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Sleeping Beauty构建对ROR1特异性识别的CAR-T,并获得多种记忆型T细胞

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发表于 2015-9-6 10:34:45 | 显示全部楼层 |阅读模式
Sleeping Beauty Transpositionof Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like OrphanReceptor-1 into Diverse Memory T Cell populations
Sleeping Beauty构建对ROR1特异性识别的CAR-T,并获得多种记忆型T细胞
T cells modified with chimeric antigen receptors (CARstargeting CD19 demonstrated clinical activity against some B-cell malignancies.However, this is often accompanied by a loss of normal CD19+ B cells andhumoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) isexpressed on sub-populations of B-cell malignancies and solid tumors, but notby healthy B cells or normal post-partum tissues. Thus, adoptive transfer of Tcells specific for ROR1 has potential to eliminate tumor cells and sparehealthy tissues. To test this hypothesis, we developed CARs targeting ROR1 inorder to generate T cells specific for malignant cells. Two Sleeping Beautytransposons were constructed with 2nd generation ROR1-specific CARs signalingthrough CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designatedROR1RCD137) and were introduced into T cells. We selected for T cellsexpressing CAR through co-culture with γ-irradiated activating and propagatingcells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numericexpansion over one month of co-culture on AaPC in presence of solubleinterleukin (IL)-2 and IL-21 occurred and resulted in a diverse memoryphenotype of CAR+ T cells as measured by non-enzymatic digital array(NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γand had specific cytotoxic activity against ROR1+ tumors. Moreover, such cellscould eliminate ROR1+ tumor xenografts, especially T cells expressingROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+T cells to specifically eliminate tumor cells while maintaining normal B-cellrepertoire.
爱康得生物编译:
靶向CD19 CAR-T被证明对某些B-细胞恶性肿瘤具有临床活性,但是,这通常伴随着正常的CD19+ B细胞和体液免疫的损失。受体酪氨酸激酶样孤儿受体1ROR1)是在B-细胞恶性肿瘤和实体瘤亚群上表达的,但不在健康B细胞或正常产后组织中表达。因此,可特异性识别ROR1T细胞具有消除肿瘤细胞和区分健康组织的潜力。为了检验这一假设,我们开发了特异性针对恶性细胞的ROR1CAR。我们利用SleepingBeauty转座子构建了两个对ROR1特异性识别的的第二代CAR分子:ROR1RCD28ROR1RCD137,并成功的构建CAR-T。我们通过将转染的细胞与表达ROR1的和共刺激因子AaPC共培养的方式,筛选表达CARsT细胞。通过NanoString和式细胞仪检测,我们发现在CAR-TAaPc在存在IL-2IL-21的环境下共培养一个月的情况下,大量扩增的CAR-T中分化出记忆型CAR-T。这种T细胞产生的γ干扰素并对ROR1+肿瘤细胞具有特异性杀伤活性。此外,这种细胞可以消除ROR1+肿瘤移植物,特别是T细胞表达的ROR1RCD137。临床试验将研究这种ROR1特异性CAR+ T细胞在维持正常B细胞数量的同时对靶细胞的特异性清除能力。。

Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor T.pdf

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