CN112074279B - 人源化bcma抗体和bcma-car-t细胞 - Google Patents
人源化bcma抗体和bcma-car-t细胞 Download PDFInfo
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Abstract
本发明涉及人源化BCMA单链可变片段(scFv)，其包含具有SEQ ID NO:3的氨基酸序列的VH和具有SEQ ID NO:5的氨基酸序列的VL。本发明还涉及BCMA嵌合抗原受体融合蛋白，其从N末端到C末端包含：(i)本发明的单链可变片段(scFv),(ii)跨膜结构域，(iii)至少一个共刺激结构域，和(iv)激活结构域。这种人源化BCMA‑CAR‑T细胞对BCMA阳性肿瘤细胞具有特异性杀伤活性。
Description
序列表、表格或计算机程序的引用
序列表以ASCII格式的文本文件与说明书一起同时通过EFS-Web提交，文件名为：序列表.txt(Sequence Listing.txt)，创建日期为2020年1月13日，大小为4千字节。通过EFS-Web提交的序列表是说明书的一部分，并且在此通过引用整体并入本文中。
发明领域
本发明涉及特异性降低多发性骨髓瘤肿瘤生长的人源化BCMA抗体(PMC306)和BCMA-CAR-T细胞，其可用于肿瘤的过继性免疫基因疗法领域。
发明背景
免疫疗法作为一种非常有希望的治疗癌症的方法而出现。T细胞或T淋巴细胞(我们免疫***的武装力量)不断寻找外源抗原，并将异常(癌症或受感染的细胞)与正常细胞区分开来。用CAR(嵌合抗原受体)构建体基因修饰T细胞是设计肿瘤特异性T细胞的最常见的方法。可以将靶向肿瘤相关抗原(TAA)的CAR-T细胞输注至患者(被称为过继性细胞转移或ACT)，这代表有效的免疫治疗方法[1,2]。与化学疗法或抗体相比，CAR-T技术的优点是经重新编程的工程化T细胞可以在患者中增殖并持续存在(“活体药物”)[1,2]。
CAR通常由在N末端部分的单克隆抗体衍生的单链可变片段(scFv)、铰链区、跨膜结构域以及与激活CD3-ζ结构域串联的许多细胞内共激活结构域组成，所述细胞内共激活结构域为：(i)CD28，(ii)CD137(4-1BB)、CD27或其它共刺激结构域(图1)。CAR的进化经历了从第一代(无共刺激结构域)发展到第二代(有一个共刺激结构域)，再到第三代CAR(有几个共刺激结构域)。产生具有两个共刺激结构域的CAR(所谓的第三代CAR)已经使得产生增加的细胞溶解CAR-T细胞活性、改善的CAR-T细胞持久性，这使得其产生增强的抗肿瘤活性。
BCMA
B细胞成熟抗原(BCMA)是细胞表面受体，也被称为CD269和肿瘤坏死因子受体超家族成员17(TNFRSF17)，其由TNFRSF17基因编码。该受体主要在成熟的B淋巴细胞中表达，并且在大多数情况下在多发性骨髓瘤(MM)中过表达[4]。在MM中靶向BCMA的当前疗法包括单克隆抗体、双特异性抗体和T细胞免疫疗法、CAR-T疗法[4,5]。
人BCMA蛋白由184个氨基酸组成：第1-54位为胞外结构域；第55-77位为跨膜结构域；第78-184位为胞浆结构域。BCMA的氨基酸序列如图2所示。BCMA缺乏信号肽并且类似于其它受体BAFF受体和跨膜激活剂以及亲环蛋白配体交互剂和钙调节剂(TACI)[4]。这些受体在B细胞成熟和分化成浆细胞中起主要作用。它们的配体包括在MM患者中表达增加的BAFF和APRIL[4]。
附图简述
图1显示了CAR的结构[3]。左图显示了第一代的结构(无共刺激结构域)。中间的图显示了第二代的结构(CD28或4-BB的一个共刺激结构域)。右图显示了第三代的结构(两个或多个共刺激结构域)。
图2显示了BCMA蛋白的氨基酸序列(SEQ ID NO:1)。胞外结构域加下划线。
图3显示了人源化BCMA CAR构建体的结构。
图4显示了用荧光标记重组BCMA蛋白，通过FACS分析检测人源化BCMA-CAR构建体。在慢病毒人源化BCMA-CAR转导T细胞后，检测人源化BCMA-CAR阳性细胞。
图5A-图5B显示了人源化BCMA-CAR-T细胞杀伤CHO-BCMA细胞，而不杀伤CHO细胞。XCelligence实时细胞毒性测定被用于检测人源化BCMA-CAR-T细胞的细胞毒性。归一化的细胞指数显示在Y轴上，时间显示在X轴上。图5A显示了CHO-BCMA靶细胞。在右侧从上到下为：模拟CAR-T细胞、T细胞、仅靶细胞和人源化CAR-T细胞。图5B显示了CHO靶细胞。在右侧从上到下为：模拟CAR-T细胞、人源化BCMA CAR-T细胞、T细胞和仅靶细胞。
图6显示了人源化BCMA-CAR-T细胞与CHO-BCMA阳性细胞一起分泌高水平的IFN-γ，而与BCMA阴性CHO对照细胞一起则没有分泌高水平的IFN-γ。p<0.05，相对于T细胞和模拟CAR-T细胞，在BCMA-CAR-T细胞的CHO-BCMA细胞中的IFN-γ分泌。
图7显示了人源化BCMA-CAR-T细胞针对多发性骨髓瘤细胞分泌高水平的IFN-γ，但针对BCMA阴性K562对照细胞则没有分泌高水平的IFN-γ。*p<0.05，相对T细胞和模拟CAR-T细胞，在BCMA-CAR-T细胞多发性骨髓瘤细胞中的IFN-γ分泌。
图8A显示了人源化BCMA-CAR-T细胞显著降低RPMI8226异种移植肿瘤的生长。在第7天和第20天通过静脉注射以1×10^7个细胞/小鼠注射CAR-T细胞。条显示平均肿瘤体积+/-标准误差。*p<0.05,BCMA CAR-T细胞相对于模拟T细胞。
图8B显示了人源化BCMA-CAR-T细胞没有降低小鼠体重。条显示平均小鼠体重+/-标准偏差。
图8C显示了用BCMA重组蛋白通过FACS在小鼠血液中检测到人源化BCMA-CAR-T细胞，而没有检测到模拟Car-T细胞。在研究结束时通过流式细胞仪分析外周血细胞与人BCMA蛋白和特异性针对人T(CD4+/CD8+)细胞的抗体的结合。与CD4抗体结合的细胞的百分比显示在图8C的左图上，也与BCMA蛋白结合的那些人T细胞的百分比显示在图8C的右图上。
发明详述
定义
如本文中所使用的“嵌合抗原受体(CAR)”是一种受体蛋白，其被工程化以赋予T细胞靶向特定蛋白的新能力。该受体是嵌合的，因为它们将抗原结合功能与T细胞激活功能结合成单个受体。CAR是包含能够结合抗原的胞外结构域、跨膜结构域和至少一个胞内结构域的融合蛋白。“嵌合抗原受体(CAR)”有时被称为“嵌合受体”、“T体(T-body)”或“嵌合免疫受体(CIR)”。“能够结合抗原的胞外结构域”意指可以结合某种抗原的任何寡肽或多肽。“胞内结构域”意指已知起结构域作用的任何寡肽或多肽，所述结构域传递信号以引起细胞中生物过程的激活或抑制。
如本文中所使用的“结构域”意指多肽中的一个区域，其独立于其它区域被折叠成特定结构。
如本文中所使用的“人源化抗体”是衍生自非人物种的抗体，其蛋白序列已经被修饰以增加它们与人中天然产生的抗体变体的相似性。例如，在开发小鼠抗体后，可以对编码该抗体的DNA进行测序。然后可以确定对应于抗体CDR的DNA序列。可以将CDR序列***含有人抗体变体DNA的构建体中以制备人源化抗体。
如本文中所使用的“单链可变片段(scFv)”意指衍生自抗体的单链多肽，其保留了与抗原结合的能力。scFv的实例包括通过重组DNA技术形成的抗体多肽，其中免疫球蛋白重链(H链)和轻链(L链)片段的Fv区域经由间隔序列连接。用于工程化scFv的各种方法是本领域技术人员已知的。
如本文中所使用的“肿瘤抗原”意指具有抗原性的生物分子，其表达引起癌症。
本发明的发明人已经从衍生自小鼠单克隆抗体克隆4C8A的小鼠单克隆抗体的重链可变区和轻链可变区开始对人源化BCMA scFv进行工程化。小鼠4C8A抗体展现出与人BCMA的强和选择性结合[6]。本发明的发明人已经产生了基于人源化BCMA抗体的BCMA-CAR-T细胞以靶向过表达BCMA肿瘤抗原的癌细胞。本发明的BCMA-CAR-T细胞对几种癌细胞系具有高细胞毒性活性。
本发明涉及人源化抗人BCMA抗体，其包含具有SEQ ID NO:3的氨基酸的VH和具有SEQ ID NO:5的氨基酸的VL。
在一个实施方案中，人源化抗人BCMA抗体是单链可变片段(scFv)。scFv可以是VH-连接子-VL或VL-连接子-VH。
本发明还涉及嵌合抗原受体融合蛋白，其从N末端到C末端包含：(i)针对BCMA的单链可变片段(scFv)，其中VH具有SEQ ID NO:3的氨基酸序列，VL具有SEQ ID NO:5的氨基酸，(ii)跨膜结构域，(iii)至少一个共刺激结构域，和(iv)激活结构域。
在一个实施方案中，在图2中显示了CAR结构。
在一个实施方案中，共刺激结构域选自：CD28、4-1BB、GITR、ICOS-1、CD27、OX-40和DAP10。优选的共刺激结构域是CD28。
优选的激活结构域是CD3ζ(CD3Z或CD3ζ)。
跨膜结构域可以衍生自天然多肽，或者可以是人工设计的。衍生自天然多肽的跨膜结构域可以从任何膜结合蛋白或跨膜蛋白获得。例如，可以使用以下的跨膜结构域：T细胞受体α或β链、CD3ζ链、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、ICOS、CD154或GITR。人工设计的跨膜结构域是主要包含疏水残基(如亮氨酸和缬氨酸)的多肽。优选在合成的跨膜结构域的每一端发现苯丙氨酸、色氨酸和缬氨酸的三联体。任选地，短寡肽连接子或多肽连接子(例如长度为2至10个氨基酸的连接子)可以排列在跨膜结构域和胞内结构域之间。在一个实施方案中，可以使用具有甘氨酸-丝氨酸连续序列的连接子序列。
本发明提供了编码BCMA-CAR的核酸。编码CAR的核酸可以通过常规方法从特定CAR的氨基酸序列制备。编码氨基酸序列的碱基序列可以从上述NCBI RefSeq ID或GenBank中针对每个结构域的氨基酸序列的登录号获得，并且本发明的核酸可以使用标准分子生物学和/或化学程序制备。例如，基于碱基序列，可以合成核酸，并且本发明的核酸可以使用聚合酶链式反应(PCR)通过组合从cDNA文库获得的DNA片段来制备。
可以将编码本发明CAR的核酸***到载体中，并且可以将载体引入到细胞中。例如，可以使用病毒载体，诸如逆转录病毒载体(包括致癌逆转录病毒载体、慢病毒载体和假型载体)、腺病毒载体，腺相关病毒(AAV)载体、猿猴病毒载体、痘苗病毒载体或仙台病毒载体、爱泼斯坦-巴尔病毒病毒(an Epstein-Barr virus，EBV)载体和HSV载体。优选使用在受感染细胞中缺乏复制能力而不能自我复制的病毒载体。
例如，当使用逆转录病毒载体时，可以选择基于LTR序列和载体所具有的包装信号序列的合适的包装细胞来使用包装细胞制备逆转录病毒颗粒。包装细胞的实例包括PG13(ATCC CRL-10686)、PA317(ATCC CRL-9078)、GP+E-86和GP+envAm-12以及Psi-Crip。还可使用具有高转染效率的293细胞或293T细胞制备逆转录病毒颗粒。基于逆转录病毒和可以被用于包装逆转录病毒载体的包装细胞产生的许多种类的逆转录病毒载体可从许多公司广泛地商购获得。
CAR-T细胞经由CAR与特异性抗原结合，从而将信号传递到细胞中，从而激活细胞。表达CAR的细胞的激活根据宿主细胞的种类和CAR的胞内结构域而变化，并且可以基于例如细胞因子的释放、细胞增殖速率的改善、细胞表面分子的变化等作为指标来确认。例如，细胞毒性细胞因子(肿瘤坏死因子、淋巴毒素等)从激活细胞中的释放引起表达抗原的靶细胞的破坏。此外，细胞因子的释放或细胞表面分子的变化刺激其它免疫细胞(例如B细胞、树突状细胞、NK细胞和巨噬细胞)。
表达CAR的细胞可以被用作疾病治疗剂。治疗剂包含表达CAR作为活性成分的细胞，并且它还可以包含合适的赋形剂。
本发明的发明人已经基于特异性靶向BCMA的人源化BCMA ScFv序列产生了CAR-T细胞。本发明的发明人已经产生了人源化BCMA-CAR-T细胞以靶向过表达BCMA肿瘤抗原的癌细胞。本发明的人源化BCMA-CAR-T细胞针对多发性骨髓瘤细胞分泌高水平的细胞因子，并杀伤CHO-BCMA阳性靶细胞，但不杀伤对照亲本CHO细胞。
本发明的人源化BCMA-ScFv相对于相应的小鼠ScFv的优点包括由于人源化BCMAscFv序列而对人的免疫原性较低。因此，本发明的人源化BCMA抗体在许多临床应用中作为治疗剂是高度有效和有利的。
本发明的人源化BCMA ScFv可以被用于免疫治疗应用：毒素/药物缀合的抗体、单克隆治疗性抗体和CAR-T细胞免疫疗法。
使用本发明的人源化BCMA ScFv的人源化BCMA-CAR-T细胞有效地靶向BCMA阳性癌细胞系(如卵巢癌、结肠癌、胰腺癌、黑素瘤、***和其它BCMA阳性癌症)中的BCMA抗原。
人源化BCMA-CAR-T细胞可以与不同的化学疗法组合使用：检查点抑制剂、靶向疗法、小分子抑制剂和抗体。
人源化BCMA-CAR-T细胞可以在临床上用于BCMA阳性癌细胞。
共激活结构域(如CD28、4-1BB等等)的修饰可以被用于增加CAR-T细胞的功效。标签缀合的人源化BCMA scFv可以被用于CAR产生。
人源化BCMA-CAR-T细胞可以与不同的安全转换剂(safety switches)一起使用，诸如t-EGFR、RQR(利妥昔单抗-CD34-利妥昔单抗)、诱导型半胱天冬酶-9等等。
第三代CAR-T或其它共激活信号传导结构域可以与人源化BCMA-scFv一起使用以制备BCMA-CAR-T。
人源化BCMA CAR可以与靶向其它肿瘤抗原或肿瘤微环境(例如VEGFR-1-3、PDL-1)的CAR组合。可以产生针对BCMA和CD3或其它抗原的双特异性抗体用于治疗。
人源化BCMA-CAR可以被用于产生可以靶向BCMA阳性癌症的其它类型的细胞，诸如CAR-自然杀伤(NK)细胞、BCMA-CAR-巨噬细胞、同种异体CAR-T细胞、基因编辑的T细胞和其它BCMA-CAR造血细胞。
本发明提供了经修饰以表达BCMA-CAR的T细胞、NK细胞、巨噬细胞或造血细胞。
BCMA-CAR-T细胞可以被用于针对癌症干细胞和循环肿瘤干细胞，其对化学疗法具有最强的抗性并形成侵袭性肿瘤。
BCMA-CAR-T细胞、BCMA-NK细胞、BCMA-巨噬细胞和其它细胞可以被用于靶向不同类型的癌症。
BCMA-CAR-T细胞可以通过肿瘤内递送给患者以提高安全性。
以下实施例进一步说明本发明。这些实施例仅旨在说明本发明，而不应解释为限制性的。
实施例
实施例1.人源化BCMA VH和VL以及scFv序列
BCMA scFv衍生自杂交瘤克隆4C8A(WO2019/195017)。测定小鼠克隆4C8A的重链可变区和轻链可变区的序列，并用于构建人源化scFv。
人源化BCMA(PMC306)scFv的结构是：VH-连接子-VL。连接子是G4Sx3。
粗体突出了人源化BCMA PMC306 ScFv克隆：VH的核苷酸序列；下划线突出了VL的核苷酸序列；在它们之间(斜体)是编码连接子的核苷酸序列。
PMC306 VH氨基酸序列：(SEQ ID NO:3)
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYIIPYNDATKYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARYNYDGYFDVWGQGTLVTVSS
连接子氨基酸序列(SEQ ID NO:4)
GGGGSGGGGSGGGGS
PMC306 VL氨基酸序列：(SEQ ID NO:5)
EIVLTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIYYASQSITGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFPPTFGGGTKVEIK
人源化BCMA(PMC306)scFv蛋白：(SEQ ID NO:6)
Q V Q L V Q S G A E V K K P G S S V K V S C K A S G Y T F T S Y V M HW V R Q A P G Q G L E W M G Y I I P Y N D A T K Y N E K F K G R V T I T A D KS T S T A Y M E L S S L R S E D T A V Y Y C A R Y N Y D G Y F D V W G Q G T LV T V S S G G G G S G G G G S G G G G S E I V L T Q S P A T L S L S P G E R A T L S C R A S Q S I S D Y L H W Y Q Q K P G Q A P R L L I Y Y A S Q S I T G I P A R F S G S G S G T D F T L T I S S L E P E D F A V Y Y C Q N G H S F P P T F G G G T K V E I K
实施例2.人源化BCMA-CAR序列
图3显示了人源化(PMC306)BCMA-CAR构建体的方案。使用具有EF1a启动子的慢病毒载体克隆人源化scFv CAR序列。
BCMA-CAR结构包括人CD8信号肽、人源化BCMA scFv(VH-连接子-VL)、CD8铰链区、CD28跨膜、激活结构域CD3ζ(图3)。
下面显示了CD8信号-BCMAscFv(VH-连接子-VL)-CD8铰链区-CD28 TM-CD28-CD3ζ的核苷酸序列和一些氨基酸序列。
<CD8前导序列>
核苷酸
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG(SEQID NO:7)
氨基酸
MALPVTALLLPLALLLHAARP(SEQ ID NO:8)
<Nhe I位点>
gctagc
<人源化BCMA，PMC 306scFv>
VH-连接子-VL，参见实施例1的核酸序列和氨基酸序列。
<XhoI限制位点>
CTCGAG
<CD8铰链区>
核苷酸
AAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGAT(SEQID NO:9)
氨基酸
KPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASD(SEQ ID NO:10)
<aagccc>
<CD28跨膜>
核苷酸
TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG(SEQ ID NO:11)
氨基酸
FWVLVVVGGVLACYSLLVTVAFIIFWV(SEQ ID NO:12)
<CD28共刺激>
核苷酸
AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(SEQ ID NO:13)
氨基酸
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:14)
<CD3ζ>终止密码子加下划线
核苷酸
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAAtag(SEQ ID NO:15)
氨基酸
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:16)
<EcoRI限制位点>
gaattc
人源化BCMA-CAR蛋白的翻译的氨基酸序列(SEQ ID NO:17)
M A L P V T A L L L P L A L L L H A A R P A S Q V Q L V Q S G A E V KK P G S S V K V S C K A S G Y T F T S Y V M H W V R Q A P G Q G L E W M G Y II P Y N D A T K Y N E K F K G R V T I T A D K S T S T A Y M E L S S L R S E DT A V Y Y C A R Y N Y D G Y F D V W G Q G T L V T V S S G G G G S G G G G S GG G G S E I V L T Q S P A T L S L S P G E R A T L S C R A S Q S I S D Y L H WY Q Q K P G Q A P R L L I Y Y A S Q S I T G I P A R F S G S G S G T D F T L TI S S L E P E D F A V Y Y C Q N G H S F P P T F G G G T K V E I K L E K P T TT P A P R P P T P A P T I A S Q P L S L R P E A S R P A A G G A V H T R G L DF A S D K P F W V L V V V G G V L A C Y S L L V T V A F I I F W V R S K R S RL L H S D Y MN M T P R R P G P T R K H Y Q P Y A P P R D F A A Y R S R V K FS R S A D A P A Y Q Q G Q N Q L Y N E L N L G R R E E Y D V L D K R R G R D PE M G G K P Q R R K N P Q E G L Y N E L Q K D K M A E A Y S E I G M K G E R RR G K G H D G L Y Q G L S T A T K D T Y D A L H M Q A L P P R(SEQ ID NO:17)
实施例3.CAR慢病毒的产生
如[7]中所述的，使用293T细胞通过标准程序产生慢病毒。本发明的发明人产生了克隆到慢病毒载体的Xba I和Eco R I位点中的慢病毒载体内部的人源化BCMA-ScFv-CAR构建体。pCD510-FMC63-28z慢病毒CAR构建体在Xba I克隆位点和Eco RI克隆位点之间包含人源化BCMA ScFv-CD28-CD3ζ***片段。
在293T细胞中产生慢病毒，并且通过RT-PCR确定滴度。然后使用相等剂量的慢病毒转导T细胞。
实施例4.从全血中分离外周血单个核细胞(PBMC)
在10mL肝素真空采血管(Becton Dickinson)中收集来自个体或混合供体(取决于所需的血液量)的全血(斯坦福医院血液中心，加利福尼亚州斯坦福(Stanford HospitalBlood Center,Stanford,CA))。在50ml离心管中，将大约10ml的全抗凝血液与无菌磷酸盐缓冲盐水(PBS)缓冲液(PBS,pH7.4，无Ca+2和Mg+2)混合，总体积为20ml。将血液/PBS(20ml)轻轻放在锥形离心管中的15mL聚蔗糖-Paque PLUS(Ficoll-Paque PLUS，GE Healthcare)顶部，然后将样品在室温下以400×g离心30-40min。除去在稀释的血浆/聚蔗糖界面处含有外周血单个核细胞(PBMC)的细胞层，洗涤，并在室温下以200×g离心10min。用血细胞计数器对细胞计数。用含有5％AB血清和1.25μg/mL两性霉素B(Gemini Bioproducts，Woodland，CA)、100U/mL青霉素和100μg/mL链霉素的CAR-T培养基(AIM V-AlbuMAX(BSA)(LifeTechnologies)洗涤PBMC一次，并用于实验或在-80℃冷冻。
实施例5.从PBMC激活T细胞
将分离的PBMC细胞重悬于含有300U/mL huIL2(来自1000×储备液；Invitrogen)的CAR-T培养基中，并以1:1的珠与细胞比与CD3-CD28珠混合。在进行病毒转导之前，将细胞在CO2存在下于37℃下孵育24小时。
实施例6.T细胞的转导和扩增
在PBMC激活后，将细胞在37℃、5％CO2下孵育24小时。向每个孔中加入1×106个细胞、5×106个慢病毒和2μL/mL的Transplus培养基(Alstem，里士满，CA)(最终稀释度为1:500)。在重复加入病毒之前，将细胞再孵育24小时。然后使细胞在连续存在含有IL-2的300U/ML IL-2新鲜培养基的情况下生长12-14天的时期(总孵育时间取决于所需的CAR-T细胞的最终数量)。每2-3天分析细胞浓度，此时加入培养基以将细胞悬浮液稀释至1×106个细胞/mL。
实施例7.人源化BCMA-CAR-T细胞表达BCMAscFv
我们设计了具有实施例2中所示的人源化BCMA-CAR构建体的人源化BCMA-CAR-T细胞。我们使用具有无关ScFv的模拟scFv并产生模拟CAR-T细胞作为阴性对照。在慢病毒人源化BCMA-CAR转导入T细胞后检测人源化BCMA-CAR阳性细胞(图4)。
实施例8.人源化BCMA-CAR-T细胞杀伤CHO-BCMA细胞，但不杀伤CHO细胞。
我们将人源化BCMA-CAR-T细胞与靶标CHO-BCMA靶细胞以及CHO(BCMA阴性)对照细胞一起孵育。人源化BCMA-CAR-T细胞特异性地杀伤CHO-BCMA细胞(图5A)，而不杀伤CHO细胞(图5B)。结果证明人源化BCMA-CAR-T细胞对靶向BCMA抗原和杀伤BCMA阳性细胞具有高度特异性。
实施例9.人源化CAR-T细胞针对靶标CHO-BCMA细胞显著分泌IFN-γ，但是针对CHO细胞不分泌IFN-γ。
我们在人源化BCMA-CAR-T细胞和靶标CHO-BCMA以及亲本CHO细胞共孵育后收集上清液并进行IFN-γ测定。BCMA-CAR-T细胞与CHO-BCMA细胞一起分泌IFN-γ，但与阴性对照CHO细胞一起不分泌IFN-γ(图6)。结果证实了人源化BCMA-CAR-T细胞的特异性。
实施例10.人源化CAR-T细胞针对BCMA阳性RPMI8226多发性骨髓瘤细胞分泌高水平的IFN-γ，但是针对BCMA阴性K562白血病细胞则不分泌高水平的IFN-γ。
我们将BCMA-CAR-T细胞与多发性骨髓瘤癌细胞RPMI8266以及BCMA阴性K562细胞(慢性骨髓性白血病细胞)一起孵育，并根据制造商的方案，使用来自Fisher的试剂盒对IFN-γ进行ELISA。人源化BCMA-CAR-T细胞针对BCMA阳性多发性骨髓瘤癌细胞分泌高水平的IFN-γ，但是针对BCMA阴性K562细胞则不分泌高水平的IFN-γ(图7)。BCMA-CAR-T细胞的IFN-γ的杀伤和分泌水平显著高于T细胞和模拟CAR-T细胞的IFN-γ的杀伤和分泌水平。这证实了人源化BCMA-CAR-T细胞针对血液学BCMA阳性细胞的特异性。
实施例11.人源化BCMA-CAR-T细胞在体内小鼠模型中显著降低了RPMI8226异种移植肿瘤的生长。
将多发性骨髓瘤RPMI8226细胞皮下注射到NSG小鼠(1×10^7个细胞/小鼠)中，然后通过静脉内注射人源化BCMA-CAR-T细胞两次(1×10^7个CAR-T细胞/小鼠)。人源化BCMA-CAR-T细胞显著降低小鼠中的RPMI8226肿瘤生长(图8A)。用人源化BCMA-CAR-T细胞处理的小鼠不引起小鼠体重降低，这表明CAR-T细胞对小鼠无毒性(图8B)。在研究期间没有观察到行为或视觉变化。
在研究结束时通过流式细胞仪分析外周血细胞与人BCMA蛋白以及特异性针对人T(CD4+/CD8+)细胞的抗体的结合。与CD4 mAb结合的细胞的百分比显示在图8C的左图上，也与BCMA蛋白结合的那些人T细胞的百分比显示在图8C的右图上。结果表明，用BCMA重组蛋白通过FACS在小鼠血液中检测到人源化BCMA-CAR-T细胞，但没有检测到模拟Car-T细胞。
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Claims (30)
1.抗BCMA抗体，其包含：
重链可变区(VH)，所述重链可变区(VH)由SEQ ID NO：3的氨基酸序列组成；和
轻链可变区(VL)，所述轻链可变区(VL)由SEQ ID NO：5的氨基酸序列组成。
2.抗BCMA单链可变片段(scFv)，其包含：
重链可变区(VH)，所述重链可变区(VH)由SEQ ID NO：3的氨基酸序列组成；和
轻链可变区(VL)，所述轻链可变区(VL)由SEQ ID NO：5的氨基酸序列组成。
3.如权利要求2所述的scFv，其还包含VH和VL之间的连接子。
4.如权利要求3所述的scFv，其中所述连接子由SEQ ID NO: 4组成。
5. 如权利要求3所述的scFv，其由SEQ ID NO: 6的氨基酸序列组成。
6.抗BCMA嵌合抗原受体(CAR)，其包含：
抗BCMA单链可变片段(scFv);
跨膜结构域；
共刺激结构域;和
激活结构域；
所述抗BCMA单链可变片段(scFv)包含：
重链可变区(VH)，所述重链可变区(VH)由SEQ ID NO：3的氨基酸序列组成；和
轻链可变区(VL)，所述轻链可变区(VL)由SEQ ID NO：5的氨基酸序列组成。
7.如权利要求6所述的CAR，其中所述跨膜结构域是选自以下的跨膜结构域：T细胞受体α链、T细胞受体β链、CD3ζ链、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、ICOS、CD154和GITR。
8.如权利要求7所述的CAR，其中所述跨膜结构包含CD8的跨膜结构域。
9.如权利要求6所述的CAR，其中所述共刺激结构域是选自以下的共刺激结构域：CD28、4-1BB、GITR、ICOS-1、CD27、OX-40和DAP10。
10.如权利要求9所述的CAR，其中所述共刺激结构域包含4-1BB共刺激结构域。
11.如权利要求6所述的CAR，其中所述激活结构域包含CD3ζ激活结构域。
12.如权利要求6所述的CAR，其中所述跨膜结构域包含CD8的跨膜结构域，所述共刺激结构域包含4-1BB共刺激结构域，并且所述激活结构域包括CD3ζ激活结构域。
13. 如权利要求6所述的CAR，其由SEQ ID NO: 17的氨基酸序列组成。
14.具有5’端和3’端的核酸，其编码权利要求6所述的CAR。
15.如权利要求14所述的核酸，其在5’端还包含前导序列。
16. 如权利要求15所述的核酸，其中所述前导序列由SEQ ID NO: 7的核酸序列组成。
17.如权利要求14所述的核酸，其在5’端还包含启动子。
18.如权利要求17所述的核酸，其中所述启动子包含EF1a启动子。
19.载体，其包含权利要求14所述的核酸。
20.如权利要求19所述的载体，其中所述载体是病毒载体。
21. T细胞或自然杀伤细胞，其经修饰以表达权利要求6所述的CAR。
22. CAR-T细胞，其包含权利要求6所述的CAR。
23. CAR-T细胞，其包含权利要求13所述的CAR。
24. CAR-T细胞在制备用于杀伤多发性骨髓瘤癌细胞的药物中的用途，其中所述CAR-T细胞包含抗BCMA嵌合抗原受体(CAR)，其包含：
抗BCMA单链可变片段(scFv)；
跨膜结构域；
共刺激结构域；和
激活结构域；
所述抗BCMA单链可变片段(scFv)包含：
重链可变区(VH)，所述重链可变区(VH)由SEQ ID NO：3的氨基酸序列组成；和
轻链可变区(VL)，所述轻链可变区(VL)由SEQ ID NO：5的氨基酸序列组成。
25.如权利要求24所述的用途，其中所述多发性骨髓瘤癌细胞包括人细胞。
26.如权利要求24所述的用途，其中所述药物是肿瘤内施用的。
27. 制备抗BCMA CAR-T细胞的方法，其包括：
将编码抗BCMA嵌合抗原受体(CAR)的核酸引入T细胞，所述抗BCMA嵌合抗原受体(CAR)包含：
抗BCMA单链可变片段(scFv)；
跨膜结构域；
共刺激结构域；和
激活结构域；
所述抗BCMA单链可变片段(scFv)包含：
重链可变区(VH)，所述重链可变区(VH)由SEQ ID NO：3的氨基酸序列组成；和
轻链可变区(VL)，所述轻链可变区(VL)由SEQ ID NO：5的氨基酸序列组成。
28.如权利要求27所述的方法，其中将所述核酸体外引入所述T细胞中。
29.如权利要求27所述的方法，其中通过病毒载体将所述核酸引入所述T细胞中。
30.如权利要求29所述的方法，其中所述病毒载体选自：逆转录病毒载体、腺病毒载体、腺相关病毒(AAV)载体、猿猴病毒载体，痘苗病毒载体、仙台病毒载体、爱泼斯坦-巴尔病毒(EBV)载体和HSV载体。
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