CN109748968A - Bcma特异性嵌合抗原受体t细胞及其应用 - Google Patents
Bcma特异性嵌合抗原受体t细胞及其应用 Download PDFInfo
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- CN109748968A CN109748968A CN201711067947.4A CN201711067947A CN109748968A CN 109748968 A CN109748968 A CN 109748968A CN 201711067947 A CN201711067947 A CN 201711067947A CN 109748968 A CN109748968 A CN 109748968A
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Abstract
本发明公开了一种针对人BCMA的特异性嵌合抗原受体(anti‑BCMA scFv‑CD8a‑41BB‑CD3ζ)及其应用，该嵌合抗原受体由单链抗体anti‑BCMA scFv，铰链区、跨膜区，胞内区串联构成，该嵌合抗原受体用于修饰人T淋巴细胞，修饰后的T淋巴细胞用于表面BCMA阳性肿瘤的预防与治疗以及肿瘤药物的制备。
Description
技术领域
本发明涉及肿瘤的细胞免疫治疗领域，具体涉及利用基因工程技术获得了一种嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ和该嵌合抗原受体修饰的T淋巴细胞，被修饰的T淋巴细胞可以特异性的识别和杀伤表面表达BCMA的肿瘤细胞，所述肿瘤细胞优选为多发性骨髓瘤细胞。
背景技术
完整抗体的相对分子质量较大。仅由抗体重链V区(VH)与轻链V区(VL)通过人工合成的连接肽(linker)连接而成的小分子抗体称为单链抗体(single chain antibodyfragment，scFv)。单链抗体相对分子质量小，易于构建和表达，而且其免疫原性弱，不易引起超敏反应和排斥反应。单链抗体是目前研究最活跃的基因工程抗体之一。由单链抗体与毒素或免疫效应分子通过DNA重组工程制备的融合蛋白以及胞内表达的单链抗体等在疾病研究与治疗中展现了良好的前景。
多发性骨髓瘤(multiple myeloma，MM)是一种由成熟浆细胞在骨髓中恶性增殖累积导致骨破坏和骨髓衰竭的常见的血液系统恶性肿瘤。据统计，MM约占所有人类恶性肿瘤的1％，占血液系统恶性肿瘤的10％，MM在美国的发病率(1/100000)为3.5～4.5，中国约为1，中位发病年龄在美国为68岁，在我国则相对较低。MM患者通常表现出骨疼痛、贫血、溶骨性病变、高钙血症、肾功能损害、血细胞减少症、症状性浆细胞产生等症状。传统的化疗、造血干细胞以及近年来使用的蛋白酶体抑制剂虽然都有一定的疗效，患者的总体生存率得到了提高，但仍无法完全治愈。
BCMA(B cell maturition antigen，B细胞成熟抗原)属于TNF受体家族成员，在成熟B细胞分化到浆细胞阶段表达，由于其具有mRNA限制性的特点，BCMA并不会表达于人体主要的器官中，这与目前所应用的大多数单抗类作用于所有B系细胞的特点相比，特异性更强，副作用更小。
嵌合抗原受体修饰的T细胞(chimeric antigen receptor，CAR-T)治疗是利用基因工程技术，借助逆转录病毒或者逆转病毒载体或者mRNA转导，使得T细胞获得可以识别肿瘤表面抗原，杀伤肿瘤的嵌合抗原受体。CAR-T治疗相比于传统的治疗方法展现出巨大的优势，具体体现在杀伤肿瘤精准性高，CAR-T细胞治疗采用抗原抗体特异性结合的技术，只有表面表达抗原的肿瘤细胞被杀伤，对正常细胞的伤害小；杀伤肿瘤范围广泛，只要表达肿瘤相关抗原，CAR-T细胞就能清除，对于转移性肿瘤、复发性肿瘤均有效；对于患者而言避免了放化疗的痛苦，恢复健康迅速。首次临床应用的报道是2008年，在Nature medicine上报道了使用CAR-T细胞治疗11例儿童成纤维神经瘤，6例好转的病例。目前的临床应用，主要集中在白血病上，针对的是CD19、CD20、CD22三个靶向位点。在2011和2013年，先后在NEJM上发表了个案报道，描述了运用CAR-T细胞治疗慢性和急性白血病的病例。2014年7月FDA授予诺华公司开发的个性化CAR-T癌症疗法CTL019突破性药物认证，2017年8月，美国FDA核准该药(商品名Kyrmiah)上市。
但是，CAR-T也并不是那么完美。鉴于目前病例数量有限，所以无法得出一个系统的疗效结论。并且已有病例结果显示，部分患者CAR-T细胞回输治疗后有较为严重的临床副反应，更有甚者，有些CAR-T的识别失误导致其攻击正常细胞，即所谓的“脱靶效应”。截止目前，已经有研究证实对靶抗原亲和力和杀伤能力强的CAR-T细胞在清除肿瘤的同时也会攻击正常组织，造成组织器官的损伤，在2010年，报道了1例结直肠癌合并肝肺转移的患者在接受人表皮生长因子受体2的CAR-T治疗后因脱靶效应而死亡。
由此可见，本领域需要特异性的胞外靶向连接区，其能够特异性的对目标抗原进行识别，避免错误识别的影响。本发明人公开了一种针对人BCMA的特异性嵌合抗原受体及其应用，所述CAR能够特异性的结合到靶抗原上，并发挥细胞毒效应。
发明内容
为解决上述问题，本发明提供了一种靶向BCMA的嵌合抗原受体(anti-BCMAscFv-CD8a-41BB-CD3ζ)，该嵌合抗原受体修饰的T淋巴细胞可以特异性的识别并杀伤表面表达BCMA分子的肿瘤细胞，以及制备治疗肿瘤药物。
本发明的第一方面，涉及一种嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ，所述嵌合抗原受体包括单链抗体anti-BCMAscFv，铰链区、跨膜区，胞内区。
优选的，所述单链抗体anti-BCMAscFv由抗人BCMA单克隆抗体的重链可变区和轻链可变区串联而成，所述的抗人BCMA单克隆抗体来源于BCMA免疫小鼠的杂交瘤细胞。
所述铰链区和所述跨膜区均来源于人的CD8a区基因。
所述胞内区由共刺激因子4-1BB和CD3ζ串联构成，即为人4-1BB胞内区及人CD3ζ胞内区。
本发明所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ还包括人的CD8α信号肽区，优选的，在所述CD8α信号肽区氨基端对应的基因序列引入Kozak序列和/或酶切位点序列。
优选的，所述抗人BCMA单克隆抗体的重链可变区的互补性决定区CDR-H1～CDR-H3的氨基酸序列如SEQ ID NO：1-5所示，所述抗人BCMA单克隆抗体的轻链可变区的互补性决定区CDR-L1～CDR-L3的氨基酸序列如SEQ ID NO：6-8所示。本发明所述的单链抗体anti-BCMAscFv的氨基酸序列如SEQ ID NO：9或SEQ ID NO：10所示，或至少与SEQ ID NO：9或SEQID NO：10有约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.4％、99.5％、99.7％、99.8％或99.9％的氨基酸序列一致性的序列。
所述的单链抗体anti-BCMAscFv的核苷酸序列如SEQ ID NO：11或SEQ ID NO：12所示。
所述的anti-BCMAscFv的轻链和重链之间由氨基酸序列为Gly Gly Gly Gly SerGly Gly Gly Gly Ser Gly Gly Gly Gly Ser的连接肽连接而成。
在本发明的一个实施例中，所述铰链区和跨膜区串联连接，所述氨基酸序列如SEQID NO：15所示，所述铰链区和跨膜区串联的核苷酸序列如SEQ ID NO：16所示。
所述胞内区4-1BB-CD3ζ的氨基酸序列如SEQ ID NO：17所示，所述胞内区4-1BB-CD3ζ的核苷酸序列如SEQ ID NO：18所示。
所述CD8α信号肽区的氨基酸序列如SEQ ID NO：13所示，所述的CD8α信号肽区的核苷酸序列如SEQ ID NO：14所示。
本发明所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ，选自下列组中的一种：
a)嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的氨基酸序列如SEQ ID NO：19或20所示的序列的部分或全部所示；
b)嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的氨基酸序列与SEQ ID NO：19或20所示氨基酸的序列同一性程度为至少大约为90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％；
c)编码嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ氨基酸的核酸序列在严格条件下，与SEQ ID NO：19或20所示的氨基酸序列的核苷酸序列杂交；
d)嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ氨基酸与SEQ ID NO：19或20所示氨基酸的序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸；
e)嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ氨基酸序列具有SEQ ID NO：19或20所示的，包括取代、缺失和/或插入一个或多个氨基酸残基的氨基酸序列；或
f)编码该嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的核苷酸序列为SEQ IDNO：21或22所示的序列的部分或全部；
g)编码该嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的核苷酸序列与SEQ IDNO：21或22所示的核苷酸序列同一性程度为至少大约为90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％；
h)编码该嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的核苷酸序列在严格条件下，与SEQ ID NO：21或22所示的核苷酸序列杂交；
i)编码该嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的核苷酸序列与SEQ IDNO：21或22所示的序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个核苷酸；
j)编码该嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的核苷酸序列具有SEQID NO：21或22所示的，包括取代、缺失和/或插入一个或多个核苷酸的核苷酸序列。
优选的，本发明所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的氨基酸序列如SEQ ID NO：19或SEQ ID NO：20所示，或与SEQ ID NO：19或SEQ ID NO：20至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.4％、99.5％、99.7％、99.8％或99.9％的氨基酸序列一致性的序列。编码上述嵌合抗原受体本发明所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的核苷酸序列，如SEQ IDNO：21或SEQ ID NO：22所示。
本发明的第二方面，涉及一种编码上述任一嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的DNA。
本发明的第三方面，涉及上述任一嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ或上述DNA在制备嵌合抗原受体T细胞的应用。
本发明的第四方面，涉及一种anti-BCMA CAR-T细胞的制备方法，其中，所述方法包括以下步骤：
1)依次按信号肽、anti-BCMAscFv、人CD8α铰链区、人CD8α跨膜区、人4-1BB胞内区、人CD3ζ胞内区序列进行连接，获得anti-BCMA-CAR；
2)将anti-BCMA-CAR片段通过EcoRI-BamHI酶切连接至骨架载体pLVX-EF1a-GFP-N1上，测序验证正确后，获得pLVX-CAR；
3)将步骤2)获得的pLVX-CAR表达载体和包装质粒pMD2.G、psPAX2共同转染293T细胞，获得含有anti-BCMA-CAR的慢病毒颗粒；
4)将步骤3)获得的含有anti-BCMA-CAR的慢病毒颗粒感染T细胞，获得BCMA特异性CAR-T细胞。
本发明的第五方面，涉及包含上述所述的嵌合抗原受体或DNA的细胞、组织、器官或构建体，所述的细胞、组织、器官或构建体表达BCMA的特异性嵌合抗原受体。
优选的，所述细胞为T细胞。更优选的，所述T细胞由上述方法制备得到。
本发明的第六方面，涉及一种上述嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ或上述DNA或上述细胞、组织、器官或构建体在制备治疗肿瘤药物组合物中的应用。
本发明的第七方面，涉及一种治疗肿瘤的药物组合物，其中，所述药物组合物包括上述任一的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ、上述DNA或上述的细胞、组织、器官或构建体。
本发明的第八方面，涉及一种上述任一的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ、DNA、或上述的细胞、组织、器官或构建体在治疗肿瘤中的应用。
以及一种治疗肿瘤的方法，所述方法包括向患者给予上述药物组合物。
优选的，所述肿瘤为任一恶性肿瘤，包括各器官的癌、肉瘤、血液系统的恶性肿瘤等等。例如白血病、淋巴瘤、多发性骨髓瘤、卵巢癌、乳腺癌、子宫内膜癌、结肠癌、直肠癌、胃癌、膀胱癌、肺癌、支气管癌、骨癌、前列腺癌、胰腺癌、肝和胆管癌、食管癌、肾癌、甲状腺癌、头颈部癌、睾丸癌、胶质母细胞瘤、星形细胞瘤、黑色素瘤、骨髓增生异常综合征、以及肉瘤。
更优选的，所述肿瘤为多发性骨髓瘤。
除非特别说明，本发明的实践将采取细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术。这些技术在以下文献中进行了详细的解释。例如：MolecularCloningALaboratoryManual，2ndEd.，ed.BySambrook，FritschandManiatis(ColdSpringHarborLaboratoryPress：1989)；DNACloning，VolumesIandII(D.N.Glovered.，1985)；OligonucleotideSynthesis(M.J.Gaited.，1984)；Mullisetal.U.S.Pat.No.4，683，195；NucleicAcidHybridization(B.D.Hames&S.J.Higginseds.1984)；TranscriptionAndTranslation(B.D.Hames&S.J.Higginseds.1984)；CultureOfAnimalCells(R.I.Freshney，AlanR.Liss，Inc.，1987)；ImmobilizedCellsAndEnzymes(IRLPress，1986)；B.Perbal，APracticalGuideToMolecularCloning(1984)；theseries，MethodsInENZYMOLOGY(J.AbelsonandM.Simon，eds.-in-chief，AcademicPress，Inc.，NewYork)，specifically，Vols.154and155(Wuetal.eds.)andVol.185，″ols.154and155(Wuetal.eds.)andVol.18，ed.)；GeneTransferVectorsForMammalianCells(J.H.MillerandM.P.Caloseds.，1987，ColdSpringHarborLaboratory)；ImmunochemicalMethodsInCellAndMolecular Biology(MayerandWalker，eds.，AcademicPress，London，1987)；HandbookOfExperimentalImmunology，VolumesV(D.M.WeirandC.C.Blackwell，eds.，1986)；andManipulatingtheMouseEmbryo，(ColdSpringHarborLaboratoryPress，ColdSpringHarbor，N.Y.，1986)。
本发明采用抗人BCMA单链抗体的基因序列，将其密码子优化。从NCBI数据库中搜索得到人CD8ɑ的信号区、铰链区、跨膜区和4-1BB及CD3ζ的胞内区基因序列，分段合成后应用重叠延伸PCR(SOE-PCR)进行拼接，然后克隆进入经改造的慢病毒表达载体pLVX-EF1a-GFP-N1中，利用该表达载体及包装质粒pMD2.G、psPAX2共同转染293T细胞获得慢病毒颗粒，然后感染人T淋巴细胞，使得T淋巴细胞表达该嵌合抗原受体。获得的CAR-T细胞在体外与RPMI8226细胞共培养，采用流式细胞仪检测细胞感染效率、ELISA法检测共培养上清中细胞因子(IL2、TNFα、γ-IFN)水平以及非放射性细胞毒性法检测CAR-T细胞对RPMI8226细胞的杀伤活性，以证实该嵌合抗原受体修饰的T淋巴细胞对肿瘤细胞的特异性杀伤作用。因此本发明所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ可在肿瘤的细胞免疫治疗中得到应用。
附图说明
图1：嵌合抗原受体anti-BCMA-CD8a-41BB-CD3ζ的结构示意图，其中，SP为CD8α信号肽区，VL为轻链可变区，VH为重链可变区，CD8hinge为铰链区，CD8TM为跨膜区，4-1BB为人的4-1BB胞内区，CD3ζ为人的CD3ζ胞内区；
图2：anti-BCMA-CD8a-41BB-CD3ζ慢病毒载体图谱；
图3：慢病毒感染T细胞96h后，用荧光显微镜观察T细胞的绿色荧光表达情况；
图4：WesternBlotting检测CAR-T细胞中外源CAR(～55KDa)的表达，其中1为Marker，2为MOI＝10，3为MOI＝20，4为MOI＝40，5为MOI＝80，6为阴性对照，30KDa的条带为内参GAPDH；
图5：流式细胞仪检测慢病毒感染T细胞72h后，CAR-T阳性细胞占总细胞数的比例，其中图5A为未经感染的T细胞，图5B为感染72h后的T细胞，框中为CAR-T阳性细胞群；
图6：ELISA检测IL-2细胞因子水平；
图7：ELISA检测IFNγ细胞因子水平；
图8：ELISA检测TNFa细胞因子水平；
图9：在不同的效:靶比例(1:4，2:4，4:4)条件下，VEC-T和CAR-T细胞对BCMA阳性细胞系RPMI8226的裂解百分率；
图10：在不同的效:靶比例(1:4，2:4，4:4)条件下，VEC-T和CAR-T细胞对BCMA阴性细胞系K562的裂解百分率；
图11：采用生物发光成像系统检测anti-BCMA CAR-T细胞对KMS-11细胞的作用，其中，转染空载体的T细胞与PBS为对照。
具体实施方式
下面结合具体实施例来进一步描述本发明。
实施例1：anti-BCMA-CD8a-41BB-CD3ζ基因序列的确定
抗人BCMA单链抗体的基因序列来源于BCMA免疫小鼠的杂交瘤细胞。从NCBI网站数据库搜索到人的CD8α信号肽区、铰链区和跨膜区、人的4-1BB胞内区和人的CD3ζ胞内区基因序列信息。将上述序列在网站http://www.jcat.de/进行密码子优化，以保证在编码氨基酸序列不变的情况下更适合人类基因表达。在信号肽基因序列氨基端引入Kozak序列和酶切位点，各段核苷酸序列送上海生工生物技术有限公司分别进行合成。各基因序列信息见SEQUENCE LISTING(SEQ ID NO：1-22)，构建成功的嵌合抗原受体的结构如图1所示，其中，SP为CD8α信号肽区，VL为轻链可变区，VH为重链可变区，CD8hinge为铰链区，CD8TM为跨膜区，4-1BB为人的4-1BB胞内区，CD3ζ为人的CD3ζ胞内区。
实施例2：anti-BCMA-CD8a-41BB-CD3ζ慢病毒载体的构建
采用重叠PCR将上述合成的序列依次按信号肽、anti-BCMAscFv、人CD8α铰链区、人CD8α跨膜区、人4-1BB胞内区、人CD3ζ胞内区序列进行连接，形成完整的anti-BCMA-CAR基因序列信息，获得CAR分子(下文称为“anti-BCMA-CAR”)。anti-BCMA-CAR的核苷酸序列如SEQID NO：21或SEQ ID NO：22所示，编码的氨基酸序列如SEQ ID NO：19或SEQ ID NO：20所示。
用EcoR I和BamH I双酶切anti-BCMA-CAR的核苷酸序列，经T4DNA连接酶连接插入经改造的慢病毒载体pLVX-EF1a-GFP-N1(Addgene)的EcoR I和BamH I酶切位点，转化到感受态DH5α大肠杆菌。将所获得的重组质粒送上海生工生物技术有限公司进行测序，将测序结果与拟合成的anti-BCMA-CAR序列比对来验证序列是否正确。anti-BCMA-CD8a-41BB-CD3ζ慢病毒载体图谱如图2所示。
实施例3：含anti-BCMA-CAR慢病毒的包装和滴度测定
用无内毒素质粒大提试剂盒内(天根生物)的操作说明书提取慢病毒包装载体pMD2.G、psPAX2和anti-BCMA-CAR慢病毒载体共转染到293T细胞中，转染后48h和72h收集细胞上清，400rcf离心10min，去除上清中的细胞及细胞碎片。用0.45μm的滤膜过滤上清液，分装冻存，备用。
按照预期的病毒滴度(MOI)，将病毒上清液梯度稀释后感染293T细胞，荧光显微镜检测GFP阳性率，根据公式推算出pLVX-CAR和pLVX-空载病毒原液的滴度分别约为2×106TU/mL和5×106TU/mL。经浓缩液浓缩后，滴度可达1×108TU/mL和2×108TU/mL。
实施例4：慢病毒感染人的T细胞
第一步：用Ficoll分离液(天津灏洋)和人T细胞富集抗体混合物分离获得较纯的CD3+T细胞，用含5％AB血清X-VIVO(LONZA)培养基调整细胞密度为1×106/mL。将细胞以1mL/孔接种到预先用抗人50ng/mLCD3抗体(北京义翘神州生物技术有限公司)和50ng/mLCD28抗体(北京义翘神州生物技术有限公司)过夜包被过的24孔板，再加入100IU/mL的白细胞介素2(PeproTech)，刺激培养48小时后用实施例3制备得到的病毒感染；
第二步：T细胞活化培养后隔天，用PBS稀释至终浓度为15μg/mL的Retronectin(Takara)包被新的24孔板，每孔250μL。避光，4℃过夜备用。
第三步：T细胞活化培养两天后，取出上一步中Retronectin包被好的24孔板，吸弃包被液，每孔加入500μL含2％BSA的HBSS，室温封闭30min后，吸弃封闭液，用含2.5％HEPES的HBSS洗板两次。
第四步：将实施例3制备得到的病毒液加入上述T细胞中，每孔500μL，再加入终浓度为8μg/mL的polybrene，混匀后32℃，1500g，离心2h。置于培养箱内培养24h。
第五步：将感染24h后的细胞1000g离心10min，小心吸去上清，加入新鲜的含10％FBS的RPMI1640培养基重悬细胞至浓度1×106/mL，以1×106/孔接种到第三步包被好的24孔板中，再加入100IU/mL的白细胞介素2，病毒原液和polybrene用量同前，32℃，1500g，离心2h。然后置于培养箱内培养。
第六步：细胞感染后，每天观察细胞的密度，适时补加含IL-2 100IU/mL的T细胞培养液，使T细胞的密度维持在5×105/mL左右，使细胞扩增。感染72h后，荧光显微镜观察T细胞的绿色荧光表达情况并拍照。结果见图3。由此获得感染了实施例3所述慢病毒的CAR-T细胞，命名为anti-BCMA CAR-T细胞，即BCMA特异性CAR-T细胞，表达实施例2的anti-BCMA-CAR。
实施例5：anti-BCMA特异性嵌合抗原受体表达鉴定
实施例4中感染的人T细胞72h收集后用PBS洗一遍，用细胞蛋白提取试剂(RIPA)裂解细胞，提取感染后的T细胞的蛋白经10％的SDS-PAGE进行分离后，恒流(300mA，1h)转印至PVDF膜上，用兔抗CD3ζ(1:400)抗体孵育，4℃孵育过夜。用TBST洗涤3遍后，用HRP山羊抗兔的二抗(1:10000)室温孵育1h。加入DAB显色后，用Azure公司的C-300多功能成像系统进行成像，其结果如图4所示。由图4可知：本发明所构建的重组质粒能够检测到目的条带的表达，大小与预期CAR(～55kDa)一致，见图4中条带2-5；而未感染的T细胞没有条带，见图4条带6。
实施例6：流式细胞仪检测感染后T淋巴细胞的比例及表面表达
实施例4中感染的T细胞于72h后，收集约106个细胞，用buffer(含5％FBS的PBS)洗涤1次，离心弃去上清，加100μL buffer重悬。加一抗BCMA-biotin1μg，于37℃孵箱中孵育40min。离心，弃上清，用300μL buffer洗涤1-2次。用100μLbuffer重悬，加入二抗streptavidin-PE(按照说明书的浓度)，4℃冰箱孵育20-30min。离心，弃去上清，用300μLbuffer洗涤1-2次。用300μLbuffer重悬，上机(BD Calibur)检测。
结果如图5所示，使用实施例4制备得到的慢病毒感染T细胞72小时后，抗BCMA+的CAR-T细胞约占总细胞数的50％。
实施例7：体外共培养测定CAR-T细胞的肿瘤杀伤能力
7.1样品的准备
1)离心收集感染一周后实施例4的anti-BCMA CAR-T细胞并重新悬浮在新鲜的含10％FBS的RPMI1640培养基中，调整浓度为2×106/mL。
2)对于实施例5anti-BCMACAR-T细胞和NT细胞，按如下所示将T细胞和靶细胞RPMI8226共培养于6孔细胞培养板进行培养：
A：2×106/孔空载体感染的T细胞，VEC-T(图例中简称VECT)
B：2×106/孔anti-BCMACAR-T(图例中简称CART)
C：2×106/孔VEC-T，1×106/孔RPMI 8226(图例中简称VECT+R)
D：2×106/孔anti-BCMACAR-T，1×106/孔RPMI 8226(图例中简称CART+R)每组设置4复孔，将共培养细胞置于37℃孵育16h。
7.2ELISA法测定细胞因子水平
ELISA试剂盒(购自上海科兴商贸有限公司，货号：F14003-A)，按试剂盒说明操作。
1)待测样品A，B，C，D组培养上清液，从-20℃取出，室温融化。
2)将浓缩洗涤液、浓缩稀释液、标准品、密封板条从冰箱中取出室温解冻，然后将浓缩洗涤液稀释至工作浓度(1×)。
3)标准品稀释：用标准品稀释液稀释标准品至2400ng/mL、1200ng/mL、600ng/mL、300ng/mL、150ng/mL。
4)加样：分别设置空白孔、标准品孔、待测样品孔。在酶标包被板上标准品孔加样50μL，待测样品孔中先加样品稀释液40μL，然后再加待测样品10μL(样品最终稀释度为5倍)。将样品加于酶标板孔底部，尽量不要触及孔壁，轻轻晃动混匀。
5)温育：用封板膜封板后置于37℃温育30min。
6)洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，甩干。
7)加酶：每孔加入酶标试剂50μL，空白孔除外。
8)温育：用封板膜封板后置于37℃温育30min。
9)洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，甩干。
10)显色：每孔先加入显色剂A 50μL，再加入显色剂B 50μL，轻轻震荡混匀，37℃避光显色15min。
11)终止：每孔先加入终止液50μL，终止反应。
测定：以空白孔调零，450nm波长依序测量各孔的吸光度值(OD值)，测定应在加终止液后15min内完成。测定结果绘出标准曲线，然后对照样品OD值查出其浓度。结果见图6-8，其中，图6为ELISA检测IL-2细胞因子水平，图7为ELISA检测γ-IFN细胞因子水平，图8为ELISA检测TNFa细胞因子水平。anti-BCMA CAR-T和RPMI 8226共同孵育后，CAR-T细胞中的IL2检测值为1876.0pg/mL，而其余3组均小于50pg/mL(见图6)；γ-IFN检测值为4070.3pg/mL，而其余3组均小于50pg/mL(见图7)；TNFa检测值为1236.8pg/mL，而其余3组均小于65pg/mL(见图8)。anti-BCMA CAR-T和RPMI 8226共孵育后，CAR-T细胞中各因子的分泌量显著增高。
7.3CytoTox 非放射性细胞毒性法检测CAR-T细胞对肿瘤细胞的杀伤作用
A.优化靶细胞数目
1)靶细胞准备：800rpm离心5min收集对数生长期RPMI 8226细胞，用含5％FBS的RPMI1640调整细胞浓度至1×106/mL。
2)乳酸脱氢酶(LDH)阳性对照配制：轻轻震荡LDH阳性对照以混匀，然后取2μL稀释到10mL
的PBS+1％BSA(1:5000稀释)。
3)在U型底96孔板设置检验孔(三复孔)：
a.培养基背景对照：110μL/孔含5％FBS的RPMI1640；
b.LDH阳性对照组：110μL/孔步骤2中稀释的LDH；
c.靶细胞最大LDH释放：设置靶细胞数目为0、5×103、1×104、2×104、3×104、4×104，用含5％FBS的RPMI1640补足每孔的体积为100μL，并在每孔加入10μL细胞裂解液(10×)。
4)将孔板置于37℃，5％CO2，饱和湿度培养箱孵育45min，250g离心4min。
5)LDH活性测定：
a.转移50μL步骤4中培养上清至另一个干净的96孔新酶标板；
b.解冻Assay Buffer，取12mL Buffer溶解LDH底物；
c.加LDH底物50μL至96孔酶标板，室温避光反应30min；
d.每孔加入50μL终止液酶促反应；
e.酶标仪检测490nm处吸光度值。
6)确定靶细胞的吸光度值至少是培养基背景对照吸光度值的两倍时的靶细胞数目为5×104。
B.细胞毒性检测
1)靶细胞准备：800rpm离心5min收集对数生长期RPMI 8226或K562细胞，用含5％FBS的RPMI1640调整细胞浓度至5×106/mL。
2)效应细胞准备：1000rpm离心10分钟收集感染后一周的CAR-T和VEC-T细胞，用含5％FBS的RPMI1640调整细胞浓度至2.5×106/mL。
3)在U型底96孔板设置对照组和实验组(三复孔)：
a.培养基背景对照：110μL/孔含5％FBS的RPMI1640；
b.体积校正对照孔：100μL 5％FBS的RPMI1640+10μL细胞裂解液(10×)；
c.靶细胞自发释放组：10μL RPMI 8226或K562细胞(5×104细胞)+100μL 5％FBS的RPMI1640；
d.靶细胞最大释放：10μL RPMI 8226或K562细胞(5×104细胞)+10μL细胞裂解液(10×)；
e.效应细胞自发释放：按不同的效：靶比例(1:4，2:4，4:4)加效应细胞5μL、10μL、20μL/孔，均用含5％FBS的RPMI1640补足体积为110μL/孔；
f.实验组：按照上述效：靶比例加入效应细胞和靶细胞，用含5％FBS的RPMI1640补足体积为110μL/孔；
4)将孔板置于37℃，5％CO2，湿润的培养箱孵育4小时。然后250g离心4min。在收集上清前45min，向靶细胞最大释放孔加入10μL细胞裂解液(10×)。
5)LDH活性测定：同前所述。
6)CTL活性计算：计算各组(3复孔)平均值(A490)，按照下列公式计算CAR-T细胞裂解靶细胞的百分率，公式如下。
如图9所示，随着E:T从1:4增加到4:4，anti-BCMA CAR-T细胞对BCMA阳性肿瘤细胞RPMI 8226的裂解率分别为34％、55％和62％，随着CAR-T细胞的数量增加，裂解效应显著增强。如图10所示，anti-BCMA CAR-T细胞对BCMA阴性细胞系K562裂解率均低于20％；综合如图9、图10所示，anti-BCMA CAR-T细胞对BCMA阳性肿瘤细胞RPMI 8226有很强的裂解效应，但对BCMA阴性细胞系K562不产生裂解。而空载体感染的T细胞对RPMI 8226和K562都不发生裂解，表明anti-BCMA CAR-T细胞对BCMA阳性肿瘤细胞具有特异性识别和杀伤作用，也就是说anti-BCMA CAR-T细胞对其他正常细胞基本没有副作用。
实施例8：anti-BCMACAR-T细胞体内药效评价
KMS-11是人多发性骨髓瘤细胞系，细胞表面表达BCMA蛋白，并且可以作为免疫受损小鼠中的异种移植物生长。用萤火虫荧光素酶标记KMS-11细胞并植入NSG小鼠，建立起测试anti-BCMACAR-T疗效的多发性骨髓瘤模型。
将1×106个荧光素酶标记的KMS-11细胞通过尾静脉注射入NSG小鼠，培养9天后，通过尾静脉再注射8×106个anti-BCMA CAR-T细胞或转染空载体的T细胞或PBS。每天检测小鼠的健康状况，分别在1天，7天，14天，21天通过生物发光成像系统监测肿瘤负荷。小鼠麻醉前在腹膜内注射D荧光素，注射后5分钟用Xenogen成像麻醉小鼠，通过肿瘤细胞的生物发光评估疾病负担。结果如图11所示，转染空载体的T细胞组和PBS组的小鼠中显示很强的生物荧光信号，相比之下，anti-BCMACAR-T细胞组在第14天产生的生物荧光信号显著降低，在第21天几乎检测不到生物荧光信号，表明在体内anti-BCMACAR-T对BCMA阳性的肿瘤细胞有很好的清除率。
以上详细描述了本发明的优选实施例，但是，本发明并不限于上述实施例中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。
序列表
<110> 西安宇繁生物科技有限责任公司
<120> BCMA特异性嵌合抗原受体T细胞及其应用
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Gly Tyr Ser Phe Thr Ser Tyr Tyr
1 5
<210> 2
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Ile Tyr Pro Glu Ser Gly Asn Thr
1 5
<210> 3
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Ile Tyr Pro Gly Ser Gly Asn Thr
1 5
<210> 4
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Ala Arg Cys Tyr Tyr Thr Tyr Asp Leu Arg Phe Ala Tyr
1 5 10
<210> 5
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Ala Arg Cys Tyr Tyr Arg Tyr Asp Leu Gly Phe Ala Tyr
1 5 10
<210> 6
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe
1 5 10
<210> 7
<211> 3
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Leu Ala Ser
1
<210> 8
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Gln Gln Asn Asn Glu Asp Pro Phe Thr
1 5
<210> 9
<211> 246
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Asn Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn
85 90 95
Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
115 120 125
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val
130 135 140
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Tyr Ile
145 150 155 160
His Trp Val Lys His Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Trp
165 170 175
Ile Tyr Pro Glu Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe Lys Gly
180 185 190
Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr Met Gln
195 200 205
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg
210 215 220
Cys Tyr Tyr Thr Tyr Asp Leu Arg Phe Ala Tyr Trp Gly Gln Gly Thr
225 230 235 240
Leu Val Thr Val Ser Ala
245
<210> 10
<211> 246
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Asn Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn
85 90 95
Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
115 120 125
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val
130 135 140
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Tyr Ile
145 150 155 160
His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Trp
165 170 175
Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe Lys Gly
180 185 190
Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr Met Gln
195 200 205
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg
210 215 220
Cys Tyr Tyr Arg Tyr Asp Leu Gly Phe Ala Tyr Trp Gly Gln Gly Thr
225 230 235 240
Leu Val Thr Val Ser Ala
245
<210> 11
<211> 738
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60
atatcctgca gagccagtga aagtgttgat agttatggca atagttttat gcactggtac 120
cagcagaaac caggacagcc acccaaactc ctcatctatc ttgcatccaa cctagaatct 180
ggggtccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattgat 240
cctgtggagg ctgatgatgc tgcaacctat tactgtcagc aaaataatga ggatccattc 300
acgttcggct cggggacaaa gttggaaata aaaggaggcg gagggagtgg gggaggcggc 360
tctggcggag gaggcagcca ggtccagctg cagcagtctg gacctgagct ggtgaagcct 420
ggggcttcag tgaagatatc ctgcaaggct tctggctaca gcttcacaag ctactatata 480
cactgggtga agcataggcc tggacaggga cttgagtgga ttggatggat ttatcctgaa 540
agtggtaata ctaagtacaa tgagaagttc aagggcaagg ccacactgac ggcagacaca 600
tcctccagca ctgcctacat gcagctcagc agcctaacat ctgaggactc tgcggtctat 660
tactgtgcaa gatgctacta tacgtacgac ctaaggtttg cttactgggg ccaagggact 720
ctggtcactg tctctgca 738
<210> 12
<211> 738
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
aacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60
atatcctgca gagccagtga aagtgttgat agttatggca atagttttat gcactggtac 120
cagcagaaac caggacagcc acccaaactc ctcatctatc ttgcatccaa cctagaatct 180
ggggtccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattgat 240
cctgtggagg ctgatgatgc tgcaacctat tactgtcagc aaaataatga ggatccattc 300
acgttcggct cggggacaaa gttggaaata aaaggaggcg gagggagtgg gggaggcggc 360
tctggcggag gaggcagcca ggtccagctg cagcagtctg gacctgagct ggtgaagcct 420
ggggcttcag tgaagatatc ctgcaaggct tctggctaca gcttcacaag ctactatata 480
cactgggtga agcagaggcc tggacaggga cttgagtgga ttggatggat ttatcctgga 540
agtggtaata ctaagtacaa tgagaagttc aagggcaagg ccacactgac ggcagacaca 600
tcctccagca ctgcctacat gcagctcagc agcctaacat ctgaggactc tgcggtctat 660
tactgtgcaa gatgctacta taggtacgac ctagggtttg cttactgggg ccaagggact 720
ctggtcactg tctctgca 738
<210> 13
<211> 21
<212> PRT
<213> 人工序列(Artificial Sequence)
<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
20
<210> 14
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccg 63
<210> 15
<211> 69
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
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 Ile Tyr Ile
35 40 45
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
50 55 60
Ile Thr Leu Tyr Cys
65
<210> 16
<211> 207
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180
ctgtcactgg ttatcaccct ttactgc 207
<210> 17
<211> 176
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
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 Arg Val Lys Phe Ser Arg
35 40 45
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
50 55 60
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
65 70 75 80
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
85 90 95
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
100 105 110
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
115 120 125
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
130 135 140
Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn
145 150 155 160
Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
165 170 175
<210> 18
<211> 529
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
aagcggggcc gcaagaagct cctgtacatc ttcaagcagc catttatgcg cccagtgcag 60
accacccagg aggaggatgg ctgctcgtgc cgcttcccag aggaggaaga gggcggctgt 120
gagctgcgcg tgaagttcag caggagcgcc gacgcccccg cgtaccagca gggccagaac 180
cagctctaca acgagctcaa cctgggccgc cgcgaggagt acgatgtgct ggacaagcgc 240
cgcggccggg accctgagat ggggggcaag ccgcgcagga agaaccctca ggagggcctg 300
tacaacgagc tgcagaagga taagatggcg gaggcctaca gcgagatcgg gatgaagggc 360
gagcgccgga ggggcaaggg gcacgatggc ctgtaccagg gcctcagcac agccaccaag 420
gacacctacg acgccctgca catgcaggcc ctgccccctc gcggaagcgg agcaacaaac 480
ttctcactac tcaaacaagc aggtgacgtg gaggagaatc ccgggcctg 529
<210> 19
<211> 512
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
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 Asn Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
20 25 30
Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu
35 40 45
Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys
50 55 60
Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu
65 70 75 80
Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe
85 90 95
Thr Leu Thr Ile Asp Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr
100 105 110
Cys Gln Gln Asn Asn Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys
115 120 125
Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
145 150 155 160
Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
165 170 175
Thr Ser Tyr Tyr Ile His Trp Val Lys His Arg Pro Gly Gln Gly Leu
180 185 190
Glu Trp Ile Gly Trp Ile Tyr Pro Glu Ser Gly Asn Thr Lys Tyr Asn
195 200 205
Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser
210 215 220
Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
225 230 235 240
Tyr Tyr Cys Ala Arg Cys Tyr Tyr Thr Tyr Asp Leu Arg Phe Ala Tyr
245 250 255
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Thr Thr Pro Ala
260 265 270
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
275 280 285
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
290 295 300
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
305 310 315 320
Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
325 330 335
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
340 345 350
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
355 360 365
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
370 375 380
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
385 390 395 400
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
405 410 415
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
420 425 430
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
435 440 445
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
450 455 460
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
465 470 475 480
Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn
485 490 495
Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
500 505 510
<210> 20
<211> 512
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
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 Asn Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
20 25 30
Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu
35 40 45
Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys
50 55 60
Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu
65 70 75 80
Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe
85 90 95
Thr Leu Thr Ile Asp Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr
100 105 110
Cys Gln Gln Asn Asn Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys
115 120 125
Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
145 150 155 160
Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
165 170 175
Thr Ser Tyr Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
180 185 190
Glu Trp Ile Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn
195 200 205
Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser
210 215 220
Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
225 230 235 240
Tyr Tyr Cys Ala Arg Cys Tyr Tyr Arg Tyr Asp Leu Gly Phe Ala Tyr
245 250 255
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Thr Thr Pro Ala
260 265 270
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
275 280 285
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
290 295 300
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
305 310 315 320
Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
325 330 335
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
340 345 350
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
355 360 365
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
370 375 380
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
385 390 395 400
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
405 410 415
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
420 425 430
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
435 440 445
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
450 455 460
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
465 470 475 480
Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn
485 490 495
Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
500 505 510
<210> 21
<211> 1536
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccgaacattg tgctgaccca atctccagct tctttggctg tgtctctagg gcagagggcc 120
accatatcct gcagagccag tgaaagtgtt gatagttatg gcaatagttt tatgcactgg 180
taccagcaga aaccaggaca gccacccaaa ctcctcatct atcttgcatc caacctagaa 240
tctggggtcc ctgccaggtt cagtggcagt gggtctagga cagacttcac cctcaccatt 300
gatcctgtgg aggctgatga tgctgcaacc tattactgtc agcaaaataa tgaggatcca 360
ttcacgttcg gctcggggac aaagttggaa ataaaaggag gcggagggag tgggggaggc 420
ggctctggcg gaggaggcag ccaggtccag ctgcagcagt ctggacctga gctggtgaag 480
cctggggctt cagtgaagat atcctgcaag gcttctggct acagcttcac aagctactat 540
atacactggg tgaagcatag gcctggacag ggacttgagt ggattggatg gatttatcct 600
gaaagtggta atactaagta caatgagaag ttcaagggca aggccacact gacggcagac 660
acatcctcca gcactgccta catgcagctc agcagcctaa catctgagga ctctgcggtc 720
tattactgtg caagatgcta ctatacgtac gacctaaggt ttgcttactg gggccaaggg 780
actctggtca ctgtctctgc aaccacgacg ccagcgccgc gaccaccaac accggcgccc 840
accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900
gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 960
gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcaa gcggggccgc 1020
aagaagctcc tgtacatctt caagcagcca tttatgcgcc cagtgcagac cacccaggag 1080
gaggatggct gctcgtgccg cttcccagag gaggaagagg gcggctgtga gctgcgcgtg 1140
aagttcagca ggagcgccga cgcccccgcg taccagcagg gccagaacca gctctacaac 1200
gagctcaacc tgggccgccg cgaggagtac gatgtgctgg acaagcgccg cggccgggac 1260
cctgagatgg ggggcaagcc gcgcaggaag aaccctcagg agggcctgta caacgagctg 1320
cagaaggata agatggcgga ggcctacagc gagatcggga tgaagggcga gcgccggagg 1380
ggcaaggggc acgatggcct gtaccagggc ctcagcacag ccaccaagga cacctacgac 1440
gccctgcaca tgcaggccct gccccctcgc ggaagcggag caacaaactt ctcactactc 1500
aaacaagcag gtgacgtgga ggagaatccc gggcct 1536
<210> 22
<211> 1536
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccgaacattg tgctgaccca atctccagct tctttggctg tgtctctagg gcagagggcc 120
accatatcct gcagagccag tgaaagtgtt gatagttatg gcaatagttt tatgcactgg 180
taccagcaga aaccaggaca gccacccaaa ctcctcatct atcttgcatc caacctagaa 240
tctggggtcc ctgccaggtt cagtggcagt gggtctagga cagacttcac cctcaccatt 300
gatcctgtgg aggctgatga tgctgcaacc tattactgtc agcaaaataa tgaggatcca 360
ttcacgttcg gctcggggac aaagttggaa ataaaaggag gcggagggag tgggggaggc 420
ggctctggcg gaggaggcag ccaggtccag ctgcagcagt ctggacctga gctggtgaag 480
cctggggctt cagtgaagat atcctgcaag gcttctggct acagcttcac aagctactat 540
atacactggg tgaagcagag gcctggacag ggacttgagt ggattggatg gatttatcct 600
ggaagtggta atactaagta caatgagaag ttcaagggca aggccacact gacggcagac 660
acatcctcca gcactgccta catgcagctc agcagcctaa catctgagga ctctgcggtc 720
tattactgtg caagatgcta ctataggtac gacctagggt ttgcttactg gggccaaggg 780
actctggtca ctgtctctgc aaccacgacg ccagcgccgc gaccaccaac accggcgccc 840
accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900
gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 960
gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcaa gcggggccgc 1020
aagaagctcc tgtacatctt caagcagcca tttatgcgcc cagtgcagac cacccaggag 1080
gaggatggct gctcgtgccg cttcccagag gaggaagagg gcggctgtga gctgcgcgtg 1140
aagttcagca ggagcgccga cgcccccgcg taccagcagg gccagaacca gctctacaac 1200
gagctcaacc tgggccgccg cgaggagtac gatgtgctgg acaagcgccg cggccgggac 1260
cctgagatgg ggggcaagcc gcgcaggaag aaccctcagg agggcctgta caacgagctg 1320
cagaaggata agatggcgga ggcctacagc gagatcggga tgaagggcga gcgccggagg 1380
ggcaaggggc acgatggcct gtaccagggc ctcagcacag ccaccaagga cacctacgac 1440
gccctgcaca tgcaggccct gccccctcgc ggaagcggag caacaaactt ctcactactc 1500
aaacaagcag gtgacgtgga ggagaatccc gggcct 1536
Claims (18)
1.一种嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ，其特征在于，所述嵌合抗原受体包括单链抗体anti-BCMAscFv，铰链区、跨膜区和胞内区。
2.根据权利要求1所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ，其特征在于，所述单链抗体anti-BCMAscFv由抗人BCMA单克隆抗体的重链可变区和轻链可变区串联而成，所述的抗人BCMA单克隆抗体来源于BCMA免疫小鼠的杂交瘤细胞。
3.根据权利要求1-2任一所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ，其特征在于，所述铰链区和所述跨膜区均来源于人的CD8a区。
4.根据权利要求1-3任一所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ，其特征在于，所述胞内区为人4-1BB胞内区及人CD3ζ胞内区。
5.根据权利要求1-4任一所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ，其特征在于，还包括人的CD8α信号肽区。
6.根据权利要求5所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ，其特征在于，在所述CD8α信号肽区氨基端对应的基因序列引入Kozak序列和/或酶切位点序列。
7.根据权利要求2所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ，其特征在于，所述抗人BCMA单克隆抗体的重链可变区的互补性决定区CDR-H1～CDR-H3的氨基酸序列如SEQ ID NO：1-5所示，所述抗人BCMA单克隆抗体的轻链可变区的互补性决定区CDR-L1～CDR-L3的氨基酸序列如SEQ ID NO：6-8所示。
8.根据权利要求1-7任一所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ，其特征在于，所述的单链抗体anti-BCMAscFv的氨基酸序列如SEQ ID NO：9或SEQ ID NO：10所示，所述的单链抗体anti-BCMAscFv的核苷酸序列如SEQ ID NO：11或SEQ ID NO：12所示。
9.根据权利要求1-8任一所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ，其特征在于，所述铰链区和跨膜区串联连接，其氨基酸序列如SEQ ID NO：15所示，所述铰链区和跨膜区串联的核苷酸序列如SEQ ID NO：16所示。
10.根据权利要求4所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ，其特征在于，所述胞内区4-1BB-CD3ζ的氨基酸序列如SEQ ID NO：17所示，编码所述胞内区4-1BB-CD3ζ的核苷酸序列如SEQ ID NO：18所示。
11.根据权利要求5所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ，其特征在于，所述的CD8α信号肽区的氨基酸序列如SEQ ID NO：13所示，编码所述CD8α信号肽区的核苷酸序列如SEQ ID NO：14所示。
12.根据权利要求1所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ，其特征在于，选自下列组中的一种：
a)嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的氨基酸序列如SEQ ID NO：19或20所示的序列的部分或全部所示；
b)嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的氨基酸序列与SEQ ID NO：19或20所示氨基酸的序列同一性程度为至少大约为90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％；
c)编码嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ氨基酸的核酸序列在严格条件下，与编码SEQ ID NO：19或20所示的氨基酸序列的核苷酸序列杂交；
d)嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ氨基酸与SEQ ID NO：19或20所示氨基酸的序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸；
e)嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ氨基酸序列具有SEQ ID NO：19或20所示的，包括取代、缺失和/或插入一个或多个氨基酸残基的氨基酸序列；或
f)编码该嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的核苷酸序列为SEQ ID NO：21或22所示的序列的部分或全部；
g)编码该嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的核苷酸序列与SEQ ID NO：21或22所示的核苷酸序列同一性程度为至少大约为90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％；
h)编码该嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的核苷酸序列在严格条件下，与SEQ ID NO：21或22所示的核苷酸序列杂交；
i)编码该嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的核苷酸序列与SEQ ID NO：21或22所示的序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个核苷酸；
j)编码该嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的核苷酸序列具有SEQ IDNO：21或22所示的，包括取代、缺失和/或插入一个或多个核苷酸的核苷酸序列。
13.一种编码权利要求1-12任一所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ的DNA。
14.权利要求1-12任一所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ或权利要求13所述的DNA在制备嵌合抗原受体T细胞的应用。
15.一种anti-BCMA CAR-T细胞的制备方法，其特征在于，所述方法包括以下步骤：
1)依次按信号肽、anti-BCMAscFv、人CD8α铰链区、人CD8α跨膜区、人4-1BB胞内区、人CD3ζ胞内区序列进行连接，获得anti-BCMA-CAR；
2)将anti-BCMA-CAR片段通过EcoRI-BamHI酶切连接至骨架载体pLVX-EF1a-GFP-N1上，经测序验证，获得pLVX-CAR；
3)将步骤2)获得的pLVX-CAR表达载体和包装质粒pMD2.G、psPAX2共同转染293T细胞，获得含有anti-BCMA-CAR的慢病毒颗粒；
4)将步骤3)获得的含有anti-BCMA-CAR的慢病毒颗粒感染T细胞，获得BCMA特异性CAR-T细胞。
16.包含权利要求1-12任一所述的嵌合抗原受体或权利要求13所述的DNA的细胞、组织、器官或构建体，其特征在于，所述的细胞、组织、器官或构建体表达BCMA的特异性嵌合抗原受体。优选的，所述细胞为T细胞。更优选的，所述T细胞由权利要求15所述的方法制备得到。
17.权利要求1-12任一所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ、权利要求13所述的DNA、或权利要求16所述的细胞、组织、器官或构建体在制备治疗肿瘤药物组合物中的应用。
18.一种治疗肿瘤的药物组合物，其特征在于，所述药物组合物包括权利要求1-12任一所述的嵌合抗原受体anti-BCMAscFv-CD8a-41BB-CD3ζ、权利要求13所述的DNA或权利要求16所述的细胞、组织、器官或构建体。
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