Section for Immunology, Radiumhospitalet, Oslo University Hospital, Oslo, Norway.
Cytotherapy. 2011 May;13(5):629-40. doi: 10.3109/14653249.2010.542461. Epub 2010 Dec 21.
T cells can be redirected to reject cancer by retroviral transduction with a chimeric antigen receptor (CAR) or by administration of a bispecific T cell engager (BiTE). We demonstrate that transfection of T cells with messenger (m) RNA coding for CAR is an alternative strategy.
We describe the pre-clinical evaluation of a method based on transient modification of expanded T cells with a CD19 CAR directed against B-cell malignancies. CAR mRNA was generated under cell-free conditions in a scalable process using recombinant RNA polymerase. Efficient and non-toxic square-wave electroporation was used to load the mRNA into the cytoplasm of T cells with no risk of insertional mutagenesis.
After transfection >80% of T cells were viable, with 94% CAR expression. Transfected T cells were cytolytic to CD19(+) targets and produced interferon (IFN)-γ in response. Killing of CD19(+) target cells was demonstrated even at day 8 with undetectable CAR expression. Increasing the concentration of mRNA resulted in higher surface CAR expression, better killing and more IFN-γ release but at the expense of increased activation-induced cell death. Finally, we demonstrated that a second transgene could be introduced by co-electroporation of CXCR4 or CCR7 with CAR to also modify chemotactic responses.
We advocate the transient redirection approach as well suited to meet safety aspects for early phase studies, prior to trials using stably transduced cells once CAR has been proven safe. The simplicity of this methodology also facilitates rapid screening of candidate targets and novel receptors in pre-clinical studies.
通过逆转录病毒转导嵌合抗原受体 (CAR) 或施用双特异性 T 细胞衔接器 (BiTE)，可以使 T 细胞重新定向以排斥癌症。我们证明，用编码 CAR 的信使 (m) RNA 转染 T 细胞是一种替代策略。
我们描述了一种基于用针对 B 细胞恶性肿瘤的 CD19 CAR 瞬时修饰扩增 T 细胞的方法的临床前评估。在无细胞条件下，使用重组 RNA 聚合酶在可扩展的过程中生成 CAR mRNA。高效且无毒的方波电穿孔用于将 mRNA 加载到 T 细胞的细胞质中，而不会有插入突变的风险。
转染后>80%的 T 细胞存活，CAR 表达率为 94%。转染的 T 细胞对 CD19(+)靶细胞具有细胞毒性，并产生干扰素 (IFN)-γ 作为响应。甚至在第 8 天，在无法检测到 CAR 表达的情况下，也证明了对 CD19(+)靶细胞的杀伤作用。增加 mRNA 的浓度会导致更高的表面 CAR 表达、更好的杀伤作用和更多的 IFN-γ 释放，但代价是增加激活诱导的细胞死亡。最后，我们证明可以通过与 CAR 共电穿孔 CXCR4 或 CCR7 将第二个转基因引入来修饰趋化反应。
我们主张短暂的重定向方法非常适合满足早期研究的安全性方面的要求，然后在使用经过稳定转导的细胞进行试验之前，证明 CAR 是安全的。这种方法的简单性还便于在临床前研究中快速筛选候选靶标和新型受体。
