Generation of tumor antigen-specific murine CD8+ T cells with enhanced anti-tumor activity via highly efficient CRISPR/Cas9 genome editing
Generation of tumor antigen-specific murine CD8+ T cells with enhanced anti-tumor activity via highly efficient CRISPR/Cas9 genome editing
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DOI：
10.1093/intimm/dxy006
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发表时间：
2018-04
影响因子：
4.4
通讯作者：
Yasuo Ouchi;Ashwini Patil;Yusuke Tamura;H. Nishimasu;Aina Negishi;S. Paul;Naoki Takemura;T. Satoh-T
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文献类型：
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作者：
Yasuo Ouchi;Ashwini Patil;Yusuke Tamura;H. Nishimasu;Aina Negishi;S. Paul;Naoki Takemura;T. Satoh-T
免疫疗法导致了癌症新疗法的成功开发。然而，人们越来越关注非肿瘤特异性免疫反应所引起的不良反应。在这里，我们报告了一种有效的策略来产生高亲和力的肿瘤抗原特异性CTL，使用Cas9/单引导RNA(SgRNA)核糖核蛋白(RNP)递送。作为原理验证，我们选择了gp100黑色素瘤相关肿瘤抗原，并从gp100免疫的小鼠中克隆了gp100特异性的高亲和力TCR。为了能够快速分析TCR的结构，我们开发了一个用于TCR/抗原-主要组织相容性复合体(PMHC)相互作用的三维蛋白质结构建模系统。结合这些技术，我们高效地产生了gp100特异性的PD-1(-)CD8+T细胞，并证明了基因工程CD8+T细胞在体外和体内都对黑色素瘤细胞具有很高的亲和力。我们的方法提供了TCR反应的计算预测，并能够有效地产生肿瘤抗原特异性CD8+T细胞，该细胞可以中和肿瘤诱导的免疫抑制，从而产生潜在的有效癌症治疗方法。
Immunotherapies have led to the successful development of novel therapies for cancer. However, there is increasing concern regarding the adverse effects caused by non-tumor-specific immune responses. Here, we report an effective strategy to generate high-avidity tumor-antigen-specific CTLs, using Cas9/single-guide RNA (sgRNA) ribonucleoprotein (RNP) delivery. As a proof-of-principle demonstration, we selected the gp100 melanoma-associated tumor antigen, and cloned the gp100-specific high-avidity TCR from gp100-immunized mice. To enable rapid structural dissection of the TCR, we developed a 3D protein structure modeling system for the TCR/antigen-major histocompatibility complex (pMHC) interaction. Combining these technologies, we efficiently generated gp100-specific PD-1(-) CD8+ T cells, and demonstrated that the genetically engineered CD8+ T cells have high avidity against melanoma cells both in vitro and in vivo. Our methodology offers computational prediction of the TCR response, and enables efficient generation of tumor antigen-specific CD8+ T cells that can neutralize tumor-induced immune suppression leading to a potentially powerful cancer therapeutic.