Construction of CD138-targeted chimeric antigen receptor-modified T cells and their effect in multiple myeloma therapy
承彩 国, 杨 卢, 克晶 唐, 海燕 邢, 征 田, 青 饶, 敏 王, 冬生 熊, 建祥 王
TL;DR
This study creates a new CAR-T cell targeting CD138 and shows it can effectively kill multiple myeloma cells in lab tests.
Contribution
A novel CD138-targeted CAR-T cell is developed and demonstrated to have potent anti-tumor activity against CD138+ myeloma cells.
Findings
CD138(5G2) CAR-T cells effectively kill CD138+ myeloma cells but not CD138− cells.
CAR-T cells show enhanced cytokine secretion when activated by CD138+ cells.
CD138(5G2) CAR-T cells preferentially differentiate into memory T cell subtypes.
Abstract
构建一种新型靶向CD138的嵌合抗原受体T（CAR-T）细胞，探索其抗恶性浆细胞肿瘤作用。 通过单克隆抗体制备及筛选技术，获得能稳定分泌CD138抗体的杂交瘤细胞株；将杂交瘤细胞接种至小鼠腹腔，收集腹水并纯化得到抗CD138抗体纯品，进一步检测抗体特异性及亲和力；RT-PCR扩增其可变区序列，以此作为抗原识别域构建CD138 CAR，并表达于T细胞表面，制备CD138 CAR-T；流式细胞术检测CAR-T细胞表型特征；体外杀伤及脱颗粒实验检测其抗肿瘤作用。 ①成功制备抗人CD138抗体杂交瘤细胞株，并筛选获得稳定分泌抗人CD138抗体的杂交瘤细胞株5G2。②CD138（5G2）抗体可以特异性识别CD138+细胞，与CD138蛋白亲和常数（KD）为6.011×10−9 mol/L，与CD138−细胞无明显交叉反应。③应用分子克隆技术扩增得到CD138（5G2）抗体可变区序列，成功构建CD138（5G2）CAR慢病毒载体，通过感染T细胞获得的CD138（5G2）CAR-T细胞，可以有效结合人CD138重组蛋白。④CD138（5G2）CAR-T可以有效大量扩增，表型检测发现CD138（5G2）CAR-T细胞更多的向中心记忆T及记忆干细胞方向分化，终末分化效应T细胞比例降低。⑤与靶细胞共培养48 h后，与Vector-T细胞相比，CD138（5G2）CAR-T细胞可以有效杀伤CD138+骨髓瘤细胞系H929［效靶比为1∶2，（12.92±8.02）％对（54.25±15.79）％，P<0.001］。但对CD138− K562细胞系无明显杀伤作用。⑥脱颗粒实验显示，H929细胞可以显著激活CD138（5G2）CAR-T细胞，但对Vector-T细胞无明显激活作用［（25.78±3.35）％对（6.13±1.30）％，P<0.001］。⑦与CD138+细胞共培养后CD138（5G2）CAR-T分泌细胞因子水平较Vector-T组明显升高［IL-2：（1 697.52±599.05）pg/ml对（5.07±1.17）pg/ml，P<0.001；IFN-γ：（3…
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Taxonomy
TopicsCAR-T cell therapy research