一种抗b7h3嵌合抗原受体及其应用
阅读说明:本技术 一种抗b7h3嵌合抗原受体及其应用 (anti-B7H3 chimeric antigen receptor and application thereof ) 是由 罗敏 李光超 周兆 王学俊 于 2020-12-14 设计创作,主要内容包括:本发明提供了一种抗B7H3嵌合抗原受体及其应用,所述抗B7H3嵌合抗原受体包括抗原结合结构域、铰链区、跨膜结构域和信号传导结构域;所述抗原结合结构域为抗人B7H3抗体。本发明的抗B7H3嵌合抗原受体对B7H3阳性肿瘤细胞具有特异性靶向作用,表达抗B7H3嵌合抗原受体的T细胞体内外杀伤作用显著,能够有效清除B7H3阳性肿瘤细胞,在肿瘤治疗领域具有重要意义。(The invention provides an anti-B7H3 chimeric antigen receptor and application thereof, wherein the anti-B7H3 chimeric antigen receptor comprises an antigen binding domain, a hinge region, a transmembrane domain and a signaling domain; the antigen binding domain is an anti-human B7H3 antibody. The anti-B7H3 chimeric antigen receptor has a specific targeting effect on B7H3 positive tumor cells, has an obvious in-vivo and in-vitro killing effect on T cells expressing the anti-B7H3 chimeric antigen receptor, can effectively remove the B7H3 positive tumor cells, and has important significance in the field of tumor treatment.)
本申请是名称为:抗B7H3嵌合抗原受体及其应用,申请号为CN202011476183.6的发明专利的分案申请,母案申请日为2020年12月14日。
技术领域
本发明属于生物医药技术领域,涉及一种抗B7H3嵌合抗原受体及其应用。
背景技术
B7H3是I型跨膜蛋白,属于B7免疫共刺激和共抑制家族,具有免疫抑制功能,可以减少T细胞释放的I型干扰素(IFN)、降低NK细胞的细胞毒性。B7H3蛋白在正常组织(例如前列腺、乳腺、胎盘、肝脏、结肠和淋巴器官)中表达有限,但是在大部分恶性肿瘤中异常高表达。在非小细胞肺癌细胞株和肿瘤组织中可以检测到B7H3的表达。在表达B7H3的肿瘤组织中,浸润性淋巴样细胞的数目显著降低,与淋巴结转移呈正相关(SunY,WangY,Zhao J,etal.B7-H3 andB7-H4 expression in non-small-cell lung cancer[J].Lung Cancer,2006,53(2):143-151.;赵文建,陈春燕,眭文妍等.B7-H3及其与肿瘤关系的研究进展[J].医学综述,2009,15(22):3430-3433.)。
B7H3在肿瘤细胞中的高表达通常与肿瘤浸润淋巴细胞减少、癌症进展加快以及恶性肿瘤(神经系统肿瘤、黑色素瘤、肺癌、头颈癌、结肠直肠癌、胰腺癌、前列腺癌、卵巢癌、肺癌和透明细胞肾癌)的临床结果密切相关。由于其在多种肿瘤中广泛表达,B7H3已成为癌症免疫疗法的潜在靶标。但目前鲜有靶向B7H3的免疫疗法报道。
发明内容
针对现有技术的不足和实际需求,本发明提供了一种抗B7H3嵌合抗原受体及其应用,所述抗B7H3嵌合抗原受体采用对人B7H3具有结合能力的抗B7H3抗体为抗原结合结构域,不仅可以结合游离的B7H3蛋白,还可以结合细胞表面的B7H3蛋白,在肿瘤治疗领域具有重要应用前景。
为达此目的,本发明采用以下技术方案:
第一方面,本发明提供了抗B7H3嵌合抗原受体,所述抗B7H3嵌合抗原受体包括抗原结合结构域、铰链区、跨膜结构域和信号传导结构域,所述抗原结合结构域为抗B7H3抗体,所述抗B7H3抗体包括SEQ ID NO:1和SEQ ID NO:2所示的氨基酸序列,或者,所述抗B7H3抗体包括SEQ ID NO:3和SEQ ID NO:4所示的氨基酸序列。
本发明中,采用对B7H3具有结合能力的抗B7H3抗体作为嵌合抗原受体的抗原结合结构域,使得嵌合抗原受体可以特异性结合B7H3阳性肿瘤细胞,实现对B7H3阳性肿瘤的特异性靶向作用。
优选地,SEQ ID NO:1和SEQ ID NO:2通过连接肽连接形成抗B7H3抗体H2B8;
SEQ ID NO:1:
DIQLTQSPSFLSASVGDRVTINCRASKTISNYLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHHEYPLTFGGGTKVEIKRTV;
SEQ ID NO:2:
QVQLVQSGAEVKKPGASVKVSCKVSGYTFTDGAMHWVRQAPGQGLEWIGIINTNSGNTNYNQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARGVFYYGYGAWFAYWGQGTLVTVSS。
优选地,SEQ ID NO:3和SEQ ID NO:4通过连接肽连接形成抗B7H3抗体2B8;
SEQ ID NO:3:
DVQITQSPSYLTASPGETIIINCRASKTISNYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSDTDFTLTISSLEPEDFAMYYCQQHHEYPLTFGAGTKLELK;
SEQ ID NO:4:
QVQLQQSGPELVRPGVSVKISCKVSGYTFTDGAMHWVKRSHAKSLEWIGIINTNSGNTNYNQKFQGKATMTVDKSSSTAYMELARLTSEDSAIYYCARGVFYYGYGAWFAYWGQGTLVTVSA。
优选地,所述抗B7H3抗体包括SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列,SEQID NO:5和SEQ ID NO:6通过连接肽连接形成抗B7H3抗体Enoblituzumab(Eno);
SEQ ID NO:5:
DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIK;
SEQ ID NO:6:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSS。
优选地,所述抗B7H3抗体包括SEQ ID NO:7和SEQ ID NO:8所示的氨基酸序列,SEQID NO:7和SEQ ID NO:8通过连接肽连接形成抗B7H3抗体huM30;
SEQ ID NO:7:
EIVLTQSPATLSLSPGERATLSCRASSRLIYMHWYQQKPGQAPRPLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWNSNPPTFGQGTKVEIK;
SEQ ID NO:8:
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYVMHWVRQAPGQGLEWMGYINPYNDDVKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARWGYYGSPLYYFDYWGQGTLVTVSS。
优选地,所述铰链区包括CD8α铰链区。
优选地,所述跨膜结构域包括CD8α跨膜区和/或CD28跨膜区。
优选地,所述信号传导结构域包括CD3ζ。
优选地,所述信号传导结构域还包括4-1BB、CD28胞内区、DAP10或OX40中的任意一种或至少两种的组合。
优选地,所述抗B7H3嵌合抗原受体还包括信号肽。
优选地,所述信号肽包括IgGκ轻链信号肽、CD8α信号肽、GM-CSF信号肽、HSA信号肽、IgG重链信号肽、IgG轻链信号肽、CD33信号肽、IL-2信号肽或胰岛素信号肽中的任意一种。
作为优选技术方案,本发明提供了抗B7H3嵌合抗原受体,所述抗B7H3嵌合抗原受体包括信号肽、抗B7H3抗体、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ。
优选地,所述抗B7H3嵌合抗原受体H2B8-CAR由IgGκ轻链信号肽、抗B7H3抗体H2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成,包括SEQ ID NO:9所示的氨基酸序列;
SEQ ID NO:9:
MDMRVPAQLLGLLLLWLRGARCDIQLTQSPSFLSASVGDRVTINCRASKTISNYLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHHEYPLTFGGGTKVEIKRTVGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKVSGYTFTDGAMHWVRQAPGQGLEWIGIINTNSGNTNYNQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARGVFYYGYGAWFAYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
优选地,所述抗B7H3嵌合抗原受体L2B8-CAR由IgGκ轻链信号肽、抗B7H3抗体2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成,包括SEQ ID NO:10所示的氨基酸序列;
SEQ ID NO:10:
MDMRVPAQLLGLLLLWLRGARCDVQITQSPSYLTASPGETIIINCRASKTISNYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSDTDFTLTISSLEPEDFAMYYCQQHHEYPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQSGPELVRPGVSVKISCKVSGYTFTDGAMHWVKRSHAKSLEWIGIINTNSGNTNYNQKFQGKATMTVDKSSSTAYMELARLTSEDSAIYYCARGVFYYGYGAWFAYWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
优选地,所述抗B7H3嵌合抗原受体2B8-CAR由CD8α信号肽、抗B7H3抗体2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成,包括SEQ ID NO:11所示的氨基酸序列;
SEQ ID NO:11:
MALPVTALLLPLALLLHAARPDVQITQSPSYLTASPGETIIINCRASKTISNYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSDTDFTLTISSLEPEDFAMYYCQQHHEYPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQSGPELVRPGVSVKISCKVSGYTFTDGAMHWVKRSHAKSLEWIGIINTNSGNTNYNQKFQGKATMTVDKSSSTAYMELARLTSEDSAIYYCARGVFYYGYGAWFAYWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
优选地,所述抗B7H3嵌合抗原受体Eno-CAR由CD8α信号肽、抗B7H3抗体Enoblituzumab、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成,包括SEQ ID NO:12所示的氨基酸序列;
SEQ ID NO:12:
MALPVTALLLPLALLLHAARPDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
优选地,所述抗B7H3嵌合抗原受体huM30-CAR由CD8α信号肽、抗B7H3抗体huM30、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成,包括SEQ ID NO:13所示的氨基酸序列;
SEQ ID NO:13:
MALPVTALLLPLALLLHAARPEIVLTQSPATLSLSPGERATLSCRASSRLIYMHWYQQKPGQAPRPLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWNSNPPTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYVMHWVRQAPGQGLEWMGYINPYNDDVKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARWGYYGSPLYYFDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
第二方面,本发明提供了核酸分子,所述核酸分子包括第一方面所述的抗B7H3嵌合抗原受体的编码基因。
优选地,所述核酸分子包括SEQ ID NO:14所示的核酸序列,为H2B8-CAR的编码基因。
优选地,所述核酸分子包括SEQ ID NO:15所示的核酸序列,为L2B8-CAR的编码基因。
优选地,所述核酸分子包括SEQ ID NO:16所示的核酸序列,为2B8-CAR的编码基因。
优选地,所述核酸分子包括SEQ ID NO:17所示的核酸序列,为Eno-CAR的编码基因。
优选地,所述核酸分子包括SEQ ID NO:18所示的核酸序列,为huM30-CAR的编码基因。
第三方面,本发明提供了一种表达载体,所述表达载体包括第二方面所述的核酸分子。
优选地,所述表达载体为含有第二方面所述的核酸分子的慢病毒载体、逆转录病毒载体或腺相关病毒载体中的任意一种,优选为慢病毒载体。
第四方面,本发明提供了一种重组慢病毒,所述重组慢病毒由转染有第三方面所述的表达载体和辅助质粒的哺乳动物细胞制备得到。
第五方面,本发明提供了一种嵌合抗原受体T细胞,所述嵌合抗原受体T细胞表达第一方面所述的抗B7H3嵌合抗原受体。
本发明中,表达抗B7H3嵌合抗原受体的T细胞利用嵌合抗原受体的抗原结合结构域靶向B7H3阳性肿瘤细胞,发挥T细胞的杀伤功能,实现了对B7H3阳性肿瘤的杀伤作用。
优选地,所述嵌合抗原受体T细胞的基因组中整合有第二方面所述的核酸分子。
优选地,所述嵌合抗原受体T细胞包括第三方面所述的表达载体和/或第四方面所述的重组慢病毒。
第六方面,本发明提供了一种药物组合物,所述药物组合物包括第五方面所述的嵌合抗原受体T细胞。
优选地,所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
第七方面,本发明提供了第一方面所述的抗B7H3嵌合抗原受体、第二方面所述的核酸分子、第三方面所述的表达载体、第四方面所述的重组慢病毒、第五方面所述的嵌合抗原受体T细胞或第六方面所述的药物组合物在制备恶性肿瘤治疗药物中的应用。
优选地,所述恶性肿瘤包括急性淋巴细胞白血病、髓性白血病、黑色素瘤、神经母细胞瘤、非小细胞肺癌、鼻咽癌、乳腺癌、结直肠癌、肝癌、胰腺癌或宫颈癌中的任意一种或至少两种的组合。
与现有技术相比,本发明具有如下有益效果:
(1)本发明采用抗人B7H3抗体作为抗原结合结构域构建CAR分子,表达抗B7H3 CAR的T细胞在不同的效靶比下对B7H3阳性肿瘤细胞均具有显著的杀伤作用;
(2)本发明的抗B7H3 CAR-T细胞与肿瘤细胞共培养后分泌大量的细胞因子IFN-γ,间接证明了CAR-T对肿瘤细胞的杀伤功效;
(3)本发明的抗B7H3 CAR-T具有显著的体内药效,向肿瘤模型小鼠给药后,可以明显抑制肿瘤细胞生长、促进肿瘤细胞凋亡、同时分泌细胞因子IFN-γ,有效清除肿瘤细胞。
附图说明
图1为抗B7H3 CAR分子的结构示意图;
图2为重组慢病毒载体pCDH-EF1-anti-B7H3-CAR图谱;
图3A为H2B8-CAR-T和L2B8-CAR-T的流式细胞图,图3B为Eno-CAR-T、huM30-CAR-T流式细胞图,图3C为另一实验的huM30-CAR-T流式细胞图,图3D为另一实验的huM30-CAR-T、L2B8-CAR-T、2B8-CAR-T流式细胞图;
图4A为H2B8-CAR-T和L2B8-CAR-T在不同的效靶比下对人肝癌细胞HepG2的杀伤效率,图4B为H2B8-CAR-T和L2B8-CAR-T在不同的效靶比下对人胰腺癌细胞PL45的杀伤效率,图4C为H2B8-CAR-T和L2B8-CAR-T在不同的效靶比下对人宫颈癌细胞SiHa的杀伤效率,图4D为huM30-CAR-T、2B8-CAR-T和T细胞在不同的效靶比下对靶细胞的杀伤效率,图4E为huM30-CAR-T、2B8-CAR-T和T细胞在不同的效靶比下对靶细胞的杀伤效率,图4F为huM30-CAR-T、2B8-CAR-T、L2B8-CAR-T和T细胞在不同的效靶比下对PL45的杀伤效率,图4G为huM30-CAR-T、2B8-CAR-T、L2B8-CAR-T和T细胞在不同的效靶比下对PC9的杀伤效率,图4H为huM30-CAR-T、2B8-CAR-T、L2B8-CAR-T和T细胞在不同的效靶比下对SiHa的杀伤效率,图4I为huM30-CAR-T、2B8-CAR-T、L2B8-CAR-T和T细胞在不同的效靶比下对HepG2的杀伤效率;
图5A为Eno-CAR-T按照不同的效靶比与靶细胞共孵育后分泌IFN-γ的情况,图5B为huM30-CAR-T按照不同的效靶比与靶细胞共孵育后分泌IFN-γ的情况。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果,以下结合实施例和附图对本发明作进一步地说明。可以理解的是,此处所描述的具体实施方式仅仅用于解释本发明,而非对本发明的限定。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道商购获得的常规产品。
实施例1CAR-T细胞的制备
本实施例选择抗B7H3抗体H2B8、2B8、Enoblituzumab(Eno)和huM30作为抗原结合结构域构建CAR分子,其中,2B8及其人源化H2B8对B7H3具有显著的结合能力,不仅可以结合游离的B7H3蛋白,还可以结合细胞表面的B7H3蛋白;huM30是日本第一三共公司(DaiichiSankyo)的人源化B7H3抗体(CN103687945B),正在开展临床I期试验用于治疗B7H3阳性的实体肿瘤(NCT02192567);Enoblituzumab(MGA271)是一款经过免疫分子优化的、针对B7H3靶点的全新单克隆抗体,由MacroGenics采用独家Fc优化技术开发,具有独特的抗体优势和治疗潜力,全球尚无此类药物获批,Enoblituzumab代表了全球领先的B7H3抗体药物。
本实施例以上述抗B7H3抗体为CAR分子的抗原结合结构域,结合铰链区、跨膜结构域和信号传导结构域,构建图1所示的抗B7H3 CAR分子。
具体地,CAR分子为:
a.IgGκ轻链信号肽、H2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:9);
b.IgGκ轻链信号肽、2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:10);
c.CD8α信号肽、2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:11);
d.CD8α信号肽、Eno、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:12);
e.CD8α信号肽、huM30、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:13)。
全基因合成以上CAR分子的编码基因,通过PCR、酶切、重组等步骤将合成的CAR分子编码基因克隆至慢病毒载体pCDH中,得到如图2所示的重组慢病毒载体pCDH-EF1-anti-B7H3-CAR。
利用293T细胞和辅助质粒将重组慢病毒质粒载体包装为重组慢病毒颗粒,感染激活的T细胞,得到表达不同CAR的CAR-T细胞H2B8-CAR-T、L2B8-CAR-T、2B8-CAR-T、Eno-CAR-T和huM30-CAR-T。
实施例2CAR-T细胞对CAR的表达效率
采用流式细胞仪检测CAR-T细胞CAR的表达率。
如图3A所示,H2B8-CAR-T、L2B8-CAR-T细胞CAR的表达率分别为31.73%和38.15%。
如图3B所示,Eno-CAR-T细胞CAR的表达率为27.3%,huM30-CAR-T细胞CAR的表达率为45.2%。
在另一实验中,如图3C所示,huM30-CAR-T细胞CAR的表达率为23.09%。
在另一实验中,如图3D所示,huM30-CAR-T细胞CAR的表达率为33.12%,L2B8-CAR-T细胞CAR的表达率为33.43%,2B8-CAR-T细胞CAR的表达率为12.55%。
实施例3CAR-T细胞的杀伤功能
将H2B8-CAR-T和L2B8-CAR-T与人肝癌细胞HepG2、人胰腺癌细胞PL45、人宫颈癌细胞SiHa,按2:1、1:1、1:4的效靶比共孵育16h,利用RTCA技术检测CAR-T的杀伤效率。
图4A、图4B、图4C结果显示,H2B8-CAR-T和L2B8-CAR-T细胞在不同的效靶比下对三种肿瘤细胞均具有杀伤作用,效靶比越大杀伤能力越强。
利用健康供者(供者1和供者2)的PBMC分别制备不同的CAR-T细胞(huM30-CAR-T和2B8-CAR-T),与靶细胞按3:1、1:1、1:3的效靶比共孵育16h,利用RTCA技术检测CAR-T的杀伤效率,同时设置T细胞对照组。
图4D和图4E结果显示,huM30-CAR-T、2B8-CAR-T和T细胞在不同的效靶比下对靶细胞均具有杀伤作用,其中,2B8-CAR-T的杀伤能力在不同的效靶比下均显著高于huM30-CAR-T和T细胞。
在另一个实验中,将huM30-CAR-T、L2B8-CAR-T和2B8-CAR-T与人胰腺癌细胞PL45、人肺癌细胞PC9、人宫颈癌细胞SiHa、人肝癌细胞HepG2按1:2、1:1、2:1的效靶比共孵育16h,利用RTCA技术检测CAR-T的杀伤效率。
图4F、图4G、图4H、图4I显示,L2B8-CAR-T、2B8-CAR-T在杀伤PL45和PC9细胞能力上与huM30-CAR-T相当,L2B8-CAR-T杀伤SiHa和HepG2的能力优于huM30-CAR-T和2B8-CAR-T。
实施例4CAR-T细胞与肿瘤细胞共培养分泌IFN-γ的能力
将Eno-CAR-T细胞用含2mM GlutaMAX、10mM HEPES、100U/mL青霉素和100μg/mL链霉素的RPMI-1640无血清培养基稀释后,按照不同的效靶比分别与1×104个靶细胞(Daudi、H929、Jurkat、Tonly、A375、A549、PC9、HCT116、SY5Y、SH、MC或293T)共培养于96孔圆底板中,每个实验设置3个复孔,37℃、5%CO2培养箱孵育16h;每孔取50μL上清液检测细胞因子IFN-γ的分泌情况。
huM30-CAR-T细胞用含2mM GlutaMAX、10mM HEPES、100U/mL青霉素和100μg/mL链霉素的RPMI-1640无血清培养基稀释后,按照效靶比为10:1分别与1×104个靶细胞(Jurkat、A375、A549、HCT116、K562、SK-N-BE(2)、HONE1或HTB20)共培养于96孔圆底板中,每个实验设置3个复孔,37℃、5%CO2培养箱孵育16h;每孔取50μL上清液检测细胞因子IFN-γ的分泌情况。
采用人IFN-γ酶联免疫试剂盒(深圳欣博盛生物科技有限公司)检测上清液中的IFN-γ含量:用试剂盒内的样品稀释液将上清液稀释20~30倍,吸取100μL加入到预包被的酶标板中,密封后37℃孵育1.5小时;孵育后的酶标板用PBST洗涤、甩干后,每孔加入100μL生物素化抗体,37℃孵育1小时,洗涤、甩干;每孔加入100μLHRP标记链霉亲和素,用铂纸包裹,37℃培养箱温育30min,洗涤、甩干;每孔加入100μLTMB底物显色液,37℃避光反应15min,加入100μL/孔终止液终止反应;用Infinite F50酶标仪(TECAN)读取450nm波长的OD值。
如图5A所示,Eno-CAR-T细胞与B7H3阳性的黑色素瘤细胞A375、肺癌细胞A549、PC9、结肠癌细胞HCT116、神经母细胞瘤SH-SY5Y、SK-N-SH、SK-N-MC细胞等共孵育,都能释放大量的IFN-γ;而与B7H3表达阴性的靶细胞Daudi、H929、jurkat共孵育不能释放明显的IFN-γ。
如图5B所示,huM30-CAR-T和Eno-CAR-T细胞都能杀伤A375、A549、HTC116、K562、HONE1和HTB20,而对Jurkat、SK-N-BE(2)无杀伤作用。
综上所述,本发明的抗B7H3 CAR-T细胞在不同的效靶比下对B7H3阳性肿瘤细胞具有显著的杀伤作用,与肿瘤细胞共培养后分泌大量的细胞因子IFN-γ,体内药效显著,能够有效清除B7H3阳性肿瘤细胞。
申请人声明,本发明通过上述实施例来说明本发明的详细方法,但本发明并不局限于上述详细方法,即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 广州百暨基因科技有限公司
<120> 一种抗B7H3嵌合抗原受体及其应用
<130> 20210804
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Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
85 90 95
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
100 105 110
Asn Asn Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
130 135 140
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
145 150 155 160
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly
165 170 175
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala
180 185 190
Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp Thr Val Lys
195 200 205
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu
210 215 220
Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys Gly
225 230 235 240
Arg Gly Arg Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp Tyr Trp Gly
245 250 255
Gln Gly Thr Thr Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg
260 265 270
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
275 280 285
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
290 295 300
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
305 310 315 320
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg
325 330 335
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
340 345 350
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
355 360 365
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
370 375 380
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
385 390 395 400
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
405 410 415
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
420 425 430
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
435 440 445
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
450 455 460
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
465 470 475 480
His Met Gln Ala Leu Pro Pro Arg
485
<210> 13
<211> 487
<212> PRT
<213> 人工序列
<400> 13
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
20 25 30
Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser
35 40 45
Arg Leu Ile Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
50 55 60
Arg Pro Leu Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn
100 105 110
Ser Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
130 135 140
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val
145 150 155 160
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Val Met
165 170 175
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Tyr
180 185 190
Ile Asn Pro Tyr Asn Asp Asp Val Lys Tyr Asn Glu Lys Phe Lys Gly
195 200 205
Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu
210 215 220
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
225 230 235 240
Trp Gly Tyr Tyr Gly Ser Pro Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln
245 250 255
Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro
260 265 270
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
275 280 285
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
290 295 300
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
305 310 315 320
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly
325 330 335
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
340 345 350
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
355 360 365
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
370 375 380
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
385 390 395 400
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
405 410 415
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
420 425 430
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
435 440 445
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
450 455 460
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
465 470 475 480
Met Gln Ala Leu Pro Pro Arg
485
<210> 14
<211> 1479
<212> DNA
<213> 人工序列
<400> 14
atggatatgc gcgttcctgc tcaactcctc ggtctgctcc tgctgtggct caggggcgcc 60
aggtgtgaca tccagctgac ccagtcacca tccttcctga gcgcctccgt gggcgacaga 120
gttactatca attgccgggc ctcaaagact atatctaatt atctggcttg gtatcagcag 180
aagccaggga aggcaccaaa gctcctgatc tactccggct ccactctgca gtccggcgtg 240
cccagtagat tttctggcag cgggtcaggg accgacttca ccctcacaat tagtagtctg 300
cagccagagg atttcgccac ttattactgc caacagcatc atgaatatcc actgaccttt 360
ggtggcggaa ccaaggtcga gatcaagcgg acagttggtg gaggaggcag tggcggaggc 420
ggcagtggag gcggaggcag tcaagtccaa ctggtgcagt ctggcgcaga agtgaagaag 480
ccaggcgcca gtgttaaggt gtcctgtaaa gtcagcggct atacctttac agacggtgca 540
atgcattggg tcagacaggc accagggcag ggtctggagt ggatcgggat catcaataca 600
aacagcggaa acactaatta caaccagaaa ttccagggaa gagtgactat gactagggac 660
acatcaatct caaccgcata tatggaactg agcaggctgc gctccgagga caccgccgtc 720
tactactgtg cacgcggcgt gttttactat ggatacggag catggttcgc ttactggggc 780
caagggactc tggttacagt ctcttcaact acgacccctg caccgcggcc gcctactcct 840
gcacctacaa tcgcaagtca gccactgagt ctcagacccg aagcatgccg ccctgctgca 900
ggcggagctg tccatacacg cggactggac tttgcatgcg atatatacat ctgggcacca 960
ctggccggca cttgcggcgt gctgctcctg tccctcgtga ttaccctgta ctgcaaacgc 1020
ggcaggaaga agctcctgta tatctttaaa cagcccttca tgaggccagt gcagaccact 1080
caagaggaag acggttgtag ctgccggttt cccgaggaag aagagggagg ctgcgagctc 1140
cgcgtgaagt tctcccgctc agccgatgca cccgcctatc agcaagggca gaaccagctg 1200
tacaatgagc tcaacctggg aagaagggag gaatatgacg ttctggataa acggcgcggt 1260
cgcgatcccg aaatgggtgg gaagcctcgc aggaagaatc ctcaggaagg gctctacaat 1320
gagctgcaga aagacaaaat ggcagaggcc tattctgaaa tcggcatgaa gggcgagcgc 1380
cgcagaggca aaggacacga cggcctgtac cagggcctgt ctacagccac caaggacacc 1440
tatgacgctc tccacatgca agccctgcca ccaaggtga 1479
<210> 15
<211> 1470
<212> DNA
<213> 人工序列
<400> 15
atggacatgc gggtgcctgc tcagctgctg ggcctgctgc tgctgtggct gagaggagcc 60
aggtgcgacg tgcaaattac tcagtcacct tcatatctca cagcatcacc aggtgaaacc 120
atcatcatta attgtcgcgc cagcaaaaca atttctaact atctggcatg gtatcaggaa 180
aagcctggaa agactaacaa gctcctcatc tatagtggct ctaccctcca gagtggtatc 240
ccatctcgct tcagcggcag cggatcagat actgatttca cactgacaat atccagcctg 300
gaacccgagg atttcgcaat gtattactgc cagcagcatc acgagtaccc tctgaccttc 360
ggcgcaggca ctaaactgga gctcaaggga ggaggaggct ccggtggagg agggagcggc 420
ggaggaggca gccaggtcca gctgcaacag tcaggccctg aactcgttcg gccaggcgtc 480
tcagtgaaga tctcctgcaa ggtgtcaggt tacactttca ccgacggcgc tatgcattgg 540
gtgaagcggt cacacgcaaa gtcactggag tggattggaa tcatcaatac caactccggt 600
aataccaatt ataaccagaa gttccagggc aaggccacta tgacagtgga caagagttca 660
agtaccgcat acatggagct ggctcgcctc acatcagagg actctgcaat ctattactgt 720
gctcgcggcg tgttctatta tggttatggc gcatggtttg catactgggg ccagggtacc 780
ctcgttaccg tgtccgccac tacgacccct gcaccgcggc cgcctactcc tgcacctaca 840
atcgcaagtc agccactgag tctcagaccc gaagcatgcc gccctgctgc aggcggagct 900
gtccatacac gcggactgga ctttgcatgc gatatataca tctgggcacc actggccggc 960
acttgcggcg tgctgctcct gtccctcgtg attaccctgt actgcaaacg cggcaggaag 1020
aagctcctgt atatctttaa acagcccttc atgaggccag tgcagaccac tcaagaggaa 1080
gacggttgta gctgccggtt tcccgaggaa gaagagggag gctgcgagct ccgcgtgaag 1140
ttctcccgct cagccgatgc acccgcctat cagcaagggc agaaccagct gtacaatgag 1200
ctcaacctgg gaagaaggga ggaatatgac gttctggata aacggcgcgg tcgcgatccc 1260
gaaatgggtg ggaagcctcg caggaagaat cctcaggaag ggctctacaa tgagctgcag 1320
aaagacaaaa tggcagaggc ctattctgaa atcggcatga agggcgagcg ccgcagaggc 1380
aaaggacacg acggcctgta ccagggcctg tctacagcca ccaaggacac ctatgacgct 1440
ctccacatgc aagccctgcc accaaggtga 1470
<210> 16
<211> 1467
<212> DNA
<213> 人工序列
<400> 16
atggctctgc cagttacagc actcctcctc cctctcgcac tcctgctcca tgcagccaga 60
cccgacgtgc aaataaccca aagtccctcc tacctcactg cttcacctgg agaaacaatt 120
atcattaact gtcgcgcctc taagacaatc tctaattacc tggcctggta tcaagagaaa 180
cccggcaaga ctaataaact gctgatctac tctgggagta ccctgcaatc cggtattcct 240
tcccgcttct ccggctccgg gagtgatacc gactttacac tgacaattag cagtctggag 300
cccgaggact ttgcaatgta ctattgccag caacatcacg agtatccact gaccttcgga 360
gccggcacca agctggaact caaaggcgga ggaggaagtg gcggcggtgg ttctggagga 420
ggaggatctc aggttcagct gcagcagagt ggaccagaac tcgtgcgccc aggcgtgagc 480
gtgaagatca gctgtaaagt gtccggctat actttcactg acggagccat gcactgggtc 540
aagaggagcc acgcaaaatc cctggagtgg attggcatta tcaatacaaa tagcggaaat 600
acaaactata accagaaatt ccagggcaaa gcaacaatga ccgtcgacaa gagctccagc 660
acagcttaca tggagctggc taggctcacc agcgaggact cagccatata ttactgcgct 720
agaggagtgt tctattacgg ctatggtgct tggtttgcct actgggggca aggaactctc 780
gtcaccgtga gcgccactac gacccctgca ccgcggccgc ctactcctgc acctacaatc 840
gcaagtcagc cactgagtct cagacccgaa gcatgccgcc ctgctgcagg cggagctgtc 900
catacacgcg gactggactt tgcatgcgat atatacatct gggcaccact ggccggcact 960
tgcggcgtgc tgctcctgtc cctcgtgatt accctgtact gcaaacgcgg caggaagaag 1020
ctcctgtata tctttaaaca gcccttcatg aggccagtgc agaccactca agaggaagac 1080
ggttgtagct gccggtttcc cgaggaagaa gagggaggct gcgagctccg cgtgaagttc 1140
tcccgctcag ccgatgcacc cgcctatcag caagggcaga accagctgta caatgagctc 1200
aacctgggaa gaagggagga atatgacgtt ctggataaac ggcgcggtcg cgatcccgaa 1260
atgggtggga agcctcgcag gaagaatcct caggaagggc tctacaatga gctgcagaaa 1320
gacaaaatgg cagaggccta ttctgaaatc ggcatgaagg gcgagcgccg cagaggcaaa 1380
ggacacgacg gcctgtacca gggcctgtct acagccacca aggacaccta tgacgctctc 1440
cacatgcaag ccctgccacc aaggtga 1467
<210> 17
<211> 1464
<212> DNA
<213> 人工序列
<400> 17
atggcactgc ctgtgactgc cctcctgctg cctctggcac tcctgctcca cgcagcccgg 60
cctgacatcc agctgactca gtcaccctct ttcctgagcg catctgtggg agacagggtt 120
accatcacct gcaaggcaag ccaaaatgtg gacaccaacg tggcctggta tcagcagaag 180
ccaggcaagg cacccaaagc cctgatctac agcgccagct accgctactc cggagtccca 240
tctcggttct ctggatcagg cagcggaacc gactttacac tgacaatctc aagcctccaa 300
ccagaggact ttgccaccta ttactgccag cagtacaaca attacccttt cacattcggg 360
cagggaacca agctggaaat taaaggcggc ggtggatctg gaggtggcgg gagtggtgga 420
ggagggtcag aggtgcagct ggtggagtct ggtggtggac tggtccaacc aggcggttct 480
ctgcgcctca gttgtgccgc ctcagggttt acattctcta gcttcggaat gcattgggtg 540
aggcaagctc caggcaaagg tctggaatgg gtggcttaca tctcttccga cagttcagcc 600
atctattacg ctgataccgt taagggccgc tttaccatca gcagagataa tgccaagaac 660
tcactgtacc tgcagatgaa tagtctccgg gatgaggaca ccgcagtcta ttattgcggt 720
agaggtcggg agaatatata ctacggctcc agactggact actggggcca agggactact 780
gtcaccgtga gctccactac gacccctgca ccgcggccgc ctactcctgc acctacaatc 840
gcaagtcagc cactgagtct cagacccgaa gcatgccgcc ctgctgcagg cggagctgtc 900
catacacgcg gactggactt tgcatgcgat atatacatct gggcaccact ggccggcact 960
tgcggcgtgc tgctcctgtc cctcgtgatt accctgtact gcaaacgcgg caggaagaag 1020
ctcctgtata tctttaaaca gcccttcatg aggccagtgc agaccactca agaggaagac 1080
ggttgtagct gccggtttcc cgaggaagaa gagggaggct gcgagctccg cgtgaagttc 1140
tcccgctcag ccgatgcacc cgcctatcag caagggcaga accagctgta caatgagctc 1200
aacctgggaa gaagggagga atatgacgtt ctggataaac ggcgcggtcg cgatcccgaa 1260
atgggtggga agcctcgcag gaagaatcct caggaagggc tctacaatga gctgcagaaa 1320
gacaaaatgg cagaggccta ttctgaaatc ggcatgaagg gcgagcgccg cagaggcaaa 1380
ggacacgacg gcctgtacca gggcctgtct acagccacca aggacaccta tgacgctctc 1440
cacatgcaag ccctgccacc aagg 1464
<210> 18
<211> 1464
<212> DNA
<213> 人工序列
<400> 18
atggctctgc ctgttactgc tctgctcctg cctctggctc tgctgctgca cgccgcacgg 60
cccgagatcg tcctgacaca gagtcctgcc accctcagcc tcagtcctgg cgaaagagcc 120
accctgtcct gtagagcaag ctcaagactg atatacatgc actggtacca acagaaaccc 180
ggacaggcac caagacctct gatatatgcc acaagcaacc tggcatcagg gataccagct 240
agattttctg gatctggtag cggcactgat ttcaccctca ctatctccag tctggagcct 300
gaagatttcg ctgtgtacta ctgccagcaa tggaactcta acccacccac tttcgggcag 360
ggcacaaagg tcgagattaa aggtggaggc gggtcaggag gaggaggatc tggaggtggc 420
gggagtcagg tccagctggt ccagtccggc gctgaagtta agaaaccagg tagttctgtt 480
aaagtctcat gcaaggccag cggctacact tttacaaatt atgtcatgca ctgggtgcgg 540
caggctccag gacaagggct cgagtggatg ggctatatta acccttataa tgacgatgtg 600
aagtataatg agaaattcaa agggagggtg accatcactg ccgacgagag tacctcaacc 660
gcatacatgg agctgtcctc cctgaggtcc gaggacaccg ccgtgtacta ttgcgcacgc 720
tgggggtatt atgggagccc actgtactac tttgactact ggggacaggg taccctcgtc 780
accgtgtcct ccactacgac ccctgcaccg cggccgccta ctcctgcacc tacaatcgca 840
agtcagccac tgagtctcag acccgaagca tgccgccctg ctgcaggcgg agctgtccat 900
acacgcggac tggactttgc atgcgatata tacatctggg caccactggc cggcacttgc 960
ggcgtgctgc tcctgtccct cgtgattacc ctgtactgca aacgcggcag gaagaagctc 1020
ctgtatatct ttaaacagcc cttcatgagg ccagtgcaga ccactcaaga ggaagacggt 1080
tgtagctgcc ggtttcccga ggaagaagag ggaggctgcg agctccgcgt gaagttctcc 1140
cgctcagccg atgcacccgc ctatcagcaa gggcagaacc agctgtacaa tgagctcaac 1200
ctgggaagaa gggaggaata tgacgttctg gataaacggc gcggtcgcga tcccgaaatg 1260
ggtgggaagc ctcgcaggaa gaatcctcag gaagggctct acaatgagct gcagaaagac 1320
aaaatggcag aggcctattc tgaaatcggc atgaagggcg agcgccgcag aggcaaagga 1380
cacgacggcc tgtaccaggg cctgtctaca gccaccaagg acacctatga cgctctccac 1440
atgcaagccc tgccaccaag gtga 1464