CN1189483C - 人源化抗cd3单克隆抗体 - Google Patents
人源化抗cd3单克隆抗体 Download PDFInfo
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- CN1189483C CN1189483C CN 01132281 CN01132281A CN1189483C CN 1189483 C CN1189483 C CN 1189483C CN 01132281 CN01132281 CN 01132281 CN 01132281 A CN01132281 A CN 01132281A CN 1189483 C CN1189483 C CN 1189483C
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- variable region
- antibody
- seq
- ser
- light chain
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Abstract
本发明提供了新人源化抗CD3单克隆抗体，其DNA编码序列及含有该编码序列的宿主细胞。本发明获得了抗排异能力强、免疫原性和毒副作用小的有效免疫抑制剂，并且提高了所述抗体在哺乳动物细胞内的表达，可广泛用于CD3相关疾病的诊断和/或治疗。
Description
发明领域
本发明涉及单克隆抗体技术，抗体人源化技术，以及基因重组技术。尤其是抗CD3的人源化单克隆抗体。
发明背景
CD3分子是广泛分布于人成熟T淋巴细胞表面的膜抗原，它与T细胞表面膜受体TCR形成复合体，在细胞内信息传递过程中起着重要的作用。已有的研究表明抗人T细胞CD3的单抗具有激活和抑制T细胞的双向功能。其疗效已在急性抗排异冲击疗法、治疗移植物抗宿主反应及器官移植前的预防性脱敏中得到充分的证实，并在抗肿瘤、抗多种器官移植排斥反应及再生障碍贫血的治疗中显示出广阔的应用前景。
mOKT3是由美国Ortho药物公司生产的鼠源抗人CD3的单抗，它对预防和治疗器官移植免疫排斥反应有良好的效果。但是，mOKT3作为鼠源性单抗，其免疫原性导致50％以上的病人产生抗mOKT3抗体，这些抗体主要有两种：抗个体型和抗同种型。前者通过中和抗原结合位点而阻断mOKT3与CD3的结合，后者除加速单抗的清除外还可能引起严重的副反应。
因而，克服鼠源性和首剂效应成为其临床广泛应用的关键。使用人源抗体或人源化抗体是克服人抗鼠源抗体反应(HAMA反应)的两种可能的方法。由于特异性强、亲和力高的人源抗体不易得到，目前主要采取鼠源单克隆抗体人源化的方法。
发明内容
本发明的目的在于提供具有潜在医学及药学价值的人源化抗人CD3单克隆抗体，其相关DNA序列和含有该序列的宿主细胞。
为了实现上述目的，本发明提供以下技术发案。
本发明提供一种人源化的抗人CD3单克隆抗体，其中原人抗体的重链可变区和轻链可变区分别被非人源重链可变区和轻链可变区所取代，所述非人源重链可变区选自SEQ ID NO：1或SEQ ID NO：5，所述非人源轻链可变区选自SEQ IDNO：2或SEQ ID NO：6。
本发明还提供一种DNA分子，其中包含上述非人源重链可变区的编码序列和/或上述非人源轻链可变区的编码序列。
本发明的实施方式之一中，上述非人源重链可变区的编码序列选自SEQ IDNO：3或SEQ ID NO：7，所述非人源轻链可变区的编码序列选自SEQ ID NO：4或SEQ ID NO：8。
本发明的实施方式之一中，所述DNA分子的形式一种载体，尤其是表达载体。
本发明还提供一种宿主细胞，它含有本发明的DNA分子，尤其是包含于宿主相容性表达系统中的所述DNA分子。所述的宿主细胞可以是从大肠杆菌等原核细胞例如到CHO等哺乳动物细胞各种适合的细胞。其选择是本领域的常规技术。
本发明还提供本发明抗体和本发明DNA分子在制备诊断和/或治疗CD3相关疾病的制剂中的用途。
本发明通过对抗CD3单抗进行人源化改造，获得了抗排异能力强、免疫原性和毒副作用小的有效免疫抑制剂，并且提高了融合抗体在哺乳动物细胞内的表达。
附图说明
图1是本发明用于合成人源化重组VH和VL编码序列的重叠PCR的示意图。
图2是本发明中使用的表达载体pMG18-3K的图谱。其中，Ck表示人抗体kappa轻链的恒定区编码序列；IgG1恒区表示人抗体IgG1的重链恒定区编码序列；pA表示SV40加poly(A)位点。
图3反映抗体接合亲合性荧光染色结果。其中纵坐标为荧光染色的细胞流量，单位是细胞数/秒，横坐标是荧光强度，单位是10-4W/cm2。分图A用的主抗体是作为阴性对照的抗CD20单克隆抗体Rituxan(购自Genentech)，第二抗体是兔抗人-FITC；分图B用的主抗体是作为阳性对照的抗CD3的mOKT3，第二抗体是兔抗鼠FITC；分图C用的主抗体是本发明的cGD3，第二抗体是兔抗人-FITC；分图D用的主抗体是本发明的huGD，第二抗体是兔抗人-FITC。
优选实施方式
一.抗CID3单抗的制备与鉴定
采用纯合CD3蛋白作为免疫原，加入完全弗氏佐剂，充分研磨至完全乳化后常规皮下多点注射10日龄Balb/c小鼠，每点注射50微升，共6点。然后以1周间隔共5次重复注射以加强免疫反应，并对血清抗体水平进行监测。最后一次静脉注射5×106新鲜细胞/PBS混合液，3日后杀死小鼠，取出脾脏。
常规方法分离脾脏单细胞，并确定细胞活性＞90％。将脾脏细胞与SP2/0小鼠骨髓瘤细胞10：1混合离心共沉淀，在37℃水浴锅中加入PEG(1400MW)进行细胞融合，融合时间为1分钟、2分钟、5分钟和10分钟。间隔5分钟加入新鲜无血清培养基1毫升、2毫升、5毫升和10毫升。离心沉淀后将细胞加入到20％FCSRPMI-1640培养液中，转入96孔板进行培养。24小时后加入1×HAT选择培养液。每3天更换新鲜培养基1次，至长出克隆。
选择单克隆形成孔，在第14天取出上清液，用ELISA法测定抗体表达量，挑选阳性孔亚克隆，在常规条件下传代，稳定至第5代。据此挑选出表达量最高的位置编号为GD3的细胞系，作为继续研究的材料。
二.抗CD3单抗可变区的扩增与测序
采用5’RACE(Rapid amplification of cDNA ends)的方法来获取抗CD3鼠源单抗mGD3的可变区编码序列。
设计并合成以下因为
1.引物设计：
GSP1-H：5’-AGC TGG GAA GGT GTG CAC ACC A CT-3’(SEQ ID NO：9)
GSP2-H：5’-CAG AGT TCC AGG TCA AGG TCA-3’(SEQ ID NO：10)
GSP3-H：5’-CTT GAC CAG GCA TCC TAG AGT-3’(SEQ ID NO：11)
GSP1-L：5’-TTG CTG TCC TGA TCA GTC CAA CT-3’(SEQ ID NO：12)
GSP2-L：5’-TGT CGT TCA CTG CCA TCA ATC TT-3’(SEQ ID NO：13)
GSP3-L：5’-TTG TTC AAG AAG CAC ACG ACT GA-3’(SEQ ID NO：14)
AAP：5’-GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3’(SEQ IDNO：15)
AUAP：5’-GGC CAC GCG TCG ACT AGT AC-3’(SEQ ID NO：16)
其中，GSP表示基因特异性引物，AAP代表5’-RACE截短锚引物，AUAP代表截短通用扩增引物。GSP1距离可变区基因最远，用于逆转录反应。GSP2和GSP3用于嵌套PCR，其中GSP3在GSP2内。
用Gibco公司的Trizol Reagent试剂分别提取2×106杂交瘤细胞mGD3的总RNA。按照5’-RACE试剂盒(Pharmacia公司产品)说明书以GSP1为引物将总RNA逆转录成cDNA，。然后给第一链cDNA的3’末端加上poly(C)尾，加尾后用GSP2和AAP为引物进行PCR扩增，将扩增产物稀释100倍再以AUAP和GSP3为引物进行嵌套PCR扩增。两次PCR反应均采用热启动，反应条件：94℃5分钟；94℃45秒，60℃45秒，72℃1分10秒，30个循环：72℃7分钟。嵌套PCR产物经1％琼脂糖凝胶电泳分离后回收纯化目的片段(轻链长度约320bp，重链长度约360bp)。克隆到pGEM-T(Promega)载体中，转化大肠杆菌TG1细胞后在IPTG/X-gal平板上进行筛选，取8个白色菌斑接种于含有氨苄青霉素的LB液体培养基中扩增。筛选阳性克隆，用QIAGEN的质粒抽提试剂盒抽提质粒并进行测序，确定了mGD3的重链和轻链可变区的DNA序列，并据此得出推断氨基酸序列
mGD3重链可变区(VH)的氨基酸序列(119氨基酸)：
QVQKSDSGGGVKQPGRSLRLSGKASGYTFTSYTAHWVRQAPGKGWNPLAYINPSSGYTKSGDKFKDRFT
ISRKKDKNTLFLQMDSLRPEDTGDYFCARWQDYDVYFDYWGQACLVTVSS(SEQ ID NO：1)
mGD3轻链可变区(VL)的氨基酸序列(107氨基酸)：
DIKLNQSPSSMNASVGDRVTISKLDSSSVSYMDDYQQTPIKAPKLLIYATSNLASGVPSTFSGSGSGTD
YTFTISSQDPEDIATYYCQQWSSDNPTFGQGTKSDKTR(SEQ ID NO：2)
mGD3 VH的DNA序列(357核苷酸)：
CAGGTTCAGAAATCTGACTCTGGTGGTGGTGTTAAACAGCCGGGTCGTTCTCTGCGTCTGTCTGGTAAA
GCTTCTGGTTACACCTTCACCTCTTACACCGCTCACTGGGTTCGTCAGGCTCCGGGTAAAGGTTGGAAC
CCGCTGGCTTACATCAACCCGTCTTCTGGTTACACCAAATCTGGTGACAAATTCAAAGACCGTTTCACC
ATCTCTCGTAAAAAAGACAAAAACACCCTGTTCCTGCAGATGGACTCTCTGCGTCCGGAAGACACCGGT
GACTACTTCTGCGCTCGTTGGCAGGACTACGACGTTTACTTCGACTACTGGGGTCAGGCTTGCCTGGTT
ACCGTTTCTTCT(SEQ ID NO：3)
mGD3 VL的DNA序列(321核苷酸)：
GACATCAAACTGAACCAGTCTCCGTCTTCTATGAACGCTTCTGTTGGTGACCGTGTTACCATCTCTAAA
CTGGACTCTTCTTCTGTTTCTTACATGGACGACTACCAGCAGACCCCGATCAAAGCTCCGAAACTGCTG
ATCTACGCTACCTCTAACCTGGCTTCTGGTGTTCCGTCTACCTTCTCTGGTTCTGGTTCTGGTACCGAC
TACACCTTCACCATCTCTTCTCAGGACCCGGAAGACATCGCTACCTACTACTGCCAGCAGTGGTCTTCT
GACAACCCGACCTTCGGTCAGGGTACCAAATCTGACAAAACCCGT(SEQ ID NO：4)
三.人源化嵌合单克隆抗体表达载体的构建
采用PCR反应分别给以上的抗体可变区加上适当的酶切位点。其中重链可变区的5′端设计XbaI酶切位点，3′端加上NheI位点，在轻链可变区编码序列5′端设计HindIII位点，3′端加上BsiWI位点。
所用的PCR引物为；
重链正向引物：5’-ctctctagaCAGGTTCAGAAATCTG-3’(SEQ ID NO：17)
重链反向引物：5’-gacgctagcAGAAGAAACGGTAAC-3’(SEQ ID NO：18)
轻链正向引物：5’-ctgaagcaaGACATCAAACTGAACCAG-3’(SEQ ID NO：19)
轻链反向引物：5’-gaccgtacgacgggttttgtc-3’(SEQ ID NO：20)
以前述含有重链可变区和轻链可变区的质粒pGEM-T为模板进行普通PCR。将所得PCR产物克隆到pGEM-T(Promega出品)中进行序列测定确证为所需的正确目的序列。然后用XbaI和NheI将抗体重链可变区编码序列从载体pGEM-T上切下插入到表达载体pMG18-3K相应位点，再用HindIII和BsiWI将抗体轻链可变区从载体pGEM-T上切下插入到表达载体pMG18-3K相应位点(参见DEVELOPMENT OF TOOLS FOR ENVIRONMENTAL MONITORING BASED ONINCP-9 PLASMIDS SEQUENCES.A.Greated，R.Krasowiak，M.Titok，C.M.Thomas Schoolof Biological Sciences，University of Birmingham，Edgbaston，Birmingham B15 2TT，UK andFaculty of Biology，Dept of Microbiology，Belarus State University Scorina Av.4，Minsk220080 Belarus)。最后构成了嵌合抗体的表达载体pcGD3。
四.人源化重组单克隆抗体VL和VH的设计
本发明对抗CD3单克隆抗体mGD3的VL和VH进行了人源化设计，同时兼顾哺乳动物细胞使用密码子的偏爱性，设计发案如下：
人源化抗体huGD3的重链可变区氨基酸序列(119氨基酸)
QVQLVQSGGGVVQPGRSLRLSCKASGYTFTSYTMHWVRQAPGKGLEWIGYINPSSGYTKYNQKFKDRFT
ISADNSKSTAFLQMDSLRPEDTAVYYCARWQDYDVYFDYWGQGTPVTVSS(SEQ ID NO：5)
人源化抗体huGD3的轻链可变区氨基酸序列(107氨基酸)
DIVLTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQTPGKAPKPWIYATSNLASGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQQWSSNPPTFGQGTKLQITR(SEQ ID NO：6)
人源化抗体huGD3的重链可变区编码序列(5′→3′，357bp)
001 CAGGTGCAGC TGGTGCAGTC TGGCGGTGGA GTGGTCCAGC CCGGCCGCAG
051 CCTGAGGCTG TCCTGCAAGG CCAGCGGCTA CACCTTCACC AGCTACACGA
101 TGCACTGGGT GCGCCAAGCC CCCGGAAAGG GCCTCGAATG GATTGGCTAC
151 ATTAATCCTA GCAGTGGTTA TACTAAGTAC AATCAGAAGT TCAAGGACAG
201 ATTTACAATA TCAGCCGACA ACAGCAAGTC CACCGCCTTC CTACAAATGG
251 ACAGCTTGCG TCCAGAGGAC ACCGCCGTAT ACTACTGTGC GCGCTGGCAG
301 GATTACGACG TCTACTTTGA CTACTGGGGC CAAGGCACTC CAGTCACCGT
351 CTCCTCT (SEQ ID NO：7)
人源化抗体huGD3的轻链可变区编码序列(5′→3′，321bp)
001 GACATCGTTC TCACTCAGAG CCCATCCAGC TTGAGCGCAT CAGTAGGCGA
051 CCGCGTAACG ATCACTTGCA GGGCCAGCTC AAGTGTAAGT TACATGCACT
101 GGTACCAGCA GACTCCCGGC AAAGCCCCAA AGCCCTGGAT TTATGCCACA
151 TCCAACCTGG CTTCTGGCGT GCCATCACGC TTTAGCGGCA GCGGGTCCGG
201 TACAGATTAC ACGTTCACCA TTAGCAGTCT GCAGCCTGAG GACATAGCCA
251 CCTACTACTG TCAGCAGTGG AGTAGTAACC CACCGACGTT TGGCCAGGGA
301 ACTAAACTGC AGATTACTCG A (SEQ ID NO：8)
五.人源化抗体可变区编码序列的合成
我们在人源化抗体重链编码序列的5′端设计XbaI酶切位点，3′端加上NheI位点，在轻链编码序列5′端设计HindIII位点，3′端加上BsiWI位点。针对以上人源化重链和轻链的DNA序列，分别合成8段长为75bp左右且相互间有20bp重叠的寡核苷酸引物，经过三次重叠PCR(见图1)：①将引物a和b，c和d，e和f，g和h分别混合进行PCR反应，得到产物ab、cd、ef、gh。②将引物a、d与上一轮PCR产物ab、cd相混合后进行扩增，同样将引物e、h与上一轮PCR产物ef、gh相混合进行扩增，分别得到产物ad和eh。③将引物a、h与上一轮PCR产物ad、eh相混合进行PCR扩增，得到产物ah。上述PCR采用Roche公司的高保真扩增系统，分别合成以上可变区编码序列。PCR反应条件均为：95C 5分钟；94C 45秒，55C 45秒，72C 55秒，30个循环；72C 7分钟。琼脂糖凝胶电泳分离扩增产物，分别回收目的条带(重链长度360bp左右，轻链长度320左右)并克隆到pGEM-T载体中，筛选阳性克隆测序，确认分别得到了上述人源化单抗huGD3的VH和VL的编码序列。
六.人源化重组单克隆抗体表达载体的构建
用XbaI和NheI将重链可变区编码序列从载体pGEM-T上切下插入到表达载体pMG18-3K的相应位点。再用HindIII和BsiWI将轻链可变区从载体pGEM-T上切下插入到表达载体pMG18-3K中相应位点(参见图2)，得到人源化重组单克隆抗体表达载体phuGD3。
七.人源化抗体的表达
用前文步骤三构建的pcGD3和以上构建的phuGD3转染大肠杆菌DH5a。接种于100ml LB培养基中，按照常规方法进行扩增。收获培养物，用Qiagen公司的UltraPure质粒DNA纯合试剂盒抽提纯化质粒DNA。将上述纯化的质粒DNA采用Invitrogen公司的脂质体法试剂盒转染CHO细胞，操作参照厂家的说明书进行。
转化的CHO细胞在选择培养基(含有0.05～10mM氨甲基蝶呤的HAT选择培养基)上进行连续9周的选择，最后在96孔板上进行极度稀释培养，连续进行3次，进行单克隆化。
挑出的单克隆细胞系在RPM1641培养基上进行培养。对上清进行Western印迹实验，根据染色反应判断表达强度。选择高表达克隆进行后续研究。结果选得分别以phuGD3转染的克隆huGD3，和以pcGD3转染的克隆cGD3。
用蛋白A亲和层析柱从上述6个细胞系的培养上清中直接分离纯化本发明的人源化单克隆抗体。SDS-PAGE电泳检查证明，所得产物纯度大于90％。
以上亲和层析的产物再次经过分子筛层析研究，获得了纯度＞98％的样品。所得抗体可用于以下的进一步研究分析。
八.人源化抗体表达量的研究
用ELISA方法对上述小鼠杂交瘤(mGD3)、嵌合单抗(cGD3)和人源化单抗(huGD3)的CHO细胞株的表达量进行了研究。将所述细胞株37度5％CO2培养箱中培养72小时后取5ml细胞，于5000RPM离心10分钟，取上清进行DOT ELISA。其第二抗体为HRP标记的兔抗人抗体。显色采用DAB系统(购自Ameresco公司)，参照厂家的说明进行染色。
多克隆抗体表达量的研究结果
表达量(mg/ml)
阳性对照1(mOKT3) 25.6
阳性对照2(Rituxan，Genetech产品) 38.8
mGD3 20.1
cGD3 66.9
huGD3 224.3
上述结果说明，我们的小鼠杂交瘤细胞的表达量不如两个对照但是差别不大，但是我们构建的嵌合细胞系的表达量比对照嵌合抗体表达量高出70％以上，我们的人源化抗体细胞系比两个阳性对分别高出8.8倍和5.8倍。
如前文第6部分所述，用Protein A亲和层析柱分离纯化杂交瘤分泌的多克隆抗体，分别获得鼠源抗体mGD3及人源化抗体cGD3和huGD3。
九.CD3人源化单克隆抗体的生物活性鉴定
人源化抗体的亲和力测定
用FITC标记鼠GD3(mGD3)，将饱和浓度的FITC-mGD3和一系列不同稀释度未标记的huGD3混合后，与靶细胞(5×105人外周血单核细胞(PBMC))在4℃温育60min。将细胞用PBS冲洗后固定，用流式细胞仪进行检测，并计算荧光阳性细胞百分率。mGD3的亲和力常数Ka用Scatchard做图法得出。人源化抗体的亲和力常数用公式[X]-[mGD3]＝(1/Kx)-(1/Ka)计算，其中Ka是mGD3的亲和力常数，[X]是标记抗体的“接合/游离”值为R0/2时竞争抗体的浓度，R0表示最大荧光细胞数。结果如下：
mGD3的亲和力常数为：1.7×109M-1
huGD3的亲和力常数为：1.6×109M-1
从结果看，本发明huGD3的亲和力常数已达到了1.6×109M-1，完全达到了鼠源抗体的亲和力。由此证明，这样，我们所得的人源化抗体保持了原鼠源抗体的亲和力。
荧光染色
以T淋巴细胞瘤细胞HPBLL或者Jarket细胞为CD3阳性靶细胞，5×106个细胞分成5管，平均每管1×106细胞。结果如图3所示，我们研制的重组嵌合和人源化CD3单克隆抗体比阳性对照mOKT3的亲和力高2个数量级，在临床上可以大大减少用量和降低可能的副反应，具有极大的经济价值。
为了确定cGD3和huGD3两种单抗与Rituxan的等效剂量，另外进行荧光染色试验。结果如下
不同单抗亲和力与其浓度的关系
| 抗体 | 抗体浓度μg/ml | ||
| 0.1 | 1 | 10 | |
| Rituxan | 57MFI | 88MFI | 101MFI |
| cGD3 | 171MFI | 224MFI | 320MFI |
| huGD3 | 55MFI | 76MFI | 97MFI |
上述结果说明，我们研制的嵌合单抗在浓度为0.1ug/ml时，即可达到阳性对照Rituxan 1μg/ml时效果的两倍，即我们的抗体亲和力相当于对照的20倍。而我们的人源化单抗的亲和力与阳性对照大体相当。
T细胞细胞膜CD3抗原调变试验
对人淋巴细胞CD3抗原的调变作用是CD3的主要免疫学功能，为了检测人源化抗体是否还具有调变CD3分子的能力，用不同稀释度的抗体与人PBMC(1ml)相混合后37℃温育24h，收获细胞用mOKT3(购自Genetech公司)-FITC(选择mOKT3-FITC是因为它结合的表位不同于mOKT3和其他的抗CD3单抗，可直观表示没有被调变的CD3分子)染色，细胞用PE标记的抗CD5单抗复染(以区别出T细胞)，然后用流式细胞仪进行分析，每次结果均为3次实验的平均值。
计算不同抗体对CD3的调变能力的公式为：
人源化抗体对人淋巴细胞的调变作用试验结果
| CD3调变(％) | |||||||
| [抗体]ng/ml | 10-1 | 10 | 101 | 102 | 103 | 104 | 105 |
| mGD3 | 10.56 | 15.45 | 16.78 | 67.67 | 78.89 | 85.86 | 85.34 |
| huGD3 | 9.52 | 13.25 | 13.57 | 56.87 | 70.00 | 85.36 | 85.64 |
从以上数据可以看出，huGD3对人淋巴细胞的调变作用已达到了鼠源抗体的强度，是一个保持了mGD3主要免疫功能的人源化抗体。
序列表
<110>上海兰生国健药业有限公司
<120>人源化抗CD3单克隆抗体
<130>016014
<160>20
<170>PatentIn version 3.0
<210>1
<211>119
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<213>小鼠(Mus musculus)
<400>1
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Ala Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Ser Gly Asp Lys Phe
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Lys Asp Arg Phe Thr Ile Ser Arg Lys Lys Asp Lys Asn Thr Leu Phe
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Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Asp Tyr Phe Cys
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Ala Arg Trp Gln Asp Tyr Asp Val Tyr Phe Asp Tyr Trp Gly Gln Ala
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Cys Leu Val Thr Val Ser Ser
115
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Asp Ile Lys Leu Asn Gln Ser Pro Ser Ser Met Asn Ala Ser Val Gly
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Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Thr Phe Ser Gly Ser
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Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Gln Asp Pro Glu
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caggttcaga aatctgactc tggtggtggt gttaaacagc cgggtcgttc tctgcgtctg 60
tctggtaaag cttctggtta caccttcacc tcttacaccg ctcactgggt tcgtcaggct 120
ccgggtaaag gttggaaccc gctggcttac atcaacccgt cttctggtta caccaaatct 180
ggtgacaaat tcaaagaccg tttcaccatc tctcgtaaaa aagacaaaaa caccctgttc 240
ctgcagatgg actctctgcg tccggaagac accggtgact acttctgcgc tcgttggcag 300
gactacgacg tttacttcga ctactggggt caggcttgcc tggttaccgt ttcttct 357
<210>4
<211>321
<212>DNA
<213>小鼠(Mus musculus)
<400>4
gacatcaaac tgaaccagtc tccgtcttct atgaacgctt ctgttggtga ccgtgttacc 60
atctctaaac tggactcttc ttctgtttct tacatggacg actaccagca gaccccgatc 120
aaagctccga aactgctgat ctacgctacc tctaacctgg cttctggtgt tccgtctacc 180
ttctctggtt ctggttctgg taccgactac accttcacca tctcttctca ggacccggaa 240
gacatcgcta cctactactg ccagcagtgg tcttctgaca acccgacctt cggtcagggt 300
accaaatctg acaaaacccg t 321
<210>5
<211>119
<212>PRT
<213>人造序列
<220>
<221>misc_feature
<223>人源化的抗CD3单克隆抗体重链可变区
<400>5
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Phe Thr Ile Ser Ala Asp Asn Ser Lys Ser Thr Ala Phe
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Trp Gln Asp Tyr Asp Val Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Pro Val Thr Val Ser Ser
115
<210>6
<211>107
<212>PRT
<213>人造序列
<220>
<221>misc_feature
<223>人源化的抗CD3单克隆抗体轻链可变区
<400>6
Asp Ile Val Leu 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 Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr
85 90 95
Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr Arg
100 105
<210>7
<211>357
<212>DNA
<213>人造序列
<220>
<221>misc_feature
<223>人源化的抗CD3单克隆抗体重链可变区编码序列
<400>7
caggtgcagc tggtgcagtc tggcggtgga gtggtccagc ccggccgcag cctgaggctg 60
tcctgcaagg ccagcggcta caccttcacc agctacacga tgcactgggt gcgccaagcc 120
cccggaaagg gcctcgaatg gattggctac attaatccta gcagtggtta tactaagtac 180
aatcagaagt tcaaggacag atttacaata tcagccgaca acagcaagtc caccgccttc 240
ctacaaatgg acagcttgcg tccagaggac accgccgtat actactgtgc gcgctggcag 300
gattacgacg tctactttga ctactggggc caaggcactc cagtcaccgt ctcctct 357
<210>8
<211>321
<212>DNA
<213>人造序列
<220>
<221>misc_feature
<223>人源化的抗CD3单克隆抗体轻链可变区编码序列
<400>8
gacatcgttc tcactcagag cccatccagc ttgagcgcat cagtaggcga ccgcgtaacg 60
atcacttgca gggccagctc aagtgtaagt tacatgcact ggtaccagca gactcccggc 120
aaagccccaa agccctggat ttatgccaca tccaacctgg cttctggcgt gccatcacgc 180
tttagcggca gcgggtccgg tacagattac acgttcacca ttagcagtct gcagcctgag 240
gacatagcca cctactactg tcagcagtgg agtagtaacc caccgacgtt tggccaggga 300
actaaactgc agattactcg a 321
<210>9
<211>24
<212>DNA
<213>引物
<400>9
agctgggaag gtgtgcacac cact 24
<210>10
<211>21
<212>DNA
<213>引物
<400>10
cagagttcca ggtcaaggtc a 21
<210>11
<211>21
<212>DNA
<213>引物
<400>11
cttgaccagg catcctagag t 21
<210>12
<211>23
<212>DNA
<213>引物
<400>12
ttgctgtcct gatcagtcca act 23
<210>13
<211>23
<212>DNA
<213>引物
<400>13
tgtcgttcac tgccatcaat ctt 23
<210>14
<211>23
<212>DNA
<213>引物
<400>14
ttgttcaaga agcacacgac tga 23
<210>15
<211>36
<212>DNA
<213>引物
<220>
<221>misc_feature
<223>n＝肌苷
<400>15
ggccacgcgt cgactagtac gggnngggnn gggnng 36
<210>16
<211>20
<212>DNA
<213>引物
<400>16
ggccacgcgt cgactagtac 20
<210>17
<211>25
<212>DNA
<213>引物
<400>17
ctctctagac aggttcagaa atctg 25
<210>18
<211>24
<212>DNA
<213>引物
<400>18
gacgctagca gaagaaacgg taac 24
<210>19
<211>27
<212>DNA
<213>引物
<400>19
ctgaagcaag acatcaaact gaaccag 27
<210>20
<211>21
<212>DNA
<213>引物
<400>20
gaccgtacga cgggttttgt c 21
Claims (9)
1.一种人源化的抗人CD3单克隆抗体，其中原本人抗体的重链可变区和轻链可变区分别被非人源重链可变区和轻链可变区所取代，所述非人源重链可变区选自SEQ ID NO：1或SEQ ID NO：5，所述非人源轻链可变区选自SEQ ID NO：2或SEQ ID NO：6。
2.一种DNA分子，其中包含权利要求1所述非人源重链可变区的编码序列和/或权利要求1所述非人源轻链可变区的编码序列。
3.根据权利要求2所述的DNA分子，所述非人源重链可变区的编码序列选自SEQ ID NO：3或SEQ ID NO：7，所述非人源轻链可变区的编码序列选自SEQID NO：4或SEQ ID NO：8。
4.根据权利要求2所述的DNA分子，它是一种载体。
5.一种宿主细胞，它含有权利要求2至4中任一项所述的DNA分子。
6.根据权利要求5所述的细胞，所含的所述DNA分子包含在宿主相容性表达系统中。
7.根据权利要求5所述的细胞，它是CHO细胞。
8.权利要求1所述抗体在制备诊断和/或治疗CD3相关疾病的制剂中的用途。
9.权利要求2所述DNA分子在制备诊断和/或治疗CD3相关疾病的制剂中的用途。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01132281 CN1189483C (zh) | 2001-11-23 | 2001-11-23 | 人源化抗cd3单克隆抗体 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01132281 CN1189483C (zh) | 2001-11-23 | 2001-11-23 | 人源化抗cd3单克隆抗体 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1421459A CN1421459A (zh) | 2003-06-04 |
| CN1189483C true CN1189483C (zh) | 2005-02-16 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 01132281 Expired - Lifetime CN1189483C (zh) | 2001-11-23 | 2001-11-23 | 人源化抗cd3单克隆抗体 |
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| Country | Link |
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| CN (1) | CN1189483C (zh) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7250494B2 (en) | 1998-06-15 | 2007-07-31 | Biosynexus Incorporated | Opsonic monoclonal and chimeric antibodies specific for lipoteichoic acid of Gram positive bacteria |
| UA94211C2 (ru) * | 2004-06-03 | 2011-04-26 | Новиммюн С.А. | Выделенное полностью человеческое моноклональное cd3 антитело |
| JP2014518615A (ja) | 2011-04-22 | 2014-08-07 | エマージェント プロダクト デベロップメント シアトル， エルエルシー | 前立腺特異的膜抗原結合タンパク質および関連組成物ならびに方法 |
| CN104098698B (zh) * | 2013-04-08 | 2019-04-05 | 中国人民解放军第二军医大学 | 一种抗cd3抗体及其制法和应用 |
| EP3352760A4 (en) | 2015-09-21 | 2019-03-06 | Aptevo Research and Development LLC | CD3 BINDING POLYPEPTIDES |
| CN109913493B (zh) | 2017-12-12 | 2021-03-16 | 百奥赛图江苏基因生物技术有限公司 | 人源化cd3基因改造动物模型的制备方法及应用 |
| WO2024152963A1 (zh) * | 2023-01-18 | 2024-07-25 | 贝达药业股份有限公司 | 抗cd3的人源化抗体及其应用 |
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2001
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| CN1421459A (zh) | 2003-06-04 |
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