CN114805605B - 一种抗间皮素嵌合抗原受体及其在制备抗肿瘤产品中的应用 - Google Patents

一种抗间皮素嵌合抗原受体及其在制备抗肿瘤产品中的应用 Download PDF

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CN114805605B
CN114805605B CN202210444507.0A CN202210444507A CN114805605B CN 114805605 B CN114805605 B CN 114805605B CN 202210444507 A CN202210444507 A CN 202210444507A CN 114805605 B CN114805605 B CN 114805605B
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mesothelin
chimeric antigen
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齐国光
湛振键
郑世鑫
刘世豪
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Guangdong Kangdun Innovation Industry Group Co ltd
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Abstract

本发明提供一种抗间皮素嵌合抗原受体及其在制备抗肿瘤产品中的应用。所述抗间皮素嵌合抗原受体，依次包括CD8α信号肽、针对间皮素的单链抗体可变区、CD8铰链区、CD8α跨膜区和胞内信号转导区，所述胞内信号转导区包括串联的CD28段、4‑1BB段和CD3ζ段，所述针对间皮素的单链抗体可变区的氨基酸序列如SEQ ID NO.3所示。本发明将特定的靶向MSLN的单链抗体可变区scFv与CD8铰链区、CD8α跨膜区和胞内信号转导区等结合构建了抗间皮素嵌合抗原受体。该scFv与肿瘤细胞表面抗原结合，将信号转导至胞内，激活共刺激因子，促使T细胞活化与增殖，提高细胞因子释放量以及T细胞对肿瘤的杀伤能力。

Description

一种抗间皮素嵌合抗原受体及其在制备抗肿瘤产品中的应用
技术领域
本发明涉及肿瘤治疗技术领域，特别涉及一种抗间皮素嵌合抗原受体及其在制备抗肿瘤产品中的应用。
背景技术
间皮素(mesothelin，MSLN)是一种通过磷脂酰肌醇区锚定在细胞质膜上的表面糖蛋白，其分子量为40kDa。由于间皮素在正常组织中不表达或低表达，而在各种实体瘤(如肺癌、胰腺癌、胆管癌、食管癌、胃癌、胸腺癌、子宫内膜癌、结肠癌和乳腺癌)中过表达，因此可用于肿瘤的特异性治疗。以间皮素为靶点的CAR-T疗法在临床试验中已经取得了有效的治疗效果。
嵌合抗原受体(CAR)是基于基因工程构造并表达于细胞表面的一种受体蛋白，主要由靶向抗原的单链抗体可变区ScFv、跨膜铰链区和胞内信号转导区组成。通过病毒感染或电转等方式可使嵌合抗原受体表达在T细胞表面从而构建成CAR-T细胞。其中ScFv决定CAR的特异性，胞内信号区域关系着T细胞活化增殖能力。当ScFv片段与肿瘤抗原特异性结合时就会刺激胞内信号转导区转导信号从而激活CAR-T细胞活化增殖并释放IL-2、IL-10等细胞因子杀伤肿瘤细胞。CN 110746508 A公开一种特异性结合间皮素的单克隆抗体及嵌合抗原受体，表达含有其单链抗体的CAR的免疫效应细胞能有效杀伤间皮素阳性肿瘤细胞。
为了提高对肿瘤的杀伤能力，研究者们还对CARs胞内信号区进行改造。第一代CARS包含的胞内区CD3ζ可引起T细胞活化，得到细胞毒性作用。第二代CARS在第一代的基础上添加一个胞内信号域，进一步提高了T细胞的效应功能。第三代CARS包含三个信号域，通常包括CD3ζ和两个串联的共刺激域(如CD28与CD137/CD134)，有利于信号放大，进一步提高抗肿瘤活性。
目前，以间皮素为靶点的CARS在抗肿瘤方面的治疗效果还有待提高，对于不同肿瘤细胞的治疗效果也表现出一定的差异。《靶向间皮素的嵌合抗原受体修饰T细胞治疗卵巢癌实验研究》(夏涌等，2017)结果显示mesoCAR-T细胞对卵巢癌肿瘤细胞的杀伤率最高为40％左右。
发明内容
基于现有技术的不足，本申请提出一种抗间皮素嵌合抗原受体及其在制备抗肿瘤产品中的应用。
本发明将CD8α信号肽、靶向MSLN的单链抗体可变区scFv、CD8铰链区、CD8α跨膜区和胞内信号转导区等结合，通过慢病毒感染T细胞获得重组MSLN-CART细胞。并通过体外检测CAR-T对高表达MSLN的肿瘤细胞的杀伤能力及细胞因子分泌能力。
本发明方案包括以下内容：
一种抗间皮素嵌合抗原受体，依次包括CD8α信号肽、针对间皮素的单链抗体可变区、CD8铰链区、CD8α跨膜区和胞内信号转导区，所述胞内信号转导区包括串联的CD28段、4-1BB段和CD3ζ段，所述针对间皮素的单链抗体可变区的氨基酸序列如SEQ ID NO.3所示。
优选的，所述针对间皮素的单链抗体可变区的碱基序列如SEQ ID NO.4所示。
优选的，所述CD28区和所述4-1BB区通过寡肽串联，所述寡肽的氨基酸序列如SEQID NO.11所示。所述寡肽的碱基序列如SEQ ID NO.12所示。
本发明还提供了一种表达载体，所述表达载体含有所述的抗间皮素嵌合抗原受体的编码基因。
本发明还提供了一种嵌合抗原受体T细胞，所述T细胞表达所述的抗间皮素嵌合抗原受体。
另一方面，本发明还提供了所述的抗间皮素嵌合抗原受体、所述的表达载体和/或所述的嵌合抗原受体T细胞在制备抗肿瘤产品中的应用。
优选的，所述肿瘤包括肺癌、胰腺癌、胆管癌、食管癌、胃癌、胸腺癌、子宫内膜癌、结肠癌和乳腺癌。
优选的，所述肿瘤为卵巢癌。
与现有技术相比，本发明的有益效果为：
本发明将特定的靶向MSLN的单链抗体可变区scFv与CD8铰链区、CD8α跨膜区和胞内信号转导区等结合构建了抗间皮素嵌合抗原受体。该scFv与肿瘤细胞表面抗原结合，将信号转导至胞内，激活共刺激因子，促使T细胞活化与增殖，提高细胞因子释放量以及T细胞对肿瘤的杀伤能力。
本发明还意外发现在CD28区和所述4-1BB区之间增加一个氨基酸序列为SLRTIY的寡肽可以进一步增强T细胞对肿瘤细胞的杀伤能力增强，细胞因子的释放量高。
附图说明
图1：本发明抗间皮素嵌合抗原受体结构示意图；
图2：细胞毒性实验结果图；图中，a代表与空白对照组相比，差异显著(p＜0.01)；b代表与MSLN-CART-1组相比，差异显著(p＜0.01)；
图3：细胞因子释放检测结果图；图中，a代表与空白对照组相比，IL-2释放量差异显著(p＜0.01)；b代表与MSLN-CART-1组相比，IL-2释放量差异显著(p＜0.01)；A代表与空白对照组相比，TNF-α释放量差异显著(p＜0.01)；B代表与MSLN-CART-1组相比，TNF-α释放量差异显著(p＜0.01)
具体实施方式
为对本发明中的技术方案进行清楚、完整地描述，以下结合实施例进行说明，但以下实施例所描述的仅仅是本发明一部分实施例，而不是全部的实施例。
实施例：
1.实验方法
1.1靶向Mesothelin的CAR载体构建
人工合成mesoCAR基因，该基因结构依次包含CD8α信号肽、靶向间皮素的scFv、CD8铰链区、CD8跨膜区、CD28、寡肽、4-1BB和CD3ζ胞内信号转导区。将目的基因克隆至载体PWPXLD中，经菌落PCR、测序鉴定，得MSLN-CAR。以CD28、4-1BB之间无上述寡肽为对照。
CD8α信号肽的氨基酸序列如SEQ ID NO.1所示，进行密码子优化后的碱基序列如SEQ ID NO.2所示；
靶向Mesothelin的scFv片段的氨基酸序列如SEQ ID NO.3所示，其中127-143位为连接重链和轻链的连接头linker；进行密码子优化后的碱基序列如SEQ ID NO.4所示；
CD8铰链区的氨基酸序列如SEQ ID NO.5所示，进行密码子优化后的碱基序列如SEQ ID NO.6所示；
CD8跨膜区的氨基酸序列如SEQ ID NO.7所示，进行密码子优化后的碱基序列如SEQ ID NO.8所示；
CD28的氨基酸序列如SEQ ID NO.9所示，进行密码子优化后的碱基序列如SEQ IDNO.10所示；
寡肽的氨基酸序列如SEQ ID NO.11所示，进行密码子优化后的碱基序列如SEQ IDNO.12所示；
4-1BB的氨基酸序列如SEQ ID NO.13所示，进行密码子优化后的碱基序列如SEQID NO.14所示；
CD3ζ的氨基酸序列如SEQ ID NO.15所示，进行密码子优化后的碱基序列如SEQ IDNO.16所示。
1.2重组慢病毒的制备、CAR-T细胞制备
转染前24h，将处于对数生长期且生长状态良好的HEK293T细胞转入培养瓶中，转染时细胞密度为70％～80％。将MSLN-CAR(5μg)、PSPAX质粒(3.75μg)、PMD2.G质粒(1.25μg)转入293T细胞，48h后收取病毒原液，保存。
用梯度离心法提取健康供者外周血单个核细胞(PBMC)，用RPMI1640完全培养基将细胞重悬(1×106个/mL)，然后将细胞接入预先用CD3/CD28抗体(各2μL/mL)铺板的细胞板中，并于培养箱中培养24h。将已添加poly-brene(8μg/mL)的病毒液加入细胞板内。感染12h后换液(含200IU/mL人IL-2的RPMI1640培养基)培养。CAR-T细胞用培养基重悬，细胞生长进入平稳期后进行后续试验。
1.3细胞毒性实验
靶细胞为表达荧光蛋白mCherry的卵巢癌细胞SNU-119(高表达MSLN)。将靶细胞(6×106/mL)接入细胞板中，30min后加入CAR-T细胞(效靶比E:T为1:1)和培养基，培养箱中培养12h。以加入感染慢病毒空载T细胞为空白对照，以CD28、4-1BB之间无所述寡肽为MSLN-CART-1组，以CD28、4-1BB之间有所述寡肽为MSLN-CART-2组。用PBS清洗细胞板，然后置于荧光显微镜下观察记录存活的卵巢癌细胞，对存活细胞累积光密度进行定量分析，计算杀伤率。
1.4细胞因子释放检测
取1.3中培养12h后所得的上清液，用ELISA试剂盒检测细胞因子IL-2和TNF-α水平。
1.5实验结果及分析：
图2结果显示：与空白对照相比，MSLN-CART-1组和MSLN-CART-2组对卵巢癌细胞杀伤效能显著增强，杀伤率达到88.3±1.66％(三次重复，均值±标准偏差)以上，杀伤率达到显著差异(p＜0.01)。与MSLN-CART-1组相比，MSLN-CART-2组的杀伤率(97.3±2.35％)显著高于MSLN-CART-1组(p＜0.01)。
图3结果显示：与空白对照相比，MSLN-CART-1组和MSLN-CART-2组的IL-2、TNF-α水平更高，且MSLN-CART-2组显著高于MSLN-CART-1组(p＜0.01)。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
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<110> 广东康盾创新产业集团股份公司
<120> 一种抗间皮素嵌合抗原受体及其在制备抗肿瘤产品中的应用
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His Ala Ala Arg Pro
20
<210> 2
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccg 65
<210> 3
<211> 258
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn
20 25 30
Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala
50 55 60
Val Ser Val Lys Ser Arg Met Ser Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Arg Gly Met Met Thr Tyr Tyr Tyr Gly Met Asp Val
100 105 110
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ile Leu Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
130 135 140
Pro Val Leu Thr Gln Ser Ser Ser Leu Ser Ala Ser Pro Gly Ala Ser
145 150 155 160
Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn Val Gly Pro Tyr
165 170 175
Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr Leu
180 185 190
Leu Asn Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val Pro
195 200 205
Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Val Leu
210 215 220
Leu Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Met
225 230 235 240
Ile Trp His Ser Ser Ala Ala Val Phe Gly Gly Gly Thr Gln Leu Thr
245 250 255
Val Leu
<210> 4
<211> 774
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
caggtgcagc tgcagcagag cggccccggc ctggtgaccc ccagccagac cctgagcctg 60
acctgcgcca tcagcggcga cagcgtgagc agcaacagcg ccacctggaa ctggatcagg 120
cagagcccca gcaggggcct ggagtggctg ggcaggacct actacaggag caagtggtac 180
aacgactacg ccgtgagcgt gaagagcagg atgagcatca accccgacac cagcaagaac 240
cagttcagcc tgcagctgaa cagcgtgacc cccgaggaca ccgccgtgta ctactgcgcc 300
aggggcatga tgacctacta ctacggcatg gacgtgtggg gccagggcac caccgtgacc 360
gtgagcagcg gcatcctggg cagcggcggc ggcggcagcg gcggcggcgg cagcggcggc 420
ggcggcagcc agcccgtgct gacccagagc agcagcctga gcgccagccc cggcgccagc 480
gccagcctga cctgcaccct gaggagcggc atcaacgtgg gcccctacag gatctactgg 540
taccagcaga agcccggcag ccccccccag tacctgctga actacaagag cgacagcgac 600
aagcagcagg gcagcggcgt gcccagcagg ttcagcggca gcaaggacgc cagcgccaac 660
gccggcgtgc tgctgatcag cggcctgagg agcgaggacg aggccgacta ctactgcatg 720
atctggcaca gcagcgccgc cgtgttcggc ggcggcaccc agctgaccgt gctg 798
<210> 5
<211> 45
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
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> 6
<211> 135
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 139
<210> 7
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
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> 8
<211> 72
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 74
<210> 9
<211> 41
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 10
<211> 123
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60
gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120
tcc 127
<210> 11
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Ser Leu Arg Thr Ile Tyr
1 5
<210> 12
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
agcctgagga ccatctac 18
<210> 13
<211> 42
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
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 Leu
35 40
<210> 14
<211> 126
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 130
<210> 15
<211> 112
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 16
<211> 336
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 346

Claims (4)

1. 一种抗间皮素嵌合抗原受体，依次包括CD8α信号肽、针对间皮素的单链抗体可变区、CD8铰链区、CD8α跨膜区和胞内信号转导区，所述胞内信号转导区包括串联的CD28段、4-1BB段和CD3ζ段，其特征在于，所述针对间皮素的单链抗体可变区的氨基酸序列如SEQ IDNO.3所示；所述CD28段和所述4-1BB段之间通过寡肽串联，所述寡肽的氨基酸序列如SEQ IDNO.11所示。
2.一种表达载体，其特征在于，所述表达载体含有权利要求1所述的抗间皮素嵌合抗原受体的编码基因。
3.一种嵌合抗原受体T细胞，其特征在于，所述T细胞表达权利要求1所述的抗间皮素嵌合抗原受体。
4.权利要求1所述的抗间皮素嵌合抗原受体、权利要求2所述的表达载体和/或权利要求3所述的嵌合抗原受体T细胞在制备抗肿瘤产品中的应用；所述肿瘤为肺癌、胰腺癌、胆管癌、食管癌、胃癌、胸腺癌、子宫内膜癌、结肠癌、乳腺癌和卵巢癌。
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