Chimeric antigen receptor-T (CAR-T) cells therapy is one of the novel immunotherapeutic approaches with significant clinical success. However, their applications are limited because of long preparation time, high cost, and interpersonal variations. Although the manufacture of universal CAR-T (U-CAR-T) cells have significantly improved, they are still not a stable and unified cell bank. Here, we tried to further improve the convenience and flexibility of U-CAR-T cells by constructing novel modular universal CAR-T (MU-CAR-T) cells. For this purpose, we initially screened healthy donors and cultured their T cells to obtain a higher proportion of stem cell-like memory T (TSCM) cells, which exhibit robust self-renewal capacity, sustainability and cytotoxicity. To reduce the alloreactivity, the T cells were further edited by double knockout of the T cell receptor (TCR) and class I human leukocyte antigen (HLA-I) genes utilizing the CRISPR/Cas9 system. The well-growing and genetically stable universal cells carrying the CAR-moiety were then stored as a stable and unified cell bank. Subsequently, the SDcatcher/GVoptiTag system, which generate an isopeptide bond, was used to covalently connect the purified scFvs of antibody targeting different antigens to the recovered CAR-T cells. The resulting CAR-T cells can perform different functions by specifically targeting various cells, such as the eradication of human immunodeficiency virus type 1 (HIV-1)-latenly-infected cells or elimination of T lymphoma cells, with similar efficiency as the traditional CAR-T cells did. Taken together, our strategy allows the production of CAR-T cells more modularization, and makes the quality control and pharmaceutic manufacture of CAR-T cells more feasible.

中文翻译：

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通过同种异体T细胞与各种抗体片段共价连接构建模块化通用嵌合抗原受体T（MU-CAR-T）细胞

嵌合抗原受体-T (CAR-T) 细胞疗法是新型免疫治疗方法之一，在临床上取得了显着的成功。然而，由于准备时间长、成本高以及人际差异，它们的应用受到限制。尽管通用CAR-T（U-CAR-T）细胞的制造已显着改善，但它们仍然不是一个稳定且统一的细胞库。在这里，我们试图通过构建新型模块化通用CAR-T（MU-CAR-T）细胞来进一步提高U-CAR-T细胞的便利性和灵活性。为此，我们首先筛选健康供体并培养他们的T细胞，以获得更高比例的类干细胞记忆T（TSCM）细胞，这些细胞表现出强大的自我更新能力、可持续性和细胞毒性。为了降低同种异体反应性，利用 CRISPR/Cas9 系统双敲除 T 细胞受体 (TCR) 和 I 类人类白细胞抗原 (HLA-I) 基因，对 T 细胞进行进一步编辑。然后将携带 CAR 部分的生长良好且遗传稳定的通用细胞储存为稳定且统一的细胞库。随后，使用产生异肽键的SDcatcher/GVoptiTag系统将针对不同抗原的纯化抗体scFv与回收的CAR-T细胞共价连接。由此产生的CAR-T细胞可以通过特异性靶向各种细胞来执行不同的功能，例如根除人类免疫缺陷病毒1型（HIV-1）潜伏感染的细胞或消除T淋巴瘤细胞，其效率与传统CAR相似-T细胞做到了。总而言之，我们的策略使CAR-T细胞的生产更加模块化，并使CAR-T细胞的质量控制和制药生产更加可行。