靶向B细胞成熟抗原的嵌合抗原受体T细胞的构建及其对肿瘤细胞的杀伤
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摘要
目的:探索通过嵌合抗原受体(chimeric antigen receptor,CAR)-T细胞靶向B细胞成熟抗原(B cell maturation antigen,BCMA)以治疗多发性骨髓瘤(multiple myeloma,MM)的方法。方法:构建基于鼠源BCMA scFv的CAR-BCMA分子,包装为慢病毒载体并感染健康人T细胞构建CAR-BCMA-T细胞;构建BCMA阳性细胞系A549-BCMA、A549-BCMAOFP和K562-BCMA作为靶细胞。将CAR-BCMA-T细胞与构建的靶细胞和人骨髓瘤细胞U266共孵育,CCK-8法和流式细胞术检测其对BCMA阳性肿瘤细胞的杀伤能力。构建MM患者来源CAR-BCMA-T细胞并检测其杀伤靶细胞A549-BCMA的能力,并采用ELISA和流式细胞术检测CAR-BCMA-T细胞IFN-γ的释放水平。结果:健康人来源的CAR-BCMA-T经过11 d培养扩增300倍,阳性率达到43%;成功构建BCMA阳性靶细胞。在5:1效靶比下,CAR-BCMA-T对A549-BCMA、K562-BCMA和U266细胞的杀伤率分别在80%、60%和80%左右,显著高于对BCMA阴性细胞的杀伤率,且杀伤力与靶细胞的BCMA表达强度相关。在效靶比20:1时,MM患者来源CAR-BCMA-T细胞对靶细胞A549-BCMA的杀伤率达到95%以上,并且大量分泌IFN-γ。结论:本研究成功构建了健康人及MM患者来源的靶向BCMA的CAR-T细胞,其能够有效特异杀伤BCMA阳性的肿瘤细胞。
Objective:To explore a novel chimeric antigen receptor(CAR)-T cell treatment to treat Multiple Myeloma(MM) via target B cell maturation antigen(BCMA). Methods:A CAR-BCMA molecular was constructed based on mouse originated BCMA scFv, and was packaged into lentiviral vector and transfected into T cells from healthy donors to construct CAR-BCMA-T cells. The BCMA positive cell lines A549-BCMA, A549-BCMAOFP and K562-BCMA were constructed as target cells. Then, the CAR-BCMA-T cells were co-incubated with the constructed target cells and human myeloma U266 cells, and the cytotoxic effects of CAR-BCMA-T cells were evaluated via CCK-8 and FACS. Finally, the CAR-BCMA-T cells originated from MM patients were constructed, and its cytotoxicity against A549-BCMA were examined; in addition, the IFN-γ release level in CAR-BCMA-T cells was evaluated by ELISA and FACS.Results: After 11 days' incubation, the CAR-BCMA-T cells originated from healthy donors amplified 300 times with a positive rate of43%. The BCMA positive target cell lines were constructed successfully. Under an effector : target ratio of 5:1, the killing rates of CARBCMA-T cells against A549-BCMA, K562-BCMA and U266 were about 80%, 60%, and 80%, respectively, which were significantly higher than those against BCMA negative cells; and the cytotoxicity was related to the BCMA expression level in target cells. What's more, at the effector : target ratio of 20:1, the CAR-BCMA-T cells originated from MM patients were demonstrated to exhibit a killing rate of more than 95% against A549-BCMA positive cells, and produced large amount of IFN-γ. Conclusion: CAR-BCMA-T cells originated from both healthy and MM donors were successfully constructed, and they can effectively and specifically kill BCMA positive tumor cells.
Objective:To explore a novel chimeric antigen receptor(CAR)-T cell treatment to treat Multiple Myeloma(MM) via target B cell maturation antigen(BCMA). Methods:A CAR-BCMA molecular was constructed based on mouse originated BCMA scFv, and was packaged into lentiviral vector and transfected into T cells from healthy donors to construct CAR-BCMA-T cells. The BCMA positive cell lines A549-BCMA, A549-BCMAOFP and K562-BCMA were constructed as target cells. Then, the CAR-BCMA-T cells were co-incubated with the constructed target cells and human myeloma U266 cells, and the cytotoxic effects of CAR-BCMA-T cells were evaluated via CCK-8 and FACS. Finally, the CAR-BCMA-T cells originated from MM patients were constructed, and its cytotoxicity against A549-BCMA were examined; in addition, the IFN-γ release level in CAR-BCMA-T cells was evaluated by ELISA and FACS.Results: After 11 days' incubation, the CAR-BCMA-T cells originated from healthy donors amplified 300 times with a positive rate of43%. The BCMA positive target cell lines were constructed successfully. Under an effector : target ratio of 5:1, the killing rates of CARBCMA-T cells against A549-BCMA, K562-BCMA and U266 were about 80%, 60%, and 80%, respectively, which were significantly higher than those against BCMA negative cells; and the cytotoxicity was related to the BCMA expression level in target cells. What's more, at the effector : target ratio of 20:1, the CAR-BCMA-T cells originated from MM patients were demonstrated to exhibit a killing rate of more than 95% against A549-BCMA positive cells, and produced large amount of IFN-γ. Conclusion: CAR-BCMA-T cells originated from both healthy and MM donors were successfully constructed, and they can effectively and specifically kill BCMA positive tumor cells.
引文
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[2]JUNE C H,SADELAIN M.Chimeric antigen receptor therapy[J].NEngl J Med,2018,379(1):64-73.DOI:10.1056/NEJMra1706169.
[3]JUNE C H,O'CONNOR R S,KAWALEKAR O U,et al.CAR T cell immunotherapy for human cancer[J].Science,2018,359(6382):1361-1365.DOI:10.1126/science.aar6711.
[4]MCHAYLEH W,BEDI P,SEHGAL R,et al.Chimeric antigen receptor T-cells:the future is now[J].J Clin Med,2019,8(2).pii:E207.DOI:10.3390/jcm8020207.
[5]ORMHOJ M,BEDOYA F,FRIGAULT M J,et al.CARs in the lead against multiple myeloma[J].Curr Hematol Malig Rep,2017,12(2):119-125.DOI:10.1007/s11899-017-0373-2.
[6]TIAN C,YANG H,ZHU L,et al.Anti-CD138 chimeric antigen receptor-modified T cell therapy for multiple myeloma with extensive extramedullary involvement[J].Ann Hematol,2017,96(8):1407-1410.DOI:10.1007/s00277-017-3029-3
[7]DRENT E R,GROEN W,NOORT W A,et al.Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma[J].Haematologica,2016,101(5):616-625.DOI:10.3324/haematol.2015.137620.
[8]DRENT E,THEMELI M,POELS R,et al.A Rational strategy for reducing on-target off-tumor effects of CD38-chimeric antigen receptors by affinity optimization[J].Mol Ther,2017,25(8):1946-1958.DOI:10.1016/j.ymthe.2017.04.024.
[9]CARPENTER R O,EVBUOMWAN M O,PITTALUGA S,et al.B-cell maturation antigen is a promising target for adoptive T-cell therapy of multiple myeloma[J].Clin Cancer Res,2013,19(8):2048-2060.DOI:10.1158/1078-0432.CCR-12-2422.
[10]COQUERY C M,ERICKSON L D.Regulatory roles of the tumor necrosis factor receptor BCMA[J].Crit Rev Immunol,2012,32(4):287-305.DOI:10.1615/CritRevImmunol.v32.i4.10.
[11]TAI Y T,ANDERSON K C.Targeting B-cell maturation antigen in multiple myeloma[J].Immunotherapy,2015,7(11):1187-1199.DOI:10.2217/imt.15.77.
[12]LEE,L.D.BOUNDS,J.PATERSON,et al.Evaluation of B cell maturation antigen as a target for antibody drug conjugate mediated cytotoxicity in multiple myeloma[J].Br J Haematol,2016,174(6):911-922.DOI:10.1111/bjh.14145.
[13]GOMEZ-EERLAND R,NUIJEN B,HEEMSKERK B,et al.Manufacture of gene-modified human T-cells with a memory stem/central memory phenotype[J].Hum Gene Ther Methods,2014,25(5):277-287.DOI:10.1089/hgtb.2014.004.
[14]GARGETT T,BROWN MP.Different cytokine and stimulation conditions influence the expansion and immune phenotype of third-generation chimeric antigen receptor T cells specific for tumor antigen GD2[J].Cytotherapy,2015,17(4):487-495.DOI:10.1016/j.jcyt.2014.12.002.
[15]LI F,ZHANG T,CAO L,et al.Chimeric antigen receptor T cell based immunotherapy for cancer[J].Curr Stem Cell Res Ther,2018,13(5):327-335.DOI:10.2174/1574888X13666180420110239.
[16]CASTELLARIN M,WATANABE K,JUNE C H,et al.Driving cars to the clinic for solid tumors[J].Gene Ther,2018,25(3):165-175.DOI:10.1038/s41434-018-0007-x.
[17]BU D X,SINGH R,CHOI E E,et al.Pre-clinical validation of Bcell maturation antigen(BCMA)as a target for T cell immunotherapy of multiple myeloma[J].Oncotarget,2018,9(40):25764-25780.DOI:10.18632/oncotarget.25359.
[18]SMITH E L,STAEHR M,MASAKAYAN R,et al.Development and evaluation of an optimal human single-chain variable fragmentderived BCMA-targeted CAR T cell vector[J].Mol Ther,2018,26(6):1447-1456.DOI:10.1016/j.ymthe.2018.03.016.
[19]ZHAO Z M,CONDOMINES S J,VAN DER STEGEN C,et al.Structural design of engineered costimulation determines tumor rejection kinetics and persistence of CAR T cells[J].Cancer Cell,2015,28(4):415-428.DOI:10.1016/j.ccell.2015.09.004.
[20]LI G,BOUCHER JC,KOTANI H,et al.4-1BB enhancement of CAR T function requires NF-κB and TRAFs[J].JCI Insight,2018,3(18).pii:121322.DOI:10.1172/jci.insight.121322.
[21]ZHONG Q,ZHU Y M,ZHENG L L,et al.Chimeric antigen receptor-T cells with 4-1BB co-stimulatory domain present a superior treatment outcome than those with CD28 domain based on bioinformatics[J].Acta Haematol,2018,140(3):131-140.DOI:10.1159/000492146.