背景:嵌合抗原受体T(CAR-T)细胞疗法是具有重大临床成功的新型免疫治疗方法之一。然而,由于准备时间长,它们的应用受到限制,高成本,和人际差异。尽管通用CAR-T(U-CAR-T)细胞的制造有了显着改善,他们仍然不是一个稳定和统一的细胞银行。
方法: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,我们最初筛选健康供体并培养他们的T细胞以获得更高比例的干细胞样记忆T(TSCM)细胞,表现出强大的自我更新能力,可持续性和细胞毒性。为了减少同种异体反应,利用CRISPR/Cas9系统,通过双敲除T细胞受体(TCR)和I类人类白细胞抗原(HLA-I)基因进一步编辑T细胞.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,SDcatcher/GVoptiTag系统,产生异肽键,用于将靶向不同抗原的抗体的纯化scFvs共价连接至回收的CAR-T细胞。
结果:所产生的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)-晚期感染的细胞或消除T淋巴瘤细胞,与传统的CAR-T细胞相似的效率。
结论:综合来看,我们的策略允许CAR-T细胞的生产更加模块化,使CAR-T细胞的质量控制和药物制造更加可行。
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.