将杂交瘤上清或纯化的抗体与293T-CTLA-4细胞(1x105细胞/孔)在U形底-96孔板中孵育1小时，加入0.1μg/mL的CD80-biotin孵育1小时，离心后用FACS缓冲液洗涤2次，加入anti-biotin-PE(Biolegend，CatNo:409004)孵育30分钟，离心洗涤后，用BDC6流式细胞仪检测293T-CTLA-4细胞的PE信号。(4)用有限稀释法，对能够阻断CTLA-4与CD80结合的杂交瘤进行亚克隆，随后再重复采用ELISA和FACS方法进行检测筛选，获得9株阳性杂交瘤单克隆。将阳性单克隆杂交瘤置50mL无血清培养基中(Invitrogen，CatNo:12045-076)培养8-9天后，离心收取上清液。用ProteinA亲和层析纯化单克隆抗体，纯化后的抗体样品经超滤离心管(Millipore，CatNo:ACS500024)换液浓缩后，用BCA方法测定蛋白浓度，用鳌试剂(厦门鲎试剂生物科技股份有限公司)检测抗体样品的内毒素含量。用ELISA和FACS检测纯化的9个抗体样品，分别为1B3D4、3D2A10、4A2B5、5D5A9、5F6A1、6A4B9、9D3B4、10F8D1、2G6A11与CTLA-4的结合，以及其阻断CTLA-4-Fc与CD80结合的活性，具体方法参考实施例2，具体结果见表1、2、3、4和图1-4.表1.用ELSIA方法检测鼠源CTLA-4单克隆抗体与CTLA-4-hFc蛋白结合的EC50。表2.用ELSIA方法检测鼠源CTLA-4单克隆抗体阻断配体结合CTLA-4-hFc的IC50。表3.用FACS方法检测鼠源CTLA-4单克隆抗体结合293T-CTLA-4细胞的EC50。表4.用FACS方法检测鼠源CTLA-4单克隆抗体阻断配体结合293T-CTLA-4细胞的IC50。根据表1和图1可以看到，用ELISA测定所得到9个抗体与人CTLA-4的结合EC50大多小于1ng/ml,表明上述抗体对人CTLA-4有高度的亲和力。根据表2和图2可以看到，用ELISA测定所得到9个抗体对人CTLA-4与其配体结合的阻断作用，阻断IC50最低可以低于60ng/ml,表明上述抗体对人CTLA-4与其配体的结合有高度的阻断作用。根据表3和图3可以看到，用FACS测定所得到9个抗体与人CTLA-4的结合EC50最低的小于8ng/ml,表明上述抗体对人CTLA-4有高度的亲和力。根据表4和图4可以看到，用ELISA测定所得到9个抗体对人CTLA-4与其配体结合的阻断作用，阻断IC50最低可以低于120ng/ml,表明上述抗体对人CTLA-4与其配体的结合有高度的阻断作用。综合表1-4和图1-4可以得出，抗体3D2A10、5F6A1、10F8D1在对人CTLA-4的亲和力和对人CTLA-4与其配体结合的阻断作用特别优异。实施例2CTLA-4杂交瘤抗体对外周血单核细胞(PBMC)分泌细胞因子的影响实验，包括以下步骤：(1)在50mL无菌离心管中加入淋巴细胞分离液Histopaque(Sigma,CatNo:1077-1)，然后加入等体积的血液，在室温用1500rpm离心30min。样品在离心管中分成四层，从上至下分别为血浆层，白细胞层，淋巴细胞分离液和红细胞层。将中间的白细胞层收集到新的离心管中，加入5倍体积的洗涤缓冲液(PBS+3％FBS)混匀洗涤，于1500rpm离心10min，共重复洗涤3次，用洗涤缓冲液重悬细胞并计数。(2)细胞计数后，用完全培养基RPMI1640+10％FCS+2ng/mLSEB重悬PBMC(1×106细胞/mL)。在U底-96孔板中，每孔加入100μLPBMC以及100μl不同浓度的CTLA-4抗体(分别为1B3D4、3D2A10、4A2B5、5D5A9、5F6A1、6A4B9、9D3B4、10F8D1、2G6A11，起始浓度为10μg/mL,4倍系列稀释,依次为2.5μg/mL，0.625μg/mL，0.156μg/mL)，将96孔细胞培养板置于37℃，5％CO2培养箱中孵育72小时，收集上清液，用IL-2ELISA试剂盒(R&DSystems,CatNo:DY202)检测细胞因子的浓度。如图5所示，CTLA-4抗体能够显著促进PBMC分泌IL-2。实施例3CTLA-4抗体可变区基因的克隆，包括以下步骤：用TRIzon(Cwbiotech,CatNo:CW0580)裂解CTLA-4单克隆杂交瘤细胞株，提取杂交瘤细胞的总RNA。用HiFiScriptcDNA合成试剂盒(Cwbiotech，CatNo:CW2569)将杂交瘤细胞的RNA反转录为cDNA。以cDNA为模板，用简并引物通过PCR方法(Kettleboroughetal.(1993)EurJImmunology23:206-211；Strebeetal.(2010)AntibodyEngineering1:3-14)扩增抗体的重链和轻链的可变区基因。将PCR扩增产物连接到T/A载体后，转化DH5a感受态细胞，涂板并置37℃过夜培养。从培养板上挑取单克隆，扩大培养后抽提质粒，测定抗体的基因序列。根据抗体的基因序列，分析其互补决定簇(CDR)和骨架区。抗体的重链和轻链的可变区基因序列和氨基酸序列见序列表。实施例4人源化CTLA-4抗体10F8D1、3D2A10、5F6A1的制备。采用互补决定区嫁接法来进行CTLA-4抗体的人源化。首先，在IMGT数据库中分别搜寻与鼠源10F8D1、3D2A10和5F6A1抗体的轻、重链可变区同源性最高的人胚系抗体(germLineantibody)序列。10F8D1抗体轻链可变区人源化选取的胚系为IGKV4-1*01，重链可变区人源化选取IGHV1-2*02。3D2A10抗体轻链可变区人源化选取的胚系为IGKV3-15*01，重链可变区人源化选取IGHV2-26*01。5F6A1抗体轻链人源化选取IGLV7-46*01，重链人源化选取IGHV5-51*01。保留鼠源抗体的CDR区，将鼠源抗体的框架区(framework)序列用人胚系抗体的框架区序列置换。其次，建立鼠源抗体的结构模型，逐个对比人源抗体与相应鼠源抗体结构模型中的每个不同氨基酸位点，如果在框架区的某个位点采用人的氨基酸序列没有导致CDR区域空间结构的破坏或改变，则该位点使用人的氨基酸序列，否则在该位点使用对应的鼠源序列(即回复突变为鼠源序列)。根据结构模拟，将10F8D1、3D2A10和5F6A1人源化抗体框架区的部分氨基酸回复突变为鼠源序列。10F8D1抗体人源化重链的第48位Met回复突变为Ile，第67位Val回复突变为Ala，第69位Met回复突变为Leu，第71位Arg回复突变为Ala，第73位Thr回复突变为Lys，第94位Arg回复突变为Thr。3D2A10抗体人源化轻链的第48位Ile回复突变为Val，第71位Phe回复突变为Tyr。3D2A10抗体人源化重链的第30位Ser回复突变为Thr，第49位Ala回复突变为Gly，第73位Thr回复突变为Asn，第78位Val回复突变为Ala。5F6A1抗体人源化轻链的第36位Phe回复突变为Val，第46位Thr回复突变为Gly，第49位Tyr回复突变为Gly，第57位Trp回复突变为Gly，第69位Gly回复突变为Asp。5F6A1抗体人源化重链的第48位的Met回复突变为Ile，第67位Val回复突变为Ala，第69位Ile回复突变为Leu，第71位Ala回复突变为Val，第94位Arg回复突变为Lys。3D2A10人源化抗体重链和轻链的可变区氨基酸序列号分别为SEQIDNO：55和SEQIDNO：56。5F6A1人源化抗体重链和轻链的可变区氨基酸序列号分别为SEQIDNO：59和SEQIDNO：60。10F8D1人源化抗体重链和轻链的可变区氨基酸序列号分别为SEQIDNO：63和SEQIDNO：64。将3D2A10、5F6A1和10F8D1人源化抗体构建为IgG1亚型。3D2A10人源化完整抗体的重链和轻链氨基酸序列号分别为SEQIDNO：57和SEQIDNO：58。5F6A1人源化完整抗体的重链和轻链氨基酸序列号分别为SEQIDNO：61和SEQIDNO：62。10F8D1人源化完整抗体的重链和轻链氨基酸序列号分别为SEQIDNO：65和SEQIDNO：66。合成编码3D2A10、5F6A1和10F8D1人源化抗体轻链和重链的核酸序列(见序列表)，并插入到表达载体pcDNA3.1。用抗体轻链和重链表达质粒各0.5mg共转染1升293细胞(细胞密度为1×106)，在37度振摇培养6天，离心收集上清液，用ProteinA纯化人源化抗体，纯化后的人源化抗体用于活性检测。实施例5人源化CTLA-4抗体10F8D1、3D2A10、5F6A1的活性检测。采用ELISA和FACS检测纯化的人源化抗体样品与CTLA-4的结合及其阻断CTLA-4-Fc与CD80结合的活性，具体方法参考实施例1。人源化抗体3D2A10、5F6A1和10F8D1的测定结果见表5-8和图6-9。表5.用ELSIA方法检测人源化CTLA-4抗体与CTLA-4-hFc蛋白结合的EC50。ng/mL3D2A105F6A110F8D1结合EC501.2032.0401.494表6.用ELSIA方法检测人源化CTLA-4抗体阻断配体结合CTLA-4-hFc的IC50。ng/mL3D2A105F6A110F8D1阻断IC50352.4807.9387.6表7.用FACS方法检测人源化CTLA-4抗体结合293T-CTLA-4细胞的EC50。ng/mL3D2A105F6A110F8D1结合EC5078.83242.623.09表8.用FACS方法检测人源化CTLA-4抗体阻断配体结合293T-CTLA-4细胞的IC50。ng/mL3D2A105F6A110F8D1阻断IC50696.115461660根据表5和图6可以看到，用ELISA测定所得到3个人源化抗体对于人CTLA-4的结合EC50在1-2ng/ml之间,表明上述抗体对人CTLA-4有高度的亲和力。根据表6和图7可以看到，用ELISA测定所得到3个人源化抗体对人CTLA-4与其配体结合的阻断作用，阻断IC50最低可以低于360ng/ml,表明上述抗体对人CTLA-4与其配体的结合有高度的阻断作用。根据表7和图8可以看到，用FACS测定所得到3个人源化抗体对人CTLA-4的结合EC50最低的小于24ng/ml,表明上述抗体对人CTLA-4有高度的亲和力。根据表8和图9可以看到，用FACS测定所得到3个人源化抗体对人CTLA-4与其配体结合的阻断作用，阻断IC50最低可以低于700ng/ml,表明上述抗体对人CTLA-4与其配体的结合有高度的阻断作用。综合表5-8和图6-9可以得出结论，人源化抗体3D2A10、5F6A1、10F8D1在对人CTLA-4的亲和力和对人CTLA-4与其配体结合的阻断作用特别优异。实施例6人源化CTLA-4抗体10F8D1、3D2A10、5F6A1外周血单核细胞(PBMC)分泌细胞因子的影响实验。CTLA-4人源化抗体对外周血单核细胞(PBMC)分泌细胞因子的影响。将CTLA-4人源化抗体以及SEB与人PBMC置37℃培养72小时，收集上清液，检测上清液中IL-2的浓度，具体测定方法参考实施例2。结果如图10，表明人源化的CTLA-4抗体能够显著促进PBMC分泌IL-2。以上所述仅是本发明的优选实施方式，应当指出，对于本
技术领域：
的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进，这些改进也应视为本发明的保护范围。SEQUENCELISTING<110>东大生物技术（苏州）有限公司<120>一组CTLA-4单克隆抗体及其医药用途<130>2019<160>66<170>PatentInversion3.3<210>1<211>342<212>DNA<213>鼠<220><223>1B3D4重链可变区DNA序列<400>1cagctgcagcagtctggggctgagctggtgaggcctgggtcttcagtgaaactgtcctgc60agggcttctggctacaccttcaccagctactggatgcattgggtgaagcagaggcctata120caaggccttgaatggattggtaacattgacccttctgatagtgaaactcactacaatgaa180gagttccaggacaaggccacattgactgtagacaactcctccagcacagcctacatgcag240ctcagcagcctgacatctgaggactctgcagtctatttctgtgcaagggggactggttac300tttgactactggggccaaggcaccactctcacagtctcctca342<210>2<211>321<212>DNA<213>鼠<220><223>1B3D4轻链可变区DNA序列<400>2gatattgtgatgacccagtctccagccaccctgtctgtgactccaggagatagcgtcagt60ctttcctgcagggccagccaaagtatcagtatcaacccacactggtatcaacaaaaatca120catgagtctccaaggcttctcatcaagtatgcttcccagtccgtctctgggatcccctcc180aggttcagtggcagtggatcagggacagatttcactctcattatcaacagtgtggagact240gaagattttggaatgtatttctgtcaacagagtaacagctggcctcacacgttcggctcg300gggacaaaattggaaataaaa321<210>3<211>114<212>PRT<213>鼠<220><223>1B3D4重链可变区氨基酸序列<400>3GlnLeuGlnGlnSerGlyAlaGluLeuValArgProGlySerSerVal151015LysLeuSerCysArgAlaSerGlyTyrThrPheThrSerTyrTrpMet202530HisTrpValLysGlnArgProIleGlnGlyLeuGluTrpIleGlyAsn354045IleAspProSerAspSerGluThrHisTyrAsnGluGluPheGlnAsp505560LysAlaThrLeuThrValAspAsnSerSerSerThrAlaTyrMetGln65707580LeuSerSerLeuThrSerGluAspSerAlaValTyrPheCysAlaArg859095GlyThrGlyTyrPheAspTyrTrpGlyGlnGlyThrThrLeuThrVal100105110SerSer<210>4<211>107<212>PRT<213>鼠<220><223>1B3D4轻链可变区氨基酸序列<400>4AspIleValMetThrGlnSerProAlaThrLeuSerValThrProGly151015AspSerValSerLeuSerCysArgAlaSerGlnSerIleSerIleAsn202530ProHisTrpTyrGlnGlnLysSerHisGluSerProArgLeuLeuIle354045LysTyrAlaSerGlnSerValSerGlyIleProSerArgPheSerGly505560SerGlySerGlyThrAspPheThrLeuIleIleAsnSerValGluThr65707580GluAspPheGlyMetTyrPheCysGlnGlnSerAsnSerTrpProHis859095ThrPheGlySerGlyThrLysLeuGluIleLys100105<210>5<211>357<212>DNA<213>鼠<220><223>3D2A10重链可变区DNA序列<400>5caggtgcagttgaagcagtcaggacctggcctagtgcagccctcacagagcctgtccatc60acctgcacagtctctggtttctcattaactaactatgctgtacactgggttcgtcagtct120ccaggaaagggtctggagtggctgggagtgatatggagtggtggatacatagactataat180gcagttttcatatccagactgagaatcagcaaggacaattccagcagccaagctttcctt240aaaatgaacagtctgcaaagtgatgacacagccatatattactgtgccagatcaggggag300actgggacctactactttgactactggggccaaggcaccattctcacagtctcctca357<210>6<211>321<212>DNA<213>鼠<220><223>3D2A10轻链可变区DNA序列<400>6gaaatccagctgtctcagtctccatccaccctatctgtatctgtgggagaaactgtcacc60atcacatgtcgcgcaagtgaaaatatttacagttatttagcatggtatcagcagaaacag120ggaaaatctcctcagctcctggtctattctgcaacaaagttagcaaatggtgtgccatca180aggttcagtggcagtggatcagggacacactattccctcaagatcaacagtctgcagtct240gaagattttgggagttattactgtcaacatttttggggtactccgtggtcgttcggtgca300ggcaccaaactggaactcaaa321<210>7<211>119<212>PRT<213>鼠<220><223>3D2A10重链可变区氨基酸序列<400>7GlnValGlnLeuLysGlnSerGlyProGlyLeuValGlnProSerGln151015SerLeuSerIleThrCysThrValSerGlyPheSerLeuThrAsnTyr202530AlaValHisTrpValArgGlnSerProGlyLysGlyLeuGluTrpLeu354045GlyValIleTrpSerGlyGlyTyrIleAspTyrAsnAlaValPheIle505560SerArgLeuArgIleSerLysAspAsnSerSerSerGlnAlaPheLeu65707580LysMetAsnSerLeuGlnSerAspAspThrAlaIleTyrTyrCysAla859095ArgSerGlyGluThrGlyThrTyrTyrPheAspTyrTrpGlyGlnGly100105110ThrIleLeuThrValSerSer115<210>8<211>107<212>PRT<213>鼠<220><223>3D2A10轻链可变区氨基酸序列<400>8GluIleGlnLeuSerGlnSerProSerThrLeuSerValSerValGly151015GluThrValThrIleThrCysArgAlaSerGluAsnIleTyrSerTyr202530LeuAlaTrpTyrGlnGlnLysGlnGlyLysSerProGlnLeuLeuVal354045TyrSerAlaThrLysLeuAlaAsnGlyValProSerArgPheSerGly505560SerGlySerGlyThrHisTyrSerLeuLysIleAsnSerLeuGlnSer65707580GluAspPheGlySerTyrTyrCysGlnHisPheTrpGlyThrProTrp859095SerPheGlyAlaGlyThrLysLeuGluLeuLys100105<210>9<211>357<212>DNA<213>鼠<220><223>4A2B5B5重链可变区DNA序列<400>9gatatacagcttcaggagtcaggacctggcctcgtgaaaccttctcagtctctgtctctc60acctgctctgtcactggctactccatcaccagtggtttttactggaactggatccggcag120tttccaggaaacaaactggaatggatgggctacataacctccgacggtatcaatgtctac180aacccatctctcaaaaatcgattctccatcactcgtgacacatctaagaaccaatttttc240ctgaagttgaattctgtgactgctgaggacacagctacatattactgtgcaagaaactat300ggtaactggggggctatggactactggggtcaaggaacctcagtcaccgtctcctca357<210>10<211>318<212>DNA<213>鼠<220><223>4A2B5B5轻链可变区DNA序列<400>10gacatccagatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcacc60atcacttgcagggcaagccaagacattaagaagtatataggttggttccaacacaagcct120ggaaaaggtcctaggcttctcatatattacacatctactttacagccaggcatcccatca180aggttcagtggaagtgggtctgggagagattattccttcagcatcaccaacctggagcct240gaagatattgcaacctattattgtctacagtatgataatatgcacacgttcggagggggg300accaagctggaaataaaa318<210>11<211>119<212>PRT<213>鼠<220><223>4A2B5B5重链可变区氨基酸序列<400>11AspIleGlnLeuGlnGluSerGlyProGlyLeuValLysProSerGln151015SerLeuSerLeuThrCysSerValThrGlyTyrSerIleThrSerGly202530PheTyrTrpAsnTrpIleArgGlnPheProGlyAsnLysLeuGluTrp354045MetGlyTyrIleThrSerAspGlyIleAsnValTyrAsnProSerLeu505560LysAsnArgPheSerIleThrArgAspThrSerLysAsnGlnPhePhe65707580LeuLysLeuAsnSerValThrAlaGluAspThrAlaThrTyrTyrCys859095AlaArgAsnTyrGlyAsnTrpGlyAlaMetAspTyrTrpGlyGlnGly100105110ThrSerValThrValSerSer115<210>12<211>106<212>PRT<213>鼠<220><223>4A2B5B5轻链可变区氨基酸序列<400>12AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerLeuGly151015GlyLysValThrIleThrCysArgAlaSerGlnAspIleLysLysTyr202530IleGlyTrpPheGlnHisLysProGlyLysGlyProArgLeuLeuIle354045TyrTyrThrSerThrLeuGlnProGlyIleProSerArgPheSerGly505560SerGlySerGlyArgAspTyrSerPheSerIleThrAsnLeuGluPro65707580GluAspIleAlaThrTyrTyrCysLeuGlnTyrAspAsnMetHisThr859095PheGlyGlyGlyThrLysLeuGluIleLys100105<210>13<211>351<212>DNA<213>鼠<220><223>5D5A9C9重链可变区DNA序列<400>13caggttcagctggaggagtctggagctgagctggtgaggcctgggtcctcagtgaagatg60tcctgcaagacttctggatatacattcacaaactacggtataaactgggtgaaacagagg120cctggacagggcctggaatggattggaaagatttatattggaaatggttatactgaatat180aatgagaagttcaagggcaaggccacactgacttcagacacatcctccagcacagcctac240atgcagctcagcagcctgacatctgacgactctgcaatctatttctgtgcacgctgggac300cactattctatggactactggggtcaaggaacctcagtcaccgtctcctca351<210>14<211>321<212>DNA<213>鼠<220><223>5D5A9C9轻链可变区DNA序列<400>14gacattgtgatgacccagtctcacaaattcatgtccacatcagtaggagacagggtcagc60atcacctgcaaggccagtcaggatgtgagtactgttgtagtctggtatcaacaaaaacca120ggacaatctcctaaagtactgatttattcggcatcttaccggtacactggagtccctgat180cgcttcgctggcagtggatctgggacggatttcactttcaccatcagcagtgtgcgggct240gaagacctggcagtttattactgtcaacaacattatagtactccgtacacgttcggaggg300gggaccaagctggaaataaaa321<210>15<211>117<212>PRT<213>鼠<220><223>5D5A9C9重链可变区氨基酸序列<400>15GlnValGlnLeuGluGluSerGlyAlaGluLeuValArgProGlySer151015SerValLysMetSerCysLysThrSerGlyTyrThrPheThrAsnTyr202530GlyIleAsnTrpValLysGlnArgProGlyGlnGlyLeuGluTrpIle354045GlyLysIleTyrIleGlyAsnGlyTyrThrGluTyrAsnGluLysPhe505560LysGlyLysAlaThrLeuThrSerAspThrSerSerSerThrAlaTyr65707580MetGlnLeuSerSerLeuThrSerAspAspSerAlaIleTyrPheCys859095AlaArgTrpAspHisTyrSerMetAspTyrTrpGlyGlnGlyThrSer100105110ValThrValSerSer115<210>16<211>107<212>PRT<213>鼠<220><223>5D5A9C9轻链可变区氨基酸序列<400>16AspIleValMetThrGlnSerHisLysPheMetSerThrSerValGly151015AspArgValSerIleThrCysLysAlaSerGlnAspValSerThrVal202530ValValTrpTyrGlnGlnLysProGlyGlnSerProLysValLeuIle354045TyrSerAlaSerTyrArgTyrThrGlyValProAspArgPheAlaGly505560SerGlySerGlyThrAspPheThrPheThrIleSerSerValArgAla65707580GluAspLeuAlaValTyrTyrCysGlnGlnHisTyrSerThrProTyr859095ThrPheGlyGlyGlyThrLysLeuGluIleLys100105<210>17<211>345<212>DNA<213>鼠<220><223>5F6A1重链可变区DNA序列<400>17gaggcccaactgcagcagtctggacctgaactggtgaagcctggagcttcaatgaagata60tcctgcaaggcttctggttactcattcactggctacaccgtgaactgggtgaagcggagc120catggagagaaccttgagtggattggacttattaatccttacaatggagttactaactat180aaccagaagttcaagggcaaggccacattaactgtagacaagtcatccagcacagcctac240atggagctcctcagtctgacatctgaggactctgcagtctattactgtgcaaaattggac300tacgttgacttctggggccaaggcaccactctcacagtctcctca345<210>18<211>327<212>DNA<213>鼠<220><223>5F6A1轻链可变区DNA序列<400>18caggctgttgtgactcaggaatctacactcaccacatcacctggtgaaacagtcacactc60acttgtcgctcaagtactggggctgttgcaactagtaactttgccaactgggtccaagaa120aagccagatcatttattcactggtctaataggtggtaccaacaaccgagctccaggtgtt180cctgccagattctcaggctccctgattggagacaaggctgccctcaccattacaggggca240cagactgaggatgaggcaatatatttctgtgctctatggtacagcacccactgggtgttc300ggtggaggaaccaaactgactgtccta327<210>19<211>115<212>PRT<213>鼠<220><223>5F6A1重链可变区氨基酸序列<400>19GluAlaGlnLeuGlnGlnSerGlyProGluLeuValLysProGlyAla151015SerMetLysIleSerCysLysAlaSerGlyTyrSerPheThrGlyTyr202530ThrValAsnTrpValLysArgSerHisGlyGluAsnLeuGluTrpIle354045GlyLeuIleAsnProTyrAsnGlyValThrAsnTyrAsnGlnLysPhe505560LysGlyLysAlaThrLeuThrValAspLysSerSerSerThrAlaTyr65707580MetGluLeuLeuSerLeuThrSerGluAspSerAlaValTyrTyrCys859095AlaLysLeuAspTyrValAspPheTrpGlyGlnGlyThrThrLeuThr100105110ValSerSer115<210>20<211>109<212>PRT<213>鼠<220><223>5F6A1轻链可变区氨基酸序列<400>20GlnAlaValValThrGlnGluSerThrLeuThrThrSerProGlyGlu151015ThrValThrLeuThrCysArgSerSerThrGlyAlaValAlaThrSer202530AsnPheAlaAsnTrpValGlnGluLysProAspHisLeuPheThrGly354045LeuIleGlyGlyThrAsnAsnArgAlaProGlyValProAlaArgPhe505560SerGlySerLeuIleGlyAspLysAlaAlaLeuThrIleThrGlyAla65707580GlnThrGluAspGluAlaIleTyrPheCysAlaLeuTrpTyrSerThr859095HisTrpValPheGlyGlyGlyThrLysLeuThrValLeu100105<210>21<211>348<212>DNA<213>鼠<220><223>6A4B9重链可变区DNA序列<400>21caggtccagctgcagcagcctggggctgagctggtgaggcctgggtcttcattgaagctg60tcctgcaaggcttctggctacaccttcaccaactactggatgcattgggtgaaacagagg120cctatacagggccttgaatggattggtaacattgacccttctgatagtgagactcactac180aatcaagacttcagggacaaggccacattgactgtagacacatcctccagcacagcctac240atgcaactcaccagcctgacatctgaggactctgcggtctatttctgtgctcgaggggga300agtttcttcgatgtctggggcacagggaccacggtcaccgtctcctca348<210>22<211>321<212>DNA<213>鼠<220><223>6A4B9轻链可变区DNA序列<400>22gatattgtgctaactcagtctccagccaccctgtctgtgactccaggagatagcgtcact60ctttcctgcagggccagccaaagtattagaaacaacctaaactggtttcaacaaaaatcg120catgagtctccaaggcttctcatcaaatatgtttcccattccatctctgggatcccctcc180aggttcagtggcagtggatcagggacagatttcactctcagtatcaacactgtggagact240gaagattttggaatgtatttctgtcaacagagtgacagctggcctcacacgttcgggggg300gggaccaagctggaaataaaa321<210>23<211>116<212>PRT<213>鼠<220><223>6A4B9重链可变区氨基酸序列<400>23GlnValGlnLeuGlnGlnProGlyAlaGluLeuValArgProGlySer151015SerLeuLysLeuSerCysLysAlaSerGlyTyrThrPheThrAsnTyr202530TrpMetHisTrpValLysGlnArgProIleGlnGlyLeuGluTrpIle354045GlyAsnIleAspProSerAspSerGluThrHisTyrAsnGlnAspPhe505560ArgAspLysAlaThrLeuThrValAspThrSerSerSerThrAlaTyr65707580MetGlnLeuThrSerLeuThrSerGluAspSerAlaValTyrPheCys859095AlaArgGlyGlySerPhePheAspValTrpGlyThrGlyThrThrVal100105110ThrValSerSer115<210>24<211>107<212>PRT<213>鼠<220><223>6A4B9轻链可变区氨基酸序列<400>24AspIleValLeuThrGlnSerProAlaThrLeuSerValThrProGly151015AspSerValThrLeuSerCysArgAlaSerGlnSerIleArgAsnAsn202530LeuAsnTrpPheGlnGlnLysSerHisGluSerProArgLeuLeuIle354045LysTyrValSerHisSerIleSerGlyIleProSerArgPheSerGly505560SerGlySerGlyThrAspPheThrLeuSerIleAsnThrValGluThr65707580GluAspPheGlyMetTyrPheCysGlnGlnSerAspSerTrpProHis859095ThrPheGlyGlyGlyThrLysLeuGluIleLys100105<210>25<211>360<212>DNA<213>鼠<220><223>9D3B4重链可变区DNA序列<400>25caggttcagctgcagcagtctgggactgtgctggcaaggcctggggcttcagtgaagatg60tcctgcaagacttctggctacacatttaccagctactggatgcactgggtaaaacagagg120cctggacagggtctggaatggataggggctatttatcctggaaatagtgatactggctac180aaccagaagttcaagggcaaggccaaactgactgcagtcacatccgccagcactgcctac240atggagctcagcagcctgacaaatgaggactctgcggtctattactgtacaagagacaaa300tttattagtacggtaatagaggactactggggccaaggcaccactctcacagtctcctca360<210>26<211>321<212>DNA<213>鼠<220><223>9D3B4轻链可变区DNA序列<400>26gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcacc60atcagttgcagggcaagtcaggacattagcaattatttaaactggtatcagcagaaacca120gatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatca180agattcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaa240gaagatattgccacttacttttgccaacaggctaatatgcttcctcggacgttcggtgga300ggcaccaacctggaaatcaaa321<210>27<211>120<212>PRT<213>鼠<220><223>9D3B4重链可变区氨基酸序列<400>27GlnValGlnLeuGlnGlnSerGlyThrValLeuAlaArgProGlyAla151015SerValLysMetSerCysLysThrSerGlyTyrThrPheThrSerTyr202530TrpMetHisTrpValLysGlnArgProGlyGlnGlyLeuGluTrpIle354045GlyAlaIleTyrProGlyAsnSerAspThrGlyTyrAsnGlnLysPhe505560LysGlyLysAlaLysLeuThrAlaValThrSerAlaSerThrAlaTyr65707580MetGluLeuSerSerLeuThrAsnGluAspSerAlaValTyrTyrCys859095ThrArgAspLysPheIleSerThrValIleGluAspTyrTrpGlyGln100105110GlyThrThrLeuThrValSerSer115120<210>28<211>107<212>PRT<213>鼠<220><223>9D3B4轻链可变区氨基酸序列<400>28AspIleGlnMetThrGlnThrThrSerSerLeuSerAlaSerLeuGly151015AspArgValThrIleSerCysArgAlaSerGlnAspIleSerAsnTyr202530LeuAsnTrpTyrGlnGlnLysProAspGlyThrValLysLeuLeuIle354045TyrTyrThrSerArgLeuHisSerGlyValProSerArgPheSerGly505560SerGlySerGlyThrAspTyrSerLeuThrIleSerAsnLeuGluGln65707580GluAspIleAlaThrTyrPheCysGlnGlnAlaAsnMetLeuProArg859095ThrPheGlyGlyGlyThrAsnLeuGluIleLys100105<210>29<211>360<212>DNA<213>鼠<220><223>10F8D1D9重链可变区DNA序列<400>29caggtccagctgcagcagtctggggctgaactggcaaaacctggggcctcagtgaagctg60tcctgcaaggcttctggctacacctttactaactactggatgcactgggtaaaacagagg120cctggacagggtctggaatggattggatacattaatcctaccactacttatactaactac180aatcagaagttcaaggacaaggccacattgactgcagacaagtcttccagcacagcctac240atgcagctgagcagcctgacatatgaggactctgcagtctattactgtgcaaccacagct300caggctacgggatatggtatggacttctggggtcaaggaacctcagtcaccgtctcctca360<210>30<211>339<212>DNA<213>鼠<220><223>10F8D1D9轻链可变区DNA序列<400>30gacattgtgatgacacagtctccatcctccctgactgtgacagcaggagagaaggtcact60atgagctgcaagtccactcagagtctgttaaacagtggaaatcaaaggaactacttgacc120tggtaccagcagaaaccagggcagcctcctaaactgttgatctactgggcatccactagg180gaatctggggtccctgatcgcttcacaggcagtggatctggaacagatttcactctcacc240atcagcagtgtgcaggctgaagacctggcagtttattactgtcagaatgattatagtttt300ccgctcacgttcggtgctgggaccaagctggagctgaaa339<210>31<211>120<212>PRT<213>鼠<220><223>10F8D1D9重链可变区氨基酸序列<400>31GlnValGlnLeuGlnGlnSerGlyAlaGluLeuAlaLysProGlyAla151015SerValLysLeuSerCysLysAlaSerGlyTyrThrPheThrAsnTyr202530TrpMetHisTrpValLysGlnArgProGlyGlnGlyLeuGluTrpIle354045GlyTyrIleAsnProThrThrThrTyrThrAsnTyrAsnGlnLysPhe505560LysAspLysAlaThrLeuThrAlaAspLysSerSerSerThrAlaTyr65707580MetGlnLeuSerSerLeuThrTyrGluAspSerAlaValTyrTyrCys859095AlaThrThrAlaGlnAlaThrGlyTyrGlyMetAspPheTrpGlyGln100105110GlyThrSerValThrValSerSer115120<210>32<211>113<212>PRT<213>鼠<220><223>10F8D1D9轻链可变区氨基酸序列<400>32AspIleValMetThrGlnSerProSerSerLeuThrValThrAlaGly151015GluLysValThrMetSerCysLysSerThrGlnSerLeuLeuAsnSer202530GlyAsnGlnArgAsnTyrLeuThrTrpTyrGlnGlnLysProGlyGln354045ProProLysLeuLeuIleTyrTrpAlaSerThrArgGluSerGlyVal505560ProAspArgPheThrGlySerGlySerGlyThrAspPheThrLeuThr65707580IleSerSerValGlnAlaGluAspLeuAlaValTyrTyrCysGlnAsn859095AspTyrSerPheProLeuThrPheGlyAlaGlyThrLysLeuGluLeu100105110Lys<210>33<211>357<212>DNA<213>鼠<220><223>2G6A11重链可变区DNA序列<400>33gaggttaagctggaggagtcaggacctggcctcgtgaaaccttctcagtctctgtctctc60acctgctctgtcactggctactccatcaccagtggttattactggagctggatccggcag120tttccaggaaacaaactggaatggatgggctacataagctacgatggtgacaataactac180aacccatctctcaaaaatcgaatctccatcactcgtgacacatctaagaaccagtttttc240ctgaagttgaattctgtgactactgaggacacagccacatattactgtgcaagaaactac300ggtacctcaggggctctggactactggggtcaaggaacctcactcaccgtctcctca357<210>34<211>318<212>DNA<213>鼠<220><223>2G6A11轻链可变区DNA序列<400>34gacatccagatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcacc60atcacttgcaaggcaagccaagacattcacaagtatataggttggtaccaacacaagcct120ggaaaaggtcctaggctgctcatatattacacatcaacattacagccaggcatcccatca180aggttcagtggaagtgggtctgggagagattattccttcagcatcagcaacctggagcct240gaagatattgcaacttattattgtcaacagtatgataatctattcacgttcggctcgggg300acaaagttggaaataaaa318<210>35<211>119<212>PRT<213>鼠<220><223>2G6A11重链可变区氨基酸序列<400>35GluValLysLeuGluGluSerGlyProGlyLeuValLysProSerGln151015SerLeuSerLeuThrCysSerValThrGlyTyrSerIleThrSerGly202530TyrTyrTrpSerTrpIleArgGlnPheProGlyAsnLysLeuGluTrp354045MetGlyTyrIleSerTyrAspGlyAspAsnAsnTyrAsnProSerLeu505560LysAsnArgIleSerIleThrArgAspThrSerLysAsnGlnPhePhe65707580LeuLysLeuAsnSerValThrThrGluAspThrAlaThrTyrTyrCys859095AlaArgAsnTyrGlyThrSerGlyAlaLeuAspTyrTrpGlyGlnGly100105110ThrSerLeuThrValSerSer115<210>36<211>106<212>PRT<213>鼠<220><223>2G6A11轻链可变区氨基酸序列<400>36AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerLeuGly151015GlyLysValThrIleThrCysLysAlaSerGlnAspIleHisLysTyr202530IleGlyTrpTyrGlnHisLysProGlyLysGlyProArgLeuLeuIle354045TyrTyrThrSerThrLeuGlnProGlyIleProSerArgPheSerGly505560SerGlySerGlyArgAspTyrSerPheSerIleSerAsnLeuGluPro65707580GluAspIleAlaThrTyrTyrCysGlnGlnTyrAspAsnLeuPheThr859095PheGlySerGlyThrLysLeuGluIleLys100105<210>37<211>5<212>PRT<213>鼠<220><223>3D2A10重链CDR1区氨基酸序列<400>37AsnTyrAlaValHis15<210>38<211>16<212>PRT<213>鼠<220><223>3D2A10重链CDR2区氨基酸序列<400>38ValIleTrpSerGlyGlyTyrIleAspTyrAsnAlaValPheIleSer151015<210>39<211>5<212>PRT<213>鼠<220><223>3D2A10重链CDR3区氨基酸序列<400>39AsnTyrAlaValHis15<210>40<211>11<212>PRT<213>鼠<220><223>3D2A10轻链CDR1区氨基酸序列<400>40ArgAlaSerGluAsnIleTyrSerTyrLeuAla1510<210>41<211>7<212>PRT<213>鼠<220><223>3D2A10轻链CDR2区氨基酸序列<400>41SerAlaThrLysLeuAlaAsn15<210>42<211>9<212>PRT<213>鼠<220><223>3D2A10轻链CDR3区氨基酸序列<400>42GlnHisPheTrpGlyThrProTrpThr15<210>43<211>5<212>PRT<213>鼠<220><223>5F6A1重链CDR1区氨基酸序列<400>43GlyTyrThrValAsn15<210>44<211>17<212>PRT<213>鼠<220><223>5F6A1重链CDR2区氨基酸序列<400>44LeuIleAsnProTyrAsnGlyValThrAsnTyrAsnGlnLysPheLys151015Gly<210>45<211>6<212>PRT<213>鼠<220><223>5F6A1重链CDR3区氨基酸序列<400>45LeuAspTyrValAspPhe15<210>46<211>14<212>PRT<213>鼠<220><223>5F6A1轻链CDR1区氨基酸序列<400>46ArgSerSerThrGlyAlaValAlaThrSerAsnPheAlaAsn1510<210>47<211>7<212>PRT<213>鼠<220><223>5F6A1轻链CDR2区氨基酸序列<400>47GlyThrAsnAsnArgAlaPro15<210>48<211>9<212>PRT<213>鼠<220><223>5F6A1轻链CDR3区氨基酸序列<400>48AlaLeuTrpTyrSerThrHisTrpVal15<210>49<211>5<212>PRT<213>鼠<220><223>10F8D1D9重链CDR1区氨基酸序列<400>49AsnTyrTrpMetHis15<210>50<211>17<212>PRT<213>鼠<220><223>10F8D1D9重链CDR2区氨基酸序列<400>50TyrIleAsnProThrThrThrTyrThrAsnTyrAsnGlnLysPheLys151015Asp<210>51<211>11<212>PRT<213>鼠<220><223>10F8D1D9重链CDR3区氨基酸序列<400>51ThrAlaGlnAlaThrGlyTyrGlyMetAspPhe1510<210>52<211>17<212>PRT<213>鼠<220><223>10F8D1D9轻链CDR1区氨基酸序列<400>52LysSerThrGlnSerLeuLeuAsnSerGlyAsnGlnArgAsnTyrLeu151015Thr<210>53<211>7<212>PRT<213>鼠<220><223>10F8D1D9轻链CDR2区氨基酸序列<400>53TrpAlaSerThrArgGluSer15<210>54<211>9<212>PRT<213>鼠<220><223>10F8D1D9轻链CDR3区氨基酸序列<400>54GlnAsnAspTyrSerPheProLeuThr15<210>55<211>119<212>PRT<213>鼠<220><223>人源化抗体3D2A10重链可变区氨基酸序列<400>55GlnValThrLeuLysGluSerGlyProValLeuValLysProThrGlu151015ThrLeuThrLeuThrCysThrValSerGlyPheSerLeuThrAsnTyr202530AlaValHisTrpIleArgGlnProProGlyLysAlaLeuGluTrpLeu354045GlyValIleTrpSerGlyGlyTyrIleAspTyrAsnAlaValPheIle505560SerArgLeuThrIleSerLysAspAsnSerLysSerGlnAlaValLeu65707580ThrMetThrAsnMetAspProValAspThrAlaThrTyrTyrCysAla859095ArgSerGlyGluThrGlyThrTyrTyrPheAspTyrTrpGlyGlnGly100105110ThrLeuValThrValSerSer115<210>56<211>107<212>PRT<213>鼠<220><223>人源化抗体3D2A10轻链可变区氨基酸序列<400>56GluIleValMetThrGlnSerProAlaThrLeuSerValSerProGly151015GluArgAlaThrLeuSerCysArgAlaSerGluAsnIleTyrSerTyr202530LeuAlaTrpTyrGlnGlnLysProGlyGlnAlaProArgLeuLeuVal354045TyrSerAlaThrLysLeuAlaAsnGlyIleProAlaArgPheSerGly505560SerGlySerGlyThrGluTyrThrLeuThrIleSerSerLeuGlnSer65707580GluAspPheAlaValTyrTyrCysGlnHisPheTrpGlyThrProTrp859095ThrPheGlyGlyGlyThrLysValGluIleLys100105<210>57<211>449<212>PRT<213>鼠<220><223>人源化抗体3D2A10重链全长氨基酸序列<400>57GlnValThrLeuLysGluSerGlyProValLeuValLysProThrGlu151015ThrLeuThrLeuThrCysThrValSerGlyPheSerLeuThrAsnTyr202530AlaValHisTrpIleArgGlnProProGlyLysAlaLeuGluTrpLeu354045GlyValIleTrpSerGlyGlyTyrIleAspTyrAsnAlaValPheIle505560SerArgLeuThrIleSerLysAspAsnSerLysSerGlnAlaValLeu65707580ThrMetThrAsnMetAspProValAspThrAlaThrTyrTyrCysAla859095ArgSerGlyGluThrGlyThrTyrTyrPheAspTyrTrpGlyGlnGly100105110ThrLeuValThrValSerSerAlaSerThrLysGlyProSerValPhe115120125ProLeuAlaProSerSerLysSerThrSerGlyGlyThrAlaAlaLeu130135140GlyCysLeuValLysAspTyrPheProGluProValThrValSerTrp145150155160AsnSerGlyAlaLeuThrSerGlyValHisThrPheProAlaValLeu165170175GlnSerSerGlyLeuTyrSerLeuSerSerValValThrValProSer180185190SerSerLeuGlyThrGlnThrTyrIleCysAsnValAsnHisLysPro195200205SerAsnThrLysValAspLysArgValGluProLysSerCysAspLys210215220ThrHisThrCysProProCysProAlaProGluLeuLeuGlyGlyPro225230235240SerValPheLeuPheProProLysProLysAspThrLeuMetIleSer245250255ArgThrProGluValThrCysValValValAspValSerHisGluAsp260265270ProGluValLysPheAsnTrpTyrValAspGlyValGluValHisAsn275280285AlaLysThrLysProArgGluGluGlnTyrAsnSerThrTyrArgVal290295300ValSerValLeuThrValLeuHisGlnAspTrpLeuAsnGlyLysGlu305310315320TyrLysCysLysValSerAsnLysAlaLeuProAlaProIleGluLys325330335ThrIleSerLysAlaLysGlyGlnProArgGluProGlnValTyrThr340345350LeuProProSerArgAspGluLeuThrLysAsnGlnValSerLeuThr355360365CysLeuValLysGlyPheTyrProSerAspIleAlaValGluTrpGlu370375380SerAsnGlyGlnProGluAsnAsnTyrLysThrThrProProValLeu385390395400AspSerAspGlySerPhePheLeuTyrSerLysLeuThrValAspLys405410415SerArgTrpGlnGlnGlyAsnValPheSerCysSerValMetHisGlu420425430AlaLeuHisAsnHisTyrThrGlnLysSerLeuSerLeuSerProGly435440445Lys<210>58<211>214<212>PRT<213>鼠<220><223>人源化抗体3D2A10轻链全长氨基酸序列<400>58GluIleValMetThrGlnSerProAlaThrLeuSerValSerProGly151015GluArgAlaThrLeuSerCysArgAlaSerGluAsnIleTyrSerTyr202530LeuAlaTrpTyrGlnGlnLysProGlyGlnAlaProArgLeuLeuVal354045TyrSerAlaThrLysLeuAlaAsnGlyIleProAlaArgPheSerGly505560SerGlySerGlyThrGluTyrThrLeuThrIleSerSerLeuGlnSer65707580GluAspPheAlaValTyrTyrCysGlnHisPheTrpGlyThrProTrp859095ThrPheGlyGlyGlyThrLysValGluIleLysArgThrValAlaAla100105110ProSerValPheIlePheProProSerAspGluGlnLeuLysSerGly115120125ThrAlaSerValValCysLeuLeuAsnAsnPheTyrProArgGluAla130135140LysValGlnTrpLysValAspAsnAlaLeuGlnSerGlyAsnSerGln145150155160GluSerValThrGluGlnAspSerLysAspSerThrTyrSerLeuSer165170175SerThrLeuThrLeuSerLysAlaAspTyrGluLysHisLysValTyr180185190AlaCysGluValThrHisGlnGlyLeuSerSerProValThrLysSer195200205PheAsnArgGlyGluCys210<210>59<211>115<212>PRT<213>鼠<220><223>人源化抗体5F6A1重链可变区氨基酸序列<400>59GluValGlnLeuValGlnSerGlyAlaGluValLysLysProGlyGlu151015SerLeuLysIleSerCysLysGlySerGlyTyrSerPheThrGlyTyr202530ThrValAsnTrpValArgGlnMetProGlyLysGlyLeuGluTrpIle354045GlyLeuIleAsnProTyrAsnGlyValThrAsnTyrAsnGlnLysPhe505560LysGlyGlnAlaThrLeuSerValAspLysSerIleSerThrAlaTyr65707580LeuGlnTrpSerSerLeuLysAlaSerAspThrAlaMetTyrTyrCys859095AlaLysLeuAspTyrValAspPheTrpGlyGlnGlyThrThrLeuThr100105110ValSerSer115<210>60<211>109<212>PRT<213>鼠<220><223>人源化抗体5F6A1轻链可变区氨基酸序列<400>60GlnAlaValValThrGlnGluProSerLeuThrValSerProGlyGly151015ThrValThrLeuThrCysArgSerSerThrGlyAlaValAlaThrSer202530AsnPheAlaAsnTrpValGlnGlnLysProGlyGlnAlaProArgGly354045LeuIleGlyGlyThrAsnAsnArgAlaProTrpThrProAlaArgPhe505560SerGlySerLeuLeuGlyGlyLysAlaAlaLeuThrLeuSerGlyAla65707580GlnProGluAspGluAlaGluTyrTyrCysAlaLeuTrpTyrSerThr859095HisTrpValPheGlyGlyGlyThrLysLeuThrValLeu100105<210>61<211>445<212>PRT<213>鼠<220><223>人源化抗体5F6A1重链全长氨基酸序列<400>61GluValGlnLeuValGlnSerGlyAlaGluValLysLysProGlyGlu151015SerLeuLysIleSerCysLysGlySerGlyTyrSerPheThrGlyTyr202530ThrValAsnTrpValArgGlnMetProGlyLysGlyLeuGluTrpIle354045GlyLeuIleAsnProTyrAsnGlyValThrAsnTyrAsnGlnLysPhe505560LysGlyGlnAlaThrLeuSerValAspLysSerIleSerThrAlaTyr65707580LeuGlnTrpSerSerLeuLysAlaSerAspThrAlaMetTyrTyrCys859095AlaLysLeuAspTyrValAspPheTrpGlyGlnGlyThrThrLeuThr100105110ValSerSerAlaSerThrLysGlyProSerValPheProLeuAlaPro115120125SerSerLysSerThrSerGlyGlyThrAlaAlaLeuGlyCysLeuVal130135140LysAspTyrPheProGluProValThrValSerTrpAsnSerGlyAla145150155160LeuThrSerGlyValHisThrPheProAlaValLeuGlnSerSerGly165170175LeuTyrSerLeuSerSerValValThrValProSerSerSerLeuGly180185190ThrGlnThrTyrIleCysAsnValAsnHisLysProSerAsnThrLys195200205ValAspLysArgValGluProLysSerCysAspLysThrHisThrCys210215220ProProCysProAlaProGluLeuLeuGlyGlyProSerValPheLeu225230235240PheProProLysProLysAspThrLeuMetIleSerArgThrProGlu245250255ValThrCysValValValAspValSerHisGluAspProGluValLys260265270PheAsnTrpTyrValAspGlyValGluValHisAsnAlaLysThrLys275280285ProArgGluGluGlnTyrAsnSerThrTyrArgValValSerValLeu290295300ThrValLeuHisGlnAspTrpLeuAsnGlyLysGluTyrLysCysLys305310315320ValSerAsnLysAlaLeuProAlaProIleGluLysThrIleSerLys325330335AlaLysGlyGlnProArgGluProGlnValTyrThrLeuProProSer340345350ArgAspGluLeuThrLysAsnGlnValSerLeuThrCysLeuValLys355360365GlyPheTyrProSerAspIleAlaValGluTrpGluSerAsnGlyGln370375380ProGluAsnAsnTyrLysThrThrProProValLeuAspSerAspGly385390395400SerPhePheLeuTyrSerLysLeuThrValAspLysSerArgTrpGln405410415GlnGlyAsnValPheSerCysSerValMetHisGluAlaLeuHisAsn420425430HisTyrThrGlnLysSerLeuSerLeuSerProGlyLys435440445<210>62<211>215<212>PRT<213>鼠<220><223>人源化抗体5F6A1轻链全长氨基酸序列<400>62GlnAlaValValThrGlnGluProSerLeuThrValSerProGlyGly151015ThrValThrLeuThrCysArgSerSerThrGlyAlaValAlaThrSer202530AsnPheAlaAsnTrpValGlnGlnLysProGlyGlnAlaProArgGly354045LeuIleGlyGlyThrAsnAsnArgAlaProTrpThrProAlaArgPhe505560SerGlySerLeuLeuGlyGlyLysAlaAlaLeuThrLeuSerGlyAla65707580GlnProGluAspGluAlaGluTyrTyrCysAlaLeuTrpTyrSerThr859095HisTrpValPheGlyGlyGlyThrLysLeuThrValLeuGlyGlnPro100105110LysAlaAsnProThrValThrLeuPheProProSerSerGluGluLeu115120125GlnAlaAsnLysAlaThrLeuValCysLeuIleSerAspPheTyrPro130135140GlyAlaValThrValAlaTrpLysAlaAspGlySerProValLysAla145150155160GlyValGluThrThrLysProSerLysGlnSerAsnAsnLysTyrAla165170175AlaSerSerTyrLeuSerLeuThrProGluGlnTrpLysSerHisArg180185190SerTyrSerCysGlnValThrHisGluGlySerThrValGluLysThr195200205ValAlaProThrGluCysSer210215<210>63<211>120<212>PRT<213>鼠<220><223>人源化抗体10F8D1D9重链可变区氨基酸序列<400>63GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGlyAla151015SerValLysValSerCysLysAlaSerGlyTyrThrPheThrAsnTyr202530TrpMetHisTrpValArgGlnAlaProGlyGlnGlyLeuGluTrpIle354045GlyTyrIleAsnProThrThrThrTyrThrAsnTyrAsnGlnLysPhe505560LysAspArgAlaThrLeuThrAlaAspLysSerIleSerThrAlaTyr65707580MetGluLeuSerArgLeuArgSerAspAspThrAlaValTyrTyrCys859095AlaThrThrAlaGlnAlaThrGlyTyrGlyMetAspPheTrpGlyGln100105110GlyThrLeuValThrValSerSer115120<210>64<211>113<212>PRT<213>鼠<220><223>人源化抗体10F8D1D9轻链可变区氨基酸序列<400>64AspIleValMetThrGlnSerProAspSerLeuAlaValSerLeuGly151015GluArgAlaThrIleAsnCysLysSerThrGlnSerLeuLeuAsnSer202530GlyAsnGlnArgAsnTyrLeuThrTrpTyrGlnGlnLysProGlyGln354045ProProLysLeuLeuIleTyrTrpAlaSerThrArgGluSerGlyVal505560ProAspArgPheSerGlySerGlySerGlyThrAspPheThrLeuThr65707580IleSerSerLeuGlnAlaGluAspValAlaValTyrTyrCysGlnAsn859095AspTyrSerPheProLeuThrPheGlyGlnGlyThrLysLeuGluIle100105110Lys<210>65<211>450<212>PRT<213>鼠<220><223>人源化抗体10F8D1D9重链全长氨基酸序列<400>65GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGlyAla151015SerValLysValSerCysLysAlaSerGlyTyrThrPheThrAsnTyr202530TrpMetHisTrpValArgGlnAlaProGlyGlnGlyLeuGluTrpIle354045GlyTyrIleAsnProThrThrThrTyrThrAsnTyrAsnGlnLysPhe505560LysAspArgAlaThrLeuThrAlaAspLysSerIleSerThrAlaTyr65707580MetGluLeuSerArgLeuArgSerAspAspThrAlaValTyrTyrCys859095AlaThrThrAlaGlnAlaThrGlyTyrGlyMetAspPheTrpGlyGln100105110GlyThrLeuValThrValSerSerAlaSerThrLysGlyProSerVal115120125PheProLeuAlaProSerSerLysSerThrSerGlyGlyThrAlaAla130135140LeuGlyCysLeuValLysAspTyrPheProGluProValThrValSer145150155160TrpAsnSerGlyAlaLeuThrSerGlyValHisThrPheProAlaVal165170175LeuGlnSerSerGlyLeuTyrSerLeuSerSerValValThrValPro180185190SerSerSerLeuGlyThrGlnThrTyrIleCysAsnValAsnHisLys195200205ProSerAsnThrLysValAspLysArgValGluProLysSerCysAsp210215220LysThrHisThrCysProProCysProAlaProGluLeuLeuGlyGly225230235240ProSerValPheLeuPheProProLysProLysAspThrLeuMetIle245250255SerArgThrProGluValThrCysValValValAspValSerHisGlu260265270AspProGluValLysPheAsnTrpTyrValAspGlyValGluValHis275280285AsnAlaLysThrLysProArgGluGluGlnTyrAsnSerThrTyrArg290295300ValValSerValLeuThrValLeuHisGlnAspTrpLeuAsnGlyLys305310315320GluTyrLysCysLysValSerAsnLysAlaLeuProAlaProIleGlu325330335LysThrIleSerLysAlaLysGlyGlnProArgGluProGlnValTyr340345350ThrLeuProProSerArgAspGluLeuThrLysAsnGlnValSerLeu355360365ThrCysLeuValLysGlyPheTyrProSerAspIleAlaValGluTrp370375380GluSerAsnGlyGlnProGluAsnAsnTyrLysThrThrProProVal385390395400LeuAspSerAspGlySerPhePheLeuTyrSerLysLeuThrValAsp405410415LysSerArgTrpGlnGlnGlyAsnValPheSerCysSerValMetHis420425430GluAlaLeuHisAsnHisTyrThrGlnLysSerLeuSerLeuSerPro435440445GlyLys450<210>66<211>220<212>PRT<213>鼠<220><223>人源化抗体10F8D1D9轻链全长氨基酸序列<400>66AspIleValMetThrGlnSerProAspSerLeuAlaValSerLeuGly151015GluArgAlaThrIleAsnCysLysSerThrGlnSerLeuLeuAsnSer202530GlyAsnGlnArgAsnTyrLeuThrTrpTyrGlnGlnLysProGlyGln354045ProProLysLeuLeuIleTyrTrpAlaSerThrArgGluSerGlyVal505560ProAspArgPheSerGlySerGlySerGlyThrAspPheThrLeuThr65707580IleSerSerLeuGlnAlaGluAspValAlaValTyrTyrCysGlnAsn859095AspTyrSerPheProLeuThrPheGlyGlnGlyThrLysLeuGluIle100105110LysArgThrValAlaAlaProSerValPheIlePheProProSerAsp115120125GluGlnLeuLysSerGlyThrAlaSerValValCysLeuLeuAsnAsn130135140PheTyrProArgGluAlaLysValGlnTrpLysValAspAsnAlaLeu145150155160GlnSerGlyAsnSerGlnGluSerValThrGluGlnAspSerLysAsp165170175SerThrTyrSerLeuSerSerThrLeuThrLeuSerLysAlaAspTyr180185190GluLysHisLysValTyrAlaCysGluValThrHisGlnGlyLeuSer195200205SerProValThrLysSerPheAsnArgGlyGluCys210215220当前第1页1 2 3