CN113045657B - 一种人源化抗人bcma单克隆抗体及其car-t细胞 - Google Patents
一种人源化抗人bcma单克隆抗体及其car-t细胞 Download PDFInfo
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Abstract
本发明涉及人源化抗BCMA抗原单克隆抗体及其单链可变片段(scFv)，包含具有SEQ ID NO：5的氨基酸序列的VH和具有SEQ ID NO：6的氨基酸序列的VL。本发明还涉及一种BCMA嵌合抗原受体融合蛋白，其从N端至C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。在体外和体内，这种人源化的抗BCMA scFv片段可用于构建本发明的CAR‑T细胞——其可针对BCMA抗原阳性靶细胞特异性分泌高水平细胞因子IFN‑γ，并对BCMA抗原阳性靶细胞具有特异性杀伤作用。该人源化抗BCMA抗原单克隆抗体、其单链可变片段(scFv)及其CAR融合蛋白以及CAR‑T可广泛应用于BCMA抗原阳性的肿瘤免疫治疗。
Description
技术领域
本发明涉及一种能特异性抑制人多发性骨髓瘤及其他BCMA阳性肿瘤细胞生长的新型人源化抗B细胞成熟抗原(BCMA)的单克隆抗体(PMC308)，以及表达含有该单克隆抗体序列的嵌合抗原受体的T免疫细胞(CAR-T细胞)，它们在肿瘤过继免疫治疗中起作用。
背景技术
细胞与免疫疗法正在成为治疗癌症的一种具有广阔发展前景的方法。T细胞或T淋巴细胞是我们人体免疫系统细胞免疫的重要组成部分，它通过不断寻找和识别外来、异己抗原，从体细胞中区分正常细胞与异常细胞(癌细胞或受病毒感染细胞)杀灭与清除异常细胞。采用表达嵌合抗原受体(Chimeric Antigen Receptor)构建体对T细胞进行基因修饰是设计肿瘤特异性T细胞的最常见方法。将靶向肿瘤相关抗原(TAA)的CAR-T细胞注入患者体内(称为过继细胞转移或ACT)是一种有效的免疫治疗方式[1,2]。与化疗或抗体疗法相比，CAR-T技术的优势在于重新编程的工程T细胞可以在患者体内增殖、持续存活并杀伤靶细胞(“活体药物”)[1,3,4]。
CAR通常由源于单克隆抗体N端的单链可变片段(scFv)、铰链区、跨膜区和许多细胞内共刺激结构域如(i)CD28，(ii)CD137(4-1BB)、CD27或其他共刺激结构域，以及与其串联的CD3zeta活化结构域组成(图1)[1,2]。在图1中，左图为第一代的CAR结构(无共刺激结构域)；中图为第二代的CAR结构(一个共刺激结构域，CD28或4-BB)；并且右图为第三代的CAR结构(两个或多个共刺激结构域)。该图来自Golubovskaya,Wu,Cancers,2016[6]。CAR经历了从第一代(无共刺激结构域)到第二代(有一个共刺激结构域)，再到第三代(有多个共刺激结构域)的发展历程。携带有两个共刺激结构域(即第三代CAR)及其转染表达的CAR-T细胞的应用使CAR-T的肿瘤杀伤活性增强，CAR-T细胞在体内的持久性延长，并且抗肿瘤疗效提高。
BCMA
B细胞成熟抗原(B cell maturation antigen；BCMA)是一种细胞表面受体，也称为CD269和肿瘤坏死因子受体超家族成员17(TNFRSF17)，它由TNFRSF17基因编码。该受体主要在成熟的B淋巴细胞中表达，在大多数情况下在多发性骨髓瘤(MM)中过表达[3]。当前针对MM中BCMA的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疗法等[3,4]。
BCMA结构和信号通路
人BCMA蛋白由184个氨基酸组成：第1-54位氨基酸为胞外结构域；第55-77位为跨膜结构域；第78-184位为胞质结构域。BCMA的氨基酸序列如图2所示，其中带下划线的是细胞外结构域。BCMA与BAFF受体、跨膜激活剂、亲环素配体相互作用剂和钙调节剂(TACI)等其他受体类似，缺乏信号肽[4]。这些受体在B细胞成熟和分化为浆细胞中起主要作用。它们的配体包括BAFF和APRIL，两者均在MM患者中表达增加[4]。单克隆抗体是通过阻断受体-配体相互作用清除MM细胞，而CAR-T细胞疗法则是通过结合BCMA进而杀死MM细胞。BCMA还能与TRAF1,2,3,5和6相互作用。本发明是基于靶向MM细胞表面BCMA的人源化BCMA-CAR-T细胞。人源化BCMA-CAR-T细胞在scFV中具有人源化抗BCMA scFv序列，使它比小鼠ScFv对人的免疫原性低，更有应用优势。
附图说明
图1.CAR的结构
图2.BCMA蛋白的氨基酸序列。
图3.人源化BCMA CAR构建体的结构。
图4.用荧光标记的重组BCMA蛋白通过FACS分析检测人源化的BCMA-CAR构建体。
图5.人源化的BCMA-CAR-T细胞杀死CHO-BCMA细胞但不杀死CHO细胞。
图6.人源化的BCMA-CAR-T细胞经CHO-BCMA阳性细胞刺激后特异性分泌高水平的IFN-γ。
图7.人源化的BCMA-CAR-T细胞经多发性骨髓瘤细胞刺激后特异性分泌高水平的IFN-γ，而经BCMA阴性的K562对照细胞刺激前后无显著差异；人源化的BCMA-CAR-T细胞在多发性骨髓瘤细胞系中检测到BCMA，但在白血病或其他癌细胞系中未检测到。
图8A.人源化的BCMA-CAR-T细胞显著抑制RPMI8226异种移植肿瘤的生长；图8B.人源化的BCMA-CAR-T细胞没有降低小鼠体重。
具体实施方式
定义
如本文所用，“嵌合抗原受体(CAR)”是指融合蛋白，其由能与抗原结合的胞外结构域、源于多肽的跨膜结构域(该多肽不同于衍生胞外结构域多肽)、以及至少一个细胞内的结构域组成。“嵌合抗原受体(CAR)”有时被称为“嵌合受体”、“T体”或“嵌合免疫受体(CIR)”，“能够与抗原结合的胞外结构域”是指能与某一抗原结合的任何寡肽或多肽。“细胞内结构域”是指已知功能为传递信号以引起细胞内生物过程的激活或抑制的任何寡肽或多肽。
如本文所用，“人源化抗体”是来自非人物种的抗体，其蛋白质序列已被修饰以增加其与人天然产生的抗体变体的相似性。
如本文所用，“结构域”是指多肽中独立于其他区域折叠成特定结构的一个区域。
如本文所用，“单链可变片段(scFv)”是指由保留结合抗原能力的抗体衍生的单链多肽。scFv的示例包括通过重组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区通过间隔序列连接。用于工程化scFv的各种方法是本领域技术人员已知的。
如本文所用，“肿瘤抗原”是指具有抗原性的生物分子，其表达引起癌症。
发明人已经基于特异性靶向BCMA的新型人源化BCMA单克隆抗体scFv序列构建了CAR-T细胞。发明人构建了人源化的BCMA-CAR-T细胞，以靶向过度表达BCMA肿瘤抗原的癌细胞。本发明的人源化的BCMA-CAR-T细胞可特异性高水平分泌针对多发性骨髓瘤癌细胞的细胞因子，并杀死CHO-BCMA阳性的靶细胞，但对BCMA阴性的CHO细胞无作用。
本发明涉及分泌人源化抗人BCMA单克隆抗体的杂交细胞克隆及其分泌产生的单克隆抗体(4C8A4或4C8A10)，其分别包含具有SEQ ID NO：5的氨基酸序列的人源化VH序列和具有SEQ ID NO：6的氨基酸序列的人源化VL序列。在一个实施方式中，人源化抗人BCMA抗体是单链可变片段(scFv)。ScFv可以是VH-接头-VL或VL-接头-VH。
本发明还涉及嵌合抗原受体的融合蛋白，该融合蛋白从N末端到C末端包含以下部分：(i)靶向BCMA的单链可变片段(scFv)(本发明)；(ii)跨膜结构域；(iii)至少一个共刺激结构域和(iv)激活结构域。
在一个具体实施方式中，CAR的共刺激结构域选自由CD28、4-1BB、GITR、ICOS-1、CD27、OX-40和DAP10组成的组。优选的共刺激结构域为CD28。
优选的激活结构域为CD3zeta(CD3Z或CD3ζ)。
跨膜结构域可以来源于天然多肽，或可以是人工设计的。来源于天然多肽的跨膜结构域可以获自任何膜结合或跨膜蛋白。例如，可以使用T细胞受体α或β链、CD3zeta链、CD28、CD3ε.、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD134、CD137、ICOS、CD154或GITR的跨膜结构域。人工设计的跨膜结构域为主要包含如亮氨酸和缬氨酸的疏水残基的多肽。优选在合成跨膜结构域的每个末端发现苯丙氨酸色氨酸和缬氨酸的三联体。任选地，短寡肽接头和多肽接头，例如具有2至10个氨基酸长度的接头可以设置在跨膜结构域和细胞内结构域之间。在一个实施方式中，可以使用具有甘氨酸-丝氨酸连续序列的接头序列。
本发明提供了编码BCMA-CAR的核酸。编码CAR的核酸可以通过传统方法由特定CAR的氨基酸序列制备。编码氨基酸序列的碱基序列可以从前述的NCBI RefSeq IDs或GenBank关于各结构域的氨基酸序列的登录号获得，并且本发明的核酸序列可以使用标准的分子生物学和/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通过使用聚合酶链反应(PCR)将从cDNA库获得的DNA片段结合来制备。
编码本发明的CAR的核酸可以插入载体中，并且该载体可以引入细胞中。例如，可以使用病毒载体，如逆转录病毒载体(包括核病毒载体、慢病毒载体、假型载体)、腺病毒载体、腺相关病毒(AAV)载体、猿猴病毒载体、牛痘病毒载体、仙台病毒载体、Epstein-Barr病毒(EBV)载体和HSV载体。优选使用缺乏复制能力的病毒载体，以便在感染细胞中不进行自我复制。
例如，当使用逆转录病毒载体时，可以基于载体所具有的LTR序列和包装信号序列选择合适的包装细胞，以使用该包装细胞制备逆转录病毒颗粒。包装细胞的示例包括PG13(ATCC CRL-10686)、PA317(ATCC CRL-9078)、GP+E-86和GP+envAm-12和Psi-Crip。还可以使用具有高转染效率的293细胞核293T细胞制备逆转录病毒颗粒。基于逆转录病毒生产的许多种类的逆转录病毒载体和可以用于包装逆转录病毒载体的包装细胞广泛地市售于许多公司。
CAR-T细胞经由CAR结合特定的抗原，从而使信号转移到细胞中，并因此激活细胞。表达CAR的细胞的激活取决于宿主细胞的种类和CAR的细胞内结构域而改变，并且可以基于例如细胞因子的释放、细胞增殖速度的改善或细胞表面分子的变化等作为指标来确认。例如，来自的激活细胞的细胞毒素细胞因子(肿瘤坏死因子、淋巴毒素等)的释放导致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他免疫细胞，例如，B细胞、树突细胞、NK细胞和巨噬细胞。
表达CAR的细胞可以用作用于癌症的治疗剂。该治疗剂包含表达CAR的细胞作为活性成分，并且其还可以包含合适的赋形剂。
本发明人已经构建了针对多发性骨髓瘤细胞(MM)的人源化的BCMA-ScFv-CD28-CD3-CAR-T(BCMA-CAR-T)细胞。BCMA-CAR-T细胞分泌高水平的细胞因子，通过LDH细胞毒性实验测定以及CHO-BCMA细胞毒性实验测定呈阳性，但是对BCMA阴性CHO细胞毒性实验测定没有显著性，这表明CAR-T细胞具有针对靶向癌细胞的特异性杀伤活性，并以该杀伤活性清除肿瘤或病毒抗原。
本发明的人源化BCMA-scFv的优点包括：由于人源化BCMA scFv而对人的免疫原性较低。因此，本发明的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可应用于多种免疫治疗：包括偶联有毒素/药物的抗体，单克隆治疗性抗体，BCMA抗体人源化和CAR-T细胞免疫治疗。
使用本发明中人源化BCMA scFv的人源化BCMA-CAR-T细胞可以有效地用于靶向BCMA阳性癌细胞系中的BCMA抗原。
该人源化BCMA-CAR-T细胞可与不同的化学疗法联合使用：检查点抑制剂、靶向疗法、小分子抑制剂、抗体等。
该人源化的BCMA-CAR-T细胞在临床上可用于治疗由BCMA阳性癌细胞导致的肿瘤。
对共刺激结构域如CD28、4-1BB及其他等的修饰可用于提高其功效。Tag-标记偶联的人源化BCMA scFv可用于CAR的制备。
该人源化BCMA-CAR-T细胞可与不同的安全开关结合应用：如t-EGFR，RQR(利妥昔单抗-CD34-利妥昔单抗)等。
第三代CAR-T或其他共激活信号结构域可同时用于CAR结构内的同一个人源化BCMA-scFv。
该人源化BCMA CAR可以与靶向其他不同肿瘤抗原或肿瘤微环境，例如VEGFR-1-3、PDL-1的CAR联合使用，也可与具有BCMA和CD3的双特异性抗体或其他抗原等联合用于疾病治疗。
该人源化BCMA-CAR-T细胞可用于针对对化疗最具耐药性的肿瘤干细胞，以及由其形成的侵袭性肿瘤。
下列实施例为对本发明的进一步说明。这些实施例仅旨在说明本发明，而不应理解为是限制性的。
实施例
本发明人在慢病毒载体内，经克隆至慢病毒载体的Xba I和Eco R I限制性酶切位点，构建了人源化的BCMA-ScFv-CAR结构体。在pCD510-FMC63-28z慢病毒CAR构建体中，包含人源化BCMA ScFv-CD28-CD3zeta片段——其插入位点在Xba I和Eco RI克隆位点之间。
慢病毒载体是通过在293T细胞中扩增生产，并通过RT-PCR方法确定其效价。然后使用等剂量的慢病毒进行T细胞的转导。
实施例1、人源化的抗BCMA抗体重链可变区VH和轻链可变区VL以及单链可变片段scFv序列
通过对BCMA阳性的杂交瘤克隆株4C8A4和4C8A10进行测序，获得BCMA scFv基因序列。人源化BCMA(PMC308)scFv的结构为：VH-接头-VL。接头为G4Sx3。
人源化的BCMA VH和VL以及scFv序列如下所示。
序列中粗体字突出显示了人源化的BCMA PMC308scFv克隆的核苷酸序列：VH(SEQID NO：1)；下划线突出显示VL的核苷酸序列(SEQ ID NO：2)；中间(斜体)是编码3xG4S接头3x(GGGGS)的核苷酸序列(SEQ ID NO：3)。
人源化BCMA(PMC308)scFv蛋白序列：(SEQ ID NO：4)
在蛋白序列中，粗体突出显示了VH的氨基酸序列(SEQ ID NO：5)；下划线突出显示了VL的氨基酸序列(SEQ ID NO：6)；中间(斜体)是3xG4S接头序列的氨基酸序列(SEQ IDNO：7)。
实施例2.人源化的BCMA-CAR序列
人源化(PMC308)BCMA-CAR构建方案如图3所示，其中使用第二代BCMA-CAR(PMC308)。在整个申请中，我们将其称为人源化BCMA。带有EF1a启动子的慢病毒载体被用于克隆人源化scFv CAR序列。以下核苷酸序列显示了本发明人源化BCMA CAR序列，包括CD8-人源化BCMA ScFv-CD8-TM28-CD28-CD3ζ的核苷酸序列。该CAR结构包括人CD8信号肽，人源化BCMA scFv(VH-接头3x(G4S)-VL)，CD8铰链结构域，CD28跨膜结构域、和CD3zeta激活结构域(图3)。
CD8前导序列--BCMA scFv(VH-接头-VL)-CD8铰链结构域-CD28TM-CD28-CD3-zeta：
<CD8前导序列>
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGgctagc
<人源化BCMA，4C8A4/4C8A10scFv克隆>
CAGGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAACCGGGCGCGAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCTATACCTTTACCAGCTATGTGATGCATTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATGGGCTATATTATTCCGTATAACGATGCGACCAAATATAACGAAAAATTTAAAGGCCGCGTGACCATGACCCGCGATACCAGCACCAGCACCGTGTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACCGCGGTGTATTATTGCGCGCGCTATAACTATGATGGCTATTTTGATGTGTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGATGTGGTGATGACCCAGAGCCCGGCGTTTCTGAGCGTGACCCCGGGCGAAAAAGTGACCATTACCTGCCGCGCGAGCCAGAGCATTAGCGATTATCTGCATTGGTATCAGCAGAAACCGGATCAGGCGCCGAAACTGCTGATTAAATATGCGAGCCAGAGCATTAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCTTTACCATTAGCAGCCTGGAAGCGGAAGATGCGGCGACCTATTATTGCCAGAACGGCCATAGCTTTCCGCCGACCTTTGGCGGCGGCACCAAAGTGGAAATTAAA
<XhoI限制位点>
CTCGAG
<CD8铰链>
AAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGATaagccc
<CD28TM/激活域>
TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC
<CD3zeta>
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAAtag
<EcoRI限制位点>
gaattc
人源化BCMA-CAR蛋白的氨基酸序列(构建体结构见图3)
(SEQ ID NO：14)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYIIPYNDATKYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYNYDGYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPAFLSVTPGEKVTITCRASQSISDYLHWYQQKPDQAPKLLIKYASQSISGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNGHSFPPTFGGGTKVEIKLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
实施例3.人源化的BCMA-CAR-T细胞能大量杀死骨髓瘤细胞并针对BCMA阳性癌细胞特异性分泌高水平的IFN-γ。
如图3所示，本发明以人源化BCMA-CAR构建并生成人源化BCMA-CAR-T细胞。同时以与BCMA scFv无关的scFv构建并生成模拟的CAR-T细胞(Mock CAR-T细胞)作为阴性对照。通过FACS检测发现，人源化BCMA-CAR-T细胞表达BCMA scFv(图4)，其中将慢病毒人源化BCMACAR导入T细胞后，检测到人源化BCMA-CAR阳性细胞。
实施例4.人源化的BCMA-CAR-T细胞杀死了CHO-BCMA细胞，但没有影响CHO细胞
我们将人源化BCMA-CAR-T细胞与表达BCMA抗原的CHO-BCMA靶细胞以及CHO(BCMA阴性)对照细胞一起孵育。人源化的BCMA-CAR-T细胞特异性杀死CHO-BCMA细胞(图5，上图)，而对CHO细胞无杀伤作用(图5，下图)。这证明了人源化的BCMA-CAR-T细胞对靶向BCMA抗原和杀死BCMA阳性细胞具有高度特异性。
在图5中，以XCelligence细胞毒性实时试验检测人源化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。上图：CHO-BCMA靶细胞。上图右侧从上到下：模拟对照CAR-T细胞、空白对照细胞(不含CAR)和效应细胞之人源化CAR-T细胞。下图为CHO靶细胞。下图右侧从上到下分别为：模拟CAR-T细胞，人源化BCMA CAR-T细胞，和空白细胞。
实施例5.人源化的CAR-T细胞能显著分泌针对靶标CHO-BCMA细胞的IFN-γ
将人源化BCMA-CAR-T细胞和靶标CHO-BCMA细胞共同孵育后，我们收集了上清液，并进行了IFN-γ分析。BCMA-CAR-T细胞与CHO-BCMA细胞共孵育后分泌高水平的IFN-γ(图6)，其中与T细胞和模拟CAR-T对照细胞相比，CHO-BCMA细胞中的IFN-γ表达显著上调(p<0.05)。对照CHO细胞没有显示出IFN-γ高水平分泌(数据未显示)。这证实了人源化的BCMA-CAR-T细胞的特异性和细胞杀伤毒性。
实施例6.人源化的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，而针对BCMA阴性白血病K562细胞很少分泌IFN-γ
我们将BCMA-CAR-T细胞与多发性骨髓瘤细胞(RPMI8226细胞)和K562细胞分别进行孵育，并使用Fisher的试剂盒根据试验方法进行ELISA检测。人源化BCMA-CAR-T细胞针对BCMA阳性多发性骨髓瘤癌细胞分泌的IFN-γ较K562细胞显著升高(图7)。在图7中，人源化的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，而针对BCMA阴性白血病K562细胞分泌较少IFN-γ。与T细胞和模拟-CAR-T细胞组，以及与K562细胞组中分泌的IFN-γ相比，人源化的CAR-T细胞与多发性骨髓瘤细胞共孵育后IFN-γ的表达显著升高，p<0.05。IFN-γ的杀伤和分泌水平明显高于对照组T细胞和模拟的CAR-T细胞。这证实了人源化BCMA-CAR-T细胞对血液学BCMA阳性细胞具有特异性。我们还将人源化BCMA与小鼠BCMA-CAR细胞以及其他来源的人源化BCMA-CAR-T细胞(用于临床试验)进行了比较，发现我们的人源化BCMA-CAR-T细胞与阳性对照CAR-T(小鼠和其他人源化BCMA-CAR-T细胞)的IFN-γ分泌相似或相当，甚至更高(数据未显示)。这支持了人源化BCMA-CAR-T细胞在未来临床应用中的高潜力。
实施例7.人源化的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小鼠中(1×10^7个细胞/小鼠)，然后通过静脉输注两次人源化的BCMA-CAR-T细胞(1×10^7个CAR-T细胞/小鼠)。人源化的BCMA-CAR-T细胞显著降低了小鼠中RPMI8226肿瘤生长(图8A)。用人源化BCMA-CAR-T细胞处理的小鼠并未引起小鼠体重降低，这表明CAR-T细胞对小鼠无毒(图8B)。此外，在研究过程中未观察到行为或视觉变化。同时通过FACS检测到小鼠血液中针对BCMA重组蛋白的人源化BCMA-CAR-T的含量(数据未显示)。
在图8中，人源化的BCMA-CAR-T细胞显著降低了RPMI8226异种移植肿瘤的生长。图8A.人源化BCMA-CAR-T细胞显著降低RPMI8226肿瘤体积。在第7天和第20天，以1×10^7个细胞/小鼠的剂量分别注射CAR-T细胞。条形图显示肿瘤平均体积+/-标准误差。*p<0.05，BCMA与模拟对照相比。图8B.人源化BCMA-CAR-T细胞并未降低小鼠体重，条形图显示小鼠平均体重+/-标准偏差。
参考文献
1.Maus,M.V.,Haas,A.R.,Beatty,G.L.,Albelda,S.M.,Levine,B.L.,Liu,X.,Zhao,Y.,Kalos,M.,和June,C.H.(2013).T cells expressing chimeric antigenreceptors can cause anaphylaxis in humans.Cancer Immunol Res 1,26-31.
2.Maus,M.V.,Grupp,S.A.,Porter,D.L.,和June,C.H.(2014).Antibody-modified T cells:CARs take the front seat for hematologic malignancies.Blood123,2625-2635.
3.Ali,S.A.,Shi,V.,Maric,I.,Wang,M.,Stroncek,D.F.,Rose,J.J.,Brudno,J.N.,Stetler-Stevenson,M.,Feldman,S.A.,Hansen,B.G.,等人(2016).T cellsexpressing an anti-B-cell maturation antigen chimeric antigen receptor causeremissions of multiple myeloma.Blood 128,1688-1700.
4.Tai,Y.T.,和Anderson,K.C.(2015).Targeting B-cell maturation antigenin multiple myeloma.Immunotherapy.
5.Boeye,A.(1986).Clonal isolation of hybridomas by manual single-cellisolation.Methods Enzymol 121,332-340.
6.Golubovskaya,V.,Wu,L(2016)Cancers,Mar 15；8(3).
Claims (12)
1.一种人源化抗人BCMA抗体，其包含由SEQ ID NO：5的氨基酸序列表示的VH和由SEQ IDNO：6的氨基酸序列表示的VL，
2.一种单链可变片段(scFv)，其包含由SEQ ID NO：5的氨基酸序列表示的VH和由SEQ IDNO：6的氨基酸序列表示的VL，
3.根据权利要求2所述的scFv，其还包含位于VH与VL之间的接头。
4.根据权利要求3所述的scFv，其具有SEQ ID NO：4的氨基酸序列，
5.一种嵌合抗原受体(CAR)融合蛋白，其从N端至C端包含：
(i)根据权利要求2-4中任一项所述的scFv；
(ii)跨膜结构域；
(iii)至少一个共刺激结构域，以及
(iv)激活结构域。
6.根据权利要求5所述的CAR融合蛋白，其具有SEQ ID NO：4的氨基酸序列，
7.根据权利要求5所述的CAR融合蛋白，其中，所述共刺激结构域为CD28。
8.根据权利要求5所述的CAR融合蛋白，其中，所述激活结构域为CD3ζ。
9.根据权利要求5所述的CAR融合蛋白，其具有SEQ ID NO：14的氨基酸序列，
10.一种核酸，其编码根据权利要求5所述的CAR融合蛋白。
11.一种T细胞，其修饰为表达根据权利要求5所述的CAR融合蛋白。
12.一种天然杀伤细胞，其修饰为表达根据权利要求5所述的CAR融合蛋白。
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