靶向CS1的第四代嵌合抗原受体T细胞治疗难治或复发性多发性骨髓瘤的初步研究

Preliminary study of the fourth-generation CAR-T cells targeting CS1 in the treatment of refractory and recurrent multiple myeloma

目的分析在第二代嵌合抗原受体(CAR)的基础上设计的同时分泌白细胞介素7(IL7)和趋化因子19(CCL19)的第四代嵌合抗原受体T细胞(CAR-T),在多发性骨髓瘤微环境中增殖、趋化、清除肿瘤细胞和持久性等方面的能力。方法通过2A自剪切肽技术构建第四代CAR载体质粒,采用第三代慢病毒包装系统制备高滴度慢病毒,通过流式细胞术检测慢病毒转导效率和CAR-T细胞亚型的变化;采用酶联免疫吸附实验(ELISA)检测CAR-T细胞中IL7和CCL19的分泌情况;手工计数法分析CAR-T细胞在培养过程中的增殖活性;Transwell迁移实验验证CAR-T细胞趋化能力;荧光素酶生物发光法检测CAR-T细胞特异性杀伤活性;小鼠异体移植模型验证CAR-T细胞体内抗骨髓瘤活性和安全性。结果流式细胞术结果显示,抗CS1 CAR-T和抗CS1-IL7-CCL19 CAR-T细胞CAR的表达率分别为(91.50±0.29)%和(46.70±0.12)%,表明CAR-T细胞构建成功。亚型分析显示,抗CS1-IL7-CCL19 CAR-T细胞中T记忆干细胞(TSCM)的比例为(67.58±0.59)%,高于抗CS1 CAR-T[(50.74±1.01)%,P=0.000 1],具有更强的免疫记忆功能,持久性更佳。抗CS1-IL7-CCL19 CAR-T细胞较对照组(MOCK-T)和抗CS1 CAR-T组能够持续分泌IL7和CCL19(P<0.000 1)。抗CS1-IL7-CCL19 CAR-T细胞在慢病毒转导后的第9天细胞数达到(22.77±0.79)×106个,高于抗CS1 CAR-T细胞[(9.40±0.79)×106个,P=0.000 1],具有更强的增殖能力。Transwell迁移实验中,抗CS1-IL7-CCL19 CAR-T细胞对于反应T细胞的趋化细胞数为(109.00±4.04)个/视野,高于对照组和抗CS1 CAR-T组[分别为(9.33±1.20)个/视野和(7.33±0.88)个/视野,均P<0.000 1],具有更强的趋化能力。杀伤实验结果显示,与对照组细胞比较,抗CS1-IL7-CCL19 CAR-T和抗CS1 CAR-T均具有特异性的杀伤效能(P<0.000 1)。动物实验表明,抗CS1-IL7-CCL19 CAR-T细胞降低了荷瘤小鼠的肿瘤负担(P<0.000 1),并延长了总生存时间(P=0.006 1)。结论第四代抗CS1-IL7-CCL19 CAR-T细胞在不影响体内外抗骨髓瘤活性的情况下,有着更强的增殖活性、趋化能力和持久性,为克服传统CAR-T细胞存活率低下、持久性差以及受肿瘤微环境抑制等缺陷提供了策略,为第四代CAR-T细胞的临床应用提供了前期实验依据。

Objective To design the fourth-generation chimeric antigen receptor-T(CAR-T)cells that secrete interleukin-7(IL7)and chemokine C legend 19(CCL19)on the basis of the second-generation CAR,and to analyze and compare the differences in proliferation,chemotaxis,tumor cell clearance and persistence in the microenvironment of multiple myeloma(MM)between them.Methods The fourth-generation CAR vector plasmid was constructed by using 2A self-cleaving peptide technology.The third-generation lentiviral packaging system was used to prepare high-titer lentivirus.Flow cytometry was used to monitor the transduction efficiency of lentivirus and the subtype changes of CAR-T cells.The enzyme-linked immunosorbent assay(ELISA)was used to quantify the IL7 and CCL19 secreted by CAR-T cells.The calculation of absolute number of CAR-T cells during culture was used to analysis cell proliferation activity.Transwell migration assay was used to verify the chemotactic ability of CAR-T cells.The specific killing activity of CAR-T cells was detected by using the luciferase bioluminescence method.The NOD-Frkdcem26Cd52I12rgem26Cd22/Nju(NOD)mouse xenograft model was used to verify the anti-myeloma activity and safety of CAR-T cells in vivo.Results Flow cytometry results showed that the stable CAR expression rates of the second-generation anti-CSl CAR-T and fourth-generation anti-CSl-IL7-CCL19 CAR-T cells were(91.50±0.29)%and(46.7±0.12)%,respectively.CAR-T cells were successfully constructed.Subtype analysis demonstrated that the ratio of stem memory T cell(TSCM)in anti-CSl-IL7-CCL19 CAR.T cells was(67.58±0.59)%,which was significantly higher than(50.74±1.01)%of anti-CSl CAR-T(P=0.0001),with more strong immune memory function and better durability.Anti-CSl-IL7-CCL19 CAR-T cells can continuously secrete IL7 and CCL19 compared to MOCK-T and anti-CSl CAR-T(P<0.0001).The number of anti-CSl-IL7-CCL19 CAR-T cells reached(22.77±0.79)x 10^(6) on the 9th day after lentivirus transduction,which was significantly higher than(9.40±0.79)x l0^(6) of anti-CSl CAR-T cells(P=0.0001),with stronger proliferation ability.The number of chemotaxis cells of anti-CSl-IL7-CCL19 CAR-T cells to reactive T cells was(109.0±4.04),which was significantly higher than(9.33±1.20)of MOCK-T(P<0.0001)and(7.33±0.88)of anti-CSl CAR-T(P<0.0001),with stronger chemotactic ability.The specific killing activity showed that both anti-CSl-IL7-CCL19 CAR-T and anti-CSl CAR-T cells had specific killing efficacies when compared with the MOCK-T cells(P<0.0001).Animal experiment indicated that anti-CSl-1L7-CCL19 CAR-T cells significantly reduced the tumor burden(P<0.0001)and extended the overall survival time(P=0.0061)of tumor-bearing mice.Conclusions The anti-CSl-IL7-CCL19 CAR-T cells designed in this study show stronger proliferative activity,chemotactic ability,and durability without affecting the anti-myeloma activity in vivo and in vivo,which provides strategies for overcoming the defects of low survival rate,poor durability and inhibition by tumor microenvironment of traditional CAR-T cells,and offers preliminary experimental basis for the clinical application of the fourth-generation CAR-T cells.