实施例4:
嵌合抗原受体LMP1-scFv-CH2CH3-CD28-CD134-CD3ζ修饰的T淋巴细胞对EB相关肿瘤细胞的杀伤作用
1、 Western Blotting检测鼻咽癌细胞中LMP1的表达
SUNE1-LMP1-TOFA和SUNE1-LMP1-TONA细胞均为鼻咽癌细胞(诺华公司赠送),前者采用Tet-off表达系统转入LMP1基因,该细胞在没有DOX刺激时能够表达LMP1蛋白,在加入DOX刺激后LMP1表达被抑制,而后采用Tet-on表达系统转入LMP1基因,该细胞在没有DOX刺激时不能够诱导表达LMP1,在DOX刺激后能够诱导细胞表达LMP1。培养SUNE1-LMP1-TOFA和SUNE1-LMP1-TONA分别用DOX刺激和未刺激,细胞用RIPA细胞裂解液(已加Roche的蛋白酶抑制剂Cocktail Tablets)冰上裂解30min后,提取细胞蛋白,进行Western Blotting检测,检测结果如图5,SUNE1-LMP1-TOFA和用DOX诱导的SUNE1-LMP1-TONA的细胞能够检测到LMP1的高表达,而SUNE1-LMP1-TONA和经DOX诱导的SUNE1-LMP1-TOFA细胞LMP1的表达明显下降。
2、 Western Blotting检测淋巴瘤细胞中LMP1的表达
选取对数生长期的不同的淋巴瘤细胞(U937,Raji,RPMI6666),提取细胞蛋白(方法同上),进行Western Blotting检测,检测结果如图6,Raji和RPMI6666细胞能够检测到LMP1的表达,而U937细胞不能检测到LMP1的表达。
3、 CART细胞对肿瘤细胞杀伤力检测
将肿瘤细胞用培养基调整到4×106/ml,加入终浓度为0.5μM calcein violet进行标记,冰上放置30min,用PBS洗涤三遍后,用1640培养基重悬细胞,计数。调整肿瘤细胞至4×106/ml,取250μl加入到24孔板内,按E:T为20:1;10:1;5:1; 1:1分别加入T细胞2×107;1×107;5×106; 1×106,共培养4h,每孔加入50μg PI,转到流式管中进行FACS检测。肿瘤细胞有鼻咽癌细胞SUNE1-LMP1-TOFA,SUNE1-LMP1-TOFA加DOX诱导,SUNE1-LMP1-TONA,SUNE1-LMP1-TONA加DOX诱导,检测结果(如图7)表明CART细胞对LMP1表达较高的SUNE1-LMP1-TOFA和DOX诱导的SUNE1-LMP1-TONA细胞杀伤力高于SUNE1-LMP1-TONA和DOX诱导的SUNE1-LMP1-TOFA细胞;淋巴瘤细胞U937,Raji,RPMI6666,检测结果(如图8)CART细胞对LMP1表达较高的Raji和RPMI6666的杀伤力高于未能检测到LMP1表达U937细胞。
4、 ELISPOT 检测CART细胞在受到LMP1抗原刺激后IFN-γ表达
用无菌ELISPOT包被液来稀释 IFN-γ抗体(1:250稀释),在 ELISPOT 板(Millipore, Cat. No. MAIPS4510) 上加入每孔 100μl, 放在4°C冰箱过夜,进行包被。第二天从板上吸掉包被液。用PBS 洗板两次, 200 μl/孔。每孔加入200 μl 含血清的细胞培养液RPMI-1640, 在37℃放置1小时。洗去含血清的细胞培养液RPMI-1640, 用PBS再洗三次。准备LMP1胞外区多肽, 稀释在含血清的细胞培养液RPMI-1640, 调至终浓度为1μg/ml。每孔加100ul。
准备CART细胞,调至1×106/ml,每孔加100ul,终浓度为1×105/孔。37℃, 5% CO2
培养24 小时。24小时后, 洗掉细胞, 用PBS洗板三次。稀释生物素标记的抗IFN-γ抗体(1:250稀释), 每孔加100μl。培养2小时。洗掉抗体, 再用PBS洗4次。准备Avidin-HRP, 加100ul进每孔。室温放置45分钟。洗掉Avidin-HRP, 用PBS-tween20洗3次,然后用PBS洗3次。准备AEC溶液,加入100ul每孔,室温10-60分钟,观察斑点形成用流水洗孔, 中止反应。等板干,计数(结果如图9),结果表明CART细胞在特异的LMP1抗原刺激下能够分泌IFN-γ。
SEQUENCE LISTING
<110> 冯, 振卿
<120> 嵌合抗原受体及其用途
<130>
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 718
<212> PRT
<213> 人工序列
<400> 1
Met Asp Trp
Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly
1 5 10 15
Ala His Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro
20 25 30
Gly Arg Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
35 40 45
Ser Tyr Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60
Trp Val Ala
Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp
65 70 75 80
Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
85 90 95
Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Ala
Ser Arg Tyr Cys Ser Ser Thr Ser Cys Tyr Phe Asp Tyr
115 120 125
Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu Leu Gln Met Thr Gln
145 150 155 160
Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
165 170 175
Cys Arg Ala
Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln
180 185 190
Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu
195 200 205
Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
210 215 220
Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
225 230 235 240
Tyr Cys Gln
Gln Ser Tyr Ser Thr Pro Tyr Thr Phe Gly Gln Gly Thr
245 250 255
Lys Leu Glu
Ile Lys Leu Glu Asp Pro Ala Glu Pro Lys Ser Pro Asp
260 265 270
Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
275 280 285
Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
290 295 300
Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
305 310 315 320
Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
325 330 335
Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
340 345 350
Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
355 360 365
Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
370 375 380
Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
385 390 395 400
Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
405 410 415
Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
420 425 430
Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
435 440 445
Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
450 455 460
Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
465 470 475 480
Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
485 490 495
Gly Lys Lys
Asp Pro Lys Phe Trp Val Leu Val Val Val Gly Gly Val
500 505 510
Leu Ala Cys
Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp
515 520 525
Val Arg Ser
Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
530 535 540
Thr Pro Arg
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala
545 550 555 560
Pro Pro Arg
Asp Phe Ala Ala Tyr Arg Ser Arg Asp Gln Arg Leu Pro
565 570 575
Pro Asp Ala
His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile
580 585 590
Gln Glu Glu
Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile Arg Val
595 600 605
Lys Phe Ser
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
610 615 620
Gln Leu Tyr
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
625 630 635 640
Leu Asp Lys
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
645 650 655
Arg Lys Asn
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
660 665 670
Met Ala Glu
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
675 680 685
Gly Lys Gly
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
690 695 700
Asp Thr Tyr
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
705 710 715
<210> 2
<211> 330
<212> DNA
<213> 人工序列
<400> 2
gctgcagatg
acacagagcc cctcctctct gtcagcaagc gtgggcgatc gagtcaccat 60
cacatgccgg
gcctcccagt ctattagttc atacctgaac tggtatcagc agaagcccgg 120
caaagcccct
aagctgctga tctacgcagc cagctccctg cagtccggag tgccatctcg 180
attcagtggg
tcaggaagcg gcacagactt tactctgacc atttctagtc tgcagcccga 240
agatttcgct
acttactatt gtcagcagag ctacagcaca ccatacacct ttgggcaggg 300
aactaaactg
gaaatcaagc tcgaggatcc 330
<210> 3
<211> 2157
<212> DNA
<213> 人工序列
<400> 3
atggattgga
tttggaggat tctgtttctg gtgggggctg ctactggggc tcacgaagtg 60
cagctgctgg
agtcaggggg cggggtggtc cagcctggcc ggtccctgag actgtcttgt 120
gccgctagtg
ggttcacctt tagctcctac ggaatgcact gggtgcgcca ggcaccaggc 180
aaaggactgg
agtgggtggc tgtcatctgg tatgacggca gcaacaaata ctatgctgat 240
agcgtcaagg
ggaggttcac catttcacgc gacaacagca agaatacact gtacctgcag 300
atgaatagcc
tgagagctga agatactgca gtgtactatt gcgcatccag gtattgttct 360
agtacctctt
gctactttga ctattgggga cagggcacac tggtgactgt ctcaagcgga 420
ggaggaggca
gtggaggagg agggtccgga ggcgggggat ctgagctgca gatgacacag 480
agcccctcct
ctctgtcagc aagcgtgggc gatcgagtca ccatcacatg ccgggcctcc 540
cagtctatta
gttcatacct gaactggtat cagcagaagc ccggcaaagc ccctaagctg 600
ctgatctacg
cagccagctc cctgcagtcc ggagtgccat ctcgattcag tgggtcagga 660
agcggcacag
actttactct gaccatttct agtctgcagc ccgaagattt cgctacttac 720
tattgtcagc
agagctacag cacaccatac acctttgggc agggaactaa actggaaatc 780
aagctcgagg
atcccgccga gcccaaatct cctgacaaaa ctcacacatg cccaccgtgc 840
ccagcacctg
aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 900
accctcatga
tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 960
gaccctgagg
tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1020
aagccgcggg
aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 1080
caccaggact
ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1140
gcccccatcg
agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1200
accctgcccc
catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 1260
aaaggcttct
atcccagcga catcgccgtg gagtgggaga gcaatgggca accggagaac 1320
aactacaaga
ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1380
ctcaccgtgg
acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1440
gaggctctgc acaaccacta
cacgcagaag agcctctccc tgtctccggg taaaaaagat
1500
cccaaatttt
gggtgctggt ggtggttggt ggagtcctgg cttgctatag cttgctagta 1560
acagtggcct
ttattatttt ctgggtgagg agtaagagga gcaggctcct gcacagtgac 1620
tacatgaaca
tgactccccg ccgccccggg cccacccgca agcattacca gccctatgcc 1680
ccaccacgcg
acttcgcagc ctatcgctcc agggaccaga ggctgccccc cgatgcccac 1740
aagccccctg
ggggaggcag tttccggacc cccatccaag aggagcaggc cgacgcccac 1800
tccaccctgg
ccaagatcag agtgaagttc agcaggagcg cagacgcccc cgcgtaccag 1860
cagggccaga
accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1920
ttggacaaga
gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1980
caggaaggcc
tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 2040
gggatgaaag
gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 2100
acagccacca
aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgctaa 2157
<210> 4
<211> 264
<212> PRT
<213> 人工序列
<400> 4
Met Asp Trp
Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly
1 5 10 15
Ala His Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro
20 25 30
Gly Arg Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
35 40 45
Ser Tyr Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60
Trp Val Ala
Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp
65 70 75 80
Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
85 90 95
Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Ala
Ser Arg Tyr Cys Ser Ser Thr Ser Cys Tyr Phe Asp Tyr
115 120 125
Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu Leu Gln Met Thr Gln
145 150 155 160
Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
165 170 175
Cys Arg Ala
Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln
180 185 190
Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu
195 200 205
Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
210 215 220
Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
225 230 235 240
Tyr Cys Gln
Gln Ser Tyr Ser Thr Pro Tyr Thr Phe Gly Gln Gly Thr
245 250 255
Lys Leu Glu
Ile Lys Leu Glu Asp
260
<210> 5
<211> 794
<212> DNA
<213> 人工序列
<400> 5
atggattgga
tttggaggat tctgtttctg gtgggggctg ctactggggc tcacgaagtg 60
cagctgctgg
agtcaggggg cggggtggtc cagcctggcc ggtccctgag actgtcttgt 120
gccgctagtg
ggttcacctt tagctcctac ggaatgcact gggtgcgcca ggcaccaggc 180
aaaggactgg
agtgggtggc tgtcatctgg tatgacggca gcaacaaata ctatgctgat 240
agcgtcaagg
ggaggttcac catttcacgc gacaacagca agaatacact gtacctgcag 300
atgaatagcc
tgagagctga agatactgca gtgtactatt gcgcatccag gtattgttct 360
agtacctctt
gctactttga ctattgggga cagggcacac tggtgactgt ctcaagcgga 420
ggaggaggca
gtggaggagg agggtccgga ggcgggggat ctgagctgca gatgacacag 480
agcccctcct
ctctgtcagc aagcgtgggc gatcgagtca ccatcacatg ccgggcctcc 540
cagtctatta gttcatacct
gaactggtat cagcagaagc ccggcaaagc ccctaagctg
600
ctgatctacg
cagccagctc cctgcagtcc ggagtgccat ctcgattcag tgggtcagga 660
agcggcacag
actttactct gaccatttct agtctgcagc ccgaagattt cgctacttac 720
tattgtcagc
agagctacag cacaccatac acctttgggc agggaactaa actggaaatc 780
aagctcgagg
atcc
794
<210> 6
<211> 18
<212> PRT
<213> 人工序列
<400> 6
Met Asp Trp
Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly
1 5 10 15
Ala His
<210> 7
<211> 36
<212> PRT
<213> 人工序列
<400> 7
Arg Asp Gln
Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly
1 5 10 15
Ser Phe Arg
Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr
20 25 30
Leu Ala Lys
Ile
35
<210> 8
<211> 108
<212> DNA
<213> 人工序列
<400> 8
agggaccaga
ggctgccccc cgatgcccac aagccccctg ggggaggcag tttccggacc 60
cccatccaag
aggagcaggc cgacgcccac tccaccctgg ccaagatc 108
<210> 9
<211> 454
<212> PRT
<213> 人工序列
<400> 9
Pro Ala Glu
Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys
1 5 10 15
Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
20 25 30
Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
35 40 45
Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
50 55 60
Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
65 70 75 80
Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
85 90 95
His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
100 105 110
Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
115 120 125
Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
130 135 140
Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
145 150 155 160
Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
165 170 175
Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
180 185 190
Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
195 200 205
Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
210 215 220
Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys Lys Asp Pro Lys Phe Trp
225 230 235 240
Val Leu Val
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
245 250 255
Thr Val Ala
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu
260 265 270
Leu His Ser
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
275 280 285
Arg Lys His
Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr
290 295 300
Arg Ser Arg
Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly
305 310 315 320
Gly Gly Ser
Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His
325 330 335
Ser Thr Leu
Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
340 345 350
Pro Ala Tyr
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
355 360 365
Gly Arg Arg
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
370 375 380
Pro Glu Met
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
385 390 395 400
Tyr Asn Glu
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
405 410 415
Gly Met Lys
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
420 425 430
Gln Gly Leu
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
435 440 445
Gln Ala Leu
Pro Pro Arg
450
<210> 10
<211> 360
<212> DNA
<213> 人工序列
<400> 10
gaagtgcagc
tgctggagtc agggggcggg gtggtccagc ctggccggtc cctgagactg 60
tcttgtgccg
ctagtgggtt cacctttagc tcctacggaa tgcactgggt gcgccaggca 120
ccaggcaaag
gactggagtg ggtggctgtc atctggtatg acggcagcaa caaatactat 180
gctgatagcg
tcaaggggag gttcaccatt tcacgcgaca acagcaagaa tacactgtac 240
ctgcagatga
atagcctgag agctgaagat actgcagtgt actattgcgc atccaggtat 300
tgttctagta
cctcttgcta ctttgactat tggggacagg gcacactggt gactgtctca 360