具体实施方式
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围;在本发明说明书和权利要求书中,除非文中另外明确指出,单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时,应理解,除非本发明另有说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明,本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明,具体可参见Sambrook等MOLECULAR CLONING:A LABORATORY MANUAL,Second edition,Cold Spring HarborLaboratory Press,1989and Third edition,2001;Ausubel等,CURRENT PROTOCOLS INMOLECULAR BIOLOGY,John Wiley&Sons,New York,1987and periodic updates;theseries METHODS IN ENZYMOLOGY,Academic Press,San Diego;Wolffe,CHROMATINSTRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998;METHODS INENZYMOLOGY,Vol.304,Chromatin(P.M.Wassarman and A.P.Wolffe,eds.),AcademicPress,San Diego,1999;和METHODS IN MOLECULAR BIOLOGY,Vol.119,ChromatinProtocols(P.B.Becker,ed.)Humana Press,Totowa,1999等。
实施例1
FMC63 scFv的重链(H)及轻链(L)的CDR1、CDR2、CDR3突变库的构建:
FMC63重链及轻链CDR区域的选择基于可变区的氨基酸计数法(模板为单克隆抗体FMC63,VH:Y14283.1,VL:Y14284.1),Kabat计数(Kabat number scheme.Bioinf.org.uk)列表如下:
表1
以pCAN-FMC63 scFv质粒(由模板序列插入pCANTAB 5E质粒(购自GE)的多克隆位点构建,模板序列为:VH:Y14283.1,VL:Y14284.1)为模板,利用随机引物PCR引入突变,引物如表2所示。获得的FMC63 scFv的重链(H)及轻链(L)的CDR1、CDR2、CDR3突变库PCR产物分别命名为H1、H2、H3、L1、L2和L3。用Sfi I和Not I对PCR产物及pCANTAB 5E酶切回收后,经T4DNA连接酶16℃连接过夜。连接产物电转至TG1感受态细胞,2xYT培养基重悬并于37℃复苏1h后,取菌液梯度稀释进行平板计数,得到各个突变库库容至少108,其余菌液全部涂布2xYT(GA)平板(葡萄糖2%,青霉素100ug/ml)。从上述突变库中分别随机挑取20个单克隆送测序,多样性均100%。
表2
实施例2
噬菌体抗体库的淘选:
加入20nM CD19-his-biotin抗原与噬菌体抗体库室温孵育2h,再将混合物转入链霉亲和素磁珠中室温共孵育15min。PBST-PBS洗去未结合的噬菌体,再加入胰酶37℃作用30min,从而洗脱下结合的噬菌体。将胰酶消化洗脱下的噬菌体感染4ml对数期的TG1菌体,37℃静置30min,取部分菌液梯度稀释进行平板计数,其余菌液全部涂布2xYT(GA)平板,用于下一轮的包装。包装后的噬菌体可用于下一轮的淘选,共进行4轮淘选富集,每轮淘选10倍稀释梯度降低抗原浓度,并逐轮增加PBST-PBS洗涤次数(四轮淘选的CD19-his-biotin抗原浓度分别为20nM、2nM、0.2nM和0.02nM,PBST-PBS洗涤次数分别为7,10,15,20次)。
实施例3
高亲和力scFv的筛选和鉴定:
经过四轮的淘选后,随机挑取单克隆,IPTG诱导后取上清进行ELISA,ELISA初步筛选后,挑取阳性信号至少2倍大于阴性信号的克隆送测序,分析测序结果,提取出富集较多的CDR区域对应的克隆。所述ELISA初步筛选的具体步骤如下:
挑取菌落克隆于4ml 2xYT培养基中,37℃200rpm过夜培养,以1:100转接至500ml2xYT培养基中,37℃200rpm培养至对数生长期;加入M13K07辅助噬菌体(MOI=20),37℃静置孵育30分钟,后于37℃200rpm孵育30分钟;离心去掉上清,菌体重悬至500ml 2xYT/Kan/Amp抗性培养基中培养过夜;将培养过夜的上清用1/5体积PEG/NaCl溶液沉淀静置1小时后离心,将沉淀重悬于含30%丙三醇PBS溶液,4℃储存。将噬菌体溶液稀释2-10倍后测定A268,具体结果如表3所示(表3中,克隆号代表挑取的菌落克隆编号),其中,EC50(A268)(EC50表示达到50%饱和信号所对应的噬菌体浓度,表3中EC50(A268)则表示EC50时所对应的A268的吸光度与噬菌体浓度成正比)越小,代表达到同样的饱和信号所需相应克隆稀释倍数越大,即相应克隆结合能力越强(A268通过Nanodrop测定(Thermofisher);阳性对照为FMC63WT噬菌体,阴性对照为表达抗非CD19单链抗体噬菌体,使用方法同其他克隆一样。
表3
克隆号 |
EC50(A268) |
3G11 |
0.07 |
阳性对照(FMC63 WT) |
7.14 |
阴性对照(SS1 WT) |
19.44 |
将Avidin包被至96孔板,将biotin标记的CD19抗原结合至96孔,加入梯度稀释的噬菌体后孵育1小时(稀释前噬菌体原始浓度A268见表4),用抗phage抗体(Anti M13 phageantibody,GE公司购买)检测phage结合CD19,读取OD450nm,具体结果如图1所示。
表4
克隆 |
A268 |
3G11 |
6.92 |
阳性对照(FMC63 WT) |
71.44 |
阴性对照(SS1 WT) |
77.76 |
实施例4
FMC63 scFv亲和力竞争性ELISA筛选:
将CDR突变重复较多的TG1克隆(3G11克隆)涂板,挑取单克隆,IPTG诱导后纯化phage,纯化的phage经浓度测定后(A268=5.4按照3倍浓度梯度稀释),稀释梯度与3ug/mlFMC63 mIgG2a竞争(孔板:将Avidin包被至96孔板,将biotin标记的CD19抗原结合至96孔),检测使用抗鼠Fc二抗,加入底物(TMB)后孵育5mins,1M硫酸终止反应并读取OD450nm,具体结果如图2所示。比较不同克隆的EC50及Emax(A268通过Nanodrop测定(Thermofisher);阳性对照为FMC63WT噬菌体,阴性对照为表达抗非CD19单链抗体噬菌体,使用方法同其他克隆一样)。EC50(A268)(EC50表示能够达到最大抑制率Emax 50%对应的噬菌体浓度,表5中EC50(A268)则表示EC50时所对应的A268的吸光度)越小代表相应克隆的单链抗体竞争FMC63 mIgG2a至50%结合能力越强;Emax(最大抑制率,所有克隆噬菌体在吸光度5.4时能够竞争野生型抗体结合能力的百分比)越大代表相应克隆的单链抗体相同A268浓度下竞争能力越强。
表5
克隆号 |
EC50(A268) |
Emax(最大抑制率) |
3G11 |
1.26 |
53% |
阳性对照(FMC63 WT) |
>5.4 |
20% |
阴性对照(SS1 WT) |
>5.4 |
19% |
实施例5
FMC63 scFv突变体Koff/Kd测定:
将FMC63 scFv突变体(突变体对应的克隆号、及克隆号对应的scFv的核苷酸序列和氨基酸序列见下文,SEQ ID No.31、SEQ ID No.32、SEQ ID No.9、SEQ ID No.10)通过普通分子生物学方法转接到pGCIgGH1(吉凯基因)(载体序列如SEQ ID No.33所示),构建可以表达分泌型FMC63 scFv突变体hIgG1 Fc融合蛋白,利用293真核表达系统表达;通过proteinA column进行纯化。
Koff测定:将CD19抗原通过氨基偶联至SA Sensor(ForteBio),将可溶的scFv-hIgG1 Fc融合蛋白作为流动相,结合时间120秒;解离时间为300s,通过Octet red 96读取scFv与CD19抗原的结合Response;通过Octet Red96软件拟合计算出Koff。
表6
|
WT |
3G11 |
Koff(1/s) |
1.06×10-3 |
1.21×10-3 |
克隆号:3G11
GAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTGGTACTACGGTGGTAGCATGGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACA(SEQ ID No.31)
其中粗体加下划线部分为连接肽,连接肽之前为重链可变区,连接肽之后为轻链可变区,下划线部分依次为CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2、CDR-L3)
EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIK DNSKSQVFLKMNSLQTDDTAIYYCAKHWYYGGSMAMDYWGQGTSVTVSS(SEQ ID No.9)
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDY SLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT(SEQ ID No.10)
载体pGCIgGH1全序列:
AGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCG
CCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGAT
GGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGG
GTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGA
GTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGG
GGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTG
GCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAG
ACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAAGATCTCTAGAAGCTGGGTACCT
TGTGCCCGGGCGCCACCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTCGCGATTCTTAAGGGTGTC
CAGTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCT
TCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG
GTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT
AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACC
GTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG
CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATC
CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA
GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC
CTCTCCCTGTCTCCGGGTAAATGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCAGACATGATAAG
ATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTT
GTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCA
TTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTG
GTATGGCTGATTATGATCCGGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGC
AGCTCCCGGATACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGC
GTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGAGGTCGACTCTAGAGGATCGATCCCCGCCC
CGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCA
GTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAA
GGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTC
ATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACTCTTGGCTG
AAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACA
CCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAG
TGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCC
AGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAA
AAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAG
GATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCAGT
GAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGT
GAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTG
GACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATA
AATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGC
CGTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATG
ATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTAT
TTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCC
CGAAAATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTT
GAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAA
GGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCA
CTAATGGCACCCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGC
GATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTC
ACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGAT
AGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTAT
CCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATAT
CTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATA
TGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAA
TATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGC
ATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCT
AATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTG
GGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGCATATACAGTCAGCATATG
ATACCCAGTAGTAGAGTGGGAGTGCTATCCTTTGCATATGCCGCCACCTCCCAAGGGGGCGTGAATTT
TCGCTGCTTGTCCTTTTCCTGCTGCTTATCGATGATAAGCTGTCAAACATGAGAATTCTTGAAGACGAA
AGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGG
CACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCG
CTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACAT
TTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTG
GTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACA
GCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTG
CTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCT
CAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAG
AATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGA
GGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGA
ACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAAC
AACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGAT
GGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATA
AATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTC
CCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTG
AGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGA
TTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATC
CCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGA
TCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTT
TGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT
ACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTC
GCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTC
AAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAG
CTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTT
CCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAG
GGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGC
GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA
CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCT
GGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATGGGGAA
GGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCCCGAGATGCGCC
GCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTCACA
GTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCCTGC
TTCATCCCCGTGGCCCGTTGCTCGCGTTTGCTGGCGGTGTCACTGGCCCCGTGGGTTAGGGACGGGGT
CCCCCATGGGGAATGGTTTATGGTTCGTGGGGGTTATTATTTTGGGCGTTGCGTGGGGTCAGGTCCAC
GACTGGACTGAGCAGACAGACCCATGGTTTTTGGATGGCCTGGGCATGGACCGCATGTACTGGCGCG
ACACGAACACCGGGCGTCTGTGGCTGCCAAACACCCCCGACCCCCAAAAACCACCGCGCGGATTTCT
GGCGTGCCAAGCTAGTCGACCAATTCTCATGTTTGACAGCTTATCATCGCAGATCCGGGCAACGTTGTTGCCATTGCTGCAGGCGCAGAACTGGTAGGTATGGAAGATCT(SEQ ID No.33)
实施例6
将高亲和力的scFv改造为CAR:
参照SEQ ID No.32、SEQ ID No.31所示scFv序列,按照CDR突变的氨基酸对FMC63-BBz嵌合抗原受体进行突变,即按照3G11相对应的氨基酸对FMC63-BBz嵌合抗原受体进行改造,具体为将scFv序列与CD8α-4-1BB-CD3zeta通过标准生物分子生物方法进行PCR连接,得到突变后的FMC63 Mutant-BBz(3G11-BBz)系列嵌合抗原受体,FMC63 Mutant-BBz(3G11-BBz)序列通过标准分子生物学方法插入CV401质粒(吉凯基因)。阳性对照FMC63 WT-BBz的改造方法相同,仅scFv序列替换为FMC63野生型。
FMC63 Mutant-BBz(3G11-BBz):
GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCA
CCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAA
ACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTC
CCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCT
GGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACG
TTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGG
TCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGC
CCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGT
GTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGG
GTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGA
CAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCC
ATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGG
CCAAGGAACCTCAGTCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCACCAACA
CCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAG
CGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTG
GGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACT
GCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGT
ACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGG
AGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCA
GGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTT
TTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAAC
CCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT
GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAG
GGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQID No.34)
3G11:
GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCA
CCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAA
ACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTC
CCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCT
GGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACG
TTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGG
TCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGC
CCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGT
GTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGG
GTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGA
CAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCC
ATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID No.31)
CD8α铰链区:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID No.35)ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCC TGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGG GGCTGGACTTCGCCTGTGAT(SEQ ID No.36)
CD8α跨膜区:IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID No.37)
ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC(SEQ ID No.38)
4-1BB:
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE(SEQ ID No.39)
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA(SEQ ID No.40)
CD3zeta:
LRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID No.41)
CTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID No.42)
实施例7
FMC63 Mutant-BBz(3G11-BBz)体外验证:
将scFv序列构建获得的FMC63 Mutant-BBz(3G11-BBz)(构建于CV401质粒,吉凯基因)通过与表达VSVg(Envelop)、gag/pol以及rev(Packaging)包装质粒共同感染293T细胞,经过48-72小时,收集培养基上清,上清中包含FMC63 Mutant-BBz(3G11-BBz)慢病毒(Lentivirus),经过感染人类原代PBMC;感染后的T细胞在体外扩增和培养,通过细胞因子释放和杀伤实验评估FMC63 mutation(3G11)能否增强T细胞杀伤肿瘤细胞的功能,具体结果如下:
细胞因子释放实验:
将人类PBMC用CD3和CD28抗体(OKT3克隆和15E8克隆,Miltenyi Biotec)刺激激活24小时后,将scFv序列构建获得的FMC63 Mutant-BBz(3G11-BBz)包装成慢病毒(Lentivirus)后感染上述PBMC,感染表达抗CD19嵌合抗原受体,PBMC继续用完全培养基(TexMACS GMP+10%FBS+200IU/ml hIL2)培养8-10天后,收集T细胞,扩增倍数如表7所示,并将T细胞和Raji细胞(表达CD19)按照比例1:1混合培养于2%血清的RPMI1640培养基16小时,感染效率如表7所示,将混合培养上清利用BD Cytometric bead array kit测定细胞因子的释放。实验表明,感染获得的PBMC(scFv序列构建获得的FMC63 Mutant-BBz(3G11-BBz)包装成慢病毒感染获得,表达FMC63 Mutant-BBz(3G11-BBz)嵌合抗原受体)与表达FMC63WT-BBz相比具有很强的与Raji细胞结合的能力并能够释放细胞因子,细胞因子分泌的实验结果如图3-6、表8所示,其中,CTL019为阳性对照(Norvatis),图3所示为IFNγ细胞因子分泌结果对比示意图,图4所示为TNFα细胞因子分泌结果对比示意图,图5所示为IL6细胞因子分泌结果对比示意图,图6所示为IL2细胞因子分泌结果对比示意图。
表7
克隆号 |
扩增倍数 |
感染效率 |
WT |
23 |
25% |
3G11 |
35 |
26% |
表8
细胞因子变化倍数 |
CTL019+Raji/CTL019 |
3G11+Raji/3G11 |
IFNγ |
13.96 |
59.29 |
TNFα |
39 |
44 |
IL6 |
6 |
4 |
IL2 |
103.75 |
104.80 |
肿瘤杀伤实验:
将上述制备获得的T细胞(PBMC继续培养8-10天后,收集T细胞)和Raji细胞按照一定的E:T比例,例如30:1,10:1,3:1,1:1,0.3:1比例混合培养于2%血清的RPMI1640培养基中培养4小时(Raji细胞为10E5/ml,T细胞按照E:T分别为3E6/ml,1E6/ml,0.3E6/ml,0.1E6/ml,0.03E6/ml)(表9中为将[E:T raito]作为横坐标,将特异性杀伤作为纵坐标,通过Prism5.0软件中非线性拟合S型曲线计算得到的EC50),将培养上清与LDH底物(CytoTox96Non-Radioactive Cytotoxicity Assay Kit,Promega)1:1体积混合,室温孵育30分钟后读取490nm光吸收。实验结果表明,感染获得的PBMC(scFv序列构建获得的FMC63Mutant-BBz(3G11-BBz)包装成慢病毒感染获得)与FMC63 WT-BBz相比均具有很强的肿瘤杀伤能力,细胞杀伤的具体实验结果如表9所示,结果显示3G11-BBz与野生型FMC63 WT-BBz相比拥有相同的EC50。
表9
实施例8
比较3G11克隆编辑T细胞(简称3G11T)与CTL019阳性对照(Norvatis)有效性体内实验:2.5x105Raji-ffluc细胞重悬于PBS,经尾静脉给予NSG(NOD-scid IL2Rγnull)小鼠(每组四只),第一天时经腹腔给予荧光素(150mg/kg)7-10分钟后活体成像(PerkinElmerIVIS Spectrum);第四天经尾静脉给予5x106 CTL019或3G11T细胞(重悬于PBS),活体成像于第十六天收集,结果如图7A-1、7A-2、7A-3所示。由图7A-1、7A-2、7A-3可见,CTL019或3G11T均可清除NSG小鼠体内Raji-ffluc肿瘤细胞,经过荧光强度统计表明(图7B),3G11与CTL019相比显示了杀伤肿瘤细胞的优势(荧光强度有统计学差异)。
实施例9
注射T细胞后第7天,所有小鼠经尾静脉采血后,离心分离血浆与血细胞,用红细胞裂解液裂解红细胞后,剩余白细胞用PBS重悬,加入mouse antihuman CD3 perCP抗体,检测小鼠外周血中循环的人类T细胞比例,结果如图8所示。由图8可见,3G11T或CTL019在注射后第七天,外周血中有人类T细胞循环,代表了3G11T或CTL019在小鼠体内清除了肿瘤细胞,并发生了扩增,与CTL019相比无显著差异(非劣性)。
实施例10
FMC63 Mutant-BBz体外验证:
将各scFv序列构建获得的FMC63 Mutant-BBz包装成慢病毒(Lentivirus)后,感染NK92细胞(培养条件:RPMI1640+20%FBS+200IU/ml hIL2);感染后的NK92细胞在体外扩增和培养,通过细胞因子释放和杀伤实验评估FMC63 mutation能否增强NK92细胞杀伤肿瘤细胞的功能,具体结果如下:
细胞因子释放实验:
NK92细胞系培养在RPMI1640+20%FBS+200IU/ml hIL2中;将FMC63 Mutant-BBz包装成慢病毒后感染NK92细胞,体外经过扩增和培养;将NK92细胞和Raji细胞(表达CD19)按照比例1:1混合培养于2%血清的RPMI1640培养基16小时,将混合培养上清利用BDCytometric bead array kit测定细胞因子的释放。实验表明,感染获得的NK92(各scFv序列构建获得的FMC63 Mutant-BBz包装成慢病毒感染获得,表达FMC63 Mutant-BBz嵌合抗原受体)与表达FMC63 WT-BBz相比均具有很强的与Raji细胞结合的能力并能够释放细胞因子。
肿瘤杀伤实验:
将上述制备获得的NK92细胞和Raji细胞按照一定的E:T比例,例如30:1,10:1,3:1,1:1比例混合培养于2%血清的RPMI1640培养基中培养4小时,将培养上清与LDH底物(CytoTox96 Non-Radioactive Cytotoxicity Assay Kit,Promega)1:1体积混合,室温孵育30分钟后读取490nm光吸收。实验结果表明,感染获得的NK92细胞(各scFv序列构建获得的FMC63 Mutant-BBz包装成慢病毒感染获得)与FMC63 WT-BBz相比均具有很强的肿瘤杀伤能力。
综上所述,本发明有效克服了现有技术中的种种缺点而具高度产业利用价值。
上述实施例仅例示性说明本发明的原理及其功效,而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下,对上述实施例进行修饰或改变。因此,举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变,仍应由本发明的权利要求所涵盖。
SEQUENCE LISTING
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<120> 一种抗CD19抗体及其制备方法和用途
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<210> 28
<211> 20
<212> DNA
<213> Artificial
<220>
<223> 引物
<400> 28
gcaactgatg gtgactctgt 20
<210> 29
<211> 20
<212> DNA
<213> Artificial
<220>
<223> 引物
<400> 29
gtagatcagg agtttaacag 20
<210> 30
<211> 20
<212> DNA
<213> Artificial
<220>
<223> 引物
<400> 30
ttggcaaaag taagtggcaa 20
<210> 31
<211> 726
<212> DNA
<213> Artificial
<220>
<223> scFv
<400> 31
gaggtgaaac tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 60
acatgcactg tctcaggggt ctcattaccc gactatggtg taagctggat tcgccagcct 120
ccacgaaagg gtctggagtg gctgggagta atatggggta gtgaaaccac atactataat 180
tcagctctca aatccagact gaccatcatc aaggacaact ccaagagcca agttttctta 240
aaaatgaaca gtctgcaaac tgatgacaca gccatttact actgtgccaa acattggtac 300
tacggtggta gcatggctat ggactactgg ggccaaggaa cctcagtcac cgtctcctca 360
ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcggacat ccagatgaca 420
cagactacat cctccctgtc tgcctctctg ggagacagag tcaccatcag ttgcagggca 480
agtcaggaca ttagtaaata tttaaattgg tatcagcaga aaccagatgg aactgttaaa 540
ctcctgatct accatacatc aagattacac tcaggagtcc catcaaggtt cagtggcagt 600
gggtctggaa cagattattc tctcaccatt agcaacctgg agcaagaaga tattgccact 660
tacttttgcc aacagggtaa tacgcttccg tacacgttcg gaggggggac caagctggag 720
atcaca 726
<210> 32
<211> 242
<212> PRT
<213> Artificial
<220>
<223> scFv
<400> 32
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Lys His Trp Tyr Tyr Gly Gly Ser Met Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr Thr Ser
130 135 140
Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala
145 150 155 160
Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp
165 170 175
Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly
180 185 190
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu
195 200 205
Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln
210 215 220
Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu
225 230 235 240
Ile Thr
<210> 33
<211> 7252
<212> DNA
<213> Artificial
<220>
<223> 载体
<400> 33
agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga 60
gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa 120
ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta 180
tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca 240
ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt 300
gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg 360
aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt 420
tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg 480
tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct 540
ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt 600
ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg 660
gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc 720
tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg 780
gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc 840
aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag 900
ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag 960
gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt 1020
ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca 1080
cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa 1140
gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gaagatctct 1200
agaagctggg taccttgtgc ccgggcgcca ccatggagtt tgggctgagc tggctttttc 1260
ttgtcgcgat tcttaagggt gtccagtgcg acaaaactca cacatgccca ccgtgcccag 1320
cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc 1380
tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc 1440
ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc aagacaaagc 1500
cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc 1560
aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc ctcccagccc 1620
ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag gtgtacaccc 1680
tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc ctggtcaaag 1740
gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagaacaact 1800
acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac agcaagctca 1860
ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg atgcatgagg 1920
ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa tgagcggccg 1980
ctcgaggccg gcaaggccgg atccagacat gataagatac attgatgagt ttggacaaac 2040
cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt 2100
atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat 2160
gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg 2220
tggtatggct gattatgatc cggctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc 2280
tgacacatgc agctcccgga tacggtcaca gcttgtctgt aagcggatgc cgggagcaga 2340
caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgaggtcg 2400
actctagagg atcgatcccc gccccggacg aactaaacct gactacgaca tctctgcccc 2460
ttcttcgcgg ggcagtgcat gtaatccctt cagttggttg gtacaacttg ccaactgggc 2520
cctgttccac atgtgacacg gggggggacc aaacacaaag gggttctctg actgtagttg 2580
acatccttat aaatggatgt gcacatttgc caacactgag tggctttcat cctggagcag 2640
actttgcagt ctgtggactg caacacaaca ttgcctttat gtgtaactct tggctgaagc 2700
tcttacacca atgctggggg acatgtacct cccaggggcc caggaagact acgggaggct 2760
acaccaacgt caatcagagg ggcctgtgta gctaccgata agcggaccct caagagggca 2820
ttagcaatag tgtttataag gcccccttgt taaccctaaa cgggtagcat atgcttcccg 2880
ggtagtagta tatactatcc agactaaccc taattcaata gcatatgtta cccaacggga 2940
agcatatgct atcgaattag ggttagtaaa agggtcctaa ggaacagcga tatctcccac 3000
cccatgagct gtcacggttt tatttacatg gggtcaggat tccacgaggg tagtgaacca 3060
ttttagtcac aagggcagtg gctgaagatc aaggagcggg cagtgaactc tcctgaatct 3120
tcgcctgctt cttcattctc cttcgtttag ctaatagaat aactgctgag ttgtgaacag 3180
taaggtgtat gtgaggtgct cgaaaacaag gtttcaggtg acgcccccag aataaaattt 3240
ggacgggggg ttcagtggtg gcattgtgct atgacaccaa tataaccctc acaaacccct 3300
tgggcaataa atactagtgt aggaatgaaa cattctgaat atctttaaca atagaaatcc 3360
atggggtggg gacaagccgt aaagactgga tgtccatctc acacgaattt atggctatgg 3420
gcaacacata atcctagtgc aatatgatac tggggttatt aagatgtgtc ccaggcaggg 3480
accaagacag gtgaaccatg ttgttacact ctatttgtaa caaggggaaa gagagtggac 3540
gccgacagca gcggactcca ctggttgtct ctaacacccc cgaaaattaa acggggctcc 3600
acgccaatgg ggcccataaa caaagacaag tggccactct tttttttgaa attgtggagt 3660
gggggcacgc gtcagccccc acacgccgcc ctgcggtttt ggactgtaaa ataagggtgt 3720
aataacttgg ctgattgtaa ccccgctaac cactgcggtc aaaccacttg cccacaaaac 3780
cactaatggc accccgggga atacctgcat aagtaggtgg gcgggccaag ataggggcgc 3840
gattgctgcg atctggagga caaattacac acacttgcgc ctgagcgcca agcacagggt 3900
tgttggtcct catattcacg aggtcgctga gagcacggtg ggctaatgtt gccatgggta 3960
gcatatacta cccaaatatc tggatagcat atgctatcct aatctatatc tgggtagcat 4020
aggctatcct aatctatatc tgggtagcat atgctatcct aatctatatc tgggtagtat 4080
atgctatcct aatttatatc tgggtagcat aggctatcct aatctatatc tgggtagcat 4140
atgctatcct aatctatatc tgggtagtat atgctatcct aatctgtatc cgggtagcat 4200
atgctatcct aatagagatt agggtagtat atgctatcct aatttatatc tgggtagcat 4260
atactaccca aatatctgga tagcatatgc tatcctaatc tatatctggg tagcatatgc 4320
tatcctaatc tatatctggg tagcataggc tatcctaatc tatatctggg tagcatatgc 4380
tatcctaatc tatatctggg tagtatatgc tatcctaatt tatatctggg tagcataggc 4440
tatcctaatc tatatctggg tagcatatgc tatcctaatc tatatctggg tagtatatgc 4500
tatcctaatc tgtatccggg tagcatatgc tatcctcatg catatacagt cagcatatga 4560
tacccagtag tagagtggga gtgctatcct ttgcatatgc cgccacctcc caagggggcg 4620
tgaattttcg ctgcttgtcc ttttcctgct gcttatcgat gataagctgt caaacatgag 4680
aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 4740
ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 4800
tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 4860
atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 4920
attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 4980
gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 5040
agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 5100
aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt 5160
cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 5220
cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 5280
actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 5340
cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 5400
ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa 5460
ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 5520
gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 5580
gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 5640
ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 5700
cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 5760
caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 5820
taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 5880
cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 5940
cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 6000
gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 6060
aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 6120
cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 6180
tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 6240
acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 6300
ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 6360
ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 6420
tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 6480
tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 6540
ctggcctttt gctggccttt tgctcacatg aagctgtccc tgatggtcgt catctacctg 6600
cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat 6660
aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc 6720
ggccccgaga tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc 6780
caagggttgg tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga 6840
gtggtgaatc cgttagcgag gtgccgccct gcttcatccc cgtggcccgt tgctcgcgtt 6900
tgctggcggt gtcactggcc ccgtgggtta gggacggggt cccccatggg gaatggttta 6960
tggttcgtgg gggttattat tttgggcgtt gcgtggggtc aggtccacga ctggactgag 7020
cagacagacc catggttttt ggatggcctg ggcatggacc gcatgtactg gcgcgacacg 7080
aacaccgggc gtctgtggct gccaaacacc cccgaccccc aaaaaccacc gcgcggattt 7140
ctggcgtgcc aagctagtcg accaattctc atgtttgaca gcttatcatc gcagatccgg 7200
gcaacgttgt tgccattgct gcaggcgcag aactggtagg tatggaagat ct 7252
<210> 34
<211> 1395
<212> DNA
<213> Artificial
<220>
<223> 3G11-BBz
<400> 34
gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60
atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120
gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180
aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240
gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300
gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360
ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420
tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480
cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540
tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600
ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660
tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720
tcctcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 780
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 840
gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 900
cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 960
atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1020
tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1080
gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1140
cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1200
aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1260
gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1320
ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1380
gccctgcccc ctcgc 1395
<210> 35
<211> 45
<212> PRT
<213> Artificial
<220>
<223> CD8α铰链区
<400> 35
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 36
<211> 135
<212> DNA
<213> Artificial
<220>
<223> CD8α铰链区
<400> 36
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 37
<211> 24
<212> PRT
<213> Artificial
<220>
<223> CD8α跨膜区
<400> 37
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 38
<211> 72
<212> DNA
<213> Artificial
<220>
<223> CD8α跨膜区
<400> 38
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 39
<211> 41
<212> PRT
<213> Artificial
<220>
<223> 4-1BB
<400> 39
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu
35 40
<210> 40
<211> 123
<212> DNA
<213> Artificial
<220>
<223> 4-1BB
<400> 40
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaa 123
<210> 41
<211> 113
<212> PRT
<213> Artificial
<220>
<223> CD3zeta
<400> 41
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln
1 5 10 15
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
20 25 30
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
35 40 45
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
50 55 60
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
65 70 75 80
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
85 90 95
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
100 105 110
Arg
<210> 42
<211> 339
<212> DNA
<213> Artificial
<220>
<223> CD3zeta
<400> 42
ctgagagtga agttcagcag gagcgcagac gcccccgcgt acaagcaggg ccagaaccag 60
ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 120
ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 180
aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 240
cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 300
acctacgacg cccttcacat gcaggccctg ccccctcgc 339